WO2024236163A1

WO2024236163A1 - T cell receptor beta constant region 2 (trbc2) antibodies

Info

Publication number: WO2024236163A1
Application number: PCT/EP2024/063649
Authority: WO
Inventors: Annika SCHMID; Lisa EBELER; Dominik ALTERAUGE; Stefanie WACHTER
Original assignee: Morphosys AG
Current assignee: Morphosys AG
Priority date: 2023-05-17
Filing date: 2024-05-17
Publication date: 2024-11-21
Anticipated expiration: 2025-11-17

Abstract

The present disclosure provides novel human antibodies that specifically bind to the TCR beta constant region 2 (TRBC2) with high affinity and selectivity.

Description

T CELL RECEPTOR BETA CONSTANT REGION 2 (TRBC2) ANTIBODIES

FIELD OF THE INVENTION

The present invention relates to novel human antibodies that bind specifically to the TCR beta constant region 2 (TRBC2) with high affinity and selectivity. The present invention also relates to nucleic acids, vectors and host cells capable of expressing said antibodies, pharmaceutical compositions comprising said antibodies and uses of said antibodies or antibody fragments thereof for the treatment of specific disorders and/or for diagnostic purposes.

BACKGROUND OF THE INVENTION

Lymphoid malignancies can largely be divided into those which are derived from either T cells or B cells. T cell malignancies are a clinically and biologically heterogeneous group of disorders, together comprising 10-20% of non-Hodgkin’s lymphomas. The most commonly identified histological subtypes are peripheral T cell lymphoma, not otherwise specified (PTCL-NOS), angio- immunoblastic T cell lymphoma (AITL) and anaplastic large cell lymphoma (ALCL).

There is currently no effective, minimally toxic immunotherapeutic available for the treatment of T cell malignancies. An important difficulty in the development of immunotherapy for T cell disorders is the considerable overlap in marker expression of clonal and normal T cells, with no single antigen clearly able to identify clonal (malignant) cells. A method for treating T cell lymphomas and leukaemias consisting in targeting the constant region of the TCR beta chain (TRBC) has been previously described in WO2015/132598. This approach is based on the finding that each T cell receptor (TCR) make use of either the TCR beta constant region 1 (TRBC1) or TCR beta constant region 2 (TRBC2), in a mutually exclusive fashion. Because T cell lymphomas and leukaemias are a clonal population of cells, each lymphoma will express one TCR, with either TRBC1 or TRBC2 on the surface.

The TCR itself is a heterodimer normally consisting of the variable alpha and beta chain expressed as part of a complex with the invariant CD3 chains. T cells expressing this receptor are referred to as ct:p (or ct|3) T cells (~95% total T cells). Each alpha and beta chain is composed of two extracellular domains: A variable (V) region and a Constant (C) region. The variable regions can exist in many millions of different sequences. In contrast, all alpha constant regions are identical. However, the beta constant region may exist in two forms known as TRBC1 and TRBC2. TRBC1 and TRBC2 differ by only 4 amino acids in the mature protein. Accordingly, each TCR will comprise, in a mutually exclusive fashion, either TRBC1 or TRBC2 and as such, each ctp T cell will express either TRBC1 or TRBC2, in a mutually exclusive manner.

The monoclonal antibody JOVI-1 (Viney et al., 1992 Hybridoma) specifically recognizes TRBC1 and has been used as a chimeric antigen receptor (CAR) binding domain for a therapy treating T- cell lymphomas (Maciocia et ah, 2017, Nat Med 23: 1416-23; WO2015/132598). This proposed therapy allows for the treatment of a subset of patients that express a TCR with the TRBC1 constant region.

However, in order to treat the entire patient population, an antibody specifically targeting TRBC2 is necessary. WO2015/132598 discloses TRBC2 specific antibodies, which were identified using a human phage display library on TRBC2-derived peptides or by immunizing animals with TRBC2 derived peptides, spanning 2 of the 4 amino acid differences between TRBC1 and TRBC2. However, no evidence was provided that any of these antibodies were able to bind to the alpha/beta T cell receptor protein complex.

Indeed, the present inventors encountered that phage selections on TRBC2 peptides using the commercially available MorphoSys Ylanthia® library resulted in multiple TRBC2 specific peptide binders with none of them being able to recognize the native protein complex. In contrast, only phage selection on the alpha/beta TCR complex (for example as soluble protein or expressed on T cells) resulted in only 1 highly TRBC2 specific cell binder. Interestingly, this antibody is also not able to bind to a linear TRBC2 peptide. In line with these findings, the inventors of the present disclosure also determined that the TRBC1 specific antibody JOVI-1 is not able to recognize linear TRBC1 peptides.

To the best knowledge, the only monoclonal antibodies with specificity for TRBC2 recognizing the TCR protein complex are described in W02020/089644. These antibodies are humanized variants of JOVI-1 , engineered to become TRBC2 selective (ONUOHA SHIMOBI ET AL., BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol 132, 29 November 2018 (2018-1 1-29), page 1661). However, these antibodies reveal only modest affinity on TRBC2 and selectivity over TRBC1.

CN115975034 describes TRBC2 antibodies. However, these antibodies also lack selectivity over TRBC1 and no evidence in provided that the disclosed antibodies can discriminate TRBC2 positive cells from TRBC1 positive cells.

W02021/191607 discloses engineered TRBC2 specific antibodies based on antibodies described in W02020/089644 with improved internalization properties but lower affinity binding. However, and as evidenced in the examples of the present disclosure, these variant TRBC2 antibodies are less superior in terms of internalization and toxin mediated killing of TRBC2+ cancer cell lines when compared to the high affinity antibodies of the present invention.

The present invention provides for the first time, fully human TRBC2 specific antibodies, which are able to bind to the native TCR expressing the TRBC2 phenotype. In particular, the antibodies of the present disclosure bind to TRBC2 with high affinity and selectivity with safety properties never observed before, thus making the antibodies of the present disclosure highly desirable for therapeutic use, such as for preventing and/or treating cancer, in particular T cell lymphomas or leukaemias.

SUMMARY OF THE INVENTION

The present disclosure provides novel human antibodies and antibody fragments specific for TRBC2.

The antibodies and antibody fragments disclosed herein can specifically bind to human TRBC2 and are able to discriminate T cells expressing T cell receptors making use of TRBC2 from T cells expressing TCRs making use of TRBC1 on their cell surface.

Accordingly, the antibodies and antibody fragments specific for human TRBC2 according to the present disclosure can be used in a variety of therapeutic formats, including monoclonal antibodies being able to induce effector function (such as ADCC or ADCP), chimeric antigen receptors (CARs), antibody-drug conjugates (ADCs) and bispecific T cell engagers, to deplete or kill malignant TRBC2 expressing T cells in a subject, without affecting healthy T cells expressing the TRBC1 phenotype.

The present disclosure provides isolated antibodies or antibody fragments that specifically bind to human TRBC2 having CDR regions according to Table 1 or Table 2 of the present specification. The present disclosure also provides isolated antibodies or antibody fragments specific for human TRBC2 having an antibody variable heavy chain region (VH) and an antibody variable light chain region (VL) comprising the amino acid sequences according to Table 1 or Table 2 of the present specification. The present disclosure also provides isolated antibodies or antibody fragments specific for TRBC2 having a heavy chain (HC) and a light chain (LC) comprising the amino acid sequences according to Table 1 or Table 2 of the present specification.

The isolated antibodies of the present disclosure may not induce effector function. In particular, the isolated antibodies of the present disclosure may comprise a variant human IgG 1 Fc region, which comprises the following amino acid substitutions: L234A, L235E, G237A, A330S and P331S with numbering according EU index. In other aspects of the present disclosure, the isolated antibodies of the present disclosure may induce effector function. In an embodiment, such effector function may comprise ADCP, ADCC and/or CDC.

The present disclosure also provides fusion molecules comprising the isolated antibody or antibody fragment of the present disclosure and at least one additional moiety. In some embodiments, such additional moiety may be a therapeutic agent, such as a drug, a cytotoxic molecule or chemotherapeutic molecule, an antibody or antibody fragment, or a T cell expressing a chimeric antigen receptor (CAR-T). In some embodiments, such additional moiety may be a detectable group or a tag suitable for detection. In preferred embodiments, such additional moiety is a cytotoxic agent or a detectable group.

The present disclosure also provides the isolated antibodies, antibody fragments or the fusion molecules of the present disclosure for use in medicine. The present disclosure also provides methods for treating a subject suffering from a disease, such as cancer, preferably TRBC2 positive lymphomas or leukaemia’s, by administering to said subject an effective amount of an antibody, antibody fragment or fusion molecule according to the present disclosure. Preferably, said fusion molecule comprises a cytotoxic molecule as the additional moiety. Preferably, said subject is human.

The present disclosure also provides pharmaceutical compositions comprising the isolated antibody, antibody fragment or fusion molecule according to the present disclosure, and a pharmaceutically acceptable carrier or excipient.

Utilization of the claimed antibodies or antibody fragments is to differentiate between T cells expressing T cell receptors making use of TRBC2 from T cells expressing T cell receptors making use of TRBC1. In particular, the claimed antibodies or antibody fragments are for therapeutic use, such as the treatment of TRBC2 positive lymphomas or leukaemias.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Cell binding of the TRBC2 specific antibodies mAb1 , mAb2. Fig. 1 A - C show binding as a function of antibody concentration as determined on TRBC2 positive primary human T cells (Fig. 1A, Fig. 1 C) or TRBC1 positive primary human T cells (Fig. 1 B) by flow cytometry. As reference control antibodies RefmAb and the neg. Ctrl antibody (Ctrl) were included. Figure 2: Depicts FACS plots of the same cell binding studied indicated in Fig. 1A and Fig. 1 B. Shown is a co-staining of human primary T cells with TRBC1 antibody JOVI-1 and mAb1 (lower panel) or mAb2 (upper panel), respectively.

Figure 3: Internalization of the TRBC2 specific antibodies mAb1 , mAb2 and RefmAb. Shown is internalization after 24h as a function of antibody concentration as determined on TRBC2 positive primary human T cells (Fig. 3A), TRBC1 positive primary human T cells (Fig. 3B), and TRBC2 positive HBP-ALL cells (Fig. 3C).

Figure 4: In vitro cytotoxicity assay for mAb1-MMAE and mAb2-MMAE and neg. Ctrl antibody- MMAE (Ctrl-MMAE) as direct toxin conjugates (ADCs) on TRBC2 positive HBP-ALL cells (Fig. 4A), TRBC1 positive H9 cells (Fig. 4B) and TRBC2 positive primary human T cells (Fig. 4C). Cytotoxic activity of ADCs was assessed by measuring CellToxGreen luminescence (ELISA) or AnnexinV fluorescence (FACS). Figure 4 graphs show the relative fluorescence or luminescence of respective cells as a function of ADC concentration.

Figure 5: In vitro cytotoxicity assay for mAb1 as secondary anti-Fab-Fc Saporin toxin conjugate on TRBC2 positive HBP-ALL cells. Cytotoxic activity of ADCs was assessed by measuring CellToxGreen luminescence (ELISA). As reference control antibodies RefmAb and the neg. Ctrl antibody (Ctrl) were included. Figure 5 graph shows the relative fluorescence of cancer cells as a function of antibody concentration.

Figure 6: In vivo efficacy study for mAb1-MMAE ADC in an HBP-ALL derived mouse xenograft model. As reference the neg. Ctrl antibody-MMAE (Ctrl-MMAE) was included. Fig. 6 shows tumor volume as a function of days after tumor cell injection. ADCs were injected at a single dose at day 14.

Figure 7: ELISA-based TNFalpha and IFNgamma cytokine release assay on whole-blood cells. Ability of effector-compromised mAb1_lgG1_AEASS and mAb2_lgG1_AEASS to induce cytokine secretion of immune cells was compared to the ability of corresponding effector-competent mAb1_lgG1 . TNFalpha (upper panel of Fig. 7) and IFNgamma release (lower panel of Fig. 7) was assessed by ELISA after 24h of cell treatment with IgG. Fig. 7 indicates the concentration of released of TNFalpha or IFNgamma, respectively, as a function of antibody concentration. As positive control, the anti-CD3 antibody OKT-3 was included. DETAILED DESCRIPTION OF THE INVENTION

The disclosure pertains to a number of human antibodies or antibody fragments, which specifically recognize human TRBC2.

DEFINITIONS

The term “TRBC2” refers to a protein known as T cell receptor beta constant region 2.

Human TRBC2 (Uniprot: A0A5B9|1-178) has the amino acid sequence of: DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLK EQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAW GRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 39)

The term “TRBC1” refers to a protein known as T cell receptor beta constant region 1 .

Human TRBC1 (Uniprot: P01850| 1 -176) has the amino acid sequence of: DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLK EQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAW GRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 40)

The term “TRAC” refers to a protein known as T cell receptor alpha constant region.

Human TRAC (Uniprot: P01848| 1 -140) has the amino acid sequence of:

IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVA WSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGF NLLMTLRLWSS (SEQ ID NO: 41)

The term “antibody” as used herein refers to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, which interacts with an antigen. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FR’s arranged from amino-terminus to carboxy- termin us in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term “antibody” includes for example, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies and chimeric antibodies. The antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.

The phrase “antibody fragment”, as used herein, refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing spatial distribution) an antigen. Examples of binding fragments include, but are not limited to, a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antibody fragment”. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136). Antibody fragments can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). Antibody fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding sites (Zapata et al., (1995) Protein Eng. 8:1057-1062; and U.S. Pat. No. 5,641 ,870).

A “human antibody”, as used herein, is an antibody and antibody fragment having variable regions in which both the framework and CDR regions are from sequences of human origin. Human antibodies can also be isolated from synthetic libraries or from transgenic mice (e.g. Xenomouse) provided the respective system yield in antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such sequences. Human origin includes, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., (2000) J Mol Biol 296:57-86). The structures and locations of immunoglobulin variable domains, e.g., CDRs, may be defined using well known numbering schemes, e.g., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, e.g. Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1991), eds. Kabat et al.; Lazikani et al., (1997) J. Mol. Bio. 273:927-948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mol. Biol. 273:927-948.

A “humanized antibody” is defined herein as an antibody molecule, which has constant antibody regions derived from sequences of human origin and the variable antibody regions or parts thereof or only the CDRs are derived from another species. For example, a humanized antibody can be CDR-grafted, wherein the CDRs of the variable domain are from a non-human origin, while one or more frameworks of the variable domain are of human origin and the constant domain (if any) is of human origin.

The term "chimeric antibody" is defined herein as an antibody molecule, which has constant antibody regions derived from, or corresponding to, sequences found in one species and variable antibody regions derived from another species. Preferably, the constant antibody regions are derived from, or corresponding to, sequences found in humans, and the variable antibody regions (e.g. VH, VL, CDR or FR regions) are derived from sequences found in a non-human animal, e.g. a mouse, rat, rabbit or hamster.

The term “synthetic antibody” is defined herein as an antibody molecule that is made outside of the human body by synthesis.

The term "isolated antibody” refers to an antibody molecule that is substantially free of other antibodies having different antigenic specificities. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. Thus, in some aspects, antibodies provided are isolated antibodies, which have been separated from antibodies with a different specificity. An isolated antibody may be a monoclonal antibody. An isolated antibody may be a recombinant monoclonal antibody. An isolated antibody that specifically binds to an epitope, isoform or variant of a target may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., species homologs).

The term "recombinant antibody", as used herein, includes all antibodies that are prepared, expressed, created or segregated by means not existing in nature. For example, antibodies isolated from a host cell transformed to express the antibody, antibodies selected and isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DNA sequences or antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom. Preferably, such recombinant antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.. A recombinant antibody may be a monoclonal antibody. In an embodiment, the antibodies and antibody fragment disclosed herein are isolated from the HuCAL library (Rothe et al, J. Mol. Biol. (2008) 376, 1182-1200).

As used herein, an antibody “binds specifically to”, “specifically binds to”, is “specific to/for” or “specifically recognizes” an antigen, such as human TRBC2, if such antibody is able to discriminate between such antigen and one or more reference antigen(s), since binding specificity is not an absolute, but a relative property. For example, a standard ELISA assay can be carried out. The scoring may be carried out by standard color development (e.g. secondary antibody with horseradish peroxide and tetramethyl benzidine with hydrogen peroxide). The reaction in certain wells is scored by the optical density, for example, at 450 nm. Typical background (=negative reaction) may be 0.1 OD; typical positive reaction may be 1 OD. This means the difference positive/negative can be more than 10-fold. Typically, determination of binding specificity is performed by using not a single reference antigen, but a set of about three to five unrelated antigens, such as milk powder, BSA, transferrin or the like. As used herein, the term "affinity" refers to the strength of interaction between the polypeptide and its target at a single site. Within each site, the binding region of the polypeptide interacts through weak non-covalent forces with its target at numerous sites; the more interactions, the stronger the affinity.

The term “K_D”, as used herein, refers to the equilibrium dissociation constant, which is obtained from the ratio of k_Off to k_on (i.e. k₀ff/k_0n) and is expressed as a molar concentration (M). K_D values for antigen binding moieties like e.g. monoclonal antibodies can be determined using methods well established in the art. Methods for determining the KD of an antigen binding moiety like e.g. a monoclonal antibody are SET (solution equilibrium titration) or surface plasmon resonance using a biosensor system such as a Biacore® system. In the present disclosure an antibody specific for TRBC2 typically has a dissociation rate constant (K_D) (k₀ff/k_0n) of less than 5x10'²M, less than 10' ²M, less than 5x10'³M, less than 10'³M, less than 5x10'⁴M, less than 10'⁴M, less than 5x10'⁵M, less than 10'⁵M, less than 5x10'⁶M, less than 10'⁶M, less than 5x10'⁷M, less than 10'⁷M, less than 5x10' ⁸M, less than 10'⁸M, less than 5x10'⁹M, less than 10'⁹M, less than 5x10'¹⁰M, less than 10'¹⁰M, less than 5x10'¹¹M, less than

less than 5x10'¹²M, less than 10'¹²M, less than 5x10'¹³M, less than 10'¹³M, less than 5x10'¹⁴M, less than 10'¹⁴M, less than 5x10'¹⁵M, or less than 10-¹⁵M or lower.

The term “epitope” includes any proteinaceous region which is specifically recognized by an antibody or otherwise interacts with a molecule. Generally, epitopes are of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally may have specific three-dimensional structural characteristics, as well as specific charge characteristics. As will be appreciated by one of skill in the art, practically anything to which an antibody can specifically bind could be an epitope.

“Compositions” of the present disclosure may be used for therapeutic or prophylactic applications. The present disclosure, therefore, includes a pharmaceutical composition containing an antibody or antibody fragment as disclosed herein and a pharmaceutically acceptable carrier or excipient therefor. In a related aspect, the present disclosure provides a method for treating cancer. Such method contains the steps of administering to a subject in need thereof an effective amount of the pharmaceutical composition that contains an antibody or antibody fragment as described herein. The present disclosure provides therapeutic methods comprising the administration of a therapeutically effective amount of an antibody or antibody fragment as disclosed herein to a subject in need of such treatment. A "therapeutically effective amount” or “effective amount”, as used herein, refers to the amount of a TRBC2 antibody necessary to elicit the desired biological response. In accordance with the subject disclosure, the therapeutic effective amount is the amount of a TRBC2 antibody necessary to treat and/or prevent a disease.

"Administered" or “administration” includes but is not limited to delivery of a drug by an injectable form, such as, for example, an intravenous, intramuscular, intradermal or subcutaneous route or mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestible solution, capsule or tablet. Preferably, the administration is by an injectable form.

As used herein, "treatment", "treat" or "treating" and the like refers to clinical intervention in an attempt to alter the natural course of a disease in the subject being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies or antibody fragments according to the preset disclosure are used to delay development of a disease or to slow the progression of a disease.

The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Non-limiting examples of antibody effector functions include C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), down regulation of cell surface receptors (e.g. B cell receptor) and B cell activation.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g. NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII, and FcyRIII.

"Complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies of the present disclosure (having the appropriate subclass), which are bound to their cognate antigen, such as TRBC2. “Antibody-dependent cellular phagocytosis” or “ADCP” refers to a mechanism of elimination of antibody-coated target cells by internalization by phagocytic cells, such as macrophages or dendritic cells.

“Preventing” or “prevention” refers to a reduction in risk of acquiring or developing a disease (i.e. causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset). “Prevention” also refers to methods which aim to prevent the onset of a disease or its symptoms or which delay the onset of a disease or its symptoms.

“Subject” or “species” as used herein, refers to any mammal, including rodents (such as mouse or rat), and primates (such as cynomolgus monkey (Macaca fascicularis), rhesus monkey (Macaca mulatta) or humans (Homo sapiens)). Preferably, the subject is a primate, most preferably a human.

Throughout this specification, unless the context requires otherwise, the words "comprise", “have” and “include” and their respective variations such as "comprises", "comprising", “has”, “having”, “includes” and “including” will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The terms "engineered" or “modified” as used herein, includes manipulation of nucleic acids or polypeptides by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, enzymatic or chemical coupling of peptides or some combination of these techniques). Preferably, the antibodies or antibody fragments according to the present disclosure are engineered or modified to improve one or more properties, such as antigen binding, stability, half-life, effector function, immunogenicity, safety and the like.

A "Variant" as used herein refers to a polypeptide or protein that differs from a reference polypeptide or protein by one or more modifications for example amino acid substitutions, insertions or deletions.

The term "amino acid mutation" as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made as long as the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor. Amino acid sequence deletions and insertions include N- and/or C-terminal deletions and insertions of amino acid residues. Particular amino acid mutations are amino acid substitutions. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PGR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid residue by methods other than genetic engineering, such as chemical modification, may also be useful. Various designations may be used herein to indicate the same amino acid mutation. For example, a substitution from glycine at position 327 of the Fc region to alanine can be indicated as 237A, G337, G337A, or Gly329Ala.

The term “EC₅o” as used herein, refers to the concentration of an antibody or antibody fragment or ligand, which induces a response in an assay half way between the baseline and maximum. It therefore represents the antibody or ligand concentration at which 50% of the maximal effect is observed.

The term “IC₅o” as used herein, refers to the concentration of an antibody or antibody fragment that inhibits a response in an assay half way between the maximal response and the baseline. It represents the antibody concentration that reduces a given response by 50%.

The terms "inhibition" or "inhibit" or “reduction” or “reduce” or “neutralization” or “neutralize” refer to a decrease or cessation of any phenotypic characteristic (such as binding or a biological activity or function) or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. The “inhibition”, “reduction” or “neutralization” needs not to be complete as long as it is detectable using an appropriate assay. In some embodiments, by "reduce" or "inhibit" or “neutralize” is meant the ability to cause a decrease of 20% or greater. In another embodiment, by "reduce" or "inhibit" or “neutralize” is meant the ability to cause a decrease of 50% or greater. In yet another embodiment, by "reduce" or "inhibit" or “neutralize” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.

A “wild-type” protein or “wt” protein is a version or variant of the protein as it is found in nature. An amino acid sequence of a wildtype protein, e.g., a Fc region of a human IgG 1 antibody, is the amino acid sequence of the protein as it occurs in nature. Due to allotypic differences, there can be more than one amino acid sequence for a wildtype protein. For example, there are several allotypes of naturally occurring human IGg1 heavy chain constant regions (see, e.g., Jeffries et al. (2009) mAbs 1 :1).

The “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

A “fusion molecule" as meant herein refers to an antibody or antibody fragment as described herein, linked or fused or conjugated to at least one further moiety. Such moiety may have a different property compared to the antibody. Such property may be a biological property, such as in vitro or in vivo activity. The property may also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction and the like. The fusion, linkage or conjugation may be covalent or non-covalent.

Embodiments

In an embodiment, the present disclosure pertains to an isolated antibody or antibody fragment specific for human TRBC2 comprising 6 CDRs defined according Kabat of any one of the antibodies disclosed in Table 1 or Table 2 of the present specification. In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2 comprising 6 CDRs defined according Chothia of any one of the antibodies disclosed in Table 1 or Table 2 of the present specification. In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2 comprising a VH and a VL comprising the 6 CDRs of any one of the antibodies disclosed in Table 1 or Table 2 of the present specification.

In an embodiment, the present disclosure pertains to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises a VH comprising the HCDR1 region of SEQ ID NO: 1 , the HCDR2 region of SEQ ID NO: 2, the HCDR3 region of SEQ ID NO: 3, and a VL comprising the LCDR1 region of SEQ ID NO: 4 or SEQ ID NO: 31 , the LCDR2 region of SEQ ID NO: 5 and the LCDR3 region of SEQ ID NO: 6.

In an embodiment, the present disclosure pertains to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 1 , the HCDR2 region of SEQ ID NO: 2, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 4 or SEQ ID NO: 31 , the LCDR2 region of SEQ ID NO: 5 and the LCDR3 region of SEQ ID NO: 6. In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises a) the HCDR1 region of SEQ ID NO: 1 , the HCDR2 region of SEQ ID NO: 2, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 4, the LCDR2 region of SEQ ID NO: 5 and the LCDR3 region of SEQ ID NO: 6, or b) the HCDR1 region of SEQ ID NO: 1 , the HCDR2 region of SEQ ID NO: 2, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 31 , the LCDR2 region of SEQ ID NO: 5 and the LCDR3 region of SEQ ID NO: 6.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises a) a VH comprising the HCDR1 region of SEQ ID NO: 1 , the HCDR2 region of SEQ ID NO: 2, the HCDR3 region of SEQ ID NO: 3, and a VL comprising the LCDR1 region of SEQ ID NO: 4, the LCDR2 region of SEQ ID NO: 5 and the LCDR3 region of SEQ ID NO: 6, or b) a VH comprising the HCDR1 region of SEQ ID NO: 1 , the HCDR2 region of SEQ ID NO: 2, the HCDR3 region of SEQ ID NO: 3, and a VL comprising the LCDR1 region of SEQ ID NO: 31 , the LCDR2 region of SEQ ID NO: 5 and the LCDR3 region of SEQ ID NO: 6.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises a VH comprising the HCDR1 region of SEQ ID NO: 7, the HCDR2 region of SEQ ID NO: 8, the HCDR3 region of SEQ ID NO: 3, and a VL comprising the LCDR1 region of SEQ ID NO: 9 or SEQ ID NO: 32, the LCDR2 region of AAS and the LCDR3 region of SEQ ID NO: 11.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 7, the HCDR2 region of SEQ ID NO: 8, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 9 or SEQ ID NO: 32, the LCDR2 region of AAS and the LCDR3 region of SEQ ID NO: 11 .

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises a) the HCDR1 region of SEQ ID NO: 7, the HCDR2 region of SEQ ID NO:8, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 9, the LCDR2 region of AAS and the LCDR3 region of SEQ ID NO: 11 , or b) the HCDR1 region of SEQ ID NO: 7, the HCDR2 region of SEQ ID NO:8, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of AAS and the LCDR3 region of SEQ ID NO: 11 .

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises a) a VH comprising the HCDR1 region of SEQ ID ID NO: 7, the HCDR2 region of SEQ ID NO:8, the HCDR3 region of SEQ ID NO: 3, and a VL comprising the LCDR1 region of SEQ ID NO: 9, the LCDR2 region of AAS and the LCDR3 region of SEQ ID NO: 11 , or b) a VH comprising the HCDR1 region of SEQ ID NO: 7, the HCDR2 region of SEQ ID NO:8, the HCDR3 region of SEQ ID NO: 3, and a VL comprising the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of AAS and the LCDR3 region of SEQ ID NO: 11.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises a) the HCDR1 region of SEQ ID NO: 1 , the HCDR2 region of SEQ ID NO: 2, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 4, the LCDR2 region of SEQ ID NO: 5 and the LCDR3 region of SEQ ID NO: 6, or b) the HCDR1 region of SEQ ID NO: 1 , the HCDR2 region of SEQ ID NO: 2, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 31 , the LCDR2 region of SEQ ID NO: 5 and the LCDR3 region of SEQ ID NO: 6, or c) the HCDR1 region of ID NO: 7, the HCDR2 region of SEQ ID NO:8, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 9, the LCDR2 region of AAS and the LCDR3 region of SEQ ID NO: 11 , or d) the HCDR1 region of SEQ ID NO: 7, the HCDR2 region of SEQ ID NO:8, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of AAS and the LCDR3 region of SEQ ID NO: 11 .

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1 , the HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, the HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, the LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 31 , the LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5 and the LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises a) the HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1 , the HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, the HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, the LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, the LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5 and the LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, or b) the HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1 , the HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, the HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, the LCDR1 region comprising the amino acid sequence of SEQ ID NO: 31 , the LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5 and the LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the HCDR1 region comprising the amino acid sequence of SEQ ID NO: 7, the HCDR2 region comprising the amino acid sequence of SEQ ID NO: 8, the HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, the LCDR1 region comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 32, the LCDR2 region comprising the amino acid sequence of AAS and the LCDR3 region comprising the amino acid sequence of SEQ ID NO: 11.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises a) the HCDR1 region comprising the amino acid sequence of SEQ ID NO: 7, the HCDR2 region comprising the amino acid sequence of SEQ ID NO: 8, the HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, the LCDR1 region comprising the amino acid sequence of SEQ ID NO: 9, the LCDR2 region comprising the amino acid sequence of AAS and the LCDR3 region comprising the amino acid sequence of SEQ ID NO: 11 , or b) the HCDR1 region comprising the amino acid sequence of SEQ ID NO: 7, the HCDR2 region comprising the amino acid sequence of SEQ ID NO: 8, the HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, the LCDR1 region comprising the amino acid sequence of SEQ ID NO: 32, the LCDR2 region comprising the amino acid sequence of AAS and the LCDR3 region comprising the amino acid sequence of SEQ ID NO: 11.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises a) the HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1 , the HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, the HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, the LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, the LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5 and the LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, or b) the HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1 , the HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, the HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, the LCDR1 region comprising the amino acid sequence of SEQ ID NO: 31 , the LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5 and the LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, or c) the HCDR1 region comprising the amino acid sequence of SEQ ID NO: 7, the HCDR2 region comprising the amino acid sequence of SEQ ID NO: 8, the HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, the LCDR1 region comprising the amino acid sequence of SEQ ID NO: 9, the LCDR2 region comprising the amino acid sequence of AAS and the LCDR3 region comprising the amino acid sequence of SEQ ID NO: 11 , or c) the HCDR1 region comprising the amino acid sequence of SEQ ID NO: 7, the HCDR2 region comprising the amino acid sequence of SEQ ID NO: 8, the HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, the LCDR1 region comprising the amino acid sequence of SEQ ID NO: 32, the LCDR2 region comprising the amino acid sequence of AAS and the LCDR3 region comprising the amino acid sequence of SEQ ID NO: 11.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 1 , the HCDR2 region of SEQ ID NO: 2, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 4, the LCDR2 region of SEQ ID NO: 5 and the LCDR3 region of SEQ ID NO: 6.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 1 , the HCDR2 region of SEQ ID NO: 2, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 31 , the LCDR2 region of SEQ ID NO: 5 and the LCDR3 region of SEQ ID NO: 6.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 7, the HCDR2 region of SEQ ID NO: 8, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 9, the LCDR2 region of AAS and the LCDR3 region of SEQ ID NO: 11.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 7, the HCDR2 region of SEQ ID NO: 8, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of AAS and the LCDR3 region of SEQ ID NO: 11.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2 comprising the variable heavy chain (VH) and the variable light chain (VL) of any one of the antibodies disclosed in Table 1 or Table 2 of the present specification.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the VH of SEQ ID NO: 12 and the VL of SEQ ID NO: 13 or SEQ ID NO: 33.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises a) the VH of SEQ ID NO: 12 and the VL of SEQ ID NO: 13 or b) the VH of SEQ ID NO: 12 and the VL of SEQ ID NO: 33.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the VH of SEQ ID NO: 12 and the VL of SEQ ID NO: 13.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the VH of SEQ ID NO: 12 and the VL of SEQ ID NO: 33.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2 comprising the heavy chain (HO) and the light chain (LC) of any one of the antibodies disclosed in Table 1 or Table 2 of the present specification. In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the HC of SEQ ID NO: 14 and the LC of SEQ ID NO: 15 or SEQ ID NO: 34.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the HC of SEQ ID NO: 14 and the LC of SEQ ID NO: 15.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the HC of SEQ ID NO: 14 and the LC of SEQ ID NO: 34.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 1 , the HCDR2 region of SEQ ID NO: 2, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 4 or SEQ ID NO: 31 , the LCDR2 region of SEQ ID NO: 5 and the LCDR3 region of SEQ ID NO: 6, and further comprises the VH of SEQ ID NO: 12 and the VL of SEQ ID NO: 13 or SEQ ID NO: 33.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises the HCDR1 region of ID NO: 7, the HCDR2 region of SEQ ID NO: 8, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 9 or SEQ ID NO: 32, the LCDR2 region of AAS and the LCDR3 region of SEQ ID NO: 11 , and further comprises the VH of SEQ ID NO: 12 and the VL of SEQ ID NO: 13 or SEQ ID NO: 33.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises a VH that has at least 80%, at least 85%, at least 90% or at least 95% identity to the VH of SEQ ID NO: 12 and a VL that has at least 80%, at least 85%, at least 90% or at least 95% identity to the VL of SEQ ID NO: 13 or SEQ ID NO: 33.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment comprises a HC that has at least 80%, at least 85%, at least 90% or at least 95% identity to the HC of SEQ ID NO: 14 and a LC that has at least 80%, at least 85%, at least 90% or at least 95% identity to the LC of SEQ ID NO: 15 or SEQ ID NO: 34 In an embodiment of the present disclosure, said isolated antibody or antibody fragment is a monoclonal antibody or antibody fragment. In an embodiment of the present disclosure, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment. In an embodiment of the present disclosure, said isolated antibody or antibody fragment is a human antibody or antibody fragment. In another embodiment of the present disclosure, said isolated antibody or antibody fragment is recombinant or synthetic antibody or antibody fragment. In an embodiment of the present disclosure, the isolated antibody or antibody fragment is of the IgG isotype. In an embodiment of the present disclosure, the isolated antibody or antibody fragment is of the lgG1 class. In another embodiment of the present disclosure, said isolated antibody or antibody fragment does not substantially induce effector function in vitro or in vivo. In another embodiment of the present disclosure, said isolated antibody or antibody fragment does not induce effector function in vitro or in vivo.

Specificity

In an embodiment, the present disclosure pertains to an isolated antibody or antibody fragment specific for human TRBC2 disclosed in Table 1 or Table 2 of the present specification. In an embodiment, the isolated antibody or antibody fragment according to the present disclosure is specific for human TRBC2. In an embodiment, the isolated antibody or antibody fragment according to the present disclosure is selective for TRBC2. In an embodiment, the isolated antibody or antibody fragment according to the present disclosure is selective for TRBC2 over TRBC1. In an embodiment, the isolated antibody or antibody fragment specific for TRBC2 according to the present disclosure binds to TRBC2 over TRBC1 in a mutually exclusive manner. In an embodiment, the isolated antibody or antibody fragment according to the present disclosure specifically binds to a T cell receptor making use of TRBC2.

In an embodiment, the isolated antibody or antibody fragment according to the present disclosure is specific for human TRBC2 encoded by the amino acid sequence of SEQ ID NO: 39. In an embodiment, the isolated antibody or antibody fragment according to the present disclosure is specific for a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 39. In an embodiment, the isolated antibody or antibody fragment according to the present disclosure is specific for a polypeptide comprising the amino acid sequence of SEQ ID NO: 45.

In an embodiment, the isolated antibody or antibody fragment according to the present disclosure is specific for a protein complex comprising or consisting of the amino acid sequences of SEQ ID NO: 42 and SEQ ID NO: 45. In an embodiment, the isolated antibody or antibody fragment according to the present disclosure is specific for a protein complex comprising or consisting of a polypeptide comprising the amino acid sequences of SEQ ID NO: 42 and a polypeptide comprising the amino acid sequences of SEQ ID NO: 45.

In an embodiment, the isolated antibody or antibody fragment according to the present disclosure does not bind to human TRBC1. In an embodiment, said TRBC1 comprises or consists of the amino acid sequence of SEQ ID NO: 40. In an embodiment, said TRBC1 comprises or consists of the amino acid sequence of SEQ ID NO: 43.

In an embodiment, the isolated antibody or antibody fragment according to the present disclosure does not bind to human TRBC1 encoded by the amino sequence of SEQ ID NO: 40. In an embodiment, the isolated antibody or antibody fragment according to the present disclosure does not bind to human TRBC1 encoded by the amino sequence of SEQ ID NO: 43.

In an embodiment, the isolated antibody or antibody fragment according to the present disclosure does not bind to a protein complex comprising or consisting of the amino acid sequences of SEQ ID NO: 42 and SEQ ID NO: 43.

In an embodiment, the isolated antibody or antibody fragment according to the present disclosure does not bind to a linear peptide comprising the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40.

In an embodiment, the TRBC2 specific antibody or antibody fragment of the present disclosure binds to TRBC2 with an at least 2-fold, 4-fold, 5-fold, 7-fold or 10-fold greater affinity than to TRBC1 . The TRBC2 specific antibody or antibody fragment of the present disclosure may be selective or specific for TRBC2 without binding or recognizing the TRBC2 specific epitope on human TRBC2. In one such embodiment, the TRBC2 specific epitope comprises or consists of one or more amino acid residues selected from the group of: K at position 3 of TRBC2; N at position 4 of TRBC2; Y at position 36 of TRBC2; and/or E at position 135 of TRBC2.

The TRBC2 specific antibody or antibody fragment of the present disclosure may be selective or specific for TRBC2 because of its inability to bind to the TRBCI specific epitope on human TRBC1. In an embodiment, such inability to bind to the TRBC1 specific epitope is caused by a steric clash. In one such embodiment, the TRBC1 specific epitope comprises or consists of one or more amino acid residues selected from the group of: N at position 3 of TRBC1 ; K at position 4 of TRBC1 ; F at position 36 of TRBC1 ; and/or V at position 135 of TRBC1. Monovalent affinity for human TRBC2

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment has a monovalent affinity for human TRBC2 with a KD of 10 nM or less, such as 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said antibody or antibody fragment has a monovalent affinity for human TRBC2 with a K_D of 1 nM or less.

In an embodiment, said monovalent affinity is determined for an IgG. In an embodiment, said monovalent affinity is determined for a Fab. In some embodiments, said monovalent affinity is determined by solution equilibrium titration (SET). In some embodiments, said monovalent affinity is determined as described herein in Example 3. In an embodiment, said TRBC2 has the amino acid sequence of SEQ ID NO: 39. In an embodiment, said monovalent affinity is determined on a protein complex comprising the amino acid sequences of SEQ ID NO: 42 and SEQ ID NO: 45.

Apparent affinity for human TRBC2

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said isolated antibody or antibody fragment has an apparent (bivalent) affinity for human TRBC2, with a K_Dof 10 nM or less, such as 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less or 0.1 nM or less.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said isolated antibody or antibody fragment has an apparent (bivalent) affinity for human TRBC2, with a Ko of 1 nM or less.

In an embodiment, said apparent affinity is determined for an IgG. In an embodiment, said apparent affinity is determined by biolayer interferometry (BLI). In an embodiment, said TRBC2 has the amino acid sequence of SEQ ID NO: 39. In an embodiment, said monovalent affinity is determined on a protein complex comprising the amino acid sequences of SEQ ID NO: 42 and SEQ ID NO: 45. Apparent affinity for TRBC2 expressed on cells

In further embodiments, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said isolated antibody or antibody fragment has an apparent (bivalent) affinity for human TRBC2 with a KD of 10 nM or less, such as 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less or 0.1 nM or less.

In an embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said isolated antibody or antibody fragment has an apparent (bivalent) affinity for human TRBC2, with a Ko of2 nM or less.

In certain embodiments, said apparent affinity is determined for an IgG. In some embodiments, said human TRBC2 is expressed on cells. In some embodiments, said human TRBC2 is expressed on HPB-ALL cells (DSMZ no.: ACC 483). In certain embodiments, said apparent affinity is determined as described herein in Example 4.

Apparent ECso on TRBC2 expressed on cells

In further embodiments, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said isolated antibody or antibody fragment binds to human TRBC2 expressed on cells with an ECso concentration of 100 nM or less, such as 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less or 0.1 nM or less.

In some embodiments, said isolated antibody or antibody fragment specific for human TRBC2, according to the present disclosure binds to human TRBC2 expressed on cells with an ECso concentration of 20 nM or less. In some embodiments, said isolated antibody or antibody fragment specific for human TRBC2, according to the present disclosure binds to human TRBC2 expressed on cells with an ECso concentration between 1 nM and 20 nM.

In some embodiments, said ECso concentration is determined for an IgG. In some embodiments, said cells are T cells. In some embodiments, said cells are primary human T cells. In certain embodiments, said ECso concentration is determined by flowcytometry. In some embodiments, said ECso concentration is determined as described herein in Example 5. Receptor internalization

In further embodiments, the present disclosure refers to an isolated antibody or antibody fragment specific for human TRBC2, wherein said isolated antibody or antibody fragment internalizes into TRBC2 expressing cells with an ECso concentration of 100 nM or less, such as 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less or 0.1 nM or less.

In some embodiments, said isolated antibody or antibody fragment according to the present disclosure internalizes into TRBC2 expressing cells with an EC₅o concentration of 20 nM or less.

In some embodiments, said isolated antibody or antibody fragment according to the present disclosure internalizes into TRBC2 expressing cells with an EC₅o concentration between 1 nM and 20 nM.

In some embodiments, said EC₅o concentration is determined for an IgG. In some embodiments, said cells are T cells. In some embodiments, said cells are primary human T cells. In some embodiments, said cells are HPB-ALL cells (DSMZ no.: ACC 483). In certain embodiments, said EC₅O concentration is determined by flowcytometry. In some embodiments, said EC₅o concentration is determined as described herein in Example 6.

Fusion molecules / Immunoconjugates

The isolated antibody or antibody fragment specific for TRBC2 according to the present disclosure may be fused or conjugated to one or more moieties for binding to other targets or target proteins of interest. The antibodies or antibody fragments may be fused, coupled or conjugated to one or more other amino acid residues, polypeptides or moieties to generate fusion molecules in accordance with any of a variety of techniques, such as genetically or chemically approaches, which are known to the person skilled in the art. Fusion molecules are generally achieved by covalent bonds between the antibodies or antibody fragments of the present disclosure and the one or more moieties. The antibody fusion molecule of the present disclosure may comprise one or more, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 additional moieties.

The fusion molecules according to the present disclosure are directed only to T cells expressing TRBC2. The T cell receptor making use of TRBC2 may internalize in response to antibody binding. The term “internalization” as used herein, refers to a process also known as receptor mediated endocytosis. “Endocytosis” is a cellular process by which molecules or substances are transported into the cell via cell membrane engulfment. Accordingly, the present disclosure provides fusion molecules comprising the TRBC2 specific antibody according to the present disclosure and at least one additional moiety.

In an embodiment, said additional moiety is a selected from the group of, but is not limited to, a therapeutic agent, a cytotoxic agent, a bacterial toxin (such as Pseudomonas exotoxin A or Diphtheria toxin, or plant toxins, such as ricin), a cytokine, an antibody or antibody fragment, a peptide, a polypeptide, an enzyme, a detectable group, a moiety being capable of binding a metal ion, a tag suitable for detection and/or purification, a targeting ligand, a homo- or heteroassociation domain, a moiety which increases solubility of a protein, or a moiety which comprises an enzymatic cleavage site, a lipid, a liposome and virus-like-particles. In an embodiment, said additional moiety is a co-stimulating domain of a chimeric antigen receptor (CAR).

The detectable group may be a fluorescent moiety, for example a fluorescent peptide. Examples of fluorescent peptides or proteins include, but are not limited to, fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), green fluorescent protein (GFP), enhanced GFP, red fluorescent protein (RFP), blue fluorescent protein (BFP). A TRBC2 specific antibody or antibody fragment according to the present disclosure conjugated to a detectable group may be used to determine or detect TRBC2 expression of a malignant or healthy T cell.

A "therapeutic agent" as used herein may be any therapeutic agent including, but not limited to radionuclide, chemotherapeutic agent, and cytotoxic agents (See, e.g., U.S. Patent No. 6,949,245), and antimicrobial peptides. The antibody fusion molecule of the present disclosure may comprise one more, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 therapeutic agents.

A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include, but is not limited to, methotrexate, daunomycin, mitomycin C, cisplatin, vincristine, epirubicin, fluorouracil, verapamil, cyclophosphamide, cytosine arabinoside, aminopterin, bleomycin, mitomycin C, democolcine, etoposide, mithramycin, chlorambucil, melphalan, daunorubicin, doxorubicin, tamosifen, paclitaxel, vincristin, vinblastine, camptothecin, actinomycin D, and cytarabine.

A "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells and/or exerts anti-proliferative effects. Examples of cytotoxic agents includes tubulin inhibitors, DNA damaging agents, topoisomerase I inhibitors, and RNA polymerase II inhibitors. Non-limiting examples of tubulin inhibitors include vincristine, vinblastine, vinflunine, halichondrin B, eribulin mesylate, cryptophycins, and dolastatins, such as the auristatins MMAF, MMAE, PF-06390101 , MMAD, auristatin E, auristatin W analogue, auristatin f-HPA, amberstatin 269, and AGD- 0182, maytansinoids and maytansinoid analogs (such as DM1 , DM4), paclitaxel, docetaxel, cyclostreptin, eleutherobin, ABI-007, ixabepilone, patupilone, and BMS-310705, colchicine, 2- methoxyestradiol, sulphonamides, and Aspergillus derivatives, laulimalide and peloruside A. DNA damaging agents include, without limitation, calicheamicins (ozogamicin), such as calicheamicin g; pyrrolobenzodiazepines, such as PDB talirine and SG3249; duocarmycins, such as DUB A; campthothecin analogues, such as SN38 and DX-8951 ; anthracyclines such as daunorubicin and doxorubicins (adriamycin). Topoisomerase I inhibitors include, without limitation, irinotecan, topotecan, and camptothecin. RNA polymerase II inhibitors include, without limitation, amatoxins, such as a-amanitin.

Further cytotoxic agents may include without limitation: taxoids, CC-1065 and CC-1065 analogs, dolastatin and dolastatin analogs, ricin, such as the ricin A chain), aclacinomycin, Diphtheria toxin, Monensin, Verrucarin A, Abrin, Tricothecenes, and Pseudomonas exotoxin A, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, anti-mitotic agents, such as the vinca alkaloids (e.g., vincristine and vinblastine), dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5- fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)), and antibiotics, including, but not limited to, dactinomycin (formerly actinomycin), bleomycin, mithramycin, calicheamicin, and anthramycin (AMC). In some embodiments, the TRBC2 specific antibody according to the present disclosure is fused to one or more Monomethyl auristatin E (MMAE) molecules.

A “radionuclide" as described herein includes, but is not limited to ⁸⁸Ga ²²⁵Ac, ²¹⁰At, Ba, ⁷⁷Br, ¹⁰⁹Cd, ⁵¹Cr, ⁶⁷Cu, ¹⁶⁵Dy, ¹⁵⁵Eu, ¹⁵³Gd, ¹⁹⁸Au, ¹⁶⁶Ho, ^113raln, ¹¹⁵¹¹¹ In, ¹²³l, ¹²⁵l, ¹³¹ l, ¹⁸⁹lr, ¹⁹¹ Ir, ¹⁹²lr, ¹⁹⁴lr, ⁵²Fe, ⁵⁵Fe, ⁵⁹Fe, ¹⁷⁷Lu, ¹⁰⁹Pd, ³²P, ²²⁶Ra, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ⁴⁶Sc, ⁴⁷Sc, ⁷²Se, ⁷⁵Se, ¹⁰⁵Ag, ⁸⁹Sr, ³⁵S, ¹⁷⁷Ta, ¹¹⁷mSn, ¹²¹Sn, ¹⁶⁶Yb, ¹⁶⁹Yb, ⁹⁰Y, ²¹²Bi, ²¹³Bi ¹¹⁹Sb, ¹⁹⁷Hg, ⁹⁷Ru, ¹⁰⁰Pd, ¹⁰¹mRh, and ²¹²Pb.

The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

A "liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as the TRBC2 specific antibodies disclosed herein and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.

"Detectable group" as used herein includes, but is not limited to, radiolabels, enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), gold beads, chemiluminescence labels, ligands (e.g., biotin, digoxin) and/or fluorescence labels (e.g., rhodamine, phycoerythrin, fluorescein, fluorescent proteins), a fluorescent protein including, but not limited to, a green fluorescent protein or one of its many modified forms, a nucleic acid segment in accordance with known techniques, and energy absorbing and energy emitting agents.

In some embodiments, the fusion molecules according to the present disclosure may be made using a variety of bi-functional protein coupling agents such as N-succinimidyl-3-(2- pyridyldithiol)propionate (SPDP), succinimidyl-4-(N- maleimidomethyl) cyclohexane-l-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p- azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). Carbon- 14-labeled 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the targeting antibody (see WO 94/11026).

1.4.7.10-Tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA (also known as tetraxetan)) or

1.4.7.10- tetraaza-l,4,7,IO-tetra(2-carbamoylmethyl)cyclododecane (TCMC) are further exemplary chelating agent for conjugation of radionucleotide to the antibodies according to the present disclosure.

The linker may be a "cleavable linker" facilitating release of the cytotoxic agent in the cell. For example, an acid-labile linker, peptidase-sensitive linker, dimethyl linker or disulfide-containing linker (Chari et al. Cancer Research 52: 127-131 (1992)) may be used. Alternatively, a fusion molecule comprising the isolated antibody or antibody fragment of the present disclosure and a cytotoxic agent may be made, e.g. by recombinant techniques or peptide synthesis. Effector function

In an embodiment, the antibody or antibody fragment according to the present disclosure does not substantially induce effector function. In an embodiment, the antibody or antibody fragment according to the present disclosure does not induce effector function. In an embodiment, the effector function is selected from the group of , but not limited to, CDC, ADCC and ADCP.

In an embodiment, the antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions in its Fc region which reduce or abolish the binding of the Fc region of the antibody to an Fc receptor and/or to C1 q and/or reduces the ability of said antibody to induce effector function when compared to the antibody comprising a wild-type Fc region. In an embodiment, the antibody or antibody fragment according to the present disclosure comprises a variant Fc region with reduced or abolished ability for binding to an Fc receptor and/or to C1q and/or reduced ability to induce effector function, when compared to a wildtype Fc region. In an embodiment, the isolated antibody or antibody fragment according to the present disclosure is of the human lgG1 class.

In an embodiment, the Fc receptor is a human Fc receptor. In an embodiment, the Fc receptor is an Fey receptor. In an embodiment, the Fc receptor is a human FcyRllla, FcyRI, FcyRlla and/or FcyRllb. In an embodiment, said wild-type Fc region is an IgG 1 Fc region. In an embodiment, said wild-type Fc region is a human lgG1 Fc region. In an embodiment, said wild-type Fc region is a human lgG1 Fc region comprising the amino acid sequence of:

APELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 50).

In an embodiment of the present disclosure, said variant Fc region comprises the amino acid sequence of: APEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 51).

In an embodiment, the antibody according to the present disclosure or the variant human I gG 1 Fc region of the antibody or antibody fragment according to the present disclosure comprises an amino acid substitution at one or more positions selected from the group of 234, 235, 237, 330 and 331 with numbering according EU index.

In an embodiment, the antibody according to the present disclosure or the variant human I gG 1 Fc region of the antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S and P331S with numbering according to EU index.

Binding of an antibody to Fc receptors via its Fc region can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a Biacore® instrument, and Fc receptors may be obtained by recombinant expression. Alternatively, the binding affinity of Fc regions may be evaluated using cell lines known to express particular Fc receptors, such as NK cells expressing Fcyllla receptor. Effector function of an antibody can be measured by methods known in the art. Suitable in vitro assays to assess ADCC activity of a molecule of interest are for instance described in WO2012130831. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652- 656 (1998). To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101 , 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)). C1q binding assays (such as ELISA) may be carried out to determine whether an antibody is able to bind C1q and hence has CDC activity (see, for example WO 2006/029879).

Therapeutic methods

In an embodiment, the present disclosure provides the use of the isolated antibody or antibody fragment specific for human TRBC2 according to the present disclosure for the manufacture of a medicament. In an embodiment, the present disclosure provides an isolated antibody or antibody fragment specific for human TRBC2 for use as a medicament. In an embodiment, the present disclosure provides an isolated antibody or antibody fragment specific for human TRBC2 for use in medicine. The isolated antibody or antibody fragment specific for human TRBC2 according to the present disclosure may be used in therapeutic methods. In an embodiment, the present disclosure provides an isolated antibody or antibody fragment specific for human TRBC2 for use in the treatment of a disease. In an embodiment, the disease is associated with the undesired presence of TRBC2, in particular human TRBC2. In an embodiment, the present disclosure provides an isolated antibody or antibody fragment specific for human TRBC2 according to the present disclosure for use in the treatment of a T cell lymphoma or leukaemia. The therapeutic method of the present disclosure may be used for the treatment of any lymphoma and/or leukaemia associated with the clonal expansion of a cell expressing a T cell receptor (TCR) comprising TRBC2. Consequently, the present disclosure pertains to a method for treating a disease which involves malignant T cells which express a TCR comprising TRBC2. The therapeutic method of the present disclosure may be used to treat a T cell lymphoma in which the malignant T cell expresses a TCR comprising a TRBC2.

“Lymphoma” refers to a cancer which typically develops in the lymph nodes, but may also affect the spleen, bone marrow, blood and other organs. Lymphoma typically presents as a solid tumor of lymphoid cells. “Leukaemia” refers to a cancer of the blood or bone marrow. In an embodiment, the present disclosure provides an isolated antibody or antibody fragment specific for human TRBC2 according to the present disclosure for use in a method of treating a subject having T cell lymphoma or leukaemia, comprising administering to the subject a therapeutically effective amount of said antibody or antibody fragment in order to reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.

In an embodiment of the present disclosure, the T cell lymphoma or leukaemia is selected from peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angio-immunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK/T cell lymphoma nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukaemia and T cell acute lymphoblastic leukaemia.

In an embodiment, the present disclosure provides an isolated antibody or antibody fragment specific for human TRBC2 according to the present disclosure for use in a method for treating a T cell lymphoma or leukaemia in a subject which comprises the step of administrating the antibody or antibody fragment of the present disclosure to a subject, to cause selective depletion of the malignant T cells, together with normal T- cells expressing TRBC2, but not to cause depletion of normal T cells expressing TRBC1.

The therapeutic method described herein may also comprise the step of investigating the TCR beta constant region (TCRB) of a malignant T cell from the subject to determine whether it expresses TRBC1 or TRBC2, preferably TRBC2.

In an embodiment, the method further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent. The subject in need of treatment is typically a mammal, more specifically a human. For use in therapeutic methods, an isolated antibody or antibody fragment according to the present disclosure would be formulated, dosed, and administered in a way consistent with good medical practice.

In an embodiment, the antibody or a fragment specific for TRBC2 according to the present disclosure may be a fusion molecule as described above, such as a conjugated antibody, which comprises a therapeutic agent. In one such embodiment, the therapeutic agent is a cytotoxic molecule. In an alternative embodiment, the therapeutic agent is an antibody or antibody fragment specific for CD3.

The therapeutic methods according to the present disclosure may comprise the step of administering the antibody or antibody fragment specific for TRBC2 discloses herein to a subject in need thereof, wherein said antibody or antibody fragment causes selective depletion of malignant T cells expressing TRBC2, together with normal T cells expressing TRBC2, but does not cause substantial depletion of normal T cells expressing TRBC1 . Retention of a proportion of the healthy T cells expressing TRBC1 results in reduced toxicity and reduced cellular and humoral immunodeficiency, thereby reducing the risk of infection.

Administration of the TRBC2 specific antibody or antibody fragment according to the present disclosure may result in a 5, 10, 20, 50, 75, 90, 95 or 99% depletion, i.e. reduction in the number of T cells expressing TRBC2. The TRBC2 specific antibody or antibody fragment of the present disclosure may cause a depletion of a greater proportion of TRBC2 expressing T cells in cell population than TRBC1 expressing T cells. For example, the ratio of depletion of TRBC2 expressing T cells overTRBCI expressing T cells may be at least 60%:40%, 70%:30%, 80%:20%, 90%: 10% or 95%:5%.

The TRBC2 specific antibody or antibody fragment of the present disclosure may cause depletion of less than 20%, 15%, 10% or 5% of the T cell population expressing TRBC1.

Pharmaceutical compositions

In an embodiment, the present disclosure provides a pharmaceutical composition comprising an isolated antibody or antibody fragment specific for human TRBC2 according to the present disclosure and a pharmaceutically acceptable carrier or excipient. The pharmaceutical compositions may further comprise at least one other pharmaceutically active compound. The pharmaceutical composition according to the present disclosure can be used in the diagnosis, prevention and/or treatment of diseases associated with the undesired presence of TRBC2, in particular human TRBC2. In particular, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure that is suitable for prophylactic, therapeutic and/or diagnostic use in a mammal, more particular in a human.

In general, an antibody or antibody fragment specific for human TRBC2 according to the present disclosure may be formulated as a pharmaceutical composition comprising at least one antibody or antibody fragment as described herein and at least one pharmaceutically acceptable carrier or excipient. Such a formulation may be suitable for oral, parenteral, topical administration or for administration by inhalation. Accordingly, a pharmaceutical composition comprising at least one antibody or antibody fragment according to the present disclosure may be administered parenterally, such as intravenously, or intramuscularly, or subcutaneously. Alternatively, an antibody or antibody fragment of the present disclosure may be administered via a non-parenteral route, such as per-orally or topically. In a preferred embodiment, a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure is administered intravenously or subcutaneously.

In an embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment specific for human TRBC2 according to the present disclosure for use in the prevention and/or treatment of a disease associated with the undesired presence of human TRBC2. In an embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according of the present disclosure for the use as a medicament. In an embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according of the present disclosure for use in the prevention and/or treatment of cancer. In an embodiment, the present disclosure provides a method for the treatment of cancer in a subject in need thereof using a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure.

In an embodiment of the present disclosure, said disease or cancer is T cell lymphoma or leukaemia. In a more specific embodiment, said T cell lymphoma or leukaemia is selected from peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angio-immunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK/T cell lymphoma nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukaemia and T cell acute lymphoblastic leukaemia.

Further provided is a method of producing an antibody or antibody fragment according to the present disclosure in a form suitable for administration in vivo, the method comprising (a) obtaining an antibody or antibody fragment by a method according to the present disclosure, and (b) formulating said antibody or antibody fragment with at least one pharmaceutically acceptable carrier or excipient, whereby a preparation of antibody or antibody fragment is formulated for administration in vivo. Pharmaceutical compositions according to the present disclosure comprise a therapeutically effective amount of one or more antibodies or antibody fragments according to the present disclosure dissolved in a pharmaceutically acceptable carrier or excipient.

Diagnostic use

In an embodiment, the present disclosure provides the use of an isolated antibody or antibody fragment specific for human TRBC2 according to the present disclosure for the diagnosis of a disease. In an embodiment, the present disclosure provides the use of an antibody or antibody fragment according to the present disclosure for the detection of TRBC2, in particular human TRBC2. In an embodiment, the present disclosure provides a method for detecting human TRBC2 in a subject or a sample, comprising the step of contacting said subject or sample with an isolated antibody or antibody fragment specific for human TRBC2 of the present disclosure.

In an embodiment, the present disclosure provides a method for diagnosing a disease in a subject, comprising the step of contacting said subject or sample with an isolated antibody or antibody fragment specific for human TRBC2 according to the present disclosure. In an embodiment, said disease is T cell lymphoma or leukaemia. In some embodiments, the present disclosure provides a method for diagnosing a T cell lymphoma or leukaemia in a subject which comprises the step of determining the percentage of total T cells in a sample which are TRBC2 positive.

A percentage of TRBC2 positive T cells which is greater than about 80% may indicate the presence of a T cell lymphoma or leukaemia. In an embodiment of the present disclosure, the percentage of total T cells which are TRBC2 positive which indicates the presence of a T cell lymphoma or leukaemia may be, for example 80, 85, 90, 95, 98 or 99% of a total population of T cells. The sample may be a peripheral blood sample or a biopsy. The agent which binds total T cells may bind to CD3. In an embodiment, said agent is an antibody or antibody fragment. The total T cells in a sample may identified by determining the number of cells in the sample which express CD3, CD4, CD8 and/or CD45. A combination of these markers may also be used.

The proportion of total T cells in a sample which express TRBC2 may be determined using methods which are known in the art, for example by PCR, Next generating sequencing (NGS), western blot, flow cytometry, immunohistochemistry or fluorescent microscopy methods. Once the TRBC expressed by a malignant T cell has been determined, the TRBC2 selective antibody or antibody fragment according to the present disclosure is administered to the subject. Kits

The present disclosure further provides a kit comprising an antibody or antibody fragment specific for TRBC2 according to the present disclosure for use in the treatment of a T cell lymphoma or leukaemia. The kit may further comprise a reagent(s) suitable for determining TRBC2 of a malignant T cell. For example the kit may comprise PCR primers or one or more antibodies which are specific for TRBC2.

Nucleic acids

In an embodiment, the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding any one of the isolated antibodies or antibody fragments specific for human TRBC2 according to the present disclosure.

In an embodiment, the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding any one of the isolated antibodies or antibody fragments disclosed in Table 1 or Table 2 of the present specification. In an embodiment, the present disclosure provides an isolated antibody or antibody fragment specific for human TRBC2, encoded by any one of the nucleic acid sequences or plurality of nucleic acid sequences disclosed in Tablel or Table 2 of the present specification.

In an embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2 according to the present disclosure, wherein the nucleic acid sequence or the plurality of nucleic acid sequences encodes the CDRs of any one of the antibodies disclosed in Table 1 or Table 2 of the present specification.

In an embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2 according to the present disclosure, wherein the nucleic acid sequence or the plurality of nucleic acid sequences encodes the VH and VL of any one of the antibodies disclosed in Table 1 or Table 2 of the present specification.

In some embodiments, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2, wherein the nucleic acid sequence or the plurality of nucleic acid sequences encodes the HC and LC of any one of the antibodies or antibody fragments disclosed in Table 1 or Table 2 of the present specification. In an embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2, wherein said isolated said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 16, the HCDR2 region of SEQ ID NO: 17, the HCDR3 region of SEQ ID NO: 18, the LCDR1 region of SEQ ID NO: 19 or SEQ ID NO: 35, the LCDR2 region of SEQ ID NO: 20 and the LCDR3 region of SEQ ID NO: 21 .

In an embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2, wherein said isolated said antibody or antibody fragment comprises a HCDR1 region comprising the nucleic acid sequence of SEQ ID NO: 16, a HCDR2 region comprising the nucleic acid sequence of SEQ ID NO: 17, a HCDR3 region comprising the nucleic acid sequence of SEQ ID NO: 18, a LCDR1 region comprising the nucleic acid sequence of SEQ ID NO: 19 or SEQ ID NO: 35, a LCDR2 region comprising the nucleic acid sequence of SEQ ID NO: 20 and a LCDR3 region comprising the nucleic acid sequence of SEQ ID NO: 21.

In an embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2, wherein said isolated said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 22, the HCDR2 region of SEQ ID NO: 23, the HCDR3 region of SEQ ID NO: 18, the LCDR1 region of SEQ ID NO: 24 or SEQ ID NO: 36, the LCDR2 region of GCCGCCAGC and the LCDR3 region of SEQ ID NO: 26.

In an embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2, wherein said isolated said antibody or antibody fragment comprises a HCDR1 region comprising the nucleic acid sequence of SEQ ID NO: 22, a HCDR2 region comprising the nucleic acid sequence of SEQ ID NO: 23, a HCDR3 region comprising the nucleic acid sequence of SEQ ID NO: 18, a LCDR1 region comprising the nucleic acid sequence of SEQ ID NO: 24 or SEQ ID NO: 36 , a LCDR2 region comprising the nucleic acid sequence of GCCGCCAGC and a LCDR3 region comprising the nucleic acid sequence of SEQ ID NO: 26.

In an embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprise the VH of SEQ ID NO: l and the VL of SEQ ID NO: 28, or a VH that has at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity to the VH of SEQ ID NO: 27 and a VL that has at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity to the VL of SEQ ID NO: 28.

In an embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprise the VH of SEQ ID NO: 27 and the VL of SEQ ID NO: 28.

In an embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprise the VH of SEQ ID NO: l and the VL of SEQ ID NO: 37, or a VH that has at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity to the VH of SEQ ID NO: 27 and a VL that has at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity to the VL of SEQ ID NO: 37.

In an embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprise the VH of SEQ ID NO: 27 and the VL of SEQ ID NO: 37

In an embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprise the HO of SEQ ID NO: 29 and the LC of SEQ ID NO: 30, or a HO and that has at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity to the HO of SEQ ID NO: 29 and a LC that has at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity of the LC of SEQ ID NO: 30.

In an embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprise the HO of SEQ ID NO: 29 and the LC of SEQ ID NO: 38, or a HO and that has at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity to the HO of SEQ ID NO: 29 and a LC that has at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity of the LC of SEQ ID NO: 38. In an embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprise the HC of SEQ ID NO: 29 and the LC of SEQ ID NO: 38.

In an embodiment, said nucleic acid composition and/or said nucleic acid sequence and/or plurality of nucleic acid sequences are isolated nucleic acids.

Vectors

In an embodiment, the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2 according to the present disclosure. In an embodiment, the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding any one of the isolated antibodies or antibody fragments specific for human TRBC2 disclosed in Table 1 or Table 2 of the present specification. In an embodiment, the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid sequence or a plurality of nucleic acid sequences disclosed in Table 1 or Table 2 of the present specification. In an embodiment, said vector composition and/or vector and/or plurality of vectors are isolated vectors.

Host cells

In an embodiment, the present disclosure provides a host cell comprising a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2 according to the present disclosure. In an embodiment, the present disclosure refers to a host cell comprising a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human TRBC2 disclosed in Table 1 or Table 2 of the present specification.

In an embodiment, the host cell according to the present disclosure is able to express the isolated antibody or antibody fragment specific for human TRBC2 encoded by the vector composition or the nucleic acid composition. In a further embodiment, the host cell is an isolated host cell. In a further embodiment, said host cell is a mammalian cell. In an embodiment, said mammalian cell is a human cell or human cell line.

The skilled man will realize that the nucleic acid sequence or the plurality of nucleic acid sequences encoding the heavy and/or light chain of an antibody or antibody fragment of the present disclosure can be cloned into different vectors or into the same vector. The vectors can be introduced into the appropriate host cells such as prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast or mammalian) cells by methods well known in the art (see e.g., "Current Protocol in Molecular Biology", Ausubel et al. (eds.), Greene Publishing Assoc, and John Wiley Interscience, New York, 1989 and 1992). Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the nucleic acid sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. Upon expression in host cells, the antibodies or antibody fragments of the present disclosure are obtained. These steps can be achieved in different ways, as will be known by the person skilled in the art. In general, such steps typically include transforming or transfecting a suitable host cell with a nucleic acid composition or vector composition or an infectious particle, which encodes the antibody, or antibody fragments. Further, such steps typically include culturing said host cells under conditions suitable for the proliferation (multiplication, growth) of said host cells and a culturing step under conditions suitable for the production (expression, synthesis) of the encoded antibody or antibody fragment. The culturing of host cells under conditions suitable for proliferation or expression is typically accomplished in the presence of media comprising components suitable for cell growth or induction of expression. In particular, embodiments, the methods for the production of the antibodies or antibody fragments of the present disclosure further comprise the step of isolating and purifying the produced antibody or antibody fragment from the host cells or medium. If the expression system secretes the protein into growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction. The selection of the appropriate growth conditions and recovery methods are within the skill of the art. The antibody or antibody fragment of the present disclosure can then be purified by a number of techniques as known to the person skilled in the art.

In an embodiment, the present disclosure refers to a method of producing an isolated antibody or antibody fragment specific for human TRBC2 of any of the antibodies disclosed in Table 1 or Table 2 of the present specification. In an embodiment, a method of producing an isolated antibody or antibody fragment according to the present disclosure is provided, wherein the method comprises culturing a host cell comprising a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment according to the present disclosure, under conditions suitable for expression of the antibody or antibody fragment, and isolating the antibody or antibody fragment from the host cell or host cell culture medium. An antibody or antibody fragment isolated as described herein may be purified techniques know in the art, such as high-performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The conditions used to purify a particular antibody or antibody fragment will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art. For affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the antibody or antibody fragment binds. For example, for affinity chromatography purification of antibody or antibody fragment according to the present disclosure, a matrix with protein A or protein G may be used. The purity of an antibody or antibody fragment can be determined by any of a variety of well-known analytical methods including gel electrophoresis, high-pressure liquid chromatography, and the like.

Antibody sequences

Table 1 : Antibody sequences of mAb1

Table 2: Antibody sequences of mAb2

WORKING EXAMPLES

Example 1: Antigens

Amino acid sequences of human TRBC2 and human TRBC1 and related TCR alpha chain and TCR beta chains were retrieved from publicly available sources (e.g. Uniprot, PDB), verified and produced in-house. The amino acid sequences of the produced TRBC1 and TRBC2 protein complexes are summarized in Table 3.

Table 3: Amino acid sequences of produced human TRBC1 and human TRBC2 alpha/beta chain complexes

FcRn receptors

Amino acid sequences of the FcRN large subunit 51 and of the FcRn small subunit p14 from various species were retrieved from publicly available sources (e.g. Uniprot), verified and produced in-house. The amino acid sequences of the produced receptors are summarized in Table 4.

Table 4: Amino acid sequences of produced FcRn proteins

Control antibodies

Various control antibodies were produced and included in experiments for comparative purposes.

RefmAb: TRBC2 benchmark antibody

Nucleotide sequences encoding the VH and VL region of the humanized TRBC2 specific antibody “MUT15" as described in International Publication W02021/191607 (Autolus) were gene synthesized as linear DNA fragments with appropriate flanking regions either in-house or by an external provider. The DNA fragments were cloned into suited mammalian lgG1 expression vectors using standard molecular biology methods. The antibody was then transiently produced in mammalian cells.

RefmAb: VH (SEQ ID NO: 46)

QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSN ERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGLMFDGAYRFFDFWGQGTMVTVS S

RefmAb: VL (SEQ ID NO: 47)

DIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGRTYLHWYLQKPGQSPRLLIYRVSNRFPGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRT

JOVI-1 : TRBC1 control antibody:

Nucleotide sequences encoding the VH and the VL of the murine TRBC1 specific antibody JOVI- 1 (Viney et al., 1992 Hybridoma) were gene synthesized as linear DNA fragments with appropriate flanking regions, either in-house or by an external provider. The DNA fragments were cloned into suited mammalian lgG1 expression vectors using standard molecular biology methods. The antibody was transiently produced in mammalian cells.

JOVI-1 VH (SEQ ID NO: 48)

EVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVKQRPGQGLEWIGFINPYNDDIQSNER F RG KATLTS DKSSTTA YM ELSS LTS E DSAVYYCARG AG YN F DG AYR F F D FWGQGTTLTVSS

JOVI-1 VL (SEQ ID NO: 49)

DWMTQSPLSLPVSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGV PDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPYTFGGGTKLEIKRT Ctr. Antibody: Negative (isotype) control antibody:

An in-house negative isotype (“Ctrl”) control antibody with specificity for hen-egg lysozyme were transiently produced in mammalian cells in the human IgG 1 or human lgG1_AEASS format.

Example 2: Selection of TRBC2 specific antibodies from the MorphoSys Ylanthia® phage library

For antibody generation, the MorphoSys Ylanthia® library was used to select Fab fragments specific for TRBC2. The MorphoSys Ylanthia® library (Tiller et al. mAbs 5:3, 1-26; May/June (2013) and U.S. Patent No. 8,728,981) is a commercially available phagemid library and employs the CysDisplay® technology for displaying the Fab on the phage surface (Lohning et al., WO2001/05950).

To identify TRBC2 specific antibodies, different panning strategies were employed. Each panning strategy comprised of at least 3 individual rounds of panning against various TRBC2 antigens (e.g. TRBC2 peptides, T cell receptor alpha/beta complexes making use of TRBC2 (either as soluble recombinant protein complex or as native protein complex expressed on cancer cells). During certain selections, varying concentrations of unlabelled TRBC1 were added to enrich for clones that do not bind TRBC1 . Selections were followed by standard screening methods such as ELISA and flow cytometry to identify individual clones that bind specifically TRBC2. This resulted in the identification of only 1 parental antibody being able to recognize TRBC2 as part of the native alpha/beta protein complex, either expressed on cells or as recombinant soluble protein. Clones which were selected using only linear peptides spanning the TRBC2 epitopes on human TRBC2 resulted in specific TRBC2 peptide binders but turned out being unable to recognize the relevant native protein complexes encoding TRBC2.

The aforementioned identified single parental antibody was further optimized through affinity maturation pannings using the same methods described above in order to improve specific TRBC2 binding. Library selections and individual clone screening were repeated as above with the modification that more stringent conditions were applied to select for clones with enhanced, but specific TRBC2 affinity. Following hit sequencing, Fab genes were reformatted into lgG1 or lgG1_AEASS antibody format for expression, purification, and characterization resulting in the identification of mAb1 as a first affinity optimized lead molecule. Additional antibody engineering efforts on mAb1 were conducted to further optimize its properties resulting in the identification of mAb2. Both candidates, mAb1 and mAb2 were nominated as potential therapeutic candidates. Since both candidates are derived from the same parental antibody, they share similar amino acid sequences and in vitro characteristics. These two antibodies are further described in the examples as outlined below.

Example 3: Monovalent affinity determination for mAb1 and mAb2 on recombinant human TRBC2 by Solution Equilibrium Titration (SET).

Method

Monovalent IgG affinity was determined by solution equilibrium titration (SET). SET was basically performed as described in the literature (Friquet et al., (1985) J. Immunol. Meth. 77: 305-19). In order to improve the sensitivity and accuracy of the SET method, it was transferred from classical ELISA to ECL based technology (Haenel et al. (2005) Anal Biochem. 339.1 : 182-84). 1 mg/mL goat-anti-human (Fab)2 fragment specific antibodies (Dianova) were labeled with MSD Sulfo TAG™ NHS-Ester (Meso Scale Discovery | Gaithersburg | MD | USA) according to the manufacturer's instructions. The experiments were carried out in polypropylene microtiter plates and PBS (GIBCO 14190 | pH 7.0-7.2) containing 0.5% BSA and 0.02% Tween20 as assay buffer. Serial dilutions of unlabeled human TRBC2 protein complex (SEQ ID NO: 42 and SEQ ID NO: 45) were prepared with 30 pL per well, starting with a concentration at least 10 times higher than the expected K_D. Wells without antigen were used to determine Bmax values; wells containing only assay buffer were used to determine background. After addition of appropriate amount of antibody (antibody concentration similar to or below the expected KD, 60 pL final volume), the mixture was incubated over night at RT. MSD plates were coated with biotinylated antigen on streptavidin plates (30 pL per well). After washing the plate with PBS with 0.05% Tween 20, the equilibrated samples were transferred to the plates and incubated for 10 min. Following incubation, 30 pL per well of the MSD-Sulfo-tag labeled detection antibody (anti-human (Fab)2 | final dilution typically 1 :2,000) was added to the washed MSD plate and incubated for 1 h at RT on an Eppendorf shaker (700 rpm). After washing the MSD plate and adding 30 pL/well MSD Read Buffer T with surfactant, electrochemiluminescence signals were detected using a Sector Imager 6000 (Meso Scale Discovery | Gaithersburg | MD | USA). The data was evaluated with XLfit (I DBS) software applying customized fitting models. For K_D determination of IgGs, the fit model according to (Haenel et al. 2005), modified according to (Abraham et al.) was used. Results

Results are summarized in Table 5. mAb1 and mAb2 revealed affinities in the mid triple digit picomolar range on the MARTI -TCR-TRBC2 protein complex.

Table 5: Affinities of mAb1 and mAb2 to human TRBC2.

Example 4: Apparent affinity (bivalent) determination for mAb1 and mAb2 on TRBC2 positive HPB-ALL cells (KinExA)

Method

Apparent K_D determination on TRBC2 expressing HPB-ALL cells was performed at RT with a KinExA 3200 instrument (Sapidyne Instruments). PBS (Gibco), supplemented with BSA (1 mg/mL) and 0.02 % (v/v) NaNs was used as assay buffer. mAb1 or mAb2, respectively, was used as analyte to generate two titration curves (for each antibody, constant cone, of 10 nM and 50 pM were applied). T acute lymphoblastic leukemia cells HPB-ALL were used as as titrant (starting cell concentrations for 2n serial dilution for mAb1 : 670 Mio cells/mL and 40 Mio cells/mL; mAb2: 700 and 80 Mio cells/mL). The mixtures were equilibrated over night at RT in an overhead shaker. After equilibration, the samples were centrifuged and the supernatants were used for analysis. Free concentration of mAb1 or mAb2, respectively, was determined using polymethylmethacrylate (PMMA) beads coated with goat anti-human Fab 2 (JacksonlmmunoResearch) and anti-human Fab2 Alexa Fluor 647 (500 ng/mL) was used for detection. The apparent K_D was obtained using KinExA software and by “n-curve analysis,” which fits all of the given curves to a single K_D value simultaneously.

Results

Results are summarized in Table 6. Both candidates, mAb1 and mAb2, revealed strong binding to cell surface expressed TRBC2 with an apparent K_D value in the triple digit pM to single digit nanomolar range. Table 6: Bivalent binding of mAb1 and mAb2 to HPB-ALL cells.

Example 5: Binding of mAb1 and mAb2 to primary human T cells (FACS)

Method

Primary human T cells were isolated from PBMCs of healthy donors using pan T cell isolation kit (StemCell) and resuspended at a concentration of 1 E6/mL in FACS buffer (PBS + 3% FCS) and 100 pL (=1 E5 cells) were transferred to a 96-well U-bottom PS plate. After centrifugation at 700g for 1 min, dilution series of mAb1 , mAb2, or RefmAb, respectively, at the indicated concentrations were added to the cells and incubated for 3h at RT on a shaker. Following 1.5x (or 3.5x for high concentration ranges up to 2000 nM) washing steps with FACS buffer, the respective fluorophore- conjugated detection antibodies were added and incubated for 30 min at 4°C on a shaker. Cells were co-stained with anti-TRBC1-FITC (JOVI-1) antibody to discriminate effects on TRBC1 and TRBC2 cells and subsequently subjected to FACS acquisition. After 1.5x washing steps with FACS buffer, the cells were resuspended in 100 pL FACS buffer and subjected to FACS acquisition.

Results

Results of the cell binding experiments are shown in Figures 1 - 2. mAb1 and mAb2 showed strong specific binding to the TRBC2+ population of primary T cells (Figure 1A, Figure 1C), with binding to almost all TRBC2+ cells (Figure 2). No binding to the TRBC1+ cell population could be observed (Figure 1 B and Figure 2), even at IgG concentrations of up to 2 pM. These results confirm specific binding of mAb1 and mAb2 to TRBC2, independent of the TCR variable region. In contrast, the TRBC2 specific antibody RefmAb revealed only very weak binding to the TRBC2+ population of primary T cells (Figure 1 C). Example 6: Antibody internalization of mAb1 and mAb2

Internalization of mAb1 and mAb2 was assessed on primary human T cells and TRBC2+ HPB- ALL cells. The irrelevant anti-hen egg lysozyme antibody was used as negative control. Internalization was also benchmarked against the TRBC2 specific antibody RefMab.

Method

1. FACS-based internalization assays on primary T cells

Primary T cells were resuspended in assay buffer (i.e. culture media of the respective cells) and 50 pL (=8E4 cells) were transferred to a 96-well U-bottom PS plate. A dilution series of targetspecific and Ctrl antibodies were incubated with a 4x molar excess of anti-Fab-Fc pHrodo conjugate (inhouse produced) for 30 min at 37°C to induce complex formation. Subsequently, the antibody complex was added to wells containing target cells and incubated for 24h. On the day of analysis, the cells were stained with anti-TRBC1-FITC (JOVI-1; Ancell Corporation) antibody to discriminate effects on TRBC1 and TRBC2 cells and subsequently subjected to FACS acquisition. The data was analyzed using the median of the pHrodo signal of TRBC1 and TRBC2 cells.

2. FACS-based internalization assays on HPB-ALL cells

HBP-ALL cells were resuspended in assay buffer (i.e. culture media of the respective cells) and 50 pL (=8E4 cells) were transferred to a 96-well U-bottom PS plate. A dilution series of targetspecific and Ctrl antibodies were incubated with a 4x molar excess of anti-Fab-Fc pHrodo conjugate (inhouse produced) for 30 min at 37°C to induce complex formation. Subsequently, the antibody complex was added to wells containing target cells and incubated for 24h. On the day of analysis, the cells were washed 1.5x with FACS buffer and resuspended in 60 pL FACS buffer and subjected to FACS acquisition. The data was analyzed using the median of the pHrodo signal.

Results

The results of the internalization experiments on primary T cells and HBP-ALL cells are shown in Figure 3. Both, mAb1 and mAb2, revealed strong internalization into the TRBC2+ subpopulation of human primary T cells (Fig. 3A) as well as into TRBC2+ HBP-ALL cells (Fig. 3C), whereas internalization into the TRBC1+ subpopulation of the human primary T cells was comparable to the isotype control IgG (Fig. 3B). In contrast, TRBC2-specific RefmAb revealed significantly less internalization into TRBC2+ HBP-ALL cells when compared to mAb1 or mAb2. Example 7: In vitro cytotoxicity of mAb1 and mAb2 direct toxin conjugates (ADCs) on cancer cell lines or primary human T cells

To test the cell killing ability of mAb1 and mAb2 as antibody drug conjugates, both antibodies were conjugated to monomethyl auristatin E (MMAE). Antibody drug conjugates were generated and quality controlled by an external service provider (Sterling Pharma Solutions Limited, UK) using the linker payload vc-PAB-MMAE. Binding of mAb1-MMAE and mAb2-MMAE to human TRBC2 was confirmed by FACS. In addition, a negative control ADC with an anti-hen egg lysozyme antibody was used (Ctrl-MMAE).

Method (ELISA)

In brief: In vitro cytotoxicity assays were performed on TRBC2+ HPB-ALL and TRBC1+ H9 cells at 1 .1 x 10⁵ cells/mL. ADC constructs were incubated with cells at different concentrations for 96h at 37°C. Subsequently supernatant was removed and cells were lysed with CellTiter Gio 2.0 (Promega) solution. Bioluminescence was measured according to manufacturer’s protocol.

Methods (FACS)

T cells were isolated from thawn PBMCs and subsequently stained with a cell division tracker (CIV; Thermo Fisher Scientific GmbH) allowing to discriminate proliferating and non-proliferating T cells. Intensity of CTV and normal distribution of TRBC1 :TRBC2 T cells were confirmed via FACS. The cells were resuspended in assay buffer (i.e. culture media) at a concentration of 1 E6/mL and transferred to an appropriate cell culture flask to which anti-CD3/anti-CD28 dynabeads (Thermo Fisher Scientific GmbH) were added at a ratio of beads:cells of 1 :5 to induce T cell activation. After 48h the beads were removed using a magnet and 1 E5 cells were transferred to a 96-well U-bottom plate in 100 pL assay buffer. A dilution series of target-specific and Ctrl ADCs at the indicated concentrations were added to the cells and incubated for 72-120h. On the day of analysis, the cells were first stained with anti-TRBC1-FITC (JOVI-1 ; Ancell Corporation) antibody to discriminate effects on TRBC1 and TRBC2 cells. In a second step, the cells were stained with AnnexinV-AF647 (BioLegend) in Annexin binding buffer and subjected to FACS acquisition. The data was analyzed by quantifying AnnexinV-negative proliferating (CTV-dim) cell counts.

Results

The results of the ELISA-based cytotoxicity assays on the cancer cell lines are shown in Fig. 4A (TRBC2+ HBP-ALL cells) and Fig. 4B (TRBC1+ H9 cells). The results of the FACS-based cytotoxicity assay on primary human T cells is shown in Fig. 4C. ADCs of mAb1 and mAb2 showed potent killing of TRBC2+ cells with IC₅o concentrations in the single digit nanomolar range.

In contrast, no killing of TRBC1+ cells was observable for both tested ADCs.

Example 8: In vitro cytotoxicity of mAb1 and RefmAb as secondary toxin conjugates (ELISA, Piggy-Back ADCs)

The ability of mAb1 non-covalently conjugated to Saporin to kill TRBC2+ cancer cells was tested in an in vitro cytotoxicity assay.

Method

Cell lines were resuspended in assay buffer (i.e. culture media of the respective cells) and 90 pL containing 1 E4 cells TRBC2+ HPB-ALL cells were transferred to a 96-well U-bottom plate. A dilution series of mAb1 or RefmAb antibodies were incubated with a constant concentration of anti-Fab-Fc Saporin (Advanced Targeting Systems) conjugate of 20 nM for 30 min at 37°C to induce complex formation. Subsequently, 10pL of the antibody conjugate complex was added to wells containing target cells and incubated for 96h. Subsequently supernatant was removed and cells were lysed with CellTiter Gio 2.0 solution (Promega). Bioluminescence was measured according to manufacturer protocol.

Results

The results of the ELISA-based cytotoxicity assay on the TRBC2-expressing cancer cell line HBP- ALL is shown in Fig. 5. mAb1 secondary toxin conjugate revealed superior cell killing of HBP-ALL cancer cells when compared to RefmAb secondary toxin conjugate.

Example 9: In vivo cancer cell killing of mAb1-ADC

The ability of mAb1-MMAE to kill cancer cells was assessed in an in vivo cell line derived xenograft model.

Method

5E6 TRBC2 HPB-ALL cells were subcutaneously injected in 100 pL into immunocompromised NOG mice (Experimental Pharmacology & Oncology Berlin-Buch GmbH Berlin). Treatment was started once tumors had reached a size of 100-150 mm³. The respective dose of the ADCs was injected in PBS intravenously. The tumor size and bodyweight were monitored over the indicated time period 2-3 times per week. Results

The results of the in vivo cancer cell killing studies are shown in Fig. 6. mAb1-MMAE ADC induced dose-dependent tumor regression or tumor growth inhibition of subcutaneously engrafted TRBC2 T cell tumors after a single administration of the treatment. Negative control Ctrl-MMAE conjugate did not exhibit significant effects on tumor size.

Final antibody format and safety

TRBC2 is expressed on various types of T cells and activation of immune cells needs to be prevented to avoid unwanted side effects. Accordingly, the final IgG format of an anti-TRBC2 antibody needs to be silent in terms of its ability to induce any effector function during therapeutic intervention.

Both, mAb1 and mAb2, contain five amino acid substitutions in the Fc region of human lgG1f, namely L234A, L235E, G237A, A330S and P331S (hlgG1f_AEASS, numbering according to EU index) to abolish antibody induced effector function. Clinical safety of this silent antibody format in the context of antibody therapy has been described in the art (Wagner F et al. Annals of the Rheumatic Diseases. 2014; 73: 499. doi: 10.1136/annrheumdis-2014-eular.2156). The lack of ability of this antibody format to induce effector functions has been further validated in various assays in the art (see i.e. W02020/182974).

Example 10: mAb1 and mAb2-induced cytokine release from whole-blood cells

Method

200 pL of whole blood were transferred to 96-well plates and 50 pL of a dilution series of mAb1_Fc_AEASS, mAb1_Fc_wild-type, mAb2_Fc_AEASS, positive control murine anti- CD3_lgG OKT3 (J Pediatr. 1982 Apr; 100(4):665-8), negative control lgG_Fc_AEASS and negative control lgG_Fc_wild-type, respectively, were added and incubated for24h. Subsequently the cell solution was resuspended and transferred to V-bottom plates to obtain stable cell pellets and allow for supernatants to be removed without disruption of the cell pellet. After a centrifuge step (8 min at 700xg and 4°C) supernatants were transferred to 96-well PP U-bottom plates and frozen at -20°C until utilization in cytokine ELISA.

Cytokine ELISAs were conducted with according to the manufacturer protocol (R&D Systems, TNFa and IFNg ELISA) using MSD plates (Meso Scale Diagnostics LLC) and MSD readout. In brief, anti-TNFa/IFNg antibodies were coated to 384-well MSD plates over night at RT. After 3 washing steps with PBS-T, the plates were blocked with 1 % BSA in PBS for 1 h at RT on a shaker. Cytokine standards were diluted 1 :2 from stock concentration. 20 pL of standard dilution or supernatants from activation assays were transferred to the blocked plates and incubated for 1h at RT on a shaker. After 3 washing steps with PBS-T, 30 pL of biotinylated mouse anti-human detection antibodies diluted in reagent diluent were added and incubated for 1 h at RT on a shaker. After 3 washes with PBS-T, 20 pL of Streptavidin-ECL were added to the MSD plate and incubated for 20 min at RT on a shaker. After 3 washes with PBS-T, 30 pL of read buffer were added to the wells and luminescence was measured with a MSD Sector Imager device (Meso Scale Diagnostics LLC).

Results

The results of the cytokine release assays on whole-blood cells are shown in Figure 7.

Both, mAb1 and mAb2, in the IgG 1 Fc-silent AEASS format, did not elicit cytokine secretion in a whole-blood assay after incubation for 24h, while the corresponding mAb1 IgG with the wild-type Fc competent hlgG 1 strongly induced TNFalpha and IFNgamma release to a similar extent as the OKT3 positive control IgG. These results confirm that the introduced mutations to the Fc region of mAb1 and mAb2 are able to efficiently abolish Fc mediated activation of immune cells.

Example 11: Binding of mAb1 and mAb2 to FcRn receptor using Octet

Method

Apparent K_D determination to immobilized neonatal Fc receptor (FcRn) from different species was performed at pH 6.0 and 7.2 at 27 °C using Octet instruments (either Octet HTX or RED384; ForteBIO, Sartorius). 0.5 nm of biotinylated human, cynomolgus, mouse or rat FcRn were bound onto streptavidin sensors (SA or SAX; ForteBIO, Sartorius). Kinetic measurements were performed using 8 different concentrations of IgGs (in the Fc compromised AEASS format) (3n serial dilution, 1000 to 0.46 nM) in Octet buffer (PBS, 0.05 % (v/v) Tween-20, 0.1 % (w/v) BSA) with 240 s association time and 180 s dissociation time. After each cycle the sensor was regenerated to remove bound antibody (2x30 s in HBS-EP+, pH 8.0). All sensorgrams were fitted using Data Analysis Software 12 (ForteBIO, Sartorius) to determine the equilibrium binding signals. For determination of the apparent affinity, the data was fitted with a steady state model. Results

Results are summarized in Table 7. mAb2 revealed apparent binding affinities to FcRn from different species in an expected pH-dependency and affinity range (comparable to isotype control antibody Ctr. Antibody lgG1f). Surprisingly, compared to mAb2, mAb1 showed increased binding at pH 7.2 on all tested species.

Table 7: Binding of mAb1 and mAb2 to FcRn via the mutated Fc region at pH 6.0 and 7.2.

*binding; incomplete titration

**nsp: no statement possible; binding only at highest assay concentration (1 pM)

Claims

We Claim

1 . An isolated antibody or antibody fragment specific for the human T cell receptor beta constant region 2 (TRBC2), wherein the antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 1 , the HCDR2 region of SEQ ID NO: 2, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 4 or SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 5 and the LCDR3 region of SEQ ID NO: 6.

2. The isolated antibody or antibody fragment according to claim 1 , wherein said antibody or antibody fragment comprises the VH of SEQ ID NO: 12 and the VL of SEQ ID NO 13 or SEQ ID NO: 33.

3. The isolated antibody or antibody fragment according to claim 1 or claim 2, wherein the antibody or antibody fragment comprises the HO of SEQ ID NO: 14 and the LC of SEQ ID NO: 15 or SEQ ID NO: 34.

4. The isolated antibody or antibody fragment according to any one of the preceding claims, wherein the antibody or antibody fragment has a monovalent affinity for human TRBC2 with a K_D of 1 nM or less.

5. The isolated antibody or antibody fragment according to any one of the preceding claims, wherein the isolated antibody or antibody fragment binds to human TRBC2 expressed on cells with an EC₅o concentration of 20 nM or less.

6. The isolated antibody or antibody fragment according to any one of the preceding claims, wherein the isolated antibody or antibody fragment internalizes into TRBC2 expressing cells with an EC₅o concentration of 20 nM or less.

7. The isolated antibody or antibody fragment according to claim 5 or claim 6, wherein the TRBC2 expressing cells are T cells.

8. The isolated antibody or antibody fragment according to any one of the preceding claims, wherein the antibody fragment is selected from the group of: Fab, scFab, Fab’, (Fab’)2, Fv, scFv, and VHH.

9. The isolated antibody or antibody fragment according to claims 1 - 7, wherein the antibody is an IgG.

10. The isolated antibody or antibody fragment according to any one of the preceding claims, wherein the antibody does not substantially induce effector function.

11 . The isolated antibody or antibody fragment according to any one of the preceding claims, wherein the antibody comprises one or more one amino acid substitutions in its Fc region which reduce or abolish the binding of the Fc region of the antibody to an Fc receptor and/or to C1q and/or reduces the ability of said antibody to induce effector function when compared to the antibody comprising a wild-type Fc region.

12. The isolated antibody or antibody fragment according to claim 10 or claim 11 , wherein the effector function is selected from the group of, but not limited to, CDC, ADCC and ADCP.

13. The isolated antibody or antibody fragment according to any one of the preceding claims, wherein the antibody comprises one or more amino acid substitution selected from the group consisting of: L234A, L235E, G237A, A330S and P331S, with numbering according to EU index.

14. The isolated antibody or antibody fragment according to any one of the preceding claims, wherein the isolated antibody or antibody fragment is a monoclonal antibody or antibody fragment.

15. The isolated antibody or antibody fragment according to any one of the preceding claims, wherein the isolated antibody or antibody fragment is a recombinant or synthetic antibody or antibody fragment.

16. The isolated antibody or antibody fragment according to any one of the preceding claims, wherein the isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

17. A fusion molecule comprising the isolated antibody or antibody fragment specific for human TRBC2 according to any one of the preceding claims and at least one additional moiety.

18. The fusion molecule according to claim 17, wherein the additional moiety is a selected from the group of: a therapeutic agent, a cytotoxic agent, a bacterial toxin, a cytokine, an antibody or antibody fragment, a peptide, a polypeptide, an enzyme, a detectable group, a moiety being capable of binding a metal ion, a tag suitable for detection and/or purification, a targeting ligand, a homo- or hetero-association domain, a moiety which increases solubility of a protein, or a moiety which comprises an enzymatic cleavage site, a lipid, a liposome, and a virus-like- particle.

19. The fusion molecule according to claim 17 or claim 18, wherein the additional moiety is a therapeutic agent or a detectable group.

20. The fusion molecule according to claim 17, wherein the additional moiety is a co-stimulating domain of a chimeric antigen receptor (CAR).

21. The fusion molecule according to claims 17 - 19, wherein the additional moiety or therapeutic agent is Monomethyl auristatin E (MMAE).

22. The isolated antibody or antibody fragment according to claims 1 - 16 or the fusion molecule according to claims 17 - 21 for use as a medicament.

23. The isolated antibody or antibody fragment according to claims 1 - 16 or the fusion molecule according to claims 17 - 22 for use in the treatment of a T cell lymphoma or leukaemia.

24. A nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding the isolated antibody or antibody fragment according to claims 1 - 16 or the fusion molecule according to claims 17 - 21.

25. A vector composition comprising a vector or a plurality of vectors comprising the nucleic acid composition of claim 24.

26. A host cell comprising the vector composition of claim 25 or the nucleic acid composition of claim 24.

27. A pharmaceutical composition comprising the isolated antibody or antibody fragment according to claims 1 - 16 or the fusion molecule according to claims 17 - 21 and a pharmaceutically acceptable carrier or excipient.

28. A method for detecting TRBC2 in a subject or a sample, comprising the step of contacting said subject or sample with an isolated antibody or antibody fragment according to claims 1 - 16 or with the fusion molecule according to claims 17 - 21.