Binding molecules for PRAME peptide-HLA complexes

Abstract

The present invention relates to binding molecules comprising a T cell receptor (TCR) variable domain capable of binding to a PRAME peptide-HLA complex. In particular, the present invention relates to binding molecules that bind to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24. The present invention also relates to the use of such molecules for the treatment of malignant diseases.

Description

[Technical Field]

[0001] The present invention relates to binding molecules comprising a T cell receptor (TCR) variable domain capable of binding to a PRAME peptide-HLA complex. The present invention also relates to the use of such molecules for the treatment of malignant diseases. [Background technology]

[0002] T cell receptor (TCR) is a receptor that binds to CD4 + T cells and CD8 + TCRs are naturally expressed by T cells. They are designed to recognize short peptide antigens presented on the surface of antigen-presenting cells in complex with major histocompatibility complex (MHC) molecules (in humans, MHC molecules are also known as human leukocyte antigens, or HLA) (Non-Patent Document 1). CD8, also known as cytotoxic T cells, + T cells have TCRs that specifically recognize peptides bound to MHC class I molecules. CD8 + T cells generally play a role in identifying and mediating the destruction of diseased cells, including cancerous and virus-infected cells. As a result of thymic selection, cancer-specific TCRs in the natural repertoire typically have low affinity for their corresponding antigens, meaning that cancerous cells often escape detection and destruction. Novel immunotherapeutic approaches aimed at promoting cancer recognition by T cells offer highly promising strategies for the development of effective anticancer treatments.

[0003] PRAME, or "Preferentially Expressed Antigen In Melanoma," was first identified as an antigen overexpressed in melanoma (Non-Patent Document 2). PRAME is also known as CT130, MAPE, and OIP-4, and its Uniprot accession number is P78395. This protein functions as a repressor of retinoic acid receptor signaling (Non-Patent Document 3). PRAME belongs to a family of germline-encoded antigens known as cancer-testis antigens. Cancer-testis antigens are attractive targets for immunotherapeutic intervention because they typically have limited or no expression in normal adult tissues. PRAME is expressed not only in leukemias and lymphomas but also in many solid tumors (Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6; Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9; Non-Patent Document 10; Non-Patent Document 11). The PRAME targeted therapy of the present invention may be particularly suitable for the treatment of cancers including, but not limited to, melanoma (skin and uveal), lung cancer (NSCLC and SCLC), breast cancer (including triple negative), ovarian cancer, endometrial cancer, esophageal cancer, bladder cancer, and head and neck cancer.

[0004] HLA-A24 has an α chain of HLA-A * It is an HLA serotype encoded by 24 alleles, with the β chain encoded by the β2-microglobulin locus. * There are 24 alleles, but the HLA-A24 serotype is A * 24:02 is dominant. Therefore, A * 24:02 is often A * In this regard, "HLA-A24" is used herein synonymously with "HLA-A24." * 24:02." HLA-A24(A * 24:02) gene frequency is HLA-A2(A * 02:01) and is relatively higher in certain populations.

[0005] The peptide PYLGQMINL (SEQ ID NO: 1) corresponds to amino acids 254-262 of the full-length PRAME protein (UniProt identification number: P78395) and is identified as HLA-A24 (i.e., "HLA-A * 24:02", which is "HLA-A * 24) and presented on the cell surface. This peptide-HLA complex provides a useful target for TCR-based immunotherapeutic intervention.

[0006] Identifying specific TCR sequences that bind with high affinity and specificity to PYLGQMINL (SEQ ID NO: 1) complexed with the HLA-A24 complex would be advantageous for the development of novel immunotherapies. Therapeutic TCRs can be used, for example, as soluble targeting agents to deliver cytotoxic agents to tumor sites or to activate immune effector functions against tumor cells (Non-Patent Document 12, Non-Patent Document 13, Non-Patent Document 14), or they can be used to engineer T cells for adoptive therapy (Non-Patent Document 15).

[0007] TCRs that bind to PYLGQMINL (SEQ ID NO: 1) in complex with HLA-A24 have been previously reported (Patent Documents 1 and 2), but these TCRs have not been engineered or characterized for use as therapeutic TCRs. As further explained herein, the production of engineered TCRs with high affinity, particularly in balance with other desirable characteristics, is not trivial and is typically associated with high attrition rates.

[0008] Therefore, the development of new immunotherapies requires binding molecules such as TCRs that can bind with high affinity and specificity to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24.

[0009] First, a person skilled in the art must identify an appropriate starting sequence, i.e., a scaffold sequence. Typically, such a sequence can be obtained from a natural source, for example, antigen-responsive T cells extracted from donor blood. Given that cancer-specific T cells are rare in the natural repertoire, it is often necessary to screen a large number of donors, for example, 20 or more donors, before a responsive T cell can be found. The screening process can take weeks or months, and even if a responsive T cell is found, it may not be suitable for immunotherapeutic use. For example, the response may be too weak and / or not specific to the target antigen. On the other hand, it may not be possible to create a clonal T cell population or to expand or maintain a given T cell lineage to generate enough material to identify the correct TCR chain sequence. A TCR sequence suitable as a starting sequence, or scaffold sequence, should have one or more of the following properties: good affinity for the target peptide-HLA complex, e.g., 200 μM or stronger; a high level of target specificity, e.g., relatively weak or no binding to other peptide-HLA complexes; suitability for use in display libraries such as phage display; ability to be refolded and purified in high yield; and maintenance of stability in purified form. Given the degenerate nature of TCR recognition, it is extremely difficult even for those skilled in the art to determine whether a particular scaffold TCR sequence has a suitable specificity profile to engineer for therapeutic use (Non-Patent Document 16).

[0010] The next challenge is to engineer TCRs to have higher affinity for target antigens while maintaining desired characteristics such as specificity and yield. Naturally occurring TCRs have weaker affinity (low micromolar range) for target antigens compared to antibodies, and TCRs directed against cancer antigens typically have weaker antigen recognition than virus-specific TCRs (Non-Patent Document 17). This weak affinity, coupled with HLA downregulation on cancer cells, means that therapeutic TCRs for cancer immunotherapy typically require engineering to increase their affinity for target antigens and thus generate stronger responses. Such increased affinity is essential for soluble TCR-based reagents. In such cases, antigen-binding affinities in the nanomolar to picomolar range, along with binding half-lives of several hours, are desirable. The improved efficacy caused by high-affinity antigen recognition with a low epitope number is illustrated in Figures 1e and 1f of Liddy et al. (Non-Patent Document 14). The affinity maturation process typically requires that a skilled artisan must engineer specific mutations and / or combinations of mutations, including but not limited to substitutions, insertions, and / or deletions, into the starting TCR sequence to enhance the strength of antigen recognition. Affinity maturation techniques, such as the use of display libraries (Non-Patent Document 18, Non-Patent Document 19), are known in the art. However, to produce a significant increase in the affinity of a given TCR for a given target, a skilled artisan may have to engineer combinations of mutations from a large pool of possible options. The specific mutations that result in a significant increase in affinity are unpredictable and exhibit high attrition rates. In many cases, a significant increase in affinity may not be achievable for a given TCR starting sequence.

[0011] The affinity maturation process must also take into account the need to maintain TCR antigen specificity. Increasing the affinity of a TCR for its target antigen increases the risk of cross-reactivity with other unintended targets as a result of the inherent degeneracy of TCR antigen recognition (Non-Patent Document 16, Non-Patent Document 20, Non-Patent Document 21). At natural affinity levels, recognition of cross-reactive antigens may be too low to trigger a response. When cross-reactive antigens are presented on normal, healthy cells, off-target binding is likely in vivo, which may manifest as clinical toxicity. Therefore, in addition to increasing antigen binding strength, those skilled in the art must also engineer mutations and / or combinations of mutations that enable the TCR to maintain high specificity for the target antigen and exhibit a favorable safety profile in preclinical trials. Again, appropriate mutations and / or combinations of mutations cannot be predicted. Attrition rates at this stage are even higher and, in many cases, may not be achievable at all from a given TCR starting sequence. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] Chinese Patent Application Publication No. 106699874 [Patent Document 2] Chinese Patent Application Publication No. 106831978 [Non-patent literature]

[0013] [Non-Patent Document 1] Davis et al., Annu Rev Immunol. 1998;16:523-44 [Non-patent document 2] Ikeda et al Immunity. 1997 Feb; 6(2):199-208 [Non-patent document 3] Epping et al., Cell. 2005 Sep 23;122(6):835-47 [Non-patent document 4] Doolan et al Breast Cancer Res Treat. 2008 May; 109(2):359-65

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Non-Patent Document 21

Summary of the Invention

[0014] As further described below, the present invention provides, for the first time, binding molecules comprising a TCR suitable for immunotherapeutic use against PRAME and HLA-A24-positive tumor cells. Many TCR immunotherapies currently under development target HLA-A2-positive cells. Thus, the present invention offers the potential to expand the scope of TCR therapy and treat diverse patient populations that would not otherwise benefit from therapies targeting HLA-A2 tumor cells. Also, as further described below, identifying the binding molecules of the present invention required an extensive engineering effort exploring over 12 TCR affinity maturation series, none of which was trivial. Even after selecting specific affinity maturation series for further study, multiple additional rounds of affinity maturation and removal of risky deamidated residues were performed. Subsequently, further stability enhancements were engineered into the molecule, all of which maintained adequate potency and affinity. These stability improvements are advantageous for enabling TCRs to be used in vivo, particularly in patients, and are also important for producing and using such TCRs.

[0015] binding molecule In a first aspect, the present invention provides a binding molecule comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the binding molecule has the property of binding to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24, wherein each of the alpha chain variable domain and the beta chain variable domain comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FRs are framework regions and CDRs are complementarity determining regions, and wherein: (a) when the binding molecule binds to PYLGQMINL (SEQ ID NO: 1) in a complex with HLA-A24, the binding molecule contacts residue P1, residue Q5, residue I7, and residue N8 of the PYLGQMINL (SEQ ID NO: 1) peptide; and / or (b) A binding molecule is provided in which the CDR3 of the α chain comprises the sequence XXXXPN / HR / HXXXXX (SEQ ID NO: 125), the CDR1 of the β chain comprises the sequence XXXL / YX (SEQ ID NO: 126), the CDR2 of the β chain comprises the sequence XYXXXX (SEQ ID NO: 127), and the CDR3 of the β chain comprises the sequence XXXV / IWSS / I / NGXXSA / SXXXX (SEQ ID NO: 128), where X is any amino acid.

[0016] The above-referenced SEQ ID NOs: 125 to 128 are consensus sequences based on the inventors' identification of the key CDR residues that contact the target peptide when the binding molecule binds to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24, as described in Example 7. These sequences are shown below in the section entitled "Sequence Description."

[0017] Surprisingly, despite the above-mentioned drawbacks, the present inventors have identified binding molecules comprising TCR variable domains with particularly high affinity (picomolar range) and high antigen specificity for the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex. When prepared as soluble reagents fused to a T cell redirecting moiety, the molecules exhibit potent killing of PRAME-positive cancer cells. Thus, the molecules of the present invention have a profile particularly suitable for therapeutic use. Certain binding molecules of the present invention were engineered from a suitable scaffold (e.g., "wild-type") TCR sequence into which multiple mutations were introduced to enhance affinity and / or stability while maintaining high specificity.

[0018] In a second aspect, the present invention provides a binding molecule comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the binding molecule has the property of binding to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24, wherein each of the alpha chain variable domain and the beta chain variable domain comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FRs are framework regions and CDRs are complementarity determining regions, and wherein: (a) the α chain CDRs have the following sequences: CDR1 - SSYSPS (SEQ ID NO: 5), optionally with 1, 2 or 3 mutations; CDR2 - YIGNVTLV (SEQ ID NO: 27), optionally with 1, 2, 3, or 4 mutations; CDR3 - VVGAPHHNDKII (SEQ ID NO: 30), optionally with 1, 2, 3, or 4 mutations; and / or (b) the CDRs of the β chain have the following sequences: CDR1 - SGDYS (SEQ ID NO: 32), optionally with 1, 2 or 3 mutations; CDR2 - YYNAEE (SEQ ID NO: 35), optionally with 1, 2 or 3 mutations; CDR3 - ASSIWSIGGASSGNLS (SEQ ID NO: 41), optionally with 1, 2, 3, 4, or 5 mutations; The present invention provides a binding molecule having the formula:

[0019] In a third aspect, the present invention provides a binding molecule comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the binding molecule has the property of binding to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24, wherein each of the alpha chain variable domain and the beta chain variable domain comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FRs are framework regions and CDRs are complementarity determining regions, and wherein: (a) the α chain CDRs have the following sequences: CDR1 - SSYSPS (SEQ ID NO: 5), optionally with 1, 2 or 3 mutations; CDR2 - YTSAATLV (SEQ ID NO: 6), optionally with 1, 2, 3, or 4 mutations; CDR3 - VVSAPNRDDKII (SEQ ID NO: 7), optionally with 1, 2, 3 or 4 mutations; and / or (b) the CDRs of the β chain have the following sequences: CDR1 - SGDLS (SEQ ID NO: 15), optionally with 1, 2 or 3 mutations; CDR2 - YYNGEE (SEQ ID NO: 16), optionally with 1, 2 or 3 mutations; CDR3 - ASSVWSSGGASAGELF (SEQ ID NO: 17), optionally with 1, 2, 3, 4, or 5 mutations; The present invention provides a binding molecule having the formula:

[0020] In a fourth aspect, the present invention provides a binding molecule that specifically binds to the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex, comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein each variable domain comprises three complementarity determining regions, termed CDR1, CDR2 and CDR3, wherein: (a) when the binding molecule binds to the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex, the binding molecule contacts residue P1, residue Q5, residue I7, and residue N8 of the PYLGQMINL (SEQ ID NO: 1) peptide; and / or (b) A binding molecule is provided in which the CDR3 of the α chain comprises XXXXPN / HR / HXXXXX (SEQ ID NO: 125), the CDR1 of the β chain comprises XXXL / YX (SEQ ID NO: 126), the CDR2 of the β chain comprises XYXXXX (SEQ ID NO: 127), and the CDR3 of the β chain comprises XXXV / IWSS / I / NGXXSA / SXXXX (SEQ ID NO: 128), where X is any amino acid.

[0021] References herein to a "binding molecule" or a "binding molecule of the invention" relate to the binding molecules of the first, second, third and fourth aspects above, respectively, unless expressly indicated otherwise.

[0022] As used herein, the term "binding molecule" generally refers to a molecule capable of binding to a target antigen. The binding molecules of the present invention comprise a TCR α chain variable domain and a TCR β chain variable domain, which associate with each other to form a TCR binding site capable of binding to the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex. As used herein, the phrase "binding to the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex" is used interchangeably with "binding to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24." The binding molecules of the present invention can take many different forms, as discussed herein. Furthermore, fragments of the binding molecules of the present invention are also contemplated. A fragment refers to a portion of a binding molecule that maintains binding to a target antigen.

[0023] The binding molecules of the present invention comprise a TCR variable domain that may correspond to a TCR variable domain derived from a native TCR, or more preferably, the TCR variable domain may be engineered (i.e., may contain mutations relative to the native sequence). A native TCR variable domain may also be referred to as a wild-type domain, natural domain, parent domain, non-mutated domain, or scaffold domain. The binding molecule may have ideal therapeutic properties, such as supraphysiological affinity for the target, a long binding half-life, high specificity for the target, and good stability. The present invention also includes multispecific (e.g., bispecific) molecules, or multifunctional (e.g., bifunctional) molecules, or fusion molecules comprising the TCR variable domains described herein and, for example, a T cell-redirecting moiety. Such molecules can mediate potent and specific responses against PRAME-positive cancer cells by redirecting and activating T cells. Furthermore, the use of binding molecules with supraphysiological affinity facilitates the recognition of cancer cells that present low levels of target peptide-HLA complexes. Alternatively, the binding molecules may further comprise (e.g., by fusion) other therapeutic and / or diagnostic agents and / or may be incorporated into engineered T cells for adoptive therapy.

[0024] The binding molecule may comprise or consist of a TCR comprising a TCR α chain variable domain and a TCR β chain variable domain. The TCR may be a soluble TCR, i.e., a TCR that does not comprise a transmembrane domain and does not comprise an intracellular / cytoplasmic domain. The TCR domain sequence may be defined in accordance with the IMGT nomenclature, which is widely known and accessible to those skilled in the art of TCRs. See, for example, LeFranc and LeFranc, (2001). "T cell Receptor Factsbook", Academic Press, Lefranc, (2011), Cold Spring Harb Protoc 2011(6): 595-603; Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 100; and Lefranc, (2003), Leukemia 17(1): 260-266. Briefly, the αβ TCR consists of two disulfide-linked chains. Each chain (α and β) is generally considered to have two domains: a variable domain and a constant domain. A short connecting region connects the variable and constant domains, which is typically considered part of the α variable region. In addition, the β chain usually contains a short diversity region adjacent to the connecting region, which is also typically considered part of the β variable region. The variable domain of each chain is located at the N-terminus and contains three complementarity-determining regions (CDRs) embedded in framework sequences (FR). The CDRs contain recognition sites for peptide-MHC binding. There are several genes encoding α chain variable (Vα) regions and several genes encoding β chain variable (Vβ) regions, which are distinguished by their framework, CDR1 and CDR2 sequences, and by a partially defined CDR3 sequence.The Vα and Vβ genes are designated by the prefixes TRAV and TRBV, respectively, in the IMGT nomenclature (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1): 42-54; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2): 83-96; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). Similarly, for the α and β chains, there are several joining or J genes, designated TRAJ or TRBJ, respectively, and for the β chain there is a diversity or D gene, designated TRBD (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(2): 107-114; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2): 97-106; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). The great diversity of T cell receptor chains arises from combinatorial rearrangements among various V, J, and D genes, including allelic variants and junctional diversity (Arstila, et al., (1999), Science 286(5441): 958-961; Robins et al., (2009), Blood 114(19): 4099-4107.). The constant or C regions of the TCR α and β chains are referred to as TRAC and TRBC, respectively (Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10).

[0025] Certain binding molecules of the invention preferably have a K for the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex that is greater (i.e., stronger) than the native TCR (also referred to as the non-mutated TCR or scaffold TCR). D K DThe binding molecules of the invention have a K for the target complex of about (i.e., ±10%) 1 pM to about 400 nM, about 1 pM to about 1000 pM, about 1 pM to about 500 pM, or about 1 pM to about 100 pM. D The binding molecule may additionally or alternatively have a binding half-life (T) for the complex within the range of about 0.5 minutes to about 50 hours, about 20 minutes to about 30 hours, or about 20 minutes to about 25 hours. 1 / 2 Preferably, the binding molecules of the invention may have a K for the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex of about 1 pM to about 500 pM. D and / or have a binding half-life of about 20 minutes to about 25 hours. Such high affinity is preferred for a soluble form of the binding molecule when associated with a therapeutic agent and / or a detectable label. The affinity of the binding molecule can be measured at 25°C. Methods for determining the affinity of a binding molecule are described herein.

[0026] Binding molecules of the invention comprising native TCR variable domains have a K for the complex of about 1 μM to about 200 μM, or about 1 μM to about 100 μM. D Such binding molecules may be preferred for adoptive therapy applications.

[0027] As described in the Examples herein below, the methionine at position 6 of the PYLGQMINL (SEQ ID NO: 1) peptide can exist in an oxidized or reduced form. The inventors have detected both forms in cancer cell lines and tumor tissues. The TCRs of the present invention can bind to both forms. The binding affinity of the binding molecules of the present invention can be evaluated using either form. Binding to the reduced form of PYLGQMINL (SEQ ID NO: 1) may be stronger. Therefore, the binding affinity of the binding molecules of the present invention can be evaluated using the reduced form of PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24.

[0028] Certain preferred mutated binding molecules have a binding affinity and / or binding half-life for the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex that is significantly higher than that of the native TCR. Increasing the binding affinity of the native TCR can reduce the specificity of the TCR for its peptide-MHC ligand, as demonstrated in Non-Patent Document 21. However, the binding molecules of the present invention surprisingly exhibit a high level of specificity for the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex despite having a binding affinity that is substantially higher than that of the native TCR.

[0029] The binding molecules of the present invention preferably have the property of specifically binding to the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex. As used herein, "specific" binding refers to a binding molecule that binds to the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex with higher affinity than other peptide-HLA complexes. Highly specific binding molecules of the present invention are particularly suitable for therapeutic use due to a reduced risk of off-target effects. Specificity in the context of binding molecules of the present invention can be determined according to their ability to recognize antigen-positive target cells while having minimal ability to recognize antigen-negative target cells, and / or to bind to peptides that are similar in sequence to the target peptide but differ by up to three amino acids.

[0030] Specificity can be measured in vitro, for example, in a cellular assay such as an ELISpot assay as described in Examples 3, 5, and 6. To test specificity, the binding molecule may be in soluble form, associated with immune effectors, and / or expressed on the surface of cells such as T cells. Specificity can 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 that is less than 20%, preferably less than 10%, preferably less than 5%, more preferably less than 1% of the level achieved in the presence of antigen-positive target cells, when measured under the same conditions (i.e., using the same lot of target and effector cells) and at a therapeutically relevant TCR concentration. In the case of soluble TCRs associated with immune effectors, a therapeutically relevant concentration is less than 10%. -9 Concentrations below M and / or the corresponding EC 50 Value or IC 50 Preferably, in the case of soluble binding molecules associated with immune effectors, the EC 50 Value or IC 50 There may be at least a 10-fold difference, at least a 100-fold difference, at least a 1000-fold difference, or at least a 10,000-fold difference in values, which may be referred to as the therapeutic window. Additionally or alternatively, the therapeutic window may be calculated based on the lowest effective concentration ("LOEL") observed for normal cells and infected cells. The antigen-positive cells may be obtained by peptide pulsing using a peptide concentration appropriate to obtain an antigen presentation level comparable to wild-type peptide presentation, or may naturally present the peptide. Preferably, both the antigen-positive and antigen-negative cells are human cells.

[0031] Additionally or alternatively, specificity may relate to the ability of a binding molecule to bind to the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex and not to a panel of other peptide-HLA complexes. Specificity may be determined, for example, by surface plasmon resonance (SPR) as described in Example 1. The panel may include at least two, at least three, at least five, or at least ten other peptide-HLA complexes. The other peptides may share a low level of sequence identity with SEQ ID NO: 1 and may be naturally or artificially presented. The other peptides are preferably derived from commonly expressed proteins and / or proteins expressed in healthy human tissues. The specific binding of a binding molecule to the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex may be at least 2-fold higher, more preferably at least 10-fold, or at least 50-fold or at least 100-fold higher, and even more preferably at least 1000-fold higher than other naturally or artificially presented peptide-HLA complexes. Naturally occurring variants of the PYLGQMINL (SEQ ID NO: 1) peptide may be excluded from the definition of alternative peptide-HLA complexes.

[0032] An alternative or additional approach to determining the specificity of a binding molecule can be to identify the peptide recognition motif of the binding molecule using serial mutagenesis of the target peptide, e.g., alanine scanning. Residues that form part of the binding motif are intolerant to substitution. An intolerant substitution can be defined as a position in the peptide where the binding affinity of the binding molecule is reduced by at least 50%, or preferably at least 80%, compared 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): 197ral03 and WO 2014 / 096803. In this case, the specificity of the binding molecule can be determined by identifying additional motif-containing peptides, particularly those within the human proteome, and testing these peptides for binding to the binding molecule. Binding of the binding molecule to one or more additional peptides can indicate a lack of specificity. In this case, further testing of the specificity of the binding molecule via a cellular assay may be necessary. The low tolerance for (alanine) substitutions in the central part of the peptide indicates that the TCR has high specificity and therefore presents a low risk of cross-reactivity with other peptides.

[0033] A binding molecule having the property of specifically binding to the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex may bind to this complex with higher affinity than other peptide-HLA-A24 complexes. The binding molecule may bind to the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex with an affinity that is at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, or at least 1000-fold higher than the affinity to the PYTGQQISL (SEQ ID NO: 81)-HLA-A24 complex and / or the PYLGQAPFL (SEQ ID NO: 82)-HLA-A24 complex and / or the PYLSTMINY (SEQ ID NO: 83)-HLA-A24 and / or the PYLGSKISL (SEQ ID NO: 84)-HLA-A24 complex. These alternative peptides are referred to herein in Example 1 as Mimetic 1 ("Mim1"), Mimetic 2 ("Mim2"), Mimetic 3 ("Mim3"), and Mimetic 4 ("Mim4"), respectively. The binding molecule may bind to the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex with an affinity that is at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, or at least 1000-fold greater than its affinity for the PYLGQAPFL (SEQ ID NO: 82)-HLA-A24 complex and / or the PYLGSKISL (SEQ ID NO: 84)-HLA-A24 complex.

[0034] Certain binding molecules of the invention can generate very strong T cell responses in vitro against antigen-positive cells, particularly cells that present antigen at low levels (i.e., on the order of 5-100). Such binding molecules may be in soluble form and linked to immune effectors such as anti-CD3 antibodies. The T cell response measured can be the release of T cell activation markers such as interferon-γ or granzyme B, or other measures of T cell activation such as target cell killing or T cell proliferation. Preferably, a very strong response will have an EC50 in the pM range, i.e., an EC50 of 1000 pM or less. 50 It has value.

[0035] The term "mutation" encompasses designed substitutions, insertions, and deletions (e.g., engineered or designed substitutions, insertions, and deletions). Mutations to a native (also referred to as parent, natural, non-mutated, wild-type, or scaffold) binding molecule may confer beneficial therapeutic properties, such as higher affinity, higher stability, higher specificity, and / or higher potency. For example, mutations may include those that increase the binding affinity (k D ) and / or binding half-life (T 1 / 2 ) can be exemplified.

[0036] The term "stability" in the context of the present invention refers to physical stability and chemical stability, and can be assessed qualitatively and / or quantitatively using a variety of analytical techniques described in the art and reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Such methods include assessing aggregate formation (e.g., using size exclusion chromatography (SEC)), by measuring turbidity (e.g., by dynamic light scattering (DLS) or light obscuration (LO)), and / or by visual inspection (e.g., by determining color and clarity).

[0037] A binding molecule of the invention may contact at least seven or at least eight peptide residues when bound to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24. As used herein, a binding "contact" refers to an (e.g., non-covalent) interaction between an atom of one molecule and an atom of another molecule when the two molecules are bound to each other. For example, a binding "contact" of a peptide residue is a binding interaction formed between an amino acid residue of the PYLGQMINL (SEQ ID NO: 1) peptide, but not HLA, and an amino acid residue of the binding molecule. A binding molecule may contact at least residues P1, Q5, I7, and N8 of the PYLGQMINL (SEQ ID NO: 1) peptide when bound to the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex. A binding molecule may contact all of the peptide residues at positions 1 through 8 of the PYLGQMINL (SEQ ID NO: 1) peptide when bound to the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex. Binding contacts can be identified using any method known in the art, including X-ray crystallography and structural modeling as described herein.

[0038] Certain angles can be used to define the binding geometry of the interaction between a binding molecule of the present invention and the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex. For example, a binding molecule may bind to the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex at a crossing angle in the range of 35° to 55°, or preferably in the range of 38° to 48°. A binding molecule may bind to the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex at a tilt angle in the range of -10° to 10°, preferably in the range of -1° to 9°. A binding molecule may bind to the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex at a roll angle in the range of -10° to 10°, preferably in the range of -6° to 4°. The binding geometry of a binding molecule, such as the crossing angle, tilt angle, and roll angle, can be determined using any method known in the art, including X-ray crystallography and structural modeling, as described herein. For example, methods for calculating these angles are described in Rudolph et al. (2006). Annu Rev Immunol. 24, 419-466.

[0039] In the binding molecules of the invention, there may be at least one mutation in the TCR α chain variable region. There may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations in the α chain CDRs (i.e., across all three CDRs in total). For example, there may be 8 mutations in the α chain CDRs. There may be no mutations in CDR1 of the α chain, and / or 4 mutations in CDR2 of the α chain, and / or 4 mutations in CDR3 of the α chain.

[0040] The binding molecule of the first aspect of the invention may comprise an alpha chain CDR3 comprising the sequence of any one of SEQ ID NO:125, SEQ ID NO:133, SEQ ID NO:134 or SEQ ID NO:135.

[0041] The binding molecule of the first aspect of the invention may comprise a CDR1 of the β chain comprising the sequence of any one of SEQ ID NO:126, SEQ ID NO:136 or SEQ ID NO:137.

[0042] The binding molecule of the first aspect of the invention may comprise a CDR2 of the β chain comprising the sequence of SEQ ID NO:127 or SEQ ID NO:138.

[0043] The binding molecule of the first aspect of the invention may comprise a CDR3 of the β chain comprising the sequence of any one of SEQ ID NO:128, SEQ ID NO:139, SEQ ID NO:140 or SEQ ID NO:141.

[0044] The binding molecule of the first aspect of the invention comprises: CDR3 of the alpha chain comprising the sequence XXS / GXPN / HR / HD / NXXXX (SEQ ID NO: 133); CDR1 of the β chain comprising the sequence S / TG / AD / EL / YS / T (SEQ ID NO: 136); CDR2 of the β chain comprising the sequence Y / W / FYN / QG / AE / DE / D (SEQ ID NO: 138); CDR3 of the beta chain comprising the sequence XXXV / IWSS / I / NGXXSA / SXE / NXF / S (SEQ ID NO: 139); may include:

[0045] The binding molecule of the first aspect of the invention comprises: and an alpha chain CDR3 comprising the sequence V / I / LV / I / LS / GA / GPN / HR / HD / ND / EK / R / HI / V / LI / V / L (SEQ ID NO: 134); CDR1 of the β chain comprising the sequence S / TG / AD / EL / YS / T (SEQ ID NO: 136); CDR2 of the β chain comprising the sequence Y / W / FYN / QG / AE / DE / D (SEQ ID NO: 138); CDR3 of the beta chain comprising the sequence A / GS / TS / TV / IWSS / I / NGG / AA / GSA / SG / AE / NL / I / VF / S (SEQ ID NO: 140); and may include:

[0046] The binding molecule of the first aspect of the invention comprises: The CDR3 of the alpha chain comprises the sequence V / I / LV / I / LGA / GPHHND / EK / R / HI / V / LI / V / L (SEQ ID NO: 135); CDR1 of the β chain comprising the sequence S / TG / AD / EYS / T (SEQ ID NO: 137); CDR2 of the β chain comprising the sequence Y / W / FYN / QG / AE / DE / D (SEQ ID NO: 138); CDR3 of the β chain comprising the sequence A / GS / TS / TIWSIGG / AA / GSSG / ANL / I / VS (SEQ ID NO: 141); may include:

[0047] The binding molecule of the first aspect of the invention comprises: CDR1 of the alpha chain comprising the sequence S / TS / TY / W / FS / TPS / T (SEQ ID NO: 130); CDR2 of the alpha chain comprising the sequence Y / W / FIGNVT / SL / I / VV / I / L (SEQ ID NO: 132); The CDR3 of the alpha chain comprises the sequence V / I / LV / I / LGA / GPHHND / EK / R / HI / V / LI / V / L (SEQ ID NO: 135); CDR1 of the β chain comprising the sequence S / TG / AD / EYS / T (SEQ ID NO: 137); CDR2 of the β chain comprising the sequence Y / W / FYN / QG / AE / DE / D (SEQ ID NO: 138); CDR3 of the β chain comprising the sequence A / GS / TS / TIWSIGG / AA / GSSG / ANL / I / VS (SEQ ID NO: 141); may include:

[0048] The binding molecule of the first aspect of the invention comprises: an alpha chain CDR3 comprising the sequence VVGAPHHNDKII (SEQ ID NO: 30), optionally with one, two, three, or four mutations at any of positions 1 to 4 or positions 8 to 12 of SEQ ID NO: 30 (i.e., these mutations exclude P5, H6, and H7 numbered according to SEQ ID NO: 30); and a CDR1 of the β chain comprising the sequence SGDYS (SEQ ID NO: 32), optionally with one, two, or three mutations at any of positions 1 to 3 or position 5 of SEQ ID NO: 32 (i.e., these mutations exclude Y5 numbered according to SEQ ID NO: 32); a CDR2 of the β chain comprising the sequence YYNAEE (SEQ ID NO: 35), optionally with one, two, or three mutations at position 1 or any of positions 3 to 6 of SEQ ID NO: 35 (i.e., these mutations exclude Y2, which are numbered according to SEQ ID NO: 35); a CDR3 of the β chain comprising the sequence ASSIWSIGGASSGNLS (SEQ ID NO: 41), optionally with 1, 2, 3, 4, or 5 mutations at any of positions 1 to 3, 9, 10, or 13 to 16 of SEQ ID NO: 41 (i.e., these mutations exclude I4, W5, S6, I7, G8, S11, and S12 numbered according to SEQ ID NO: 41); may include:

[0049] The binding molecule of the first aspect of the invention comprises: an alpha chain CDR3 comprising the sequence VVGAPHHNDKII (SEQ ID NO: 30), optionally with one, two, or three mutations at any of positions 1 to 4 or positions 8 to 12 of SEQ ID NO: 30 (i.e., these mutations exclude P5, H6, and H7 numbered according to SEQ ID NO: 30); a CDR1 of the β chain comprising the sequence SGDYS (SEQ ID NO: 32), optionally with one or two mutations at any of positions 1 to 3 or position 5 of SEQ ID NO: 32 (i.e., these mutations exclude Y5 numbered according to SEQ ID NO: 32); a CDR2 of the β chain comprising the sequence YYNAEE (SEQ ID NO: 35), optionally with one or two mutations at position 1 or any of positions 3 to 6 of SEQ ID NO: 35 (i.e., these mutations exclude Y2, which are numbered according to SEQ ID NO: 35); a CDR3 of the β chain comprising the sequence ASSIWSIGGASSGNLS (SEQ ID NO: 41), optionally with one, two, or three mutations at any of positions 1 to 3, 9, 10, or 13 to 16 of SEQ ID NO: 41 (i.e., these mutations exclude I4, W5, S6, I7, G8, S11, and S12 numbered according to SEQ ID NO: 41); may include:

[0050] The binding molecule of the first aspect of the invention comprises: an alpha chain CDR3 comprising the sequence VVGAPHHNDKII (SEQ ID NO: 30), optionally with one or two mutations at positions 1 to 4 or positions 8 to 12 of SEQ ID NO: 30 (i.e., these mutations exclude P5, H6, and H7 numbered according to SEQ ID NO: 30); CDR1 of the β chain comprising the sequence SGDYS (SEQ ID NO: 32), optionally with one mutation at any of positions 1 to 3 or 5 of SEQ ID NO: 32 (i.e., these mutations exclude Y5 numbered according to SEQ ID NO: 32); a CDR2 of the β chain comprising the sequence YYNAEE (SEQ ID NO: 35), optionally with one mutation at position 1 or any of positions 3 to 6 of SEQ ID NO: 35 (i.e., these mutations exclude Y2, which are numbered according to SEQ ID NO: 35); a CDR3 of the β chain comprising the sequence ASSIWSIGGASSGNLS (SEQ ID NO: 41), optionally with one or two mutations at positions 1 to 3, 9, 10, or 13 to 16 of SEQ ID NO: 41 (i.e., these mutations exclude I4, W5, S6, I7, G8, S11, and S12 numbered according to SEQ ID NO: 41); may include:

[0051] The binding molecule of the first aspect of the invention comprises: an alpha chain CDR3 comprising the sequence VVGAPHHNDKII (SEQ ID NO: 30), optionally with one mutation at any of positions 1 to 4 or positions 8 to 12 of SEQ ID NO: 30 (i.e., these mutations exclude P5, H6, and H7 numbered according to SEQ ID NO: 30); CDR1 of the β chain comprising the sequence SGDYS (SEQ ID NO: 32), optionally with one mutation at any of positions 1 to 3 or 5 of SEQ ID NO: 32 (i.e., these mutations exclude Y5 numbered according to SEQ ID NO: 32); a CDR2 of the β chain comprising the sequence YYNAEE (SEQ ID NO: 35), optionally with one mutation at position 1 or any of positions 3 to 6 of SEQ ID NO: 35 (i.e., these mutations exclude Y2, which are numbered according to SEQ ID NO: 35); a CDR3 of the β chain comprising the sequence ASSIWSIGGASSGNLS (SEQ ID NO: 41), optionally with one mutation at any of positions 1 to 3, 9, 10, or 13 to 16 of SEQ ID NO: 41 (i.e., these mutations exclude I4, W5, S6, I7, G8, S11, and S12 numbered according to SEQ ID NO: 41); may include:

[0052] The binding molecule of the first aspect of the invention comprises: CDR1 of the alpha chain comprising the sequence SSYSPS (SEQ ID NO: 5), optionally with 1, 2 or 3 mutations; an alpha chain CDR2 comprising the sequence YIGNVTLV (SEQ ID NO: 27), optionally with 1, 2, 3 or 4 mutations; may include:

[0053] The binding molecule of the first aspect of the invention comprises: CDR1 of the alpha chain comprising the sequence SSYSPS (SEQ ID NO: 5), optionally with one or two mutations; CDR2 of the alpha chain comprising the sequence YIGNVTLV (SEQ ID NO: 27), optionally with one or two mutations; may include:

[0054] The binding molecule of the first aspect of the invention comprises: CDR1 of the alpha chain comprising the sequence SSYSPS (SEQ ID NO: 5), optionally with one mutation; CDR2 of the alpha chain comprising the sequence YIGNVTLV (SEQ ID NO: 27), optionally with one mutation; may include:

[0055] The binding molecule of the first aspect of the invention may comprise an alpha chain CDR1 comprising the sequence S / TS / TY / W / FS / TP / GS / T (SEQ ID NO: 129) and an alpha chain CDR2 comprising the sequence Y / W / FT / IS / GA / NA / D / VT / SL / I / VV / I / L (SEQ ID NO: 131). The binding molecule of the first aspect of the invention may comprise an alpha chain CDR1 comprising the sequence S / TS / TY / W / FS / TPS / T (SEQ ID NO: 130) and an alpha chain CDR2 comprising the sequence Y / W / FIGNVT / SL / I / VV / I / L (SEQ ID NO: 132).

[0056] In the sequences specified herein, "X" represents any amino acid. A slash (" / ") represents "or," e.g., "S / T" indicates that the amino acid at the specified position in the sequence can be S (Ser) or T (Thr).

[0057] In the binding molecule of the third aspect of the invention, the mutations in the CDRs of the alpha chain may be selected from T51I, S52G, A53N, A54V, A54D, S94G, N97H, R98H, and D99N, numbered according to SEQ ID NO: 3. Thus, any or all of these mutations may be present, optionally in combination with other mutations. In particular, the binding molecule may have the following mutations in the CDRs of the alpha chain, numbered according to SEQ ID NO: 3: (a) S94G, N97H, and D99N, (b) T51I, S52G, A53N, A54D, S94G, N97H, and D99N; (c) T51I, S52G, A53N, A54V, S94G, N97H, and D99N, or (d) T51I, S52G, A53N, A54V, S94G, N97H, R98H, and D99N; may include:

[0058] Preferred are the T51I, S52G, A53N, A54V, S94G, N97H, R98H, and D99N mutations listed in (d) above.

[0059] In the binding molecule of the second aspect of the invention, the mutations in the CDRs of the α chain may be conservative, semi-conservative, permissive, or other phenotypically silent mutations as described herein. The mutations may be selected from I51T, G52S, N53A, V54A, D54A, G94S, H97N, H98R, and N99D, numbered according to SEQ ID NO: 29. Other suitable conservative, semi-conservative, permissive, or other phenotypically silent mutations will be apparent to those skilled in the art.

[0060] The mutated α chain variable domain may be combined with any β chain variable domain defined herein.

[0061] There may be at least one mutation in the TCR β chain variable region of a binding molecule of the invention. There may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations in the CDRs of the β chain (i.e., across all three CDRs in total). For example, there may be 7 mutations in the CDRs of the β chain. There may be 1 mutation in CDR1 of the β chain and / or 1 mutation in CDR2 of the β chain and / or 5 mutations in CDR3 of the β chain.

[0062] In the binding molecule of the third aspect of the invention, the mutation(s) in the CDRs of the β-chain may be selected from L30Y, G52A, V95I, S98I, A103S, E105N, and F107S, numbered according to SEQ ID NO: 13. Thus, any or all of these mutations may be present, optionally in combination with other mutations. In particular, the binding molecule may have the following mutations in the CDRs of the β-chain, numbered according to SEQ ID NO: 13: (a) L30Y, S98N, A103S, E105N, and F107S, (b) L30Y, G52A, S98I, A103S, E105N, and F107S, or (c) L30Y, G52A, V95I, S98I, A103S, E105N, and F107S, may include:

[0063] Preferred are the L30Y, G52A, V95I, S98I, A103S, E105N, and F107S mutations listed in (c) above.

[0064] In the binding molecule of the second aspect of the present invention, the mutations in the CDRs of the β-chain may be conservative, semi-conservative, permissive, or other phenotypically silent mutations, as described herein. For example, the mutations may be selected from Y30L, A52G, I95V, I98S, S103A, N105E, and S107F, numbered according to SEQ ID NO: 40. Other suitable conservative, semi-conservative, permissive, or other phenotypically silent mutations will be apparent to those skilled in the art.

[0065] The mutated β chain variable domain may be combined with any α chain variable domain defined herein.

[0066] Mutation(s) within a CDR relative to the native sequence may improve the binding affinity or stability of a binding molecule of the invention, but may additionally or alternatively confer other benefits, such as improved specificity or improved potency, when fused to an immune effector. Mutations may also reduce the risk of destabilizing post-translational modifications, such as deamidation. Mutations at one or more positions may additionally or alternatively affect the interaction with adjacent positions and cognate pMHC complexes, for example, by providing a more favorable angle for the interaction. Mutations may include mutations that result in reduced nonspecific binding, i.e., reduced binding to other antigens compared to the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex. Mutations may include mutations that increase folding efficiency and / or stability and / or manufacturability. Some mutations may contribute to each of these attributes, while others may contribute, for example, to affinity but not specificity, or specificity but not affinity, or stability but not affinity, etc.

[0067] A total of at least three, at least five, or at least ten CDR mutations may be required compared to the wild-type sequence to obtain a binding molecule with pM affinity for a target antigen. Binding molecules with pM affinity for a target antigen are particularly suitable as soluble therapeutic agents. Binding molecules used in adoptive therapy applications may have lower affinity for the target antigen and therefore may have fewer CDR mutations, for example, a total of up to one, up to two, up to five, or more CDR mutations. In some cases, a native (also referred to as non-mutated) binding molecule may have a sufficiently high affinity for a target antigen without the need for mutations. The binding molecules of the invention in their native form also advantageously have high affinity and specificity.

[0068] The binding molecules of the invention comprise the following combinations of α-chain CDRs and β-chain CDRs: (a) the amino acid sequences of CDR1, CDR2, and CDR3 of the α chain of SSYSPS (SEQ ID NO: 5), YTSAATLV (SEQ ID NO: 6), and VVSAPNRDDKII (SEQ ID NO: 7), respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the β chain of SGDLS (SEQ ID NO: 15), YYNGEE (SEQ ID NO: 16), and ASSVWSSGGASAGELF (SEQ ID NO: 17), respectively; (b) the amino acid sequences of CDR1, CDR2, and CDR3 of the α chain of SSYSPS (SEQ ID NO: 5), YTSAATLV (SEQ ID NO: 6), and VVGAPHRNDKII (SEQ ID NO: 23), respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the β chain of SGDLS (SEQ ID NO: 15), YYNGEE (SEQ ID NO: 16), and ASSVWSSGGASAGELF (SEQ ID NO: 17), respectively; (c) the amino acid sequences of CDR1, CDR2, and CDR3 of the α chain of SSYSPS (SEQ ID NO: 5), YIGNDTLV (SEQ ID NO: 25), and VVGAPHRNDKII (SEQ ID NO: 23), respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the β chain of SGDYS (SEQ ID NO: 32), YYNGEE (SEQ ID NO: 16), and ASSVWSNGGASSGNLS (SEQ ID NO: 33), respectively; (d) the amino acid sequences of CDR1, CDR2, and CDR3 of the α chain of SSYSPS (SEQ ID NO: 5), YIGNVTLV (SEQ ID NO: 27), and VVGAPHRNDKII (SEQ ID NO: 23), respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the β chain of SGDYS (SEQ ID NO: 32), YYNAEE (SEQ ID NO: 35), and ASSVWSIGGASSGNLS (SEQ ID NO: 36), respectively; or (e) the amino acid sequences of CDR1, CDR2, and CDR3 of the α chain of SSYSPS (SEQ ID NO: 5), YIGNVTLV (SEQ ID NO: 27), and VVGAPHHNDKII (SEQ ID NO: 30), respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the β chain of SGDYS (SEQ ID NO: 32), YYNAEE (SEQ ID NO: 35), and ASSIWSIGGASSGNLS (SEQ ID NO: 41), respectively; It may include one of:

[0069] Preferably, the binding molecule comprises the amino acid sequences of CDR1, CDR2, and CDR3 of the α chain of SSYSPS (SEQ ID NO: 5), YIGNVTLV (SEQ ID NO: 27), and VVGAPHHNDKII (SEQ ID NO: 30), respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the β chain of SGDYS (SEQ ID NO: 32), YYNAEE (SEQ ID NO: 35), and ASSIWSIGGASSGNLS (SEQ ID NO: 41), respectively. These are the CDR sequences of the TCR referred to in the examples as "a90b152."

[0070] Mutations may additionally or alternatively occur within framework regions outside the CDRs, and such mutations may result in improved therapeutic properties, such as improved affinity and / or specificity and / or stability of the binding molecule and / or yield of its purified, soluble form. For example, a binding molecule of the present invention may additionally or alternatively contain one or more mutations at the N-terminus of FR1 of one of the chains compared to the classical framework sequences for a given TRAV and TRBV chain. Such mutations may improve the efficiency of N-terminal methionine cleavage. Removal of the N-terminal start methionine is often important for protein function and stability. Insufficient cleavage may be harmful for therapeutic purposes, as it may result in heterogeneous protein products and / or the presence of the start methionine may be immunogenic in humans. In some cases, an start methionine may be present in a binding molecule of the present invention.

[0071] The framework regions of the alpha chain variable domain of the binding molecules of the invention have the following sequence: FR1 - AQSVTQLDSHVSVSEGTPVLLRCNYS (SEQ ID NO: 8), optionally with 1, 2, or 3 mutations; FR2 - LFWYVQHPNKGLQLLLK (SEQ ID NO: 9), optionally with 1, 2, or 3 mutations; FR3—KGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFC (SEQ ID NO: 10), optionally with 1, 2, or 3 mutations; FR4—FGKGTRLHILP (SEQ ID NO: 11), optionally with 1, 2, or 3 mutations; and / or The framework regions of the β chain variable domain have the following sequence: FR1 - DSGVTQTPKHLITATGQRVTLRCSPR (SEQ ID NO: 18), optionally with 1, 2, or 3 mutations; FR2—VYWYQQSLDQGLQFLIQ (SEQ ID NO: 19), optionally with 1, 2, or 3 mutations; FR3 - RAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFC (SEQ ID NO: 20), optionally with 1, 2, or 3 mutations; FR4—FGEGSRLTVL (SEQ ID NO: 21), optionally with 1, 2, or 3 mutations; may include:

[0072] The framework regions FR1, FR2, and FR3 of the α chain are * and / or the framework regions FR1, FR2, and FR3 of the β chain may comprise an amino acid sequence corresponding to that of the TRBV9 * It may contain an amino acid sequence corresponding to the amino acid sequence of the O1 chain.

[0073] The FR4 region may contain the junction region of the α and β variable chains (TRAJ and TRBJ, respectively). * The TRBJ region may contain an amino acid sequence corresponding to the amino acid sequence of TRBJ2-2. * It may contain an amino acid sequence corresponding to the amino acid sequence of 01.

[0074] The framework regions of the α chain variable domain may have a total of 1, 2, 3, 4, 5 or more mutations relative to the above sequence. The framework regions of the α chain variable domain may have one mutation relative to the above sequence. The framework regions of the α chain variable domain may comprise an N61Q mutation numbered according to SEQ ID NO: 3. The framework regions of the α chain variable domain may not comprise other mutations (other than N61Q).

[0075] The framework regions of the β-chain variable domain may have a total of 1, 2, 3, 4, 5, or more mutations relative to the above sequence. The framework regions of the β-chain variable domain may have 5 mutations relative to the above sequence. The framework regions of the β-chain variable domain comprise one or more or all of the following mutations, numbered according to SEQ ID NO: 13: T13K, L43P, 147F, L61P, and F90I. The framework regions of the β-chain variable domain may comprise the following mutations, numbered according to SEQ ID NO: 13: T13K, L43P, 147F, L61P, and F90I. The framework regions of the β-chain variable domain may not comprise other mutations relative to the above sequence.

[0076] The α-chain variable domain of a binding molecule of the invention may comprise a respective framework amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. The β-chain variable domain of a binding molecule of the invention may comprise a respective framework amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. Alternatively, the specified percentage of identity may be across the framework sequences when considered as a whole.

[0077] The alpha chain variable domain may comprise any one of the amino acid sequences of SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:118, or SEQ ID NO:119, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity to any one of SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:118, or SEQ ID NO:119. The β chain variable domain may comprise any one of the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NOs: 106 to 108, SEQ ID NOs: 112 to 117, or SEQ ID NOs: 120 to 124, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity to any one of SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NOs: 106 to 108, SEQ ID NOs: 112 to 117, or SEQ ID NOs: 120 to 124. Because all α chain and β chain variable domains are derived from the same scaffold TCR sequence (i.e., SEQ ID NO: 3 and SEQ ID NO: 13, respectively), all α chain variable domain sequences are expected to be compatible with all β chain variable domain sequences. Thus, the α chain variable domain may comprise the amino acid sequence set forth in any one of SEQ ID NO: 3, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 118, or SEQ ID NO: 119, or an amino acid sequence having at least 90% identity to that amino acid sequence, and the β chain variable domain may comprise the amino acid sequence set forth in any one of SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 106 to SEQ ID NO: 108, SEQ ID NO: 112 to SEQ ID NO: 117, or SEQ ID NO: 120 to SEQ ID NO: 124, or an amino acid sequence having at least 90% identity to that amino acid sequence.

[0078] The α chain variable domain may comprise any one of the amino acid sequences of SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:29, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity to any one of SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:29. The β chain variable domain may comprise any one of the amino acid sequences of SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:40, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity to any one of SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:40. Because all α chain and β chain variable domains are derived from the same scaffold TCR sequence (i.e., SEQ ID NO:3 and SEQ ID NO:13, respectively), all α chain variable domain sequences are expected to be compatible with all β chain variable domain sequences. Thus, the alpha chain variable domain may comprise the amino acid sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:29, or an amino acid sequence having at least 90% identity to said amino acid sequence, and the beta chain variable domain may comprise the amino acid sequence set forth in any one of SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:40, or an amino acid sequence having at least 90% identity to said amino acid sequence.

[0079] The binding molecules may comprise the following combinations of alpha and beta chain variable domains: (a) an α chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 22 and a β chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 13; (b) an α chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 24 and a β chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 31; (c) an α chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 26 and a β chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 34, or (d) an α chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 29 and a β chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 40; It may include one of:

[0080] Preferably, the α chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 29 and the β chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 40. In this regard, the present invention provides a binding molecule having the property of binding to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24, the binding molecule comprising an α chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 29, and a β chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 40 or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 40.

[0081] In the binding molecules of the invention, the variable domains, and, if present, the constant domains, and / or any other domains, may be organized in any suitable format / configuration that allows antigen binding. As used herein, the "format" of a binding molecule designates a defined spatial arrangement of the domains, particularly the variable domains and, optionally, the constant domains. Characteristics of such protein formats are the number of polypeptide chains (single-chain, double-chain, or multiple-chain), the type and length of linkers connecting different domains, the number of antigen-binding moieties (and thus the valency), the number of different antigen-binding moieties (and thus the number of specificities for different antigens, e.g., bispecific, multispecific), and the order and orientation of the variable domains (e.g., crossover, parallel). For example, the variable domains may be arranged in a monoclonal TCR format, in which two chains are linked by disulfide bonds either within the constant domains or within the variable domains, or in which the variable domains are fused to one or more dimerization domains. Alternatively, the variable domains may be arranged in a single-chain format, with or without one or more constant domains, or in a diabody format. Other suitable formats are described herein.

[0082] The binding molecules of the present invention may comprise at least one TCR constant domain or fragment thereof, such as the α-chain TRAC constant domain and / or the β-chain TRBC1 or TRBC2 constant domain. As will be recognized by those skilled in the art, the terms TRAC and TRBC1 / 2 also include naturally occurring polymorphic variants, such as the N to K variant at position 4 of TRAC (Bragado et al. International Immunology. 1994 Feb; 6(2):223-30).

[0083] If present, one or both of the constant domains may contain mutations, substitutions, or deletions relative to the native constant domain sequence. The constant domains may be truncated, i.e., may lack the transmembrane and cytoplasmic domains. Thus, a binding molecule of the invention may comprise the extracellular region of a TCR α chain constant domain and / or the extracellular region of a TCR β chain constant domain. Alternatively, the constant domains may be full-length, meaning that the extracellular, transmembrane, and cytoplasmic domains are all present. The TRAC and TRBC domain sequences may be modified by truncation or substitution to eliminate the native disulfide bond between Cys4 in exon 2 of TRAC and Cys2 in exon 2 of TRBC1 or TRBC2. The α chain constant domain sequence and / or β chain constant domain sequence(s) may have disulfide bonds introduced between residues of the respective constant domains, e.g., as described in WO 03 / 020763. Thus, the binding molecule may contain a non-natural covalent disulfide bond linking a residue of the TCR α chain constant domain to a residue of the TCR β chain constant domain. Preferably, the α and β constant domains may be modified by substitution of a cysteine ​​residue at position Thr48 of TRAC and position Ser57 of TRBC1 or TRBC2, where the cysteine ​​forms a non-natural disulfide bond between the TCR α and β constant domains. TRBC1 or TRBC2 may further contain a cysteine ​​to alanine mutation at position 75 of the constant domain and an asparagine to aspartic acid mutation at position 89 of the constant domain. For example, one or both of the extracellular constant domains present in the αβ heterodimer may be further truncated at one or more C-termini, for example, by up to 15, or up to 10, or up to 8, or fewer amino acids. For example, one or both of the extracellular constant domains present in the αβ heterodimer may be truncated at one or more C-termini, for example by up to 15, or up to 10, or up to 8 amino acids. The C-terminus of the α chain extracellular constant domain may be truncated by 8 amino acids.

[0084] Binding molecules of the invention may comprise the extracellular region of a TCR α chain constant domain, optionally truncated at the C-terminus by up to 15 amino acids, and / or the extracellular region of a TCR β chain constant domain, optionally truncated at the C-terminus by up to 15 amino acids. Binding molecules of the invention may comprise the extracellular region of a TCR β chain constant domain comprising an L3M mutation numbered according to SEQ ID NO: 14. Such mutations have been identified by the inventors to result in increased stability of the TCR, herein designated a77150 and a90b152, compared to the native TCR sequence.

[0085] the extracellular region of the TCR alpha chain constant domain may comprise the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 4; and / or The extracellular region of the TCR β chain constant domain may comprise the amino acid sequence set forth in SEQ ID NO:48, or an amino acid sequence having at least 90% identity to the sequence set forth in SEQ ID NO:48.

[0086] The binding molecule may comprise the extracellular region of a TCR alpha chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 4 and the extracellular region of a TCR beta chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 48. The binding molecule may not comprise the transmembrane or cytoplasmic domain of the TCR.

[0087] Alternatively, rather than having a full-length or truncated constant domain, the TCR constant domain may be absent. Thus, a binding molecule of the invention may consist of the variable domains of the TCR α and β chains, and optionally include additional domains as described herein. Additional domains include, but are not limited to, immune effector domains (e.g., antibody domains), Fc domains or albumin-binding domains, therapeutic agents, or detectable labels.

[0088] The binding molecule may comprise a TCR alpha chain variable domain and a TCR beta chain variable domain in a single chain format. Single-chain formats include, but are not limited to, αβ TCR polypeptides of the Vα-L-Vβ type, Vβ-L-Vα type, Vα-Cα-L-Vβ type, Vα-L-Vβ-Cβ type, or Vα-Cα-L-Vβ-Cβ type, where Vα and Vβ are the TCR α variable region and TCR β variable region, respectively, Cα and Cβ are the TCR α constant region and TCR β constant region, respectively, and L is a linker sequence (Weidanz et al., (1998) J Immunol Methods. Dec 1;221(1-2):59-76, Epel et al., (2002), Cancer Immunol Immunother. Nov;51(10):565-73, WO 2004 / 033685, WO 9918129).

[0089] The term "linker" as used herein refers to one or more amino acid residues inserted between domains or elements, e.g., between domains and an agent, of a binding molecule of the present invention, providing sufficient flexibility for the domains to fold correctly and form an antigen-binding site. At the amino acid sequence level, linkers may be inserted at the transition between variable domains or between a variable domain and a constant domain (or other domain), respectively. The approximate sizes of TCR domains, as well as antibody domains, are well understood by those skilled in the art, allowing the determination of the transition between domains. The precise location of the domain transition can be determined by finding peptide stretches that do not form secondary structure elements, such as β-sheets or α-helices, as demonstrated by experimental data or as can be assumed by modeling or secondary structure prediction techniques.

[0090] Linker sequences are typically flexible because they are primarily composed of amino acids such as glycine, alanine, and serine, which lack bulky side chains that can limit flexibility. Alternatively, a linker with greater rigidity may be desirable. A usable or optimal length for a linker sequence can be easily determined. Often, linker sequences will be less than about 12 amino acids in length, e.g., less than 10 amino acids, or between 2 and 10 amino acids in length. Linkers 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 that may be used in the binding molecules of the invention include, but are not limited to, GGGGS (SEQ ID NO: 64), GGGSG (SEQ ID NO: 70), GGSGG (SEQ ID NO: 71), GSGGG (SEQ ID NO: 72), GSGGGP (SEQ ID NO: 73), GGEPS (SEQ ID NO: 74), GGEGGGP (SEQ ID NO: 75), GGEGGGSEGGGS (SEQ ID NO: 76), GGGSGGGG (SEQ ID NO: 77), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 59), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 78), EAAAK (SEQ ID NO: 79), and EAAAKEAAAKEAAAK (SEQ ID NO: 80). If present, one or both of the constant domains may be full length or they may be truncated and / or contain the mutations described above. Single-chain TCRs may be soluble, i.e., they do not contain a transmembrane domain. In certain embodiments, the single chain TCRs of the invention may have disulfide bonds introduced between residues of each constant domain, as described in WO 2004 / 033685.Single-chain TCRs are further described in WO 2004 / 033685, WO 98 / 39482, WO 01 / 62908, Weidanz et al. (1998) J Immunol Methods 221 (1-2): 59-76, Hoo et al. (1992) Proc Natl Acad Sci USA 89(10): 4759-4763, Schodin (1996) Mol Immunol 33(9): 819-829.

[0091] Alternatively, the binding molecule comprises two or more polypeptide chains, wherein the TCR α chain variable domain and the TCR β chain variable domain are comprised in separate polypeptide chains.

[0092] TCR variable domains can be arranged in a diabody format, in which two single-chain fragments dimerize in a head-to-tail configuration, resulting in a compact molecule with a molecular weight similar to that of a tandem scFv (approximately 50 kDa).

[0093] Particularly suitable TCR alpha chain sequences include, but are not limited to, any one of SEQ ID NO:2, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:49. Particularly suitable TCR beta chain sequences include, but are not limited to, any one of SEQ ID NO:12, SEQ ID NO:45, SEQ ID NO:47, or SEQ ID NO:50. Such sequences do not contain transmembrane or cytoplasmic domains. All alpha chain sequences (i.e., SEQ ID NO:2, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:49) are expected to be compatible with all beta chain sequences (i.e., SEQ ID NO:12, SEQ ID NO:45, SEQ ID NO:47, or SEQ ID NO:50), since they are all derived from the same native (scaffold) TCR sequence (SEQ ID NO:2 and SEQ ID NO:12, respectively). Therefore, the binding molecules of the invention may be a TCR alpha chain comprising an amino acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:49, or an amino acid sequence having at least 90% identity, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:49; a TCR beta chain comprising an amino acid sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 50, or an amino acid sequence having at least 90% identity, for example at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 50; may include:

[0094] More specifically, the binding molecule is (a) a TCR α chain comprising the amino acid sequence of SEQ ID NO: 42, and a TCR β chain comprising the amino acid sequence of SEQ ID NO: 12; (b) a TCR α chain comprising the amino acid sequence of SEQ ID NO: 44, and a TCR β chain comprising the amino acid sequence of SEQ ID NO: 45; (c) a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 46 and a TCR beta chain comprising the amino acid sequence of SEQ ID NO: 47; or (d) a TCR α chain comprising the amino acid sequence of SEQ ID NO: 49, and a TCR β chain comprising the amino acid sequence of SEQ ID NO: 50; may include:

[0095] Preferably, the binding molecule comprises a TCR alpha chain comprising the amino acid sequence of SEQ ID NO:49 and a TCR beta chain comprising the amino acid sequence of SEQ ID NO:50. In this regard, the present invention provides a binding molecule having the property of binding to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24, the binding molecule comprising a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 49 or an amino acid sequence having at least 90% identity, for example at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 49, and a TCR beta chain comprising the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having at least 90% identity, for example at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 50.

[0096] The binding molecules of the invention are useful for delivering detectable labels or therapeutic agents to antigen-presenting cells and tissues containing antigen-presenting cells. Thus, they may comprise or be associated (covalently or otherwise) with detectable labels (for diagnostic purposes, in which the binding molecules are used to detect the presence of cells presenting the cognate antigen), and / or with therapeutic agents, including immune effectors, and / or pharmacokinetic (PK)-modifying moieties.

[0097] Examples of PK-modifying moieties include, but are not limited to, PEG (Dozier et al., (2015) Int J Mol Sci. Oct 28;16(10):25831-64 and Jevsevar et al., (2010) Biotechnol J. Jan; 5(1):113-28), PAS (Schlapschy et al., (2013) Protein Eng Des Sel. Aug; 26(8):489-501), albumin and albumin binding domains (Dennis et al., (2002) J Biol Chem. Sep 20; 277(38):35035-43), and / or unstructured polypeptides (Schellenberger et al., (2009) Nat Biotechnol. Dec; 27(12):1186-90). Further PK-modifying moieties include immunoglobulin Fc domains, which may serve to extend the in vivo half-life of the binding molecules of the invention.

[0098] When an immunoglobulin Fc domain is used, it may be the Fc region of any antibody. The Fc region is the tail region of an antibody that interacts with cell surface Fc receptors and several proteins of the complement system. The Fc region typically contains two polypeptide chains, each of which has two or three heavy chain constant domains (termed CH2, CH3, and CH4) and a hinge region. The two chains are linked by a disulfide bond within the hinge region. The Fc domains from the immunoglobulin subclasses IgG1, IgG2, and IgG4 bind to FcRn and undergo FcRn-mediated recycling, resulting in a long circulatory half-life (3 to 4 weeks). The interaction between IgG and FcRn is localized in the Fc region spanning the CH2 and CH3 domains. Immunoglobulin Fc domains particularly suitable for use in the present invention include, but are not limited to, the Fc domains from IgG1 or IgG4. The Fc domain may be derived from a human sequence. The Fc region also preferably contains KiH mutations that promote dimerization, as well as mutations that prevent interaction with activating receptors, i.e., functionally silent molecules. The Fc domain of an immunoglobulin may be fused to the C-terminus or N-terminus of other domains (i.e., TCR variable domains and / or TCR constant domains and / or immune effector domains) in any suitable order or configuration. The immunoglobulin Fc may be fused to one or more of the other domains (i.e., TCR variable domains and / or TCR constant domains and / or immune effector domains) via a linker. Suitable linker sequences are known in the art and include those described herein. When the Fc of an immunoglobulin is fused to a TCR, it may be fused to either the α chain or the β chain, with or without a linker. Furthermore, individual chains of the Fc may be fused to individual chains of the TCR.

[0099] The Fc region, if present, may contain mutations compared to the WT sequence. Mutations include substitutions, insertions, and deletions. Such mutations may be made for the purpose of introducing desirable therapeutic properties. For example, knob-into-hole (KiH) mutations may be engineered into the CH3 domain to promote heterodimerization. In this case, one chain is engineered to contain a large protruding residue (i.e., knob), such as Y, and the other chain is engineered to contain a complementary pocket (i.e., hole). Suitable locations for KiH mutations are known in the art. Additionally or alternatively, mutations may be introduced that abrogate or reduce binding to Fcγ receptors and / or increase binding to FcRn and / or prevent Fab arm exchange or remove protease sites. Additionally or alternatively, mutations may improve manufacturability, for example, by removing or altering glycosylation sites.

[0100] The PK-modifying moiety may also be albumin or an albumin-binding domain, which may also act to extend half-life. As known in the art, albumin has a long circulating half-life of 19 days, in part due to its size above the renal threshold and its specific interaction and recycling through FcRn. Attachment to albumin is a known strategy to improve the in vivo circulating half-life of therapeutic molecules. Albumin can be attached non-covalently through the use of specific albumin-binding domains or covalently by conjugation or direct gene fusion. Examples of therapeutic molecules utilizing attachment to albumin to improve half-life are provided in Sleep et al., Biochim Biophys Acta. 2013 Dec; 1830(12):5526-34.

[0101] The albumin binding domain may be any moiety capable of binding to albumin, including any known albumin binding moiety, and may be selected from endogenous or exogenous ligands, small organic molecules, fatty acids, peptides, and proteins that specifically bind to albumin. Examples of preferred albumin binding domains include short peptides such as those described in Dennis et al., J Biol Chem. 2002 Sep 20; 277(38):35035-43 (e.g., the peptide QRLMEDICLPRWGCLWEDDF), proteins engineered to bind albumin, such as antibodies, antibody fragments and antibody-like scaffolds, such as Albudab™ marketed by GSK (O'Connor-Semmes et al., Clin Pharmacol Ther. 2014 Dec; 96(6):704-12) and Nanobody™ marketed by Ablynx (Van Roy et al., Arthritis Res Ther. 2015 May 20; 17:135), and proteins based on albumin binding domains found in nature, such as the streptococcal protein G protein (Stork et al., Eng Des Sel. 2007 Nov; 20(11):569-76), for example, Albumod™ marketed by Affibody. Preferably, the albumin is human serum albumin (HSA). The affinity of the albumin-binding domain for human albumin can be in the picomolar to micromolar range. Considering the very high concentration of albumin in human serum (35 mg / ml to 50 mg / ml, approximately 0.6 mM), it is calculated that substantially all of the albumin-binding domain will bind to albumin in vivo.

[0102] The albumin binding moiety may be fused to the C-terminus or N-terminus of the other domains (i.e., TCR variable domain and / or TCR constant domain and / or immune effector domain) in any suitable order or configuration. The albumin binding moiety may be fused to one or more of the other domains (i.e., TCR variable domain and / or TCR constant domain and / or immune effector domain) via a linker. Suitable linkers are known in the art and include those described herein. When the albumin binding moiety is linked to the TCR, it may be linked to either the α chain or the β chain, with or without a linker.

[0103] Detectable labels for diagnostic purposes include, for example, fluorescent labels, radioactive labels, enzymes, nucleic acid probes and imaging agent reagents.

[0104] For some purposes, the binding molecules of the present invention may be aggregated into complexes containing several binding molecules to form multivalent binding molecule complexes. Several human proteins contain multimerization domains that can be used to produce multivalent binding molecule complexes. For example, the tetramerization domain of p53 has been utilized to produce tetramers of scFv antibody fragments that exhibit increased serum persistence and significantly reduced off-rates compared to monomeric scFv fragments (Willuda et al. (2001) J. Biol. Chem. 276 (17) 14385-14392). Hemoglobin also has a tetramerization domain that can be used for this type of application. The multivalent binding molecule complexes of the present invention may have enhanced binding affinity to the complex compared to the non-multimeric native (also referred to as parent, natural, non-mutated wild-type, or scaffold) T cell receptor heterodimers of the present invention. Thus, multivalent complexes of the binding molecules of the present invention are also encompassed within the present invention. Such multivalent binding molecule complexes according to the invention are particularly useful for tracking or targeting cells presenting specific antigens in vitro or in vivo, and are also useful as intermediates for the production of additional multivalent binding molecule complexes having such uses.

[0105] Therapeutic agents that may be associated with or contained in the binding molecules of the invention include immunomodulators and effectors, radioactive compounds, enzymes (e.g., perforin), or chemotherapeutic agents (e.g., cisplatin). To ensure that the therapeutic effect is exerted at the desired location, the therapeutic agent may be within a liposome or other nanoparticulate structure linked to the binding molecule, so that the compound is released slowly. This will prevent damaging effects during transport within the body and ensure that the therapeutic agent has its maximum effect after the binding molecule binds to the appropriate antigen-presenting cells.

[0106] Examples of suitable therapeutic agents include, but are not limited to: antibodies or fragments thereof, including anti-T cell or NK cell determinant antibodies (e.g., anti-CD3, anti-CD28, or anti-CD16); alternative protein scaffolds with antibody-like binding properties (e.g., DARPins); Immunostimulators, i.e., immune effector molecules that stimulate the immune response, such as cytokines such as IL-2 and IFN-γ; chemokines, such as IL-8, platelet factor 4, and melanoma growth stimulatory protein; complement pathway activators or Fc receptors; checkpoint inhibitors, such as those targeting PD1 or PD-L1; Small molecule cytotoxic agents, i.e., compounds with a molecular weight of less than 700 daltons and the ability to kill mammalian cells. These compounds may also contain toxic metals that can have cytotoxic effects. Furthermore, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that break down or are converted under physiological conditions to release a cytotoxic agent. Examples of such cytotoxic agents include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate arbourate, auristatin E, vincristine, and doxorubicin; Peptide cytotoxic agents, i.e., proteins or fragments thereof capable of killing mammalian cells, such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNase and RNase; Radionuclides, i.e., unstable isotopes of elements that decay with the simultaneous emission of one or more alpha or beta particles, or gamma rays, such as 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 radionuclides with the TCR or its multimers; Superantigens and their mutants; Heterologous protein domains, homologous protein domains, viral / bacterial protein domains, viral / bacterial peptides; Includes:

[0107] The binding molecules of the present invention may be multispecific. As used herein, the term "multispecific" refers to a binding molecule comprising two or more antigen-binding moieties, including a TCR antigen-binding moiety formed by a TCR α chain variable domain and a TCR β chain variable domain. Such a binding molecule can bind to the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex and also to one or more different antigens. For example, a binding molecule may be bispecific. Such a binding molecule comprises a TCR antigen-binding moiety (formed by an α chain variable domain and a β chain variable domain) that binds to the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex and another antigen-binding moiety (e.g., an antibody antigen-binding moiety) that binds to a different antigen. This other antigen-binding moiety may be referred to herein as a "second antigen-binding moiety," and the antigen bound by the second antigen-binding moiety may be referred to herein as a "second antigen." The term "antigen-binding moiety" refers to a protein or a region thereof that can bind to an antigen. For example, the term encompasses antigen-binding sites of antibodies, including antigen-binding sites from conventional antibodies and engineered antibodies.

[0108] A multispecific binding molecule may comprise an antigen-binding portion of an antibody capable of binding to an antigen (i.e., a second antigen). In this regard, the binding molecule may comprise an antibody or a functional fragment or variant thereof. The term "antibody" as used herein is intended to include conventional / natural antibodies and engineered antibodies, in particular functional antibody fragments, single-chain antibodies, single-domain antibodies, bispecific antibodies, or multispecific antibodies. "Natural" or "conventional" refers to an antibody that has domains and domain arrangements identical to those of antibodies found in nature and contains CDR and FR sequences derived from the antibody. In natural / conventional antibodies, 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 the light chain (VL) and the heavy chain (VH) determine binding recognition and specificity to the antigen. Conventional antibody binding sites are composed primarily of residues from the "antibody complementarity-determining regions" (CDRs) or hypervariable regions. In some cases, residues from non-hypervariable regions or framework regions (FR) may influence the overall domain structure and thus the binding site. CDR refers to the amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a natural antibody binding site. The light and heavy chains of a conventional antibody each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, the antigen-binding site of a conventional antibody contains six CDRs, including a complete set of CDRs from each of the VH and VL.

[0109] "Engineered" antibody formats include functional antibody fragments, single-chain antibodies, single-domain antibodies, and chimeric, humanized, bispecific, or multispecific antibodies. Engineered antibody formats further include constructs in which a TCR-derived CDR, optionally including three, two, or one additional N-terminal and / or C-terminal framework residues, or an entire variable domain from a TCR is grafted onto an antibody heavy or light chain. A "functional antibody fragment" refers to a portion of a full-length antibody, or a protein similar to a portion of a full-length antibody, particularly the antigen-binding or variable region of a full-length antibody, that retains the ability to bind to a target antigen. Examples of functional antibody "fragments" include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies. For example, a binding molecule of the present invention may comprise an scFv. A functional antibody fragment may also be a single-domain antibody, such as a heavy-chain antibody. Thus, a binding molecule may comprise, for example, a VHH. As is known in the art, the antigen-binding site of a single-domain antibody, such as a VHH, may contain three CDRs (as opposed to six in conventional antibodies). As used herein, the term "antigen-binding portion of an antibody" encompasses such binding sites. Alternatively or additionally, the binding molecule may comprise a Fab fragment or an Fv fragment. The term "Fab" refers to an antibody fragment obtained by treating IgG with a protease, such as papain, in which approximately the N-terminal half of the H chain and the entire L chain are linked to each other via disulfide bonds, and has a molecular weight of approximately 50,000 daltons and antigen-binding activity. An Fv fragment is the N-terminal portion of an antibody Fab fragment and consists of the variable portion of one light chain and one heavy chain.

[0110] Binding molecules comprising the antigen-binding portion of an antibody described above may be referred to as bispecific TCR antibody molecules (i.e., binding molecules comprising at least two antigen-binding portions, one derived from an antibody and the other derived from a TCR). Such binding molecules may comprise an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), which associate to form the antigen-binding portion of an antibody capable of binding to an antigen. Thus, the antigen-binding portion may comprise a VH and a VL. For example, the binding molecule may comprise an scFv comprising a VH and a VL. Alternatively, the antigen-binding portion of an antibody may comprise a single variable domain (e.g., a VHH).

[0111] In such bispecific TCR antibody molecules, the variable domains may be arranged, for example, as described for the various bispecific antibody formats discussed above. Techniques for producing such bispecific molecules are also disclosed in the prior art cited above, and therefore, one skilled in the art can readily use the CDRs or variable domains defined herein to create and produce the antigen-binding proteins of the present invention in the formats disclosed herein. Furthermore, further formats are possible, for example, in which the variable domains on each chain are separated by a constant domain that mediates dimerization, such that in the final molecule, two antigen-binding sites are located on either side of the dimerized constant domain. One skilled in the art is fully capable of selecting an appropriate linker to ensure folding in the desired conformation.

[0112] The second antigen-binding portion (e.g., an antigen-binding portion of an antibody comprising a VH and a VL) can bind to an antigen of an effector cell. Such a binding molecule may be referred to as a "recruiter" because it recruits effector cells to tumors. In the context of the present invention, "effector cell" refers to a T cell or a natural killer cell (NK cell). In particular, the antigen (i.e., the second antigen) may be a T cell surface antigen.

[0113] Antigens include CD2, CD3 (CD3γ chain, CD3δ chain, and CD3ε chain, etc.), CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD The antigen may be selected from the group consisting of CD56, CD61, CD64, CD68, CD90, CD94, CD95, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, FcεRI, TCRα / β, TCRγ / δ, HLA-DR, and 4-1 BB, or a combination thereof. "Combination thereof" refers to a complex of two or more of the above antigens, such as a TCRα / β CD3 complex. Preferably, the antigen is CD3.

[0114] Antigen-binding portions suitable for binding to CD3 include binding domains derived from the CD3-specific humanized antibody hUCHT1 (Zhu et al., "Identification of heavy chain residues in a humanized anti-CD3 antibody important for efficient antigen binding and T cell activation." J. Immunol., 1995, 155, 1903-1910). In particular, the VH and VL domains derived from the UCHT1 variants UCHT1-V17, UCHT1-V17opt, UCHT1-V21, or UCHT1-V23 can be used. Alternatively, the VH and VL domains derived from the antibody BMA031, which targets the TCRα / β CD3 complex, and humanized versions thereof (Shearman et al., "Construction, expression and characterization of humanized antibodies directed against the human alpha / beta T cell receptor", J Immunol, 1991, 147, 4366-73), in particular the VH and VL domains derived from the BMA031 variants BMA031(V36) or BMA031(V10), can be used. Suitable BMA031 antibody variant sequences are described in WO 2022 / 233957. Alternatively, the VH and VL domains derived from the CD3-specific antibody H2C (described in EP 2 155 783) can be used.

[0115] Other suitable CD3 binding portions can be derived from the anti-CD3 scFv designated herein as "U0" (SEQ ID NO: 51) or "U28" (SEQ ID NO: 60). For example, the binding molecule can be an antigen-binding portion of an antibody comprising a VH and a VL and capable of binding to CD3, (a) VH has the following sequence: CDR1-GYSFTGYT (SEQ ID NO: 56) or GYSFTGYA (SEQ ID NO: 62), CDR2-INPYKGVS (SEQ ID NO: 57), CDR3-ARSGYYGDSDWYFDV (SEQ ID NO: 58), and (b) VL has the following sequence: CDR1-QDIRNY (SEQ ID NO: 52), CDR2-YTS and CDR3-QQGNTLPWT (SEQ ID NO: 54), The antibody may comprise an antigen-binding portion comprising a CDR having the following structure:

[0116] The binding molecule may comprise an antigen-binding portion of an antibody comprising a VH and a VL and capable of binding to CD3, VH comprises an amino acid sequence set forth in SEQ ID NO:55 or SEQ ID NO:61, or an amino acid sequence having at least 90% identity, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:55 or SEQ ID NO:61; and The VL comprises the amino acid sequence set forth in SEQ ID NO: 85 or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 85, for example at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

[0117] The binding molecule may comprise an scFv capable of binding to CD3. The scFv may comprise a VH comprising the amino acid sequence set forth in SEQ ID NO: 55 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 85. Such an scFv may comprise the amino acid sequence set forth in SEQ ID NO: 51.

[0118] Alternatively, the scFv may comprise a VH comprising the amino acid sequence set forth in SEQ ID NO: 61 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 85. Such an scFv may comprise the amino acid sequence set forth in SEQ ID NO: 60.

[0119] In the case of binding molecules comprising the antigen-binding portion of an antibody, the VH or VL may be covalently linked to the C-terminus or N-terminus of the TCR α chain or TCR β chain, optionally via a linker sequence. Suitable linker sequences are known in the art. Linker sequences are typically flexible because they are primarily composed of amino acids such as glycine, alanine, and serine, which lack bulky side chains that may limit their flexibility. Alternatively, a linker with greater rigidity may be desirable. A usable or optimal length for the linker sequence can be easily determined. In many cases, the linker sequence will be less than about 12, e.g., less than 10, or between 2 and 10 amino acids in length. For example, the VH or VL may be covalently linked to the C-terminus or N-terminus of the TCR alpha chain or TCR beta chain via a linker sequence selected from GGGGS (SEQ ID NO: 64), GGGSG (SEQ ID NO: 70), GGSGG (SEQ ID NO: 71), GSGGG (SEQ ID NO: 72), GSGGGP (SEQ ID NO: 73), GGEPS (SEQ ID NO: 74), GGEGGGP (SEQ ID NO: 75), GGEGGGSEGGGS (SEQ ID NO: 76), GGGSGGGG (SEQ ID NO: 77), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 59), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 78), EAAAK (SEQ ID NO: 79), and EAAAKEAAAKEAAAK (SEQ ID NO: 80).

[0120] In the case of a binding molecule comprising an antigen-binding portion of an antibody, preferably the C-terminus of the VH is covalently linked to the N-terminus of the TCR β chain, optionally via a linker comprising the amino acid sequence set forth in SEQ ID NO: 64. Preferably, such a binding molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises a TCR alpha chain (the alpha chain itself comprises the extracellular regions of the variable and constant domains); and The second polypeptide chain (also referred to herein as the "β chain-anti-CD3" chain) comprises a TCR β chain (the β chain itself comprising the extracellular regions of the variable and constant domains) and an scFv comprising a VH and a VL, wherein the C-terminus of the VH is covalently linked to the N-terminus of the TCR β chain, optionally via a linker comprising the amino acid sequence set forth in SEQ ID NO: 64.

[0121] Binding molecules in the above format include ImmTAC™ molecules. Examples of such molecules include tebentafusp, sold under the trade name KIMMTRAK™, as well as the binding molecules described in, for example, WO 2010 / 133828, WO 2019 / 012138, and WO 2019 / 012141. Exemplary binding molecules of the invention in this format include a9bwt-U0 (consisting of SEQ ID NOs: 42 and 63), a18b49-U0 (consisting of SEQ ID NOs: 44 and 65), a77b150-U0 (consisting of SEQ ID NOs: 46 and 66), a77b150-U28 (consisting of SEQ ID NOs: 46 and 67), a90b152-U0 (consisting of SEQ ID NOs: 49 and 68), and a90b152-U28 (consisting of SEQ ID NOs: 49 and 69).

[0122] The binding molecule in the above format is an alpha chain amino acid sequence set forth in any one of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:49, or an alpha chain amino acid sequence having at least 90% identity, for example at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:49; a beta chain-anti-CD3 amino acid sequence set forth in any one of SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69, or a beta chain amino acid sequence having at least 90% identity, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69; may include:

[0123] More particularly, the binding molecule in the above format is (a) an α chain amino acid sequence shown in SEQ ID NO: 42, and a β chain-anti-CD3 amino acid sequence shown in SEQ ID NO: 63; (b) an α chain amino acid sequence set forth in SEQ ID NO: 44, and a β chain-anti-CD3 amino acid sequence set forth in SEQ ID NO: 65; (c) an α chain amino acid sequence set forth in SEQ ID NO: 46 and a β chain anti-CD3 amino acid sequence set forth in SEQ ID NO: 66 or SEQ ID NO: 67, or (d) an α chain amino acid sequence set forth in SEQ ID NO: 49 and a β chain-anti-CD3 amino acid sequence set forth in SEQ ID NO: 68 or SEQ ID NO: 69; may include:

[0124] Preferably, the binding molecule comprises the α chain amino acid sequence set forth in SEQ ID NO: 49 and the β chain-anti-CD3 amino acid sequence set forth in SEQ ID NO: 69. Thus, the present invention provides a binding molecule having the property of binding to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24, the binding molecule comprising a TCR α chain and a TCR β chain covalently linked to an anti-CD3 scFv, wherein the α chain comprises the amino acid sequence set forth in SEQ ID NO: 49 and the β chain-anti-CD3 chain comprises the amino acid sequence set forth in SEQ ID NO: 69.

[0125] The binding molecule is a soluble F that contains the antigen-binding portion of a TCR and the antigen-binding portion of an antibody.c The antigen-binding portion of an antibody may be formed by the heavy and light chain variable domains of the antibody. TCER™ comprises two polypeptide chains, where the antigen-binding site is formed by the variable domains arranged in a crossover orientation on different polypeptide chains. Thus, the binding molecule is a first polypeptide chain comprising a TCR alpha chain variable domain and an antibody VH or VL; a second polypeptide chain comprising a TCR β chain variable domain and the other of the antibody VH and VL; wherein: The individual polypeptide chains associate in such a way that the binding molecule can simultaneously bind to the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex and the antigen of the antibody.

[0126] The binding molecules of the present invention preferably comprise proteins. The binding molecules may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts, and / or optionally dimerized or multimerized or conjugated. All such forms are encompassed by the present invention.

[0127] Binding molecules may be synthetic, recombinant, isolated, engineered, and / or purified. "Purified," when referring to a polypeptide or nucleotide sequence, means the presence of the referenced molecule but substantially absence of other biological macromolecules of the same type. As used herein, the term "purified" means that at least 75%, 85%, 95%, or 98% by weight of the biological macromolecules of the same type are the referenced molecule. A purified nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide of interest, although the molecule may contain some additional bases or moieties that do not adversely affect the essential characteristics of the composition.

[0128] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but a nucleic acid or peptide partially or completely separated from the materials that coexist in the natural state of the same nucleic acid or peptide is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or it may exist in a non-native environment, such as a host cell. An isolated binding molecule may be substantially free of other binding molecules with different antigen specificities. Furthermore, an isolated binding molecule may be substantially free of other cellular material and / or chemicals.

[0129] A "recombinant" molecule is a molecule that is prepared, expressed, produced, or isolated by recombinant means. In this regard, it does not occur in nature.

[0130] Amino acid sequence Phenotypically silent variants of any molecule disclosed herein are within the scope of the present invention. As used herein, the term "phenotypically silent variant" is understood to refer to variants that contain one or more additional amino acid changes, including substitutions, insertions, and deletions, in addition to those set forth above, and that have a phenotype similar to the corresponding molecule without said change(s). For purposes of the present invention, phenotype refers to binding affinity (K D and / or binding half-life) and / or specificity. Phenotypes for soluble binding molecules can include binding affinity and specificity, as well as immunostimulatory potency and purification yield. Phenotypically silent variants are those that have a measured K of a corresponding binding molecule that does not have the above alteration(s) when measured under identical conditions (e.g., 25°C and / or on the same SPR chip). D and / or a K for the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex within 50%, or more preferably within 30%, 25%, or 20% of the binding half-life. D and / or binding half-life. Suitable conditions are further illustrated in the Examples.

[0131] Furthermore, phenotypically silent variants may maintain the same or substantially the same therapeutic window between binding to the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex and binding to one or more additional peptide-HLA complexes. Phenotypically silent variants may maintain the same or substantially the same therapeutic window between the strength of immune cell activation in response to cells presenting the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex and the strength of immune cell activation in response to cells presenting one or more additional off-target peptide-HLA complexes. The therapeutic window may be calculated based on the lowest effective concentration ("LOEL") observed for normal cells and indication-relevant cell lines. The therapeutic window may differ by at least 10-fold, at least 100-fold, at least 1000-fold, or more. Phenotypic variants may share the same or substantially the same recognition motif as determined by serial mutagenesis techniques, discussed further below.

[0132] As known to those skilled in the art, it may be possible to produce binding molecules whose variable domains are altered compared to those detailed above without significantly altering the affinity of the interaction with the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex and / or other functional characteristics. In particular, such silent mutations may be incorporated into parts of the sequence known not to be directly involved in antigen binding (e.g., framework regions and / or parts of the CDRs that do not contact the antigen). Such variants are included within the scope of the present invention.

[0133] A phenotypically silent variant may contain one or more conservative substitutions and / or one or more permissive substitutions. Permissive substitutions refer to substitutions that do not fall within the definition of conservative as provided below, but are nevertheless phenotypically silent. Those skilled in the art know that various amino acids have similar properties and are therefore "conservative." One or more such amino acids of a protein, polypeptide, or peptide can often be substituted with one or more other such amino acids without eliminating the desired activity of the protein, polypeptide, or peptide.

[0134] Thus, the amino acids glycine, alanine, valine, leucine, and isoleucine can often be substituted for one another (amino acids with aliphatic side chains). Of these possible substitutions, glycine and alanine are preferably used to substitute for one another (because they have relatively short side chains), and valine, leucine, and isoleucine are preferably used to substitute for one another (because they have larger, hydrophobic aliphatic side chains). Other amino acids that are often substituted for one another include phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains), lysine, arginine, and histidine (amino acids with basic side chains), aspartic acid and glutamic acid (amino acids with acidic side chains), asparagine and glutamine (amino acids with amide side chains), cysteine ​​and methionine (amino acids with sulfur-containing side chains), and serine and threonine (amino acids with hydroxyl-containing side chains). It should be recognized that amino acid substitutions within the scope of the present invention can be made with naturally occurring or non-naturally occurring amino acids. For example, it is contemplated herein that the methyl group on alanine may be replaced with an ethyl group and / or minor changes may be made to the peptide backbone. Whether natural or synthetic amino acids are used, it is preferred that only L-amino acids be present.

[0135] Substitutions of this nature are often referred to as "conservative" or "semi-conservative" amino acid substitutions. The present invention therefore extends to the use of molecules comprising either an amino acid sequence as described above, but comprising one or more conservative substitutions and / or one or more permissive substitutions in the sequence, such that the amino acid sequence of the molecule, or any domain or region thereof, has at least 90% identity, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to a sequence disclosed herein.

[0136] "Identity," as known in the art, is a 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 sequences or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Although several methods exist for measuring identity between two polypeptide sequences or two polynucleotide sequences, commonly used methods for determining identity are codified in computer programs. Preferred computer programs for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).

[0137] To compare amino acid sequences, one may use a program such as the CLUSTAL program. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. For optimal alignment, it is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type). Programs such as BLASTx align the longest stretch of similar sequences and assign a value to the match. It is therefore possible to obtain a comparison in which several regions of similarity are found, each with a different score. Both types of identity analysis are contemplated in the present invention.

[0138] For optimal comparison purposes, the percent identity of two amino acid sequences or two nucleic acid sequences is determined by aligning the sequences (e.g., gaps may be introduced in the first sequence to optimally align the sequences) and comparing the amino acid residues or nucleotides at corresponding positions. An "optimal alignment" is the alignment of two sequences that 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).

[0139] The determination of percent identity between two sequences can be achieved using a mathematical algorithm known to those skilled in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The BLASTn and BLASTp programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 incorporate such an algorithm. The determination of percent identity between two nucleotide sequences can be performed using the BLASTn program. The determination of percent identity between two protein sequences can be performed using the BLASTp program. To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast may be used to perform an iterated search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTp and BLASTp) may be used. See http: / / www.ncbi.nlm.nih.gov. Default general parameters may include, for example, word size=3 and expectation threshold=10. For short input sequences, parameters may be selected to adjust automatically. Another example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0), which is part of the CGC sequence alignment software package, incorporates such an algorithm.Other algorithms for sequence analysis known in the art include ADVANCE and ADAM, as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5, and FASTA, as described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. For purposes of assessing percent identity in this disclosure, BLASTp with default parameters is used as the comparison methodology. Furthermore, if the listed percent identity provides a non-integer number of amino acids, i.e., a sequence of 25 amino acids with 90% sequence identity would provide a value of "22.5," the resulting value is truncated to the next integer, thus "22." Thus, in the example provided, a sequence with 22 matches out of 25 amino acids would be within 90% sequence identity.

[0140] As will be apparent to those skilled in the art, it may be possible to shorten or extend the sequences provided at the C-terminus and / or N-terminus by one, two, three, four, five or more residues without substantially affecting the functional properties of the molecule, e.g., the TCR portion. The sequences provided at the C-terminus and / or N-terminus may be shortened or extended by one, two, three, four, or five residues. All such variants are encompassed by the present invention.

[0141] Mutations, including conservative and permissive substitutions, insertions, and deletions, can be introduced into the provided sequences using any suitable method, including, but not limited to, polymerase chain reaction (PCR)-based, restriction enzyme-based cloning, or ligation-independent cloning (LIC) methods. These methods are detailed in many standard molecular biology textbooks. For further details regarding polymerase chain reaction (PCR) and restriction enzyme-based cloning, see Sambrook & Russell, (2001) Molecular Cloning - A Laboratory Manual (3rd (Ed.) CSHL Press. Further information regarding ligation-independent cloning (LIC) procedures can be found in Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6. The protein sequences provided by the present invention can be obtained from recombinant expression, solid phase synthesis, or any other suitable method known in the art.

[0142] Assessment of binding properties and activity of binding molecules Binding affinity (equilibrium constant K D ) and binding half-life (T 1 / 2 Methods for determining binding affinity (represented as ΛΛΛΛ) are known to those skilled in the art. Binding affinity and binding half-life can be determined using surface plasmon resonance (SPR) or biolayer interferometry (BLI), for example, using a BIAcore instrument or an Octet instrument, respectively. For example, the binding affinity of a binding molecule to a peptide-HLA complex can be determined using SPR at 25°C, where the peptide-HLA complex is immobilized on a solid support (e.g., a sensor chip) and contacted with a solution containing the binding molecule. Suitable experimental conditions and methods for determining binding parameters are described in Example 1.

[0143] Those skilled in the art will appreciate that the higher the affinity, the greater the K D It will be appreciated that a lower value for K indicates stronger binding. In other words, a doubling of affinity results in a D It indicates that the value of T will be halved. 1 / 2 is the dissociation rate constant (k off ) is calculated by dividing by T 1 / 2 When doubled, k off is halved. K for TCR D value and k offValues ​​are usually measured for soluble forms of TCRs, i.e., truncated forms of TCRs so that cytoplasmic and transmembrane domain residues are removed. To account for variations between independent measurements, particularly interactions with dissociation times greater than 20 hours, the binding affinity and / or binding half-life of a given protein can be measured several times, for example, three or more times, using the same assay protocol, and the results are averaged. To compare binding data between two samples (i.e., two different proteins and / or two preparations of the same protein), it is preferable to perform measurements using the same assay conditions (e.g., temperature). The measurement methods described for TCRs can also be applied to the binding molecules described herein.

[0144] Certain binding molecules of the invention can generate very strong T cell responses in vitro against antigen-positive cells, particularly cells that present low levels of antigens typical of cancer cells (i.e., 5-100 antigens per cell, e.g., around 50 antigens per cell) (Bossi et al., (2013) Oncoimmunol. 1;2(11):e26840; Purbhoo et al., (2006). J Immunol 176(12):7308-7316.). Such TCRs may be suitable for incorporation into the binding molecules described herein. The measured T cell response may be the release of T cell activation markers such as interferon-γ or granzyme B, or other measures of T cell activation, such as target cell killing or T cell proliferation. A very strong response would be in the nM-pM range, e.g., an EC of 500 nM or less, preferably 1 nM or less, or 500 pM or less. 50 It may have a value.

[0145] Molecules encompassed by the present invention may have improved half-lives. Methods for determining whether a protein has an improved half-life will be apparent to those skilled in the art. For example, the protein's ability to bind to fetal Fc receptor (FcRn) is evaluated. In this regard, increased binding affinity to FcRn increases the serum half-life of the protein (see, for example, Kim et al. Eur J Immunol., 24:2429, 1994).

[0146] The half-life of the proteins disclosed herein can also be measured by pharmacokinetic studies, for example, according to the method described by Kim et al. Eur J of Immunol 24: 542, 1994. According to this method, a radiolabeled protein is intravenously injected into mice, and its plasma concentration is measured periodically over time, for example, from 3 minutes to 72 hours after injection. Alternatively, an unlabeled protein of the present disclosure can be injected, and its plasma concentration can be measured periodically using ELISA. The resulting clearance curve should be biphasic, i.e., have an α-phase and a β-phase. To determine the in vivo half-life of a protein, the clearance rate in the β-phase is calculated and compared to the clearance rate of the wild-type or unmodified protein.

[0147] Structural properties of the binding molecules described herein, such as crossing angles, tilt angles, roll angles, and binding contacts of peptide residues, can be determined. Methods for determining such structural properties can include, for example, determining the three-dimensional atomic structure of the binding molecule bound to the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex.

[0148] As used herein, the term "three-dimensional atomic structure" refers to a model of the three-dimensional arrangement of atoms of a protein or protein complex. A 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 ​​that define the position of one or more atoms in a protein relative to an axis system. Atomic coordinates can be used in a computer to generate a representation, e.g., an image of the three-dimensional structure of the protein, which can be displayed by a computer and / or represented in an electronic file. Such atomic structures can be determined using techniques well known in the art, including X-ray crystallography, nuclear magnetic resonance (NMR), or cryo-electron microscopy (cryo-EM). For example, the three-dimensional atomic structure can be an X-ray crystallography. An X-ray crystallography is a three-dimensional atomic structure of a protein or protein complex obtained using X-ray crystallography. X-ray crystallography techniques are well known in the art. Suitable techniques are described in Example 7 under the heading "X-ray Crystallography."

[0149] X-ray crystal structures can be obtained by an X-ray crystallography technique known as molecular replacement. Molecular replacement methods are generally known to those skilled in the art and can be implemented using publicly available software packages. Generally, molecular replacement involves the following steps: i) collecting X-ray diffraction data from the crystal of a crystallized target protein complex; ii) converting the X-ray diffraction data to calculate a Patterson function; iii) comparing the Patterson function of the crystallized target structure with the Patterson function calculated from one or more known structures (referred to in the art as a "query structure" or "query model"); iv) rotating the Patterson function of the query structure on the Patterson function of the target structure to determine the correct orientation of the query structure in the crystal to obtain a rotation function; and v) calculating a translation function to determine the position of the query structure relative to the crystal axes. Alternatively, likelihood-based molecular replacement methods can also be used to determine the position of the query structure. Once the query structure is correctly positioned within the unit cell, initial phases for the experimental data can be calculated. These phases are necessary for calculating an electron density map, which determines and refines the initial three-dimensional atomic structure. Preferably, the structural features of the search model (e.g., amino acid sequence, conserved disulfide bonds, and β-strands or β-sheets) are related to the crystallized target complex. Suitable search models can be obtained from protein structure databases such as the RCSB Protein Data Bank (RCSB PDB). Suitable search models for determining the three-dimensional atomic structure of the complex formed by the pHLA and the binding molecule include the atomic coordinates of known TCR and pHLA structures. The electron density map can then be subjected to any well-known model building and structure refinement techniques to obtain the final, accurate structure of the unknown (i.e., target) crystallized molecular structure.

[0150] Once the three-dimensional atomic structure of the binding molecule bound to the pHLA complex is obtained, structural features such as bond geometry (e.g., crossing angle, roll angle, and tilt angle) and binding contacts of peptide residues can be determined based on the positions of the atoms in the structure.

[0151] 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). Specifically, the crossing angle is the angle formed between two vectors: the HLA groove vector and the TCR interdomain vector (also referred to herein as the "TCR cysteine ​​vector").

[0152] The HLA groove vector (also referred to as the "HLA peptide-binding groove vector") is a directed line segment (i.e., vector) corresponding to a peptide positioned across the HLA peptide-binding groove, i.e., from the N-terminus to the C-terminus of the peptide. In this regard, the HLA groove vector follows the two parallel HLA groove helices in the N-terminus to C-terminus direction of HLA helix 1 and passes through the HLA center of gravity. The TCR inter-domain vector is a directed line segment (i.e., vector) connecting the intrachain disulfide bond in the TCR α chain variable domain with the intrachain disulfide bond in the TCR β chain variable domain (in the α chain to β chain direction).

[0153] Similarly, the "tilt angle" of a binding molecule is the angle between the "TCR symmetry vector" and the HLA groove vector. The TCR symmetry vector corresponds to the pseudo-twofold symmetry axis of the TCR variable subunit, points toward its CDRs, and passes through the center of mass of the TCR.

[0154] The "roll angle" of a binding molecule is the angle formed between the "second HLA vector" or "HLA v2 vector" and the TCR symmetry vector. The second HLA vector (HLA v2 vector) is generated perpendicular to the HLA groove vector and points from HLA helix 1 towards HLA helix 2. The two vectors intersect at the center of gravity of the HLA helices.

[0155] Methods for calculating the above angles 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.

[0156] The binding molecules of the present invention can be evaluated to determine the binding contacts of their peptide residues. The phrase "binding contacts of peptide residues" refers to the binding interactions formed between the amino acid residues of the peptide in the peptide-HLA complex (not HLA) and the amino acid residues of the binding molecule. Each peptide residue contact by the binding molecule is considered to be a binding contact of the peptide residue when the binding molecule binds to the pHLA complex. For example, there may be 4, 5, 6, 7, or 8 or more binding contacts of the peptide residues. There may be a minimum number (e.g., 4, 5, 6, 7, or 8) of these binding contacts of the peptide residues that is sufficient for the binding molecule to specifically bind to the pHLA complex.

[0157] Binding contacts of amino acid residues may be determined using any method known in the art, including measuring the distance between atoms in the three-dimensional atomic structure of the binding molecule bound to the pHLA complex. For example, if the distance between any atom from the binding molecule residue and any atom from the peptide residue is 4.1 Å or less, the residues in the binding molecule and peptide may be considered to be in binding contact. Alternatively or additionally, binding interactions (e.g., binding contacts of peptide residues) can be identified from the three-dimensional atomic structure based on known atomic interaction geometries for various types of interactions, such as hydrogen bonds (H-bonds), electrostatic interactions, and van der Waals (vdW) interactions. For example, an H-bond bonding contact may be defined as an interaction between a donor atom and an acceptor atom, where the donor-acceptor distance in the three-dimensional atomic structure is approximately 3.0 Å or less and the donor-hydrogen acceptor angle is within 45° to 180°. A vdW bonding contact may be defined as an interaction between two heavy atoms that are within approximately 4 Å of each other in the three-dimensional atomic structure. Methods for identifying binding contacts of peptide residues may include performing molecular dynamics simulations using publicly available software packages.

[0158] Nucleic acids, vectors, and host cells The present invention provides nucleic acids encoding the binding molecules of the present invention. The TCR α chain variable domain and the TCR β chain variable domain of the binding molecule may be encoded in a single open reading frame or in two separate open reading frames. Alternatively, the TCR α chain variable domain and the TCR β chain variable domain of the binding molecule may be encoded on separate nucleic acids. The term "nucleic acid" includes, but is not limited to, ribonucleic acid (RNA) molecules and deoxyribonucleic acid (DNA) molecules, which may be single-stranded or double-stranded. Nucleic acids may be present in whole cells, cell lysates, or in isolated, partially purified, or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it has been purified and separated from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques. The nucleic acid may be recombinant, non-naturally occurring, and / or engineered. The nucleic acid sequence may be codon-optimized depending on the expression system used. As known to those skilled in the art, expression systems can include bacterial cells such as E. coli, or yeast cells, or mammalian cells, or insect cells, or the expression system can be a cell-free expression system.

[0159] The present invention also provides constructs in the form of plasmids, vectors, transcription cassettes, or expression cassettes comprising at least one nucleic acid as described above. In particular, the present invention provides expression vectors comprising the nucleic acids of the invention. The terms "vector," "cloning vector," and "expression vector" refer to vehicles capable of introducing DNA or RNA sequences (e.g., foreign genes) into a host cell to transform the host and, optionally, promote expression (e.g., transcription and translation) of the introduced sequences.

[0160] The present invention also provides recombinant host cells comprising one or more of the constructs described above. As noted above, nucleic acids encoding binding molecules of the invention constitute one aspect of the invention, as do methods for producing binding molecules of the invention comprising expression from nucleic acids encoding the binding molecules of the invention. Expression can be conveniently achieved by culturing recombinant host cells comprising the nucleic acid under appropriate conditions. Following production by expression, the binding molecules can be isolated and / or purified using any suitable technique and then used as appropriate.

[0161] Systems for cloning and expression of polypeptides in a wide variety of host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines available in the art for expressing heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, and many others. A commonly preferred bacterial host is E. coli. Expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see, e.g., Plueckthun, Bio / Technology 9:545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for producing binding molecules; for recent reviews, see, e.g., Reff, Curr. Opinion Biotech. 4:573-576 (1993) and Trill et al., Curr. Opinion Biotech. 6:553-560 (1995).

[0162] 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 vector known in the art, including plasmids or viral vectors (e.g., phage or phagemid), as appropriate. For further details, see, e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual," 2nd ed., Cold Spring Harbor Laboratory Press (1989). For example, many known techniques and protocols for preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells, and manipulating nucleic acids in gene expression and protein analysis are described in detail in Ausubel et al., ed., "Short Protocols in Molecular Biology," 2nd ed., John Wiley & Sons (1992).

[0163] The present invention also provides a host cell comprising the nucleic acid disclosed herein. (a) an expression vector of the present invention, or (b) a first expression vector comprising a nucleic acid encoding a first polypeptide comprising a TCR α chain variable domain of a binding molecule of the invention; and a second expression vector comprising a nucleic acid encoding a second polypeptide comprising a TCR β chain variable domain of a binding molecule of the invention; Also provided are non-naturally occurring and / or purified and / or engineered cells, preferably T cells, presenting a binding molecule of the invention.

[0164] The present invention further provides methods comprising introducing such nucleic acids into host cells. Any available technique can be used for introduction. For eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia, or, in the case of insect cells, baculovirus. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophages. Following introduction, expression from the nucleic acid can be caused or permitted, for example, by culturing the host cells under conditions for gene expression.

[0165] Suitable host cells for cloning or expressing the polynucleotides and / or vectors of the present invention are known in the art. Suitable host cells for expressing (glycosylated) proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension may be useful. Other examples of useful mammalian host cell lines are SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (e.g., 293 cells or 293T cells described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), 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 (e.g., Mather, JP et al., Annals NY Acad. Sci. 383 (1982) 44-68), MRC5 cells, and FS4 cells.Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268. Host cells can be eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). Alternatively, the host cell may be a prokaryotic cell, such as an E. coli cell.

[0166] The nucleic acids of the invention may be integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by the inclusion of sequences that facilitate recombination with the genome, according to standard techniques.

[0167] How to make binding molecules Further provided herein are methods for producing the binding molecules of the invention. In one embodiment, the methods comprise: a) maintaining a cell of the invention under conditions suitable for expression of the binding molecule; and b) isolating the binding molecule. In another embodiment, the methods comprise: a) providing a first cell capable of expressing a first polypeptide comprising a TCR alpha chain variable domain of a binding molecule of the invention, and a second cell capable of expressing a second polypeptide comprising a TCR beta chain variable domain of a binding molecule of the invention; b) maintaining the first cells under conditions suitable for expression of a first polypeptide and the second cells under conditions suitable for expression of a second polypeptide; c) isolating the first polypeptide and the second polypeptide from the cell; d) forming a complex between the first polypeptide and the second polypeptide to form a binding molecule of the invention; Includes.

[0168] Methods for producing recombinant proteins are well known in the art. After cloning a nucleic acid encoding a protein into an expression construct or expression vector, these can be transfected into host cells that do not otherwise produce the protein, such as E. coli cells, yeast cells, insect cells, or mammalian cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells. Exemplary mammalian cells used to express proteins are CHO cells, myeloma cells, or HEK cells. Preferred cells for producing the binding molecules of the invention are E. coli cells. Molecular cloning techniques to achieve these goals are known in the art and are described, for example, in Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification techniques are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art. See, e.g., U.S. Patent No. 4,816,567 or U.S. Patent No. 5,530,101.

[0169] Nucleic acids can be operably linked to a promoter and inserted into an expression construct or expression vector for further cloning (DNA amplification) or expression in a cell-free system or in cells. As used herein, the term "promoter" should be interpreted in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activation sequences, transcription factor binding sites, enhancers, and silencers) that alter expression of the nucleic acid, for example, in response to developmental and / or external stimuli or in a tissue-specific manner. In this context, the term "promoter" is also used to describe a recombinant, synthetic, or fusion nucleic acid, or derivative, that confers, activates, or enhances expression of an operably linked nucleic acid. Exemplary promoters may include additional copies of one or more specific regulatory elements that further enhance expression and / or alter the spatial and / or temporal expression of the nucleic acid. As used herein, the term "operably linked" means that the promoter is positioned relative to the nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0170] Many vectors for intracellular expression are commercially available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a protein-coding sequence (e.g., a sequence obtained from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. Those skilled in the art will be aware of sequences suitable for protein expression. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, alpha-factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

[0171] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), the human elongation factor 1 alpha promoter (EF1), the small nuclear RNA promoters (U1a and U1b), the alpha-myosin heavy chain promoter, the simian virus 40 promoter (SV40), the Rous sarcoma virus promoter (RSV), the adenovirus major late promoter, the beta-actin promoter, hybrid regulatory elements comprising the CMV enhancer / beta-actin promoter or immunoglobulin promoter, or active fragments thereof. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), the 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).

[0172] Exemplary promoters suitable for expression in yeast cells, such as yeast cells selected from the group including 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.

[0173] Host cells used to produce proteins can be cultured in a variety of media depending on the cell type used. Commercially available media such as Ham's F10 (Sigma), Minimum 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 the other cell types discussed herein are known in the art.

[0174] Methods for isolating proteins are known in the art. If the protein is secreted into the culture medium, the supernatant from such expression systems can first be concentrated using a commercially available protein concentration filter, such as an Amicon ultrafiltration unit or a Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants. Alternatively or additionally, the supernatant can be filtered and / or separated from the cells expressing the protein, for example, using continuous centrifugation.

[0175] Proteins prepared from 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 above.

[0176] These methods are known in the art and are described, for example, in WO 99 / 57134 or in "Antibodies: A Laboratory Manual," Ed Harlow and David Lane (editors), Cold Spring Harbor Laboratory, (1988). Those skilled in the art will also recognize that proteins can be modified to include tags that facilitate purification or detection, such as polyhistidine tags, hexahistidine tags, influenza virus hemagglutinin (HA) tags, simian virus 5 (V5) tags, LLAG tags, or glutathione S-transferase (GST) tags. The resulting proteins are then purified using methods known in the art, such as affinity purification. For example, proteins containing hexa-His tags are purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA), which specifically binds to the hexa-His tag immobilized on a solid or semi-solid support, washing the sample to remove unbound proteins, and subsequently eluting the bound proteins. Alternatively or additionally, a ligand or antibody that binds to the tag is used in affinity purification methods.

[0177] The molecules of the present invention may be suitable for high-yield purification. The 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 solubilized material obtained before refolding) and / or the 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 a yield of more than 1%, or more than 5%, or higher. High yield means a yield of more than 1 mg / ml, or more than 3 mg / ml, or more than 5 mg / ml, or higher.

[0178] Pharmaceutical compositions and medical methods For administration to a patient, 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 (e.g., buffers, also known as "buffers"). The pharmaceutical composition may be in any suitable form (e.g., depending on the desired method of administration to a patient). The pharmaceutical composition may be provided in unit dosage form, generally in a hermetically sealed container, and may be provided as part of a kit. Such kits will usually (but not necessarily) include instructions for use. Such kits may include a plurality of the unit dosage forms described above.

[0179] Pharmaceutical compositions may be adapted for administration by any suitable route, such as parenteral (including subcutaneous, intramuscular, intrathecal, or intravenous), enteral (including oral or rectal), inhalation, or intranasal. Such compositions can be prepared by any method known in the art of pharmacy, for example, by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions. Methods for preparing proteins in a form suitable for administration to a subject (e.g., pharmaceutical compositions) are known in the art and include, for example, those described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, PA, 1990) and the US Pharmacopeia: National Formulary (Mack Publishing Company, Easton, PA, 1984).

[0180] Pharmaceutical compositions typically comprise a solution of the binding molecules of the invention (or nucleic acids, cells, or vectors of the invention) dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. Pharmaceutical compositions may contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, as needed to approximate physiological conditions, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the molecules of the invention in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, and the like, depending on 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. Non-aqueous vehicles, such as mixed oils and ethyl oleate, may also be used. Liposomes may also be used as carriers. The vehicle may contain minor amounts of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0181] The binding molecules, pharmaceutical compositions, vectors, nucleic acids, and cells of the invention may be provided in substantially pure form, e.g., 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.

[0182] The binding molecules of the present invention may have an ideal safety profile for use as therapeutic reagents. As used herein, "safety profile" refers to the ability to distinguish tumor cells, particularly tumor cells displaying the PYLGQMINL (SEQ ID NO: 1)-HLA-A24 complex, from healthy cells. This ability is often expressed in terms of a safety window. In this case, the binding molecule may be in a soluble form and preferably fused to an immune effector. Suitable immune effectors are described herein, and include, but are not limited to, cytokines such as IL-2 and IFN-γ; superantigens and their mutants; chemokines such as IL-8, platelet factor 4, and melanoma growth stimulatory protein; antibody-like scaffolds, including antibodies and their fragments, derivatives, and variants that bind to antigens on immune cells such as T cells or NK cells (e.g., anti-CD3, anti-CD28, or anti-CD16); and Fc receptors or complement activators. In addition to exhibiting good specificity, an ideal safety profile means that the binding molecules of the present invention can pass further preclinical safety testing. Examples of such tests include whole blood assays to confirm minimal cytokine release in the presence of whole blood, and therefore low risk of causing potential cytokine release syndrome in vivo, and alloreactivity tests to confirm low ability to recognize alternative HLA types.

[0183] Appropriate dosages of the molecules of the invention can vary between wide limits, depending on the disease or disorder being treated, the age and condition of the subject being treated, etc. Preferably, the subject is human. Ultimately, a physician will be able to determine the appropriate dosage to use. Administration of the TCR anti-CD3 fusion molecules can be in a "therapeutically effective amount," which is an amount sufficient to provide benefit to the patient.

[0184] The binding molecules of the present invention may also be associated with a therapeutic agent. Therapeutic agents that can be associated with the molecules of the present invention include immunomodulators and immune effectors, radioactive compounds, enzymes (e.g., perforin), or chemotherapeutic agents (e.g., cisplatin). To ensure that the toxic effect is exerted at the desired location, the toxin can be in a liposome linked to the binding molecules described herein, thereby allowing the compound to be released slowly. This prevents harmful effects during transport within the body and ensures that the toxin exerts its maximum effect after the binding molecules described herein bind to the relevant antigen-presenting cells.

[0185] The binding molecules, nucleic acids, vectors, pharmaceutical compositions, and cells of the present invention can be used to treat diseases such as cancer. The cancer to be treated can be a cancer associated with PRAME expression. "Associated with PRAME expression" means that the cancer contains cancer cells that express PRAME. In this regard, the cancer can be a PRAME-positive cancer. The cancer may be known to be associated with PRAME expression. For example, PRAME expression may be known to be elevated in cancer, so PRAME expression may not be assessed or may be assessed retrospectively. Alternatively, PRAME expression can be assessed using any method known in the art, including, for example, histological methods or other quantitative or qualitative measurements, including PCR and RNA expression analysis, and / or kits or sequence panels designed to measure PRAME expression levels. It is not intended that the present invention be limited to the treatment of cancers in which PRAME expression can be detected by histological methods. Rather, the binding molecules of the present invention may be useful in treating cancers and tumor types thought to be associated with PRAME expression.

[0186] When PRAME expression is detected by histological methods such as immunohistochemistry (IHC), it can be quantified using an H-score. First, PRAME expression in individual cells or their subcellular compartments within a tumor is detected and classified as either positive or negative. Positive cells can be further classified as high, medium, or low based on the intensity of the IHC signal. The H-score captures both the intensity and proportion of a biomarker of interest from an IHC image and includes values ​​between 0 and 300, providing a dynamic range for quantifying the abundance of a particular marker or gene.

[0187] Cancers associated with PRAME expression include, but are not limited to, melanoma, lung cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, bladder cancer, head and neck cancer, uterine cancer, acute myeloid leukemia, chronic myeloid leukemia, and Hodgkin's lymphoma. For example, the cancer associated with PRAME expression can be melanoma. The melanoma can be uveal melanoma or cutaneous melanoma. The lung cancer can be non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). The breast cancer can be triple-negative breast cancer (TNBC). The bladder cancer can be urothelial carcinoma. The esophageal cancer can be gastroesophageal junction (GEJ) adenocarcinoma. The ovarian cancer can be epithelial ovarian cancer, such as high-grade serous ovarian cancer. In particular, the cancer can be cutaneous melanoma, ovarian cancer, NSCLC, or endometrial cancer.

[0188] The antigen PRAME constitutes a cancer marker and may therefore be used to indicate the effectiveness of anti-cancer therapy or to detect disease recurrence. Thus, in another aspect, the present invention provides a binding molecule, nucleic acid, vector, host cell, or pharmaceutical composition of the present invention for use as a diagnostic agent, in particular for use as an in vivo diagnostic agent. In a preferred embodiment, the diagnostic agent is for diagnosing a proliferative disease. In a more preferred embodiment, the diagnostic agent is for diagnosing a cancer presenting a peptide comprising or consisting of the amino acid sequence PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24.

[0189] The present invention also provides the following: a binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the invention for use in medicine, preferably for use in a human subject and / or preferably for use in a method for the treatment of cancer or tumors; 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 tumors; a method of treating cancer or tumor in a patient, the method comprising administering to the patient a binding molecule, nucleic acid, vector, pharmaceutical composition, or cell of the invention; An injectable formulation for administration to a human subject comprising a binding molecule, nucleic acid, vector, pharmaceutical composition, or cell of the invention.

[0190] Kits and manufactured products In another aspect, kits or articles of manufacture are provided containing materials useful for the treatment and / or prevention of the above diseases.

[0191] The kit may include (a) a container containing a binding molecule, nucleic acid, vector, or cell of the invention, optionally in a pharmaceutically acceptable carrier or diluent, and (b) a package insert containing instructions for treating a disease (e.g., cancer) in a subject. The kit may further include (c) at least one additional therapeutically active compound or drug.

[0192] The package insert may be on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials, such as glass or plastic. The container holds or contains a composition comprising the molecule, nucleic acid, vector, or cell of the invention and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a binding molecule, nucleic acid, vector, or cell of the invention. The label or package insert indicates that the composition is used for treating a suitable subject, e.g., a subject suffering from or predisposed to developing a disease described herein, and provides specific instructions regarding dosage and dosing intervals of the composition and other medications. The kit may further comprise an additional container with 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.

[0193] The present invention also includes particles displaying the binding molecules of the present invention, and including such particles within a library of particles. Such particles include, but are not limited to, phage, yeast cells, ribosomes, or mammalian cells. Methods for producing such particles and libraries are known in the art (see, e.g., WO 2004 / 044004, WO 01 / 48145, Chervin et al. (2008) J. Immuno. Methods 339.2: 175-184).

[0194] 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. Preferred features of each aspect of the invention are applicable to each of the other aspects mutatis mutandis. All documents referred to in this specification are incorporated herein by reference to the fullest extent permitted by law. [Brief explanation of the drawings]

[0195] [Figure 1] FIG. 1 shows a graph depicting the binding affinity of an exemplary native TCR to the target (PYLGQMINL, SEQ ID NO: 1) and mimetic peptide-HLA complexes described in Example 1. [Figure 2] 1 is a bar graph showing the results of an alanine scanning experiment of the native S11 TCR, where relative binding to the wild-type target peptide (PYLGQMINL, SEQ ID NO: 1) is shown for each mutant peptide with an alanine mutation at the indicated position, as described in Example 1. [Figure 3] FIG. 1 shows a series of line graphs showing T cell activation for selected mutant TCRs when fused to anti-CD3 scFv, as described in Example 3. [Figure 4] FIG. 1 shows the affinity window (difference in affinity between the target peptide complex and the similar mimetic peptide) for selected mutant TCRs, as described in Example 4. [Figure 5] FIG. 1 is a series of bar graphs showing the levels of T cell activation induced by selected TCR-anti-CD3 fusion molecules on normal cells from healthy tissue, as described in Example 5. [Figure 6] FIG. 1 is a series of line graphs showing the levels of T cell activation induced by selected TCR-anti-CD3 fusion molecules for various cancer cell lines, as described in Example 6. [Figure 7]

[0023] Figure 1 shows an image of the X-ray crystal structure of the TCR-peptide interface formed by TCR "S11" when bound to PYLGQMINL (SEQ ID NO: 1) in complex with HLA-A24, showing key residue changes introduced during affinity maturation. [Figure 8] Figure 1 shows an image of the computer-modeled structure of the TCR-peptide interface formed by TCR mutant a90b152 when bound to PYLGQMINL (SEQ ID NO: 1) in complex with HLA-A24, with key residue changes introduced during affinity maturation labeled. DETAILED DESCRIPTION OF THE INVENTION

[0196] Array Description HLA-A24 restricted peptides The genes for the original proteins are shown in brackets below.

[0197] SEQ ID NO: 1 (PRAME): PYLGQMINL SEQ ID NO: 81 (PLEC): PYTGQQISL SEQ ID NO: 82 (SYNRG): PYLGQAPFL SEQ ID NO: 83 (PLCB4): PYLSTMINY SEQ ID NO: 84 (LSM14B): PYLGSKISL

[0198] Exemplary scaffold TCR alpha chain (SEQ ID NO: 2) SEQ ID NO:2 is the amino acid sequence of the α chain of an exemplary wild-type (e.g., "scaffold") TCR (comprising an α chain of SEQ ID NO:2 and a β chain of SEQ ID NO:12) that binds to PYLGQMINL (SEQ ID NO:1) complexed with HLA-A24. This TCR is referred to herein as "S11." The α chain comprises a variable domain (SEQ ID NO:3) and a constant domain (SEQ ID NO:4, italics). The CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and the framework regions (FR1, FR2, FR3, and FR4) are in standard text and are designated SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively. The constant domain contains the T48C (numbered according to SEQ ID NO:4) mutation (relative to the wild-type constant domain), shown in bold, to introduce a non-native covalent disulfide bond between the α and β chains.

[0199] SEQ ID NO:2: TIFF2026501721000001.tif18170

[0200] Exemplary scaffold TCR beta chain (SEQ ID NO: 12) SEQ ID NO:12 is the amino acid sequence of the β chain of an exemplary wild-type (e.g., "scaffold") TCR (comprising an α chain of SEQ ID NO:2 and a β chain of SEQ ID NO:12) that binds to PYLGQMINL (SEQ ID NO:1) complexed with HLA-A24. This TCR is referred to herein as "S11." The β chain comprises a variable domain (SEQ ID NO:13) and a constant domain (SEQ ID NO:14, in italics). The CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NOs:15, 16, and 17, respectively, and the framework regions (FR1, FR2, FR3, and FR4) are in standard text and are designated SEQ ID NOs:18, 19, 20, and 21, respectively. The constant domain contains the S57C (numbered according to SEQ ID NO:14) mutation (relative to the wild-type constant domain), shown in bold, to introduce a non-native covalent disulfide bond between the α and β chains. Also shown in bold is the C75A mutation (numbered according to SEQ ID NO: 14), which removes a native cysteine ​​and reduces spurious disulfide formation.

[0201] SEQ ID NO: 12: TIFF2026501721000002.tif24170

[0202] Exemplary Mutant TCR Alpha Chain Variable Domains The following sequence is an exemplary alpha chain variable domain containing mutations relative to the wild-type sequence in SEQ ID NO: 3. The CDRs are underlined and the mutations are shown in bold.

[0203] Alpha chain variable domain "a9" (SEQ ID NO: 22) comprising CDRs (CDR1, CDR2, and CDR3) designated in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 23, respectively, and framework regions (FR1, FR2, FR3, and FR4) designated in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively. This sequence contains the following mutations: S94G, N97H, D99N, which the inventors have found to increase affinity. SEQ ID NO: 22: TIFF2026501721000003.tif12170

[0204] Alpha chain variable domain "a18" (SEQ ID NO: 24) comprising CDRs (CDR1, CDR2, and CDR3) designated in SEQ ID NO: 5, SEQ ID NO: 25, and SEQ ID NO: 23, respectively, and framework regions (FR1, FR2, FR3, and FR4) designated in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively. This sequence contains the following mutations: T51I, S52G, A53N, A54D, S94G, N97H, D99N, which have been found by the inventors to increase affinity. SEQ ID NO: 24: TIFF2026501721000004.tif12170

[0205] Alpha chain variable domain "a77" (SEQ ID NO: 26) comprising CDRs (CDR1, CDR2, and CDR3) designated in SEQ ID NO: 5, SEQ ID NO: 27, and SEQ ID NO: 23, respectively, and framework regions (FR1, FR2, FR3, and FR4) designated in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 28, and SEQ ID NO: 11, respectively. This sequence contains the following mutations: T51I, S52G, A53N, S94G, N97H, D99N, which the inventors found to increase affinity, A54V and N61Q, which reduce the risk of deamidation. SEQ ID NO: 26: TIFF2026501721000005.tif12170

[0206] The alpha chain variable domain "a90" (SEQ ID NO: 29) comprises the CDRs (CDR1, CDR2, and CDR3) designated in SEQ ID NO: 5, SEQ ID NO: 27, and SEQ ID NO: 30, respectively, and the framework regions (FR1, FR2, FR3, and FR4) designated in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 28, and SEQ ID NO: 11, respectively. This sequence contains the following mutations: T51I, S52G, A53N, S94G, N97H, D99N, which the inventors have found to increase affinity, A54V and N61Q, which reduce the risk of deamidation, and R98H, which has been found to increase affinity and stability. SEQ ID NO: 29: TIFF2026501721000006.tif12170

[0207] Exemplary Mutant TCR β Chain Variable Domains The following sequence is an exemplary beta chain variable domain containing mutations relative to the wild-type sequence in SEQ ID NO: 13. The CDRs are underlined and the mutations are shown in bold.

[0208] A beta chain variable domain "b49" (SEQ ID NO: 31) comprising the CDRs (CDR1, CDR2, and CDR3) designated in SEQ ID NO: 32, SEQ ID NO: 16, and SEQ ID NO: 33, respectively, and the framework regions (FR1, FR2, FR3, and FR4) designated in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively. This sequence contains the following mutations: L30Y, S98N, A103S, E105N, F107S, which have been found by the inventors to increase affinity. SEQ ID NO: 31: TIFF2026501721000007.tif12170

[0209] A beta chain variable domain "b150" (SEQ ID NO: 34) comprising the CDRs (CDR1, CDR2, and CDR3) designated in SEQ ID NO: 32, SEQ ID NO: 35, and SEQ ID NO: 36, respectively, and the framework regions (FR1, FR2, FR3, and FR4) designated in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 21, respectively. This sequence contains the following mutations: L30Y, A103S, E105N, F107S, which the inventors have found to increase affinity; G52A and S98I, which reduce the risk of deamidation; and T13K, L43P, I47F, L61P, F90I, which have been found to increase stability. SEQ ID NO: 34: TIFF2026501721000008.tif12170

[0210] Beta chain variable domain "b152" (SEQ ID NO: 40) comprising CDRs (CDR1, CDR2, and CDR3) designated in SEQ ID NO: 32, SEQ ID NO: 35, and SEQ ID NO: 41, respectively, and framework regions (FR1, FR2, FR3, and FR4) designated in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 21, respectively. This sequence contains the following mutations: L30Y, A103S, E105N, F107S, which have been found by the inventors to increase affinity; G52A and S98I, which reduce the risk of deamidation; T13K, L43P, I47F, L61P, F90I, which have been found to increase stability; and V95I, which has been found to increase affinity and stability. SEQ ID NO: 40: TIFF2026501721000009.tif12170

[0211] Exemplary TCRs The following sequences are TCRs containing exemplary combinations of α and β chain variable domains shown above. The TCRs are designated "aXXbXX" (e.g., "a90b152"), where "aXX" indicates the α chain and "bXX" indicates the β chain. The constant domains are shown in italics. The CDRs are underlined, and mutations relative to the scaffold TCR sequence (i.e., SEQ ID NO:2 or SEQ ID NO:12) are shown in bold.

[0212] a9bwt TCR The TCR "a9bwt" alpha chain sequence (SEQ ID NO:42) comprises the a9 variable domain (SEQ ID NO:22) described above and the constant domain (SEQ ID NO:4) from the scaffold TCR described above: TIFF2026501721000010.tif18170

[0213] The TCR "a9bwt" beta chain sequence (SEQ ID NO: 12) comprises the wild-type variable domain (SEQ ID NO: 13) and the constant domain (SEQ ID NO: 14) from the scaffold TCR described above: TIFF2026501721000011.tif24170

[0214] a18b49 TCR The TCR "a18b49" alpha chain sequence (SEQ ID NO:44) comprises the a18 variable domain (SEQ ID NO:24) described above and the constant domain (SEQ ID NO:4) from the scaffold TCR described above: TIFF2026501721000012.tif18170

[0215] The TCR "a18b49" beta chain sequence (SEQ ID NO:45) comprises the b49 variable domain (SEQ ID NO:31) and the constant domain (SEQ ID NO:14) from the scaffold TCR described above: TIFF2026501721000013.tif24170

[0216] a77b150 TCR The TCR "a77b150" alpha chain sequence (SEQ ID NO:46) comprises the a77 variable domain (SEQ ID NO:26) described above and the constant domain (SEQ ID NO:4) from the scaffold TCR described above: TIFF2026501721000014.tif18170

[0217] The TCR "a77b150" beta chain sequence (SEQ ID NO:47) comprises the b150 variable domain (SEQ ID NO:34) and the constant domain containing the L3M mutation to the scaffold TCR (SEQ ID NO:48): TIFF2026501721000015.tif25170

[0218] a90b152 TCR The TCR "a90b152" alpha chain sequence (SEQ ID NO:49) comprises the a90 variable domain (SEQ ID NO:29) described above and the constant domain (SEQ ID NO:4) from the scaffold TCR described above: TIFF2026501721000016.tif19170

[0219] The TCR "a90b152" beta chain sequence (SEQ ID NO:50) comprises the b152 variable domain (SEQ ID NO:40) and the constant domain (SEQ ID NO:48) containing the L3M mutation to the scaffold TCR: TIFF2026501721000017.tif24170

[0220] Exemplary Anti-CD3 Antibody Sequences Anti-CD3 scFv:U0 SEQ ID NO:51 is the amino acid sequence of an exemplary anti-CD3 scFv designated herein as "U0." The light chain variable domain (VL) is in italics and is designated SEQ ID NO:85. The light chain CDRs (CDR1, CDR2, and CDR3) are underlined, with CDR1 and CDR3 designated as SEQ ID NO:52 and SEQ ID NO:54, respectively. The heavy chain variable domain (VH) is shown in bold and is designated as SEQ ID NO:55. The heavy chain CDRs (CDR1, CDR2, and CDR3) are underlined and are designated as SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58. The glycine-serine linker connecting VL and VH is shown in plain text and is designated as SEQ ID NO:59.

[0221] SEQ ID NO:51: TIFF2026501721000018.tif25170

[0222] Anti-CD3 scFv:U28 SEQ ID NO:60 is the amino acid sequence of another exemplary anti-CD3 scFv, designated herein as "U28." This sequence is identical to SEQ ID NO:51 above, except for two substitutions (T164A and I201F) that are double-underlined. The light chain variable domain (VL) is in italics and is designated SEQ ID NO:85. The light chain CDRs (CDR1, CDR2, and CDR3) are underlined, with CDR1 and CDR3 designated as SEQ ID NO:52 and SEQ ID NO:54, respectively. 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 (CDR1 contains the double-underlined alanine residue) and are designated as SEQ ID NO:62, SEQ ID NO:57, and SEQ ID NO:58. The glycine-serine linker connecting VL and VH is shown in plain text and is designated as SEQ ID NO:59.

[0223] SEQ ID NO:60: TIFF2026501721000019.tif25170

[0224] Exemplary TCR-anti-CD3 fusion sequences a9bwt-U0 "a9bwt-U0" is a binding molecule comprising the TCR "a9bwt" α chain (SEQ ID NO: 42) and a TCR β chain-anti-CD3 fusion (SEQ ID NO: 63). The β chain-anti-CD3 fusion sequence (SEQ ID NO: 63) is shown below. This sequence comprises the U0 anti-CD3 scFv (SEQ ID NO: 51, italics, CDRs underlined) fused to the TCR "a9bwt" β chain (SEQ ID NO: 43, bold, CDRs underlined). The TCR β chain and anti-CD3 scFv sequence are covalently linked via a glycine-serine linker (normal text) designated SEQ ID NO: 64.

[0225] SEQ ID NO:63: TIFF2026501721000020.tif49170

[0226] a18b49-U0 "a18b49-U0" is a binding molecule comprising the TCR "a18b49" α chain (SEQ ID NO: 44) and a TCR β chain-anti-CD3 fusion (SEQ ID NO: 65). The β chain-anti-CD3 fusion sequence (SEQ ID NO: 65) is shown below. This sequence comprises the U0 anti-CD3 scFv (SEQ ID NO: 51, italics, CDRs underlined) fused to the TCR "a18b49" β chain (SEQ ID NO: 45, bold, CDRs underlined). The TCR β chain and anti-CD3 scFv sequence are covalently linked via a glycine-serine linker (plain text) designated SEQ ID NO: 64.

[0227] SEQ ID NO:65: TIFF2026501721000021.tif49170

[0228] a77b150-U0 "a77b150-U0" is a binding molecule comprising the TCR "a77b150" α chain (SEQ ID NO: 46) and a TCR β chain-anti-CD3 fusion (SEQ ID NO: 66). The β chain-anti-CD3 fusion sequence (SEQ ID NO: 66) is shown below. This sequence comprises the U0 anti-CD3 scFv (SEQ ID NO: 51, italics, CDRs underlined) fused to the TCR "a77b150" β chain (SEQ ID NO: 47, bold, CDRs underlined). The TCR β chain and anti-CD3 scFv sequence are covalently linked via a glycine-serine linker (normal text) designated SEQ ID NO: 64.

[0229] SEQ ID NO:66: TIFF2026501721000022.tif48170

[0230] a77b150-U28 "a77b150-U28" is a binding molecule comprising the TCR "a77b150" α chain (SEQ ID NO: 46) and a TCR β chain-anti-CD3 fusion (SEQ ID NO: 67). The β chain-anti-CD3 fusion sequence (SEQ ID NO: 67) is shown below. This sequence comprises the U28 anti-CD3 scFv (SEQ ID NO: 60, italics, CDRs underlined) fused to the TCR "a77b150" β chain (SEQ ID NO: 47, bold, CDRs underlined). The TCR β chain and anti-CD3 scFv sequence are covalently linked via a glycine-serine linker (plain text) designated SEQ ID NO: 64.

[0231] SEQ ID NO:67: TIFF2026501721000023.tif49170

[0232] a90b152-U0 "a90b152-U0" is a binding molecule comprising the TCR "a90b152" α chain (SEQ ID NO: 49) and a TCR β chain-anti-CD3 fusion (SEQ ID NO: 68). The β chain-anti-CD3 fusion sequence (SEQ ID NO: 68) is shown below. This sequence comprises the U0 anti-CD3 scFv (SEQ ID NO: 51, italics, CDRs underlined) fused to the TCR "a90b152" β chain (SEQ ID NO: 50, bold, CDRs underlined). The TCR β chain and anti-CD3 scFv sequence are covalently linked via a glycine-serine linker (normal text) designated SEQ ID NO: 64.

[0233] SEQ ID NO:68: TIFF2026501721000024.tif50170

[0234] a90b152-U28 "a90b152-U28" is a binding molecule comprising the TCR "a90b152" α chain (SEQ ID NO: 49) and a TCR β chain-anti-CD3 fusion (SEQ ID NO: 69). The β chain-anti-CD3 fusion sequence (SEQ ID NO: 69) is shown below. This sequence comprises the U28 anti-CD3 scFv (SEQ ID NO: 60, italics, CDRs underlined) fused to the TCR "a90b152" β chain (SEQ ID NO: 50, bold, CDRs underlined). The TCR β chain and anti-CD3 scFv sequence are covalently linked via a glycine-serine linker (plain text) designated SEQ ID NO: 64.

[0235] SEQ ID NO:69: TIFF2026501721000025.tif50170

[0236] Exemplary Amino Acid Linker Sequences GGGGS (SEQ ID NO: 64), GGGSG (SEQ ID NO: 70), GGSGG (SEQ ID NO: 71), GSGGG (SEQ ID NO: 72), GSGGGP (SEQ ID NO: 73), GGEPS (SEQ ID NO: 74), GGEGGGP (SEQ ID NO: 75), GGEGGGSEGGGS (SEQ ID NO: 76), GGGSGGGG (SEQ ID NO: 77), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 59), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 78), EAAAK (SEQ ID NO: 79), and EAAAKEAAAKEAAAK (SEQ ID NO: 80).

[0237] PYLGQMINL (SEQ ID NO: 1) TCR CDR amino acid residues important for binding to the HLA-A24 complex Using the structures of TCR "S11" and mutant "a90b152" bound to the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex (described in Example 7 and shown in Figures 7 and 8), the amino acid sequences of the CDRs that confer specific binding of the TCR to the pHLA complex were determined based on the key CDR residues that contact the peptide in the complex. In the table below, "X" represents any amino acid. A slash (" / ") represents "or," e.g., "S / T" indicates that the amino acid at the specified position in the sequence can be S (Ser) or T (Thr).

[0238] [Table 1]

[0239] [Table 2]

[0240] [Table 3]

[0241] [Table 4]

[0242] [Table 5]

[0243] [Table 6]

[0244] Other amino acid sequences mentioned herein Other naturally occurring TCR sequences SEQ ID NOs: 86 to 105 are the CDR3 sequences of the α and β chains of native TCRs, namely S1 to S10, described in Example 1. S11 is an exemplary native TCR as described above. The CDR3 sequences for SEQ ID NOs: 86 to 105 are shown in the table in Example 1 below.

[0245] Other mutant TCR sequences containing mutations relative to the exemplary native TCR "S11" The CDRs are underlined and mutations relative to the exemplary native TCR "S11" (alpha chain of SEQ ID NO: 2 and beta chain of SEQ ID NO: 12) are shown in bold.

[0246] a19 alpha chain variable domain (SEQ ID NO: 109): TIFF2026501721000032.tif12170

[0247] a20 alpha chain variable domain (SEQ ID NO: 110): TIFF2026501721000033.tif12170

[0248] a23 alpha chain variable domain (SEQ ID NO: 111): TIFF2026501721000034.tif12170

[0249] a25 alpha chain variable domain (SEQ ID NO: 118): TIFF2026501721000035.tif12170

[0250] a26 alpha chain variable domain (SEQ ID NO: 119): TIFF2026501721000036.tif12170

[0251] b10 β chain variable domain (SEQ ID NO: 106): TIFF2026501721000037.tif12170

[0252] b12 β chain variable domain (SEQ ID NO: 107): TIFF2026501721000038.tif13170

[0253] b13 β chain variable domain (SEQ ID NO: 108): TIFF2026501721000039.tif12170

[0254] b16 β chain variable domain (SEQ ID NO: 112): TIFF2026501721000040.tif13170

[0255] b17 β chain variable domain (SEQ ID NO: 113): TIFF2026501721000041.tif12170

[0256] b19 β chain variable domain (SEQ ID NO: 114): TIFF2026501721000042.tif12170

[0257] b20 β chain variable domain (SEQ ID NO: 115): TIFF2026501721000043.tif12170

[0258] b29 beta chain variable domain (SEQ ID NO: 116: TIFF2026501721000044.tif12170

[0259] b32 β chain variable domain (SEQ ID NO: 117): TIFF2026501721000045.tif13170

[0260] b39 β chain variable domain (SEQ ID NO: 120): TIFF2026501721000046.tif13170

[0261] b42 β chain variable domain (SEQ ID NO: 121): TIFF2026501721000047.tif13170

[0262] b50 β chain variable domain (SEQ ID NO: 123): TIFF2026501721000048.tif12170

[0263] b51 β chain variable domain (SEQ ID NO: 124): TIFF2026501721000049.tif12170 [Example]

[0264] The present invention will be more fully understood by reference to the following examples. However, these examples should not be construed as limiting the scope of the present invention. The examples and embodiments described herein are for illustrative purposes only, and it is understood that various modifications or changes therein will be suggested to those skilled in the art and are included within the scope of this application and the scope of the appended claims.

[0265] Example 1 - Identification of natural TCRs with suitable therapeutic properties PYLGQMINL-HLA-A *TCRs that bind to the 24 complex were isolated from T cell clones obtained from human donors or from TCR phage libraries, and the amino acid sequences of the α and β variable regions were determined. Construction of naive TCR phage libraries has been described previously (e.g., in WO 2015 / 136072, WO 2017 / 046198, and WO 2017 / 046201). Soluble forms of the TCRs were generated by fusing the variable regions to truncated versions of the respective α and β chain constant domains, incorporating non-native interchain disulfide bonds between constant domain residues, as previously described (WO 2003 / 020763).

[0266] The α and β chains were expressed separately in E. coli inclusion bodies. The solubilized inclusion bodies containing the α and β chains were combined. The refolded TCR was then purified by anion exchange and size exclusion chromatography using established methods (Non-Patent Document 13, Non-Patent Document 14).

[0267] To assess the ability of native TCR to recognize the target pHLA complex, binding parameters were obtained by surface plasmon resonance (SPR). Binding to both oxidized and reduced forms of the target peptide was tested separately (position M6 of the target peptide PYLGQMINL can exist in both oxidized and reduced forms). SPR measurements were performed on a BIAcore 8K instrument, a BIAcore 3000 instrument, or a BIAcore T200 instrument. Briefly, biotinylated class I HLA-A complexes were bound to the target peptide. *The 24 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). Biotinylated peptide-HLA monomers were immobilized on a streptavidin-coupled CM-5 sensor chip. Equilibrium binding constants were determined using serial dilutions of soluble TCR. K D The values ​​were calculated using Prism software and the Langmuir binding isotherm equation bound = C × Max / (C + K D ) where "bound" is the equilibrium binding in response units at the injected TCR concentration C and Max is the maximal binding. Unless otherwise indicated, measurements were performed at 25°C in Dulbecco's PBS buffer supplemented with 0.005% P20.

[0268] Eleven native TCRs were identified that bind to the target pHLA complex with affinities in the low μM range. The chain usage and CDR3 sequence of each TCR were determined. The results are summarized in the table below.

[0269] [Table 7]

[0270] To determine the specificity of each native TCR for its target pHLA complex, binding to mimetic peptides was assessed. Potential mimetic peptides were first identified by searching the human proteome for sequences that differed from the target peptide by up to three amino acids. In each case, the mimetic peptides were identified as binding to HLA-A. * 24. Four mimetic peptides were identified, which are shown in the table below. The positions that differ from the target peptide are underlined. The interaction between each native TCR and the mimetic peptides was investigated using the SPR method described above.

[0271] [Table 8]

[0272] Ten of the 11 native TCRs showed detectable recognition of one or more mimetic pHLA complexes (Figure 1). TCR S11 did not bind to any of the four mimetics.

[0273] The recognition profile of the native TCR S11 was further characterized using single alanine substitutions of the target peptide and testing for binding using SPR. The data showed that substitutions with alanine at eight of the nine peptide positions abolished TCR binding, indicating that the S11 TCR has a high level of specificity for the target pHLA (Figure 2).

[0274] Furthermore, the S11 TCR was stimulated with 18 naturally presented irrelevant peptides from HLA-A * Binding to a panel of 24 complexes was assessed. For SPR measurements, irrelevant pHLA was divided into three groups and loaded onto one of three flow cells. Soluble TCR was injected at concentrations of 85.8 μM, 42.9 μM, and 21.4 μM across all flow cells. No significant binding was detected at either concentration, suggesting that soluble wild-type TCR binds peptide-HLA-A. * These results demonstrate that the antibody has a high degree of specificity for the 24 complex.

[0275] Example 2 - Identification of TCR mutants with improved binding As previously described (Non-Patent Document 18), TCR S11 was used as a template to identify mutations with higher affinity. Briefly, a TCR phage library was created using NNK oligonucleotides to generate mutations in the complementarity-determining regions (CDRs). Further improvement of TCR affinity was achieved in a second-generation library using TCRs isolated from the first round. Subsequently, combinations of mutated α and β chains were selected.

[0276] To assess binding, soluble TCRs containing mutated α and β chains were first prepared as bispecific molecules by fusing an anti-CD3 scFv fragment to the N-terminus of the TCR β chain. Binding molecules in this format are, for example, ImmTAC™ molecules containing tebentafusp, sold under the trade name KIMMTRAK™. Such molecules are hereinafter referred to as "TCR-anti-CD3 fusions."

[0277] TCR-anti-CD3 fusion molecules containing the mutated α and β chains were expressed in E. coli and purified as previously described. Yields were calculated from the concentration of purified material, determined by absorbance at 280 nm using a Nanodrop spectrophotometer.

[0278] Binding to the target was assessed by SPR using the same method as described above. For high-affinity interactions, binding parameters were determined by single-cycle kinetic analysis. Five different concentrations of TCR-anti-CD3 fusion were applied in 50 μl min onto a flow cell coated with approximately 100 RU to 200 RU (or 50 RU to 100 RU for a Biacore 8K instrument) of peptide-HLA complex. -1 ~60μl min -1 A flow rate of 1000 bp was used for injections. Typically, 60 μl to 120 μl (or approximately 240 μl for a Biacore 8K instrument) of TCR-anti-CD3 fusion was injected at a highest concentration between 50 nM and 100 nM (or 2 nM to 50 nM for a Biacore 8K instrument), where serial two-fold dilutions were used for the other four injections. The lowest concentration was injected first. To measure the dissociation phase, buffer was injected until 10% or more dissociation occurred, typically after 1 to 3 hours. Kinetic parameters were calculated using BIAevaluation™ software. The dissociation phase was fitted to a monoexponential decay equation, which allowed for the calculation of the half-life. The equilibrium constant K D k off / k on It was calculated from.

[0279] In the first round of mutagenesis, TCR mutants were identified that had higher (i.e., stronger) binding affinities for the target pHLA (reduced form) than the wild type, ranging from 25 nM to 171 nM, as shown in the table below.

[0280] [Table 9]

[0281] The mutant a9bwt was selected as a template for further manipulation.

[0282] In further rounds of mutagenesis, TCR mutants were identified with binding affinities for the target pHLA (reduced form) in the range of 3 nM to 37 nM, as shown in the table below.

[0283] [Table 10]

[0284] Further combinations of the mutated TCR chains resulted in TCR variants with binding affinities for the target pHLA (reduced form) in the range of 101 pM to 1.3 nM, as shown in the table below.

[0285] [Table 11]

[0286] The TCR α and TCR β chains a18 and b19 were selected for combination with additional mutant chains. The table below shows TCR variants with binding affinities to the target pHLA (reduced form) ranging from 17.7 pM to 817 pM, as shown in the table below.

[0287] [Table 12]

[0288] These data demonstrate that soluble, high-affinity TCR variants of the S11 TCR can be generated that have supraphysiological affinity and binding half-life for the target.

[0289] Example 3 - TCR-anti-CD3 fusion molecules exhibit potent T cell activation in the presence of antigen-positive target cells T cell activation was determined by measuring IFNγ secretion using an ELISpot assay. The assay was performed using a human IFN-γ ELISPOT kit (BD Biosciences) according to the manufacturer's instructions. Peripheral blood mononuclear cells (PBMCs) isolated from fresh donor blood were used as effector cells.

[0290] In this example, the following cancer cell lines were used as antigen-positive target cells: CHP-212 (neuroblastoma), HT144 (melanoma), and RMGI (ovarian cancer). Additionally, the following antigen-negative cell lines were used: SW620 (colon adenocarcinoma) and NUDUL1 (anaplastic lymphoma). Data were plotted using PRISM software, and EC values ​​were calculated from the curves. 50 values ​​were calculated.

[0291] In both cases, concentration-dependent T cell reactivity was observed in the presence of antigen-positive cells, as shown in the table below.

[0292] [Table 13]

[0293] Figure 3 shows graphical data for four exemplary TCR-anti-CD3 fusion molecules (a18b19, a18b49, a25b19, and a26b19) from the table above. In each case, T cell reactivity against antigen-positive cells was observed in the low pM range. At TCR-anti-CD3 fusion concentrations below 1 nM, no detectable reactivity against antigen-negative cell lines was observed, indicating a wide therapeutic window between on-target and nonspecific binding. These data demonstrate that TCR-anti-CD3 fusion molecules can mediate potent redirection of T cells against antigen-positive cell lines.

[0294] Example 4 - TCR-anti-CD3 fusion molecules exhibit a wide affinity window between recognition of target and mimic pHLA complexes To further confirm the specificity of the TCR-anti-CD3 fusion molecule, SPR was performed as described above to compare target binding with recognition to the four mimetic peptides identified above.

[0295] No binding to Mim2 and Mim4 was observed. Binding to Mim1 and Mim3 was detected, but the interactions were weak, especially compared to target recognition. The data are shown in the table below. Figure 4 shows a graphical representation of the data.

[0296] [Table 14]

[0297] These data demonstrate a wide affinity window of at least two to three orders of magnitude between binding to the target and the mimetic.

[0298] Example 5 - TCR-anti-CD3 fusion molecules do not activate T cells at therapeutic concentrations in the presence of cells from normal tissues To further explore their suitability for therapeutic use, the TCR-anti-CD3 fusion molecules were tested for nonspecific activation in the presence of normal cells derived from healthy human tissues using the same ELISPOT methodology described above. The panel of normal cells used in this example was derived from lung, heart, and muscle tissue: lung-bronchial epithelial cells (HBEpiC14), muscle-skeletal myoblasts (HSkMM21), heart-aortic endothelial cells (HAoEC3a), and lung-fibroblasts (HPF18). HT144 and SW620 cells were used as target-positive and target-negative cell lines, respectively.

[0299] Results for three TCR-anti-CD3 fusion molecules: a18b19, a25b19, and a18b49 are shown in Figure 5. The data indicate minimal reactivity against normal cells at concentrations below 1 nM. Further analysis showed an approximately 200-fold spread between the lowest concentration eliciting reactivity against antigen-positive cells (5 pM) and the lowest concentration at which reactivity against normal cells was observed (1 nM).

[0300] Molecule a18b49 was selected for further optimization.

[0301] Example 6 - Further amino acid substitutions reduce manufacturing burden while maintaining other desirable therapeutic properties Additional amino acid substitutions were made in the TCR α and β chain variable domains of the a18 and b49 chains to improve manufacturability while maintaining the affinity, potency, and specificity of the corresponding TCR-anti-CD3 fusion molecules.

[0302] First, based on sequence analysis, five residues prone to deamidation were identified: three in the α-chain and two in the β-chain. Deamidation was abundant at four of the five sites, and four of the five sites showed an increased abundance of deamidation after two weeks at +30°C, as shown in the table below.

[0303] [Table 15]

[0304] Various amino acid substitutions were tested at each site and adjacent positions to reduce the risk of deamidation. Incorporation of V at position 54 and Q at position 61 of the α chain, and A at position 52 and I at position 98 of the β chain (based on a18 and b49 numbering) was found to significantly reduce the occurrence of deamidation while maintaining other desirable properties.

[0305] Next, we identified amino acid substitutions that contribute to the increased thermal stability of the molecule, as determined by differential scanning fluorimetry (DSF). Using DSF, the melting temperature (Tm) of a18b49 was determined to be 53.7°C. Five mutations were identified, one α-chain mutation and four β-chain framework mutations that increase Tm: R98H in the α-chain based on the a18 numbering, and T13K, L43P, I47F, and F90I in the β-chain based on the b49 numbering.

[0306] Molecules combining these mutations were prepared and tested.

[0307] join Binding to the target was determined at both 25°C and 37°C by SPR as described above.

[0308] The results, shown in the table below, show that the molecule retained pM affinity.

[0309] [Table 16]

[0310] Potency - T cell activation Potency was determined in a cellular assay. In this example, the TCR-anti-CD3 fusion molecule contains a mutant anti-CD3 sequence designated U28, previously described for molecules a77b150 and a90b152 (WO 2020 / 157210). T cell activation was determined by the IFNγ ELISPOT assay described above. The following cancer cell lines were used as antigen-positive target cells: HT144 (melanoma), RMGI (ovarian cancer), and SKNAS (neuroblastoma). Additionally, the following antigen-negative cell line was used: SW620 (colon adenocarcinoma). Data were plotted using PRISM software, and EC values ​​were extracted from the curves. 50 values ​​were calculated.

[0311] The data obtained is shown in FIG.

[0312] Efficacy - Cell Killing To examine the ability of a77b150U28, a90b152U0, and a90b152U28 to kill antigen-positive cancer cells, a killing assay was performed using the xCelligence platform (Agilent) equipped with an appropriate 96-well plate for impedance reading (xCELLigence E-plate 96 PET, part number 300600900). PBMCs were used as effector cells. Target cells were grown in E-plates for 24 hours before adding the effector cells and test molecules. In this example, HT144 (melanoma) cells and HEC-6 (endometrial adenocarcinoma) cells were used as target-positive cancer cells. The cell lines were titrated and optimized to reach a cell index of approximately 1 after 24 hours of culture in E-plates (HT144: 10,000 cells per well, HEC6: 1 5,000 cells per well). To avoid too rapid depletion of nutrients in the medium, effectors are plated at different ratios depending on the number of target cells (HT144 E:T ratio = 5:1, HEC6 E:T ratio = 10:1). The percentage of cell lysis was determined using normalized cell index (impedance measurement) at various time points over 73 hours. The % of cell lysis at 73 hours was used to plot a dose-response curve. EC 50Values ​​were calculated from the curve and are shown in the table below.

[0313] [Table 17]

[0314] These data demonstrate that TCR-anti-CD3 fusion molecules mediate potent T cell activation and cancer cell killing, with EC 50 It indicates that it has a value.

[0315] Normal cell reactivity Reactivity against normal cells was performed as described above using the TCR-anti-CD3 fusion molecules a77b150U28, a90b152U0, and a90b152U28. The table below shows the lowest concentrations at which reactivity was observed against cells from normal tissues: lung fibroblasts (HPF18), muscle skeletal myoblasts (HSkMM21), and heart aortic endothelial cells (HAoEC3a).

[0316] [Table 18]

[0317] No substantial reactivity was observed at concentrations below 1 nM.

[0318] Binding to mimetic peptides The TCR-anti-CD3 fusion molecule a90b152U28 was evaluated for binding to the mimetic peptide at both 25°C and 37°C using SPR as described above.

[0319] [Table 19]

[0320] Again, no binding was observed to peptides Mim2 and Mim4, whereas binding to Mim1 and Mim3 was in the nM range, thus maintaining a wide affinity window between the target peptide and the mimetic peptides.

[0321] Serum stability TCR-anti-CD3 fusion molecules were diluted in human serum (10 μg / ml) and incubated at 37° C. for up to 168 hours. The presence of high and low molecular weight species (HMW / LMW) and % monomer were determined by SEC UPLC. The data are shown in the table below.

[0322] [Table 20]

[0323] These data indicate that a77b150U28 and a90b152U28 have acceptable stability in human serum.

[0324] Overall, these data confirm that the TCR-anti-CD3 fusion molecules of the present invention have desirable therapeutic properties.

[0325] Example 7 - Structural analysis of TCR-pHLA binding Structural analysis of the S11 TCR bound to the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex was performed using X-ray crystallography. Unique structural features that confer potency and specificity were identified.

[0326] method X-ray crystallography. Crystals were grown by vapor diffusion via the sitting drop method using MRC 2-well crystallization plates. Using a Gryphon dispensing robot (Art Robbins), 150 nL of 10 mg / mL to 15 mg / mL TCR-pHLA complexes (mixed at a 1:1.1 molar ratio) was added to 150 nL of reservoir solution. Plates were then incubated at 20 °C and imaged using a ROCK IMAGER 1000 (Formulatrix). Crystals selected for further analysis were cryoprotected using 30% ethylene glycol and then flash-cooled in liquid nitrogen. Diffraction data were collected at Diamond Light Source (Didcot, UK) on several different beamlines and processed through automated pipelines: xia2 DIALS (Winter (2010). J Appl Crystallogr. 43, 186-190; Winter et al. (2018). Acta Crystallogr Sect D Struct Biology. 74, 85-97), 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). The structure of the TCR-pHLA complex was solved by molecular replacement using Phaser (McCoy et al. (2007). J. Appl. Crystallogr. 40, 658-674), a model was 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 of this was done 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 various TCR molecules, protein structures with high sequence similarity to the α and β sequences were individually searched in the PDB and used as models. For HLA-A2-B2m, PDB 6RPA (with the TCR removed) was used. Computational modeling of the TCR, referred to herein as "a90b152," was performed using the "S11" TCR as a template using the Molecular Operating Environment (MOE) package (version 2022.02, Chemical Computing Group ULC, Canada), with refinement using QuickPrep.

[0327] TCR docking geometry angle calculations: The following angle calculations were adopted from Rudolph et al. (2006). Annu Rev Immunol. 24, 419-466.

[0328] a. Crossing angle. This angle was calculated by generating two vectors: the HLA groove vector and the TCR cysteine ​​vector (i.e., the "TCR inter-domain vector"). The HLA groove vector follows the two parallel HLA helices in the direction from the N-terminus to the C-terminus of HLA helix 1 and passes through the HLA center of mass. The TCR cysteine ​​vector (i.e., the "TCR inter-domain vector") connects characteristic cysteines within the two variable regions (i.e., intrachain disulfide bonds) and points from the intrachain disulfide bond in the α chain variable region toward the intrachain disulfide bond in the β chain variable region. The crossing angle was defined as the angle between the TCR cysteine ​​vector and the HLA groove vector.

[0329] b. Tilt angle. The TCR symmetry vector corresponds to the pseudo-twofold symmetry axis of the TCR variable subunit, points toward its CDRs, and passes through the center of mass of the TCR. The tilt angle was calculated between the TCR symmetry axis and the HLA groove vector.

[0330] c. Roll angle. A second HLA vector (HLA v2) was generated perpendicular to the HLA groove vector and points from HLA helix 1 towards HLA helix 2. The two vectors intersect at the center of gravity of the HLA helices. The roll angle was calculated between HLA v2 and the TCR symmetry vector.

[0331] Interaction analysis. Residues between the TCR and pHLA were considered to be in contact if the measured distance between any atom from the TCR residue and any atom from the peptide or HLA residue was within 4.1 Å.

[0332] result The crystal structure revealed that S11 binds to its cognate pHLA (i.e., the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex) at a crossing angle of approximately 43 degrees. Four of the six CDRs (αCDR3, βCDR1, βCDR2, and βCDR3) directly interacted with the PYLGQMINL (SEQ ID NO: 1) peptide, contacting peptide residues between positions 1 and 8. The TCR β chain, particularly CDR3β, dominated the interaction between the TCR and the peptide. The following table summarizes the buried surface area and binding geometry calculations for the interaction between S11 TCR and the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex.

[0333] [Table 21]

[0334] [Table 22]

[0335] Identification of peptide and TCR CDR residues important for binding The crystal structure of the S11 TCR bound to PYLGQMINL (SEQ ID NO: 1) in complex with HLA-A24 was further analyzed to identify key residues within the interface between the peptide and the TCR CDRs. As can be seen from the crystal structure shown in Figure 7, peptide positions P1, Q5, I7, and N8 of PYLGQMINL (SEQ ID NO: 1) form the major antigen contact points on the peptide, with residues at these positions having exposed side chains facing the TCR interface. The high-affinity mutants of the S11 TCR described in Examples 1-6 have enhanced contacts with these four exposed peptide positions due to the inclusion of bulkier residues within the TCR CDRs. For example, the TCR variant referred to herein as "a90b152" contains bulkier residues at key CDR positions near the peptide (i.e., R98H and R97H in aCDR3, A103S, S98I, and V95I in bCDR3, and L30Y in bCDR1) and has affinity for the pHLA complex in the low pM range. The interface between a90b152 and the PYLGQMINL (SEQ ID NO: 1) peptide was computationally modeled based on the TCR crystal structure of S11 and is shown in Figure 8.

[0336] The CDR residues of the S11 TCR that are important for binding to the PYLGQMINL (SEQ ID NO: 1) peptide were also determined from the crystal structure based on inter-residue proximity, and are shown in the table below as bold, double-underlined letters.

[0337] [Table 23]

[0338] To demonstrate that the high-affinity TCR mutants described in Examples 1-6 maintain the same or similar peptide contacts as the S11 TCR, the crystal structure of the S11 TCR bound to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24 was used as a template to determine the CDR residues in contact with the peptide of the TCR mutant a90b152 by computer modeling. The CDR positions of a90b152 important for binding to the PYLGQMINL (SEQ ID NO: 1) peptide were similar to those of the S11 TCR. These are shown in the table below.

[0339] [Table 24]

[0340] Comparison of the S11 TCR structure with TCRs on alternative scaffolds Additional crystal structures were obtained for TCRs derived from various native TCRs and used to compare close contact points and interaction profiles with those of the S11 TCR. As shown in the table below, direct contact between the S11 TCR and the PYLGQMINL (SEQ ID NO: 1) peptide resulted in the highest number of peptide contacts (8 residues), with no other TCR having contact with more than six peptide residues. Furthermore, only the S11 TCR showed contact with the exposed proline residue at position 1 of the peptide. These data indicate that the binding geometry appears to be crucial for generating the largest interface with the peptide and resulting in a highly specific interaction.

[0341] The following table shows the binding geometry of the S11 TCR compared to other TCRs.

[0342] [Table 25]

Claims

1. 1. A binding molecule comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, said binding molecule having the property of binding to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24, wherein each of said alpha chain variable domain and said beta chain variable domain comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR is a framework region and CDR is a complementarity determining region, and wherein: (a) when the binding molecule binds to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24, the binding molecule contacts at least residue P1, residue Q5, residue 17, and residue N8 of the PYLGQMINL (SEQ ID NO: 1) peptide; and / or (b) a binding molecule wherein CDR3 of the α chain comprises the sequence X-X-X-X-P-N / H-R / H-X-X-X-X (SEQ ID NO: 125), CDR1 of the β chain comprises the sequence X-X-X-L / Y-X (SEQ ID NO: 126), CDR2 of the β chain comprises the sequence X-Y-X-X-X-X (SEQ ID NO: 127), and CDR3 of the β chain comprises the sequence X-X-X-V / I-W-S-S / I / N-G-X-X-S-A / S-X-X-X (SEQ ID NO: 128), where X is any amino acid.

2. CDR3 of the alpha chain comprises the sequence V / I / L-V / I / L-S / G-A / G-P-N / H-R / H-D / N-D / E-K / R / H-I / V / L-I / V / L (SEQ ID NO: 134); CDR1 of the beta chain comprises the sequence S / T-G / A-D / E-L / Y-S / T (SEQ ID NO: 136); CDR2 of the beta chain comprises the sequence Y / W / F-Y-N / Q-G / AE / DE / D (SEQ ID NO: 138), and 2. The binding molecule of claim 1, wherein the CDR3 of the beta chain comprises the sequence A / G-S / T-S / T-V / I-W-S-S / I / N-G-G / A-A / G-S-A / S-G / A-E / N-L / I / V-F / S (SEQ ID NO: 140).

3. the CDR3 of the alpha chain comprises the sequence VVGAPHHNDKII (SEQ ID NO: 30), optionally with 1, 2, 3, or 4 mutations at any of positions 1 to 4 or positions 8 to 12 of SEQ ID NO: 30; CDR1 of the β chain comprises the sequence SGDYS (SEQ ID NO:32), optionally with one, two, or three mutations at any of positions 1 to 3 or position 5 of SEQ ID NO:32; CDR2 of the β chain comprises the sequence YYNAEE (SEQ ID NO:35), optionally with one, two, or three mutations at position 1 or any of positions 3 through 6 of SEQ ID NO:35; and 3. The binding molecule of claim 1 or 2, wherein the CDR3 of the beta chain comprises the sequence ASSIWSIGGASSGNLS (SEQ ID NO: 41), optionally with 1, 2, 3, 4, or 5 mutations at any of positions 1 to 3, 9, 10, or 13 to 16 of SEQ ID NO:

41.

4. CDR1 of the alpha chain comprises the sequence SSYSPS (SEQ ID NO: 5), optionally with 1, 2 or 3 mutations, and 4. The binding molecule of claim 1, wherein the CDR2 of the alpha chain comprises the sequence YIGNVTLV (SEQ ID NO: 27), optionally with 1, 2, 3 or 4 mutations.

5. CDR1 of the alpha chain comprises the sequence S / T-S / TY / W / FS / TP / GS / T (SEQ ID NO: 129), and 5. The binding molecule of any one of claims 1 to 4, wherein CDR2 of the alpha chain comprises the sequence Y / W / F-T / I-S / G-A / N-A / D / V-T / S-L / I / V-V / I / L (SEQ ID NO: 131).

6. 1. A binding molecule comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, said binding molecule having the property of binding to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24, wherein each of said alpha chain variable domain and said beta chain variable domain comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR is a framework region and CDR is a complementarity determining region, and wherein: (a) the α chain CDRs have the following sequences: CDR1 - SSYSPS (SEQ ID NO: 5) optionally with 1, 2 or 3 mutations; CDR2 - YIGNVTLV (SEQ ID NO: 27), optionally with 1, 2, 3, or 4 mutations; CDR3 - VVGAPHHNDKII (SEQ ID NO: 30), optionally with 1, 2, 3, or 4 mutations; and / or (b) the CDRs of the β chain have the following sequences: CDR1 - SGDYS (SEQ ID NO: 32), optionally with 1, 2 or 3 mutations; CDR2 - YYNAEE (SEQ ID NO: 35), optionally with 1, 2, or 3 mutations; CDR3 - ASSIWSIGGASSGNLS (SEQ ID NO: 41), optionally with 1, 2, 3, 4, or 5 mutations; A binding molecule having the formula:

7. The binding molecule of claim 6, wherein the mutation(s) in the CDRs of the alpha chain are selected from I51T, G52S, N53A, V54A, D54A, G94S, H97N, H98R, and N99D, numbered according to SEQ ID NO:

29.

8. 8. The binding molecule of claim 6 or 7, wherein the mutation(s) in the CDRs of the beta chain are selected from Y30L, A52G, I95V, I98S, S103A, N105E, and S107F numbered according to SEQ ID NO:

40.

9. 9. The binding molecule of any one of claims 1 to 8, wherein the binding molecule contacts at least 7, or at least 8, peptide residues when binding to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24.

10. 10. The binding molecule of any one of claims 1 to 9, wherein the binding molecule contacts all of the peptide residues at positions 1 to 8 of PYLGQMINL (SEQ ID NO: 1) when binding to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24.

11. 11. The binding molecule of any one of claims 1 to 10, wherein the binding molecule binds to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24 at a crossing angle in the range of 35° to 55°, or preferably in the range of 38° to 48°.

12. 12. The binding molecule of any one of claims 1 to 11, wherein the binding molecule binds to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24 at a tilt angle in the range of -10° to 10°, preferably in the range of -1° to 9°.

13. 13. The binding molecule of any one of claims 1 to 12, wherein the binding molecule binds to PYLGQMINL (SEQ ID NO: 1) complexed with HLA-A24 at a roll angle in the range of -10° to 10°, preferably in the range of -6° to 4°.

14. The following combinations of α-chain CDRs and β-chain CDRs: (a) the amino acid sequences of CDR1, CDR2, and CDR3 of the α chain of SSYSPS (SEQ ID NO: 5), YTSAATLV (SEQ ID NO: 6), and VVSAPNRDDKII (SEQ ID NO: 7), respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the β chain of SGDLS (SEQ ID NO: 15), YYNGEE (SEQ ID NO: 16), and ASSVWSSGGASAGELF (SEQ ID NO: 17), respectively; (b) the amino acid sequences of CDR1, CDR2, and CDR3 of the α chain of SSYSPS (SEQ ID NO: 5), YTSAATLV (SEQ ID NO: 6), and VVGAPHRNDKII (SEQ ID NO: 23), respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the β chain of SGDLS (SEQ ID NO: 15), YYNGEE (SEQ ID NO: 16), and ASSVWSSGGASAGELF (SEQ ID NO: 17), respectively; (c) the amino acid sequences of CDR1, CDR2, and CDR3 of the α chain of SSYSPS (SEQ ID NO: 5), YIGNDTLV (SEQ ID NO: 25), and VVGAPHRNDKII (SEQ ID NO: 23), respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the β chain of SGDYS (SEQ ID NO: 32), YYNGEE (SEQ ID NO: 16), and ASSVWSNGGASSGNLS (SEQ ID NO: 33), respectively; (d) the amino acid sequences of CDR1, CDR2, and CDR3 of the α chain of SSYSPS (SEQ ID NO: 5), YIGNVTLV (SEQ ID NO: 27), and VVGAPHRNDKII (SEQ ID NO: 23), respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the β chain of SGDYS (SEQ ID NO: 32), YYNAEE (SEQ ID NO: 35), and ASSVWSIGGASSGNLS (SEQ ID NO: 36), respectively; or (e) the amino acid sequences of CDR1, CDR2, and CDR3 of the α chain of SSYSPS (SEQ ID NO: 5), YIGNVTLV (SEQ ID NO: 27), and VVGAPHHNDKII (SEQ ID NO: 30), respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the β chain of SGDYS (SEQ ID NO: 32), YYNAEE (SEQ ID NO: 35), and ASSIWSIGGASSGNLS (SEQ ID NO: 41), respectively; The binding molecule of any one of claims 1 to 13, comprising one of:

15. The framework regions of the alpha chain variable domain have the following sequence: FR1 - AQSVTQLDSHVSVSEGTPVLLRCNYS (SEQ ID NO: 8), optionally with 1, 2, or 3 mutations; FR2 - LFWYVQHPNKGLQLLLK (SEQ ID NO: 9), optionally with 1, 2, or 3 mutations; FR3 - KGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFC (SEQ ID NO: 10), optionally with 1, 2, or 3 mutations; FR4—FGKGTRLHILP (SEQ ID NO: 11), optionally with 1, 2, or 3 mutations; and / or The framework regions of the β chain variable domain have the following sequence: FR1 - DSGVTQTPKHLITATGQRVTLRCSPR (SEQ ID NO: 18), optionally with 1, 2, or 3 mutations; FR2—VYWYQQSLDQGLQFLIQ (SEQ ID NO: 19), optionally with 1, 2, or 3 mutations; FR3 - RAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFC (SEQ ID NO: 20), optionally with 1, 2, or 3 mutations; FR4—FGEGSRLTVL (SEQ ID NO: 21), optionally with 1, 2, or 3 mutations; The binding molecule of any one of claims 1 to 14, comprising:

16. 16. The binding molecule of claim 15, wherein the framework region of the alpha chain variable domain comprises an N61Q mutation numbered according to SEQ ID NO:

3.

17. 17. The binding molecule of claim 15 or 16, wherein the framework regions of the beta chain variable domain comprise one or more of the following mutations, numbered according to SEQ ID NO: 13: T13K, L43P, I47F, L61P, and F90I.

18. 17. The binding molecule of claim 15 or 16, wherein the framework regions of the beta chain variable domain comprise the following mutations numbered according to SEQ ID NO: 13: T13K, L43P, I47F, L61P, and F90I.

19. 19. The binding molecule of claim 1, wherein the α chain variable domain comprises an amino acid sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:29, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity to any one of SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:29, and the β chain variable domain comprises an amino acid sequence set forth in any one of SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:40, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity to any one of SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:

40.

20. The following combinations of α-chain variable domains and β-chain variable domains: (a) an α chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 22 and a β chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 13; (b) an α chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 24 and a β chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 31; (c) an α chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 26 and a β chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 34; or (d) an α chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 29 and a β chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 40; The binding molecule of any one of claims 1 to 19, comprising one of:

21. 21. The binding molecule of any one of claims 1 to 20, wherein the alpha chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 29 and the beta chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:

40.

22. 22. The binding molecule of any one of claims 1 to 21, comprising the extracellular region of the TCR alpha chain constant domain, optionally truncated at the C-terminus by up to 15 amino acids, and / or the extracellular region of the TCR beta chain constant domain, optionally truncated at the C-terminus by up to 15 amino acids.

23. 23. The binding molecule of claim 22, comprising the extracellular region of a TCR beta chain constant domain comprising the L3M mutation numbered according to SEQ ID NO:

14.

24. 24. The binding molecule of claim 22 or 23, wherein the residues of the TCR alpha chain constant domain and the residues of the TCR beta chain constant domain are linked by a non-natural covalent disulfide bond.

25. the extracellular region of the TCR alpha chain constant domain comprises the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 4; and / or 25. The binding molecule of any one of claims 22 to 24, wherein the extracellular region of the TCR β chain constant domain comprises the amino acid sequence set forth in SEQ ID NO: 48, or an amino acid sequence having at least 90% identity to the sequence set forth in SEQ ID NO:

48.

26. 26. The binding molecule of any one of claims 1 to 25, in a single chain format of the type Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, where Vα and Vβ are the TCR alpha and TCR beta variable regions, respectively, Cα and Cβ are the TCR alpha and TCR beta constant regions, respectively, and L is a linker sequence.

27. 27. The binding molecule of any one of claims 1 to 26, comprising two or more polypeptide chains, wherein the TCR alpha chain variable domain and the TCR beta chain variable domain are contained in separate polypeptide chains.

28. 28. The binding molecule of any one of claims 1 to 27, comprising or consisting solely of a TCR comprising the TCR alpha chain variable domain and the TCR beta chain variable domain.

29. 29. The binding molecule of claim 28, wherein the TCR is a soluble TCR.

30. The binding molecule a TCR alpha chain comprising an amino acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:49, or an amino acid sequence having at least 90% identity, for example at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:49; a TCR β chain comprising an amino acid sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 50, or an amino acid sequence having at least 90%, for example at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 50; 30. The binding molecule of claim 28 or 29, comprising:

31. (a) a TCR α chain comprising the amino acid sequence of SEQ ID NO: 42, and a TCR β chain comprising the amino acid sequence of SEQ ID NO: 12; (b) a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 44, and a TCR beta chain comprising the amino acid sequence of SEQ ID NO: 45; (c) a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 46 and a TCR beta chain comprising the amino acid sequence of SEQ ID NO: 47; or (d) a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 49, and a TCR beta chain comprising the amino acid sequence of SEQ ID NO: 50; The binding molecule of any one of claims 28 to 30, comprising:

32. 32. The binding molecule of any one of claims 1 to 31, which is multispecific, optionally the binding molecule is bispecific.

33. 33. The binding molecule of any one of claims 1 to 32, comprising an antigen-binding portion of an antibody capable of binding to an antigen.

34. 34. The binding molecule of claim 33, wherein the antigen-binding portion comprises a heavy chain variable region (VH) and an antibody light chain variable region (VL).

35. 35. The binding molecule of claim 33 or 34, wherein the antigen is a T cell surface antigen.

36. The binding molecule of any one of claims 33 to 35, wherein the antigen is CD3.

37. The binding molecule of any one of claims 34 to 36, wherein the binding molecule comprises a single chain variable fragment (scFv) comprising the VH and VL.

38. (a) the VH has the following sequence: CDR1 - GYSFTGYT (SEQ ID NO: 56) or GYSFTGYA (SEQ ID NO: 62), CDR2-INPYKGVS (SEQ ID NO: 57), and CDR3 - ARSGYYGDSDWYFDV (SEQ ID NO: 58), and (b) the VL has the following sequence: CDR1-QDIRNY (SEQ ID NO: 52), CDR2-YTS, and CDR3-QQGNTLPWT (SEQ ID NO: 54), 38. The binding molecule of any one of claims 34 to 37, comprising a CDR having:

39. the VH comprises an amino acid sequence set forth in SEQ ID NO:55 or SEQ ID NO:61, or an amino acid sequence having at least 90% identity, for example at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:55 or SEQ ID NO:61; and 39. The binding molecule of any one of claims 34 to 38, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 85 or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 85, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

40. 40. The binding molecule of any one of claims 34 to 39, wherein the VH or VL is covalently linked to the C-terminus or N-terminus of the TCR alpha chain or the TCR beta chain, optionally via a linker sequence.

41. 41. The binding molecule of claim 40, wherein the VH or VL is covalently linked to the C-terminus or N-terminus of the TCR alpha chain or the TCR beta chain via a linker sequence selected from GGGGS (SEQ ID NO: 64), GGGSG (SEQ ID NO: 70), GGSGG (SEQ ID NO: 71), GSGGG (SEQ ID NO: 72), GSGGGP (SEQ ID NO: 73), GGEPS (SEQ ID NO: 74), GGEGGGP (SEQ ID NO: 75), GGEGGGSEGGGGS (SEQ ID NO: 76), GGGSGGGG (SEQ ID NO: 77), GGGGSGGGGGSGGGGGSGGGGS (SEQ ID NO: 59), GGGGSGGGGGSGGGGGSGGGGS (SEQ ID NO: 78), EAAAK (SEQ ID NO: 79), and EAAAKEAAAKEAAAK (SEQ ID NO: 80).

42. 42. The binding molecule of claim 40 or 41, wherein the C-terminus of the VH is covalently linked to the N-terminus of the TCR beta chain, optionally via a linker comprising the amino acid sequence set forth in SEQ ID NO:

64.

43. an alpha chain amino acid sequence set forth in any one of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:49, or an alpha chain amino acid sequence having at least 90% identity, for example at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:49; and a beta chain-anti-CD3 amino acid sequence set forth in any one of SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69, or a beta chain amino acid sequence having at least 90% identity, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69; 43. The binding molecule of claim 42, comprising:

44. (a) an α chain amino acid sequence set forth in SEQ ID NO: 42, and a β chain-anti-CD3 amino acid sequence set forth in SEQ ID NO: 63; (b) an α chain amino acid sequence set forth in SEQ ID NO: 44, and a β chain-anti-CD3 amino acid sequence set forth in SEQ ID NO: 65; (c) an α chain amino acid sequence set forth in SEQ ID NO: 46 and a β chain-anti-CD3 amino acid sequence set forth in SEQ ID NO: 66 or SEQ ID NO: 67, or (d) an α chain amino acid sequence set forth in SEQ ID NO: 49, and a β chain-anti-CD3 amino acid sequence set forth in SEQ ID NO: 68 or SEQ ID NO: 69; 43. The binding molecule of claim 42, comprising:

45. 43. The binding molecule of claim 42, comprising an alpha chain amino acid sequence shown in SEQ ID NO:49 and a beta chain-anti-CD3 amino acid sequence shown in SEQ ID NO:

69.

46. a first polypeptide chain comprising the TCR alpha chain variable domain and a VH or VL of the antibody; a second polypeptide chain comprising the TCR β chain variable domain and the other of the VH and VL of the antibody; where:

40. The binding molecule of any one of claims 34 to 36, claim 38, or claim 39, wherein each polypeptide chain associates such that the binding molecule is capable of simultaneously binding to the PYLGQMINL (SEQ ID NO: 1) HLA-A24 complex and the antigen of the antibody.

47. 47. The binding molecule of any one of claims 1 to 46, associated with a detectable label, and / or a therapeutic agent, and / or a pharmacokinetic-modifying moiety.

48. 48. The binding molecule of any one of claims 1 to 47, comprising an Fc domain.

49. 49. A nucleic acid encoding the binding molecule of any one of claims 1 to 48, wherein the TCR alpha chain variable domain and the TCR beta chain variable domain are encoded in a single open reading frame or in two separate open reading frames.

50. 50. An expression vector comprising the nucleic acid of claim 49.

51. (a) an expression vector according to claim 50, or (b) a first expression vector comprising a nucleic acid encoding a first polypeptide comprising the TCR alpha chain variable domain of the binding molecule of any one of claims 1 to 48, and a second expression vector comprising a nucleic acid encoding a second polypeptide comprising the TCR beta chain variable domain of the binding molecule of any one of claims 1 to 48; Cells having

52. A non-naturally occurring and / or purified and / or engineered cell, preferably a T cell, presenting a binding molecule according to any one of claims 1 to 48.

53. 52. A pharmaceutical composition comprising a binding molecule according to any one of claims 1 to 48, a nucleic acid according to claim 49, an expression vector according to claim 50, and / or a cell according to claim 51 or 52, together with one or more pharmaceutically acceptable carriers or excipients.

54. 54. A binding molecule according to any one of claims 1 to 48, a nucleic acid according to claim 49, an expression vector according to claim 50, a cell according to claim 51 or 52 and / or a pharmaceutical composition according to claim 53 for use as a medicament, preferably in a human subject.

55. 55. The binding molecule of claim 54, wherein the binding molecule is used in a method for treating cancer.

56. 56. The binding molecule of claim 55, wherein the cancer is melanoma, ovarian cancer, lung cancer, or endometrial cancer.

57. 52. A method for producing a binding molecule according to any one of claims 1 to 48, comprising: a) maintaining a cell according to claim 51 under conditions suitable for expression of said binding molecule; and b) isolating said binding molecule.

58. A method for producing a binding molecule according to any one of claims 1 to 48, comprising the steps of: a) providing a first cell capable of expressing a first polypeptide comprising a TCR alpha chain variable domain of a binding molecule of any one of claims 1 to 48, and a second cell capable of expressing a second polypeptide comprising a TCR beta chain variable domain of a binding molecule of any one of claims 1 to 48; b) maintaining the first cells under conditions suitable for expression of the first polypeptide and the second cells under conditions suitable for expression of the second polypeptide; c) isolating the first polypeptide and the second polypeptide from the cell; and d) forming a complex between the first polypeptide and the second polypeptide to form a binding molecule according to any one of claims 1 to 48; A method comprising:

59. 52. A method of treating cancer in a subject, comprising administering to the subject a binding molecule of any one of claims 1 to 48, a nucleic acid of claim 49, an expression vector of claim 50, a cell of claim 51 or 52, and / or a pharmaceutical composition of claim 53.

Images