Binding molecules for PIWIL1 peptide-HLA complexes

Abstract

The present invention relates to binding molecules comprising a T-cell receptor (TCR) variable domain capable of binding to a PIWIL1 peptide-HLA complex. 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 PIWIL1 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] PIWIL1 (also known as piwi-like protein 1 or HIWI, with Uniprot accession number Q96J94) is associated with meiosis and plays a central role in spermatogenesis. PIWIL and PIWIL-like proteins are known to be involved in oncogenic processes such as cell renewal, cell migration, and cell invasion, as well as disease progression. PIWIL1 expression has been reported in various tumors, including those in the colon and esophagus, whereas its expression in normal tissues is restricted to the testis (Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6, Patent Document 1). PIWIL overexpression is associated with various tumor types, and PIWIL expression is associated with poor prognosis in colorectal and gastric cancer (Non-Patent Document 7, Non-Patent Document 8).

[0004] The peptide SLSNRLYYL (SEQ ID NO: 1) corresponds to amino acids 853 to 861 of the full-length PIWIL protein and binds to HLA-A2 ("HLA-A"). * As used herein, "HLA-A" is a complex with HLA-A (also referred to as "HLA-A 02") and presented on the cell surface. * "HLA-A02" or its synonym "HLA-A2" is generally used to refer to HLA-A * 02:01. This peptide-HLA complex provides a useful target for TCR-based immunotherapeutic intervention.

[0005] Identifying specific TCR sequences that bind with high affinity and specificity to SLSNRLYYL (SEQ ID NO: 1) complexed with HLA-A2 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 9, Non-Patent Document 10, Non-Patent Document 11), or they can be used to engineer T cells for adoptive therapy (Non-Patent Document 12).

[0006] TCRs that bind to SLSNRLYYL (SEQ ID NO: 1) complexed with HLA-A2 have previously been reported (Patent Document 2). However, these TCRs have not been engineered or characterized for use as therapeutic TCRs, and no affinity data has actually been provided. 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.

[0007] Therefore, the development of new immunotherapies requires binding molecules such as TCRs that can bind with high affinity and specificity to SLSNRLYYL (SEQ ID NO: 1) complexed with HLA-A2.

[0008] 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, suitable for use in a display library such as phage display, capable of being refolded and purified in high yield, and maintained 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 13).

[0009] 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 14). 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 11). The affinity maturation process typically requires that a skilled artisan must engineer specific mutations, including but not limited to substitutions, insertions, and / or deletions, into the starting TCR sequence to increase the strength of antigen recognition. Affinity maturation techniques, such as the use of display libraries (Non-Patent Document 15, Non-Patent Document 16), 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 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.

[0010] 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 13, Non-Patent Document 17, Non-Patent Document 18). 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 to occur 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]

[0011] [Patent Document 1] International Publication No. 2000 / 032039 [Patent Document 2] International Publication No. 2017 / 089771 [Non-patent literature]

[0012] [Non-Patent Document 1] Davis et al., Annu Rev Immunol. 1998;16:523-44 [Non-patent document 2] He et al. BMC Cancer. 2009 Dec 8;9:426 [Non-patent document 3] Grochola et al. Br J Cancer. 2008 Oct 7;99(7):1083-8 [Non-patent document 4] Taubert et al. Oncogene. 2007 Feb 15;26(7):1098-100 [Non-patent document 5] Li et al. Oncol Rep. 2010 Apr;23(4):1063-8 [Non-patent document 6] Zeng et al. Chin Med J (Engl). 2011 Jul;124(14):2144-9 [Non-Patent Document 7] Dong et al. (2021) "Critical Roles of PIWIL1 in Human Tumors: Expression, Functions, Mechanisms, and Potential Clinical Implications," Front. Cell Dev. Biol., 9:1-11, doi.org / 10.3389 / fcell.2021.656993 [Non-patent document 8] Gao et al. (2018) "PIWI-like protein 1 upregulation promotes gastric cancer invasion and metastasis." Onco Targets Ther. 11, 8783-8789. doi: 10.2147 / OTT.S186827 [Non-Patent Document 9] "High-Affinity Monoclonal T-cell receptor (mTCR) Fusions" by Lissin et al. in "Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges" (2013, SR Schmidt, Wiley)

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Summary of the Invention

[0013] As further described below, the present invention provides novel and potent binding molecules containing TCR variable domains suitable for immunotherapeutic use against PIWIL1-positive tumor cells expressing HLA-A2, thus providing opportunities for the treatment of related cancers, including those such as colon cancer. The identification of the binding molecules of the present invention required an extensive engineering effort exploring more than 15 TCR affinity maturation series. Even after selecting specific affinity maturation series for further study, multiple additional rounds of affinity maturation were performed. Following this, further stability enhancements were engineered into the molecules, all of which maintained adequate potency and affinity. These stability improvements are advantageous for enabling TCR-based binding molecules to be used in vivo, particularly in patients, and are also important for the manufacture and use of such molecules. Furthermore, the present inventors found low PIWIL1 peptide-HLA-A2 density in primary tumor tissues. Surprisingly, despite this low target density, the binding molecules of the present invention exhibited good killing efficacy against PIWIL1-positive cell lines.

[0014] 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 is HLA-A * 02, wherein each of the α chain variable domain and the β 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 (a) wherein the binding molecule contacts at least N4, R5, Y7, and Y8 of SLSNRLYYL (SEQ ID NO: 1); and / or (b) where: The CDR1 of the α chain has the sequence TIFF2026501720000001.tif5170 (SEQ ID NO: 98), The CDR3 of the α chain has the sequence TIFF2026501720000002.tif5170 (SEQ ID NO: 104), and The CDR3 of the β chain has the sequence TIFF2026501720000003.tif6170 (SEQ ID NO: 112), wherein X is an amino acid.

[0015] The above-mentioned SEQ ID NO: 98, SEQ ID NO: 104 and SEQ ID NO: 112 are HLA-A, as described in Example 7. * These sequences are consensus sequences based on the inventors' identification of the key CDR residues that contact the target peptide when the binding molecule binds to SLSNRLYYL (SEQ ID NO: 1) in complex with 02. These sequences are shown below in the section entitled "Sequence Description."

[0016] Surprisingly, the present inventors have found that despite the above-mentioned drawbacks, SLSNRLYYL (SEQ ID NO: 1) / HLA-A * We have identified binding molecules containing TCR variable domains that have particularly high affinity (picomolar range) and high antigen specificity for the O2 complex. When prepared as soluble reagents fused to a T cell redirecting moiety, these molecules exhibit potent killing of PIWIL-positive cancer cells. Thus, the molecules of the invention have a profile particularly suited for therapeutic use. Certain binding molecules of the invention were engineered from an appropriate scaffold (e.g., "wild-type") TCR sequence into which multiple mutations were introduced to enhance affinity and / or stability while maintaining high specificity.

[0017] 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 is HLA-A * 02 and SLSNRLYYL (SEQ ID NO: 1) in complex, wherein each of the α chain variable domain and the β chain variable domain comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR is a framework region and CDR is a complementarity determining region, (a) the α chain CDRs have the following sequences: CDR1 - YIAANDF (SEQ ID NO: 23), optionally with 1, 2 or 3 mutations; CDR2 - GYKTN (SEQ ID NO: 24), optionally with 1, 2 or 3 mutations; CDR3 - LAWGGTDLLP (SEQ ID NO: 29), optionally with 1, 2, 3, or 4 mutations; and / or (b) the CDRs of the β chain have the following sequences: CDR1 - SGHGT (SEQ ID NO: 37), optionally with 1, 2 or 3 mutations; CDR2 - FHEEGV (SEQ ID NO: 45), optionally with 1, 2 or 3 mutations; CDR3 - ASSVDWVGDGERQY (SEQ ID NO: 41), optionally with 1, 2, 3, 4, or 5 mutations; The present invention provides a binding molecule comprising or having:

[0018] 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 is HLA-A * 02 and SLSNRLYYL (SEQ ID NO: 1) in complex, wherein each of the α chain variable domain and the β chain variable domain comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR is a framework region and CDR is a complementarity determining region; where: (a) the α chain CDRs have the following sequences: CDR1 - NIATNDY (SEQ ID NO: 5), optionally with 1, 2 or 3 mutations; CDR2 - GYKTK (SEQ ID NO: 6), optionally with 1, 2 or 3 mutations; CDR3 - LAWGGTDKLI (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 - SGHAT (SEQ ID NO: 15), optionally with 1, 2 or 3 mutations; CDR2 - FQNNGV (SEQ ID NO: 16), optionally with 1, 2 or 3 mutations; CDR3 - ASSLDWVGSGETQY (SEQ ID NO: 17), optionally with 1, 2, 3, 4, or 5 mutations; The present invention provides a binding molecule comprising or having:

[0019] In a fourth aspect, the present invention provides SLSNRLYYL (SEQ ID NO: 1)-HLA-A * 1. A binding molecule having the property of specifically binding to or binding to a TCR α chain variable domain and a TCR β chain variable domain, wherein each variable domain contains three complementarity determining regions, designated CDR1, CDR2, and CDR3; (a) wherein the binding molecule contacts N4, R5, Y7, and Y8 of SLSNRLYYL (SEQ ID NO: 1); and / or (b) where: The CDR1 of the α chain has the sequence TIFF2026501720000004.tif5170 (SEQ ID NO: 98), The CDR3 of the α chain has the sequence TIFF2026501720000005.tif4170 (SEQ ID NO: 104), The CDR3 of the β chain has the sequence TIFF2026501720000006.tif5170 (SEQ ID NO: 112), wherein X is any amino acid.

[0020] As used herein, "binding molecule" or "binding molecule of the invention" relates to the binding molecules of the first, second, third and fourth aspects above, respectively, unless expressly indicated otherwise.

[0021] As used herein, the term "binding molecule" generally refers to a molecule capable of binding to a target antigen. The binding molecule of the present invention comprises a TCR α chain variable domain and a TCR β chain variable domain, which associate with each other to form the SLSNRLYYL (SEQ ID NO: 1)-HLA-A * As used herein, "SLSNRLYYL (SEQ ID NO: 1)-HLA-A" forms a TCR binding site capable of binding to the HLA-A 02 complex. * The phrase "binds to the HLA-A complex" is used to describe * The terms "binding to SLSNRLYYL (SEQ ID NO: 1) complexed with 02" are used interchangeably. The binding molecules of the invention can take many different forms, as discussed herein. Additionally, fragments of the binding molecules of the invention are also contemplated. A fragment refers to a portion of a binding molecule that retains binding to a target antigen.

[0022] 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 molecules of the present invention may possess 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, multifunctional (e.g., bifunctional) molecules, or fusion molecules comprising a TCR variable domain described herein and a therapeutic moiety, such as a T cell-redirecting moiety. Such molecules can mediate potent and specific responses against PIWIL-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.

[0023] The binding molecules of the present invention may be in the form 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 domain. 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 domain. 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) domains and several genes encoding β chain variable (Vβ) domains, 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 domains or C domains of the TCR α and β chains are referred to as TRAC and TRBC, respectively (Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10).

[0024] Certain binding molecules of the invention preferably have a larger (i.e., stronger) SLSNRLYYL (SEQ ID NO: 1)-HLA-A TCR than the native TCR (also referred to as the non-mutated TCR or scaffold TCR). * K for O2 complex D The higher the affinity, the higher the KD A low value for K indicates strong binding. D The 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 about 1 pM to about 500 pM of SLSNRLYYL (SEQ ID NO: 1)-HLA-A. * K for O2 complex 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.

[0025] 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.

[0026] Certain preferred mutated binding molecules have a SLSNRLYYL (SEQ ID NO: 1)-HLA-A binding affinity that is significantly higher than that of the native TCR. * 02 complex. Increasing the binding affinity of a native TCR can decrease the specificity of the TCR for its peptide-MHC ligand, as demonstrated in Non-Patent Document 18. However, certain binding molecules of the present invention surprisingly exhibit substantially higher binding affinity than native TCRs, yet exhibit a binding affinity similar to that of SLSNRLYYL (SEQ ID NO: 1)-HLA-A. * It shows a high level of specificity for the O2 complex.

[0027] The binding molecule of the invention preferably comprises SLSNRLYYL (SEQ ID NO: 1)-HLA-A * As used herein, "specific" binding refers to binding to the SLSNRLYYL (SEQ ID NO: 1)-HLA-A complex with higher affinity than other peptide-HLA complexes. * Specificity refers to a binding molecule that binds to the 02 complex. Highly specific binding molecules of the present invention are particularly suitable for therapeutic use because they have 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 minimizing their ability to recognize antigen-negative target cells.

[0028] Specificity can be measured in vitro, for example, in a cellular assay such as an ELISpot assay as described in Examples 4, 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 50There 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.

[0029] Additionally or alternatively, the specificity may be determined by determining whether the binding molecule is SLSNRLYYL (SEQ ID NO: 1)-HLA-A * Specificity may relate to the ability to bind to the HLA-A102 complex and not to a panel of other peptide-HLA complexes. Specificity can be determined, for example, by surface plasmon resonance (SPR) techniques described herein, e.g., in Example 2. 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. SLSNRLYYL(SEQ ID NO: 1)-HLA-A * The binding of the binding molecule to the O2 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 SLSNRLYYL (SEQ ID NO: 1) peptide may be excluded from the definition of a separate peptide-HLA complex.

[0030] 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 to (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.

[0031] SLSNRLYYL (SEQ ID NO: 1)-HLA-A * Binding molecules with specific binding properties to the O2 complex are also useful for other peptide-HLA-A * MLDKYSHYL (SEQ ID NO: 97)-HLA-A can bind to this complex with higher affinity than the 02 complex. * The O2 complex contains the target antigen (SLSNRLYYL (SEQ ID NO: 1)-HLA-A * Certain binding molecules of the invention have particularly high affinity (picomolar range) yet surprisingly retain a high degree of antigen specificity, binding to MLDKYSHYL (SEQ ID NO: 97)-HLA-A. *The binding molecule does not bind to the HLA-A 02 complex or binds to the complex with significantly lower affinity. In particular, the affinity window between binding to the target and binding to the mimetic can be at least 100-fold, at least 500-fold, at least 1000-fold, at least 2000-fold, at least 3000-fold, at least 4000-fold, and preferably at least 5000-fold. The binding molecule is MLDKYSHYL (SEQ ID NO: 97)-HLA-A 02 complex. * SLSNRLYYL (SEQ ID NO: 1)-HLA-A with an affinity that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 10-fold, at least 100-fold, or at least 1000-fold higher than the affinity for SLSNRLYYL (SEQ ID NO: 1)-HLA-A. * Surprisingly, this specificity is achieved by mutating the natural S residue at position 101 of the β-chain, which can be mutated to D.

[0032] 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.

[0033] 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, a mutation may be SLSNRLYYL (SEQ ID NO: 1)-HLA-A * The binding affinity (k D) and / or binding half-life (T 1 / 2 ) can be exemplified.

[0034] 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).

[0035] The binding molecules of the present invention are * The SLSNRLYYL (SEQ ID NO: 1) can form contacts, such as hydrogen bond contacts, with at least N4, R5, Y7, and Y8 of SLSNRLYYL (SEQ ID NO: 1) complexed with O2. 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 SLSNRLYYL (SEQ ID NO: 1) peptide, but not HLA, and an amino acid residue of the binding molecule. The binding molecule is a SLSNRLYYL (SEQ ID NO: 1) HLA-A * When bound to the HLA-A 02 complex, the binding molecule may contact at least residues N4, R5, Y7, and Y8 of the SLSNRLYYL (SEQ ID NO: 1) peptide. *When bound to the O2 complex, the ATP may contact all of the peptide residues at positions 1 through 9 of the SLSNRLYYL (SEQ ID NO: 1) peptide. Binding contacts (i.e., non-covalent interactions between atoms of one molecule and atoms of another molecule when the two molecules are bound to each other) may be identified using any method known in the art, including X-ray crystallography and structural modeling, as described herein.

[0036] Using a specific angle, the binding molecules of the present invention and HLA-A * The binding geometry of the interaction between HLA-A202 and complexed SLSNRLYYL (SEQ ID NO: 1) can be defined. For example, the binding molecule can bind to HLA-A202 with a crossing angle in the range of 53° to 75°, or preferably in the range of 59° to 68°. * The binding molecule can bind to HLA-A SLSNRLYYL (SEQ ID NO: 1) complexed with HLA-A SEQ ID NO: 02 at a tilt angle in the range of -38° to -18°, preferably in the range of -32° to -24°. * The binding molecule can bind to SLSNRLYYL (SEQ ID NO: 1) complexed with HLA-A 02. The binding molecule binds to HLA-A 02 at a roll angle in the range of -25° to -5°, preferably in the range of -20° to -10°. * The binding geometry of the 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.

[0037] In the binding molecules of the invention, there may be at least one mutation in the TCR alpha chain variable domain. There may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations in the alpha chain CDRs (i.e., across all three CDRs in total). For example, there may be 6 mutations in the alpha chain CDRs. There may be 3 mutations in CDR1 of the alpha chain, and / or 1 mutation in CDR2 of the alpha chain, and / or 2 mutations in CDR3 of the alpha chain.

[0038] The binding molecule of the first aspect of the invention may comprise an alpha chain CDR1 comprising the sequence of any one of SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101.

[0039] 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:104, SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:107.

[0040] 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:112, SEQ ID NO:113, SEQ ID NO:114 and SEQ ID NO:115.

[0041] The binding molecule of the first aspect of the invention comprises: CDR1 of the alpha chain comprising the sequence of SEQ ID NO: 99; CDR3 of the alpha chain comprising the sequence of SEQ ID NO: 105; CDR3 of the β chain comprising the sequence of SEQ ID NO: 113; may include:

[0042] The binding molecule of the first aspect of the invention comprises: CDR1 of the alpha chain comprising the sequence Y / NI / V / LA / GA / TND / EY / F (SEQ ID NO: 100); The CDR3 of the alpha chain comprises the sequence V / I / LA / GY / W / FGGTDK / V / LV / I / LI / P (SEQ ID NO: 106); CDR3 of the beta chain comprising the sequence A / GS / TS / TVDY / W / FVGS / DA / GD / ERQ / NY / W / F (SEQ ID NO: 114); may include:

[0043] The binding molecule of the first aspect of the invention comprises: CDR1 of the alpha chain comprising the sequence YI / V / LA / GAND / EF (SEQ ID NO: 101); The CDR3 of the alpha chain comprises the sequence V / I / LA / GY / W / FGGTDLV / I / LP (SEQ ID NO: 107); CDR3 of the beta chain comprising the sequence A / GS / TS / TVDY / W / FVGDA / GD / ERQ / NY / W / F (SEQ ID NO: 115); may include:

[0044] The binding molecule of the first aspect of the invention comprises: CDR1 of the alpha chain comprising the sequence Y / NI / V / LA / GA / TND / EY / F (SEQ ID NO: 100); CDR2 of the alpha chain comprising the sequence G / AY / W / FK / R / HT / SN (SEQ ID NO: 103); The CDR3 of the alpha chain comprises the sequence V / I / LA / GY / W / FGGTDK / V / LV / I / LI / P (SEQ ID NO: 106); CDR1 of the β chain comprising the sequence SA / GK / R / HGS / T (SEQ ID NO: 109); CDR2 of the β chain comprising the sequence Y / W / FHEEA / GV / I / L (SEQ ID NO: 111); CDR3 of the beta chain comprising the sequence A / GS / TS / TVDY / W / FVGS / DA / GD / ERQ / NY / W / F (SEQ ID NO: 114); may include:

[0045] The binding molecule of the first aspect comprises: array TIFF2026501720000007.tif5170 (SEQ ID NO: 23), optionally with one, two, or three mutations at any of positions 1 to 4 and position 6 of SEQ ID NO: 23 (i.e., these mutations exclude the N at position 7 and the F at position 8 numbered according to SEQ ID NO: 3); array TIFF2026501720000008.tif6170 (SEQ ID NO: 29), optionally with 1, 2, 3, or 4 mutations at any of positions 1 to 3 and positions 8 to 10 of SEQ ID NO: 29 (i.e., these mutations exclude G at position 4, G at position 5, T at position 6, and D at position 7), and / or array CDR3 of the β chain comprising TIFF2026501720000009.tif6170 (SEQ ID NO: 41), optionally having 1, 2, 3, or 4 mutations at any of positions 1 to 4, 6, and 10 to 14 of SEQ ID NO: 41 (i.e., these mutations exclude D at position 5, V at position 7, G at position 8, and D at position 9); may include:

[0046] The binding molecule of the first aspect comprises: array TIFF2026501720000010.tif6170 (SEQ ID NO: 23), optionally with one or two mutations at any of positions 1 to 4 and 6 of SEQ ID NO: 23; array TIFF2026501720000011.tif5170 (SEQ ID NO: 29), optionally with one, two, or three mutations at any of positions 1 to 3 and positions 8 to 10 of SEQ ID NO: 29, and / or array TIFF2026501720000012.tif5170 (SEQ ID NO: 41), optionally with one, two, or three mutations at any of positions 1 to 4, 6, and 10 to 14 of SEQ ID NO: 41; may include:

[0047] The binding molecule of the first aspect comprises: array TIFF2026501720000013.tif5170 (SEQ ID NO: 23), optionally with one mutation at any of positions 1 to 4 and 6 of SEQ ID NO: 23; array TIFF2026501720000014.tif4170 (SEQ ID NO: 29), optionally with one or two mutations at positions 1 to 3 and positions 8 to 10 of SEQ ID NO: 29, and / or array TIFF2026501720000015.tif5170 (SEQ ID NO: 41), optionally with one or two mutations at any of positions 1 to 4, 6, and 10 to 14 of SEQ ID NO: 41; may include:

[0048] The binding molecule of the first aspect comprises: array TIFF2026501720000016.tif4170 (SEQ ID NO: 23), optionally with one mutation at any of positions 1 to 4 and 6 of SEQ ID NO: 23; array TIFF2026501720000017.tif5170 (SEQ ID NO: 29), optionally with one mutation at any of positions 1 to 3 and positions 8 to 10 of SEQ ID NO: 29, and / or array TIFF2026501720000018.tif4170 (SEQ ID NO: 41), optionally with one mutation at any of positions 1 to 4, 6, and 10 to 14 of SEQ ID NO: 41; may include:

[0049] The binding molecule of the first aspect comprises: CDR2 of the alpha chain comprising the sequence GYKTN (SEQ ID NO: 24), optionally with one, two or three mutations; CDR1 of the β chain comprising the sequence SGHGT (SEQ ID NO: 37), optionally with 1, 2 or 3 mutations; CDR2 of the β chain comprising the sequence FHEEGV (SEQ ID NO: 45), optionally with 1, 2 or 3 mutations; may include:

[0050] The binding molecule of the first aspect comprises: CDR2 of the alpha chain comprising the sequence GYKTN (SEQ ID NO: 24), optionally with one or two mutations; CDR1 of the β chain comprising the sequence SGHGT (SEQ ID NO: 37), optionally with one or two mutations; CDR2 of the β chain comprising the sequence FHEEGV (SEQ ID NO: 45), optionally with one or two mutations; may include:

[0051] The binding molecule of the first aspect comprises: CDR2 of the alpha chain comprising the sequence GYKTN (SEQ ID NO: 24), optionally with one mutation; CDR1 of the β chain comprising the sequence SGHGT (SEQ ID NO: 37), optionally with one mutation; CDR2 of the β chain comprising the sequence FHEEGV (SEQ ID NO: 45), optionally with one mutation; may include:

[0052] The binding molecule of the first aspect of the invention may comprise an α-chain CDR2 comprising the sequence G / AY / W / FK / R / HT / SN / K (SEQ ID NO: 102), a β-chain CDR1 comprising the sequence S / TA / GK / R / HA / GS / T (SEQ ID NO: 108), and a β-chain CDR2 comprising the sequence Y / W / FH / QN / EN / EA / GV / I / L (SEQ ID NO: 110). The binding molecule of the first aspect of the invention may comprise an α-chain CDR2 comprising the sequence G / AY / W / FK / R / HT / SN (SEQ ID NO: 103), a β-chain CDR1 comprising the sequence SA / GK / R / HGS / T (SEQ ID NO: 109), and a β-chain CDR2 comprising the sequence Y / W / FHEEA / GV / I / L (SEQ ID NO: 111).

[0053] 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).

[0054] In the binding molecule of the third aspect of the invention, the mutations in the CDRs of the alpha chain are numbered according to SEQ ID NO: 3: TIFF2026501720000019.tif30170. Thus, any or all of these mutations may be present, optionally in combination with other mutations. In particular, the binding molecule may have in the CDRs of the alpha chain the following mutations, numbered according to SEQ ID NO: 3: CDR1:N26Y, T29A, Y32F; CDR2:K54N; and CDR3:K96V, I98P; Or, CDR1:N26Y, T29A, Y32F; CDR2:K54N; and CDR3:K96L, I98P; may include:

[0055] The second set of mutations listed above is preferred.

[0056] In the binding molecules of the present invention, mutations in the CDRs of the α chain may be conservative, semi-conservative, permissive, or other phenotypically silent mutations, as described herein. Other suitable conservative, semi-conservative, permissive, or other phenotypically silent mutations will be apparent to those skilled in the art. Additional mutations may correspond to residues within the α chain sequences disclosed herein, including, for example, Y26N, A29T, F32Y, N54K, L96K, and P98I, numbered according to SEQ ID NO: 34.

[0057] The mutated α chain variable domain may be combined with any β chain variable domain defined herein.

[0058] There may be at least one mutation in the TCR β chain variable domain 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 6 or 7 mutations in the CDRs of the β chain. There may be 1 or 2 mutations in CDR1 of the β chain, and / or 2 or 3 mutations in CDR2 of the β chain, and / or 5 mutations in CDR3 of the β chain.

[0059] In the binding molecule of the third aspect of the invention, the mutation(s) in the CDRs of the β chain are numbered according to SEQ ID NO: 13: TIFF2026501720000020.tif40170. Thus, any or all of these mutations may be present, optionally in combination with other mutations. In particular, the binding molecule may have, in the CDRs of the β chain, the following mutations, numbered according to SEQ ID NO: 13: CDR1:A30G; CDR2: Q50H, N51E; and CDR3:L96V, S101D, T104R; Or, CDR1:A30G; CDR2: Q50H, N51E; and CDR3:L96W, S101D, T104R; Or, CDR1:S27T, A30G; CDR2: Q50H, N51E; and CDR3:L96V, S101D, T104R; Or, CDR1:A30G; CDR2: Q50H, N51E, N52E; and CDR3:L96V, S101D, T104R; may include:

[0060] The set of mutations listed above, A30G, Q50H, N51E, N52E, L96V, S101D, and T104R, is preferred.

[0061] In the binding molecules of the present invention, mutations in the CDRs of the β-chain may be conservative, semi-conservative, permissive, or other phenotypically silent mutations, as described herein. Other suitable conservative, semi-conservative, permissive, or other phenotypically silent mutations will be apparent to those skilled in the art. Additional mutations may correspond to residues within the α-chain sequences disclosed herein, including, for example, G30A, H50Q, E51N, E52N, V96L, and D101S, numbered according to SEQ ID NO: 48.

[0062] The mutated β chain variable domain may be combined with any α chain variable domain defined herein.

[0063] Mutation(s) within the CDR(s) relative to the native sequence may improve the binding affinity or stability of the 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. An example of such a mutation is N52E in the β-chain variable domain of SEQ ID NO: 13. 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 include those that reduce non-specific binding, i.e., SLSNRLYYL(SEQ ID NO: 1)-HLA-A *Mutations may include those that result in decreased binding to another antigen compared to the O2 complex. Mutations may include those that increase folding efficiency and / or stability and / or manufacturability. Some mutations may contribute to each of these attributes, while others may, for example, contribute to affinity but not specificity, or specificity but not affinity, or stability but not affinity, etc.

[0064] 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.

[0065] A binding molecule of the invention may comprise one of the following combinations of α-chain CDRs and β-chain CDRs:

[0066] TIFF2026501720000021.tif45170

[0067] A preferred combination is combination 6 in the table above. These are the CDR sequences of the TCR referred to in the examples as "a67b72".

[0068] 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 the binding molecules of the present invention. Mutations that remove or modify glycosylation sites may be performed.

[0069] The framework regions of the alpha chain variable domain of the binding molecules of the invention have the following sequence: FR1 - LAKTTQPISMDSYEGQEVNITCSHN (SEQ ID NO: 8), optionally with 1, 2, or 3 mutations; FR2 - ITWYQQFPSQGPRFIIQ (SEQ ID NO: 9), optionally with 1, 2, or 3 mutations; FR3—VTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYC (SEQ ID NO: 10), optionally with 1, 2, or 3 mutations; FR4—FGTGTRLQVFP (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 - EAGVAQSPRYKIIEKRQSVAFWCNPI (SEQ ID NO: 18), optionally with 1, 2 or 3 mutations; FR2—LYWYQQILGQGPKLLIQ (SEQ ID NO: 19), optionally with 1, 2, or 3 mutations; FR3 - VDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLC (SEQ ID NO: 20), optionally with 1, 2, or 3 mutations; FR4—FGPGTRLLVL (SEQ ID NO: 21), optionally with 1, 2, or 3 mutations; Includes.

[0070] The framework regions FR1, FR2, and FR3 of the α chain may contain amino acid sequences corresponding to the TRAV4 chain, and / or the framework regions FR1, FR2, and FR3 of the β chain may contain amino acid sequences corresponding to the amino acid sequences of the TRBV11-2 chain.

[0071] The FR4 region may contain the junction region of the α and β variable chains (TRAJ and TRBJ, respectively). The TRAJ region may contain an amino acid sequence corresponding to the amino acid sequence of TRAJ34-1. The TRBJ region may contain an amino acid sequence corresponding to the amino acid sequence of TRBJ5-1.

[0072] 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 sequence above. The framework regions of the α chain variable domain may have 5 mutations relative to the sequence above. The framework regions of the α chain variable domain may have the following mutations numbered according to SEQ ID NO: 3: TIFF2026501720000022.tif25170. Mutations at I47 and T56 may improve affinity. Mutations at L1, T21, and P65 may improve manufacturability and / or stability. The framework regions of the α chain variable domain may contain one or more of the following mutation combinations, numbered according to SEQ ID NO:3: (a) I47F, T56Q, (b) L1A, I47F, T56Q, (c)T21P, I47F, T56Q, P65S, (d)L1A, T21P, I47F, T56Q, P65S, A preferred combination is combination (d) above. The framework regions of the α chain variable domain may contain no other mutations (other than those listed above).

[0073] 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 one mutation relative to the above sequence. The framework regions of the β-chain variable domain contain the following mutation, numbered according to SEQ ID NO: 13: R16G. This mutation may improve stability. The framework regions of the β-chain variable domain may not contain other mutations relative to the above sequence.

[0074] 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.

[0075] 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:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, or SEQ ID NO:90, 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:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, or SEQ ID NO:90. The β chain variable domain may comprise any one of the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 91, or SEQ ID NO: 92, 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: 36, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 91, or SEQ ID NO: 92. Because all α chain and β chain variable domains are derived from the same scaffold TCR sequence (i.e., SEQ ID NO: 2 and SEQ ID NO: 12, 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:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, or SEQ ID NO:90, 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:36, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:91, or SEQ ID NO:92, or an amino acid sequence having at least 90% identity to said amino acid sequence.

[0076] 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:28, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:34, 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:28, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:34. The β chain variable domain may comprise any one of the amino acid sequences of SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48, 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:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48. Because all α chain and β chain variable domains are derived from the same scaffold TCR sequence (i.e., SEQ ID NO:2 and SEQ ID NO:12, 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:28, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:34, or an amino acid sequence having at least 90% identity to that 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:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48, or an amino acid sequence having at least 90% identity to that amino acid sequence.

[0077] The binding molecule may comprise one of the following combinations of alpha and beta chain variable domains:

[0078] TIFF2026501720000023.tif35170

[0079] Preferably, the α chain variable domain comprises the amino acid sequence of SEQ ID NO: 34 and the β chain variable domain comprises the amino acid sequence of SEQ ID NO: 48. In this regard, the present invention relates to an HLA-A *The present invention provides a binding molecule having the property of binding to SLSNRLYYL (SEQ ID NO: 1) complexed with 02, the binding molecule comprising an alpha chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 34, and a beta chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 48, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 48.

[0080] The α chain variable domain may comprise the amino acid sequence of SEQ ID NO: 28 [a40], and the β chain variable domain comprises the amino acid sequence of SEQ ID NO: 36 [b23]. In this regard, the present invention relates to HLA-A * The present invention provides a binding molecule having the property of binding to SLSNRLYYL (SEQ ID NO: 1) complexed with 02, the binding molecule comprising an alpha chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 28, and a beta chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 36.

[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 may also be used.

[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] The α and / or β constant domains may additionally or alternatively contain mutations that improve manufacturability, e.g., remove or alter glycosylation sites. Such mutations are typically required for production in mammalian systems such as CHO cells. SEQ ID NO:116 and SEQ ID NO:117 provide α and β constant domain sequences, respectively, that are suitable for production in mammalian cells.

[0085] A binding molecule of the invention may comprise the extracellular region of a TCR alpha chain constant domain, optionally truncated at the C-terminus by up to 15 amino acids, and / or the extracellular region of a TCR beta chain constant domain, optionally truncated at the C-terminus by up to 15 amino acids.

[0086] 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 TCR beta chain constant domain may comprise the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 14.

[0087] 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: 14. The binding molecule may not comprise the transmembrane or cytoplasmic domain of the TCR.

[0088] 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.

[0089] 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 domain and TCR β variable domain, respectively, Cα and Cβ are the TCR α constant domain and TCR β constant domain, 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).

[0090] 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.

[0091] 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:72), GGGSG (SEQ ID NO:78), GGSGG (SEQ ID NO:79), GSGGG (SEQ ID NO:80), GSGGGP (SEQ ID NO:81), GGEPS (SEQ ID NO:82), GGEGGGP (SEQ ID NO:83), GGEGGGSEGGGS (SEQ ID NO:84), GGGSGGGG (SEQ ID NO:85), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO:86), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:87), EAAAK (SEQ ID NO:88), and EAAAKEAAAKEAAAK (SEQ ID NO:89). 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.

[0092] 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.

[0093] 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).

[0094] Particularly suitable TCR alpha chain sequences include, but are not limited to, any one of SEQ ID NO:2, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:58. Particularly suitable TCR beta chain sequences include, but are not limited to, any one of SEQ ID NO:12, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, and SEQ ID NO:59. Such sequences do not contain transmembrane or cytoplasmic domains. All alpha chain sequences (i.e., SEQ ID NO:2, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:58) are expected to be compatible with all beta chain sequences (i.e., SEQ ID NO:12, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, and SEQ ID NO:59) 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:49, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:58, 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:49, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:58; a TCR β chain comprising an amino acid sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, and SEQ ID NO: 59, 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: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, and SEQ ID NO: 59; may include:

[0095] More specifically, the binding molecule is (a) 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; (b) a TCR α chain comprising the amino acid sequence of SEQ ID NO: 51, and a TCR β chain comprising the amino acid sequence of SEQ ID NO: 52; (c) 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: 53; (d) a TCR α chain comprising the amino acid sequence of SEQ ID NO: 54, and a TCR β chain comprising the amino acid sequence of SEQ ID NO: 55; (e) a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 56 and a TCR beta chain comprising the amino acid sequence of SEQ ID NO: 57; or (f) a TCR α chain comprising the amino acid sequence of SEQ ID NO: 58, and a TCR β chain comprising the amino acid sequence of SEQ ID NO: 59; may include.

[0096] Preferably, the binding molecule includes a TCRα chain comprising the amino acid sequence of SEQ ID NO: 58 and a TCRβ chain comprising the amino acid sequence of SEQ ID NO: 59. In this regard, the present invention relates to HLA-A * A binding molecule having the property of binding to SLSNRLYYL (SEQ ID NO: 1) complexed with 02, the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 58, 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, and the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 59, 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, is provided.

[0097] The binding molecules of the present invention are useful for delivering a detectable label or a therapeutic agent to antigen-presenting cells and tissues containing antigen-presenting cells. Thus, these can be used with a detectable label (for diagnostic purposes of detecting the presence of cells presenting cognate antigen using the binding molecule), and / or a therapeutic agent comprising an immune effector, and / or may comprise a pharmacokinetic (PK) modifying moiety or be associated (in a covalent or other manner) with them.

[0098] 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 regions, which may serve to increase the in vivo half-life of the binding molecules of the invention.

[0099] When an immunoglobulin Fc region is used, it may be the Fc region of any antibody. The Fc region is the tail portion of an antibody that interacts with Fc receptors on cell surfaces 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 regions from 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 regions particularly suitable for use in the present invention include, but are not limited to, the Fc regions from IgG1 or IgG4. The Fc region may be derived from a human sequence.

[0100] The first Fc domain may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 95, and the second Fc domain may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 96. The first Fc domain may comprise or consist of an amino acid sequence at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 95, and the domain Fc region may comprise or consist of an amino acid sequence at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 96. Preferably, the first Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 95, and the second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 96. As will be appreciated by those skilled in the art, the sequences set forth above for the first Fc domain and the second Fc domain are also suitable vice versa. For example, the first Fc domain may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 96, and the second Fc domain may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 95.

[0101] The Fc region may also preferably contain KiH mutations that promote dimerization, as well as mutations that prevent interaction with activating receptors, i.e., functionally silent molecules. The Fc region 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.

[0102] The Fc region, if present, may contain mutations compared to a wild-type or unmodified Fc sequence. Mutations include substitutions, insertions, and deletions. Such mutations may be made for the purpose of introducing desirable therapeutic properties. The Fc region may contain one or more amino acid substitutions that promote dimerization of each Fc domain. 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). Without wishing to be bound by theory, this is thought to stabilize heterodimers of each Fc domain by favoring the formation of heterodimers over other species, such as homomultimers, thereby enhancing stability and manufacturability. Suitable locations for KiH mutations and other mutations that promote dimerization of Fc domains are known in the art and include those described in Merchant et al., Nat Biotechnol 16:677 (1998), Ridgway et al., Prot Engineering 9:617 (1996), and Atwell et al. J Mol Biol 270,1 (1997): 26-35. For example, the substitutions that form corresponding knobs and holes in the two Fc domains can correspond to one or more pairs shown in the following table:

[0103] TIFF2026501720000024.tif54170

[0104] Substitutions in the table above are indicated by the original residue, followed by its position using the EU numbering system, then the incorporated residue (all residues are indicated by the single-letter amino acid code). Multiple substitutions are separated by colons.

[0105] The first Fc domain and the second Fc domain may comprise one or more substitutions in the table above. For example: (i) one of the first Fc domain and the second Fc domain may comprise one or more amino acid substitutions selected from the group consisting of T366S, L368A, T394S, F405A, Y407A, Y407T, and Y407V according to the EU numbering scheme; and (ii) The other of the first Fc domain and the second Fc domain may comprise one or more amino acid substitutions selected from the group consisting of T366W, T366Y, T366W, T394W, and F405W according to the EU numbering scheme. The substitutions in (i) and (ii) are hole-forming and knob-forming substitutions, respectively. The first Fc domain may comprise one or more of the substitutions in (i), and the second Fc domain may comprise one or more of the substitutions in (ii).

[0106] for example, (i) one of the first Fc domain and the second Fc domain may comprise one or more amino acid substitutions selected from the group consisting of T366S, L368A, and Y407V according to the EU numbering scheme; and (ii) The other of the first Fc domain and the second Fc domain may comprise an amino acid substitution of T366W according to the EU numbering scheme. The first Fc domain may comprise one or more of the substitutions in (i), and the second Fc domain may comprise the substitution in (ii).

[0107] Preferably, (i) one of the first Fc domain and the second Fc domain comprises amino acid substitutions of T366S, L368A, and Y407V according to the EU numbering scheme, and (ii) the other of the first Fc domain and the second Fc domain comprises amino acid substitutions of T366W according to the EU numbering scheme. For example, the first Fc domain may comprise amino acid substitutions of T366S, L368A, and Y407V according to the EU numbering scheme, and the second Fc domain may comprise amino acid substitutions of T366W according to the EU numbering scheme.

[0108] 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.

[0109] The Fc region may contain one or more mutations that attenuate an effector function of the Fc region. Exemplary effector functions include, but are not limited to, complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC). A modification that attenuates effector function may be a modification that alters the glycosylation pattern of the Fc region, for example, a modification that results in a deglycosylated Fc region. Alternatively, a modification that attenuates effector function may be a modification that does not alter the glycosylation pattern of the Fc region. A modification that attenuates effector function may reduce or eliminate binding to human effector cells, binding to one or more Fc receptors, and / or binding to cells expressing Fc receptors. For example, the Fc domain may comprise one or more amino acid substitutions selected from the group consisting of S228P, E233P, L234A, L235A, L235E, L235P, G236R, G237A, P238S, F241A, V264A, D265A, H268A, D270A, N297A, N297G, N297Q, E318A, K322A, L328R, P329G, P329A, A330S, A330L, P331A, and P331S according to the EU numbering scheme. Particular modifications include the N297G or N297A substitution in the Fc region of human IgG1 (EU numbering). Other suitable modifications include substitutions of L234A, L235A, and P329G (EU numbering) in the Fc region of human IgG1, which result in attenuated effector function. The Fc domain may contain a substitution at residue N297, numbered according to the EU index. For example, the substitution may be N297G or N297A. Other suitable mutations (e.g., at residue N297) are known to those skilled in the art.

[0110] An Fc variant with reduced effector function refers to an Fc variant in which effector function (e.g., activity such as CDC, ADCC, and / or FcR binding) is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or more, compared to the effector function achieved by a wild-type Fc region (e.g., an Fc region without mutations that reduce effector function, but which may have other mutations). An Fc variant with reduced effector function may be an Fc variant in which all detectable effector function is abolished compared to the wild-type Fc region. Assays for measuring effector function are known in the art and are described below. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / impaired CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that an Fc region, Fc domain, or fusion protein lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985) and U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).

[0111] Substitutions that abolish or reduce binding to Fcγ receptors, and / or increase binding to FcRn, and / or prevent Fab arm exchange, and / or remove protease sites can be introduced into the first and second Fc domains. In this regard, the one or more Fc regions may contain one or more amino acid substitutions that prevent or reduce binding to activating receptors. The half-life extending domain may contain one or more amino acid substitutions that prevent or reduce binding to FcγRs. For example, the first and / or second Fc domain may contain an N297G amino acid substitution according to the EU numbering scheme. Both the first and second Fc domains may contain an N297G amino acid substitution.

[0112] The one or more Fc domains may contain one or more amino acid substitutions that enhance binding to FcRn. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward, Immunol. Today 18: (12): 592-8 (1997); Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton et al., J. Biol. Chem. 279 (8): 6213-6 (2004); WO 2004 / 92219 (Hinton et al.)). The in vivo FcRn binding and serum half-life of human FcRn high-affinity-binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides having mutant Fc regions. WO 2004 / 42072 (Presta) describes antibody substitutions that improve or reduce binding to FcR. See also, e.g., Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001). In particular, Mackness et al., MAbs. 11:1276-1288 (2019) describe amino acid substitutions in the Fc region of antibodies suitable for enhancing binding to FcRn.

[0113] Additionally or alternatively, for manufacturing reasons, mutations may be made to remove or replace amino acids that may be subject to post-translational modifications, such as glycosylation, as described herein. The immunoglobulin Fc may be fused to other domains in the molecules of the invention via linker and / or hinge sequences as described herein. Alternatively, no linker may be used.

[0114] When present, the two Fc domains in the molecules of the invention may comprise a CH2 constant domain and a CH3 constant domain and all or part of a hinge sequence. The hinge sequence may substantially or partially correspond to a hinge region from IgG1, IgG2, IgG3, or IgG4. The hinge sequence may be an IgG1 hinge sequence, such as the amino acid sequence set forth in SEQ ID NO: 94. The hinge may comprise all or part of the core hinge domain and all or part of the lower hinge region.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] Detectable labels for diagnostic purposes include, for example, fluorescent labels, radioactive labels, enzymes, nucleic acid probes and imaging agent reagents.

[0119] 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 dissociation rate constants 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.

[0120] 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.

[0121] 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:

[0122] The binding molecules of the present invention may be multispecific. As used herein, the term "multispecific" refers to a binding molecule that comprises two or more antigen-binding moieties, including a TCR antigen-binding moiety formed by a TCR alpha chain variable domain and a TCR beta chain variable domain. Such a binding molecule may comprise SLSNRLYYL (SEQ ID NO: 1)-HLA-A *The binding molecule may further bind to one or more different antigens. For example, the binding molecule may be bispecific. Such a binding molecule may be SLSNRLYYL (SEQ ID NO: 1)-HLA-A * The TCR antigen-binding portion (formed by the α chain variable domain and the β chain variable domain) that binds to the TCR 02 complex and another antigen-binding portion (e.g., an antibody antigen-binding portion) that binds to a different antigen. This other antigen-binding portion may be referred to herein as a "second antigen-binding portion," and the antigen bound by the second antigen-binding portion may be referred to herein as the "second antigen." The second antigen-binding portion may be an immune cell engager. The term "antigen-binding portion" refers to a protein or a region or domain thereof that is capable of binding to an antigen. For example, the term encompasses antigen-binding portions of antibodies, including antigen-binding portions from conventional antibodies and engineered antibodies.

[0123] 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 (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The binding site of a conventional antibody is primarily composed 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.

[0124] "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 only the variable portion of one light chain and one heavy chain.

[0125] Binding molecules comprising the antigen-binding portion of an antibody as 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 domain (VH) and an antibody light chain variable domain (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).

[0126] 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.

[0127] 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 cells" refer to T cells or natural killer cells (NK cells). The second antigen-binding portion may be an immune cell engager. In particular, the antigen (i.e., the second antigen) may be a T cell surface antigen.

[0128] 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.

[0129] Antigen-binding portions suitable for binding to CD3 include binding domains derived from the CD3-specific humanized antibody hUCHU (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, 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.

[0130] Other suitable CD3 binding portions can be derived from the anti-CD3 scFv designated herein as "U0" (SEQ ID NO: 60) or "U28" (SEQ ID NO: 70). 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: 66) or GYSFTGYA (SEQ ID NO: 71), CDR2-INPYKGVS (SEQ ID NO: 67), CDR3-ARSGYYGDSDWYFDV (SEQ ID NO: 68), and (b) VL has the following sequence: CDR1-QDIRNY (SEQ ID NO: 62), CDR2-YTS (SEQ ID NO: 63), CDR3-QQGNTLPWT (SEQ ID NO: 64), The antibody may comprise an antigen-binding portion comprising a CDR having the following structure:

[0131] 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 the amino acid sequence set forth in SEQ ID NO:65 or SEQ ID NO:69, 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:65 or SEQ ID NO:69; and The VL comprises the amino acid sequence set forth in SEQ ID NO: 61 or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 61, 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.

[0132] The binding molecule may comprise an scFv capable of binding to CD3. The scFv may preferably comprise a VH comprising the amino acid sequence set forth in SEQ ID NO: 69 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61. Such an scFv may comprise the amino acid sequence set forth in SEQ ID NO: 70.

[0133] Alternatively, the scFv may comprise a VH comprising the amino acid sequence set forth in SEQ ID NO: 65 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61. Such an scFv may comprise the amino acid sequence set forth in SEQ ID NO: 60.

[0134] 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 composed primarily of amino acids such as glycine, alanine, and serine, without bulky side chains that may limit their flexibility. Alternatively, a linker with greater rigidity may be desirable. The usable or optimal length of 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: 72), GGGSG (SEQ ID NO: 78), GGSGG (SEQ ID NO: 79), GSGGG (SEQ ID NO: 80), GSGGGP (SEQ ID NO: 81), GGEPS (SEQ ID NO: 82), GGEGGGP (SEQ ID NO: 83), GGEGGGSEGGGS (SEQ ID NO: 84), GGGSGGGG (SEQ ID NO: 85), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 86), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 87), EAAAK (SEQ ID NO: 88), and EAAAKEAAAKEAAAK (SEQ ID NO: 89).

[0135] 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: 72. 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 comprising the extracellular region of a variable domain and optionally a constant domain); 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 region of a variable domain and optionally a constant domain) 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: 72.

[0136] 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 a40b23U28 (consisting of SEQ ID NOs: 49 and 119), a36b38U28 (consisting of SEQ ID NOs: 51 and 73), a40b37U28 (consisting of SEQ ID NOs: 49 and 74), a58b63U28 (consisting of SEQ ID NOs: 54 and 75), a61b68U28 (consisting of SEQ ID NOs: 56 and 76), and a67b72U28 (consisting of SEQ ID NOs: 58 and 77).

[0137] The binding molecule in the above format comprises an alpha chain amino acid sequence set forth in any one of SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:58, or an alpha 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:49, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:58; a beta chain-anti-CD3 amino acid sequence set forth in any one of SEQ ID NO:119, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, 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:119, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77; may include:

[0138] More particularly, the binding molecule in the above format is (a) 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: 119; (b) an α chain amino acid sequence set forth in SEQ ID NO: 51, and a β chain-anti-CD3 amino acid sequence set forth in SEQ ID NO: 73; (c) 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: 74; (d) an α chain amino acid sequence set forth in SEQ ID NO: 54, and a β chain-anti-CD3 amino acid sequence set forth in SEQ ID NO: 75; (e) an α chain amino acid sequence set forth in SEQ ID NO: 56 and a β chain-anti-CD3 amino acid sequence set forth in SEQ ID NO: 76; or (f) an α chain amino acid sequence set forth in SEQ ID NO: 58, and a β chain-anti-CD3 amino acid sequence set forth in SEQ ID NO: 77; may include:

[0139] Preferably, the binding molecule comprises the α chain amino acid sequence shown in SEQ ID NO: 58 and the β chain-anti-CD3 amino acid sequence shown in SEQ ID NO: 77. Thus, the present invention relates to a method for the treatment of HLA-A *02 in complex with SLSNRLYYL (SEQ ID NO: 1), the binding molecule comprising a TCR alpha chain and a TCR beta chain covalently linked to an anti-CD3 scFv, wherein the alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 58, and the beta chain-anti-CD3 chain comprises the amino acid sequence set forth in SEQ ID NO: 77.

[0140] 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 each polypeptide chain is selected from the group consisting of SLSNRLYYL (SEQ ID NO: 1) HLA-A * The O2 complex and the antibody associate so that they can simultaneously bind to the antigen.

[0141] The binding molecules of the invention comprise: i) a peptide-major histocompatibility complex (pMHC) binding region comprising a first variable domain-constant domain linkage (VC1) and a second variable domain-constant domain linkage (VC2), wherein VC1 and VC2 dimerize to form a pMHC binding region; ii) an antigen-binding region comprising an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH); iii) a half-life extending region comprising a first IgG Fc domain (FC1) and a second IgG Fc domain (FC2), wherein the FC1 and FC2 domains dimerize to form an Fc region; A multi-domain single chain binding molecule comprising: the first variable domain comprises one of (a) a TCR alpha chain variable domain and (b) a TCR beta chain variable domain, and the second variable domain comprises the other of (a) a TCR alpha chain variable domain and (b) a TCR beta chain variable domain; the antigen-binding region is linked to the N-terminus of VC1, VC1 is linked via its C-terminus to the N-terminus of the FC1 domain, the FC1 domain is linked via its C-terminus to the N-terminus of VC2, and VC2 is linked via its C-terminus to the N-terminus of the FC2 domain; and The pMHC binding domain and antigen binding domain are SLSNRLYYL (SEQ ID NO: 1) HLA-A * The antibody may be in the form of a multi-domain single chain binding molecule capable of binding to the antigen of the O2 complex and antibody.

[0142] The antigen-binding region may be a T cell-engaging immune effector, which may be an ScFv. The antigen-binding region may be a CD3 effector that activates T cells through interaction with CD3 and / or the TCR / CD3 complex. The T cell-engaging immune effector may be an anti-CD3 scFv.

[0143] VC1 may comprise either (a) a TCR alpha variable domain and a TCR alpha constant domain or (b) a TCR beta variable domain and a TCR beta constant domain, and VC2 may comprise the other of (a) and (b). It is preferred that VC1 comprises a TCR beta variable domain and a TCR beta constant domain, and VC2 comprises a TCR alpha variable domain and a TCR alpha constant domain.

[0144] The VL domain may be linked via its C-terminus to the N-terminus of the VH domain, and the VH domain may be linked via its C-terminus to the N-terminus of VC1.

[0145] Two or more of the VH domain, VL domain, VC1 domain, VC2 domain, FC1 domain, and FC2 domain may be linked to each other via a linker and / or an IgG hinge sequence, wherein the one or more linkers may have a sequence described herein.

[0146] VC1 may be linked to the FC1 domain via a sequence comprising an IgG hinge sequence, and / or VC2 may be linked to the FC2 domain via a sequence comprising an IgG hinge sequence, which IgG hinge sequence is preferably at least 80% identical to SEQ ID NO:94.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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 SLSNRLYYL (SEQ ID NO: 1) HLA-A within 50%, or more preferably within 30%, 25%, or 20% of the binding half-life * K for O2 complex D and / or binding half-life. Suitable conditions are further illustrated in the Examples.

[0152] Additionally, a phenotypically silent variant is SLSNRLYYL (SEQ ID NO: 1) HLA-A * The phenotypically silent variants may retain the same or substantially the same therapeutic window between binding to the HLA-A 02 complex and binding to one or more additional peptide-HLA complexes. * The therapeutic window may maintain the same or substantially the same therapeutic window between the strength of immune cell activation in response to cells presenting the 02 complex and 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.

[0153] As known to those skilled in the art, SLSNRLYYL (SEQ ID NO: 1) HLA-A * It may be possible to produce binding molecules whose variable domains are altered compared to those detailed above without significantly altering the affinity of their interaction with the O2 complex and / or other functional properties. In particular, such silent mutations may be incorporated within 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.

[0154] A phenotypically silent variant may contain one or more conservative substitutions and / or one or more permissive substitutions. Permissive substitutions refer to substitutions that are phenotypically silent, even though they do not fall within the definition of conservative as provided below. 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.

[0155] 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.

[0156] 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.

[0157] "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)).

[0158] 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.

[0159] 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).

[0160] 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.

[0161] As used herein, when a sequence is said to have sequence identity to another sequence, the sequence retains a function of the other sequence, e.g., in the case of peptides, a general binding property.

[0162] 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.

[0163] 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 (3 rd (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.

[0164] 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 Λ ...

[0165] 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 DIt 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 off Values ​​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.

[0166] 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.

[0167] 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).

[0168] 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.

[0169] Structural characteristics 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 characteristics include, for example, HLA-A * The method may include determining the three-dimensional atomic structure of a binding molecule bound to SLSNRLYYL (SEQ ID NO: 1) complexed with 02.

[0170] 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."

[0171] 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.

[0172] 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.

[0173] 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 cystine vector").

[0174] 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).

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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, 8, or 9 or more binding contacts of the peptide residues. There may be a minimum number (e.g., 4, 5, 6, 7, 8, or 9) of these peptide residue binding contacts sufficient for the binding molecule to specifically bind to the pHLA complex.

[0179] 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, residues between a binding molecule and a peptide may be considered to be in binding contact if the distance between any atom from the binding molecule residue and any atom from the peptide residue is 4.1 Å or less. 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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).

[0184] 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).

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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).

[0193] 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).

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] Pharmaceutical compositions and medical methods For administration to a patient, the molecules, nucleic acids, expression vectors, and / or cells of the invention may be provided as part of a pharmaceutical composition along with one or more pharmaceutically acceptable carriers or excipients (e.g., buffering agents, 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.

[0201] 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).

[0202] 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, e.g., 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.

[0203] 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.

[0204] 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 of the binding molecules to bind to tumor cells, particularly SLSNRLYYL (SEQ ID NO: 1)-HLA-A. *The term "antibody-specific binding" refers to the ability of a binding molecule to distinguish tumor cells displaying the .O2 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, including, but 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 molecule 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, thus reducing the risk of potential cytokine release syndrome in vivo, and alloreactivity tests to confirm a reduced ability to recognize alternative HLA types.

[0205] 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.

[0206] 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.

[0207] 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 PIWIL1 expression. "Associated with PIWIL1 expression" means that the cancer contains cancer cells that express PIWIL1. In this regard, the cancer can be a PIWIL1-positive cancer. The cancer may be known to be associated with PIWIL1 expression. For example, PIWIL1 expression may be known to be elevated in cancer, so PIWIL1 expression may not be assessed or may be assessed retrospectively. Alternatively, PIWIL1 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 PIWIL1 expression levels. It is not intended that the present invention be limited to the treatment of cancers in which PIWIL1 expression can be detected by histological methods. Rather, the binding molecules of the present invention may be useful in treating types of cancers and tumors thought to be associated with PIWIL1 expression.

[0208] When PIWIL1 expression is detected by histological methods such as immunohistochemistry (IHC), it can be quantified using an H-score. First, PIWIL1 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.

[0209] Cancers associated with PIWIL1 expression include, but are not limited to, gastric cancer, pancreatic cancer, colorectal cancer, esophageal cancer, or thyroid cancer. For example, the cancer associated with PIWIL1 expression can be colorectal cancer. Alternatively or additionally, the cancer associated with PIWIL1 expression can be pancreatic cancer, such as pancreatic adenocarcinoma. The esophageal cancer can be gastroesophageal junction (GEJ) adenocarcinoma. The cancer can be microsatellite instability-high (MSI-high).

[0210] The antigen PIWIL1 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 or diagnostic method, in particular for use as an in vivo diagnostic agent or in vivo diagnostic method. In a preferred embodiment, the diagnostic agent / method is for diagnosing a proliferative disease. In a more preferred embodiment, the diagnostic agent / method is for diagnosing a proliferative disease. * The present invention is for diagnosing cancer by presenting a peptide comprising or consisting solely of the amino acid sequence of SLSNRLYYL (SEQ ID NO: 1) complexed with 02.

[0211] The present invention also provides the following: a binding molecule, a nucleic acid, a vector, a pharmaceutical composition or a 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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).

[0216] 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]

[0217] [Figure 1] Figure 1 shows T cell activation measured by IFNγ release against antigen-positive and -negative cancer cell lines using selected TCR variants ("a6b3" in the left graph and "a6b6" in the right graph) obtained with or without negative selection during affinity maturation. [Figure 2] FIG. 1 shows a plot showing affinity as determined by SPR versus potency as determined by IFNγ release for selected TCR variants obtained during affinity maturation. [Figure 3] Figure 1 shows T cell activation measured by IFNγ release for the TCR anti-CD3 fusion molecule a40b23U28 in the presence of A) antigen-positive and antigen-negative cancer cell lines and B) normal tissue. [Figure 4] A) T cell activation measured by IFNγ and GrB release for the TCR anti-CD3 fusion molecule a67b72U28 in the presence of antigen-positive and antigen-negative cancer cell lines. B) Redirected T cell killing of antigen-positive cancer cell lines by the TCR anti-CD3 fusion molecule a67b72U28. [Figure 5] 1 shows a schematic diagram of an exemplary multidomain single-chain binding molecule of the invention, where A) shows a representation of the domain arrangement from N-terminus to C-terminus, and B) shows a hypothetical representation of the folded structure of the molecule. [Figure 6]FIG. 1 shows T cell activation measured by IFNγ release for the TCR anti-CD3 fusion molecule a40b23U28-mol93 in the presence of antigen-positive and antigen-negative cancer cell lines. [Figure 7] FIG. 1 shows T cell activation measured by IFNγ release for the half-life extended TCR anti-CD3 fusion molecules a40b23U28-mol93 and a40b23U28-mol14 in the presence of antigen-positive cells. [Figure 8]

[0023] Figure 1 shows an image of the X-ray crystal structure model of the TCR-peptide interface formed by TCR "S8" when bound to SLSNRLYYL (SEQ ID NO: 1) in complex with HLA-A*02, showing key residue changes introduced during affinity maturation. [Figure 9] Figure 1 shows an image of the computer-modeled structure of the TCR-peptide interface formed by TCR mutant a67b72 when bound to SLSNRLYYL (SEQ ID NO: 1) in complex with HLA-A*02, with key residue changes introduced during affinity maturation labeled. [Figure 10] 1 shows an overlay of X-ray crystal structures showing the conformation of the SLSNRLYYL (SEQ ID NO: 1) peptide when bound to various scaffold TCRs, showing the position of peptide residue Y7 when bound to the S8 TCR. DETAILED DESCRIPTION OF THE INVENTION

[0218] Array Description HLA-A * 02 Restrictive peptide The genes for the original proteins are shown in brackets below. SEQ ID NO: 1: (PIWIL1): SLSNRLYYL SEQ ID NO: 97: (DOCK11): MLDKYSHYL

[0219] Exemplary scaffold TCR alpha chain (SEQ ID NO: 2) TIFF2026501720000025.tif24170

[0220] 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 SLSNRLYYL (SEQ ID NO:1) complexed with HLA-A2. This TCR is referred to herein as "S8." The α chain comprises a variable domain (SEQ ID NO:3) and a constant domain (SEQ ID NO:4, in 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.

[0221] Exemplary scaffold TCR beta chain (SEQ ID NO: 12) TIFF2026501720000026.tif33170

[0222] 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 SLSNRLYYL (SEQ ID NO:1) complexed with HLA-A2. This TCR is referred to herein as "S8." 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.

[0223] 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.

[0224] Alpha chain variable domain "a36" (SEQ ID NO: 22) comprising the CDRs (CDR1, CDR2, and CDR3 - underlined) designated in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively, and the framework regions (FR1, FR2, FR3, and FR4 - plain text) designated in SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 11, respectively: TIFF2026501720000027.tif15170

[0225] Alpha chain variable domain "a40" (SEQ ID NO: 28) comprising the CDRs (CDR1, CDR2, and CDR3 - underlined) designated in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 29, respectively, and the framework regions (FR1, FR2, FR3, and FR4 - plain text) designated in SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 11, respectively: TIFF2026501720000028.tif15170

[0226] Alpha chain variable domain "a58" (SEQ ID NO: 30) comprising the CDRs (CDR1, CDR2, and CDR3 - underlined) designated in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 29, respectively, and the framework regions (FR1, FR2, FR3, and FR4 - plain text) designated in SEQ ID NO: 31, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 11, respectively: TIFF2026501720000029.tif17170

[0227] Alpha chain variable domain "a61" (SEQ ID NO: 32) comprising the CDRs (CDR1, CDR2, and CDR3 - underlined) designated in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 29, respectively, and the framework regions (FR1, FR2, FR3, and FR4 - plain text) designated in SEQ ID NO: 33, SEQ ID NO: 26, SEQ ID NO: 118, and SEQ ID NO: 11, respectively: TIFF2026501720000030.tif17170

[0228] Alpha chain variable domain "a67" (SEQ ID NO: 34) comprising the CDRs (CDR1, CDR2, and CDR3 - underlined) designated in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 29, respectively, and the framework regions (FR1, FR2, FR3, and FR4 - plain letters) designated in SEQ ID NO: 35, SEQ ID NO: 26, SEQ ID NO: 118, and SEQ ID NO: 11, respectively: TIFF2026501720000031.tif17170

[0229] 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: 12. The CDRs are underlined and the mutations are shown in bold.

[0230] Beta chain variable domain "b23" (SEQ ID NO: 36) comprising the CDRs (CDR1, CDR2, and CDR3 - underlined) designated in SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 41, respectively, and the framework regions (FR1, FR2, FR3, and FR4 - plain text) designated in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively: TIFF2026501720000032.tif17170

[0231] Beta chain variable domain "b37" (SEQ ID NO:40) comprising the CDRs (CDR1, CDR2, and CDR3 - underlined) designated in SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively, and the framework regions (FR1, FR2, FR3, and FR4 - plain text) designated in SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively: TIFF2026501720000033.tif17170

[0232] Beta chain variable domain "b38" (SEQ ID NO:42) comprising the CDRs (CDR1, CDR2, and CDR3 - underlined) designated in SEQ ID NO:43, SEQ ID NO:38, and SEQ ID NO:41, respectively, and the framework regions (FR1, FR2, FR3, and FR4 - plain text) designated in SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively: TIFF2026501720000034.tif17170

[0233] Beta chain variable domain "b63" (SEQ ID NO:44) comprising the CDRs (CDR1, CDR2, and CDR3 - underlined) designated in SEQ ID NO:37, SEQ ID NO:45, and SEQ ID NO:41, respectively, and the framework regions (FR1, FR2, FR3, and FR4 - plain text) designated in SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively: TIFF2026501720000035.tif17170

[0234] Beta chain variable domain "b68" (SEQ ID NO:46) comprising the CDRs (CDR1, CDR2, and CDR3 - underlined) designated in SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:41, respectively, and the framework regions (FR1, FR2, FR3, and FR4 - plain text) designated in SEQ ID NO:47, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively: TIFF2026501720000036.tif17170

[0235] Beta chain variable domain "b72" (SEQ ID NO:48) comprising the CDRs (CDR1, CDR2, and CDR3 - underlined) designated in SEQ ID NO:37, SEQ ID NO:45, and SEQ ID NO:41, respectively, and the framework regions (FR1, FR2, FR3, and FR4 - plain text) designated in SEQ ID NO:47, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively: TIFF2026501720000037.tif17170

[0236] Exemplary TCRs The following sequences are TCRs containing exemplary combinations of the α and β chain variable domains shown above. 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.

[0237] a40b23 TCR TCR "a40b23" alpha chain sequence (SEQ ID NO:49), comprising the a40 variable domains described above (SEQ ID NO:28 - plain text) and the constant domains from the scaffold TCR described above (SEQ ID NO:4 - italics): TIFF2026501720000038.tif25170

[0238] TCR "a40b23" beta chain sequence (SEQ ID NO:50), comprising the b23 variable domain (SEQ ID NO:36 - plain text) and the constant domain from the scaffold TCR above (SEQ ID NO:14 - italics): TIFF2026501720000039.tif33170

[0239] a36b38 TCR TCR "a36b38" alpha chain sequence (SEQ ID NO:51) comprising the a36 variable domains described above (SEQ ID NO:22 - plain text) and the constant domains from the scaffold TCR described above (SEQ ID NO:4 - italics): TIFF2026501720000040.tif28170

[0240] TCR "a36b38" beta chain sequence (SEQ ID NO:52), comprising the b38 variable domain (SEQ ID NO:42 - plain text) and the constant domain from the scaffold TCR above (SEQ ID NO:14 - italics): TIFF2026501720000041.tif35170

[0241] a40b37 TCR TCR "a40b23" alpha chain sequence (SEQ ID NO:49), comprising the a40 variable domains described above (SEQ ID NO:28 - plain text) and the constant domains from the scaffold TCR described above (SEQ ID NO:4 - italics): TIFF2026501720000042.tif27170

[0242] TCR "a40b37" beta chain sequence (SEQ ID NO:53), comprising the b37 variable domain (SEQ ID NO:40 - plain text) and the constant domain from the scaffold TCR above (SEQ ID NO:14 - italics): TIFF2026501720000043.tif32170

[0243] a58b63 TCR TCR "a58b63" alpha chain sequence (SEQ ID NO:54), comprising the a58 variable domains described above (SEQ ID NO:30 - plain text) and the constant domains from the scaffold TCR described above (SEQ ID NO:4 - italics): TIFF2026501720000044.tif27170

[0244] TCR "a58b63" beta chain sequence (SEQ ID NO:55), comprising the b63 variable domain (SEQ ID NO:44 - plain text) and the constant domain from the scaffold TCR above (SEQ ID NO:14 - italics): TIFF2026501720000045.tif33170

[0245] a61b68 TCR TCR "a61b68" alpha chain sequence (SEQ ID NO:56), comprising the a61 variable domains described above (SEQ ID NO:32 - plain text) and the constant domains from the scaffold TCR described above (SEQ ID NO:4 - italics): TIFF2026501720000046.tif27170

[0246] TCR "a61b68" beta chain sequence (SEQ ID NO:57), comprising the b68 variable domain (SEQ ID NO:46 - plain text) and the constant domain from the scaffold TCR above (SEQ ID NO:14 - italics): TIFF2026501720000047.tif32170

[0247] a67b72 TCR "a67b72" alpha chain sequence (SEQ ID NO:58), comprising the a67 variable domain (SEQ ID NO:34 - plain text) above and the constant domain (SEQ ID NO:4 - italics) from the scaffold TCR above: TIFF2026501720000048.tif27170

[0248] TCR "a67b72" beta chain sequence (SEQ ID NO:59), comprising the b72 variable domain (SEQ ID NO:48) and the constant domain from the scaffold TCR above (SEQ ID NO:14): TIFF2026501720000049.tif33170

[0249] Exemplary Anti-CD3 Antibody Sequences Anti-CD3 scFv:U0 SEQ ID NO:60 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:61. The light chain CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO:65. The heavy chain CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68. The glycine-serine linker connecting VL and VH is shown in plain text and is designated SEQ ID NO:86.

[0250] SEQ ID NO:60: TIFF2026501720000050.tif33170

[0251] Anti-CD3 scFv:U28 SEQ ID NO:70 is the amino acid sequence of another exemplary anti-CD3 scFv, designated herein as "U28." This sequence is identical to SEQ ID NO:60 above, except for two substitutions (T164A and I201F), which are double-underlined. The light chain variable domain (VL) is in italics and is designated SEQ ID NO:61. The light chain CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO:69. The heavy chain CDRs (CDR1, CDR2, and CDR3) are underlined (CDR1 contains the double-underlined alanine residue) and are designated SEQ ID NO:71, SEQ ID NO:67, and SEQ ID NO:68. The glycine-serine linker connecting VL and VH is shown in plain text and is designated SEQ ID NO:86.

[0252] SEQ ID NO:70: TIFF2026501720000051.tif34170

[0253] Exemplary TCR-anti-CD3 fusion sequences a40b23U28 "a40b23U28" is a binding molecule comprising the TCR "a40" alpha chain (SEQ ID NO: 49) described above and a TCR beta chain-anti-CD3 fusion (SEQ ID NO: 119). The beta chain-anti-CD3 fusion sequence (SEQ ID NO: 119) is shown below. This sequence comprises the U28 anti-CD3 scFv (SEQ ID NO: 70, italics) described above fused to the TCR "b23" beta chain (SEQ ID NO: 50) described above. The TCR beta chain and anti-CD3 scFv sequences are linked via a glycine-serine linker (underlined) designated SEQ ID NO: 72.

[0254] SEQ ID NO:119: TIFF2026501720000052.tif58170

[0255] a36b38U28 "a36b38U280" is a binding molecule comprising the TCR "a36b38" α chain (SEQ ID NO: 51) and a TCR β chain-anti-CD3 fusion (SEQ ID NO: 73). The β chain-anti-CD3 fusion sequence (SEQ ID NO: 73) is shown below. This sequence comprises the U28 anti-CD3 scFv (SEQ ID NO: 70, italics) fused to the TCR "a36b38" β chain (SEQ ID NO: 52). The TCR β chain and anti-CD3 scFv sequences are covalently linked via a glycine-serine linker (underlined) as specified in SEQ ID NO: 72.

[0256] SEQ ID NO:73: TIFF2026501720000053.tif55170

[0257] a40b37U28 "a40b37U28" is a binding molecule comprising the TCR "a40b37" α chain (SEQ ID NO: 49) and a TCR β chain-anti-CD3 fusion (SEQ ID NO: 74). The β chain-anti-CD3 fusion sequence (SEQ ID NO: 74) is shown below. This sequence comprises the U28 anti-CD3 scFv (SEQ ID NO: 70, italics) fused to the TCR "a40b37" β chain (SEQ ID NO: 53). The TCR β chain and anti-CD3 scFv sequences are covalently linked via a glycine-serine linker (underlined) designated SEQ ID NO: 72.

[0258] SEQ ID NO:74: TIFF2026501720000054.tif55170

[0259] a58b63U28 "a58b63U28" is a binding molecule comprising the TCR "a58b63" α chain (SEQ ID NO: 54) and a TCR β chain-anti-CD3 fusion (SEQ ID NO: 75). The β chain-anti-CD3 fusion sequence (SEQ ID NO: 75) is shown below. This sequence comprises the U28 anti-CD3 scFv (SEQ ID NO: 70, italics) fused to the TCR "a58b63" β chain (SEQ ID NO: 55). The TCR β chain and anti-CD3 scFv sequences are covalently linked via a glycine-serine linker (underlined) designated SEQ ID NO: 72.

[0260] SEQ ID NO:75: TIFF2026501720000055.tif54170

[0261] a61b68U28 "a61b68U28" is a binding molecule comprising the TCR "a61b68" α chain (SEQ ID NO: 56) and a TCR β chain-anti-CD3 fusion (SEQ ID NO: 75). The β chain-anti-CD3 fusion sequence (SEQ ID NO: 76) is shown below. This sequence comprises the U28 anti-CD3 scFv (SEQ ID NO: 70, italics) fused to the TCR "a61b68" β chain (SEQ ID NO: 57). The TCR β chain and anti-CD3 scFv sequences are covalently linked via a glycine-serine linker (underlined) designated SEQ ID NO: 72.

[0262] SEQ ID NO:76: TIFF2026501720000056.tif59170

[0263] a67b72U28 "a67b72U28" is a binding molecule comprising the TCR "a67b72" α chain (SEQ ID NO: 58) and a TCR β chain-anti-CD3 fusion (SEQ ID NO: 77). The β chain-anti-CD3 fusion sequence (SEQ ID NO: 77) is shown below. This sequence comprises the U28 anti-CD3 scFv (SEQ ID NO: 70, italics) fused to the TCR "a67b72" β chain (SEQ ID NO: 59). The TCR β chain and anti-CD3 scFv sequences are covalently linked via a glycine-serine linker (underlined) designated SEQ ID NO: 72.

[0264] SEQ ID NO:77: TIFF2026501720000057.tif59170

[0265] a40b23U28-mol93 "a40b23U28-mol93" is an a40b23U28 binding molecule in a single-chain format and with an Fc half-life extending domain. From N- to C-terminus, this molecule comprises the U28 anti-CD3 scFv (SEQ ID NO:70, italics), a b23 TCR β chain sequence (double underlined, plain text) comprising the b23 variable domain (SEQ ID NO:36) described above and a constant domain with reduced glycosylation (SEQ ID NO:117), a glycine-serine linker (SEQ ID NO:85, underlined, italics), an Fc hinge region (SEQ ID NO:94, bold italics), a first Fc domain (SEQ ID NO:95, plain text), a glycine-serine linker (SEQ ID NO:85, underlined, italics), and an a40 U28 binding molecule comprising the a40 variable domain (SEQ ID NO:28) described above and a constant domain with reduced glycosylation (SEQ ID NO:116). It comprises the TCR alpha chain sequence (double underlined, bold), a glycine-serine linker (SEQ ID NO: 85, underlined, italic), an Fc hinge region (SEQ ID NO: 94, bold italic), and a second Fc domain (SEQ ID NO: 96, bold).

[0266] SEQ ID NO:93: TIFF2026501720000058.tif133170

[0267] Exemplary Amino Acid Linker Sequences GGGGS (SEQ ID NO: 72), GGGSG (SEQ ID NO: 78), GGSGG (SEQ ID NO: 79), GSGGG (SEQ ID NO: 80), GSGGGP (SEQ ID NO: 81), GGEPS (SEQ ID NO: 82), GGEGGGP (SEQ ID NO: 83), GGEGGGSEGGGS (SEQ ID NO: 84), GGGSGGGG (SEQ ID NO: 85), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 86), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 87), EAAAK (SEQ ID NO: 88), and EAAAKEAAAKEAAAK (SEQ ID NO: 89).

[0268] Additional mutant α chain variable domains a6 (SEQ ID NO: 90) TIFF2026501720000059.tif12170

[0269] Additional mutated β-chain variable domains b6 (SEQ ID NO: 91) TIFF2026501720000060.tif12170

[0270] b3 (SEQ ID NO: 92) TIFF2026501720000061.tif12170

[0271] Constant domain sequences with reduced glycosylation Alpha chain constant domain with three N→Q mutations (SEQ ID NO: 116) TIFF2026501720000062.tif13170

[0272] β-chain constant domain with one N→Q mutation (SEQ ID NO: 117) TIFF2026501720000063.tif20170

[0273] SLSNRLYYL (SEQ ID NO: 1) HLA-A * TCR CDR amino acid residues important for binding to the O2 complex HLA-A *Using the structures of TCR "S8" and mutant "a67b72" bound to SLSNRLYYL (SEQ ID NO: 1) complexed with 02 (described in Example 7 and shown in Figures 8 and 9), 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).

[0274] TIFF2026501720000064.tif44170

[0275] TIFF2026501720000065.tif27170

[0276] TIFF2026501720000066.tif37170

[0277] TIFF2026501720000067.tif25170

[0278] TIFF2026501720000068.tif25170

[0279] TIFF2026501720000069.tif37170 [Example]

[0280] 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.

[0281] Example 1 - Expression of PIWIL in cancer PIWIL and PIWIL-like proteins are known to be involved in carcinogenic processes such as cell reproduction, cell migration, and cell invasion, as well as disease progression. Overexpression of PIWIL is associated with various tumor types, and PIWIL expression is associated with poor prognosis in colorectal and gastric cancers (Non-Patent Document 7, Non-Patent Document 8).

[0282] Publicly available PIWIL1 mRNA expression data obtained from The Cancer Genome Atlas (TCGA, http: / / cancergenome.nih.gov / ) showed a relatively higher frequency of PIWIL expression in colon cancer, especially in the high-microsatellite instability (MSI) and BRAF-mutated subsets, where expression was also observed in esophageal and gastric tumors. Gene expression values ​​for the TCGA dataset were normalized by FPKM (fragments per thousand bases of transcript per million mapped reads). PIWIL1 expression was also assessed in the samples by routine quantitative reverse transcription PCR (RTqPCR). RTqPCR results validated the frequencies observed from the TCGA dataset. Exemplary frequency values ​​are shown in the table below.

[0283] TIFF2026501720000070.tif59170

[0284] PIWIL1 gene expression data were supplemented by analysis from a dataset corresponding to TempusxT (http: / / www.tempus.com / oncology / genomic-profiling / ), a next-generation sequencing (NGS) panel of 648 genes containing whole-transcriptome RNA-Seq data. PIWIL1 gene expression was observed within the top 25 most common solid tumor types in the Tempus dataset. Gene expression values ​​were normalized by transcripts per million (TPM) and presented in log2-transformed (TPM + 1) units. Each 1-unit increase in expression corresponds to a doubling of gene expression. The table below shows the frequency of PIWIL1 expression in the 10 most highly expressed cancer types.

[0285] TIFF2026501720000071.tif59170

[0286] Further analysis of the Tempus data showed enrichment for PIWIL expression in a small subset of patients with high-frequency MSI and BRAF mutations, consistent with the TCGA and RTqPCR data, as well as slightly higher PIWIL1 expression in female patients and an increase in PIWIL1 expression with age. No significant correlation was found between PIWIL1 expression and disease progression.

[0287] Immunohistochemistry (IHC) studies were also performed on colon adenocarcinoma tumor samples. These analyses demonstrated that PIWIL1 was uniformly expressed in colon adenocarcinoma samples and expression was maintained in metastatic samples. IHC analysis also confirmed enrichment of PIWIL1 expression in MSI-H (microsatellite instability-high) mutant and BRAF mutant subsets.

[0288] Example 2 - Identification of TCRs specific for PIWIL1 peptide-HLA complexes To generate a soluble, potent TCR-anti-CD3 fusion cancer therapy protein (Immunomobilizing Monoclonal TCR against Cancer, or ImmTAC™) that targets PIWIL1, SLSNRLYYL (SEQ ID NO: 1)-HLA-A with high affinity and specificity. * We first identified TCR candidates capable of binding to the 02 complex. In a first step, 16 natural, i.e., wild-type, TCR candidates with binding affinities in the low μM range were isolated from T cell clones obtained from human donors or from natural TCR phage libraries. The construction of natural TCR phage libraries has been previously described (e.g., in WO 2015 / 136072, WO 2017 / 046198, and WO 2017 / 046201). The chain usage and CDR3 sequence of each natural TCR were determined. Next, each natural TCR was used as a template to identify TCR mutants with higher affinity binding. Affinity maturation was performed on 16 series, where each series represented an affinity optimization run starting from a natural TCR.

[0289] SLSNRLYYL (SEQ ID NO: 1)-HLA-A * Selection of higher affinity TCRs that bind to the O2 complex was performed using a TCR phage display library as previously described (Non-Patent Document 15). For each affinity maturation run associated with a single lineage, typically at least two rounds of affinity maturation were performed to iteratively select higher affinity TCR variants. The amino acid sequences of the α and β variable domains were then determined.

[0290] Soluble forms of the TCR were generated by fusing the variable domains to truncated versions of the respective α- and β-chain constant domains, incorporating non-native interchain disulfide bonds between the constant domain residues, as previously described (WO 2003 / 020763).

[0291] 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 10, Non-Patent Document 11).

[0292] To assess the ability of each native TCR of each lineage to recognize the target pHLA complex, binding parameters were obtained by surface plasmon resonance (SPR). SPR measurements were performed on a BIAcore 8K instrument, a BIAcore 3000 instrument, or a BIAcore T200 instrument. Briefly, biotinylated class I HLA-A * O2 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.

[0293] From the 16 series analyzed, a TCR identified from series 8 (S8) with a chain combination corresponding to TRAV4 / TRVB11-2 was selected for further optimization.

[0294] The affinity of the soluble native S8 TCR for the peptide-HLA complex was determined to be 225 nM.

[0295] The specificity of the native S8 TCR for the target pHLA complex was determined by screening a peptide library with different peptide-HLA-A. * Binding to the O2 complex was assessed. One particular peptide from the protein DOCK11 was identified as a potential mimetic. The positions within DOCK11 that differ from the target PIWIL peptide are shown in bold and underlined below.

[0296] TIFF2026501720000072.tif15170

[0297] The interaction between each native TCR and the mimetic peptide was performed using the SPR method described above.

[0298] The affinity of the native S8 TCR for the mimetic peptide-HLA complex was determined to be 1540 nM, indicating a 6.8-fold affinity window between binding to the target and the mimetic.

[0299] TCR S8 was then used as a template in the first round of affinity maturation to identify TCR variants with higher affinities, as previously described (Non-Patent Document 15). Briefly, a TCR phage library was created using NNK oligonucleotides to generate mutations in the complementarity-determining regions (CDRs). For affinity maturation of S8, negative selection was also incorporated in the first round of affinity maturation.

[0300] Negative selection with mimetic peptides during affinity maturation improved the affinity window with the mimetic peptides. TCR variants identified using negative selection exhibited improved specificity profiles for antigen-negative cells, as determined by the ability of the TCR to drive T cell activation when fused to anti-CD3 scFv. As shown in Figure 1, TCR variants fused to anti-CD3 and carrying additional mutations identified only by negative selection exhibited improved specificity profiles for antigen-positive cells (Ag +), whereas TCR mutants that had not undergone negative selection exhibited specific binding to antigen-negative (Ag - ) cells.

[0301] From the first round of affinity maturation of the S8 lineage, the highest affinity TCR variant (a6b6U) selected using negative selection had a K of 365 pM. d Other TCR variants were identified with relatively higher binding affinities, which is an important factor in selecting TCR therapeutic candidates for development. - The cross-reactivity to cells was not very specific or clear.

[0302] Example 3 - Identification of potent high affinity TCRs specific for PIWIL peptide Further improvements in TCR affinity were achieved in a second round of affinity maturation using a second generation library prepared from TCRs isolated from the first round. Mutated α and β chain combinations from this second round were selected and identified.

[0303] Following the second round of selection, an additional (third) round of selection was performed using a library generated by shuffling the selected CDRs of interest. For example, α and β chains were generated by randomly shuffling the three CDRs of the target α chain and the three CDRs of the target β chain from the first two rounds of affinity maturation to generate new α and β chains. These new α and β chains were utilized in the TCR phage display library method in additional rounds of selection. Several TCR variants obtained from this additional shuffled library selection generated TCR variants with further enhanced affinity and increased potency in cellular assays, as shown in Figure 2. The a40b23U28 TCR variant, highlighted by an arrow in Figure 2, combined the strongest potency with very high affinity. The a40b23U28 TCR variant was selected for further characterization and optimization.

[0304] To evaluate the binding of these TCR mutants, 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. A binding molecule in this format is, for example, the ImmTAC™ molecule, which contains tebentafusp, sold under the trade name KIMMTRAK™. Such molecules are hereinafter referred to as "TCR-anti-CD3 fusions." The TCR-anti-CD3 fusion molecules described herein contain a mutant anti-CD3 sequence designated U28 (SEQ ID NO: 70), previously described (WO 2020 / 157210).

[0305] 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.

[0306] 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. 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.

[0307] A summary of the binding parameters of selected TCR-anti-CD3 fusion molecules is shown in the table below:

[0308] TIFF2026501720000073.tif30170

[0309] Further evaluation of the binding of a40b23U28 to a mimetic peptide from DOCK11 determined that the resulting affinity window was greater than 5000-fold.

[0310] Example 4 - Affinity-selected TCR-anti-CD3 fusion molecules exhibit potent and specific T cell activation The potency of the TCR-anti-CD3 fusion molecules selected from multiple rounds of affinity maturation was evaluated. 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.

[0311] In this example, the following cancer cell lines were used as antigen-positive target cells compared to the antigen-negative cells NCI-H1755: KATOIII (gastric cancer), CL11 (colon cancer), COLO201 (colorectal adenocarcinoma), COLO205 (colorectal adenocarcinoma), and COLO206F (colorectal adenocarcinoma). Data were plotted using PRISM software, and EC values ​​were extracted from the curves. 50 values ​​were calculated.

[0312] As shown in Figure 3A, the a40b23U28 TCR-anti-CD3 fusion molecule exhibited good potency in a T cell activation assay via IFN-γ release, where EC against antigen-positive target cells. 50 The values ​​fell within the range between 10 pM and 200 pM.

[0313] EC for three TCR-anti-CD3 fusion molecules 50 The values ​​are shown in the table below:

[0314] TIFF2026501720000074.tif43170

[0315] To further explore their suitability for therapeutic use, the a40b23U28 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: (pulmonary-bronchial epithelial cells HRCE) pic12 and HRCE pic11 ), cardiac-aortic endothelial cells (HAoEC2), muscle-skeletal myoblasts (SkMC2), and lung fibroblasts (InMycFib1). As shown in Figure 3B, reactivity against these normal cells was undetectable below 3 nM.

[0316] In additional studies, no widespread release of pro-inflammatory cytokines was observed below 10 nM.

[0317] Example 5 - Further optimization improves manufacturing yield and stability while maintaining other desirable therapeutic properties Additional amino acid substitutions were made in the TCR α and β chain variable domains of the a40 and b23 chains of the a40b23U28 TCR anti-CD3 fusion molecule to improve manufacturability and yield while maintaining affinity, potency, and specificity. One methionine cleavage site and one deamidation-prone residue were identified in the α chain and one in the β chain.

[0318] An L1A substitution in the α-chain of a40 was found to be beneficial for improving N-terminal methionine cleavage during production in E. coli. Furthermore, an N52E substitution in the β-chain of b23 was found to reduce the risk of deamidation. Incorporation of these mutations resulted in a four-fold improvement in the yield of the a40b23U28 TCR anti-CD3 fusion molecule. The resulting TCR anti-CD3 fusion molecule was designated a58b63U28.

[0319] In parallel, amino acid substitutions responsible for increasing the thermal stability of the molecule, as determined by differential scanning fluorometry (DSF), were identified. Three mutations were identified, two α-chain mutations and one β-chain mutation that increased Tm: T21P and P65S in the α-chain based on the numbering of a40 (SEQ ID NO: 28), and R16G in the β-chain based on the numbering of b23 (SEQ ID NO: 36). These mutations were found to increase Tm by more than 10°C. The resulting TCR-anti-CD3 fusion molecule was designated a61b68U28.

[0320] Combinations of these mutations were introduced into a40b23U28 to create a TCR-anti-CD3 fusion molecule designated a67b72U28.

[0321] join The SPR binding analysis described above with a67b72U28 and two intermediates (a58b63U28 and a61b68U28) at both 25°C and 37°C confirmed that the a67b72U28 TCR anti-CD3 fusion molecule retained pM affinity.

[0322] The binding data are shown in the table below:

[0323] TIFF2026501720000075.tif30170

[0324] Potency - T cell activation The potency of a67b72U28 was determined in a cellular assay. T cell activation was assessed by the release of interferon-γ (IFNγ) or granzyme B (GrB) using the ELISPOT assay described above. In both cases, peripheral blood mononuclear cells (PBMCs) isolated from fresh donor blood were used as effector cells. The cancer cell lines KATOIII and C11 were used as antigen-positive target cells in comparison with the antigen-negative cell lines NCI-1755 and IM95. Data were plotted using PRISM software, and EC values ​​were extracted from the curves. 50 values ​​were calculated.

[0325] The resulting T cell activation data is shown in Figure 4A.

[0326] Efficacy - Cell Killing To examine the ability of the TCR-anti-CD3 fusion molecule a67b72U28 to redirect T cells to kill antigen-positive cancer cells, immune cell killing assays were performed using the Incucyte live-cell imaging platform with the CellPlayer 96-well Caspase-3 / 7 Apoptosis Assay Kit (Essen BioScience, Catalog No. 4440) according to the manufacturer's instructions. PBMCs were used as effector cells. Tumor cell death was detected by measuring apoptosis using Incucyte™ Caspase 3 / 7 reagent or by counting nuclear-labeled cells using the manufacturer's protocol and reagents. The number of apoptotic cells in each image was determined. In both cases, assays were performed in triplicate, with measurements taken every 2 hours over a 96-hour period. The percentage of cell lysis was calculated at various concentrations of the test molecule. In this example, KATO III (gastric cancer), CL11, and COLO206F (colon adenocarcinoma) cells were used as target-positive cancer cells. EC 50 Values ​​were calculated from the curves and are shown in Figure 4B.

[0327] These data demonstrate that the a67b72U28 TCR-anti-CD3 fusion molecule inhibits inflammatory cytokines with EC2s in the low pM range. 50 These results demonstrate that IL-16 mediates potent T cell activation and cancer cell killing.

[0328] Stability and Yield For the a67b72U28 TCR-anti-CD3 fusion molecule, a typical refolding yield of approximately 17.5 mg / L was obtained from 2 x 20 L batches. The onset Tm was determined to be 50.45°C.

[0329] Overall, these data confirm that the TCR-anti-CD3 fusion molecules of the present invention have desirable therapeutic properties.

[0330] Example 6 - Multidomain single-chain binding molecules targeting the PIWIL1 peptide-MHC complex exhibit potent and specific T cell activation A multidomain single-chain binding molecule containing a pMHC-binding domain that targets the PIWIL1 peptide (SLSNRLYYL, SEQ ID NO: 1)-MHC complex was designed. The full sequence of the resulting molecule, designated "a40b23U28-mol93," is shown in SEQ ID NO: 93. The TCR alpha and TCR beta variable domains of a40b23U28-mol93 correspond to SEQ ID NO: 28 ("a40") and SEQ ID NO: 36 ("b23"), respectively.

[0331] In the multi-domain binding molecule format shown in Figure 5a, the U28 scFv ("TCE-VL" and "TCE-VH") are linked via a linker to the N-terminus of a concatenation of a TCR β chain variable domain and a TCR β chain constant domain (collectively "VC1"). The TCR β chain constant domain is linked at its C-terminus to the N-terminus of a first IgG Fc domain ("FC1") via a linker, and the first IgG Fc domain is linked at its C-terminus to the N-terminus of a TCR α chain variable domain via a linker. The TCR α chain constant domain is linked at its C-terminus to the N-terminus of a second IgG Fc domain ("FC2") via a linker. The domains are covalently linked as a single polypeptide chain. A format referred to as "Mol93" is disclosed in U.S. Patent Application No. 63 / 371,861, filed August 18, 2022, the contents of which are incorporated herein by reference.

[0332] As shown in Figure 5b, the variable light chain and variable heavy chain of U28 dimerize to form the anti-CD3 scFv, the TCR α chain and TCR β chain dimerize to form the pMHC-binding region, and the first IgG Fc domain and the second IgG Fc domain dimerize to form the half-life-extending region.

[0333] Expression a40b23U28-mol93 was expressed in Cho cells using Thermo's ExpiCHO™ transient expression protocol. Briefly, 6×10 cells were cultured. 6 Transfection was performed after dilution to a concentration of 100 μg. Cells were harvested 14 days after transfection and the temperature was shifted to 32°C on day 1 after transfection. Feed additions were performed on days 1 and 5 after transfection. Clarification was performed by two consecutive centrifugation steps at 300 × g and 17,500 × g. The resulting supernatant was passed through 0.45 μm and 0.2 μm membrane filters.

[0334] purification The clarified supernatant was purified using Protein A, followed by a size-exclusion chromatography step. A 15 cm bed height MabSelect Extra Protein A resin column was prepared. Fifty column volumes of the supernatant were loaded onto the column and eluted using pH 3.0 sodium citrate buffer. The eluted product was collected over three column volumes, filtered through a 0.2 μm membrane filter, and neutralized by adding 2 M Tris. The Protein A eluate was concentrated to at least 2 mg / mL using tangential flow filtration (Pellicon™ XL50 with Ultracel™ 30 kDa membrane) and then loaded onto HiLoad 26 / 600 Superdex SEC resin. 5% of the column volume was loaded onto the column. The product was eluted in phosphate-citrate buffer, and the relevant fraction was filtered through a 0.22 μm membrane filter.

[0335] join a40b23U28-mol93 and SLSNRLYYL (SEQ ID NO: 1)-HLA-A * Binding to the 02 complex was determined using SPR as described above in Examples 2 and 3. Binding affinity (K D ) is 50 pM, and the binding half-life (t 1 / 2 ) was 11.8 hours. These results indicate a binding affinity (K D) and a binding half-life (t 1 / 2) was comparable to the equivalent a40b23U28 TCR-anti-CD3 fusion molecule lacking the half-life extending (i.e., Fc) domain.

[0336] The ability of a40b23U28-mol93 to stimulate 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. In this assay, KATOIII (gastric cancer) and CL11 (colon cancer) cells were used as antigen-positive target cells. NCI-H1755 cells were used as antigen-negative cells. Data were plotted using PRISM software, and EC values ​​were calculated from the curves. 50 values ​​were calculated.

[0337] Figure 6 shows that a40b23U28-mol93 has EC in the low pM range against two antigen-positive cell lines. 50 values ​​(42.1 pM for KATO-III and 164.0 pM for CL11), indicating little to no response in the presence of antigen-negative cells (at concentrations of a40b23U28-mol93 less than 1 nM). An equivalent TCR-anti-CD3 fusion molecule (a40b23U28) lacking the half-life-extending (i.e., Fc) domain exhibited EC values ​​of 12.9 pM and 52.7 pM for KATO-III and CL11 cells, respectively, as shown in the table in Example 4. 50 had.

[0338] These data demonstrate that the multidomain single-chain binding molecules of the invention containing a pMHC-binding region that targets the PIWIL1 peptide-MHC complex retain the same high affinity and potency as comparable TCR-anti-CD3 fusion molecules lacking the half-life extending (i.e., Fc) region, and retain specificity for antigen-positive cells.

[0339] In further experiments, a40b23U28-mol93 was compared with another multidomain molecular format designated a40b23U28-mol14. a40b23U28-mol14 has the same amino acid sequences of the individual domains as a40b23U28-mol93, except that it is arranged in a two-chain format, as shown in Figure 7. In this two-chain format, the first chain (the left chain in Figure 7) contains, from N- to C-terminus, a TCR α chain variable domain, a TCR α chain constant domain, and an Fc domain. The second chain (the right chain in Figure 7) contains, from N- to C-terminus, an anti-CD3 scFv, a TCR β chain variable domain, a TCR β chain constant domain, and an Fc domain.

[0340] T cell activation against the KATOIII cell line was compared between the two molecules. As shown in Figure 7, both molecules drive T cell activation. However, Mol93 elicits a stronger response than Mol14.

[0341] The results described in this example demonstrate that the multi-domain single chain format was effective in extending half-life (by providing an Fc region) without significantly affecting affinity for the target or potency of T cell activation.

[0342] Example 7 - Structural analysis of TCR-pHLA binding forms SLSNRLYYL (SEQ ID NO: 1) HLA-A * Structural analysis of the S8 TCR bound to the O2 complex was performed using X-ray crystallography, and unique features that confer potency and specificity were identified.

[0343] 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 performed 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 "a67b72" was performed using the "S8" TCR as a template using the Molecular Operating Environment (MOE) package (version 2022.02, Chemical Computing Group ULC, Canada), with refinement using QuickPrep.

[0344] TCR docking geometry angle calculations: The following angle calculations were adopted from Rudolph et al. (2006). Annu Rev Immunol. 24, 419-466.

[0345] a. Crossing angle. This angle was calculated by generating two vectors: the HLA groove vector and the TCR cystine 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 cystine vector (i.e., the "TCR inter-domain vector") connects the characteristic cystines within the two variable regions (i.e., the intra-chain disulfide bonds) and points from the intra-chain disulfide bond in the α chain variable region to the intra-chain disulfide bond in the β chain variable region. The crossing angle was defined as the angle between the TCR cystine vector and the HLA groove vector.

[0346] 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.

[0347] 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.

[0348] 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.

[0349] result The crystal structure reveals that the S8 TCR interacts with its cognate HLA-A (i.e., SLSNRLYYL (SEQ ID NO: 1)) * It was revealed that the S8 TCR bound to the SLSNRLYYL (SEQ ID NO: 1) HLA-A complex at a crossing angle of approximately 63°. Three of the six CDRs (αCDR1, αCDR3, and βCDR3) directly interacted with the SLSNRLYYL (SEQ ID NO: 1) peptide, contacting peptide residues at positions 4 to 8. The table below shows the interaction between the S8 TCR and the SLSNRLYYL (SEQ ID NO: 1) HLA-A complex. * Figure 1 shows an overview of buried surface area and binding geometry calculations for interactions between the 02 complex.

[0350] TIFF2026501720000076.tif92170

[0351] Identification of peptide and TCR CDR residues important for binding HLA-A *Further analysis of the crystal structure of the S8 TCR bound to SLSNRLYYL (SEQ ID NO: 1) complexed with 02 identified key residues within the interface between the peptide and the TCR CDR. As can be seen from the crystal structure shown in Figure 8, the peptide side chains at positions N4, R5, Y7, and Y8 of SLSNRLYYL (SEQ ID NO: 1) form the major antigen contact points on the peptide, with the residues at these positions having exposed side chains facing the TCR interface. In the case of the high-affinity mutants described in the Examples above, the combined introduction of an S→D mutation in CDR3β and an F→Y mutation in CDR1α resulted in improved shape complementarity between the peptide and the CDR. Furthermore, the S→D mutation in CDR3β improved specificity by expanding the window for the mimetic peptide DOCK11. For example, the TCR mutant referred to herein as "a67b72" contains both of these mutations and has affinity for pHLA complexes in the pM range and a wide affinity window for mimetic peptides. The interface between a67b72 and the SLSNRLYYL (SEQ ID NO: 1) peptide was computationally modeled based on the TCR crystal structure of S11 and is shown in FIG.

[0352] The CDR residues of the S8 TCR that are important for binding to the SLSNRLYYL (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.

[0353] TIFF2026501720000077.tif10170

[0354] To demonstrate that the high affinity TCR mutants described in Examples 1-6 maintain the same or similar peptide contacts with the S8 TCR, HLA-A *Using the crystal structure of the S8 TCR bound to SLSNRLYYL (SEQ ID NO: 1) in complex with O2 as a template, the CDR residues in contact with the peptide of the TCR variant a67b72 SLSNRLYYL (SEQ ID NO: 1) were determined by computer modeling. The CDR positions of a67b72 important for binding to SLSNRLYYL (SEQ ID NO: 1) were the same as those of S8. These are shown in the table below.

[0355] TIFF2026501720000078.tif10170

[0356] Comparison of the S8 TCR structure with TCRs from other scaffolds Additional crystal structures were obtained for TCRs that bind to the same pHLA complex but are derived from different scaffolds. Of the 10 TCRs analyzed, only the S8 TCR (and its high-affinity variants described above) showed hydrogen-bonding contacts with all four exposed peptide residues, and this was the only TCR to show hydrogen-bonding contacts with position Y7. In several other TCRs, the peptide Y7 side chain was in a partially buried conformation, preventing contact with the TCR. An overlay of the peptide conformations when bound to each TCR showed that in the case of the S8 TCR, peptide position Y7 was pulled upward to make contact (Figure 10).

[0357] These data demonstrate the major antigenic features of the peptide and suggest that engagement of Y7 directly contributes to both the affinity and specificity of the interaction.

Claims

1. A binding molecule comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the binding molecule is ＊ 02, wherein each of the α chain variable domain and the β 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 (a) wherein the binding molecule contacts at least N4, R5, Y7, and Y8 of SLSNRLYYL (SEQ ID NO: 1); and / or (b) where: The CDR1 of the α chain has the sequence (SEQ ID NO: 98), and the CDR3 of the alpha chain comprises the sequence (SEQ ID NO: 104), and The CDR3 of the β chain has the sequence (SEQ ID NO: 112), wherein X is any amino acid.

2. CDR1 of the alpha chain comprises the sequence Y / N-I / V / L-A / G-A / T-N-D / E-Y / F (SEQ ID NO: 100); the CDR3 of the alpha chain comprises the sequence V / I / L-A / G-Y / W / F-G-G-T-D-K / V / L-V / I / L-I / P (SEQ ID NO: 106); and / or The binding molecule of claim 1, wherein the CDR3 of the beta chain comprises the sequence A / G-S / T-S / T-V-D-Y / W / F-V-G-S / DA / G-D / E-R-Q / N-Y / W / F (SEQ ID NO: 114).

3. The CDR1 of the α chain has the sequence (SEQ ID NO:23), optionally with one, two, or three mutations at any of positions 1 to 4 and 6 of SEQ ID NO:23; The CDR3 of the α chain has the sequence (SEQ ID NO:29), optionally with 1, 2, 3, or 4 mutations at any of positions 1 to 3 and positions 8 to 10 of SEQ ID NO:29; and / or The CDR3 of the β chain has the sequence (SEQ ID NO: 41), optionally with 1, 2, 3, or 4 mutations at any of positions 1 to 4, 6, and 10 to 14 of SEQ ID NO:

41.

4. CDR2 of the alpha chain comprises the sequence GYKTN (SEQ ID NO: 24), optionally with 1, 2 or 3 mutations; CDR1 of the β chain comprises the sequence SGHGT (SEQ ID NO: 37), optionally with 1, 2 or 3 mutations; and / or 4. The binding molecule of claim 1, wherein CDR2 of the beta chain comprises the sequence FHEEGV (SEQ ID NO: 45), optionally with 1, 2 or 3 mutations.

5. CDR2 of the alpha chain comprises the sequence G / A-Y / W / F-K / R / H-T / S-N / K (SEQ ID NO: 102); CDR1 of the beta chain comprises the sequence S / T-A / G-K / R / H-A / G-S / T (SEQ ID NO: 108), and 4. The binding molecule of claim 1, wherein CDR2 of the beta chain comprises the sequence Y / W / FH / QN / EN / EA / GV / I / L (SEQ ID NO: 110).

6. A binding molecule comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the binding molecule is ＊ 02 in complex with SLSNRLYYL (SEQ ID NO: 1), wherein each of the α chain variable domain and the β chain variable domain comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR is a framework region and CDR is a complementarity determining region; where: (a) the α chain CDRs have the following sequences: CDR1 - YIAANDF (SEQ ID NO: 23), optionally with 1, 2 or 3 mutations; CDR2 - GYKTN (SEQ ID NO: 24) optionally with 1, 2 or 3 mutations; CDR3 - LAWGGTDLLP (SEQ ID NO: 29), optionally with 1, 2, 3, or 4 mutations; and / or (b) the CDRs of the β chain have the following sequences: CDR1 - SGHGT (SEQ ID NO: 37), optionally with 1, 2 or 3 mutations; CDR2 - FHEEGV (SEQ ID NO: 45), optionally with 1, 2 or 3 mutations; CDR3 - ASSVDWVGDGERQY (SEQ ID NO: 41), optionally with 1, 2, 3, 4, or 5 mutations; A binding molecule comprising:

7. The one or more mutations in the CDRs of the alpha chain are numbered according to SEQ ID NO: 34: The binding molecule of claim 6, selected from:

8. The one or more mutations in the CDRs of the β chain are numbered according to SEQ ID NO: 48: The binding molecule of claim 6 or 7, selected from:

9. The following combinations of α-chain CDRs and β-chain CDRs: The binding molecule of any one of claims 1 to 8, comprising one of:

10. The binding molecule of any one of claims 6 to 9, which contacts at least N4, R5, Y7, and Y8 of SLSNRLYYL (SEQ ID NO: 1).

11. HLA-A at a crossing angle in the range of 53° to 75°, or preferably in the range of 59° to 68° ＊ 11. The binding molecule of any one of claims 1 to 10, which binds to SLSNRLYYL (SEQ ID NO: 1) complexed with 02.

12. HLA-A at a tilt angle in the range of -38° to -18°, preferably in the range of -32° to -24° ＊ 12. The binding molecule of any one of claims 1 to 11, which binds to SLSNRLYYL (SEQ ID NO: 1) complexed with 02.

13. The binding molecule binds to HLA-A at a roll angle in the range of -25° to -5°, preferably in the range of -20° to -10°. ＊ 13. The binding molecule of any one of claims 1 to 12, which binds to SLSNRLYYL (SEQ ID NO: 1) complexed with 02.

14. The framework regions of the alpha chain variable domain have the following sequence: FR1 - LAKTTQPISMDSYEGQEVNITCSHN (SEQ ID NO: 8), optionally with 1, 2, or 3 mutations; FR2 - ITWYQQFPSQGPRFIIQ (SEQ ID NO: 9), optionally with 1, 2, or 3 mutations; FR3 - VTNEVASLFIPADRKSSTTLSLPRVSLSDTAVYYC (SEQ ID NO: 10), optionally with 1, 2, or 3 mutations; FR4—FGTGTRLQVFP (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 - EAGVAQSPRYKIIEKRQSVAFWCNPI (SEQ ID NO: 18), optionally with 1, 2, or 3 mutations; FR2—LYWYQQILGQGPKLLIQ (SEQ ID NO: 19), optionally with 1, 2, or 3 mutations; FR3—VDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLC (SEQ ID NO: 20), optionally with 1, 2, or 3 mutations; FR4—FGPGTRLLVL (SEQ ID NO: 21), optionally with 1, 2, or 3 mutations; The binding molecule of any one of claims 1 to 13, comprising:

15. The framework regions of the α chain variable domain contained the following mutations, numbered according to SEQ ID NO:3: The binding molecule of claim 14, comprising one or more of:

16. The framework regions of the α chain variable domain contain the following combinations of mutations numbered according to SEQ ID NO:3: (a) I47F, T56Q, (b) L1A, I47F, T56Q, (c) T21P, I47F, T56Q, P65S, (d) L1A, T21P, I47F, T56Q, P65S, 16. The binding molecule of claim 15, comprising one of:

17. The framework regions of the beta chain variable domain contained the following mutations, numbered according to SEQ ID NO: 13: The binding molecule of any one of claims 14 to 16, comprising:

18. 18. The binding molecule of any one of claims 1 to 17, comprising one of the following alpha chain variable domains: SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:34, 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:22, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:34, in combination with one of the following beta chain variable domains: SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48, 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:36, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:

48.

19. The following combinations of α-chain variable domains and β-chain variable domains:

20. The coupling element of claim 18, comprising one of:

20. 20. The binding molecule of any one of claims 1 to 19, wherein the alpha chain variable domain comprises the amino acid sequence of SEQ ID NO: 28 and the beta chain variable domain comprises the amino acid sequence of SEQ ID NO:

36.

21. 20. The binding molecule of any one of claims 1 to 19, wherein the alpha chain variable domain comprises the amino acid sequence of SEQ ID NO: 34 and the beta chain variable domain comprises the amino acid sequence of SEQ ID NO:

48.

22. 22. The specific binding molecule of any one of claims 1 to 21, comprising at least a portion of a TCR alpha chain constant domain and / or at least a portion of a TCR beta chain constant domain.

23. 23. The binding molecule of any one of claims 1 to 22, which is an alpha-beta heterodimer having an alpha chain TRAC constant domain sequence and a beta chain TRBC1 constant domain sequence or a beta chain TRBC2 constant domain sequence.

24. 24. The binding molecule of claim 23, wherein residues of the constant domain of the α chain and residues of the constant domain of the β chain are linked by a non-natural covalent disulfide bond.

25. 23. The binding molecule of any one of claims 1 to 22, in 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 TCR alpha and TCR beta variable domains, respectively, Cα and Cβ are TCR alpha and TCR beta constant domains, respectively, and L is a linker sequence.

26. 26. The binding molecule of any one of claims 1 to 25, comprising or consisting solely of a TCR comprising the alpha chain variable domain and the beta chain variable domain.

27. The binding molecule of claim 20, wherein the TCR is a soluble TCR.

28. 28. The binding molecule of any one of claims 1 to 27, further comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), which associate to form the site of an antigen-binding moiety capable of binding to an antigen.

29. 29. The binding molecule of claim 28, wherein the specific binding molecule comprises a single-chain variable fragment (scFv) comprising the VH and VL.

30. 30. The binding molecule of claim 28 or 29, wherein the antigen is a T cell surface antigen.

31. The binding molecule of any one of claims 28 to 30, wherein the antigen is CD3.

32. (a) the VH has the following sequence: CDR1 - GYSFTGYT (SEQ ID NO: 66) or GYSFTGYA (SEQ ID NO: 71), CDR2-INPYKGVS (SEQ ID NO: 67), and CDR3 - ARSGYYGDSDWYFDV (SEQ ID NO: 68), and / or (b) the VL has the following sequence: CDR1-QDIRNY (SEQ ID NO: 62), CDR2-YTS (SEQ ID NO: 63), and CDR3-QQGNTLPWT (SEQ ID NO: 64), 32. The binding molecule of any one of claims 28 to 31, comprising a CDR having:

33. the VH comprises an amino acid sequence set forth in SEQ ID NO:65 or SEQ ID NO:69, 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:65 or SEQ ID NO:69; and 33. The binding molecule of any one of claims 28 to 32, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 61, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 61, 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.

34. 34. The binding molecule of any one of claims 28 to 33, wherein the VH or the VL is covalently linked to the C-terminus or N-terminus of the α-chain or β-chain of the TCR, optionally via a linker sequence, and optionally the linker sequence is selected from SEQ ID NO: 72 and SEQ ID NOs: 78 to 89.

35. an alpha chain amino acid sequence set forth in any one of SEQ ID NOs:51, 49, 54, 56, and 58, 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 NOs:51, 49, 54, 56, and 58; and a beta chain-anti-CD3 amino acid sequence set forth in any one of SEQ ID NO:73, SEQ ID NO:119, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, 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:73, SEQ ID NO:119, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77; The binding molecule of any one of claims 28 to 34, comprising:

36. (a) an α chain amino acid sequence corresponding to SEQ ID NO: 51, and a β chain anti-CD3 amino acid sequence corresponding to SEQ ID NO: 73; (b) an α chain amino acid sequence corresponding to SEQ ID NO: 49, and a β chain anti-CD3 amino acid sequence corresponding to SEQ ID NO: 119; (c) an α chain amino acid sequence corresponding to SEQ ID NO: 49, and a β chain anti-CD3 amino acid sequence corresponding to SEQ ID NO: 74; (d) an α chain amino acid sequence corresponding to SEQ ID NO: 54, and a β chain anti-CD3 amino acid sequence corresponding to SEQ ID NO: 75; (e) an α chain amino acid sequence corresponding to SEQ ID NO: 56 and a β chain anti-CD3 amino acid sequence corresponding to SEQ ID NO: 76; or (f) an alpha chain amino acid sequence corresponding to SEQ ID NO: 58, and a beta chain anti-CD3 amino acid sequence corresponding to SEQ ID NO: 77; 36. The binding molecule of claim 35, comprising:

37. a first polypeptide chain comprising the α chain variable domain and a first binding domain of a variable region of an antibody; a second polypeptide chain comprising the beta chain variable domain and a second binding domain of the variable region of the antibody; wherein each said polypeptide chain comprises: ＊ 33. The binding molecule of any one of claims 28 to 32, which associates with SLSNRLYYL (SEQ ID NO: 1) complexed with 02 so as to be capable of simultaneously binding to the antigen of the antibody.

38. 38. The binding molecule of any one of claims 1 to 37, associated with a detectable label, and / or a therapeutic agent, and / or a pharmacokinetic-modifying moiety, optionally with an Fc domain.

39. 39. A nucleic acid encoding the binding molecule of any one of claims 1 to 38, 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.

40. 40. An expression vector comprising the nucleic acid of claim 39.

41. (a) an expression vector according to claim 39, 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 38, 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 38; Cells having

42. A non-naturally occurring and / or purified and / or engineered cell, in particular a T cell, presenting a binding molecule according to any one of claims 1 to 38.

43. 41. A pharmaceutical composition comprising a binding molecule according to any one of claims 1 to 38, a nucleic acid according to claim 39, an expression vector according to claim 40, and / or a cell according to claim 41 or 42, together with one or more pharmaceutically acceptable carriers or excipients.

44. 44. A binding molecule according to any one of claims 1 to 38, a nucleic acid according to claim 39, an expression vector according to claim 40, a cell according to claim 41 or 42 and / or a pharmaceutical composition according to claim 43 for use in medicine, preferably in a human subject.

45. 44. A binding molecule according to any one of claims 1 to 38, a nucleic acid according to claim 39, an expression vector according to claim 40, a cell according to claim 41 or 42, and / or a pharmaceutical composition according to claim 43 for use in a method of treating cancer, preferably in a human subject.

46. 46. ​​The binding molecule, nucleic acid, expression vector, cell and / or pharmaceutical composition used according to claim 45, wherein the cancer is gastric cancer, colon cancer or esophageal cancer.

47. 10. A method for producing a binding molecule according to any one of claims 1 to 38, comprising: a) maintaining a cell according to claim 41 or 42 under conditions optimal for expression of said binding molecule chain; and b) isolating said binding molecule chain.

Images