WO2025073803A1

WO2025073803A1 - Peptide-hla binding molecules

Info

Publication number: WO2025073803A1
Application number: PCT/EP2024/077801
Authority: WO
Inventors: Tristan Vaughan; Stephen Harper; Vijaykumar KARUPPIAH; Peter RAPALI
Original assignee: Immunocore Ltd
Current assignee: Immunocore Ltd
Priority date: 2023-10-03
Filing date: 2024-10-02
Publication date: 2025-04-10
Anticipated expiration: 2026-04-03
Also published as: GB202315181D0

Abstract

The present invention relates generally to binding molecules comprising T cell receptor (TCR) mimics that specifically bind to a disease associated peptide-human leukocyte antigen complex (DA-pHLA), compositions comprising such binding molecules, methods of producing such binding molecules, and uses thereof. The TCR mimic may comprise an antibody or an antigen-binding fragment thereof, and may (i) bind to the DA-pHLA with a crossing angle in the range of 150-250 degrees; (ii) with 4 or more peptide residue binding contacts; and (iii) with an affinity of 20 nM or stronger.

Description

PEPTIDE-HLA BINDING MOLECULES

Field of the invention

The present invention relates generally to binding molecules comprising T cell receptor (TOR) mimics that specifically bind to a disease associated peptide-human leukocyte antigen complex (DA-pHLA), compositions comprising such binding molecules, methods of producing such binding molecules, and uses thereof.

Background to the invention

Monoclonal antibodies can be potent and successful cancer therapeutic agents such as those targeting the checkpoint inhibitors like PD-1. Antibodies typically recognize cell-surface targets or extracellular antigens whereas many cancer-associated antigens, such as overexpressed or dysregulated self-proteins, activated oncogenes, mutated tumour suppressors, and translocated gene products, are found only intracellularly. This has presented a significant challenge for antibody-based therapeutics. However, short regions of these intracellular antigens may also be presented as peptides on the cell surface by human leukocyte antigens (pHLA) making them potentially accessible to an antibody therapeutic. Targeting peptides from these cancer-associated proteins, presented by HLA, is difficult for several reasons. First, the natural presentation levels of such target peptides can be very low (often below 100 copies of each specific peptide per cell). Secondly, these peptides need to be co-recognized in the context of HLA, a molecule expressed by almost every nucleated cell in the body. Hence, strong peptide selectivity is absolutely critical to ensure on-target efficacy with minimal potential off-target toxicity, but could be lost if the antibody HLA interaction dominates the binding surface.

The immune system naturally overcomes these hurdles via selective T cell receptor (TOR), but not antibody-driven, recognition of pHLA. While the mechanisms that determine peptide selectivity of TCRs are not fully understood, it has been observed that the binding mode of TCRs is conserved for virtually all TCR-pHLA structures solved to date. TCRs are selected in the thymus to avoid having specificities in common with abundant self-epitopes to maintain self-tolerance. More recently, this ability to target cancer-specific peptides presented by HLA has been utilized to develop tebentafusp, a soluble TCR bispecific therapeutic targeting gp100, a first-in-class soluble TCR therapeutic approved for the treatment of uveal melanoma.

Monoclonal antibodies, relative to TCRs, are more readily able to be engineered as soluble reagents and therapeutics. Moreover, they typically have a much higher affinity for their target antigen, making them attractive for development as soluble therapeutics. However, high hurdles remain to develop monoclonal antibodies as pHLA targeting therapeutics. In addition to the challenges that already exist for engineering soluble TCRs, significant additional hurdles must be overcome to engineer pHLA targeting antibodies (also referred to as a “TOR mimic” or “TCRm” ) with sufficient specificity, in the context of a vast landscape of potential self-antigens. Indeed, it has been observed that in response to very common human pathogens, natural antibodies recognizing pHLA have not been detected. In addition, in cellular testing, pHLA targeting antibodies have been shown to demonstrate insufficient specificity compared to HLA-targeting TCR bispecifics engineered from natural TCR scaffolds. This relatively poor specificity could translate to off-target binding, and potentially unwanted side-effects, when such molecules are attempted to be developed as therapeutics. The high specificity required for a particular pHLA can be difficult to achieve with TCR mimics because a significant portion of the surface area of the antibody paratope must be dedicated to recognizing the largely invariant (for a given subject) HLA molecule found on virtually all cells.

Thus, there exists a real need for developing TCR mimics that are sufficiently potent but have the exquisite specificity profile of a soluble engineered TCR such as tebentafusp for use as therapeutic agents.

Summary of the invention

The inventors have identified, for the first time, the structural binding characteristics of TCR mimics that correlate with optimal therapeutic properties, such as docking angle, location and number of pHLA peptide contact residues, affinity threshold, and high specificity for target pHLA. Such TCR mimics can be used to develop therapeutics for the treatment of diseases, including cancer. Such TCR mimics have the structural attributes as described below.

In one aspect, the present invention provides a binding molecule comprising a TCR mimic (TCRm) that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA), wherein the TCRm comprises an antibody or an antigen-binding fragment thereof, and wherein the TCRm:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the DA-pHLA with an affinity of 20 nM or stronger.

In a further aspect, there is provided a multispecific binding molecule comprising a TCRm that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA) and an immune cell engaging domain, wherein the TCRm comprises an antibody or antigen binding fragment thereof, and wherein the TCRm:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the pHLA complex with an affinity of 20 nM or stronger. In a further aspect, there is provided a multispecific binding molecule comprising a TCRm that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA) and an immune cell engaging domain, wherein

(a) the immune cell engaging domain binds to CD3 and comprises:

(i) an antibody light chain variable region (VL) comprising a CDR1 comprising the amino acid sequence provided in SEQ ID NO: 33, a CDR2 comprising the amino acid sequence provided in SEQ ID NO: 34 and a CDR3 comprising the amino acid sequence provided in SEQ ID NO: 35; and

(ii) an antibody heavy chain variable region (VH) comprising a CDR1 comprising the amino acid sequence provided in SEQ ID NO: 36 or 42, a CDR2 comprising the amino acid sequence provided in SEQ ID NO: 37 and a CDR3 comprising the amino acid sequence provided in SEQ ID NO: 38, and

(b) the TCRm comprises an antibody or antigen binding fragment thereof and:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the DA-pHLA complex with an affinity of 20 nM or stronger.

In a further aspect, there is provided a multispecific binding molecule comprising a TCRm that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA) and an immune cell engaging domain, wherein

(a) the immune cell engaging domain is a PD-1 agonist and comprises a single domain antibody comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID Nos: 43, 44 and 45 respectively; and

(b) the TCRm comprises an antibody or antigen binding fragment thereof and:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts;

(iii) binds to the DA-pHLA with a binding affinity for the pHLA of 20 nM or stronger.

In a further aspect, there is provided a binding molecule comprising a TCR mimic (TCRm) that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA), wherein the TCRm comprises an antibody or an antigen-binding fragment thereof, and wherein the TCR mimic:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the DA-pHLA with an affinity of 20 nM or stronger; and

(iv) binds to a DA-pHLA positive cell, but does not detectably bind to a DA-pHLA negative cell. In a further aspect, there is provided an antibody or antigen binding fragment thereof that specifically targets a disease associated peptide human leukocyte antigen (DA-pHLA) complex, comprising: a means to dock to said DA-pHLA complex and to have an affinity of 20 nM or stronger.

In a further aspect, there is provided a molecule that redirects immune cells to a DA-pHLA presenting cell, wherein the molecule comprises an antibody or antigen binding fragment thereof that specifically targets a disease associated peptide HLA (DA-pHLA) complex and comprising: (a) a means for said antibody or antigen binding fragment thereof to dock to said DA-pHLA complex and to have an affinity of 20 nM or stronger; and (b) a means to engage said immune cells.

In yet a further aspect, there is provided a nucleic acid encoding a binding molecule, antibody or antigen binding fragment thereof or molecule provided herein. There is also provided an expression vector comprising the nucleic acid of this aspect. In addition, there is provided a host cell comprising the nucleic acid or the vector of this aspect.

Also provided, in a further aspect, is a method of making a binding molecule, antibody or antigen binding fragment thereof or molecule provided herein, comprising maintaining the host cell described above under optimal conditions for expression of the nucleic acid and isolating the binding molecule, antibody or antigen binding fragment thereof or molecule.

In a further aspect, there is provided a pharmaceutical composition comprising a binding molecule, antibody or antigen binding fragment thereof or molecule provided herein.

The binding molecule, antibody, or antigen binding fragment thereof, or molecule, nucleic acid, vector, host cell or pharmaceutical composition of any of the above aspects may be used in the treatment of diseases such as cancer, infectious diseases and autoimmune diseases. Thus, in a further aspect, also provided is the binding molecule, antibody, or antigen binding fragment thereof, or molecule, nucleic acid, vector, host cell or pharmaceutical composition provided herein for use in medicine. In a still further aspect there is provided a method of treatment comprising administering the binding molecule, antibody, or antigen binding fragment thereof, or molecule, nucleic acid, vector, host cell or pharmaceutical composition to a patient in need thereof.

In a further aspect, there is provided a method of producing a binding molecule comprising a TOR mimic (TCRm) that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA), wherein the TCRm comprises an antibody or an antigen-binding fragment thereof, and wherein the method comprises:

(a) providing a plurality of candidate binding molecules that bind to the DA-pHLA; and

(b) identifying from the plurality of candidate binding molecules a binding molecule comprising a TCRm that:

(i) binds to the DA-pHLA complex with a crossing angle in the range of 150-250 degrees; (ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the pHLA complex with an affinity of 20 nM or stronger.

Description of the invention

Binding molecules

As used herein, the term “binding molecule” generally refers to a molecule capable of binding to one or more target antigen(s). Statements herein such as “the binding molecule” or “the binding molecule of the invention”, “binding molecule provided herein” or “binding molecule described herein” are references to all binding molecules in any one or more of the above aspects, unless indicated otherwise. The binding molecules comprise a T cell receptor (“TOR”) mimic (“TCRm”), where the TCRm comprises an antibody or antigen binding fragment thereof. Such antibodies or antigen binding fragments thereof can comprise, for example, an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). The VH and VL dimerise to form the binding site of the TCRm. The antibody or antigen binding fragment of the TCRm thus comprises the binding site that specifically recognises and binds to the pHLA complex. Such molecules may adopt a number of different formats as discussed herein.

The binding molecule may comprise one or more polypeptide chain(s). As used herein, the term “polypeptide chain” refers to a polymer (i.e., a chain) of amino acids, typically twenty or more amino acids, linked by peptide bonds and having an N- and C- terminus. As is known in the art, a protein may comprise multiple polypeptide chains assembled together by non-covalent or covalent interactions.

In addition to the TCRm, the binding molecule may comprise one or more other antigen-binding moieties. Thus, the binding molecule may be multispecific. In this regard, the term “multispecific” refers to a binding molecule comprising two or more antigen binding moieties, including the TCRm. Such binding molecules are able to simultaneously bind to the pHLA and a further one or more different antigens. For example, the binding molecule may be bispecific. The one or more other (i.e., other than the TCRm) antigen-binding moieties may be a second antibody or antigen-binding fragment thereof. The one or more other antigen-binding moieties in a multispecific binding molecule may comprise an immune cell engaging domain described herein. For example, the binding molecule may be a Bi-specific T-cell engager (BiTE). As is known in the art, BiTEs comprise a first scFv (e.g.., an scFv corresponding to a TCRm) which binds to a target antigen (in the case of a TCRm, to a target pHLA complex) and a second scFv (e.g.., an immune cell engaging domain) which binds to a T cell surface antigen, such as CD3.

The binding molecule may be soluble. Thus, the binding molecule may not comprise any transmembrane regions. Such binding molecules may be used as soluble therapeutic or diagnostic agents, for example when the TCRm is linked to a therapeutic or diagnostic agent, such as an immune cell engaging domain as described below.

Therapeutic agents which may be associated with or comprised in (e.g., fused to) the binding molecules described herein include immune-modulators and effectors, radioactive compounds, enzymes (perforin for example) or chemotherapeutic agents (cis-platin for example). To ensure that the therapeutic effects are exercised in the desired location the agent could be inside a liposome or other nanoparticle structure linked to the binding molecule so that the compound is released slowly. This will prevent damaging effects during the transport in the body and ensure that the agent has maximum effect after binding of the binding molecule to the relevant antigen presenting cells.

Examples of suitable therapeutic agents include, but are not limited to:

• antibodies, or fragments thereof, including immune cell engaging domains in this format, such as anti-T cell or NK cell determinant antibodies (e.g. anti-CD3, anti-CD28 or anti-CD16)

• alternative protein scaffolds with antibody-like binding characteristics (e.g. DARPins)

• immuno-stimulants, i.e. immune effector molecules which stimulate immune response. For example, cytokines such as IL-2 and IFN-y,

• chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein, etc.

• activators of the complement pathway or Fc receptors

• checkpoint inhibitors, such as those that target PD1 or PD-L1

• small molecule cytotoxic agents, i.e. compounds with the ability to kill mammalian cells having a molecular weight of less than 700 Daltons. Such compounds could also contain toxic metals capable of having a cytotoxic effect. Furthermore, it is to be understood that these small molecule cytotoxic agents also include pro-drugs, i.e. compounds that decay or are converted under physiological conditions to release cytotoxic agents. Examples of such agents include cis-platin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II, temozolomide, topotecan, trimetreate arbourate, auristatin E vincristine and doxorubicin

• peptide cytotoxins, i.e., proteins or fragments thereof with the ability to kill mammalian cells. For example, ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, Dnase and Rnase;

• radio-nuclides, i.e. unstable isotopes of elements which decay with the concurrent emission of one or more of a or p particles, or y rays. For example, iodine 131 , rhenium 186, indium 111 , yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213; chelating agents may be used to facilitate the association of these radio-nuclides to TCRs, or multimers thereof;

• superantigens and mutants thereof

• xenogeneic protein domains, allogeneic protein domains, viral/bacterial protein domains, viral/bacterial peptides. The binding molecule may constitute one or more fusion proteins. The TCRm and other protein domains in such a fusion protein may be covalently linked via one or more linker sequence(s).

The term "linker" as used herein refers to one or more amino acid residues inserted between domains, or a domain and an agent, to provide sufficient mobility for the domains or elements, for example the domains of the binding molecules described herein to fold correctly to form the antigen binding sites. A linker may be inserted at the transition between variable domains or between variable domains and constant domains (or other domains), respectively, at the amino acid sequence level. The transition between domains can be identified because the approximate size of antibody domains as well as TOR domains is well understood by those skilled in the art. The precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be assumed by techniques of modelling or secondary structure prediction.

Linker sequences are usually flexible, in that they are made up primarily of amino acids such as glycine, alanine and serine, which do not have bulky side chains likely to restrict flexibility. Alternatively, linkers with greater rigidity may be desirable. Usable or optimum lengths of linker sequences may be easily determined. Often the linker sequence will be less than about 12, such as less than 10, or from 2-10 amino acids in length, The linker may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. Examples of suitable linkers are known in the art.

Binding molecules described herein may also be used as diagnostic reagents to detect cells presenting the target pHLA. In this case the molecules may be fused to a detectable label. Detectable labels for diagnostic purposes include for instance, fluorescent labels, radiolabels, enzymes, nucleic acid probes and contrast reagents.

For some purposes, the binding molecules described herein may be aggregated into a complex comprising several binding molecules to form a multivalent binding molecule complex. There are a number of human proteins that contain a multimerisation domain that may be used in the production of multivalent binding molecule complexes. For example the tetramerisation domain of p53 which has been utilised to produce tetramers of scFv antibody fragments which exhibited increased serum persistence and significantly reduced off-rate compared to the monomeric scFv fragment. Haemoglobin also has a tetramerisation domain that could be used for this kind of application. A multivalent binding molecule complex described herein may have enhanced binding capability for the complex compared to a non-multimeric native (also referred to as parental, natural, unmutated wild type, or scaffold) binding molecule described herein. Thus, multivalent complexes of binding molecules described herein are also included within the invention. Such multivalent binding molecule complexes according to the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent binding molecule complexes having such uses.

Binding molecules described herein can also be used in a treatment process known as adoptive therapy, for example chimeric antigen receptor (CAR) T cell therapy. In such therapies, cells expressing a nucleic acid encoding the binding molecule are administered to a patient in need thereof. Thus, the binding molecule may be a CAR. As is known in the art, CARs comprise an extracellular targeting domain (e.g., a pHLA-binding domain described herein), an extracellular linker/hinge domain, a transmembrane domain, and intracellular T-cell-activating and co-stimulatory signaling domains.

Peptide-human leukocyte antigen complex (pHLA) binding domains

A “pHLA-binding domain” (also referred to herein as a “pMHC-binding domain”), as used herein, is a protein domain capable of binding to a peptide-HLA complex (pHLA). A pHLA-binding domain comprising an antibody or antigen binding fragment thereof, or a molecule derived from an antibody or antigen binding fragment thereof, that is capable of binding to a peptide-HLA complex is referred to herein as a TCR mimic (“TCR mimic” or “TCRm”).

The term “antibody” as used herein is meant to include conventional/native antibodies and engineered antibodies, in particular functional antibody fragments, single chain antibodies, and bispecific or multispecific antibodies. “Native” or “conventional”, in this context, refers to an antibody that has the same type of domains and domain arrangements as an antibody found in nature and comprises antibody-derived CDR and FR seguences. In a native/conventional four-chain, e.g. human, antibody, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. The variable domains of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. Conventional antibody binding sites are made up of residues that are primarily from the CDRs or hypervariable regions. As described herein, CDRs refer to amino acid seguences that together define the antigen binding site, where binding affinity and specificity of the antibody is generally associated with the antibody or antigen binding site.

The terms "full-length antibody," "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen binding fragment of an antibody. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be wild-type seguence constant domains (e.g., human wild-type seguence constant domains) or amino acid seguence variants thereof.

“Engineered” antibody formats include functional antibody fragments, single chain antibodies, single domain antibodies, and chimeric, humanized, bispecific or multispecific antibodies. A “functional antibody fragment” or “antigen-binding fragment” (used interchangeably herein) refers to a portion of a full-length antibody, or a protein that resembles a portion of a full-length antibody, that retains the ability to bind to its target antigen, in particular the antigen binding region or variable region of the full- length antibody. Examples of “antigen binding fragments” include Fv, Fab, F(ab’)2, Fab’, dsFv, (dsFv)2, scFv, sc(Fv)2 and diabodies. The TCRm in the binding molecules described herein may comprise any one or more of the above described antibodies or antibody formats. For example, the TCRm may comprise an scFv comprising the VH and VL. The scFv may comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

One or more disulphide bonds may be incorporated between VH and VL domains to e.g., enhance stability of the TCRm. Suitable positions of such bonds are known in the art.

As used herein, “variable region" refers to the portions of the light and/or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and includes amino acid sequences of complementarity determining regions (CDRs); i.e., CDR1 , CDR2, and CDR3, and framework regions (FRs). Exemplary variable regions comprise three or four FRs (e.g., FR1 , FR2, FR3 and optionally FR4) together with three CDRs.

As used herein, the term "complementarity determining regions” (syn. CDRs; i.e., CDR1 , CDR2, and CDR3) refers to the amino acid residues of an antibody variable region which define the antigen specificity of an antibody. "Framework regions" (FRs) are those variable region residues other than the CDR residues. Each variable region typically has three CDR regions identified as CDR1 , CDR2 and CDR3. The amino acid positions assigned to CDRs and FRs can be defined according to Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 or other numbering systems, e.g., the canonical numbering system of Chothia; the IMGT numbering system; or the AHO numbering system of. For example, according to the numbering system of Kabat, VH framework regions (FRs) and CDRs are positioned as follows: residues 1-30 (FR1 ), 31-35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3) and 103- 113 (FR4). According to the numbering system of Kabat, VL FRS and CDRs are positioned as follows: residues 1-23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3) and 98-107 (FR4). The present disclosure is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including those discussed above.

The VL and VH of the TCRm in the binding molecules described herein each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. The antigenbinding site of the pHLA-binding domain, therefore, includes six CDRs, comprising the CDR set from each of a VH and VL.

The TCRm may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) which dimerise to form an antibody variable fragment (“Fv”). In this regard, the pHLA-binding domain in the binding molecules described herein may be considered a “TOR mimic” (TCRm) because it is an antibody-derived domain that recognizes an epitope similar to a TCR (i.e., a pHLA).

Thus, the TCRm described herein can comprise an Fv. As used herein, the term “Fv” shall be taken to mean any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a VL and a VH associate and form a complex having an antigen binding site, i.e., capable of specifically binding to an antigen. The VH and the VL which form the antigen binding site can be in a single polypeptide chain or in different polypeptide chains. The term “Fv” shall be understood to encompass fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means. In some examples, the VH is not linked to a heavy chain constant domain (CH) 1 and/or the VL is not linked to a light chain constant domain (CL). Exemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab' fragment, a F(ab') fragment, a scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, e.g., CH2 or CH3 domain, e.g., a minibody. The pHLA-binding domain in the binding molecules described herein may comprise any one or more of these Fv containing proteins.

Alternatively, or additionally, the TCRm may comprise a Fab. The term “Fab” (“fragment antigenbinding”) denotes an antigen-binding fragment of an antibody, which comprises the antibody light chain (VL-CL) and the variable and CH1 domain (VH-CH1 ) of the antibody heavy chain. Fab fragments typically have a molecular weight of about 50,000 Dalton. The Fv fragment is the N- terminal part of the Fab fragment of an antibody and consists of the variable portions of one light chain (VL) and one heavy chain (VH).

A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain or can be produced using recombinant means. A "Fab¹ fragment" of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain comprising a VH and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. A Fab' fragment can also be produced by recombinant means. A "F(ab')2 fragment” of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A “Fab2” fragment is a recombinant fragment comprising two Fab fragments linked using, for example a leucine zipper or a CH3 domain. A “single chain Fv” or “scFv” is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker. An antibody may also comprise constant domains, some of which can be arranged into a constant region, which includes a constant fragment or fragment crystallizable (Fc) region, in the case of a heavy chain. Generally, a light chain from mammals is either a k light chain or a I light chain and a heavy chain from mammals is a, d, e, g, or m. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGi, lgG2, IgGa, lgG4, IgAi and lgA2) or subclass. The term “antibody” also encompasses humanized antibodies, primatized antibodies, human antibodies and chimeric antibodies.

The TCRm of the binding molecules described herein specifically binds to a disease associated peptide antigen in complex with HLA (i.e., a DA-pHLA). As used herein, the term “disease associated peptide antigen in complex with HLA” or “DA-pHLA” refers to any pHLA that is associated with a disease. For example these terms include a pHLA that is presented on a diseased cell (e.g., a cancerous cell or virally infected cell) but not presented on, or presented at a reduced level on, a healthy cell. Presentation of the DA-pHLA on cells may also be tissue-specific. For example, the DA- pHLA may be a PLA which is presented on cells in one tissue in a disease, but the same pHLA may be presented on healthy cells in another tissue. These terms also include a pHLA that is targeted by T cells in an autoimmune disease. The DA-pHLA that is specifically bound by the TCRm is occasionally referred to herein as the “target pHLA”, and the peptide within that pHLA may be referred to herein as the “target peptide”, as a way of distinguishing these from other (non-target or nonspecific) pHLA and peptides therein. The terms “MHO” and “HLA” as used herein are used interchangeably.

The human leukocyte antigen (HLA) may be a class I HLA. As is known in the art, class I HLA molecules present peptide epitopes to cytotoxic T lymphocytes (CTLs), also referred to as CD8+ T cells. HLA class I molecules are heterodimers; they have a polymorphic heavy a-subunit whose gene is present inside the HLA locus and a small invariant p2 microglobulin subunit whose gene is located usually outside of the HLA locus. The polymorphic heavy chain of a class I HLA molecule contains (i) an N-terminal extra-cellular region comprising three domains, a1 , a2, and a3, (ii) a transmembrane helix to the hold the HLA molecule on the cell surface and (iii) a short cytoplasmic tail. The a1 and a2 domains form a deep peptide-binding groove between two long a-helices and the floor of the groove is formed by eight p-strands. Immunoglobulin-like domain a3 is involved in the interaction with the CD8 co-receptor on a T cell. The p2 microglobulin subunit provides stability to the complex and participates in the interaction with the CD8 co-receptor. The peptide is non-covalently bound to a class I HLA molecule. It is held by several pockets on the floor of the peptide-binding groove. Amino acid side-chains that are most polymorphic in human alleles tend to fill up the central and widest portion of the binding groove, while conserved side-chains tend to be clustered at the narrower ends of the groove.

The human leukocyte antigen (HLA) may be of any suitable serotype. Preferably the HLA is a HLA- A*02 serotype or HLA-A*24 serotype HLA molecule. The peptide in the DA-pHLA may be a human peptide. The peptide antigen may be a tumour associated antigen peptide. Alternatively, the peptide antigen may be an autoimmune disease associated peptide antigen. The peptide antigen may be a peptide from a protein selected from the group consisting of PRAME, GP100, PIWIL1 , MAGEA4, WT1 and pre-pro-insulin. The peptide antigen may be a peptide from a protein selected from the group consisting of PRAME, GP100, PIWIL1 , MAGEA4 and pre-pro-insulin. For example, the peptide antigen may be a peptide antigen described in WO2011001152, WO2017109496, WO2017175006 or WO2018234319.

The peptide antigen may be from GP100. For example, the peptide in the DA-pHLA may have the amino acid sequence provided in SEQ ID NO: 1 (ALDGGNKHFL). Such a peptide forms a complex with HLA molecules of the HLA-A*02 serotype. Thus, the TCRm may bind to a ALDGGNKHFL (SEQ ID NO: 1)-HLA-A*02 complex.

The peptide antigen may be from PRAME. For example, the peptide in the DA-pHLA may have the amino acid sequence provided in SEQ ID NO: 2 (PYLGQMINL). Such a peptide forms a complex with HLA molecules of the HLA-A*24 serotype. Thus, the TCRm may bind to a PYLGQMINL (SEQ ID NO: 2)-HLA-A*24 complex.

The peptide antigen may be from MAGEA4. For example, the peptide in the DA-pHLA may have the amino acid sequence provided in SEQ ID NO: 74 (GVYDGREHTV). Such a peptide forms a complex with HLA molecules of the HLA-A*02 serotype. Thus, the TCRm may bind to a GVYDGREHTV (SEQ ID NO: 74)-HLA-A*02 complex.

The peptide antigen may be from WT1. For example, the peptide in the DA-pHLA may have the amino acid sequence provided in SEQ ID NO: 76 (VLDFAPPGA). Such a peptide forms a complex with HLA molecules of the HLA-A*02 serotype. Thus, the TCRm may bind to a VLDFAPPGA (SEQ ID NO: 76)-HLA-A*02 complex.

The binding molecule comprising a TCRm may therefore specifically bind to a DA-pHLA where the peptide in the DA-pHLA has the amino acid sequence provided in SEQ ID NO: 1 , 2, 74 or 76, or the amino acid sequence provided in SEQ ID NO: 1 , 2 or 74.

Alternatively, the peptide in the pHLA may be a peptide from a pathogen, such as a bacterial, fungal or viral peptide. For example, the peptide may be a viral peptide, such as a peptide from human immunodeficiency virus (HIV) or hepatitis B virus (HBV).

The TCRm may comprise amino acid sequences derived from any one of the exemplary TCR mimics described herein. The TCRm may comprise (a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 4, 5 and 6 respectively and a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 8, 9 and 10 respectively;

(b) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 14, 15 and 16 respectively and a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 18, 19 and 20 respectively;

(c) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 23, 24 and 25 respectively and a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 27, 28 and 29 respectively;

(d) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 52, 53 and 25 respectively and a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 55, 28 and 29 respectively;

(e) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 58, 59 and 60 respectively and a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 62, 63 and 64 respectively; or

(f) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 67, 68 and 69 respectively and a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 71 , 72 and 73 respectively.

The TCRm may comprise

(a) a VL comprising the amino acid sequence provided in SEQ ID NO: 47 and a VH comprising the amino acid sequence provided in SEQ ID NO: 7;

(b) a VL comprising the amino acid sequence provided in SEQ ID NO: 13 and a VH comprising the amino acid sequence provided in SEQ ID NO: 17;

(c) a VL comprising the amino acid sequence provided in SEQ ID NO: 22 and a VH comprising the amino acid sequence provided in SEQ ID NO: 26;

(d) a VL comprising the amino acid sequence provided in SEQ ID NO: 51 and a VH comprising the amino acid sequence provided in SEQ ID NO: 54;

(e) a VL comprising the amino acid sequence provided in SEQ ID NO: 57 and a VH comprising the amino acid sequence provided in SEQ ID NO: 61 ; or

(f) a VL comprising the amino acid sequence provided in SEQ ID NO: 66 and a VH comprising the amino acid sequence provided in SEQ ID NO: 70.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL043_AB011”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 4, 5 and 6 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 4, 5 and 6; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 8, 9 and 10 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 8, 9 and 10 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e. weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 4, 5 and 6 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 8, 9 and 10 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL043_AB011”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 47 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 7. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 47 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 7.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 47 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 7, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 4, 5 and 6 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 8, 9 and 10 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 47 and a VH comprising the amino acid sequence provided in SEQ ID NO: 7.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 3. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 3. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 3.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL043-AB035”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 14, 15 and 16 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 14, 15 and 16; and (b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 18, 19 and 20 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 18, 19 and 20 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 14, 15 and 16 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 18, 19 and 20 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL043-AB035”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 13 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 17. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 13 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 17.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 13 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 17, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 14, 15 and 16 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 18, 19 and 20 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 13 and a VH comprising the amino acid sequence provided in SEQ ID NO: 17.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 12. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 12. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 12.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL043-AB004 L05 Hwt”. In this regard, the TCRm may comprise (a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 179, 180 and 181 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 179, 180 and 181 ; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 183, 184 and 185 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 183, 184 and 185 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e. weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 179, 180 and 181 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 183, 184 and 185 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL043-AB004 L05 Hwt”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 178 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 182. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 178 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 182.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 178 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 182, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 179, 180 and 181 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 183, 184 and 185 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 178 and a VH comprising the amino acid sequence provided in SEQ ID NO: 182.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 177. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 177. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 177. The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL043-AB004 Lwt H09”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 189, 190 and 191 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 189, 190 and 191 ; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 193, 194 and 195 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 193, 194 and 195 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e. weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 189, 190 and 191 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 193, 194 and 195 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL043-AB004 Lwt H09”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 188 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 192. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 188 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 192.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 188 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 192, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 189, 190 and 191 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 193, 194 and 195 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 188 and a VH comprising the amino acid sequence provided in SEQ ID NO: 192.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 187. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 187. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 187.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL043-AB035 L06 H06”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 199, 200 and 201 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 199, 200 and 201 ; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 203, 204 and 205 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 203, 204 and 205 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e. weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 199, 200 and 201 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 203, 204 and 205 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL043-AB035 L06 H06”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 198 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 202. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 198 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 202.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 198 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 202, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 199, 200 and 201 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 203, 204 and 205 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 198 and a VH comprising the amino acid sequence provided in SEQ ID NO: 202. The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 197. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 197. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 197.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL043-AB035 L11 H 11 ”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 209, 210 and 211 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 209, 210 and 211 ; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 213, 214 and 215 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 213, 214 and 215 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e. weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 209, 210 and 211 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 213, 214 and 215 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL043-AB035 L11 H 11 ”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 208 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 212. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 208 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 212.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 208 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 212, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 209, 210 and 211 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 213, 214 and 215 respectively. More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 208 and a VH comprising the amino acid sequence provided in SEQ ID NO: 212.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 207. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 207. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 207.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL043-AB035 L17 H17”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 219, 220 and 221 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 219, 220 and 221 ; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 223, 224 and 225 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 223, 224 and 225 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e. weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 219, 220 and 221 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 223, 224 and 225 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL043-AB035 L17 H17”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 218 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 222. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 218 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 222.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 218 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 222, wherein (a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 219, 220 and 221 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 223, 224 and 225 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 218 and a VH comprising the amino acid sequence provided in SEQ ID NO: 222.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 217. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 217. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 217.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL29_AB003”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 23, 24 and 25 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 23, 24 and 25; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 27, 28 and 29 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 27, 28 and 29 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 23, 24 and 25 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 27, 28 and 29 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL29_AB003”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 22 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 26. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 22 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 26. The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 22 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 26, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 23, 24 and 25 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 27, 28 and 29 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 22 and a VH comprising the amino acid sequence provided in SEQ ID NO: 26.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 21. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 21. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 21.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL029-AB003 L2H2”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 52, 53 and 25 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 52, 53 and 25 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 55, 28 and 29 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 55, 28 and 29 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 52, 53 and 25 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 55, 28 and 29 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL029-AB003 L2H2”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 51 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 54. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 51 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 54.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 51 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 54, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 52, 53 and 25 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 55, 28 and 29 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 51 and a VH comprising the amino acid sequence provided in SEQ ID NO: 54.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 50. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 50. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 50.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL029-AB002”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 79, 80 and 81 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 79, 80 and 81 ; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 83, 84 and 85 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 83, 84 and 85 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 79, 80 and 81 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 83, 84 and 85 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL029-AB002”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 78 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 82. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 78 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 82.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 78 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 82, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 79, 80 and 81 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 83, 84 and 85 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 78 and a VH comprising the amino acid sequence provided in SEQ ID NO: 82.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 77. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 77. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 77.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL029-AB003 L19 H20”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 89, 90 and 91 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 89, 90 and 91 ; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 93, 94 and 95 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 93, 94 and 95 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 89, 90 and 91 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 93, 94 and 95 respectively. The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL029-AB003 L19 H20”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 88 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 92. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 88 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 92.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 88 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 92, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 89, 90 and 91 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 93, 94 and 95 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 88 and a VH comprising the amino acid sequence provided in SEQ ID NO: 92.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 87. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 87. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 87.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL029-AB003 L02 H21”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 99, 100 and 101 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 99, 100 and 101 ; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 103, 104 and 105 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 103, 104 and 105 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 99, 100 and 101 respectively; and (b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 103, 104 and 105 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL029-AB003 L02 H21”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 98 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 102. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 98 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 102.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 98 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 102, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 99, 100 and 101 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 103, 104 and 105 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 98 and a VH comprising the amino acid sequence provided in SEQ ID NO: 102.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 97. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 97. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 97.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL029-AB003 L26 H02”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 109, 110 and 111 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 109, 110 and 111 ; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 113, 114 and 115 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 113, 114 and 115 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA. More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 109, 110 and 111 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 113, 114 and 115 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL029-AB003 L26 H02”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 108 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 112. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 108 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 112.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 108 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 112, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 109, 110 and 111 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 113, 114 and 115 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 108 and a VH comprising the amino acid sequence provided in SEQ ID NO: 112.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 107. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 107. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 107.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL030-AB002”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 58, 59 and 60 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 58, 59 and 60 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 62, 63 and 64 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 62, 63 and 64 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 58, 59 and 60 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 62, 63 and 64 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL030-AB002”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 57 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 61. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 57 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 61.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 57 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 61 , wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 58, 59 and 60 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 62, 63 and 64 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 57 and a VH comprising the amino acid sequence provided in SEQ ID NO: 61.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 56. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 56. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 56.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL030-AB102”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 67, 68 and 69 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 67, 68 and 69 respectively; and (b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 71 , 72 and 73 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 71 , 72 and 73 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 67, 68 and 69 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 71 , 72 and 73 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL030-AB102”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 66 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 70. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 66 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 70.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 66 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 70, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 67, 68 and 69 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 71 , 72 and 73 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 66 and a VH comprising the amino acid sequence provided in SEQ ID NO: 70.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 65. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 65. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 65.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL030-AB002 Lwt H03”. In this regard, the TCRm may comprise (a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 119, 120 and 121 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 119, 120 and 121 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 123, 124 and 125 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 123, 124 and 125 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 119, 120 and 121 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 123, 124 and 125 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL030-AB002 Lwt H03”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 118 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 122. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 118 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 122.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 118 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 122, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 119, 120 and 121 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 123, 124 and 125 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 118 and a VH comprising the amino acid sequence provided in SEQ ID NO: 122.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 117. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 117. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 117. The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL030-AB102 H02 L06”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 129, 130 and 131 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 129, 130 and 131 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 133, 134 and 135 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 133, 134 and 135 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 129, 130 and 131 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 133, 134 and 135 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL030-AB102 H02 L06”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 128 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 132. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 128 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 132.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 128 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 132, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 129, 130 and 131 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 133, 134 and 135 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 128 and a VH comprising the amino acid sequence provided in SEQ ID NO: 132.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 127. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 127. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 127.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL030-AB102 H07 L07”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 139, 140 and 141 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 139, 140 and 141 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 143, 144 and 145 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 143, 144 and 145 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 139, 140 and 141 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 143, 144 and 145 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL030-AB102 H07 L07”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 138 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 142. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 138 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 142.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 138 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 142, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 139, 140 and 141 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 143, 144 and 145 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 138 and a VH comprising the amino acid sequence provided in SEQ ID NO: 142. The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 137. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 137. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 137.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL036-AB002 L02 Hwt”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 149, 150 and 151 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 149, 150 and 151 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 153, 154 and 155 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 153, 154 and 155 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 149, 150 and 151 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 153, 154 and 155 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL036-AB002 L02 Hwt”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 148 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 152. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 148 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 152.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 148 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 152, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 149, 150 and 151 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 153, 154 and 155 respectively. More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 148 and a VH comprising the amino acid sequence provided in SEQ ID NO: 152.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 147. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 147. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 147.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL036-AB002 L03 Hwt”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 159, 160 and 161 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 159, 160 and 161 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 163, 164 and 165 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 163, 164 and 165 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 159, 160 and 161 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 163, 164 and 165 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL036-AB002 L03 Hwt”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 158 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 162. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 158 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 162.

The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 158 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 162, wherein (a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 159, 160 and 161 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 163, 164 and 165 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 158 and a VH comprising the amino acid sequence provided in SEQ ID NO: 162.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 157. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 157. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 157.

The TCRm may comprise CDR sequences that are substantially the same as those in the exemplary TCRm referred to herein as “FL036-AB002 L04 Hwt”. In this regard, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 169, 170 and 171 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 169, 170 and 171 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 173, 174 and 175 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 173, 174 and 175 respectively. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the affinity of the TCRm for the target pHLA.

More specifically, the TCRm may comprise

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 169, 170 and 171 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 173, 174 and 175 respectively.

The TCRm may comprise variable region sequences that are similar to those in the exemplary TCRm referred to herein as “FL036-AB002 L04 Hwt”. The TCRm may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 168 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 172. For example, the TCRm may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 168 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 172. The TCRm may comprise a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 168 and a VH comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 172, wherein

(a) the VL comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 169, 170 and 171 respectively; and

(b) the VH comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 173, 174 and 175 respectively.

More specifically, the TCRm may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 168 and a VH comprising the amino acid sequence provided in SEQ ID NO: 172.

The TCRm may comprise an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 167. For example, the TCRm may comprise an scFv comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 167. More specifically, the TCRm may comprise an scFv comprising the amino acid sequence provided in SEQ ID NO: 167.

Immune cell engaging domains

A binding molecule described herein may comprise an immune cell engaging domain. An “Immune cell engaging domain”, as used herein, is a protein domain that is capable of binding to a target on an immune cell and/or modifying an immune response, for example promoting or suppressing an immune response such as T cell activation. The immune cell engaging domain may comprise an antibody, or antigen-binding fragment thereof. Thus, the immune cell engaging domain may comprise an antigen-binding site. Suitable types of antibodies, and fragments thereof, are described herein above in relation to TCR mimics.

The immune cell engaging domain may be a T cell engaging immune effector domain. A “T cell engaging immune effector domain”, as used herein, is a protein domain that is capable of binding to a target on a T cell to promote an immune response (e.g., promote T cell recruitment and/or activation).

The T cell engaging immune effector domain may bind to a protein expressed on a cell surface of a T cell to promote activation of the T cell. For example, the T cell engaging immune effector domain may be a CD3 effector domain. The T cell engaging immune effector domain may bind to, for example specifically bind to, CD3 (i.e., the T cell engaging immune effector domain may be a CD3-binding protein). The T cell engaging immune effector may be an antibody, or an antigen-binding fragment thereof, for example a single-chain variable fragment (scFv), or a similar sized antibody-like scaffold, or any other binding protein that activates a T cell through interaction with CD3 and/or the TCR/CD3 complex. The antibody may also be a single domain antibody, such as the variable region of a heavy chain antibody (e.g., a VHH). Alternatively, the immune cell engaging domain may be an immune suppressor. As used herein, the term “immune suppressor” refers to any molecule, e.g., a protein, that is capable of inhibiting an immune response, such as inhibiting T cell activation. The immune suppressor may bind to a target (e.g. antigen). For example, the immune suppressor may be an immune checkpoint agonist, i.e., a molecule that induces immune checkpoint signalling (such as a PD-1 agonist). The immune suppressor may comprise an antigen-binding moiety that is capable of binding to an antigen. The antigen of the immune suppressor may be located on an immune cell, such as a T cell. The binding molecule may comprise an antibody or antigen binding fragment thereof, for example, the antibody may also be a single domain antibody, such as the variable region of a heavy chain antibody (VHH). Alternatively, the antibody may be a single-chain variable fragment (scFv), or a similar sized antibodylike scaffold, or any other binding protein that suppresses a T cell through induction of immune checkpoint signalling. Such immune suppressors are described below.

The immune cell engaging domain may comprise an antigen-binding moiety that is capable of binding to an antigen. The antigen of the immune cell engaging domain may be located on an immune cell, such as a T cell.

The antigen may be selected from the group consisting of CD2, CD3 (such as the CD3y, CD35 and CD3E chains), CD4, CD5, CD7, CD8, CD10, CD11 b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41 , CD41 b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61 , CD64, CD68, CD90, CD94, CD95, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, FcsRI, TCRa/p, TCRy/5, HLA-DR and 4-1 BB, or combinations thereof. “Combinations thereof” refers to complexes of two or more of said antigens, e.g. a TCRa/p CD3 complex. Preferably, the antigen is CD3.

The immune cell engaging domain may comprise an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), which associate to form the antigen-binding moiety that is capable of binding to the antigen, as described above in relation to TCR mimics. For example, the immune cell engaging domain may comprise a scFv comprising the VH and VL.

Alternatively, the immune cell engaging domain may comprise a single domain antibody, such as the variable region of a heavy chain antibody. In this regard, the term “single domain antibody” refers to an antibody that consists of a single antibody variable domain (e.g., a heavy chain variable domain). Thus, the immune cell engaging domain may comprise a VHH (i.e., the variable domain of a heavy chain antibody), for example. As is known in the art, the antigen binding site of a single domain antibody, such as a VHH, may comprise three CDRs (as opposed to six in a conventional four-chain antibody). The term “antigen binding moiety of an antibody”, as used herein, encompasses such binding sites. Other suitable antigen binding moieties are heavy chain antibodies (hcAb), single domain antibodies (sdAb), minibodies), the variable domain of camelid heavy chain antibodies (VHH), the variable domain of the new antigen receptors (VNAR), affibodies, alphabodies, designed ankyrin-repeat domains (DARPins), anticalins, knottins and engineered CH2 domains (nanoantibodies).

The immune cell engaging domain may be, or comprise, a heavy chain variable domain that comprises, consists or essentially consists of four framework regions (FR1 to FR4 respectively) and three complementarity determining regions (CDR1 to CDR3 respectively); or any suitable fragment of such a heavy chain variable domain (which retains the antigen binding site). The immune cell engaging domain may thus comprise a heavy chain antibody. The immune cell engaging domain may comprise a heavy chain variable domain sequence of an antibody that is derived from a conventional four-chain antibody, such as, without limitation, a VH sequence that is derived from a human antibody. The immune cell engaging domain may comprise the variable domain of a heavy chain antibody (e.g., a camelid antibody), such as a VHH (also referred to herein as a “VHH domain”).

As described herein, the immune cell engaging domain may comprise an antigen binding moiety (e.g., an antibody antigen binding moiety) that binds to an antigen located on an immune cell. In the context of the present invention, “immune cell” may refer to, for example, a T cell or a B cell. In particular, the antigen of the antigen-binding moiety may be a T cell surface antigen.

The immune cell engaging domain may be a single-chain variable fragment (scFv). “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. The scFv polypeptide may further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

The immune cell engaging domain may be a CD3 effector. Thus, the immune cell engaging domain may bind to CD3. CD3 effectors include but are not limited to anti-CD3 antibodies or antibody fragments, in particular an anti-CD3 scFv or antibody-like scaffolds. The immune cell engaging domain may be a T cell engaging immune effector domain which may be an anti-CD3 scFv. Further immune effectors include but are not limited to antibodies, including fragments, derivatives and variants thereof, that bind to antigens on T cells. Such antigens include CD28, 4-1 bb (CD137) or CD16 or any molecules that exert an effect at the immune synapse. A particularly preferred immune effector is an anti-CD3 antibody, or a functional fragment or variant of said anti-CD3 antibody. As used herein, the term “antibody” encompasses such fragments and variants. Examples of anti-CD3 antibodies include but are not limited to OKT3, UCHT-1 , BMA-031 and 12F6. Antibody fragments and variants/analogues which are suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab’)2 fragments, dsFv and scFv fragments.

Suitable antigen binding moieties for binding to CD3 include binding domains derived from the CD3- specific, humanized antibody hUCHTI . In particular VH and VL domains derived from the UCHT1 variants UCHT1-V17, UCHT1-V17opt, UCHT1-V21 or UCHT1-V23 may be used. Alternatively, VH and VL domains derived from the antibody BMA031 , which targets the TCRa/p CD3 complex, and humanized versions thereof may be used, in particular VH and VL domains derived from BMA031 variants BMA031 (V36) or BMA031(V10). Suitable BMA031 antibody variant sequences are described in WO 2022/233957. As another alternative, VH and VL domains derived from the CD3- specific antibody H2C (described in EP 2155783) may be used.

Other suitable immune cell engaging domains that bind to CD3 include domains derived from the amino acid sequences of the exemplary T cell engaging immune effector domains referred to herein as “DO” and “U28”. In this regard, the immune cell engaging domain may comprise:

(a) a VL comprising a CDR1 comprising the amino acid sequence provided in SEQ ID NO: 33, a CDR2 comprising the amino acid sequence provided in SEQ ID NO: 34 and a CDR3 comprising the amino acid sequence provided in SEQ ID NO: 35; and

(b) a VH comprising a CDR1 comprising the amino acid sequence provided in SEQ ID NO: 36 or 42, a CDR2 comprising the amino acid sequence provided in SEQ ID NO: 37 and a CDR3 comprising the amino acid sequence provided in SEQ ID NO: 38.

The VL and VH CDR sequences above may each optionally have one, two, three, or four amino acid substitutions relative to the sequences recited above. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the binding affinity of the immune cell engaging domain.

The immune cell engaging domain may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 31 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 32 or 41. For example, the immune cell engaging domain may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 31 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 32 or 41.

The immune cell engaging domain may comprise a VL comprising the amino acid sequence provided in SEQ ID NO: 31 and a VH comprising the amino acid sequence provided in SEQ ID NO: 32 or 41.

As described above, the immune cell engaging domain may be an scFv. The immune cell engaging domain may be an scFv comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 30 or 40. The scFv may comprise an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 30 or 40. The scFv may comprise the amino acid sequence provided in SEQ ID NO: 30. Alternatively, the scFv may comprise the amino acid sequence provided in SEQ ID NO: 40. The immune cell engaging domain may alternatively be an immune suppressor. For example, the target of the immune suppressor may be an immune checkpoint molecule, such as PD-1 (Programmed Death 1 receptor), A2AR (Adenosine A2A receptor), A2BR (Adenosine A2B receptor), B7-H3 (B7 Homolog 3, also called CD276) B7-H4 (B7 Homolog 4, also called VTCN1), BTLA (B and T Lymphocyte Attenuator, also called CD272), CTLA-4 (Cytotoxic T-Lymphocyte-Associated protein 4, also called CD152), IDO (Indoleamine 2,3-dioxygenase), CD200 Receptor, KIR (Killer-cell Immunoglobulin-like Receptor), TIGIT (T cell Immunoreceptor with Ig and ITIM domains), LAG3 (Lymphocyte Activation Gene-3), NOX2 (nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2), TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3), VISTA (V-domain Ig suppressor of T cell activation), SIGLEC7 (Sialic acid-binding immunoglobulin-type lectin 7, also called CD328), and SIGLEC9 (Sialic acid-binding immunoglobulin-type lectin 9, also called CD329).

In this regard, the immune suppressor may be an agonist of one or more of the above immune checkpoint molecules. Thus, the immune suppressor may be an immune checkpoint agonist (i.e., to inhibit immune activation). Suitable immune checkpoint agonists, including native ligands and antibodies, are reviewed in Paluch et al Front Immunol, 2018, 9:2306, for example.

Alternatively the immune suppressor may comprise an agonist antibody that binds to, and may stimulate signalling of, an immune checkpoint molecule. For example, the immune suppressor may be, or comprise, a PD-1 agonist antibody (e.g., single domain antibody). Such PD-1 agonists preferably do not compete with PD-L1 for binding to PD-1. The PD-1 agonist may a full-length antibody or fragment thereof, such as a scFv antibody or a Fab fragment, or a single domain antibody. Examples of such antibodies are provided in WO2011110621 (US 9102728), WO2010029434 (US9181342) and WO2018024237(US11274153), the contents of which are herein incorporated by reference. Thus, the antigen of the immune suppressor may be PD-1 and the antigen binding moiety of the immune suppressor may be a PD-1 agonist. The antigen binding moiety of the immune suppressor may comprise a single domain antibody, optionally a VHH. For example, the immune suppressor may be a PD-1 agonist VHH.

As described above, the immune suppressor may be a PD-1 agonist. As used herein, the term “PD-1 agonist” refers to any molecule that is capable of binding to PD-1 and activating PD-1 signalling, including e.g., the PD-1 ligand, PD-L1 , and PD-1 agonist antibodies. Activation of the PD-1 pathway down-reg ulates immune activity, promoting peripheral immune tolerance and preventing autoimmunity.

Suitable PD-1 agonist antibody sequences may be derived from the exemplary PD1 agonist VHH sequences provided herein. For example, the immune cell engaging domain may comprise a single domain antibody comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 43, 44 and 45 respectively. The immune cell engaging domain may comprise a single domain antibody comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 75 or 46. For example, the immune cell engaging domain may comprise a single domain antibody comprising an amino acid sequence that is at least 80%, at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 75 or 46. The immune cell engaging domain may comprise the amino acid sequence provided in SEQ ID NO: 75 or 46.

The immune cell engaging domain may comprise a single domain antibody comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 75 or 46, wherein the single domain antibody comprises a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 43, 44 and 45 respectively.

Instead of comprising an antigen binding moiety of an antibody, the immune cell engaging domain may comprise one of a receptor-ligand pair, whereby the immune cell engaging domain is capable of binding to the other of the receptor-ligand pair. The target ligand or receptor may be located on an immune cell. For example, the immune cell engaging domain may be an immune suppressor comprising a ligand of an immune checkpoint molecule described above. In particular, the immune suppressor may comprise a portion (e.g., a soluble extracellular region) of PD-L1 that is capable of binding to PD-1. Such an immune suppressor may engage an immune cell by binding to PD-1 and stimulate PD-1 signalling.

Half-life extending domains

The binding molecules described herein may comprise a half-life extending domain. A “half-life extending domain”, as used herein, refers to a protein domain for extending the half-life of the binding protein, relative to a binding protein lacking the half-life extending domain.

The term “half-life”, as used herein, refers to a pharmacokinetic property of a binding molecule that is a measure of the mean survival time of binding molecules following their administration. Binding molecule half-life can be expressed as the time required to eliminate 50 percent of a known quantity of a binding molecule from the patient's body (or other mammal) or a specific compartment thereof, for example, as measured in serum, i.e., circulating half-life, or in other tissues.

An increase in half-life allows for the reduction in amount of binding molecule given to a patient as well as reducing the frequency of administration. An increase in half-life can be beneficial, for example, for treatment of cancer, infectious disease or an autoimmune disease or condition. Binding molecules with increased half-lives may also be generated by modifying amino acid residues identified as involved in the interaction between the Fc and the FcRn receptor. Binding molecules comprising Fc regions that comprise one or more modifications which promote binding to FcRn may have an increased half-life of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150% or more as compared to a binding molecule comprising a native Fc region. Binding molecules comprising Fc regions that comprise one or more modifications which promote binding to FcRn may have an increased half-life of about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 20 fold, about 50 fold or more, or is between 2 fold and 10 fold, or between 5 fold and 25 fold, or between 15 fold and 50 fold, as compared to binding molecules comprising a native Fc region.

The half-life extending domain may comprise an immunoglobulin Fc domain. The term “Fc domain”, as used herein, refers to a dimer of two Fc regions. As used herein, the term “Fc region” is used to refer to a region of a single polypeptide chain comprising at least a CH2 region and a CH3 region sequence. The Fc regions may comprise all or part of a hinge sequence. The hinge sequence may correspond substantially or partially to a hinge region from lgG1 , lgG2, lgG3 or lgG4.

The immunoglobulin Fc domain may be any antibody Fc domain. The Fc domain is the tail region of an antibody that interacts with cell surface Fc receptors and some proteins of the complement system. The Fc domain comprises two polypeptide chains (i.e., two Fc “regions”) both having two or three heavy chain constant domains (termed CH2, CH3 and CH4), and optionally a hinge region. The two Fc region chains may be linked by one or more disulphide bonds within the hinge region. Fc domains from immunoglobulin subclasses lgG1 , lgG2 and lgG4 bind to and undergo FcRn mediated recycling, affording a long circulatory half-life (3 - 4 weeks), thus extending the half-life of the binding molecule described herein. The interaction of IgG with FcRn has been localized in the Fc region covering parts of the CH2 and CH3 domains. Preferred immunoglobulin Fc domains for use in the present invention include, but are not limited to Fc domains from IgG 1 or lgG4. For example, the IgG 1 Fc domain.

The Fc regions may comprise mutations relative to a wild-type or unmodified Fc sequence. Mutations include substitutions, insertions and deletions. Such mutations may be made for the purpose of introducing desirable therapeutic properties such as to enhance dimerisation of the Fc regions and/or enhance binding to FcRn and/or to attenuate an effector function of the Fc domain. Additionally or alternatively, mutations may be made for manufacturing reasons, for example to remove or replace amino acids that may be subject to post-translational modifications such as glycosylation, as described herein. The immunoglobulin Fc may be fused to the other domains (i.e., TCRm or immune cell engaging domain, if present) in the molecule of the invention via a linker, and/or a hinge sequence as described herein. Alternatively no linker may be used.

The half-life extending domain may alternatively comprise albumin or an albumin-binding domain. As is known in the art, albumin has a long circulatory half-life of 19 days, due in part to its size, being above the renal threshold, and by its specific interaction and recycling via FcRn. Attachment to albumin is a well-known strategy to improve the circulatory half-life of a therapeutic molecule in vivo. Albumin may be attached non-covalently, through the use of a specific albumin binding domain, or covalently, by conjugation or direct genetic fusion.

The Fc regions may comprise mutations made to facilitate hetero-dimerisation, for example, knobs into holes (KiH) mutations maybe engineered into the CH3 domain. Thus, the half-life extending domain may comprise one or more amino acid substitutions which facilitate dimerisation of the FC1 region and the FC2 region. Such substitutions include “Knob-in-hole” substitutions. In this case, one chain (i.e. one of the FC1 or FC2 regions) is engineered to contain a bulky protruding residue (i.e. the knob), such as Y, and the other chain (i.e., the other of the FC1 and FC2 regions) is engineered to contain a complementary pocket (i.e. the hole). For example, a knob may be constructed by replacing a small amino acid side chain with a larger side chain. A hole may be constructed by replacing a large amino acid side chain with a smaller side chain. Without wishing to be bound to theory, this is thought to stabilize a hetero-dimer of the FC1 and FC2 regions by favouring formation of the hetero-dimer over other species, for example homomultimers of FC1 and FC2, thereby enhancing the stability and manufacturability of the multi-domain binding molecule of the invention. Suitable positions and substitutions for KiH mutations, and other mutations for facilitating dimerisation of Fc regions, are known in the art.

The Fc domain may also comprise one or more mutations that attenuate an effector function of the Fc domain. Exemplary effector functions include, without limitation, complement-dependent cytotoxicity (CDC) and/or antibody-dependent cellular cytotoxicity (ADCC). The modification to attenuate effector function may be a modification that alters the glycosylation pattern of the Fc domain, e.g., a modification that results in an aglycosylated Fc domain. Alternatively, the modification to attenuate effector function may be a modification that does not alter the glycosylation pattern of the Fc domain. The modification to attenuate effector function may reduce or eliminate binding to human effector cells, binding to one or more Fc receptors, and/or binding to cells expressing an Fc receptor. For example, the half-life extending domain may comprise one or more amino acid substitutions selected from the group consisting of S228P, E233P, L234A, L235A, L235E, L235P, G236R, G237A, P238S, F241A, V264A D265A, H268A, D270A, N297A, N297G, N297Q, E318A, K322A, L328R, P329G, P329A, A330S, A330L, P331A and P331S, according to the EU numbering scheme. Particular modifications include a N297G or N297A substitution in the Fc region of human IgG 1 (EU numbering). Other suitable modifications include L234A, L235A and P329G substitutions in the Fc region of human lgG1 (EU numbering), that result in attenuated effector function. The Fc regions in the multidomain binding molecule of the invention may comprise a substitution at residue N297, numbering according to EU index. For example, the substitution may be an N297G or N297A substitution. Other suitable mutations (e.g., at residue N297) are known to those skilled in the art.

Fc variants having reduced effector function refers to Fc variants that reduce effector function (e.g., CDC, ADCC, and/or binding to FcR, etc. activities) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more as compared to the effector function achieved by a wild- type Fc region (e.g., an Fc region not having a mutation to reduce effector function, although it may have other mutations). The Fc variants having reduced effector function may be Fc variants that eliminate all detectable effector function as compared to a wild-type Fc region. Assays for measuring effector function are known in the art and described below.

In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the Fc region or fusion protein lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRI I and FcyRIII.

Substitutions may be introduced into the FC1 and FC2 regions that abrogate or reduce binding to Fey receptors and/or increase binding to FcRn, and/or prevent Fab arm exchange, and/or remove protease sites. In this regard, the half-life extending domain may also comprise one or more amino acid substitutions which prevent or reduce binding to activating receptors. The half-life extending domain may comprise one or more amino acid substitutions which prevent or reduce binding to FcyR.

Amino acid sequences

Within the scope of the invention are phenotypically silent variants of any binding molecule disclosed herein. As used herein the term “phenotypically silent variants” is understood to refer to a variant which incorporates one or more further amino acid changes, including substitutions, insertions and deletions, in addition to those set out above, and which variant has a similar phenotype to the corresponding molecule without said change(s). For the purposes of this application, phenotype comprises binding affinity (KD and/or binding half-life) and specificity. The phenotype for a soluble binding molecule may include potency of immune activation and purification yield, in addition to binding affinity and specificity.

Phenotypically silent variants may contain one or more conservative substitutions and/or one or more tolerated substitutions. By tolerated substitutions it is meant those substitutions which do not fall under the definition of conservative as provided below but are nonetheless phenotypically silent. The skilled person is aware that various amino acids have similar properties and thus are ‘conservative’. One or more such amino acids of a protein, polypeptide or peptide can often be substituted by one or more other such amino acids without eliminating a desired activity of that protein, polypeptide or peptide.

Thus the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains). It should be appreciated that amino acid substitutions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. For example, it is contemplated herein that the methyl group on an alanine may be replaced with an ethyl group, and/or that minor changes may be made to the peptide backbone. Whether or not natural or synthetic amino acids are used, it is preferred that only L- amino acids are present.

Substitutions of this nature are often referred to as “conservative” or “semi-conservative” amino acid substitutions. The present invention therefore extends to use of a molecule comprising any of the amino acid sequences described above but with one or more conservative substitutions and or one or more tolerated substitutions in the sequence, such that the amino acid sequence of the molecule, or any domain or region thereof, has at least 90% identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to the sequences disclosed herein.

“Identity” as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polypeptide or two polynucleotide sequences, methods commonly employed to determine identity are codified in computer programs. Preferred computer programs to determine identity between two sequences include, but are not limited to, GCG program package , BLASTP, BLASTN, and FASTA.

One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention.

The percent identity of two amino acid sequences or of two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The “best alignment” is an alignment of two sequences which results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., % identity = number of identical positions/total number of positions x 100).

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. . Determination of percent identity between two nucleotide sequences can be performed with the BLASTn program. Determination of percent identity between two protein sequences can be performed with the BLASTp program. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilised . Alternatively, PSI- Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilising BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTp and BLASTp) can be used. See http://www.ncbi.nlm.nih.gov. Default general parameters may include for example, Word Size = 3, Expect Threshold = 10. Parameters may be selected to automatically adjust for short input sequences. Another example of a mathematical algorithm utilised for the comparison of sequences is the algorithm CABIOS . The ALIGN program (version 2.0) which is part of the CGC sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM, and FASTA. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. For the purposes of evaluating percent identity in the present disclosure, BLASTp with the default parameters is used as the comparison methodology. In addition, when the recited percent identity provides a non-whole number value for amino acids (i.e., a sequence of 25 amino acids having 90% sequence identity provides a value of “22.5”, the obtained value is rounded down to the next whole number, thus “22”). Accordingly, in the example provided, a sequence having 22 matches out of 25 amino acids is within 90% sequence identity.

As will be obvious to those skilled in the art, it may be possible to truncate, or extend, the sequences provided at the C-terminus and/or N-terminus thereof, by 1 , 2, 3, 4, 5 or more residues, without substantially affecting the functional characteristics of the binding molecule. The sequences provided at the C-terminus and/or N-terminus thereof may be truncated or extended by 1 , 2, 3, 4 or 5 residues. All such variants are encompassed by the present invention.

Mutations, including conservative and tolerated substitutions, insertions and deletions, may be introduced into the sequences provided using any appropriate method including, but not limited to, those based on polymerase chain reaction (PCR), restriction enzyme-based cloning, or ligation independent cloning (LIC) procedures. These methods are detailed in many of the standard molecular biology texts. The protein sequences provided herein may be obtained from recombinant expression, solid state synthesis, or any other appropriate method known in the art.

As is well-known in the art, protein sequences, such as those in the binding molecules described herein, may be subject to post-translational modifications. Glycosylation is one such modification, which comprises the covalent attachment of oligosaccharide moieties to certain amino acids in a protein sequence. For example, asparagine residues, or serine/threonine residues are well-known locations for oligosaccharide attachment. The glycosylation status of a particular protein depends on a number of factors, including protein sequence, protein conformation and the availability of certain enzymes. Furthermore, glycosylation status (i.e. oligosaccharide type, covalent linkage and total number of attachments) can influence protein function. Therefore, when producing recombinant proteins, controlling glycosylation is often desirable. Controlled glycosylation has been used to improve antibody based therapeutics. . Glycosylation may be controlled, by using particular cell lines for example (including but not limited to mammalian cell lines such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells), or by chemical modification. Such modifications may be desirable, since glycosylation can improve pharmacokinetics, reduce immunogenicity and more closely mimic a native human protein. Alternatively, glycosylation can lead to a lack of consistency in manufacturing which is not desirable for a therapeutic molecule. Residues at high risk of glycosylation, such as asparagine, may be substituted with an alternative amino acid, such as glutamine.

Determining activity of binding molecules and structural characteristics of their interaction with pHLA

The methods described herein may comprise screening a plurality of candidate binding molecules to determine certain structural binding characteristics of their TCRm domains, such as pHLA crossing angle, pHLA residue binding contacts and binding affinity. These methods may comprise determining a three-dimensional atomic structure of the complex formed by the pHLA and the TCRm of a candidate binding molecule.

As used herein, the term “three-dimensional atomic structure” refers to a model of the three- dimensional arrangement of atoms in a protein or protein complex. Such atomic structures may be determined using any method known in the art, including x-ray crystallography, nuclear magnetic resonance (NMR) or cryo-electron microscopy (cryo-EM).

The three-dimensional atomic structure may be based on a set of atomic coordinates. As used herein, the term "atomic coordinates" or "set of coordinates" refers to a set of values which define the position of one or more atoms in a protein with reference to a system of axes. The atomic coordinates may be used in a computer to generate a representation, e.g. an image of the three-dimensional structure of proteins which can be displayed by the computer and/or represented in an electronic file.

The three-dimensional atomic structure may be an x-ray crystal structure. An x-ray crystal structure is a three-dimensional atomic structure of a protein or protein complex that is obtained using x-ray crystallography. X-ray crystallography techniques are well known in the art. A suitable technique is described in Example 1 under the heading “X-ray crystallography”. The x-ray crystal structure may be obtained by an x-ray crystallography technique known as molecular replacement. Methods of molecular replacement are generally known by those skilled in the art and can be performed using publicly available software packages. Generally, molecular replacement involves the following steps: i) X-ray diffraction data are collected from a crystal of a crystallized target protein complex, then ii) the X-ray diffraction data are transformed to calculate a Patterson function, then iii) the Patterson function of the crystallized target structure is compared with a Patterson function calculated from one or more known structures (referred to in the art as a “search structure” or “search model”), iv) the Patterson function of the search structure is rotated on the target structure Patterson function to determine the correct orientation of the search structure in the crystal to obtain a rotation function, v) a translation function is then calculated to determine the location of the search structure with respect to the crystal axes. Alternatively, likelihood-based molecular replacement methods can be used to determine the location of the search structure. Once the search structure has been correctly positioned in the unit cell, initial phases for the experimental data can be calculated. These phases are necessary for calculation of an electron density map from which an initial three-dimensional atomic structure is determined and refined. Preferably, the structural features (e.g., amino acid sequence, conserved disulphide bonds, and beta-strands or beta-sheets) of the search models are related to the crystallized target complex. Suitable search models can be obtained from a protein structure database such as the RCSB Protein Data Bank (RCSB PDB). Suitable search models for determining the three-dimensional atomic structure of a complex formed by the pHLA and the TCRm include the atomic coordinates of known pHLA-binding domain and pHLA structures. The electron density map can, in turn, be subjected to any well-known model building and structure refinement techniques to provide a final, accurate structure of the unknown (i.e. target) crystallized molecular structure.

Additionally, or alternatively, the three dimensional structure may be a predicted structure obtained by molecular modelling. In this case the predicted structure is typically a high affinity variant of a WT TOR mimic, and the starting model for prediction is the experimentally determined structure of the corresponding WT TOR mimic. Examples of software packages that may be used for such molecular modelling include the Schrodinger BioLuminate software, MOE (Molecular Operating Environment), Discovery Studio and Rosetta. The Schrodinger BioLuminate software may be preferred.

Once a three-dimensional atomic structure of the complex formed by the pHLA and the TCRm of a candidate binding molecule is obtained, the pHLA crossing angle and peptide residue binding contacts can be determined, based on the positions of the atoms in the structure.

The “pHLA crossing angle” or “crossing angle” (also known in the art as “docking angle”) is a parameter known in the art for TCRs (see Rudolph et al. (2006) Annu. Rev. Immunol. 24:419) and can be applied analogously to TCR mimics (in which the TCRm VL is analogous to the TCR alpha chain variable region and the TCRm VH is analogous to the TCR beta chain variable region). Specifically, the crossing angle is the angle formed between two vectors: a HLA groove vector and a TCRm interdomain vector (also referred to herein as the “TCRm cystine vector”). See Figure 1 for a schematic illustration of calculating a crossing angle.

In the present invention, the crossing angle is in the range of 150-250, preferably in the range of 170- 240 degrees, and more preferably in the range 190-240 degrees.

The HLA groove vector (also referred to as the “HLA peptide-binding groove vector”) is the directed line segment (or vector) corresponding to the peptide positioned across the HLA peptide binding groove, i.e., from N to C-terminus of the peptide. In this regard, the HLA groove vector follows the two parallel HLA groove helices with the direction N-terminus to C-terminus of HLA helix 1 and passing through the HLA centroid. The TCRm interdomain vector is the directed line segment (or vector) connecting the intrachain disulfide bond in the TCRm VL domain to the intrachain disulfide bond in the TCRm VH domain (in the direction of VL to VH). Similarly, for TCRs (as opposed to TCR mimics) the interdomain vector is generally calculated using the centroids of the conserved intra-chain disulfide bonds in the alpha chain and beta chain variable domains.

The “pHLA crossing angle” thus corresponds to the twist, or orientation, of the TCR/TCRm over the pHLA. A schematic illustrating the pHLA crossing angle is shown in Figure 1. Methods for calculating the crossing angle are known in the art and include those described by Rudolph et al. (2006) Annu. Rev. Immunol. 24:419 and Mareeva et al. (2006) JBC 283:29053. A suitable method for calculating a pHLA crossing angle is also described in Example 1.

The plurality of candidate binding molecules may be screened to determine their pHLA residue binding contacts. The phrase “DA-pHLA residue binding contacts” or “DA-pHLA residue binding contacts of the TCRm”, as used herein, refers to binding interactions formed between amino acid residues of the pHLA (either in the peptide sequence or the HLA protein) and amino acid residues of the TCRm of the candidate binding molecule. Each amino acid residue in the pHLA that forms an interaction with the TCRm is considered an individual pHLA residue binding contact of the TCRm. In this regard, as used herein, the phrase “DA-pHLA residues bound by the TCRm” is synonymous with “DA-pHLA residue binding contacts”.

The phrase “peptide residue binding contacts” refers to binding interactions formed between amino acid residues of the peptide (not the HLA) and amino acid residues of the TCRm. There may be 4, 5 or 6 or more, peptide binding contacts. For example, there may be 4 or 5 or more peptide binding contacts across the whole length of the peptide. Alternatively, or additionally, there may be 4 or more peptide binding contacts across the central region of the peptide, excluding residues at position 1 and the two anchor residues. Preferably, the peptide residues binding contacts exclude the residues at position 1 in the peptide and the two anchor positions. In this regard, a percentage of pHLA residue binding contacts of the TCRm which are peptide residue binding contacts (i.e, percentage of the DA- pHLA residues bound by the TCRm that are peptide residues) is a reference to the number of peptide residue binding contacts as a proportion of all residue binding contacts that the pHLA makes with the TCRm. For example, a hypothetical TCRm may bind to its target pHLA with a total of 20 pHLA residue binding contacts, of which 6 are peptide residue binding contacts and 14 are HLA residue binding contacts. The percentage of pHLA residue binding contacts of the TCRm which are peptide residue binding contacts for this hypothetical TCRm is therefore 30%. Without wishing to be bound by theory, a higher percentage or number of peptide residue binding contacts may be associated with greater pHLA target specificity.

Amino acid residue binding contacts can be determined using any method known in the art and may comprise measuring distances between atoms in the three-dimensional atomic structure of the complex formed by the pHLA and the TCRm. For example, residues between TCRm and pHLA may be identified to be in binding contact if the distance between any atom from a TCRm residue and any atom from a peptide or a HLA residue is within or equal to 4 A. Alternatively, or additionally, binding interactions (e.g., peptide residue binding contacts) can be identified from the three-dimensional atomic structure based on known atomic interaction geometry for different types of interactions, e.g., hydrogen bonds (H-bond), electrostatic interactions, van de Waals (vdW) interactions. For example, an H-bond binding contact can be defined as an interaction between donor atom and acceptor atom, where the donor-acceptor distance in the three-dimensional atomic structure is about 3.0 A or less and the donor hydrogen acceptor angle is within 45° to 180°. A vdW binding contact can defined as an interaction between two heavy atoms which are within about 4 A of one another in the three- dimensional atomic structure. Methods of identifying peptide residue binding contacts may comprise performing molecular dynamics simulations using publicly available software packages.

The phrase “HLA binding contacts” refers to binding interactions formed between amino acid residues of the HLA (i.e., not including the peptide) and amino acid residues of the TCRm. In other words, the phrase “HLA binding contacts” is synonymous with “residues of the HLA bound by the TCRm”. Preferably the TCRm contacts at least one amino acid located on helix 1 and at least one amino acid located on helix 2 of the HLA binding groove. Preferably, the at least one amino acid on helix 1 is located in positions 65, 69, 72 and/or 76 and in helix 2 at positions 155 158 163 and/or 167. HLA binding contacts may, additionally or alternatively, be located at one or more of positions 44, 57, 58, 59, 61 , 62, 64, 66, 68, 73, 75, 79, 80, 82, and 89 on helix 1 and 146, 147, 149, 150, 151 , 152, 154, 156, 157, 159, 161 , 162, 166, 169, 170, and 173 on helix 2. The TCRm may contact 2, 3, 4, 5, 6, 7, 8,

9, 10, 11 , or more, amino acids located on helix 1 and/or the TCRm may contact 2, 3, 4, 5, 6, 7, 8, 9,

10, 11 , 12, 13, 14 or more, amino acids located on helix 2. The HLA binding contacts may differ between different HLA types, such as between HLA-A*02 and HLA-A24.

Methods of screening candidate binding molecules may comprise determining a binding affinity of a TCRm for a pHLA. The affinity of the TCRm for the DA-pHLA may be 20 nM or stronger, 10 nM or stronger, 5 nM or stronger, 1 nM or stronger, 500 pM or stronger, or 100 pM or stronger. For example, the affinity may be 500 pM or stronger. Methods to determine binding affinity (inversely proportional to the equilibrium constant KD) and binding half-life (expressed as T¹/₂) are known to those skilled in the art. Binding affinity and binding half-life may be determined using Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI), for example using a BIAcore instrument or Octet instrument, respectively. For example, binding affinity may be determined at 25°C or 37°C. Binding affinity of a TCRm for a pHLA complex may be determined using SPR at 25°C, wherein the pHLA complex is immobilised on a solid support (e.g., a sensor chip) and is contacted with a solution comprising a binding molecule comprising the TCRm (or vice-versa). Suitable experimental conditions and methods for determining binding parameters are described in Example 1. As is known to those skilled in the art binding affinity may be influenced by changes in either or both the on rate (k_on) and the off rate (kotf) of the interaction.

It will be appreciated by those skilled in the art that a higher affinity refers to a lower numerical value for Koand indicates stronger binding. Therefore doubling the affinity results in halving the numerical value of the KD. In this regard, reference herein to an affinity of “20 nM or stronger” is a reference to a KD of 20 nM or a KD of lower numerical value, e.g., 10 nM. The phrase “affinity of 20 nM or stronger” is used synonymously herein with an “affinity of at least 20 nM”. T¹/z is calculated as In2 divided by the off-rate (kotf). Therefore, doubling of T¹/z results in a halving in k_Off. KD and k_Off values for binding molecules are usually measured for soluble forms of the molecule, i.e. those forms which are truncated to remove cytoplasmic and transmembrane domain residues. To account for variation between independent measurements, and particularly for interactions with dissociation times in excess of 20 hours, the binding affinity and or binding half-life of a given protein may be measured several times, for example 3 or more times, using the same assay protocol, and an average of the results taken. To compare binding data between two samples (i.e. two different proteins and or two preparations of the same protein) it is preferable that measurements are made using the same assay conditions (e.g. temperature). Measurement methods described in relation to TCRs may also be applied to the binding molecules described herein.

The binding molecules described herein comprise a TCRm that preferably has the property of specifically binding to the pHLA. As used herein, “specific” binding refers to a binding molecule that binds to the target pHLA complex with higher affinity than to other pHLA complexes. Similarly, as used herein the term “dock” refers to binding of a molecule to the pHLA with an orientation (i.e., position of the molecule relative to the HLA) that provides specific binding. Highly specific binding molecules of the invention are particularly suitable for therapeutic use due to the reduced risk of off- target effects. Specificity in the context of binding molecules of the invention can be determined according to their ability to recognise target cells that are antigen positive, whilst having minimal, or preferably no detectable, ability to recognise target cells that are antigen negative, and/or to bind to peptides that are similar in sequence to the target peptide but differ by up to three amino acids.

A TCRm of a binding molecule described herein may not bind (e.g., not detectably bind) to cells that do not express the target peptide (i.e., “antigen-negative” or “DA-pHLA negative” cells). Specificity, in this context, can be measured in vitro, for example, in cellular assays, such as ELISpot assays. Such cellular assays (e.g. ELISpot or ELISA assays) can measure the release of T cell activation-related cytokines. To test specificity, the TCRm may be in soluble form and fused with (or otherwise attached to) a T cell engaging immune effector domain (e.g., anti-CD3 antibody), and/or may be expressed on the surface of cells, such as T cells. Specificity may be determined by measuring the level of T cell activation in the presence of antigen positive and antigen negative target cells as defined above. Minimal recognition of antigen negative target cells is defined as a level of T cell activation of less than 20%, preferably less than 10%, preferably less than 5%, and more preferably less than 1%, of the level produced in the presence of antigen positive target cells, when measured under the same conditions (i.e. using the same lots of target and effector cells) and at a therapeutically relevant TCRm concentrations. For soluble binding molecules comprising a T cell engaging immune effector domain, a therapeutically relevant concentration may be defined as a concentration of 10^-9 M or below, and/or a concentration of up to 100, preferably up to 1000, fold greater than the corresponding ECso or IC50 value. Preferably, for soluble binding molecules comprising a T cell engaging immune effector domain there is at least a 10 fold difference, at least a 100 fold, at least 1000 fold, at least 10000 fold difference in EC50 or IC50 value between T cell activation against antigen positive cells relative to antigen negative cells. This difference may be referred to as a therapeutic window. As will be appreciated a 100 fold window is equivalent to 2 log window. Additionally or alternatively the therapeutic window may be calculated based on lowest effective concentrations (“LOEL”) observed for normal cells and a target pHLA positive cell. Additionally or alternatively the therapeutic window may be calculated based on LOEL observed for antigen negative cells and a target pHLA positive cell. Antigen (i.e., target pHLA) positive cells may be obtained by peptide-pulsing using a suitable peptide concentration to obtain a level of antigen presentation comparable to wt peptide presentation, or, they may naturally present said peptide. Antigen positive cells may be T2 cells that are pulsed with target peptide. Cells may be pulsed with excess peptide to obtain a high level of peptide presentation (i.e. high target density). Preferably, both antigen positive and antigen negative cells are human cells. Preferably, both antigen positive and antigen negative cells are primary or cultured cells from human origin.

Specificity may additionally, or alternatively, relate to the ability of a TCRm in a binding molecule to bind to the target pHLA complex and not to a panel of alternative pHLA complexes. This may, for example, be determined by the Surface Plasmon Resonance (SPR) method described in Examples 1 . Said panel may contain at least 2, at least 3, at least 5, or at least 10, alternative pHLA complexes. The alternative peptides may share a low level of sequence identity with the target peptide antigen and may be naturally or artificially presented. The alternative peptides may share a low level of sequence similarity with the target peptide antigen and may be naturally or artificially presented. Such alternative peptides (with low level sequence identity or sequence similarity) may be termed mimetic peptides. Alternative peptides are preferably derived from commonly expressed proteins and or proteins expressed in healthy human tissues. Binding of the binding molecule to the target pHLA complex may be at least 2 fold greater than to other naturally or artificially-presented pHLA complexes, more preferably at least 10 fold, or at least 50 fold or at least 100 fold greater, even more preferably at least 1000 fold greater. Natural variants of the target peptide may be excluded from the definition of alternative pHLA complexes.

An alternative or additional approach to determine TCRm specificity may be to identify the peptide recognition motif of the TCRm using sequential mutagenesis, e.g. alanine scanning, of the target peptide. Residues that form part of the binding motif are those that are not permissible to substitution. Non-permissible substitutions may be defined as those peptide positions in which the binding affinity of the TCRm is reduced (i.e., weakened) by at least 50%, or preferably at least 80% relative to the binding affinity for the non-mutated peptide. Such an approach is further described in Cameron et al., (2013), Sci Transl Med. 2013 Aug 7; 5 (197): 197ra103 and W02014096803. Binding molecule specificity in this case may be determined by identifying alternative motif containing peptides, particularly alternative motif containing peptides in the human proteome, and testing these peptides for binding to the TCRm. Binding of the TCRm to one or more alternative peptides may indicate a lack of specificity. In this case further testing of TCRm specificity via cellular assays may be required. A low tolerance for (alanine) substitutions in the central part of the peptide indicate that the TCRm has a high specificity and therefore presents a low risk for cross-reactivity with alternative peptides.

The TCRm in the binding molecules of the invention binds to a DA-pHLA positive cell, but preferably does not detectably bind to a DA-pHLA negative cell (i.e., a cell that does not naturally present the target DA-pLHA complex). This can be assessed using any suitable method known in the art, including those described above. In particular, the TCRm may be deemed to not detectably bind to a DA-pHLA negative cell if there is at least a 1000 fold difference in response Ec50 (or lc50) between a DA-pHLA positive cell and a DA-pHLA negative cell, as measured in an in vitro cell-based functional assay, such as an IFNy ELISpot assay, or other suitable assay.

Certain binding molecules of the invention are able to generate a highly potent T cell response in vitro against antigen positive cells (also referred to herein as “pHLA positive” cells), in particular those cells presenting low levels of antigen typical of cancer cells (i.e. in the order of 5-100, for example 50, antigens per cell (Bossi et al., (2013) Oncoimmunol. 1 ;2 (11 ) :e26840; Purbhoo et a/., (2006). J Immunol 176(12): 7308-7316.). The T cell response that is measured may be the release of T cell activation markers such as Interferon y or Granzyme B, or target cell killing, or other measure of T cell activation, such as T cell proliferation. A highly potent response may be one with an EC50 value in the nM - pM range, for example 500 nM or lower, preferably 1 nM or lower, or 500 pM or lower.

Alternatively, certain binding molecules of the invention may generate a highly potent antiinflammatory response, such as inhibition of CD8+ cell killing and/or CD4+ inflammation. Such binding molecules may be in soluble form and may comprise an immune cell engaging domain which is an immune suppressor such as a PD-1 agonist or an interleukin or cytokine such as IL-2, IL-4, IL- 10 or IL-13. The anti-inflammatory response that is measured may be inhibition of CD8+ cell killing and/or CD4+ inflammation, and or inhibition of CD8+ T cell signalling pathways. Suitable methods for assessing an anti-inflammatory response will be known in the art and include Jurkat NFAT cell reporter assays. Preferably a highly potent response is one with IC50 value in the pM range, i.e. 1000 pM or lower. Preferably the maximum inhibition obtained in reporter assays is greater than 50%, for example 80% or more.

Molecules encompassed by the present invention may have an improved half-life. Methods for determining whether a protein has an improved half-life will be apparent to the skilled person. For example, the ability of a protein to bind to a neonatal Fc receptor (FcRn) is assessed. In this regard, increased binding affinity for FcRn increases the serum half-life of the protein.

The half-life of a molecule described herein can also be measured by pharmacokinetic studies. According to this method radiolabeled protein is injected intravenously into mice and its plasma concentration is periodically measured as a function of time, for example at 3 minutes to 72 hours after the injection. Alternatively, unlabeled protein of the disclosure can be injected and its plasma concentration periodically measured using an ELISA. The clearance curve thus obtained should be biphasic, that is, an alpha phase and beta phase. For the determination of the in vivo half-life of the protein, the clearance rate in beta-phase is calculated and compared with that of the wild type or unmodified protein.

Nucleic acids, vectors and host cells

The present invention provides a nucleic acid encoding a binding molecule described herein. The nucleic acid may be cDNA. The nucleic acid may be mRNA. The nucleic acid may be non-naturally occurring and/or purified and/or engineered. The nucleic acid sequence may be codon optimised, in accordance with the expression system utilised. As is known to those skilled in the art, expression systems may include bacterial cells such as E. coli, or yeast cells, or mammalian cells, or insect cells, or they may be cell free expression systems.

The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one nucleic acid as described above. The present invention also provides a recombinant host cell which comprises one or more constructs as above. As mentioned, a nucleic acid encoding a specific binding molecule of the invention forms an aspect of the present invention, as does a method of production of the specific binding molecule comprising expression from a nucleic acid encoding a specific binding molecule of the invention. Expression may conveniently be achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. Following production by expression, a binding molecule may be isolated and/or purified using any suitable technique, then used as appropriate. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common, preferred bacterial host is E. coli. The expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. . Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of a specific binding molecule.

Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be any suitable vectors known in the art, including plasmids or viral vectors (e.g. ‘phage, or phagemid), as appropriate. . Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins.

The present invention also provides a host cell containing a nucleic acid as disclosed herein. Further, the invention provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.

Suitable host cells for cloning or expression of nucleic acids and/or vectors of the present invention are known in the art. Suitable host cells for the expression of (glycosylated) proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts.). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described,); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described; monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells); and myeloma cell lines such as YO, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see,. The host cell may be eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20 cell).

The nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.

Methods of producing binding molecules

The methods described herein for producing a binding molecule that specifically binds to a target pHLA comprise identifying the binding molecule from a plurality of candidate binding molecules that bind to the pHLA. This initial plurality of candidate binding molecules can be obtained by any means. For example the plurality of candidate binding molecules can be obtained from a display library using well known techniques, such as a phage display library as described in, for example. For example, the display library may be contacted with the target pHLA, e.g., in a soluble form immobilized on a solid support, and then non-binders are washed away. Several rounds of this panning procedure may be performed to identify a plurality of candidate binding molecules that bind to the pHLA. The plurality of candidate binding molecules can then be assessed to identify a binding molecule that has the pHLA binding characteristics defined herein.

Methods of producing a binding molecule described herein may comprise recombinantly expressing the binding molecule in a host cell and subsequently purifying the binding molecule. The methods may comprise maintaining the host cell under optimal conditions for expression of a nucleic acid or expression vector encoding the binding molecule and isolating the binding molecule.

Methods of producing recombinant proteins are well known in the art. Nucleic acids encoding the protein can be cloned into expression constructs or vectors, which are then transfected into host cells, such as E. coli cells, yeast cells, insect cells, or mammalian cells, such as simian COS cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not otherwise produce the protein. Exemplary mammalian cells used for expressing a protein are CHO cells, myeloma cells or HEK cells. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Methods of producing recombinant antibodies are also known in the art.

The nucleic acid may be inserted operably linked to a promoter in an expression construct or expression vector for further cloning (amplification of the DNA) or for expression in a cell-free system or in cells. As used herein, the term “promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and/or external stimulus, or in a tissue specific manner. In the present context, the term “promoter” is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or enhances the expression of a nucleic acid to which it is operably linked. Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter the spatial expression and/or temporal expression of said nucleic acid. As used herein, the term “operably linked to" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

Many vectors for expression in cells are commercially available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding a protein (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. The skilled person will be aware of suitable sequences for expression of a protein. Exemplary signal sequences include prokaryotic secretion signals (e.g., pe1 B, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, a factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal).

Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-a promoter (EF1 ), small nuclear RNA promoters (Ula and Ulb), a-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, p-actin promoter; hybrid regulatory element comprising a CMV enhancer/p-actin promoter or an immunoglobulin promoter or an active fragment thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GALA promoter, the CUP1 promoter, the PH05 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

The host cells used to produce the protein may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art. Methods for isolating a protein are known in the art. Where a protein is secreted into culture medium, supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. Alternatively, or additionally, supernatants can be filtered and/or separated from cells expressing the protein, e.g., using continuous centrifugation.

The protein prepared from the cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the foregoing. These methods are known in the art.

The skilled person will also be aware that a protein can be modified to include a tag to facilitate purification or detection, e.g., a poly-histidine tag, a hexa-histidine tag, an influenza virus hemagglutinin (HA) tag, a Simian Virus 5 (V5) tag, a LLAG tag, or a glutathione S-transferase (GST) tag. The resulting protein is then purified using methods known in the art, such as, affinity purification. For example, a protein comprising a hexa-his tag is purified by contacting a sample comprising the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein. Alternatively, or in addition a ligand or antibody that binds to a tag is used in an affinity purification method.

Binding molecules described herein may be amenable to high yield purification. Yield may be determined based on the amount of material retained during the purification process (i.e. the amount of correctly folded material obtained at the end of the purification process relative to the amount of solubilised material obtained prior to refolding), and or yield may be based on the amount of correctly folded material obtained at the end of the purification process, relative to the original culture volume. High yield means greater than 1 %, or greater than 5%, or higher yield. High yield means greater than 1 mg/ml, or greater than 3 mg/ml, or greater than 5 mg/ml, or higher yield.

Pharmaceutical compositions and medical methods

For administration to patients, the molecules of the invention, nucleic acids, expression vectors or cells of the invention may be provided as part of a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. This pharmaceutical composition may be in any suitable form, (e.g. depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, and will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

The pharmaceutical composition may be adapted for administration by any appropriate route, such as parenteral (including subcutaneous, intramuscular, intrathecal or intravenous), enteral (including oral or rectal), inhalation or intranasal routes. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carriers) or excipient(s) under sterile conditions. Methods for preparing a protein into a suitable form for administration to a subject (e.g. a pharmaceutical composition) are known in the art.

The pharmaceutical compositions will commonly comprise a solution of a binding molecule of the invention (or the nucleic acid, cell, or vector of the invention) dissolved in a pharmaceutically acceptable carrier, for example an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of the binding molecules in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as mixed oils and ethyl oleate may also be used. Liposomes may also be used as carriers. The vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

Binding molecules described herein may have an ideal safety profile for use as therapeutic agents. An ideal safety profile means that in addition to demonstrating good specificity, the binding molecules described herein may have passed further preclinical safety tests. Examples of such tests include whole blood assays to confirm minimal cytokine release in the presence of whole blood and thus low risk of causing a potential cytokine release syndrome in vivo, and alloreactivity tests to confirm low potential for recognition of alternative HLA types.

Dosages of the binding molecules described herein can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. A physician will ultimately determine appropriate dosages to be used.

Binding molecules, pharmaceutical compositions, vectors, nucleic acids and cells of the invention may be provided in substantially pure form, for example, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure. The binding molecule, nucleic acid, vector, pharmaceutical composition and cell of the invention may be used for treating diseases such as cancer, particularly cancers which are associated with expression of a tumour-associated antigen. The cancer may be associated with expression of GP100, PRAME, PIWIL1 , MAGEA4, WT1 or PPI. For example, the cancer may be associated with expression of GP100, PRAME, PIWIL1 , MAGEA4, PRAME or PPI. For example, the cancer may be associated with expression of an antigen described in, for example, WO2011001152 (and US 2012-0225481 A1 ), WO2017109496 (and US 2019-0002523 A1 ), WO2017175006 (and US 2019-0092834 A1 ) or WO2018234319 (and US 2021 - 0355188 A1 ), which are all incorporated herein by reference in their entirety.

Alternatively, the binding molecule, nucleic acid, vector, pharmaceutical composition and cell of the invention may be used for treating an infectious disease. The infectious disease may be caused by a bacterial, viral, fungal or parasitic pathogen. Any infection with a pathogen which results in antigen- presenting cells presenting HLA bound to a peptide from the pathogen may be suitable for treatment with the binding molecule of the invention. The binding molecule of the invention is particularly well suited to infections where antigen-presenting cells present levels of pathogen peptide that are lower than optimal for the natural immune system to clear the infection without additional treatment. The infectious disease may be a chronic infection. Exemplary infectious diseases include Hepatitis B virus (HBV) infection and human immunodeficiency virus (HIV) infection. Thus, the peptide in the DA-pHLA may be a peptide from a HBV or HIV protein.

The binding molecule of the invention may alternatively be used in a method of treating an autoimmune disease, such as type 1 diabetes. Organ-specific immune suppression, rather than systemic immunosuppression, may be a beneficial route for treatment given the potential significant adverse events associated with systemic immunosuppression. In autoimmunity, there is also mounting evidence that PD-1 pathway impairment plays an important role in disease pathogenesis. PD-1 , PD-L1 and PD-L2 gene polymorphisms are associated with several autoimmune diseases. Abnormally low PD-L1 expression has been observed in samples from type 1 diabetes and Crohn’s disease patients. Activating PD-1 on autoreactive lymphocytes thus may serve as a mechanism to treat autoimmune diseases. Effective therapeutics for the treatment of autoimmune diseases include those having an advantageous risk profile (e.g., a high level of target and tissue specificity) and are capable of being administered with less frequency.

Also provided by the invention are:

• a binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the invention for use in medicine, preferably for use in a method of treating cancer or a tumour or an autoimmune disease or an infectious disease;

• the use of a binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the invention in the manufacture of a medicament for treating cancer or a tumour or an autoimmune disease or an infectious disease; • a method of treating cancer or a tumour or an autoimmune disease or an infectious disease in a patient, comprising administering to the patient a binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the invention; and

• an injectable formulation for administering to a human subject comprising a binding molecule, nucleic acid, vector pharmaceutical composition or cell of the invention.

The method of treatment may further include administering separately, in combination, or sequentially, an additional anti-neoplastic agent. Example of such agents are known in the art and may include immune activating agents and/or T cell modulating agents.

Kits and articles of manufacture

In another aspect, a kit or an article of manufacture containing materials useful for the treatment and/or prevention of the diseases described above is provided.

The kit may comprise (a) a container comprising the binding molecule, nucleic acid, vector or cell of the invention, optionally in a pharmaceutically acceptable carrier or diluent; and (b) a package insert with instructions for treating a disease (e.g., cancer, immune disease or autoimmune disease) in a subject. The kit may further comprise (c) at least one further therapeutically active compound or drug.

The package insert may be on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds or contains a composition that comprises the molecule, nucleic acid, vector or cell of the invention and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the molecule, nucleic acid, vector or cell of the invention. The label or package insert indicates that the composition is used for treating a subject eligible for treatment, e.g., one having or predisposed to developing a disease described herein, with specific guidance regarding dosing amounts and intervals of the composition and any other medicament being provided. The kit may further comprise an additional container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and/or dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The kit optionally further comprises a container comprising a second medicament, wherein the molecule, nucleic acid, vector or cell of the invention is a first medicament, and which kit further comprises instructions on the package insert for treating the subject with the second medicament, in an effective amount. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. Features of each aspect of the invention are as for each of the other aspects mutatis mutandis. For example, any features disclosed herein in the context of a binding molecule of the invention, equally apply to the methods, and other aspects, of the invention. The documents referred to herein are incorporated by reference to the fullest extent permitted by law.

Description of the drawings

Figure 1 is a schematic illustrating a pHLA crossing angle of a hypothetical TCRm bound to a target pHLA. The top left image depicts a pHLA, showing the peptide, with N- to C- terminal direction indicated, bound in the peptide-binding groove of the HLA. The top right image depicts a crosssection of a TCRm comprising an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). The middle bottom image depicts the position of a TCRm bound to a pHLA. The vector designating the peptide (the “HLA groove vector”) is shown in the N- to C- terminal direction. The vector that connects the intrachain disulfide bonds in the VH and VL domains of the pHLA-binding domain is also shown (the “TCRm interdomain vector”). The pHLA crossing angle is the angle formed between these two vectors and is indicated by the curved arrow. The pHLA crossing angle in this image would be approximately 235 degrees.

Figure 2 shows that TCRm bispecifics mediate a potent and specific redirected T cell response against antigen positive cells in cell-based assays. Antigen positive and antigen negative cells are indicated.

Figure 3 shows that TCRm bind to cognate pHLA with a reverse crossing angle compared to canonical TCRs. (A) Box and whiskers plot showing calculated crossing angles for TCRm and TCRs bound to pHLA. Data is shown for structures obtained from PDB and in-house data. A selection of 5 in-house TCRm structures with reverse binding polarity is labelled ‘IMCR5’ (B) Modelling of crossing angle within pHLA binding groove for 5 in-house TCRm.

Figure 4 shows an analysis of TCRm binding contacts to the peptide component of pHLA. (A) schematic showing numbering of anchor positions in three different length peptides, 8mer, 9mer and 10mer. (B) No. of contacts between select TCRm and peptide based on a contact distance threshold of <4A. Black squares indicate contact positions, blank squares indicate non-contact positions, hashed squares indicate no amino acid is present.

Figure 5 shows analysis of TCRm binding contacts to helix 1 and helix 2 of the HLA binding groove. (A) schematic of HLA binding grove showing positions on helix 1 and helix 2 that are important for engaging with TCRm. (B) No. of contacts between select TCRm and helix 1 and Helix 2 based on a contact distance threshold of <4A. Black squares indicate contact positions. Figure 6 shows dose response curves obtained for the indicated molecule in the presence of antigen positive and antigen negative cells. T cell response was determined by IFNy release. EC50 values against antigen positive cells were determined from the curves. # indicates that at least one of the triplicate data values for that data point has been extrapolated above the standard curve.

Figure 7 shows contact positions between TCRm and cognate peptide determined from corresponding 3D structure data. Peptide residue numbers 1-10 are shown across the top. Contact sites to the peptide are shown in black.

Figure 8 shows contact positions between each TCRm and HLA determined from corresponding 3D structure data. HLA residue positions in helix 1 and 2 are indicated across the top. Contact sites to HLA are shown in black.

Figure 9 shows total number of contact sites between TCRm and HLA determined from corresponding 3D structure data.

Description of the sequences

Exemplary peptide antigens

Exemplary TCRm sequences

FL043_AB011

SEQ ID NO: 3 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL043_AB011”. FL043_AB011 is an scFv that binds to a peptide having the sequence ALDGGNKHFL (SEQ ID NO: 1) in complex with HLA-A*02. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 47. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 4, 5 and 6. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 7. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 8, 9 and 10. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 11. SEQ ID NO: 3:

DIVMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSG

TDFTL TISSLQPEDFATYYCQQSYSTPRTFGQGTKLEIKR TS S GGGGS GGGGS GGGRSEVQLVQSGAE

VKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYVQKFQGRVTMTRDTSTST

VYMELSSLRSEDTAVYYCTRDLGGSYNDYWGQGTLVTVSS

FL043-AB035

SEQ ID NO: 12 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL043- AB035”. FL043-AB035 is an scFv that binds to a peptide having the sequence ALDGGNKHFL (SEQ ID NO: 1) in complex with HLA-A*02. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 13. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 14, 15 and 16. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 17. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 18, 19 and 20. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 48.

SEQ ID NO: 12:

QSALTQPASVSGSPGQSITISCSGSRIDVGSNDFVSWYQQHPGEVPKLILYDVHKRPSGVPDRFSGSK SDNTAYLTISGLQAEDEAYYYCSSYTSSSTLVFGGGTELTVLSGGGGGSGGGGSGGGRSQVQLQ'ESGP GLVKPSETLSLTCTVSGGSISSSNWWSWVRQPPGKGLEWIGEIYYSGSTYYNPSLKSRVTISVDTSKN QFSLKLSSVTAADTAVYYCARERAATPGALDYWGQGTLVTVSS

FL029_AB003

SEQ ID NO: 21 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL029_AB003”. FL029_AB003 is an scFv that binds to a peptide having the sequence PYLGQMINL (SEQ ID NO: 2) in complex with HLA-A*24. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 22. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 23, 24 and 25. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 26. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 27, 28 and 29. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 11.

SEQ ID NO: 21 :

EIVLTQSPGTLSLSPGERATLSCRASQSVTSGYLAWYQQRPGQAPRLLIYGPSTRATGIPDRFSGSGS GTDFTLSISRLEPEDSAVYYCQQFGSSP TFGGGTKLEIKRTSSGGGGSGGGGSGGGRSQVQLQ'ESGP GLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSK

NQFSLKLSSVTAADTAVYYCARGSYYFDYWGQGTLVTVSS

FL029-AB003 L2H2

SEQ ID NO: 50 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL029- AB003 L2H2”. FL029-AB003 L2H2 is an affinity matured variant of FL029-AB003. FL029-AB003 L2H2 is an scFv that binds to a peptide having the sequence PYLGQMINL (SEQ ID NO: 2) in complex with HLA-A*24. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 51. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 52, 53 and 25. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 54. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 55, 28 and 29. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 11.

SEQ ID NO: 50:

EIVLTQSPGTLSLSPGERATLSCRASQSVTSGTLAWYQQRPGQAPRLLIYRYSTRATGIPDRFSGSGS GTDFTLSISRLEPEDSAVYYCQQFGSSPVTFGGGTKLEIKRTSSGGGGSGGGGSGGGRSQVQLQ'ESGP GLVKPSETLSLTCTVSGGSISRSSHYWGWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSK NQFSLKLSSVTAADTAVYYCARGSYYFDYWGQGTLVTVSS

FL030-AB002

SEQ ID NO: 56 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL030- AB002”. FL030-AB002 is an scFv that binds to a peptide having the sequence GVYDGREHTV (SEQ ID NO: 74) in complex with HLA-A*02. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 57. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 58, 59 and 60. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 61. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 62, 63 and 64. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 11.

SEQ ID NO: 56

DIQLTQS PS SL SAS VGDR VI I TCRAS QD VFHHLAWYQQKPGKVPKLL I YAASSL QSGVPSR FR GS GS G TDFTLTISSLQPEDFATYYCQQSYSTPQTFGQGTKVEIKRTSSGGGGSGGGGSGGGRSEVQLVQSGA.'E VKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWINPNSGGTIYAQKFQGRVTMTRDTSIST

AYMELSSLRSEDTAVYYCASSYCSSTSCYLGPYYYGMDVWGQGTLVTVSS FL030-AB102

SEQ ID NO: 65 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL030- AB102”. FL030-AB102 is an scFv that binds to a peptide having the sequence GVYDGREHTV (SEQ ID NO: 74) in complex with HLA-A*02. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 66. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 67, 68 and 69. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 70. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 71 , 72 and 73. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 49.

SEQ ID NO: 65

QSVKESGGGLVTPGGTLTLTCTASGFSLTNYYMNWVRQAPGKGLEWIGIIGGSGTTYYANWAQGRFVI SKTSSTTVTLQMTSLTTGDTATYFCARDSAYLTLWGQGTLVTVSSGQPKAPSVGGGGS GGGGS S S S RS SGGELVMTQTPSSVSKPVGGTVTIKCQASQDIGSNLAWYQQKPGQPPKLLIYQASILESGVPSRFSGS GSGTQLTLTISGVQCEDAATYYCQGTYWDSGWLGAFGGGTEVWK

FL029-Ab002

SEQ ID NO: 77 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL029-AB002”. FL029-AB002 is an scFv that binds to a peptide having the sequence PYLGQMINL (SEQ ID NO: 2) in complex with HLA-A*24. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 78. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 79, 80 and 81. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 82. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 83, 84 and 85. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 86.

SEQ ID NO: 77

EIVMTQSPGTLSLSPGERATLSCRASQSVSNNLAWYQQQPGQAPRLLIYGASRRATGIPDRFSGSGSG TDFTLSISRLEPEDFAVYYCQQYGSSPPYTFGQGTKLEIKRTSSGGGGSGGGGSGGGRSQLQLQ'EESG PGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTS KNQFSLKLSSVTAADTAVYYCARGSYYFDYWGQGTLVTVSS

FL029-Ab003 L19 H20

SEQ ID NO: 87 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL029-AB003 L19 H20”. FL029-AB003 L19 H20 is an scFv that binds to a peptide having the sequence PYLGQMINL (SEQ ID NO: 2) in complex with HLA-A*24. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 88. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 89, 90 and 91. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 92. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 93, 94 and 95. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 96.

SEQ ID NO: 87

EIVLTQSPGTLSLSPGERATLSCRASQSVTSGTLAWYQQRPGQAPRLLIYRYSTRATGIPDRFSVSGS GTDFTLSISRLEPEDSAVYYCQQFGSSPVTFGCGTKLEIKRTSSGGGGSGGGGSGGGRSQVQLQ'ESGP GLVKPSETLSLTCTVSGGSISRSSYYWGWIRQPPGKCLEWIGYIYYSGSTNYNPSLKSRVTISVDTSK NQFSLKLSSVTAADTAVYYCVRGSYYFDYWGQGTLVTVSS

FL029-Ab003 L02 H21

SEQ ID NO: 97 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL029- AB003 L02 H21”. FL029-AB003 L02 H21 is an scFv that binds to a peptide having the sequence PYLGQMINL (SEQ ID NO: 2) in complex with HLA-A*24. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 98. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 99, 100 and 101. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 102. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 103, 104 and 105. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 106.

SEQ ID NO: 97

EIVLTQSPGTLSLSPGERATLSCRASQSVTSGTLAWYQQRPGQAPRLLIYRYSTRATGIPDRFSGSGS GTDFTLS I SRLEPEDSAVYYCQQFGSSPVTFGCGTKLEIKRTS SGGGGSGGGGSGGGRSQVQLQESGP GLVKPSETLSLTCTVSGGSISRSSHYWGWIRQPPGKCLEWIGYIYYSGSTNYNPSLKSRVTISVDTSK NQFSLKLSSVTAADTAVYYCARGSYFFENWGQGTLVTVSS

FL029-Ab003 L26 H02

SEQ ID NO: 107 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL029-Ab003 L26 H02”. FL029-Ab003 L26 H02 is an scFv that binds to a peptide having the sequence PYLGQMINL (SEQ ID NO: 2) in complex with HLA-A*24. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 108. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 109, 110 and 111. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 112. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 113, 114 and 115. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 116.

SEQ ID NO: 107

EIVLTQSPGTLSLSPGERATLSCRASQSVPEGTLAWYQQRPGQAPRLLIYRYTSRATGIPDRFSGSGS GTDFTLS I SRLEPEDSAVYYCQQFGHVPVTFGCGTKLEIKRTS SGGGGSGGGGSGGGRSQVQLQESGP GLVKPSETLSLTCTVSGGSISRSSHYWGWIRQPPGKCLEWIGYIYYSGSTNYNPSLKSRVTISVDTSK NQFSLKLSSVTAADTAVYYCARGSYYFDYWGQGTLVTVSS

FL030-Ab002 Lwt H03

SEQ ID NO: 117 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL030-Ab002 Lwt H03”. FL030-Ab002 Lwt H03 is an scFv that binds to a peptide having the sequence GVYDGREHTV (SEQ ID NO: 74) in complex with HLA-A*02. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 118. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 119, 120 and 121. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 122. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 123, 124 and 125. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 126.

SEQ ID NO: 117

DIQLTQSPSSLSASVGDRVIITCRASQDVFHHLAWYQQKPGKVPKLLIYAASSLQSGVPSRFRGSGSG TDFTL TISSLQPEDFATYYCQQSYSTPQTFGQGTKVEIKR TSSGGGGS GGGGS GGGRSEVQLVQSGAE VKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWINPNSGGTIYAQKFQGRVTMTRDTSIST AYMELSSLRSEDTAVYYCASSYCSSHSCYLGPYYYGMDVWGQGTLVTVSS

FL030-Ab102 H02 L06

SEQ ID NO: 127 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL030-Ab102 H02 L06”. FL030-Ab102 H02 L06 is an scFv that binds to a peptide having the sequence GVYDGREHTV (SEQ ID NO: 74) in complex with HLA-A*02. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 128. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 129, 130 and 131. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 132. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 133, 134 and 135. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 136.

SEQ ID NO: 127 QSVKESGGGLVTPGGTLTLTCTASGFSLHNYYMNWVRQAPGKGLEWIGIIGGSGTTYYANWAQGRFVI

SKTSSTTVTLQMTSLTTGDTATYFCARDSAYLTLWGQGTLVTVSSGQPKAPSVGGGGSGGGGSSSSRS

SGGELVMTQTPSSVSKPVGGTVTIKCQASQDIFSNLAWYQQKPGQPPKLLIYQASILEDGVPSRFSGS

GSGTQLTLT I SGVQCEDAAT YYCQGTYWDSGWLGAFGGGTEWVK

FL030-Ab102 H07 L07

SEQ ID NO: 137 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL030- Ab102 H07 L07”. FL030-Ab102 H07 L07 is an scFv that binds to a peptide having the sequence GVYDGREHTV (SEQ ID NO: 74) in complex with HLA-A*02. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 138. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 139, 140 and 141. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 142. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 143, 144 and 145. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 146.

SEQ ID NO: 137

QSVKESGGGLVTPGGTLTLTCTASGFDLRNYYMNWVRQAPGKGLEWIGI IGGPGTTYYANWAQGRFVI SKTSSTTVTLQMTSLTTGDTATYFCARDSAYLTLWGQGTLVTVSSGQPKAPSVGGGGSGGGGSSSSRS SGGELVMTQTPSSVSKPVGGTVTIKCQASQDIYSNLAWYQQKPGQPPKLLIYQASILEFGVPSRFSGS

NSGTQLTLT I SGVQCEDAAT YYCQGTYWDSGWLGAFGGGTEWVK

FL036-Ab002 L02 Hwt

SEQ ID NO: 147 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL036-Ab002 L02 Hwt ”. FL036-Ab002 L02 Hwt is an scFv that binds to a peptide having the sequence VLDFAPPGA (SEQ ID NO: 76) in complex with HLA-A*02. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 148. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 149, 150 and 151. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 152. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 153, 154 and 155. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 156.

SEQ ID NO: 147

DIQL TQSPSSLSASVGDR VTI TCQASQDISNYLNWYQQKPGKAPNLLIYDAENLATGVPSRFSGSASG TDFTL TISSLQPEDFATYYCQQYSSYPLTFGGGTKVEIKR TSSGGGGS GGGGSGGGRSQVQLVQSGAE VKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTST AYMELSSLRSEDTAVYYCARVSVTHFAFDIWGQGTMVTVSS FL036-Ab002 L03 Hwt

SEQ ID NO: 157 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL036-Ab002 L03 Hwt”. FL036-Ab002 L03 Hwt is an scFv that binds to a peptide having the sequence VLDFAPPGA (SEQ ID NO: 76) in complex with HLA-A*02. The light chain variable domain () is in italics and is designated SEQ ID NO: 158. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 159, 160 and 161. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 162. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 163, 164 and 165. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 166.

SEQ ID NO: 157

DIQL TQSPSSLSASVGDR VTI TCQASQDISNYLNWYQQKPGKAPNLLIYDASNLADGVPSRFSGSASG TDFTL TISSLQPEDFATYYCQQYSSYPLTFGGGTKVEIKR TSSGGGGS GGGGSGGGRSQVQLVQSGAE VKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTST AYMELSSLRSEDTAVYYCARVSVTHFAFDIWGQGTMVTVSS

FL036-Ab002 L04 Hwt

SEQ ID NO: 167 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL036-Ab002 L04 Hwt”. FL036-Ab002 L04 Hwt is an scFv that binds to a peptide having the sequence VLDFAPPGA (SEQ ID NO: 76) in complex with HLA-A*02. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 168. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 169, 170 and 171. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 172. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 173, 174 and 175. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 176.

SEQ ID NO: 167

DIQL TQSPSSLSASVGDR VTI TCQASQDISNYLNWYQQKPGKAPNLLIYDAENLADGVPSRFSGSASG TDFTL TISSLQPEDFATYYCQQYSSYPLTFGGGTKVEIKR TSSGGGGS GGGGSGGGRSQVQLVQSGAE VKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTST AYMELSSLRSEDTAVYYCARVSVTHFAFDIWGQGTMVTVSS

FL043-Ab004 L05 Hwt

SEQ ID NO: 177 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL043-Ab004 L05 Hwt”. FL043-Ab004 L05 Hwt is an scFv that binds to a peptide having the sequence ALDGGNKHFL (SEQ ID NO: 1 ) in complex with HLA-A*02. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 178. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 179, 180 and 181. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 182. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 183, 184 and 185. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 186.

SEQ ID NO: 177

DIQMTQSPSSLSASIGDRVTITCQASQDISNYLNWYQQKPGKAPQLLIYAASTLQSGVPSRFSGSGSG

TEFTL TISSLQPEDFAAYYCQQSYSDARTFGQGTKLEIKR TSSGGGGS GGGGSGGGRSEVQLVQSGAE

VKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSIST AYMELSRLRSGDTAVYYCARDLGGSYSDVWGQGTLVTVSS

FL043-Ab004 Lwt H09

SEQ ID NO: 187 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL043- Ab004 Lwt H09 ”. FL043-Ab004 Lwt H09 is an scFv that binds to a peptide having the sequence ALDGGNKHFL (SEQ ID NO: 1) in complex with HLA-A*02. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 188. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 189, 190 and 191. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 192. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 193, 194 and 195. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 196.

SEQ ID NO: 187

DIQMTQSPSSLSASIGDRVTITCQASQDISNYLNWYQQKPGKAPQLLIYAASSLQSGVPSRFSGSGSG TEFTL TISSLQPEDFAAYYCQQSYSTPNTFGQGTKLEIKR TSSGGGGS GGGGSGGGRSEVQLVQSGAE VKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPLNGWFNYAQKFQGRVTMTRDTSIST AYMELSRLRSGDTAVYYCARDLGGSYSDVWGQGTLVTVSS

FL043-Ab035 L06 H06

SEQ ID NO: 197 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL043- Ab035 L06 H06”. FL043-Ab035 L06 H06 is an scFv that binds to a peptide having the sequence ALDGGNKHFL (SEQ ID NO: 1) in complex with HLA-A*02. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 198. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 199, 200 and 201. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 202. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 203, 204 and 205. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 206. SEQ ID NO: 197

QSAL TQPASVSGS PGQS IT I SCSGSRID GSNDFVSWYQQHPGEVPKLIL YDVHKRPSGVPDRFSGSK

SDNTAYLTISGLQAEDEAYYYCSSYTTSNTLVFGGGTELTVLSGGGGGSGGGGSGGGRSQVQLQ'ESGP

GLVKPSETLSLTCTVSGGSISSMNWWSWVRQPPGKGLEWIGEIYYSGSTYYNPSLKSRVTISVDTSKN

QFSLKLSSVTAADTAVYYCARERIATPGALDYWGQGTLVTVSS

FL043-Ab035 L11 H11

SEQ ID NO: 207 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL043-Ab035 L11 H11 FL043-Ab035 L11 H11 is an scFv that binds to a peptide having the sequence ALDGGNKHFL (SEQ ID NO: 1 ) in complex with HLA-A*02. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 208. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 209, 210 and 211. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 212. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 213, 214 and 215. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 216.

SEQ ID NO: 207

QSAL TQPASVSGS PGQS ITI SCSGSRIDVGSNDFVSWYQQHPGEVPKLIL YDVHKRPSGVPDRFSGSK SDNTAYLTISGLQAEDEAYYYCSSYTFSQTLVFGGGTELTVLSGGGGGSGGGGSGGGRSQVQLQ'ESGP GLVKPSETLSLTCTVSGGSISSMNWWSWVRQPPGKGLEWIGEIYYSGSTYYNPSLKSRVTISVDTSKN QFSLKLSSVTAADTAVYYCAREYMATPGALDYWGQGTLVTVSS

FL043-Ab035 L17 H17

SEQ ID NO: 217 is the full amino acid sequence of an exemplary TCRm referred to herein as “FL043-Ab035 L17 H17 ”. FL043-Ab035 L17 H17 is an scFv that binds to a peptide having the sequence ALDGGNKHFL (SEQ ID NO: 1 ) in complex with HLA-A*02. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 218. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 219, 220 and 221. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 222. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 223, 224 and 225. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 226.

SEQ ID NO: 217

QSAL TQPASVSGS PGQS ITI SCSGSRIDVGSNDFVSWYQQHPGEVPKLIL YDVHKRPSGVPDRFSGSK SDNTAYLTISGLQAEDEAYYYCSSYTPSNTLVFGGGTELTVLSGGGGGSGGGGSGGGRSQVQLQ'ESGP GLVKPSETLSLTCTVSGGSISSMNWWSWVRQPPGKGLEWIGEIYYSGSTYYNPSLKSRVTISVDTSKN QFSLKLSSVTAADTAVYYCAREQLATPGALDYWGQGTLVTVSS Exemplary immune cell engaging domain sequences

U0 anti-CD3 scFv

SEQ ID NO: 30 is an exemplary anti-CD3 scFv (T cell engaging immune effector domain) referred to herein as “U0”. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 31 . The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 33, 34 and 35. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 32. The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 36, 37 and 38. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 39.

SEQ ID NO: 30:

AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRF SGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGG GGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYK GVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTL VTVSS

U28 anti-CD3 scFv

SEQ ID NO: 40 is another exemplary anti-CD3 scFv (T cell engaging immune effector domain) referred to herein as “U28”. This sequence is the same as SEQ ID NO: 30 above, except for two substitutions that are double underlined (T164A and 1201 F). The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 31. The light chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 33, 34 and 35. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 41 . The heavy chain CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 42, 37 and 38. A glycine-serine linker, linking the VL and VH, is shown in plain text and is designated SEQ ID NO: 39.

SEQ ID NO: 40:

AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRF SGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGG GGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYK GVSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTL VTVSS Camelid PD1 agonist

SEQ ID NO: 75 is the amino acid sequence of an exemplary immune suppressor (immune cell engaging domain) which is a PD1 agonist VHH. The CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 43, 44 and 45 respectively. Positions different from the human lgHV3-23 consensus are shown in bold.

SEQ ID NO: 75:

EVQLVE S GGALVQPGGS LRLS CAAS GFTFSSYAMTWVRQAPGKGPEWVSAIASDGASTS YLD SVKGRFTVSRDNAKNTLYLQMNSLKPEDTAVYYCARGGYLTYDRYGQGTQVTVSS

Humanised PD1 agonist

SEQ ID NO: 46 is the amino acid sequence of a humanised version of the exemplary PD1 agonist VHH of SEQ ID NO: 75 above. CDR sequences are the same as SEQ ID NO: 75. Mutations relative to SEQ ID NO: 75 are shown in bold.

SEQ ID NO: 46:

AVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMTWVRQAPGKGPEWVSAIASDGASTSYAD

SVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCARGGYLTYDRYGQGTLVTVSS

Examples

The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the purview of this application and scope of the appended claims.

Example 1 - Materials and Methods

TCRm antibody library screening. TCRm were isolated from either a human naive antibody phage library (kappa and lambda) or isolated from rabbit immunisation. The human TCRm libraries were generated from cDNA obtained from 426 male/female donors’ peripheral blood leukocytes (PBL) (Clontech cat.: 636592). The phagemid libraries were generated in a VL-VH format using a human kappa interdomain linker sequence SSGGGGSGGGGSGGGRS (SEQ ID NO: 11) or a human lambda interdomain linker sequence SGGGGGSGGGGSGGGRS (SEQ ID NO: 48). To obtain rabbit derived TCRm, animals were immunized with recombinant peptide-HLA complex using a standard immunization protocol. Spleen and bone marrow samples were subsequently collected for RNA isolation. After cDNA preparation with oligo dT primers, heavy and light variable domains were specifically amplified and stitched together for library building. Rabbit scFv's have a VH-VL orientation with a GGGGSGGGGSSSSRSSGG (SEQ ID NO: 49) linker between the variable domains.

Human and rabbit antibodies were isolated with the same panning and screening process.

TCRm were selected from the library in three rounds of bio-panning with a disease associated peptide-HLA (pHLA). Prior to antigen specific enrichment using paramagnetic beads coated with the pHLA antigen, a stringent depletion panning approach was performed using a mixture of 20 irrelevant pHLA containing common peptides. At the end of each round, phage particles were eluted by trypsin and used to infect early log phase TG1 E. coli cells for phage amplification for the next round. At the end of the bio-panning procedure, phage infected TOP10 F’ E. coli colonies were randomly selected and TCRm were expressed in autoinduction media. Crude periplasm extract was used for screening ELISA against cognate pHLA and a mixture of 20 irrelevant pHLA common peptides. Affinity variants of native TCRm were generated using phage mutagenesis libraries and screening.

Construct design, protein expression and purification. Soluble TCRm bispecific molecules were constructed using an TCRm scFv targeting arm, with a linker between the variable heavy and light chains, fused to an antiCD3 scFv effector arm via a linker. The dual-scFv expression cassette was cloned into pCEP4 and protein expressed in mammalian cells using the ExpiCHO Expression System (Thermo Fisher Scientific). Molecules were purified using Protein L chromatography and size exclusion chromatography. HLA molecules were refolded with the peptide of interest and purified using available methods (O’Callaghan et al. (1999), Anal Biochem 266(1 ): 9-15; Garboczi, et al. (1992), Proc Natl Acad Sci USA 89(8): 3429-3433).

SPR single-cycle kinetic analysis. TCRm bispecfic molecules were subjected to SPR analysis using a BIAcore 8K with single cycle kinetic analysis. Kinetic parameters were calculated using BIAevaluation® software. The dissociation phase was fitted to a single exponential decay equation enabling calculation of half-life. The equilibrium constant KD was calculated from koff/kon. Measurements were performed at 25°C, unless otherwise indicated, in Dulbecco’s PBS buffer, supplemented with 0.005% P20.

X-ray crystallography. Crystals were grown by vapor diffusion via the sitting drop technique using the MRC 2-well crystallisation plates. 150 nL of 10 to 15 mg/mL scFv-pHLA complex (mixed at a 1 :1 molar ratio and purified by size exclusion chromatography) was added to 150 nL of reservoir solution using the Gryphon dispensing robot (Art Robbins). The plates were then incubated at 20°C and imaged using ROCK IMAGER 1000 (Formulatrix). Crystals selected for further analysis were cryoprotected with 30% ethylene glycol and then flash-cooled in liquid nitrogen in Litho loops (Molecular Dimensions). Diffraction data were collected at several different beamlines at the Diamond Light Source (Didcot, UK) and processed through xia2 DIALS (Winter (2010). J Appl Crystallogr. 43, 186-190, Winter et al. (2018). Acta Crystallogr Sect D Struct Biology. 74, 85-97) or xia2 3dii (.Kabsch (2010). Acta Crystallogr Sect D Biological Crystallogr. 66, 125-132) or autoPROC (Vonrhein et al. (2011 ). Acta Crystallogr Sect D Biological Crystallogr. 67, 293-302) automated pipelines. scFv-pHLA complex structures were solved with molecular replacement using Phaser (McCoy et al. (2007). J. Appl. Crystallogr. 40, 658-674), models built using Coot (Emsley et al. (2010). Acta Crystallogr Sect D Biological Crystallogr. 66, 486- 501 ), and refined using refmac (Kovalevskiy et al. (2018). Acta Crystallogr. Sect. D, Struct. Biol. 74, 215-227), all within the CCP4 suite (Agirre et al. (2023). Acta Crystallogr. Sect. D, Struct. Biol. 79, 449- 461 ). Molecular replacement search models were identified as follows: for the different TCRm (scFv) molecules, the PDB was searched for structures of proteins with high sequence similarity to variable heavy and light sequences separately and these were used as models. For HLA-B2m, the PDB 6RPA (for HLA-A2, TCR removed) and 3VXN (for HLA-A24) were used. Where experimental structures for high affinity variants were not available, structures were obtained by introducing mutations to the crystal structures of WT TCRm-pHLA complexes. This was carried out using the Schrodinger BioLuminate software and loading the WT crystal structure as a template for the Residue Scanning tool. Mutations in the variants were then defined and the tool was run with a refinement setting cut-off of 4 A for sidechain prediction with backbone minimization.

Crossing angle calculation. The crossing angle was calculated by generating two vectors: a HLA groove vector and a TCRm cystine vector (or “TCRm interdomain vector”) see Figure 1 for a schematic illustration of calculating a crossing angle. The HLA groove vector follows the two parallel HLA helices with the direction N-terminus to C-terminus of HLA helix 1 and passing through the HLA centroid. The TCRm cystine vector (or “TCRm interdomain vector”) connects the characteristic cystines (i.e., the intrachain disulfide bonds) in the two variable regions and points from the intrachain disulfide bond in the light chain variable region (VL) to the intrachain disulfide bond in the heavy chain variable region (VH). The crossing angle was defined by the angle between the TCRm cysteine vector and the HLA vector. This calculation was adopted from the crossing angle calculation described for the TCRs (Rudolph et al. (2006). Annu Rev Immunol. 24, 419-466), where the TCRm VL is comparable to the TCR alpha chain variable region and the TCRm VH is comparable to the TCR beta chain variable region.

Interaction analysis. Residues between TCRm and pHLA were identified to be in contact if the distance measurement between any atom from a TCRm residue was within or equal to 4 A to any atom from a peptide or a HLA residue.

Cell assays. The activity of the TCRm scFv-aCD3 scFv bispecific molecules was tested through their ability to redirect T cells against antigen-positive and antigen-negative cell lines. Cells were cultured in RPMI supplemented with 10% FCS, 2 mM l-glutamine, and 1 % (v/v) penicillin/streptomycin. IFNy ELISpot assays were performed according to the manufacturer’s instructions (BD Biosciences). Briefly, target cells were plated at ~5x10⁴ cells/per well and incubated with PBMC effector cells at a donor-dependant density. Test molecules were added at the indicated concentration and plates were incubated overnight at 37°C/5% CO2. IFNy-release was quantified using the BD ELISpot reader (Immunospot Series 5 Analyzer, Cellular Technology Ltd). Alternatively, IFNy ELISA cell assays were per performed according to manufacturer instructions, using a working concentration of capture antibody 2pg/mL (DuoSet ELISA R&D systems), Briefly, target cells, PBMC effector cells and TCRm BsAb molecules were added at the indicated concentration and plates were incubated for 48 hrs at 37°C/5% CO2. IFNy release was detected using was detected using LumiGLO Peroxidase Chemiluminescent Substrate Kit (R&D systems) and quantified against an [IFNy] standard curve. Data were plotted using PRISM software and EC50 values were calculated using the non-linear regression analysis and “log(agonist) vs. response (three parameters)” setting.

Example 2 - soluble TCRm bispecific molecules with high affinity for target pHLA

TCRm antibodies were obtained from phage libraries following panning with disease associated pHLA. Three different pHLAs were used in this example: HLA-A*02 restricted peptide ALDGGNKHFL from gp100; HLA-A*024 restricted peptide PYLGQMINL from PRAME; and HLA-A*02 restricted peptide GVYDGREHTV from MAGEA4. TCRm scFv fragments were prepared as bispecific molecules fused to an antiCD3 scFv (SEQ ID NO: 30) via a short linker sequences, and binding affinity to cognate pHLA was determined using Surface Plasmon Resonance. Amino acid sequences of the corresponding TCRm scFVs are indicated in Table 1 . Data show native TCRm bound to cognate antigen in the low nanomolar range, and one affinity matured variant recognising PYLGQMINL bound with pM affinity. These data demonstrate that TCRm with high affinity for target pHLA can be obtained. Strong binding to target antigen is particularly important for therapeutic molecules targeting cells with a low density of target pHLA.

Table 1 - TCRm bispecfic molecules bind to cognate pHLA with high affinity

Example 3 - Potency and specificity of soluble TCRm bispecific molecules

The ability of TCRm bispecific molecules to specifically redirect T cells against antigen positive cells was determined using cell assays as described in Example 1. Figure 2 shows the resulting response curves obtained with 5 TCRm bispecific molecules provided in Table 1. These data confirm that TCRm bispecific molecules were able to redirect a potent redirected T cell response in the presence of antigen positive cells, with minimal or no response in the absence of antigen positive cells. Example 4 - TCRm binds to pHLA with reverse polarity interaction

Structural features of the TCRm-pHLA interaction were investigated using crystal structures obtained from both the Protein Data Bank (PDB) and in-house data, including the 5 TCRm described in Table 1 (except that for FL029-AB003-L2H2 structural analysis was performed using the non-affinity matured version of the TCRm (SEQ ID NO: 21). The crossing angle of the interaction between TCRm and pHLA and was determined by molecular modelling (Figure 1). The equivalent crossing angle between a TCR and pHLA was also analysed for comparison, using both PDB and in-house TCR structures. Data in Figure 3A show that for the TCR structures analysed the crossing angle of the interaction was typically in the region of 45 degrees, whereas for TCRm the crossing angle varied more widely (mean +/-1SD). The crossing angle obtained for the 5 TCRm shown in Table 1 was between 150-250 degrees (Figure 3B), meaning that the orientation of VL and VH was reversed compared to a typical TCR pHLA interaction (where VL is comparable to the TCR v alpha chain and VH is comparable to the TCR v beta chain). These data indicate TCRm that bind pHLA with reverse polarity also demonstrate both high affinity and specificity.

Example 5 - Peptide Contact Characteristics

Further molecular modelling was carried out using both known and in-house TCRm crystal structures to probe the amino acid contacts between TCRm and pHLA. In a first analysis, the number of contacts between TCRm and peptide was probed. Figure 4 shows data obtained from 28 TCRm with a reverse crossing angle, 11 from the PDB and 17 in-house, including the 5 TCRm used in Example 4. The number of direct peptide contacts across the central region of the peptide (i.e. excluding residues at position 1 and the two anchor positions) shows the number of contacts range between 1 and 6 amino acids. For the 5 TCRm exemplified here, 4 have 4 of more contacts with peptide. These data suggests that contacts in the central region of the peptide could be important for generating potent and specific TCRm. In a second analysis, the number of contacts between TCRm and defined positions on helix 1 and helix 2 of the HLA binding groove (positions 65, 69, 72 and 76 on helix 1 and positions 155 158 163 and 167 on helix 2) was probed. Figure 5 shows TCRm interact with positions on one or both helicies. For the 5 TCRm exemplified here, all make contact with at least 1 position on helix 1 and at least one position on helix 2. These data suggest that a broad interaction footprint, involving interactions to both helix 1 and helix 2 could be important for generating potent and specific TCRm.

Table 2 - Summary of structural features of TCRm described in this study

In summary these data demonstrate that TCRm bispecific molecules can be produced which demonstrate both high affinity and specificity for target pHLA and are therefore suitable for therapeutic development. In addition, the structural features that are desirable for such a TCRm pHLA interaction include a crossing in the opposite or ‘reverse’ orientation to a TCR pHLA interaction as well as an extensive interface involving 4 or more contacts across the central region of the peptide and contacts to defined positions within both helix 1 and helix 2 of the HLA.

Example 6 - Identification of further TCRm bispecific molecules with optimal structural and functional properties

Further TCRm antibodies were obtained from libraries as described in Example 1 and subjected to affinity maturation to produce high affinity variants. A further pHLA was used in this example in addition to the three described in Example 1 (HLA-A*02 restricted peptide VLDFAPPGA (SEQ ID NO: 76) from the antigen WT1 ). TCRm scFv fragments were prepared as bispecific molecules fused to an antiCD3 scFv (U28 antiCD3 scFv of SEQ ID NO: 40). Sequences of the TCRm scFvs are provided in SEQ ID NOs: 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207 and 217. Binding affinity to cognate antigen was determined by SPR using single cycle kinetics. EC50 values were determined using ELISA assays as previously described. Cell assays were performed using both antigen positive and antigen negative cells.

The table below provides the binding affinity and EC50 values obtained for 15 TCRm bispecific molecules. Figure 6 provides corresponding graphical data used to determine EC50 values. In each case minimal recognition of target negative cells was observed at concentrations below 1 nM.

Three dimensional structures of each molecule were obtained using X-ray crystallography and molecular modelling. Crossing angle, peptide contacts and HLA contacts were determined as previously described.

The structural data are summarised in the table below. The positions of the contacts sites on the peptide are shown in Figure 7. Contacts to HLA helix 1 and helix 2 are provided in Figures 8 and total HLA contacts are provided in Figure 9. In Figures 7-9 an asterisk indicates data were obtain directly from the crystal structure, for the remainder data were obtained from molecular modelling using the crystal structure of the corresponding WT TCRm.

Further aspects of the invention:

1 . A binding molecule comprising a TOR mimic (TCRm) that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA), wherein the TCRm comprises an antibody or an antigen-binding fragment thereof, and wherein the TCRm:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the DA-pHLA with an affinity of 20 nM or stronger.

2. The binding molecule of clause 1 , wherein the TCRm binds to the DA-pHLA with a crossing angle in the range of 170-240 degrees.

3. The binding molecule of clause 1 or clause 2, wherein the TCRm binds to the DA-pHLA with 5 or 6 or more peptide residue binding contacts.

4. The binding molecule of any preceding clause, wherein at least 30%, or at least 35%, of the DA-pHLA residues bound by the TCRm are peptide residues.

5. The binding molecule of any preceding clause, wherein the TCRm binds to at least one residue in helix 1 in the peptide-binding groove of the HLA and at least one residue in helix 2 in the peptide-binding groove of the HLA.

6. The binding molecule of any preceding clause, wherein the affinity of the TCRm for the DA-pHLA is 10 nM or stronger, 5 nM or stronger, 1 nM or stronger, 500 pM or stronger, or 100 pM or stronger.

7. The binding molecule of any preceding clause, wherein the human leukocyte antigen (HLA) is a class I HLA.

8. The binding molecule of any preceding clause, wherein the human leukocyte antigen (HLA) is a HLA-A*02 serotype or HLA-A*24 serotype.

9. The binding molecule of any preceding clause, wherein the peptide in the DA-pHLA is a human peptide.

10. The binding molecule of any preceding clause, wherein the peptide in the DA-pHLA is a peptide from a tumour associated antigen.

11. The binding molecule of any preceding clause, wherein the peptide is from PRAME, gp100, MAGEA4, PIWIL1 , or pre-pro-insulin. 12. The binding molecule of any one of clauses 1 to 9, wherein the peptide in the DA-pHLA is a viral peptide.

13. The binding molecule of any one of clauses 1 to 9 or 12, wherein the peptide in the DA-pHLA is from HIV or HBV.

14. The binding molecule of any preceding clause, wherein the binding molecule is multispecific, optionally wherein the binding molecule is bispecific.

15. The binding molecule of any preceding clause, further comprising a half-life extending domain.

16. The binding molecule of clause 15, wherein the half-life extending domain is an immunoglobulin Fc domain.

17. The binding molecule of any preceding clause, wherein the TCRm binds to a DA-pHLA positive cell, but does not detectably bind to a DA-pHLA negative cell.

18. The binding molecule of any preceding clause, wherein the peptide in the DA-pHLA has the amino acid sequence provided in SEQ ID NO: 1 , 2 or 74.

19. The binding molecule of any preceding clause, wherein the TCRm comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

20. The binding molecule of any preceding clause, wherein the peptide in the DA-pHLA has the amino acid sequence provided in SEQ ID NO: 1.

21 . The binding molecule of clause 20, wherein the TCRm comprises

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 4, 5 and 6 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 4, 5 and 6 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 8, 9 and 10 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 8, 9 and 10 respectively.

22. The binding molecule of clause 20 or clause 21 , wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 47 and a VH region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95% or is 100% identical to the sequence of SEQ ID NO: 7. 23. The binding molecule of clause 20, wherein the TCRm comprises

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 14, 15 and 16 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 14, 15 and 16 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 18, 19 and 20 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 18, 19 and 20 respectively.

24. The binding molecule of any of clause 20 or clause 23, wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 13 and a VH region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 17.

25. The binding molecule of any one of clauses 1 to 19, wherein the peptide in the pHLA has the amino acid sequence provided in SEQ ID NO: 2.

26. The binding molecule of clause 25, wherein the TCRm comprises

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 23, 24 and 25 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 23, 24 and 25 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 27, 28 and 29 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 27, 28 and 29 respectively.

27. The binding molecule of clause 25 or clause 26, wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 22 and a VH region at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 26.

28. The binding molecule of clause 25, wherein the TCRm comprises

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 55, 28 and 29 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 55, 28 and 29 respectively.

29. The binding molecule of clause 25 or clause 28, wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 51 and a VH region at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 54.

30. The binding molecule of any one of clauses 1 to 19, wherein the peptide in the DA-pHLA has the amino acid sequence provided in SEQ ID NO: 74.

31. The binding molecule of clause 30, wherein the TCRm comprises

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 62, 63 and 64 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 62, 63 and 64 respectively.

32. The binding molecule of clause 30 or clause 31 , wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 57 and a VH region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95% or is 100% identical to the sequence of SEQ ID NO: 61.

33. The binding molecule of clause 30, wherein the TCRm comprises

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 67, 68 and 69 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 67, 68 and 69 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 71 , 72 and 73 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 71 , 72 and 73 respectively.

34. The binding molecule of clause 30 or clause 33, wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 66 and a VH region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95% or is 100% identical to the sequence of SEQ ID NO: 70.

35. The binding molecule of any preceding clause, further comprising an immune cell engaging domain.

36. The binding molecule of clause 35, wherein the immune cell engaging domain binds to a T cell surface antigen.

37. The binding molecule of clause 36, wherein the T cell surface antigen is CD3. 38. The binding molecule of clause 36, wherein the T cell surface antigen is PD-1.

39. The binding molecule of any one of clauses 35 to 37, wherein the immune cell engaging domain comprises:

(a) a VL comprising a CDR1 comprising the amino acid sequence provided in SEQ ID NO:

33, a CDR2 comprising the amino acid sequence provided in SEQ ID NO: 34 and a CDR3 comprising the amino acid sequence provided in SEQ ID NO: 35; and

40. The binding molecule of any one of clauses 35 to 37 or clause 39, wherein the immune cell engaging domain comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 31 and a VH region at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 32 or 41.

41. The binding molecule of any one of clauses 35, 36 or 38, wherein the immune cell engaging domain comprises a single domain antibody comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 43, 44 and 45 respectively.

42. The binding molecule of any one of clauses 35, 36, 38 or 41 , wherein the immune cell engaging domain comprises a single domain antibody comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 75 or 46.

43. The binding molecule of any preceding clause, which is soluble.

44. A multispecific binding molecule comprising a TCRm that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA) and an immune cell engaging domain, wherein the TCRm comprises an antibody or antigen binding fragment thereof, and wherein the TCRm:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the pHLA complex with an affinity of 20 nM or stronger.

45. A multispecific binding molecule comprising a TCRm that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA) and an immune cell engaging domain, wherein

(a) the immune cell engaging domain binds to CD3 and comprises:

(b) the TCRm comprises an antibody or antigen binding fragment thereof and:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the DA-pHLA complex with an affinity of 20 nM or stronger.

46. A multispecific binding molecule comprising a TCRm that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA) and an immune cell engaging domain, wherein

(b) the TCRm comprises an antibody or antigen binding fragment thereof and:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts;

47. A binding molecule comprising a TCR mimic (TCRm) that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA), wherein the TCRm comprises an antibody or an antigen-binding fragment thereof, and wherein the TCR mimic:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the DA-pHLA with an affinity of 20 nM or stronger; and

(iv) binds to a DA-pHLA positive cell, but does not detectably bind to a DA-pHLA negative cell.

48. The binding molecule of any one of clauses 44 to 47, which is further characterised by the features of any one or more of clauses 2 to 43.

49. An antibody or antigen binding fragment thereof that specifically targets a disease associated peptide human leukocyte antigen (DA-pHLA) complex, comprising: a means to dock to said DA- pHLA complex and to have an affinity of 20 nM or stronger.

50. A molecule that redirects immune cells to a DA-pHLA presenting cell, wherein the molecule comprises an antibody or antigen binding fragment thereof that specifically targets a disease associated peptide HLA (DA-pHLA) complex and comprising: (a) a means for said antibody or antigen binding fragment thereof to dock to said DA-pHLA complex and to have an affinity of 20nM or stronger; and (b) a means to engage said immune cells.

51. The antibody or antigen binding fragment of clause 49 or the molecule of clause 50, wherein the means to dock comprises binding to the DA-pHLA complex with a crossing angle in the range of 150- 250 degrees and binding to the DA-pHLA complex with 4 or more peptide residue binding contacts.

52. The antibody or antigen binding fragment, or molecule, of clause 51 , wherein the means to dock comprises binding to the DA-pHLA with a crossing angle in the range of 170-240 degrees.

53. The antibody or antigen binding fragment, or molecule, of any one of clauses 49 to 52, wherein the means to dock comprises binding to the DA-pHLA with 5 or 6 or more peptide residue binding contacts.

54. The antibody or antigen binding fragment, or molecule, of any one of clauses 49 to 53, wherein the means to dock comprises binding DA-pHLA wherein at least 30%, or at least 35%, of the DA-pHLA residues bound by the antibody or antigen binding fragment are peptide residues.

55. The antibody or antigen binding fragment, or molecule, of any one of clauses 49 to 54, wherein the means to dock comprises binding to at least one residue in helix 1 in the peptide-binding groove of the HLA and at least one residue in helix 2 in the peptide-binding groove of the HLA.

56. The antibody or antigen binding fragment, or molecule, of any one of clauses 49 to 55, configured to have an affinity for the DA pHLA of 10 nM or stronger, 5 nM or stronger, 1 nM or stronger, 500 pM or stronger, or 100 pM or stronger.

57. The antibody or antigen binding fragment, or molecule, of any one of clauses 49 to 56, further characterised by the features of any one or more of clauses 1 to 48.

58. A nucleic acid encoding the binding molecule of clauses 1 to 48 or the antibody or antigen binding fragment, or molecule, of any one of clauses 49 to 57.

59. An expression vector comprising the nucleic acid of clause 58.

60. A host cell comprising the nucleic acid of clause 58, or the expression vector of clause 59.

61. A method of making the binding molecule of any one of clauses 1 to 48, or the antibody or antigen binding fragment or molecule of any one of clauses 49 to 57, comprising maintaining the host cell of clause 60 under optimal conditions for expression of the nucleic acid of clause 58, or the expression vector of clause 59 and isolating the binding molecule, the antibody, or antigen binding fragment, or molecule.

62. A pharmaceutical composition comprising the binding molecule of any one of clauses 1 to 48, or the antibody or antigen binding fragment or molecule of any one of clauses 49 to 57, the nucleic acid of clause 58, the expression vector of clause 59 or the cell of clause 60, and one or more pharmaceutically acceptable excipients.

63. The binding molecule of any one of clauses 1 to 48, or the antibody or antigen binding fragment or molecule of any one of clauses 49 to 57, the nucleic acid of clause 58, the expression vector of clause 59 or the cell of clause 60, or the pharmaceutical composition of clause 62, for use in medicine.

64. The binding molecule of any one of clauses 1 to 48, or the antibody or antigen binding fragment or molecule of any one of clauses 49 to 57, the nucleic acid of clause 58, the expression vector of clause 59 or the cell of clause 60, or the pharmaceutical composition of clause 62, for use in treating cancer, an autoimmune disease or an infectious disease.

65. A method of treating cancer, an autoimmune disease or an infectious disease in a subject, the method comprising administering the binding molecule of any one of clauses 1 to 48, or the antibody or antigen binding fragment or molecule of any one of clauses 49 to 57, the nucleic acid of clause 58, the expression vector of clause 59 or the cell of clause 60, or the pharmaceutical composition of clause 62 to the subject.

66. A method of producing a binding molecule comprising a TOR mimic (TCRm) that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA), wherein the TCRm comprises an antibody or an antigen-binding fragment thereof, and wherein the method comprises:

(i) binds to the DA-pHLA complex with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the pHLA complex with an affinity of 20 nM or stronger.

67. The method of clause 66, comprising, subsequent to step (a) and prior to step (b), screening the plurality of candidate binding molecules to determine the (i) DA-pHLA crossing angle, (ii) DA- pHLA residue binding contacts, and (iii) binding affinity for the DA-pHLA, for the TCRm of each of the plurality of candidate binding molecules. 68. The method of clause 67, wherein screening the plurality of candidate binding molecules for their (i) DA-pHLA crossing angle and/or (ii) DA-pHLA residue binding contacts is based on a three- dimensional atomic structure of the complex formed by the DA-pHLA and the TCRm of each of the candidate binding molecules.

69. The method of any one of clauses 66 to 68, wherein the method comprises determining the three-dimensional atomic structure of the complex formed by the DA-pHLA and the TCRm of each of the candidate binding molecules.

70. The method of any one of clauses 66 to 69, wherein the three-dimensional atomic structure is an x-ray crystal structure.

71. The method of any one of clauses 66 to 70, wherein the TCRm identified in step (b) does not detectably bind to DA-pHLA negative cells.

72. The method of any one of clauses 66 to 71 , wherein the method further comprises recombinantly expressing the binding molecule identified in step (b) in a host cell and subsequently purifying the binding molecule.

73. The method of any one of clauses 66 to 72, wherein the method further comprises formulating the binding molecule with one or more pharmaceutically acceptable excipients, thereby providing a pharmaceutical formulation comprising the binding molecule.

74. The method of any one of clauses 66 to 73, wherein the binding molecule has any one or more features defined in any one of clauses 1 to 48.

75. A binding molecule produced according to the method of any one of clauses 66 to 74.

Claims

1. A binding molecule comprising a TCR mimic (TCRm) that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA), wherein the TCRm comprises an antibody or an antigen-binding fragment thereof, and wherein the TCRm:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the DA-pHLA with an affinity of 20 nM or stronger.

2. The binding molecule of claim 1 , wherein the TCRm binds to the DA-pHLA with a crossing angle in the range of 170-240 degrees, optionally in the range of 190-240 degrees.

3. The binding molecule of claim 1 or claim 2, wherein the TCRm binds to the DA-pHLA with 5 or 6 or more peptide residue binding contacts.

4. The binding molecule of any preceding claim, wherein at least 30%, or at least 35%, of the DA-pHLA residues bound by the TCRm are peptide residues.

5. The binding molecule of any preceding claim, wherein the TCRm binds to at least one residue in helix 1 in the peptide-binding groove of the HLA and at least one residue in helix 2 in the peptide- binding groove of the HLA.

6. The binding molecule of any preceding claim, wherein the affinity of the TCRm for the DA-pHLA is 10 nM or stronger, 5 nM or stronger, 1 nM or stronger, 500 pM or stronger, or 100 pM or stronger.

7. The binding molecule of any preceding claim, wherein the human leukocyte antigen (HLA) is a class I HLA.

8. The binding molecule of any preceding claim, wherein the human leukocyte antigen (HLA) is a HLA-A*02 serotype or HLA-A*24 serotype.

9. The binding molecule of any preceding claim, wherein the peptide in the DA-pHLA is a human peptide.

10. The binding molecule of any preceding claim, wherein the peptide in the DA-pHLA is a peptide from a tumour associated antigen.

11. The binding molecule of any preceding claim, wherein the peptide is from PRAME, gp100, MAGEA4, PIWIL1 , WT1 , or pre-pro-insulin.

12. The binding molecule of any one of claims 1 to 9, wherein the peptide in the DA-pHLA is a viral peptide.

13. The binding molecule of any one of claims 1 to 9 or 12, wherein the peptide in the DA-pHLA is from HIV or HBV.

14. The binding molecule of any preceding claim, wherein the binding molecule is multispecific, optionally wherein the binding molecule is bispecific.

15. The binding molecule of any preceding claim, further comprising a half-life extending domain.

16. The binding molecule of claim 15, wherein the half-life extending domain is an immunoglobulin Fc domain.

17. The binding molecule of any preceding claim, wherein the TCRm binds to a DA-pHLA positive cell, but does not detectably bind to a DA-pHLA negative cell.

18. The binding molecule of any preceding claim, wherein the peptide in the DA-pHLA has the amino acid sequence provided in SEQ ID NO: 1 , 2, 74 or 76.

19. The binding molecule of any preceding claim, wherein the TCRm comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

20. The binding molecule of any preceding claim, wherein the peptide in the DA-pHLA has the amino acid sequence provided in SEQ ID NO: 1.

21. The binding molecule of claim 20, wherein the TCRm comprises

22. The binding molecule of claim 20 or claim 21 , wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 47 and a VH region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95% or is 100% identical to the sequence of SEQ ID NO: 7.

23. The binding molecule of claim 20, wherein the TCRm comprises

24. The binding molecule of any of claim 20 or claim 23, wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 13 and a VH region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 17.

25. The binding molecule of any one of claims 1 to 19, wherein the peptide in the pHLA has the amino acid sequence provided in SEQ ID NO: 2.

26. The binding molecule of claim 25, wherein the TCRm comprises

27. The binding molecule of claim 25 or claim 26, wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 22 and a VH region at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 26.

28. The binding molecule of claim 25, wherein the TCRm comprises

29. The binding molecule of claim 25 or claim 28, wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 51 and a VH region at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 54.

30. The binding molecule of claim 25, wherein the TCRm comprises

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 79, 80 and 81 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 79, 80 and 81 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 83, 84 and 85 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 83, 84 and 85 respectively.

31. The binding molecule of claim 25 or claim 30, wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 78 and a VH region at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 82.

32. The binding molecule of claim 25, wherein the TCRm comprises

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 89, 90 and 91 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 89, 90 and 91 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 93, 94 and 95 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 93, 94 and 95 respectively.

33. The binding molecule of claim 25 or claim 32, wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 88 and a VH region at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 92.

34. The binding molecule of claim 25, wherein the TCRm comprises

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 99, 100 and 101 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 99, 100 and 101 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 103, 104 and 105 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 103, 104 and 105 respectively.

35. The binding molecule of claim 25 or claim 34, wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 98 and a VH region at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 102.

36. The binding molecule of claim 25, wherein the TCRm comprises

(a) a VL comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 109, 110 and 111 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 109, 110 and 111 respectively; and

(b) a VH comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 113, 114 and 115 respectively, or amino acid sequences having no more than one, two or three substitutions relative to SEQ ID NOs: 113, 114 and 115 respectively.

37. The binding molecule of claim 25 or claim 36, wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 108 and a VH region at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 112.

38. The binding molecule of any one of claims 1 to 19, wherein the peptide in the DA-pHLA has the amino acid sequence provided in SEQ ID NO: 74.

39. The binding molecule of claim 38, wherein the TCRm comprises

40. The binding molecule of claim 38 or claim 39, wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 57 and a VH region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95% or is 100% identical to the sequence of SEQ ID NO: 61.

41. The binding molecule of claim 38, wherein the TCRm comprises

42. The binding molecule of claim 38 or claim 41 , wherein the TCRm comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 66 and a VH region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95% or is 100% identical to the sequence of SEQ ID NO: 70.

43. The binding molecule of any preceding claim, further comprising an immune cell engaging domain.

44. The binding molecule of claim 43, wherein the immune cell engaging domain binds to a T cell surface antigen.

45. The binding molecule of claim 44, wherein the T cell surface antigen is CD3.

46. The binding molecule of claim 44, wherein the T cell surface antigen is PD-1.

47. The binding molecule of any one of claims 43 to 45, wherein the immune cell engaging domain comprises:

48. The binding molecule of any one of claims 43 to 45 or claim 47, wherein the immune cell engaging domain comprises a VL region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 31 and a VH region at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 32 or 41.

49. The binding molecule of any one of claims 43, 44 or 46, wherein the immune cell engaging domain comprises a single domain antibody comprising a CDR1 , CDR2 and CDR3 comprising the amino acid sequences provided in SEQ ID NOs: 43, 44 and 45 respectively.

50. The binding molecule of any one of claims 43, 44, 46 or 49, wherein the immune cell engaging domain comprises a single domain antibody comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 75 or 46.

51. The binding molecule of any preceding claim, which is soluble.

52. A multispecific binding molecule comprising a TCRm that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA) and an immune cell engaging domain, wherein the TCRm comprises an antibody or antigen binding fragment thereof, and wherein the TCRm:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the pHLA complex with an affinity of 20 nM or stronger.

53. A multispecific binding molecule comprising a TCRm that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA) and an immune cell engaging domain, wherein

(a) the immune cell engaging domain binds to CD3 and comprises:

(b) the TCRm comprises an antibody or antigen binding fragment thereof and:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the DA-pHLA complex with an affinity of 20 nM or stronger.

54. A multispecific binding molecule comprising a TCRm that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA) and an immune cell engaging domain, wherein

(b) the TCRm comprises an antibody or antigen binding fragment thereof and:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees;

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts;

55. A binding molecule comprising a TCR mimic (TCRm) that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA), wherein the TCRm comprises an antibody or an antigen-binding fragment thereof, and wherein the TCR mimic:

(i) binds to the DA-pHLA with a crossing angle in the range of 150-250 degrees; (ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the DA-pHLA with an affinity of 20 nM or stronger; and

56. The binding molecule of any one of claims 52 to 55, which is further characterised by the features of any one or more of claims 2 to 51.

57. An antibody or antigen binding fragment thereof that specifically targets a disease associated peptide human leukocyte antigen (DA-pHLA) complex, comprising: a means to dock to said DA- pHLA complex and to have an affinity of 20 nM or stronger.

58. A molecule that redirects immune cells to a DA-pHLA presenting cell, wherein the molecule comprises an antibody or antigen binding fragment thereof that specifically targets a disease associated peptide HLA (DA-pHLA) complex and comprising: (a) a means for said antibody or antigen binding fragment thereof to dock to said DA-pHLA complex and to have an affinity of 20nM or stronger; and (b) a means to engage said immune cells.

59. The antibody or antigen binding fragment of claim 57 or the molecule of claim 58, wherein the means to dock comprises binding to the DA-pHLA complex with a crossing angle in the range of 150- 250 degrees and binding to the DA-pHLA complex with 4 or more peptide residue binding contacts.

60. The antibody or antigen binding fragment, or molecule, of claim 59, wherein the means to dock comprises binding to the DA-pHLA with a crossing angle in the range of 170-240 degrees, optionally in the range 190-240 degrees.

61. The antibody or antigen binding fragment, or molecule, of any one of claims 57 to 60, wherein the means to dock comprises binding to the DA-pHLA with 5 or 6 or more peptide residue binding contacts.

62. The antibody or antigen binding fragment, or molecule, of any one of claims 57 to 61 , wherein the means to dock comprises binding DA-pHLA wherein at least 30%, or at least 35%, of the DA- pHLA residues bound by the antibody or antigen binding fragment are peptide residues.

63. The antibody or antigen binding fragment, or molecule, of any one of claims 57 to 62, wherein the means to dock comprises binding to at least one residue in helix 1 in the peptide-binding groove of the HLA and at least one residue in helix 2 in the peptide-binding groove of the HLA.

64. The antibody or antigen binding fragment, or molecule, of any one of claims 57 to 63, configured to have an affinity for the DA pHLA of 10 nM or stronger, 5 nM or stronger, 1 nM or stronger, 500 pM or stronger, or 100 pM or stronger.

65. The antibody or antigen binding fragment, or molecule, of any one of claims 57 to 64, further characterised by the features of any one or more of claims 1 to 56.

66. A nucleic acid encoding the binding molecule of claims 1 to 56 or the antibody or antigen binding fragment, or molecule, of any one of claims 57 to 65.

67. An expression vector comprising the nucleic acid of claim 66.

68. A host cell comprising the nucleic acid of claim 66, or the expression vector of claim 67.

69. A method of making the binding molecule of any one of claims 1 to 56, or the antibody or antigen binding fragment or molecule of any one of claims 57 to 65, comprising maintaining the host cell of claim 68 under optimal conditions for expression of the nucleic acid of claim 66, or the expression vector of claim 67 and isolating the binding molecule, the antibody, or antigen binding fragment, or molecule.

70. A pharmaceutical composition comprising the binding molecule of any one of claims 1 to 56, or the antibody or antigen binding fragment or molecule of any one of claims 57 to 65, the nucleic acid of claim 66, the expression vector of claim 67 or the cell of claim 68, and one or more pharmaceutically acceptable excipients.

71. The binding molecule of any one of claims 1 to 56, or the antibody or antigen binding fragment or molecule of any one of claims 57 to 65, the nucleic acid of claim 66, the expression vector of claim 67 or the cell of claim 68, or the pharmaceutical composition of claim 70, for use in medicine.

72. The binding molecule of any one of claims 1 to 56, or the antibody or antigen binding fragment or molecule of any one of claims 57 to 65, the nucleic acid of claim 66, the expression vector of claim 67 or the cell of claim 68, or the pharmaceutical composition of claim 70, for use in treating cancer, an autoimmune disease or an infectious disease.

73. A method of treating cancer, an autoimmune disease or an infectious disease in a subject, the method comprising administering the binding molecule of any one of claims 1 to 56, or the antibody or antigen binding fragment or molecule of any one of claims 57 to 65, the nucleic acid of claim 66, the expression vector of claim 67 or the cell of claim 68, or the pharmaceutical composition of claim 70 to the subject.

74. A method of producing a binding molecule comprising a TCR mimic (TCRm) that specifically binds to a disease associated peptide-human leukocyte antigen complex (DA-pHLA), wherein the TCRm comprises an antibody or an antigen-binding fragment thereof, and wherein the method comprises:

(ii) binds to the DA-pHLA with 4 or more peptide residue binding contacts; and

(iii) binds to the pHLA complex with an affinity of 20 nM or stronger.

75. The method of claim 74, comprising, subsequent to step (a) and prior to step (b), screening the plurality of candidate binding molecules to determine the (i) DA-pHLA crossing angle, (ii) DA- pHLA residue binding contacts, and (iii) binding affinity for the DA-pHLA, for the TCRm of each of the plurality of candidate binding molecules.

76. The method of claim 75, wherein screening the plurality of candidate binding molecules for their (i) DA-pHLA crossing angle and/or (ii) DA-pHLA residue binding contacts is based on a three- dimensional atomic structure of the complex formed by the DA-pHLA and the TCRm of each of the candidate binding molecules.

77. The method of any one of claims 74 to 76, wherein the method comprises determining the three-dimensional atomic structure of the complex formed by the DA-pHLA and the TCRm of each of the candidate binding molecules.

78. The method of any one of claims 74 to 77, wherein the three-dimensional atomic structure is an x-ray crystal structure.

79. The method of any one of claims 74 to 78, wherein the TCRm identified in step (b) does not detectably bind to DA-pHLA negative cells.

80. The method of any one of claims 74 to 79, wherein the method further comprises recombinantly expressing the binding molecule identified in step (b) in a host cell and subsequently purifying the binding molecule.

81. The method of any one of claims 74 to 80, wherein the method further comprises formulating the binding molecule with one or more pharmaceutically acceptable excipients, thereby providing a pharmaceutical formulation comprising the binding molecule.

82. The method of any one of claims 74 to 81 , wherein the binding molecule has any one or more features defined in any one of claims 1 to 56.

83. A binding molecule produced according to the method of any one of claims 74 to 82.