TW202342536A - Multispecific anti-tcr delta variable 1 antibodies - Google Patents

Abstract

The present invention provides multispecific antibodes comprising a Fab region and an Fc region, wherein the Fab region comprises a binding site specific for an epitope of the variable delta 1 (Vδ1) chain of a γδ T cell receptor (TCR); and the Fc region comprises an EGFR binding site. The present invention also provides compositions and pharmaceutical compositions comprising such multispecific antibodies, and method of making such multispecific antibodies. The present invention also provides methods of treatment and medical uses involving the multispecific antibodies.

Description

Multispecific anti-TCR delta variable 1 antibody

The present invention relates to multispecific antibodies and fragments and variants thereof that specifically bind to the T cell receptor of γδ T cells and specifically bind to EGFR.

Attention to T cell immunotherapy for cancer has increasingly focused on the apparent ability of CD8+ and CD4+ αβ (αβ) T cell subsets to recognize cancer cells and mediate functional potential to protect the host, especially in clinically mediated treatment of PD. -1. When the inhibition is released by antagonizing the inhibitory pathways played by CTLA-4 and other receptors. However, αβ T cells are MHC restricted, which can lead to graft-versus-host disease.

γδ T cells (γδ T cells) represent a subset of T cells that express on their surface a uniquely defined γδ T cell receptor (TCR). This TCR consists of a γ (γ) chain and a δ (δ) chain. Each chain undergoes chain reconfiguration, but has a limited number of V genes compared to αβ T cells. The main TRGV gene segments encoding Vγ are TRGV2, TRGV3, TRGV4, TRGV5, TRGV8, TRGV9 and TRGV11 and the non-functional genes TRGV10, TRGV11, TRGVA and TRGVB. The most common TRDV gene segments encode Vδ1, Vδ2, and Vδ3, plus several V segments with Vδ and Vα designations (Adams et al., 296:30-40 (2015) Cell Immunol .). Human gamma delta T cells can be broadly classified based on their TCR chains, as certain gamma and delta types are more commonly (but not exclusively) found on cells in one or more tissue types. For example, most γδ T cells present in the blood express the Vδ2 TCR, usually Vγ9Vδ2, whereas it is less common among γδ T cells present in tissues, such as those in the skin, which are more frequently used Pairs with gamma chains, such as the Vδ1 TCR that often pairs with Vγ4 in the intestine.

γδ T cells play an important role in immune surveillance, recognizing malignant or transformed cells (such as cancer cells) via stress marker patterns and subsequently exerting potent and selective cytotoxicity. Therefore, γδ T cells can coordinate the immune response. Modulating these cells in situ offers the possibility of increasing immunogenicity, even in tumors with low mutational burden that have proven challenging to other immunotherapies. Tumor recognition by γδ T cells is not dependent on any single tumor antigen, so modulators of γδ T cells have potential in a range of disease indications, including hematological and solid malignancies. The recognition mechanism of γδ T cells is not limited to MHC.

The authors of WO2019147735 hypothesized that some γδ cells have tumor-promoting activity or inhibit anti-cancer immune responses mediated by αβ T cells. The authors hypothesized that γδ T cells are immunosuppressive and therefore suggested that they should be depleted, inhibited or blocked with the aid of antibodies in the context of cancer.

However, although the prevailing view is that anti-γδ antibodies will negatively regulate γδ cell function by blocking or killing these cells, a positive correlation has been found between γδ T cell infiltration and patient prognosis and/or survival.

Compared to αβ TCR receptor/ligand interactions, knowledge of vδ1 TCR receptor/ligand interactions is limited. In the absence of such understanding, antibodies recognizing the vδ1 TCR have so far been the primary exploratory tool for probing this interaction. Such tools are typically crude blocking antibodies, which demonstrate that TCR receptor/ligand interactions result in blockade, inhibition, or removal of vδ1+ cells. For example, the tool antibodies TS8.2 and TS-1 were used as anti-γδ blocking antibodies in studies, and studies have shown that these antibodies reduce the cytotoxicity of vδ1 cells. These studies, combined with others, suggest that it is implausible to use such anti-vδ1 antibodies to favorably modulate vδ1 cell cytotoxicity in an orthotopic disease setting, and that antibodies that increase rather than decrease vδ1 cytotoxicity are needed.

To utilize γδ T cells for immunotherapy, a means is needed to expand the cells in situ or to harvest the cells and expand them ex vivo before reinfusion. The latter approach, which adds exogenous interleukins, has been previously described, see for example WO2017/072367 and WO2018/212808. Methods have been described for expanding a patient's own γδ T cells using pharmacologically modified forms of but-2-enyl hydroxy-methyl pyrophosphate (HMBPP) or clinically approved aminobisphosphonates. More than 250 cancer patients have been treated with this method, and it appears to be safe, but complete remission is rarely achieved. However, there is still a need for activators proven to expand large numbers of γδ T cells.

Furthermore, there is a great need for a binding agent or activator as a pharmaceutical agent that can preferentially target or bind or recognize or specifically modulate or increase the number of Vδ1+ cells in situ.

However, although there are agents that may modulate Vδ2+ cells, including aminobisphosphonates such as Zometa® (zoledronic acid), these drugs are primarily designed to slow bone resorption. Regardless of this Vδ2+ regulation, there is a need to develop drugs specifically designed to bind, target, modulate, activate or increase the number of Vδ1+ cells. This is because, for example, repeated Vδ2+ regulation can induce a persistent and progressive depletion phenotype.

Additionally, and given the predominantly tissue-resident nature of Vδ1+ cells, an ideal agent capable of modulating Vδ1+ would also exhibit fewer undesirable "off-target" effects and rapid renal clearance. Often, these undesirable effects may manifest when working with small molecule chemicals. For example, aminobisphosphonates shown above to modulate another type of Vδ2+ cells (with a minor effect relative to their primary modulation of bone) cause nephrotoxicity, manifested by worsening of renal function and potential renal failure (e.g., Markowitz et al. (2003) Kidney Int. 64(1):281-289). Additional adverse effects listed by the European Medicines Agency for Zometa include anemia, allergic reactions, hypertension, arterial fibrosis, myalgia, general pain, malaise, increased blood urea, vomiting, joint swelling, chest pain, etc.

The in situ environment in which the vδ1+ cells themselves exist must also be considered. For example, it has previously been shown that non-hematopoietic γδ T cells present in tissue exhibit a strong proliferative response when first separated from the tissue, but only when they are not in direct cell contact with autologous fibroblasts. It was found that non-hematopoietic T cells (γδ T cells) present in tissues must be separated from non-hematopoietic cells (such as stromal cells, especially fibroblasts) in order to function. This is because direct contact of lymphocytes with stromal or epithelial cells appears to inhibit the expansion of γδ T cells present in the tissue. The observation that preactivated cells in situ exist in an alternative inhibitory state is another reason why vδ1 cells have not so far been considered as a promising therapeutic target; indeed, until the findings described here, it had not been conceived how this could be advantageously selected. These cells are regulated in situ, among which blood and tissue vδ1+ cells are usually considered "dormant", "pre-activated" or "unactivated".

Therefore, there is a need for improved agents specifically designed to target Vδ1+ cells and for the treatment of infections, autoimmune conditions, and cancer. Specifically, it is desirable to administer by specifically binding to Vδ1+ cells, targeting Vδ1+ cells, specifically activating Vδ1+ cells, specifically enhancing proliferation and/or cytotoxic activity of Vδ1+ cells, or specifically blocking activation of Vδ1+ cells. Medications to improve the signs and symptoms of disease.

A variety of bispecific and multispecific antibody formats have been developed for a variety of therapeutic uses. Bispecific and multispecific antibodies can be divided into overlapping but independent categories based on the type of biological target and mode of action. For example, such multispecific antibodies can be classified into categories such as cytotoxic effector cell redirecting agents (also known as bispecifics, T cell recruiting antibodies, bispecific T cell engaging molecules, TCEs or BiTEs) and dual immunomodulators Category of agent (DI).

TCE (T Cell Engagement Molecule) is intended to enhance the patient's immune response to tumors by targeting T cells to tumor cells, or by targeting tumor cells to T cells, and by targeting the third component of the T cell receptor complex of T cells. One epitope (usually CD3) and a second epitope (which is a cancer antigen or a cancer-associated antigen, such as a tumor-associated antigen (TAA)) act. Such antibodies co-localize to tumor cells and T cells to promote tumor cell killing. Examples of BiTEs include the CD3×CD19 bispecific antibody blinatumomab, the CD3×EpCAM bispecific antibody catumaxomab, and the CD3×HER2 bispecific antibody ertumaxomab. ). TCEs such as BiTE are usually provided in scFv format, although other forms are available. For example, BiKE is similar to BiTE but targets CD16 on NK cells rather than CD3.

T cell receptors have been described as the most intricate receptor structures of the mammalian immune system. It consists of a transmembrane multiprotein receptor complex containing T cell receptors in close proximity to multiple CD3 chains. For example, in mammals, a typical such complex contains a T cell receptor, a CD3 gamma chain, a CD3 delta chain, and two CD3 epsilon chains. These chains associate with the T cell receptor (TCR) along the ζ-chain (ζ-chain), which then combines in T lymphocytes to produce the classic activation signal. However, alternative complexes have also been reported. For example, T cell receptor complexes containing T cell receptors and zeta chain homodimers have been described. Additional co-receptors such as CD4 and CD8 can also assist TCR function.

Regardless of receptor complex composition, it is recognized that these complexes convert cell surface binding events into intracellular phosphorylation signaling cascades. These phosphorylation events culminate activation of transcription factors (such as NFAT and NFkB), causing increased expression of interleukins and effector proteins (such as granzymes and perforin).

However, while the use of such TCEs to treat cancer remains a compelling concept, to date and even after 30 years of concerted efforts to advance TCEs in early clinical development, many of these bispecific antibodies have demonstrated dismal safety profiles, Efficacy and manufacturability overview. In fact, as of January 2020, blinatumomab remains the only approved TCE that has not been subsequently withdrawn. This TCE multispecific antibody fragment binds to the T cell receptor complex on the first binding arm and the CD19 target on the second binding arm.

Bispecific antibodies that recruit T cells are discussed in Lejeune et al., 2020, Front Immunol., 11:762. However, given the potency of the CD3 antigen as a signal transducer and its ubiquity in patient T cell populations, existing bispecific antibodies in this class, especially those that recruit T cells via CD3 binding, suffer from significant off-target effect, may result in serious adverse effects. Therefore, for bispecific examples targeting CD3, such as catomosimab (now withdrawn), systemic delivery (eg intravenously) is not realistically possible. The reality is that more inclusive deliveries are more often covered, such as intraoperative, intraperitoneal, intraabdominal, etc. This limits the selectivity and use of these bispecific antibodies as pharmaceutical agents. Indeed, even for anti-CD3 antibodies with diminished efficacy (i.e., T cell complex engagement molecules that target CD3, but are not bispecific), associated toxicities make intravenous delivery challenging. For example, to limit exposure and reduce toxicity, the anti-CD3 antibody Foralumab is now most commonly considered for oral delivery (eg, to treat intestinal disease).

It is often said that much of the hindrance currently observed with such TCEs in early clinical trials is attributable to the high-affinity T-cell complex binding domains used. Furthermore, it has been proposed that because the design of these TCEs has not properly considered the low affinity of the native TCR-complex binding event, they are hampered by severe dose-limiting toxicities, resulting in an overly narrow therapeutic range. In this regard, it has been highlighted that many early TCE drug developers relied on three anti-CD3 T cell complex binding domains derived from OKT3, SP34 and UCHT1. And these original binding domains all bind with relatively high affinity, ranging from single digits to low double digits nM, which is roughly equivalent to an affinity that is approximately 1,000 times higher than the natural binding time. It has also been suggested that this can lead to extremely different (and often detrimental) effects on T cell activation compared to native binding of T cell receptor complexes. For example, TCE developers using higher-affinity OKT3-based platforms may be confused by the fact that OKT3 causes T cells to undergo apoptosis in the presence of IL-2.

For these reasons, it is apparent that lower affinity T cell complex binding is an important consideration in determining design parameters for T cell-engaged bispecific antibody therapeutics.

Another issue when designing these TCEs is the need to weaken the Fc functionality. Indeed, TCEs generally need to completely inhibit Fc-mediated effector functions in order to maximize therapeutic efficacy and minimize off-target toxicity, since the binding of Fc to Fcγ receptors (FcγR) results in activation of immune effector cells. In fact, most bispecific antibodies targeting CD3 in current clinical practice have an Fc domain with reduced binding activity to FcγR, or are bispecific fragments intentionally devoid of an Fc region. TCEs with undiminished Fc function are generally expected to induce antibody-dependent cell-mediated cytotoxicity (ADCC), thereby depleting the γδ T cell population recognized by the antibody. However and in addition, toxicity/safety complications can be avoided by attenuating such functionality, for example by engaging CD16+ or CD32+ or CD64+ immune cells or by shortening the half-life of bispecific antibodies (e.g. if smaller bispecific antibodies are used). Specific antibody fragments, such as BITE) to attenuate potentially important efficacy aspects. It is desirable that methods that reduce the interaction of FcyRs with TCE, such as using IgG formats designed to reduce this interaction, reduce Fc-mediated TCE immobilization and reduce TCR clustering due to cross-linking to immobilized TCE.

To address some, but not all, of these complexities, a number of companies, such as Xencor (Pasadena, CA), Macrogenics (Gaithersburg, MD), and Genentech (San Francisco, CA), have recently reported in their respective TCE platforms reduced T cell Binding affinity of the binding arm of the receptor complex. However, reducing the affinity of this binding may result in less effective efficacy and less selectivity with respect to TCE design and functionality. For example, it is now demonstrated that the affinity-driven distribution profile of binding domains in such TCEs in vivo is demonstrated. Specifically, TCE distributions are typically observed to be biased toward their highest affinity targets. Therefore, by reducing the affinity of the TCE binding domain to the T cell complex, the distribution is often shifted away from the T cells that are required to drive the efficacy of such TCEs. This is part of the reason why the therapeutic scope of TCE is said to be "too narrow."

Furthermore, and particularly for solid tumors, there remain significant additional barriers to immunomodulatory therapeutics. For example, current state-of-the-art approaches include bispecific antibodies targeting CD3, where the first domain binds CD3 and the second domain targets TAA. However, this often proves problematic. For example, Middelburg J et al., Overcoming Challenges for CD3-Bispecific Antibody Therapy in Solid Tumors. Cancers . 2021; 13(2):287 summarizes the part of the solid tumor space regarding this type of multispecificity, T cell engagement, and immunomodulation Some of these barriers include; i. On-target/off-tumor toxicity issues - for example, the authors emphasize that solid tumor TAA often also manifest on the tissue of healthy organs, which in turn can lead to potentially fatal immune pathology and organ failure. , as shown in preclinical mouse studies using CD3 bispecific antibodies. ii. Availability of effector cells in the tumor microenvironment (TME) - for example, it is known that CD3+ "unprocessed" T cells mainly exist in the blood and lymphatic system without any additional dendritic cell-mediated activation. iii. Quality of tumor-infiltrating lymphocytes (TILs) (assuming these could include CD3+ Tregs and/or depleted CD3+ cells) - for example, bispecific antibodies targeting CD3 could induce TIL apoptosis via activation-induced cell death death, which hinders the strong anti-tumor response.

Therefore, there is a need for improved multispecific immunomodulatory agents in which at least one binding domain binds T cells and at least a second binding domain targets TAA.

There is also a need for improved agents specifically designed to target Vδ1+ cells and for the treatment of infections, autoimmune conditions and cancer. Specifically, it is desirable to administer by specifically binding to Vδ1+ cells, targeting Vδ1+ cells, specifically activating Vδ1+ cells, specifically enhancing proliferation and/or cytotoxic activity of Vδ1+ cells, or specifically blocking activation of Vδ1+ cells. Medications to improve the signs and symptoms of disease.

The present invention relates to high-affinity antibodies ("multispecific antibodies") containing multiple antigen-binding sites, including the antigen-binding site of TCR delta variable 1 (Vδ1) and the antigen-binding site of EGFR. point. More specifically, the invention relates to multispecific antibodies comprising a Fab region and an Fc region, wherein the Fab region contains an epitope specific for the variable delta 1 (Vdelta) chain of the gamma delta T cell receptor (TCR) The binding site; and the Fc region contains a binding site specific for EGFR, wherein the EGFR binding site is provided by the following CH3 domain, where: 1. Residues 359 to 362 (EU numbering) contain EEGP (SEQ ID NO: 523), residues 384 to 386 (EU numbering) contain TYG (SEQ ID NO: 511), and residues 413 to 415 (EU numbering) Contains SYW(SEQ ID NO: 533); 2. Residues 359 to 362 (EU numbering) contain EEGP (SEQ ID NO: 523), and residues 413 to 419 (EU numbering) contain SYWRWYK (SEQ ID NO: 512); 3. Residues 358 to 361, 361.1 and 362 (EU numbering) contain LDEGGP (SEQ ID NO: 542), residues 384 to 386 (EU numbering) contain TYG (SEQ ID NO: 511), and residues 413 to 419 (EU number) contains SYWRWVK (SEQ ID NO: 543); 4. Residues 384 to 386 (EU numbering) contain TYG (SEQ ID NO: 511), and residues 413 to 419 (EU numbering) contain SYWRWYK (SEQ ID NO: 512); or 5. Residues 358 to 362 (EU numbering) contain TESGP (SEQ ID NO: 553), residues 384 to 386 (EU numbering) contain KFG (SEQ ID NO: 554), and residues 413 to 421 (EU numbering) contain SNLRWTKGH (SEQ ID NO: 555), and residue 378 is valine.

The multispecific antibodies of the invention are in a format called "mAb ² antibodies" or "mAb square antibodies", which are antibodies containing an Fc region engineered to contain an antigen-binding loop in the CH3 domain - this modified Fc The region is called "Fcab" (Fc with antigen binding). The obtained Fcab can be quickly inserted into the natural IgG antibody format to create a quadrivalent mAb ² bispecific antibody that binds to two different antigens. Therefore, the mAb ² antibody further comprises a Fab region comprising a VH-VL domain pair that provides the antigen binding site. The mAb ² molecules of the present invention include EGFR-binding Fcab and vδ1-binding Fab.

The antibodies of the invention have advantageous functional profiles. Specifically, unlike prior art anti-Vδ1 antibodies that focus on depleting Vδ1 T cells, the antibodies of the present invention can be used to activate Vδ1 T cells. Although it can cause TCR downregulation on the T cells it binds, it does not cause Vδ1 T cell depletion, but in fact it stimulates T cells and can therefore be used in therapeutic contexts that would benefit from activation of this T cell compartment. Activation of Vδ1 T cells is evident through TCR downregulation, activation markers such as CD25, Ki67, degranulation marker CD107a, NCR (natural cytotoxicity receptor) and/or changes in 4-1BB. Activation of Vδ1 T cells in turn triggers the release of inflammatory cytokines such as INFγ and TNFα, thereby promoting immune licencing. Surprisingly, antibodies with appropriately high affinity for TRDV1 caused increased Vδ1 T cell killing and, unlike antibodies targeting CD3, for example, could provide high affinity antibodies without the disadvantages associated with large-scale activation via CD3 effect. High-affinity antibodies are in turn capable of inducing strong immunostimulatory effects via tumor-infiltrating lymphocytes (TILs). This can be achieved with minimal depletion or killing of Vδ1 cells. Therefore, the multispecific antibodies of the present invention can be regarded as agonist antibodies.

The binding interface of the Fcab region contained in the multispecific antibody of the invention is smaller than the binding site of a typical monoclonal antibody. Where the Fab arms of a typical mAb are separated by a flexible hinge region, the binding sites of Fcab form a compact antibody fragment in which the two binding sites are close together. Compared with the two antigen-binding sites of typical mAbs, the two antigen-binding sites of Fcab are also spatially close to each other. Based on this smaller binding interface and the reduced flexibility of the two binding sites, unexpectedly, the multispecific antibody (including the Fcab region) of the present invention is able to bind to and inhibit EGFR with similar affinity and potency as the monoclonal antibody basis. .

According to a first aspect of the present invention, a multispecific antibody comprising a Fab region and an Fc region is provided, wherein the Fab region includes an epitope specific to the variable δ1 (Vδ1) chain of the γδ T cell receptor (TCR). Specific binding site; and the Fc region contains an EGFR binding site, where the EGFR binding site is provided by the following CH3 domain, where: a. Residues 359 to 362 (EU numbering) comprise EEGP (SEQ ID NO: 523), residues 384 to 386 (EU numbering) comprise TYG (SEQ ID NO: 511), and residues 413 to 415 (EU numbering) Contains SYW(SEQ ID NO: 533); b. Residues 359 to 362 (EU numbering) contain EEGP (SEQ ID NO: 523), and residues 413 to 419 (EU numbering) contain SYWRWYK (SEQ ID NO: 512); c. Residues 358 to 361, 361.1 and 362 (EU numbering) contain LDEGGP (SEQ ID NO: 542), residues 384 to 386 (EU numbering) contain TYG (SEQ ID NO: 511), and residues 413 to 419 (EU number) contains SYWRWVK (SEQ ID NO: 543); d. Residues 384 to 386 (EU numbering) contain TYG (SEQ ID NO: 511), and residues 413 to 419 (EU numbering) contain SYWRWYK (SEQ ID NO: 512); e. Residues 358 to 362 (EU numbering) contain TESGP (SEQ ID NO: 553), residues 384 to 386 (EU numbering) contain KFG (SEQ ID NO: 554), and residues 413 to 421 (EU numbering) contain SNLRWTKGH (SEQ ID NO: 555), and residue 378 is valine.

In a second aspect of the invention, a polynucleotide sequence encoding a multispecific antibody of the invention is provided. For example, a polynucleotide sequence encoding an anti-Vδ1 first binding domain is provided, comprising at least 70% sequence identity to SEQ ID NO: 199 to 222, 224 to 247, 249 to 259, or 261 to 271 sequence.

In a third aspect of the invention, there is provided an expression vector comprising the polynucleotide sequence of the invention. Also provided is a host cell comprising the polynucleotide sequence of the invention or the expression vector of the invention. Also provided is a method for producing any multispecific antibody or antigen-binding fragment thereof of the invention, comprising culturing a host cell of the invention in a cell culture medium.

In another aspect of the invention, there is provided a composition comprising the multispecific antibody of the invention. A pharmaceutical composition is also provided, which includes the multispecific antibody of the present invention and a pharmaceutically acceptable diluent or carrier. The compositions and pharmaceutical compositions may optionally further comprise one or more additional therapeutically active agents.

In another aspect of the invention, there is provided a kit comprising a multispecific antibody of the invention or a pharmaceutical composition of the invention, optionally including instructions for use and/or additional therapeutically active agents.

In another aspect of the invention, there is provided a method of treating a disease or condition in an individual, comprising administering to the individual a multispecific antibody of the invention or a pharmaceutical composition of the invention. Also provided is a method of modulating an immune response in an individual, comprising administering to the individual a multispecific antibody of the invention or a pharmaceutical composition of the invention. The antibody may be administered to an individual in a therapeutically effective amount.

In another aspect of the invention, there is provided a multispecific antibody of the invention or a pharmaceutical composition of the invention for use in medicine. Also provided is the use of the multispecific antibodies of the invention for the manufacture of medicaments.

The present invention provides multispecific antibodies and fragments and variants thereof that specifically bind to the T cell receptor of γδ T cells and specifically bind to EGFR.

More specifically, the present invention relates to high-affinity antibodies ("multispecific antibodies") that contain multiple antigen-binding sites, including the antigen-binding site of TCR delta variable 1 (Vδ1) and Antigen binding site of EGFR.

The multispecific antibodies of the invention are in mAb ² format and comprise an Fc region engineered to contain an antigen-binding loop in the CH3 domain - this modified Fc region is termed an "Fcab". The mAb ² antibody further comprises a Fab region comprising a VH-VL domain pair that provides the antigen binding site. The mAb ² molecules of the present invention include EGFR-binding Fcab and vδ1-binding Fab.

More specifically, the present invention relates to providing and characterizing optimized multispecific antibodies, e.g., from parental anti-Vδ1 antibodies, such as referred to herein as G04, E07, C08, B07, C05, Antibodies prepared initially from the parent antibodies of E04, F07, G06, G09, B09, G10 and E01. In particular, the present invention relates to optimized multispecific antibodies derived from G04 and E07. definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms have the meanings assigned to them below.

γδ (γδ) T cells represent a small subset of T cells that ostensibly express a unique, defined T cell receptor (TCR). This TCR consists of a γ (γ) chain and a δ (δ) chain. Each chain contains a variable (V) region, a constant (C) region, a transmembrane region and a cytoplasmic tail. The V region contains the antigen-binding site. There are two main subtypes of human γδ T cells: one mainly in peripheral blood and one mainly in non-hematopoietic tissues. Both subtypes can be defined by the delta and/or gamma types present on the cell. For example, γδ T cells predominantly in peripheral blood express the delta variable 2 chain (Vδ2). γδ T cells mainly in non-hematopoietic tissues (that is, present in tissues) mainly express the δ variable 1 chain. References to " Vδ1 T cells " or " Vδ1+ T cells " refer to γδ T cells possessing a Vδ1 chain, that is, Vδ1 ⁺ cells.

References to " delta variable 1 " may also be referred to as Vδ1 or Vd1, and the nucleotide encoding a TCR chain containing this region or the TCR protein complex containing this region may be referred to as "TRDV1." Antibodies or antigen-binding fragments thereof that interact with the Vδ1 chain of γδ TCR and also interact with EGFR are effective antibodies or antigen-binding fragments thereof that bind to Vδ1, and can be called "anti-TCR δ variable 1 antibodies or antigens thereof "binding fragment" or "anti-Vδ1 antibody or antigen-binding fragment thereof" or "anti-TRDV1 antibody or antigen-binding fragment thereof" or "anti-Vδ1×EGFR antibody or antigen-binding fragment thereof".

Other delta chains are additionally mentioned herein, such as " delta variable 2 " chains. These may be mentioned in a similar manner. For example, the delta variable 2 chain can be called Vδ2, and the nucleotide encoding the TCR chain containing this region or the TCR protein complex containing this region can be called "TRDV2." In preferred embodiments, multispecific antibodies or antigen-binding fragments thereof that interact with the Vδ1 chain of a γδ TCR do not interact with other δ chains such as Vδ2. In the present invention, the antibodies are specific for TRDV1 and do not bind to TRDV2 (SEQ ID NO: 310) or other antigens present on the γδ T cell receptor, such as TRDV3 (SEQ ID NO: 311).

" γ variable chains " are also mentioned in this article. These may be termed γ-chains or Vγ, and the nucleotides encoding TCR chains containing this region or the TCR protein complex containing this region may be termed TRGV. For example, TRGV4 refers to the Vγ4 chain. In a preferred embodiment, the multispecific antibody or antigen-binding fragment thereof that interacts with the Vδ1 chain of the γδ TCR does not interact with the γ chain such as Vγ4 (e.g., SEQ ID NO: 309). Nor does the antibody interact with the Vδ1 chain of the γδ TCR. Other domains found within the γδ TCR, such as TRDJ, TRDC, TRGJ or TRGC bind or interact.

The term " T cell receptor complex " refers to a protein complex containing the "T cell receptor" (or "TCR") found on the surface of T cells responsible for recognizing a variety of antigens. The T cell receptor complex contains the alpha and beta chains of the T cell receptor, or in the case of gamma delta T cells, the gamma and delta chains of the T cell receptor, and up to 6 additional chains or more, such as CD3δ , CD3γ, CD3ε, and CD3ζ, although the precise composition of the T cell receptor complex can vary. T cell receptor complexes mediate intracellular signaling in T cells, which can lead to T cell activation.

The term " antibody " includes any antibody protein construct comprising at least one antibody variable domain including at least one antigen binding site (ABS). Antibodies include, but are not limited to, immunoglobulins of types IgA, IgG, IgE, IgD, IgM (and subtypes thereof). The overall structure of immunoglobulin G (IgG) antibodies assembled from two identical heavy (H) chains and two identical light (L) chain polypeptides is generally recognized and highly conserved in mammals (Padlan (1994) Mol . Immunol. 31:169-217).

It is generally known that antibodies or immunoglobulins (Ig) are proteins containing four polypeptide chains: two heavy (H) chains and two (L) light chains. Each chain is divided into constant and variable domains. The heavy (H) chain variable domain is abbreviated herein as VH, and the light (L) chain variable domain is abbreviated herein as VL. These domains, domains related thereto and domains derived therefrom may be referred to herein as immunoglobulin chain variable domains. The VH and VL domains (also known as VH and VL regions) can be further divided into regions called "complementarity determining regions" (CDRs), which are interspersed with more conservative regions called "framework regions" (FRs). The framework regions and complementarity-determining regions have been precisely defined (Kabat et al. Sequences of Proteins of Immunological Interest, 5th ed. US Department of Health and Human Services , (1991) NIH Publication No. 91-3242). Alternative numbering conventions for CDR sequences also exist, such as those set forth in Chothia et al. (1989) Nature 342: 877-883 or as summarized at IMGT.org. In conventional antibodies, each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino end to the carboxyl end: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Conventional antibody tetramers of two immunoglobulin heavy chains and two immunoglobulin light chains are formed with the immunoglobulin heavy and light chains interconnected, for example, by disulfide bonds, and the heavy chains are similarly linked. Therefore, a full antibody contains two Fabs, each Fab containing a VH-VL domain pair. The heavy chain constant region includes three domains, CH1, CH2 and CH3. The light chain constant region consists of one domain, CL. The heavy chain variable domain and the light chain variable domain are the binding domains that interact with antigens. The constant region of an antibody typically mediates binding of the antibody to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

" EU Numbering " means the numbering convention used for numbering amino acid residues in antibodies. Each residue of an antibody is assigned a number to allow comparison of antibodies. In cases where the variant antibody contains residues that are not present in the wild-type antibody (eg, insertional mutations), decimals are used to indicate insertions. For example, if a residue is inserted between the residues assigned numbers 361 and 362, the new residue is given the EU number 361.1. EU numbers are alternatives to other numbering schemes such as Kabat (Kabat et al. Sequences of Proteins of Immunological Interest, 5th ed. US Department of Health and Human Services , (1991) NIH Publication No. 91-3242), Chothia (Chothia et al. (1989) Nature 342: 877-883) or as summarized at IMGT.org., as presented in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). (10.1073 /pnas.63.1.78) EU number for further cross-reference. Figure 33 shows the correspondence between EU numbering, IMGT numbering and Kabat numbering of CH3 domain residues. Figure 32 shows how the residues of the multispecific antibodies of the invention are numbered according to the EU numbering scheme.

As used herein, a " Fab region " refers to a portion of an antibody (or a construct containing such portion) that includes a VH-VL domain pair that provides an antigen-binding site (also known as a CDR-based antigen-binding site). The Fab region may further include CL and CH1 domains. A conventional antibody with two heavy chains and two light chains therefore contains two Fabs, each Fab containing a VH-VL domain pair.

As used herein, " Fc region " (fragment crystallizable region) refers to the portion of an antibody (or construct containing such portion) that includes the CH2 and CH3 domains. The Fc region is the C-terminal region of the immunoglobulin heavy chain, including wild-type sequence Fc region and modified Fc region. The Fc region is dimeric and therefore contains pairs of heavy chain constant regions, each containing a CH2 and CH3 domain.

As used herein, a " fragment " of an antibody (which may also be referred to as an "antibody fragment", "immunoglobulin fragment", "antigen-binding fragment" or "antigen-binding polypeptide") refers to one that specifically binds to a first target (also called an "antigen-binding polypeptide"). i.e., the delta variable 1 (Vδ1) chain of the gamma delta T cell receptor) or a portion (or a construct containing that portion) of an antibody to the second target (i.e., EGFR) (for example, one or more immunoglobulin chains are not full length, but specifically binds to the target molecule). Examples of binding fragments encompassed within the term antibody fragment include: (i) Fab fragments (monovalent fragments consisting of VL, VH, CL and CH1 domains); (ii) F(ab')2 fragments (composed of two hinge regions) (a bivalent fragment composed of Fab fragments linked by a disulfide bridge); (iii) Fd fragment (composed of VH and CH1 domains); (iv) Fv fragment (composed of VL and VH domains of a single arm of an antibody); (v) Single chain variable fragment scFv (consisting of VL and VH domains joined using recombinant methods by a synthetic linker that enables the manufacture of a single protein chain in which the VL and VH domains pair to form a monovalent molecule); (vi) VH (variable domain of immunoglobulin chain consisting of VH domain); (vii) VL (variable domain of immunoglobulin chain consisting of VL domain); (viii) Domain antibody (dAb, consisting of VH or VL domain) ; (ix) minibodies (consisting of a pair of scFV fragments linked via the CH3 domain); and (x) diabodies (consisting of non-covalent dimers of scFV fragments, consisting of the VH domain from an antibody via a small peptide The linker is connected to the VL domain from another antibody). (xi) Fc fragment (consisting of CH2 and CH3 domains) (xii) Fcab fragment (consisting of an Fc fragment engineered to contain an antigen-binding loop in the CH3 domain) (xiii) Antigen-binding CH3 domain of Fcab.

" Human antibody " refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Human individuals administered these human antibodies do not develop a cross-species antibody response (eg, known as a HAMA reaction - human anti-mouse antibody) against one of the primary amino acids contained in the antibodies. Such human antibodies may include amino acids not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random or site-specific mutagenesis or introduced by somatic mutations), for example, in the CDRs and in particular CDR3. residue. However, the term is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant combinatorial human antibody libraries, antibodies directed against human immunoglobulin genes Antibodies isolated from transgenic animals (e.g., mice) or prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences may also be referred to as " Recombinant human antibodies ".

Substitution of at least one amino acid residue in the framework region of a non-human immunoglobulin variable domain with the corresponding residue from a human variable domain is referred to as " humanization ." Humanization of variable domains may reduce immunogenicity in humans.

" Specificity " refers to the number of different types of antigens or antigenic determinants that a particular antibody or antigen-binding fragment thereof can bind. Antibody specificity is the ability of an antibody to recognize a specific antigen as a unique molecular entity and distinguish it from another antigen. An antibody that "specifically binds" to (or has "specificity" for) an antigen or epitope is a term well understood in the art. A molecule is said to exhibit "specific binding" if it reacts with a specific target antigen or epitope more frequently, more rapidly, for longer duration, and/or with greater affinity than with an alternative target. An antibody "specifically binds" to a target antigen or epitope if it binds to the target antigen or epitope with greater affinity, affinity, easier and/or longer duration than to other substances. .

The antibodies of the invention are multispecific antibodies. " Multispecific antibodies " are antibodies capable of binding to multiple different epitopes simultaneously or sequentially. Generally speaking, the epitopes are not on the same antigen. Therefore, multispecific antibodies have the ability to selectively bind to epitopes present on different antigens via multiple different binding domains. This is in contrast to conventional monospecific antibodies that do not have this ability. Specifically, a " monospecific antibody " specifically binds only one antigen, although it may have multiple binding sites for one antigen (for example, a fully human IgG antibody has a valency of 2, and other antibodies may have a valency of more High, but if the antibody recognizes only one antigen, it is still classified as a monospecific antibody). Therefore, the multispecific antibodies of the invention bind multiple different antigens simultaneously and/or sequentially.

In some embodiments of the invention, the antibody is a bispecific antibody. " Bispecific antibodies " are antibodies that are capable of binding to two different epitopes simultaneously and/or sequentially. Generally speaking, the epitopes are not on the same antigen. Therefore, a bispecific antibody has the ability to selectively bind to two different epitopes present on two different antigens via two different binding domains. This is in contrast to conventional monospecific antibodies that do not have this ability. Therefore, the bispecific antibodies of the invention bind two different antigens simultaneously and/or sequentially.

As used herein, " mAb ² antibody " or " mAb 2 antibody " refers to a bispecific antibody format according to which the bispecific antibody includes an Fc region engineered to contain an antigen-binding loop in the CH3 domain. This modified Fc region is called " Fcab ". The mAb ² antibody further comprises a Fab region comprising a VH-VL domain pair that provides the antigen binding site.

The CH3 domain of human IgG1 starts at residue number 341 and ends at residue number 447 (EU numbering). Residue number 447 (EU numbering) is sometimes cleaved during manufacturing, so it is not always present in the final molecule. The CH3 domain of human IgG1 (SEQ ID NO: 563) is composed of two β-sheets, which are formed by three and four β-strands respectively. Three structural loops connect these β-strands to the C-terminus of the CH3 domain. In the antibodies of the invention, these loops are modified to form the EGFR binding site. Residues 355 to 362 are defined as the loop connecting strands A and B of the CH3 domain, also known as the " AB loop ", and residues 383 to 391 are defined as the loop connecting strands C and D of the CH3 domain, also known as the " CD loop ". And residues 413 to 422 are defined as the loop connecting strands E and F of the CH3 domain, that is, the " EF loop " uses EU numbering (see Figure 32).

" Affinity " expressed by the dissociation equilibrium constant (KD) of an antigen and an antigen-binding polypeptide is a measure of the binding strength between the antigenic determinant and the antigen-binding site on the antibody (or its antigen-binding fragment): the smaller the KD value, the The stronger the binding strength between the antigenic determinant and the antigen-binding polypeptide. Alternatively, affinity can be expressed as the affinity constant (KA), which is 1/KD. Affinity can be determined by known methods, depending on the particular antigen of interest. For example, KD can be measured by surface plasmon resonance.

Any KD value less than 10 ^-6 is considered to indicate binding. The specific binding of the antibody or antigen-binding fragment thereof to the antigen or antigenic determinant can be determined in any suitable known manner, including, for example, Scatchard analysis and/or competition binding assays, such as radioimmunoassay (RIA), Enzyme immunoassays (EIA) and sandwich competition assays, equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance or spectroscopy (for example using fluorescence analysis) and different variations thereof known in the art.

" Avidity " is a measure of the strength of the binding between an antibody or its antigen-binding fragment and a related antigen. Affinity is related to both the affinity between the antigenic determinant and its antigen-binding site on the antibody and the number of relevant binding sites present on the antibody.

" Original " means the natural or original position, instead of being moved to another position. For example, in situ Vδ1+ cells in a patient refer to vδ1 cells in vivo as opposed to ex vivo or ex vivo cells.

" Human tissue V δ 1+ cells " and " hematopoietic and blood V δ 1+ cells " and " tumor-infiltrating lymphocytes (TIL) V δ 1+ cells " are respectively defined as those in human tissue or hematopoietic blood system or human tumors. Contains or originates from Vδ1+ cells. All such cell types can be identified by their (i) localization or site of origin and (ii) the expression of their Vδ1+ TCR.

A " modulating antibody " is an antibody that confers a measurable change upon contact or binding to a cell that exhibits a target for antibody binding. The measurable change includes (but is not limited to) cell cycle and/or cell number and/or cell number. Viability, and/or secretion of one or more cell surface markers, and/or one or more secreted molecules (e.g., interleukins, chemokines, leukotrienes, etc.), and/or function (such as on target cells or measurable changes in the cytotoxicity of diseased cells). A method of "modulating" a cell or population thereof is a method that triggers at least one measurable change in the cell or cells or their secretions to produce one or more "modulated cells."

An " immune response " is a measurable change in at least one cell or cell type or an endocrine pathway or an exocrine pathway of the immune system upon the addition of a modulating antibody (including but not limited to cell-mediated responses, humoral responses, interleukins reaction, chemokine reaction).

" Immune cells " are defined as cells of the immune system, including (but not limited to) CD34+ cells, B cells, CD45+ (lymphocyte common antigen) cells, α-β T cells, cytotoxic T cells, helper T cells, plasma cells, Neutrophils, monocytes, macrophages, erythrocytes, platelets, dendritic cells, phagocytes, granulocytes, innate lymphoid cells, natural killer (NK) cells and γδ T cells. Typically, immune cells are identified or grouped or clustered by means of combined cell surface molecular analysis (eg, via flow cytometry) to differentiate immune cells into subpopulations. These can then be further broken down using additional analysis. For example, CD45+ lymphocytes can be further subdivided into vδ-positive populations and vδ-negative populations.

A " model system " is a biological model or biological representation designed to facilitate understanding of how a drug, such as an antibody or antigen-binding fragment thereof, acts as an agent that ameliorates signs or symptoms of disease. Such models usually include the use of diseased cells, non-diseased cells, healthy cells, effector cells and tissues in vitro, ex vivo and in vivo, in which the efficacy of such agents is studied and compared.

A " pathological cell " exhibits a phenotype associated with a disease such as cancer, an infection such as a viral infection, or an inflammatory condition or progression of an inflammatory disease. For example, the diseased cells may be tumor cells, autoimmune tissue cells, or virus-infected cells. Therefore, these diseased cells can be defined as tumorous or virally infected or inflamed.

" Healthy cells " refer to normal cells that are not diseased. They may also be called " normal " or " non-disease " cells. Non-disease cells include non-cancerous, or non-infected, or non-inflammatory cells. These cells are often used together with relevant diseased cells to determine the diseased cell specificity conferred by an agent and/or to better understand the therapeutic index of the agent.

" Disease cell specificity " is a measure of how effectively effector cells or populations thereof (eg, Vδ1+ cell populations) distinguish and kill diseased cells, such as cancer cells, while avoiding non-diseased or healthy cells. This potential can be measured in model systems and may involve comparing the potential of such effector cells to kill or lyse non-diseased or healthy cells, and comparing the propensity of effector cells or populations of effector cells to selectively kill or lyse diseased cells. This disease cell specificity may convey the potential therapeutic index of the agent.

" Enhanced disease cell specificity " describes the phenotype of effector cells, such as Vδ1+ cells, or populations thereof, that are modulated to further increase the ability to specifically kill disease cells. This enhancement can be measured in a variety of ways, including disease cell killing specificity. The fold change or percentage increase in selectivity or selectivity.

" ADCC " or " antibody-dependent cell-mediated cytotoxicity " describes an immune response against cells coated with antibodies that bind to surface antigens on the cells. It is a cell-mediated process whereby immune effector cells (such as NK cells) recognize cell-bound antibodies, triggering degranulation and lysis of target cells. Typically, this is mediated via Fc-Fcγ interactions. The Fc region of cell-bound antibodies recruits effector cells (such as NK cells) expressing Fcγ receptors, causing effector cell degranulation and target cell death.

" Having Fc function " means that the antibody contains a functional Fc region (crystallizable fragment region), that is, an Fc region that has not been disabled by mutation or other factors. Antibodies with Fc function exhibit undiminished Fc function. Antibodies with Fc functionality may comprise human IGHC heavy chain sequences as listed by IMGT that have not been modified or engineered or constructed to reduce binding to one or more Fcγ receptors. For example, via IGHC hinge mutations or by constructing antibodies containing heavy chain constant domains that are chimeric or hybridized with IgG1/IgG2A or IgG1/IgG4 IGHC sequences.

Suitably, the antibodies of the invention or antigen-binding fragments thereof (ie, polypeptides) are isolated. An " isolated " polypeptide is a polypeptide that has been removed from its original environment. The term "isolated" may be used to mean that the antibody is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody or fragment thereof that specifically binds Vδ1 is substantially free of antibodies that specifically bind antigens other than Vδ1). . The term "isolated" may also be used to refer to an antibody wherein the isolated antibody is sufficiently pure or at least 70-80% (w/w) pure, and more preferably, at least 80-90% (w) pure when formulated as an active ingredient in a pharmaceutical composition. /w) pure, even more preferably 90-95% pure, and optimally, at least 95%, 96%, 97%, 98%, 99% or 100% (w/w) pure and therapeutically acceptable Preparations for administration.

Suitably, the polynucleotides used in the invention are isolated. An "isolated" polynucleotide is one that has been removed from its original environment. For example, a naturally occurring polynucleotide is isolated if it is separated from some or all of the coexisting species in the natural system. A polynucleotide is considered isolated if, for example, it is cloned into a vector that is not part of its natural environment or if the polynucleotide is contained within cDNA.

Antibodies or antigen-binding fragments thereof may be " functionally active variants " that also include naturally occurring allele variants, as well as mutants or any other non-naturally occurring variants. As is known in the art, an allelogenic variant is an alternative form of a (poly)peptide characterized by the substitution, deletion or addition of one or more amino acids that does not substantially alter the biological function of the polypeptide. By way of non-limiting example, these functionally active variants occur when the framework containing the CDRs is modified, when the CDRs themselves are modified, when the CDRs are grafted onto an alternative framework, or when N-terminal or C-terminal extensions are incorporated The body can still function. Furthermore, a CDR-containing binding domain can be paired with a different partner chain, such as a binding domain shared with another antibody. These binding domains can still function when shared with a so-called "common" light chain or a "common" heavy chain. Additionally, these binding domains may function in multimerization. In addition, "antibodies or antigen-binding fragments thereof" may also include functional variants in which the VH or VL or constant domain has been modified or modified to a different typical sequence (for example, as listed at IMGT.org) and still functions.

For the purpose of comparing two closely related polypeptide sequences, the " % sequence identity " between the first polypeptide sequence and the second polypeptide sequence can be determined using NCBI BLAST v2.0 using the peptide sequence benchmark setting (BLASTP ) to calculate. For the purpose of comparing two closely related polynucleotide sequences, the "% sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be determined using NCBI BLAST v2.0, using nucleotide The sequence standard setting (BLASTN) is used to calculate.

A polypeptide or polynucleotide sequence is said to be identical or " identical " to another polypeptide or polynucleotide sequence if it shares 100% sequence identity throughout its length. Sequence residues are numbered from left to right, that is, from the N-terminus to the C-terminus for polypeptides and from the 5' to 3' end for polynucleotides.

In some embodiments, the % sequence identity for any given sequence is calculated without the sequence of all 6 CDRs of the antibody. For example, an anti-Vδ1 antibody or antigen-binding fragment thereof can comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% similarity to a specified heavy chain variable region sequence. , a heavy chain variable region sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical and/or is identical to a specified light chain variable region The sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, A light chain variable region sequence that is at least 98%, at least 99% or 100% identical, in which any amino acid variation occurs only in the framework regions of the heavy chain and light chain variable region sequences. In such embodiments, the anti-Vδ1 antibody or antigen-binding fragment thereof with certain sequence identity retains the complete heavy and light chain CDR1, CDR2, and CDR3 sequences of the corresponding anti-Vδ1 antibody or antigen-binding fragment thereof. In a more specific example, although in no way limiting and merely further illustrating these embodiments of the present invention, an anti-Vδ1 antibody or an antigen-binding fragment thereof is provided, comprising: at least 90% of the amino acid sequence of SEQ ID NO: 15 % identical amino acid sequence or a VH consisting of the amino acid sequence and a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 40 or consisting of the amino acid sequence, Any amino acid variation occurs only in the framework regions of the heavy chain and light chain variable region sequences. Therefore, the antibody of this particular example further includes: VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53, and VHCDR2 comprising the amino acid sequence of SEQ ID NO: 68 VHCDR3, VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 95.

In addition, the multispecific antibodies provided herein may comprise a kappa light chain variable sequence and retain an amino acid residue that is not serine at position 74 according to the IMGT numbering system, such as non-polar and/or non-human germline The residue, for example, may comprise a leucine residue at this position. For example, although in no way limiting and only further illustrating these embodiments of the present invention, an anti-Vδ1 antibody or an antigen-binding fragment thereof is provided, which includes: an amino acid sequence that is at least 90% identical to SEQ ID NO: 15 The amino acid sequence or the VH consisting of the amino acid sequence and the amino acid sequence containing at least 90% identity with the amino acid sequence of SEQ ID NO: 40 or the VL consisting of the amino acid sequence, any of which Amino acid variation occurs only in the framework regions of the heavy and light chain variable region sequences, and wherein the antibody contains a non-human germline and/or non-polar amino acid residue at position 74 according to the IMGT numbering system A kappa light chain variable sequence (e.g., containing a leucine residue at this position). Antibodies of this specific example further include VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53, VHCDR3 comprising the amino acid sequence of SEQ ID NO: 68, VLCDR1 having the amino acid sequence of SEQ ID NO: 79, VLCDR2 having the amino acid sequence of SEQ ID NO: 80 and VLCDR3 having the amino acid sequence of SEQ ID NO: 95.

A " difference " between sequences refers to the insertion, deletion or substitution of a single amino acid residue at a position in the second sequence compared to the first sequence. Two polypeptide sequences may contain one, two, or more such amino acid differences. Otherwise, insertions, deletions, or substitutions in the second sequence that are identical to the first sequence (100% sequence identity) result in a decrease in % sequence identity. For example, if the consensus sequence is 9 amino acid residues long, then one substitution in the second sequence results in 88.9% sequence identity. If the first and second polypeptide sequences are 9 amino acid residues long and share 6 identical residues, then the first and second polypeptide sequences share greater than 66% identity (the first and second polypeptide sequences share 66.7% consistency).

Alternatively, for the purpose of comparing a first reference polypeptide sequence to a second comparative polypeptide sequence, the number of additions, substitutions and/or deletions to the first sequence to produce the second sequence can be determined. " Addition " means the addition of an amino acid residue to the sequence of the first polypeptide (including addition to either end of the first polypeptide). " Substitution " means the substitution of one amino acid residue in the sequence of the first polypeptide with a different amino acid residue. The substitution can be conservative or non-conservative. A " deletion " is the deletion of an amino acid residue in the sequence of the first polypeptide (including deletion at either terminus of the first polypeptide).

Using three-letter and one-letter codes, naturally occurring amino acids may be mentioned as follows: glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), proline (P or Pro), phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp), ion Amino acid (K or Lys), arginine (R or Arg), histidine (H or His), aspartic acid (D or Asp), glutamic acid (E or Glu), aspartic acid (N or Asn), glutamine (Q or Gln), cysteine (C or Cys), methionine (M or Met), serine (S or Ser) and threonine (T or Thr). Where the residue may be aspartic acid or asparagine, the symbol Asx or B may be used. Where the residue can be any amino acid, the symbol Xaa or X can be used. Where the residue may be glutamic acid or glutamic acid, the notation Glx or Z may be used. Unless the context dictates otherwise, references to aspartic acid include aspartate esters, and glutamic acid includes glutamate esters.

As used herein, the numbering of polypeptide sequences and the definitions of CDRs and FRs are as defined according to the EU and/or IMGT numbering systems, as indicated in the context. The " corresponding " amino acid residue between a first polypeptide sequence and a second polypeptide sequence is the affinity of the first sequence to the amine group in the second sequence according to the EU and/or IMGT numbering system as indicated in the context. The acid residues share the same position as the amino acid residues, while the amino acid residues in the second sequence differ in identity from the first sequence. If the framework and CDR lengths are the same as defined by EU or IMGT, the appropriately corresponding residues will share the same number (and letter). Alignment can be done manually or by using, for example, known computer algorithms for sequence alignment, such as NCBI BLAST v2.0 (BLASTP or BLASTN) using standard settings.

References herein to " epitope " refer to the target portion of an antibody or antigen-binding fragment thereof that specifically binds. Antigenic determinants may also be called " antigenic determinants ". When one antibody and another antibody recognize identical or spatially overlapping epitopes, the two bind to " substantially the same epitope ." A common method for determining whether two antibodies bind to identical or overlapping epitopes is a competition assay, which can be configured in a number of different formats (e.g., using radioactive or enzyme-labeled well plates, or flow cytometry on cells expressing the antigen ), using labeled antigen or labeled antibody. When one antibody recognizes the same epitope as another antibody (that is, all points of contact between the antigen and the antibody are the same), the two bind to the " same epitope ."

Epitopes found on protein targets can be defined as " linear epitopes " or " conformational epitopes ". Linear epitopes are formed by contiguous amino acid sequences in protein antigens. Conformational epitopes are formed from amino acids that are discontinuous in the protein sequence but are bound together after the protein folds into its three-dimensional structure.

As used herein, the term " vector " is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a " plastid ," which is a circular double-stranded DNA ring into which other DNA segments can be linked. Another type of vector is a viral vector, in which additional DNA segments can be ligated to the viral genome. Certain vectors are capable of spontaneous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian and yeast vectors). Other vectors (eg, non-episomal mammalian vectors), when introduced into a host cell, can integrate into the host cell's genome and then replicate together with the host genome. In addition, certain vectors are capable of directing the expression of the gene to which they are operably linked. Such vectors are referred to herein as " recombinant expression vectors " (or simply " expression vectors "). Generally speaking, expression vectors useful in recombinant DNA technology are usually in plastid form. In the present invention, since plastid is the most commonly used carrier form, "plastid" and "carrier" can be used interchangeably. However, the present invention is intended to include other forms of expression vectors that provide equivalent functionality, such as viral vectors (eg, replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), as well as phage and phagemid systems. As used herein, the term " recombinant host cell " (or simply " host cell ") is intended to refer to a cell into which a recombinant expression vector has been introduced. It is intended that such terms refer not only to the specific cells examined, but also to the progeny of such cells, such as when the progeny are used to generate cell lines or cell banks, which are then stored, provided, sold, transferred, or used to make, as appropriate, The antibody or antigen-binding fragment thereof.

References to " subject " , " patient " or " individual " refer to the individual to be treated, especially the mammalian individual. Mammalian subjects include humans, non-human primates, farm animals (such as cows), sporting animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, or mice. In some embodiments, the individual is a human. In alternative embodiments, the subject is a non-human mammal, such as a mouse.

The term " sufficient amount " means an amount sufficient to produce the desired effect. The term " therapeutically effective amount " is an amount effective to ameliorate the symptoms of a disease or condition. A therapeutically effective amount may be a " prophylactically effective amount " because prophylaxis may be considered therapy.

A disease or condition is defined as a disease or condition if the severity of the signs or symptoms, the frequency of such signs or symptoms experienced by the individual, or both (compared to an earlier time point, such as before administration of any antibody) is reduced. " improve ".

As used herein, " treating a disease or condition " means reducing the frequency and/or severity of at least one sign or symptom of a disease or condition experienced by an individual (compared to an earlier time point, such as before administration of any antibody).

As used herein, " cancer " refers to abnormal growth or division of cells. Generally speaking, cancer cells grow and/or live longer and uncoordinatedly than normal cells and their surrounding tissues. Cancer can be benign, premalignant, or malignant. Cancer occurs in a variety of cells and tissues, including the oral cavity (e.g., mouth, tongue, pharynx, etc.), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gallbladder, pancreas, etc.), respiratory system (e.g., larynx, lungs, bronchi, etc.), bones, joints, skin (e.g., basal cells, squamous cells, meningiomas, etc.), breasts, reproductive system (e.g., uterus, ovaries, prostate, testicles, etc.), urinary system (e.g., bladder, kidney, ureter, etc.), eyes, nervous system (e.g., brain, etc.), endocrine system (e.g., thyroid, etc.), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphoblastic leukemia, Chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, etc.).

As used herein, the term " about " when used herein includes up to and including 10% greater than the specified value and up to and including 10% less than the specified value, appropriately, up to and including 5% greater than the specified value and less than the specified value. Values are up to and including 5%, especially the specified value. The term "between" includes values specifying boundaries. Multispecific antibodies and antigen-binding fragments thereof

Provided herein are multispecific antibodies that are capable of specifically binding to the delta variable 1 chain (Vδ1) of the γδ T cell receptor (TCR) and are also capable of specifically binding to EGFR. The present invention relates to the use of such antibodies as medicaments for administration to an individual to be treated.

In one embodiment, the antibody or antigen-binding fragment thereof is a scFv, Fab, Fab', F(ab')2, Fv, variable domain (eg VH or VL), diabody, minibody or monoclonal antibody. In another embodiment, the antibody or antigen-binding fragment thereof is a scFv.

Multispecific antibodies of the invention can be of any class, such as IgG, IgA, IgM, IgE, IgD or isotypes thereof, and can comprise kappa or lambda light chains. In one embodiment, the antibody is an IgG antibody, such as at least one of the isotypes IgG1, IgG2, IgG3, or IgG4. In another example, the antibody can be in a format such as an IgG format that is modified to confer desired properties, such as mutating the Fc to reduce effector function, extend half-life, alter ADCC, or increase hinge stability. Such modifications are well known in the art.

In one embodiment, the antibody or antigen-binding fragment thereof is human. Thus, antibodies or antigen-binding fragments thereof may be derived from human immunoglobulin (Ig) sequences. The CDRs, framework and/or constant regions of the antibody (or antigen-binding fragment thereof) may be derived from human Ig sequences, especially human IgG sequences. The CDRs, framework and/or constant regions of human Ig sequences, especially human IgG sequences, may be substantially identical. The advantage of using human antibodies is that they have low or no immunogenicity in humans.

Antibodies or antigen-binding fragments thereof may also be chimeric, such as mouse-human antibody chimeras.

Alternatively, the antibody or antigen-binding fragment thereof is derived from a non-human species, such as mouse. Such non-human antibodies can be modified to increase their similarity to naturally occurring antibody variants in humans, so that the antibody or antigen-binding fragment thereof can be partially or fully humanized. Thus, in one embodiment, the antibody or antigen-binding fragment thereof is humanized. This article provides a summary of specific Fab areas

A summary of some of the specific antigen-binding molecules (i.e., antibodies) used in the invention is provided below, identifying the assigned SEQ ID NO. in the accompanying sequence listing. Antigen-binding variants, derivatives and fragments thereof are also provided as part of the invention. The sequences are provided in the accompanying sequence listing and figures. In the event of any deviation between the sequences in the sequence listing and the sequences in Figures 22 to 25, the sequences in the figures shall prevail. Table 1. Summary of Fab regions ( heavy chains ) derived from ADT1-4 and related SEQ ID NOs antibody VH HFR1 VHCDR1 HFR2 VHCDR2 HFR3 VHCDR3 HFR4 Parental ADT1-4 1 170 51 172 53 173 54 174 ADT1-4-105 2 170 51 172 53 173 55 174 ADT1-4-107 3 170 51 172 53 173 56 174 ADT1-4-110 4 170 51 172 53 173 57 174 ADT1-4-112 5 170 51 172 53 173 58 174 ADT1-4-117 6 170 51 172 53 173 59 174 ADT1-4-19 7 170 51 172 53 173 60 174 ADT1-4-21 8 170 52 172 53 173 61 174 ADT1-4-31 9 170 51 172 53 173 62 174 ADT1-4-139 10 170 51 172 53 173 63 174 ADT1-4-4 11 170 51 172 53 173 64 174 ADT1-4-143 12 171 51 172 53 173 65 174 ADT1-4-53 13 170 52 172 53 173 66 174 ADT1-4-173 14 170 51 172 53 173 67 174 ADT1-4-2 15 170 51 172 53 173 68 174 ADT1-4-8 16 170 51 172 53 173 69 174 ADT1-4-82 17 170 51 172 53 173 70 174 ADT1-4-83 18 170 51 172 53 173 71 174 ADT1-4-3 19 170 51 172 53 173 72 174 ADT1-4-84 20 170 51 172 53 173 73 174 ADT1-4-86 twenty one 170 52 172 53 173 74 174 ADT1-4-95 twenty two 170 51 172 53 173 75 174 ADT1-4-1 twenty three 170 51 172 53 173 76 174 ADT1-4-6 twenty four 170 51 172 53 173 77 174 ADT1-4-138 25 170 51 172 53 173 78 174 Table 2. Summary of Fab regions ( light chains ) derived from ADT1-4 and related SEQ ID NOs antibody VL LFR1 VLCDR1 LFR2 VLCDR2 LFR3 VLCDR3 LFR4 Parental ADT1-4 26 175 79 176 80 178 81 179 ADT1-4-105 27 175 79 176 80 177 82 179 ADT1-4-107 28 175 79 176 80 177 83 179 ADT1-4-110 29 175 79 176 80 177 84 180 ADT1-4-112 30 175 79 176 80 177 85 179 ADT1-4-117 31 175 79 176 80 177 86 179 ADT1-4-19 32 175 79 176 80 177 87 179 ADT1-4-21 33 175 79 176 80 177 88 179 ADT1-4-31 34 175 79 176 80 177 89 179 ADT1-4-139 35 175 79 176 80 177 90 179 ADT1-4-4 36 175 79 176 80 177 91 179 ADT1-4-143 37 175 79 176 80 177 92 181 ADT1-4-53 38 175 79 176 80 177 93 179 ADT1-4-173 39 175 79 176 80 177 94 179 ADT1-4-2 40 175 79 176 80 177 95 179 ADT1-4-8 41 175 79 176 80 177 96 179 ADT1-4-82 42 175 79 176 80 177 97 179 ADT1-4-83 43 175 79 176 80 177 98 179 ADT1-4-3 44 175 79 176 80 177 99 179 ADT1-4-84 45 175 79 176 80 177 100 179 ADT1-4-86 46 175 79 176 80 177 101 179 ADT1-4-95 47 175 79 176 80 177 102 182 ADT1-4-1 48 175 79 176 80 177 103 179 ADT1-4-6 49 175 79 176 80 177 104 179 ADT1-4-138 50 175 79 176 80 178 105 179 Table 3. Summary of Fab regions ( heavy chains ) derived from ADT1-7 and related SEQ ID NOs antibody VH HFR1 VHCDR1 HFR2 VHCDR2 HFR3 VHCDR3 HFR4 Parental ADT1-4 106 189 130 190 131 191 132 192 ADT1-7-10 107 189 130 190 131 191 133 192 ADT1-7-15 108 189 130 190 131 191 134 192 ADT1-7-17 109 189 130 190 131 191 135 192 ADT1-7-18 110 189 130 190 131 191 136 192 ADT1-7-19 111 189 130 190 131 191 137 192 ADT1-7-20 112 189 130 190 131 191 138 192 ADT1-7-22 113 189 130 190 131 191 139 192 ADT1-7-23 114 189 130 190 131 191 140 192 ADT1-7-42 115 189 130 190 131 191 141 192 ADT1-7-3 116 189 130 190 131 191 142 192 ADT1-7-61 117 189 130 190 131 191 143 192 Table 4. Summary of Fab regions ( light chains ) derived from ADT1-7 and related SEQ ID NOs antibody VL LFR1 VLCDR1 LFR2 VLCDR2 LFR3 VLCDR3 LFR4 Parental ADT1-7 118 193 144 195 145 196 146 197 ADT1-7-10 119 193 144 195 145 196 147 197 ADT1-7-15 120 193 144 195 145 196 148 197 ADT1-7-17 121 193 144 195 145 196 149 197 ADT1-7-18 122 194 144 195 145 196 150 197 ADT1-7-19 123 193 144 195 145 196 151 197 ADT1-7-20 124 193 144 195 145 196 152 197 ADT1-7-22 125 193 144 195 145 196 153 197 ADT1-7-23 126 193 144 195 145 196 154 197 ADT1-7-42 127 193 144 195 145 196 155 197 ADT1-7-3 128 193 144 195 145 196 156 197 ADT1-7-61 129 193 144 195 145 196 157 197 Table 5. Summary of other parental Fab regions and related SEQ ID NOs Parental antibody VH VL C08 273 282 B07 274 283 C05 275 284 E04 276 285 F07 277 286 G06 278 287 G09 279 288 B09 280 289 G10 281 290 E01 312 313 Fab area where ADT1-4 and ADT1-7 come from

The invention provides multispecific antibodies, wherein the Fab region is derived from the parent antibody ADT1-4 (having a heavy chain variable region sequence according to SEQ ID NO: 1 and a light chain variable region sequence according to SEQ ID NO: 26), And an antibody derived from the parent antibody ADT1-7 (having a heavy chain variable region sequence according to SEQ ID NO: 106 and a light chain variable region sequence according to SEQ ID No: 118). ADT1-4 is also referred to as G04 herein, and ADT1-4 and G04 are used interchangeably. ADT1-7 is also referred to as E07 herein, and ADT1-7 and E07 are used interchangeably.

In some embodiments, the invention provides multispecific antibodies, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence to or from the VHCDR3 and/or a light chain variable region, which includes at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence or by This amino acid sequence constitutes VLCDR3. Certain amino acid substitutions can be made to provide one or more variant antibodies as described herein.

In some embodiments, the invention provides multispecific antibodies, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: Heavy chain variable region, which contains: VHCDR1 comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, An amino acid sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or consists of: VHCDR2 comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or consisting of an amino acid sequence: and VHCDR3 comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92 similarity to a sequence selected from the group consisting of SEQ ID NOs: 55 to 78 and 133 to 143 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to or consisting of an amino acid sequence; and A light chain variable region comprising: VLCDR1 comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence or consisting of: VLCDR2 comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% similarity to a sequence selected from the group consisting of SEQ ID NO: 80 and 145 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or consisting of an amino acid sequence: and VLCDR3 comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92 similarity to a sequence selected from the group consisting of SEQ ID NOs: 82 to 105 and 147 to 157 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence or consisting of the amino acid sequence.

Certain amino acid substitutions can be made to provide one or more variable Fab regions as described herein.

In some embodiments, the invention provides a multispecific antibody, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising and selected from the group consisting of SEQ ID NOs: 2 to 25 and 107 The sequences of the group consisting of to 117 have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99% or 100% identical sequences or consisting of the sequence; and/or a light chain variable region comprising and selected from SEQ ID NOs: 27 to 50 and 119 to 129 sequences consisting of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , or consists of a sequence that is at least 97%, at least 98%, at least 99% or 100% identical. ADT1-4 source Fab area

The invention provides multispecific antibodies comprising Fab derived from the parent antibody ADT1-4 (having a heavy chain variable region sequence according to SEQ ID NO: 1 and a light chain variable region sequence according to SEQ ID NO: 26) area, for example as explained below. ADT1-4 is also referred to as G04 herein, and ADT1-4 and G04 are used interchangeably. Antibodies containing specific CDR sequences derived from ADT1-4

Multispecific antibodies provided herein include the following Fab regions having specific sequences derived from ADT1-4.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising a polypeptide selected from the group consisting of SEQ ID NOs: 55 to 78 and/or a light chain variable region, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 82 to 105 or a VHCDR3 consisting of the amino acid sequence; This amino acid sequence constitutes VLCDR3. Certain amino acid substitutions can be made to provide one or more variant antibodies as described herein.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising a polypeptide selected from the group consisting of SEQ ID NO: 55 to 77 An amino acid sequence or a VHCDR3 consisting of the amino acid sequence; and/or a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 82 to 104 or consisting of the amino acid sequence Sequence composed of VLCDR3. Certain amino acid substitutions can be made to provide one or more variant antibodies as described herein.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising a polypeptide selected from the group consisting of SEQ ID NO: 58, 60, 61, 62 , 65, 66, 68, 74, 76 and 77, or a VHCDR3 consisting of the amino acid sequence; and/or a light chain variable region, which includes a sequence selected from the group consisting of SEQ ID NO: 85 , 87, 88, 89, 92, 93, 95, 101, 103 and 104 or VLCDR3 composed of the amino acid sequence. Certain amino acid substitutions can be made to provide one or more variant antibodies as described herein.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: Heavy chain variable region, which contains: VHCDR1, which includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 51 and 52; VHCDR2, which includes or consists of the amino acid sequence of SEQ ID NO: 53; and VHCDR3, which includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 55 to 78; and A light chain variable region comprising: VLCDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 79; VLCDR2, which includes or consists of the amino acid sequence of SEQ ID NO: 80; and VLCDR3, which includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 82 to 105.

Certain amino acid substitutions can be made to provide one or more variant antibodies as described herein.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: Heavy chain variable region, which contains: VHCDR1, which includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 51 and 52; VHCDR2, which includes or consists of the amino acid sequence of SEQ ID NO: 53; and VHCDR3, which includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 55 to 77; and A light chain variable region comprising: VLCDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 79; VLCDR2, which includes or consists of the amino acid sequence of SEQ ID NO: 80; and VLCDR3, which includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 82 to 104.

Certain amino acid substitutions can be made to provide one or more variant antibodies as described herein.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: Heavy chain variable region, which contains: VHCDR1, which includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 51 and 52; VHCDR2, which includes or consists of the amino acid sequence of SEQ ID NO: 53; and VHCDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 58, 60, 61, 62, 65, 66, 68, 74, 76 and 77; and A light chain variable region comprising: VLCDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 79; VLCDR2, which includes or consists of the amino acid sequence of SEQ ID NO: 80; and VLCDR3, which includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 85, 87, 88, 89, 92, 93, 95, 101, 103 and 104.

Certain amino acid substitutions can be made to provide one or more variant antibodies as described herein.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 55 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 82 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; b) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 56 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 83 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; c) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 57 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 84 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; d) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 58 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 85 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; e) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 59 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 86 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; f) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 60 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 87 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; g) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 52, 53 and 61 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 88 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; h) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 62 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 89 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; i) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 63 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 90 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; j) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 64 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 91 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; k) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 65 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 92 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; l) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 52, 53 and 66 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 93 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; m) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 67 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 94 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; n) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 68 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 95 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; o) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 69 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 96 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; p) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 70 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 97 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; q) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 71 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 98 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; r) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 72 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 99 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; s) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 73 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 100 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; t) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 52, 53 and 74 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 101 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; u) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 75 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 102 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; v) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 76 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 103 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; w) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 77 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 104 respectively or VLCDR1, VLCDR2 and VLCDR3 consisting of the amino acid sequence; or; x) VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 78 or consisting of the amino acid sequences, and comprising the amino acid sequences of SEQ ID NO: 79, 80 and 105 respectively Or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence.

The multispecific antibody may comprise a kappa light chain variable sequence (or comprise a variable light chain derived from a kappa light chain variable sequence), wherein residue at position 74 of the kappa light chain variable sequence according to the IMGT numbering system Not serine, for example a non-human germline residue at position 74 and/or a non-polar residue, for example the residue at position 74 is a leucine residue.

Additional examples are provided below. ADT1-4-105 and its fragments and variants

Certain embodiments relate to antibody ADT1-4-105 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 55 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 55 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 82. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 55; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 82.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-105, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 55, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 82 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 55 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 82 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 55 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 82 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 55; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 82.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-105, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and optionally VHCDR3 of the amino acid sequence of SEQ ID NO: 55 containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 82 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-107 and its fragments and variants

Certain embodiments relate to antibody ADT1-4-107 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 56 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 56 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 83. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 56; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 83.

Amino acid substitutions can be made to provide variant antibodies comprising a Fab region derived from ADT1-4-107, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 56, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 83 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 56 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 with 100% identity and the amino acid sequence of SEQ ID NO: 83 includes at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 56 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 83 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 56; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 83.

Amino acid substitutions can be made to provide variant antibodies comprising a Fab region derived from ADT1-4-107, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and optionally VHCDR3 of the amino acid sequence of SEQ ID NO: 56 containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 83 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-110 and its fragments and variants

Certain embodiments relate to antibody ADT1-4-110 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 57 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 with an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 57 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 84. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 57; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 84.

Amino acid substitutions can be made to provide variant antibodies comprising a Fab region derived from ADT1-4-110, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 57, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 84 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 57 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 84 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 57 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 84 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 57; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 84.

Amino acid substitutions can be made to provide variant antibodies comprising a Fab region derived from ADT1-4-110, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vδ1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and VHCDR3 of the amino acid sequence of SEQ ID NO: 57 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 84 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-112 and its fragments and variants

Certain embodiments relate to antibody ADT1-4-112 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 58 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 58 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 85. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 58; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 85.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-112, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 58, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 85 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 58 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 85 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 58 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 85 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 58; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 85.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-112, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and optionally VHCDR3 of the amino acid sequence of SEQ ID NO: 58 containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 85 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-117 and its fragments and variants

Certain embodiments relate to antibody ADT1-4-117 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 59 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 with an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 59 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 86. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 59; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 86.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-117, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 59, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 86 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 59 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 86 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 59 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 86 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 59; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 86.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-117, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and optionally VHCDR3 of the amino acid sequence of SEQ ID NO: 59 containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 86 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-19 and its fragments and variants

Certain embodiments relate to antibody ADT1-4-19 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 60 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 60 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 87. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 60; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 87.

Amino acid substitutions can be made to provide variant antibodies comprising a Fab region derived from ADT1-4-19, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 60, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 87 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 60 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 87 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 60 contains at least 90% identity to VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 87 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 60; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 87.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-19, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and VHCDR3 of the amino acid sequence of SEQ ID NO: 60 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 87 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-21 and its fragments and variants

Certain embodiments relate to antibody ADT1-4-21 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 61 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 with an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 61 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 88. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 61; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 88.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-21, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 61, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 88 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 52 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 61 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 88 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 52, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 61 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 88 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 52, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 61; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 88.

Amino acid substitutions can be made to provide variant antibodies comprising a Fab region derived from ADT1-4-21, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 52 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 61 and VHCDR3 of the amino acid sequence of SEQ ID NO: 61 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 88 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-31 and its fragments and variants

Certain embodiments relate to antibody ADT1-4-31 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 62 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 with an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 62 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 89. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 62; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 89.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-31, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 62, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 89 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 62 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 89 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 62 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 89 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 62; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 89.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-31, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and VHCDR3 of the amino acid sequence of SEQ ID NO: 62 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 89 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-139 and its fragments and variants

Certain embodiments relate to antibody ADT1-4-139 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 63 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 63 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 90. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 63; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 90.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-139, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 63, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 90 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 63 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 90 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 63 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 90 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 63; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 90.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-139, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and VHCDR3 of the amino acid sequence of SEQ ID NO: 63 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 90 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-4 and its fragments and variants

Certain embodiments relate to the antibody ADT1-4-4 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 64 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 64 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 91. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 64; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 91.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-4, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 64, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 91 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 64 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 91 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 64 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 91 contains at least 90% identity of VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 64; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 91.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-4, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and VHCDR3 of the amino acid sequence of SEQ ID NO: 64 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 91 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-143 and its fragments and variants

Certain embodiments relate to antibody ADT1-4-143 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 65 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 65 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 92. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 65; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 92.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-143, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 65, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 92 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 65 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 92 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 65 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 92 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 65; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 92.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-143, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and VHCDR3 of the amino acid sequence of SEQ ID NO: 65 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 92 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-53 and its fragments and variants

Certain embodiments relate to the antibody ADT1-4-53 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 66 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 66 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 93. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 66; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 93.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-53, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 66, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 93 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 52 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 66 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 52, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 66 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 93 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 52, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 66; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 93.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-53, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 52 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 66 and VHCDR3 of the amino acid sequence of SEQ ID NO: 66 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 93 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-173 and its fragments and variants

Certain embodiments relate to antibody ADT1-4-173 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 67 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 67 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 94. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 67; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 94.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-173, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 67, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 94 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 67 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 94 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 67 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 94 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 67; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 94.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-173, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 67 and VHCDR3 of the amino acid sequence of SEQ ID NO: 67 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 94 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-2 and its fragments and variants

Certain embodiments relate to the antibody ADT1-4-2 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 68 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 68 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 95. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 68; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 95.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-2, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 68, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 95 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 68 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 95 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 68 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 95 contains at least 90% identity of VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 68; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 95.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-2, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and VHCDR3 of the amino acid sequence of SEQ ID NO: 68 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 95 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-8 and its fragments and variants

Certain embodiments relate to the antibody ADT1-4-8 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 69 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 69 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 96. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 69; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 96.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-8, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 69, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 96 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 69 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 96 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 69 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 96 contains at least 90% identity of VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 69; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 96.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-8, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 69 and VHCDR3 of the amino acid sequence of SEQ ID NO: 69, optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 96 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-82 and its fragments and variants

Certain embodiments relate to antibody ADT1-4-82 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 70 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 70 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 97. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 70; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 97.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-82, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 70, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 97 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 70 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 97 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 70 contains at least 90% identity to VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 97 contains at least 90% identity of VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 70; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 97.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-82, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and optionally VHCDR3 of the amino acid sequence of SEQ ID NO: 70 containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 97 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-83 and its fragments and variants

Certain embodiments relate to the antibody ADT1-4-83 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 71 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 71 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 98. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 71; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 98.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-83, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 71, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 98 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 71 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 98 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 71 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 98 contains at least 90% identity of VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 71; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 98.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-83, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 71 and VHCDR3 of the amino acid sequence of SEQ ID NO: 71 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 98 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-3 and its fragments and variants

Certain embodiments relate to the antibody ADT1-4-3 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 72 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 with an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 72 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 99. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 72; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 99.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-3, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 72, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 99 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 72 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 99 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 72 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 99 contains at least 90% identity of VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 72; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 99.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-3, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and VHCDR3 of the amino acid sequence of SEQ ID NO: 72 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 99 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-84 and its fragments and variants

Certain embodiments relate to antibody ADT1-4-84 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 73 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 with an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 73 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 100. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 73; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 100.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-84, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 73, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amine group that includes SEQ ID NO: 100 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 73 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 100 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 73 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 100 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 73; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80, and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 100.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-84, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and VHCDR3 of the amino acid sequence of SEQ ID NO: 73 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 100 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-86 and its fragments and variants

Certain embodiments relate to the antibody ADT1-4-86 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 74 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 with an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 74 A VHCDR3 having an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 101. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 74; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 101.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-86, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 74, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amine group that includes SEQ ID NO: 101 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 52 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 74 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 101 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 52, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 74 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 101 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 52, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 74; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80, and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 101.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-86, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 52, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and VHCDR3 of the amino acid sequence of SEQ ID NO: 74 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 101 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-95 and its fragments and variants

Certain embodiments relate to the antibody ADT1-4-95 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 75 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 75 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 102. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 75; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 102.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-95, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 75, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amine group that includes SEQ ID NO: 102 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 75 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 102 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 75 contains at least 90% identity to VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 102 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 75; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 102.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-95, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and VHCDR3 of the amino acid sequence of SEQ ID NO: 75 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 102 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-1 and its fragments and variants

Certain embodiments relate to the antibody ADT1-4-1 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 76 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 76 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 103. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 76; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 103.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-1, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 76, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amine group that includes SEQ ID NO: 103 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 76 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 103 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 76 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 103 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 76; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 103.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-1, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and VHCDR3 of the amino acid sequence of SEQ ID NO: 76 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 103 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-6 and its fragments and variants

Certain embodiments relate to the antibody ADT1-4-6 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 77 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 77 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 104. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 77; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 104.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-6, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 77, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amine group that includes SEQ ID NO: 104 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 77 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 104 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 77 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 104 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 77; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 104.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-6, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and VHCDR3 of the amino acid sequence of SEQ ID NO: 77 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 104 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-4-138 and its fragments and variants

Certain embodiments relate to antibody ADT1-4-138 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 78 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 with an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 78 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 105. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 78; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 105.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-4-138, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 78, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 105 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 51 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 53 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 78 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 79 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 80, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 105 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. The amino acid sequence of ID NO: 78 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. The amino acid sequence of ID NO: 105 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 53 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 78; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 80 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 105.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-4-138, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 51 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 53 and VHCDR3 of the amino acid sequence of SEQ ID NO: 78 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 79, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 80 and VLCDR3 of the amino acid sequence of SEQ ID NO: 105 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). Antibodies comprising heavy chain and / or light chain variable regions derived from ADT1-4

Provided herein are multispecific antibodies, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92 similarity to a sequence selected from the group consisting of SEQ ID NO: 2 to 25 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical sequence or consisting of the sequence; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92 % similarity to a sequence selected from the group consisting of SEQ ID NO: 27 to 50 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical sequence or consisting of the sequence.

Also provided herein are multispecific antibodies, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92 similarity to a sequence selected from the group consisting of SEQ ID NO: 2 to 24 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical sequence or consisting of the sequence; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92 % similarity to a sequence selected from the group consisting of SEQ ID NO: 27 to 49 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical sequence or consisting of the sequence.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, or at least 80% similarity to a sequence selected from the group consisting of SEQ ID NO: 5, 7, 8, 9, 12, 13, 15, 21, 23 and 24. %, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% consistency sequence of or consisting of; and/or A light chain variable region comprising at least 70%, at least 75%, or at least 80% similarity to a sequence selected from the group consisting of SEQ ID NO: 30, 32, 33, 34, 37, 38, 40, 46, 48 and 49. %, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% consistency sequence or consists of this sequence.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: A heavy chain variable region comprising or consisting of a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 2 to 25; and/or A light chain variable region comprising or consisting of a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 27 to 50.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: A heavy chain variable region comprising or consisting of a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2 to 24; and/or A light chain variable region comprising or consisting of a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 27 to 49.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: A heavy chain variable region comprising or consisting of a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 5, 7, 8, 9, 12, 13, 15, 21, 23 and 24. Sequence composition; and/or A light chain variable region comprising or consisting of a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 30, 32, 33, 34, 37, 38, 40, 46, 48 and 49 sequence composition.

Optionally, the above antibodies retain the corresponding CDR sequences such that any variability in VH and VL sequences occurs in the framework regions.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: A heavy chain variable region comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 2 to 25; and/or A light chain variable region comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 27 to 50.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: A heavy chain variable region comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 2 to 24; and/or A light chain variable region comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 27 to 49.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: A heavy chain variable region comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 5, 7, 8, 9, 12, 13, 15, 21, 23 and 24; and/or A light chain variable region comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 30, 32, 33, 34, 37, 38, 40, 46, 48 and 49.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 27 The amino acid sequence or the VL composed of the amino acid sequence; b) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 28 The amino acid sequence or the VL composed of the amino acid sequence; c) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 29 The amino acid sequence or the VL composed of the amino acid sequence; d) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 30 The amino acid sequence or the VL composed of the amino acid sequence; e) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 31 The amino acid sequence or the VL composed of the amino acid sequence; f) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 32 The amino acid sequence or the VL composed of the amino acid sequence; g) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 33 The amino acid sequence or the VL composed of the amino acid sequence; h) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 34 The amino acid sequence or the VL composed of the amino acid sequence; i) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 35 The amino acid sequence or the VL composed of the amino acid sequence; j) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 36 The amino acid sequence or the VL composed of the amino acid sequence; k) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 37 The amino acid sequence or the VL composed of the amino acid sequence; l) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 38 The amino acid sequence or the VL composed of the amino acid sequence; m) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 39 The amino acid sequence or the VL composed of the amino acid sequence; n) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 40 The amino acid sequence or the VL composed of the amino acid sequence; o) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 41 The amino acid sequence or the VL composed of the amino acid sequence; p) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 42 The amino acid sequence or the VL composed of the amino acid sequence; q) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 18 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 43 The amino acid sequence or the VL composed of the amino acid sequence; r) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 44 The amino acid sequence or the VL composed of the amino acid sequence; s) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 45 The amino acid sequence or the VL composed of the amino acid sequence; t) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 21 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 46 The amino acid sequence or the VL composed of the amino acid sequence; u) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 47 The amino acid sequence or the VL composed of the amino acid sequence; v) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 23 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 48 The amino acid sequence or the VL composed of the amino acid sequence; w) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 24 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 49 or a VL consisting of the amino acid sequence; or x) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 25 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 50 The amino acid sequence or the VL composed of the amino acid sequence.

Optionally, the above antibodies retain the corresponding CDR sequences such that any variability in VH and VL sequences occurs in the framework regions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 2 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to SEQ ID NO: 27 The amino acid sequence or the VL composed of the amino acid sequence; b) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 3 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 28 The amino acid sequence or the VL composed of the amino acid sequence; c) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 4 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 29 The amino acid sequence or the VL composed of the amino acid sequence; d) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 5 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 30 The amino acid sequence or the VL composed of the amino acid sequence; e) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 6 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 31 The amino acid sequence or the VL composed of the amino acid sequence; f) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 7 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 32 The amino acid sequence or the VL composed of the amino acid sequence; g) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 8 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 33 The amino acid sequence or the VL composed of the amino acid sequence; h) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 9 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 34 The amino acid sequence or the VL composed of the amino acid sequence; i) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 10 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 35 The amino acid sequence or the VL composed of the amino acid sequence; j) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 11 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 36 The amino acid sequence or the VL composed of the amino acid sequence; k) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 12 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 37 The amino acid sequence or the VL composed of the amino acid sequence; l) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 13 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 38 The amino acid sequence or the VL composed of the amino acid sequence; m) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 14 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 39 The amino acid sequence or the VL composed of the amino acid sequence; n) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 15 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 40 The amino acid sequence or the VL composed of the amino acid sequence; o) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 16 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 41 The amino acid sequence or the VL composed of the amino acid sequence; p) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 17 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 42 The amino acid sequence or the VL composed of the amino acid sequence; q) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 18 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 43 The amino acid sequence or the VL composed of the amino acid sequence; r) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 19 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 44 The amino acid sequence or the VL composed of the amino acid sequence; s) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 20 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 45 The amino acid sequence or the VL composed of the amino acid sequence; t) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 21 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 46 The amino acid sequence or the VL composed of the amino acid sequence; u) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 22 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 47 The amino acid sequence or the VL composed of the amino acid sequence; v) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 23 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 48 The amino acid sequence or the VL composed of the amino acid sequence; w) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 24 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 49 or a VL consisting of the amino acid sequence; or x) Contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 25 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 50 The amino acid sequence or the VL composed of the amino acid sequence.

Optionally, the above antibodies retain the corresponding CDR sequences such that any variability in VH and VL sequences occurs in the framework regions.

Optionally, the above antibodies retain the corresponding CDR sequences such that any variability in the VH and VL sequences occurs in the framework regions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a) VH comprising the amino acid sequence of SEQ ID NO: 2 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 27 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; b) VH comprising the amino acid sequence of SEQ ID NO: 3 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 28 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; c) VH comprising the amino acid sequence of SEQ ID NO: 4 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 29 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; d) VH comprising the amino acid sequence of SEQ ID NO: 5 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 30 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; e) VH comprising the amino acid sequence of SEQ ID NO: 6 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 31 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; f) VH comprising the amino acid sequence of SEQ ID NO: 7 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 32 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; g) VH comprising the amino acid sequence of SEQ ID NO: 8 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 33 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; h) VH comprising the amino acid sequence of SEQ ID NO: 9 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 34 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; i) VH comprising the amino acid sequence of SEQ ID NO: 10 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 35 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; j) VH comprising the amino acid sequence of SEQ ID NO: 11 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 36 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; k) VH comprising the amino acid sequence of SEQ ID NO: 12 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 37 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; l) VH comprising the amino acid sequence of SEQ ID NO: 13 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 38 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; m) VH comprising the amino acid sequence of SEQ ID NO: 14 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 39 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; n) VH comprising the amino acid sequence of SEQ ID NO: 15 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 40 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; o) VH comprising the amino acid sequence of SEQ ID NO: 16 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 41 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; p) VH comprising the amino acid sequence of SEQ ID NO: 17 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 42 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; q) VH comprising the amino acid sequence of SEQ ID NO: 18 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 43 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; r) VH comprising the amino acid sequence of SEQ ID NO: 19 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 44 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; s) VH comprising the amino acid sequence of SEQ ID NO: 20 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 45 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; t) VH comprising the amino acid sequence of SEQ ID NO: 21 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 46 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; u) VH comprising the amino acid sequence of SEQ ID NO: 22 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 47 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; v) VH comprising the amino acid sequence of SEQ ID NO: 23 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 48 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; w) VH comprising the amino acid sequence of SEQ ID NO: 24 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 49 or consisting of the amino acid sequence, in two possible The variable region contains up to 5, up to 4, up to 3, up to 2 or 1 amino acid substitutions, as appropriate; or x) VH comprising the amino acid sequence of SEQ ID NO: 25 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 50 or consisting of the amino acid sequence, in two possible The variable region may optionally contain up to 5, up to 4, up to 3, up to 2 or 1 amino acid substitutions.

"On both variable regions" means that, collectively, the antibody may contain up to a total of the specified number of substitutions when considering both the heavy and light chain variable regions. Amino acid substitutions may be conservative amino acid substitutions. In some embodiments, substitutions, if present, can occur anywhere in the variable region sequence. In preferred embodiments, substitutions, if present, may be limited to framework regions. Thus in some embodiments, amino acid substitutions do not occur in CDR sequences.

Optionally, the above antibodies retain the corresponding CDR sequences such that any variability in the VH and VL sequences occurs in the framework regions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a) VH comprising the amino acid sequence of SEQ ID NO: 2 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 27 or consisting of the amino acid sequence; b) VH comprising the amino acid sequence of SEQ ID NO: 3 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 28 or consisting of the amino acid sequence; c) VH comprising the amino acid sequence of SEQ ID NO: 4 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 29 or consisting of the amino acid sequence; d) VH comprising the amino acid sequence of SEQ ID NO: 5 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 30 or consisting of the amino acid sequence; e) VH comprising the amino acid sequence of SEQ ID NO: 6 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 31 or consisting of the amino acid sequence; f) VH comprising the amino acid sequence of SEQ ID NO: 7 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 32 or consisting of the amino acid sequence; g) VH comprising the amino acid sequence of SEQ ID NO: 8 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 33 or consisting of the amino acid sequence; h) VH comprising the amino acid sequence of SEQ ID NO: 9 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 34 or consisting of the amino acid sequence; i) VH comprising the amino acid sequence of SEQ ID NO: 10 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 35 or consisting of the amino acid sequence; j) VH comprising the amino acid sequence of SEQ ID NO: 11 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 36 or consisting of the amino acid sequence; k) VH comprising the amino acid sequence of SEQ ID NO: 12 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 37 or consisting of the amino acid sequence; l) VH comprising the amino acid sequence of SEQ ID NO: 13 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 38 or consisting of the amino acid sequence; m) VH comprising the amino acid sequence of SEQ ID NO: 14 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 39 or consisting of the amino acid sequence; n) VH comprising the amino acid sequence of SEQ ID NO: 15 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 40 or consisting of the amino acid sequence; o) VH comprising the amino acid sequence of SEQ ID NO: 16 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 41 or consisting of the amino acid sequence; p) VH comprising the amino acid sequence of SEQ ID NO: 17 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 42 or consisting of the amino acid sequence; q) VH comprising the amino acid sequence of SEQ ID NO: 18 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 43 or consisting of the amino acid sequence; r) VH comprising the amino acid sequence of SEQ ID NO: 19 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 44 or consisting of the amino acid sequence; s) VH comprising the amino acid sequence of SEQ ID NO: 20 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 45 or consisting of the amino acid sequence; t) VH comprising the amino acid sequence of SEQ ID NO: 21 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 46 or consisting of the amino acid sequence; u) VH comprising the amino acid sequence of SEQ ID NO: 22 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 47 or consisting of the amino acid sequence; v) VH comprising the amino acid sequence of SEQ ID NO: 23 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 48 or consisting of the amino acid sequence; w) VH comprising or consisting of the amino acid sequence of SEQ ID NO: 24 and VL comprising or consisting of the amino acid sequence of SEQ ID NO: 49; or x) VH comprising or consisting of the amino acid sequence of SEQ ID NO: 25 and VL comprising or consisting of the amino acid sequence of SEQ ID NO: 50.

Certain amino acid substitutions can be made to provide one or more variant antibodies as described herein.

In any embodiment associated with a Fab region having a defined VH and/or VL sequence (eg, any VH and/or VL sequence defined as having a certain identity and/or substitution percentage), the VH sequence is preferably not is SEQ ID NO: 1 and the VL sequence is not SEQ ID NO: 26. Other antibodies containing Fab regions derived from ADT1-4

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a) a sequence comprising GDSVSSKSX ₁ A (SEQ ID NO: 158) or an HCDR1 consisting of the sequence Sequence; b) HCDR2 sequence containing or consisting of SEQ ID NO: 53 c) Sequence containing or consisting of X ₂ WX ₃ X ₄ X ₅ X ₆ DX ₇ (SEQ ID NO: 162) HCDR3 sequence, wherein the HCDR3 sequence is not SEQ ID NO: 54; d) LCDR1 sequence containing or consisting of SEQ ID NO: 79; e) Sequence containing or consisting of SEQ ID NO: 80 LCDR2 _sequence ; and _f ) _a sequence comprising _{QQX 8} YX ₉ And each of X ₁ to X ₁₃ is a naturally occurring amino acid.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a) a sequence comprising GDSVSSKSX ₁ A (SEQ ID NO: 159) or an HCDR1 consisting of the sequence Sequence; b) The sequence of SEQ ID NO: 53 or the HCDR2 sequence consisting of the sequence c) The sequence of X ₂ WX ₃ X ₄ X ₅ X ₆ DX ₇ (SEQ ID NO: 163) or the sequence consisting of the sequence HCDR3 sequence, wherein the HCDR3 sequence is not SEQ ID NO: 54; d) LCDR1 sequence containing or consisting of SEQ ID NO: 79; e) Sequence containing or consisting of SEQ ID NO: 80 LCDR2 _sequence ; and _f ) _a sequence comprising _{QQX 8} YX ₉ Among them: X ₁ is selected from the group consisting of A and V; X ₂ is selected from the group consisting of S and T; X ₃ is selected from the group consisting of V, A and L; X ₄ is selected from the group consisting of G, E and X ₅ is selected from the group consisting of Y and N; X ₆ is selected from the group consisting of V, A and P; X ₇ is selected from the group consisting of V, Y and R; X ₈ is selected from the group consisting of X ₉ is selected from the group consisting of S and K; X ₁₀ is selected from the group consisting of T, Q, A, E and D; X ₁₁ is selected from the group consisting of P, H and D X ₁₂ is selected from the group consisting of Q, R, K, W, P, E and I; and X ₁₃ is selected from the group consisting of I, V and L. The antibody may further comprise: an HFR1 sequence comprising the sequence of SEQ ID NO: 170 or 171; an HFR2 sequence comprising the sequence of SEQ ID NO: 172; an HFR3 sequence comprising the sequence of SEQ ID NO: 173; The HFR4 sequence of the sequence; the LFR1 sequence comprising the sequence of SEQ ID NO: 175; the LFR2 sequence comprising the sequence of SEQ ID NO: 176; the LFR3 sequence comprising the sequence of SEQ ID NO: 177 or 178; and the LFR3 sequence comprising the sequence of SEQ ID NO: 179 , 180, 181 or 182 sequence of LFR4 sequence.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a) a sequence comprising GDSVSSKSX ₁ A (SEQ ID NO: 160) or an HCDR1 consisting of the sequence Sequence; b) HCDR2 sequence containing or consisting of SEQ ID NO: 53 c) Sequence containing or consisting of X ₂ WX ₃ X ₄ X ₅ X ₆ DX ₇ (SEQ ID NO: 164) HCDR3 sequence, wherein the HCDR3 sequence is not SEQ ID NO: 54; d) LCDR1 sequence containing or consisting of SEQ ID NO: 79; e) Sequence containing or consisting of SEQ ID NO: 80 LCDR2 _sequence ; and _f ) _a sequence comprising _{QQX 8} YX ₉ Among them, X ₁ is selected from the group consisting of A and V; X ₂ is selected from the group consisting of S and T; X ₃ is selected from the group consisting of V, A and L; X ₄ is selected from the group consisting of G, E and D X ₅ is selected from the group consisting of Y and N; X ₆ is selected from the group consisting of V, A and P; X ₇ is selected from the group consisting of V, Y and R; X ₈ is selected from the group K , R and G; X ₉ is selected from the group composed of S and K; X ₁₀ is selected from the group composed of T, Q, A, E and D; X ₁₁ is selected from the group composed of P and H; X ₁₂ is selected from the group consisting of Q, R, K, W, P, E, and I; and X ₁₃ is selected from the group consisting of I, V, and L. The antibody may further comprise: an HFR1 sequence comprising the sequence of SEQ ID NO: 170 or 171, an HFR2 sequence comprising the sequence of SEQ ID NO: 172, an HFR3 sequence comprising the sequence of SEQ ID NO: 173, an HFR3 sequence comprising the sequence of SEQ ID NO: 174 The HFR4 sequence of the sequence, the LFR1 sequence comprising the sequence of SEQ ID NO: 175, the LFR2 sequence comprising the sequence of SEQ ID NO: 176, the LFR3 sequence comprising the sequence of SEQ ID NO: 177 and the sequence of SEQ ID NO: 179, 180, The LFR4 sequence of sequence 181 or 182.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a) a sequence comprising GDSVSSKSX ₁ A (SEQ ID NO: 161) or an HCDR1 consisting of the sequence Sequence; b) The sequence of SEQ ID NO: 53 or the HCDR2 sequence consisting of the sequence c) The sequence of X ₂ WX ₃ X ₄ X ₅ X ₆ DX ₇ (SEQ ID NO: 165) or the sequence consisting of the sequence HCDR3 sequence, wherein the HCDR3 sequence is not SEQ ID NO: 54; d) LCDR1 sequence containing or consisting of SEQ ID NO: 79; e) Sequence containing or consisting of SEQ ID NO: 80 LCDR2 _sequence ; and _f ) _a sequence comprising _{QQX 8} YX ₉ Among them, X ₁ is selected from the group consisting of A and V; X ₂ is selected from the group consisting of S and T; X ₃ is selected from the group consisting of V, A and L; X ₄ is selected from the group consisting of G and D. Group; X ₅ is selected from the group consisting of Y and N; X ₆ is selected from the group consisting of V, A and P; X ₇ is selected from the group consisting of V, Y and R _; X ₉ is selected from the group consisting of S and K; X ₁₀ is selected from the group consisting of T, Q, A and E; X ₁₁ is selected from the group consisting of P and H; X ₁₂ is selected from the group Q , K, W, P and I; and X ₁₃ is selected from the group consisting of V and L. The antibody may further comprise: an HFR1 sequence comprising the sequence of SEQ ID NO: 170 or 171; an HFR2 sequence comprising the sequence of SEQ ID NO: 172; an HFR3 sequence comprising the sequence of SEQ ID NO: 173; The HFR4 sequence of the sequence; the LFR1 sequence comprising the sequence of SEQ ID NO: 175; the LFR2 sequence comprising the sequence of SEQ ID NO: 176; the LFR3 sequence comprising the sequence of SEQ ID NO: 177; and the LFR3 sequence comprising the sequence of SEQ ID NO: 179 or 181 The sequence of LFR4 sequence. ADT1-7 source Fab area

The present invention provides multispecific antibodies comprising a Fab region derived from the parent antibody ADT1-7 (having a heavy chain variable region sequence according to SEQ ID NO: 106 and a light chain variable region sequence according to SEQ ID No: 118) , for example as explained below. ADT1-7 is also referred to as E07 herein, and ADT1-7 and E07 are used interchangeably. Antibodies containing specific CDR sequences derived from ADT1-7

Multispecific antibodies provided herein include the following Fab regions having specific sequences derived from ADT1-7.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising a polypeptide selected from the group consisting of SEQ ID NO: 133 to 143 and/or a light chain variable region, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 147 to 157 or a VHCDR3 consisting of the amino acid sequence; This amino acid sequence constitutes VLCDR3. Certain amino acid substitutions can be made to provide one or more variant antibodies as described herein.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising a polypeptide selected from the group consisting of SEQ ID NOs: 138, 142 and 143 The amino acid sequence of the group or a VHCDR3 consisting of the amino acid sequence; and/or the light chain variable region, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 152, 156 and 157 or consisting of The VLCDR3 composed of this amino acid sequence can undergo certain amino acid substitutions to provide one or more variant antibodies as described herein.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: Heavy chain variable region, which contains: VHCDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 130; VHCDR2, which includes or consists of the amino acid sequence of SEQ ID NO: 131; and VHCDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 133 to 143; and A light chain variable region comprising: VLCDR1, which includes or consists of the amino acid sequence of SEQ ID NO: 144; VLCDR2, which includes or consists of the amino acid sequence of SEQ ID NO: 145; and VLCDR3, which includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 147 to 157.

Certain amino acid substitutions can be made to provide one or more variant antibodies as described herein.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: Heavy chain variable region, which contains: VHCDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 130; VHCDR2, which includes or consists of the amino acid sequence of SEQ ID NO: 131; and VHCDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 138, 142 and 143; and A light chain variable region comprising: VLCDR1, which includes or consists of the amino acid sequence of SEQ ID NO: 144; VLCDR2, which includes or consists of the amino acid sequence of SEQ ID NO: 145; and VLCDR3, which includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 152, 156 and 157.

Certain amino acid substitutions can be made to provide one or more variant antibodies as described herein.

Additional examples are provided below. ADT1-7-10 and its fragments and variants

Certain embodiments relate to antibody ADT1-7-10 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 133 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 133 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 147. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 133; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 147.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-10, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 133, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amine group that includes SEQ ID NO: 147 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 130 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 131 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 133 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 144 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 145, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 147 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 130, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131. The amino acid sequence of ID NO: 133 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 144, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145. The amino acid sequence of ID NO: 147 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 131 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 133; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 145 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 147.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-10, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 131 and optionally VHCDR3 of the amino acid sequence of SEQ ID NO: 133 containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 145 and VLCDR3 of the amino acid sequence of SEQ ID NO: 147 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-7-15 and its fragments and variants

Certain embodiments relate to antibody ADT1-7-15 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 134 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 134 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 148. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 134; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 148.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-15, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 134, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 148 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 130 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 131 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 134 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 144 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 145, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 148 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 130, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131. The amino acid sequence of ID NO: 134 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 144, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145. The amino acid sequence of ID NO: 148 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 131 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 134; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 145 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 148.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-7-15, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 131 and optionally VHCDR3 of the amino acid sequence of SEQ ID NO: 134 containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 145 and VLCDR3 of the amino acid sequence of SEQ ID NO: 148 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-7-17 and its fragments and variants

Certain embodiments relate to antibody ADT1-7-17 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 135 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 135 A VHCDR3 having an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 149. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 135; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 149.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-17, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 135, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 149 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 130 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 131 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 135 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 144 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 145, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 149 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 130, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131. The amino acid sequence of ID NO: 135 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 144, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145. The amino acid sequence of ID NO: 149 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 131 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 135; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 145 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 149.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-17, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 131 and optionally VHCDR3 of the amino acid sequence of SEQ ID NO: 135 containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 145 and VLCDR3 of the amino acid sequence of SEQ ID NO: 149 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-7-18 and its fragments and variants

Certain embodiments relate to antibody ADT1-7-18 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 136 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 with an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 136 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 150. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 136; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 150.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-18, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 136, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amine group that includes SEQ ID NO: 150 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 130 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 131 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 136 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 144 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 145, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 150 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 130, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131. The amino acid sequence of ID NO: 136 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 144, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145. The amino acid sequence of ID NO: 150 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 131 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 136; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 145, and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 150.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-18, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 131 and VHCDR3 of the amino acid sequence of SEQ ID NO: 136 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. VLCDR2 of the amino acid sequence of SEQ ID NO: 145 and VLCDR3 of the amino acid sequence of SEQ ID NO: 150 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-7-19 and its fragments and variants

Certain embodiments relate to antibody ADT1-7-19 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 137 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 137 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 151. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 137; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 151.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-19, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 137, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 151 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 130 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 131 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 137 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 144 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 145, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 151 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 130, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131. The amino acid sequence of ID NO: 137 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 144, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145. The amino acid sequence of ID NO: 151 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 131 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 137; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 145, and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 151.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-19, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 131 and optionally VHCDR3 of the amino acid sequence of SEQ ID NO: 137 containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 145 and VLCDR3 of the amino acid sequence of SEQ ID NO: 151 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-7-20 and its fragments and variants

Certain embodiments relate to antibody ADT1-7-20 and fragments and variants thereof.

For example, in some embodiments, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group comprising SEQ ID NO: 138 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 138 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 152. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 138; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 152.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-20, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 138, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amine group that includes SEQ ID NO: 152 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 130 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 131 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 138 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 144 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 145, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 152 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 130, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131. The amino acid sequence of ID NO: 138 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 144, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145. The amino acid sequence of ID NO: 152 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 131 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 138; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 145 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 152.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-20, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 131 and optionally VHCDR3 of the amino acid sequence of SEQ ID NO: 138 containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. VLCDR2 of the amino acid sequence of SEQ ID NO: 145 and VLCDR3 of the amino acid sequence of SEQ ID NO: 152 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-7-22 and its fragments and variants

Certain embodiments relate to antibody ADT1-7-22 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 139 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 139 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 153. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 139; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 153.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-22, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 139, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amine group that includes SEQ ID NO: 153 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 130 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 131 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 139 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 144 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 145, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 153 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 130, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131. The amino acid sequence of ID NO: 139 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 144, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145. The amino acid sequence of ID NO: 153 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 131 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 139; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 145, and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 153.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-22, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 131 and optionally VHCDR3 of the amino acid sequence of SEQ ID NO: 139 containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 145 and VLCDR3 of the amino acid sequence of SEQ ID NO: 153 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-7-23 and its fragments and variants

Certain embodiments relate to the antibody ADT1-7-23 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 140 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 140 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 154. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 140; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 154.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-23, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 140, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amine group that includes SEQ ID NO: 154 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 130 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 131 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 140 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 144 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 145, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 154 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 130, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131. The amino acid sequence of ID NO: 140 contains at least 90% identity to VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 144, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145. The amino acid sequence of ID NO: 154 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 131 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 140; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 145 and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 154.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-23, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 131 and optionally VHCDR3 of the amino acid sequence of SEQ ID NO: 140 containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 145 and VLCDR3 of the amino acid sequence of SEQ ID NO: 154 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-7-42 and its fragments and variants

Certain embodiments relate to antibody ADT1-7-42 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 141 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 141 A VHCDR3 having an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 155. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 141; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 155.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-42, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 141, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amino group that includes SEQ ID NO: 155 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 130 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 131 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 141 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 144 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 145, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 155 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 130, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131. The amino acid sequence of ID NO: 141 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 144, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145. The amino acid sequence of ID NO: 155 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 131 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 141; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 145, and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 155.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-42, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 131 and optionally VHCDR3 of the amino acid sequence of SEQ ID NO: 141 containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 145 and VLCDR3 of the amino acid sequence of SEQ ID NO: 155 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-7-3 and its fragments and variants

Certain embodiments relate to the antibody ADT1-7-3 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 142 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region, which includes a VHCDR3 that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 142 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 156. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 142; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 156.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-3, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 142, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amine group that includes SEQ ID NO: 156 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 130 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 131 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 142 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 144 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 145, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 156 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 130, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131. The amino acid sequence of ID NO: 142 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 144, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145. The amino acid sequence of ID NO: 156 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 131 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 142; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 145, and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 156.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-3, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 131 and VHCDR3 of the amino acid sequence of SEQ ID NO: 142 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VLCDR2 of the amino acid sequence of SEQ ID NO: 145 and VLCDR3 of the amino acid sequence of SEQ ID NO: 156 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). ADT1-7-61 and its fragments and variants

Certain embodiments relate to antibody ADT1-7-61 and fragments and variants thereof.

For example, in some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a heavy chain variable region comprising an amine group containing SEQ ID NO: 143 The acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , a VHCDR3 that has an amino acid sequence that is at least 98%, at least 99%, or 100% identical; and/or a light chain variable region that includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR3 with 100% identical amino acid sequence. In one embodiment, an antibody or an antigen-binding fragment or variant thereof is provided, comprising: a heavy chain variable region comprising an amine group having at least 90% identity with the amino acid sequence of SEQ ID NO: 143 A VHCDR3 of an acid sequence; and/or a light chain variable region, which includes a VLCDR3 comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 157. In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a heavy chain variable region comprising VHCDR3 comprising the amino acid sequence of SEQ ID NO: 143; And/or a light chain variable region comprising VLCDR3 comprising the amino acid sequence of SEQ ID NO: 157.

Amino acid substitutions can be made to provide variant antibodies with Fab regions derived from ADT1-7-61, for example to provide multispecific antibodies where the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region includes: heavy chain variable A region that includes a VHCDR3 that includes the amino acid sequence of SEQ ID NO: 143, optionally including 1 or 2 amino acid substitutions; and/or a light chain variable region that includes an amine group that includes SEQ ID NO: 157 The acid sequence of VLCDR3 contains 1 or 2 amino acid substitutions as appropriate. Amino acid substitutions may be conservative amino acid substitutions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 130 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VHCDR1, and the amino acid sequence of SEQ ID NO: 131 contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VHCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 143 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% conformance to VHCDR3; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 144 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical VLCDR1 to the amino acid sequence of SEQ ID NO: 145, including at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or VLCDR2 that is 100% identical and contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 157 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% consistent VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising a VHCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 130, a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131, and a VHCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 131. The amino acid sequence of ID NO: 143 contains at least 90% identity of VHCDR3; and/or A light chain variable region comprising a VLCDR1 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 144, a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145, and a VLCDR2 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 145. The amino acid sequence of ID NO: 157 contains at least 90% identity to VLCDR3.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: The heavy chain variable region includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 131 and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 143; and The light chain variable region includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 145, and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 157.

Amino acid substitutions can be made to provide variant antibodies with a Fab region derived from ADT1-7-61, for example to provide a multispecific antibody where the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region includes: Heavy chain variable region, which includes VHCDR1 comprising the amino acid sequence of SEQ ID NO: 130 optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. : VHCDR2 of the amino acid sequence of SEQ ID NO: 131 and VHCDR3 of the amino acid sequence of SEQ ID NO: 143 optionally containing 1 or 2 amino acid substitutions; and/or Light chain variable region, which includes VLCDR1 comprising the amino acid sequence of SEQ ID NO: 144, optionally comprising 1 or 2 amino acid substitutions, optionally comprising 1 or 2 amino acid substitutions comprising SEQ ID NO. VLCDR2 of the amino acid sequence of SEQ ID NO: 145 and VLCDR3 of the amino acid sequence of SEQ ID NO: 157 optionally containing 1 or 2 amino acid substitutions. Amino acid substitutions may be conservative amino acid substitutions.

Alternatively, the antibody may consist of a specified sequence (with or without amino acid substitutions). Antibodies comprising heavy chain and / or light chain variable regions derived from ADT1-7

Provided herein are multispecific antibodies, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92 similarity to a sequence selected from the group consisting of SEQ ID NO: 107 to 117 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical sequence or consisting of the sequence; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92 similarity to a sequence selected from the group consisting of SEQ ID NO: 119 to 129 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical sequence or consisting of the sequence.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: A heavy chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, with a sequence selected from the group consisting of SEQ ID NO: 112, 116 and 117. or consisting of a sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical; and/or A light chain variable region comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, a sequence selected from the group consisting of SEQ ID NO: 124, 128 and 129. or consists of a sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: A heavy chain variable region comprising or consisting of a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 107 to 117; and/or A light chain variable region comprising or consisting of a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 119 to 129.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: A heavy chain variable region comprising or consisting of a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 112, 116 and 117; and/or A light chain variable region comprising or consisting of a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 124, 128 and 129.

Optionally, the above antibodies retain the corresponding CDR sequences such that any variability in the VH and VL sequences occurs in the framework regions.

Multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: A heavy chain variable region comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 107 to 117; and/or A light chain variable region comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 119 to 129.

Multispecific antibodies are also provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: A heavy chain variable region comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 112, 116 and 117; and/or A light chain variable region comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 124, 128 and 129.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 107 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 119 The amino acid sequence or the VL composed of the amino acid sequence; b) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 108 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 120 The amino acid sequence or the VL composed of the amino acid sequence; c) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 109 or a VH composed of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 121 The amino acid sequence or the VL composed of the amino acid sequence; d) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 110 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 122 The amino acid sequence or the VL composed of the amino acid sequence; e) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 111 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 123 The amino acid sequence or the VL composed of the amino acid sequence; f) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 112 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 124 The amino acid sequence or the VL composed of the amino acid sequence; g) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 113 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 125 The amino acid sequence or the VL composed of the amino acid sequence; h) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 114 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 126 The amino acid sequence or the VL composed of the amino acid sequence; i) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 115 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 127 The amino acid sequence or the VL composed of the amino acid sequence; j) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 116 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 128 or a VL consisting of the amino acid sequence; or k) Contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 117 or a VH consisting of this amino acid sequence and contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 129 The amino acid sequence or the VL composed of the amino acid sequence.

Optionally, the above antibodies retain the corresponding CDR sequences such that any variability in the VH and VL sequences occurs in the framework regions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a) VH comprising the amino acid sequence of SEQ ID NO: 107 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 119 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; b) VH comprising the amino acid sequence of SEQ ID NO: 108 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 120 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; c) VH comprising the amino acid sequence of SEQ ID NO: 109 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 121 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; d) VH comprising the amino acid sequence of SEQ ID NO: 110 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 122 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; e) VH comprising the amino acid sequence of SEQ ID NO: 111 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 123 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; f) VH comprising the amino acid sequence of SEQ ID NO: 112 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 124 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; g) VH comprising the amino acid sequence of SEQ ID NO: 113 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 125 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; h) VH comprising the amino acid sequence of SEQ ID NO: 114 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 126 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; i) VH comprising the amino acid sequence of SEQ ID NO: 115 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 127 or consisting of the amino acid sequence, in two possible The variable region may contain at most 5, at most 4, at most 3, at most 2 or 1 amino acid substitutions, as appropriate; j) VH comprising the amino acid sequence of SEQ ID NO: 116 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 128 or consisting of the amino acid sequence, in two possible The variable region contains up to 5, up to 4, up to 3, up to 2 or 1 amino acid substitutions, as appropriate; or k) VH comprising the amino acid sequence of SEQ ID NO: 117 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 129 or consisting of the amino acid sequence, in two possible The variable region may optionally contain up to 5, up to 4, up to 3, up to 2 or 1 amino acid substitutions.

"On both variable regions" means that, collectively, the antibody may contain up to a total of the specified number of substitutions when considering both the heavy and light chain variable regions. Amino acid substitutions may be conservative amino acid substitutions. In some embodiments, substitutions, if present, can occur anywhere in the variable region sequence. In preferred embodiments, substitutions, if present, may be limited to framework regions. Thus in some embodiments, amino acid substitutions do not occur in CDR sequences.

Optionally, the above antibodies retain the corresponding CDR sequences such that any variability in the VH and VL sequences occurs in the framework regions.

In some embodiments, multispecific antibodies are provided, the anti-TCR delta variable 1 (anti-Vdelta 1) binding Fab region comprising: a) VH comprising the amino acid sequence of SEQ ID NO: 107 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 119 or consisting of the amino acid sequence; b) VH comprising the amino acid sequence of SEQ ID NO: 108 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 120 or consisting of the amino acid sequence; c) VH comprising the amino acid sequence of SEQ ID NO: 109 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 121 or consisting of the amino acid sequence; d) VH comprising the amino acid sequence of SEQ ID NO: 110 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 122 or consisting of the amino acid sequence; e) VH comprising the amino acid sequence of SEQ ID NO: 111 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 123 or consisting of the amino acid sequence; f) VH comprising the amino acid sequence of SEQ ID NO: 112 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 124 or consisting of the amino acid sequence; g) VH comprising the amino acid sequence of SEQ ID NO: 113 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 125 or consisting of the amino acid sequence; h) VH comprising the amino acid sequence of SEQ ID NO: 114 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 126 or consisting of the amino acid sequence; i) VH comprising the amino acid sequence of SEQ ID NO: 115 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 127 or consisting of the amino acid sequence; j) VH comprising or consisting of the amino acid sequence of SEQ ID NO: 116 and VL comprising or consisting of the amino acid sequence of SEQ ID NO: 128; or k) VH comprising or consisting of the amino acid sequence of SEQ ID NO: 117 and VL comprising or consisting of the amino acid sequence of SEQ ID NO: 129.

Certain amino acid substitutions can be made to provide one or more variant antibodies as described herein.

In any embodiment associated with a Fab region having a defined VH and/or VL sequence (eg, any VH and/or VL sequence defined as having a certain identity and/or substitution percentage), the VH sequence is preferably not is SEQ ID NO: 106 and the VL sequence is not SEQ ID NO: 118. Other antibodies containing Fab regions derived from ADT1-7

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a) a sequence comprising SEQ ID NO: 130 or an HCDR1 sequence consisting of the sequence; b) comprising _The sequence of SEQ _ID NO: 131 or the HCDR2 sequence consisting of the sequence _; c) The sequence of X ₁ is not SEQ ID NO: 132; d) contains the sequence of SEQ ID NO: 144 or the LCDR1 sequence consisting of the sequence; e) contains the sequence of SEQ ID NO: 145 or the LCDR2 sequence consisting of the sequence; and f) contains _{The sequence of QQX 5 _ _} Amino acids present.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a) a sequence comprising SEQ ID NO: 130 or an HCDR1 sequence consisting of the sequence; b) comprising The sequence of SEQ ID NO: 131 or the HCDR2 sequence consisting of the sequence; c) The sequence of X ₁ X ₂ YX ₃ X ₄ AFDI (SEQ ID NO: 184) or the HCDR3 sequence consisting of the sequence, wherein the HCDR3 sequence is not SEQ ID NO: 132; d) contains the sequence of SEQ ID NO: 144 or the LCDR1 sequence consisting of the sequence; e) contains the sequence of SEQ ID NO: 145 or the LCDR2 sequence consisting of the sequence; and f) contains _{The sequence of QQX 5 _} and V; X ₂ is selected from the group consisting of D and S; X ₃ is selected from the group consisting of N, E, D, Q and A; _{X 4} is selected from the group consisting of D and E; X 6 is selected from the group consisting of T _and S; X ₆ is selected from the group consisting of A, G, Y and S; X ₇ is selected from the group consisting of S and D; group; and X ₉ is selected from the group consisting of L and D. The antibody may further comprise: an HFR1 sequence comprising the sequence of SEQ ID NO: 189; an HFR2 sequence comprising the sequence of SEQ ID NO: 190; an HFR3 sequence comprising the sequence of SEQ ID NO: 191; HFR4 sequence; LFR1 sequence comprising the sequence of SEQ ID NO: 193 and 194; LFR2 sequence comprising the sequence of SEQ ID NO: 195; LFR3 sequence comprising the sequence of SEQ ID NO: 196; and SEQ ID NO: 197 sequence The LFR4 sequence.

In one embodiment, a multispecific antibody is provided, the anti-TCR delta variable 1 (anti-V delta 1) binding Fab region comprising: a) a sequence comprising SEQ ID NO: 130 or an HCDR1 sequence consisting of the sequence; b) comprising _The sequence of SEQ _ID NO: 131 or the HCDR2 sequence consisting of the sequence _; c) The sequence of X ₁ is not SEQ ID NO: 132; d) contains the sequence of SEQ ID NO: 144 or the LCDR1 sequence consisting of the sequence; e) contains the sequence of SEQ ID NO: 145 or the LCDR2 sequence consisting of the sequence; and f) contains _{The sequence of QQX 5 _} X ₂ is selected from the group consisting of D and S; X ₃ is selected from the group consisting of D, Q and A; X ₄ is selected from the group consisting of D and E; _{X 5 is} S; X 7 is selected from the group consisting of A and Y; X ₇ is S; X ₈ is selected from the group consisting of E and G; and X ₉ is selected from the group consisting of L and D. The antibody may further comprise: an HFR1 sequence comprising the sequence of SEQ ID NO: 189; an HFR2 sequence comprising the sequence of SEQ ID NO: 190; an HFR3 sequence comprising the sequence of SEQ ID NO: 191; HFR4 sequence; LFR1 sequence comprising the sequence of SEQ ID NO: 193; LFR2 sequence comprising the sequence of SEQ ID NO: 195; LFR3 sequence comprising the sequence of SEQ ID NO: 196; and LFR4 comprising the sequence of SEQ ID NO: 197 sequence. Antibodies with sequence substitutions

The skilled artisan is aware that a variety of amino acids have similar properties. One or more of such amino acids of a substance can often be substituted with one or more other such amino acids without eliminating the desired activity of the substance.

Thus, the amino acids glycine, alanine, valine, leucine and isoleucine can often substitute for each other (amino acids with aliphatic side chains). Of these possible substitutions, glycine and alanine are preferred for each other (because they have relatively short side chains), and valine, leucine, and isoleucine are preferred for each other (because they have relatively short side chains). It has larger aliphatic side chains that are hydrophobic). Other amino acids that often substitute for each other include: phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (with basic side chains) of amino acids); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamate (amino acids with amide side chains); and cysteamine acid and methionine (an amino acid with sulfur-containing side chains).

A "conservative" amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue of similar chemical structure and is expected to have minimal effect on the function, activity, or other biological properties of the polypeptide. Such conservative substitutions are preferably substitutions of one amino acid within the following group with another amino acid residue from the same group. group amino acid residue nonpolar aliphatic glycine alanine Valine methionine Leucine isoleucine Aromatic Phenylalanine tyrosine Tryptophan Polarity is uncharged Serine threonine cysteine proline aspartic acid Glutamine negatively charged aspartic acid glutamate Positively charged lysine Arginine Histidine

Suitably, the hydrophobic amino acid residue is a non-polar amino acid. More suitably, the hydrophobic amino acid residues are selected from V, I, L, M, F, W or C. In some embodiments, the hydrophobic amino acid residue is selected from glycine, alanine, valine, methionine, leucine, isoleucine, phenylalanine, tyrosine, or tryptamine acid.

Thus, reference to a "conservative" amino acid substitution means that one or more amino acids in an antibody sequence (e.g., in a CDR or in a VH or VL sequence) is substituted by another amino acid in the same class as indicated above. Acid substituted amino acid substitution. Conservative amino acid substitutions in the CDR regions may be preferred to minimize adverse effects on antibody function. However, conservative amino acid substitutions can also occur in framework regions. Thus, in some embodiments, any substitution in a CDR may be a conservative substitution, while a substitution in a framework region may be a naturally occurring amino acid substituted by another naturally occurring amino acid.

Amino acid deletions or insertions may also be made relative to the amino acid sequences provided for the antibodies described herein. Thus, for example, amino acids may be deleted that have no substantial effect on the activity of the polypeptide or at least do not eliminate such activity. Such deletions can be advantageous because the overall length and molecular weight of the polypeptide can be reduced while still retaining activity. This may result in a reduction in the amount of polypeptide required to achieve a particular purpose, eg, lower dosage levels.

Amino acid changes can be made relative to the sequences provided herein using any suitable technique, such as by using site-directed mutagenesis or solid state synthesis.

It will be understood that amino acid substitutions or insertions within the scope of the present invention may be made using naturally occurring or non-naturally occurring amino acids, although naturally occurring amino acids are preferred. Whether natural amino acids or synthetic amino acids are used, it is preferred that only L-amino acids are present.

Provided herein are various examples that include optional amino acid substitutions in the sequences provided. Furthermore, in one embodiment of the invention, there is provided an antibody of the invention or an antigen-binding fragment thereof, which comprises up to 10, suitably up to 5, or suitably up to 2 amino acid substitutions in the antibody-binding domain or antigen-binding domain. . For example, in one embodiment of the present invention, a Vδ1 antibody or an antigen-binding fragment thereof is provided, wherein the antibody or an antigen-binding fragment thereof comprises 6 CDR regions selected from an antibody consisting of: ADT1-4- 105. ADT1-4-107, ADT1-4-110, ADT1-4-112, ADT1-4-117, ADT1-4-19, ADT1-4-21, ADT1-4-31, ADT1-4-139, ADT1-4-4, ADT1-4-143, ADT1-4-53, ADT1-4-173, ADT1-4-2, ADT1-4-8, ADT1-4-82, ADT1-4-83, ADT1- 4-3, ADT1-4-84, ADT1-4-86, ADT1-4-95, ADT1-4-1, ADT1-4-6, ADT1-4-138, ADT1-7-10, ADT1-7- 15. ADT1-7-17, ADT1-7-18, ADT1-7-19, ADT1-7-20, ADT1-7-22, ADT1-7-23, ADT1-7-42, ADT1-7-3 and ADT1-7-61, wherein the antibody or antigen-binding fragment thereof has up to 10 amino acid substitutions in all its CDR regions, suitably up to 5 amino acid substitutions or up to 2 amino acid substitutions. In another embodiment of the present invention, an anti-Vδ1 antibody or an antigen-binding fragment thereof is provided, wherein the antibody or an antigen-binding fragment thereof comprises the VH and VL sequences of an antibody selected from the group consisting of: ADT1-4-105, ADT1-4-107, ADT1-4-110, ADT1-4-112, ADT1-4-117, ADT1-4-19, ADT1-4-21, ADT1-4-31, ADT1-4-139, ADT1- 4-4, ADT1-4-143, ADT1-4-53, ADT1-4-173, ADT1-4-2, ADT1-4-8, ADT1-4-82, ADT1-4-83, ADT1-4- 3. ADT1-4-84, ADT1-4-86, ADT1-4-95, ADT1-4-1, ADT1-4-6, ADT1-4-138, ADT1-7-10, ADT1-7-15, ADT1-7-17, ADT1-7-18, ADT1-7-19, ADT1-7-20, ADT1-7-22, ADT1-7-23, ADT1-7-42, ADT1-7-3 and ADT1- 7-61, wherein the antibody has up to 10 amino acid substitutions in its VH and VL sequences, suitably up to 5 amino acid substitutions or up to 2 amino acid substitutions. In another embodiment of the present invention, an anti-Vδ1 antibody or an antigen-binding fragment thereof is provided, wherein the antibody or an antigen-binding fragment thereof is an antibody selected from the group consisting of: ADT1-4-105, ADT1-4-107 , ADT1-4-110, ADT1-4-112, ADT1-4-117, ADT1-4-19, ADT1-4-21, ADT1-4-31, ADT1-4-139, ADT1-4-4, ADT1 -4-143, ADT1-4-53, ADT1-4-173, ADT1-4-2, ADT1-4-8, ADT1-4-82, ADT1-4-83, ADT1-4-3, ADT1-4 -84, ADT1-4-86, ADT1-4-95, ADT1-4-1, ADT1-4-6, ADT1-4-138, ADT1-7-10, ADT1-7-15, ADT1-7-17 , ADT1-7-18, ADT1-7-19, ADT1-7-20, ADT1-7-22, ADT1-7-23, ADT1-7-42, ADT1-7-3 and ADT1-7-61, among which The antibody has up to multiple amino acid substitutions, suitably up to 5 amino acid substitutions or up to 2 amino acid substitutions. Substitutions are, of course, substitutions relative to the original CDR or variable chain sequence of the starting antibody.

In some embodiments, one or more amino acids are substituted in the CDR region. In other embodiments, one or more amino acid substitutions are in the framework regions, that is, in the variable heavy and light chains, but not in the CDR regions. In other embodiments, one or more amino acid substitutions can be at any position in the variable heavy chain and/or variable light chain region. In some embodiments, amino acid substitutions do not occur in the CDR sequence.

In some embodiments, amino acid substitutions do not adversely affect the binding specificity and/or affinity of the antibody. Thus, a variant antibody may have the same (or substantially the same) or superior functional profile as the antibody from which it is derived.

In some embodiments, amino acid substitutions can be made to increase the binding affinity of the antibody for a particular antigen. For example, in embodiments of the present invention, regarding mutagenesis of serine at position 74 of the light chain (variable region), substitutions may be made to increase the cynomolgus homology of the antibody to the human antigen against which the antibody was prepared. cross-reactivity of substances. Different from other substitutions mentioned above, this substitution may preferably be a non-conservative substitution. In some embodiments, the substitution may be a serine at position 74 with a non-polar amino acid (e.g., selected from the group consisting of glycine, alanine, valine, methionine, leucine, and isoleucine). A group of amino acids composed of acids). Alternatively, serine may be substituted with a non-human germline amino acid (e.g., substituted with one selected from the group consisting of arginine, cysteine, glutamic acid, glutamic acid, glycine, histidine, isoleucine A group of amino acids composed of acid, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine). In some embodiments, serine may be derived from an amino acid that is neither human germline nor polar, that is, selected from the group consisting of glycine, valine, methionine, leucine, and isoleucine. group of amino acid substitutions. In some embodiments, serine can be substituted with leucine. epitope

Provided herein are multispecific antibodies (or antigen-binding fragments thereof) that bind to epitopes of the Vδ1 chain of a γδ TCR. Such binding may optionally have an effect on γδ TCR activity, such as activation. The multispecific antibodies of the invention are specific for the Vδ1 chain of γδ TCR and do not bind to epitopes of other antigens, such as the Vδ2 chain of γδ TCR or the Vδ3 chain of γδ TCR. The antibodies of the invention may be considered agonist antibodies, at least with respect to the agonistic effects conferred upon Vδ1 cells upon binding.

In one embodiment, the epitope may be an activating epitope of a γδ T cell. "Activating" epitopes may include, for example, stimulation of TCR functions such as cell degranulation, TCR downregulation, cytotoxicity, proliferation, motility, increased survival or resistance to depletion, intracellular signaling, interleukin or growth factor secretion, Phenotypic changes or changes in gene expression. For example, binding of an activating epitope may stimulate expansion (i.e., proliferation) of a population of γδ T cells, preferably a population of Vδ1+ T cells. Therefore, these antibodies can be used to modulate gamma delta T cell activation, thereby modulating the immune response. Thus, in one embodiment, binding of the activating epitope downregulates the γδ TCR. In another or alternative embodiment, binding of the activating epitope activates degranulation of γδ T cells. In another additional or alternative embodiment, binding of the activating epitope promotes gamma delta T cell-mediated killing.

In some embodiments, the activating epitope of TRDV1 is an epitope that when bound by the antibody causes receptor downregulation and optional activation of Vδ1 cells. In some embodiments, the activation epitope is one that upregulates the expression of activation markers, such as CD107a, CD25, CD69 and/or Ki67, on Vδ1 cells. In some embodiments, the activation epitope is one that upregulates the expression of activation markers on Vδ1 cells, such as CD107 and CD25, and optionally CD69 and/or Ki67. In some embodiments, upregulation of one or more activation markers, such as CD107a, can be upregulated in the presence of cancer cells. In a preferred embodiment of the invention, the antibody binds to the activation epitope of TRDV1, particularly via the TRDV1 binding domain.

Because T cell receptors are often complexed with other proteins, downregulation of the T cell receptor via Vδ1 antibody binding may result in downregulation of other proteins associated with the T cell receptor (i.e., binding of the Vδ1 antibody results in downregulation of the T cell receptor complex). For example, in some embodiments, the activating epitope of TRDV1 is an epitope that upon binding downregulates the TCR/CD3 receptor complex. In this manner, the antibodies of the invention can cause indirect down-regulation of cell surface proteins that are not bound by the antibodies but that are complexed with T cell receptors. Given that T cells expressing the γδ1 chain (i.e., Vδ1 cells) represent only a minority of the total T cell population, the multispecific antibodies of the invention can be used to selectively (and Indirectly) downregulate these proteins in the TCR complex.

In some embodiments, the T cell receptor complex activating epitope is an epitope that downregulates the T cell receptor complex upon activation without downregulating CD3 molecules not associated with the TRDV1 TCR complex.

The epitope preferably consists of or consists of at least one extracellular, soluble, hydrophilic or external portion of the Vδ1 chain of the γδ TCR.

Specifically, the epitope does not include or consists of the epitope found in the hypervariable region of the Vδ1 chain of the γδ TCR, especially the CDR3 of the Vδ1 chain. In a preferred embodiment, the epitope is within the non-variable region of the Vδ1 chain of the γδ TCR. It will be appreciated that such binding allows for unique recognition of the Vδ1 chain without limitation on highly variable TCR sequences (especially CDR3). Various γδ TCR complexes that recognize antigen can be recognized in this manner solely by the presence of the Vδ1 chain. Therefore, it is understood that any γδ TCR comprising a Vδ1 chain can be recognized using a multispecific antibody as defined herein, regardless of the specificity of the γδ TCR. In one embodiment, the epitope comprises amino acid regions 1-24 and/or 35-90 of SEQ ID NO: 272, such as one or more within the portion of the Vδ1 chain that is not part of the CDR1 and/or CDR3 sequence. amino acid residues or consisting of them. In one embodiment, the epitope does not comprise or consists of amino acid residues within amino acid region 91-105 (CDR3) of SEQ ID NO:272.

In some embodiments, the epitope comprises or consists of amino acids in the TRDV-1 CDR2 sequence.

In a similar manner to well-characterized αβ T cells, γδ T cells utilize a unique set of somatically rearranged variable (V), diverse (D), conjugative (J), and constant (C) genes, except that γδ T cells Cells contain fewer V, D, and J segments than αβ T cells. In one embodiment, the Vδ1 epitope bound by the multispecific antibody (or antigen-binding fragment thereof) is not comprised in the J region of the Vδ1 chain (e.g., one of the four J regions encoded in the germline of the human δ1 chain: SEQ ID NO: 301(J1*0) or 302(J2*0) or 303(J3*0) or 304(J4*0)) or in the C region of the Vδ1 chain (for example, containing the C-terminal juxtamembrane/transmembrane region SEQ ID NO: 305 (C1*0)) or consists of the epitope found in SEQ ID NO: 305 (C1*0)). In one embodiment, the Vδ1 epitope bound by the multispecific antibody (or antigen-binding fragment thereof) does not comprise an epitope found in the N-terminal leader sequence of the Vδ1 chain (e.g., SEQ ID NO: 299) or consists of its composition. Thus, the antibody or fragment may bind only the V region of the Vδ1 chain (eg, SEQ ID NO: 300). Thus, in one embodiment, the epitope consists of an epitope in the V region of a γδ TCR (eg, amino acid residues 1-90 of SEQ ID NO: 272).

The epitope is mentioned with respect to the Vδ1 sequence derived from the sequence described in Luoma et al. (2013) Immunity 39: 1032-1042 and RCSB Protein Data Bank Accession: 3OMZ, shown as SEQ ID NO: 272: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALISALQLEDSAKYFCAL GESLTRADKLIFGKGTRVTVEPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS(SEQ ID NO: 272)

SEQ ID NO: 272 represents a soluble TCR that includes or consists of a V region (also known as a variable domain), a D region, a J region, and a TCR constant region. The V region contains or consists of amino acid residues 1-90, the D region contains or consists of amino acid residues 91-104, and the J region contains or consists of amino acid residues 105-115, and is constant Region (derived from T cell receptor alpha) contains or consists of amino acid residues 116-209. Within the V region, CDR1 is defined as amino acid residues 25-34 of SEQ ID NO: 272, CDR2 is defined as amino acid residues 50-54 of SEQ ID NO: 272, and CDR3 is defined as SEQ ID NO: 272 Amino acid residues 93-104 (Xu et al., PNAS USA 108(6):2414-2419 (2011)).

Therefore, according to one aspect of the present invention, there is provided an isolated antibody or antigen-binding fragment thereof that binds to an epitope of the variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR), the epitope comprising One or more amino acid residues within or consisting of the following amino acid regions: (i) SEQ ID NO: 272-3-20; and/or (ii) SEQ ID NO: 272-37-77.

In another embodiment, the multispecific antibody additionally recognizes a polymorphic V region comprising or consisting of an epitope of amino acid residues 1-90 of SEQ ID NO: 306. Therefore, when defining the epitopes described herein, amino acids 1-90 of SEQ ID NO: 272 and the polymorphic germline variant sequence (amino acids 1-90 of SEQ ID NO: 306) may be considered. Swap. Studies presented herein have demonstrated that the multispecific antibodies of the invention recognize both variants of this germline sequence. By way of example, it is stated that a multispecific antibody or antigen-binding fragment thereof as defined herein recognizes one or more amino acids within amino acid regions 1-24 and/or 35-90 comprising SEQ ID NO: 272 When a residue or an epitope consisting thereof, it additionally recognizes the equivalent epitope in SEQ ID NO: 306 (ie, at the same position).

In one embodiment, the multispecific antibody or antigen-binding fragment thereof recognizes one or more amino acid residues within the amino acid region 1-90 of SEQ ID NO: 272. More specifically, in one embodiment, a multispecific antibody or antigen-binding fragment thereof as defined herein recognizes a human germline epitope, wherein the germline encodes alanine at position 71 of SEQ ID NO: 272 (A) or valine (V).

In one embodiment, the epitope comprises one or more within the desired region, such as two, three, four, five, six, seven, eight, nine, ten or more amino acid residues or consisting of them.

In another embodiment, the epitope comprises one or more (such as 5 or more, such as 10 or more) amine groups within 3-20 within the amino acid region of SEQ ID NO: 272 acid residue or consisting of. In an alternative embodiment, the epitope comprises one or more (such as 5 or more) within amino acid regions 37-77 (such as amino acid regions 50-54) of SEQ ID NO: 272, such as 10 or more) amino acid residues or consisting of. In yet another embodiment, the epitope comprises one or more (such as 5 or more, such as 5 or more) within amino acid regions 3-20 (such as 5-20 or 3-17) of SEQ ID NO: 272 10 or more) amino acid residues and one or more (such as 5 or more) within amino acid regions 37-77 (such as 62-77 or 62-69) of SEQ ID NO: 272 , such as 10 or more) or consisting of amino acid residues.

It is further understood that the antibody (or antigen-binding fragment thereof) need not bind all amino acids within the defined range. Such epitopes may be called linear epitopes. For example, an antibody that binds to an epitope consisting of or consisting of amino acid residues within the amino acid region 5-20 of SEQ ID NO: 272 may only bind to one or more amino groups within this range. Acid residue combinations, such as amino acid residues at each end of the range (i.e., amino acids 5 and 20), optionally include amino acids within the range (i.e., amino acids 5, 9, 16 and 20).

In one embodiment, the epitope comprises amino acid residues 3, 5, 9, 10, 12, 16, 17, 20, 37, 42, 50, 53, 59, 62, 64 of SEQ ID NO: 272 , 68, 69, 72 or 77, or at least one of them. In other embodiments, the epitope comprises amino acid residues 3, 5, 9, 10, 12, 16, 17, 20, 37, 42, 50, 53, 59, 62 selected from SEQ ID NO: 272 , one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve amino acids of , 64, 68, 69, 72 or 77 or consisting of.

In one embodiment, the epitope comprises or consists of one or more amino acid residues within the following amino acid region of SEQ ID NO: 272: (i) 3-17; (ii) 5-20; (iii) 37-53; (iv) 50-64; (v) 59-72; (vi) 59-77; (vii) 62-69; and/or (viii) 62-77.

In other embodiments, the epitope includes or consists of one or more amino acid residues within the following amino acid region of SEQ ID NO: 272: 5-20 and 62-77 (such as, but not limited to ) about antibodies derived from the parent pure line E07, such as embodiments of affinity matured variants thereof); 50-64 (for example (but not limited to) implementations regarding antibodies derived from the parent pure line C08, such as affinity matured variants thereof) Examples); 37-53 and 59-72 (for example, but not limited to, examples regarding antibodies derived from the parent pure line G04, such as affinity matured variants thereof); 59-77 (for example, but not limited to, regarding sources Examples of antibodies derived from the parent pure line C05, such as affinity matured variants thereof); or 3-17 and 62-69 (for example (but not limited to) for antibodies derived from the parent pure line E01, such as affinity matured variants thereof) embodiment). In another embodiment, the epitope consists of one or more amino acid residues within the following amino acid region of SEQ ID NO: 272: 5-20 and 62-77; 50-64; 37-53 and 59-72; 59-77; or 3-17 and 62-69.

In another embodiment, the epitope comprises or consists of the following amino acid residues of SEQ ID NO: 272: 3, 5, 9, 10, 12, 16, 17, 62, 64, 68 and 69, Or suitably composed of the following amino acid residues of SEQ ID NO: 272: 3, 5, 9, 10, 12, 16, 17, 62, 64, 68 and 69 (for example, but not limited to) derived from the parent Examples of pure E01 antibodies, such as affinity matured variants thereof). In another embodiment, the epitope comprises or consists of the following amino acid residues of SEQ ID NO: 272: 5, 9, 16, 20, 62, 64, 72 and 77, or suitably consists of SEQ ID NO: 272 : 272 consisting of the following amino acid residues: 5, 9, 16, 20, 62, 64, 72 and 77 (for example (but not limited to) for antibodies derived from the parent pure line E07, such as affinity matured variants thereof Example). In yet another embodiment, the epitope comprises or consists of the following amino acid residues of SEQ ID NO: 272: 37, 42, 50, 53, 59, 64, 68, 69, 72, 73 and 77, Or suitably consists of the following amino acid residues of SEQ ID NO: 272: 37, 42, 50, 53, 59, 64, 68, 69, 72, 73 and 77 (for example, but not limited to) derived from the parent Examples of pure G04 antibodies, such as affinity matured variants thereof). In another embodiment, the epitope comprises or consists of the following amino acid residues of SEQ ID NO: 272: 50, 53, 59, 62 and 64, or suitably consists of the following amino acid residues of SEQ ID NO: 272 Acid residue composition: 50, 53, 59, 62 and 64 (for example, but not limited to, examples with respect to antibodies derived from the parent pure line C08, such as affinity matured variants thereof). In another embodiment, the epitope comprises or consists of the following amino acid residues of SEQ ID NO: 272: 59, 60, 68 and 72, or suitably consists of the following amino acid residues of SEQ ID NO: 272 Base composition: 59, 60, 68 and 72 (for example, but not limited to, examples regarding antibodies derived from the parent pure line C05, such as affinity matured variants thereof).

In one embodiment, the epitope comprises or consists of one or more amino acid residues within amino acid regions 37-53 and/or 59-77 of SEQ ID NO: 272. In another embodiment, the epitope consists of one or more amino acid residues within amino acid regions 37-53 and 59-77 of SEQ ID NO: 272. In another alternative embodiment, the epitope comprises or consists of one or more amino acid residues within amino acid regions 37-53 or 59-77 of SEQ ID NO: 272. Multispecific antibodies or antigen-binding fragments thereof having such epitopes may have some or all of the sequence of G04, or such antibodies or antigen-binding fragments thereof may be derived from G04. For example, a multispecific antibody or antigen-binding fragment thereof having one or more CDR sequences of G04 or one or both of the VH and VL sequences of G04 can bind such epitopes.

In one embodiment, the epitope comprises or consists of one or more amino acid residues within amino acid regions 5-20 and/or 62-77 of SEQ ID NO: 272. In another embodiment, the epitope consists of one or more amino acid residues within amino acid regions 5-20 and 62-77 of SEQ ID NO: 272. In another alternative embodiment, the epitope comprises or consists of one or more amino acid residues within amino acid regions 5-20 or 62-77 of SEQ ID NO: 272. Multispecific antibodies or antigen-binding fragments thereof having such epitopes may have some or all of the sequence of E07, or such antibodies or antigen-binding fragments thereof may be derived from E07. For example, a multispecific antibody or antigen-binding fragment thereof having one or more CDR sequences of E07 or one or both of the VH and VL sequences of E07 can bind such epitopes.

In one embodiment, the epitope comprises or consists of one or more amino acid residues within amino acid regions 50-64 of SEQ ID NO: 272. In another embodiment, the epitope consists of one or more amino acid residues within amino acid region 50-64 of SEQ ID NO: 272. Multispecific antibodies or antigen-binding fragments thereof having such epitopes may have some or all of the sequence of C08, or such antibodies or antigen-binding fragments thereof may be derived from C08. For example, a multispecific antibody or antigen-binding fragment thereof having one or more CDR sequences of C08 or one or both of the VH and VL sequences of C08 can bind such epitopes.

In one embodiment, the epitope comprises or consists of one or more amino acid residues within amino acid regions 59-72 of SEQ ID NO: 272. In another embodiment, the epitope consists of one or more amino acid residues within amino acid regions 59-72 of SEQ ID NO: 272. Multispecific antibodies or antigen-binding fragments thereof having such epitopes may have some or all of the sequence of C05, or such antibodies or antigen-binding fragments thereof may be derived from C05. For example, a multispecific antibody or antigen-binding fragment thereof having one or more CDR sequences of C05 or one or both of the VH and VL sequences of C05 can bind such epitopes.

In one embodiment, the epitope does not comprise or consists of amino acid residues within amino acid regions 11-21 of SEQ ID NO: 272. In one embodiment, the epitope does not comprise or consists of amino acid residues within amino acid regions 21-28 of SEQ ID NO: 272. In one embodiment, the epitope does not comprise or consists of amino acid residues within amino acid regions 59 and 60 of SEQ ID NO: 272. In one embodiment, the epitope does not comprise or consists of the amino acid residues within amino acid regions 67-82 of SEQ ID NO: 272.

The epitopes of the affinity matured antibodies will generally be the same as those identified herein for the parent clones. For those epitopes on cynomolgus monkey TRDV1, the position of the epitope of the affinity matured antibody will generally be the same position as the epitope identified against the corresponding parental pure line. The reference to "position" is necessary because the skilled artisan will understand that the identity of some of the amino acids in the epitope is different from that of human TRDV1. Despite these variations between human and macaque TRDV1, these antibodies are still able to specifically bind to both antigens.

In one embodiment, the epitope is not the same epitope bound by a commercially available anti-Vδ1 antibody, such as TS-1 or TS8.2. As described in WO2017197347, binding of TS-1 and TS8.2 to soluble TCR was detected when the δ1 chain included Vδ1 J1 and Vδ1 J2 sequences, but not the Vδ1 J3 chain, indicating that TS-1 and TS8.2 Binding involves key residues in the delta J1 and delta J2 regions.

References herein to "within" include the ends of the defined range. For example, "within the amino acid region 5-20" refers to all amino acid residues from and including residue 5 up to and including residue 20.

Various techniques are known in the art to determine which epitope is bound by an antibody. Exemplary techniques include, for example, conventional cross-blocking analysis, alanine scanning mutational analysis, peptide blot analysis, peptide fragmentation analysis crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction and antigen chemical modification can be used. Another method that can be used to identify amino acids within polypeptides that interact with antibodies is hydrogen/deuterium exchange detected by mass spectrometry (as described in Example 9). Generally speaking, hydrogen/deuterium exchange methods involve deuterium labeling the protein of interest and then binding the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water and the exchangeable protons in the amino acids protected by the antibody complex undergo exchange of deuterium back to hydrogen at a slower rate than the exchangeable protons in the amino acids that are not part of the interface. . Thus, amino acids that form part of the protein/antibody interface may retain deuterium and therefore exhibit relatively higher mass than amino acids that are not included in the interface. After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry analysis, which reveals deuterium-labeled residues corresponding to specific amino acids that interact with the antibody.

The multispecific antibodies and their antigen-binding fragments preferably specifically bind to human TRDV1 (polymorphic variants of SEQ ID NO: 272 and SEQ ID NO: 306) and the ortholog in the cynomolgus macaque, cynomolgus monkey TRDV1 (SEQ ID NO: 308, see also UniProtKB-G7P9S6(G7P9S6_MACFA)). In some embodiments, the multispecific antibody or antigen-binding fragment thereof is a G04-derived antibody that specifically binds to both human and cynomolgus monkey TRDV1. Epitopes hypothesized to be bound by a multispecific antibody of the invention (e.g. binding to the region within amino acid residues 37 to 77 of any of SEQ ID NO: 272, 306 or 308) may be particularly advantageous because This allows the provision of anti-Vδ1 antibodies specific for Vδ1 (i.e., which do not bind similar antigens such as Vδ2 or Vδ3), and also the provision of polymorphic variants with Vδ1 (i.e., TRDV1 SEQ ID NO: 272 and TRDV1 SEQ ID NO: 306, despite the polymorphism at residue position 20 and this identification as a possible contact residue or close to a contact residue identified for some antibodies) and provides cross-reactivity between human and cynomolgus Vδ1 Reaction (although residues 42, 50, 54, 59, 60, 68, 73, 50, 54, 59, 60, 68, 73, 75 and 76 are different). Epitopes of antibodies derived from ADT1-4

The ADT1-4 derived antibody binds to the same or substantially the same epitope as the ADT1-4 parent antibody. Accordingly, in some embodiments, particularly with respect to embodiments derived from or related to an ADT1-4 parent antibody, an anti-Vδ1 antibody or antigen-binding fragment binds an antibody comprising SEQ ID NO: 272 (or SEQ ID NO: 306) within the amino acid regions 37 to 77, for example, one or more amino acid residues or epitopes composed thereof within the amino acid regions 37-53 and/or 59-77. In some embodiments, the epitope comprises amino acid residues 37, 42, 50, 53, 59, 64, 68, 69, 72, 73 and 77 of SEQ ID NO: 272 (or SEQ ID NO: 306) or consisting of. In some embodiments, the epitope consists of amino acid residues 37, 42, 50, 53, 59, 64, 68, 69, 72, 73 and 77 of SEQ ID NO: 272 (or SEQ ID NO: 306) composition.

Antibodies derived from ADT1-4 also bind to the epitope of the cynomolgus variable δ1 (Vδ1) chain (also known as cynomolgus TRDV1) (SEQ ID NO: 308) of the γδ T cell receptor (TCR). Accordingly, in some embodiments, particularly with respect to embodiments derived from or related to a G04 parent antibody, an anti-Vδ1 antibody or antigen-binding fragment binds amino acid region 37 to 1 of SEQ ID NO: 308. 77, for example, one or more amino acid residues or epitopes composed thereof within the amino acid regions 37-53 and/or 59-77. In some embodiments, the epitope comprises or consists of amino acid residues 37, 42, 50, 53, 59, 64, 68, 69, 72, 73, and 77 of SEQ ID NO: 308. In some embodiments, the epitope consists of amino acid residues 37, 42, 50, 53, 59, 64, 68, 69, 72, 73, and 77 of SEQ ID NO: 308.

Taking into account the cross-reactivity of antibodies derived particularly from ADT1-4, as well as the ability of the antibodies disclosed herein to bind to polymorphic variants of TRDV1, in some embodiments, in particular with respect to antibodies derived from or related to the G04 parent Examples of antibodies related to the present antibody include anti-Vδ1 antibodies or antigen-binding fragments that bind to amino acid regions 37 to 77 including SEQ ID NO: 272, 306 and 308, such as amino acid regions 37-53 and/or 59- One or more amino acid residues within 77 or an epitope composed thereof. In some embodiments, the epitope comprises or consists of amino acid residues 37, 42, 50, 53, 59, 64, 68, 69, 72, 73 and 77 of SEQ ID NO:: 272, 306 and 308. composition. In some embodiments, the epitope consists of amino acid residues 37, 42, 50, 53, 59, 64, 68, 69, 72, 73, and 77 of SEQ ID NOs: 272, 306, and 308.

In view of the positioning of amino acid variants between such sequences from the two species, those that comprise or consist of epitopes such as those described above but provide cross-reactivity between human and cynomolgus monkey TRDV1 sequences Antibodies were provided unexpectedly. Epitopes of antibodies derived from ADT1-7

The ADT1-7 derived antibody binds to the same or substantially the same epitope as the ADT1-7 parent antibody. Thus, in some embodiments, particularly with respect to embodiments derived from or related to an ADT1-7 parent antibody, the anti-Vδ1 antibody or antigen-binding fragment binds an epitope comprising SEQ ID NO: 272 (or SEQ ID NO: 306) one or more amino acid residues within or consisting of amino acid residues 5-20 and/or 62-77. In some embodiments, the epitope comprises or consists of amino acid residues 5, 9, 16, 20, 62, 64, 72 and 77 of SEQ ID NO: 272 (or SEQ ID NO: 306). In some embodiments, the epitope consists of amino acid residues 5, 9, 16, 20, 62, 64, 72, and 77 of SEQ ID NO: 272 (or SEQ ID NO: 306). Architecture and other sequences

Suitably, the VH and VL regions of the multispecific antibody or antigen-binding fragment of the invention each comprise four framework regions (FR1-FR4). In one embodiment, the antibody or antigen-binding fragment thereof includes a framework region comprising a sequence that has at least 80% sequence identity to a framework region in any of the following (e.g., FR1, FR2, FR3, and/or FR4): SEQ ID NO: 2 to 25 (for example, in the case of a light chain variable sequence derived from G04), SEQ ID NO: 27 to 50 (for example, in the case of a heavy chain variable sequence derived from G04), SEQ ID NO: 107 to 117 (eg in the case of a light chain variable sequence derived from E07) or SEQ ID NO: 119 to 129 (eg in the case of a light chain variable sequence derived from E07). In one embodiment, the antibody or antigen-binding fragment thereof includes a framework region comprising a sequence that has at least 90%, such as at least 95%, 97% or 99% sequence identity to a framework region in any of the following (e.g. FR1, FR2, FR3 and/or FR4): SEQ ID NO: 2 to 25 (e.g. in the case of a light chain variable sequence derived from G04), SEQ ID NO: 27 to 50 (e.g. in the case of a heavy chain derived from G04 SEQ ID NO: 107 to 117 (e.g., in the case of a light chain variable sequence derived from E07), or SEQ ID NO: 119 to 129 (e.g., in the case of a light chain variable sequence derived from E07) In the case of changing the sequence). In one embodiment, the antibody or antigen-binding fragment thereof includes a framework region (e.g., FR1, FR2, FR3, and/or FR4) comprising the sequence of any one of: SEQ ID NOs: 2 to 25 (e.g., in the source In the case of the light chain variable sequence derived from G04), SEQ ID NOs: 27 to 50 (e.g. in the case of the heavy chain variable sequence derived from G04), SEQ ID NOs: 107 to 117 (e.g. derived from E07 (for example, in the case of a light chain variable sequence derived from E07) or SEQ ID NO: 119 to 129 (for example, in the case of a light chain variable sequence derived from E07). In one embodiment, the antibody or antigen-binding fragment thereof comprises a framework region (e.g., FR1, FR2, FR3, and/or FR4) consisting of the sequence of any one of: SEQ ID NOs: 2 to 25 (e.g., in In the case of a light chain variable sequence derived from G04), SEQ ID NOs: 27 to 50 (for example, in the case of a heavy chain variable sequence derived from G04), SEQ ID NOs: 107 to 117 (for example, in the case of a heavy chain variable sequence derived from in the case of a light chain variable sequence derived from E07) or SEQ ID NOs: 119 to 129 (eg in the case of a light chain variable sequence derived from E07).

In some embodiments, an anti-Vδ1 antibody or an antigen-binding fragment thereof may comprise: a HFR1 (i.e., heavy chain framework 1 region) sequence comprising or consisting of the sequence of SEQ ID NO: 170 or 171; a sequence comprising SEQ ID NO: 170 or 171. : The sequence of SEQ ID NO: 172 or the HFR2 sequence consisting of the sequence; the sequence of SEQ ID NO: 173 or the HFR3 sequence consisting of the sequence; the sequence of SEQ ID NO: 174 or the HFR4 sequence consisting of the sequence; the HFR4 sequence comprising SEQ ID NO: 174 The sequence of NO: 175 or the LFR1 sequence consisting of the sequence; the sequence of SEQ ID NO: 176 or the LFR2 sequence consisting of the sequence; the sequence of SEQ ID NO: 177 or 178 or the LFR3 sequence consisting of the sequence; and/or an LFR4 sequence comprising or consisting of the sequence of SEQ ID NO: 179, 180, 181 or 182.

In some embodiments, an anti-Vδ1 antibody or antigen-binding fragment thereof may comprise a HFR1 (i.e., heavy chain framework 1 region) sequence comprising or consisting of the sequence of SEQ ID NO: 170 or 171; comprising SEQ ID NO: The sequence of 172 or the HFR2 sequence consisting of the sequence; the sequence of SEQ ID NO: 173 or the HFR3 sequence consisting of the sequence; the sequence of SEQ ID NO: 174 or the HFR4 sequence consisting of the sequence; the HFR4 sequence comprising SEQ ID NO. : The sequence of SEQ ID NO: 175 or the LFR1 sequence consisting of the sequence; the sequence of SEQ ID NO: 176 or the LFR2 sequence consisting of the sequence; the sequence of SEQ ID NO: 177 or the LFR3 sequence consisting of the sequence; and/or An LFR4 sequence comprising or consisting of the sequence of SEQ ID NO: 179, 180, 181 or 182.

In some embodiments, an anti-Vδ1 antibody or antigen-binding fragment thereof may comprise a HFR1 (i.e., heavy chain framework 1 region) sequence comprising or consisting of the sequence of SEQ ID NO: 170 or 171; comprising SEQ ID NO: The sequence of 172 or the HFR2 sequence consisting of the sequence; the sequence of SEQ ID NO: 173 or the HFR3 sequence consisting of the sequence; the sequence of SEQ ID NO: 174 or the HFR4 sequence consisting of the sequence; the HFR4 sequence comprising SEQ ID NO. : The sequence of SEQ ID NO: 175 or the LFR1 sequence consisting of the sequence; the sequence of SEQ ID NO: 176 or the LFR2 sequence consisting of the sequence; the sequence of SEQ ID NO: 177 or the LFR3 sequence consisting of the sequence; and/or An LFR4 sequence comprising or consisting of the sequence of SEQ ID NO: 179 or 181.

In some embodiments, an anti-Vδ1 antibody or an antigen-binding fragment thereof may comprise: an HFR1 sequence comprising or consisting of the sequence of SEQ ID NO: 189; an HFR2 sequence comprising or consisting of the sequence of SEQ ID NO: 190 ; HFR3 sequence comprising or consisting of the sequence of SEQ ID NO: 191; HFR4 sequence comprising or consisting of the sequence of SEQ ID NO: 192; Sequence comprising or consisting of SEQ ID NO: 193 and 194 The LFR1 sequence of SEQ ID NO: 195; the LFR2 sequence of SEQ ID NO: 195; the LFR3 sequence of SEQ ID NO: 196; and the LFR3 sequence of SEQ ID NO: 197. The sequence consists of the LFR4 sequence.

In some embodiments, an anti-Vδ1 antibody or an antigen-binding fragment thereof may comprise: an HFR1 sequence comprising or consisting of the sequence of SEQ ID NO: 189; an HFR2 sequence comprising or consisting of the sequence of SEQ ID NO: 190 ; HFR3 sequence comprising the sequence of SEQ ID NO: 191 or consisting of the sequence; HFR4 sequence comprising the sequence of SEQ ID NO: 192 or consisting of the sequence; LFR1 comprising the sequence of SEQ ID NO: 193 or consisting of the sequence Sequence; an LFR2 sequence comprising or consisting of the sequence of SEQ ID NO: 195; an LFR3 sequence comprising or consisting of the sequence of SEQ ID NO: 196; and a sequence comprising or consisting of the sequence of SEQ ID NO: 197 The LFR4 sequence.

For fragments that include both VH and VL regions, these regions may be associated covalently (eg, via disulfide bonds or linkers) or non-covalently. Antibody fragments described herein may comprise scFv, that is, a fragment comprising a VH region and a VL region joined by a linker. In one embodiment, the VH and VL regions are joined by a polypeptide linker (eg, synthetic). The polypeptide linker may comprise a (Gly ₄ Ser) _n linker, where n = 1 to 8, such as 2, 3, 4, 5 or 7. The polypeptide linker may comprise a [(Gly ₄ Ser) _n (Gly ₃ AlaSer) _m ] _p linker, wherein n = 1 to 8, such as 2, 3, 4, 5 or 7, and m = 1 to 8, such as 0, 1, 2 or 3, and p = 1 to 8, such as 1, 2 or 3. In another embodiment, the linker comprises SEQ ID NO: 291. In another embodiment, the linker consists of SEQ ID NO: 291.

Those skilled in the art will understand that scFv constructs can be designed and made to include N- and C-terminal modifications to aid translation, purification and detection. For example, at the N-terminus of the scFV sequence, additional methionine and/or alanine amino acid residues may be included before the typical VH sequence (eg, starting QVQ or EVQ). At the C-terminus (i.e., the C-terminus where the typical mature VL domain sequence ends according to the IMGT definition), additional sequences may be included, such as (i) partial sequences of the constant domains, and/or (ii) additional synthetic sequences, including tags, such as His-tags and Flag-tags to aid purification and detection (eg tags of any of SEQ ID NO: 292 to 295).

As described herein, the antibodies can be in any format. In a preferred embodiment, the antibody is in IgG1 (eg, human IgG1) format (i.e., the antibody is a human IgG1 antibody).

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a light chain constant region comprising the sequence of SEQ ID NO: 296 or SEQ ID NO: 307 (or having at least the same sequence as SEQ ID NO: 296 or SEQ ID NO: 307). 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or a sequence with at least 99% sequence identity); and/or a heavy chain constant region comprising the sequence of SEQ ID NO: 297 or SEQ ID NO: 298 (or having at least 99% sequence identity with SEQ ID NO: 297 or SEQ ID NO: 298 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or a sequence with at least 99% sequence identity). In some embodiments, the effector function of the heavy chain constant region may be reduced or disabled (effector function-disabling mutations). Suitable mutations that reduce the effector function are known to the skilled person. For example, the L235A and/or G237A mutation ("LAGA") or the L234A and/or L235A mutation ("LALA") according to EU numbering. For example, in one embodiment, the antibody or antigen-binding fragment thereof includes a light chain constant region comprising the sequence of SEQ ID NO: 296 and/or a heavy chain comprising the sequence of SEQ ID NO: 297 or SEQ ID NO: 298 constant region. competing antibodies

In one embodiment, the antibody binds to or competes with the same or substantially the same epitope as an antibody as defined herein, or an antigen-binding fragment thereof. By using routine methods known in the art, it can be readily determined whether the antibody binds to or competes with the same epitope as a reference anti-Vδ1 antibody. For example, to determine whether a test antibody binds to the same epitope as a reference anti-Vδ1 antibody of the invention, the reference antibody is allowed to bind to the Vδ1 protein or peptide under saturating conditions. Subsequently, the test antibody is assessed for its ability to bind to the Vδ1 chain. If the test antibody is able to bind to Vδ1 after saturating binding with the reference anti-Vδ1 antibody, it can be inferred that the test antibody binds to a different epitope than the reference anti-Vδ1 antibody. On the other hand, if the test antibody is unable to bind to the Vδ1 chain after saturation binding with the reference anti-Vδ1 antibody, the test antibody can bind to the same epitope to which the reference anti-Vδ1 antibody of the invention binds.

The invention also includes anti-Vδ1 antibodies that compete for binding to Vδ1 with an antibody as defined herein, or an antigen-binding fragment thereof, or an antibody having the CDR sequence of any of the exemplary antibodies described herein. For example, competition assays can be performed using the antibodies of the invention to determine which proteins, antibodies and other antagonists compete with the antibodies of the invention for binding to the Vδ1 chain and/or shared epitopes. Such assays are readily apparent to those skilled in the art; they assess competition between antagonists or ligands for a limited number of binding sites on a protein such as Vδ1. The antibody (or antigen-binding fragment thereof) is immobilized or insolubilized before or after competition, and the binding to the Vδ1 chain is removed, for example, by decantation (in which the antibody is not previously insolubilized) or by centrifugation (in which the antibody is precipitated after the competition reaction). The sample is separated from the unbound sample. In addition, competitive binding can be determined by whether the binding or lack of binding of the antibody to the protein is altered, e.g., the antibody molecule inhibits or enhances, e.g., the activity of a labeled enzyme. ELISA and other functional assays can be used, as is known in the art and described herein.

Two antibodies bind to the same or overlapping epitopes if they each competitively inhibit (block) the other from binding to the target antigen. That is, a 1, 5, 10, 20, or 100-fold excess of an antibody inhibits the binding of another antibody by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competition binding assay. Alternatively, two antibodies have the same epitope if substantially all amino acid mutations in the target antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

Other routine experiments (e.g., peptide mutagenesis and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or to steric blocking (or another phenomenon) resulting in the observed lack of union. Such experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in this technology. Antibody binding and pharmacological properties of antibodies

Multispecific antibodies of the invention may have advantageous binding and/or pharmacological properties, for example, as described below.

In some embodiments, the binding affinity of the antibody or antigen-binding fragment thereof is determined by coating the antibody or antigen-binding fragment thereof directly or indirectly (e.g., by capture with an anti-human IgG Fc) to a sensor (e.g., an amine high-capacity chip). or equivalent), where the target to which the antibody or antigen-binding fragment thereof binds (i.e., the Vδ1 chain of the γδ TCR or the EGFR) flows across the chip to detect binding. Appropriately, use the MASS-2 instrument (which may also be called the Sierra SPR-32) at 30 µl/min in PBS + 0.02% Tween 20 operating buffer at 25°C.

This article describes other assays that can be used to define antibody function. For example, the multispecific antibodies described herein, or antigen-binding fragments thereof, can be assessed by γδ TCR engagement, e.g., measuring γδ TCR downregulation upon antibody binding. The surface expression of the γδ TCR after application of the antibody or antigen-binding fragment thereof, as appropriate, presented on the cell surface, can be measured, for example, by flow cytometry. The antibodies or antigen-binding fragments thereof described herein can also be evaluated by measuring γδ T cell degranulation. For example, expression of CD107a (a marker of cellular degranulation) can be measured upon application of the antibody or antigen-binding fragment thereof (optionally presented on the cell surface) to γδ T cells, for example by flow cytometry. The antibodies or antigen-binding fragments thereof described herein can also be evaluated by measuring γδ T cell-mediated killing activity (to test whether the antibody has an effect on the killing activity of γδ T cells). For example, target cells can be incubated with γδ T cells in the presence of antibodies or antigen-binding fragments thereof, optionally presented on the cell surface. After incubation, the culture can be stained with a cell viability dye to differentiate between live and dead target cells. The proportion of dead cells can then be measured, for example by flow cytometry.

Additionally, described herein are assays that can be used to define the function of multispecific antibodies as assessed in an assay containing a mixture of at least two different cell types that include separate γδ TCR positive cells and EGFR positive cells. These analyzes help to further highlight some of the surprising technical effects of the multispecific antibodies described herein, whereby further enhanced effects (e.g., increased TCR downregulation). Binding affinity to human TRDV1 (K _D )

Generally speaking, affinity matured clones (and multispecific antibodies derived from or related to such clones) have a higher affinity for their antigens than the parent clones. For example, the affinity for human TRDV1 (SEQ ID NO 272 or 306) will be at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 500% greater than the affinity of the parent antibody.

The multispecific antibodies or antigen-binding fragments _thereof of the invention can bind to human TRDV1 (SEQ ID NO 272 or 306).

The multispecific antibody, or antigen-binding fragment thereof, may be further defined as binding to human _TRDV1 (SEQ ID NO 272 or 306).

In some embodiments, for example, a multispecific antibody or antigen-binding fragment thereof that is related to or derived from the ADT1-4 repertoire (e.g., generated by affinity maturation or otherwise) has a function related to or derived from the ADT1-4 repertoire Multispecific antibodies of the present invention derived from the Fab region of the ADT1-4 lineage), the multispecific antibodies of the present invention or antigen-binding fragments thereof can bind to human TRDV1 with a K _D of less than about 100 nM, preferably less than about 50 nM. .

In some embodiments, such as multispecific antibodies or antigen-binding fragments thereof related to or derived from the ADT1-4 repertoire (but excluding ADT1-4-138), the multispecific antibodies or antigens thereof of the invention The binding fragment may bind to human TRDV1 with a _KD of less than about 10 nM, preferably less than about 5 nM.

In some embodiments, for example, with ADT1-4 lineage members ADT1-4-19, ADT1-4-21, ADT1-4-31, ADT1-4-53, ADT1-4-2, ADT1-4-86, ADT1 -4-112, ADT1-4-143 and/or multispecific antibodies or antigen-binding fragments thereof related to or derived from members of these lineages. The multispecific antibodies or antigen-binding fragments thereof of the present invention can be Binds to human TRDV1 with a _KD of less than about 10 nM, preferably less than about 1 nM.

The multispecific antibodies or antigen-binding fragments thereof of the present invention may bind to cynomolgus monkey _TRDV1 ( SEQ ID NO 308).

In some embodiments, such as multispecific antibodies or antigen-binding fragments thereof related to or derived from the ADT1-4 repertoire, the multispecific antibodies or antigen-binding fragments thereof of the invention can be less than about 100 nM, less than Preferably, the K _D is less than about 50 nM for binding to cynomolgus monkey TRDV1.

In some embodiments, for example, with ADT1-4 lineage members ADT1-4-19, ADT1-4-21, ADT1-4-31, ADT1-4-53, ADT1-4-2, ADT1-4-86, ADT1 -4-112, ADT1-4-143 and/or multispecific antibodies or antigen-binding fragments thereof related to or derived from members of these lineages. The multispecific antibodies or antigen-binding fragments thereof of the present invention can be Binds to cynomolgus monkey TRDV1 with a _KD of less than about 50 nM.

In some embodiments, such as embodiments related to or derived from the ADT1-4 repertoire, the multispecific antibodies or antigen-binding fragments thereof of the invention may have a K of less than about 100 nM, preferably less than about 50 nM. _D binds to human TRDV1 and binds to cynomolgus monkey TRDV1 with a binding affinity (K _D , eg, as measured by surface plasmon resonance) of less than about 100 nM, preferably less than about 50 nM.

In some embodiments, such as multispecific antibodies or antigen-binding fragments thereof related to or derived from the ADT1-4 repertoire (but excluding ADT1-4-138), the multispecific antibodies or antigens thereof of the invention The binding fragment can bind to human TRDV1 with a _KD of less than about 10 nM and to cynomolgus monkey TRDV1 with a binding affinity ( _KD , eg, as measured by surface plasmon resonance) of less than about 100 nM. Preferably in this embodiment, the multispecific antibody or antigen-binding fragment thereof of the invention can bind to human TRDV1 with a K _D of less than about 5 nM and with a binding affinity (K _{D )} of less than about 50 nM, e.g., via a surface Plasmon resonance measurements) bound to cynomolgus macaque TRDV1.

In some embodiments, for example, with ADT1-4 lineage members ADT1-4-19, ADT1-4-21, ADT1-4-31, ADT1-4-53, ADT1-4-2, ADT1-4-86, ADT1 -4-112, ADT1-4-143 and/or multispecific antibodies or antigen-binding fragments thereof related to or derived from members of these lineages. The multispecific antibodies or antigen-binding fragments thereof of the present invention can be Binds to human TRDV1 with a _K of less than about 10 nM and binds to human TRDV1 with a _K of less than 50 nM. Preferably in this embodiment, the multispecific antibodies or antigen-binding fragments thereof of the invention can bind to human TRDV1 with a K _D of less than about 1 nM and with a binding affinity (K _{D )} of less than about 50 nM, e.g., via a surface Plasmon resonance measurements) bound to cynomolgus macaque TRDV1.

In some embodiments, for example, a multispecific antibody or antigen-binding fragment thereof that is related to or derived from the ADT1-7 lineage (e.g., generated by affinity maturation or otherwise) has a gene that is related to or derived from the ADT1-7 lineage. Multispecific antibodies of the present invention derived from the Fab region of the ADT1-7 lineage), the multispecific antibodies of the present invention or antigen-binding fragments thereof can bind to human TRDV1 with a K _D of less than about 10 nM, preferably less than about 5 nM. . Table 6 : Summary of human TRDV1 affinities of exemplary multispecific antibodies of the invention as determined by SPR antibody Affinity for V δ1 ( nM) ADT1-4-2×LEE 0.416 ADT1-4-6×LEE 3.24 ADT1-4-138×LEE 55

Increasing binding affinity to less than about 10 nM for human TRDV1 was shown to deliver beneficial technical effects such as increased binding (MFI), improved vδ1 TCR downregulation, improved CD107A upregulation, increased cytotoxicity, and increased proliferation (see Figure 35L). Binding affinity to EGFR (K _D )

In some embodiments, the multispecific antibodies of the invention can bind to EGFR with a _KD of less than about 150 nM, such as ADT1-4-2×FS1-65, ADT1-4-2×FS1-67, ADT1-4- 2×LEE, ADT1-4-2×747, ADT1-4-2×LEE3, ADT1-4-2×LEE2 and ADT1-4-2×LEE1.

In some embodiments, the multispecific antibodies of the invention can bind to EGFR with a _KD of less than about 20 nM, such as ADT1-4-2×FS1-65, ADT1-4-2×FS1-67, ADT1-4- 2×LEE, ADT1-4-2×747 and ADT1-4-2×LEE3.

In some embodiments, the multispecific antibodies of the invention can bind to EGFR with a _KD of less than about 10 nM, such as ADT1-4-2×FS1-65, ADT1-4-2×FS1-67, ADT1-4- 2×LEE and ADT1-4-2×747.

In some embodiments, the multispecific antibodies of the invention can bind to EGFR with a _KD of less than about 5 nM, such as ADT1-4-2×FS1-65, ADT1-4-2×FS1-67, ADT1-4- 2×LEE and ADT1-4-2×747.

In any embodiment, the EGFR can be human EGFR.

Table 7 provides a summary of the EGFR affinities of exemplary multispecific antibodies of the invention as determined by SPR. Table 7 : Summary of TRDV1 and EGFR affinity Example affinity results for molecules as indicated (note; inter-assay variation is typically <2-fold) antibody Alias Affinity for human EGFR (nM) Affinity for human TRDV1 (nM) ADT1-4-2/ADT3-3 ADT1-4-2×FS1-65 1.35 0.464 ADT1-4-2/ADT3-1 ADT1-4-2×FS1-67 1.37 0.378 ADT1-4-2/ADT3-2 ADT1-4-2×LEE 1.49 0.416 ADT1-4-2/ADT3-4 ADT1-4-2×747 2.35 0.511 ADT1-4-2/ADT3-7 ADT1-4-2×LEE3 8.63 0.462 ADT1-4-2/ADT3-6 ADT1-4-2×LEE2 19.2 0.818 ADT1-4-2/ADT3-5 ADT1-4-2×LEE1 142 0.883 ADT1-4/ADT3-1 G04×FS1-67 1.14 152 ADT1-4-138/ADT3-2 ADT1-4-138×LEE 2.78 55 ADT1-4-6/ADT3-2 ADT1-4-6×LEE 0.655 3.24 ADT1-4 G04 - 115 ADT1-4-2 - - 0.522

In some embodiments, the multispecific antibodies of the invention can bind to human TRDV1 with a _KD of less than about 5 nM and can bind to EGFR with a _KD of less than about 150 nM, such as ADT1-4-2×FS1-65, ADT1 -4-2×FS1-67, ADT1-4-2×LEE, ADT1-4-2×747, ADT1-4-2×LEE3, ADT1-4-2×LEE2 and ADT1-4-2×LEE1.

In some embodiments, the multispecific antibodies of the invention can bind to human TRDV1 with a _KD of less than about 5 nM and can bind to EGFR with a _KD of less than about 20 nM, such as ADT1-4-2×FS1-65, ADT1 -4-2×FS1-67, ADT1-4-2×LEE, ADT1-4-2×747 and ADT1-4-2×LEE3.

In some embodiments, the multispecific antibodies of the invention can bind to human TRDV1 with a _KD of less than about 5 nM and can bind to EGFR with a _KD of less than about 10 nM, such as ADT1-4-2×FS1-65, ADT1 -4-2×FS1-67, ADT1-4-2×LEE and ADT1-4-2×747.

In some embodiments, the multispecific antibodies of the invention can bind to human TRDV1 with a _K of less than about 5 nM and can bind to EGFR with a _K of less than about 5 nM, such as ADT1-4-2×FS1-65, ADT1 -4-2×FS1-67, ADT1-4-2×LEE and ADT1-4-2×747.

Increased binding affinity to EGFR was shown to deliver beneficial technical effects such as increased binding (MFI), improved vδ1 TCR downregulation, improved degranulation, increased cytotoxicity, increased 4-1BB upregulation, and increased proliferation (see Figure 36). A clustering effect was observed with multispecific antibodies of the invention (ADT1-4-2×LEE, ADT1-4-2×LEE3) with moderate to high affinity for EGFR in the affinity range of about 1 nM to about 20 nM. , ADT1-4-2LEE2) functionally clustered together, whereas the lower affinity molecule (ADT1-4-2×LEE1) with an affinity of approximately 142 nM was significantly attenuated and equivalent to non-binding in this assay (to the TCR combination) comparison. Interestingly, highest affinity EGFR binding (less than about 2 nM) did not confer the highest saturating effects of improved vδ1 TCR downregulation, improved degranulation, increased cytotoxicity, increased 41BB upregulation, and increased proliferation. Specifically, molecules with mid-range affinity of approximately 9 nM-19 nM (ADT1-4-2×LEE3 and ADT1-4-2×LEE2, respectively) conferred the highest effects at saturating concentrations of the antibody. This superior effect conferred by the mid-range affinity multispecific antibodies of the invention is discussed below. The results are shown in Figure 36. Increase and decrease the affinity of the binding domain

Multispecific antibodies as presented herein comprise at least one first binding domain that binds the TRDV1 domain of the Vδ1 TCR and at least one second binding domain that binds EGFR. Each binding domain binds to its target with a different binding affinity (Kd). These binding affinities can vary independently of each other. Slightly increasing or decreasing the affinity for the first and second binding domains affects functionality and confers technical effects.

Affinity of the first binding domain (TRDV1 domain of Vδ1 TCR): 1) In some cases, increasing the affinity for the first binding domain (to greater than about 10 nM) can further enhance the effect of these multispecific antibodies. This is surprising given that all naturally occurring human Vδ1 TCR ligands reported to date exhibit much lower affinities in the range of 2.9 uM (MART1) to 900 uM (MICA). 2) Increasing the concentration of multispecific antibodies with lower affinity for TRDV1 (less than about 10 nM) can overcome some of the lower observed activities (e.g., proliferation) conferred by these molecules, but interestingly, not all Both effects can be replicated by increasing the concentration of the lower affinity variant. 3) Cancer cells with higher TAA copy numbers may further enhance the effects observed by these multispecific antibodies; especially the lower affinity (for TRDV1) variants.

Affinity of the second binding domain (for EGFR) 1) Multispecific antibodies of the invention with higher affinity for EGFR are more effective in many ongoing exploratory studies (see Examples 13 to 17). 2) Threshold effects are observed, where the affinity for EGFR can range between 1 nM and 20 nM without significant impact on many functional effects (see Examples 13 to 17). This clustering or threshold effect observed for multispecific antibodies of high to moderate affinity was unexpected. 3) Higher TAA copy numbers on cancer cell lines may overcome some of the reducing effects observed with lower affinity (to EGFR) multispecific antibodies - however and moreover, only in some cases (see Examples 13 to 17 ). 4) Some multispecific antibodies of the invention with mid-range affinities for EGFR (9 nm to 20 nM) are in some cases more potent than the highest affinity variants (less than 2 nM) at saturating concentrations. For example, when operating at nM antibody concentrations, it was observed in the assay that EGFR had a lower affinity for EGFR compared to the highest affinity variant (less than 2 nM, preferably less than 1.5 nM) and the lowest affinity variant (approximately 142 nM). Multispecific antibodies of intermediate range affinities (approximately 8 nM to 20 nM) confer enhanced proliferation and 41BB activation.

Increasing and decreasing affinity provides a "toolbox" of multispecific antibodies, some of which are optimal for lower replica number TAA cases, others in some cases superior in the nM concentration/dose range, and some only for high Copy number TAA cancers are more selective. Affinity for TRDV1 Affinity for EGFR Example molecules potential utility higher affinity higher affinity ADT1-4-2×FS1-65 ADT1-4-2×LEE ADT1-4-2×FS1-67 ADT1-4-2×747 Treating EGFR-positive cancers; the most effective format across multiple measures. By many measures, it was very effective against lower and higher EGFR copy number cancer cells. higher affinity medium affinity ADT1-4-2×LEE2 ADT1-4-2×LEE3 Treatment of EGFR-positive cancers; shown by several measures to cluster high-affinity molecules (see above), but at saturating concentrations possibly in a more efficient format (such as conferring proliferation on Vδ1+ cells) higher affinity lower affinity ADT1-4-2×LEE1 Treating EGFR-positive cancers. There may be greater selectivity for cancer cells with higher EGFR copy numbers. This format will likely bias distribution to Vδ1+ cells. lower affinity higher affinity ADT1-4-6×LEE Treating EGFR-positive cancers. There may be greater selectivity for cancer cells with higher EGFR copy numbers. By some measures, equivalent to higher/higher molecules at saturation (e.g. conferring a proliferative effect on Vδ1+ cells). This format will more likely favor distribution to EGFR-loaded cells. Inhibitory concentration (IC50) TCR downregulation

TCR downregulation can be measured according to the assay described herein. For example, the antibody to be tested can be incubated with a culture of γδ T cells at different concentrations and down-regulation measured. If cell killing is measured (eg, THP-1 cell killing or A375 cell killing), the γδ T cells are co-cultured with an appropriate cell line, such as THP-1 cells or A375 cells. TCR downregulation can be measured by flow cytometry. Cell killing can be achieved by any suitable means, such as by flow cytometry.

For initial TCR down-regulation studies using parental antibodies such as ADT1-7 and ADT1-4, the TCR down-regulation assay involved "loading" the antibodies onto Fcγ receptor-positive THP-1 cells (see Example 1 of WO2021/032963, Example 6 and Table 5). Therefore, in this example, the antibodies are presented on the cell surface and subsequently incubated with γδ T cells. Such loading thus provides the greatest opportunity to exploit the cross-linking effect upon TCR engagement. In addition to loading antibodies onto Fc receptor-positive cells, alternative similar methods of presenting antibodies on solid surfaces may include pre-incubating the antibodies on a disk (so-called disk binding), or using beads to present the antibodies. In all such assays and once presented on a solid surface, the technical effect conferred by the antibody upon engagement of the target receptor (in this case the gamma delta T cell receptor) is typically studied and measured. It is common to present antibodies in this manner, particularly when exploring the role of antibody engagement of immune cell receptor targets and complexes, such as targets such as CD3, CD28, etc.

In contrast, for affinity matured antibodies of the invention as described herein, it is desirable to maximize the analysis and comparison of the effects of affinity maturation, and therefore explore the ability of these antibodies in a more "soluble" TCR downregulation assay format . Assessing effects in solution may also be more physiologically relevant. For these reasons and unless otherwise indicated, all cell-based TCR down-regulation experiments in which the effects of affinity matured antibodies, multispecific antibodies, or fragments thereof are measured or characterized and compared to the parent antibody (e.g., see Figure 35)), explore soluble analysis formats. The IC50 differences using this more stringent solubility analysis are summarized below, where the EC50 values for TCR downregulation of example parent molecules (ADT1-4) are summarized by both methods. TCR downregulation: ‘Present’ versus ‘Soluble’ analysis format: ADT1-4 IC50 (loaded on THP-1 cells): 0.01-0.05 ug/ml = 0.065 nM to 0.325 nM (See Example 1, Example 6 and Table 5 of WO2021/032963) Contrast ADT1-4 IC50 (soluble analysis, added as solution) = 38.28 nM (See Figure 12)

The IC50 of the multispecific antibody or antigen-binding fragment thereof of the present invention for down-regulating TCR may be less than about 50 nM, less than about 10 nM, or less than about 1 mM. Preferably, the IC50 is less than about 1 nM.

In some embodiments, for example, a multispecific antibody or antigen-binding fragment thereof that is related to or derived from the ADT1-4 repertoire (e.g., generated by affinity maturation or otherwise) has a function related to or derived from the ADT1-4 repertoire Multispecific antibodies of the present invention derived from the Fab region of the ADT1-4 lineage), the multispecific antibodies of the present invention or antigen-binding fragments thereof can downregulate TCR with an IC50 of less than about 50 nM, less than about 10 nM, or less than about 1mM. Preferably, the IC50 is less than about 1 nM.

In some embodiments, such as multispecific antibodies or antigen-binding fragments thereof related to or derived from the ADT1-4 repertoire (but excluding ADT1-4-138), the multispecific antibodies or antigens thereof of the invention The IC50 for TCR downregulation by binding fragments can be less than approximately 1 nM. Preferably, the IC50 is less than about 0.5 nM.

In some embodiments, for example, with ADT1-4 lineage members ADT1-4-19, ADT1-4-21, ADT1-4-31, ADT1-4-53, ADT1-4-2, ADT1-4-86, ADT1 -4-112, ADT1-4-143 and/or multispecific antibodies or antigen-binding fragments thereof related to or derived from members of these lineages. The multispecific antibodies or antigen-binding fragments thereof of the present invention allow The IC50 for TCR downregulation can be less than approximately 1 nM. Preferably, the IC50 is less than about 0.5 nM.

In some embodiments, for example, a multispecific antibody or antigen-binding fragment thereof that is related to or derived from the ADT1-7 lineage (e.g., generated by affinity maturation or otherwise) has a gene that is related to or derived from the ADT1-7 lineage. Multispecific antibodies of the present invention derived from the Fab region of the ADT1-7 lineage), the multispecific antibodies of the present invention or antigen-binding fragments thereof can down-regulate TCR with an IC50 of less than about 50 nM, less than about 10 nM, or less than about 1 mM. Preferably, the IC50 is less than about 1 nM.

In some embodiments, for example, a multispecific antibody related to or derived from ADT1-7 lineage member ADT1-7-20 or ADT1-7-3 or Its antigen-binding fragment, the multispecific antibody of the present invention or its antigen-binding fragment can down-regulate TCR with an IC50 of less than about 5 nM. Preferably, the IC50 is less than about 10 nM. cell killing

Cell killing can be measured according to the assay described herein. For example, the antibodies to be tested can be incubated with co-cultures of γδ T cells and tumor cells (eg, THP-1 A375 cells) at varying concentrations. Cell killing can be measured by any suitable means, such as by flow cytometry.

The IC50 of the multispecific antibody or antigen-binding fragment thereof of the present invention for killing A375 cells may be less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.1 nM or less than about 0.01 nM. Preferably, the IC50 is less than about 1 nM.

In some embodiments, for example, a multispecific antibody or antigen-binding fragment thereof that is related to or derived from the ADT1-4 repertoire (e.g., generated by affinity maturation or otherwise) has a function related to or derived from the ADT1-4 repertoire The multispecific antibody of the present invention derived from the Fab region of the ADT1-4 lineage), the IC50 of the multispecific antibody of the present invention or its antigen-binding fragment for killing A375 cells can be less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 1 nM, less than about 0.1 nM, or less than about 0.01 nM. Preferably, the IC50 is less than about 1 nM.

In some embodiments, for example, a multispecific antibody or antigen-binding fragment thereof that is related to or derived from the ADT1-7 lineage (e.g., generated by affinity maturation or otherwise) has a gene that is related to or derived from the ADT1-7 lineage. The multispecific antibody of the present invention derived from the Fab region of the ADT1-7 lineage), the IC50 of the multispecific antibody of the present invention or its antigen-binding fragment for killing A375 cells can be less than about 5 nM, less than about 1 nM, or less than About 0.1 nM. Preferably, the IC50 is less than about 1 nM.

In some embodiments, for example, a multispecific antibody related to or derived from ADT1-7 lineage member ADT1-7-20 or ADT1-7-3 or The IC50 of its antigen-binding fragment, the multispecific antibody or its antigen-binding fragment of the present invention for killing A375 cells may be less than about 10 nM, less than about 5 nM, less than about 1 nM, or less than about 0.1 nM. Preferably, the IC50 is less than about 1 nM.

Of course, the favorable pharmacological profiles of multispecific antibodies can be combined so that the antibodies exhibit favorable _KD and favorable IC50 values for various tested properties.

For example, in some embodiments, the multispecific antibodies or antigen-binding fragments thereof of the invention can: ● With a binding affinity (K _D ) of less than about 100 nM (preferably less than about 50 nM), e.g., via a surface Plasmon resonance measurement) binds to human TRDV1 (SEQ ID NO 272 or 306); ● Optionally with a binding affinity (K _D ) of less than about 100 nM (preferably less than about 50 nM), e.g., by surface plasmon Sub-resonance measurement) binds to cynomolgus monkey TRDV1 (SEQ ID NO 308); ● The IC50 for down-regulating TCR is less than about 50 nM (preferably less than about 1 nM); ● The IC50 for killing A375 cells is less than about 10 nM (more preferably less than about 10 nM) Preferably less than about 5 nM).

In some embodiments, particularly those related to the ADT1-4 repertoire, the multispecific antibody or antigen-binding fragment thereof can: ● bind to human TRDV1 with a K _D of less than about 100 nM (preferably less than about 50 nM); ● Binds to cynomolgus monkey TRDV1 with a K _D of less than about 100 nM (preferably less than about 50 nM); ● The IC50 for down-regulating TCR is less than about 50 nM (preferably less than about 1 nM); ● The IC50 for killing A375 cells Less than about 10 nM (preferably less than about 5 nM).

In some embodiments, such as multispecific antibodies or antigen-binding fragments thereof related to or derived from the ADT1-4 repertoire (but excluding ADT1-4-138), the multispecific antibodies or antigens thereof of the invention The binding fragment may: ● bind to human TRDV1 with a K _D of less than about 10 nM (preferably less than about 5 nM); ● bind to cynomolgus monkey TRDV1 with a K _D of less than about 100 nM (preferably less than about 50 nM); ● The IC50 for down-regulating TCR is less than about 1 nM (preferably less than about 0.5 nM); ● The IC50 for killing A375 cells is less than about 10 nM (preferably less than about 5 nM).

In some embodiments, for example, with ADT1-4 lineage members ADT1-4-19, ADT1-4-21, ADT1-4-31, ADT1-4-53, ADT1-4-2, ADT1-4-86, ADT1 -4-112, ADT1-4-143 and/or multispecific antibodies or antigen-binding fragments thereof related to or derived from members of these lineages. The multispecific antibodies or antigen-binding fragments thereof of the present invention can : ● Binds to human TRDV1 with a K _D of less than about 10 nM (preferably less than about 1 nM); ● Binds to cynomolgus monkey TRDV1 with a K _D of less than about 50 nM; ● The IC50 for down-regulating TCR is less than about 1 nM ( Preferably less than about 0.5 nM); ● The IC50 for killing A375 cells is less than about 10 nM (preferably less than about 5 nM).

In some embodiments, particularly those related to the ADT1-7 lineage, the multispecific antibody or antigen-binding fragment thereof can: ● bind to human TRDV1 with a _KD of less than about 10 nM (preferably less than about 5 nM); ● The IC50 for down-regulating TCR is less than about 50 nM (preferably less than about 10 nM); ● The IC50 for killing A375 cells is less than about 5 nM (preferably less than about 1 nM).

In some embodiments, particularly those related to the ADT1-7 lineage ADT1-7-20 or ADT1-7-3, the multispecific antibody or antigen-binding fragment thereof can: ● be present at less than about 10 nM (preferably less than about Binds to human TRDV1 with a _KD of 5 nM); ● The IC50 for down-regulating TCR is less than about 5 nM (preferably less than about 1 nM); ● The IC50 for killing A375 cells is less than about 10 nM (preferably less than about 5 nM). Pharmacological properties of antibodies containing Fab regions derived from the ADT1-4 lineage

The pharmacological properties of these antibodies with respect to their binding to TRDV1 (via the Fab region) may be within certain parameters. For example, an antibody belonging to or derived from ADT1-4-105 (e.g., a fragment thereof, a variant thereof having one or more amino acid substitutions, or a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus TRDV1 may be less than about 50 nM.

Antibodies belonging to or derived from ADT1-4-107 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM.

Antibodies belonging to or derived from ADT1-4-110 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM.

Antibodies belonging to or derived from ADT1-4-112 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM. Alternatively or additionally, such antibodies can downregulate TCR with an IC50 of less than about 1 nM (preferably less than about 0.5 nM).

Antibodies belonging to or derived from ADT1-4-117 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM.

Antibodies belonging to or derived from ADT1-4-19 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 1 nM (preferably less than about 0.5 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 10 nM (preferably less than about 5 nM). Alternatively or additionally, such antibodies can downregulate TCR with an IC50 of less than about 1 nM (preferably less than about 0.5 nM).

Antibodies belonging to or derived from ADT1-4-21 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM (preferably less than about 10 nM). Alternatively or additionally, such antibodies can downregulate TCR with an IC50 of less than about 1 nM (preferably less than about 0.5 nM).

Antibodies belonging to or derived from ADT1-4-31 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM (preferably less than about 10 nM). Alternatively or additionally, such antibodies can downregulate TCR with an IC50 of less than about 1 nM (preferably less than about 0.5 nM).

Antibodies belonging to or derived from ADT1-4-139 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM.

Antibodies belonging to or derived from ADT1-4-4 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM.

Antibodies belonging to or derived from ADT1-4-143 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM (preferably less than about 10 nM). Alternatively or additionally, such antibodies can downregulate TCR with an IC50 of less than about 1 nM (preferably less than about 0.5 nM).

Antibodies belonging to or derived from ADT1-4-53 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM (preferably less than about 10 nM). Alternatively or additionally, such antibodies can downregulate TCR with an IC50 of less than about 1 nM (preferably less than about 0.5 nM).

Antibodies belonging to or derived from ADT1-4-173 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM.

Antibodies belonging to or derived from ADT1-4-2 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 1 nM (preferably less than about 0.5 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 10 nM (preferably less than about 5 nM). Alternatively or additionally, such antibodies can downregulate TCR with an IC50 of less than about 1 nM (preferably less than about 0.5 nM).

Antibodies belonging to or derived from ADT1-4-8 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM.

Antibodies belonging to or derived from ADT1-4-82 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM.

Antibodies belonging to or derived from ADT1-4-83 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM.

Antibodies belonging to or derived from ADT1-4-3 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM.

Antibodies belonging to or derived from ADT1-4-84 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM.

Antibodies belonging to or derived from ADT1-4-86 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM (preferably less than about 10 nM). Alternatively or additionally, such antibodies can downregulate TCR with an IC50 of less than about 1 nM (preferably less than about 0.5 nM).

Antibodies belonging to or derived from ADT1-4-95 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than about 1 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 50 nM.

Antibodies belonging to or derived from ADT1-4-1 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 1 nM (preferably less than about 0.5 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 10 nM (preferably less than about 1 nM). Alternatively or additionally, such antibodies can downregulate TCR with an IC50 of less than about 1 nM (preferably less than about 0.5 nM).

Antibodies belonging to or derived from ADT1-4-6 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than about 5 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 10 nM (preferably less than about 1 nM). Alternatively or additionally, such antibodies can downregulate TCR with an IC50 of less than about 1 nM (preferably less than about 0.5 nM).

Antibodies belonging to or derived from ADT1-4-138 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 100 nM (preferably less than about 50 nM) and/or the K _D for cynomolgus monkey TRDV1 may be less than about 100 nM (preferably less than about 50 nM). Alternatively or additionally, such antibodies can downregulate TCR with an IC50 of less than about 50 nM (preferably less than about 10 nM). Pharmacological properties of antibodies containing Fab regions derived from the ADT1-7 lineage

The pharmacological properties of these antibodies with respect to their binding to TRDV1 (via the Fab region) may be within certain parameters. For example, an antibody belonging to or derived from ADT1-7-10 (e.g., a fragment thereof, a variant thereof having one or more amino acid substitutions, or a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than about 5 nM).

Antibodies belonging to or derived from ADT1-7-15 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than approximately 5 nM).

Antibodies belonging to or derived from ADT1-7-17 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than approximately 5 nM).

Antibodies belonging to or derived from ADT1-7-18 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than approximately 5 nM).

Antibodies belonging to or derived from ADT1-7-19 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than approximately 5 nM).

Antibodies belonging to or derived from ADT1-7-20 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than approximately 5 nM). Alternatively or additionally, such antibodies can downregulate TCR with an IC50 of less than about 5 nM (preferably less than about 1 nM).

Antibodies belonging to or derived from ADT1-7-22 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than approximately 5 nM).

Antibodies belonging to or derived from ADT1-7-23 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than approximately 5 nM).

Antibodies belonging to or derived from ADT1-7-42 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than approximately 5 nM).

Antibodies belonging to or derived from ADT1-7-3 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a K _D for human TRDV1 of less than about 10 nM (preferably less than approximately 1 nM). Alternatively or additionally, such antibodies can downregulate TCR with an IC50 of less than about 5 nM (preferably less than about 1 nM).

Antibodies belonging to or derived from ADT1-7-61 (e.g., fragments thereof, variants having one or more amino acid substitutions, or having a certain percent identity thereto) may have a _K for human TRDV1 of less than about 10 nM (preferably less than approximately 1 nM). Alternatively or additionally, such antibodies can downregulate TCR with an IC50 of less than about 50 nM (preferably less than about 10 nM). Other functional properties of antibodies

The antibodies of the invention have advantageous functional profiles. Specifically, unlike prior art anti-Vδ1 antibodies that focus on depleting Vδ1 T cells, the antibodies of the present invention can be used to activate Vδ1 T cells. Although it can cause TCR downregulation on the T cells it binds, it does not cause Vδ1 T cell depletion, but in fact it stimulates T cells and can therefore be used in therapeutic contexts that would benefit from activation of this T cell compartment. Activation of Vδ1 T cells is evident through TCR downregulation, changes in activation markers such as CD25 and Ki67, and the degranulation marker CD107a. Activation of Vδ1 T cells in turn triggers the release of inflammatory cytokines such as INFγ and TNFα, thereby promoting immune permissiveness. Surprisingly, antibodies with appropriately high affinity for TRDV1 caused increased Vδ1 T cell killing and, unlike antibodies targeting CD3, for example, could provide high affinity antibodies without the disadvantages associated with large-scale activation via CD3 effect. High-affinity antibodies are in turn capable of inducing strong immunostimulatory effects via tumor-infiltrating lymphocytes (TILs). This can be achieved with minimal depletion or killing of Vδ1 cells. Therefore, the antibodies of the invention can be regarded as agonist antibodies.

In one embodiment of the present invention, an anti-Vδ1 antibody or an antigen-binding fragment thereof is provided, which is characterized in that: a) Causes TCR downregulation on Vδ1 T cells; b) CDC or ADCC is not displayed; and c) Vδ1 T cells are not depleted.

In some embodiments, multispecific anti-Vδ1 antibodies or antigen-binding fragments also stimulate Vδ1 T cell proliferation.

In some embodiments, multispecific antibodies of the invention confer one or more of the following on Vδ1 T cells when they are in the presence (mixed, close, or adjacent) of EGFR-positive cells, such as EGFR+ cancer cells: (a) Enhanced TCR down-regulation effect; (b) Enhanced Vδ1 T cell proliferation; and/or (c) Enhanced Vδ1 T cell activation.

The multispecific antibody, or antigen-binding fragment thereof, may be further defined as binding to human _TRDV1 (SEQ ID NO 272 or 306).

Multispecific antibodies or antigen-binding fragments thereof may be further defined as having favorable _KD and/or IC50 values as discussed above.

T cell exhaustion is the process of removing or reducing T cells through death. Reference to an antibody or antigen-binding fragment that does not deplete Vδ1 T cells means when incubated with one or more multispecific antibodies of the invention as described herein (e.g. when the antibody is provided as an IgG1 antibody) and as in a control Less than about 30% or less than about 20% as measured by any suitable means in the study (e.g., via control flow cytometry or via other established control assays such as those described in Figures 18 and 29) (Preferably less than about 10%) the viable Vδ1 T+ cell population is depleted.

ADCC and CDC are mechanisms by which T cell exhaustion may occur. References herein to an antibody or antigen-binding fragment that does not cause ADCC or CDC means that when raised with one or more multispecific antibodies of the invention as described herein (e.g. when the antibody is provided as an IgG1 antibody), as by Below about 30% or below about 20% (preferably below about 10%) of the viable Vδ1 T+ cell population was depleted via ADCC and/or CDC.

In one embodiment, an anti-Vδ1 antibody or antigen-binding fragment thereof is provided, characterized in that it does not induce IL-17A secretion. IL-17A (interleukin-17A) is a tumor-promoting interleukin produced by activated T cells. IL-17A enhances tumor growth and weakens anti-cancer immune responses. As shown in Figure 40 , anti-vδ1 antibodies did not induce IL-17A secretion when added to a cell population containing human lymphocytes including Vδ1-positive cells, whereas comparative anti-CD3 antibodies induced IL17A in such cases such as OKT 3 . Therefore, reference herein to an antibody or antigen-binding fragment that does not induce IL-17A secretion refers to the induction ratio in such cases by comparing anti-CD3 antibodies (represented by OKT 3; the anti-CD3 comparison used in Figure 63 G to I substance)-induced IL-17A secretion is about 30% lower, or about 20% lower, or about 10% lower. Antibody modification

Antibodies and fragments thereof may be otherwise modified using known methods. Sequence modifications to the antibody molecules described herein will be readily incorporated by those skilled in the art. The following examples are non-limiting.

During antibody discovery and sequence recovery from phage libraries, the desired antibody variable domains can be reformatted into full-length IgG by sub-population. To speed up the process, restriction enzymes are often used to transfer the variable domains. These unique restriction sites can introduce additional/substitute amino acids away from the canonical sequence (such canonical sequences can be found, for example, in the International ImMunoGeneTics [IMGT] information system, see http://www.imgt.org). These can be introduced as kappa or lambda light chain sequence modifications. light chain modification

The variable light chain variable sequences can be selected using restriction sites (eg Nhe1-Not1) during reformatting to full-length IgG. More specifically, at the N-terminus of the light chain, additional Ala-Ser sequences were introduced in the parental (non-affinity matured) antibody to support selection. Preferably, this additional AS sequence is then removed during further development of such antibodies to generate a canonical N-terminal sequence. Therefore, in some embodiments, the light chain-containing antibodies described herein do not contain an AS sequence at their N-terminus, that is, SEQ ID NO: 26, 118, 282 to 290, or 313 does not contain the initial AS sequence. The N-terminus of the light chain sequence of affinity matured antibodies no longer contains this AS motif.

Additional amino acid changes can be made to support selective breeding. For example, for the parent antibodies described herein having a kappa light chain (i.e., B07, C05, E04, F07, G06, G09, B09, G10, G04, and E07), in the kappa light chain variable domain/constant domain boundaries, a valine to alanine change was introduced when preparing the full-length sequence to support selective colonization. This results in kappa constant domain modification. Specifically, this resulted in a constant domain starting with RTAAAPS (from the Notl restriction site). Preferably, such sequences can be modified during further development to generate a typical kappa light chain constant region starting with RTVAAPS. Such modifications do not alter the functional properties of the antibody. Thus, in some embodiments, a kappa light chain-containing antibody described herein contains a constant domain beginning with the sequence RTV (eg, as in SEQ ID NO: 296).

As another example, for the antibodies described herein (specifically, E01 and C08), a lysine to alanine sequence change was introduced at the lambda light chain variable domain/constant domain boundary to support selection. This results in a lambda constant domain modification. Specifically, this resulted in a constant domain starting with GQPAAAPS (from the Notl restriction site). Preferably, this sequence can be modified during further development of such antibodies to generate a typical lambda light chain constant region starting with GQPKAAPS. Thus, in some embodiments, lambda light chain-containing antibodies described herein contain a constant domain beginning with the sequence GQPK. Heavy chain modification

Typically, human variable heavy chain sequences begin with either basic glutamine (Q) or acid glutamine (E). However, it was subsequently known that such sequences are converted into the acidic amino acid residue, pyroglutamic acid (pE). The transition from Q to pE causes a change in the antibody charge, while the transition from E to pE does not change the antibody charge. Therefore, to avoid charge variability - changing over time, one option is to modify the starting heavy chain sequence from Q to E in the first case. Thus, in one embodiment, the heavy chain of an antibody described herein having a Q residue at the N-terminus of the heavy chain may contain a Q to E modification at the N-terminus. In detail, the initial residue of any one of SEQ ID NO: 1, 106, 276 to 279 or 312 may be modified from Q to E. It will be appreciated that this embodiment also applies to any embodiment in which such sequences are incorporated into, for example, a full-length multispecific antibody or an antigen-binding fragment thereof. In some embodiments, it is desirable to replace the E residue at the N-terminus of the heavy chain with an E residue. Therefore, in some embodiments, the E residue at the N-terminus of any of SEQ ID NOs: 2 to 25, 107 to 117, 273 to 275, 280, or 281 may be substituted with a Q residue.

In addition, the C-terminus of the IgG1 constant domain is terminated with PGK. However, the subsequent terminal basic lysine (K, EU position 447) is often cleaved during expression (eg in CHO cells). This, in turn, changes the charge of the antibody through the loss of the C-terminal lysine residue. Therefore, one option would be to remove the lysine in the first case, resulting in a uniform and consistent heavy chain C-terminal sequence ending in PG. Another option is to also remove the terminal G (EU position 446). Thus, in one embodiment, the heavy chain of an antibody described herein has a terminal K or terminal GK removed from its C-terminus.

In some embodiments, the antibody or antigen-binding fragment thereof contains an effector function modified by changing to a sugar linked to Asn 297 (EU numbering scheme). In another such modification, Asn 297 is unfucosylated or exhibits reduced fucosylation (ie, an afucosylated antibody or an afucosylated antibody). Fucosylation involves adding the sugar fucose to molecules, such as attaching fucose to N-glycans, O-glycans, and glycolipids. Therefore, in an afucosylated antibody, fucose is not attached to the carbohydrate chain of the constant region. Antibodies can be modified to prevent or inhibit antibody fucosylation. Typically, glycosylation modification involves expression of the antibody or antigen-binding fragment thereof via targeted engineering or via targeted or incidental host or lineage selection (eg, see Example 13) in host cells containing alternative glycosylation processing capabilities. These and other effector modifications are further discussed in recent reviews such as Xinhua Wang et al. (2018) Protein & Cell 9: 63-73 and Pereira et al. (2018) mAbs 10(5): 693-711 and are incorporated herein. middle. Special modifications depending on the situation

During antibody discovery, specific human isotypes can be employed. Optionally, antibodies may switch to different human isotypes during development. By way of non-limiting example, for the kappa chain there are three human isoforms, called Km1, Km1,2 and Km3, which define three Km alleles (using allotype numbering): Km1 and Valine 153 (IMGT V45. 1) is related to leucine 191 (IMGT L101); Km1,2 is related to alanine 153 (IMGT A45.1) and leucine 191 (IMGT L101); and Km3 is related to alanine 153 (IMGT A45.1) and Valine 191 (IMGT V101) related. Optionally, the sequence can therefore be modified from one isotype to another by standard selective breeding methods. For example, the L191V (IMGT L101V) change converts the Km1,2 allotype into the Km3 allotype. For further reference to such abnormalities, see Jefferis and Lefranc (2009) MAbs 1(4):332-8, which is incorporated herein by reference.

Thus, in one embodiment, the antibodies described herein contain amino acid substitutions derived from another human allotype of the same gene. In another embodiment, the antibody contains an L191V (L101V) substitution of the kappa chain to convert the c-domain from km1,2 to km3 isoform. multispecific antibodies

Antibodies of the invention are multispecific. It can be bispecific or multispecific. Multispecific antibodies can be specific for different epitopes of one target polypeptide or can be specific for more than one target polypeptide. Thus, in one embodiment, the antibody or antigen-binding fragment thereof comprises a first binding specificity for Vδ1 and a second binding specificity for EGFR.

Specifically, the present invention provides a class of novel high-affinity antibodies ("multispecific antibodies") that contain multiple antigen-binding sites, including the antigen-binding site of TCR delta variable 1 (Vδ1) and Antigen binding site of EGFR.

The multispecific antibodies of the invention are in a format called "mAb ² antibodies" or "mAb square antibodies", which are antibodies containing an Fc region engineered to contain an antigen-binding loop in the CH3 domain - this modified Fc The area is called "Fcab". The mAb ² antibody further comprises a Fab region comprising a VH-VL domain pair that provides the antigen binding site. The mAb ² molecules of the present invention include EGFR-binding Fcab and vδ1-binding Fab.

The TCE of the present invention provides several advantages over prior art TCEs. Specifically, TCE can solve many of the challenges associated with prior art TCEs by targeting T cell receptor complexes through a completely novel and discrete mechanism. Indeed, by specifically targeting (and activating) T cell receptor complexes only via epitopes bound to the TRDV1 domain, several advantages are achieved, including: Engaging only a subset of T cells rather than all T cells cells (e.g. T-reg engagement in a cancer context may not be desired); ● Engage only a subset of T cells that are primarily “present in the tissue” and whose presence in the cancer/tumor context is generally positively associated with good prognosis (TRDV1+ T cells) ; ● Activating the T cell receptor complex via TRDV1 engagement, thereby providing more selectivity (for example, increasing the affinity of this binding domain, such as in the antibody of the present invention). For example, by developing recombinant TCEs that engage T cell receptor complexes only via the TRDV1 domain and not via CD3, increased affinity may drive more favorable functionality. For example, high-affinity TRDV1 binding to TCE activates but does not deplete T cells; and/or Engaging TCE complexes in this novel manner and via recombinant TRDV1 binding domains may have less detrimental effects, thus reducing the potential for attenuation of these TCE moieties. The functional requirements of Fc. TCEs with undiminished Fc function are generally expected to induce antibody-dependent cell-mediated cytotoxicity (ADCC), thereby depleting the γδ T cell population recognized by the antibody. However and in addition, toxicity/safety complications can be avoided by attenuating such functionality, for example by engaging CD16+ or CD32+ or CD64+ immune cells or by shortening the half-life of bispecific antibodies (e.g. if smaller bispecific antibodies are used). Specific antibody fragments, such as BITE) to attenuate potentially important efficacy aspects. It is desirable that methods that reduce the interaction of FcyRs with TCE, such as using IgG formats designed to reduce this interaction, reduce Fc-mediated TCE immobilization and reduce TCR clustering due to cross-linking to immobilized TCE. Reducing the need to reduce Fc functionality in these TCE moieties could then provide additional selectivity, such as allowing the TRDV1 TCE to engage TRDV1+ cells via one binding domain and a second cell type (such as cancer cells) via a second binding arm. , and engage other effector cells, such as CD16+ or CD32+ or CD64+ immune cells via a functional Fc domain. ● Activating vδ1 TCR without depleting vδ1+ T cells. Therefore, the multispecific antibodies described herein can engage and activate the CD3/γδ TCR complex and confer downregulation and loss of surface expression without unnecessary depletion of vδ1 T cells (see Figure 29 ). This enables the multispecific antibodies described herein to be used as agents to treat a disease or disorder in order to ameliorate at least one sign or symptom of the disease or disorder via mechanisms involving activation of Vδ1+ T cells present in blood, tissue and tumors.

Therefore, through the discoveries described herein, the present inventors have generated a novel class of recombinant TCEs. Specifically, the present inventors have discovered a new class of TCEs that engage T cell receptors via the TRDV1 domain, but not other domains in these T cell receptor signaling complexes. More specifically, the inventors have discovered a new class of TCEs that bind to this complex via the activation epitope on TRDV1 and can bind with higher affinity without causing binding to the high-affinity T cell receptor complex. Some pre-reported adverse effects. Additionally, this new class of TCEs can be conjugated in a manner that also allows for wild-type Fc functionality, thereby also providing additional efficacy potential.

Multispecific antibodies of the invention may also exhibit improved properties compared to equivalent monospecific antibodies. For example, multispecific antibodies of the invention may also exhibit improved properties compared to monospecific antibodies having an antigen-binding domain that is partially identical to a component of the multispecific antibody. In some embodiments, for example, the recombinant multispecific antibody increases γδ T cell-mediated targeting of diseased cells expressing a second epitope compared to the cytotoxicity conferred by an equivalent amount of the first monospecific antibody. Cytotoxicity. The multispecific antibodies of the present invention can also exhibit improved cytotoxicity against diseased cells while also avoiding healthy cells.

In some embodiments, the multispecific antibody is a human recombinant antibody encoded by a recombinant nucleic acid open reading framework expressed from a recombinant host cell. In some embodiments, the multispecific antibodies are not rodent or other non-human antibodies derived from B cell fusion tumor technology. In some embodiments, the multispecific antibody does not comprise non-human IgG constant domain sequences found only in non-human animal species, such as sequences found in fusionomas of rodent origin.

In preferred embodiments, the multispecific antibodies (suitably bispecific antibodies) of the invention do not specifically bind (or interact with) CD3.

Reference herein to an antigen "on" a cell refers to an antigen expressed on or associated with the cell surface membrane (extracellular side) of such cells.

The second target epitope is EGFR. EGFR (epidermal growth factor receptor) is a cancer-associated antigen and is an example of a receptor tyrosine kinase in the ErbB subfamily. EGFR is expressed in multiple organs and plays an important role in initiating signaling that directs the behavior of epithelial cells and tumors derived from epithelial cells. EGFR-mediated signaling is also related to the control of cell proliferation, migration, survival and cancer metastasis by regulating a variety of cellular pathways. As with other receptor tyrosine kinases, mutations that affect EGFR activity or cause EGFR upregulation are associated with many cancers. Indeed, genetic alterations in EGFR are observed in up to 30% of solid tumors and are often associated with poor prognosis. Disrupting EGFR signaling by inhibiting EGF binding to the extracellular domain or by inhibiting intracellular tyrosine kinase activity may limit the growth of EGFR-expressing tumors. Therefore, EGFR inhibitors can be anticancer agents. Indeed, some tumor cells depend on EGFR signaling and thus have "oncogene addiction," making this receptor an attractive target for therapy. Monoclonal antibodies that specifically bind to epitopes of EGFR are well known in the art. For example, cetuximab is a monoclonal antibody that specifically binds to the epitope of EGFR. Avoid healthy cells

Although the mechanism by which T cells recognize antigens and distinguish healthy cells from diseased cells is not yet fully understood (Ming Heng and Madalene Heng, Antigen Recognition by γδ T-Cells . Madame Curie Bioscience Database [Internet], Austin(TX): Landes Bioscience; 2000- 2013), but the fact that γδ T cells are able to distinguish healthy cells from diseased cells and exhibit significant multicytotoxicity of diseased cells (see non-limiting example cell types in Table 8) means that they can be used to provide Improved potion with improved healing range. Furthermore, by taking advantage of such γδ T cell capabilities, by colocalizing γδ T cells with diseased cells, even when a specific cancer antigen, inflammatory antigen, or pathogen antigen or antigen is not known to be present on healthy cells in a particular patient, May offer the opportunity to treat disease while sparing healthy cells. Table 8. Example cancer cells killed by multicytotoxic human Vδ1+ cells breast cancer M-CSF7, T47D, MDA-MB-231 Mahvi et al. (1993) Cancer Imm. Immunother. (1993) 37:181 - 186 Dutta I et al. (2017). Front. Immunol. 8:776 lung cancer GLC1, N592 Ferrarini et al (1996) Jn of Nat. Cancer Inst., Issue 88(7) pp. 436-441 pancreatic cancer panc89, QGP-1, PANC-1 Maeurer et al. (1996) JEM 183 (4) 1681-1696 Kitayama 1993 Clin Exp Imm 93 (3) 442-7 gastrointestinal cancer HT29, HCT116, Y, SKCO1, Caco2, HCT116, Lovo, DLD-1, SW480 Wu et al. (2015), OncoImmunology, 4:3, e992749 Mikulak, et al. (2019) JCI Insight. 4(24):e125884 Groh et al. (1999) PNAS 96 (12) 6879-6884 Neuroblastoma LAN1、KELLY Fisher et al(2014) Clin Cancer Res; 20(22); 5720-32 melanoma A375 Cordova (2012) Plos ONE 7 (11) e49878 ovarian cancer OV-1063, SW626 Groh et al (1999) PNAS 96 (12) 6879-6884 liver cancer HepG2 Groh et al (1999) PNAS 96 (12) 6879-6884 cervical cancer HeLa Groh et al (1999) PNAS 96 (12) 6879-6884 prostate cancer DV145, PC-3 Groh et al (1999) PNAS 96 (12) 6879-6884 multiple myeloma ARH77, U266 Knight et al(2012) Cytotherapy, 14:9, 1110-1118, AML KG-1, KASUMI-1, OCl-AML3, U937, HL60, MV4 11, AML193, HEL, THP-1 Lorenzo et al. (2019) Canc Imm Res 7 (4) CLL MEC-1 Almeida et al (2016) Clin Can Res 22 (23)

By way of a non-limiting example, recent studies on CD3×EGFR multispecific antibodies highlight the challenges of current or conventional approaches. In particular, the use of such conventional methods can result in a less favorable toxicity profile. This is because, like many other tumor-associated antigens (TAAs), the EGFR antigen is expressed not only in cancers such as breast, colorectal, and head and neck cancers, but also on healthy tissues such as epithelial cells. Therefore, the use of CD3×EGFR agents that engage all T cells and colocalize all T cells with EGFR-positive cells may result in a less favorable therapeutic range or therapeutic index. This is because such agents will engage all T cells, the vast majority of which will be αβ T cells in circulation (CD4+ positive, CD8+ positive, etc.). And once αβ T cells co-localize with EGFR-positive cells, such conventional αβ T cells exhibit limited ability to avoid EGFR+ healthy cells and limited ability to kill only diseased EGFR+ diseased cells. These disorders were further highlighted in an exemplary toxicology study in cynomolgus macaques (males only). In a study by Lutterbuese et al. 2010 (doi: 10.1073/pnas.1000976107), small BITE®-formatted CD3×EGFR bispecific antibodies were used. And because these molecules exhibit a reduced half-life/systemic exposure profile relative to larger antibody-like molecules (>70 Kda), this class of molecules is expected to be relatively well tolerated. However, even at moderately "higher" doses by continuous intravenous infusion of 31 and 154 mcg/kg/day, signs of severe toxicity were observed within 56 hours of starting the infusion. This resulted in the animal being terminated for welfare reasons. Histopathological analysis of dosed animals showed signs of liver and kidney toxicity, which may be the result of lysis of cells that express low levels of EGFR in these organs. Additionally, animals in both high-dose groups showed increased levels of inflammatory cytokines (i.e., TNF-α, IFN-γ, IL-6, IL-5, and IL-2) in serum, presumably due to T cells are released upon encountering EGFR-positive cells. Histopathological changes including lymphocyte infiltration and cell death are noted in all tissues known to express EGFR (i.e., salivary glands, liver, stomach, small intestine, colon, rectum, kidneys, adrenal glands, ureters, bladder, prostate, and epididymis).

Therefore, instead of employing such conventional methods, multispecific antibodies are provided herein, wherein at least one first binding domain is capable of specifically binding to Vδ1+ cells and at least one second binding domain is capable of specifically binding to EGFR (which is present on diseased tissues and cells). Target). Use of such multispecific antibodies in this manner allows Vδ1+ cells to co-localize to diseased cells expressing a secondary target. Furthermore, and given that such disease-relevant targets are often not 100% disease-specific, this method of specifically targeting and co-localizing Vδ1+ effector cells may be superior to conventional methods. This is because Vδ1+ effector cells recognize stress patterns in diseased or infected cells and are therefore able to selectively kill diseased cells while avoiding healthy cells that also express the same target.

Therefore, the multispecific antibodies presented here are able to engage the TCR of vδ1 cells, but full activation does not occur unless tumor cells are also present. Full engagement of the presently presented antibodies on the TCR results in partial down-regulation, and it is believed that vδ1 cells bound by the presently presented antibodies only become fully activated and become cellular when in the presence of stress cells such as tumor cells. toxicity. This is shown in figures such as Figure 18 and Figure 40. This represents another important safety advantage of the approach presented here, since off-target cytotoxicity is reduced and the full potency of multispecific antibodies to activate vδ1 cells is only released in the presence of tumor cells, meaning that healthy cells (even those expressing EGFR) are avoided of healthy cells).

It is believed that one mechanism behind the ability of γδ T cells to detect stress signals on tumor cells is caused by their expression of NCR (natural cytotoxicity receptor). NCR can bind to NCR ligands on tumor cells. A dual activation mechanism may therefore be employed, in which γδ T cells are activated via TCR stimulation, including via NPC, which can sense tumor cells to enable full activation and cytotoxicity.

This contrasts with stimulation of αβ T cells via CD3, for example where all stimulation is via the TCR. Therefore, such cells are virtually indistinguishable between healthy or transformed cells because they do not possess mechanisms such as sensing of tumor cells via NCR, independent of antigen presentation. Therefore, if a CD3 antibody has Fc function, it attracts other immune cells that can trigger a cascade of unpredictable and desirable events, such as interleukin storm, exhaustion and even overactivation of immune cells, leading to, for example, NK cell killing of T cells etc. In the method of the present invention, stimulation of γδ T cells with the multispecific antibodies presented in the invention does not cause such problems because γδ T cells are able to differentiate between healthy cells and tumor cells, including via their NCR sensing mechanism, and therefore due to this Diseased cells specifically and selectively kill stressed cells, such as cancer cells or virus-infected cells.

The second target epitope can be on different cells, including different T cells, B cells, tumor cells, autoimmune tissue cells or virus-infected cells.

Various multispecific formats derived from antibodies have been described previously and are often empirically constructed from component binding moieties. Generally, once constructed, the efficacy of such multispecific or multitarget binding formats as described herein can be demonstrated in one or more of the aforementioned model systems (cell killing, cell proliferation, healthy cell avoidance/diseased cell specificity model, etc.). Where appropriate, it is also compared to those component parts and other comparators.

Although not limited by this method, generally speaking, when constructing an antibody as a multispecific antibody of the invention, the binding domain module of each target (first and second) is bound by Fab and Fcab combined into a full-length antibody. Mod building. Modification and production of multispecific antibodies and desired Fab and Fcab binding domains can be performed by conventional molecular biology methods well known in the art. As a non-limiting example, oligonucleotide-based methods with or without PCR amplification can be used to construct synthetic DNA sequences encoding the desired CH3 domain and/or other elements of the desired bispecific antibody. Optionally, these can then be correctly cloned via restriction enzyme-based subcloning methods into the open reading frame (ORF) encoding the starting antibody heavy chain (such as the antibody heavy chain described herein), so as to use the desired The modified CH3 sequence replaces the starting CH3 sequence. Traditionally, one can use oligonucleotide-based methods with or without PCR amplification to generate DNA ORF elements designed to encode a desired VH region, such as the VH regions described herein, and subsequently select or Switch to a pre-existing heavy chain ORF cassette that already represents the desired CH3 domain coding region. Alternatively, oligonucleotide and/or PCR methods can be used to reconstruct larger elements or ORFs that actually encode the entire antibody heavy chain from the starting in silico sequence to generate the entire final ORF that encodes the preferred full-length heavy chain. The chain includes a VH binding arm and a modified CH3 domain such as a multispecific antibody described herein. This can then be expressed, recovered and purified in combination with a preferred cognate kappa light chain (such as the cognate kappa light chain described herein) to produce a final purified multispecific antibody that binds to the TRDV1 domain of the human Vδ1 TCR and can Operably linked to a modified CH3 binding domain that binds human EGFR.

Notably, when the first binding domain targeting the Vδ1 chain of the γδ TCR is formatted into a multispecific antibody format comprising at least one second binding domain directed against EGFR, the binding domain comprising at least one (first) binding domain is further enhanced. Multispecific antibodies. Multispecific antibodies - non-limiting examples:

To outline the applicability of this approach, a series of non-limiting example multispecific antibodies were constructed. These multispecific antibodies comprise at least one (first) binding domain targeting the Vδ1 chain of the γδ TCR and at least one (second) binding domain targeting EGFR: Vδ1-EGFR multispecific antibodies (LEE)

In this aspect of the invention, there is provided a multispecific antibody comprising a Fab region and an Fc region, wherein the Fab region comprises an epitope having a variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR). Specific binding site; and the Fc region contains an EGFR binding site. Specifically, the Fab region contains VH and homologous VL domains. The Fc region contains the binding domain within the heavy chain constant domain (CH1-CH2-CH3), specifically the CH3 domain. SEQ ID NO: describe 389 ADT1-4-2×LEE(LAGA) 391 EGFR(LEE) binding module CH1-CH2-CH3(LAGA) 392 EGFR(LEE) binding module CH1-CH2-CH3(IgG1 wt) 400 ADT1-4-2×LEE(wt) 432 ADT1-4-2 HC(LAGA)EGFR LEE 436 ADT1-4-2 HC(wt)EGFR LEE 562 EGFR LEE CH3 combination module 523 EGFR LEE AB substituted residues 359 to 362 (EU numbering) 511 EGFR LEE CD substituted residues 384 to 386 (EU numbering) 512 EGFR LEE EF substitutes residues 413 to 419 (EU numbering) 524 EGFR LEE AB loop residues 355 to 362 (EU numbering) 514 EGFR LEE CD loop residues 383 to 391 (EU numbering) 515 EGFR LEE EF loop residues 413 to 422 (EU numbering) 563 IgG1 wt CH3 (residues 341 to 446 (EU numbering)

In this aspect, the EGFR binding site may be provided by the IgG1 CH3 domain, in which residues 359 to 362 (EU numbering) comprise EEGP (SEQ ID NO: 523) and residues 384 to 386 (EU numbering) comprise TYG (SEQ ID NO: 523). NO: 511), and residues 413 to 419 (EU numbering) comprise SYWRWYK (SEQ ID NO: 512).

In this aspect, residues 355 to 362 (EU numbering) form an AB loop containing RDELEEGP (SEQ ID NO: 524) and residues 383 to 391 (EU numbering) form a CD loop containing STYGPENNY (SEQ ID NO: 514 ) and residues 413 to 422 (EU numbering) form the EF loop containing SYWRWYKGNV (SEQ ID NO: 515).

In this aspect, the IgG1 CH3 domain may contain the following mutations (EU numbering). In the AB loop, the following mutations can exist: T359E.K360E.N361G.Q362P. In the CD loop, the following mutations can be present: N384T.G385Y.Q386G. In the EF loop, the following mutations can be present: D413S.K414Y.S415W.Q418Y.Q419K.

In this aspect, the amino acid sequence of the CH3 domain may be at least 90% identical, or at least 95% identical, or 100% identical to SEQ ID NO: 562.

In this aspect, the heavy chain constant domain may comprise SEQ ID NO: 391 or SEQ ID NO: 392.

In this aspect, the multispecific antibody can comprise SEQ ID NO: 389; or SEQ ID NO: 414 and SEQ ID NO: 432. In this aspect, the multispecific antibody can comprise SEQ ID NO: 400; or SEQ ID NO: 414 and SEQ ID NO: 436.

In this aspect, the Fab region can be any Fab region described herein (eg, see Table 9). Specifically, the Fab region of this aspect may comprise VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 68, and the amine groups comprising SEQ ID NO: 79, 80 and 95 respectively. Acid sequences of VLCDR1, VLCDR2 and VLCDR3. A Fab region in this aspect may include a VH including SEQ ID NO: 15 and a VL including SEQ ID NO: 40.

In one embodiment of this aspect, the multispecific antibody comprises: a Fab region including a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 40; and an Fc region comprising a CH3 domain comprising the amino acid sequence of SEQ ID NO: 562. In some embodiments, multispecific antibodies of the invention may comprise an additional lysine (K) residue immediately C-terminal to the CH3 domain sequence (K447, EU numbering). In some embodiments, multispecific antibodies of the invention can remove the terminal glycine (G) residue (G446, EU numbering) immediately C-terminus of the CH3 domain sequence. Table 9. LEE - Exemplary multispecific vδ1-EGFR human IgG1 antibodies with CDRs derived from antibodies of ADT1-4 and ADT1-7 and associated SEQ ID NOs Fab area Fc area VH VL AB ring contains CD ring contains EF ring contains derived from antibodies CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 Parental ADT1-4 51 53 54 79 80 81 Residues 359 to 362 (EU numbering) comprise EEGP SEQ ID NO: 523 Residues 384 to 386 (EU numbering) comprise TYG SEQ ID NO:511 Residues 413 to 419 (EU numbering) contain SYWRWYK SEQ ID NO: 512 ADT1-4-105 51 53 55 79 80 82 ADT1-4-107 51 53 56 79 80 83 ADT1-4-110 51 53 57 79 80 84 ADT1-4-112 51 53 58 79 80 85 ADT1-4-117 51 53 59 79 80 86 ADT1-4-19 51 53 60 79 80 87 ADT1-4-21 52 53 61 79 80 88 ADT1-4-31 51 53 62 79 80 89 ADT1-4-139 51 53 63 79 80 90 ADT1-4-4 51 53 64 79 80 91 ADT1-4-143 51 53 65 79 80 92 ADT1-4-53 52 53 66 79 80 93 ADT1-4-173 51 53 67 79 80 94 ADT1-4-2 51 53 68 79 80 95 ADT1-4-8 51 53 69 79 80 96 ADT1-4-82 51 53 70 79 80 97 ADT1-4-83 51 53 71 79 80 98 ADT1-4-3 51 53 72 79 80 99 ADT1-4-84 51 53 73 79 80 100 ADT1-4-86 52 53 74 79 80 101 ADT1-4-95 51 53 75 79 80 102 ADT1-4-1 51 53 76 79 80 103 ADT1-4-6 51 53 77 79 80 104 ADT1-4-138 51 53 78 79 80 105 Parental ADT1-7 130 131 132 144 145 146 ADT1-7-10 130 131 133 144 145 147 ADT1-7-15 130 131 134 144 145 148 ADT1-7-17 130 131 135 144 145 149 ADT1-7-18 130 131 136 144 145 150 ADT1-7-19 130 131 137 144 145 151 ADT1-7-20 130 131 138 144 145 152 ADT1-7-22 130 131 139 144 145 153 ADT1-7-23 130 131 140 144 145 154 ADT1-7-42 130 131 141 144 145 155 ADT1-7-3 130 131 142 144 145 156 ADT1-7-61 130 131 143 144 145 157

The present disclosure expressly includes antibodies having the sequences listed in the above table. For example, the present disclosure includes a multispecific antibody comprising a Fab region and an Fc region, wherein the Fab region includes an epitope specific for the variable delta 1 (Vdel) chain of the gamma delta T cell receptor (TCR). Binding site, wherein the Fab region includes VHCDR1 comprising the sequence of SEQ ID NO: 51, VHCDR2 comprising the sequence of SEQ ID NO: 53, VHCDR3 comprising the sequence of SEQ ID NO: 68, and VHCDR3 comprising the sequence of SEQ ID NO: 79 VLCDR1, VLCDR2 comprising the sequence of SEQ ID NO: 80 and VLCDR3 comprising the sequence of SEQ ID NO: 95; and the Fc region comprises an EGFR binding site, wherein the EGFR binding site is provided by the following CH3 domain, wherein residues 359 to 362 (EU numbering) contains SEQ ID NO: 523, residues 384 to 386 (EU numbering) contain SEQ ID NO: 511 and residues 413 to 419 (EU numbering) contain SEQ ID NO: 512. Each column of the table above is revealed in the same manner. Such antibodies are human IgG1 antibodies. Accordingly, a human IgGl antibody may comprise a CH3 domain comprising SEQ ID NO: 562. Table 10. LEE - Exemplary multispecific vδ1-EGFR human IgG1 antibodies with variable regions derived from antibodies of ADT1-4 and ADT1-7 and associated SEQ ID NOs Fab area Fc area derived from antibodies VH VL AB ring contains CD ring contains EF ring contains Parental ADT1-4 1 26 Residues 359 to 362 (EU numbering) comprise EEGP SEQ ID NO: 523 Residues 384 to 386 (EU numbering) comprise TYG SEQ ID NO:511 Residues 413 to 419 (EU numbering) contain SYWRWYK SEQ ID NO: 512 ADT1-4-105 2 27 ADT1-4-107 3 28 ADT1-4-110 4 29 ADT1-4-112 5 30 ADT1-4-117 6 31 ADT1-4-19 7 32 ADT1-4-21 8 33 ADT1-4-31 9 34 ADT1-4-139 10 35 ADT1-4-4 11 36 ADT1-4-143 12 37 ADT1-4-53 13 38 ADT1-4-173 14 39 ADT1-4-2 15 40 ADT1-4-8 16 41 ADT1-4-82 17 42 ADT1-4-83 18 43 ADT1-4-3 19 44 ADT1-4-84 20 45 ADT1-4-86 twenty one 46 ADT1-4-95 twenty two 47 ADT1-4-1 twenty three 48 ADT1-4-6 twenty four 49 ADT1-4-138 25 50 Parental ADT1-7 106 118 ADT1-7-10 107 119 ADT1-7-15 108 120 ADT1-7-17 109 121 ADT1-7-18 110 122 ADT1-7-19 111 123 ADT1-7-20 112 124 ADT1-7-22 113 125 ADT1-7-23 114 126 ADT1-7-42 115 127 ADT1-7-3 116 128 ADT1-7-61 117 129 C08 273 282 B07 274 283 C05 275 284 E04 276 285 F07 277 286 G06 278 287 G09 279 288 B09 280 289 G10 281 290 E01 312 313

The present disclosure expressly includes antibodies having the sequences listed in the above table. For example, the present disclosure includes a multispecific antibody comprising a Fab region and an Fc region, wherein the Fab region includes an epitope specific for the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR). Binding site, wherein the Fab region includes a VH region including the sequence of SEQ ID NO: 15 and a VL region including the sequence of SEQ ID NO: 40; and the Fc region includes an EGFR binding site, wherein the EGFR binding site is composed of the following CH3 domain Provided, wherein residues 359 to 362 (EU numbering) comprise SEQ ID NO: 523, residues 384 to 386 (EU numbering) comprise SEQ ID NO: 511 and residues 413 to 419 (EU numbering) comprise SEQ ID NO: 512 . Each column of the table above is revealed in the same manner. Such antibodies are human IgG1 antibodies. Thus, a human IgGl antibody may comprise a CH3 domain comprising SEQ ID NO: 562. Vδ1-EGFR multispecific antibody (LEE1)

In this aspect of the invention, a multispecific antibody is provided that includes a Fab region and an Fc region, wherein the Fab region includes an epitope specific for the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR). specific binding site; and the Fc region contains an EGFR binding site. Specifically, the Fab region contains VH and homologous VL domains. The Fc region contains the binding domain within the heavy chain constant domain (CH1-CH2-CH3), specifically the CH3 domain. SEQ ID NO: describe 504 ADT1-4-2×LEE1(LAGA) 505 ADT1-4-2×LEE1(wt) 506 ADT1-4-2 HC EGFR LEE1(LAGA) 507 ADT1-4-2 HC EGFR LEE1(wt) 508 EGFR LEE1 binding module CH1-CH2-CH3 (LAGA) 509 EGFR LEE1 binding module CH1-CH2-CH3(wt) 510 EGFR LEE1 CH3 binding module 511 EGFR LEE1 CD substituted residues 384 to 386 (EU numbering) 512 EGFR LEE1 EF substituted residues 413 to 419 (EU numbering) 513 EGFR LEE1 AB loop (WT) residues 355 to 362 (EU numbering) 514 EGFR LEE1 CD loop residues 383 to 391 (EU numbering) 515 EGFR LEE1 EF loop residues 413 to 422 (EU numbering)

In this aspect, the EGFR binding site may be provided by the IgG1 CH3 domain, in which residues 384 to 386 (EU numbering) comprise TYG (SEQ ID NO: 511) and residues 413 to 419 (EU numbering) comprise SYWRWYK (SEQ ID NO: 511). NO: 512).

In this aspect, residues 355 to 362 (EU numbering) form the AB loop containing RDELTKNQ (SEQ ID NO: 513), and residues 383 to 391 (EU numbering) form the CD loop containing STYGPENNY (SEQ ID NO: 514 ) and residues 413 to 422 (EU numbering) form the EF loop containing SYWRWYKGNV (SEQ ID NO: 515).

In this aspect, the IgG1 CH3 domain may contain the following mutations (EU numbering). The AB loop can be wild type (no mutations present). In the CD loop, the following mutations can be present: N384T.G385Y.Q386G. In the EF loop, the following mutations can be present: D413S.K414Y.S415W.Q418Y.Q419K.

In this aspect, the amino acid sequence of the CH3 domain may be at least 90% identical, or at least 95% identical, or 100% identical to SEQ ID NO: 510.

In this aspect, the heavy chain constant domain may comprise SEQ ID NO: 508 or SEQ ID NO: 509.

In this aspect, the multispecific antibody can comprise SEQ ID NO: 504; or SEQ ID NO: 414 and SEQ ID NO: 506. In this aspect, the multispecific antibody can comprise SEQ ID NO: 505; or SEQ ID NO: 414 and SEQ ID NO: 507.

In this aspect, the Fab region can be any Fab region described herein (see, eg, Table 11). Specifically, the Fab region of this aspect may comprise VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 68, and the amine groups comprising SEQ ID NO: 79, 80 and 95 respectively. Acid sequences of VLCDR1, VLCDR2 and VLCDR3. A Fab region in this aspect may include a VH including SEQ ID NO: 15 and a VL including SEQ ID NO: 40.

In one embodiment of this aspect, the multispecific antibody comprises: a Fab region including a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 40; and an Fc region comprising a CH3 domain comprising the amino acid sequence of SEQ ID NO: 510. In some embodiments, multispecific antibodies of the invention may comprise an additional lysine (K) residue immediately C-terminal to the CH3 domain sequence (K447, EU numbering). In some embodiments, multispecific antibodies of the invention can remove the terminal glycine (G) residue (G446, EU numbering) immediately C-terminus of the CH3 domain sequence. Table 11. LEE1 - Exemplary multispecific vδ1-EGFR human IgG1 antibodies with CDRs derived from antibodies derived from ADT1-4 and ADT1-7 and associated SEQ ID NOs Fab area Fc area VH VL derived from antibodies CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 AB ring CD ring EF ring Parental ADT1-4 51 53 54 79 80 81 Residues 355 to 362 (EU numbering) contain RDELTKNQ SEQ ID NO: 513 Residues 384 to 386 (EU numbering) comprise TYG SEQ ID NO:511 Residues 413 to 419 (EU numbering) contain SYWRWYK SEQ ID NO: 512 ADT1-4-105 51 53 55 79 80 82 ADT1-4-107 51 53 56 79 80 83 ADT1-4-110 51 53 57 79 80 84 ADT1-4-112 51 53 58 79 80 85 ADT1-4-117 51 53 59 79 80 86 ADT1-4-19 51 53 60 79 80 87 ADT1-4-21 52 53 61 79 80 88 ADT1-4-31 51 53 62 79 80 89 ADT1-4-139 51 53 63 79 80 90 ADT1-4-4 51 53 64 79 80 91 ADT1-4-143 51 53 65 79 80 92 ADT1-4-53 52 53 66 79 80 93 ADT1-4-173 51 53 67 79 80 94 ADT1-4-2 51 53 68 79 80 95 ADT1-4-8 51 53 69 79 80 96 ADT1-4-82 51 53 70 79 80 97 ADT1-4-83 51 53 71 79 80 98 ADT1-4-3 51 53 72 79 80 99 ADT1-4-84 51 53 73 79 80 100 ADT1-4-86 52 53 74 79 80 101 ADT1-4-95 51 53 75 79 80 102 ADT1-4-1 51 53 76 79 80 103 ADT1-4-6 51 53 77 79 80 104 ADT1-4-138 51 53 78 79 80 105 Parental ADT1-7 130 131 132 144 145 146 ADT1-7-10 130 131 133 144 145 147 ADT1-7-15 130 131 134 144 145 148 ADT1-7-17 130 131 135 144 145 149 ADT1-7-18 130 131 136 144 145 150 ADT1-7-19 130 131 137 144 145 151 ADT1-7-20 130 131 138 144 145 152 ADT1-7-22 130 131 139 144 145 153 ADT1-7-23 130 131 140 144 145 154 ADT1-7-42 130 131 141 144 145 155 ADT1-7-3 130 131 142 144 145 156 ADT1-7-61 130 131 143 144 145 157 Table 12. LEE1 - Exemplary multispecific vδ1-EGFR human IgG1 antibodies with CDRs derived from antibodies of ADT1-4 and ADT1-7 and associated SEQ ID NOs Fab area Fc area derived from antibodies VH VL AB ring CD ring EF ring Parental ADT1-4 1 26 Residues 355 to 362 (EU numbering) contain RDELTKNQ SEQ ID NO: 513 Residues 384 to 386 (EU numbering) comprise TYG SEQ ID NO:511 Residues 413 to 419 (EU numbering) contain SYWRWYK SEQ ID NO: 512 ADT1-4-105 2 27 ADT1-4-107 3 28 ADT1-4-110 4 29 ADT1-4-112 5 30 ADT1-4-117 6 31 ADT1-4-19 7 32 ADT1-4-21 8 33 ADT1-4-31 9 34 ADT1-4-139 10 35 ADT1-4-4 11 36 ADT1-4-143 12 37 ADT1-4-53 13 38 ADT1-4-173 14 39 ADT1-4-2 15 40 ADT1-4-8 16 41 ADT1-4-82 17 42 ADT1-4-83 18 43 ADT1-4-3 19 44 ADT1-4-84 20 45 ADT1-4-86 twenty one 46 ADT1-4-95 twenty two 47 ADT1-4-1 twenty three 48 ADT1-4-6 twenty four 49 ADT1-4-138 25 50 Parental ADT1-7 106 118 ADT1-7-10 107 119 ADT1-7-15 108 120 ADT1-7-17 109 121 ADT1-7-18 110 122 ADT1-7-19 111 123 ADT1-7-20 112 124 ADT1-7-22 113 125 ADT1-7-23 114 126 ADT1-7-42 115 127 ADT1-7-3 116 128 ADT1-7-61 117 129 C08 273 282 B07 274 283 C05 275 284 E04 276 285 F07 277 286 G06 278 287 G09 279 288 B09 280 289 G10 281 290 E01 312 313 Vδ1-EGFR multispecific antibody (LEE2)

In this aspect of the invention, a multispecific antibody is provided that includes a Fab region and an Fc region, wherein the Fab region includes an epitope specific for the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR). specific binding site; and the Fc region contains an EGFR binding site. Specifically, the Fab region contains VH and homologous VL domains. The Fc region contains the binding domain within the heavy chain constant domain (CH1-CH2-CH3), specifically the CH3 domain. SEQ ID NO: describe 516 ADT1-4-2×LEE2(LAGA) 517 ADT1-4-2×LEE2(wt) 518 ADT1-4-2 HC EGFR LEE2(LAGA) 519 ADT1-4-2 HC EGFR LEE2(wt) 520 EGFR LEE2 binding module CH1-CH2-CH3 (LAGA) 521 EGFR LEE2 binding module CH1-CH2-CH3(wt) 522 EGFR LEE2 CH3 binding module 523 EGFR LEE2 AB substituted residues 359 to 362 (EU numbering) 512 EGFR LEE2 EF substituted residues 413 to 419 (EU numbering) 524 EGFR LEE2 AB loop residues 355 to 362 (EU numbering) 525 EGFR LEE2 CD loop (WT) residues 383 to 391 (EU numbering) 515 EGFR LEE2 EF loop residues 413 to 422 (EU numbering)

In this aspect, the EGFR binding site may be provided by the IgG1 CH3 domain, in which residues 359 to 362 (EU numbering) comprise EEGP (SEQ ID NO: 523) and residues 413 to 419 (EU numbering) comprise SYWRWYK (SEQ ID NO: 512).

In this aspect, residues 355 to 362 (EU numbering) form an AB loop containing RDELEEGP (SEQ ID NO: 524), and residues 383 to 391 (EU numbering) form a CD loop containing SNGQPENNY (SEQ ID NO: 525 ) and residues 413 to 422 (EU numbering) form the EF loop containing SYWRWYKGNV (SEQ ID NO: 515).

In this aspect, the IgG1 CH3 domain may contain the following mutations (EU numbering). In the AB loop, the following mutations can exist: T359E.K360E.N361G.Q362P. The CD loop can be wild type (no mutations present). In the EF loop, the following mutations can be present: D413S.K414Y.S415W.Q418Y.Q419K.

In this aspect, the amino acid sequence of the CH3 domain may be at least 90% identical, or at least 95% identical, or 100% identical to SEQ ID NO: 522.

In this aspect, the heavy chain constant domain may comprise SEQ ID NO: 520 or SEQ ID NO: 521. In this aspect, the multispecific antibody can comprise SEQ ID NO: 516; or SEQ ID NO: 414 and SEQ ID NO: 518. In this aspect, the multispecific antibody can comprise SEQ ID NO: 517; or SEQ ID NO: 414 and SEQ ID NO: 519.

In this aspect, the Fab region can be any Fab region described herein (see, eg, Table 13). Specifically, the Fab region of this aspect may comprise VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 68, and the amine groups comprising SEQ ID NO: 79, 80 and 95 respectively. Acid sequences of VLCDR1, VLCDR2 and VLCDR3. A Fab region in this aspect may include a VH including SEQ ID NO: 15 and a VL including SEQ ID NO: 40.

In one embodiment of this aspect, the multispecific antibody comprises: a Fab region including a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 40; and an Fc region comprising a CH3 domain comprising the amino acid sequence of SEQ ID NO: 522. In some embodiments, multispecific antibodies of the invention may comprise an additional lysine (K) residue immediately C-terminal to the CH3 domain sequence (K447, EU numbering). In some embodiments, multispecific antibodies of the invention can remove the terminal glycine (G) residue (G446, EU numbering) immediately C-terminus of the CH3 domain sequence. Table 13. LEE2 - Exemplary multispecific vδ1-EGFR human IgG1 antibodies with CDRs derived from antibodies of ADT1-4 and ADT1-7 and associated SEQ ID NOs Fab area Fc area VH VL derived from antibodies CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 AB ring CD ring EF ring Parental ADT1-4 51 53 54 79 80 81 Residues 359 to 362 (EU numbering) comprise EEGP SEQ ID NO: 523 Wild-type residues 383 to 391 (EU numbering) form a CD loop containing SNGQPENNY SEQ ID NO: 525 Residues 413 to 419 (EU numbering) contain SYWRWYK SEQ ID NO: 512 ADT1-4-105 51 53 55 79 80 82 ADT1-4-107 51 53 56 79 80 83 ADT1-4-110 51 53 57 79 80 84 ADT1-4-112 51 53 58 79 80 85 ADT1-4-117 51 53 59 79 80 86 ADT1-4-19 51 53 60 79 80 87 ADT1-4-21 52 53 61 79 80 88 ADT1-4-31 51 53 62 79 80 89 ADT1-4-139 51 53 63 79 80 90 ADT1-4-4 51 53 64 79 80 91 ADT1-4-143 51 53 65 79 80 92 ADT1-4-53 52 53 66 79 80 93 ADT1-4-173 51 53 67 79 80 94 ADT1-4-2 51 53 68 79 80 95 ADT1-4-8 51 53 69 79 80 96 ADT1-4-82 51 53 70 79 80 97 ADT1-4-83 51 53 71 79 80 98 ADT1-4-3 51 53 72 79 80 99 ADT1-4-84 51 53 73 79 80 100 ADT1-4-86 52 53 74 79 80 101 ADT1-4-95 51 53 75 79 80 102 ADT1-4-1 51 53 76 79 80 103 ADT1-4-6 51 53 77 79 80 104 ADT1-4-138 51 53 78 79 80 105 Parental ADT1-7 130 131 132 144 145 146 ADT1-7-10 130 131 133 144 145 147 ADT1-7-15 130 131 134 144 145 148 ADT1-7-17 130 131 135 144 145 149 ADT1-7-18 130 131 136 144 145 150 ADT1-7-19 130 131 137 144 145 151 ADT1-7-20 130 131 138 144 145 152 ADT1-7-22 130 131 139 144 145 153 ADT1-7-23 130 131 140 144 145 154 ADT1-7-42 130 131 141 144 145 155 ADT1-7-3 130 131 142 144 145 156 ADT1-7-61 130 131 143 144 145 157 Table 14. LEE2 - Exemplary multispecific vδ1-EGFR human IgG1 antibodies with variable regions derived from antibodies of ADT1-4 and ADT1-7 and associated SEQ ID NOs Fab area Fc area derived from antibodies VH VL AB ring CD ring EF ring Parental ADT1-4 1 26 Residues 359 to 362 (EU numbering) comprise EEGP SEQ ID NO: 523 Wild-type residues 383 to 391 (EU numbering) form a CD loop containing SNGQPENNY SEQ ID NO: 525 Residues 413 to 419 (EU numbering) contain SYWRWYK SEQ ID NO: 512 ADT1-4-105 2 27 ADT1-4-107 3 28 ADT1-4-110 4 29 ADT1-4-112 5 30 ADT1-4-117 6 31 ADT1-4-19 7 32 ADT1-4-21 8 33 ADT1-4-31 9 34 ADT1-4-139 10 35 ADT1-4-4 11 36 ADT1-4-143 12 37 ADT1-4-53 13 38 ADT1-4-173 14 39 ADT1-4-2 15 40 ADT1-4-8 16 41 ADT1-4-82 17 42 ADT1-4-83 18 43 ADT1-4-3 19 44 ADT1-4-84 20 45 ADT1-4-86 twenty one 46 ADT1-4-95 twenty two 47 ADT1-4-1 twenty three 48 ADT1-4-6 twenty four 49 ADT1-4-138 25 50 Parental ADT1-7 106 118 ADT1-7-10 107 119 ADT1-7-15 108 120 ADT1-7-17 109 121 ADT1-7-18 110 122 ADT1-7-19 111 123 ADT1-7-20 112 124 ADT1-7-22 113 125 ADT1-7-23 114 126 ADT1-7-42 115 127 ADT1-7-3 116 128 ADT1-7-61 117 129 C08 273 282 B07 274 283 C05 275 284 E04 276 285 F07 277 286 G06 278 287 G09 279 288 B09 280 289 G10 281 290 E01 312 313 Vδ1-EGFR multispecific antibody (LEE3)

In this aspect of the invention, a multispecific antibody is provided that includes a Fab region and an Fc region, wherein the Fab region includes an epitope specific for the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR). specific binding site; and the Fc region contains an EGFR binding site. Specifically, the Fab region contains VH and homologous VL domains. The Fc region contains the binding domain within the heavy chain constant domain (CH1-CH2-CH3), specifically the CH3 domain. SEQ ID NO: describe 526 ADT1-4-2×LEE3(LAGA) 527 ADT1-4-2×LEE3(wt) 528 ADT1-4-2 HC EGFR LEE3(LAGA) 529 ADT1-4-2 HC EGFR LEE3(wt) 530 EGFR LEE3 binding module CH1-CH2-CH3 (LAGA) 531 EGFR LEE3 binding module CH1-CH2-CH3(wt) 532 EGFR LEE3 CH3 binding module 523 EGFR LEE3 AB substituted residues 359 to 362 (EU numbering) 511 EGFR LEE3 CD substituted residues 384 to 386 (EU numbering) 533 EGFR LEE3 EF substituted residues 413 to 415 (EU numbering) 524 EGFR LEE3 AB loop residues 355 to 362 (EU numbering) 514 EGFR LEE3 CD loop residues 383 to 391 (EU numbering) 534 EGFR LEE3 EF loop residues 413 to 422 (EU numbering)

In this aspect, the EGFR binding site may be provided by the IgG1 CH3 domain, in which residues 359 to 362 (EU numbering) comprise EEGP (SEQ ID NO: 523) and residues 384 to 386 (EU numbering) comprise TYG (SEQ ID NO: 523). NO: 511), and residues 413 to 415 (EU numbering) comprise SYW (SEQ ID NO: 533).

In this aspect, residues 355 to 362 (EU numbering) form an AB loop containing RDELEEGP (SEQ ID NO: 524) and residues 383 to 391 (EU numbering) form a CD loop containing STYGPENNY (SEQ ID NO: 514 ), and residues 413 to 422 (EU numbering) form the EF loop containing SYWRWQQGNV (SEQ ID NO: 534).

In this aspect, the IgG1 CH3 domain may contain the following mutations (EU numbering). In the AB loop, the following mutations can exist: T359E.K360E.N361G.Q362P. In the CD loop, the following mutations can be present: N384T.G385Y.Q386G. In the EF loop, the following mutations can be present: D413S.K414Y.S415W.

In this aspect, the amino acid sequence of the CH3 domain may be at least 90% identical, or at least 95% identical, or 100% identical to SEQ ID NO: 532.

In this aspect, the heavy chain constant domain may comprise SEQ ID NO: 530 or SEQ ID NO: 531.

In this aspect, the multispecific antibody can comprise SEQ ID NO: 526; or SEQ ID NO: 414 and SEQ ID NO: 528. In this aspect, the multispecific antibody can comprise SEQ ID NO: 527; or SEQ ID NO: 414 and SEQ ID NO: 529.

In this aspect, the Fab region can be any Fab region described herein (see, eg, Table 15). Specifically, the Fab region of this aspect may comprise VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 68, and the amine groups comprising SEQ ID NO: 79, 80 and 95 respectively. Acid sequences of VLCDR1, VLCDR2 and VLCDR3. A Fab region in this aspect may include a VH including SEQ ID NO: 15 and a VL including SEQ ID NO: 40.

In one embodiment of this aspect, the multispecific antibody comprises: a Fab region including a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 40; and an Fc region comprising a CH3 domain comprising the amino acid sequence of SEQ ID NO: 532. In some embodiments, multispecific antibodies of the invention may comprise an additional lysine (K) residue immediately C-terminal to the CH3 domain sequence (K447, EU numbering). In some embodiments, multispecific antibodies of the invention can remove the terminal glycine (G) residue (G446, EU numbering) immediately C-terminus of the CH3 domain sequence. Table 15. LEE3 - Exemplary multispecific vδ1-EGFR human IgG1 antibodies with CDRs derived from antibodies of ADT1-4 and ADT1-7 and associated SEQ ID NOs Fab area Fc area VH VL derived from antibodies CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 AB ring CD ring EF ring Parental ADT1-4 51 53 54 79 80 81 Residues 359 to 362 (EU numbering) comprise EEGP SEQ ID NO: 523 Residues 384 to 386 (EU numbering) comprise TYG SEQ ID NO:511 Residues 413 to 415 (EU numbering) comprise SYW SEQ ID NO: 533 ADT1-4-105 51 53 55 79 80 82 ADT1-4-107 51 53 56 79 80 83 ADT1-4-110 51 53 57 79 80 84 ADT1-4-112 51 53 58 79 80 85 ADT1-4-117 51 53 59 79 80 86 ADT1-4-19 51 53 60 79 80 87 ADT1-4-21 52 53 61 79 80 88 ADT1-4-31 51 53 62 79 80 89 ADT1-4-139 51 53 63 79 80 90 ADT1-4-4 51 53 64 79 80 91 ADT1-4-143 51 53 65 79 80 92 ADT1-4-53 52 53 66 79 80 93 ADT1-4-173 51 53 67 79 80 94 ADT1-4-2 51 53 68 79 80 95 ADT1-4-8 51 53 69 79 80 96 ADT1-4-82 51 53 70 79 80 97 ADT1-4-83 51 53 71 79 80 98 ADT1-4-3 51 53 72 79 80 99 ADT1-4-84 51 53 73 79 80 100 ADT1-4-86 52 53 74 79 80 101 ADT1-4-95 51 53 75 79 80 102 ADT1-4-1 51 53 76 79 80 103 ADT1-4-6 51 53 77 79 80 104 ADT1-4-138 51 53 78 79 80 105 Parental ADT1-7 130 131 132 144 145 146 ADT1-7-10 130 131 133 144 145 147 ADT1-7-15 130 131 134 144 145 148 ADT1-7-17 130 131 135 144 145 149 ADT1-7-18 130 131 136 144 145 150 ADT1-7-19 130 131 137 144 145 151 ADT1-7-20 130 131 138 144 145 152 ADT1-7-22 130 131 139 144 145 153 ADT1-7-23 130 131 140 144 145 154 ADT1-7-42 130 131 141 144 145 155 ADT1-7-3 130 131 142 144 145 156 ADT1-7-61 130 131 143 144 145 157 Table 16. LEE3 - Exemplary multispecific vδ1-EGFR human IgG1 antibodies with variable regions derived from antibodies of ADT1-4 and ADT1-7 and associated SEQ ID NOs Fab area Fc area derived from antibodies VH VL AB ring CD ring EF ring Parental ADT1-4 1 26 Residues 359 to 362 (EU numbering) comprise EEGP SEQ ID NO: 523 Residues 384 to 386 (EU numbering) comprise TYG SEQ ID NO:511 Residues 413 to 415 (EU numbering) comprise SYW SEQ ID NO: 533 ADT1-4-105 2 27 ADT1-4-107 3 28 ADT1-4-110 4 29 ADT1-4-112 5 30 ADT1-4-117 6 31 ADT1-4-19 7 32 ADT1-4-21 8 33 ADT1-4-31 9 34 ADT1-4-139 10 35 ADT1-4-4 11 36 ADT1-4-143 12 37 ADT1-4-53 13 38 ADT1-4-173 14 39 ADT1-4-2 15 40 ADT1-4-8 16 41 ADT1-4-82 17 42 ADT1-4-83 18 43 ADT1-4-3 19 44 ADT1-4-84 20 45 ADT1-4-86 twenty one 46 ADT1-4-95 twenty two 47 ADT1-4-1 twenty three 48 ADT1-4-6 twenty four 49 ADT1-4-138 25 50 Parental ADT1-7 106 118 ADT1-7-10 107 119 ADT1-7-15 108 120 ADT1-7-17 109 121 ADT1-7-18 110 122 ADT1-7-19 111 123 ADT1-7-20 112 124 ADT1-7-22 113 125 ADT1-7-23 114 126 ADT1-7-42 115 127 ADT1-7-3 116 128 ADT1-7-61 117 129 C08 273 282 B07 274 283 C05 275 284 E04 276 285 F07 277 286 G06 278 287 G09 279 288 B09 280 289 G10 281 290 E01 312 313 Vδ1-EGFR multispecific antibody (FS1-67)

In this aspect of the invention, a multispecific antibody is provided that includes a Fab region and an Fc region, wherein the Fab region includes an epitope specific for the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR). specific binding site; and the Fc region contains an EGFR binding site. Specifically, the Fab region contains VH and homologous VL domains. The Fc region contains the binding domain within the heavy chain constant domain (CH1-CH2-CH3), specifically the CH3 domain. SEQ ID NO: describe 377 Anti-chicken lysozyme×FS1-67 (EGFR binding domain) 378 Parent C08×FS1-67(LAGA) 379 Parental G04×FS1-67(LAGA) 380 Parent E07×FS1-67(LAGA) 385 EGFR(FS-167) binding module CH1-CH2-CH3(IgG1 wt) 386 EGFR(FS-167) combined module CH1-CH2-CH3(LAGA) 388 ADT1-4-2×FS1-67 EGFR(LAGA) 399 ADT1-4-2×FS1-67 EGFR(wt) 426 C08 HC(LAGA)EGFR FS1-67 427 E07 HC(LAGA)EGFR FS1-67 431 ADT1-4-2 HC(LAGA)EGFR FS1-67 435 ADT1-4-2 HC(wt)EGFR FS1-67 437 G04 HC(LAGA)EGFR FS1-67 559 EGFR FS1-67 CH3 binding module 560 EGFR FS1-67 AB substituted residues 358 to 362 (EU numbering) 511 EGFR FS1-67 CD substituted residues 384 to 386 (EU numbering) 512 EGFR FS1-67 EF substituted residues 413 to 419 (EU numbering) 561 EGFR FS1-67 AB loop residues 355 to 362 (EU numbering) 514 EGFR FS1-67 CD loop residues 383 to 391 (EU numbering) 515 EGFR FS1-67 EF loop residues 413 to 422 (EU numbering)

In this aspect, the EGFR binding site may be provided by the IgG1 CH3 domain, in which residues 358 to 362 (EU numbering) comprise TDDGP (SEQ ID NO: 560) and residues 384 to 386 (EU numbering) comprise TYG (SEQ ID NO: 560). NO: 511), and residues 413 to 419 (EU numbering) comprise SYWRWYK (SEQ ID NO: 512).

In this aspect, residues 355 to 362 (EU numbering) form an AB loop containing RDETDDGP (SEQ ID NO: 561), and residues 383 to 391 (EU numbering) form a CD loop containing STYGPENNY (SEQ ID NO: 514 ), and residues 413 to 422 (EU numbering) form the EF loop containing SYWRWYKGNV (SEQ ID NO: 515).

In this aspect, the IgG1 CH3 domain may contain the following mutations (EU numbering). In the AB loop, the following mutations can exist: L358T.T359D.K360D.N361G.Q362P. In the CD loop, the following mutations can be present: N384T.G385Y.Q386G. In the EF loop, the following mutations can be present: D413S.K414Y.S415W.Q418Y.Q419K.

In this aspect, the amino acid sequence of the CH3 domain may be at least 90% identical, or at least 95% identical, or 100% identical to SEQ ID NO: 559.

In this aspect, the heavy chain constant domain may comprise SEQ ID NO: 385 or SEQ ID NO: 386.

In this aspect, the multispecific antibody can comprise SEQ ID NO: 388; or SEQ ID NO: 414 and SEQ ID NO: 431. In this aspect, the multispecific antibody can comprise SEQ ID NO: 399; or SEQ ID NO: 414 and SEQ ID NO: 435.

In this aspect, the Fab region can be any Fab region described herein (see, eg, Table 17). Specifically, the Fab region of this aspect may comprise VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 68, and the amine groups comprising SEQ ID NO: 79, 80 and 95 respectively. Acid sequences of VLCDR1, VLCDR2 and VLCDR3. A Fab region in this aspect may include a VH including SEQ ID NO: 15 and a VL including SEQ ID NO: 40.

In one embodiment of this aspect, the multispecific antibody comprises: a Fab region including a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 40; and an Fc region comprising a CH3 domain comprising the amino acid sequence of SEQ ID NO: 559. In some embodiments, multispecific antibodies of the invention may comprise an additional lysine (K) residue immediately C-terminal to the CH3 domain sequence (K447, EU numbering). In some embodiments, multispecific antibodies of the invention can remove the terminal glycine (G) residue (G446, EU numbering) immediately C-terminus of the CH3 domain sequence.

In one embodiment, the multispecific antibody of the invention is not ADT1-4xFS1-67. Table 17. FS1-67 - Exemplary multispecific vδ1-EGFR human IgG1 antibody with CDRs derived from antibodies derived from ADT1-4 and ADT1-7 and associated SEQ ID NOs Fab area Fc area VH VL derived from antibodies CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 AB ring CD ring EF ring Parental ADT1-4 51 53 54 79 80 81 Residues 358 to 362 (EU numbering) comprise TDDGP SEQ ID NO: 560 Residues 384 to 386 (EU numbering) comprise TYG SEQ ID NO:511 Residues 413 to 419 (EU numbering) contain SYWRWYK SEQ ID NO: 512 ADT1-4-105 51 53 55 79 80 82 ADT1-4-107 51 53 56 79 80 83 ADT1-4-110 51 53 57 79 80 84 ADT1-4-112 51 53 58 79 80 85 ADT1-4-117 51 53 59 79 80 86 ADT1-4-19 51 53 60 79 80 87 ADT1-4-21 52 53 61 79 80 88 ADT1-4-31 51 53 62 79 80 89 ADT1-4-139 51 53 63 79 80 90 ADT1-4-4 51 53 64 79 80 91 ADT1-4-143 51 53 65 79 80 92 ADT1-4-53 52 53 66 79 80 93 ADT1-4-173 51 53 67 79 80 94 ADT1-4-2 51 53 68 79 80 95 ADT1-4-8 51 53 69 79 80 96 ADT1-4-82 51 53 70 79 80 97 ADT1-4-83 51 53 71 79 80 98 ADT1-4-3 51 53 72 79 80 99 ADT1-4-84 51 53 73 79 80 100 ADT1-4-86 52 53 74 79 80 101 ADT1-4-95 51 53 75 79 80 102 ADT1-4-1 51 53 76 79 80 103 ADT1-4-6 51 53 77 79 80 104 ADT1-4-138 51 53 78 79 80 105 Parental ADT1-7 130 131 132 144 145 146 ADT1-7-10 130 131 133 144 145 147 ADT1-7-15 130 131 134 144 145 148 ADT1-7-17 130 131 135 144 145 149 ADT1-7-18 130 131 136 144 145 150 ADT1-7-19 130 131 137 144 145 151 ADT1-7-20 130 131 138 144 145 152 ADT1-7-22 130 131 139 144 145 153 ADT1-7-23 130 131 140 144 145 154 ADT1-7-42 130 131 141 144 145 155 ADT1-7-3 130 131 142 144 145 156 ADT1-7-61 130 131 143 144 145 157 Table 18. FS1-67 - Exemplary multispecific vδ1-EGFR human IgG1 antibodies with variable regions derived from antibodies of ADT1-4 and ADT1-7 and associated SEQ ID NOs Fab area Fc area derived from antibodies VH VL AB ring CD ring EF ring Parental ADT1-4 1 26 Residues 358 to 362 (EU numbering) comprise TDDGP SEQ ID NO: 560 Residues 384 to 386 (EU numbering) comprise TYG SEQ ID NO:511 Residues 413 to 419 (EU numbering) contain SYWRWYK SEQ ID NO: 512 ADT1-4-105 2 27 ADT1-4-107 3 28 ADT1-4-110 4 29 ADT1-4-112 5 30 ADT1-4-117 6 31 ADT1-4-19 7 32 ADT1-4-21 8 33 ADT1-4-31 9 34 ADT1-4-139 10 35 ADT1-4-4 11 36 ADT1-4-143 12 37 ADT1-4-53 13 38 ADT1-4-173 14 39 ADT1-4-2 15 40 ADT1-4-8 16 41 ADT1-4-82 17 42 ADT1-4-83 18 43 ADT1-4-3 19 44 ADT1-4-84 20 45 ADT1-4-86 twenty one 46 ADT1-4-95 twenty two 47 ADT1-4-1 twenty three 48 ADT1-4-6 twenty four 49 ADT1-4-138 25 50 Parental ADT1-7 106 118 ADT1-7-10 107 119 ADT1-7-15 108 120 ADT1-7-17 109 121 ADT1-7-18 110 122 ADT1-7-19 111 123 ADT1-7-20 112 124 ADT1-7-22 113 125 ADT1-7-23 114 126 ADT1-7-42 115 127 ADT1-7-3 116 128 ADT1-7-61 117 129 C08 273 282 B07 274 283 C05 275 284 E04 276 285 F07 277 286 G06 278 287 G09 279 288 B09 280 289 G10 281 290 E01 312 313 Vδ1-EGFR multispecific antibody (FS1-65)

In this aspect of the invention, a multispecific antibody is provided that includes a Fab region and an Fc region, wherein the Fab region includes an epitope specific for the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR). specific binding site; and the Fc region contains an EGFR binding site. Specifically, the Fab region contains VH and homologous VL domains. The Fc region contains the binding domain within the heavy chain constant domain (CH1-CH2-CH3), specifically the CH3 domain. SEQ ID NO: describe 535 ADT1-4-2×FS1-65(LAGA) 536 ADT1-4-2×FS1-65(wt) 537 ADT1-4-2 HC EGFR FS1-65(LAGA) 538 ADT1-4-2 HC EGFR FS1-65(wt) 539 EGFR FS1-65 combined module CH1-CH2-CH3 (LAGA) 540 EGFR FS1-65 combined module CH1-CH2-CH3(wt) 541 EGFR FS1-65 CH3 binding module 542 EGFR FS1-65 AB replaces residues 358 to 362 ( inclusive of 361.1) (EU numbering) 511 EGFR FS1-65 CD substituted residues 384 to 386 (EU numbering) 543 EGFR FS1-65 EF substituted residues 413 to 419 (EU numbering) 544 EGFR FS1-65 AB loop residues 355 to 362 (including 361.1) (EU numbering) 514 EGFR FS1-65 CD loop residues 383 to 391 (EU numbering) 545 EGFR FS1-65 EF loop residues 413 to 422 (EU numbering)

In this aspect, the EGFR binding site may be provided by the IgG1 CH3 domain, in which residues 358 to 361, 361.1 and 362 (EU numbering) comprise LDEGGP (SEQ ID NO: 542) and residues 384 to 386 (EU numbering) comprise TYG (SEQ ID NO: 511), and residues 413 to 419 (EU numbering) contain SYWRWVK (SEQ ID NO: 543). Among them, residue 361.1 represents an additional residue (EU numbering) between residues 361 and 362.

In this aspect, residues 355 to 362, including residue 361.1 (EU numbering) form an AB loop containing RDELDEGGP (SEQ ID NO: 544), and residues 383 to 391 (EU numbering) form a CD loop containing STYGPENNY ( SEQ ID NO: 514), and residues 413 to 422 (EU numbering) form the EF loop containing SYWRWVKGNV (SEQ ID NO: 545). Among them, residue 361.1 represents an additional residue (EU numbering) between residues 361 and 362.

In this aspect, the IgG1 CH3 domain may contain the following mutations (EU numbering). In the AB ring, the following mutations can exist: T359D.K360E.N361G.361-1G.Q362P. In the CD loop, the following mutations can be present: N384K.G385F.Q386G. In the EF loop, the following mutations can be present: D413S.K414Y.S415W.Q418V.Q419K. Among them, residue 361-1 represents an additional residue (EU numbering) between residues 361 and 362.

In this aspect, the amino acid sequence of the CH3 domain may be at least 90% identical, or at least 95% identical, or 100% identical to SEQ ID NO: 541.

In this aspect, the heavy chain constant domain may comprise SEQ ID NO: 539 or SEQ ID NO: 540.

In this aspect, the multispecific antibody can comprise SEQ ID NO: 535; or SEQ ID NO: 414 and SEQ ID NO: 537. In this aspect, the multispecific antibody can comprise SEQ ID NO: 536; or SEQ ID NO: 414 and SEQ ID NO: 538.

In this aspect, the Fab region can be any Fab region described herein (see, eg, Table 19). Specifically, the Fab region of this aspect may comprise VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 68, and the amine groups comprising SEQ ID NO: 79, 80 and 95 respectively. Acid sequences of VLCDR1, VLCDR2 and VLCDR3. A Fab region in this aspect may include a VH including SEQ ID NO: 15 and a VL including SEQ ID NO: 40.

In one embodiment of this aspect, the multispecific antibody comprises: a Fab region including a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 40; and an Fc region comprising a CH3 domain comprising the amino acid sequence of SEQ ID NO: 541. In some embodiments, multispecific antibodies of the invention may comprise a complete lysine (K) residue (K447, EU numbering) immediately C-terminus of the CH3 domain sequence. In some embodiments, multispecific antibodies of the invention can remove the terminal glycine (G) residue (G446, EU numbering) immediately C-terminus of the CH3 domain sequence. Table 19. FS1-65 - Exemplary multispecific vδ1-EGFR human IgG1 antibody with CDRs derived from antibodies of ADT1-4 and ADT1-7 and associated SEQ ID NOs Fab area Fc area VH VL derived from antibodies CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 AB ring CD ring EF ring Parental ADT1-4 51 53 54 79 80 81 Residues 358 to 361, 361.1 and 362 (EU numbering) comprise LDEGGP SEQ ID NO: 542 Residues 384 to 386 (EU numbering) comprise TYG SEQ ID NO:511 Residues 413 to 419 (EU numbering) comprise SYWRWVK SEQ ID NO: 543 ADT1-4-105 51 53 55 79 80 82 ADT1-4-107 51 53 56 79 80 83 ADT1-4-110 51 53 57 79 80 84 ADT1-4-112 51 53 58 79 80 85 ADT1-4-117 51 53 59 79 80 86 ADT1-4-19 51 53 60 79 80 87 ADT1-4-21 52 53 61 79 80 88 ADT1-4-31 51 53 62 79 80 89 ADT1-4-139 51 53 63 79 80 90 ADT1-4-4 51 53 64 79 80 91 ADT1-4-143 51 53 65 79 80 92 ADT1-4-53 52 53 66 79 80 93 ADT1-4-173 51 53 67 79 80 94 ADT1-4-2 51 53 68 79 80 95 ADT1-4-8 51 53 69 79 80 96 ADT1-4-82 51 53 70 79 80 97 ADT1-4-83 51 53 71 79 80 98 ADT1-4-3 51 53 72 79 80 99 ADT1-4-84 51 53 73 79 80 100 ADT1-4-86 52 53 74 79 80 101 ADT1-4-95 51 53 75 79 80 102 ADT1-4-1 51 53 76 79 80 103 ADT1-4-6 51 53 77 79 80 104 ADT1-4-138 51 53 78 79 80 105 Parental ADT1-7 130 131 132 144 145 146 ADT1-7-10 130 131 133 144 145 147 ADT1-7-15 130 131 134 144 145 148 ADT1-7-17 130 131 135 144 145 149 ADT1-7-18 130 131 136 144 145 150 ADT1-7-19 130 131 137 144 145 151 ADT1-7-20 130 131 138 144 145 152 ADT1-7-22 130 131 139 144 145 153 ADT1-7-23 130 131 140 144 145 154 ADT1-7-42 130 131 141 144 145 155 ADT1-7-3 130 131 142 144 145 156 ADT1-7-61 130 131 143 144 145 157 Table 20. FS1-65 - Exemplary multispecific vδ1-EGFR human IgG1 antibodies with variable regions derived from antibodies of ADT1-4 and ADT1-7 and associated SEQ ID NOs Fab area Fc area derived from antibodies VH VL AB ring CD ring EF ring Parental ADT1-4 1 26 Residues 358 to 361, 361.1 and 362 (EU numbering) comprise LDEGGP SEQ ID NO: 542 Residues 384 to 386 (EU numbering) comprise TYG SEQ ID NO:511 Residues 413 to 419 (EU numbering) comprise SYWRWVK SEQ ID NO: 543 ADT1-4-105 2 27 ADT1-4-107 3 28 ADT1-4-110 4 29 ADT1-4-112 5 30 ADT1-4-117 6 31 ADT1-4-19 7 32 ADT1-4-21 8 33 ADT1-4-31 9 34 ADT1-4-139 10 35 ADT1-4-4 11 36 ADT1-4-143 12 37 ADT1-4-53 13 38 ADT1-4-173 14 39 ADT1-4-2 15 40 ADT1-4-8 16 41 ADT1-4-82 17 42 ADT1-4-83 18 43 ADT1-4-3 19 44 ADT1-4-84 20 45 ADT1-4-86 twenty one 46 ADT1-4-95 twenty two 47 ADT1-4-1 twenty three 48 ADT1-4-6 twenty four 49 ADT1-4-138 25 50 Parental ADT1-7 106 118 ADT1-7-10 107 119 ADT1-7-15 108 120 ADT1-7-17 109 121 ADT1-7-18 110 122 ADT1-7-19 111 123 ADT1-7-20 112 124 ADT1-7-22 113 125 ADT1-7-23 114 126 ADT1-7-42 115 127 ADT1-7-3 116 128 ADT1-7-61 117 129 C08 273 282 B07 274 283 C05 275 284 E04 276 285 F07 277 286 G06 278 287 G09 279 288 B09 280 289 G10 281 290 E01 312 313 Vδ1-EGFR multispecific antibody (747)

In this aspect of the invention, a multispecific antibody is provided that includes a Fab region and an Fc region, wherein the Fab region includes an epitope specific for the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR). specific binding site; and the Fc region contains an EGFR binding site. Specifically, the Fab region contains VH and homologous VL domains. The Fc region contains the binding domain within the heavy chain constant domain (CH1-CH2-CH3), specifically the CH3 domain. SEQ ID NO: describe 546 ADT1-4-2×747(LAGA) 547 ADT1-4-2×747(wt) 548 ADT1-4-2 HC EGFR 747(LAGA) 549 ADT1-4-2 HC EGFR 747(wt) 550 EGFR 747 binding module CH1-CH2-CH3 (LAGA) 551 EGFR 747 binding module CH1-CH2-CH3(wt) 552 747 CH3 combination module-plus additional structural mutation A378V 553 EGFR 747 AB replaces residues 358 to 362 (EU numbering) 554 EGFR 747 CD substituted residues 384 to 386 (EU numbering) 555 EGFR 747 EF substituted residues 413 to 421 (EU numbering) 556 EGFR 747 AB loop residues 355 to 362 (EU numbering) 557 EGFR 747 CD loop residues 383 to 391 (EU numbering) 558 EGFR 747 EF loop residues 413 to 422 (EU numbering)

In this aspect, the EGFR binding site may be provided by the IgG1 CH3 domain, in which residues 358 to 362 (EU numbering) comprise TESGP (SEQ ID NO: 553) and residues 384 to 386 (EU numbering) comprise KFG (SEQ ID NO: 553). NO: 554), residues 413 to 421 (EU numbering) comprise SNLRWTKGH (SEQ ID NO: 555), and residue 378 is valine.

In this aspect, residues 355 to 362 (EU numbering) form an AB loop containing RDETESGP (SEQ ID NO: 556) and residues 383 to 391 (EU numbering) form a CD loop containing SKFGPENNY (SEQ ID NO: 557 ), residues 413 to 422 (EU numbering) form the EF loop containing SNLRWTKGHV (SEQ ID NO: 558) and residue 378 is valine.

In this aspect, the IgG1 CH3 domain may contain the following mutations (EU numbering). In the AB loop, the following mutations can exist: L358T.T359E.K360S.N361G.Q362P. In the CD loop, the following mutations can be present: N384K.G385F.Q386G. In the EF loop, the following mutations can be present: D413S.K414N.S415L.Q418T.Q419K.N421H. The following mutation also exists: A378V.

In this aspect, the amino acid sequence of the CH3 domain may be at least 90% identical, or at least 95% identical, or 100% identical to SEQ ID NO: 552.

In this aspect, the heavy chain constant domain may comprise SEQ ID NO: 550 or SEQ ID NO: 551.

In this aspect, the multispecific antibody can comprise SEQ ID NO: 546; or SEQ ID NO: 414 and SEQ ID NO: 548. In this aspect, the multispecific antibody can comprise SEQ ID NO: 547; or SEQ ID NO: 414 and SEQ ID NO: 549.

In this aspect, the Fab region can be any Fab region described herein (see, eg, Table 21). Specifically, the Fab region of this aspect may comprise VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 68, and the amine groups comprising SEQ ID NO: 79, 80 and 95 respectively. Acid sequences of VLCDR1, VLCDR2 and VLCDR3. A Fab region in this aspect may include a VH including SEQ ID NO: 15 and a VL including SEQ ID NO: 40.

In one embodiment of this aspect, the multispecific antibody comprises: a Fab region including a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 40; and an Fc region comprising a CH3 domain comprising the amino acid sequence of SEQ ID NO: 552. In some embodiments, multispecific antibodies of the invention may comprise an additional lysine (K) residue immediately C-terminal to the CH3 domain sequence (K447, EU numbering). In some embodiments, multispecific antibodies of the invention can remove the terminal glycine (G) residue (G446, EU numbering) immediately C-terminus of the CH3 domain sequence. Table 21. 747 - Exemplary multispecific vδ1-EGFR human IgG1 antibodies with CDRs derived from antibodies of ADT1-4 and ADT1-7 and associated SEQ ID NOs Fab area Fc area VH VL derived from antibodies CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 AB ring CD ring EF ring Parental ADT1-4 51 53 54 79 80 81 Residues 358 to 362 (EU numbering) comprise TESGP SEQ ID NO: 553 and residue 378 is valine Residues 384 to 386 (EU numbering) comprise KFG SEQ ID NO: 554 Residues 413 to 421 (EU numbering) contain SNLRWTKGH SEQ ID NO: 555 ADT1-4-105 51 53 55 79 80 82 ADT1-4-107 51 53 56 79 80 83 ADT1-4-110 51 53 57 79 80 84 ADT1-4-112 51 53 58 79 80 85 ADT1-4-117 51 53 59 79 80 86 ADT1-4-19 51 53 60 79 80 87 ADT1-4-21 52 53 61 79 80 88 ADT1-4-31 51 53 62 79 80 89 ADT1-4-139 51 53 63 79 80 90 ADT1-4-4 51 53 64 79 80 91 ADT1-4-143 51 53 65 79 80 92 ADT1-4-53 52 53 66 79 80 93 ADT1-4-173 51 53 67 79 80 94 ADT1-4-2 51 53 68 79 80 95 ADT1-4-8 51 53 69 79 80 96 ADT1-4-82 51 53 70 79 80 97 ADT1-4-83 51 53 71 79 80 98 ADT1-4-3 51 53 72 79 80 99 ADT1-4-84 51 53 73 79 80 100 ADT1-4-86 52 53 74 79 80 101 ADT1-4-95 51 53 75 79 80 102 ADT1-4-1 51 53 76 79 80 103 ADT1-4-6 51 53 77 79 80 104 ADT1-4-138 51 53 78 79 80 105 Parental ADT1-7 130 131 132 144 145 146 ADT1-7-10 130 131 133 144 145 147 ADT1-7-15 130 131 134 144 145 148 ADT1-7-17 130 131 135 144 145 149 ADT1-7-18 130 131 136 144 145 150 ADT1-7-19 130 131 137 144 145 151 ADT1-7-20 130 131 138 144 145 152 ADT1-7-22 130 131 139 144 145 153 ADT1-7-23 130 131 140 144 145 154 ADT1-7-42 130 131 141 144 145 155 ADT1-7-3 130 131 142 144 145 156 ADT1-7-61 130 131 143 144 145 157 Table 22. 747 - Exemplary multispecific vδ1-EGFR human IgG1 antibodies with CDRs derived from antibodies of ADT1-4 and ADT1-7 and associated SEQ ID NOs Fab area Fc area derived from antibodies VH VL AB ring CD ring EF ring Parental ADT1-4 1 26 Residues 358 to 362 (EU numbering) comprise TESGP SEQ ID NO: 553 and residue 378 is valine Residues 384 to 386 (EU numbering) comprise KFG SEQ ID NO: 554 Residues 413 to 421 (EU numbering) comprise SNLRWTKGH SEQ ID NO: 555 ADT1-4-105 2 27 ADT1-4-107 3 28 ADT1-4-110 4 29 ADT1-4-112 5 30 ADT1-4-117 6 31 ADT1-4-19 7 32 ADT1-4-21 8 33 ADT1-4-31 9 34 ADT1-4-139 10 35 ADT1-4-4 11 36 ADT1-4-143 12 37 ADT1-4-53 13 38 ADT1-4-173 14 39 ADT1-4-2 15 40 ADT1-4-8 16 41 ADT1-4-82 17 42 ADT1-4-83 18 43 ADT1-4-3 19 44 ADT1-4-84 20 45 ADT1-4-86 twenty one 46 ADT1-4-95 twenty two 47 ADT1-4-1 twenty three 48 ADT1-4-6 twenty four 49 ADT1-4-138 25 50 Parental ADT1-7 106 118 ADT1-7-10 107 119 ADT1-7-15 108 120 ADT1-7-17 109 121 ADT1-7-18 110 122 ADT1-7-19 111 123 ADT1-7-20 112 124 ADT1-7-22 113 125 ADT1-7-23 114 126 ADT1-7-42 115 127 ADT1-7-3 116 128 ADT1-7-61 117 129 C08 273 282 B07 274 283 C05 275 284 E04 276 285 F07 277 286 G06 278 287 G09 279 288 B09 280 289 G10 281 290 E01 312 313

Notably, in all such examples comprising at least one (first) binding domain of the Vδ1 chain targeting the γδ TCR and at least one (second) binding domain targeting EGFR, comparing the control and component parts, it was observed to functional enhancements (see Examples 2 and 6 to 10 herein).

Overall, these non-limiting examples highlight the flexibility of multispecific antibodies or antigen-binding fragments thereof as described herein. These non-limiting examples outline a multispecific antibody approach in which antibodies or fragments thereof targeting the germline Vδ1 chain (e.g., amino acids 1-90 of SEQ ID NO: 272) can be formed by combining with an EGFR binding domain to form a polypeptide. Specific antibodies are further enhanced.

In one embodiment, a multispecific antibody binding domain targeting the Vδ1 chain of a γδ TCR (the first target) may comprise (i) one or two or more each comprising a heavy chain (VH-CH1-CH2-CH3 ) and an antibody binding domain of a cognate light chain partner (VL-CL) and/or (ii) one or two or more each comprising a heavy chain variable domain (VH or VH-CH1) and a cognate light chain The antibody binding domain of the variable domain partner (VL or VL-VC) and/or (iii) one or two or more antibody binding domains each comprising a CDR-containing antibody fragment.

In one embodiment, a multispecific antibody is provided comprising at least one binding domain derived from a first antibody targeting the Vδ1 chain of a γδ TCR and operably linked to at least one second antibody binding domain targeting EGFR. . Optionally, these binding domains include at least one or more VH and cognate VL binding domains, or one or more VH-CH1-CH2-CH3 and cognate VL-CL binding domains, or one or more antibody fragments. area. The multispecific antibody comprising at least one first binding domain derived from the antibody targeting the Vδ1 chain of the γδ TCR is operably linked to the EGF receptor and comprises one or more of the following heavy chain modifications according to EU numbering. Two binding domains; L358T and/or (T359D or T359E) and/or (K360D or K360E ["LEE"] or K360S) and/or N361G and/or Q362P and/or 361.1G (where 361.1 is between 361 and 362 additional residues between) and/or A378V and/or (N384T or N384K) and/or (G385Y or G385F) and/or Q386G and/or D413S and/or (K414Y or K414N) and/or (S415W or S415L ) and/or (Q418Y or Q418T or Q418V) and/or Q419K and/or N421H.

Such heavy chain modifications have been found to be advantageous. For example, a multispecific antibody comprising at least one first binding domain derived from an antibody that targets the Vδ1 chain of a γδ TCR operably linked to a second binding domain that binds EGFR and includes wild-type L358 and T359E and K360E heavy chain modifications Has several advantages. First, such heavy chain modifications eliminate the risk of double isomerization present in the unmodified heavy chain sequence. Isomerizing motifs tend to have the potential to reduce the affinity, potency, stability and homogeneity of the antibody, which can complicate product development or manufacturing. Additionally, the T358L change in the AB ring of molecules such as ADT1-4-2×FS1-67 reverts threonine to leucine. Leucine corresponds to the typical wild-type human sequence at this position and results in a more conserved AB modified loop sequence. Another surprising finding is that at least one first binding domain derived from an antibody comprising a Vδ1 chain targeting a γδ TCR is operably linked to a second binding domain that binds the EGF receptor and includes L358 and T359E and K360E heavy chain modifications. Multispecific antibodies improve performance in transient CHO cells relative to an otherwise identical highly related antibody containing T358 and D359 and D360. This improved performance profile means that antibodies containing heavy chain modifications such as T358, D359, and D360 are incorporated into SEQ ID NO: 388 and are otherwise identical compared to antibodies containing heavy chain modifications such as L358 and E359 and E360, such as are incorporated into SEQ ID NO: 389 The harvesting efficiency of the CH3-modified multispecific antibodies of the present invention from CHO cells can be greatly increased. This is unexpected because removing isomerization risk was not expected to improve performance. See Figure 34A. In any embodiment, the multispecific antibodies of the invention may not include the additional DD and DG higher risk isomerization diamino acid motifs spanning positions 359, 360 and 361 (EU numbering). Indeed, and more specifically, in any embodiment, the multispecific antibodies of the invention may comprise modified AB loops including modifications T359E, K360E, N361G. More specifically, in any embodiment, a multispecific antibody of the invention may comprise a CH3 modified AB ring containing a double negative charge at positions 359 and 360, wherein the double negative charge is formed by glutamic acid (E, Glu) rather than aspartic acid (D, Asp).

A multispecific antibody comprising at least one antibody-derived first binding domain targeting a Vδ1 chain targeting a γδ TCR operably linked to a polypeptide comprising SEQ ID NO: 385 or SEQ ID NO: 386 or SEQ ID NO: 391 or SEQ ID NO: 392 or SEQ ID NO: 520 or SEQ ID NO: 521 or SEQ ID NO: 530 or SEQ ID NO: 531 or SEQ ID NO: 539 or SEQ ID NO: 540 or SEQ ID NO: 508 or SEQ ID NO: 509 or SEQ ID NO: 550 or SEQ ID NO: 551 or a functionally equivalent binding variant thereof and targets the second binding domain of EGFR. Optionally, the multispecific antibody includes SEQ ID NO: 389; or SEQ ID NO: 414 and SEQ ID NO: 432; or SEQ ID NO: 400; or SEQ ID NO: 414 and SEQ ID NO: 436; or SEQ ID NO: 516; or SEQ ID NO: 414 and SEQ ID NO: 518; or SEQ ID NO: 517; or SEQ ID NO: 414 and SEQ ID NO: 519; or SEQ ID NO: 526; or SEQ ID NO: 414 and SEQ ID NO: 528; or SEQ ID NO: 527; or SEQ ID NO: 414 and SEQ ID NO: 529; or SEQ ID NO: 535; or SEQ ID NO: 414 and SEQ ID NO: 537; or SEQ ID NO: 536; or SEQ ID NO: 414 and SEQ ID NO: 538; or SEQ ID NO: 504; or SEQ ID NO: 414 and SEQ ID NO: 506; or SEQ ID NO: 505; or SEQ ID NO: 414 and SEQ ID NO: 507; or SEQ ID NO: 388; or SEQ ID NO: 414 and SEQ ID NO: 431; or SEQ ID NO: 399; or SEQ ID NO: 414 and SEQ ID NO: 435; or SEQ ID NO: 414 and SEQ ID NO: 435; NO: 546; or SEQ ID NO: 414 and SEQ ID NO: 548; or SEQ ID NO: 547; or SEQ ID NO: 414 and SEQ ID NO: 549; or SEQ ID NO: 378; or SEQ ID NO: 425 and SEQ ID NO: 426; or SEQ ID NO: 379; or SEQ ID NO: 421 and SEQ ID NO: 437; or SEQ ID NO: 380; or SEQ ID NO: 423 and SEQ ID NO: 427.

In one aspect of the invention, the multispecific antibodies of the invention can be used in a therapeutically effective amount to treat a disease or disorder so as to ameliorate at least one sign or symptom of the disease or disorder.

In one embodiment, a method of selecting or characterizing or comparing an antibody as described herein that binds to the Vδ1 chain of a γδ TCR, or an antigen-binding fragment thereof, in a multispecific antibody format is provided, wherein the multispecific antibody is applied to Vδ1+ cells to measure the effects conferred by the multispecific entities on Vδ1+ cells (eg, specificity and/or enhancement of the Vδ1+ phenotype and/or its cytotoxicity and/or diseased cells). combination therapy

The multispecific antibodies of the invention may be used in certain combination therapies. In some embodiments, the multispecific antibody can be combined with a modulator of a cancer antigen or cancer-associated antigen, for example, selected from the group consisting of: AFP, AKAP-4, ALK, alpha-fetoprotein, androgen receptor, B7H3 , BAGE, BCA225, BCAA, Bcr-abl, β-catenin, β-HCG, β-human chorionic gonadotropin, BORIS, BTAA, CA 125, CA 15-3, CA 195, CA 19-9, CA 242, CA 27.29, CA 72-4, CA-50, CAM 17.1, CAM43, carbonic anhydrase IX, carcinoembryonic antigen, CD22, CD33/IL3Ra, CD68\P1, CDK4, CEA, chondroitin sulfate proteoglycan 4( CSPG4), c-Met, CO-029, CSPG4, cyclin B1, cyclophilin C-related protein, CYP1B1, E2A-PRL, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, pteridin B2, Epstein-Barr II Epstein Barr virus antigen EBVA, ERG (TMPRSS2ETS fusion gene), ETV6-AML, FAP, FGF-5, Fos-related antigen 1, fucosyl GM1, G250, Ga733\EpCAM, GAGE-1, GAGE- 2. GD2, GD3, glioma-related antigen, GloboH, glycolipid F77, GM3, GP 100, GP 100 (Pmel 17), H4-RET, HER-2/neu, HER-2/Neu/ErbB-2, High molecular weight melanoma-associated antigen (HMW-MAA), HPV E6, HPV E7, hTERT, HTgp-175, human telomerase reverse transcriptase, ontogeny, IGF-I receptor, IGF-II, IGH-IGK, Insulin growth factor (IGF)-I, intestinal carboxyl esterase, K-ras, LAGE-1a, LCK, lectin-responsive AFP, legumin, LMP2, M344, MA-50, Mac-2 binding protein, MAD-CT -1, MAD-CT-2, MAGE, MAGE A1, MAGE A3, MAGE-1, MAGE-3, MAGE-4, MAGE-5, MAGE-6, MART-1, MART-1/MelanA, M-CSF , Melanoma-associated chondroitin sulfate proteoglycan (MCSP), mesothelin, MG7-Ag, ML-IAP, MN-CA IX, MOV18, MUC1, Mum-1, hsp70-2, MYCN, MYL-RAR, NA17, NB/70K, neuronal-glial antigen 2 (NG2), neutrophil elastase, nm-23H1, NuMa, NY-BR-1, NY-CO-1, NY-ESO, NY-ESO-1, NY-ESO-1, OY-TES1, p15, p16, p180erbB3, p185erbB2, p53, p53 mutant, Page4, PAX3, PAX5, PDGFR-β, PLAC1, polysialic acid, prostate cancer tumor antigen-1 (PCTA-1 ), prostate-specific antigen, prostatic acid phosphatase (PAP), protease 3 (PR1), PSA, PSCA, PSMA, RAGE-1, Ras, Ras-mutant, RCAS1, RGS5, RhoC, ROR1, RU1, RU2 ( AS), SART3, SDCCAG16, sLe(a), sperm protein 17, SSX2, STn, survivin, TA-90, TAAL6, TAG-72, telomerase, thyroglobulin, Tie 2, TLP, Tn, TPS , TRP-1, TRP-2, TRP-2, TSP-180, tyrosinase, VEGF, VEGFR2, VISTA, WT1, XAGE 1, 43-9F, 5T4 and 791Tgp72.

In some embodiments, the multispecific antibody can be combined with a modulator of an immunomodulatory antigen selected from, for example, the group consisting of: B7-1 (CD80), B7-2 (CD86), B7-DC (CD273), B7 -H1(CD274), B7-H2(CD275), B7-H3(CD276), B7-H4(VTCN1), B7-H5(VISTA), BTLA(CD272), CD137, CD137L, CD24, CD27, CD28, CD38 , CD40, CD40L(CD154), CD54, CD59, CD70, CTLA4(CD152), CXCL9, GITR(CD357), HVEM(CD270), ICAM-1(CD54), ICOS(CD278), LAG-3(CD223), OX40(CD134), OX40L(CD252), PD-1(CD279), PD-L1(CD274), TIGIT, CD314, CD334, CD335, CD337 and TIM-3(CD366).

In some embodiments, the multispecific antibody can be combined with a modulator of a CD antigen, for example, selected from the group consisting of: CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5 , CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CD13, CD14, CD15, CD16, CD16a, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24 , CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32A, CD32B, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44 , CD45, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E , CD62L, CD62P, CD63, CD64a, CD65, CD65s, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79A, CD79B, CD80 , CD81, CD82, CD83, CD84, CD85A, CD85B, CD85C, CD85D, CD85F, CD85G, CD85H, CD85I, CD85J, CD85K, CD85M, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95 、CD96、CD97、CD98、CD99、CD100、CD101、CD102、CD103、CD104、CD105、CD106、CD107、CD107a、CD107b、CD108、CD109、CD110、CD111、CD112、CD113、CD114、CD115、CD116、CD117、CD118 . 0A, CD140B . 2. CD158C, CD158D . CD169, CD170, CD171, CD172a , CD172b, CD172g, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179a, CD179b, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD187, CD188, CD189, CD190, CD191, CD1 92.CD193 , CD194, CD195, CD196, CD197, CDw198, CDw199, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CDw210a, CDw210b, CD211, CD212, CD213a1, CD2 13a2, CD214, CD215 , CD216, CD217, CD218a, CD218b, CD219, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235b, CD236, CD23 7. CD238 . CD262 , CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD285, CD286, CD2 87 , CD288, CD289, CD290, CD291, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300A, CD300C, CD301, CD302, CD303, CD304, CD305, CD306, CD307, CD307a, CD307b, CD3 07c, CD307d , CD307e, CD308, CD309, CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD323, CD324, CD325, CD326, CD327, CD328, CD329, CD330, 331 . 66 , CD367, CD368, CD369, CD370 and CD371.

In some embodiments, modulators may have antagonistic or agonistic properties. Suitable modulators include antibodies, fusion proteins or small molecules. immunoconjugate

The antibodies of the invention, or antigen-binding fragments thereof, may be bound to therapeutic moieties, such as cytotoxic or chemotherapeutic agents. Such conjugates may be termed immunoconjugates. As used herein, the term "immunoconjugate" means a chemically or biologically linked substance such as a cytotoxin, radioactive agent, interleukin, interferon, target or reporter moiety, enzyme, toxin, peptide or protein, or therapeutic agent. of antibodies. Antibodies can be linked to cytotoxins, radioactive agents, interleukins, interferons, target or reporter moieties, enzymes, toxins, peptides or therapeutic agents at any position along the molecule so long as they are capable of binding to their target. Examples of immunoconjugates include antibody drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent can be a second different antibody directed against Vδ1. In certain embodiments, antibodies can be combined with agents specific for tumor cells or virus-infected cells. The type of therapeutic moiety that can be combined with the anti-Vδ1 antibody will take into account the condition to be treated and the desired therapeutic effect to be achieved. In one embodiment, the agent may be a second antibody or antigen-binding fragment thereof that binds to a molecule other than Vδ1. Crab-eating macaque cross-reactivity

Suitably, the multispecific antibody exhibits cross-reactivity with human TRDV1 SEQ ID NO: 272 (including polymorphic variants, namely SEQ ID NO: 306) and cynomolgus monkey TRDV1 (SEQ ID NO: 308). Cross-reactivity can be clearly used to provide antibodies that can be used during preclinical evaluation in in vivo animal studies.

The inventors have unexpectedly identified a framework mutation that increases the binding of antibodies (eg, anti-Vδ1×EGFR antibodies and antigen-binding fragments thereof) to cynomolgus monkey antigens. The framework mutation does not adversely affect the affinity of the antibody or antigen-binding fragment thereof for the corresponding human form of the antigen. A mutation is a mutation from a serine residue at position 74 of the kappa light chain variable sequence to a residue that is not serine (eg, a non-human germline and/or non-polar residue) according to the IMGT numbering system. The non-polar amino acid may be selected from the group consisting of glycine, alanine, valine, methionine, leucine and isoleucine. Non-reproductive amino acids can be selected from arginine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, and methionine. A group of amino acids, phenylalanine, threonine, tryptophan, tyrosine and valine. Non-polar and non-germ amino acids (i.e., amino acids that are neither polar nor germ-line) may be selected from the group consisting of glycine, valine, methionine, leucine and isoleucine. group). In some embodiments, the mutation is a mutation from a serine residue to a leucine residue at position 74 of the kappa light chain variable sequence according to the IMGT numbering system. Mutation is a direct substitution so that the total chain length does not change. Therefore, the serine at position 74 according to the IMGT numbering system is removed and directly replaced by another amino acid, such as a non-human germline and/or a non-polar amino acid, such as leucine. Replacement may be made according to any suitable method known to the skilled person.

Reference herein to an antibody comprising a kappa light chain variable sequence comprising a residue that is not serine at position 74 according to IMGT numbering may alternatively be defined as comprising an LFR1 region, an LCDR1 region, an LFR2 region, an LCDR2 region, an LFR3 region Antibodies of the light chain variable sequence of the region, the LCDR3 region and the LFR4 region, wherein the LFR3 region contains a residue at position 74 that is not serine (e.g., non-human germline and/or non-polar amino acids according to IMGT numbering , such as leucine residues). Such alternative definitions may apply to those disclosed herein that include a residue that is not serine at position 74 according to IMGT numbering (e.g., a non-human germline and/or non-polar amino acid, such as a leucine residue) All antibodies with kappa light chain variable sequences.

Accordingly, the present invention provides anti-TCR delta variable 1 (anti-Vdelta 1)×EGFR multispecific antibodies or antigen-binding fragments thereof comprising a kappa light chain variable sequence, wherein the light chain variable sequence is at position 74 according to the IMGT numbering system The residue at is not serine (eg, a non-human germline and/or non-polar amino acid, such as a leucine residue). In some embodiments, residue at position 74 of the light chain variable sequence according to the IMGT numbering system is leucine. Suitably, the antibody may be an IgG antibody. For example, the antibody can be an IgG1 antibody.

The anti-Vδ1×EGFR multispecific antibodies and antigen-binding fragments thereof provided herein may possess a substitution at position 74 (according to the IMGT numbering system) of the light chain variable sequence. For example, anti-Vδ1×EGFR multispecific antibodies and antigen-binding fragments and variants thereof described herein may be comprised at position 74 according to the IMGT numbering system (e.g., non-human germline and/or non-polar amino acids, Kappa light chain variable sequences containing residues other than serine, such as leucine residues. In some embodiments, the anti-Vδ1×EGFR multispecific antibodies and antigen-binding fragments and variants thereof described herein may comprise a kappa light chain variable sequence comprising a leucine residue at position 74 according to the IMGT numbering system . Embodiments comprising a mutation at position 74 of the kappa light chain according to the IMGT numbering system may be particularly relevant to antibodies derived from ADT1-4, although the mutations may be equally applicable to other antibodies. For example, it was observed that light chain position 74 is not highly conserved among different kappa/lambda light chain germlines. It is further noted that the different light chain germ lines at this position range from nonpolar (e.g., alanine) to polar neutral (e.g., serine) to negatively or positively charged (e.g., aspartate or aspartate, respectively). Differently polarized and/or negatively charged amino acids within the range of amines. It is understood that amino acid polarity/charge at any given position can affect protein structure. For example, it is understood that changes in polarity and charge can affect hydrophobicity and the tendency of amino acids to be more surface exposed or more buried. Regardless, this remarkable finding highlights that a non-conservative amino acid change at position 74 of the light chain can alter the affinity to a selected target by more than 10-fold. Therefore, use of this knowledge may provide new ways to increase or decrease affinity more broadly, in addition to improving affinity as outlined herein. For example, methods involving changing the residue at position 74 from a more polar to a non-polar, or, for example, from a non-polar to a more charged method when it is desired to increase or decrease the affinity for any given antibody are more complex/tedious. Mutagenesis methods such as saturation mutagenesis may be preferred.

In other embodiments of the invention, a method of mutating an antibody or an antigen-binding fragment thereof is also provided, which includes providing a kappa light chain comprising a serine at position 74 of the light chain variable sequence according to the IMGT numbering system. Antibodies, and mutating serine to different residues, such as non-human germline and/or non-polar amino acids, such as leucine. In some embodiments, the antibody is an anti-TCR delta variable 1 (anti-V delta 1) antibody or antigen-binding fragment thereof. In some embodiments, the antibody is an antibody having at least 70%, at least 75%, at least 80%, at least 85% SEQ ID NO: 1 before and after the introduction of the mutation at position 74 of the light chain variable sequence. , at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical amino acid sequence VH and comprising At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A VL with an amino acid sequence that is at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody is a VH having an amino acid sequence that is 100% identical to SEQ ID NO: 1, except for a mutation at position 74, and a VH that is 100% identical to SEQ ID NO: 26, except for a mutation at position 74. Antibodies to VL with identical amino acid sequences.

In some embodiments, the antibody is an antibody that has at least 70%, at least 75%, at least 80%, at least 85% SEQ ID NO: 106 before and after the introduction of the mutation at position 74 of the light chain variable sequence. , at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical amino acid sequence VH and comprising At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A VL with an amino acid sequence that is at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody is a VH having an amino acid sequence that is 100% identical to SEQ ID NO: 106, except for a mutation at position 74, and a VH that is 100% identical to SEQ ID NO: 118, except for a mutation at position 74. Antibodies to VL with identical amino acid sequences.

In some embodiments, the antibody is an antibody comprising: Containing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, the same amino acid sequence as SEQ ID NO: 273 before and after introducing the mutation. A VH that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence and contains at least 70%, at least 75% of the amino acid sequence identical to SEQ ID NO: 282 %, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% consistent The VL of the amino acid sequence; Containing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, the same amino acid sequence as SEQ ID NO: 274 before and after introducing the mutation. A VH that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence and contains at least 70%, at least 75 of the amino acid sequence of SEQ ID NO: 283 %, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% consistent The VL of the amino acid sequence; Containing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, the same amino acid sequence as SEQ ID NO: 275 before and after introducing the mutation. A VH that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence and contains at least 70%, at least 75% of the amino acid sequence identical to SEQ ID NO: 284 %, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% consistent The VL of the amino acid sequence; Containing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, the same amino acid sequence as SEQ ID NO: 276 before and after introducing the mutation. A VH that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence and contains at least 70%, at least 75 of the amino acid sequence of SEQ ID NO: 285 %, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% consistent The VL of the amino acid sequence; Containing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, the same amino acid sequence as SEQ ID NO: 277 before and after introducing the mutation. A VH that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence and contains at least 70%, at least 75% of the amino acid sequence identical to SEQ ID NO: 286 %, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% consistent The VL of the amino acid sequence; Containing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, the same amino acid sequence as SEQ ID NO: 278 before and after introducing the mutation. A VH that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence and contains at least 70%, at least 75% of the amino acid sequence identical to SEQ ID NO: 287 %, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% consistent The VL of the amino acid sequence; Containing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, the same amino acid sequence as SEQ ID NO: 279 before and after introducing the mutation. A VH that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence and contains at least 70%, at least 75% of the amino acid sequence identical to SEQ ID NO: 288 %, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% consistent The VL of the amino acid sequence; Containing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, the same amino acid sequence as SEQ ID NO: 280 before and after introducing the mutation. A VH that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence and contains at least 70%, at least 75% of the amino acid sequence identical to SEQ ID NO: 289 %, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% consistent The VL of the amino acid sequence; Containing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, the same amino acid sequence as SEQ ID NO: 281 before and after introducing the mutation. A VH that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence and contains at least 70%, at least 75% of the amino acid sequence identical to SEQ ID NO: 290 %, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% consistent VL of the amino acid sequence; or Containing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, the same amino acid sequence as SEQ ID NO: 312 before and after introducing the mutation. A VH that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence and contains at least 70%, at least 75% of the amino acid sequence identical to SEQ ID NO: 313 %, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% consistent The amino acid sequence of VL.

In some embodiments, the antibody is an antibody comprising: VH comprising the amino acid sequence of SEQ ID NO: 273 and VL comprising the amino acid sequence of SEQ ID NO: 282; VH comprising the amino acid sequence of SEQ ID NO: 274 and VL comprising the amino acid sequence of SEQ ID NO: 283; VH comprising the amino acid sequence of SEQ ID NO: 275 and VL comprising the amino acid sequence of SEQ ID NO: 284; VH comprising the amino acid sequence of SEQ ID NO: 276 and VL comprising the amino acid sequence of SEQ ID NO: 285; VH comprising the amino acid sequence of SEQ ID NO: 277 and VL comprising the amino acid sequence of SEQ ID NO: 286; VH comprising the amino acid sequence of SEQ ID NO: 278 and VL comprising the amino acid sequence of SEQ ID NO: 287; VH comprising the amino acid sequence of SEQ ID NO: 279 and VL comprising the amino acid sequence of SEQ ID NO: 288; VH comprising the amino acid sequence of SEQ ID NO: 280 and VL comprising the amino acid sequence of SEQ ID NO: 289; VH comprising the amino acid sequence of SEQ ID NO: 281 and VL comprising the amino acid sequence of SEQ ID NO: 290; or VH comprising the amino acid sequence of SEQ ID NO: 312 and VL comprising the amino acid sequence of SEQ ID NO: 313.

Except for the mutation at position 74, antibodies generated by such methods are 100% identical to the specified VH and VL sequences.

In some embodiments, mutation at position 74 increases the affinity of the antibody, or antigen-binding fragment thereof, for a homologous cynomolgus monkey (cynomolgus macaque) antigen. The increase in affinity of the antibody or antigen-binding fragment thereof is relative to the affinity of the antibody or antigen-binding fragment thereof before the mutation was introduced (when measured under the same or substantially the same conditions). In some embodiments, the antibody, or antigen-binding fragment thereof, is an antibody that binds to the human variable delta 1 (Vdel) chain (e.g., SEQ ID NO: 272 and 306) of the γδ T cell receptor (TCR) before and after introduction of the mutation. A TCR delta variable 1 (anti-Vδ1) antibody, or an antigen-binding fragment thereof, which after introducing mutations is directed against the cynomolgus macaque variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR) (e.g., SEQ ID NO: 308) affinity increases. Polynucleotide sequences and expression vectors

In one aspect of the invention, a polynucleotide encoding a multispecific antibody of the invention is provided. In one embodiment, the polynucleotide comprises at least 70%, such as at least 80%, such as at least 90%, any one of SEQ ID NOs: 199 to 222, 224 to 247, 249 to 259, or 261 to 271 , such as or consisting of a sequence with at least 95%, such as at least 99% sequence identity. In another embodiment, the polynucleotide comprises or consists of any one of SEQ ID NOs: 199 to 222, 224 to 247, 249 to 259, or 261 to 271. In another aspect, a cDNA comprising the polynucleotide is provided.

In one aspect of the invention, there is provided a polynucleotide comprising at least 70% identical to a portion of any one of SEQ ID NO: 199 to 222, 224 to 247, 249 to 259, or 261 to 271, Such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to or consisting of a sequence encoding the CDR1, CDR2 and/or CDR3 of the encoded immunoglobulin chain variable domain .

In one aspect of the invention, there is provided a polynucleotide comprising at least 70% identical to a portion of any one of SEQ ID NOs: 199 to 222, 224 to 247, 249 to 259, or 261 to 271, such as Sequences with or consisting of at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity, encoding FR1, FR2, FR3 and/or of the encoded immunoglobulin chain variable domain FR4.

The invention also provides expression vectors and plasmids comprising the polynucleotide sequences of the invention. In some embodiments, the expression vector comprises at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, any one of SEQ ID NO: 199 to 222 or 249 to 259. Sequence identity or 100% identity of the sequence (encoding the heavy chain variable region). In some embodiments, the expression vector comprises at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, any one of SEQ ID NOs: 224 to 247 or 261 to 271 Sequence identity or 100% identity of the sequence (encoding the light chain variable region). Such expression vectors can be used in pairs, and the heavy chain and light chain variable sequences are appropriately paired according to the pairing of various amino acid sequences to provide the multispecific antibodies of the invention disclosed herein. In some embodiments, the expression vector comprises at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99, any one of SEQ ID NOs: 199 to 222 or 249 to 259. % sequence identity or 100% identity to a sequence (encoding a heavy chain variable region) and further comprising at least 70%, such as at least 80%, with any one of SEQ ID NO: 224 to 247 or 261 to 271, such as A sequence (encoding the light chain variable region) of at least 90%, such as at least 95%, such as at least 99% sequence identity or 100% identity. Again, sequences may be provided in specific pairs as described herein to encode multispecific antibodies of the invention.

The invention also provides polynucleotide sequences and expression vectors and plasmids encoding all of the antibody sequences disclosed herein, including any variant antibody sequences disclosed herein, optionally including one or more amino acid substitutions.

The polynucleotides and expression vectors of the invention may also be described with reference to the encoded amino acid sequences. Thus, in one embodiment, the polynucleotide comprises or consists of a sequence encoding the amino acid sequence of any of SEQ ID NO: 197.

To express multispecific antibodies or antigen-binding fragments thereof, polynucleotides encoding partial or full-length light and heavy chains as described above are inserted into expression vectors such that the genes are operably linked to transcriptional and translational control sequences. Accordingly, in one aspect of the invention there is provided an expression vector comprising a polynucleotide sequence as defined herein. In one embodiment, the expression vector comprises the VH region of any one of SEQ ID NOs: 199 to 222 or 249 to 259. In another embodiment, the expression vector comprises the VL region of any one of SEQ ID NOs: 224 to 247 or 261 to 271.

It is understood that the nucleotide sequences described herein may contain additional sequences encoding amino acid residues to aid translation, purification and detection, however, alternative sequences may be used depending on the expression system used. If alternative design, translation, purification or detection strategies are employed, these optional sequences may be removed, modified or replaced.

The DNA or cDNA encoding a polypeptide can be mutated such that the amino acid sequence for the polypeptide is silent but provides preferred codons for translation in a particular host. Preferred codons are known for translation of nucleic acids in, for example, E. coli and S. cerevisiae , as well as in mammals, especially humans.

Mutation of a polypeptide can be achieved, for example, by substitution, addition or deletion of the nucleic acid encoding the polypeptide. Substitutions, additions or deletions to the nucleic acid encoding a polypeptide can be introduced by a number of methods, including, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis Induction, recursive global mutation induction, exponential global mutation induction, site-specific mutation induction, gene recombination, artificial gene synthesis, gene site saturation mutation induction (GSSM), synthetic ligation reassembly (SLR) or other methods combination. Modifications, additions or deletions to nucleic acids can also be introduced by methods including: recombination, recursive sequence recombination, phosphorothioate modified DNA mutagenesis, uracil-containing template mutagenesis, gapped double strands Gapped duplex mutagenesis, point mismatch repair mutation induction, repair-deficient host strain mutagenesis, chemical mutation induction, radiation mutation induction, deletion mutation induction, restricted selection mutation induction, Restriction-purification mutagenesis, global mutagenesis, chimeric nucleic acid multimer creation, or combinations thereof.

In detail, artificial gene synthesis can be used. Genes encoding polypeptides of the invention can be produced synthetically, for example, by solid-phase DNA synthesis. The entire gene can be synthesized de novo without the need for precursor template DNA. To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required for the sequence of the product. After chain assembly is complete, the product is released from the solid phase into solution, deprotected, and collected. The product can be separated by high performance liquid chromatography (HPLC) to obtain the desired oligonucleotide with high purity.

Expression vectors include, for example, episomes derived from plastids, retroviruses, myxoplasts, yeast artificial chromosomes (YAC), and Epstein-Barr virus (EBV). The polynucleotide is conjugated into the vector so that the transcription and translation control sequences within the vector provide its desired function of regulating DNA transcription and translation. Expression and/or control sequences may include promoters, enhancers, transcription terminators, start codons 5' of the coding sequence (i.e., ATG), splicing signals for introns, and stop codons. Select expression vectors and expression control sequences that are compatible with the expression host cell used. For example, the sequence may comprise a nucleotide sequence encoding single-chain variable fragment versions of the antibodies of the invention, which single-chain variable fragment versions comprise VH linked by a synthetic linker (e.g., encoding SEQ ID NO: 291) area and VL area. It will be appreciated that the polynucleotides or expression vectors of the invention may comprise VH regions, VL regions, or both (optionally including linkers). Thus, the polynucleotides encoding the VH and VL regions can be inserted into separate vectors, or the sequences encoding both regions can be inserted into the same expression vector. The polynucleotide is inserted into the expression vector by standard methods (eg, joining complementary restriction sites on the polynucleotide and the vector, or joining blunt ends if no restriction sites are present).

A suitable vector system encodes a fully functional human CH or CL immunoglobulin sequence and has appropriate restriction sites engineered to allow easy insertion and expression of any VH or VL sequence, as described above. The expression vector may also encode a signal peptide that promotes secretion of the antibody (or antigen-binding fragment thereof) from the host cell. The polynucleotide can optionally be cloned into a vector such that the signal peptide is linked in frame to the amino terminus of the antibody. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

In one aspect of the invention there is provided a cell (eg a host cell, such as a recombinant host cell) comprising a polynucleotide or expression vector as defined herein. It will be appreciated that the cell may comprise a first vector encoding the light chain of the antibody or antigen-binding fragment thereof, and a second vector encoding the heavy chain of the antibody or antigen-binding fragment thereof. Alternatively, both the heavy and light chains are encoded on the same expression vector introduced into the cell.

In one embodiment, the polynucleotide or expression vector encodes a membrane anchor or transmembrane domain fused to an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is presented on the extracellular surface of the cell.

Transformation can be performed by any method known for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include polydextrose-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation perforation, encapsulation of polynucleotides in liposomes, gene gun injection, and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells via viral vectors.

Mammalian cell lines that can serve as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include inter alia Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg Hep G2), A549 cells, 3T3 cells and many other cell lines. Mammalian host cells include human, mouse, rat, canine, monkey, porcine, goat, bovine, equine and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that can be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. Antigen-binding fragments of antibodies, such as scFv and Fv fragments, can be isolated and expressed in E. coli using methods known in the art.

Antibodies are produced by culturing a host cell for a period of time sufficient to allow expression of the antibody in the host cell or, more preferably, secretion of the antibody into the medium in which the host cell is grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

The antibodies (or fragments) of the invention can be obtained and manipulated using, for example, techniques disclosed in Green and Sambrook, Molecular Cloning: A Laboratory Manual (2012) 4th Edition Cold Spring Harbor Laboratory Press.

Monoclonal antibodies can be produced using fusionoma technology by fusing specific antibody-producing B cells with myeloma (B-cell cancer) cells selected for their ability to grow in tissue culture and the absence of antibody chain synthesis.

Monoclonal antibodies against a defined antigen can be obtained, for example, by: a) immortalize lymphocytes obtained from the peripheral blood of an animal previously vaccinated with a defined antigen using immortalized cells, preferably myeloma cells, to form fusion tumors, b) Cultivate the formed immortalized cells (fusionoma) and recover cells that produce antibodies with the desired specificity.

Alternatively, the use of fusion tumor cells is not required. Antibodies capable of binding to an antigen of interest as described herein can be isolated from a suitable antibody library by routine practice, such as using phage display, yeast display, ribosome display, or mammalian display techniques known in the art. Therefore, monoclonal antibodies can be obtained, for example, by a method comprising the following steps: a) Selection of DNA or cDNA sequences obtained from lymphocytes, especially peripheral blood lymphocytes, of animals (preferably immunized with a predetermined antigen) into vectors, especially phages and more particularly filamentous phages, b) Use the above vector to transform prokaryotic cells under conditions that allow the production of antibodies, c) Selection of antibodies by antigen affinity selection of antibodies, d) Recover antibodies with the desired specificity.

Optionally, isolated polynucleotides encoding an antibody as described herein, or an antigen-binding fragment thereof, that binds to the Vδ1 chain of γδ are also readily produced in amounts sufficient to be used as a medicament to ameliorate signs or symptoms of disease. When used as a medicament in this manner, the polynucleotide of interest is typically first operably linked to an expression vector or expression cassette designed to express the antibodies or antigen-binding fragments thereof in an individual or patient. Such expression cassettes and methods of delivering polynucleotides, or what are sometimes referred to as "nucleic acid-based" agents, are well known in the art. For a recent review, see Hollevoet and Declerck (2017) J. Transl. Med. 15(1): 131.

Also provided is a method for producing an anti-Vδ1 antibody, or an antigen-binding fragment or variant thereof, comprising culturing a host cell of the invention in a cell culture medium under conditions that allow intracellular expression of the nucleic acid sequence encoding the plasmid or vector. The method may further comprise obtaining an anti-Vδ1 antibody or antigen-binding fragment or variant thereof from the cell culture supernatant. The antibodies obtained can then be formulated into pharmaceutical compositions. In addition, a method of generating cells expressing anti-Vδ1 antibodies or antigen-binding fragments or variants thereof is provided, which method includes transfecting the cells with the plasmid or vector of the invention. The cells can then be cultured to produce anti-Vδ1 antibodies or antigen-binding fragments or variants thereof. Pharmaceutical composition

According to another aspect of the invention there is provided a composition comprising a multispecific antibody as defined herein. In such embodiments, the composition may comprise the antibody, optionally combined with other excipients. Also included are compositions containing one or more additional active agents (eg, active agents suitable for treating the diseases mentioned herein).

According to another aspect of the present invention, a pharmaceutical composition is provided, which includes an antibody or an antigen-binding fragment thereof as defined herein, and a pharmaceutically acceptable diluent or carrier. The multispecific antibodies of the invention may be incorporated into pharmaceutical compositions suitable for administration to an individual. Typically, the pharmaceutical composition includes the antibody of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are physiologically compatible things. Examples of pharmaceutically acceptable carriers include one or more of water, saline, salt, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases it will be preferable to include an isotonic agent, such as a sugar, a polyol (such as mannitol, sorbitol) or sodium chloride in the composition. Pharmaceutically acceptable substances, such as wetting agents or minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, enhance the shelf life or effectiveness of the antibody or antigen-binding fragment thereof.

The compositions of the present invention may take a variety of forms. Such forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form will depend on the intended mode of administration and therapeutic application. Typically preferred compositions are in the form of injectable or infusible solutions.

Preferred modes of administration are parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular, intrathecal) administration. In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high drug concentrations.

It is within the scope of the invention that the pharmaceutical compositions of the invention are used in methods of treatment of diseases as described herein as adjunctive therapy or in combination with other established therapies commonly used for the treatment of such diseases.

In another aspect of the invention, the antibody, composition or pharmaceutical composition and at least one active agent are administered sequentially, simultaneously or separately. Treatment

According to another aspect of the invention there is provided an isolated multispecific antibody as defined herein for use as a medicament. References herein to an antibody being "suitable" for use as a medicament or "suitable" for use in therapy are limited to administration of the antibody to an individual.

In one embodiment, the multispecific antibodies may be used to treat cancer. In one embodiment, the invention is a method of treating a disease or condition in an individual in need thereof, comprising the step of administering to the individual a multispecific antibody. In various embodiments, the disease or condition is cancer. In one embodiment, multispecific antibodies are used to treat cancer, causing diseased cells to die while sparing healthy cells. In another embodiment, the antibody or antigen-binding fragment thereof is used to treat cancer.

In one embodiment, the antibody or antigen-binding fragment thereof is used to treat cancer. In another embodiment, the antibody or antigen-binding fragment thereof is used to treat cancer.

According to another aspect of the invention there is provided a pharmaceutical composition as defined herein, which is suitable for use as a medicament. In one embodiment, the pharmaceutical composition is used to treat cancer. In another embodiment, the pharmaceutical composition is used to treat cancer.

According to another aspect of the invention, there is provided a method of modulating an immune response in an individual in need thereof, comprising administering a therapeutically effective amount of an isolated multispecific antibody as defined herein. In various embodiments, modulating an immune response in an individual comprises binding or targeting γδ T cells, activating γδ T cells, causing or increasing proliferation of γδ T cells, causing or increasing expansion of γδ T cells, causing or increasing γδ T cells Degranulation, causing or increasing gamma delta T cell-mediated killing activity, causing or increasing gamma delta T cell-mediated killing activity while avoiding healthy cells, causing or increasing gamma delta T cell toxicity, causing or increasing gamma delta T cell toxicity while avoiding Open healthy cells, cause or increase γδ T cell mobilization, increase γδ T cell survival or increase resistance to γδ T cell exhaustion. Modulating an immune response in an individual may further comprise binding or targeting a second antigen. For example, in some embodiments, in addition to immunomodulation via binding of the multispecific antibody to TRDV1, binding of the second antigen, especially if it is an immunomodulatory antigen, may stimulate immunomodulation via the second antigen. . Thus, modulation of an immune response may comprise modulation via two different signaling pathways, a first signaling pathway via engagement of a TRDV1 antigen and a second signaling pathway via engagement of a second immunomodulatory antigen.

According to another aspect of the invention, there is provided a method of treating cancer in an individual in need thereof, comprising administering a therapeutically effective amount of an isolated multispecific antibody as defined herein. Alternatively, a therapeutically effective amount of the pharmaceutical composition is administered.

According to other aspects of the invention there is provided the use of an antibody as defined herein, or an antigen-binding fragment thereof, for the manufacture of, for example, the treatment of cancer.

In one embodiment, the antibody or antigen-binding fragment thereof is administered to an individual, wherein the individual has cancer.

According to another aspect of the invention there is provided a pharmaceutical composition as defined herein, which is suitable for use as a medicament. In one embodiment, a pharmaceutical composition is administered to an individual, wherein the individual has cancer.

According to another aspect of the invention, there is provided a method of administering a therapeutically effective amount of an isolated multispecific antibody as defined herein to an individual, wherein the individual has cancer. Alternatively, a therapeutically effective amount of the pharmaceutical composition is administered.

According to other aspects of the invention there is provided the use of an antibody as defined herein, or an antigen-binding fragment thereof, for the manufacture of a medicament, for example for administration to an individual, wherein the individual suffers from cancer.

In various embodiments, cancers treatable by the disclosed methods and compositions include, but are not limited to, acute lymphoblastic, acute myeloid leukemia, adrenocortical cancer, appendiceal cancer, basal cell carcinoma, cholangiocarcinoma, bladder cancer, Bone cancer, osteosarcoma and malignant fibrous histiocytoma, brainstem glioma, brain tumor, brain tumor, brainstem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumor, Cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, moderately differentiated pineal parenchyma Tumors, supratentorial primitive neuroectodermal tumors and pinealoblastomas, visual pathway and hypothalamic gliomas, brain and spinal cord tumors, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumors, Gastrointestinal carcinoid tumors, central nervous system atypical teratoid/rhabdoid tumors, central nervous system embryonal tumors, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, Cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colorectal cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, esophageal cancer, Ewing family tumors of tumor), extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, intraocular melanoma, retinoblastoma, gallbladder cancer, stomach (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), germ cell tumor , gestational trophoblastic tumor, glioma, brainstem glioma, glioma brain astrocytoma, visual pathway and hypothalamic glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Langerhans Langerhans cell histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamus and visual pathway glioma, intraocular melanoma, islet cell tumor, kidney (renal cell) Carcinoma, Langerhan's cell histiocytosis, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cancer, liver cancer, non-small cell lung cancer cell lung cancer, small cell lung cancer, aids-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Waldenstrom's macroglobulinemia Waldenstrom macroglobulinemia, malignant fibrous histiocytoma of bone and osteosarcoma, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cervical squamous cell carcinoma with occult primary tumor Cancer, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma/plasmacytoma, mycosis, mycosis fungoides, myelodysplasia syndrome, myelodysplasia/myeloproliferative disorders, myeloid leukemia, myeloid leukemia , acute myeloid leukemia, multiple myeloma, myeloproliferative disorders, nasal cavity and sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and bone malignancy Fibrous histiocytoma, ovarian cancer, epithelial ovarian cancer, ovarian germ cell tumors, ovarian tumors of low malignant potential, pancreatic cancer, pancreatic cancer, papillomatosis, parathyroid cancer, penile cancer, pharyngeal cancer, chromaffin cells tumors, moderately differentiated parenchymal pineal tumors, pinealoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumors, plasma cell tumors/multiple myeloma, pleuropulmonary blastoma, primary central nervous system Lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, respiratory cancer involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Ewing family tumor, Kaposi Kaposi sarcoma, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell skin, small cell lung cancer, small bowel cancer, Soft tissue sarcoma, squamous cell carcinoma, neck squamous cell carcinoma, stomach (stomach) cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymus cancer, thyroid cancer, renal pelvis and ureter Transitional cell cancer, gestational trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia and Wilms tumor. In various embodiments, cancer treatable by the disclosed methods and compositions is treated while sparing healthy cells.

In various embodiments, cancers treatable by the disclosed methods and compositions include EGFR-positive (EGFR+) cancers.

In one embodiment, the invention is a method of activating at least one γδ T cell in an individual, comprising the step of administering a multispecific antibody as defined herein.

In one embodiment, the invention is a method of causing or increasing γδ T cell proliferation in an individual, comprising the step of administering to the individual a multispecific antibody as defined herein.

In one embodiment, the invention is a method of causing or increasing degranulation of γδ T cells in an individual, comprising the step of administering to the individual a multispecific antibody as defined herein.

In one embodiment, the invention is a method of causing or increasing γδ T cell killing activity (e.g., T cell mediated killing activity) in an individual, comprising administering to the individual an anti-Vδ1 as defined herein Antibodies or antigen-binding fragments thereof. In one embodiment, the invention is a method of causing or increasing gamma delta T cell killing activity (e.g., T cell mediated killing activity) in an individual while sparing healthy cells, comprising administering to the individual a compound as described herein Anti-Vδ1 antibody or antigen-binding fragment thereof as defined.

In one embodiment, the invention is a method of inducing or increasing γδ T cell toxicity in an individual, comprising the step of administering to the individual an anti-Vδ1 antibody, or an antigen-binding fragment thereof, as defined herein. In one embodiment, the invention is a method of inducing or increasing γδ T cell toxicity in an individual while sparing healthy cells, comprising the step of administering to the individual an anti-Vδ1 antibody or an antigen-binding fragment thereof as defined herein .

In one embodiment, the invention is a method of causing or increasing γδ T cell mobilization in an individual, comprising the step of administering to the individual a multispecific antibody as defined herein.

In one embodiment, the invention is a method of increasing gamma delta T cell survival in an individual, comprising the step of administering to the individual a multispecific antibody as defined herein.

In one embodiment, the invention is a method of increasing resistance to γδ T cell depletion in an individual, comprising the step of administering to the individual a multispecific antibody as defined herein.

According to another aspect of the invention, a method of stimulating an immune response in an individual is provided, the method comprising administering to the individual a multispecific antibody in an amount effective to stimulate the immune response. Uses of multispecific antibodies or antigen-binding fragments thereof

According to another aspect of the invention, there is provided the use of a multispecific antibody as described herein for studying antigen recognition, activation, signal transduction or function of γδ T cells, in particular Vδ1 T cells. As described herein, the antibodies have been shown to be active in assays useful in studying gamma delta T cell function. Such antibodies can also be used to induce the proliferation of γδ T cells and thus can be used in methods of expanding γδ T cells, such as Vδ1 T cells.

Antibodies that bind to the Vδ1 chain can be used to detect γδ T cells. For example, the antibody can be labeled with a detectable label or reporter molecule, or used as a capture ligand to selectively detect and/or isolate Vδ1 T cells in a sample. Labeled antibodies can be used in many methods known in the art, such as immunohistochemistry and ELISA.

The detectable label or reporter molecule can be a radioactive isotope, such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I; a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or if rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase or luciferase. Fluorescent labels applied to the multispecific antibodies of the invention can then be used in fluorescent activated cell sorting (FACS) methods.

Accordingly, in various embodiments, the invention includes methods of modulating γδ T cells in vivo, methods of binding γδ T cells, methods of targeting γδ T cells, methods of activating γδ T cells, methods of proliferating γδ T cells, methods of expanding γδ T cells, Methods for increasing γδ T cells, methods for detecting γδ T cells, methods for causing degranulation of γδ T cells, methods for inducing γδ T cell-mediated killing activity, and methods for selecting antibodies or antigen-binding fragments thereof, the methods comprising The step of administering to an individual a multispecific antibody, or antigen-binding fragment thereof, as described herein. Agents used to modulate gamma delta T cells

Multispecific antibodies as described herein can be used to modulate in situ (i.e., in vivo) or are suitable for modulating delta variable 1 chain (Vδ1) T cells in a patient. Multispecific antibodies or antigen-binding fragments thereof may be included in a medicament for such purposes.

Modulation of Vδ1 T cells may include: - For example, expanding Vδ1 T cells by selectively increasing the number of Vδ1 T cells or improving the survival of Vδ1 T cells; - For example, stimulating Vδ1 T cells by increasing Vδ1 T cell potency, i.e. increasing target cell killing; - For example, preventing Vδ1 T cell exhaustion by increasing Vδ1 T cell persistence; - Degranulate Vδ1 T cells; - Increase NCR performance; - For example, immunomodulating Vδ1 T cells by downregulating Vδ1 TCR cell surface expression, that is, by causing Vδ1 TCR internalization or reducing the expression of Vδ1 TCR protein or blocking Vδ1 TCR binding; and/or - Downregulate TCR/CD3 complex.

Unlike prior art anti-Vδ1 antibodies that focus on depleting Vδ1 T cells, the antibodies of the invention can be used to activate Vδ1 T cells via the TRDV1 binding domain. Although it can cause TCR downregulation on the T cells it binds, it does not cause Vδ1 T cell depletion, but in fact it stimulates T cells and can therefore be used in therapeutic contexts that would benefit from activation of this T cell compartment. Activation of Vδ1 T cells is evident through TCR downregulation, CD3 downregulation, changes in activation markers such as CD25 and Ki67, and the degranulation marker CD107a. Activation of Vδ1 T cells in turn triggers the release of inflammatory cytokines such as INFγ and TNFα, thereby promoting immune permissiveness. Agents that modulate immune cell markers on Vδ1+ cells

The antibody or antigen-binding fragment thereof modulates immune cell markers of Vδ1+ cells after administration to a patient.

The suitability of the antibodies or antigen-binding fragments thereof described herein for their therapeutic use can also be assessed by measuring γδ T modulation. For example, by measuring changes in the levels of CD25 or CD69 or CD107a present on Vδ1+ T cells in a model system. Such markers are commonly used as markers of lymphocyte regulation (eg proliferation or degranulation) and can be measured, for example by flow cytometry, following application of an antibody or antigen-binding fragment thereof as described herein. Unexpectedly, during such evaluations (see, e.g., Examples 7, 17, 18), it was observed that antibodies as described herein conferred measurably higher levels of CD25 or CD69 or CD107a on target Vδ1+ T cells. Optionally, the phenotypic changes of the Vδ1+ cells or populations thereof tested in the model system can then be compared to the phenotypic changes when alternative comparative antibodies (e.g., OKT-3, TS8.2, etc.) are applied to these equivalent γδ T cells. .

Accordingly, in one aspect of the present invention, there is provided a method of evaluating the therapeutic use of an antibody or antigen-binding fragment thereof that binds to the Vδ1 chain of a γδ TCR, comprising administering the antibody or antigen-binding fragment thereof to a cell population comprising Vδ1+ cells. fragments and determine the effect on the levels of CD25 and/or CD69 and/or CD107a on the surface of Vδ1+ cells. The effect on CD25, CD69 and/or CD107a levels can be determined/measured over a period of time. It will be appreciated that the effect can be measured compared to the levels of CD25 and/or CD69 and/or CD107a on the surface of Vδ1+ cells when the antibody is not applied to the cells for the same period of time. In another aspect of the invention, a method of selecting or characterizing or comparing multispecific antibodies or antigen-binding fragments thereof that bind to the Vδ1 chain of a γδ TCR as described herein is provided by adding such antibodies to A cell population is comprised of Vδ1+ cells and the level (or expression) of CD25 or CD69 or CD107a on the surface of these Vδ1+ cells is then measured. Agents that modulate the growth characteristics or number of Vδ1+ cells

The antibody or antigen-binding fragment thereof can modulate the growth characteristics of Vδ1+ cells after administration to a patient. For example, antibodies or antigen-binding fragments thereof can expand Vδ1+ cells.

An alternative method of measuring γδ T proliferation may include measuring changes in the relative number of Vδ1+ cells over time when an antibody or antigen-binding fragment thereof as described herein is applied to a model system containing such cells. Unexpectedly, during such evaluation, it was observed that antibodies as described herein were able to measurably increase the number of such Vδ1+ T cells (see, eg, Examples 10, 17, and 18). Optionally, this number change can then be compared to the number change observed when an alternative comparison antibody (eg, anti-OKT3) is applied to the model system.

Accordingly, in another aspect of the invention, there is provided a method of assessing an antibody or an antigen-binding fragment thereof that binds to a Vδ1 chain of a γδ TCR, comprising administering an antibody or an antigen-binding fragment thereof to a cell population comprising Vδ1+ cells and The effect on the number of Vδ1+ cells in the population was determined. The effect on cell numbers can be determined/measured over a period of time. It will be appreciated that the effect can be measured in comparison to the effect on cell number observed when the antibody is not applied to the cell population for the same period of time. In another aspect of the invention, there is provided a method of selecting or characterizing or comparing antibodies, or antigen-binding fragments thereof, that bind to the Vδ1 chain of a γδ TCR as described herein, by applying the antibodies to a solution containing Vδ1+ Cell populations of cells are then measured over time. Agents that regulate the proliferation ability and number of Vδ1+ cells

An ideal therapeutic antibody or antigen-binding fragment thereof that binds to the Vδ1 chain of a γδ TCR as described herein would be an antibody or antigen-binding fragment thereof that is capable of enhancing the proliferation of Vδ1+ cells in vivo. Such antibodies can then be used as agents designed to specifically increase the number of Vδ1+ cells in an individual or patient. For example: Cancer:

A relative increase in the number of Vδ1+ cells has been reported as a positive prognostic indicator associated with improved outcome in many cancers (see, e.g., Gentles et al. (2015) Nature Immunology 21: 938-945; Wu et al. (2019) Sci. Trans. Med . 11(513): eaax9364; Catellani et al. (2007) Blood 109(5): 2078-2085). In one embodiment, presented herein is an agent capable of increasing the relative or absolute number of Vδ1+ cells in situ in a cancer patient. Pathogenic / parasitic / viral infections:

Enrichment of Vδ1+ cells is observed during host defense against many acquired pathogenic/parasitic/viral infections. For a recent general review, see Zhao et al. (2018) Immunol. Res. 2018:5081634. In addition, an increase in the number of Vδ1+ is also seen as preventing infection by a variety of DNA and RNA viruses. For example, increased numbers are also considered protective during CMV infection associated with allogeneic transplantation (see van Dorp et al. (2011) Biology of Blood and Marrow Transplantation 17(2): S217). Additionally, the number of Vδ+ cells is increased in patients with coronavirus infection (Poccia et al. (2006) J. Infect. Dis. 193(9): 1244-1249).

In one embodiment, presented herein is an agent capable of increasing the relative or absolute number of Vδ1+ cells in an individual or patient with a pathogenic infection. Stem cell transplant :

Generally during hematopoietic stem cell transplantation, increased Vδ1+ cell numbers are also associated with fewer disease recurrences, fewer viral infections, higher overall and disease-free survival, and favorable clinical outcomes (see, for example, Aruda et al. (2019) Blood 3( 21): 3436-3448 and see Godder et al. (2007) Bone Marrow Transplantation 39: 751-757). Accordingly, in another embodiment, presented herein is an agent capable of increasing the relative or absolute number of Vδ1+ cells in an individual as part of a treatment regimen to support stem cell transplantation.

Therefore, there is a particular need for agents that can preferentially or specifically increase the number of Vδ1+ cells in situ. Agents that maintain, induce or increase interleukin secretion from Vδ1+ cells

Interleukins are a large group of proteins, peptides, or glycoproteins secreted by specialized cells of the immune system. It is a type of signaling molecule that mediates and regulates immunity, inflammation and blood cell production. A variety of interleukins participate in improving the signs and symptoms of disease by directly or indirectly regulating tumor and cell microenvironments, autoimmune tissues and related microenvironments, or virus-infected tissues or cell environments. Exemplary pro-inflammatory cytokines include tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ).

However, many of these interleukins exhibit adverse toxicity when administered systemically. For example, although TNFα can induce hemorrhagic necrosis in transplanted tumors and has been reported to exert synergistic antitumor effects when combined with other chemotherapeutic agents, multiple clinical trials utilizing systemic recombinant human TNFα (rhTNFα) have highlighted that Significant dose-limiting side effects include hypotension, stiffness, phlebitis, thrombocytopenia, leukopenia and hepatotoxicity, fever, fatigue, nausea/vomiting, malaise and weakness, headache, chest tightness, low back pain, diarrhea and shortness of breath.

The use of recombinant IFNγ also faces similar systemic toxicity challenges. For example, although IFNγ can exert beneficial pleiotropic effects in the context of cancer, including MHC class I and class II upregulation to stimulate anti-tumor immunity, increase T cell infiltration, confer anti-angiogenic effects, induce chemokines/ Cytokines secrete and exert direct anti-proliferative effects on cancer cells, but harmful side effects have also been observed. These side effects include fever, headache, chills, fatigue, diarrhea, nausea, vomiting, anorexia, transient increases in liver transaminases, and transient decreases in granulocyte and white blood cell counts.

For a recent review of the potential and limitations of systemic recombinant TNFα and IFNγ, see Shen et al. (2018) Cell Prolif. 51(4): e12441.

Therefore, there is a need for more in situ control of such cytokines, more localized production of such cytokines, and more tissue- or cell-specific production of such cytokines. For example, it is proposed that the induction of more controlled expression of pro-inflammatory cytokines may be a way to turn "cold" tumors into "hot" tumors. Because an increase in the number or density of CD45+ T cells is also observed, thermal tumors are sometimes referred to as "T cell inflammation." For a recent review, see Bonaventura et al. (2019) Front. Immunol. 10: 168.

For this reason, an ideal therapeutic antibody or antigen-binding fragment thereof that binds to the Vδ1 chain of a γδ TCR as described herein would be an antibody or antigen-binding fragment thereof that can maintain or enhance or induce interleukin secretion in Vδ1+ cells in vivo. Antigen-binding fragments. Such antibodies can then be used as agents designed to specifically increase or induce interleukins in an individual or patient in a manner that is more localized, less systemic, and consistent with Vδ1+ in that individual or patient. The distribution of cells is more relevant.

Notably, when antibodies binding to the Vδ1 chain of the γδ TCR as described herein were applied to Vδ1+ cells, significantly higher levels of secreted interleukin were observed. More specifically and as a non-limiting example, significantly higher levels of TNFα and IFNγ were observed. See, for example, Example 15.

Accordingly, in another aspect of the invention, there is provided a method of assessing an antibody or an antigen-binding fragment thereof that binds to a Vδ1 chain of a γδ TCR, comprising administering an antibody or an antigen-binding fragment thereof to a cell population comprising Vδ1+ cells and The amount of at least one interleukin produced by a population of cells is determined. The amount of interleukin produced can be determined/measured over a period of time and optionally compared to the amount observed over the same period of time when the antibody is not applied to the cell population. In one embodiment, the observed level of interleukin produced when the antibody is administered to a population of cells is greater than about 10%, greater than about 20%, greater than about 20% relative to the level of interleukin produced when no antibody is applied. 30%, more than about 50%, more than about 100%, more than about 150%, more than about 200%, more than about 250%, more than about 300%, more than about 350%, more than about 400%, more than about 450%, more than about 500%, more than about 1000%. In another aspect of the invention, the interleukin is a pro-inflammatory cytokine. In another aspect of the invention, the interleukin is TNF-alpha interleukin. In another aspect of the invention, it is IFN-γ interleukin.

In another aspect of the invention, there is provided a method of selecting or characterizing or comparing antibodies, or antigen-binding fragments thereof, that bind to the Vδ1 chain of a γδ TCR as described herein, by applying the antibodies to a solution containing Vδ1+ The cell population of cells is then measured and the level of the at least one interleukin produced is measured. In another aspect of the invention, the interleukin measured is TNF-alpha interleukin and/or IFN-gamma interleukin.

In another aspect of the invention, there is provided a method of assessing an antibody or antigen-binding fragment thereof that binds to the Vδ1 chain of a γδ TCR by applying the antibody or antigen-binding fragment thereof to a cell population comprising Vδ1+ cells, And the effect of antibodies on modulating tumors from cooler or colder to warmer or hotter is measured by measuring the amount of pro-inflammatory cytokines produced and/or the number or density of CD45+ T cells present in the tumor or tumor microenvironment. The role of tumors. Agents that maintain, induce or increase granzyme B activity in Vδ1+ cells

Granzyme B is a serine protease commonly found in the granules of natural killer cells (NK cells) and cytotoxic T cells. Together with the pore-forming protein perforin, it is secreted by these cells to mediate apoptosis in target cells, such as diseased cells.

When Vδ1+ cells are cultured in co-culture with target disease cells (such as cancer cells) in a model system, the level and activity of granzyme B can be measured in the target disease cells prior to lysis. Notably, when an antibody or antigen-binding fragment thereof that binds to the Vδ1 chain of a γδ TCR as described herein is administered to such co-cultures of Vδ1+ cells and cancer cells in such model systems, prior cell death is observed Granzyme B levels and activities are higher in diseased cancer cells (see, eg, Example 16).

Accordingly, in another aspect of the present invention, a method for assessing an antibody or antigen-binding fragment thereof that binds to the Vδ1 chain of a γδ TCR is provided, which comprises adding to a common cell including Vδ1+ cells and diseased cells (such as cancer cells). The cultures are dosed with antibodies or antigen-binding fragments thereof and the effect on the amount of granzyme B produced by diseased cells in the co-culture is measured. The amount of interleukin produced can be determined/measured over a period of time and optionally compared to the amount observed over the same period of time when the antibody is not applied to the co-culture. In one embodiment, the level of granzyme B measured when the antibody is applied to the co-culture is greater than about 10%, greater than about 10% relative to the level of granzyme B observed when the antibody is not applied. 20%, more than about 30%, more than about 40%, more than about 50%, more than about 70%, more than about 80%, more than about 90%, more than about 100%, more than about 200%.

In another aspect of the invention, there is provided a method of selecting or characterizing or comparing antibodies, or antigen-binding fragments thereof, that bind to the Vδ1 chain of a γδ TCR as described herein, by applying the antibodies to a solution containing Vδ1+ Cell populations of cells are then measured for the amount or activity of granzyme B in the diseased cells. Agent to expand multiple Vδ1+ cell populations

The ideal antibody agent would also be one designed to ensure that the expanded Vδ1+ cells do not become too homogeneously concentrated at the level of hypervariable CDR3 sequences. Therefore, an ideal antibody agent could be designed to avoid inducing proliferating Vδ1+ cells by binding to specific or “dedicated” δ1+ CDR3 sequence paratopes. Specifically, the antibody binds via conserved germline sequences present on all Vδ1+ T cell receptors and in a γ-chain-independent manner, rather than only to sequences presented by a subset of Vδ1+ cells.

Therefore, an ideal antibody agent would stimulate the expansion of Vδ1+ cells to generate multiple Vδ1+ cells containing a mixture of CDR3 sequences. This in turn will generate an in vivo expanded heterogeneous multi-strain population of Vδ1+ cells exhibiting different CDR3 sequences on the delta variable 1 chain. Notably, during the analysis of the expanded Vδ1+ cell population generated by the method of adding an antibody as described herein or an antigen-binding fragment thereof to a starting population of immune cells containing Vδ1+ cells, by RNAseq-based Extensive polygenicity was observed with methods designed to sequence via the CDR3 hypervariable regions of extracted RNA (see, eg, Example 10).

Accordingly, in one aspect, a method of assessing an antibody or an antigen-binding fragment thereof that binds to a Vδ1 chain of a γδ TCR is provided, comprising administering an antibody or an antigen-binding fragment thereof to a cell population comprising Vδ1+ cells and determining the amplified Polythallicity of Vδ1+ cells. Antibody agents are expected to generate amplified multi-strain populations containing a plurality of Vδ1+ CDR3 sequences. Polyclonalism can be determined using methods known in the art, such as by nucleic acid sequencing methods capable of analyzing the Vδ1 chain hypervariable CDR3 content of the Vδ1+ cells. A drug that can expand multiple strains of Vδ1+ cells for a long time

An ideal antibody agent would be able to enhance or promote or stimulate the proliferation of primary Vδ1+ cells without depleting such cells in vivo. By way of example and by way of comparison, anti-CD3 agents such as OKT3 (eg, Muronomab), while capable of expanding CD3-positive T cells, may also deplete or induce anergy. To evaluate the ability of antibodies as described herein that bind to the Vδ1 chain of the γδ TCR to drive sustained cell division of viable Vδ1+ cells, long-term proliferation studies were performed. Notably, these studies show that antibodies as described herein that bind to the Vδ1 chain of the γδ TCR are able to drive cell division/proliferation of viable and functionally cytotoxic Vδ1+ cells for up to 40 days (see, e.g., Example 10) .

In one embodiment, a method of assessing an antibody or antigen-binding fragment thereof that binds to the Vδ1 chain of a γδ TCR is provided, comprising administering the antibody or antigen-binding fragment thereof to a population of cells and monitoring the length of time Vδ1+ cell division occurs. Ideally, the antibody is capable of stimulating Vδ1+ cell division for a period of 5 to 60 days, such as at least 7 to 45 days, 7 to 21 days, or 7 to 18 days.

In another embodiment, there is provided an antibody, or antigen-binding fragment thereof, as described herein, which binds to the Vδ1 chain of a γδ TCR and, when administered to a patient, is capable of stimulating Vδ1+ cell division to increase number, such that the number is increased by at least 2 times, the number increased at least 5 times, the number increased at least 10 times, the number increased at least 25 times, the number increased at least 50 times, the number increased at least 60 times, the number increased at least 70 times, the number increased at least 80 times, the number increased at least 90 times , the number is increased by at least 100 times, the number is increased by at least 200 times, the number is increased by at least 300 times, the number is increased by at least 400 times, the number is increased by at least 500 times, the number is increased by at least 600 times, or the number is increased by at least 1,000 times.

In another aspect of the invention, there is provided a method of selecting or characterizing or comparing antibodies, or antigen-binding fragments thereof, that bind to the Vδ1 chain of a γδ TCR as described herein, by applying the antibodies to Vδ1+ cells or a mixed cell population containing Vδ1+ cells and then measuring Vδ1+ cell numbers over time. Agents that regulate non- Vδ1+ immune cells by targeting Vδ1+ immune cells

Antibodies or antigen-binding fragments thereof as described herein may also be evaluated by measuring Vδ1+ cell-mediated modulation of other immune cells. For example, the expression of non-γδ T cells can be measured after administration of an antibody or antigen-binding fragment thereof as described herein to a model system comprising a mixed immune cell population, such as a model system comprising human tissue αβ cells and γδ T cells. Changes observed in "Part". Additionally, the effects on non-γδ cell types in these models can be measured by flow cytometry. For example, by measuring the relative change in the number of CD8+ αβ T cells upon addition of an antibody or antigen-binding fragment thereof as described herein to a mixed culture comprising γδ T cells and non-γδ T cells. Optionally, the observed changes in the number or phenotype of the non-γδ T cell CD8+ lymphocyte population can then be compared to the changes in number when an alternative comparison antibody (eg, OKT-3) is applied to this mixed population.

Therefore, in another aspect of the present invention, there is provided a method of assessing antibodies or antigen-binding fragments thereof that bind to the Vδ1 chain of a γδ TCR, which comprises introducing a mixed immune cell population or tissue containing Vδ1+ cells and Vδ1-negative immune cells. The antibody or antigen-binding fragment thereof is administered and the effect on Vδ1 negative immune cells is measured. The effect can be determined/measured over a period of time and optionally compared to the effect observed in Vδ1 negative cells over the same period of time when the antibody is not applied. This effect can be measured as a change in the number of Vδ1 negative immune cells. For example, an antibody can increase the number of Vδ1 negative immune cells by more than about 10%, more than about 20%, more than about 30%, more than about 40%, more than about 40%, more than about 10%, more than about 40%, more than about 40%, or more than about 40%, relative to the level observed when the antibody is not applied. 50%, more than about 70%, more than about 80%, more than about 90%, more than about 100%, more than about 500%.

In another aspect of the invention, the modulated Vδ1 negative cells are CD45+ cells. In another aspect of the invention, the modulating cells are αβ T cells. In another aspect of the invention, the αβ+ cells modulated are CD8+ lymphocytes. In another aspect of the invention, the modulated αβ T cells or populations thereof exhibit evidence of enhanced cell division. In another aspect of the invention, there is provided a method of selecting or characterizing or comparing antibodies, or antigen-binding fragments thereof, that bind to the Vδ1 chain of a γδ TCR as described herein, by immunizing cells comprising Vδ1+ and Vδ1 negative A mixed immune cell population of cells is administered the antibodies and the effect on the Vδ1 negative cell population conferred by the Vδ1+ cells modulated by the multispecific antibodies or antigen-binding fragments thereof is then measured.

Optionally, and during "Vδ1+ cell-mediated modulation of the immune system" conferred by an antibody or antigen-binding fragment thereof as described herein, a concomitant increase in the number of Vδ1+ cells is also observed. While not bound by this theory, this increase in Vδ1+ cell numbers may be responsible for driving the consequent expansion of co-existing Vδ1 negative immune cells, such as αβ T cells. An alternative hypothesis could be that antibody-induced secretion of interleukins from Vδ1+ T cells stimulates the expansion of Vδ1-negative immune cells.

In another aspect of the invention, the observed increase in the population of αβ+ CD8+ lymphocytes is compared to a comparator antibody such as an OKT3 antibody or a surrogate anti-Vδ1 antibody. In another aspect of the invention, there is provided a method of selecting or characterizing or comparing antibodies, or antigen-binding fragments thereof, that bind to the Vδ1 chain of a γδ TCR as described herein, by applying the antibodies to a solution containing Vδ1+ Mixed immune cell populations of T cells and αβ T cells and then measure the number of CD8+ αβ+ T cell lymphocytes over time. Agents that modulate tumor-infiltrating lymphocytes (TILs)

Antibodies or antigen-binding fragments thereof as described herein may also be evaluated by measuring the effects conferred on tumor-infiltrating populations (TILs) in model systems. Unexpectedly (see, eg, Example 18), antibodies as described herein measurably modulate TIL populations in human tumors during such assessments. For example, measuring changes in the number or phenotype of a γδ+ lymphocyte TIL population or a non-γδ lymphocyte TIL population following application of an antibody as described herein, or an antigen-binding fragment thereof, to a human tumor, such as human renal cell carcinoma. . Optionally, the observed changes in the number or phenotype of the γδ+ lymphocyte TIL population or the non-γδ lymphocyte TIL population can then be compared to the changes observed when an alternative comparative antibody (e.g., OKT-3) is applied to the model system. Make a comparison.

Accordingly, in another aspect of the invention, there is provided a method of assessing an antibody or an antigen-binding fragment thereof that binds to the Vδ1 chain of a γδ TCR, comprising administering an antibody to a TIL located in or derived from a human tumor or Its antigen-binding fragments and the effect on the number of TILs were determined. The effect can be determined/measured over a period of time and optionally compared to the number of TILs observed over the same period of time when the antibody is not applied. This effect can be an increase in the number of TILs. For example, an antibody can increase the number of TILs by more than about 10%, by more than about 20%, by more than about 30%, by more than about 40%, by more than about 50%, by more than about 50%, relative to the number of TILs observed when the antibody is not applied. About 70%, more than about 80%, more than about 90%, more than about 100%. In another aspect, the observed number of TILs are γδ+ lymphocyte TIL cells and/or non-γδ lymphocyte TIL cells.

In another aspect of the invention, there is provided a method of selecting or characterizing or comparing antibodies, or antigen-binding fragments thereof, that bind to the Vδ1 chain of a γδ TCR cell antibody as described herein, by applying the antibodies to a region located TILs in or derived from human tumors and then measuring changes in TIL cell numbers over time. Agents that modulate human Vδ1+ cytotoxicity

Antibodies or antigen-binding fragments thereof as described herein can also be evaluated by measuring the effect conferred on Vδ1+-mediated cytotoxicity. Unexpectedly, during such evaluation of antibodies as described herein (eg, see Examples 19 and 27), measurably enhanced Vδl+-mediated cytotoxicity to cells was observed. For example, a decrease in the number of cancer cells or an increase in the number of killed cancer cells was observed after the antibody or antigen-binding fragment thereof was applied to a model system including a mixed culture of Vδ1+ cells and such cancer cells. Optionally, the reduction in the number of cancer cells or the increase in the number of killed cancer cells can then be compared to the results when an alternative comparative antibody (eg, OKT-3) is applied to the model systems.

Therefore, in another aspect of the invention, there is provided a method of assessing an antibody or an antigen-binding fragment thereof that binds to a Vδ1 chain of a γδ TCR, which comprises applying the antibody or an antigen-binding fragment thereof to a cell comprising Vδ1+ cells and cancer cells. Cell populations were mixed and the cytotoxicity of Vδ1+ cells against cancer cells was measured. Cytotoxicity can be measured by the increase in the number of dead cancer cells over a period of time, optionally compared to the number of dead cancer cells observed over the same time period when the antibody is not applied to the mixed cell population. For example, the increase in dead cells observed when the antibody is applied can be more than about 10%, more than about 20%, more than about 30%, more than about 40% relative to the number of dead cells observed when the antibody is not applied. , more than about 50%, more than about 70%, more than about 80%, more than about 90%, more than about 100%, more than about 200%, more than about 500%.

In another aspect of the invention, there is provided a method of selecting or characterizing or comparing antibodies, or antigen-binding fragments thereof, that bind to the Vδ1 chain of a γδ TCR cell as described herein, by adding the antibodies to a compound containing This mixed immune cell population of human Vδ1+ cells and cancer cells was then measured over time for the increase in dead cancer cells. Agents that modulate Vδ1+ cell target to effector cell ratio (T:E ratio )

Antibodies or antigen-binding fragments thereof as described herein can also be evaluated by measuring how the antibodies enhance Vδ1+-mediated cytotoxicity against cancer cells by determining the target cell to effector cell ratio, where killing in a model system 50% target cells (EC50) to evaluate these antibodies as potential agents. For example, a mixed culture contains target cancer cells and human Vδ1+ effector cells. Unexpectedly, during such assessment (eg, see, eg, Example 19), antibodies as described herein favorably modulate the EC50 T:E ratio in a model system. Such regulation can be measured as the number of Vδ1+ cells required to observe 50% killing of cancer cells within a set time. This may also be reported as a change or fold improvement or percentage improvement in cytotoxicity to these cancer cells. Optionally, the T:E ratio conferred by the antibodies of the invention can then be compared to the T:E ratio when an alternative comparison antibody (eg, OKT-3) is applied to such model systems. In some cases, multispecific antibodies of the invention present opportunities for improved cytotoxicity against cancer cells compared to monospecific antibodies even at lower E:T ratios.

Accordingly, in another aspect of the invention, a method of assessing an antibody or an antigen-binding fragment thereof that binds to the Vδ1 chain of a γδ TCR is provided, comprising applying the antibody or antigen-binding fragment thereof to a cell comprising human Vδ1+ cells and cancer cells. Mix the cell populations and measure the number of Vδ1+ cells required to kill 50% of cancer cells. This can be measured relative to the number of Vδ1+ cells required to kill 50% of cancer cells in the same time period without the application of the antibody, optionally. For example, the number of Vδ1+ cells required to kill 50% of cancer cells when the antibody is applied can be reduced by greater than about 10%, relative to the number of Vδ1+ cells required to kill 50% of cancer cells when the antibody is not applied. Greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 100%, greater than about 200%, greater than about 500%.

In another aspect of the invention, there is provided a method of selecting or characterizing or comparing antibodies that bind to a Vδ1 chain, or antigen-binding fragments thereof, as described herein, by adding the antibodies to a solution containing Vδ1+ cells plus cancer cells. of this cell population and then measured the number of Vδ1+ cells required to kill 50% of cancer cells. Agents that enhance EC50 cytotoxicity of Vδ1+ cells

An alternative way of measuring the observed enhanced cytotoxicity of human Vδ1+ cells or populations thereof is to measure the number of cells required to kill 50% of cancer cells over a set period of time under Condition A (such as a starting control), And compare this to the number of cells required to kill 50% of cancer cells over a set period of time under Condition B, such as when an antibody of the invention as described herein is applied.

While it is recognized that there are many ways to measure such parameters, to aid understanding, the following non-limiting hypothetical examples will be outlined:

Hypothetically, the enhancement of effector cell cytotoxicity can be measured as follows: Under condition A (control treatment), it can be observed that 1000 Vδ1+ cells are required to kill 50% of cancer cells within a set time period (e.g., 5 hours). Under Condition B (eg, application of an antibody of the invention as described herein), it was observed that 500 Vδ1+ cells were required to kill 50% of cancer cells in the same time period. Thus, in this example, application of the antibody enhanced the cytotoxicity of the Vδ1+ cell population by 200%: (1000/500) × 100= 200%

For example, (see, eg, Examples 19 to 21), unexpectedly, such percentage enhancements have been observed for multispecific antibodies of the invention as described herein.

In another aspect of the invention, there is provided a method of selecting or characterizing or comparing antibodies, or antigen-binding fragments thereof, that bind to the Vδ1 chain of a γδ TCR as described herein, by adding the antibodies to cells containing Vδ1+ and cancer cells and determine the relative change or percentage change in cytotoxicity relative to an equivalent or control experiment in which the antibody was not applied to the cell mixture. Agents that enhance the disease cell specificity of Vδ1+ cells while avoiding healthy cells

Another method of evaluating multispecific antibodies or antigen-binding fragments thereof as described herein is to measure how these antibodies modulate disease cell-specific cytotoxicity. Unexpectedly, during such studies, it was found that such antibodies can specifically enhance the specific killing of diseased cells such as cancer cells by Vδ1+ cells (see, e.g., Examples 19 and 27), while sparing healthy or non-pathological cells . An ideal antibody agent administered to patients to ameliorate cancer symptoms would confer enhanced cytotoxicity specific to diseased cells while sparing healthy cells. And it can be said that an agent that enhances the cytotoxicity of effector cells specifically against diseased cells exhibits an enhanced therapeutic index (TI) compared to an agent that does not selectively enhance the cytotoxicity of effector cells specifically against such diseased cells. The therapeutic index is also called the therapeutic ratio and is a quantitative measure of the relative safety of a drug. It is the amount of a therapeutic agent that causes a therapeutic effect compared to the amount that causes toxicity, for example, by causing inappropriate death of an associated or associated healthy cell population. An antibody or antigen-binding fragment thereof as described herein may be assessed by measuring its ability to alter or enhance or fold the ability of Vδ1+ cells to selectively kill diseased cells relative to and over healthy cells in a model system. For example, the model system can include Vδ1+ effector cells, cancer cells, and control cells (such as healthy cells). Optionally, the fold increase in selective killing of diseased cells conferred by the multispecific antibodies of the invention can then be compared to the fold increase observed when an alternative comparative antibody (eg, OKT-3) is applied to such model systems.

The enhancement of disease cell specificity and disease cell specificity of Vδ1+ cells can be measured in cultures containing Vδ1+ cells, diseased cells, and healthy cells. For example, Vδ1+ specificity against diseased cells can be measured by observing the number of cancer cells killed by Vδ1+ cells and then comparing the number of healthy cells killed by Vδ1+ cells. Such comparisons can be controlled by including equal numbers of diseased and healthy cells in a model system that also contains Vδ1+ cells (eg, "triple cultures"). Alternative comparison methods may also be considered - for example when assay or equipment limitations reduce the ability to simultaneously distinguish and track all three cell types or more cell types (including Vδ1+ cells, diseased cells, and non-diseased cells) in a single analysis. In these cases, comparing the cytotoxicity of Vδ1+ cells against diseased cells in one experiment and then comparing the cytotoxicity of Vδ1+ cells against healthy cells in another equivalent experiment provides an alternative to such studies.

In another aspect of the invention, there is provided a method of assessing an antibody or antigen-binding fragment thereof that binds to the Vδ1 chain of a γδ TCR, comprising administering the antibody or antigen-binding fragment thereof to a cell population comprising Vδ1+ cells and target cells. And measure the cytotoxicity to target cells. In one embodiment, the cytotoxicity specificity for a first target cell type can be compared to the observed cytotoxicity for a second target cell type, and thus the method can be repeated using different target cell types. In another aspect of the invention, the first target cell type is a diseased cell and the second target cell type is a control cell, such as a healthy cell or a cell with a different disease than the first target cell type.

In another aspect of the invention, there is provided a method for selecting or characterizing or comparing antibodies or antigen-binding fragments thereof that bind to the Vδ1 chain of a γδ TCR as described herein, wherein the response conferred by the antibody is measured. Effects of Vδ1+ cells on cytotoxicity of (i) first cell type and (ii) second cell type and comparison. In another aspect of the invention, an antibody is selected that enhances specific cytotoxicity toward a first cell type more than specific cytotoxicity toward a second cell type. In another aspect of the invention, the first cell type is diseased cells and the second cell type is healthy cells.

As described herein, the multispecific antibodies or antigen-binding fragments thereof used in the assay can be presented on a surface, for example, a cell, such as a cell containing an Fc receptor. For example, multispecific antibodies or antigen-binding fragments thereof can be presented on the surface of THP-1 cells, such as TIB-202™ cells (available from the American Type Culture Collection (ATCC)). Alternatively, multispecific antibodies or antigen-binding fragments thereof can be used directly in the assay.

In this type of functional analysis, the output can be measured by calculating the half-maximal concentration, also known as the "EC50" or "50% effective concentration." The term "IC50" refers to the inhibitory concentration. Both EC50 and IC50 can be measured using methods known in the art, such as flow cytometry. In some cases, EC50 and IC50 are the same value or can be considered equivalent. For example, the effective concentration (EC) of effector cells required to inhibit (e.g., kill) 50% of a certain cell type can also be regarded as the 50% inhibitory concentration (IC). For the avoidance of doubt, when referring to antibodies, the EC50 values in this application are provided using IgGl formatted antibodies. To find equivalent values, it is easy to convert such values based on molecular weight in antibody format, as follows: (µg/ml) / (MW in kDa) = µM

For the parent pure line described herein, the EC50 for down-regulation of γδ TCR when the antibody (or fragment) binds can be less than 0.50 µg/ml, such as less than 0.40 µg/ml, 0.30 µg/ml, 0.20 µg/ml, 0.15 µg/ml , 0.10 µg/ml, 0.06 µg/ml or 0.05 µg/ml. In a preferred embodiment, the EC50 of down-regulation of γδ TCR when the antibody (or fragment) binds is less than 0.10 μg/ml. Specifically, the EC50 for down-regulation of γδ TCR upon binding of the antibody (or fragment) may be less than 0.06 μg/ml, such as less than 0.05 μg/ml, 0.04 μg/ml, or 0.03 μg/ml. Specifically, these EC50 values are when the antibody is measured in IgG1 format. For example, EC50 γδ TCR downregulation can be measured using flow cytometry (eg, as described in the assays of Examples 6 and 27, for example).

The EC50 for degranulation of γδ T cells upon binding of the antibody (or fragment) may be less than 0.050 µg/ml, such as less than 0.040 µg/ml, 0.030 µg/ml, 0.020 µg/ml, 0.015 µg/ml, 0.010 µg/ml or 0.008 µg /ml. In particular, the EC50 for degranulation of γδ T cells upon binding of the antibody (or fragment) may be less than 0.005 μg/ml, such as less than 0.002 μg/ml. In a preferred embodiment, the EC50 of degranulating γδ T cells when the antibody (or fragment) binds is less than 0.007 μg/ml. Specifically, these EC50 values are when the antibody is measured in IgG1 format. For example, the γδ T cell degranulation EC50 value can be measured by detecting CD107a expression (ie, a marker of cell degranulation) using flow cytometry (eg, as described in the analysis of Example 7). In one example, CD107a performance is measured using anti-CD107a antibodies, such as anti-human CD107a BV421 (clone H4A3) (BD Biosciences).

The EC50 for killing γδ T cells when the antibody (or fragment) binds can be less than 0.50 µg/ml, such as less than 0.40 µg/ml, 0.30 µg/ml, 0.20 µg/ml, 0.15 µg/ml, 0.10 µg/ml or 0.07 µg /ml. In a preferred embodiment, the EC50 of killing γδ T cells when the antibody (or fragment) binds is less than 0.10 μg/ml. Specifically, the EC50 for killing γδ T cells when the antibody (or fragment) binds may be less than 0.060 μg/ml, such as less than 0.055 μg/ml, especially less than 0.020 μg/ml. Specifically, these EC50 values are when the antibody is measured in IgG1 format. For example, the EC50 γδ T cell killing value can be measured by using flow cytometry to detect the proportion of dead cells (i.e., using a cell viability dye) after incubation of antibodies, γδ T cells, and target cells ( For example, as described in the analysis of Example 8). In one embodiment, a cell viability dye, Viability DyeeFluor™ 520 (ThermoFisher), is used to measure target cell death.

In the assays described in these aspects, the antibody or antigen-binding fragment thereof can be presented on the surface of cells, such as THP-1 cells, eg, TIB-202™ (ATCC). THP-1 cells are optionally labeled with a dye, such as CellTracker™ Orange CMTMR (ThermoFisher). Agents that downregulate CD3 molecules associated with V δ 1 TCR

Presented herein are antibodies that engage T cell/CD3 complexes in different ways. Specifically, these antibodies can engage via the TRDV1 domain of the Vδ1 TCR expressed only on Vδ1+ cells. Thereby, such agents work in different ways. This conjugation event can in turn downregulate the TCR-associated CD3 molecular complex. Such CD3 downregulation may be synonymous with T cell activation. However, by engaging the T cell/CD3 complex via the TRDV1 domain in this manner, only CDR3 molecules associated with the Vδ1 TCR are subsequently downregulated. This mechanism is clearly demonstrated in Figure 29.

Accordingly, in one embodiment, a method of using antibodies to downregulate TRDV1-containing Vδ1 TCR and associated CD3 molecule complexes on the cell surface is provided, and the use of such antibodies for this purpose.

In some embodiments, multispecific TCEs capable of engaging T cell/CD3 complexes via TRDV1 domains are also presented herein. Current multispecific TCE-formatting agents typically engage and activate T cells via CD3 binding events. This can cause downregulation of CD3 molecular complexes from the T cell surface. However, it is also well understood that TCEs can also overstimulate T cells through such engagement and down-regulation. The CD3 molecular complex is not specific to one type of T cell, so it is not a precise target. Stimulation of all T cells (the main αβ subtype) via CD3 can in turn cause overproduction of interleukins, leading to an acute interleukin burst (the so-called interleukin storm). Additionally, in non-targeted approaches that engage and activate all T cells via CD3, paradoxically, T cells can become hyperactivated, which leads to chronic T cell exhaustion and/or T cell death. In fact, this non-specific pan-T cell activation results in the activation of both effector and regulatory T cells, and the methods presented in this invention specifically interact with the effector population. Therefore, the "sledgehammer" approach is far from ideal when one wishes only to upregulate selective T cells.

In contrast, multispecific antibodies, in particular T cell engagement molecules, are presented herein that engage T cell/CD3 complexes in different ways. Specifically, these TCEs can engage via the TRDV1 domain of the Vδ1 TCR expressed only on Vδ1+ cells. Thereby, such TCE-based agents work in different ways. First, these TCEs are able to downregulate the TCR by engaging TRDV-1 epitopes. This conjugation event in turn downregulates the TCR-associated CD3 molecular complex. Such CD3 downregulation may be synonymous with T cell activation. However, by engaging the T cell/CD3 complex via the TRDV1 domain in this manner, only CDR3 molecules associated with the Vδ1 TCR are subsequently downregulated. This method of specifically targeting and activating Vδ1 cells allows avoiding many of the above-mentioned problems (eg, interleukin storm, T cell depletion/exhaustion, and ADCC). Again, this mechanism is clearly demonstrated in Figure 29.

Stimulation of T cells via the CD3 "sledgehammer" approach can also lead to T cell depletion via Fcγ receptor-driven mechanisms such as ADCC. Therefore, most bispecific antibodies targeting CD3 in current clinical practice have an Fc domain with reduced binding activity to FcγR, or are bispecific fragments intentionally devoid of an Fc region. Therapies targeting CD3 can also reduce the binding affinity of the binding arm of the T cell receptor complex.

This reduction in affinity can lead to reduced efficacy and less choice in TCE design and functionality. For example, it is known that the affinity of the binding domain in such TCEs drives the distribution profile in vivo. Specifically, TCE distributions are typically observed to be biased toward their highest affinity targets. Therefore, by reducing the affinity of the TCE binding domain to the T cell complex, the distribution is often shifted away from the T cells that are required to drive the efficacy of such TCEs. This is part of the reason why the therapeutic scope of TCE is said to be "too narrow."

The method presented here specifically targets and activates vδ1 cells, avoiding the need to remove Fc function or reduce affinity. This is shown for example in Figure 37 and Example 15, demonstrating the potential to further reduce dose-limiting skin toxicity, for example relative to the higher affinity EGFR variants of the invention.

Additionally, in the approach presented here, antibodies bind to the TCR of vδ1 cells, but full activation does not occur unless tumor cells are also present. Complete engagement of the presently presented antibodies on the TCR causes partial downregulation, and only vδ1 cells bound by the presently presented antibodies in the tumor microenvironment become fully activated and become cytotoxic. This represents another important safety advantage of the method of the invention, since off-target cytotoxicity is reduced and the full potency of the antibodies of the invention to activate vδ1 cells is only released in the presence of tumor cells.

It is believed that one mechanism behind the ability of γδ T cells to detect stress signals on tumor cells is caused by their expression of NCR (natural cytotoxicity receptor). NCR can bind to NCR ligands on tumor cells. A dual activation mechanism is therefore employed, in which γδ T cells are activated via TCR stimulation, and the NCR senses tumor cells to enable their full activation and cytotoxicity.

This is in contrast to stimulation of αβ T cells via CD3, where all stimulation is via the TCR. Therefore, such cells are virtually indistinguishable between healthy or transformed cells because they do not have the ability to sense tumor cells independent of antigen presentation, such as via NCR. Therefore, if a CD3 antibody has Fc function, it attracts other immune cells that can trigger a cascade of unpredictable and desirable events, such as interleukin storm, exhaustion and even overactivation of immune cells, leading to, for example, NK cell killing of T cells etc. In the methods of the present invention, stimulation of γδ T cells with the antibodies presented herein does not cause such problems because γδ T cells (and other immune cells, such as NK cells) are able to differentiate between healthy cells and tumor cells, including via their NCR sensing detection mechanism, and therefore selectively kills stressed cells, such as cancer cells or virus-infected cells, due to this diseased cell specificity.

For example, in the use of the Fc-functional anti-vδ1 multispecific antibodies presented herein that are specific for human and cynomolgus vδ1 antigens (SEQ ID NO: 1 and SEQ D NO: 172, respectively) In the initial cynomolgus macaque study, it was found to be safe and well tolerated by all parameters measured in an in vivo study with repeated doses of escalating doses. No side effects commonly associated with T cell activation, such as interleukin release or weight loss, were observed.

These findings also highlight another advantage of the approach described here. Specifically, and different from TCE represented by CD3 conjugating molecules and their analogs, the TCE bispecific antibody of the present invention can be designed as a full-length antibody, for example, including a heavy chain with a VH-CH1-CH2-CH3 format and Homologous light chain in VL-CL format. Unlike smaller bispecific formats (eg, less than 70 KDa), such full-length bispecific formats may exhibit longer in vivo half-lives, thereby requiring less frequent dosing regimens. The longer half-life observed in this format is due to several reasons, including increased size (>70 KDa), which means that this format cannot be filtered by the kidneys (glomerular pore size cutoff 60-70 KDa). In one embodiment, the multispecific antibody can be greater than about 70 KDa and can comprise human IGHC sequences as listed by IMGT (eg, IGHA, IGHD, IGHD, IGHM, IGHG sequences). Such IgG1 formats can also be recycled by the FcRn mechanism. Obvious disadvantages of such full-length antibody formats of TCE bispecific antibodies are, inter alia, that such formats exhibit an unfavorable safety profile due to reduced clearance rates, increased and more chronic exposure.

Thus, the method of the present invention allows for Fc functionality without any issues with off-target effects, such as NK cells killing γδ T cells or vice versa. Therefore, the method of the present invention is superior to CD3-directed approaches, which are hampered by the need for circumvention strategies such as reducing CD3 affinity, eliminating Fc function, etc. to limit collateral damage outside the tumor environment. The multispecific antibodies presented here, especially T cell engagement molecules, are able to bind to vδ1 cells without any possibility of damage. They are fully activated and enhance cytotoxicity only when vδ1 cells are in close contact with tumor cells.

Therefore, in one embodiment, a method of down-regulating TRDV1-containing Vδ1 TCR and associated CD3 molecular complexes on the cell surface using TCE multispecific antibodies is provided, and the use of such multispecific antibodies for this purpose .

Other features and advantages of the invention will be apparent from the description provided herein. It is to be understood, however, that this specification and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, as various changes and modifications will become apparent to those skilled in the art. The invention will now be described using the following non-limiting examples. Example Example 1 : Table

Parent antibody clones were prepared as described in Examples 1 to 19 in WO2021/032963.

The sequences of the CDRs of the parental homogeneous lines are shown in Table 24 below. Table 23. DV1 binding agents for IgG conversion Pure ID Heavy CDR1 SEQ ID NO. Heavy CDR2 SEQ ID NO. Heavy CDR3 SEQ ID NO. Light CDR1 SEQ ID NO. Light CDR2 SEQ Light CDR3 SEQ ID NO. 100 nM L1 1245_P02_G04 GDSVSSKAA 51 TYYRSKWST 53 TWSGYVDV 54 QDINDW 79 DAS 80 QQSYSTPQVT 81 5896 1245_P01_E07 GFTFSDYY 130 ISSSGSTI 131 VDYADAFDI 132 QSIGTY 144 VAS 145 QQSYSTLLT 146 162591 1252_P01_C08 GFTVSSNY 317 IYSGGST 318 PIELGAFDI 319 NIGSQS 347 YDS 348 QVWDSSSDHVV 349 1977 1245_P01_B07 GFTFSDYY 320 ISSSGSTI 321 ENYLNAFDI 322 QSLSNY 350 AAS 351 QQSYSTPLT 352 64271 1251_P02_C05 GFTFSSYA 323 ISGGGGTT 324 DSGVAFDI 325 QNIRTW 353 DAS 354 QQFKRYPPT 355 65269 1139_P01_E04 GDSVSSNSAA 326 TYYRSKWYN 327 SWNDAFDI 328 QSISTW 356 DAS 357 QQSYSTPLT 358 23786 1245_P02_F07 GDSVSSNSAA 329 TYYRSKWYN 330 DYYYSMDV 331 QSISSW 359 DAS 360 QQSHSHPPT 361 10450 1245_P01_G06 GFTFSDYY 332 ISSSGSTI 333 HSWNDAFDV 334 QSISSY 362 AAS 363 QQSYSTPDT 364 22474 1245_P01_G09 GDSVSSNSAA 335 TYYGSKWYN 336 DYYYSMDV 337 QSISTW 365 DAS 366 QQSYSTPVT 367 18430 1138_P01_B09 GFTFSDYY 338 ISSSGSTI 339 HSWSDAFDI 340 QDISNY 368 DAS 369 QQSYSTPLT 370 29193 1251_P02_G10 GFTFSDYY 341 ISSSGSTI 342 HSWNDAFDI 343 QSISSH 371 AAS 372 QQSYSTLLT 373 17053 1141_P01_E01 GYSFTSYW 344 IYPGDSDT 345 HQVDTRTADY 346 RSDVGGYNY 374 EVS 375 SSYTSTSTLV 376 136780

The K _D values of the parental pure lines as determined by SPR analysis are as follows: Table 24. IgG capture results Analyte Pure ID K _D (nM) K _D (M) L1(DV1-GV4) 1245_P01_E07 12.4 1.24e-08 L1(DV1-GV4) 1252_P01_C08 100 1.00e-07 L1(DV1-GV4) 1245_P02_G04 126 1.26e-07 L1(DV1-GV4) 1245_P01_B07 341 3.41e-07 L1(DV1-GV4) 1251_P02_C05 1967* 1.97e-06 L1(DV1-GV4) 1139_P01_E04 251 2.51e-07 L1(DV1-GV4) 1245_P02_F07 193 1.93e-07 L1(DV1-GV4) 1245_P01_G06 264 2.64e-07 L1(DV1-GV4) 1245_P01_G09 208 2.08e-07 L1(DV1-GV4) 1138_P01_B09 290 2.90e-07 L1(DV1-GV4) 1251_P02_G10 829 8.29e-07 L1(DV1-GV4) TS8.2 (commercial anti-Vδ1 antibody) 44 4.40e-08 *The combination of 1252_P02_C05 has not reached saturation, so the data is extrapolated.

Epitope mapping of five parental lines using hydrogen-deuterium exchange. A summary of the epitope mapping results is presented in Table 25 . Table 25. Epitope mapping results of antigen / antibody complexes Pure ID Epitope location , amino acid number of SEQ ID NO: 272 1245_P01_E07 5, 9, 16, 20, 62, 64, 72, 77 1252_P01_C08 50, 53, 59, 62, 64 1245_P02_G04 37, 42, 50, 53, 59, 64, 68, 69, 72, 73, 77 1251_P02_C05 59, 60, 68, 72 1141_P01_E01 3, 5, 9, 10, 12, 16, 17, 62, 64, 68, 69 Example 2 : Multispecific antibodies enhance Vδ1+ effector cell-mediated cytotoxicity; target tissue-centered disease-associated antigens

Cytotoxicity/potency assay studies were performed to explore the effects of multispecific antibodies on co-cultures of Vδ1+ effector cells and A-431 cancer cells. A-431 (EGFR ⁺⁺ ; ATCC) target cells were seeded at 1,000 cells/well in 384-well imaging dishes (Perkin Elmer) and incubated overnight at 37°C in DMEM (10% FCS). Dilute antibodies and multispecific antibodies as indicated to 10 µg/ml and add to assay plate (2 µg/ml final assay concentration). Expanded skin-derived Vδ1 γδ T cells were isolated from tissue culture flasks and serially diluted to obtain a range of E:T ratios (maximum E:T ratio 60:1), then added to assay plates. A-431 cells were incubated with Vδ1 γδ T cells at 30°C, 5% CO in the presence of antibody or _control . After 24 hours of incubation, Hoechst 33342 (ThermoFisher) was added to stain cells (final 2 µM). To determine the number of viable A-431 cells, confocal images were acquired using the Opera Phenix high-content platform, which captures nine fields of view at 10x magnification. Quantitative viable cell counting based on size, morphology, texture, and intensity of viable cell staining. Effect/target (E:T) time course studies were performed to determine ET ratios in which 50% of target cells were killed in model systems +/- controls, comparators, antibodies and multispecific antibodies as indicated. The results are presented in Figure 31 .

First, Figure 31 (AD) presents example co-culture results in which a Vδ1 +/A-431 co-culture was studied, +/- a multispecific antibody containing an anti-Vδ1 × anti-TAA (EGFR) bispecific binding moiety, in which anti-Vδ1 The VL+VH binding domain (binds to the first target) is combined with the CH1-CH2-CH3 domain of the anti-EGFR binding portion (binds to the second target). The controls and comparators used are as indicated; from left to right: no mAb = no antibody added; D1.3 = D1.3 control; D1.3 IgG LAGA = D1.3 + L235A, G237A; D1.3 FS1- 67 = D1.3 variable domain with EGFR binding constant domain plus L235A, G237A; cetuximab (in-house produced). More specifically, Figure 31 (A) presents the results of five hours of co-culture with the above controls, comparators and the following test articles: C08-LAGA = 1252_P01_C08 with L235A and G237A; C08 FS1-67 = with L235A and G237A EGFR binding domain combination 1252_P01_C08. Figure 31(B) presents the equivalent data after five hours of co-culture with the above controls, comparators and the following test products: G04-LAGA = 1245_P02_G04 with L235A and G237A; G04 FS1-67 = with the EGFR binding domain containing L235A and G237A Combination 1245_P02_G04. Figure 31 (C) presents the equivalent data after five hours of co-culture with control, comparator and the following test products: E07-LAGA = 1245_P01_E07 with L235A, G237A; E07 FS1-67 = combined with the EGFR binding domain containing L235A, G237A of 1245_P01_E07. Figure 31 (D) presents a table summarizing the % increase in cytotoxicity of Vδ1 γδ T cells in the presence of control, comparator and test article over 5, 12 and 24 hours. When an antibody or antigen-binding fragment thereof as described herein is presented in a multispecific format, an enhancement of more than 450% is observed.

Next, Figure 31 (EH) presents example results in which Vδ1+/A-431 co-cultures +/- multispecific antibodies containing anti-Vδ1×anti-TAA (EGFR) bispecific binding moieties were studied, in which the anti-Vδ1 binding domain ( (binding to the first target) comprises a full-length antibody (VH-CH1-CH2-CH3/VL-CL), followed by combination with an anti-EGFR scFv binding portion (binding to the second target). Controls and comparators used are as indicated; from left to right: no mAb = no antibody added; D1.3 = control; D1.3 IgG LAGA = D1.3 + L235A, G237A; D1.3 LAGA cetuximab Anti = D1.3 with L235A, G237A plus C-terminal cetuximab-derived scFv; cetuximab (in-house generated). More specifically, Figure 31(E) presents five hours of co-culture with the above controls, comparators and the following test articles: C08-LAGA = 1252_P01_C08 with L235A, G237A; C08 LAGA cetuximab = with L235A, G237A and 1252_P01_C08 with C-terminal cetuximab-derived scFV. Figure 31(F) shows co-culture with the above control, comparator and the following test products for five hours: G04-LAGA = 1245_P02_G04 with L235A, G237A; G04 LAGA cetuximab = with L235A, G237A and C-terminal cetuximab Ximab-derived scFV 1245_P02_G04. Figure 31 (G) shows co-culture with control, comparator and the following test products for five hours: E07-LAGA = 1245_P01_E07 with L235A, G237A; E07 LAGA cetuximab = with L235A, G237A and C-terminal cetuximab Monoclonal antibody-derived scFV 1245_P01_E07. Figure 31 (H) presents a table summarizing the % increase in potency of Vδ1 γδ T cells in the presence of control, comparator and test article over 5, 12 and 24 hours. When an antibody, or antigen-binding fragment thereof, as described herein is presented in a multispecific format, an enhancement of over 300% is observed. Example 3 : Mammalian presentation

Two pure lines were selected for affinity maturation. The above example describes the preparation and characterization of affinity matured pure lines derived from pure line ADT1-4 (G04) and pure line ADT1-7 (E07). Affinity maturation of human anti -Vδ1 monoclonal antibodies

Phage display was used to generate parental anti-Vd1 monoclonal antibodies, producing antibodies with affinities in the range of 10 nM-1 μM, as described above. Subsequently, the parent antibodies pure line ADT1-4 (G04) and pure line ADT1-7 (E07) underwent affinity maturation in vitro, which unexpectedly increased the affinity by 100 times and achieved excellent target engagement. In vitro affinity maturation of parent antibodies is achieved through a two-step approach: diversifying the parent antibody sequence using targeted CDR3 mutagenesis, followed by selective enrichment of antibodies with improved affinity using phage and mammalian display platforms. Kunkel mutagenesis (Kunkel et al., 1987; Sidhu and Weiss, 2004) and RCA amplification were used to establish VH and VL CDR3 2-mer libraries of pure lines of ADT1-4 and ADT1-7. Agilent primer synthesis technology was used to incorporate all combinations of single and double amino acid substitutions at specified positions in VH and VL CDR3. Specify the number of different amino acids to be incorporated at a specific position. Ignore cysteine and methionine. The changes made in the CDR of the pure lines are summarized in Table 26 below. Table 26. CDR3 positions and designated residue changes

The dimensions of each library are shown in Table 27. Table 27. Library dimensions ADT1-7 5.4×10 ⁹ ADT1-4 5.5×10 ⁹ Generation of mutation-induced VH and VL CDR3 libraries

Mutagenesis-induced libraries are prepared from appropriate culture volumes covering appropriate excess amounts of library members. Affinity maturation was performed using solution phase selection using phage display technology as described in Schofield et al. (2007). Selection was performed using human and cynomolgus monkey antigens in solution. To isolate higher affinity binders from mutant libraries, control of antigen concentration results in a series of increasingly stringent selections. As presented in Figure 1 , each pure line was subjected to several rounds of phage display selection with human and cynomolgus monkey antigens. For the ADT1-4 lineage, the antigen concentrations used in the selection were in the range of 100 nM to 10 pM for human and between 100 nM and 10 nM for the cynomolgus monkey antigen. For the ADT1-7 lineage, concentrations of 10 nM to 1 pM for human antigen and 100 nM to 100 pM for cynomolgus monkey antigen were used. Figure 1 shows phage selection rounds for the ADT1-7 library (iA), the ADT1-4 library (iB), and the ADT1-7 library (iC) using a selection strategy for isolated cynomolgus cross-reactive binders.

Selection progress was assessed using a multi-strain phage ELISA. Human DV1/GV4 and cynomolgus monkey DV1/GV76 were coated overnight at 150 ng/well and 50 μL/well. Crab-eating macaque DV2/GV76 and HSA were used as controls. Only output from the ADT4-1 library cross-reacted with human and cynomolgus DV1 antigens. Output from ADT1-7 is reactive only to human antigens. Selected outputs were further characterized by single strain phage ELISA and are summarized in Table 28. Light gray boxes and dark gray boxes indicate selection using human antigen or cynomolgus monkey antigen, respectively. The arrows provide an indication of the percentage of homogeneous binders classified as human or cynomolgus VD1 (pointing up is high and pointing down is low). Table 28. Summary of single strain phage ELISA G04 Phage round combine select #1 #2 #3 huDV1 cyDV1 2378 10nM 1nM 79% 25% 2409 100pM 75% 100% 2412 10nM 72% 100% 2413 10nM 1nM 84% 100% 2385 100nM 10nM 71% 75% 2418 1nM 100% 94% 2420 1nM 94% 94% 2415 10nM 10nM 100% 94% E07 Phage round select #1 #2 #3 huDV1 cyDV1 2370 1nM 100pM 96% 0% 2404 10pM 83% 0%

Sequence diversity is summarized in Table 29. Light gray boxes and dark gray boxes indicate selection using human antigen or cynomolgus monkey antigen, respectively. Arrows provide an indication of the degree of diversity (high when pointing upward and low when pointing downward). Table 29. Summary of single strain phage sequencing G04 Phage round Diversity select #1 #2 #3 Heavy CDR3 Light CDR3 Heavy-Light CDR3 2378 10nM 1nM 83% 96% 100% 2409 100pM 50% 53% 72% 2412 10nM 63% 97% 97% 2413 10nM 1nM 71% 97% 100% 2385 100nM 10nM 83% 88% 100% 2418 1nM 66% 81% 88% 2420 1nM 63% 73% 80% 2415 10nM 10nM 63% 78% 88% E07 Phage round heavy-light select #1 #2 #3 Heavy CDR3 Light CDR3 CDR3 2370 1nM 100pM 88% 96% 100% 2404 10pM 75% 67% 96% Generation of IgG mammalian presentation libraries

To form the final library for mammalian presentation, the following ADT1-4 selections were assembled: - SEL2409, 2412 and 2413 to create ADT1-4 library 1 (human) - SEL 2418, 2420 and 2415 to establish ADT1-4 library 2 (cynomolgus macaque) - SEL2370 and 2404 to create the ADT1-7 library.

The pool was then advanced to mammal presentation. The entire single-chain variable fragment antibody (scFv) population is converted into IgG format, maintaining the original variable heavy chain (VH) and variable light chain (VL) pairing. The IgG-formatted antibodies were then selected in a mammalian presentation donor vector that was co-transfected with a plasmid encoding a TALE nuclease pair to enable nuclease-directed targeting at a single chromosomal locus. Antibody gene integration. A mammalian display antibody library covering phage output diversity (>10 ⁶ pure lines) was established in HEK293 cells. Two days after transfection (dpt), stable cell populations expressing antibodies on the cell surface were selected by adding blasticidin. Cells expressing antibodies on the cell surface were enriched by magnet-activated cell sorting (MACS) sorting (7 dpt); cells were labeled with anti-Fc-PE, followed by anti-PE beads and using Midi MACS magnets (Miltenyi Biotec) and LS column sorting. These cell populations proceed to selection. Selection of mature antibodies by mammalian presentation

After performing MACS to enrich for antibody-expressing cells, two strategies were employed to identify TRDV1 binders. The main strategy involves dual-color fluorescence sorting based on Fc expression and antigen binding. Another involves two-color fluorescent sorting based on the binding of both cynomolgus and human antigens to maximize the chance of isolating high-affinity cross-reactive binders. A schematic overview of this process is presented in Figure 2. Sequence, specificity, affinity sequencing and characterization

Genomic DNA was extracted from eight different sorted populations. DNA encoding the selected IgG is amplified and cloned into a soluble IgGl expression vector. Since 8 different selections, a total of 1472 pure lines have been selected. pDNA was transfected into Expi293 cells. Supernatants were collected on day 5 after transfection and antibody affinity ranked against human and cynomolgus TRDV1 binding pairs in a capture ELISA. A total of 93 anti-DV1 antibodies were selected from each selection line for sequence and SPR off-rate analysis. Additionally, these pure lines were examined by direct ELISA for binding to: human TRDV1, human TRDV1(A->V), human TRDV2, cynomolgus monkey TRDV1 and BSA. Example 4 : Binding Affinity for Human and Cynomolgus Antigens Binding to Recombinant Antigens and vδ1 TCR Expressed on Cells : ADT1-4 Lineage

Studies were conducted to explore the binding of anti-vδ1 antibodies to their target antigens. Anti-vd1 mAb binding to vd1 TCR antigen was tested by ELISA. 1 ug human antigen or 1 ug cynomolgus monkey antigen per well was immobilized onto a 96-well immunoassay plate (SLS #475904) and then blocked with BSA to prevent non-specific binding. 1.3 pmol (20 ng) of each mAb was added and incubated for 1 hour at room temperature. Binding of mAb to antigen was detected by measuring the absorbance at 450 nM using Protein A-HRP (Abcam #Ab7456) with TMB substrate (Fisher #12750000) and stop solution (Biolegend #423001). Parental controls were included as positive controls for analysis and inter-plate variation. Hits were identified as those mAbs that resulted in an absorbance reading higher than that of the parent mAb. Figure 5A shows the fold increase in the absorbance of the mature ADT1-4 pure line compared to the absorbance of the parental ADT1-4 when 1.3 pmol of antibody is bound to the human vδ1 antigen. Figure 5B shows the fold increase in the absorbance of the mature ADT1-4 pure line compared to the absorbance of the parental ADT1-4 when 1.3 pmol of antibody was bound to the cynomolgus vδ1 antigen.

Binding of mAb to endogenous vd1-TCR was tested using flow cytometry. Skin vd1 cells (donor ATS006; ADT-amplified E0000113) or PEER vδ1 cell line were seeded at 3 × 10 ⁵ cells/well in a 96-well round bottom plate, and resuspended in 50 ul containing 3 ug/ml test mAb in FACS buffer (v/v: 2% FCS, 0.1% sodium azide, and 1 mM EDTA in PBS) for 15 minutes. Cells were pelleted and secondary anti-hmIgG-APC antibody (Miltenyi # 130-119-772) was added at 1/100 in FACS buffer and incubated for an additional 20 minutes at 4°C. Cells were washed and fixed in CellFix (BD #340181) and analyzed by flow cytometry. Calculate vd1 phenotype % and average fluorescence intensity (Inivai Technologies, Flowlogicv7.2). Parental antibody was included as a positive control. Figure 5C shows the fold increase in average fluorescence intensity of mature ADT1-4 pure lines compared to parental ADT1-4 combined with primary skin-derived human vδ1 cells. Figure 5D shows the fold increase in the average fluorescence intensity of the mature ADT1-4 pure line compared with the parental ADT1-4 combined-transformed PEER vδ1 cell line. Binding to recombinant antigens and vδ1 TCR expressed on cells : ADT1-7 lineage:

Studies were conducted to explore the binding of ADT1-7 mature anti-vδ1 antibodies to their target antigens. Anti-vd1 mAb binding to vδ1 TCR antigen was tested by ELISA. 1 ug of antigen per well was immobilized onto a 96-well immunoassay plate (SLS #475904) and then blocked with BSA to prevent non-specific binding. Titrations of 6.7 pmol (100 ng), 1.3 pmol (20 ng) and 0.27 pmol (4 ng) of mAb were added and incubated for 1 hour at room temperature. Binding of mAb to antigen was detected by measuring absorbance at 450 nM using Protein A-HRP (Abcam #Ab7456) with TMB substrate (Fisher #12750000) and stop solution (Biologend #423001). Parental controls were included as positive controls for analysis and inter-plate variation. Hits were identified as those mAbs that resulted in an absorbance reading higher than that of the parent mAb. Figure 6A shows the fold increase in the absorbance of the mature ADT1-4 pure line compared to the absorbance of the parental ADT1-4 when 1.3 pmol of antibody is bound to the human vδ1 antigen.

Binding of mAb to endogenous vd1-TCR was tested using flow cytometry. Skin vδ1 cells (donor ATS006; ADT-amplified E0000113) or PEER vδ1 cell lines were seeded at 3 × 10 ⁵ cells/well in a 96-well round bottom plate, and resuspended in 50 ul containing 3 ug/ml test mAb in FACS buffer (v/v: 2% FCS, 0.1% sodium azide, and 1 mM EDTA in PBS) for 15 minutes. Cells were pelleted and secondary anti-hmIgG-APC antibody (Miltenyi # 130-119-772) was added at 1/100 in FACS buffer and incubated for an additional 20 minutes at 4°C. Cells were washed and fixed in CellFix (BD #340181) and analyzed by flow cytometry. Calculate vd1 phenotype % and average fluorescence intensity (Inivai Technologies, Flowlogicv7.2). Parental antibody was included as a positive control. Figure 6B shows the fold increase in average fluorescence intensity of mature ADT1-4 homogeneous lines compared to parental ADT1-4 combined with primary skin-derived human vδ1 cells. Figure 6C shows the fold increase in the average fluorescence intensity of the mature ADT1-7 pure line compared with the parental ADT1-7 combined-transformed PEER vδ1 cell line. Improved dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) relative to the parent pure line

To demonstrate improved binding of affinity matured mAbs to human and cynomolgus monkey antigens, antigen (3 µg/mL antigen in 50 µL PBS, overnight at 4°C) was coated directly onto plates (Nunc #437111). Perform DELFIA immunoassay. For detection, DELFIA Eu-N1 anti-human IgG (Perkin Elmer # 1244-330) was used as secondary antibody in 50 µL 3% MPBS (PBS + 3 % (w/V) skim milk powder) 1/500 Dilute. Visualize with 50 µL DELFIA enhancement solution (Perkin Elmer #4001-0010).

Antibodies of interest were affinity ranked using a DELFIA immunoassay, in which antibodies were captured via protein G coated on plates and supplemented with 0.4 nM human soluble biotinylated L1 (DV1-GV4) antigen and 10 nM cynomolgus monkey antigen DV1/ GV77 (3MPBS). For detection, 50 µL of Streptavidin-Eu (1:500 in assay buffer, Perkin Elmer) was used and the signal was visualized with DELFIA enhancement solution. D1.3 hlgG1 (described in England et al. (1999) J. Immunol. 162: 2129-2136) was used as a negative control. The results are provided in Figures 7A to C. SPR analysis -IgG capture

SPR analysis is used to compare the KD values of affinity mature pure lines compared to parental pure lines. Instrument used: MASS-2 (Sierra Sensors); Chip: Amine High Capacity (Sierra Sensors); Operating buffer PBS + 0.02% Tween 20. Experiments were performed at room temperature or 37°C with protein G coupled to the wafer. Antigen was flowed through the cells in a dilution series ranging from 50 nM to 0.2 nM for human DV1-GV4 and 100 nM to 1 nM for cynomolgus monkey DV1-77. 120 seconds association, 600 seconds dissociation, 50 μL/min flow rate, regeneration with 10 mM glycine pH 1.5, kinetic fitting based on Langmuir 1:1 binding using a software Sierra analyzer. The results are shown in Figures 8A to C. Binding affinity analysis ( by SPR , KD) Binding affinity of Vδ1 monoclonal antibody to Vδ1 antigen

The binding affinity of the antibody to the target (ie, the Vδ1 chain of the γδ TCR) was established by SPR analysis using a Reichert 4SPR instrument (Reichert Technologies). Antibody (1.5 ug/mL) was coated onto a planar protein A sensor chip (Reichert Technologies), resulting in a baseline increase of approximately 500 uRIU. Antigens (e.g., L1(DV1-GV4)) are flowed through cells in a 1:3 dilution series from 300 nM to 3.7 nM with the following parameters: 180 s association, 600 s dissociation, flow rate 25 μL/min, working buffer PBS +0.05% Tween 20. All experiments were performed at room temperature. Steady-state fitting was determined based on Langmuir 1:1 binding using the software TraceDrawer (Reichert Technologies). human antigen

The affinity-matured Vδ1 mAb exhibited greatly enhanced affinity for the human Vδ1 antigen compared to either parent, as determined by surface plasmon resonance (SPR) analysis (Figure 9). (A , B) Surface plasmon resonance (SPR) analysis using ADT1-4 (A) or ADT1-7 (B) parental mAbs and their affinity matured derivatives to determine binding affinity for the human Vδ1 antigen. Binding interaction of Vδ1 with affinity matured mAb based on Langmuir 1:1 binding model. (C , D) Table summarizing the equilibrium dissociation constants (KD) of antibodies targeting Vδ1 as derived from the sensor patterns depicted in (A) and (B) . Data are expressed as the average of two replicates performed on two different SPR instruments. ( E , F ) Bar graphs showing the fold increase in the equilibrium dissociation constant (KD) of affinity mature derivatives for Vδ1 antigen compared to their parent ADT1-4 or ADT1-7.

Conclusion : This data demonstrates that affinity matured derivatives of ADT1-4 and ADT1-7 exhibit significantly higher affinity for the human Vδ1 antigen compared to the parent antibodies. crab-eating macaque antigen

Surface plasmon resonance analysis demonstrated that the affinity mature Vδ1 mAb of the ADT1-4 lineage displayed greatly enhanced affinity for the cynomolgus monkey Vδ1 antigen compared with the parental antibody (Figure 10). (A) Surface plasmon resonance (SPR) analysis using ADT1-4 parental antibody and affinity matured derivatives to determine binding affinity for the cynomolgus monkey Vδ1 antigen. Binding interaction of Vδ1 with affinity matured mAb based on Langmuir 1:1 binding model. (B) Table summarizing the equilibrium dissociation constants (KD) of antibodies targeting Vδ1, as derived from the sensor patterns depicted in (A) . Data are expressed as the average of two replicates performed on two different SPR instruments. The results are shown in Figures 10A and 10B.

Conclusion : This data demonstrates that affinity matured derivatives of ADT1-4 exhibit significantly higher affinity for the cynomolgus monkey Vδ1 antigen compared to the parent antibody. Binding affinity to cell surface Vδ1 TCR ( IC50 binding to cell surface Vδ1 )

Affinity-matured Vδ1 mAb exhibited greatly enhanced affinity for Vδ1-positive γδ T cells and no significant binding to Vδ1-deficient cells, as shown in Figure 11A-D. (A , B) Binding levels of Vδ1 mAb to two γδ T cell donors, ATS006 (A) and TS164 (B). γδ T cells were stained with varying concentrations of Vδ1 mAb, followed by fluorescent anti-human IgG detection antibody (xxx). All incubation steps were performed at 4°C and mAb binding was determined using flow cytometry to measure the median fluorescence level. Logarithmic four-parameter dose-response curves were fitted using GraphPad Prism 9. (C) Bar graph showing the average EC50 of ADT1-4 and ADT1-7 pure lines binding to Vδ1-positive γδ T cells, expressed as the average of two donors. (D) Table summarizing the EC50 and Vδ1 negative cell types mapped in (A) and (B) , including HEK293A, Raji cells, and multiple leukocyte subsets with primary blood monocytes. For Vδ1-positive γδ T cells, data are expressed as the average of two donors.

Conclusion : This data demonstrates that affinity mature derivatives of ADT1-4 and ADT1-7 exhibit significantly higher affinity to Vδ1-positive γδ T cells than their parent mAbs, and also show affinity to Vδ1-negative cells HEK293T, Raji or PBMC. It does not bind to CD8, CD4, NK, CD19 or monocytes. Target cell binding of Vδ1 monoclonal antibody

A range of target cells were analyzed to determine the specificity and affinity of Vδ1 mAb binding. This includes expanded skin-derived Vδ1 γδ T cells, HEK293T cells, Raji cells, and multiple leukocyte subsets within human primary blood monocytes. Adherent or semi-adherent cells (skin-derived Vδ1 γδ T cells, HEK293T) were isolated from tissue culture bottles and resuspended in PBS. Similarly, non-adherent cell types (Raji, PBMC) were harvested and resuspended in PBS. Cells were seeded into v-bottom 96-well plates at a final density of 100,000 cells/well. Cells were centrifuged and the cell pellet was resuspended in FcR blocking reagent and incubated at 4°C for 20 minutes before further washing according to the manufacturer's instructions. Vδ1 mAb, anti-RSV IgG control and anti-CD3 OKT3 were diluted to 500 nM in PBS and serially diluted 1:5 in PBS to 6.4 pM and added to the cells followed by incubation at 4°C for 20 min. To determine the amount of mAb bound to the cell surface, cells were then stained with a murine anti-human IgG secondary antibody conjugated to APC (product code, dilution: 1:100). For Vδ1 γδ T cells, HEK293T and Raji, cells were also stained with viability dye only. For PBMC, binding antibodies against CD4, CD8, CD56, CD11b, and CD19 were also included (all at 1:100), allowing identification of the αβ subset, NK cells, B cells, and monocytes. After incubation for 20 minutes at 4°C, cells were washed twice, and fluorescence was measured using MACSQuant. IC50 is shown in Figure 11D. Summary of KD values of ADT1-4 lineage ( human and crab-eating macaque )

The following table provides the KD values of 24 pure lines in the ADT1-4 lineage and the ADT1-4 parental pure line (G04) (combined with human TRDV1 and cynomolgus monkey TRDV1): human human crab-eating macaque crab-eating macaque Pure ID K _D [M] K _D (nM) K _D [M] K _D (nM) ADT1-4 1.26E-07 126 nM N/A N/A ADT1-4-19 1.63E-10 0.163 nM 3.40E-09 3.40 nM ADT1-4-21 4.41E-10 0.441 nM 6.79E-09 6.79 nM ADT1-4-31 5.22E-10 0.522 nM 9.43E-09 9.43 nM ADT1-4-53 6.08E-10 0.608 nM 5.34E-09 5.34 nM ADT1-4-2 1.95E-10 0.195 nM 3.08E-09 3.08 nM ADT1-4-8 5.41E-10 0.541 nM 1.51E-08 15.1 nM ADT1-4-82 2.49E-10 0.249 nM 1.67E-08 16.7 nM ADT1-4-83 2.41E-10 0.241 nM 1.58E-08 15.8 nM ADT1-4-84 2.27E-10 0.227 nM 1.16E-08 11.6 nM ADT1-4-86 9.39E-10 0.939 nM 9.49E-09 9.49 nM ADT1-4-95 6.42E-10 0.642 nM 1.50E-08 15.0 nM ADT1-4-105 3.81E-10 0.381 nM 1.01E-08 10.1 nM ADT1-4-107 1.77E-10 0.177 nM 1.05E-08 10.5 nM ADT1-4-110 3.33E-10 0.333nM 1.19E-08 11.9 nM ADT1-4-112 7.84E-10 0.784 nM 1.11E-08 11.1 nM ADT1-4-117 2.45E-10 0.245 nM 1.01E-08 10.1 nM ADT1-4-139 4.99E-10 0.499 nM 2.69E-08 26.9 nM ADT1-4-4 5.19E-10 0.519 nM 1.62E-08 16.2 nM ADT1-4-143 4.63E-10 0.463 nM 9.39E-09 9.39 nM ADT1-4-173 9.07E-10 0.907 nM 2.40E-08 24.0 nM ADT1-4-3 9.35E-10 0.935 nM 2.85E-08 28.5 nM ADT1-4-1 8.00E-11 0.08 nM 4.1E-10 0.41 nM ADT1-4-6 1.06E-9 1.06 nM 1.01E-9 1.01 nM ADT1-4-138 4.38E-8 43.8 nM 3.47E-8 34.7 nM Summary of KD values for ADT1-7 lineage ( human only )

The following table provides the KD values of 11 pure lines in the ADT1-7 lineage and the ADT1-7 parental pure line (E07) (only binds to human TRDV1): Pure ID K _D [M] K _D (nM) ADT1-7 1.24E-08 12.4 nM ADT1-7-10 1.30E-09 1.30 nM ADT1-7-15 1.42E-09 1.42 nM ADT1-7-17 1.26E-09 1.26 nM ADT1-7-18 1.61E-09 1.61 nM ADT1-7-19 1.19E-09 1.19 nM ADT1-7-20 1.17E-09 1.17 nM ADT1-7-22 1.18E-09 1.18 nM ADT1-7-23 1.29E-09 1.29 nM ADT1-7-42 1.51E-09 1.51 nM ADT1-7-3 8.31E-10 0.831 nM ADT1-7-61 5.57E-10 0.557 nM Summary of pharmacological data on preferred members of the ADT1-4 spectrum human human crab-eating macaque crab-eating macaque IC50 TCR DR ( without THP-1) IC90 TCR DR ( without THP-1) IC50 kill IC90 kill Pure ID K_D[M] K_D(nM) K_D[M] K_D(nM) ADT1-4 1.26E-07 126 nM N/A N/A N/A N/A 2.603E-08 (26.03 nM) 7.048E-08 ADT1-4-19 1.63E-10 0.163 nM 3.40E-09 3.40 nM 3.67E-10 (0.367 nM) 1.583E-9 (1.583 nM) 1.12E-09 (1.12 nM) 8.38E-09 (8.38 nM) ADT1-4-21 4.41E-10 0.441 nM 6.79E-09 6.79 nM 2.66E-10 (0.266 nM) 8.92E-10 (0.892 nM) 9.4E-10 (0.94mM) 5.88E-09 (5.88 nM) ADT1-4-31 5.22E-10 0.522 nM 9.43E-09 9.43 nM 2.45E-10 (0.245 nM) 1.091E-9 (1.091 nM) 1.63E-09 (1.63 nM) 1.658E-08 (16.58 nM) ADT1-4-53 6.08E-10 0.608 nM 5.34E-09 5.34 nM 2.54E-10 (0.254 nM) 6.98E-10 (0.698 nM) 1.05E-09 (1.05 nM) 4.23E-09 (4.23 nM) ADT1-4-2 1.95E-10 0.195 nM 3.08E-09 3.08 nM 3.1E-10 (0.31 nM) 8.34E-10 (0.834 nM) 8.7E-10 (0.87 nM) 3.12E-09 (3.12 nM) ADT1-4-86 9.39E-10 0.939 nM 9.49E-09 9.49 nM 3.07E-10 (0.307 nM) 1.001E-9 (1.001 nM) 1.05E-09 (1.05 nM) 4.3E-09 (4.3 nM) ADT1-4-112 7.84E-10 0.784 nM 1.11E-08 11.1 nM 2.95E-10 (0.295 nM) 1.093E-9 (1.093 nM) 9E-10 (0.9 nM) 3.8E-09 (3.8 nM) ADT1-4-143 4.63E-10 0.463 nM 9.39E-09 9.39 nM 3.13E-10 (0.313 nM) 8.53E-10 (0.853 nM) 1.78E-09 (1.78 nM) 1.42E-08 (14.2 nM) ADT1-4-1 8.00E-11 0.08 4.1E-10 0.41 3.604E-10 (0.3604 nM) 6.702E-10 (0.6702 nM) 1.4E-10 (0.14 nM) 2.8E-10 (0.28 nM) ADT1-4-6 1.06E-9 1.06 1.01E-9 1.01 4.796E-10 (0.4796 nM) 1.911E-9 (1.911 nM) 1.4E-10 (0.14 nM) 2E-10 (0.2 nM) ADT1-4-138 4.38E-8 43.8 3.47E-8 34.7 8.384E-9 (8.384 nM) 3.83E-8 (38.3 nM) 3.8E-10 (0.38 nM) 6.44E-09 (6.44 nM) Summary of pharmacological data on preferred members of the ADT1-7 lineage Pure ID K_D[M] K_D(nM) IC50 TCR DR ( without THP-1) IC90 TCR DR ( without THP-1) IC50 kill IC90 kill ADT1-7 1.24E-08 12.4 nM - - 5.53E-09 (5.53 nM) 7.48E-08 (74.8 nM) ADT1-7-20 1.17E-09 1.17 nM 1.64E-10(0.164 nM) 1.036E-9 (1.036 nM) 2.80E-10 (0.28 nM) 2.47E-09 (2.47 nM) ADT1-7-3 8.31E-10 0.831 nM 1.61E-10(0.161 nM) 7.90E-10 (0.79 nM) 1.60E-10 (0.16 nM) 1.06E-09 (1.06 nM) ADT1-7-61 5.57E-10 0.557 nM 6.264E-9(6.264 nM) 1.704E-6*(1704.445 nM) 4.20E-10 (0.42 nM) 1.66E-08 (16.6 nM) *Data artifacts attributed to this pure line not achieving 100% inhibition Example 5. Functional characterization of TCR downregulation by Vδ1 monoclonal antibody

The ability of the ADT mAb to engage the gamma delta T cell receptor and thereby downregulate the gamma delta T cell receptor was assessed by measuring TCR performance. Skin γδ T cells (from donors ATS006 and TS164) were seeded in 96-well round bottom plates at 3 × 10 ⁵ cells/ml in γδ medium with increasing concentrations of test mAb diluted in PBS (in 0.00067 to 67 nM range) or the corresponding isotype control (hlgG1, RSV) at the highest concentration (67 nM). Incubate cells in a humidified _CO2 chamber at 37 °C for 2 h. Cells were washed and stained for dead cells (Thermo Fisher #15580607) and their VD1 TCR (Miltenyi #130-117-697) for 30 minutes at 4°C. Cells were washed in FACS buffer and resuspended in cell fixative (BD sciences #340181) and then incubated overnight at 4°C in the dark. VD1 TCR expression levels as measured by median fluorescence intensity (MFI) were measured by flow cytometry using a MACS Quant Analyzer 16 the next day.

The results are shown in Figure 12, which shows that the affinity matured Vδ1 mAb binds and downregulates the Vδ1 TCR (A and B) more efficiently than its parental clone and OKT3. After γδ cells were incubated with Vδ1 mAb and OKT3 for 2 hours, γδ TCR expression was quantified, followed by staining of Vδ1 TCR with a-Vδ1 TCR-PE-Vio770. The MFI of a-Vδ1 staining was obtained using flow cytometry and calculated from the TCR-positive gate, and then the %MFI was calculated from the MFI of TCR-positive untreated γδ cells. The upper panel represents data from the ADT1-4 lineage and the lower panel represents data from the ADT1-7 lineage. (C) Bar graph showing IC50 values calculated from (A). (D and E). Bar graph showing IC50 values in (B) presented as fold improvement relative to parental ADT1-4 (left) and ADT1-7 (right). (F) Table representing IC50 values from (B) as percent improvement calculated from the corresponding parents of ADT1-4 (top) and ADT1-7 (bottom). (G) Table representing IC50 values from (B) as fold improvement calculated from the corresponding parents of ADT1-4 (top) and ADT1-7 (bottom). Activation of γδ by Vδ1 monoclonal antibody as measured by CD107a expression

The ability of the ADT mAb to engage and activate VD1 γδ cells was assessed by measuring CD107a expression. Skin γδ T cells (from donor ATS006) were seeded in 96-well round bottom plates at 6×10 ⁵ cells/ml in γδ medium with 1.2×10 ⁶ cells/ml of THP-1 cells ( ATCC-TIB-202) and increasing concentrations of test mAb (in the range 0.00067 to 67 nM) or the corresponding isotype control (hlgG1, RSV) at the highest concentration (67 nM) diluted in PBS. Incubate cells in a humidified _CO2 chamber at 37 °C for 2 h. Cells were washed and stained for dead cells (Thermo Fisher #15580607), VD1 TCR (Miltenyi #130-117-697) and aCD107a (Miltenyi #130-112-610) for 30 minutes at 4°C. Cells were washed in FACS buffer and subsequently incubated overnight at 4°C in the dark. VD1 TCR and CD107a expression levels determined by median fluorescence intensity (MFI) were measured by flow cytometry using a MACS Quant Analyzer 16 on the next day.

The results are presented in Figure 13, which shows that affinity matured Vδ1 mAb induces activation only in the presence of target cells. (A and B) Quantification of γδ TCR expression (black) and CD107a expression after 2 hours of incubation of γδ cells with ADT1-4-2 alone (top) and with THP-1 cells at a 1:2 target to effector ratio (bottom) (grey), and then stained Vδ1 TCR and CD107a with a-Vδ1 TCR-PE-Vio770 and a-CD107a-VioBlue respectively. The MFI of Vδ1 staining was obtained using flow cytometry and calculated from the TCR-positive gate, and then the %MFI was calculated from the MFI of TCR-positive untreated and non-cocultured γδ cells. The MFI of CD107a staining was obtained using flow cytometry and calculated from the 'non-THP-1' gate, and then the %MFI was calculated from the CD107a MFI of untreated, non-cocultured γδ cells. (C and D) Quantification of γδ TCR expression (black) and CD107a expression after 2 hours of incubation of γδ cells with ADT1-7-3 alone (top) and with THP-1 cells at a 1:2 target to effector ratio (bottom) (grey), and then stained Vδ1 TCR and CD107a with a-Vδ1 TCR-PE-Vio770 and a-CD107a-VioBlue respectively. The MFI of Vδ1 staining was obtained using flow cytometry and calculated from the TCR-positive gate, and then the %MFI was calculated from the MFI of TCR-positive untreated and non-cocultured γδ cells. The MFI of CD107a staining was obtained using flow cytometry and calculated from the 'non-THP-1' gate, and then the %MFI was calculated from the CD107a MFI of untreated, non-cocultured γδ cells. (E) Table showing the percent increase in γδ CD107a performance from cocultured cells treated with the highest concentration of Vδ1 mAb compared to untreated, non-cocultured γδ cells. (F) Table showing the percent increase in γδ CD107a performance from co-cultured cells treated with the highest concentration of Vδ1 mAb compared to untreated co-cultured and non-co-cultured γδ cells. Activation of γδ by Vδ1 monoclonal antibody as measured by CD25 expression

The ability of the ADT mAb to engage and activate VD1 γδ cells was assessed by measuring CD25 expression. Skin γδ T cells (from donor ATS006) were seeded in 96-well round bottom plates at 6×10 ⁵ cells/ml in γδ medium with 1.2×10 ⁶ cells/ml of THP-1 cells ( ATCC-TIB-202) and increasing concentrations of test mAb (in the range 0.00067 to 67 nM) or the corresponding isotype control (hlgG1, RSV) at the highest concentration (67 nM) diluted in PBS. Cells were incubated in a humidified _CO2 chamber at 37 °C for 24 h. Cells were washed and stained for dead cells (Thermo Fisher #15580607), VD1 TCR (Miltenyi #130-117-697) and CD25 (Miltenyi #130-113-280) for 30 minutes at 4°C. Cells were washed in FACS buffer and subsequently incubated overnight at 4°C in the dark. VD1 TCR and CD107a expression levels determined by median fluorescence intensity (MFI) were measured by flow cytometry using a MACS Quant Analyzer 16 on the next day.

The results are presented in Figure 14, which shows that affinity matured Vδ1 mAb induces activation only in the presence of target cells. (A and B) Quantification of γδ after 24 hours of incubation of γδ cells with RSV (added at 67 nM) and ADT1-4-2 alone (top) and with THP-1 cells at a 1:2 target to effector ratio (bottom) TCR expression (black) and CD25 expression (grey), and then stained Vδ1 TCR and CD25 with a-Vδ1 TCR-PE-Vio770 and a-CD25-VioBlue respectively. The MFI of Vδ1 staining was obtained using flow cytometry and calculated from the TCR-positive gate, and then the %MFI was calculated from the MFI of TCR-positive untreated and non-cocultured γδ cells. The MFI of CD25 staining was obtained using flow cytometry and calculated from the 'non-THP-1' gate, and then the %MFI was calculated from the CD25 MFI of untreated, non-cocultured γδ cells. (C and D) Quantification of γδ after 2 hours of incubation of γδ cells with RSV (added at 67 nM) and ADT1-7-3 alone (top) and with THP-1 cells at a 1:2 target to effector ratio (bottom) TCR expression (black) and CD25 expression (grey), and then stained Vδ1 TCR and CD25 with a-Vδ1 TCR-PE-Vio770 and a-CD25-VioBlue respectively. The MFI of Vδ1 staining was obtained using flow cytometry and calculated from the TCR-positive gate, and then the %MFI was calculated from the MFI of TCR-positive untreated and non-cocultured γδ cells. The MFI of CD25 staining was obtained using flow cytometry and calculated from the 'non-THP-1' gate, and then the %MFI was calculated from the CD25 MFI of untreated, non-cocultured γδ cells. (E) Table showing the percent increase in γδ CD25 expression from cocultured cells treated with the highest concentration of Vδ1 mAb compared to untreated, non-cocultured γδ cells. (F) Table showing the percent increase in γδ CD25 expression from co-cultured cells treated with the highest concentration of Vδ1 mAb compared to untreated co-cultured and non-co-cultured γδ cells. TCR downregulation in crab-eating macaques

Studies were conducted to explore the ability of ADT1-4-2 to bind to cynomolgus monkey vδ1 γδ T cells and induce their TCR downregulation. For these studies, cynomolgus monkey PBMC were collected from fresh blood and placed in culture for 14 days to expand γδ-T cells. Alternatively, magnetic beads coated with anti-αβ antibody (clone R73) were used to deplete αβ-T cells from the total PBMC population prior to ex vivo expansion. Amplify as follows: 250,000 cells per well in a U-bottomed 96-well plate in RPMI-1640 medium containing 10% FCS, antibiotics (P/S), and a cocktail of interleukins (IFNγ, IL21, IL4, IL1β, and IL15) . Seven days later, 10 μl of fresh medium containing 10% FCS, antibiotics (P/S), and interleukins IL21 and IL15 were added to the cells. Then on day 11, 100 μl of medium was replaced with fresh medium containing 10% FCS, antibiotics (P/S) and IL15. On day 14, cells were harvested and analyzed for TCR downregulation. The assay consists of mixing cells with various concentrations of antibodies for 2 hours. After the incubation time, cells were stained for flow cytometry analysis for the following markers: CD3, TCR-αβ, TCR-γδ, and VD1. Live/dead cell dyes are also included to distinguish viable cell populations. Flow cytometry analysis was performed by gating CD3+, TCR-γδ+, and VD1+ cell populations, and measuring the average VD1 fluorescence intensity inside the VD1+ gate. Isotype control (anti-RSV) or no antibody was used as a negative control only at the highest concentration. Data are normalized to controls without any antibodies. Figure 15(A) shows a comparison of the ability of ADT1-4-2 and ADT1-4 to engage VD1 on cynomolgus monkey γδ-T cells and reduce VD1 expression. The results showed a dose-response reduction in VD1 expression (up to 76% at 33.3 nM) after treatment with ADT1-4-2, but not with ADT1-4 antibodies. In this particular analysis, γδ-T cells expanded from an αβ-T cell-depleted population. Use 500x concentration range. Figure 15(B) shows the cell surface expression percentage of VD1 after treatment with ADT1-4-2. In this analysis, γδ-T cells were expanded from all PBMC populations. Use 10,000x concentration range. As an example, the data showed that VD1 cells showed a dose-response decrease in 2 different donors (AD8170 and AM945BA). A total of 4 donors were tested and EC50s were extracted individually using the GraphPad Prism nonlinear fit curve function. Figure 15(C) shows the individual EC50 values of different donors, as well as the average and standard deviation. The average value is 3.83 ± 2.23 nM. Antibodies: CD3-AF700 (BD Bioscience, pure line SP34-2, reference: 557917) TCR-αβ-AF647 (BioLegend, pure line R73, reference: 201116) TCR-γδ-BV421 (BioLegend, pure line B1, reference: 331218 VD1-PE (eBioscience, pure line TS8.2, reference: 12-5679-42) Enhanced cytotoxicity of affinity mature pure lines

Affinity maturation of ADT1-4 and ADT1-7 Vδ1 pure lines significantly enhanced the cytotoxic effect of Vδ1 γδ T cells in an in vitro THP-1 killing assay. THP-1 cell killing assay

Vδ1 mAb and anti-RSV IgG control were diluted to 1 µg/ml in PBS and serially diluted 1:10 in PBS before addition to a 384-well Ultra Imaging assay plate (Perkin Elmer). THP-1 cells (ATCC) cultured in RPMI, 10% FCS (Invitrogen) were stained with [0.5 µM] CellTrace CFSE live cell dye for 20 minutes. Expanded skin-derived Vδ1 γδ T cells were isolated from tissue culture bottles and resuspended in basal growth medium and subsequently mixed 1:1 with THP-1 cells in the suspension. The cell suspension was seeded into a 384-well assay plate to obtain a final cell seeding density of 1,000 THP-1 cells/well and 2,000 Vδ1 γδ T cells/well. Dilute the mAb to a concentration ranging from 200 ng/ml to 0.2 pg/ml in the final assay volume. THP-1 and Vδ1 γδ T cells were cultured in the _presence of Vδ1 mAb at 37°C, 5% CO for 24 hours. To determine the number of viable THP-1 cells after 24 hours, confocal images were acquired using the Opera Phenix high-content platform, which captures nine fields of view at 10x magnification. Quantitative viable cell counting based on size, morphology, texture, and intensity of viable cell staining.

Figure 16 shows that affinity matured Vδ1 mAb exhibits enhanced potency compared to the parental pure line in a THP-1 high content cytotoxicity assay. (A and B) Quantification of viable THP-1 cell numbers after 24 hours of coculture with γδ T cells in the presence of Vδ1 mAb or control [effect:target ratio 2:1]. Cell numbers were normalized as percentage of no γδ T cell control (100%). OKT3 (anti-CD3 antibody) and anti-RSV isotype controls were included as analytical controls. Use high-content confocal microscopy on live cells to count cell numbers. The figure shows the parental ADT1-4 (A) and ADT1-7 (B) pure lines. (C) Bar graph showing the average EC50 of OKT3 control and ADT1-4 and ADT1-7 pure lines. Data are expressed as the mean ± standard deviation of n = 3 biological replicates. (D) Table summarizing the EC50 plotted in (C) . The data are expressed as the average value, standard deviation, change fold and improvement percentage compared with the ADT1-4 and ADT1-7 parental pure lines. Vδ1 monoclonal antibodies on antibody-dependent cellular cytotoxicity (ADCC)

Expanded skin-derived Vδ1 γδ T cells from three donors were isolated from tissue culture flasks and resuspended in basal growth medium and plated in white 96-well plates at a final density of 20,000 cells/well. Raji cells were similarly seeded at 20,000/well to serve as a positive control for ADCC induced by the CD20-targeting antibody rituximab. mAb was diluted and added to each well at a final concentration ranging from 1 to 100 nM. FcγRIIIa+ADD Bioassay effector cells were thawed, and 62,500 cells were added to each well, followed by incubation at 37°C, 5% CO _for 4.5 hours. Bio-Glo reagent containing luciferin was added to each well, followed by incubation for an additional 10 minutes, and luminescence was measured using a disk reader.

Figure 17 shows that mAbs targeting Vδ1 induce negligible antibody-dependent cellular cytotoxicity (ADCC). ADCC was measured using a surrogate reporter assay in which downstream NFAT signaling was measured by induction of luciferase activity in FcγRIIIa+ effector cells. Effector cells were co-cultured with three γδ T cell donors (effector: target ratio 3) and different concentrations of monoclonal antibodies. CD20 ⁺ Raji cells and rituximab were included as positive controls. Bioluminescence was measured after 4.5 hours of co-culture. Data are expressed as the mean ± SD of three biological replicates (three γδ T cell donors).

Conclusion : This data demonstrates that the ADT1-4 parent and the affinity mature derivative ADT1-4-2 do not induce ADCC and that the FcγRIIIa+ effector reporter cell line induces luciferase activity via NFAT signaling. However, OKT3 appears to induce high levels of ADCC-specific signaling. Vδ1 monoclonal antibody on complement-dependent cytotoxicity (CDC)

Expanded skin-derived Vδ1 γδ T cells were isolated from tissue culture flasks and resuspended in basal growth medium and plated in white 96-well plates at a final density of 75,000 cells/well. Raji cells were similarly seeded at 75,000/well to serve as a positive control for ADCC induced by the CD20-targeting antibody rituximab. mAb was diluted and added to each well at a final concentration ranging from 1 to 100 nM. Add fresh or heat-inactivated human serum to a final concentration of 40%. Heat inactivation was performed by heating the serum at 56° C. for 30 minutes in a water bath and then cooling it on ice. Cells were then incubated at 37°C, 5% CO _for 24 hours. To determine the number of viable cells after 24 hours, cells were harvested and stained with xx viability dye at a 1:1000 dilution for 20 minutes. Fluorescence was measured using MACSQuant and viable cell counts were quantified based on negative staining with viability dyes.

The results are presented in Figure 18, which demonstrates that mAbs targeting Vδl do not induce complement-dependent cytotoxicity (CDC). Number of viable γδ T cells after incubation with 40% human serum and 50 nM anti-CD3 OKT3 or anti-Vδ1 ADT1-4-2 and various concentrations of monoclonal antibodies. Use fresh or previously heated serum. CD20 ⁺ Raji cells and rituximab were included as positive controls. Viability was measured via viability dye after 24 hours of co-culture. Data are expressed as mean ± SD of three biological replicates.

Conclusion : This data demonstrates that ADT1-4-2 does not induce complement-dependent cytotoxicity (CDC) and that cytotoxicity of Vδ1-positive cells is not increased in the presence of complement-containing serum. However, rituximab demonstrated strong CDC stimulation against CD20-positive Raji cells. Avoid healthy cells

Studies were conducted to explore the effects of stimulating/activating vδ1 cells with anti-vδ1 antibodies relative to cytotoxicity against healthy cells. This was tested by incubating anti-vδ1 pure line ADT1-4-2 with PBMC and subsequently assessing monocytotoxicity and apoptosis.

Cryopreserved human peripheral blood mononuclear cells (PBMC) were commercially derived from 3 healthy donors. PBMC were seeded into round bottoms at 250,000 cells/well in 250 ul of complete medium with 10 ng/ml IL15 (RPMI supplemented with 10% FCS, pen/strep, non-essential amino acids, sodium pyruvate, and HEPES) 96-well tissue culture plate. Add titrated vδ1 antibody ADT1-4-2 to a final concentration of 6.6 nM. RSV IgG, IgG2α and OKT3 antibodies were included as controls. Stimulate for 20 hours. Flow cytometry analysis was performed at the endpoint to determine the monocyte phenotype and apoptotic monocytes on vδ1 cells under each condition. Targeting first live singlets and then CD14 (Miltenyi 130-110-523) gated cells to identify mononuclear spheres. Apoptotic mononuclear spheres were then identified via positive ApoTracker green staining (Biolegend 427402).

Figure 19A (upper left panel) shows vδ1 TCR stained MFI after antibody stimulation, indicating target engagement. Figures 19B (upper right panel) and 19C (lower panel) show the endpoint monocyte phenotype and apoptotic monocytes (respectively) after antibody stimulation. Stimulation with the vδ1 antibody pure line ADT1-4-2 engages Vδ1 cells but does not cause cytotoxicity in healthy monocytes. Example 6. Effect of anti -Vδ1 antibodies on TIL populations from primary tumor biopsies Anti -Vδ1 antibodies promote activation, proliferation and cytotoxicity of tumor-infiltrating lymphocytes

Studies were conducted to explore the modulation of human tumor-infiltrating lymphocytes (TILs) conferred by anti-Vδ1 antibodies. For these studies, human renal cell carcinoma (RCC) tumor biopsies were shipped fresh and processed upon receipt. The biopsies were cut into pieces measuring approximately ² mm, and TILs were obtained using a modification of the method originally described by Kupper and Clarke (Clarke et al., 2006). Specifically, up to four 2 mm ^biospecimens were placed on 9 mm × 9 mm × 1.5 mm Cellfoam matrix, with one matrix per well placed on a 24-well plate. The biopsies were then cultured in 2 ml of Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 4% human plasma, β-mercaptoethanol (50 μM), penicillin ( 100 U/ml), streptomycin (100 μg/ml), amphotericin B (2.5 μg/ml), HEPES (10 mM), sodium pyruvate (1 mM), MEM non-essential amino acid solution (1X ) and IL-15 (2 ng/ml, Miltenyi Biotech). Every 3-4 days 1 ml of medium was aspirated and replaced with 1 ml of complete medium containing 2x concentrated IL-15. TILs were harvested after 10 or 11 days, passed through a 70 μM nylon cell strainer, centrifuged at 300×g for 5 min and resuspended in complete medium for counting. Next, 400,000 cells/well were plated in 96-well plates and subsequently stimulated with anti-Vδ1 antibodies. TILs were stimulated with ADT1-4, ADT1-4-2, ADT1-7-3, or RSV IgG1 isotype control antibodies in the presence of IL-15 at concentrations of 2 or 50 ng/ml.

Figure 20 (A and B) shows the % reduction in Vδ1 TCR expression on total tumor-infiltrating γδ T cells after 48 (A) or 72 (B) hours of mAb stimulation in two independent donors, confirming that in each case the target Engagement. Tumor-infiltrating Vδ1+ cells were analyzed for expression of CD25 and Ki67. Figure 20(C) shows that the expression of CD25 and Ki67 on Vδ1 ⁺ T cells was enhanced after 48 hours of stimulation with ADT1-4-2 compared with stimulation with IgG1 isotype control or ADT1-4. Supernatants from cultures of mAb-stimulated TIL were analyzed for interleukin production by MSD. Figure 20(D) shows the substantial fold increase in IFN-γ production from TILs stimulated with ADT1-4-2 or ADT1-7-3 for 72 hours in the presence of 50 ng/ml IL-15. Figure 20(E) shows that stimulation of TILs with ADT-1-4-2 or ADT1-7-3 did not enhance secretion of type 17-related interleukins IL-6 or IL-17 at this time point.

Alternatively, biological specimens from different donors are enzymatically digested upon receipt to obtain single cell suspensions. Specifically, up to 1 g of tissue was placed in a Miltenyi C tube along with 4.7 ml of RPMI and enzymes from Miltenyi's Tumor Dissociation Kit at concentrations recommended by the manufacturer, except for Enzyme R, which Use at 0.2x concentration to prevent cleavage of relevant cell surface molecules. The C-tube was placed on a gentleMACS ^™ Octo dissociator with a heating block attached. The program 37C_h_TDK_1 for small tumor dissociation was selected. After 1 hour, the digests were filtered through a 70 μM filter and complete IMDM containing 4% human plasma was added to quench the enzyme activity. Cells were then washed twice and resuspended in complete IMDM for counting. Depending on the number of cells, 2 × 10 ⁶ or 4 × 10 ⁶ cells/well were plated in a 48-well plate and incubated with ADT1-4, ADT1-4-2 in the presence of IL-15 at a concentration of 2 ng/ml. or RSV IgG1 isotype control antibody stimulation. TILs isolated by enzymatic digestion were analyzed by flow cytometry 24-72 hours after mAb stimulation. Figure 20 ( F and G ) shows the % reduction in Vδ1 TCR expression on total tumor-infiltrating γδ T cells after 24 (F) or 72 (G) hours of mAb stimulation in two individual donors, confirming that by enzymatic digestion Target engagement in detached TIL. Figure 20(H) shows the dose-dependent enhancement of Ki67 on γδ T cells after stimulation with ADT1-4-2 for 72 hours. Supernatants from cultures of mAb-stimulated TIL were analyzed for interleukin production by MSD. Figure 20 (I and J) shows at two time points 24 hours (I) and 72 hours (J), isolated from two individual donors by enzymatic digestion and used in the presence of 2 ng/ml IL-15 6. The fold increase in IFN-γ production by TILs stimulated by ADT1-4-2 at a concentration of 6.66 nM. Taken together, these combined results highlight the ability of affinity-matured anti-Vδ1 antibodies to drive tumor-infiltrating γδ T cell activation, proliferation, and anti-tumorigenic interleukin production within 72 hours of stimulation.

In another experiment, lung tumor-derived TILs were isolated on grid matrices and stimulated with ADT1-4, ADT1-4-2, or IgG1 isotype control in the presence of 2 ng/ml IL-15 for 10 days. Figure 21(A) shows the enhanced expression of CD25 and Ki67 in γδ T cells stimulated with ADT1-4-2 for 10 days. Figure 21(B) shows a substantial increase in perforin ⁺ granzyme B ⁺ γδ T cells after 10 days of stimulation with ADT1-4-2. Stimulation with mAbs over a longer period of time allows immune permissiveness of αβ T cells via analysis of Vδ1 T cells stimulated with anti-Vδ1 antibodies. Figure 21(C) shows that the expression of granzyme B and perforin in CD8 ⁺ and CD8 ^- αβ T cells was significantly enhanced after stimulating tumor-infiltrating Vδ1 ⁺ T cells with ADT1-4-2 for 10 days. Supernatants from TIL cultured with mAb for 10 days were analyzed by MSD. Figure 21(D) shows a significant increase in IFN-γ production and a moderate increase in IL-17 and IL-6 production by lung tumor-derived TILs after stimulation with ADT1-4-2. Figure 21(E) demonstrates that the production of chemokines CCL2, CCL4 and CXCL10 in TILs was enhanced after 10 days of stimulation with ADT1-4-2. Taken together, these results highlight that anti-Vδ1 antibodies can drive activation and cytotoxicity of tumor-infiltrating γδ T cells and subsequently promote cytotoxicity of tumor-infiltrating αβ T cells. Example 7. Binding affinity of Vδ1-EGFR bispecific antibodies to human Vδ1 and human EGFR antigens

The binding affinity of the antibody to the target (ie, the Vδ1 chain of the γδ TCR and the EGFR) was established by SPR analysis using a Reichert 4SPR instrument (Reichert Technologies). Antibody (1.5 ug/mL) was coated onto a planar protein A sensor chip (Reichert Technologies), resulting in a baseline increase of approximately 500 uRIU. Recombinant human Vδ1 heterodimer or human EGFR flows through cells at a concentration of 100 nM, with the following parameters: 180 s association, 480 s dissociation, flow rate 25 μL/min, operating buffer PBS+0.05% Tween 20. All experiments were performed at room temperature. The results are shown in Figures 26A and 26B.

Conclusion : This data demonstrates that, despite the introduction of human eGFR binding capacity, the Vδ1/EGFR bispecific antibody still exhibits similar binding to human Vδ1 as the monospecific anti-Vδ1 antibody used in its preparation. Example 8. Target cell binding of Vδ1-EGFR bispecific antibodies

EGFR-positive A431 and Vδ1-positive primary γδ T cells were analyzed to determine the specificity and affinity of EGFR/Vδ1 bispecific antibody binding. Target cells were isolated from tissue culture flasks, resuspended in PBS and seeded into v-bottom 96-well plates at a final density of 100,000 cells/well. Cells were centrifuged and the cell pellet was resuspended in FcR blocking reagent and incubated at 4°C for 20 minutes before further washing according to the manufacturer's instructions. Antibodies were diluted to 500 nM in PBS and serially diluted 1:10 in PBS to 50 pM and added to cells followed by incubation at 4°C for 20 minutes. To determine the amount of mAb bound to the cell surface, cells were then stained with an APC-conjugated murine anti-human IgG secondary antibody (dilution: 1:100) in addition to the viability dye. After incubation for 20 minutes at 4°C, cells were washed twice, and fluorescence was measured using MACSQuant. The results are shown in Figure 27.

Conclusion : This data demonstrates that despite the introduction of binding to the EGFR-positive A431 cell line, the Vδ1/EGFR bispecific antibody still exhibits similar binding to Vδ1-positive γδ T cells as the monospecific anti-Vδ1 antibody used in its preparation. Example 9. In vitro assessment of γδ T cell activation and target cell cytotoxicity

Expanded skin-derived Vδ1 γδ T cells and A431 cells were isolated from tissue culture flasks and resuspended in basal growth medium and plated in 96-well plates at relevant cell dilutions depending on the desired effect:target ratio. mAb was diluted and added to each well at the indicated concentration. Cultures were then incubated at 37°C, 5% CO2 for 4 hours (D) or 24 hours (A-C, E). To determine the number of viable cells, cells were harvested and stained with viability dye at a 1:1000 dilution for 20 minutes. To determine CD25 status, cells were surface stained with anti-CD25 antibodies after cell harvest. To measure degranulation, fluorophore-conjugated anti-CD107α antibody was added directly to the cell-antibody mixture at the beginning of the co-culture. After two washes and cell fixation, fluorescence was measured using MACSQuant and viable cell count and median fluorescence intensity were determined. The results are shown in Figure 28.

Conclusion : This data demonstrates that Vδ1/EGFR bispecific antibodies induce activation and degranulation of primary Vδ1-positive γδ T cells, resulting in increased cell-mediated lysis of the EGFR-positive A431 cell line. Example 10. Further evaluation of γδ T cell activation and non-depletion effects conferred by the antibodies of the invention

Studies were performed to explore the effects of stimulation/activation of vδ1 cells with anti-vδ1 antibodies relative to CD3 downregulation on vδ1 cells. This was tested by incubating anti-vδ1 pure line ADT1-4-2 with PBMC and subsequently analyzing the TCR by phenotyping.

Cryopreserved human peripheral blood mononuclear cells (PBMC) were of commercial origin and cultured in 250 μl of complete medium with 10 ng/ml IL15 (supplemented with 10% FCS, pen/strep, non-essential amino acids, sodium pyruvate and HEPES (RPMI) were seeded into round-bottom 96-well tissue culture plates at 250,000 cells/well. Add titrated vδ1 antibody ADT1-4-2 to a final concentration of 1 ug/ml (6.67 nM), 0.01 ug/ml (0.067 nM), or 0.0001 ug/ml (0.00067 nM). RSV IgG antibody was included as a control for matching concentrations. Cultures were grown for 14 days, with medium and antibodies replenished every 3 days. Flow cytometric analysis was performed at endpoint to phenotype vδ1 cells and TCR expression in each condition. First, it targets the living singlet state, and then it targets the pan-γδ (Miltenyi REA592; 130-113-508) gated cells, which is the parent gate of vδ1 (Miltenyi REA173; 130-100-553) and itself targets CD3 (Miltenyi REA613 ;130-113-142)'s parent gate. Cell populations were identified via positive staining, and then the relative expression levels of each marker between samples were identified via MFI.

Figure 29A shows vδ1 TCR MFI after antibody stimulation as an indication of mAb target engagement. Figure 29B shows the MFI of CD3 expression on positively gated vδ1 cells. Stimulation with the vδ1 antibody pure line ADT1-4-2 engages vδ1 cells and causes downregulation of vδ1 and CD3 on vδ1 cells. Example 11 : Anti -vδ1 antibodies do not induce ADCC

ADCC reporter bioassay (Promega) was used to assess the level of ADCC (antibody-dependent cell-mediated cytotoxicity) induced by anti-vδ1 antibodies compared to control antibodies.

ADCC refers to the biological phenomenon in which effector cells kill target cells labeled by antibodies. Effector cells bind to the antibody via their Fcγ receptors and subsequently kill the target cells. By detecting the early onset of ADCC via activation of gene transcription via the NFAT (nuclear factor of activated T cells) pathway, the ADCC reporter bioassay presented here uncovers the potential ADCC mechanistic effects of the antibodies tested within the assay. The reporter assay is an engineered system that utilizes effector cells (Jurkats) expressing high-affinity FcγRIIIa receptors linked to the NFAT pathway, which are further engineered to induce further activation of firefly luciferase upon their activation . Luciferase activity is quantified using luminescence readings, which can be correlated to the level at which ADCC occurs.

Use this analysis to understand whether anti-vδ1 antibodies, or the anti-vδ1 arm of an appropriate multispecific antibody, drive ADCC responses. The target cells utilized are γδ cells, which bind to anti-vδ1 antibodies via the vδ1 γδ TCR. If the ADCC mechanism of action exists, the anti-vδ1 antibody will bind to the Fcγ receptor on the effector cell and generate a luminescent signal; if no signal is generated, this will indicate that ADCC has not occurred.

ADCC Reporter Bioassay Kit (Promega) was used for this analysis. Thaw the vial of Bio-Glo Luciferase Assay Buffer and transfer to the matrix vial. Keep the mixture at room temperature for 4-6 hours. Prepare dilution plates using antibody concentrations (3X concentrations) ranging from 10 nM to 0.01 nM (final concentration) for the following antibodies: anti-vδ1 antibody (ADT1-4-2), identical but Fc-disabled (L235A, G237A) antibodies vδ1 antibody (ADT1-4-2 LAGA), rituximab, RSV and OKT3. Target cells (γδ cells) were seeded into 2 assay plates at 25 μl per well. Then transfer 25 μl of the appropriate antibody solution from the antibody dilution tray to the appropriate wells. Effector cells (engineered Jurkat cells) were thawed into warm assay buffer, resuspended in 4 ml of assay buffer, and 25 μl of effector cell solution was pipetted into each well. The plates were then incubated at 37°C for 4.5 hours. After the incubation period, the plate was allowed to equilibrate to room temperature before 75 μl of Bio-Glo Luciferase Assay Reagent was added to each well and the plate was incubated at room temperature for 10 minutes. The disc is then read using a Biotek H4 disc reader, and the luminescence signal (in relative light units (RLU)) from the disc is collected. Calculate the induction fold using the following equation: induction fold = RLU (induction - background) / RLU (no antibody control - background).

OKT3 (anti-CD3 antibody) was used as a positive control. As an additional positive control, control wells were seeded with Raji cells instead of γδ cells. Raji cells are a generally recognized cell line for demonstrating strong ADCC responses when used with the anti-CD20 antibody rituximab. As an internal control and to understand whether anti-vδ1 antibodies drive ADCC in the absence of vδ1 binding on γδ cells, the anti-vδ1 antibodies of the invention and the same anti-vδ1 antibodies but with Fc inactivation (L235A, G237A) were also added along with effector cells alone.

The results are shown in Figure 30 .

Conclusion : Demonstrated strong ADCC response in positive control using rituximab with Raji cells and even stronger ADCC response with OKT3 against γδ cells. In contrast, no ADCC response was detected under any condition using the anti-vδ1 antibody of the invention, the same anti-vδ1 antibody that also had Fc disabled (L235A, G237A), or the RSV negative control. This demonstrates that, in this system, antibodies of the invention that bind vδ1 (such as anti-vδ1 mAb or anti-vδ1 multispecific antibodies) show no evidence of an ADCC mechanism of action. Notably, even anti-Vδ1 antibodies with Fc function do not deplete γδ T cells, which provides the option of maintaining Fc function in the anti-Vδ1 antibodies presented herein, such as increasing functionality in high Fcγ tumor environments. This further emphasizes that such anti-Vδ1 antibodies are suitable for inclusion in bispecific antibody formats as described herein. Example 12 : Manufacturability / Stability Assessment of Vδ 1×EGFR Multispecific Antibodies

To better understand the manufacturability/stability of novel Vδ1×EGFR multispecific molecules as described herein, the inventors evaluated a panel of these molecules via a variety of methods. Research shows surprising differences and is now summarized. (A) Yield in CHO

Methods : Multispecific antibodies were first generated in a standard CHO performance platform ("CHO Platform 1"). This platform includes standard expression vectors containing heavy chain and light chain cassettes. Suspension-adapted CHO K1 cells (originally from ATCC and adapted for serum-free growth in suspension culture) were used for manufacturing. Seed cells are grown in chemically defined animal component-free, serum-free medium. The cells are then transfected with vectors and transfection reagents, and the cells are allowed to grow further. Supernatants were harvested by centrifugation and antibodies were purified using MabSelect™ SuRe™ prior to formulation.

Conclusion : Comparative yields of the two molecules performed in this platform are indicated in Figure 34A and highlighted for ADT1-4-2×LEE compared to ADT1-4-2×FS1-67 (indicated by arrows) Observed superior performance and recycling. Further comparisons were then performed in an alternative CHO platform ("CHO Platform 2"). In this alternative platform, the yields of individual molecules are more comparable. (B) Stability under accelerated storage conditions

Methods : Antibodies generated in the 'CHO Platform 2' above were then formulated at 2 mg/ml in typical antibody formulation buffer and studied under accelerated stress conditions (37°C). Molecules were then analyzed by standard icIEF methods using the Uncle platform and standard protocols (Unchained Labs, CA, USA).

Conclusion : Figure 34B shows that although most of the molecules appeared relatively stable, for the ADT1-4-2×FS1-67 molecule a significant increase in basic species was noted after only 48 hours (indicated by the arrow). Such an increase was not observed for other molecules, as exemplified by the ADT1-4-2×LEE molecule in Figure 34B. This finding demonstrates that the ADT1-4-2×LEE molecule is a more stable (eg, more thermostable) Vδ1×EGFR multispecific antibody. Assuming that the only sequence difference between ADT1-4-2×FS1-67 and ADT1-4-2×LEE is at position L358.T359.K360, this suggests that in some cases at least one of the Vδ1 chains containing the γδ TCR-targeting A Vδ1×EGFR multispecific antibody derived from the first binding domain of the antibody and the second binding domain that binds the EGF receptor and containing L358.E359E.E360 relative to one that actually contains T358.D359.D360 at these positions Vδ1×EGFR bispecific antibody is the better variant (all numbers are according to EU). (C) Aggregation tendency

Methods : The melting profile of multispecific antibodies was assessed using Uncle and standard protocols (Unchained Labs, CA, USA).

Conclusion : Figure 34C highlights that for the ADT1-4-2×FS1-65 molecule, aggregation begins before melting, causing significant differences (indicated by arrows). This is in contrast to other molecules where the onset of aggregation is more simultaneous with the onset of melting. Simultaneous melting/aggregation usually indicates a more stable molecule. (D) Decay profile at higher concentrations under accelerated conditions ( non-reducing analysis )

Methods : Although studies exploring aggregation propensity at 2 mg/ml did not show significant differences, at 5 mg/ml when analyzed by non-reducing CE-SDS according to Maurice and standard protocols (Unchained Labs, CA, USA) The following studies (18 days at 37°C under accelerated conditions) showed significant changes in different molecules.

Conclusion : With this type of analysis, a significant increase in the occurrence of non-monomeric impurities was noted in a large number (but not all) of the bispecific molecules analyzed. Exemplary results of this study and tabulated summary results are presented in Figure 34D (significant changes are indicated by arrows). (E) Decay profile at high concentrations under accelerated conditions ( reducibility analysis )

Although the study in (D) above highlights that ADT1-4-2×FS1-67 exhibits a more stable profile under accelerated conditions when analyzed under non-reducing conditions, when analyzed under reducing CE-SDS conditions (again using Uncle and standard protocols; Unchained Labs, CA, USA), this same ADT1-4-2×FS1-67 molecule relative to other multispecific molecules exemplified by ADT1-4-2×LEE in Figure 34E Show a unique and atypical profile. ADT1-4-2×LEE did not show an increased “shoulder” after 18 days at 37°C (only unique to ADT1-4-2×FS1-67), which may indicate the more favorable properties of ADT1-4-2×LEE . Example 13 : Increase / decrease the affinity of the VD1 binding arm of the VD1×EGFR multispecific antibody

In these studies, a series of multispecific antibodies were created, among which (i) The affinity for Vδ1+ increases/decreases between approximately 0.2 nM and 20 nM, while (ii) The affinity for EGFR remains constant at approximately 1 nM.

The effects of these molecules were then studied in a series of experiments. The control D1.3×EGFR multispecific molecule was also included in this series of studies. This control included the first binding domain of mAb D1.3 that bound chicken lysozyme (but not the Vδ1 TCR), and the second binding domain that bound EGFR with an affinity of approximately 1 nM. (A) Affinity summary .

The nomenclature and affinities of this set of multispecific molecules are presented.

SPR method of Example 13 : The binding affinity of the antibody to the target (human Vδ1) was determined by SPR analysis using a 4SPR instrument (Reichert Technologies). Antibody (1.5 µg/mL) was coated onto a planar protein A sensor chip (Reichert Technologies), resulting in a baseline increase of approximately 500 uRIU. Human Vδ1 antigen was flowed through the cells in a 1:3 dilution series with a maximum concentration of 50 nM, with the following parameters: 180 s association, 900 s dissociation, flow rate 25 μL/min, operating buffer PBS+0.05% Tween-20. The wafer surface was regenerated after each injection using 20 mM phosphoric acid. All experiments were performed at room temperature. Steady-state fits were determined based on Langmuir 1:1 binding using TraceDrawer software (Reichert Technologies). (B) (C) Effect of v δ 1 TCR affinity on the binding of multispecific antibodies to V δ 1+ cells present in tissues

In this first experiment using this set of molecules, the role of affinity for the Vδ1 TCR was explored.

Methods : Skin tissue-derived Vδ1+ effector cells were co-incubated with antibodies serially diluted in PBS to concentrations ranging from 10 nM to 0.13 pM. Cells were incubated at 4°C for 20 minutes. Cells were washed and stained with anti-human Fc secondary antibody (Jackson Immunoresearch) and dead cell viability dye (eFlour 780, Invitrogen) for 20 min at 4°C. Cells were washed in FACS buffer and resuspended in cell fixative (BD sciences) before analysis on a MACSQuant Analyzer 10 flow cytometer (Miltenyi).

Conclusion : The binding data presented indicate a strong correlation between increased Vδ1 affinity and increased levels of multispecific antibody binding to Vδ1+ cells present in tissues. See Figure 35B and Figure 35C showing the list results of Figure 35B. This data highlights that higher affinity molecules confer enhanced effects at saturating concentrations. Notably, higher concentrations of lower affinity variants of the antibody are unable to mimic or reproduce the effects conferred by higher affinity variants. This suggests that molecules with (unnatural) high affinity for the Vδ1 TCR confer different effects that lower affinity molecules cannot mimic, even at significantly higher concentrations (eg 100-fold). This finding is further discussed in this article.

The role of these molecules was then further studied in the following series of experiments. Skin tissue-derived Vδ1+ effector cells were co-cultured with EGFR+ A375 melanoma cancer cell lines as follows: ● First, seed EGFR+ target cells into multi-well tissue culture plates approximately 16 hours before assay setup. ● The next day, serially dilute the antibody to a series of basal growth concentrations and then add it to the EGFR+ target cells. ● Isolate skin-derived Vδ1 γδ T cells from culture bottles, inoculate the cell suspension into assay plates and incubate the co-culture at 37°C, 5% CO2 for the specified duration. ● Depending on the readout selected, cells are processed and analyzed as described in each case.

The results of these studies are presented below: (D)(E) Effect of v δ 1 TCR affinity on V δ 1 TCR internalization by V δ 1+ cells present in tissues

In this first analysis of co-incubation of Vδ1+ responsive cells with EGFR+ cancer cells, the focus was on the induction of Vδ1 TCR internalization following multispecific antibody engagement.

Methods : Co-cultures were established as above, where 2.5×10 ⁴ Vδ1 γδ T cells were added to 2.5×10 ⁴ EGFR+ target cells/well in a U-bottom 96-well plate. Vδ1 γδ T cells were stained with [0.5 µM] CellTrace purple live cell dye for 20 minutes before addition. Final assay antibody concentrations ranged from 10 nM to 0.13 pM. After incubation for 4 hours at 37°C, 5% CO2, cells were harvested and stained for surface expression of dead cells (eFlour 780, Invitrogen) and Vδ1 TCR (Miltenyi Biotec) for 20 minutes at 4°C. Cells were washed in FACS buffer and resuspended in cell fixative (BD sciences) before analysis on a MACSQuant Analyzer 10 flow cytometer (Miltenyi).

Conclusion : The reduction in MFI is presented in Figure 35D. These results indicate increased TCR downregulation upon antibody engagement. It was also found that the higher the affinity, the greater the reduction in MFI signal (and therefore TCR downregulation). Notably, even adding 100-fold more of the lower affinity molecule (ADT1-4-138×ADT3-2) failed to induce equivalent signal reduction. This suggests that unnaturally high affinity for the TRDV1 target may further enhance TCR downregulation. It is also shown that extremely high affinity binding confers different effects on TCR function. One can only speculate as to why such high affinity is required to achieve better saturation downregulation. Possibly this is a stochastic effect on a homogeneous Vδ1+ population, where longer residence times of higher affinity molecules (via reduced off-rates) confer a higher saturation effect, possibly via higher levels of TCR cross-linking. A possible alternative is that a subset of Vδ1+ cells is unresponsive to TCR downregulation conferred by lower affinity molecules. A summary of the findings in Figure 35D is presented in tabular form (see Figure 35E). This further emphasizes the fact that high affinity delivers different effects at saturating concentrations and that the reduced effects conferred by lower affinity molecules cannot be compensated by administration at higher concentrations. (F)(G) Effect of v δ 1 TCR affinity on degranulation of V δ 1+ cells present in tissues

This subsequent analysis focused on the degranulation effect conferred by the multispecific antibodies of the invention.

Methods : Co-cultures were established as above, where 2.5×10 ⁴ Vδ1 γδ T cells were added to 2.5×10 ⁴ EGFR+ target cells/well in a U-bottom 96-well plate. Vδ1 γδ T cells were stained with [0.5 µM] CellTrace purple live cell dye for 20 minutes before addition. Final analytical multispecific antibody concentrations ranged from 10 nM to 0.13 pM. Additionally, a fluorophore-conjugated antibody (MiltenyiBiotec) specific for CD107a was added at the beginning of the co-culture. After incubation for 4 hours at 37°C, 5% CO2, cells were harvested and stained for dead cells (eFlour 780, Invitrogen) for 20 minutes at 4°C. Cells were washed in FACS buffer and resuspended in cell fixative (BD sciences) before analysis on a MACSQuant Analyzer 10 flow cytometer (Miltenyi). See Figure 35F.

Conclusion : These studies again demonstrate that increased affinity confers optimal effects. A direct correlation between increased CD107a upregulation and increased affinity was observed. Again, this data suggests that the non-native high affinity for Vδ1 TCR drives significantly different, more favorable effects that cannot be reproduced with molecules with lower affinity for Vδ1, even when administered at much higher concentrations. The summary results of Figure 35F are presented in tabular form. (See Figure 35G) Again, this summary highlights the finding that higher affinity molecules at saturating concentrations impart a different, more enhanced saturation effect relative to lower affinity molecules even when employed at much higher concentrations. (H)(I) Effect of v δ 1 TCR affinity on the cytolytic function of multispecific antibodies on EGFR+ cancer cells

This subsequent analysis focused on the cytolytic effect conferred by the multispecific antibodies of the invention.

Methods : Co-cultures were established as above, where 4×10 ³ Vδ1 γδ T cells were added to 4×10 ³ EGFR+ target cells/well in 384-well CellCarrier Ultra plates (PerkinElmer). Final assay antibody concentrations ranged from 5 nM to 0.03 pM. After incubation for 24 hours at 37°C, 5% CO2, cells were stained with Hoechst 33342 (Invitrogen) and for dead cells (DRAQ7, Abcam) for 1 hour at 37°C. Cells were imaged on the Opera Phenix system (PerkinElmer), and the number of viable target cells was determined using a linear regression model (Harmony 4.9) based on the size, morphology, texture, and intensity of viable cell staining and the absence of DRAQ7 staining.

Conclusion : The data presented here demonstrate that molecules with the highest Vδ1 affinity are most effective in achieving cytolysis of EGFR+ cancer cells. See Figure 35H. Furthermore, qualitative differences were observed between the lowest Vδ1 affinity multispecific antibody (20 nM) and the medium/high Vδ1 affinity multispecific antibody (1.2 nM and 0.2 nM, respectively), where significantly different saturation effects were observed. This indicates that the unnatural high affinity confers a significantly different potentiation effect. Using molecules with lower affinity for the Vδ1 TCR cannot mimic these effects, even when administered at much higher (eg, 100-fold) concentrations. Summary results from Figure 35H are presented in tabular form (see Figure 35I). This underscores the finding that higher affinity molecules at saturating concentrations impart a different, more enhanced saturation effect relative to lower affinity molecules even when delivered at much higher concentrations. (J)(K) Effect of v δ 1 TCR affinity on the proliferation of V δ 1+ cells present in tissues

This subsequent analysis focused on the induction of Vδ1+ cell proliferation conferred by the multispecific antibodies of the invention.

Methods : Co-cultures were established as above, where 3×10 ⁴ Vδ1 γδ T cells were added to 1.5×10 ⁴ EGFR+ target cells/well in a U-bottom 96-well plate. Stain target cells with [0.5 µM] CellTrace Purple Live Cell Dye for 20 minutes before addition. Final analytical multispecific antibody concentrations ranged from 2 nM to 0.25 pM. Cells were incubated at 37 °C in 5% _CO for 5 or 7 days. Complete medium supplemented with interleukins (IL-2, IL-15, IL-4, and IL-21; essential units) was added on days 3 and 5. Cells were washed and stained for dead cells (eFlour 780, Invitrogen) for 20 min at 4°C. Cells were washed in FACS buffer and resuspended in cell fixative (BD sciences). The number of events was measured by flow cytometry the next day using a MACS Quant Analyzer 10.

Conclusion : These studies further strengthen the finding that increased affinity confers a more potent effect, with a direct correlation observed between increased induction of Vδ1 T cell proliferation and increased affinity. See Figure 35J (and summarized in tabular form in Figure 35K). This was most evident in the highest affinity iteration that exhibited the lowest EC50 and EC95 and the highest maximum cell number. Higher affinity molecules are more effective in driving Vδ1 T cell proliferation. Interestingly, and slightly different from other technical effects conferred by higher affinity molecules, lower affinity molecule variants can confer similar effects when delivered in this case at 100-fold higher concentrations. (L) Summary of this study

See Figure 35L.

This summary highlights (i) the advantageous technical effects conferred by the antibodies of the invention with higher affinity (less than 10 nM) for Vδ1+TCR; and (ii) the subsequent inability to compensate for the higher affinity by increasing the concentration 100-fold or more Low affinity molecules (more than 10 nM) confer a reducing effect. (M) Effect of number of TAA replicas

The above findings, as summarized in Figure 35L, highlight some interesting results. Specifically, (i) Enhanced delivery of high-affinity molecules (to the Vδ1 TCR) relative to lower-affinity molecule variants, and (ii) The reduced effect conferred by lower affinity variants can never be compensated for by increasing the concentration of these molecules.

However, the inventors then explored the effect of replica number on a second target, in this case EGFR, in such an analysis. (See Figure 35M). The study used the same approach as outlined above, but replaced a lower replica number TAA cancer cell line (A375) with a cancer cell line showing extremely high replica numbers (A431). Interestingly, in this equivalent study, which therefore involved co-culture of primary Vδ1+ cells with a unique high-replicate EGFR+ cancer cell line, different findings were revealed. Specifically, in some cases, the poorer effects observed with lower affinity variants can be overcome by increasing the concentration of these variants in the assay when combined with higher TAA copy number cancer cell lines. Although high-affinity molecules exhibit similar effects at similar concentrations regardless of the number of EGFR replicas in the cancer cell lines used in the analysis, for lower-affinity molecules, increasing the concentration of the molecules in combination with using higher replica number cancer cell lines The combination produces a compensating effect. When higher concentrations of low-affinity variants are used in such cases, the saturating CD107a up-regulation and saturating TCR down-regulation conferred by lower affinity molecules can for the first time mimic the saturation effects of higher affinity molecules.

This suggests that the reduced effects of lower affinity molecules can be "restored" under the following conditions: (a) Analysis using extremely high TAA target cell lines and (b) Use molecules whose concentration is increased by a factor of 100 (or more).

Although the mechanisms behind these findings are only speculative, they once again highlight the advantages of molecules of the invention with higher affinity for the Vδ1 TCR. For example, if the target cancer cell line has normal EGFR TAA levels, the efficacy of the lower affinity variant is significantly reduced. Higher dosing cannot overcome this. Additionally, in the case of EGFR TAA, it should be understood that very high copy number EGFR cancers are a minority among most cancers, see for example regarding some breast cancers (e.g. Kim et al. 201310.1371/journal.pone.0079014) or head and neck cancers (e.g. Licitra et al. 2011 DOI: 10.1093/annonc/mdq588) or gastric cancer (e.g. Zhang et al. 2015 doi.org/10.3892/ol.2015.3875). Thus, the increased utility and surprising advantages of the non-natural higher affinity antibodies of the present invention over and over lower affinity variants are very clear. Example 14 : Increase / decrease the affinity of the EGFR binding arm of VD1×EGFR multispecific antibody

Create a series of multispecific antibodies in which (i) Increase/decrease affinity for EGFR, while (ii) The affinity for Vδ1 remains constant at approximately 0.2 nM.

The starting EGFR binding domain for this search (FS1-67) has been described previously (WO2018015448). One motivation for further modification of this binding domain is to attempt to further fine-tune the affinity of this domain when used in the multispecific antibodies of the invention. In this context, the inventors have discovered that returning certain amino acids in the EGFR binding domain to wild-type human immunoglobulin sequence affects affinity for the EGFR target more than other amino acids. This exploration uncovered a more finely tuned set of binding domains with affinity selection ranging from about 1 nM to about 140 nM for EGFR binding. When incorporated into the multispecific antibodies of the invention, different EGFR binding domains confer multiple distinguishing properties. Therefore, this set of multispecific antibodies will also prove extremely useful when exploring and measuring affinity effects.

Also included in this study (where indicated) was a multispecific D1.3×FS1-67 control (containing a 1 nM affinity EGFR second binding domain and a first binding domain containing a binding domain to chicken lysozyme) (A ) Affinity Summary

The nomenclature and affinities of this set of multispecific molecules are presented.

SPR method of Example 14 : For human EGFR, a recombinant protein (Stratech; 10001-H08H-SIB-100ug) was coupled to a carboxymethylpolydextrose sensor chip (Reichert Technologies) to increase the baseline by approximately 500 uRIU. Inject antibodies in the concentration range (7.5-200 nM) in PBS-T buffer with the following parameters: 180 s association, 660 s dissociation, flow rate 25 μL/min, operating buffer PBS-T. The wafer surface was regenerated after each injection using 20 mM phosphoric acid. All experiments were performed at room temperature. Steady-state fits were determined based on Langmuir 1:1 binding using TraceDrawer software (Reichert Technologies).

Conclusion : Summary results are presented in Figure 36A. For this study, the starting CH3-based FS1-67 binding module contained 5 modifications in the AB loop, 3 modifications in the CD loop, and 5 modifications in the EF loop. When trying to vary the affinity of this molecule to varying degrees, some interesting findings were made. For example, restoring all 5 amino acids in the AB loop to wild-type sequence was found to have the most significant impact on EGFR binding affinity (as indicated by ADT1-4-2×LEE1, dashed black rectangle). Of course, compared with the complete restoration of the CD loop or the (partial) restoration of the EF loop to the wild-type immunoglobulin sequence (ADT1-4-2×LEE2 and ADT1-4-2×LEE3 respectively), the changes in the AB loop are the most influential . Indeed, by some measures (see below), restoring the AB loop to the wild-type sequence results in a loss of binding affinity and specificity. Therefore, unexpectedly, when the non-conserved amino acid found in the AB ring of FS1-67 was modified from threonine (polar) back to leucine (non-polar), the affinity was not affected. This was achieved by comparing the ADT1-4-2 LEE molecule (highlighted by the solid black rectangle, 1 change - negligible impact on affinity/function) with the ADT1-4-2 LEE1 molecule (highlighted by the dashed black rectangle, 5 changes that have the greatest impact on affinity/function) are highlighted by comparison. As mentioned above, the resulting ADT1-4-2×LEE, which now contains only 4 changes to the AB loop, also incorporates additional modifications. Therefore, the possibility that these other (conservative) changes in the binding loop (D3659E, D360E) also contribute to maintaining high affinity cannot be ruled out; however, this was unexpected. Regardless, it is postulated that reverting such modifications to wild-type would reduce the immunogenicity risk of the multispecific antibodies of the invention, with the wild-type leucine at position 358 and as a four amino acid change in the AB loop part, deemed to be, for example, the starting FS1-67, FS1-65 or 747 incorporated into ADT1-4-2×FS1-67, ADT1-4-2×FS1-65 and ADT1-4-2×747 respectively. Changes in the 5 (or more) amino acids found in the EGFR binding domain are even better. (B) (C) Effect of EGFR affinity on the binding of multispecific antibodies to EGFR+ colorectal cancer cell line HT-29

Methods : Exemplary binding of EGFR×Vδ1 multispecific antibodies was determined by incubating EGFR+HT-29 target cells with antibodies serially diluted from 500 nM to 0.13 pM in PBS for 20 min at 4°C. concentration range. After washing, cells were incubated with anti-human IgG secondary antibody (Jackson Immunoresearch) and live/dead dye (eFluor 780, Invitrogen) for an additional 20 minutes, followed by washing and fixation using CellFix. The amount of antibody bound to each cell type was determined using flow cytometry (MACSQuant Analyzer 10, Miltenyi Biotech).

Conclusions : The data presented demonstrate a strong, direct, and graded correlation observed between increased EGFR affinity and increased levels of multispecific antibody binding to EGFR+HT-29 cancer cells. See Figure 36B. Figure 36C presents the results of Figure 36B in a list form.

The role of these molecules was then further studied in the following series of experiments. Briefly, skin tissue-derived Vδ1+ effector cells were co-cultured with EGFR+ A375 melanoma cancer cell lines as follows: EGFR+ target cells were seeded into multi-well tissue culture dishes approximately 16 hours before assay setup. The next day, the antibody was serially diluted to a concentration series for basal growth and then added to EGFR+ target cells. Skin-derived Vδ1 γδ T cells were isolated from culture flasks, cell suspensions were seeded in assay plates and co-cultures were incubated at 37°C, 5% CO2 for the indicated durations. Depending on the readout selected, cells were processed and analyzed as described in each case. The results of these studies are presented below: (D) (E) Effect of EGFR affinity on V δ 1 TCR downregulation in V δ 1 + cells present in tissues

This subsequent analysis focused on the induction of Vδ1 TCR internalization following multispecific antibody engagement.

Methods : Co-cultures were established as above, where 2.5×10 ⁴ Vδ1 γδ T cells were added to 2.5×10 ⁴ A375 EGFR+ target cells/well in a U-bottom 96-well plate. Vδ1 γδ T cells were stained with [0.5 µM] CellTrace purple live cell dye for 20 minutes before addition. Final assay antibody concentrations ranged from 10 nM to 0.13 pM. After incubation for 4 hours at 37°C, 5% CO2, cells were harvested and stained for surface expression of dead cells (eFlour 780, Invitrogen) and Vδ1 TCR (Miltenyi Biotec) for 20 minutes at 4°C. Cells were washed in FACS buffer and resuspended in cell fixative (BD sciences) before analysis on a MACSQuant Analyzer 10 flow cytometer (Miltenyi).

Conclusions : Decreases in Vδ1 TCR MFI indicate increased TCR downregulation, and specifically, it was found that the higher the affinity for EGFR, the greater the decrease in this signal. Notably, however, the relationship between EGFR affinity and induction of TCR downregulation does not appear to be a graded correlation, but rather depends on the threshold affinity value. Specifically, three molecules (ADT1-4-2×LEE, ADT1-4-2×LEE2, and ADT1-4-2×LEE3) exhibit affinities for EGFR of about 1 nM, about 9 nM, and about 19 nM, respectively. ) cluster together and confer similar TCR down-regulation effects and associated EC50 values. These three molecules contrast significantly with ADT1-4-2×LEE1. As discussed previously, this molecule contains the wild-type immunoglobulin AB ring and exhibits a minimum affinity of approximately 140 nM. In contrast to the other three molecules, this molecule did not confer the required TCR downregulation even in combination with the high-affinity anti-Vδ1 TCR ADT1-4-2 binding domain, as was the case here. This would suggest that there is a threshold effect or steep gradient effect that clusters multispecific antibodies such as ADT1-4-2×LEE, ADT1-4-2×LEE2 and ADT1-4-2×LEE3 together with those of ADT1 -4-2× LEE1 separation of lower EGFR affinity molecules. This contrasts with the shallower, more graded affinity/binding events seen previously (see Figure 36B above), where the binding conferred by these multispecific antibodies was graded by their corresponding affinity to the EGFR secondary binding target. Rather than clustering into two contrasting groups. See Figure 36D. Figure 36E presents the results of Figure 36D in a list form. (F) (G) Effect of EGFR affinity on degranulation of V δ 1+ cells present in tissues

This subsequent analysis focused on the degranulation effect conferred by the multispecific antibodies of the invention.

Methods : Co-cultures were established as above, where 2.5×10 ⁴ Vδ1 γδ T cells were added to 2.5×10 ⁴ EGFR+ target cells/well in a U-bottom 96-well plate. Vδ1 γδ T cells were stained with [0.5 µM] CellTrace purple live cell dye for 20 minutes before addition. Final analytical multispecific antibody concentrations ranged from 10 nM to 0.13 pM. Additionally, a fluorophore-conjugated antibody (MiltenyiBiotec) specific for CD107a was added at the beginning of the co-culture. After incubation for 4 hours at 37°C, 5% CO2, cells were harvested and stained for dead cells (eFlour 780, Invitrogen) for 20 minutes at 4°C. Cells were washed in FACS buffer and resuspended in cell fixative (BD sciences) before analysis on a MACSQuant 16 flow cytometer (Miltenyi).

Conclusions : These studies again demonstrate that increased affinity for EGFR confers optimal effects, with a correlation between increased CD107a upregulation and increased affinity observed. This relationship depends on the threshold affinity value. As with TCR downregulation, multispecific antibodies with EGFR affinities in the range of about 1 to about 19 nM functionally clustered together and induced significantly higher levels of EGFR relative to lower affinity molecules (ADT1-4-2×LEE1). CD107a. This data demonstrates that, under certain circumstances, multispecific antibodies of the invention with high to moderate affinity for EGFR drive significantly different and more beneficial effects than molecules with lower affinity for EGFR even at much higher concentrations. This effect cannot be replicated when administered (e.g., 1,000 times). The results are shown in Figure 36F and summarized in tabular form in Figure 36G. (H) (I) Effect of EGFR affinity on the activation status of 41BB in V δ 1+ cells present in tissues

This subsequent analysis focused on the increase in the activation-induced marker 41BB of Vδ1+ cells conferred by the multispecific antibodies of the invention.

Methods : Co-cultures were established as above, where 2×10 ⁴ Vδ1 γδ T cells were added to 2×10 ⁴ EGFR+ target cells/well in a U-bottomed 96-well plate. Vδ1+ γδ T cells were stained with [0.5 µM] CellTrace purple live cell dye for 20 minutes before addition. Final analytical multispecific antibody concentrations ranged from 100 pM to 0.13 pM. After 24 hours of incubation at 37°C, 5% CO2, cells were harvested and stained for dead cells (eFlour 780, Invitrogen) and 41BB (Miltenyi Biotec) surface expression at 4°C for 20 minutes. Cells were washed in FACS buffer and resuspended in cell fixative (BD sciences) before analysis on a MACSQuant Analyzer 10 flow cytometer (Miltenyi).

Conclusion : The data presented here (see Figure 36H and tabulated summary in Figure 36I) demonstrate that multispecific antibodies of the invention with higher affinity for EGFR are most effective in stimulating increased 41BB activation in Vδ1+ cells. As in some other studies previously discussed, significant qualitative differences were again observed between the lowest EGFR affinity multispecific antibodies at approximately 140 nM and the intermediate to high EGFR affinity multispecific antibodies (which were below the approximately 20 nM threshold), where Lower affinity was unable to induce significant Vδ1+ cell activation. This indicates a threshold for functional clustering of moderate to high affinities as measured by 41BB upregulation in this case. Third, using molecules with lower affinity for EGFR, these effects cannot be replicated even when administered at much higher (eg, 1,000-fold) concentrations. Interestingly, however, it was also observed that the 41BB level increase upon saturation with the highest affinity molecule (ADT1-4-2×LEE, approximately 1 nM) was less than that for the intermediate range multispecificity of the present invention between approximately 9 nM-19 nM. The observed 41BB level increase for molecules (ADT1-4-2×LEE2 and ADT1-4-2 LEE3). This observation, discussed further below, is that the highest affinity may not always be optimal. (J)(K) Effect of EGFR affinity on the cytolytic function of multispecific antibodies on EGFR+ cancer cells

This subsequent analysis focused on the cytolytic effect conferred by the multispecific antibodies of the invention.

Methods : Co-cultures were established as above, where 4×10 ³ Vδ1 γδ T cells were added to 4×10 ³ EGFR+ target cells/well in 384-well CellCarrier Ultra plates (PerkinElmer). Final assay antibody concentrations ranged from 5 nM to 0.03 pM. After incubation for 24 hours at 37°C, 5% CO2, cells were stained with Hoechst 33342 (Invitrogen) and for dead cells (DRAQ7, Abcam) for 1 hour at 37°C. Cells were imaged on the Opera Phenix system (PerkinElmer), and the number of viable target cells was determined using a linear regression model (Harmony 4.9) based on the size, morphology, texture, and intensity of viable cell staining and the absence of DRAQ7 staining.

Conclusion : This data demonstrates that higher EGFR affinity molecules are most effective in achieving cytolysis of EGFR+ cancer cells. Again, significant qualitative differences were observed between the lowest EGFR affinity multispecific antibodies (approximately 140 nM) and the moderate to high EGFR affinity multispecific antibodies (approximately 1 nM to 19 nM). Indeed, the low EGFR affinity molecule (ADT1-4-2×LEE1) induced no measurable target killing. Thus, this is another example in which a threshold effect is observed in which about 1 nM to about 19 nM of molecules (ADT1-4-2×LEE, ADT1-4-2×LEE3, ADT1-4-2×LEE2) confer Better functional effects of clustering. Using molecules with lower affinity for EGFR, these effects cannot be replicated even when administered at much higher (eg, 10,000-fold) concentrations. The results are shown in Figure 36J and summarized in tabular form in Figure 36K. (L)(M) Effect of EGFR affinity on the proliferation of V δ 1+ cells present in tissues

This subsequent analysis focused on the induction of Vδ1+ cell proliferation conferred by the multispecific antibodies of the invention.

Methods : Co-cultures were established as above, where 3×10 ⁴ Vδ1 γδ T cells were added to 1.5×10 ⁴ EGFR+ target cells/well in a U-bottom 96-well plate. Stain target cells with [0.5 µM] CellTrace Purple Live Cell Dye for 20 minutes before addition. Final analytical multispecific antibody concentrations ranged from 2 nM to 0.25 pM. Cells were incubated at 37°C, 5% CO _for 5 or 7 days. Complete medium supplemented with interleukins (IL-2, IL-15, IL-4, and IL-21; essential units) was added on days 3 and 5. Cells were washed and stained for dead cells (eFlour 780, Invitrogen) for 20 min at 4°C. Cells were washed in FACS buffer and resuspended in cell fixative (BD sciences). The number of events was measured by flow cytometry the next day using a MACS Quant Analyzer 10.

Conclusions : These studies demonstrate once again that increased affinity confers superior effects, with a direct correlation observed between increased induction of Vδ1 T cell proliferation and increased affinity. Again, a clustering effect was observed with multispecific antibodies of the invention (ADT1-4-2×LEE, ADT1-4-2) with moderate to high affinity for EGFR in the affinity range of about 1 nM to about 19 nM. ×LEE3, ADT1-4-2LEE2) functionally clustered together, whereas the lower affinity molecule (ADT1-4-2×LEE1) with an affinity of approximately 140 nM was significantly attenuated and equivalent to non-binding in this assay ( For TCR binding) control. Interestingly and similar to the 41BB effect (see Figure 36H), the highest affinity for EGFR at approximately 1 nM affinity did not confer the highest saturating effect. Specifically, molecules with mid-range affinity of approximately 9 nM-19 nM (ADT1-4-2×LEE3 and ADT1-4-2×LEE2, respectively) conferred the highest proliferative effects at saturating concentrations of the antibody. This superior effect conferred by the mid-range affinity multispecific antibodies of the invention is discussed below. The results are shown in Figure 36L and summarized in tabular form in Figure 36M. (N) Effect on the affinity of EGFR and the number of TAA copies in cancer cells

These studies highlight the impact of the multispecific antibodies of the invention on the affinity for EGFR.

These effects were then compared to those observed in Vδ1+ cell/EGFR+ cancer cell co-culture studies in which the very high-replicate A431 cell line was actually used instead of the low-replicate A375 cell line.

A summary of this comparison is presented in Figure 36N and found that for extremely high EGFR copy number cancers such as those represented by A431, approximately 140 nM of the lower affinity multispecific antibody (ADT1-4-2×LEE1) can be "recovered" Some reducing effects were observed. However, this recovery is still graded, and the lower affinity molecules still do not "cluster" with the moderate to high affinity variants ADT1-4-2×LEE, ADT1-4-2×LEE2, and ADT1-4-2×LEE3. Nonetheless, this observation is interesting and suggests that lower affinity variants are more functional (and therefore more selective) for cancer cells bearing higher copy numbers of EGFR.

Suitably, the functional properties of the antibody when provided in a monospecific format are shared by a further multispecific antibody of the invention that specifically binds to a second antigen. Example 15 : Effect of affinity on preferential binding to V δ 1 cells over EGFR cells

This subsequent experiment explored how increasing and decreasing the affinity at respective binding domains affects molecular distribution and preferential binding. Specifically, this study explores how changing the binding affinity for respective Vδ1 and EGFR targets affects the binding preference of the multispecific antibodies of the invention.

Methods : Co-cultures were established in which 2 × ¹⁰ Vδ1 T cells were added to 2 × ¹⁰ EGFR+ target cells/well in a U-bottomed 96-well plate. A "limited" amount (16 pM) of each multispecific antibody as indicated was then added and the cells were incubated at 4°C for 20 minutes. Cells were incubated with anti-human IgG secondary antibody (Jackson Immunoresearch) and live/dead dye (eFluor 780, Invitrogen) for an additional 20 minutes, followed by washing and fixation (BD Cell Fixative). The amount of antibody bound to each cell type was determined using flow cytometry (MACS Quant Analyzer 10, Miltenyi Biotech).

Conclusions : Figure 37(A) shows the effect of affinity on preferential cellular binding to Vδ1 over EGFR, in bar chart format: showing the relative distribution of each antibody between the two cell types as indicated. This data demonstrates that changes in Vδ1 affinity have only marginal effects on binding ratios and distribution profiles. In contrast, there appear to be more profound effects when changing the affinity for the EGFR binding domain. Specifically, multispecific antibodies with lower EGFR affinity showed considerable preference for Vδ1 binding, with the lowest EGFR binder ADT1-4-2×LEE1 (Kd, 140 nM) showing the greatest preference for Vδ1 cells. Taken together, these results demonstrate that by increasing and decreasing the affinity of respective binding domains, different cellular target/tissue distribution profiles can be selected and directed. Furthermore, these findings, when combined with other findings herein, demonstrate that although the EGFR intermediate affinity antibodies of the invention (between about 9 nM and about 19 nM) exhibit in multiple ways the higher affinity variants (having a higher affinity for EGFR (approximately 1 nM affinity) have a similar effect, but such intermediate affinity multispecific antibodies of the invention vary in other ways and in some cases may provide a more favorable tumor-mediated antibody clearance, distribution and EGF blocking profile. This in turn may provide greater potential to further reduce dose-limiting skin toxicities relative to the higher affinity EGFR variants of the invention. Figure 37B presents the results for graph (A) in a tabular format, and Figure 37C presents the results for part (A/B) as a ratio. Example 16 : Affinity for EGFR ; Effect on internalization of multispecific antibodies in A431 tumor cells

(A) (B) This analysis focuses on the internalization of certain multispecific antibodies (and comparators). A series of multispecific antibodies were established as previously described, in which (i) Increase/decrease the affinity to EGFR, the affinity selection range is from about 1 nM to about 140 nM and the binding range to EGFR, while (ii) The affinity for Vδ1 remains constant at approximately 0.2 nM.

Also included in this study (where indicated) were a monospecific RSV IgG comparator (containing a binding domain to the RSV protein) and a multispecific D1.3×FS1-67 control (containing a 1 nM affinity EGFR second binding domain). and a first binding domain that includes a binding domain to chicken lysozyme).

Methods : Cultures were established at 4×10 ³ EGFR+ A431 tumor cells/well in 384-well CellCarrier Ultra plates (PerkinElmer). Antibodies were covalently labeled with Cy5 fluorophore using a Lightning Link antibody conjugation kit (Abcam) according to the manufacturer's instructions. Before adding antibodies, cells were imaged on an Opera Phenix system (PerkinElmer) using a Cy5 filter set. Labeled antibodies were then added at a final concentration of 1 nM, and cells were imaged by Opera Phenix at 15-minute intervals for an additional 8.5 hours. The intensity of Cy5 in the intracellular region was determined using Harmony 4.9 analysis.

Conclusion : This data demonstrates that lower EGFR affinity multispecific molecules exhibit minimal EGFR-mediated internalization in target A431 tumor cells. Significant qualitative differences were observed between the lowest EGFR affinity multispecific antibody (approximately 140 nM; equivalent to no antibody control) and the high EGFR affinity multispecific antibody (approximately 1 nM). Specifically, a positive correlation was found between EGFR target affinity and the observed level of antibody internalization. This represents an example of a graded effect associated with EGFR affinity of molecules of the invention for EGFR in the range of about 1 nM to about 140 nM. The results are shown in Figure 38A and tabulated in Figure 38B.

(C) (D) This analysis focuses on the clearance of multispecific antibodies described in this invention. In this case, clearance refers to the removal of the antibody from the culture medium of the A431 cell culture by EGFRA-mediated internalization of the antibody.

Methods : Cultures were established at 2×10 ⁴ EGFR+ A431 tumor cells/well in 96-well flat-bottomed TC-treated plates. Final assay antibody concentrations ranged from 5 nM to 1.6 pM. After incubation for 24 hours at 37°C, 5% CO2, the supernatant was harvested and stored for further analysis. Multispecific antibody concentrations were determined by in-house ELISA. Briefly, ELISA was performed by coating plates directly with 1 ug of Vδ1 antigen, followed by blocking with blocking buffer. Harvested supernatant was added to the plate, washed and anti-human Fc-HRP was used as secondary antibody at 1/2000 dilution. Develop with 50 µL of TMB solution, and measure absorbance at 450 nM using a microdisk reader (Tecan).

Conclusion : This data demonstrates that lower EGFR affinity multispecific molecules exhibit minimal EGFR-mediated antibody clearance. Significant differences were observed between the lowest EGFR affinity multispecific antibody (approximately 140 nM; equivalent to no antibody control) and the high EGFR affinity multispecific antibody (approximately 1 nM). Furthermore, this demonstrates a positive correlation between EGFR target affinity and the observed level of antibody clearance. Again, these findings highlight a direct graded correlation between the affinity of multispecific antibodies for EGFR and EGFR-mediated internalization for molecules in the range of about 1 nM to about 140 nM targeting EGFR. The results are shown in Figure 38C and tabulated in Figure 38D. Example 17 : Effect of EGFR affinity on the inhibition of EGF ligand binding by multispecific antibodies and tumor cell proliferation

(A) (B) This analysis focuses on the inhibition (or blocking) of the binding of EGF to EGFR-positive tumor cells by the multispecific antibodies described in the invention (and comparators). Also included in this study (where indicated) were monospecific RSV IgG comparators (containing binding domains to RSV proteins).

Methods : EGFR×Vδ1 multispecific antibody-mediated EGF blockade was determined by incubating 3× ¹⁰ EGFR+ A431 target cells with antibodies serially diluted to 250 in PBS for 30 min at 4°C. nM to 3.2 pM concentration. Cells were washed and a final concentration of 40 ug/mL EGF (EGF-AF647, Life Tech) was added to each well. Cells were then incubated at 4°C for 1 hour, washed and stained with live/dead dye (eFluor 520, Fisher) and incubated at 4°C for a further 20 minutes. Cells were washed in FACS buffer and resuspended in cell fixative (BD sciences) and then incubated overnight at 4°C in the dark. EGF blockade was measured by flow cytometry the next day using a MACS Quant Analyzer 10.

Conclusion : The decrease in MFI shown in Figures 39A and 39B indicates increased blocking of EGF binding to EGFRA-positive A431 cells. For the multispecific antibodies described, there is a direct correlation between EGFR target affinity and the level of EGF blockade observed. This represents another example of the graded effects conferred by molecules with affinities for EGFR ranging from about 1 nM to about 140 nM.

(C) (D) This subsequent analysis focuses on the inhibition of tumor cell proliferation conferred by the multispecific antibodies of the invention.

Method : Briefly, EGFR+ A431 cells were seeded into a 384-well imaging dish (Perkin Elmer) to obtain a final seeding density of 2×10 ³ target cells/well, and then incubated overnight at 37°C and 5% CO2. Antibodies were diluted in basal growth medium and serially diluted 1:5 before being added to assay plates to give final assay concentrations of 500 nM to 0.05 pM. Cells were cultured for 24 hours and subsequently stained with Hoechst (1:1000 final, Invitrogen) and DRAQ7 (1:300 final, Abcam). To determine the number of viable target cells, confocal images were acquired using the Opera Phenix high-content platform, which captures nine fields of view at 10x magnification. Quantitative viable cell counts based on size, morphology, texture, and intensity of viable cell staining and lack of DRAQ7 staining.

Conclusion : The data presented in Figures 39C and 39D demonstrate that multispecific antibodies of the invention with higher affinity for EGFR are most effective in inhibiting the proliferation of EGFR-positive tumor cells. Furthermore, this demonstrates a clear positive correlation between EGFR target affinity and the observed level of tumor proliferation inhibition. Thus, this is another example of highlighting the effects of fractionation related to affinity for the EGFR target (ranging from about 1 nM to about 140 nM). Example 18 : Avoiding Healthy EGFR+ Cells - VD1×EGFR Multispecific Antibody

Methods : High-content confocal imaging in Opera Phenix (Perkin Elmer) was used to determine the cytotoxic effect of EGFR-Vδ1 bispecific antibodies on EGFR+ target cells. Briefly, EGFR+ A375 cells (melanoma) and HUVEC (human umbilical vein endothelial cells) were seeded into a 384-well imaging dish (Perkin Elmer) to obtain a final seeding density of 5000 target cells/well, and then incubated at 37°C. Incubate overnight under 5% CO2. Antibodies were diluted in basal growth medium and serially diluted 1:5 before being added to assay plates to give final assay concentrations of 1 nM to 0.0005 pM. Expanded skin-derived Vδ1 γδ T cells were isolated from tissue culture bottles and resuspended in basal growth medium and subsequently added to assay plates at 5000 cells/well at a 1:1 effect:target ratio. Cells were co-cultured for 24 hours and subsequently stained with Hoechst 33342 (1:1000 final, Invitrogen) and DRAQ7 (1:300 final, Abcam). To determine the number of viable target cells, confocal images were acquired using the Opera Phenix high-content platform, which captures nine fields of view at 10x magnification. Quantitative viable cell counts based on size, morphology, texture, and intensity of viable cell staining and lack of DRAQ7 staining. The results are shown in Figure 40 AC.

The effects of dose titration are presented in Figure 40A. This data helps highlight the inherent ability of Vδ1 T cells to differentiate between healthy and malignant cells. The multispecific antibodies of the invention then direct Vδ1 T cells to kill EGFR+ tumor cells while "avoiding" healthy cells that exhibit comparable EGFR copy numbers. The tabulated results of (A) are presented in Figure 40C.

Representative microscopy images from the experiment described in (A) are presented in Figure 40B, demonstrating T cell-induced cytotoxicity on A375 tumor cells while "avoiding" healthy HUVEC cells. Images are shown in grayscale with nuclei stained with Hoechst 33342. Example 19 : VD1×EGFR multispecific antibody : Dose-response comparison between ADT1-4-2×FS1-67 and the lower affinity “parent” ADT1-4×FS1-67 ( alias G04 FS1-67)

The inventors have previously demonstrated that the "parental" (non-affinity matured) G04 FS1-67 multispecific antibody confers a variety of desired technical effects in co-cultures containing Vδ1+ cells and target A431 cells (WO2021032963).

Specifically, the inventors observed that this multispecific antibody enhanced cytotoxicity against A431 cells by more than 200%. In this series of experiments, G04 FS1-67 (SEQ ID NO: 379), which contains the lower affinity "parent" first Vδ1 binding domain (approximately 126 nM) and high affinity (approximately 1 nM) EGFR binding, was compared with an equivalent but Compared to multispecific antibody ADT1-4-2 (affinity approximately 0.2 nM) containing the first Vδ1 binding domain of an affinity mature variant (SEQ ID NO: 414)

Methods : In this assay, Vδ1+ effector cells were co-cultured with EGFR+ cancer cells, in which induction of Vδ1 TCR internalization following multispecific antibody engagement was measured. Co-cultures were established in which 2.5×10 ⁴ Vδ1 γδ T cells were added to 2.5×10 ⁴ EGFR+ target cells/well in a U-bottom 96-well plate. Vδ1 γδ T cells were stained with [0.5 µM] CellTrace purple live cell dye for 20 minutes before addition. Final assay antibody concentrations ranged from 10 nM to 0.13 pM. After incubation for 4 hours at 37°C, 5% CO2, cells were harvested and stained for surface expression of dead cells (eFlour 780, Invitrogen) and Vδ1 TCR (Miltenyi Biotec) for 20 minutes at 4°C. Cells were washed in FACS buffer and resuspended in cell fixative (BD sciences) before analysis on a MACSQuant Analyzer 10 flow cytometer (Miltenyi).

Conclusion : The results of this comparison now presented in Figure 41A (and in tabular form in Figure 41B) are consistent with the findings described elsewhere herein. showed reduced MFI (and therefore TCR downregulation) and this comparative study further confirms that (i) the non-native higher Vδ1 affinity of the invention is more specific than the lower affinity variant (in this case G04 FS1-67) specific antibodies confer enhanced effects; and (ii) these enhanced effects are particularly evident in studies utilizing lower copy number TAA target cancer cells such as A375. This data also highlights again that for a molecule with lower vd1 affinity (in this case the G04 FS1-67 comparator), even when 100-fold more of this molecule is added, this is not achieved in the Vδ1/A375 co-culture Equivalent TCR down-regulation effect. Example 20 : VD1×EGFR multispecific antibody : comparison with CD3×EGFR multispecific antibody

Establish comparative multi-specific antibodies, among which (i) The affinity for EGFR remains constant at approximately 0.2 nM and (ii) The variable domain targeting Vδ1 was replaced with a variable domain specific for CD3ε (derived from OKT3).

This molecule (termed CD3/ADT3-2) was then compared to the Vδ1×EGFR multispecific ADT1-4-2/ADT3-2 (alias ADT1-4-2×LEE) previously described elsewhere herein. One motivation for this comparison was to compare the difference in T cell activation conferred by multispecific TCEs (T cell engagement molecules) targeting the Vδ1 TCR versus multispecific TCEs engaging via the CD3ε coreceptor. In this context, the inventors have discovered that multispecific TCE engaging T cells via the Vδ1 TCR confer a differential effect compared to multispecific TCE engaging T cells via CD3ε. These effects are presented in the next set of experiments. Also included in these studies was a multispecific D1.3×FS1-67 control (comprising a 1 nM affinity EGFR second binding domain and a first binding domain comprising a binding domain to chicken lysozyme).

(A) (B) Differentiation from CD3×EGFR multispecific antibody ; Effect of T cell target receptors on V δ 1 TCR and CD3 ε downregulation. This subsequent experiment determined how targeting different T cell receptors (Vδ1 or CD3ε) after multispecific antibody engagement affects the induction of Vδ1 TCR internalization.

Methods : A series of T cell populations were analyzed as indicated in the legend shown in Figure 42A. These include PBMC and purified Vδ1 γδ T cells isolated from skin tissue or directly from peripheral blood. Co-cultures were established as above, where 2×10 ⁴ T cells were added to 2×10 ⁴ A375 EGFR+ A375 tumor cells/well in a U-bottom 96-well plate. Antibody was then added to a final concentration of 1 nM and the culture was incubated at 37°C, 5% CO2 4. Cells were harvested and stained for dead cells (eFlour 780, Invitrogen), Vδ1 TCR (Miltenyi), CD3ε (Miltenyi) surface expression for 20 min at 4°C. Cells were washed in FACS buffer and resuspended in cell fixative (BD sciences) before analysis on a MACSQuant Analyzer 10 flow cytometer (Miltenyi).

Conclusion : The reduction in MFI is presented in Figure 42A. Notably, antibodies engaging the TCR complex via Vδ1 were observed to downregulate the TCR more efficiently than via CD3ε, as demonstrated using Vδ1 T cell populations purified from skin and peripheral blood. In contrast, T cell engagement via CD3ε induced more potent CD3 downregulation in PBMCs containing only a minority Vδ1 cell population (0.08% in this donation). The significant role of CD3-based TCE in PBMC highlights the sledgehammer approach of this known strategy. Specifically, CD3-based TCE will affect all CD3+ cells present in PBMC, including Tregs, CD4+, and CD8+ T cells. In contrast, multispecific antibodies of the invention that engage T cells via a first Vδ1 binding domain are selective only for the highly required Vδ1+ CD3 subset of cells that are typically more prevalent in tissues than in blood.

(B) Effect of T cell target receptors on V δ 1 TCR downregulation. The tabulated results of (A) are presented in Figure 42B, which highlight the superior induction of TCR downregulation by direct targeting of the Vδ1 TCR by a multispecific antibody compared to targeting this complex via the CD3 epsilon co-receptor.

(C) Effect of T cell target receptors on CD3 epsilon downregulation. The tabulated results of (A) are presented in Figure 42C, which highlight the more pronounced impact of CD3-based TCE on CD3 levels; especially in unfractionated PBMC.

(D) (E) Effect of multispecific antibodies on the cytolytic function of EGFR+ cancer cells. This subsequent experiment determined how engaging different T cell receptors (Vδ1 or CD3ε) affects the stimulation of T cell populations to lyse tumor target cells.

Methods : As before, a range of T cell populations were evaluated, including PBMC or purified γδ T cells isolated from skin tissue or directly from peripheral blood. Cultures were established by seeding 4×10 ³ A375 EGFR+ A375 tumor cells/well in 384-well CellCarrier Ultra plates (PerkinElmer). Different numbers of T cells were added to vary the effect-to-target ratio between 10 and 0.08. Antibody was then added to a final concentration of 1 nM and the culture was incubated at 37°C, 5% CO2 for 24 hours. Cells were stained with Hoechst 33342 (Invitrogen) and for dead cells (DRAQ7, Abcam) for 1 hour at 37°C and imaged on an Opera Phenix system (PerkinElmer). The number of viable tumor cells was determined using a linear regression model (Harmony 4.9) based on the size, morphology, texture, and intensity of viable cell staining and the absence of DRAQ7 staining.

Conclusions : The data presented in Figure 42D demonstrate the unique ability of multispecific antibodies targeting Vδ1 to specifically and exclusively redirect Vδ1 T cell populations to kill EGFR+ tumor cells. This contrasts with CD3-based TCEs, which are more promiscuous in function, as demonstrated by the observations here: these TCEs also redirect unfractionated PBMC to EGFR+ cells. The tabulated results of (D) are presented in Figure 42E. This further highlights the superior specificity of Vδ1-based TCE relative to CD3-based TCE.

(F) Effect on interleukin secretion induced by activated T cells. This subsequent experiment determined how targeting different T cell receptors (Vδ1 or CD3ε) or target populations affects the interleukin secretion profile of activated T cells.

Methods : A range of T cell populations were analyzed, including PBMC or purified γδ T cells derived directly from peripheral blood of two healthy donors. Co-cultures were established as above, where 2×10 ⁴ T cells were added to 2×10 ⁴ A375 EGFR+ A375 tumor cells/well in a U-bottom 96-well plate. Antibody was then added at a final concentration of 1 nM and the culture was incubated at 37°C, 5% CO2 for 48 hours. The supernatant was harvested, cells removed, and interleukin concentration determined using a LEGENDplex interleukin bead array kit (BioLegend, CA, USA).

Conclusion : The data presented in Figures 42F and 42G demonstrate significant quantitative and qualitative differences between specific activation of Vδ1+ T cells and broader activation of all CD3+ T cells within unfractionated PBMC. One notable difference was the 248-fold increase in IL-17A levels induced by CD3-based TCE in PBMCs alone. In contrast, γδ T cells stimulated with CD3-based or Vδ1-based TCE did not produce IL17A. IL17A is an interleukin that enhances tumor growth and weakens anti-cancer immune responses. This highlights the unique interleukin secretion profile of the Vδ1+ T cell subset. Additionally, CD3-based TCE induced significantly higher levels of multiple interleukins, most notably TNFa, across all T cell populations. Again, this highlights that CD3-based TCEs confer less tailored, greater hammer-like effects involving more global induction of CD3+ cells.

(H) Effect on the proliferation of V δ 1+ cells present in tissues . This subsequent analysis focused on the induction of Vδ1+ cell proliferation conferred by the multispecific antibodies of the invention.

Methods : Co-cultures were established as above, where 3×10 ⁴ Vδ1 γδ T cells were added to 1.5×10 ⁴ EGFR+ target cells/well in a U-bottom 96-well plate. Stain target cells with [0.5 µM] CellTrace Purple Live Cell Dye for 20 minutes before addition. Final analytical multispecific antibody concentrations ranged from 2 nM to 0.25 pM. Cells were incubated at 37°C, 5% CO2 for 5 or 7 days. Complete medium supplemented with interleukins (IL-2, IL-15, IL-4, and IL-21; essential units) was added on days 3 and 5. Cells were washed and stained for dead cells (eFlour 780, Invitrogen) for 20 min at 4°C. Cells were washed in FACS buffer and resuspended in cell fixative (BD sciences). The number of events was measured by flow cytometry the next day using a MACS Quant Analyzer 10.

Conclusion : This study demonstrates that compared to equivalent CD3-based TCEs, Vδ1-based TCEs are significantly superior in inducing the proliferation of Vδ1 T cells, in which the Vδ1 bispecific protein induces proliferation at a 10-fold lower dose and also at saturating concentrations. Show a more obvious effect. See Figures 42H and I, which highlight that antibody-mediated stimulation of Vδ1 T cells confers increased proliferation compared to stimulation via CD3ε. Example

The present invention includes at least the following numbered embodiments: 1. A multispecific antibody comprising a Fab region and an Fc region, wherein the Fab region contains an epitope for the variable δ1 (Vδ1) chain of the γδ T cell receptor (TCR) Has a specific binding site; and the Fc region contains an EGFR binding site, wherein the EGFR binding site is provided by the following CH3 domain, wherein a. Residues 359 to 362 (EU numbering) comprise EEGP (SEQ ID NO: 523 ), residues 384 to 386 (EU numbering) comprise TYG (SEQ ID NO: 511), and residues 413 to 415 (EU numbering) comprise SYW (SEQ ID NO: 533); b. Residues 359 to 362 (EU Numbering) contains EEGP (SEQ ID NO: 523), and residues 413 to 419 (EU numbering) contain SYWRWYK (SEQ ID NO: 512); c. Residues 358 to 361, 361.1 and 362 (EU numbering) contain LDEGGP ( SEQ ID NO: 542), residues 384 to 386 (EU numbering) comprise TYG (SEQ ID NO: 511), and residues 413 to 419 (EU numbering) comprise SYWRWVK (SEQ ID NO: 543); d. Residues e. Residues 358 to 362 (EU numbering) contain TESGP (SEQ ID NO: 553), residues 384 to 386 (EU numbering) comprise KFG (SEQ ID NO: 554), residues 413 to 421 (EU numbering) comprise SNLRWTKGH (SEQ ID NO: 555), and residues 378 is valine. 2. The multispecific antibody of any preceding embodiment, wherein: a. Residues 355 to 362 (EU numbering) form an AB loop comprising RDELEEGP (SEQ ID NO: 524), and residues 383 to 391 (EU numbering) form an AB loop comprising RDELEEGP The CD loop of STYGPENNY (SEQ ID NO: 514) and residues 413 to 422 (EU numbering) form an EF loop containing SYWRWQQGNV (SEQ ID NO: 534); b. Residues 355 to 362 (EU numbering) form an EF loop containing RDELEEGP AB loop (SEQ ID NO: 524), residues 383 to 391 (EU numbering) form the CD loop containing SNGQPENNY (SEQ ID NO: 525) and residues 413 to 422 (EU numbering) form the EF loop containing SYWRWYKGNV (SEQ ID NO: 515); c. Residues 355 to 362 (EU numbering) form an AB loop containing RDELDEGGP (SEQ ID NO: 544), and residues 383 to 391 (EU numbering) form a CD loop containing STYGPENNY (SEQ ID NO : 514), and residues 413 to 422 (EU numbering) form an EF loop containing SYWRWVKGNV (SEQ ID NO: 545); d. Residues 355 to 362 (EU numbering) form an AB loop containing RDELTKNQ (SEQ ID NO: or e. Residues 355 to 362 (EU numbering) form an AB loop containing RDETESGP (SEQ ID NO: 556), residues 383 to 391 (EU numbering) form a CD loop containing SKFGPENNY (SEQ ID NO: 557), residues 413 to 422 (EU numbering) form the EF loop containing SNLRWTKGHV (SEQ ID NO: 558). 3. The antibody according to embodiment 2, wherein the Fc region is a human IgG Fc region comprising the CH3 domain engineered to comprise the EGFR binding site. 4. The antibody according to any preceding embodiment, wherein the amino acid sequence of the CH3 domain is at least 90% identical, or at least 95% identical, or 100% identical to: a. SEQ ID NO: 532; b. SEQ ID NO : 522; c. SEQ ID NO: 541; d. SEQ ID NO: 510; or e. SEQ ID NO: 552. 5. The multispecific antibody of any preceding embodiment, wherein the antibody has Fc function. 6. The multispecific antibody of any preceding embodiment, wherein the heavy chain constant domain comprises: a. SEQ ID NO: 530 or SEQ ID NO: 531; b. SEQ ID NO: 520 or SEQ ID NO: 521; c. SEQ ID NO: 539 or SEQ ID NO: 540; d. SEQ ID NO: 508 or SEQ ID NO: 509; or e. SEQ ID NO: 550 or SEQ ID NO: 551. 7. The multispecific antibody of any preceding embodiment, comprising: a. SEQ ID NO: 526; or SEQ ID NO: 414 and SEQ ID NO: 528; b. SEQ ID NO: 527; or SEQ ID NO: 414 and SEQ ID NO: 529; c. SEQ ID NO: 516; or SEQ ID NO: 414 and SEQ ID NO: 518; d. SEQ ID NO: 517; or SEQ ID NO: 414 and SEQ ID NO: 519; e. . SEQ ID NO: 535; or SEQ ID NO: 414 and SEQ ID NO: 537; f. SEQ ID NO: 536; or SEQ ID NO: 414 and SEQ ID NO: 538; g. SEQ ID NO: 504; or SEQ ID NO: 414 and SEQ ID NO: 506; h. SEQ ID NO: 505; or SEQ ID NO: 414 and SEQ ID NO: 507; i. SEQ ID NO: 546; or SEQ ID NO: 414 and SEQ ID NO: 548; or j. SEQ ID NO: 547; or SEQ ID NO: 414 and SEQ ID NO: 549. 8. The multispecific antibody of any preceding embodiment, wherein the Fc region binds to human EGFR with a binding affinity ( _KD , eg, as measured by surface plasmon resonance) of less than about 150 nM. 9. The multispecific antibody of any preceding embodiment, wherein the Fc region binds to human EGFR with a binding affinity ( _KD , eg, as measured by surface plasmon resonance) of less than about 20 nM. 10. The multispecific antibody of any preceding embodiment, wherein the Fc region binds to human EGFR with a binding affinity ( _KD , eg, as measured by surface plasmon resonance) of less than about 10 nM. 11. The multispecific antibody of any preceding embodiment, wherein the Fc region binds to human EGFR with a binding affinity ( _KD , eg, as measured by surface plasmon resonance) of less than about 5 nM. 12. The multispecific antibody of any preceding embodiment, wherein the Fc region binds to EGFR with a binding affinity ( _KD , eg, as measured by surface plasmon resonance) of less than about 3 nM. 13. The multispecific antibody of any preceding embodiment, wherein the Fab region comprises: a heavy chain variable region comprising at least 70% similarity to a sequence selected from the group consisting of SEQ ID NOs: 55 to 78 and 133 to 143 , at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least An amino acid sequence with 99% or 100% identity or a VHCDR3 consisting of the amino acid sequence; and/or a light chain variable region comprising and selected from SEQ ID NOs: 82 to 105 and 147 to 157 The group of sequences has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least An amino acid sequence that is 97%, at least 98%, at least 99% or 100% identical or a VLCDR3 consisting of the amino acid sequence. 14. The multispecific antibody of embodiment 13, wherein the Fab region comprises: a heavy chain variable region comprising: VHCDR1 comprising at least 70 Å with a sequence selected from the group consisting of SEQ ID NO: 51, 52 and 130 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, An amino acid sequence or a composition of an amino acid sequence that is at least 99% or 100% identical; VHCDR2, which includes at least 70%, at least 75%, or at least a sequence selected from the group consisting of SEQ ID NO: 53 and 131 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% consistent or consisting of a specific amino acid sequence; and VHCDR3, which includes at least 70%, at least 75%, and at least 80% similarity with a sequence selected from the group consisting of SEQ ID NO: 55 to 78 and 133 to 143 %, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% consistency The amino acid sequence of or consisting of the amino acid sequence; and/or the light chain variable region, which includes: VLCDR1, which includes at least 70% of the sequence selected from the group consisting of SEQ ID NO: 79 and 144, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % or 100% identical amino acid sequence or consisting of the amino acid sequence; VLCDR2, which includes at least 70%, at least 75%, at least 80% identity with a sequence selected from the group consisting of SEQ ID NO: 80 and 145 %, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% consistency The amino acid sequence of or consisting of the amino acid sequence; and VLCDR3, which includes at least 70%, at least 75%, at least 80% with a sequence selected from the group consisting of SEQ ID NO: 82 to 105 and 147 to 157 , at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% consistency an amino acid sequence or consisting of an amino acid sequence. 15. The multispecific antibody of embodiment 13 or 14, wherein the Fab region comprises: a heavy chain variable region comprising at least 70% similarity to a sequence selected from the group consisting of SEQ ID NOs: 2 to 25 and 107 to 117 , at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least A sequence that is 99% or 100% identical or consists of the sequence; and/or a light chain variable region comprising at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % or 100% identical sequence or consists of this sequence. 16. The multispecific antibody of one of embodiments 1 to 7, wherein the Fab region comprises: a. VHCDR3 comprising at least 90% sequence identity to any one of SEQ ID NO: 55 to 78 an amino acid sequence or consisting of an amino acid sequence; and/or b. VLCDR3 comprising or consisting of an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 82 to 105 Amino acid sequence composition. 17. The multispecific antibody of any preceding embodiment, wherein the Fab region comprises: a. VHCDR3, which comprises or consists of the amino acid sequence of any one of SEQ ID NO: 55 to 78; and/or b. VLCDR3, which comprises or consists of the amino acid sequence of any one of SEQ ID NO: 82 to 105. 18. The multispecific antibody of any preceding embodiment, wherein the Fab region comprises: a. VHCDR1 comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 51 or SEQ ID NO: 52 or consisting of The amino acid sequence consists of; b. VHCDR2, which contains or consists of an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 53; c. VLCDR1, which contains the same amino acid sequence as SEQ ID NO: 53 : 79 has an amino acid sequence with at least 90% sequence identity or consists of the amino acid sequence; and/or d. VLCDR2, which contains an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 80 Or consisting of this amino acid sequence. 19. The multispecific antibody of any preceding embodiment, wherein the Fab region includes: a. VHCDR1, which includes or consists of the amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 52; b. . VHCDR2, which includes or consists of the amino acid sequence of SEQ ID NO: 53; c. VLCDR1, which includes or consists of the amino acid sequence of SEQ ID NO: 79; and / or d. VLCDR2, which contains or consists of the amino acid sequence of SEQ ID NO: 80. 20. The multispecific antibody of any preceding embodiment, wherein the Fab region comprises: a. VHCDR1, VHCDR2 and VHCDR3 respectively comprising or consisting of the amino acid sequences of SEQ ID NO: 51, 53 and 55. , and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 82 or consisting of the amino acid sequences; b. comprising the amino acids of SEQ ID NO: 51, 53 and 56 respectively The sequence or VHCDR1, VHCDR2 and VHCDR3 consisting of the amino acid sequence, and the amino acid sequence respectively comprising SEQ ID NO: 79, 80 and 83 or VLCDR1, VLCDR2 and VLCDR3 consisting of the amino acid sequence; c. VHCDR1, VHCDR2 and VHCDR3 respectively comprising or consisting of the amino acid sequences of SEQ ID NO: 51, 53 and 57, and VHCDR1, VHCDR2 and VHCDR3 respectively comprising or consisting of the amino acid sequences of SEQ ID NO: 79, 80 and 84. VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; d. respectively comprising the amino acid sequence of SEQ ID NO: 51, 53 and 58 or VHCDR1, VHCDR2 and VHCDR3 consisting of the amino acid sequence, and respectively comprising SEQ The amino acid sequences of ID NO: 79, 80 and 85 or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; e. The amino acid sequences of SEQ ID NO: 51, 53 and 59 respectively or composed of the amine VHCDR1, VHCDR2 and VHCDR3 consisting of amino acid sequences, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 86 or consisting of the amino acid sequences; f. respectively comprising SEQ ID NO. : VHCDR1, VHCDR2 and VHCDR3 consisting of the amino acid sequences of 51, 53 and 60 or consisting of the amino acid sequences, and the amino acid sequences of SEQ ID NO: 79, 80 and 87 respectively or consisting of the amino acid sequences VLCDR1, VLCDR2 and VLCDR3 composed of; g. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 52, 53 and 61 or consisting of the amino acid sequences, and respectively comprising SEQ ID NO: 79, VLCDR1, VLCDR2 and VLCDR3 consisting of the amino acid sequences of SEQ ID NO: 51, 53 and 62 or consisting of the amino acid sequences of SEQ ID NO: 51, 53 and 62, respectively. VHCDR1, VHCDR2 and VHCDR3, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising or consisting of the amino acid sequences of SEQ ID NOs: 79, 80 and 89; i. respectively comprising SEQ ID NOs: 51, 53 and The amino acid sequence of 63 or VHCDR1, VHCDR2 and VHCDR3 composed of the amino acid sequence, and the amino acid sequences of SEQ ID NO: 79, 80 and 90 respectively or VLCDR1, VLCDR2 composed of the amino acid sequence and VLCDR3; j. VHCDR1, VHCDR2 and VHCDR3 respectively comprising or consisting of the amino acid sequences of SEQ ID NO: 51, 53 and 64, and amines comprising SEQ ID NO: 79, 80 and 91 respectively. The amino acid sequence or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; k. The amino acid sequence of SEQ ID NO: 51, 53 and 65 respectively or the VHCDR1, VHCDR2 and VHCDR3 composed of the amino acid sequence , and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 92 or consisting of the amino acid sequences; l. comprising the amino acids of SEQ ID NO: 52, 53 and 66 respectively The sequence or VHCDR1, VHCDR2 and VHCDR3 consisting of the amino acid sequence, and the amino acid sequence respectively comprising SEQ ID NO: 79, 80 and 93 or VLCDR1, VLCDR2 and VLCDR3 consisting of the amino acid sequence; m. VHCDR1, VHCDR2 and VHCDR3 respectively comprising or consisting of the amino acid sequences of SEQ ID NO: 51, 53 and 67, and VHCDR1, VHCDR2 and VHCDR3 respectively comprising or consisting of the amino acid sequences of SEQ ID NO: 79, 80 and 94. VLCDR1, VLCDR2 and VLCDR3 consisting of the amino acid sequence; n. respectively comprising the amino acid sequence of SEQ ID NO: 51, 53 and 68 or VHCDR1, VHCDR2 and VHCDR3 consisting of the amino acid sequence, and respectively comprising SEQ VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequences of ID NO: 79, 80 and 95 or composed of the amino acid sequence; o. Comprising the amino acid sequence of SEQ ID NO: 51, 53 and 69 respectively or composed of the amine VHCDR1, VHCDR2 and VHCDR3 consisting of amino acid sequences, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising or consisting of the amino acid sequences of SEQ ID NO: 79, 80 and 96; p. respectively comprising SEQ ID NO. : VHCDR1, VHCDR2 and VHCDR3 consisting of the amino acid sequences of 51, 53 and 70 or consisting of the amino acid sequences, and the amino acid sequences of SEQ ID NO: 79, 80 and 97 respectively or consisting of the amino acid sequences VLCDR1, VLCDR2 and VLCDR3 composed of; q. VHCDR1, VHCDR2 and VHCDR3 composed of the amino acid sequences of SEQ ID NO: 51, 53 and 71 respectively or composed of the amino acid sequences, and including SEQ ID NO: 79, VLCDR1, VLCDR2 and VLCDR3 containing the amino acid sequences of SEQ ID NO: 51, 53 and 72 or consisting of the amino acid sequences; r. VHCDR1, VHCDR2 and VHCDR3, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising or consisting of the amino acid sequences of SEQ ID NOs: 79, 80 and 99; s. comprising SEQ ID NOs: 51, 53 and The amino acid sequence of 73 or VHCDR1, VHCDR2 and VHCDR3 composed of the amino acid sequence, and the amino acid sequences of SEQ ID NO: 79, 80 and 100 respectively or VLCDR1, VLCDR2 composed of the amino acid sequence and VLCDR3; t. VHCDR1, VHCDR2 and VHCDR3 respectively comprising or consisting of the amino acid sequences of SEQ ID NO: 52, 53 and 74, and amines comprising SEQ ID NO: 79, 80 and 101 respectively. The amino acid sequence or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; u. The amino acid sequence of SEQ ID NO: 51, 53 and 75 respectively or the VHCDR1, VHCDR2 and VHCDR3 composed of the amino acid sequence , and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 102 or consisting of the amino acid sequences; v. comprising the amino acids of SEQ ID NO: 51, 53 and 76 respectively Sequence or VHCDR1, VHCDR2 and VHCDR3 consisting of the amino acid sequence, and amino acid sequences respectively comprising SEQ ID NO: 79, 80 and 103 or VLCDR1, VLCDR2 and VLCDR3 consisting of the amino acid sequence; w. VHCDR1, VHCDR2 and VHCDR3 respectively comprising or consisting of the amino acid sequences of SEQ ID NO: 51, 53 and 77, and VHCDR1, VHCDR2 and VHCDR3 respectively comprising or consisting of the amino acid sequences of SEQ ID NO: 79, 80 and 104. VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; or The amino acid sequences of SEQ ID NO: 79, 80 and 105 or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequences. 21. The multispecific antibody of any preceding embodiment, wherein the Fab region comprises a. a VH comprising an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2 to 25; and/ or b. A VL comprising or consisting of an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 27 to 50. 22. The multispecific antibody of any of the preceding embodiments, wherein the Fab region comprises: a. comprising the amino acid sequence of SEQ ID NO: 2 to 25 or a VH consisting of the amino acid sequence; and/or b. comprising The amino acid sequence of SEQ ID NO: 27 to 50 or the VL composed of the amino acid sequence. 23. The multispecific antibody of any preceding embodiment, wherein the Fab region comprises: a. A VH comprising or consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2 and Contains an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 27 or a VL consisting of the amino acid sequence; b. Contains an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 3 Sequence or VH consisting of the amino acid sequence and VL comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 28 or consisting of the amino acid sequence; c. Comprising SEQ ID NO: 4. An amino acid sequence having at least 90% sequence identity or a VH consisting of the amino acid sequence and comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 29 or consisting of the amino acid sequence A VL consisting of; d. A VH consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5 or consisting of the amino acid sequence and having at least 90% sequence identity with SEQ ID NO: 30 An amino acid sequence or a VL consisting of the amino acid sequence; e. An amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 6 or a VH consisting of the amino acid sequence and a VH consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 6 SEQ ID NO: 31 has an amino acid sequence with at least 90% sequence identity or a VL consisting of the amino acid sequence; f. contains an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 7 or VH consisting of the amino acid sequence and comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 32 or VL consisting of the amino acid sequence; g. Comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8 An amino acid sequence with at least 90% sequence identity or a VH consisting of an amino acid sequence and a VH containing an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 33 or consisting of an amino acid sequence VL; h. A VH comprising or consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9 and an amine having at least 90% sequence identity with SEQ ID NO: 34 An amino acid sequence or a VL consisting of the amino acid sequence; i. An amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10 or a VH consisting of the amino acid sequence and a VH consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10 NO: 35 has an amino acid sequence with at least 90% sequence identity or a VL consisting of the amino acid sequence; j. contains an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 11 or consists of the amino acid sequence VH consisting of an amino acid sequence and VL comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 36 or consisting of the amino acid sequence; k. comprising at least 90% sequence identity with SEQ ID NO: 12 % sequence identity of the amino acid sequence or a VH consisting of the amino acid sequence and an amino acid sequence containing at least 90% sequence identity with SEQ ID NO: 37 or a VL consisting of the amino acid sequence; l. A VH comprising or consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 13 and an amino acid having at least 90% sequence identity with SEQ ID NO: 38 Sequence or VL consisting of the amino acid sequence; m. Comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 14 or VH consisting of the amino acid sequence and comprising SEQ ID NO: 39 An amino acid sequence with at least 90% sequence identity or a VL consisting of the amino acid sequence; n. Containing an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 15 or consisting of the amino acid sequence VH consisting of an acid sequence and VL comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 40 or consisting of the amino acid sequence; o. Comprising a sequence having at least 90% sequence identity with SEQ ID NO: 16 An identical amino acid sequence or a VH consisting of the amino acid sequence and a VL comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 41 or consisting of the amino acid sequence; p. A VH comprising or consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 17 and comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 42 or A VL consisting of the amino acid sequence; q. A VL consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18 or a VH consisting of the amino acid sequence and comprising an amino acid sequence identical to SEQ ID NO: 43 An amino acid sequence with at least 90% sequence identity or a VL consisting of the amino acid sequence; r. Containing an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 19 or consisting of the amino acid sequence VH consisting of and VL comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 44 or consisting of the amino acid sequence; s. comprising at least 90% sequence identity with SEQ ID NO: 20 An amino acid sequence or a VH consisting of the amino acid sequence and a VL comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 45 or a VL consisting of the amino acid sequence; t. SEQ ID NO: 21 has an amino acid sequence with at least 90% sequence identity or a VH consisting of the amino acid sequence and contains an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 46 or consists of the amino acid sequence VL consisting of an amino acid sequence; u. Comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 22 or a VH consisting of this amino acid sequence and comprising at least 90% sequence identity with SEQ ID NO: 47 % sequence identity of an amino acid sequence or a VL consisting of such an amino acid sequence; v. Containing an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 23 or consisting of an amino acid sequence VH and VL comprising or consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48; w. containing an amine having at least 90% sequence identity with SEQ ID NO: 24 The amino acid sequence or a VH consisting of the amino acid sequence and the amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49 or the VL consisting of the amino acid sequence; or x. ID NO: 25 has an amino acid sequence with at least 90% sequence identity or a VH consisting of the amino acid sequence and contains an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 50 or consists of the amine VL composed of amino acid sequences. 24. The multispecific antibody of any preceding embodiment, wherein the Fab region comprises: a. a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 2 and an amine comprising SEQ ID NO: 27 The amino acid sequence or VL consisting of the amino acid sequence; b. The amino acid sequence comprising SEQ ID NO: 3 or the VH consisting of the amino acid sequence and the amino acid sequence comprising SEQ ID NO: 28 or VL consisting of the amino acid sequence; c. VH comprising the amino acid sequence of SEQ ID NO: 4 or consisting of the amino acid sequence and VH comprising the amino acid sequence of SEQ ID NO: 29 or consisting of the amino acid sequence VL consisting of the amino acid sequence; d. VH comprising the amino acid sequence of SEQ ID NO: 5 or consisting of the amino acid sequence and VH comprising the amino acid sequence of SEQ ID NO: 30 or consisting of the amino acid sequence VL; e. VH comprising the amino acid sequence of SEQ ID NO: 6 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 31 or consisting of the amino acid sequence; f. A VH comprising the amino acid sequence of SEQ ID NO: 7 or consisting of the amino acid sequence and a VL comprising the amino acid sequence of SEQ ID NO: 32 or consisting of the amino acid sequence; g. Comprising SEQ ID NO : The amino acid sequence of 8 or VH consisting of the amino acid sequence and the amino acid sequence comprising SEQ ID NO: 33 or the VL consisting of the amino acid sequence; h. The amine comprising SEQ ID NO: 9 The amino acid sequence or the VH consisting of the amino acid sequence and the amino acid sequence comprising SEQ ID NO: 34 or the VL consisting of the amino acid sequence; i. The amino acid sequence comprising SEQ ID NO: 10 or VH consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 35 or consisting of the amino acid sequence; j. Comprising the amino acid sequence of SEQ ID NO: 11 or consisting of the amino acid sequence VH consisting of the acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 36 or consisting of the amino acid sequence; k. Comprising the amino acid sequence of SEQ ID NO: 12 or consisting of the amino acid sequence VH and VL comprising the amino acid sequence of SEQ ID NO: 37 or consisting of the amino acid sequence; l. VH comprising the amino acid sequence of SEQ ID NO: 13 or consisting of the amino acid sequence and VL comprising SEQ The amino acid sequence of ID NO: 38 or the VL consisting of the amino acid sequence; m. The amino acid sequence of SEQ ID NO: 14 or the VH consisting of the amino acid sequence and the VH consisting of SEQ ID NO: 39 The amino acid sequence of SEQ ID NO: 15 or the VL composed of the amino acid sequence; n. The amino acid sequence of SEQ ID NO: 15 or the VH composed of the amino acid sequence and the amino acid of SEQ ID NO: 40 Sequence or VL consisting of the amino acid sequence; o. VH comprising the amino acid sequence of SEQ ID NO: 16 or consisting of the amino acid sequence and VH comprising the amino acid sequence of SEQ ID NO: 41 or consisting of the amino acid sequence VL consisting of an amino acid sequence; p. VH comprising or consisting of the amino acid sequence of SEQ ID NO: 17 and VH comprising or consisting of the amino acid sequence of SEQ ID NO: 42 VL consisting of; q. VH comprising the amino acid sequence of SEQ ID NO: 18 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 43 or consisting of the amino acid sequence; r. Contains the amino acid sequence of SEQ ID NO: 19 or a VH composed of the amino acid sequence and includes the amino acid sequence of SEQ ID NO: 44 or a VL composed of the amino acid sequence; s. Contains SEQ The amino acid sequence of ID NO: 20 or VH consisting of the amino acid sequence and the amino acid sequence comprising SEQ ID NO: 45 or the VL consisting of the amino acid sequence; t. Comprising SEQ ID NO: 21 The amino acid sequence or the VH consisting of the amino acid sequence and the amino acid sequence comprising SEQ ID NO: 46 or the VL consisting of the amino acid sequence; u. The amino acid comprising SEQ ID NO: 22 The sequence or VH consisting of the amino acid sequence and the amino acid sequence comprising SEQ ID NO: 47 or the VL consisting of the amino acid sequence; v. The amino acid sequence comprising SEQ ID NO: 23 or the VL consisting of the amino acid sequence VH consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 48 or consisting of the amino acid sequence; w. Comprising the amino acid sequence of SEQ ID NO: 24 or consisting of the amino acid sequence VH consisting of and VL comprising the amino acid sequence of SEQ ID NO: 49 or consisting of the amino acid sequence; or x. VH comprising the amino acid sequence of SEQ ID NO: 25 or consisting of the amino acid sequence And a VL comprising the amino acid sequence of SEQ ID NO: 50 or consisting of the amino acid sequence. 25. The multispecific antibody of any preceding embodiment, wherein the Fab region comprises an amino acid residue other than serine at position 74 according to the IMGT numbering system. 26. The multispecific antibody of embodiment 25, wherein the residue at position 74 according to the IMGT numbering system is selected from the group consisting of glycine, alanine, valine, methionine, leucine and isoleucine. Amino acids are a group of amino acids. 27. The multispecific antibody of embodiment 25, wherein the residue at position 74 according to the IMGT numbering system is selected from the group consisting of arginine, cysteine, glutamic acid, glutamic acid, glycine, A group of amino acids composed of histamine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine. 28. The multispecific antibody of embodiment 25, wherein the residue at position 74 according to the IMGT numbering system is selected from the group consisting of glycine, valine, methionine, leucine and isoleucine. group of amino acids. 29. The multispecific antibody of embodiment 25, wherein the residue at position 74 according to the IMGT numbering system is a leucine residue. 30. The multispecific antibody of any preceding embodiment, wherein the Fab region is selected from the group consisting of ADT1-4-105, ADT1-4-107, ADT1-4-110, ADT1-4-112, ADT1-4-117, ADT1 -4-19, ADT1-4-21, ADT1-4-31, ADT1-4-139, ADT1-4-4, ADT1-4-143, ADT1-4-53, ADT1-4-173, ADT1-4 -2. ADT1-4-8, ADT1-4-82, ADT1-4-83, ADT1-4-3, ADT1-4-84, ADT1-4-86, ADT1-4-95, ADT1-4-1 , ADT1-4-6 and ADT1-4-138. 31. The multispecific antibody of embodiment 30, wherein the Fab region comprises 1 to 10, 1 to 5, or 1 to 2 amino acid substitutions on all 6 CDR regions. 32. The multispecific antibody of embodiment 30 or 31, wherein the Fab region contains at most 2 or at most 1 amino acid substitutions on all 6 CDR regions. 33. The multispecific antibody of any one of embodiments 30 to 32, wherein the Fab region contains 1 or 2 amino acid substitutions on all 6 CDR regions. 34. The multispecific antibody of any one of embodiments 30 to 33, wherein the Fab region comprises 1 to 10, 1 to 5, or 1 to 2 amino acid substitutions in one or more framework regions. 35. The multispecific antibody of any one of embodiments 30 to 34, wherein the Fab region contains up to 2 or up to 1 amino acid substitution in one or more framework regions. 36. The multispecific antibody of any one of embodiments 34 to 35, wherein the Fab region contains 1 or 2 amino acid substitutions in one or more framework regions. 37. The multispecific antibody of any one of embodiments 30 to 37, wherein the amino acid substitutions are conservative amino acid substitutions. 38. The multispecific antibody of any preceding embodiment, wherein the Fab region comprises: a. a HCDR1 sequence comprising or consisting of the sequence GDSVSSKSX ₁ A; b. comprising or consisting of the sequence of SEQ ID NO: 53 The HCDR2 sequence c. contains the sequence of X _{2 WX 3} X _{4 X 5} sequence or an LCDR1 sequence consisting of the sequence; e. a sequence comprising SEQ ID NO: 80 or an LCDR2 sequence consisting of the sequence; and f. a sequence comprising QQX ₈ YX ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ T or consisting of The LCDR3 sequence composed of this sequence, wherein the LCDR3 sequence is not SEQ ID NO: 81; wherein X ₁ to X ₁₃ are each a naturally occurring amino acid. 39. The multispecific antibody of embodiment 38, wherein: a. _X1 is selected from the group consisting of A and V; b. _X2 is selected from the group consisting of S and T; c. _X3 is selected from the group consisting of V, X ₄ is selected from the group consisting of G, E and D; e. X ₅ is selected from the group consisting of Y and N; f. X ₆ is selected from the group consisting of V, A and P g. X ₇ is selected from the group consisting of V, Y and R; h. X ₈ is selected from the group consisting of K, R and G; i. X ₉ is selected from the group consisting of S and K; j . X ₁₀ is selected from the group consisting of T, Q, A, E and D; k. X ₁₁ is selected from the group consisting of P, H and D; l. X ₁₂ is selected from the group consisting of Q, R, K, W, The group consisting of P, E and I; and m. X ₁₃ is selected from the group consisting of I, V and L. 40. The multispecific antibody of embodiment 38 or embodiment 39, wherein the Fab region further comprises: n. a sequence comprising SEQ ID NO: 170 or 171 or an HFR1 sequence consisting of the sequence; o. comprising SEQ ID NO: The sequence of SEQ ID NO: 172 or the HFR2 sequence consisting of the sequence; p. The sequence of SEQ ID NO: 173 or the HFR3 sequence consisting of the sequence; q. The sequence of SEQ ID NO: 174 or the HFR4 sequence consisting of the sequence; r. The sequence containing SEQ ID NO: 175 or the LFR1 sequence consisting of this sequence; s. The sequence containing SEQ ID NO: 176 or the LFR2 sequence consisting of this sequence; t. The sequence containing SEQ ID NO: 177 or 178 or an LFR3 sequence consisting of the sequence; and u. a LFR4 sequence comprising or consisting of the sequence of SEQ ID NO: 179, 180, 181 or 182. 41. The multispecific antibody of any of the preceding embodiments, wherein the Fab region: a. With a binding affinity ( _KD ) of less than about 100 nM (preferably less than about 50 nM), for example, as by surface plasmon resonance (measured) binding to human TRDV1 (SEQ ID NO 272 or 306); b. Optionally with a binding affinity (K _D ) of less than about 100 nM (preferably less than about 50 nM), for example, as measured by surface plasmon resonance ) binds to cynomolgus monkey TRDV1 (SEQ ID NO 308); and/or c. downregulates TCR with an IC50 of less than about 50 nM (preferably less than about 10 nM). 42. The multispecific antibody of any preceding embodiment, wherein the Fab region: a. binds to human TRDV1 with a KD of less than about 100 nM (preferably less than about 50 nM); b. binds to human TRDV1 with a KD of less than about 100 nM (preferably less than about 50 nM); Binds to cynomolgus monkey TRDV1 with a KD of less than about 50 nM); and/or c. The IC50 for down-regulating TCR is less than about 50 nM (preferably less than about 10 nM) and the IC90 for down-regulating TCR is less than about 100 nM (less than about 50 nM). 43. The multispecific antibody of any of the preceding embodiments, wherein: a. the Fab region binds to the Fab region with a binding affinity ( _KD ) of less than about 100 nM (preferably less than about 50 nM), for example, as determined by surface plasmon resonance (tested) binds to human TRDV1 (SEQ ID NO 272 or 306); b. Optionally, the Fab region binds to human TRDV1 (SEQ ID NO 272 or 306) with a binding affinity (K _D ) of less than about 100 nM (preferably less than about 50 nM), for example, by surface plasmon (sub-resonance measurement) binds to cynomolgus monkey TRDV1 (SEQ ID NO 308); and/or c. its Fab region binds to cynomolgus monkey TRDV1 (SEQ ID NO 308) with a binding affinity (K _D ) of less than about 150 nM (preferably less than about 50 nM), for example, as (measured by surface plasmon resonance) binds to human EGFR. 44. The multispecific antibody of any preceding embodiment, wherein the multispecific antibody specifically binds to human TRDV1 with a K of less than about ₅ nM and with a K of less than about 150 nM, less than about 20 nM, less than about 10 nM, or Binds specifically to human EGFR with a _KD of less than about 5 nM. 45. The multispecific antibody of any preceding embodiment, wherein the multispecific antibody specifically binds to human TRDV1 with a _K of less than about 5 nM and specifically binds to human EGFR with a _K of less than about 5 nM. 46. The multispecific antibody of embodiment 38, wherein: a. _X1 is selected from the group consisting of A and V; b. _X2 is selected from the group consisting of S and T; c. _X3 is selected from the group consisting of V, X ₄ is selected from the group consisting of G, E and D; e. X ₅ is selected from the group consisting of Y and N; f. X ₆ is selected from the group consisting of V, A and P g. X ₇ is selected from the group consisting of V, Y and R; h. X ₈ is selected from the group consisting of K, R and G; i. X ₉ is selected from the group consisting of S and K; j . X ₁₀ is selected from the group consisting of T, Q, A, E and D; k. X ₁₁ is selected from the group consisting of P and H; l. X ₁₂ is selected from the group consisting of Q, R, K, W, P, The group consisting of E and I; and m. X ₁₃ is selected from the group consisting of I, V and L. 47. The multispecific antibody of embodiment 46, wherein the Fab region further comprises: a. a sequence comprising SEQ ID NO: 170 or 171 or an HFR1 sequence consisting of the sequence; b. a sequence comprising SEQ ID NO: 172 or The HFR2 sequence consisting of the sequence; c. The sequence of SEQ ID NO: 173 or the HFR3 sequence consisting of the sequence; d. The sequence of SEQ ID NO: 174 or the HFR4 sequence consisting of the sequence; e. The sequence of SEQ ID NO: 174 or the HFR4 sequence consisting of the sequence; e. The sequence of ID NO: 175 or the LFR1 sequence consisting of the sequence; f. The sequence of SEQ ID NO: 176 or the LFR2 sequence consisting of the sequence; g. The sequence of SEQ ID NO: 177 or the LFR1 sequence consisting of the sequence LFR3 sequence; and h. An LFR4 sequence comprising or consisting of the sequence of SEQ ID NO: 179, 180, 181 or 182. 48. The multispecific antibody of any preceding embodiment, wherein the Fab region: a. binds to human TRDV1 with a KD of less than about 10 nM (preferably less than about 5 nM); b. binds to human TRDV1 with a _KD of less than about 100 nM (more preferably less than about 5 nM). The K _D of preferably less than about 50 nM) binds to cynomolgus monkey TRDV1; c. The IC50 for down-regulating TCR is less than about 1 nM (preferably less than about 0.5 nM). 49. The multispecific antibody of embodiment 38, wherein: a. _X1 is selected from the group consisting of A and V; b. _X2 is selected from the group consisting of S and T; c. _X3 is selected from the group consisting of V, d. X ₄ is selected from the group consisting of G and D; e. X ₅ is selected from the group consisting of Y and N; f. X ₆ is selected from the group consisting of V, A and P ; g. X ₇ is selected from the group consisting of V, Y and R; h. X ₈ is selected from the group consisting of K and R; i. X ₉ is selected from the group consisting of S and K; j. X ₁₀ is selected from the group is selected from the group consisting of T, Q, A and E; k. X ₁₁ is selected from the group consisting of P and H; l. X ₁₂ is selected from the group consisting of Q, K, W, P and I; and m. X ₁₃ is selected from the group consisting of V and L. 50. The multispecific antibody of embodiment 47, wherein the Fab region further comprises: a. a sequence comprising SEQ ID NO: 170 or 171 or an HFR1 sequence consisting of the sequence; b. a sequence comprising SEQ ID NO: 172 or The HFR2 sequence consisting of the sequence; c. The sequence of SEQ ID NO: 173 or the HFR3 sequence consisting of the sequence; d. The sequence of SEQ ID NO: 174 or the HFR4 sequence consisting of the sequence; e. The sequence of SEQ ID NO: 174 or the HFR4 sequence consisting of the sequence; e. The sequence of ID NO: 175 or the LFR1 sequence consisting of the sequence; f. The sequence of SEQ ID NO: 176 or the LFR2 sequence consisting of the sequence; g. The sequence of SEQ ID NO: 177 or the LFR1 sequence consisting of the sequence LFR3 sequence; and h. An LFR4 sequence comprising or consisting of the sequence of SEQ ID NO: 179 or 181. 51. The multispecific antibody of any preceding embodiment, wherein the Fab region: a. binds to human TRDV1 with a K of less than about 10 nM (preferably less than about 1 nM); b. binds to human _TRDV1 with a K of less than about 50 nM _D binds to cynomolgus monkey TRDV1; c. The IC50 for down-regulating TCR is less than about 1 nM (preferably less than about 0.5 nM). 52. The multispecific antibody of any preceding embodiment, wherein the epitope bound by the Fab region comprises one or more amino groups within amino acid regions 37-53 and/or 59-77 of SEQ ID NO: 272 acid residue. 53. The multispecific antibody of any preceding embodiment, wherein the epitope bound by the Fab region comprises amino acid residues 37, 42, 50, 53, 59, 64, 68, 69, of SEQ ID NO: 272. 72, 73 and 77. 54. The multispecific antibody of any preceding embodiment, wherein the epitope bound by the Fab region consists of amino acid residues 37, 42, 50, 53, 59, 64, 68, 69, of SEQ ID NO: 272. Composed of 72, 73 and 77. 55. The multispecific antibody of one of embodiments 1 to 7, wherein the Fab region comprises: a. VHCDR3 comprising at least 90% sequence identity to any one of SEQ ID NOs: 133 to 143 an amino acid sequence or consisting of an amino acid sequence; and/or b. VLCDR3 comprising or consisting of an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 147 to 157 Amino acid sequence composition. 56. The multispecific antibody of embodiment 55, the Fab region includes: c. VHCDR3, which includes or consists of the amino acid sequence of any one of SEQ ID NO: 133 to 143; and/ or d. VLCDR3, which comprises or consists of the amino acid sequence of any one of SEQ ID NO: 147 to 157. 57. The multispecific antibody of any one of embodiments 55 to 56, the Fab region further comprising: a. VHCDR1 comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 130 or consisting of Composed of an amino acid sequence; b. VHCDR2, which contains or consists of an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 131; c. VLCDR1, which contains an amino acid sequence consistent with SEQ ID NO: 131; 144 has or consists of an amino acid sequence with at least 90% sequence identity; and/or d. VLCDR2, which contains an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 145, or Composed of this amino acid sequence. 58. The multispecific antibody of any preceding embodiment, the Fab region further comprises: a. VHCDR1, which comprises or consists of the amino acid sequence of SEQ ID NO: 130; b. VHCDR2, which comprises SEQ The amino acid sequence of ID NO: 131 or consisting of the amino acid sequence; c. VLCDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 144; and/or d. VLCDR2, It contains or consists of the amino acid sequence of SEQ ID NO: 145. 59. The multispecific antibody of any one of embodiments 55 to 58, the Fab region comprising: a. VHCDR1 comprising or consisting of the amino acid sequences of SEQ ID NO: 130, 131 and 133 respectively. , VHCDR2 and VHCDR3, and VLCDR1, VLCDR2 and VLCDR3 respectively containing the amino acid sequence of SEQ ID NO: 144, 145 and 147 or consisting of the amino acid sequence; b. containing SEQ ID NO: 130, 131 and 134 respectively The amino acid sequence of or VHCDR1, VHCDR2 and VHCDR3 composed of the amino acid sequence, and the amino acid sequence of SEQ ID NO: 144, 145 and 148 respectively or VLCDR1, VLCDR2 and the amino acid sequence composed of the amino acid sequence VLCDR3; c. VHCDR1, VHCDR2 and VHCDR3 respectively containing or consisting of the amino acid sequences of SEQ ID NO: 130, 131 and 135, and containing the amino groups of SEQ ID NO: 144, 145 and 149 respectively. The acid sequence or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequence; d. The amino acid sequence of SEQ ID NO: 130, 131 and 136 respectively or VHCDR1, VHCDR2 and VHCDR3 composed of the amino acid sequence, And VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 144, 145 and 150 or consisting of the amino acid sequences; e. respectively comprising the amino acid sequences of SEQ ID NO: 130, 131 and 137 Or VHCDR1, VHCDR2 and VHCDR3 consisting of the amino acid sequence, and respectively comprising the amino acid sequence of SEQ ID NO: 144, 145 and 151 or VLCDR1, VLCDR2 and VLCDR3 consisting of the amino acid sequence; f. respectively VHCDR1, VHCDR2 and VHCDR3 comprising or consisting of the amino acid sequences of SEQ ID NO: 130, 131 and 138, and VHCDR1, VHCDR2 and VHCDR3 respectively comprising or consisting of the amino acid sequences of SEQ ID NO: 144, 145 and 152. VLCDR1, VLCDR2 and VLCDR3 composed of amino acid sequences; g. VHCDR1, VHCDR2 and VHCDR3 composed of the amino acid sequences of SEQ ID NO: 130, 131 and 139 respectively or composed of the amino acid sequences, and respectively containing SEQ ID The amino acid sequences of NO: 144, 145 and 153 or VLCDR1, VLCDR2 and VLCDR3 composed of the amino acid sequences; h. The amino acid sequences of SEQ ID NO: 130, 131 and 140 respectively or composed of the amino acid sequences VHCDR1, VHCDR2 and VHCDR3 consisting of acid sequences, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 144, 145 and 154 or consisting of the amino acid sequences; i. respectively comprising SEQ ID NO: VHCDR1, VHCDR2 and VHCDR3 which contain or consist of the amino acid sequences of 130, 131 and 141, and the amino acid sequences of SEQ ID NO: 144, 145 and 155 respectively or which consist of the amino acid sequences. VLCDR1, VLCDR2 and VLCDR3; j. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequence of SEQ ID NO: 130, 131 and 142 or consisting of the amino acid sequence, and respectively comprising SEQ ID NO: 144 and 145 and 156 amino acid sequences or VLCDR1, VLCDR2 and VLCDR3 consisting of the amino acid sequences; or k. respectively comprising the amino acid sequences of SEQ ID NO: 130, 131 and 143 or consisting of the amino acid sequences VHCDR1, VHCDR2 and VHCDR3, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising or consisting of the amino acid sequences of SEQ ID NO: 144, 145 and 157. 60. The multispecific antibody of any one of embodiments 55 to 59, the Fab region comprising a. comprising an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 107 to 117, or VH consisting of the amino acid sequence; and/or b. comprising or consisting of an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 119 to 129 VL. 61. The multispecific antibody of any one of embodiments 55 to 60, the Fab region comprising: a. a VH comprising the amino acid sequence of SEQ ID NO: 107 to 117 or consisting of the amino acid sequence; and/ or b. VL comprising or consisting of the amino acid sequence of SEQ ID NO: 119 to 129. 62. The multispecific antibody of any one of embodiments 55 to 61, the Fab region comprising: a. comprising or consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 107 VH consisting of and VL comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 119 or consisting of the amino acid sequence; b. Containing at least 90% sequence identity with SEQ ID NO: 108 An amino acid sequence or a VH consisting of the amino acid sequence and a VL comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 120 or a VL consisting of the amino acid sequence; c. SEQ ID NO: 109 has an amino acid sequence with at least 90% sequence identity or a VH consisting of the amino acid sequence and contains an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 121 or consists of the amino acid sequence A VL consisting of an amino acid sequence; d. A VL consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 110 or a VH consisting of the amino acid sequence and comprising at least 90% sequence identity with SEQ ID NO: 122 % sequence identity of an amino acid sequence or a VL consisting of such an amino acid sequence; e. Containing or consisting of an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 111 VH and VL comprising or consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 123; f. containing an amine having at least 90% sequence identity with SEQ ID NO: 112 The amino acid sequence or the VH consisting of the amino acid sequence and the amino acid sequence having at least 90% sequence identity with SEQ ID NO: 124 or the VL consisting of the amino acid sequence; g. Comprising the amino acid sequence with SEQ ID NO: 124 NO: 113 has an amino acid sequence with at least 90% sequence identity or a VH consisting of the amino acid sequence and contains an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 125 or consists of the amino acid sequence A VL consisting of an acid sequence; h. A VL consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 114 or a VH consisting of this amino acid sequence and a VH having at least 90% sequence identity with SEQ ID NO: 126 An identical amino acid sequence or a VL composed of such an amino acid sequence; i. An amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 115 or a VH composed of such an amino acid sequence and Contains an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 127 or a VL consisting of the amino acid sequence; j. Contains an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 116 sequence or a VH consisting of the amino acid sequence and a VL comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 128 or consisting of the amino acid sequence; or k. comprising a sequence identical to SEQ ID NO: 128 : 117 has an amino acid sequence with at least 90% sequence identity or a VH consisting of the amino acid sequence and contains an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 129 or consists of the amino acid sequence VL composed of sequences. 63. The multispecific antibody of any one of embodiments 55 to 62, the Fab region comprising a. a VH comprising the amino acid sequence of SEQ ID NO: 107 or consisting of the amino acid sequence and comprising SEQ ID NO: The amino acid sequence of SEQ ID NO: 119 or a VL composed of the amino acid sequence; b. The amino acid sequence of SEQ ID NO: 108 or the VH composed of the amino acid sequence and the amino group of SEQ ID NO: 120 Acid sequence or VL consisting of the amino acid sequence; c. VH comprising the amino acid sequence of SEQ ID NO: 109 or consisting of the amino acid sequence and VH comprising the amino acid sequence of SEQ ID NO: 121 or consisting of VL consisting of the amino acid sequence; d. VH comprising the amino acid sequence of SEQ ID NO: 110 or consisting of the amino acid sequence and VH comprising the amino acid sequence of SEQ ID NO: 122 or consisting of the amino acid VL consisting of the sequence; e. VH comprising the amino acid sequence of SEQ ID NO: 111 or consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 123 or consisting of the amino acid sequence ; f. Contains the amino acid sequence of SEQ ID NO: 112 or a VH composed of the amino acid sequence and includes the amino acid sequence of SEQ ID NO: 124 or a VL composed of the amino acid sequence; g. Contains The amino acid sequence of SEQ ID NO: 113 or a VH composed of the amino acid sequence and the amino acid sequence of SEQ ID NO: 125 or a VL composed of the amino acid sequence; h. Containing SEQ ID NO: The amino acid sequence of 114 or VH consisting of the amino acid sequence and the amino acid sequence comprising SEQ ID NO: 126 or the VL consisting of the amino acid sequence; i. Comprising the amino group of SEQ ID NO: 115 The acid sequence or VH consisting of the amino acid sequence and the amino acid sequence comprising SEQ ID NO: 127 or the VL consisting of the amino acid sequence; j. The amino acid sequence comprising SEQ ID NO: 116 or consisting of VH consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 128 or consisting of the amino acid sequence; or k. comprising the amino acid sequence of SEQ ID NO: 117 or consisting of the amino acid sequence VH consisting of an acid sequence and VL comprising or consisting of the amino acid sequence of SEQ ID NO: 129. 64. The multispecific antibody of any one of embodiments 55 to 63, wherein the Fab region comprises a kappa light chain variable sequence having an amino acid residue other than serine at position 74 according to the IMGT numbering system . 65. The multispecific antibody of embodiment 64, wherein the residue at position 74 of the light chain variable sequence according to the IMGT numbering system is selected from the group consisting of glycine, alanine, valine, methionine A group of amino acids composed of , leucine and isoleucine. 66. The multispecific antibody of embodiment 65, wherein the residue at position 74 of the light chain variable sequence according to the IMGT numbering system is selected from the group consisting of arginine, cysteine, glutamine, gluten A group of amino acids, glycine, histamine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine of amino acids. 67. The multispecific antibody of embodiment 65, wherein the residue at position 74 of the light chain variable sequence according to the IMGT numbering system is selected from the group consisting of glycine, valine, methionine, and leucine A group of amino acids composed of acid and isoleucine. 68. The multispecific antibody of embodiment 65, wherein the residue at position 74 of the light chain variable sequence according to the IMGT numbering system is a leucine residue. 69. The multispecific antibody of any one of embodiments 55 to 68, wherein the Fab region is selected from the group consisting of ADT1-7-10, ADT1-7-15, ADT1-7-17, ADT1-7-18, ADT1- 7-19, ADT1-7-20, ADT1-7-22 ADT1-7-23, ADT1-7-42, ADT1-7-3 and ADT1-7-61. 70. The multispecific antibody of embodiment 69, wherein the Fab region comprises 1 to 10, 1 to 5, or 1 to 2 amino acid substitutions on all 6 CDR regions. 71. The multispecific antibody of embodiment 69 or 70, wherein the Fab region contains at most 2 or at most 1 amino acid substitutions on all 6 CDR regions. 72. The multispecific antibody of any one of embodiments 69 to 71, wherein the Fab region contains 1 or 2 amino acid substitutions on all 6 CDR regions. 73. The multispecific antibody of any one of embodiments 69 to 72, wherein the Fab region comprises 1 to 10, 1 to 5, or 1 to 2 amino acid substitutions in one or more framework regions. 74. The multispecific antibody of any one of embodiments 69 to 73, wherein the Fab region contains up to 2 or up to 1 amino acid substitution in one or more framework regions. 75. The multispecific antibody of any one of embodiments 69 to 74, wherein the Fab region contains 1 or 2 amino acid substitutions in one or more framework regions. 76. The multispecific antibody of any one of embodiments 69 to 75, wherein the amino acid substitution is a conservative amino acid substitution. 77. The multispecific antibody of one of embodiments 1 to 12, wherein the Fab region comprises: a. an HCDR1 sequence comprising or consisting of the sequence of SEQ ID NO: 130 b. comprising SEQ ID NO: 131 The sequence or the HCDR2 _sequence consisting of _the sequence c. The sequence comprising _{X 1} : The sequence of SEQ ID NO: 144 or the LCDR1 sequence composed of the sequence; e. The sequence of SEQ ID NO: 145 or the LCDR2 sequence composed of the sequence; and f. The sequence of QQX ₅ X ₆ X ₇ X ₈ LX ₉ T or An LCDR3 sequence consisting of this sequence, wherein the LCDR3 sequence is not SEQ ID NO: 146; wherein X ₁ to X ₉ are each a naturally occurring amino acid. 78. The multispecific antibody of embodiment 77, wherein: a. _X1 is selected from the group consisting of D, I and V; b. _X2 is selected from _the group consisting of D and S; c. X 4 is selected from the group consisting of N, E, D, Q and A; _d . X ₄ is selected from the group consisting of D and E; e. X ₅ is selected from the group consisting of T and S; f. g. X ₇ is selected from the group consisting of S and D; h. X ₈ is selected from the group consisting of T, E and G; and i. X ₉ is selected from the group consisting of L and D form a group. 79. The multispecific antibody of embodiment 77 or embodiment 78, wherein the Fab region further comprises: a. a sequence comprising SEQ ID NO: 189 or an HFR1 sequence consisting of the sequence; b. comprising SEQ ID NO: 190 The sequence or the HFR2 sequence consisting of the sequence; c. The sequence comprising SEQ ID NO: 191 or the HFR3 sequence consisting of the sequence; d. The sequence comprising SEQ ID NO: 192 or the HFR4 sequence consisting of the sequence; e. The sequence of SEQ ID NO: 193 and 194 or the LFR1 sequence consisting of the sequence; f. The sequence of SEQ ID NO: 195 or the LFR2 sequence consisting of the sequence; g. The sequence of SEQ ID NO: 196 or the LFR2 sequence consisting of the sequence The LFR3 sequence consisting of the sequence; and h. The LFR4 sequence comprising or consisting of the sequence of SEQ ID NO: 197. 80. The multispecific antibody of any one of embodiments 55 to 79, wherein the antibody or antigen-binding fragment thereof: a. binds to human TRDV1 with a _KD of less than about 10 nM (preferably less than about 5 nM); b . The IC50 for down-regulating TCR is less than about 50 nM (preferably less than about 10 nM). 81. The multispecific antibody of embodiment 80, wherein: a. _X1 is selected from the group consisting of D and V; b. _X2 is selected from the group consisting of D and S; c. _X3 is selected from the group consisting of D, Q and A; d. X ₄ is selected from the group consisting of D _and E; e. X ₅ is S; f. X ₆ is selected from the group consisting of A and Y; g. . X ₈ is selected from the group consisting of E and G; and i. X ₉ is selected from the group consisting of L and D. 82. The multispecific antibody of embodiment 81, wherein the Fab region further comprises: a. a HFR1 sequence comprising or consisting of the sequence of SEQ ID NO: 189; b. a sequence comprising or consisting of SEQ ID NO: 190 The HFR2 sequence consisting of the sequence; c. The sequence of SEQ ID NO: 191 or the HFR3 sequence consisting of the sequence; d. The sequence of SEQ ID NO: 192 or the HFR4 sequence consisting of the sequence; e. The sequence of SEQ ID NO: 192 or the HFR4 sequence consisting of the sequence; e. : The sequence of SEQ ID NO: 193 or the LFR1 sequence consisting of the sequence; f. The sequence of SEQ ID NO: 195 or the LFR2 sequence consisting of the sequence; g. The sequence of SEQ ID NO: 196 or the LFR3 sequence consisting of the sequence ; and h. An LFR4 sequence comprising or consisting of the sequence of SEQ ID NO: 197. 83. The multispecific antibody of any one of embodiments 55 to 82, wherein the Fab region: a. binds to human TRDV1 with a _KD of less than about 10 nM (preferably less than about 5 nM); and/or b. The IC50 for down-regulating TCR is less than about 5 nM (preferably less than about 1 nM). 84. The multispecific antibody of any one of embodiments 55 to 83, wherein the epitope bound by the Fab region comprises one of amino acid regions 5-20 and/or 62-77 of SEQ ID NO: 271 or Multiple amino acid residues. 85. The multispecific antibody of any one of embodiments 55 to 83, wherein the epitope bound by the Fab region comprises amino acid residues 5, 9, 16, 20, 62, 64, of SEQ ID NO: 272. 72 and 77. 86. The multispecific antibody of any one of embodiments 55 to 85, wherein the epitope bound by the Fab region consists of amino acid residues 5, 9, 16, 20, 62, 64, of SEQ ID NO: 272. Composed of 72 and 77. 87. The multispecific antibody of any one of embodiments 1 to 12, wherein the Fab region is derived from an affinity matured variant of a parent anti-Vδ1 antibody, wherein the parent anti-Vδ1 antibody includes an amine comprising SEQ ID NO: 1 The VH of the amino acid sequence and the VL sequence of the amino acid sequence of SEQ ID NO: 26. 88. The multispecific antibody of embodiment 87, wherein the Fab region is as defined in any preceding embodiment. 89. The multispecific antibody of any preceding embodiment, wherein the Fab region: a. binds to human TRDV1 with a KD of less than about 100 nM (preferably less than about 50 nM); b. binds to human TRDV1 with a KD of less than about 100 nM (preferably less than about 50 nM) Binds to cynomolgus monkey TRDV1 with a KD less than about 50 nM); and/or c. Downregulates TCR with an IC50 less than about 50 nM (preferably less than about 10 nM). 90. The multispecific antibody of any preceding embodiment, wherein the Fab region: a. binds to human TRDV1 with a KD of less than about 10 nM (preferably less than about 5 nM); b. binds to human TRDV1 with a _KD of less than about 100 nM (preferably less than about 5 nM) Binds to cynomolgus monkey TRDV1 with a _KD of about 50 nM); and/or c. Downregulates TCR with an IC50 of less than about 1 nM (preferably less than about 0.5 nM). 91. The multispecific antibody of any preceding embodiment, wherein the Fab region: a. binds to human TRDV1 with a _K of less than about 10 nM (preferably less than about 1 nM); b. binds with a _K of less than about 50 nM In cynomolgus monkey TRDV1; and/or c. The IC50 for down-regulating TCR is less than about 1 nM (preferably less than about 0.5 nM). 92. The multispecific antibody of any one of embodiments 1 to 12, wherein the Fab region is derived from an affinity matured variant of a parent anti-Vδ1 antibody, wherein the parent anti-Vδ1 antibody or antigen-binding fragment thereof comprises SEQ ID The VH of the amino acid sequence of NO: 106 and the VL sequence of the amino acid sequence of SEQ ID NO: 118. 93. The multispecific antibody of embodiment 92, wherein the Fab region is as defined in any preceding embodiment. 94. The multispecific antibody of any preceding embodiment, wherein the antibody or antigen-binding fragment thereof: a. binds to human TRDV1 with a _KD of less than about 10 nM (preferably less than about 5 nM); and/or b. causes The IC50 for TCR downregulation is less than about 50 nM (preferably less than about 10 nM). 95. The multispecific antibody of any preceding embodiment, wherein the Fab region: a. binds to human TRDV1 with a _KD of less than about 10 nM (preferably less than about 5 nM); and/or b. has an IC50 that downregulates TCR Less than about 5 nM (preferably less than about 1 nM). 96. The multispecific antibody of any one of embodiments 1 to 12, wherein the Fab region is an affinity matured variant of a parent anti-Vδ1 antibody, wherein the parent anti-Vδ1 antibody or antigen-binding fragment thereof comprises: a. comprising The amino acid sequence of SEQ ID NO: 273 or a VH composed of the amino acid sequence and the amino acid sequence of SEQ ID NO: 282 or a VL composed of the amino acid sequence; b. Containing SEQ ID NO: The amino acid sequence of 274 or VH consisting of the amino acid sequence and the amino acid sequence comprising SEQ ID NO: 283 or the VL consisting of the amino acid sequence; c. The amino group comprising SEQ ID NO: 275 acid sequence or VH consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 284 or consisting of the amino acid sequence; d. comprising the amino acid sequence of SEQ ID NO: 276 or consisting of VH consisting of the amino acid sequence and VL comprising the amino acid sequence of SEQ ID NO: 285 or consisting of the amino acid sequence; e. comprising the amino acid sequence of SEQ ID NO: 277 or consisting of the amino acid VH consisting of the sequence and VL comprising the amino acid sequence of SEQ ID NO: 286 or consisting of the amino acid sequence; f. VH comprising the amino acid sequence of SEQ ID NO: 278 or consisting of the amino acid sequence and a VL comprising the amino acid sequence of SEQ ID NO: 287 or consisting of the amino acid sequence; g. comprising the amino acid sequence of SEQ ID NO: 279 or a VH consisting of the amino acid sequence and comprising SEQ ID The amino acid sequence of NO: 288 or the VL consisting of the amino acid sequence; h. The amino acid sequence of SEQ ID NO: 280 or the VH consisting of the amino acid sequence and the VL comprising SEQ ID NO: 289 Amino acid sequence or VL consisting of the amino acid sequence; i. Comprising the amino acid sequence of SEQ ID NO: 281 or VH consisting of the amino acid sequence and comprising the amino acid sequence of SEQ ID NO: 290 or VL consisting of the amino acid sequence; or j. VH comprising the amino acid sequence of SEQ ID NO: 312 or consisting of the amino acid sequence and comprising the amino acid sequence of SEQ ID NO: 313 or consisting of the amino acid sequence VL composed of amino acid sequences. 97. The multispecific antibody of any preceding embodiment, wherein the affinity mature variant binds to the variable δ1 (Vδ1) chain of the γδ T cell receptor (TCR) with an affinity greater than the affinity, e.g., as measured by Kd. The antibody is at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 500% or preferably at least about 1000% larger. 98. The multispecific antibody of any preceding embodiment, wherein the affinity matured variant comprises VH and VL sequences that are at least 80%, at least 90%, at least 95%, or at least 96% identical to the corresponding parent VH and VL sequences. 99. The multispecific antibody of any preceding embodiment, wherein the affinity mature variant comprises up to 20, such as up to 15, such as up to 10, amino acid substitutions compared to the parent antibody sequence. 100. The multispecific antibody of any one of embodiments 1 to 12, the Fab region comprising a kappa light chain variable sequence, wherein the residue at position 74 according to the IMGT numbering system is not a serine residue. 101. The multispecific antibody of embodiment 100, wherein the residue at position 74 according to the IMGT numbering system is selected from the group consisting of glycine, alanine, valine, methionine, leucine and isoleucine. Amino acids are a group of amino acids. 102. The multispecific antibody of embodiment 100, wherein the residue at position 74 according to the IMGT numbering system is selected from the group consisting of arginine, cysteine, glutamic acid, glutamic acid, glycine, A group of amino acids composed of histamine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine. 103. The multispecific antibody of embodiment 100, wherein the residue at position 74 according to the IMGT numbering system is selected from the group consisting of glycine, valine, methionine, leucine and isoleucine group of amino acids. 104. The multispecific antibody of embodiment 100, wherein the residue at position 74 according to the IMGT numbering system is a leucine residue. 105. The multispecific antibody of any preceding embodiment, wherein the antibody is an IgG antibody. 106. The multispecific antibody of any preceding embodiment, wherein the antibody is an IgG1 antibody. 107. The multispecific antibody of any preceding embodiment, wherein the Fab region includes a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 40; and the Fc region A CH3 domain comprising the amino acid sequence of SEQ ID NO: 532 is included. 108. The multispecific antibody of any preceding embodiment, wherein the Fab region includes a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 40; and the Fc region A CH3 domain comprising the amino acid sequence of SEQ ID NO: 522 is included. 109. The multispecific antibody of any preceding embodiment, wherein the Fab region includes a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 40; and the Fc region A CH3 domain comprising the amino acid sequence of SEQ ID NO: 541 is included. 110. The multispecific antibody of any preceding embodiment, wherein the Fab region includes a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 40; and the Fc region A CH3 domain comprising the amino acid sequence of SEQ ID NO: 510 is included. 111. The multispecific antibody of any preceding embodiment, wherein the Fab region includes a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 40; and the Fc region A CH3 domain comprising the amino acid sequence of SEQ ID NO: 552 is included. 112. The multispecific antibody of any preceding embodiment, wherein the Fab region specifically binds to the variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR) and competes with the antibody or antigen-binding fragment thereof of any preceding embodiment The variable delta 1 (Vδ1) chain binds to the γδ T cell receptor (TCR). 113. The multispecific antibody of any preceding embodiment, wherein the antibody has Fc function. 114. The multispecific antibody of any of the preceding embodiments, characterized in that it: a. causes TCR downregulation on Vδ1 T cells to which it binds; b. does not exhibit CDC or ADCC; and c. does not deplete Vδ1 T cells. 115. The multispecific antibody of embodiment 114, wherein the antibody causes depletion of less than about 30%, or less than about 20%, or less than about 10% of the viable Vδ1 T+ cell population via ADCC and/or CDC. 116. The multispecific antibody of embodiment 114, wherein the antibody does not induce IL-17A secretion. 117. The multispecific antibody of embodiment 114, wherein the IL-17A secretion induced by the antibody is less than about 30%, or less than about 20%, or less than about 10% of the IL-17A secretion induced by an equivalent CD3 antibody. 118. The multispecific antibody of any preceding embodiment, wherein the Fab region binds to human TRVD1 with a KD of less than about 10 nM. 119. The multispecific antibody of any preceding embodiment, wherein the Fab region binds to human TRVD1 with a KD of less than about 5 nM. 120. The multispecific antibody of any preceding embodiment, wherein the Fab region binds to cynomolgus monkey TRVD1 with a KD of less than about 100 nM. 121. The multispecific antibody of embodiment 120, wherein the Fab region binds to cynomolgus monkey TRVD1 with a KD of less than about 50 nM. 122. The multispecific antibody according to any preceding embodiment, wherein the Fab region specifically binds to the human variable delta 1 (Vδl) chain of the gamma delta T cell receptor (TCR) with a KD of less than about 10 nM. 123. The multispecific antibody of any preceding embodiment, wherein the Fab region is as defined in any one of embodiments 1 to 126. 124. The multispecific antibody of any preceding embodiment, wherein the Fab region binds to the cynomolgus monkey variable δ1 (Vδ1) chain of the γδ T cell receptor (TCR) with a KD of less than about 100 nM or less than about 50 nM. 125. The multispecific antibody of any preceding embodiment, wherein the Fab region downregulates TCR with an IC50 of less than about 50 nM, preferably less than about 10 nM. 126. The multispecific antibody of any preceding embodiment, wherein the multispecific antibody kills A375 cells with an IC50 of less than about 10 nM, preferably less than about 5 nM. 127. The multispecific antibody of any preceding embodiment, wherein the Fab region stimulates Vδ1 T cell proliferation. 128. The multispecific antibody of any preceding embodiment, wherein the Fab region binds human TVRD1 with a KD of less than about 100 nM. 129. The multispecific antibody of any preceding embodiment, wherein the Fab region binds human TVRD1 with a KD of less than about 50 nM. 130. The multispecific antibody of any preceding embodiment, wherein the Fab region binds human TRDV1 with a _KD of less than about 10 nM. 131. The multispecific antibody of any preceding embodiment, wherein the Fab region binds human TRDV1 with a _KD of less than about 5 nM. 132. The multispecific antibody of any preceding embodiment, wherein the Fab region binds human TRDV1 with a _KD of less than about 1 nM. 133. The multispecific antibody of any preceding embodiment, wherein the Fab region binds cynomolgus monkey TRDV1 with a _KD of less than about 100 nM. 134. The multispecific antibody of any preceding embodiment, wherein the Fab region binds cynomolgus monkey TRDV1 with a _KD of less than about 50 nM. 135. The multispecific antibody of any preceding embodiment, wherein the Fab region downregulates TCR with an IC50 of less than about 50 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, or less than about 0.5 nM. 136. The multispecific antibody of any preceding embodiment, wherein the Fab region: a. has a binding affinity ( _KD ) of less than about 100 nM (preferably less than about 50 nM), e.g., as measured by surface plasmon resonance ) binds to human TRDV1 (SEQ ID NO 272 or 306); b. Optionally with a binding affinity (K _D , as measured, for example, by surface plasmon resonance) of less than about 100 nM (preferably less than about 50 nM) Binds to cynomolgus monkey TRDV1 (SEQ ID NO 308); c. The IC50 for down-regulating TCR is less than about 50 nM (preferably less than about 10 nM). 137. The multispecific antibody of any preceding embodiment, wherein the Fab region: a. binds to human TRDV1 with a KD of less than about 100 nM (preferably less than about 50 nM); b. binds to human TRDV1 with a _KD of less than about 100 nM (preferably less than about 50 nM) The K _D of less than about 50 nM) binds to cynomolgus monkey TRDV1; c. The IC50 for down-regulating TCR is less than about 50 nM (preferably less than about 10 nM) and the IC90 for down-regulating TCR is less than about 100 nM (less than about 50 nM). . 138. The multispecific antibody of any preceding embodiment, wherein the Fab region: a. binds to human TRDV1 with a _KD of less than about 10 nM (preferably less than about 5 nM); and/or b. with a KD of less than about 100 nM (preferably less than about 50 nM) binds to cynomolgus _monkey TRDV1. 139. The multispecific antibody of any preceding embodiment, wherein the Fab region: a. binds to human TRDV1 with a _KD of less than about 10 nM (preferably less than about 1 nM); b. binds to human TRDV1 with a _KD of less than about 50 nM Binds to cynomolgus monkey TRDV1; c. The IC50 for down-regulating TCR is less than about 1 nM (preferably less than about 0.5 nM). 140. The multispecific antibody of any preceding embodiment, wherein the Fab region: a. Binds to human TRDV1 with a _KD of less than about 10 nM (preferably less than about 5 nM); b. Downregulates the TCR with an IC50 of less than about 50 nM (preferably less than about 10 nM). 141. The multispecific antibody of any preceding embodiment, wherein the Fab region: a. binds to human TRDV1 with a _KD of less than about 10 nM (preferably less than about 5 nM); and b. causes TCR downregulation with an IC50 of less than about 141. 5 nM (preferably less than about 1 nM). 142. The multispecific antibody of any preceding embodiment, wherein the Fab region is a human Fab region. 143. The multispecific antibody of any preceding embodiment, wherein the antibody is ^mAb2 . 144. The multispecific antibody of any preceding embodiment, wherein the antibody is a monoclonal antibody. 145. The multispecific antibody of any preceding embodiment, wherein the Fab region binds only to epitopes in the V region of the Vδ1 chain of the γδ TCR. 146. The multispecific antibody of any preceding embodiment, wherein the Fab region binds only to epitopes within amino acid residues 1-90 of SEQ ID NO: 272, SEQ ID NO: 306, or SEQ ID NO: 308 . 147. The multispecific antibody of any preceding embodiment, wherein the Fab region does not bind to an epitope found in CDR3 of the Vδ1 chain of the γδ TCR. 148. The multispecific antibody of any preceding embodiment, wherein the Fab region does not bind to an epitope within amino acid regions 91-105 (CDR3) of SEQ ID NO: 272. 149. The multispecific antibody of any preceding embodiment, wherein the Fab region binds to an epitope within the following amino acid region: (i) 3-20 of SEQ ID NO: 272; and/or (ii) SEQ ID NO: 272-37-77. 150. The multispecific antibody of embodiment 149, wherein the epitope bound by the Fab region comprises one or more amino acid residues within amino acid regions 37-53 and/or 59-77 of SEQ ID NO: 272 base. 151. The multispecific antibody of embodiment 149, wherein the epitope bound by the Fab region comprises amino acid residues 37, 42, 50, 53, 59, 64, 68, 69, 72 of SEQ ID NO: 272. 73 and 77. 152. The multispecific antibody of embodiment 149, wherein the epitope bound by the Fab region consists of amino acid residues 37, 42, 50, 53, 59, 64, 68, 69, 72, of SEQ ID NO: 272. Composed of 73 and 77. 153. The multispecific antibody of embodiment 149, wherein the epitope bound by the Fab region comprises one or more amino acid residues within amino acid regions 5-20 and/or 62-77 of SEQ ID NO: 272 base. 154. The multispecific antibody of embodiment 149, wherein the epitope bound by the Fab region comprises amino acid residues 5, 9, 16, 20, 62, 64, 72 and 77 of SEQ ID NO: 272. 155. The multispecific antibody of embodiment 149, wherein the epitope bound by the Fab region consists of amino acid residues 5, 9, 16, 20, 62, 64, 72 and 77 of SEQ ID NO: 272. 156. The multispecific antibody of embodiment 149, wherein the epitope bound by the Fab region is an activation epitope of a γδ T cell. 157. The multispecific antibody of embodiment 156, wherein binding of the activating epitope: (i) downregulates the γδ TCR; (ii) activates degranulation of the γδ T cell; and/or (iii) promotes γδ T cell mediation Lead it to death. 158. The multispecific antibody of any preceding embodiment, wherein binding of the activating epitope upregulates expression of CD107a, CD25, CD69 and/or Ki67. 159. The multispecific antibody of any preceding embodiment, wherein the multispecific antibody is greater than about 70 KDa. 160. The multispecific antibody of any preceding embodiment, wherein the antibody does not comprise the VHCDR3 with SEQ ID NO: 54 and the VLCDR3 with SEQ ID NO: 81, and/or wherein the antibody does not comprise the VHCDR3 with SEQ ID NO: 132 VHCDR3 and VLCDR3 with SEQ ID NO: 146. 161. The multispecific antibody of any preceding embodiment, wherein the antibody does not comprise a VH region having SEQ ID NO: 1 and a VL region having SEQ ID NO: 26, and/or wherein the antibody does not comprise a VH region having SEQ ID NO: 26. The VH area of 106 and the VL area with SEQ ID NO: 118. 162. A polynucleotide sequence encoding a multispecific antibody as defined in any preceding embodiment. 163. A polynucleotide sequence encoding an anti-Vδ1 first binding domain, comprising a sequence having at least 70% sequence identity with SEQ ID NO: 199 to 222, 224 to 247, 249 to 259, or 261 to 271. 164. An expression vector comprising a polynucleotide sequence as defined in Example 162 or Example 163. 165. A host cell comprising a polynucleotide sequence as defined in Example 162 or Example 163 or an expression vector as defined in Example 164, optionally wherein the host cell is a recombinant host cell. 166. The host cell of embodiment 165, comprising a first polynucleotide sequence encoding a VH sequence and a second polynucleotide sequence encoding a VL sequence. 167. The host cell of embodiment 166, comprising a first expression vector comprising a first polynucleotide sequence encoding a VH sequence, and a second expression vector comprising a second polynucleotide sequence encoding a VL sequence. 168. The host cell of embodiment 166, comprising an expression vector comprising a first polynucleotide sequence encoding a VH sequence and a second polynucleotide sequence encoding a VL sequence. 169. The host cell as defined in any one of embodiments 166 to 168, wherein the polynucleotide or expression vector encodes a membrane anchor or transmembrane domain fused to the antibody or antigen-binding fragment thereof, wherein the antibody Or its antigen-binding fragment region is displayed on the extracellular surface of the cell. 170. A method for producing any of the multispecific antibodies of any one of embodiments 1 to 161, comprising culturing the host cell of any one of embodiments 165 to 169 in a cell culture medium. 171. The method of embodiment 170, further comprising isolating the multispecific antibody from the culture medium. 172. A composition comprising a multispecific antibody or an antibody-binding fragment thereof as defined in any one of embodiments 1 to 161. 173. A pharmaceutical composition comprising a multispecific antibody as defined in any one of embodiments 1 to 161 and a pharmaceutically acceptable diluent or carrier. 174. A kit comprising the multispecific antibody of any one of embodiments 1 to 161 or the pharmaceutical composition of embodiment 173. 175. The kit of embodiment 174, further comprising an additional therapeutically active agent. 176. The kit of embodiment 174 or embodiment 175, further comprising instructions for use. 177. A method of treating a disease or condition in an individual, comprising administering to the individual the multispecific antibody of any one of Embodiments 1 to 161 or the pharmaceutical composition of Embodiment 173. 178. The method of embodiment 177, wherein the disease or condition is cancer. 179. The method of embodiment 178, wherein the cancer is an EGFR-positive cancer. 180. The method of any one of embodiments 177 to 179, further comprising administering a second agent to the individual simultaneously or sequentially in any order. 181. A method of modulating an immune response in an individual, comprising administering to the individual the multispecific antibody of any one of embodiments 1 to 161 or the pharmaceutical composition of embodiment 173. 182. The method of embodiment 181, wherein the individual has cancer. 183. The method of embodiment 181 or 182, wherein modulating the immune response of the individual comprises at least one selected from the group consisting of: activating γδ T cells, causing or increasing the proliferation of γδ T cells, causing or increasing the expansion of γδ T cells. Increase, cause or increase the degranulation of γδ T cells, cause or increase the killing activity mediated by γδ T cells, cause or increase the toxicity of γδ T cells, cause or increase the mobilization of γδ T cells, increase the survival of γδ T cells and increase the response to γδ T cells. Resistance to cell exhaustion. 184. The method of any one of embodiments 177 to 183, wherein diseased cells are killed while avoiding healthy cells. 185. The multispecific antibody of any one of embodiments 1 to 161, or the pharmaceutical composition of embodiment 173, or the set of any one of embodiments 174 to 176, for use in medicine. 186. The multispecific antibody of any one of embodiments 1 to 161, or the pharmaceutical composition of embodiment 177, or the set of any one of embodiments 174 to 176, for use in the treatment of cancer. 187. Use of the multispecific antibody of any one of embodiments 1 to 161 for the manufacture of a medicament. 188. Use of the multispecific antibody of any one of embodiments 1 to 161 for the manufacture of a medicament for the treatment of cancer.

Figure 1 : Phage selection rounds . (A) Phage selection rounds 1 to 3 of the ADT1-7 library. (B) The first to third rounds of phage selection in the ADT1-4 library. (C) Rounds 1 to 3 of phage selection of the ADT1-4 library using a selection strategy for isolating cynomolgus cross-reactive binders. Figure 2 : Schematic overview of selection of mature antibodies by mammalian presentation. (A) Schematic diagram of flow sorting in ADT1-7 library. (B) Schematic diagram of flow sorting of ADT1-4 library 1 (human). (C) Schematic diagram of flow sorting of ADT1-4 library 2 (cynomolgus macaque). Figure 3 : Effect of S74L changes on affinity. (A) ADT1-4 lineage mammalian presentation output: human off-rate (SPR) versus human antigen binding (Delfia ELISA, 0.4 nM human antigen), with use of LC 74S (open circles) or 74L (filled circles) indicated. The results highlight a good agreement between affinity improvement by SPR and antigen binding by Delfia ELISA. These results also highlight that variable domain S74L modification can improve binding. (B) ADT1-4 lineage mammalian presentation output: cynomolgus dissociation rate (SPR) versus cynomolgus antigen binding (Delfia ELISA, 10 nM cynomolgus antigen), with indicated LC 74S (open circles) or 74L (filled circles) circle). (C) ADT1-4 lineage mammals present output: correlation between increased human antigen off-rate (SPR) and cynomolgus macaque antigen off-rate (SPR), plus indication of LC 74S (open circles) or 74L (filled circles) ) use. Figure 4 : Usage and cross-sharing of CDR3 . (A) Example heavy and light chain cross-sharing between affinity matured antibodies in the ADT1-4(G04) lineage (heat map binning of 0.4 nM human TRDV1 ELISA results). This "heat map" demonstrates the cross-sharing of CDR3 sequences between different affinity matured antibodies in the ADT1-4 lineage, showing that the affinity maturation process provides antibody sequences that do not necessarily provide specific heavy and light chain pairs. The results highlight the potential for affinity-matured antibodies to cross-share light and heavy chains, and that different LC/HC combinations produce equivalent or improved results in high-stringency antigen binding studies. Also included in the figure is the ADT1-4(G04) parental pure line (lower left). (B) ADT1-4 lineage final selection: using CDR3 and cross-shared + cynomolgus monkey antigen binding "heat map" compared to the starting parent ADT1-4 parent G04 mAb (lower left). (C) Final selection of the ADT1-7 lineage: using CDR3 and cross-shared + human antigen binding “heat maps” compared to the starting ADT1-7 parental E07 mAb (lower left). Figure 5 : Fold enhancement in binding of the ADT1-4 lineage compared to the ADT1-4 parent G04 . (A) Fold enhancement of recombinant human Vδ1 antigen binding. (B) Fold enhancement of recombinant cynomolgus monkey Vδ1 antigen binding. (C) Enhancement factor of primary Vδ1 MFI. (D) Enhancement fold of MFI in PEER Vδ1 cell line. Figure 6 : Fold enhancement in binding of the ADT1-1 lineage compared to the ADT1-4 parent E07 . (A) . Fold enhancement of recombinant human Vδ1 antigen binding. (B) .Enhancement factor of primary Vδ1 MFI. (C). Enhancement fold of MFI of PEER Vδ1 cell line. Figure 7. The fold increase in antigen binding in humans ( and cynomolgus monkeys ) relative to the parental pure line. (A) The fold increase in binding to human antigens. DELFIA ELISA signal of affinity matured pure line compared to parent ADT1-4 (G04), 0.4 nM recombinant human L1 antigen (containing human Vδ1 TCR antigen). (B) Fold improvement in binding to cynomolgus antigen (DV1/GV77, containing cynomolgus SEQ ID NO: 308 (mature, minus leader sequence)). DELFIA ELISA signal of affinity matured pure line compared with parental ADT1-4 (G04), 0.4 nM recombinant human L1 antigen. (C) The fold increase in binding to human antigens. DELFIA ELISA signal of affinity matured pure line compared with parental ADT1-7(E07), 0.4 nM recombinant human L1 antigen. Figure 8. Fold increase in KD for human ( and cynomolgus monkey ) antigens relative to the parental pure line . (A) Fold increase in KD of ADT1-4(G04) lineage by human antigens. (B) Fold increase in KD of ADT1-4(G04) lineage by cynomolgus monkey antigen. (C) Fold increase in KD of ADT1-7(E07) lineage by human antigens. Figure 9. Binding affinity analysis ( KD by SPR ) for human Vδ1 antigen . (A) Surface plasmon resonance of the ADT1-4 spectrum. (B) Surface plasmon resonance of the ADT1-7 spectrum. (C) KD value of ADT1-4 lineage and fold change relative to the parent pure line. (D) KD value of ADT1-7 lineage and fold change relative to the parent pure line. ( E ) The fold change of the KD value of the ADT1-4 lineage relative to the parental pure line. (F) Fold change in KD of ADT1-7 lineage relative to the parent pure line. Figure 10. Binding affinity analysis ( KD by SPR ) for cynomolgus monkey antigens . (A) Surface plasmon resonance of ADT1-4 lineage against cynomolgus monkey antigen. (B) KD value of ADT1-4 lineage against cynomolgus monkey antigen. Figure 11. Binding affinity to cell surface V δ 1 TCR ( EC50 for binding to cell surface V δ 1 ) . (A , B) Binding levels of Vδ1 mAb to two γδ T cell donors, ATS006 (A) and TS164 (B). (C) Bar graph showing the average 50% binding values of ADT1-4 and ADT1-7 pure lines to Vδ1-positive γδ T cells, expressed as the average of two donors, where these values are presented as % improvement in table. (D) Table summarizing the IC50 and Vδ1 negative cell types plotted in (A) and (B), including HEK293A, Raji cells, and multiple leukocyte subsets with primary blood monocytes. For Vδ1-positive γδ T cells, data are expressed as the average of two donors. Figure 12. TCR downregulation. (A , B) ADT1-4 lineage (A) and ADT1-7 lineage (B). (C) Average TCR downregulation - IC50 results from two GD cell donors. (D , E) TCR down-regulation fold increased from the parental ADT1-4 pure line (D) and ADT1-7 pure line (E). (F) 50% effect value from (C), using percent improvement calculated from the corresponding parents of ADT1-4 (top) and ADT1-7 (bottom). (G) 50% effect value from (C), using fold improvement calculated from the corresponding parents of ADT1-4 (top) and ADT1-7 (bottom). Figure 13. Activation of γδ by Vδ1 monoclonal antibodies as measured by CD107a expression. (A , B) ADT1-4-2 homogeneous lines in GD cells alone (A) or together with THP-1 cells (B). (C , D) ADT1-7-3 homogeneous lines in GD cells alone (C) or together with THP-1 cells (D). (E) Table showing the percent increase in γδ CD107a performance from cocultured cells treated with the highest concentration of Vδ1 mAb compared to untreated, non-cocultured γδ cells. (F) Table showing the percent increase in γδ CD107a performance from co-cultured cells treated with the highest concentration of Vδ1 mAb compared to untreated co-cultured and non-co-cultured γδ cells. Figure 14. γδ activation by Vδ1 monoclonal antibodies as measured by CD25 expression . (A , B) ADT1-4-2 homogeneous lines in GD cells alone (A) or together with THP-1 cells (B). (C , D) ADT1-7-3 homogeneous lines in GD cells alone (C) or together with THP-1 cells (D). (E) Table showing the percent increase in γδ CD25 expression from cocultured cells treated with the highest concentration of Vδ1 mAb compared to untreated, non-cocultured γδ cells. (F) Table showing the percent increase in γδ CD25 expression from co-cultured cells treated with the highest concentration of Vδ1 mAb compared to untreated co-cultured and non-co-cultured γδ cells. Figure 15. Downregulation of TCR in crab-eating macaques . (A) Comparison of the ability of ADT1-4-2 and ADT1-4 to engage VD1 on cynomolgus macaque γδ-T cells and reduce VD1 expression. (B) Percent cell surface expression of VD1 after treatment with ADT1-4-2. (C) Individual EC50 values of different donors, as well as the average and standard deviation. Figure 16. Quantification of viable THP-1 cell numbers after 24 hours of co-culture with γδ T cells in the presence of V δ 1 mAb or control. (A) THP-1 cell killing analysis of ADT1-4 pure lines. (B) THP-1 cell killing assay of ADT1-7 pure line. (C) Average EC50 of THP-1 killing assay for ADT1-4 and ADT1-7 pure lines. (D) Table summarizing the EC50 plotted in (C). Figure 17. Vδ1 monoclonal antibodies on antibody-dependent cellular cytotoxicity ( ADCC) . Figure 18. Vδ1 monoclonal antibody on complement-dependent cytotoxicity (CDC) . Figure 19. Pure line ADT1-4-2 avoids healthy cells. Figure 20. Effect of anti - Vδ1 antibodies on TIL populations from primary tumor sections . (A , B) Shown is the % reduction in Vδ1 TCR expression on total tumor-infiltrating γδ T cells after 48 (A) or 72 (B) hours of mAb stimulation in two separate donors, confirming target engagement in each case. (C) Demonstrates enhanced expression of CD25 and Ki67 on Vδ1+ T cells after 48 hours of stimulation with ADT1-4-2 compared with stimulation with IgG1 isotype control or ADT1-4. (D) Shows the substantial fold increase in IFN-γ production from TILs stimulated with ADT1-4-2 or ADT1-7-3 for 72 hours in the presence of 50 ng/ml IL-15. (E) Shows that stimulation of TILs with ADT-1-4-2 or ADT1-7-3 at this time point does not enhance secretion of the type 17-related interleukins IL-6 or IL-17. (F , G) Show the % reduction in Vδ1 TCR expression on total tumor-infiltrating γδ T cells after 24 (F) or 72 (G) hours of mAb stimulation in two individual donors, confirmed in TIL isolated by enzymatic digestion Target engagement. (H) Demonstrates dose-dependent enhancement of Ki67 expression on γδ T cells after 72 hours of stimulation with ADT1-4-2. (I , J) Display isolated from two individual donors by enzymatic digestion and stimulated with ADT1-4-2 at a concentration of 6.66 nM in the presence of 2 ng/ml IL-15 for 24 (I) or 72 (J) hours IFN-γ production by TILs increased fold. In all cases, the control anti-RSV and parental ADT1-4 concentrations were the highest concentration used in the study (i.e., A, C, F, G, H, I, J were at a concentration of 6.66 nM, and B and E for a concentration of 66.6 nM). Figure 21. Effect of anti - Vδ1 antibodies on TIL populations from primary tumor sections . (A) Shows the enhanced expression of CD25 and Ki67 in γδ T cells stimulated with ADT1-4-2 for 10 days. (B) Demonstrates substantial increase in perforin ⁺ granzyme B ⁺ γδ T cells after 10 days of stimulation with ADT1-4-2. (C) Shows that granzyme B and perforin expression of CD8 ⁺ and CD8 ^- αβ T cells were significantly enhanced after stimulating tumor-infiltrating Vδ1 ⁺ T cells with ADT1-4-2 for 10 days. (D) Demonstrates a significant increase in IFN-γ production and a moderate increase in IL-17 and IL-6 production by lung tumor-derived TILs after stimulation with ADT1-4-2. (E) Demonstrates that the production of chemokines CCL2, CCL4 and CXCL10 in TIL is enhanced after 10 days of stimulation with ADT1-4-2. In all cases, control anti-RSV and parental ADT1-4 were matched to the same concentrations as affinity matured ADT1-4-2. Figure 22 : Sequence of the ADT1-4 lineage pure line ( light chain ) . Figure 23 : Sequence of the pure line ( heavy chain ) of the ADT1-4 lineage . Figure 24 : Sequence of the ADT1-7 lineage pure line ( light chain ) . Figure 25 : Sequence of the pure line ( heavy chain ) of the ADT1-7 lineage . Figure 26 : V δ 1 /EGFR bispecific antibody exhibits high affinity binding to human EGFR and similar human V δ 1 binding affinity to its parent mAb . (A , B) Surface plasmon resonance (SPR) analysis was performed with Vδ1/EGFR bispecific variants to assess binding to human Vδ1 (A) and human EGFR antigen (B) . Parental mAb, cetuximab, and negative control mAb were included for comparison purposes. Figure 27 : V δ 1/EGFR bispecific antibody binds to EGFR ⁺ A431 target cells and V δ 1 γδ T cells (A) Cell surface expression of EGFR and Vδ1 on A431 cell lines and primary Vδ1 γδ T cells. (B) Binding level of Vδ1/EGFR bispecific antibody to A431 cell line or primary Vδ1 γδ T cells. Target cells were stained with varying concentrations of antibodies, followed by fluorescent anti-human IgG detection antibodies. All incubation steps were performed at 4°C and mAb binding was determined using flow cytometry to measure the median fluorescence level. Logarithmic four-parameter dose-response curves were fitted using GraphPad Prism 9. Figure 28 : Vδ1 /EGFR bispecific antibody induces EGFR -specific T cell activation and degranulation, resulting in increased γδ T cell - mediated cytotoxicity of A431 target cells. (A) Cell surface expression of γδ TCR on primary Vδ1 γδ T cells after 24 hours of incubation with bispecific antibodies in the presence or absence of A431 cells. (B , C) The number of viable A431 cells (B) and the activation status of primary Vδ1 γδ T cells after co-culture with different concentrations of antibodies at a 1:1 ratio for 24 hours. Viability was measured by viability dye and activation status was measured using CD25 antibody. (D) Degranulation of primary Vδ1 γδ T cells after four hours of co-culture with A431 cells at a 1:1 ratio with different concentrations of antibodies. Degranulation was measured by adding fluorophore-conjugated anti-CD107α antibody directly to the cell-antibody mixture at the beginning of co-culture. (E) Number of viable A431 cells after 24 hours of co-culture with 10 pM antibody and different numbers of primary Vδ1 γδ T cells. (AE) In all cases, fluorescence was measured using flow cytometry to measure the median fluorescence level. Logarithmic four-parameter dose-response curves were fitted using GraphPad Prism 9. Data are expressed as the mean ± SD of two biological replicates. Figure 29 : Further evidence of non-exhaustion and CD3 downregulation in blood-derived and tumor-associated Vδ1 T cells . (A) Shows vδ1 TCR MFI after antibody stimulation as an indicator of mAb target engagement on blood-derived Vδ1 T cells. (B) MFI showing CD3 expression on positively gated blood-derived vδ1 cells. Stimulation with vδ1 antibodies engages vδ1 cells and causes downregulation of vδ1 and CD3 on vδ1 cells. ( A and B ) Demonstrate activation and non-depletion of tumor-associated Vδ1 T cells conferred by exemplary anti-Vδ1 antibodies. Figure 30 : ADCC reporter bioassay does not demonstrate ADCC caused by anti - vδ1 antibodies . Target cells, namely γδ cells, were incubated with ADCC bioassay effector cells in the presence of anti-vδ1 antibody, anti-vδ1 LAGA antibody (Fc-inactivated), and RSV isotype control. The luminescence signal was recorded as relative light units (RLU) and the induction fold was calculated as described in Methods. γδ donors of "anti-vδ1 antibody", "anti-vδ1 LAGA antibody", "RSV", and "OKT3" (technically performed in duplicate) N=2. The Raji cell line N=1 for the "Rituximab + Raji" condition (technically duplicated), and the γδ supply for the "anti-vδ1 antibody + effector" and "anti-vδ1 LAGA antibody + effector" conditions Body n=1 (techniques are performed in duplicate and in duplicate respectively). Effect:Target ratio is 3:1. Figure 31 : Multispecific antibodies enhance V δ 1+ effector cell-mediated cytotoxicity. Targeting of Tissue Center Disease-Associated Antigens: (AD) Example of Vδ1+ effector cells co-cultured with A-431 cancer cells +/- a multispecific antibody containing an anti-Vδ1 × anti-TAA (EGFR) bispecific binding moiety, where the The anti-Vδ1 VL+VH binding domain (binding to the first target) is paired with the CH1-CH2-CH3 domain of the anti-EGFR binding portion (binding to the second target). (EH) Example of Vδ1+ effector cells co-cultured with A-431 cancer cells +/- a multispecific antibody containing an anti-Vδ1 × anti-TAA (EGFR) bispecific binding moiety, wherein the anti-Vδ1 binding domain (binding to the first Target) contains a full-length antibody (VH-CH1-CH2-CH3/VL-CL), which is then combined with an anti-EGFR scFv binding portion derived from cetuximab (which binds to the second target). Figure 32 : Sequence of EGFR binding domain (IgG1 CH1-CH2-CH3) , showing sequence alignment and changes relative to IgG1 CH3 (EU numbering ) . Figure 33 : Table showing cross-referenced CH3 residue numbers. This table is modified from IMGT.org. The positions of modified amino acids according to the IMGT (standard), IMGT (exon), EU and Kabat numbering mentioned elsewhere in this article are highlighted by shading. Note that a necessary but not conventional positional nomenclature is also indicated to describe the additional amino acid insert found in an example EGFR binding construct - see below positions 21.1 (IMGT exon row)/361.1 (EU row) /384.1(Kabat line)/17.1(IMGT standard line). Figure 34 : Manufacturability / stability assessment of Vδ 1×EGFR multispecific antibodies : performance and analytical studies on six molecules. (A) Yield in CHO. (B) Stability under accelerated storage conditions. (C) Aggregation tendency. (D) Decay profile under accelerated conditions (non-reductive analysis). (E) Decay profile under accelerated conditions (reducibility analysis). (F) Summary/Conclusion Figure 35 : Extended study on the VD1×EGFR multispecific antibody and the different effects conferred by increasing / decreasing the affinity on the VD1 binding arm . (A) Affinity summary. (B) (C) Effect on binding of multispecific antibodies to Vδ1+ cells present in tissues. (D) (E) Effect on Vδ1 TCR internalization by Vδ1+ cells present in tissue. (F) (G) Effect on degranulation of Vδ1+ cells present in tissues. (H) (I) Effect of multispecific antibodies on the cytolytic function of EGFR+ cancer cells. (J) (K) Effect on the proliferation of Vδ1+ cells present in tissues. (L) Summary of this study. (M) Effect of number of TAA replicas. Figure 36 : Extended study on the VD1×EGFR multispecific antibody and the different effects conferred by increasing / decreasing the affinity on the EGFR binding arm. (A) Affinity summary. (B) (C) Effect on the binding of multispecific antibodies to EGFR+ colorectal cancer cell line HT-29. (D) Effect of (E) on downregulation of Vδ1 TCR in Vδ1+ cells present in tissues. (F) (G) Effect on degranulation of Vδ1+ cells present in tissues. (H) (I) Effect on the 41BB activation status of Vδ1+ cells present in tissues. (J) (K) Effect of multispecific antibodies on the cytolytic function of EGFR+ cancer cells. (L) Effect of (M) on the proliferation of Vδ1+ cells present in tissues. (N) Effect on the affinity of EGFR and the number of TAA copies in cancer cells. Figure 37 : Effect of affinity on preferential cellular binding to V δ 1 over EGFR . The effects of EGFR cells' preferential binding affinity to Vδ1 are compared in bar graph (A) , table (B) and ratio (C) . Figure 38 : Effect of affinity for EGFR on internalization of multispecific antibodies by A431 tumor cells. (A) (B) Affinity to EGFR; effect on internalization of multispecific antibodies in A431 tumor cells. (C) (D) Effect on the clearance rate of multispecific antibodies in A431 tumor cells. Figure 39 : Effect of affinity to EGFR on the inhibition of EGF ligand binding and tumor cell proliferation by multispecific antibodies . (A) (B) Effect of affinity to EGFR on multispecific antibody binding to EGF ligand and inhibition of tumor cell proliferation. (C) (D) Effect of affinity to EGFR on the inhibition of EGFR+ tumor cell proliferation by multispecific antibodies. Figure 40 : VD1×EGFR multispecific antibody : avoiding healthy EGFR+ cells. (A) (C) shows a dose titration effect. (B) Representative microscopic images from the experiment described in (A). Figure 41 : VD1×EGFR multispecific antibody : ADT1-4-2×FS1-67 compared with the lower affinity “parent” ADT1-4×FS1-67 ( alias G04 FS1-67) . (A) Dose-response comparison of TCR downregulation-ADT1-4-2×FS1-67 and ADT1-4×FS1-67. (B) TCR downregulation comparison to summary of results. Figure 42 : VD1×EGFR multispecific antibody : comparison with CD3×EGFR multispecific antibody. (A) (B) (C) Effect of T cell target receptors on the down-regulation of Vδ1 TCR and CD3ε (D) (E) Effect of multispecific antibodies on the cytolytic function of EGFR+ cancer cells. (F) (G) Effect on interleukin secretion induced by activated T cells. (H) (I) Effect on the proliferation of Vδ1+ cells present in tissues.

TW202342536A_112105895_SEQL.xml

Claims (41)

A multispecific antibody comprising a Fab region and an Fc region, wherein the Fab region contains a binding site specific for the epitope of the variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR); and the Fc The region contains an EGFR binding site, wherein the EGFR binding site is provided by a CH3 domain, where: a. Residues 359 to 362 (EU numbering) comprise EEGP (SEQ ID NO: 523), residues 384 to 386 (EU numbering) comprise TYG (SEQ ID NO: 511), and residues 413 to 415 (EU numbering) Contains SYW(SEQ ID NO: 533); b. Residues 359 to 362 (EU numbering) contain EEGP (SEQ ID NO: 523), and residues 413 to 419 (EU numbering) contain SYWRWYK (SEQ ID NO: 512); c. Residues 358 to 361, 361.1 and 362 (EU numbering) contain LDEGGP (SEQ ID NO: 542), residues 384 to 386 (EU numbering) contain TYG (SEQ ID NO: 511), and residues 413 to 419 (EU number) contains SYWRWVK (SEQ ID NO: 543); d. Residues 384 to 386 (EU numbering) contain TYG (SEQ ID NO: 511), and residues 413 to 419 (EU numbering) contain SYWRWYK (SEQ ID NO: 512); or e. Residues 358 to 362 (EU numbering) contain TESGP (SEQ ID NO: 553), residues 384 to 386 (EU numbering) contain KFG (SEQ ID NO: 554), and residues 413 to 421 (EU numbering) contain SNLRWTKGH (SEQ ID NO: 555), and residue 378 is valine. The multispecific antibody of claim 1, wherein the EGFR binding site is provided by the following CH3 domain, wherein: a. Residues 355 to 362 (EU numbering) form an AB loop containing RDELEEGP (SEQ ID NO: 524), residues 383 to 391 (EU numbering) form a CD loop containing STYGPENNY (SEQ ID NO: 514) and residues 413 to 422 (EU numbering) form an EF loop containing SYWRWQQGNV (SEQ ID NO: 534); b. Residues 355 to 362 (EU numbering) form an AB loop containing RDELEEGP (SEQ ID NO: 524), residues 383 to 391 (EU numbering) form a CD loop containing SNGQPENNY (SEQ ID NO: 525) and residues 413 to 422 (EU numbering) form an EF loop containing SYWRWYKGNV (SEQ ID NO: 515); c. Residues 355 to 362 (EU numbering) form an AB loop containing RDELDEGGP (SEQ ID NO: 544), residues 383 to 391 (EU numbering) form a CD loop containing STYGPENNY (SEQ ID NO: 514), and residues Bases 413 to 422 (EU numbering) form an EF loop containing SYWRWVKGNV (SEQ ID NO: 545); d. Residues 355 to 362 (EU numbering) form an AB loop containing RDELTKNQ (SEQ ID NO: 513), residues 383 to 391 (EU numbering) form a CD loop containing STYGPENNY (SEQ ID NO: 514) and residues 413 to 422 (EU numbering) form an EF loop containing SYWRWYKGNV (SEQ ID NO: 515); e. Residues 355 to 362 (EU numbering) form an AB loop containing RDETESGP (SEQ ID NO: 556), residues 383 to 391 (EU numbering) form a CD loop containing SKFGPENNY (SEQ ID NO: 557), residues 413 to 422 (EU numbering) form the EF loop containing SNLRWTKGHV (SEQ ID NO: 558) and residue 378 is valine. The multispecific antibody according to any one of the preceding claims, wherein the EGFR binding site is not provided by the following CH3 domain, wherein: a. Residues 358 to 362 (EU numbering) comprise TDDGP (SEQ ID NO: 560), residues 384 to 386 (EU numbering) comprise TYG (SEQ ID NO: 511), and residues 413 to 419 (EU numbering) Contains SYWRWYK (SEQ ID NO: 512); or b. Residues 355 to 362 (EU numbering) form an AB loop containing RDETDDGP (SEQ ID NO: 561), residues 383 to 391 (EU numbering) form a CD loop containing STYGPENNY (SEQ ID NO: 514), residues 413 to 422 (EU numbering) form the EF loop containing SYWRWYKGNV (SEQ ID NO: 515). The multispecific antibody of claim 1, wherein the Fc region is a human IgG Fc region comprising the CH3 domain engineered to include the EGFR binding site. The multispecific antibody of any one of the preceding claims, wherein the amino acid sequence of the CH3 domain is at least 90% identical, or at least 95% identical, or 100% identical to the following: a. SEQ ID NO: 532; b. SEQ ID NO: 522; c. SEQ ID NO: 541; d. SEQ ID NO: 510; or e. SEQ ID NO: 552. The multispecific antibody according to any one of the preceding claims, wherein the antibody has an Fc function. The multispecific antibody according to any one of the preceding claims, wherein the Fc region includes: a. SEQ ID NO: 530 or SEQ ID NO: 531; b. SEQ ID NO: 520 or SEQ ID NO: 521; c. SEQ ID NO: 539 or SEQ ID NO: 540; d. SEQ ID NO: 508 or SEQ ID NO: 509; or e. SEQ ID NO: 550 or SEQ ID NO: 551. The multispecific antibody according to any one of the preceding claims, comprising: a. SEQ ID NO: 526; or SEQ ID NO: 414 and SEQ ID NO: 528; b. SEQ ID NO: 527; or SEQ ID NO: 414 and SEQ ID NO: 529; c. SEQ ID NO: 516; or SEQ ID NO: 414 and SEQ ID NO: 518; d. SEQ ID NO: 517; or SEQ ID NO: 414 and SEQ ID NO: 519; e. SEQ ID NO: 535; or SEQ ID NO: 414 and SEQ ID NO: 537; f. SEQ ID NO: 536; or SEQ ID NO: 414 and SEQ ID NO: 538; g. SEQ ID NO: 504; or SEQ ID NO: 414 and SEQ ID NO: 506; h. SEQ ID NO: 505; or SEQ ID NO: 414 and SEQ ID NO: 507; i. SEQ ID NO: 546; or SEQ ID NO: 414 and SEQ ID NO: 548; or j. SEQ ID NO: 547; or SEQ ID NO: 414 and SEQ ID NO: 549. The multispecific antibody of any of the preceding claims does not include: a. SEQ ID NO: 385 or SEQ ID NO: 386; b. SEQ ID NO: 378; or SEQ ID NO: 425 and SEQ ID NO: 426; c. SEQ ID NO: 379; or SEQ ID NO: 421 and SEQ ID NO: 437; or d. SEQ ID NO: 380; or SEQ ID NO: 423 and SEQ ID NO: 427. The multispecific antibody of any one of the preceding claims, wherein the multispecific antibody has a binding affinity (K _D ) of less than about 150 nM, less than about 20 nM, less than about 10 nM, or less than about 5 nM, for example, as (measured by surface plasmon resonance) binds to human EGFR. The multispecific antibody according to any one of the preceding claims, wherein the Fab region includes: Heavy chain variable region, which contains: VHCDR1 comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, a sequence selected from the group consisting of SEQ ID NO: 51, 52 and 130. or consists of an amino acid sequence that is at least 97%, at least 98%, at least 99% or 100% identical; VHCDR2 comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98%, at least 99% or 100% identity to or consisting of an amino acid sequence; and VHCDR3 comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or consists of an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical; and/or A light chain variable region comprising: VLCDR1 comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98%, at least 99% or 100% identical amino acid sequence or consisting of the amino acid sequence; VLCDR2 comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98%, at least 99% or 100% identity to or consisting of an amino acid sequence; and VLCDR3 comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or consists of an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical. For example, the multispecific antibody according to any one of the preceding claims, the Fab region includes: a. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 68, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 95; b. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 55, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 82; c. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 56, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 83; d. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 57, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 84; e. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 58, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 85; f. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 59, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 86; g. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 60, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 87; h. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 52, 53 and 61, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 88; i. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 62, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 89; j. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 63, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 90; k. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 64, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 91; l. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 65, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 92; m. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 52, 53 and 66, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 93; n. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 67, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 94; o. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 69, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 96; p. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 70, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 97; q. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 71, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 98; r. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 72, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 99; s. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 73, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 100; t. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 52, 53 and 74, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 101; u. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 75, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 102; v. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 76, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 103; w. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 77, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 104; x. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 51, 53 and 78, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 79, 80 and 105; y. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 130, 131 and 133, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 144, 145 and 147; z. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 130, 131 and 134, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 144, 145 and 148; aa. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 130, 131 and 135, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 144, 145 and 149; bb. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 130, 131 and 136, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 144, 145 and 150; cc. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 130, 131 and 137, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 144, 145 and 151; dd. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 130, 131 and 138, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 144, 145 and 152; ee. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 130, 131 and 139, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 144, 145 and 153; ff. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 130, 131 and 140, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 144, 145 and 154; gg. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 130, 131 and 141, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 144, 145 and 155; hh. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 130, 131 and 142, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 144, 145 and 156; or ii. VHCDR1, VHCDR2 and VHCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 130, 131 and 143, and VLCDR1, VLCDR2 and VLCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 144, 145 and 157. For example, the multispecific antibody according to any one of the preceding claims, the Fab region includes: A heavy chain variable region comprising at least 90%, at least 91%, at least 92%, at least 93%, or consists of a sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical; and/or A light chain variable region comprising at least 90%, at least 91%, at least 92%, at least 93%, or consists of a sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical. For example, the multispecific antibody according to any one of the preceding claims, the Fab region includes: a. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 40 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; b. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 27 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; c. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 28 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; d. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or VH and amino acid sequence having 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 29 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; e. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or VH and amino acid sequence having 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 30 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; f. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 31 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; g. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 32 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; h. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or VH and amino acid sequence having 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 33 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; i. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 34 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; j. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 35 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; k. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 36 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; l. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or VH and amino acid sequence having 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 37 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; m. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 38 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; n. Comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 39 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; o. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 41 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; p. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or VH and amino acid sequence having 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 42 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; q. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 43 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; r. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 44 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; s. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 45 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; t. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 46 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; u. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 47 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; v. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 48 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; w. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 49 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; x. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or VH and amino acid sequence having 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 50 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; y. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or VH and amino acid sequence having 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 119 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; z. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or VH and amino acid sequence having 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 120 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; aa. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 121 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; bb. Comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or VH and amino acid sequence having 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 122 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; cc. contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 123 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; dd. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 124 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; ee. Comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 125 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; ff. Comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or VH and amino acid sequence having 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 126 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; gg. Comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or VH and amino acid sequence having 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 127 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; hh. Comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or VH and amino acid sequence having 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 128 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity; or ii. Contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or A VH having an amino acid sequence of 100% sequence identity and comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least SEQ ID NO: 129 A VL with an amino acid sequence that has 97%, at least 98%, at least 99% or 100% sequence identity. The multispecific antibody of any one of the preceding claims, wherein the Fab region contains an amino acid residue other than serine at position 74 according to the IMGT numbering system, optionally at position 74 according to the IMGT numbering system The residue at 74 is a leucine residue. The multispecific antibody of any one of the preceding claims, wherein the Fab region includes: a. an HCDR1 sequence including the sequence of GDSVSSKSX ₁ A (SEQ ID NO: 158); b. including the sequence of SEQ ID NO: 53 HCDR2 sequence c _{. An} HCDR3 _sequence containing the sequence _{of X 2} W The LCDR1 sequence of the sequence; e. The LCDR2 sequence of the sequence of SEQ ID NO: 80; and f. The LCDR3 sequence of the sequence of QQX ₈ YX ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ T (SEQ ID NO: 166), wherein the LCDR3 sequence is not SEQ ID NO: 81; wherein X ₁ to X ₁₃ are each a naturally occurring amino acid, or wherein the antibody or antigen-binding fragment thereof comprises: a. HCDR1 comprising the sequence of SEQ ID NO: 130 Sequence b. HCDR2 _sequence comprising the sequence of SEQ ID NO: 131 _c . HCDR3 sequence comprising the sequence of _{X 1} : LCDR1 sequence of the sequence 144; e. LCDR2 sequence including the sequence of SEQ ID NO: 145; and f. LCDR3 sequence including the sequence of QQX ₅ X ₆ X ₇ X ₈ LX ₉ T, wherein the LCDR3 sequence is not SEQ ID NO: 146; wherein X ₁ to X ₉ are each a naturally occurring amino acid. The multispecific antibody of any one of the preceding claims, wherein the Fab region is an affinity matured variant of a parent anti-Vδ1 Fab region, wherein the parent anti-Vδ1 Fab region includes: a. VH including the amino acid sequence of SEQ ID NO: 1 and VL sequence including the amino acid sequence of SEQ ID NO: 26; b. VH comprising the amino acid sequence of SEQ ID NO: 106 and VL sequence comprising the amino acid sequence of SEQ ID NO: 118; c. VH comprising the amino acid sequence of SEQ ID NO: 273 and VL comprising the amino acid sequence of SEQ ID NO: 282; d. VH comprising the amino acid sequence of SEQ ID NO: 274 and VL comprising the amino acid sequence of SEQ ID NO: 283; e. VH comprising the amino acid sequence of SEQ ID NO: 275 and VL comprising the amino acid sequence of SEQ ID NO: 284; f. VH comprising the amino acid sequence of SEQ ID NO: 276 and VL comprising the amino acid sequence of SEQ ID NO: 285; g. VH comprising the amino acid sequence of SEQ ID NO: 277 and VL comprising the amino acid sequence of SEQ ID NO: 286; h. VH comprising the amino acid sequence of SEQ ID NO: 278 and VL comprising the amino acid sequence of SEQ ID NO: 287; i. VH comprising the amino acid sequence of SEQ ID NO: 279 and VL comprising the amino acid sequence of SEQ ID NO: 288; j. VH comprising the amino acid sequence of SEQ ID NO: 280 and VL comprising the amino acid sequence of SEQ ID NO: 289; k. VH comprising the amino acid sequence of SEQ ID NO: 281 and VL comprising the amino acid sequence of SEQ ID NO: 290; or l. VH comprising the amino acid sequence of SEQ ID NO: 312 and VL comprising the amino acid sequence of SEQ ID NO: 313; Depending on the situation: For example, the affinity mature antibody binds to the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR) with an affinity that is at least 20%, at least 30%, or at least 40% greater than the parent antibody, as measured by Kd , at least 50%, at least 100%, at least 500% or better at least about 1000%; The affinity matured anti-Vδ1 antibody or antigen-binding fragment thereof comprises VH and VL sequences that are at least 80%, at least 90%, at least 95%, or at least 96% identical to the corresponding parental VH and VL sequences; and/or The affinity matured anti-Vδ1 antibody or antigen-binding fragment contains up to 20, such as up to 15, such as up to 10, amino acid substitutions compared to the parent antibody sequence. The multispecific antibody of any one of the preceding claims, wherein the Fab region includes a VH including the amino acid sequence of SEQ ID NO: 15 and a VL sequence including the amino acid sequence of SEQ ID NO: 40; and the The Fc region includes a CH3 domain comprising the amino acid sequence of SEQ ID NO: 532. The multispecific antibody of any one of the preceding claims, wherein the Fab region includes a VH including the amino acid sequence of SEQ ID NO: 15 and a VL sequence including the amino acid sequence of SEQ ID NO: 40; and the The Fc region includes a CH3 domain comprising the amino acid sequence of SEQ ID NO: 522. The multispecific antibody of any one of the preceding claims, wherein the Fab region includes a VH including the amino acid sequence of SEQ ID NO: 15 and a VL sequence including the amino acid sequence of SEQ ID NO: 40; and the The Fc region includes a CH3 domain comprising the amino acid sequence of SEQ ID NO: 541. The multispecific antibody of any one of the preceding claims, wherein the Fab region includes a VH including the amino acid sequence of SEQ ID NO: 15 and a VL sequence including the amino acid sequence of SEQ ID NO: 40; and the The Fc region includes a CH3 domain comprising the amino acid sequence of SEQ ID NO: 510. The multispecific antibody of any one of the preceding claims, wherein the Fab region includes a VH including the amino acid sequence of SEQ ID NO: 15 and a VL sequence including the amino acid sequence of SEQ ID NO: 40; and the The Fc region includes a CH3 domain comprising the amino acid sequence of SEQ ID NO: 552. The multispecific antibody according to any one of the preceding claims, characterized in that: a. Cause down-regulation of TCR on the Vδ1 T cells to which it binds; b. No CDC or ADCC is displayed; and c. Vδ1 T cells are not depleted. The multispecific antibody of any one of the preceding claims, wherein the multispecific antibody causes depletion of less than about 30%, or less than about 20%, or less than about 10% of the viable Vδ1 T+ cell population via ADCC and/or CDC. The multispecific antibody of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof binds to human TRVD1 with a KD of less than about 10 nM, optionally wherein the antibody or antigen-binding fragment thereof binds to human TRVD1 with a KD of less than about 100 nM. KD binds to cynomolgus monkey TRVD1. The multispecific antibody of any one of the preceding claims, which specifically binds to the human variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR) with a KD of less than about 10 nM, optionally wherein the anti-Vδ1 The antibody or antigen-binding fragment thereof binds to the cynomolgus variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR) with a KD of less than about 100 nM, and/or specifically binds to the cynomolgus monkey variable delta 1 (Vδ1) chain with a _KD of less than about 150 nM. human EGFR. The multispecific antibody of any one of the preceding claims, which specifically binds to the human variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR) with a KD of less than about 10 nM, optionally wherein the anti-Vδ1 The antibody or antigen-binding fragment thereof binds to the cynomolgus variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR) with a K of less than about 100 nM, and/or specifically binds to the cynomolgus monkey variable delta 1 ( _Vδ1 ) chain with a K of less than about 20 nM. Human EGFR. The multispecific antibody of any one of the preceding claims, which: a. has a binding affinity (K _D , for example, as measured by surface plasmon resonance) of less than about 100 nM (preferably less than about 50 nM) Binds to human TRDV1 (SEQ ID NO 272 or 306); b. Optionally binds with a binding affinity (K _D , e.g., as measured by surface plasmon resonance) of less than about 100 nM (preferably less than about 50 nM) In cynomolgus monkey TRDV1 (SEQ ID NO 308); c. The IC50 for down-regulating TCR is less than about 50 nM (preferably less than about 10 nM). The multispecific antibody of any one of the preceding claims, wherein the Fab region only binds to amino acid residues 1-90 of SEQ ID NO: 272, SEQ ID NO: 306 and/or SEQ ID NO: 308 of epitopes. The multispecific antibody of any one of the preceding claims, wherein the Fab region binds to an epitope within the following amino acid region: (i) SEQ ID NO: 272, SEQ ID NO: 306 and/or SEQ ID NO: 308-3-20; and/or (ii) SEQ ID NO: 272, SEQ ID NO: 306 and/or 37-77 of SEQ ID NO: 308. The multispecific antibody of any one of the preceding claims, wherein the Fab region binds to an activating epitope of a γδ T cell, and optionally the binding of the activating epitope: (i) downregulates the γδ TCR; (ii) ) activates degranulation of the γδ T cell; and/or (iii) promotes γδ T cell-mediated killing. The multispecific antibody of claim 13, wherein binding of the activating epitope upregulates the expression of CD107a, CD25, CD69 and/or Ki67. The multispecific antibody according to any one of the preceding claims, wherein the multispecific antibody is a bispecific antibody. The multispecific antibody according to any one of the preceding claims, wherein the multispecific antibody is not a bispecific antibody. A pharmaceutical composition comprising the multispecific antibody according to any one of claims 1 to 33 and a pharmaceutically acceptable diluent or carrier. A method of treating a disease or condition in an individual or a method of modulating an immune response in an individual, comprising administering to the individual a multispecific antibody according to any one of claims 1 to 33 or a pharmaceutical composition according to claim 39 . The method of claim 35, wherein the disease or condition is cancer, or wherein the individual modulating the immune response has cancer. The method of claim 36, wherein the cancer is an EGFR positive cancer. The method of claim 35, wherein regulating the immune response of the individual includes at least one selected from the group consisting of: activating γδ T cells, causing or increasing the proliferation of γδ T cells, causing or increasing the expansion of γδ T cells, causing Or increase γδ T cell degranulation, cause or increase γδ T cell-mediated killing activity, cause or increase γδ T cell toxicity, cause or increase γδ T cell mobilization, increase the survival of γδ T cells, and increase the response to γδ T cell exhaustion. Resistance. The method of any one of claims 36 to 38, wherein diseased cells are killed while healthy cells are avoided. The multispecific antibody according to any one of claims 1 to 33 or the pharmaceutical composition according to claim 23, which is used to treat cancer.

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