MAGE-A4 peptide dual T cell adapter

Abstract

Described herein are antigen binding proteins having specificity for melanoma associated antigen A4 (MAGE-A4) peptide-MHC (pMHC). Also described are multispecific antigen binding proteins comprising an antigen binding domain having specificity for CD3, and at least one MAGE-A4pMHC antigen binding domain. Methods of treating cancer with the substances are also described.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 406,475, filed Sep. 14, 2022, the entire disclosure of which is hereby incorporated herein by reference. Background of the Invention

[0003] Melanoma-associated antigen A4 (MAGE-A4) peptide-major histocompatibility complex (pMHC) expression is present in many cancers, including non-small cell lung cancer (NSCLC), melanoma, bladder cancer, head and neck cancer, and gastroesophageal cancer (Grossman et al. N Engl J Med. 2016. 375:1109-1112). Thus, it represents an attractive target for TCR-based T cell therapies. Unfortunately, TCR molecules have low affinity for their pMHC targets. In addition, TCR-based T cell therapies are laborious and expensive to develop and use. In contrast, isolated monoclonal antibodies provide substantially higher binding affinity for their targets and may have reduced off-target activity. However, due to the small epitopes of binding peptides in HLA, it is difficult to generate monoclonal antibodies against pMHC targets.

[0004] Accordingly, there is a need in the art for novel antigen-binding proteins that are suitable as therapeutic agents and specifically recognize the target MAGE-A4 pMHC with high affinity while maintaining high specificity (i.e., low or even no off-target effects on healthy tissues), have favorable drug-like properties, and can be produced in sufficient quantity and quality at a reasonable cost. Summary of the Invention

[0005] The present disclosure relates to antigen-binding proteins and multispecific antigen-binding proteins that specifically bind to melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC). The antigen-binding proteins and multispecific antigen-binding proteins of the invention comprise enhanced binding affinity for and / or enhanced stability to target MAGE-A4 pMHC, as well as low off-target binding to non-target MAGE-A4 pMHC or other molecules. The multispecific antigen-binding proteins may further comprise a Fab domain that specifically binds to a cell surface protein (such as CD3) of an immune cell, the Fab domain comprising a heavy chain and a light chain; at least a first pMHC-binding domain operably linked to the heavy chain, wherein the first pMHC-binding domain binds to a first target peptide-MHC (pMHC) complex; and c) at least a second pMHC-binding domain operably linked to the light chain, wherein the second pMHC-binding domain binds to a second pMHC complex. The bivalent targeting of pMHC with the bispecific antigen-binding proteins of the invention results in increased cancer cell killing compared to their monovalent bispecific counterparts, while the overall specificity for cells carrying the same HLA but not expressing the target protein is substantially unaffected.

[0006] Preferably, the antigen-binding proteins of the invention lack an Fc domain and thus are not recognized by Fc receptors on effector cells (such as FcγRIII on macrophages and activated neutrophils) or inhibitory receptors (such as FcγRIIb) and the FcγRIIa complex on non-cytotoxic cells (such as platelets and B cells). For bispecific T cell engagers, to avoid non-antigen-dependent cytokine release syndrome (CRS) resulting from cross-linking of CD3 and Fc receptors followed by non-specific immune cell activation, Fc-mediated immune functions are not required. Instead, the Fab domain of the antigen-binding protein serves as a specific heterodimerization scaffold linked to other pMHC-binding domains. The native and efficient heterodimerization properties of the heavy chain (Fd fragment) and light chain (L) of the Fab fragment make the Fab fragment a useful scaffold. The other binding domains can be in several different formats, including but not limited to another Fab domain, scFv, or sdAb. Additionally, in some cases, Fc-containing antigen-binding proteins may be disadvantageous due to increased half-life. The extended half-life may lead to increased toxicity, especially the release of excessive cytokines from immune cells. The extended half-life may also promote T cell exhaustion. The antigen-binding proteins of the present disclosure lacking an Fc domain may have reduced cytotoxicity, in part due to a shorter half-life compared to Fc-containing antigen-binding proteins.

[0007] In addition to being highly specific for their targets, the antigen-binding proteins of the invention also exhibit favorable drug-like properties, such as intrinsic stability and / or general physical and chemical stability.

[0008] In one aspect, the present disclosure provides an antigen-binding protein that specifically binds to a melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), the antigen-binding protein comprising: (i) an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 15; (ii) a VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 20, and a VL domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 25; (iii) a VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, wherein the VH domain comprises a C amino acid at position 44 of SEQ ID NO: 30, and a VL domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 35, wherein the VL domain comprises a C amino acid at position 102 of SEQ ID NO: 35; or (iv) a VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 40, wherein the VH domain comprises a Y amino acid at position 47, an R amino acid at position 71, and an N amino acid at position 73 of SEQ ID NO: 40, and a VL domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 45.

[0009] In certain embodiments, the MAGE-A4 pMHC complex is the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex. In certain embodiments, the MAGE-A4 pMHC complex is the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02:01 complex.

[0010] In certain embodiments, as determined by SEC-HPLC, during storage in PBS at 4°C for at least two weeks at a concentration of about 1 mg / mL (e.g., 0.90 to 1.10 mg / mL) to about 10 mg / mL (e.g., 9.9 to 10.1 mg / mL), the antigen-binding protein remains at least 94% monomeric, such as 94%, 95%, 96%, 97%, 98%, or 99%. In certain embodiments, as determined by SEC-HPLC, during storage in PBS at 37°C for at least two weeks at a concentration of about 1 mg / mL (e.g., 0.90 to 1.10 mg / mL) to about 10 mg / mL (e.g., 9.9 to 10.1 mg / mL), the antigen-binding protein remains at least 80% monomeric. In certain embodiments, as determined by SEC-HPLC, during storage in PBS at 37°C for at least two weeks at a concentration of about 1 mg / mL (e.g., 0.90 to 1.10 mg / mL) to about 10 mg / mL (e.g., 9.9 to 10.1 mg / mL), the antigen-binding protein remains at least 95% monomeric. In certain embodiments, as determined by SEC-HPLC, during storage in PBS at 4°C for at least two weeks at concentrations of about 1 mg / mL and about 10 mg / mL, the antigen-binding protein remains at least 94% monomeric, such as 94%, 95%, 96%, 97%, 98%, or 99%.

[0011] In certain embodiments, the antigen-binding protein comprises a full-length immunoglobulin or an antibody fragment, such as a Fab, Fab', F(ab')2, scFv, or Fv fragment.

[0012] In certain embodiments, the VH domain comprises the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), the HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6) (where X1 corresponds to the amino acid S or D, X2 corresponds to the amino acid W or S, and X3 corresponds to the amino acid A or V), and the HCDR3 amino acid sequence of DLYYGPX4TX5YX6X7X8NL (SEQ ID NO: 7) (where X4 corresponds to the amino acid T, N, or S, X5 corresponds to the amino acid D or is absent, X6 corresponds to the amino acid S or F, X7 corresponds to the amino acid A or V, and X8 corresponds to the amino acid F or A); and the VL domain comprises the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and ATX9X 10 X 11 SGSNFQX 12The LCDR3 amino acid sequence of (SEQ ID NO: 8), wherein X9 corresponds to the amino acid S or R, X 10 corresponds to the amino acid D or P, X 11 corresponds to the amino acid G, S or F, and X 12 corresponds to the amino acid L or A.

[0013] In certain embodiments, the antigen-binding protein is linked to or combined with a detectable label, a therapeutic agent, or a PK modifying moiety.

[0014] In certain embodiments, the antigen-binding protein is chemically or biologically modified. In certain embodiments, the antigen-binding protein is glycosylated, PEGylated, PASylated, XTENylated, or HESylated.

[0015] In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising the antigen-binding protein described above.

[0016] In another aspect, the present disclosure provides an immune cell expressing the CAR described above. In certain embodiments, the immune cell is a T cell.

[0017] In another aspect, the present disclosure provides a multispecific antigen-binding protein comprising the antigen-binding protein described above.

[0018] In certain embodiments, the multispecific antigen-binding protein is bispecific or trispecific.

[0019] In certain embodiments, the multispecific antigen-binding protein further comprises at least one other binding domain. In certain embodiments, the other binding domain is an immune cell engager, particularly a CD3 binding domain or a CD16a binding domain.

[0020] In certain embodiments, the multispecific antigen-binding protein further comprises a third antigen-binding domain. In certain embodiments, the third antigen-binding domain binds to HLA-A*02 / MAGE-A4. In certain embodiments, the third antigen-binding domain is the same as the first MAEG-A4 pMHC antigen-binding domain as defined above.

[0021] In certain embodiments, the third antigen-binding domain comprises: an antibody heavy chain variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), the HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6) (where X1 corresponds to the amino acid S or D, X2 corresponds to the amino acid W or S, and X3 corresponds to the amino acid A or V), and the HCDR3 amino acid sequence of DLYYGP X4TX5YX6X7X8NL (SEQ ID NO: 7) (where X4 corresponds to the amino acid T, N or S, X5 corresponds to the amino acid D or is absent, X6 corresponds to the amino acid S or F, X7 corresponds to the amino acid A or V, and X8 corresponds to the amino acid F or A); and an antibody light chain variable (VL) domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and the LCDR3 amino acid sequence of 10 X 11 SGSNFQX 12 (SEQ ID NO: 8), where X9 corresponds to the amino acid S or R, X 10 corresponds to the amino acid D or P, X 11 corresponds to the amino acid G, S or F, and X 12 corresponds to the amino acid L or A.

[0022] In certain embodiments, the third antigen-binding domain comprises: i) an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 15; ii) a VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 20, and a VL domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 25; iii) a VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, and a VL domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 35; or iv) a VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 40, and a VL domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 45.

[0023] In one aspect, the present disclosure provides a multispecific antigen-binding protein comprising: a) a first antigen-binding domain that specifically binds to CD3; b) a second antigen-binding domain that specifically binds to a melanoma-associated antigen A4 (MAGE-A4) peptide-major histocompatibility complex (pMHC), the second antigen-binding domain comprising: b1) an antibody heavy chain variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), the HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6) (where X1 corresponds to the amino acid S or D, X2 corresponds to the amino acid W or S, and X3 corresponds to the amino acid A or V), and the HCDR3 amino acid sequence of DLYYGPX4TX5YX6X7X8NL (SEQ ID NO: 7) (where X4 corresponds to the amino acid T, N or S, X5 corresponds to the amino acid D or is absent, X6 corresponds to the amino acid S or F, X7 corresponds to the amino acid A or V, and X8 corresponds to the amino acid F or A); and b2) an antibody light chain variable (VL) domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and 10 X 11 SGSNFQX 12 (SEQ ID NO: 8) of the LCDR3 amino acid sequence, where X9 corresponds to the amino acid S or R, X 10 corresponds to the amino acid D or P, X 11 corresponds to the amino acid G, S or F, and X 12 corresponds to the amino acid L or A.

[0024] In certain embodiments, the MAGE-A4 peptide-MHC (pMHC) is the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex.

[0025] In certain embodiments, as determined by SEC-HPLC, after incubation in PBS at 4 °C for 14 days, the multispecific antigen-binding protein remains at least 94%, 95%, 96%, 97%, 98%, 99% or 100% monomeric.

[0026] In certain embodiments, the multispecific antigen-binding protein further comprises a third antigen-binding domain, particularly, the third binding domain is the same as the second binding domain.

[0027] In certain embodiments, the multispecific antigen-binding protein comprises: c) a third antigen-binding domain that specifically binds to MAGE-A4 pMHC, the third antigen-binding domain comprising: c1) a VH domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), the HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6) (where X1 corresponds to the amino acid S or D, X2 corresponds to the amino acid W or S, and X3 corresponds to the amino acid A or V), and the HCDR3 amino acid sequence of DLYYGPX4TX5YX6X7X8NL (SEQ ID NO: 7) (where X4 corresponds to the amino acid T, N or S, X5 corresponds to the amino acid D or is absent, X6 corresponds to the amino acid S or F, X7 corresponds to the amino acid A or V, and X8 corresponds to the amino acid F or A); and c2) a VL domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and the LCDR3 amino acid sequence of ATX9X 10 X 11 SGSNFQX 12 (SEQ ID NO: 8), where X9 corresponds to the amino acid S or R, X 10 corresponds to the amino acid D or P, X 11 corresponds to the amino acid G, S or F, and X 12 corresponds to the amino acid L or A.

[0028] In certain embodiments, the second and third antigen-binding domains comprise: (i) a VH that comprises an HCDR1 sequence having the amino acid sequence containing SNYAMS (SEQ ID NO: 11), an HCDR2 sequence having the amino acid sequence containing IVSSGGTTYYADSVKG (SEQ ID NO: 12), and an HCDR3 sequence having the amino acid sequence containing DLYYGPNTDYSAANL (SEQ ID NO: 13); and a VL that comprises an LCDR1 sequence having the amino acid sequence containing TA DTLSRSYAS (SEQ ID NO: 16), an LCDR2 sequence having the amino acid sequence containing RDTSRPS (SEQ ID NO: 17), and an LCDR3 sequence having the amino acid sequence containing ATRPSSGSNFQA (SEQ ID NO: 18); (ii) a VH that comprises an HCDR1 sequence having the amino acid sequence containing SNYAMS (SEQ ID NO: 21), an HCDR2 sequence having the amino acid sequence containing IVSSGGTTYYADSVKG (SEQ ID NO: 22), and an HCDR3 sequence having the amino acid sequence containing DLYYGPSTYFVANL (SEQ ID NO: 23); and a VL that comprises an LCDR1 sequence having the amino acid sequence containing TADTLSRSYAS (SEQ ID NO: 26), an LCDR2 sequence having the amino acid sequence containing RDTSRPS (SEQ ID NO: 27), and an LCDR3 sequence having the amino acid sequence containing ATRPSSGSNFQL (SEQ ID NO: 28); (iii) a VH that comprises an HCDR1 sequence having the amino acid sequence containing SNYAMS (SEQ ID NO: 31), an HCDR2 sequence having the amino acid sequence containing IVSSGGTTYYASWAKG (SEQ ID NO: 32), and an HCDR3 sequence having the amino acid sequence containing DLYYGPTTYSAANL (SEQ ID NO: 33); and a VL that comprises an LCDR1 sequence having the amino acid sequence containing TADTLSRSYAS (SEQ ID NO: 36), an LCDR2 sequence having the amino acid sequence containing RDTSRPS (SEQ ID NO: 37), and an LCDR3 sequence having the amino acid sequence containing ATRDFSGSNFQL (SEQ ID NO: 38);or (iv) a VH that comprises an HCDR1 sequence having the amino acid sequence of SNYAMS (SEQ ID NO: 41), an HCDR2 sequence having the amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 42), and an HCDR3 sequence having the amino acid sequence of DLYYGPTTYSAFNL (SEQ ID NO: 43); and a VL that comprises an LCDR1 sequence having the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 46), an LCDR2 sequence having the amino acid sequence of RDTSRPS (SEQ ID NO: 47), and an LCDR3 sequence having the amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 48).;

[0029] In certain embodiments, the second and third antigen-binding domains comprise: (i) an antibody heavy chain variable (VH) domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and an antibody light chain variable (VL) domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 15; (ii) a VH domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 20, and a VL domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 25; (iii) a VH domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, and a VL domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 35; or (iv) a VH domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 40, and a VL domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 45.

[0030] In certain embodiments, any one or more of the first, second, and third antigen-binding domains comprise an antibody fragment. In certain embodiments, the antibody fragment comprises a Fab fragment, an F(ab′)2 fragment, a Fab′ fragment, an Fv fragment, a single-chain variable fragment (scFv), and a single-domain antibody fragment.

[0031] In certain embodiments, the immune cell or CD3 antigen-binding domain is a Fab fragment, wherein the Fab fragment comprises a heavy chain containing a CH1 domain and a VH, and a light chain containing a Cl domain and a VL.

[0032] In certain embodiments, the CH1 domain comprises at least 5 amino acids of the antibody hinge region. In certain embodiments, the CH1 domain comprises the amino acid sequence EPKSC (SEQ ID NO: 88) of the antibody hinge region.

[0033] In certain embodiments, the MAGE-A4 pMHC antigen-binding domain comprises an scFv.

[0034] In certain embodiments, the second antigen-binding domain is operably linked to the C-terminus or the N-terminus of the heavy chain of the Fab domain. In certain embodiments, the third antigen-binding domain is operably linked to the C-terminus or the N-terminus of the heavy chain of the Fab domain.

[0035] In certain embodiments, a) the second antigen-binding domain comprises an scFv linked to the C-terminus of the heavy chain of the Fab domain, and the third antigen-binding domain comprises an scFv linked to the C-terminus of the light chain of the Fab domain; b) the second antigen-binding domain comprises an scFv linked to the N-terminus of the heavy chain of the Fab domain, and the third antigen-binding domain comprises an scFv linked to the N-terminus of the light chain of the Fab domain; c) the second antigen-binding domain comprises an scFv linked to the N-terminus of the heavy chain of the Fab domain, and the third antigen-binding domain comprises an scFv linked to the C-terminus of the light chain of the Fab domain; or d) the second antigen-binding domain comprises an scFv linked to the C-terminus of the heavy chain of the Fab domain, and the third antigen-binding domain comprises an scFv linked to the N-terminus of the light chain of the Fab domain.

[0036] In certain embodiments, the scFv is linked to the Fab domain by an amino acid linker. In certain embodiments, the amino acid linker comprises (GGGGS)n (SEQ ID NO: 73), where n is an integer between 1 and 5. In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGS (SEQ ID NO: 74), GGGGSGGGGSGGGGS (SEQ ID NO: 75), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 76), GGGGSGGGGSGGGGSGGGGAS (SEQ ID NO: 77), or GGGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 78).

[0037] In certain embodiments, the second and / or third antigen-binding domains VH and VL are linked by an amino acid linker. In certain embodiments, the amino acid linker comprises (GGGGS)n (SEQ ID NO: 73), where n is an integer between 1 and 5. In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGS (SEQ ID NO: 74), GGGGSGGGGSGGGGS (SEQ ID NO: 75), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 76), GGGGSGGGGSGGGGSGGGGAS (SEQ ID NO: 77), or GGGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 78).

[0038] In certain embodiments, the multispecific antigen-binding protein does not comprise an Fc domain.

[0039] In certain embodiments, the multispecific antigen-binding protein comprises (scFv)2, (scFv)3, BiTE, BIKE, Dart, diabody, triabody, Fab2, Fab3, Fab4, scFv-Fab-scFv, or minibody-scFv.

[0040] In certain embodiments, the multispecific antigen-binding protein has a molecular weight of about 75 kDa to about 100 kDa or about 110 kDa. In certain embodiments, the antigen-binding protein has an extended serum half-life relative to an antigen-binding protein having a molecular weight of < about 75 kDa.

[0041] In certain embodiments, the CD3 antigen-binding domain comprises: a1) a VH that comprises an HCDR1 sequence having the amino acid sequence containing STYAMN (SEQ ID NO: 51), an HCDR2 sequence having the amino acid sequence containing RIRSKYNNYATYYADSVKG (SEQ ID NO: 52), and an HCDR3 sequence having the amino acid sequence containing HGNFGDSYVSWFAY (SEQ ID NO: 53); and a2) a VL that comprises an LCDR1 sequence having the amino acid sequence containing GSSTGAVTTSNYAN (SEQ ID NO: 56), an LCDR2 sequence having the amino acid sequence containing GTNKRAP (SEQ ID NO: 57), and an LCDR3 sequence having the amino acid sequence containing ALWYSNHWV (SEQ ID NO: 58).

[0042] In certain embodiments, the CD3 antigen-binding domain comprises: a VH that comprises an amino acid sequence that is at least about 90% identical (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence of SEQ ID NO: 50, and a VL that comprises an amino acid sequence that is at least about 90% identical (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence of SEQ ID NO: 55.

[0043] In certain embodiments, the CD3 antigen-binding domain comprises: a heavy chain that comprises an amino acid sequence that is at least about 90% identical (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence of SEQ ID NO: 49, and a light chain that comprises an amino acid sequence that is at least about 90% identical (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence of SEQ ID NO: 54.

[0044] In certain embodiments, the multispecific antigen-binding protein comprises: (i) a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 9, and a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 14; (ii) a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 19, and a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 24; (iii) a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 29, and a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 34; or (iv) a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 39; and a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 44, or a variant of the sequence, the variant being at least 90%, 95%, 96%, 97%, 98% or 99% identical thereto while retaining antigen specificity (i.e., retaining specificity for the (SEQ ID NO: 3) HLA-A*02 complex and for CD3).

[0045] In certain embodiments, particularly in the scFv format, the MAGE-A4 pMHC-binding domain (such as the second and / or third antigen-binding domain above) comprises a variable heavy chain having polar amino acids at positions 11, 89 and / or 108 according to Kabat numbering. In certain embodiments, the Fab domain comprises a variable heavy chain having polar amino acids at positions 11, 89 and / or 108 according to Kabat numbering.

[0046] In certain embodiments, the variable heavy chain comprises: leucine (L) or serine (S) at amino acid position 11 according to Kabat numbering; valine (V), serine (S) or threonine (T) at amino acid position 89 according to Kabat numbering; and / or leucine (L), serine (S) or threonine (T) at amino acid position 108 according to Kabat numbering.

[0047] In certain embodiments, the polar amino acids are serine (S) and / or threonine (T).

[0048] In certain embodiments, the variable heavy chain comprises serine (S) at amino acid position 11, serine (S) or threonine (T) at amino acid position 89 and serine (S) or threonine (T) at amino acid position 108 according to Kabat numbering.

[0049] In certain embodiments, the variable heavy chain comprises serine (S) at amino acid position 11, serine (S) at amino acid position 89, and serine (S) at amino acid position 108 according to Kabat numbering.

[0050] In one aspect, the present disclosure provides a multispecific antigen-binding protein comprising: a) a first antigen-binding domain that specifically binds to CD3, the first antigen-binding domain comprising: a1) a VH that comprises an HCDR1 sequence having an amino acid sequence containing STYAMN (SEQ ID NO: 51), an HCDR2 sequence having an amino acid sequence containing RIRSKYNNYATYYADSVKG (SEQ ID NO: 52), and an HCDR3 sequence having an amino acid sequence containing HGNFGDSYVSWFAY (SEQ ID NO: 53); and a2) a VL that comprises an LCDR1 sequence having an amino acid sequence containing GSSTGAVTTSNYAN (SEQ ID NO: 56), an LCDR2 sequence having an amino acid sequence containing GTNKRAP (SEQ ID NO: 57), and an LCDR3 sequence having an amino acid sequence containing ALWYSNHWV (SEQ ID NO: 58); and b) a second antigen-binding domain that specifically binds to melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC).

[0051] In certain embodiments, the second antigen-binding domain comprises: b1) an antibody heavy chain variable (VH) domain that comprises an HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), an HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6) (where X1 corresponds to amino acid S or D, X2 corresponds to amino acid W or S, and X3 corresponds to amino acid A or V), and an HCDR3 amino acid sequence of DLYYGPX4TX5YX6X7X8NL (SEQ ID NO: 7) (where X4 corresponds to amino acid T, N or S, X5 corresponds to amino acid D or absent, X6 corresponds to amino acid S or F, X7 corresponds to amino acid A or V, and X8 corresponds to amino acid F or A); and b2) an antibody light chain variable (VL) domain that comprises an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and ATX9X 10 X 11 SGSN FQX 12The LCDR3 amino acid sequence of (SEQ ID NO: 8), where X9 corresponds to amino acid S or R, X 10 corresponds to amino acid D or P, X 11 corresponds to amino acid G, S or F, and X 12 corresponds to amino acid L or A.

[0052] In certain embodiments, the CD3 binding domain comprises: -VH, wherein the VH comprises an amino acid sequence that is at least about 90% identical (such as 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO: 50, and -VL, wherein the VL comprises an amino acid sequence that is at least about 90% identical (such as 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO: 55.

[0053] In certain embodiments, the CD3 binding domain comprises: -VH, wherein the VH comprises an amino acid sequence that is at least about 90% identical (such as 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO: 49, and -VL, wherein the VL comprises an amino acid sequence that is at least about 90% identical (such as 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO: 54.

[0054] In certain embodiments, the multispecific antigen-binding protein further comprises a third antigen-binding domain.

[0055] In certain embodiments, the multispecific antigen-binding protein comprises: c) a third antigen-binding domain that specifically binds to MAGE-A4 pMHC, the third antigen-binding domain comprising: c1) a VH domain, the VH domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), the HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6) (where X1 corresponds to amino acid S or D, X2 corresponds to amino acid W or S, and X3 corresponds to amino acid A or V), and the HCDR3 amino acid sequence of DLYYGPX4TX5YX6X7X8NL (SEQ ID NO: 7) (where X4 corresponds to amino acid T, N or S, X5 corresponds to amino acid D or absent, X6 corresponds to amino acid S or F, X7 corresponds to amino acid A or V, and X8 corresponds to amino acid F or A); and c2) a VL domain, the VL domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and ATX9X 10 X11 SGSNFQX 12 (SEQ ID NO: 8) The LCDR3 amino acid sequence, wherein X9 corresponds to the amino acid S or R, X 10 corresponds to the amino acid D or P, X 11 corresponds to the amino acid G, S or F, and X 12 corresponds to the amino acid L or A.

[0056] In one aspect, the present disclosure provides a multispecific antigen-binding protein that binds to MAGE-A4-pMHC and CD3, the multispecific antigen-binding protein comprising: (i) a first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 9, and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 14; (ii) a first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 19, and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 24; (iii) a first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 29, and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 34; or (iv) a first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 39; and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 44, or a variant of the sequence, the variant being at least 90%, 95%, 96%, 97%, 98% or 99% identical thereto, while retaining antigen specificity (i.e., retaining specificity for the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex and for CD3).

[0057] In one aspect, the present disclosure provides a multispecific antigen-binding protein that binds to the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex and CD3, the multispecific antigen-binding protein comprising: (i) a first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 9, and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 14; (ii) a first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 19, and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 24; (iii) a first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 29, and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 34; or (iv) a first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 39; and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 44.

[0058] In certain embodiments of the multispecific antigen-binding proteins described herein, the MAGE-A4 peptide-MHC (pMHC) is the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex.

[0059] In certain embodiments of the multispecific antigen-binding proteins described herein, as determined by SEC-HPLC, after incubation in PBS at 4°C for 14 days at 1 mg / mL and / or 10 mg / mL, the multispecific antigen-binding protein remains at least 94%, 95%, 96%, 97%, 98%, 99% or 100% monomeric.

[0060] In certain embodiments of the multispecific antigen-binding proteins described herein, as determined by LDH cytotoxicity assay, the multispecific antigen-binding protein exhibits efficacy (e.g., cytotoxicity) against target-positive tumor cells.

[0061] In certain embodiments of the multispecific antigen-binding proteins described herein, in a cell line-derived murine non-small cell lung cancer (NSCLC) xenograft model, the multispecific antigen-binding protein exhibits tumor growth inhibition and tumor eradication.

[0062] In certain embodiments, the multispecific antigen-binding protein is chemically or biologically modified. In certain embodiments, the multispecific antigen-binding protein is glycosylated, PEGylated, HESylated, PASylated or XTENylated.

[0063] In certain embodiments, the multispecific antigen-binding protein is linked to or combined with a functional entity such as a detectable label, a therapeutic agent, or a PK-modifying moiety.

[0064] In certain embodiments, the functional entity is a toxin.

[0065] In certain embodiments of the multispecific antigen-binding proteins described herein, the light chain and / or heavy chain comprises an N-terminal and / or C-terminal truncation of 1, 2, 3, 4, or 5 amino acids.

[0066] In certain embodiments of the multispecific antigen-binding proteins described herein, the light chain comprises an N-terminal truncation of 1 or 2 amino acids.

[0067] In certain embodiments of the multispecific antigen-binding proteins described herein, the multispecific antigen-binding protein comprises pyroglutamic acid (pE) at position 1 of the light chain and / or heavy chain instead of glutamine (Q) or glutamic acid (E).

[0068] In certain embodiments of the multispecific antigen-binding proteins described herein, the multispecific antigen-binding protein comprises pyroglutamic acid (pE) at position 1 of the light chain instead of glutamine (Q) or glutamic acid (E).

[0069] In certain embodiments, the antigen-binding protein is used for diagnosis.

[0070] In certain embodiments, the multispecific antigen-binding proteins described above are used in methods for inhibiting cancer cell growth or proliferation.

[0071] In certain embodiments, the multispecific antigen-binding proteins described above are used in methods for redirecting T cells to cancer cells expressing MAGE-A4.

[0072] In certain embodiments, the antigen-binding protein described above or the multispecific antigen-binding proteins described above are used as medicaments.

[0073] In one aspect, the present disclosure provides a nucleic acid encoding the antigen-binding protein described above or the multispecific antigen-binding proteins described above.

[0074] In one aspect, the present disclosure provides a vector comprising the nucleic acid described above.

[0075] In one aspect, the present disclosure provides a population of host cells comprising the vector described above.

[0076] In one aspect, the present disclosure provides a kit comprising the antigen-binding protein described above or the multispecific antigen-binding proteins described above.

[0077] In one aspect, the present disclosure provides a method for manufacturing the antigen-binding protein or the multispecific antigen-binding protein described above, the method comprising the steps of: (i) culturing the host cell described above under conditions allowing the expression of the antigen-binding protein or the multispecific antigen-binding protein; (ii) recovering the antigen-binding protein or the multispecific antigen-binding protein; and optionally (iii) further purifying and / or modifying and / or formulating the antigen-binding protein or the multispecific antigen-binding protein.

[0078] In one aspect, the present disclosure provides a pharmaceutical composition comprising the antigen-binding protein or the multispecific antigen-binding protein described above and a pharmaceutically acceptable buffer.

[0079] In one aspect, the present disclosure provides the use of the antigen-binding protein, the multispecific antigen-binding protein or the pharmaceutical composition described above in the manufacture of a medicament.

[0080] In one aspect, the present disclosure provides the use of the antigen-binding protein, the CAR, the immune cell, the multispecific antigen-binding protein, the cell or the pharmaceutical composition described above in the treatment of a disease, particularly cancer.

[0081] In one aspect, the present disclosure provides a method for treating cancer expressing MAGE-A4 pMHC in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the antigen-binding protein, the CAR, the immune cell, the multispecific antigen-binding protein, the cell or the pharmaceutical composition described above.

[0082] In certain embodiments, the cancer is selected from the group consisting of: head and neck squamous cell carcinoma (HNSC), non-small cell lung cancer (NSCLC), triple-negative breast cancer, urothelial carcinoma, high-grade endometrial cancer (including uterine carcinosarcoma (UCS; particularly the UCEC subgroup)), myxoid / round cell liposarcoma, gastric or gastroesophageal junction (GEJ) adenocarcinoma, epithelial ovarian cancer (such as high-grade serous ovarian cancer), synovial sarcoma, bladder urothelial carcinoma (BLCA) (particularly transitional cell carcinoma), testicular germ cell tumor (TGCT), and cervical squamous cell carcinoma (CESC).

[0083] In certain embodiments, the cancer is of squamous origin, such as head and neck squamous cell carcinoma (HNSCC) or squamous NSCLC.

[0084] In one aspect, the present disclosure provides a method of selecting a patient suitable for treatment with a MAGE-A4 antagonist, the method comprising the following steps in sequence: (i) obtaining a tumor sample from the patient; (ii) adding an anti-MAGEA4 detection antibody to the sample; (iii) incubating the detection antibody and the sample; (iv) detecting the detection antibody bound to the sample; and (v) if the detection antibody is bound by the sample, then selecting the patient for treatment with a MAGE-A4 antagonist.

[0085] In certain embodiments, the detection antibody is OTI1F9, E7O1U or an antigen-binding protein described herein.

[0086] In certain embodiments, the method further comprises the step of performing RNA sequencing to detect total MAGE-A4.

[0087] In certain embodiments, in the treatment of a disease (particularly cancer), the MAGE-A4 antagonist is an antigen-binding protein described herein, a CAR described herein, an immune cell described herein, a multispecific antigen-binding protein described herein or a pharmaceutical composition described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] The foregoing and other features and advantages of the invention will be more fully understood from the following detailed description of illustrative embodiments in conjunction with the accompanying drawings. This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0089] FIG. 1 shows the binding of an antibody to HLA-A*02:01 / MAGE-A4 ( Figure 1A and Figure 1B ) or the control complex HLA-A*02:01 / peptide mixture ( Figure 1C and Figure 1D ), as determined by direct ELISA.

[0090] FIG. 2 shows the binding of antibodies M0709 ( Figure 2A ) and M0763 ( Figure 2B ) to T2 cells presenting MAGE-A4 or control peptides 1, 2, and 3.

[0091] FIG. 3 shows the binding of a humanized M0763 variant to HLA-A*02:01 / MAGE-A4 ( Figure 3A ) or the control complex HLA-A*02:01 / peptide mixture ( Figure 3B ), as determined by direct ELISA.

[0092] Figure 4Schematic representation of an embodiment of the bispecific antibody of the invention. The presented embodiment comprises an anti-CD3 Fab fragment and two single-chain antibody fragments (scFv) that specifically bind to a target peptide presented on an MHC complex. The scFv that binds pMHC can be linked to the C-terminus of the CH1-domain and the CL-domain via a glycine-serine flexible linker.

[0093] Figure 5 shows the cytotoxicity of the dual T cell engager M1048 and its monovalent counterpart M1041 in MAGE-A4-positive HLA-A*02:01-positive U2OS cancer cells ( Figure 5A ), MAGE-A4-negative HLA-A*02:01-positive SK-MEL-30 ( Figure 5B ), MDA-MB-231 ( Figure 5C ), and PANC-1 ( Figure 5D ) cancer cells.

[0094] Figure 6 shows the stability of the dual engagers M1397 and M1403 in PBS over 0, 3, 7, and 14 days as determined by SEC-HPLC. Figure 6A Data shown are for incubation at 4 °C, Figure 6B and for incubation at 37 °C.

[0095] Figure 7 shows the stability of the dual engagers M1397 ( Figure 7A ) and M1403 ( Figure 7B ) in human serum at 37 °C.

[0096] Figure 8 shows the cytotoxicity of the dual engagers M1397 and M1403 or comparator 1 in MAGE-A4-positive HLA-A*02:01-positive cancer cells ( Figures 8A to 8E ) and MAGE-A4-negative HLA-A*02:01-positive cancer cells ( Figures 8F to 8K ). Figure 8A : Dual engager M1397 and comparator 1 in U2OS; Figure 8B : Dual engager M1403 and comparator 1 in U2OS; Figure 8C : Dual engager M1397 and comparator 1 in NCI-H1703; Figure 8D : Dual engager M1403 and comparator 1 in NCI-H1703; Figure 8E : Dual engagers M1397 and M1403 and comparator 1 in A375; Figure 8F : Dual engager M1397 and comparator 1 in PANC-1; Figure 8G : Dual engager M1403 and comparator 1 in PANC-1; Figure 8H: Dual adaptor M1397 and comparator 1 in MDA-MB-231; Figure 8I : Dual adaptor M1397 and comparator 1 in MDA-MB-231; Figure 8J : Dual adaptor M1397 and comparator 1 in NCI-H441; Figure 8K : Dual adaptor M1403 and comparator 1 in NCI-H441.

[0097] Figure 9 shows the cancer cell killing mediated by dual adaptor M1397, comparator 1, or comparator 2 in MAGE-A4 positive HLA-A*02:01 positive cancer cell lines U2OS and NCI-H1703 ( Figure 9A and Figure 9B ), respectively, and MAGE-A4 negative HLA-A*02:01 positive cancer cell lines PANC-1 and SKMEL-30 ( Figure 9C and Figure 9D ), respectively.

[0098] Figure 10 shows IFNγ release in MAGE-A4 positive HLA-A*02:01 positive cancer cells ( Figure 10A : M1397 and comparator 1 in U2OS; Figure 10B : M1403 and comparator 1 in U2OS; Figure 10C : M1397 and comparator 1 in NCI-H1703; Figure 10D : M1403 and comparator 1 in NCI-H1703) and MAGE-A4 negative HLA-A*02:01 positive cancer cells ( Figure 10E : M1397 and comparator 1 in PANC-1; Figure 10F : M1403 and comparator 1 in PANC-1; Figure 10G : M1397 and comparator 1 in MDA-MB-231; Figure 10H : M1403 and comparator 1 in MDA-MB-231; Figure 10I : M1397 and comparator 1 in NCI-H441; Figure 10J : M1403 and comparator 1 in NCI-H441) after treatment with dual T cell adaptors M1397 and M1403 or comparator 1.

[0099] Figure 11 shows in MAGE-A4 positive HLA-A*02:01 positive cancer cell NCI-H1703 ( Figure 11A and Figure 11C ), respectively, and MAGE-A4 negative HLA-A*02:01 positive cancer cell SKMEL-30 ( Figure 11B and Figure 11D) Granzyme B ([ Figures 11A to 11B ]) and IFNγ release ([ Figures 11C to 11D ]) as a measure of T cell activation after treatment with dual adaptor M1397, comparator 1, or comparator 2. Figures 11A to 11B ) and IFNγ release ([ Figures 11C to 11D ]) Figures 11C to 11D .

[0100] Figure 12 shows cytokine release in MAGE-A4-positive HLA-A*02:01-positive U2OS and MAGE-A4-negative HLA-A*02:01-positive PANC-1 cells after treatment with dual T cell adaptor M1397 or M1403 and comparator 1. Quantification of IL-2 ([ Figures 12A to 12D ]), IL-6 ([ Figures 12G to 12H ]), and TNFα ([ Figures 12I to 12L ]). Figures 12A to 12D ) and IL-6 ([ Figures 12G to 12H ]) Figures 12G to 12H ) and TNFα ([ Figures 12I to 12L ]) Figures 12I to 12L .

[0101] Figure 13 shows IFN-γ expression in TAP-deficient T2 cells pulsed with MAGE-A4 or physiologically relevant highly similar peptide controls 1 or 2 and incubated with PBMC as effector cells and dual adaptor M1397 ([ Figure 13A ]), comparator 1 ([ Figure 13B ]), or comparator 2 ([ Figure 13C ]). Controls used T2 cells plus PBMC only, PBMC plus test compound, and PBMC only. Figure 13A ) and comparator 1 ([ Figure 13B ]) Figure 13B ) or comparator 2 ([ Figure 13C ]) Figure 13C .

[0102] Figure 14 shows granzyme B release in antigen-negative cancer cell lines KLE, LNCaP, KMRC-2, KMRC-3, 639-V, EKVX, and HCT116 ([ Figures 14B to 14H ]) after treatment with dual T cell adaptor M1397, comparator 1, or comparator 2. The antigen-positive cell line NCI-H1703 served as a positive control ([ Figure 14A ]). Figures 14B to 14H ) and the antigen-positive cell line NCI-H1703 served as a positive control ([ Figure 14A ]) Figure 14A .

[0103] Figure 15 shows the safety of dual adaptors M1397 and M1403 or comparator 1 in HLA-A*02:01-positive primary cells. Figure 15A : HMVEC-C cells; Figure 15B : NHBE cells; Figure 15C : NHA cells.

[0104] Figure 16 shows the safety of dual adaptor M1397, comparator 1, and comparator 2 in various HLA-A*02:01-positive primary cells. Figure 16A : In HCM_679 cells; Figure 16B : In HCM_639 cells; Figure 16C : In HCM_745 cells; Figure 16D : In HCM_746 cells; Figure 16E : In HMVEC-L_73809 cells;Figure 16F : In HCF_251 cells; Figure 16G : In RPTEC_82573 cells; Figure 16H : In NHLF_19232 cells; Figure 16I : In NHLF_76039 cells; Figure 16J : In NHA_72445 cells; Figure 16K : In NHBE_35497 cells; Figure 16L : In RPTEC_49985 cells; Figure 16M : In HaoSMC_735 cells.

[0105] Figure 17 Shows the in vivo efficacy of M1397 in a cell line-derived murine NSCLC xenograft model detailed in Example 14. The mean tumor volume at various doses of the active compound during the study period is shown. Detailed Description

[0106] In general, the nomenclatures used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. Unless otherwise indicated, the methods and techniques provided herein are generally carried out according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed in this specification. Enzymatic reactions and purification techniques are carried out as commonly done in the art or as described herein according to the manufacturer's instructions. The nomenclatures, as well as the laboratory procedures and techniques, used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and treatment of patients are used.

[0107] Unless otherwise defined, in this document, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definition. Unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. Unless otherwise stated, the use of "or" means "and / or". The use of the terms "including" and other forms (such as "includes" and "included") is not restrictive.

[0108] To more easily understand the present invention, certain terms are first defined.

[0109] Antigen-binding protein

[0110] "MAGE-A4" represents "melanoma-associated antigen 4", which is a member of the MAGE family of cancer-testis antigens (CTAs). MAGE A family proteins encompass 12 highly homologous genes that cluster at Xq28 and are characterized by the presence of a conserved domain (MAGE homology domain, MHD). Human MAGE-A4 is described in UniProt (www.uniprot.org) accession number P43358 (entry version 163). "MAGE-A4" p230-239 " or "203-239 peptide" refers to a MAGE-A4-derived peptide having the amino acid sequence GVYDGREHTV (SEQ ID NO: 3) at positions 230-239 of the MAGE-A4 protein.

[0111] The cluster of differentiation 3 co-receptor (or co-receptor complex) of the T cell receptor, "CD3", is a complex composed of four distinct chains. In mammals, the complex contains the CD3γ (γ) chain / subunit, the CD3δ (δ) chain / subunit, and two CD3ε (ε) chain / subunits. CD3 is referred to throughout this text as a cell surface protein of immune cells. The term "CD3" refers to any native CD3 from any vertebrate source, including primates. In certain embodiments, the antigen-binding proteins of the present disclosure specifically bind to human CD3, particularly the CD3ε (ε) chain / subunit of CD3 (see, for example, UniProt (www.uniprot.org) accession number P07766 (version 189), or NCBI (www.ncbi.nlm.nih.gov / ) NP_000724.1.) The CD3 molecule can be a full-length, unprocessed CD3 molecule or a fragment or variant thereof, such as those produced by processing in cells. By way of example, such variants can be naturally occurring variants, such as splice variants or allelic variants. In certain embodiments, the antigen-binding proteins disclosed herein bind to CD3 epitopes that are conserved among CD3 antigens from different species, such as non-human primates (e.g., cynomolgus monkeys) or rodents (e.g., mice, rats). In certain embodiments, the antigen-binding proteins do not cross-react with CD3 antigens from rodents (e.g., mice or rats) or miniature pigs.

[0112] As used herein, the terms "antibody" or "antigen-binding protein" refer to an immunoglobulin molecule or immunoglobulin-derived molecule that specifically binds to or is immunoreactive with an antigen or epitope, and includes polyclonal and monoclonal antibodies as well as functional antibody fragments, including but not limited to antigen-binding (Fab) fragments, F(ab′)2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain variable fragments (scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody, VHH) fragments. Thus, an antibody can be a single-domain antibody or comprise at least one variable light chain and at least one variable heavy chain. In one embodiment, the at least one variable light chain and the at least one variable heavy chain are presented as a single polypeptide chain. The terms "antibody" or "antigen-binding protein" include germline-derived antibodies. The terms "antibody" or "antigen-binding protein" include immunoglobulins in genetically engineered or otherwise modified forms, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetra-bodies, tandem diabodies, tandem triabodies), etc. Unless otherwise stated, the terms "antibody" or "antigen-binding protein" should be understood to encompass their functional antibody fragments.

[0113] In certain embodiments, the antigen-binding protein is multispecific (i.e., binds to two or more different target molecules or two or more epitopes on the same target molecule). In certain embodiments, the antigen-binding protein is bispecific and, for example, binds to two different target molecules or two epitopes on the same target molecule. In certain embodiments, the antibody is trispecific and, for example, binds to at least three different target molecules.

[0114] An antigen-binding protein can be monovalent or multivalent, i.e., having one or more antigen-binding sites. Non-limiting examples of monovalent antigen-binding proteins include scFv, Fab, scFab, dAb, VHH, V(NAR), DARPin, affilin, and nanobodies. Multivalent antigen-binding proteins can have two, three, four, or more antigen-binding sites. Non-limiting examples of multivalent antigen-binding proteins include full-length immunoglobulins, F(ab′)2 fragments, bis-scFv (or tandem scFv or BiTE), DART, diabodies, scDb, DVD-Ig, IgG-scFab, scFab-Fc-scFab, IgG-scFv, scFv-Fc, scFv-fc-scFv, Fv2-Fc, FynomAB, tetrabody, CrossMab, DuoBody, triabody, and tetrabody. In some embodiments, the multivalent antigen-binding protein is divalent, i.e., there are two binding sites. In some embodiments, the multivalent antigen-binding protein is bispecific, i.e., the antigen-binding protein is directed against two different targets or two different target sites on one target molecule. In some embodiments, the multivalent antigen-binding protein includes more than two different binding sites, such as three or four different binding sites directed against three or four different antigens, respectively. Such antigen-binding proteins are multivalent and multispecific, specifically trispecific or tetra-specific, respectively.

[0115] In some embodiments, the antigen-binding protein is multispecific (e.g., bispecific), such as but not limited to diabodies, single-chain diabodies, DART, BiTE, BIKE, tandem scFv, or IgG-like asymmetric heterobispecific antibodies. In certain embodiments, one of the binding specificities of the multispecific antigen-binding protein is an immune cell engager (i.e., includes binding affinity for a cell surface protein of an immune cell). Examples of immune cells that can be recruited include but are not limited to T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, monocytes, and macrophages. Examples of surface proteins that can be used to recruit immune cells include but are not limited to CD3, TCRα, TCRβ, CD16, NKG2D, CD94 / NKG2C, NKp30, NKp46, CD89, CD64, and CD32. In certain embodiments, the immune cell target antigen is CD3.

[0116] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., antibodies that bind to the same epitope and / or have the same sequence. In contrast, a polyclonal antibody population will bind to multiple epitopes and contain antibodies of different sequences. A monoclonal antibody preparation may or may not contain a minor degree of variant antibodies, e.g., due to naturally occurring mutations. Such variants can be produced, for example, by post-translational modifications such as cleavage at the N-terminus and / or C-terminus of the light and / or heavy chains, or formation of pyroglutamic acid at the N-terminus of the polypeptide chain (see, e.g., Liu YD et al. J Biol Chem. April 1, 2011; 286(13):11211-7). Depending on the method used and the antibody, the percentage of variants in the mixture will vary and can range from substantially all of the antibodies produced to a very low percentage.

[0117] As used herein, "single-chain variable fragment" (scFv) is an antigen-binding protein that comprises a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL). The VH and VL domains of the scFv are linked via any suitable linker recognized in the art. Such linkers include, but are not limited to, the repeated GGGGS (SEQ ID NO:74) amino acid sequence or variants thereof. ScFvs generally do not contain antibody constant domain regions, although the scFvs of the present disclosure can be linked or attached to antibody constant domain regions (e.g., the antibody Fc domain) to alter various properties of the scFv, including but not limited to increased serum or tissue half-life. ScFvs generally have a molecular weight of approximately 25 kDa and a hydrodynamic radius of approximately 2.5 nm.

[0118] As used herein, "Fab fragment" or "Fab" or "Fab domain" is an antibody fragment that comprises a light-chain fragment that includes the variable light-chain (VL) domain and the constant domain (CL) of the light chain, and the variable heavy-chain (VH) domain and the first constant domain (CH1) of the heavy chain. F(ab′)2 contains two antigen-binding regions linked by a disulfide bond at the hinge.

[0119] As used herein, "VHH", "nanobody", "heavy-chain only antibody", "single-domain antibody", or "sdAb" is an antigen-binding protein that comprises a single heavy-chain variable domain derived from a species of the camelid family, which species include camel, llama, and alpaca. VHHs generally have a molecular weight of approximately 15 kDa.

[0120] The antigen-binding proteins of the present disclosure can include one or more linkers for linking the domains of the antigen-binding protein (e.g., linking VH and VL to form an scFv, or linking multiple binding domains to form a multispecific antigen-binding protein).

[0121] Illustrative examples of linkers include glycine polymers (Gly)n ; glycine-serine polymer (Gly n Ser) n , where n is an integer of at least one, two, three, four, five, six, seven or eight; glycine-alanine polymer; alanine-serine polymer; and other flexible linkers known in the art.

[0122] Glycine and glycine-serine polymers are relatively unstructured and can therefore potentially act as neutral tethers between domains of a fusion protein such as an antigen-binding protein described herein. Glycine accesses significantly more phi-psi space than other small side-chain amino acids and is much less restricted than residues with longer side chains (Scheraga, Rev. Computational Chem. 1:1173-142 (1992)). Those skilled in the art will recognize that in certain embodiments, the design of an antigen-binding protein can include all or part of a flexible linker such that the linker can include a flexible linker segment and one or more segments that confer less flexibility to provide the desired structure.

[0123] However, a linker sequence similar to a native linker sequence can be selected, for example, using the starting amino acid segments corresponding to human CH1 and Cκ sequences or the lower amino acid segment corresponding to the human IgG hinge region.

[0124] The design of the peptide linker that connects the VL and VH domains in the scFv portion is a flexible linker that is typically composed of small non-polar or polar residues such as Gly, Ser, and Thr. A specific exemplary linker that connects the variable domains of the scFv portion is the (Gly4Ser)4 linker (SEQ ID NO: 76), where 4 is the exemplary number of repeats of the motif.

[0125] Linkers that connect the scFv antigen-binding protein to the Fab domain are also contemplated. In certain embodiments, the scFv antigen-binding protein is linked to the CH1 and CL domains of the Fab with a Gly-Ser linker. In certain embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 74). In certain embodiments, the amino acid linker comprises the amino acid sequences GGGGSGGGGSGGGGS (SEQ ID NO: 75), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 76), GGGGSGGGGSGGGGSGGGGAS (SEQ ID NO: 77), or GGGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 78).

[0126] Other exemplary linkers include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 79); TGEKP (SEQ ID NO: 80) (Liu et al., Proc. Natl. Acad. Sci. 94: 5525-5530 (1997)); GGRR (SEQ ID NO: 81); (GGGGS) n (SEQ ID NO: 73), where n = 1, 2, 3, 4 or 5 (Kim et al., Proc. Natl. Acad. Sci. 93: 1156-1160 (1996)); EGKSSGSGSESKVD (SEQ ID NO: 82) (Chaudhary et al., Proc. Natl. Acad. Sci. 87: 1066-1070 (1990)); KESGSVSSEQLAQFRSLD (SEQ ID NO: 83) (Bird et al., Science 242: 423-426 (1988)), GGRRGGGS (SEQ ID NO: 84); LRQRDGERP (SEQ ID NO: 85); LRQKDGGGSERP (SEQ ID NO: 86); and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 87) (Cooper et al., Blood, 101(4): 1637-1644 (2003)). Alternatively, computer programs capable of mimicking the 3D structure of proteins and peptides or rational design of flexible linkers by phage display methods can be used.

[0127] An antibody can comprise a variable light chain (VL) domain and a variable heavy chain (VH) domain. Each VL and VH domain also comprises a set of three CDRs.

[0128] As used herein, the term "complementary determining region" or "CDR" refers to the discontinuous amino acid sequences within the variable regions of an antibody that confer antigen specificity and affinity. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) present in each heavy chain variable domain, and three CDRs (LCDR1, LCDR2, LCDR3) present in each light chain variable domain. "Framework region" or "FR" is known in the art to refer to the non-CDR portions of the variable domains of the heavy and light chains. Generally, there are four FRs (HFR1, HFR2, HFR3, and HFR4) present in each heavy chain variable domain, and four FRs (LFR1, LFR2, LFR3, and LFR4) present in each light chain variable domain. Thus, the amino acid sequence of an antibody variable region can be represented by the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Each segment of the formula (i.e., FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4) represents a discrete amino acid sequence (or polynucleotide sequence encoding it) that can be mutated, including one or more amino acid substitutions, deletions, and insertions. In certain embodiments, the amino acid sequence of an antibody variable light chain can be represented by the formula LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4. In certain embodiments, the amino acid sequence of an antibody variable heavy chain can be represented by the formula HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0129] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745. ("Contact" numbering scheme); Lefranc M P et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 January; 27(1):55-77 ("IMGT" numbering scheme); and Honegger A and Plückthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun. 8; 309(3):657-70 ("AHo" numbering scheme).

[0130] The boundaries of a given CDR or FR can vary according to the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. The numbering of the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, where inserted letters (e.g., "30a") are used to accommodate insertions, and deletions occur in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects.

[0131] Table 1 below lists exemplary position boundaries of LCDR1, LCDR2, LCDR3, and HCDR1, HCDR2, HCDR3 of antibodies identified by the Kabat, Chothia, and Contact schemes, respectively. For HCDR1, residue numbers are listed using the Kabat and Chothia numbering schemes. The CDRs are located between the FRs, for example where LCDR1 is located between LFR1 and LFR2, etc. It should be noted that since the Kabat numbering scheme shown places insertions at H35A and H35B, the end of the Chothia HCDR1 loop varies between H32 and H34 depending on the length of the loop when numbered using the Kabat numbering convention shown.

[0132] Table 1 - Exemplary Position Boundaries of CDRs

[0133]

[0134] 1 - Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD

[0135] 2 - Al - Lazikani et al. (1997), J Mol. Biol. 273:927 - 948

[0136] Thus, unless otherwise specified, the “CDR” or “complementary - determining region” or an individually - specified CDR (e.g., HCDR1, HCDR2) of a given antibody or its fragment (such as its variable domain) should be understood to encompass one (or a particular) complementary - determining region as defined by any of the known schemes. Similarly, unless otherwise specified, the “FR” or “framework region” or an individually - specified FR (e.g., “HFR1,” “HFR2”) of a given antibody or its fragment (such as its variable domain) should be understood to encompass one (or a particular) framework region as defined by any of the known schemes. In some cases, the scheme used to identify a particular CDR or FR is specified, such as the CDR as defined by the Kabat, Chothia, or Contact methods. In other cases, the specific amino - acid sequence of the CDR or FR is given.

[0137] In certain embodiments, the antigen-binding proteins disclosed herein are rabbit-derived antigen-binding proteins. In certain embodiments, the antigen-binding proteins are humanized. As used herein, the term "humanized" or "humanization" means that the antigen-binding protein has been altered to make it more like a human antibody. Non-human antigen-binding proteins, such as rabbit antigen-binding proteins, can elicit negative immune responses if administered to humans for therapeutic purposes. Thus, it is advantageous to humanize rabbit antigen-binding proteins for subsequent therapeutic use.

[0138] In certain embodiments, the antigen-binding protein is humanized by surface resurfacing (i.e., reconstructing the solvent-accessible residues of the non-human framework such that they become more human-like). Surface resurfacing strategies are described in more detail in WO2004 / 016740, WO2008 / 144757, and WO2005 / 016950, each of which is incorporated herein by reference.

[0139] In certain embodiments, the antigen-binding protein is humanized by CDR grafting (i.e., inserting rabbit antigen-binding protein CDRs into a human antibody acceptor framework). Grafting strategies and human acceptor frameworks are described in more detail in WO2009 / 155726, which is incorporated herein by reference.

[0140] As used herein, "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with the sequence of a second polypeptide. Similarly, "sequence identity" between two polynucleotides is determined by comparing the nucleotide sequence of one polynucleotide with the sequence of a second polynucleotide. The terms "% identity", "percent identity", or like terms are intended to specifically refer to the percentage of identical nucleotides or amino acids (as applicable) after the sequences to be compared have been aligned to yield maximum identity (potentially introducing gaps). The percentage may be purely statistical, and the differences between two sequences may or may not be randomly distributed across the entire length of the sequences to be compared. Comparison of two sequences is typically carried out by comparing the sequences relative to a segment or "comparison window" after optimal alignment to identify local regions of corresponding sequences. By way of example, optimal alignment for comparison can be conducted manually or by means of the local homology algorithm of Smith and Waterman, 1981, AdsApp.Math.2, 482, by means of the local homology algorithm of Needleman and Wunsch, 1970, J.Mol.Biol.48, 443, by means of the similarity search algorithm of Pearson and Lipman, 1988, Proc.Natl Acad.Sci.USA 88, 2444, or by means of computer programs using algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis., or Clustal Omega). In some embodiments, the percent identity between two sequences is determined using the BLASTN or BLASTP algorithms available at the website of the United States National Center for Biotechnology Information (NCBI) (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). Typically, the percent identity is determined over the entire length of the reference sequence being analyzed.

[0141] Variant polypeptides, such as antigen-binding proteins, may contain one or more substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence. In certain embodiments, the variant polypeptide contains one, two, or three substitutions, insertions, and / or deletions relative to the reference sequence. The substitutions, insertions, or deletions may result in a change in one or more biophysical parameters, but particularly preferred are tolerated changes such that the polypeptide retains the desired activity. In some embodiments, the variant polypeptide contains one or more tolerated substitutions, such as conservative substitutions. In certain embodiments, such variant polypeptides maintain the properties of the corresponding reference sequence physically, biologically, chemically, and / or functionally.

[0142] "Specifically recognize" or "specifically bind" refers to the ability of an antigen-binding protein to selectively bind to an antigen as compared to non-specific interactions with unrelated proteins that do not contain the binding epitope. Suitable assays for determining specific binding are described below. In certain embodiments, as determined, for example, by SPR, the equilibrium dissociation constant (K D ) of the antigen-binding protein for an unrelated protein is less than about 50-fold lower than the equilibrium dissociation constant of the antigen-binding protein for its antigen.

[0143] As used herein, the term "affinity" (or "binding affinity," which may be used interchangeably herein) refers to the strength of the interaction between the antigen-binding site of an antibody and the epitope to which it binds. As will be readily understood by those skilled in the art, antibody or antigen-binding protein affinity can be reported as the equilibrium dissociation constant (K D ) in molar concentration (M). The equilibrium dissociation constant K D is calculated from the association rate constant k a (units of M -1 s -1 ) and the dissociation rate constant k d (units of s -1 ) by k d / k a . The antibodies of the present disclosure may have a K -8 value in the range of 10 -14 to 10 D M.

[0144] The ability of an antibody to bind to a specific epitope (e.g., a target peptide-MHC) can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., surface plasmon resonance (SPR) techniques (e.g., using a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000))) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Generally, the kinetic rate constants can be determined at temperatures in the range of 15°C to 37°C. Throughout this specification, reference is made to kinetic rate constants determined by SPR. Generally, in embodiments related to each mention of SPR in this specification, the association rate constant values, dissociation rate constant values, and equilibrium dissociation constant values described herein are determined by SPR at 25°C based on the SPR of monovalent antigen-binding proteins. Preferably, the SPR-based system used is a Biacore SPR system. Those skilled in the art will understand that binding parameters can be measured in the context of monovalent or divalent bispecific, trispecific, or multispecific constructs.

[0145] In certain embodiments, the antigen-binding protein is not a T cell receptor (TCR), including but not limited to soluble TCRs. As used herein, the term "T cell receptor" or "TCR" refers to a heterodimeric protein composed of two different chains (TCRα and TCRβ), which structurally belongs to the immunoglobulin (Ig) superfamily. The extracellular portion of each chain consists of variable ("Vα" and "Vβ") and constant ("Cα" and "Cβ") domains, as well as a hinge region in which stable disulfide bonds are formed. The intracellular region forms non-covalent interactions with another transmembrane protein, CD3, which, upon correct target recognition, leads to a series of conformational changes and the first T cell activation signal. The recognition and binding of TCR to peptide-MHC (pMHC) are controlled by six hypervariable loops called complementarity-determining regions (CDRs) located on the variable domains of TCRα (CDRαl, CDRα2, CDRα3) and TCRβ (CDRβ1, CDRβ2, CDRβ3). The CDR3 loops (CDRα3 and CDRβ3), supported by CDRα1 and CDRβ1, mediate the recognition of processed antigens, with CDRα1 and CDRβ1 participating in the recognition of the N-terminal and C-terminal amino acids of the presented peptide, respectively (Rudolph et al. Annu Rev Immunol. 24:419-66. 2006). The recognition of MHC is typically achieved through interactions with CDRα2 and CDRβ2. The high sequence diversity of TCRs is achieved through the V(D)J recombination process, in which the variable domains are generated by combinations of the following genes: V (variable) and J (joining) for TCRα and TCRβ, and an additional D (diversity) gene for TCRβ. The high antigen specificity of TCRs is controlled by the thymic maturation process, in which autoreactive T cells are negatively selected. The affinity and functional avidity of TCRs for specific pMHCs are key factors controlling T cell activation. However, the key role in antigen recognition is played by affinity (i.e., the strength of binding between the TCR and the pMHC presented by the cell) (Tian et al. J Immunol. 179:2952-2960. 2007). The physiological affinity of TCRs ranges from 1 μM to 100 μM (Davis et al. Annu Rev Immunol. 16:523-544. 1998), which is relatively low compared to antibodies.

[0146] As used herein, the terms "peptide-MHC" or "pMHC" or "pMHC complex" are used interchangeably herein to refer to a major histocompatibility complex (MHC) molecule (MHC-I or MHC-II) having an antigenic peptide bound in the peptide-binding pocket of the MHC. As is known in the art, MHC molecules present peptides, particularly antigenic peptides, on the cell surface for recognition by immune cells. Thus, as will be understood by one of ordinary skill in the art, the term "pMHC" as used herein refers to a complex of an MHC molecule and a peptide (particularly an antigenic peptide) presented by the MHC molecule. This is generally referred to as MHC-restricted antigen presentation. Thus, the peptides targeted by a pMHC-binding domain are MHC-restricted peptides. Thus, the peptides can be considered target peptides or target antigenic peptides. In addition, according to the present disclosure, the terms "target pMHC-binding domain" and "pMHC-binding domain" are used interchangeably herein and in any case refer to at least a first and at least a second pMHC-binding domain referred to throughout this text. The terms "target peptide / antigen presented by an MHC molecule / complex" and "MHC-restricted target peptide / antigen" or similar expressions used in this specification are used interchangeably herein.

[0147] In certain embodiments, the MHC is a human MHC. Although MHCs are present in all vertebrates, the MHC in humans is called HLA (human leukocyte antigen). HLA is highly polygenic and can be broadly classified into three classes of MHC molecules: class I, class II, and class III. In addition, HLA genes have the highest level of polymorphism in the human genome. Target peptides can be presented on MHC class I complexes (such as serotypes HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L, or their corresponding subtypes) or MHC class II complexes (such as serotypes HLA-DP, HLA-DQ, HLA-DR, DM, or DO, or their corresponding subtypes). The HLA-A protein constitutes the α chain of the corresponding class I MHC (major histocompatibility complex) protein, which also contains a β2-microglobulin subunit. As used interchangeably herein, "HLA-A2", "HLA-A*02", "HLA-A02", or "HLA-A*2" refers to the human leukocyte antigen serotype in the HLA-A serotype group 2. Each serotype contains different subtypes. HLA-A*02:01 (also referred to as HLA-A0201, HLA-A02.01, or HLA-A201) is a specific subtype of the HLA-A*02 protein. "MAGE-A4 pMHC" refers to the complex of an HLA-A*02 molecule and a MAGE-A4-derived peptide (also referred to herein as the "MAGE-A4 peptide"), specifically GVYDGREHTV (SEQ ID NO: 3). "MAGE-A8 pMHC" refers to the complex of an HLA-A*02 molecule and a MAGE-A8-derived peptide (also referred to herein as the "MAGE-A8 peptide"), specifically GLYDGREHSV (SEQ ID NO: 71). "MAGE-B4 pMHC" refers to the complex of an HLA-A*02 molecule and a MAGE-B4-derived peptide (also referred to herein as the "MAGE-B4 peptide"), specifically GIYDGKRHLI (SEQ ID NO: 72).

[0148] As used herein, the term "PBS" refers to phosphate buffered saline. PBS is an admixture of a pH-adjusted phosphate buffer and a saline solution. In certain embodiments, PBS contains about 100 - 150 mM NaCl, about 1 - 5 mM KCl, about 1 - 10 mM Na2HPO4, and 1 - 5 mM KH2PO4. In certain embodiments, PBS contains 130 mM NaCl, 10 mM Na2HPO4, and has a pH of 6.0.

[0149] MAGE-A4 peptide-MHC and its antigen-binding protein

[0150] The antigen-binding proteins and multispecific antigen-binding proteins described herein have binding specificity for MAGE-A4 peptide-MHC.

[0151] In certain embodiments, there is provided an isolated antigen-binding protein that binds to GVYDGREHTV (SEQ ID NO: 3) presented by HLA, i.e., the isolated antigen-binding protein does not associate or bind to the surface of cells such as T cells. The isolated antigen-binding protein is separated from the components of its natural environment. In certain embodiments, the isolated antigen-binding protein is purified to greater than 95% or 99% purity as determined, for example, by electrophoresis (such as SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (such as ion exchange or reverse-phase HPLC, affinity chromatography, size exclusion chromatography). In certain embodiments, the antigen-binding protein is not a soluble TCR (e.g., a TCR lacking one or more of a transmembrane domain, an intracellular signaling domain, and a constant domain). In certain embodiments, the antigen-binding protein is a monoclonal antibody, particularly an antibody fragment such as an scFv.

[0152] The target peptide can be presented on MHC class I complexes (such as serotypes HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L, or their corresponding subtypes) or MHC class II complexes (such as serotypes HLA-DP, HLA-DQ, HLA-DR, DM, or DO, or their corresponding subtypes). Each of the serotypes contains different subtypes. In one embodiment, the antigen-binding protein targets and binds to a peptide of the HLA-A2 pMHC complex (also known as HLA-A*02, particularly HLA-A*02:01).

[0153] The antigen-binding protein has a surprisingly high binding affinity while retaining high specificity for the target (i.e., having low affinity or even no binding to pMHC presenting an unrelated peptide or β-2-microglobulin). As used herein, an "unrelated peptide" corresponds to a peptide that does not contain the binding epitope of the antigen-binding protein of the present disclosure. In certain embodiments, as determined, for example, by SPR, the affinity of the antigen-binding protein for an unrelated peptide MHC is less than about 50-fold lower than the binding of the antigen-binding protein to GVYDGREHTV (SEQ ID NO: 3) presented by HLA.

[0154] In certain embodiments, the antigen-binding protein comprises specificity for the MAGE-A4 peptide amino acid sequence shown in SEQ ID NO: 3 (GVY DGREHTV), which corresponds to amino acids 230-239 of MAGE-A4.

[0155] In certain embodiments, the antigen-binding protein binds to MAGE-A4, MAGE-A8, and MAGE-B4 peptides presented by related HLAs with a similar affinity range. Accordingly, the present disclosure provides an antigen-binding protein that comprises binding specificity for MAGE-A4 pMHC, MAGE-A8 pMHC, and MAGE-B4 pMHC, particularly GVYDGREHTV (SEQ ID NO: 3) derived from MAGE-A4, GLYD GREHSV (SEQ ID NO: 71) derived from MAGE-A8, and GIYDGKRHLI (SEQ ID NO: 72) derived from MAGE-B4.

[0156] In certain embodiments, the MAGE-A4 peptide, the MAGE-A8 peptide, and / or the MAGE-B4 peptide are complexed with an HLA-A*02 polypeptide.

[0157] In certain embodiments, the HLA-A*02 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1.

[0158] In certain embodiments, the β-2-microglobulin polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2.

[0159] Accordingly, in certain embodiments, the antigen-binding protein comprises specificity for HLA-A*02 / MAGE-A4, particularly for HLA-A*02, and more particularly for the peptide GVYDGREHTV (SEQ ID NO: 3) presented by the HLA-A*02 subtype HLA-A*02:01.

[0160] In one aspect, the present disclosure provides an antigen-binding protein that specifically binds to a melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), the antigen-binding protein comprising: an antibody heavy chain variable (VH) domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10 or consists of the amino acid sequence of SEQ ID NO: 10; and an antibody light chain variable (VL) domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 or consists of the amino acid sequence of SEQ ID NO: 15.

[0161] In certain embodiments, such antigen-binding proteins are scFvs that comprise or consist of SEQ ID NO: 61 or a variant thereof that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 61.

[0162] In one aspect, the present disclosure provides an antigen-binding protein that specifically binds to a melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), the antigen-binding protein comprising: a VH domain that comprises or consists of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20; and a VL domain that comprises or consists of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25.

[0163] In certain embodiments, the antigen-binding protein is an scFv that comprises or consists of SEQ ID NO: 62 or a variant thereof that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62.

[0164] In one aspect, the present disclosure provides an antigen-binding protein that specifically binds to a melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), the antigen-binding protein comprising: a VH domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, wherein the VH domain comprises a C amino acid at position 44 of SEQ ID NO: 30; and a VL domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 35, wherein the VL domain comprises a C amino acid at position 102 of SEQ ID NO: 35.

[0165] In certain embodiments, the antigen-binding protein comprises a VH domain consisting of the amino acid sequence of SEQ ID NO: 30 and a VL domain consisting of the amino acid sequence of SEQ ID NO: 35.

[0166] In certain embodiments, the antigen-binding protein is a scFv that comprises or consists of SEQ ID NO: 63 or a variant thereof that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 63.

[0167] In one aspect, the present disclosure provides an antigen-binding protein that specifically binds to melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), the antigen-binding protein comprising: a VH domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40, wherein the VH domain comprises a Y amino acid at position 47, an R amino acid at position 71, and an N amino acid at position 73 of SEQ ID NO: 40, and a VL domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45.

[0168] In certain embodiments, the antigen-binding protein comprises a VH domain consisting of the amino acid sequence of SEQ ID NO: 40 and a VL domain consisting of the amino acid sequence of SEQ ID NO: 45.

[0169] In certain embodiments, the antigen-binding protein is a scFv that comprises or consists of SEQ ID NO: 64 or a variant thereof that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62.

[0170] Other exemplary MAGE-A4 pMHC antigen-binding proteins are described in PCT / IB2022 / 052117 filed on March 9, 2022 and PCT / IB2022 / 052119 filed on March 9, 2022, the content of each patent being incorporated herein by reference.

[0171] The antigen-binding protein can be monovalent or multivalent, such as bispecific or trispecific. In certain embodiments, the antigen-binding protein is monovalent. In certain aspects, monovalent antigen-binding proteins comprising the CDRs of the above-described VL and / or VH sequences are provided. Such monovalent antigen-binding proteins include, but are not limited to, scFv, Fab, scFab, dAb, VHH, V(NAR), DARPin, affilin, and nanobody.

[0172] MAGE-A4 peptide-MHC-immune cell bridging antigen-binding protein

[0173] The multispecific antigen-binding proteins described herein have at least one MAGE-A4 peptide-MHC binding domain and a binding domain that has binding specificity for a cell surface protein of an immune cell (e.g., CD3 on the surface of a T cell or CD16a expressed on the surface of an NK cell). Alternatively, the multispecific antigen-binding proteins described herein have at least two MAGE-A4 peptide-MHC binding domains and optionally a binding domain that has binding specificity for a cell surface protein of an immune cell (e.g., CD3 on the surface of a T cell or CD16a expressed on the surface of an NK cell). In certain embodiments, the multispecific antigen-binding protein comprises the MAGE-A4 peptide-MHC antigen-binding protein described above.

[0174] For dual pMHC engagement, targeting two MAGE-A4 peptide-MHCs on the surface of a target cell (e.g., a cancer cell) improves target cell engagement through enhanced binding affinity. The enhanced binding affinity, in turn, can contribute to improved target cell killing relative to an antigen-binding protein that has only one pMHC binding domain. The enhanced binding affinity formed by at least two pMHC binding domains can be particularly useful when targeting low copy number pMHC complexes on the surface of target cells (e.g., cancer cells). Dual pMHC engagement multispecific binding proteins are described in further detail in U.S. 63 / 289,380, filed Dec. 14, 2021, and U.S. 63 / 317,256, filed Mar. 7, 2022, the contents of each patent being incorporated herein by reference.

[0175] In certain embodiments, the multispecific antigen-binding protein is bispecific or trispecific.

[0176] In certain embodiments, the multispecific antigen-binding protein is bivalent or multivalent, such as trivalent.

[0177] In certain embodiments, the multispecific antigen-binding protein further comprises at least one other binding domain.

[0178] In certain embodiments, the other binding domain is an immune cell adaptor, particularly a CD3 binding domain or a CD16a binding domain.

[0179] In certain embodiments, the multispecific antigen-binding protein further comprises a third antigen-binding domain.

[0180] In certain embodiments, the third antigen-binding domain binds to HLA-A*02 / MAGE-A4.

[0181] In certain embodiments, the third antigen-binding domain is the same as the first HLA-A*02 / MAGE-A4 antigen-binding domain described above.

[0182] In certain embodiments, the multispecific antigen-binding protein can bind to two (or more) different epitopes of HLA-A2 / MAGE-A4.

[0183] In one aspect, the present disclosure provides a multispecific antigen-binding protein comprising: a) a first antigen-binding domain that specifically binds to CD3; b) a second antigen-binding domain that specifically binds to a melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), the second antigen-binding domain comprising: b1) an antibody heavy chain variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), the HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6) (where X1 corresponds to the amino acid S or D, X2 corresponds to the amino acid W or S, and X3 corresponds to the amino acid A or V), and the HCDR3 amino acid sequence of DLYYGPX4TX5YX6X7X8NL (SEQ ID NO: 7) (where X4 corresponds to the amino acid T, N or S, X5 corresponds to the amino acid D or is absent, X6 corresponds to the amino acid S or F, X7 corresponds to the amino acid A or V, and X8 corresponds to the amino acid F or A); and b2) an antibody light chain variable (VL) domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and the LCDR3 amino acid sequence of ATX9X 10 X 11 SGSNFQX 12 (SEQ ID NO: 8), where X9 corresponds to the amino acid S or R, X 10 corresponds to the amino acid D or P, X 11 corresponds to the amino acid G, S or F, and X 12 corresponds to the amino acid L or A.

[0184] In certain embodiments, the MAGE-A4 peptide-MHC (pMHC) is the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex.

[0185] In certain embodiments, as determined by SEC-HPLC, after incubation in PBS at 4 °C for 14 days at 1 mg / ml and / or 10 mg / ml, the multispecific antigen-binding protein remains at least 94%, 95%, 96%, 97%, 98%, 99% or 100% monomeric.

[0186] In certain embodiments, the multispecific antigen-binding protein further comprises a third antigen-binding domain, specifically, the third binding domain is identical to the second binding domain (i.e., the MAGE-A4 pMHC binding domain).

[0187] In certain embodiments, such multispecific antigen-binding proteins comprise: c) a third antigen-binding domain that specifically binds to MAGE-A4 pMHC, the third antigen-binding domain comprising: c1) a VH domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), the HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6) (where X1 corresponds to the amino acid S or D, X2 corresponds to the amino acid W or S, and X3 corresponds to the amino acid A or V), and the HCDR3 amino acid sequence of DLYY GPX4TX5YX6X7X8NL (SEQ ID NO: 7) (where X4 corresponds to the amino acid T, N or S, X5 corresponds to the amino acid D or is absent, X6 corresponds to the amino acid S or F, X7 corresponds to the amino acid A or V, and X8 corresponds to the amino acid F or A); and c2) a VL domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and 10 X 11 SGSNFQX 12 (SEQ ID NO: 8) of the LCDR3 amino acid sequence, where X9 corresponds to the amino acid S or R, X 10 corresponds to the amino acid D or P, X 11 corresponds to the amino acid G, S or F, and X 12 corresponds to the amino acid L or A.

[0188] In certain embodiments, the second and third antigen-binding domains comprise:

[0189] (i) VH, wherein the VH comprises an HCDR1 sequence having the amino acid sequence of SNYAMS (SEQ ID NO: 11), an HCDR2 sequence having the amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 12), and an HCDR3 sequence having the amino acid sequence of DLYYGPNTDYSAANL (SEQ ID NO: 13); and VL, wherein the VL comprises an LCDR1 sequence having the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), an LCDR2 sequence having the amino acid sequence of RDTSRPS (SEQ ID NO: 17), and an LCDR3 sequence having the amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 18);

[0190] (ii) VH, wherein the VH comprises an HCDR1 sequence having the amino acid sequence of SNYAMS (SEQ ID NO: 21), an HCDR2 sequence having the amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 22), and an HCDR3 sequence having the amino acid sequence of DLYYGPSTYFVANL (SEQ ID NO: 23); and VL, wherein the VL comprises an LCDR1 sequence having the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 26), an LCDR2 sequence having the amino acid sequence of RDTSRPS (SEQ ID NO: 27), and an LCDR3 sequence having the amino acid sequence of ATRPSSGSNFQL (SEQ ID NO: 28);

[0191] (iii) VH, wherein the VH comprises an HCDR1 sequence having the amino acid sequence of SNYAMS (SEQ ID NO: 31), an HCDR2 sequence having the amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 32), and an HCDR3 sequence having the amino acid sequence of DLYYGPTTYSAANL (SEQ ID NO: 33); and VL, wherein the VL comprises an LCDR1 sequence having the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 36), an LCDR2 sequence having the amino acid sequence of RDTSRPS (SEQ ID NO: 37), and an LCDR3 sequence having the amino acid sequence of ATRDFSGSNFQL (SEQ ID NO: 38); or

[0192] (iv) VH, wherein the VH comprises an HCDR1 sequence having an amino acid sequence containing SNYAMS (SEQ ID NO: 41), an HCDR2 sequence having an amino acid sequence containing IVSSGGTTYYASWAKG (SEQ ID NO: 42), and an HCDR3 sequence having an amino acid sequence containing DLYYGPTTYSAFNL (SEQ ID NO: 43); and VL, wherein the VL comprises an LCDR1 sequence having an amino acid sequence containing TADTLSRSYAS (SEQ ID NO: 46), an LCDR2 sequence having an amino acid sequence containing RDTSRPS (SEQ ID NO: 47), and an LCDR3 sequence having an amino acid sequence containing ATRPSSGSNFQA (SEQ ID NO: 48).

[0193] In certain embodiments, the second and third antigen-binding domains comprise:

[0194] (i) an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15;

[0195] (ii) a VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20, and a VL domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25;

[0196] (iii) a VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 30, and a VL domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35; or

[0197] (iv) A VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40, and a VL domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45.

[0198] In certain embodiments, any one or more of the first, second, and third antigen-binding domains comprise an antibody fragment. In certain embodiments, the antibody fragment comprises a Fab fragment, an F(ab’)2 fragment, a Fab' fragment, an Fv fragment, a single-chain variable fragment (scFv), and a single-domain antibody fragment.

[0199] In certain embodiments, the MAGE-A4 pMHC antigen-binding domain comprises an scFv. In certain embodiments, the multispecific antigen-binding protein comprises at least one scFv, the at least one scFv comprising the amino acid sequence of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and / or SEQ ID NO: 64 or a variant thereof, the variant being at least about 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and / or SEQ ID NO: 64, respectively.

[0200] In certain embodiments, the immune cell or CD3 antigen-binding domain is a Fab fragment, wherein the Fab fragment comprises a heavy chain containing a CH1 domain and a VH, and a light chain containing a Cl domain and a VL.

[0201] In certain embodiments, the CH1 domain comprises at least 5 amino acids of the antibody hinge region. In certain embodiments, the CH1 domain comprises the amino acid sequence EPKSC (SEQ ID NO: 88) of the antibody hinge region.

[0202] In certain embodiments, the second antigen-binding domain is operably linked to the C-terminus or the N-terminus of the heavy chain.

[0203] In certain embodiments, the third antigen-binding domain is operably linked to the C-terminus or the N-terminus of the heavy chain.

[0204] In certain embodiments, the second antigen-binding domain is operably linked to the C-terminus or the N-terminus of the light chain.

[0205] In certain embodiments, the third antigen-binding domain is operably linked to the C-terminus or the N-terminus of the light chain.

[0206] In certain embodiments, a) the second antigen-binding domain comprises an scFv linked to the C-terminus of the heavy chain of the Fab domain and the third antigen-binding domain comprises an scFv linked to the C-terminus of the light chain of the Fab domain; b) the second antigen-binding domain comprises an scFv linked to the N-terminus of the heavy chain of the Fab domain and the third antigen-binding domain comprises an scFv linked to the N-terminus of the light chain of the Fab domain; c) the second antigen-binding domain comprises an scFv linked to the N-terminus of the heavy chain of the Fab domain and the third antigen-binding domain comprises an scFv linked to the C-terminus of the light chain of the Fab domain; or d) the second antigen-binding domain comprises an scFv linked to the C-terminus of the heavy chain of the Fab domain and the third antigen-binding domain comprises an scFv linked to the N-terminus of the light chain of the Fab domain.

[0207] In certain embodiments, the scFv is linked to the Fab domain with an amino acid linker.

[0208] In certain embodiments, the amino acid linker comprises (GGGGS)n (SEQ ID NO: 73), where n is an integer between 1 and 5. In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGS (SEQ ID NO: 74), GGGGSGGGGSGGGGS (SEQ ID NO: 75), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 76), GGGGSGGGGSGGGGSGGGGAS (SEQ ID NO: 77), or GGGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 78).

[0209] In certain embodiments, the VH and VL of the second and / or third antigen-binding domains are linked with an amino acid linker.

[0210] In certain embodiments, the amino acid linker comprises (GGGGS)n (SEQ ID NO: 73), where n is an integer between 1 and 5. In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGS (SEQ ID NO: 74), GGGGSGGGGSGGGGS (SEQ ID NO: 75), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 76), GGGGSGGGGSGGGGSGGGGAS (SEQ ID NO: 77), or GGGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 78).

[0211] In certain embodiments, the multispecific antigen-binding protein does not comprise an Fc domain.

[0212] In certain embodiments, the multispecific antigen-binding protein comprises (scFv)2, (scFv)3, BiTE, BIKE, Dart, diabody, triabody, Fab2, Fab3, Fab4, scFv-Fab-scFv, or minibody-scFv.

[0213] In certain embodiments, the multispecific antigen-binding protein has a molecular weight of from about 75 kDa to about 110 kDa.

[0214] In certain embodiments, the antigen-binding protein has a longer serum half-life relative to an antigen-binding protein having a molecular weight of < about 75 kDa.

[0215] Anti-CD3 binding domains suitable for use with the multispecific antigen-binding domains disclosed herein are known in the art, particularly T cell-activating CD-ε binding domains. Exemplary CD3 binding domains are disclosed in US6750325, WO2008079713, US7635475, WO2005040220, US7728114, WO9404679, US7381803, WO2008119567, WO2014110601, WO2014145806, WO2016086189, and / or WO2019195535A1, each of which is incorporated herein by reference. In certain embodiments, the CD3 domain does not cross-react with miniature pig CD3 or rodent CD3, particularly rat or mouse CD3.

[0216] In certain embodiments, the CD3 antigen-binding domain comprises: a1) VH, wherein the VH comprises an HCDR1 sequence having an amino acid sequence containing STYAMN (SEQ ID NO: 51), an HCDR2 sequence having an amino acid sequence containing RIRSKYNNYATYYADSVKG (SEQ ID NO: 52), and an HCDR3 sequence having an amino acid sequence containing HGNFGDSYVSWFAY (SEQ ID NO: 53); and a2) VL, wherein the VL comprises an LCDR1 sequence having an amino acid sequence containing GSSTGAVTTSNYAN (SEQ ID NO: 56), an LCDR2 sequence having an amino acid sequence containing GTNKRAP (SEQ ID NO: 57), and an LCDR3 sequence having an amino acid sequence containing ALWYSNHWV (SEQ ID NO: 58).

[0217] In certain embodiments, the CD3 antigen-binding domain comprises: a VH that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 50, and a VL that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55.

[0218] In certain embodiments, the CD3 antigen-binding domain comprises: a VH consisting of the amino acid sequence of SEQ ID NO: 50, and a VL consisting of the amino acid sequence of SEQ ID NO: 55.

[0219] In certain embodiments, the CD3 antigen-binding domain comprises: a heavy chain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49, and a light chain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54.

[0220] In certain embodiments, the CD3 antigen-binding domain comprises: a VH consisting of the amino acid sequence of SEQ ID NO: 49, and a VL consisting of the amino acid sequence of SEQ ID NO: 54.

[0221] In certain embodiments, the multispecific antigen-binding protein comprises: a) a first antigen-binding domain that specifically binds to CD3, the first antigen-binding domain comprising: a1) a VH that comprises an HCDR1 sequence having the amino acid sequence containing STYAMN (SEQ ID NO: 51), an HCDR2 sequence having the amino acid sequence containing RIRSKYNNYATYYADSVKG (SEQ ID NO: 52), and an HCDR3 sequence having the amino acid sequence containing HGNFGDSYVSWFAY (SEQ ID NO: 53); and a2) a VL that comprises an LCDR1 sequence having the amino acid sequence containing GSSTGAVTTSNYAN (SEQ ID NO: 56), an LCDR2 sequence having the amino acid sequence containing GTNKRAP (SEQ ID NO: 57), and an LCDR3 sequence having the amino acid sequence containing ALWYSNHWV (SEQ ID NO: 58); and b) a second antigen-binding domain that specifically binds to a melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC). Embodiments of suitable second antigen-binding domains have been described in detail above. Such multispecific antigen-binding proteins may comprise a third binding domain that can be the same as the second binding domain. Possible modes and modifications have also been described in detail above.

[0222] In one aspect, the present disclosure provides a multispecific antigen-binding protein, the antigen-binding protein comprising: a) a single Fab domain that specifically binds CD3 on T cells, the Fab domain comprising a heavy chain and a light chain and a CD3 antigen-binding domain, the CD3 antigen-binding domain comprising a VH containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 50, and a VL containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 55; b) a MAGE-A4 pMHC-binding domain operably linked to the C-terminus of the Fab heavy chain, the MAGE-A4 pMHC-binding domain comprising a VH containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10 and a VL containing an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 15; and c) a MAGE-A4 pMHC-binding domain operably linked to the C-terminus of the Fab light chain, the MAGE-A4 pMHC-binding domain comprising a VH containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10 and a VL containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the multispecific antigen-binding protein is, for example, Figure 4 the bispecific Fab(scFv)2 shown in

[0223] In one aspect, the present disclosure provides a multispecific antigen-binding protein, the antigen-binding protein comprising: a) a single Fab domain that specifically binds CD3 on a T cell, the Fab domain comprising a heavy chain and a light chain and a CD3 antigen-binding domain, the CD3 antigen-binding domain comprising a VH containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 50, and a VL containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 55; b) a MAGE-A4 pMHC-binding domain operably linked to the C-terminus of the Fab heavy chain, the MAGE-A4 pMHC-binding domain comprising a VH containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20 and a VL containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25; and c) a MAGE-A4 pMHC-binding domain operably linked to the C-terminus of the Fab light chain, the MAGE-A4 pMHC-binding domain comprising a VH containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20 and a VL containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25. In certain embodiments, the multispecific antigen-binding protein is a bispecific Fab(scFv)2 as shown, for example, in Figure 4 as shown in

[0224] In one aspect, the present disclosure provides a multispecific antigen-binding protein comprising: a) a single Fab domain that specifically binds CD3 on a T cell, the Fab domain comprising a heavy chain, a light chain, and a CD3 antigen-binding domain, the CD3 antigen-binding domain comprising a VH having an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 50, and a VL having an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55; b) a MAGE-A4 pMHC-binding domain operably linked to the C-terminus of the Fab heavy chain, the MAGE-A4 pMHC-binding domain comprising a VH having an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30 and a VL having an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 35; and c) a MAGE-A4 pMHC-binding domain operably linked to the C-terminus of the Fab light chain, the MAGE-A4 pMHC-binding domain comprising a VH having an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30 and a VL having an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 35. In certain embodiments, the multispecific antigen-binding protein is a bispecific Fab(scFv)2 as shown, for example, Figure 4 as in

[0225] In one aspect, the present disclosure provides a multispecific antigen-binding protein comprising: a) a single Fab domain that specifically binds CD3 on T cells, the Fab domain comprising a heavy chain, a light chain, and a CD3 antigen-binding domain, the CD3 antigen-binding domain comprising a VH containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 50, and a VL containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55; b) a MAGE-A4 pMHC-binding domain operably linked to the C-terminus of the Fab heavy chain, the MAGE-A4 pMHC-binding domain comprising a VH containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40 and a VL containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45; and c) a MAGE-A4 pMHC-binding domain operably linked to the C-terminus of the Fab light chain, the MAGE-A4 pMHC-binding domain comprising a VH containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40 and a VL containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45. In certain embodiments, the multispecific antigen-binding protein is a bispecific Fab(scFv)2 as shown, for example, Figure 4 as in

[0226] In certain embodiments, the multispecific antigen-binding protein is a Fab(scFv)2, the Fab(scFv)2 comprising (a) a single Fab domain targeting CD3, the Fab domain comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49 and the light chain comprises or consists of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54; and (b) two scFvs targeting SEQ ID NO: 3 presented by HLA, each scFv comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and / or SEQ ID NO: 64. In certain embodiments, one scFv is linked to the C-terminus of the Fab heavy chain and the second is linked to the C-terminus of the light chain, see Figure 4 exemplary embodiments.

[0227] In one aspect, the present disclosure provides a multispecific antigen-binding protein that binds to the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex and CD3, the multispecific antigen-binding protein comprising:

[0228] (i) a first polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9, and a second polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14;

[0229] (ii) a first polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 19, and a second polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 24;

[0230] (iii) a first polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 29, and a second polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 34; or

[0231] (iv) a first polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 39; and a second polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 44;

[0232] or a variant of said sequence, said variant being at least 90%, 95%, 96%, 97%, 98% or 99% identical thereto while retaining antigen specificity (i.e., retaining specificity for the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex and CD3). In certain embodiments, the pMHC binding domain, particularly when in the scFv mode, comprises a variable heavy chain having polar amino acids at positions 11, 89 and / or 108 according to Kabat numbering.

[0233] In certain embodiments, the Fab domain comprises a variable heavy chain having polar amino acids at positions 11, 89 and / or 108 according to Kabat numbering.

[0234] In certain embodiments, the variable heavy chain comprises: leucine (L) or serine (S) at amino acid position 11 according to Kabat numbering; valine (V), serine (S) or threonine (T) at amino acid position 89 according to Kabat numbering; and / or leucine (L), serine (S) or threonine (T) at amino acid position 108 according to Kabat numbering.

[0235] In certain embodiments, according to Kabat numbering, when leucine (L) is present at amino acid position 11, then serine (S) or threonine (T) is present at amino acid position 89, and serine (S) or threonine (T) is present at amino acid position 108.

[0236] In certain embodiments, according to Kabat numbering, when valine (V) is present at amino acid position 89, then serine (S) is present at amino acid position 11, and serine (S) or threonine (T) is present at amino acid position 108.

[0237] In certain embodiments, according to Kabat numbering, when leucine (L) is present at amino acid position 108, then serine (S) or threonine (T) is present at amino acid position 11, and serine (S) or threonine (T) is present at amino acid position 89.

[0238] In certain embodiments, the polar amino acids are serine (S) and / or threonine (T).

[0239] In certain embodiments, the variable heavy chain comprises serine (S) at amino acid position 11, serine (S) or threonine (T) at amino acid position 89, and serine (S) or threonine (T) at amino acid position 108 according to Kabat numbering.

[0240] In certain embodiments, the variable heavy chain comprises serine (S) at amino acid position 11 according to Kabat numbering, serine (S) at amino acid position 89, and serine (S) at amino acid position 108.

[0241] In certain embodiments, the Fab domain comprises a variable heavy chain lacking serine (S) at position 113 according to Kabat numbering.

[0242] In certain embodiments, the pMHC binding domain (such as the first and / or second pMHC binding domain) comprises a variable heavy chain lacking serine (S) at position 113 according to Kabat numbering.

[0243] In certain embodiments, the Fab domain comprises a variable heavy chain lacking serine (S) at position 112 and lacking serine (S) at position 113 according to Kabat numbering.

[0244] In certain embodiments, the pMHC binding domain (such as the first and / or second pMHC binding domain) comprises a variable heavy chain lacking serine (S) at position 112 and lacking serine (S) at position 113 according to Kabat numbering.

[0245] In certain embodiments, the antigen-binding protein comprises an S113A, S113G, or S113T substitution according to Kabat numbering.

[0246] In certain embodiments, the antigen-binding protein comprises an S113A, S113G, or S113T substitution according to Kabat numbering, and wherein S112 is absent.

[0247] In certain embodiments, the antigen-binding protein comprises an S112A, S112G, or S112T substitution according to Kabat numbering.

[0248] In certain embodiments, the antigen-binding protein comprises an S112A, S112G, or S112T substitution according to Kabat numbering, and wherein S113 is absent.

[0249] In certain embodiments, the immune cell to which the multispecific antigen-binding molecule binds via the immunocyte linker binding arm is selected from the group consisting of: T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, monocytes, and macrophages. In certain embodiments, the immune cell is a T cell.

[0250] In certain embodiments, as determined by SPR, the Fab domain specifically binds to CD3 with a binding affinity between about 1 nM and about 100 nM (such as 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 31 nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41 nM, 42 nM, 43 nM, 44 nM, 45 nM, 46 nM, 47 nM, 48 nM, 49 nM, 50 nM, 51 nM, 52 nM, 53 nM, 54 nM, 55 nM, 56 nM, 57 nM, 58 nM, 59 nM, 60 nM, 61 nM, 62 nM, 63 nM, 64 nM, 65 nM, 66 nM, 67 nM, 68 nM, 69 nM, 70 nM, 71 nM, 72 nM, 73 nM, 74 nM, 75 nM, 76 nM, 77 nM, 78 nM, 79 nM, 80 nM, 81 nM, 82 nM, 83 nM, 84 nM, 85 nM, 86 nM, 87 nM, 88 nM, 89 nM, 90 nM, 91 nM, 92 nM, 93 nM, 94 nM, 95 nM, 96 nM, 97 nM, 98 nM, 99 nM or 100 nM). In certain embodiments, as determined by SPR, the Fab domain specifically binds to CD3 with a binding affinity between about 1 nM and about 50 nM. In certain embodiments, as determined by SPR, the Fab domain specifically binds to CD3 with a binding affinity between about 20 nM and about 50 nM.

[0251] In certain embodiments, as determined by SPR, the Fab domain specifically binds to CD3 with a binding affinity of about 1 nM, about 10 nM or about 50 nM.

[0252] In some embodiments, the association rate constant ka of the anti-CD3 binding domain is between about 1×10 5 and about 1×10 7 M -1 s -1 , such as at least 1×10 6 M -1 s -1 or at least 2×10 6 M -1 s -1 . In some embodiments, the dissociation rate constant k dbetween about 1×10 -1 and about 1×10 -6 s -1 such as at least 2×10 -3 s -1 or at least 3×10 -3 s -1 or at least 4×10 -3 s -1 . Without being bound by theory, a fast dissociation rate (e.g., a k -3 s -1 value of 2 - 3×10 d s

[0253] In one embodiment, for both the CD3 - heterodimers CD3εγ (ε / γ) and CD3εδ (ε / δ), the association rate constant k a and / or the dissociation rate constant k d are equivalent or similar, i.e., when measured under the same conditions, particularly when determined by SPR at 25°C, the k a or k d or both of the anti - CD3 binding domains to CD3εγ (ε / γ) and CD3εδ (ε / δ) have no significant difference. In some of its embodiments, the association rate constant k a and / or the dissociation rate constant k d values are within 1 - fold, 1.5 - fold, 2 - fold, 2.5 - fold, or 3 - fold of each other, i.e., the association rate constant k a value is 1×10 5 M -1 s -1 and 3×10 5 M -1 s -1 .

[0254] In certain embodiments, the pMHC binding domain binds to the target MAGE-A4-pMHC complex with a binding affinity of from about 100 pM to about 5 nM (such as about 100 pM, about 150 pM, about 200 pM, about 250 pM, about 300 pM, about 350 pM, about 400 pM, about 450 pM, about 500 pM, about 550 pM, about 600 pM, about 650 pM, about 700 pM, about 750 pM, about 800 pM, about 850 pM, about 900 pM, about 950 pM, about 1 nM (1,000 pM), about 2 nM, about 3 nM, about 4 nM or about 5 nM). In certain embodiments, the pMHC binding domain binds to the target pMHC complex with a binding affinity of from about 100 pM to about 1 nM. In certain embodiments, the pMHC binding domain binds to the target pMHC complex with a binding affinity of from about 100 pM to about 400 pM.

[0255] In certain embodiments, the pMHC binding domains disclosed herein bind to MAGE-A8 and MAGE-B4 peptides presented by pMHC with a binding affinity similar to that of the MAGE-A4 peptide presented by pMHC. In certain embodiments, the pMHC binding domain binds to the MAGE-A8 peptide presented by pMHC and / or the MAGE-B4 peptide presented by pMHC with a binding affinity of from about 1.5 nM to about 2.5 nM (such as 1.6 nM, 1.7 nM, 1.8 nM, 1.9 nM, 2 nM, 2.1 nM, 2.2 nM, 2.3 nM, 2.4 nM or 2.5 nM). In certain embodiments, as measured by SPR, the pMHC binding domain binds to the MAGE-A8 peptide presented by pMHC with a binding affinity of about 2.2 nM, and / or binds to the MAGE-B4 peptide presented by pMHC with a binding affinity of about 1.9 nM. In certain embodiments, the pMHC binding domain binds to MAGE-A4, MAGE-A8, and MAGE-B4 peptides presented by pMHC with a similar binding affinity in the range of about 1.9 nM to about 2.2 nM. In certain embodiments, the MAGE-A8 peptide is GLYDGREHSV (SEQ ID NO: 71). In certain embodiments, the MAGE-B4 peptide is GIYDGKRHLI (SEQ ID NO: 72).

[0256] In some embodiments, the pMHC binding domain comprises between about 1×10 5 to about 1×10 7 M -1 s -1 preferably about 0.5×10 6 M -1 s -1 to about 3×10 6 M-1 s -1 between, such as at least 0.5×10 6 M -1 s -1 , at least 1×M -1 s -1 , at least 2×10 6 M -1 s -1 or at least 3×10 6 M -1 s -1 ) for the association rate constant ka of MAGE-A4 pMHC. In some embodiments, the pMHC binding domain comprises between about 1×10 -1 to about 1×10 -6 s -1 between, such as about 1×10 -2 to about 1×10 -5 s -1 between, such as at least 2×10 -3 s -1 , at least 4×10 -3 s -1 , at least 6×10 -3 s -1 , at least 8×10 -3 s -1 , at least 2×10 -4 s -1 , at least 4×10 -4 s -1 , at least 6×10 -4 s -1 or at least 8×10 -4 s -1 ) for the dissociation rate constant k d .

[0257] In certain embodiments, the antigen-binding protein has a molecular weight of from about 75 kDa to about 110 kDa (such as about 75 kDa, about 80 kDa, about 85 kDa, about 90 kDa, about 95 kDa, about 100 kDa, about 105 kDa or about 110 kDa). In certain embodiments, the antigen-binding protein has an extended serum half-life relative to an antigen-binding protein having a molecular weight of < about 75 kDa.

[0258] The advantages of the antigen-binding protein scaffolds of the present disclosure are an intermediate molecular size of about 75 - 110 kDa. Blinatumomab, a bispecific T cell engager (BiTE), has shown excellent results in patients with relapsed or refractory acute lymphoblastic leukemia. Due to its small size (54 kDa), Blinatumomab is characterized by a short serum half-life of a few hours and thus requires continuous infusion (see U.S. 7,112,324 B1). It is expected that the antigen-binding proteins of the present disclosure will have a significantly longer half-life compared to smaller bispecific antibodies such as BiTEs like Blinatumomab and thus will not require continuous infusion due to their favorable half-life. Medium-sized molecules can avoid renal clearance and provide a half-life sufficient to improve tumor accumulation. Although the antigen-binding proteins of the present disclosure have an increased plasma half-life compared to other small bispecific formats, they still retain tumor penetration ability.

[0259] The Fab domains of the antigen-binding proteins of the present disclosure can act as specific heterodimerization scaffolds linked to other pMHC-binding domains. The native and efficient heterodimerization properties of the heavy (Fd fragment) and light (L) chains of the Fab fragment make the Fab fragment an available scaffold. The other binding domains can be in several different formats, including but not limited to another Fab domain, scFv, or sdAb.

[0260] Each chain of the Fab fragment can be extended at the N-terminus or C-terminus with other binding domains. These chains can be co-expressed in mammalian cells where the host cell-binding immunoglobulin protein (BiP) chaperone drives the formation of heavy chain-light chain heterodimers (Fd:L). These heterodimers are stable and each binder retains its specific affinity. Then the two remaining pMHC-binding domains can be fused as scFvs or sdAbs to different Fab chains, where each chain can be extended, for example, at the C-terminus with another scFv or sdAb domain (see, for example, Schoonjans et al. J. Immunology, 165(12):7050 - 7057, 2000; Schoonjans et al. Biomolecular Engineering, 17:193 - 202, 2001). Another advantage of using Fab as a heterodimerization unit is that Fab molecules are abundant in serum and thus can be non-immunogenic when administered to a subject.

[0261] In certain embodiments, the (multispecific) antigen-binding proteins of the present disclosure have one or more of the following properties: (i) it is capable of inhibiting tumor growth and eradicating tumors, for example, in a cell line-derived murine NSCLC xenograft model such as that exemplarily shown in Example 14; and / or (ii) it shows efficacy against target-positive tumor cells as determined by LDH cytotoxicity; and / or (iii) it shows efficacy against target-positive tumor cells as determined by, for example, the IncuCyte S3 system; and / or (iv) it induces lower levels of the pro-inflammatory cytokine IFN γ in antigen-positive and antigen-negative cell lines compared to Comparator 1 (exemplary assays are described in, for example, Example 12); and / or (v) it induces lower levels of the pro-inflammatory cytokine IL-2, IL-6, and / or TNFα cytokine release compared to Comparator 1 (exemplary assays are described in, for example, Example 12); (vi) it has T cell activation properties, exemplary assays are described in, for example, Example 12; and / or (vii) safety as measured by, for example, granzyme B release (e.g., in antigen-negative cancer cell lines KLE (endometrial cancer), LNCaP (prostate lymph node cancer), KMRC-2 (clear cell renal cell carcinoma), KMRC-3 (clear cell renal cell carcinoma), 639-V (urothelial bladder cancer), EKVX (lung adenocarcinoma), and / or HCT 116 (colorectal cancer), see, for example, Example 12) and / or reactivity in healthy tissues as determined by, for example, T cell activation assays, exemplary assays are described in Examples 12 and 13; and / or (viii) it remains at least 94% monomeric as determined by SEC-HPLC during storage in PBS at 4°C at 1 mg / ml and / or 10 mg / ml for at least two weeks.

[0262] Also encompasses variants of the sequences disclosed herein. Variant amino acid or nucleic acid sequences differ from their parental sequences by the insertion (including addition), deletion, and / or substitution of one or more amino acid residues or nucleobases, respectively, while retaining at least one desired property of the parental sequences disclosed herein, such as specific antigen binding, efficacy against target positive tumor cells, stability (e.g., serum stability, thermal stability, and / or storage stability), producibility (e.g., expression level), safety (e.g., reactivity in healthy tissues, granzyme B release, pro-inflammatory cytokine IFNγ antigen positive or antigen negative cell lines, and / or low-level induction or even non-induction of pro-inflammatory cytokine IL-2, IL-6, and TNFα cytokine release), efficacy (e.g., tumor growth inhibition, tumor eradication, and / or T cell activation properties as determined, for example, in vitro), or binding to the MAGE-A4 GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex, MAGE-A8-derived GLYDGREHSV (SEQ ID NO: 71) HLA-A*02 complex, and MAGE-B4-derived GIYDGKRHLI (SEQ ID NO: 72) HLA-A*02 complex) within a similar affinity range. In certain embodiments, the variant antigen-binding protein maintains binding to the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02:01 complex at least 50%, such as 60%, 70%, 80%, 90%, or 95% of the equilibrium dissociation constant K D of a reference antigen-binding protein (i.e., the corresponding antigen-binding protein without said substitution, insertion, and / or deletion). Variants can be engineered or naturally occurring, such as allelic or splice variants. In some embodiments, the variant antigen-binding protein comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences disclosed herein.

[0263] Thus, in certain embodiments, the variant antigen-binding protein maintains specific binding to a target (i.e., to MAGE-A4 pMHC, particularly the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02:01 complex, or CD3, respectively) and / or competes with the antigen-binding proteins disclosed herein for binding to its target. In certain embodiments, as determined by SEC-HPLC, after incubation in PBS at 4°C for 14 days at 1 mg / ml and / or 10 mg / ml, the antigen-binding protein maintains stability and remains at least 94%, 95%, 96%, 97%, 98%, 99%, or 100% monomeric.

[0264] Reduction of anti-drug antibody binding

[0265] Anti-drug antibodies (ADAs) can affect the risk profile and efficacy of biopharmaceuticals. If neutralizing, they can block the ability of the drug to bind to its target. Therefore, testing for anti-drug antibody binding and its neutralizing potential for biopharmaceuticals is a regulatory requirement. Anti-drug antibody assays are described in detail, for example, in WO2007101661A1 (Hoffmann La Roche), WO2018178307A1 (Ablynx), WO2021046316A2 (Adverum Biotechnologies, Charles River), and US20180088140A1 (Genzyme Corporation), each of which is incorporated herein by reference.

[0266] Binding of an anti-drug antibody to the tumor-targeting domain of an antigen-binding protein can cause aggregation of the antigen-binding protein when each variable domain of the ADA binds to one tumor-targeting domain of two antigen-binding proteins. Two or more CD3-binding domains on the antigen-binding protein aggregate in the absence of target engagement and overstimulate the targeted T cells, resulting in off-target toxicity. Nonspecific stimulation of T cells can cause systemic cytokine release.

[0267] Generally speaking, there is a need in the art to develop safer and more effective bispecific antibodies for cancer immunotherapy.

[0268] The inventors have found that certain mutations in the tumor antigen-binding domain of a T cell engager reduce the ADA response and, at the same time, reduce nonspecific T cell stimulation in the absence of target engagement. Thus, a highly effective and safe method for cancer immunotherapy is provided.

[0269] For this purpose, the variable heavy chain amino acids at positions 11, 89, and / or 108 according to Kabat numbering are replaced with polar amino acids; and / or the serine (S) at position 113 according to Kabat numbering is deleted. Such substitutions are particularly advantageous when the binding domain is in the scFv mode. In the case of Fab(scFv)2, one or both scFvs can contain such substitutions or deletions.

[0270] In certain embodiments, the polar amino acids are serine (S) and / or threonine (T).

[0271] In certain embodiments, according to Kabat numbering, the heavy chain amino acid at heavy chain amino acid position 11 is replaced with serine (S), the heavy chain amino acid at heavy chain amino acid position 89 is replaced with serine (S) or threonine (T), and / or the heavy chain amino acid at heavy chain amino acid position 108 is replaced with serine (S) or threonine (T).

[0272] In certain embodiments, according to Kabat numbering, the heavy chain amino acids are substituted with serine (S) at heavy chain amino acid position 11, serine (S) at heavy chain amino acid position 89, and serine (S) at heavy chain amino acid position 108.

[0273] In certain embodiments, according to Kabat numbering, in addition to the deletion of serine at position 113, serine (S) at position 112 is also deleted.

[0274] In certain embodiments, the method further comprises adding alanine (A), glycine (G), or threonine (T), particularly alanine (A), at Kabat amino acid position 112 or 113.

[0275] In certain embodiments, the method further comprises adding alanine (A) at Kabat amino acid position 112 or 113.

[0276] Expression of antigen-binding protein

[0277] In one aspect, polynucleotides or nucleic acids encoding the antigen-binding proteins (including multispecific antigen-binding proteins) disclosed herein are provided. Such polynucleotides or nucleic acids are generally isolated and synthetic. Methods for preparing antigen-binding proteins are also provided, the methods comprising expressing these polynucleotides.

[0278] Generally, the polynucleotides encoding the antigen-binding proteins disclosed herein are inserted into a cloning vector or an expression vector for introduction into a host cell that can be used to produce a desired amount of the antigen-binding protein. Thus, in certain aspects, the present invention provides expression vectors comprising the polynucleotides disclosed herein and host cells comprising these vectors and polynucleotides.

[0279] The term "vector" or "expression vector" is used herein to refer to a vehicle used according to the present invention for introducing a desired gene into a cell and expressing it in the cell. As known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, vectors compatible with the present invention will contain selectable markers, appropriate restriction sites for facilitating the cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0280] Many expression vector systems can be used for the purposes of the present invention. For example, one class of vectors uses DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retroviruses (e.g., RSV, MMTV, MoMLV, etc.) or SV40 virus. Others involve the use of polycistronic systems with internal ribosome entry sites. Additionally, cells that have had DNA integrated into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. The markers can provide prototrophy to auxotrophic hosts, provide antimicrobial resistance (e.g., antibiotics), or provide resistance to heavy metals such as copper. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cells by co-transformation. Optimal synthesis of mRNA may also require other elements. These elements can include signal sequences, splice signals, and transcriptional promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (e.g., human constant region genes) synthesized as described above.

[0281] In other embodiments, polycistronic constructs can be used to express antigen-binding proteins. In such expression systems, polygenic products of interest such as the heavy and light chains of an antibody can be produced from a single polycistronic construct. These systems advantageously use internal ribosome entry sites (IRESs) to provide relatively high levels of polypeptides in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is incorporated herein by reference in its entirety for all purposes. Those skilled in the art will appreciate that such expression systems can be used to effectively produce the full range of polypeptides disclosed in this application.

[0282] More generally, once a vector or DNA sequence encoding an antigen-binding protein has been prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. Introduction of the plasmid into the host cell can be achieved by a variety of techniques well known to those skilled in the art. These methods include, but are not limited to, transfection (including electroporation and electrotransfer), protoplast fusion, calcium phosphate precipitation, cell fusion with encapsulated DNA, microinjection, and infection with intact virus. See Ridgway, A.A.G. "Mammalian Expression Vectors" Chapter 24.2, pages 470-472 in Vectors, edited by Rodriguez and Denhardt (Butterworths, Boston, Mass. 1988). The plasmid can be introduced into the host by electroporation. The transformed cells are grown under conditions suitable for the production of the light and heavy chains, and the synthesis of the heavy and / or light chain proteins is determined. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorter analysis (FACS), immunohistochemistry, and the like.

[0283] As used herein, the term "transformation" is used in a broad sense to refer to the introduction of DNA into a recipient host cell, thereby altering the genotype and thus causing the recipient cell to change.

[0284] Along the same lines, a "host cell" refers to a cell that has been transformed with a vector that has been constructed using recombinant DNA techniques and encodes at least one heterologous gene. Unless otherwise expressly stated, in the description of methods for isolating polypeptides from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to denote the source of the antibody. In other words, recovering a polypeptide from a "cell" can mean recovering from centrifuged whole cells or from a cell culture containing both the culture medium and the suspended cells.

[0285] In one embodiment, the host cell line for antibody expression is of mammalian origin. Those skilled in the art can determine the particular host cell line most suitable for expressing the desired gene product. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary line, DHFR-), HELA (human cervical carcinoma), CV-1 (monkey kidney line), COS (a derivative of CV-1 with SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte), 293 (human kidney), and the like. In one embodiment, the cell line provides altered glycosylation of the antibody expressed by it, such as non-fucosylated (e.g., PER. (Crucell) or FUT8-knockout CHO cell line ( cells) (Biowa, Princeton, N.J.)). Host cell lines are generally available from commercial services (such as the American Tissue Culture Collection) or from published literature.

[0286] In vitro preparation allows for amplification to obtain large amounts of the desired polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include homogeneous suspension culture, such as in an airlift reactor or in a continuously stirred reactor; or immobilized or entrapped cell culture, such as in hollow fibers, microcapsules, on agarose microbeads or in ceramic cartridges. If desired and / or necessary, the polypeptide solution can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, DEAE-cellulose chromatography and / or (immuno)affinity chromatography.

[0287] The genes encoding the antigen-binding proteins provided by the present invention can also be expressed in non-mammalian cells, such as bacteria, yeast or plant cells. In this regard, it should be understood that various single-celled non-mammalian microorganisms, such as bacteria (i.e., those capable of growing in culture or fermentations), can also be transformed. Bacteria sensitive to transformation include members of the Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; the Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. It should also be understood that when expressed in bacteria, the protein can become part of inclusion bodies. The protein must be isolated, purified and then assembled into a functional molecule.

[0288] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms, although many other strains are generally available. For expression in the genus Saccharomyces, plasmid Yrp7 is commonly used (e.g., Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)). This plasmid already contains the TRP1 gene, which provides a selectable marker for yeast mutants lacking the ability to grow in tryptophan (e.g., ATCC No. 44076 or PEP4-1) (Jones, Genetics, 85:12 (1977)). The presence of a trp1 lesion, which is characteristic of the yeast host cell genome, provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0289] Engineering and optimization of antigen-binding protein

[0290] The antigen-binding proteins of the present disclosure, including multispecific antigen-binding proteins, can be engineered or optimized. As used herein, "optimized" or "optimizing" refers to altering an antigen-binding protein to improve one or more functional properties. Alterations include, but are not limited to, deletions, substitutions, additions, and / or modifications of one or more amino acids within the antigen-binding protein.

[0291] As used herein, the term "functional property" is a property of an antigen-binding protein, the improvement of which (e.g., relative to a conventional antigen-binding protein, such as an antibody) is desirable and / or advantageous to one of ordinary skill in the art, e.g., to improve the manufacturing properties or therapeutic efficacy of the antigen-binding protein. In one embodiment, the functional property is stability (e.g., thermal stability). In another embodiment, the functional property is solubility (e.g., under cellular conditions). In another embodiment, the functional property is aggregation behavior. In another embodiment, the functional property is protein expression (e.g., in prokaryotic cells). In another embodiment, the functional property is the refolding behavior after inclusion body solubilization during the manufacturing process. In certain embodiments, the functional property is not an improvement in antigen-binding affinity. In another embodiment, the improvement of one or more functional properties has no substantial effect on the binding affinity of the antigen-binding protein.

[0292] In certain embodiments, the antigen-binding proteins of the present disclosure comprise scFvs and are optimized by identifying preferred amino acid residues that are substituted, deleted, and / or added at amino acid positions of interest (e.g., identified by comparing a database of scFv sequences having at least one desired property, such as selected by a quality control (QC) assay, with a database of mature antibody sequences, such as the Kabat database). Accordingly, the present disclosure also provides an “enrichment / exclusion” method for selecting specific amino acid residues. Additionally, the present disclosure provides methods for engineering antigen-binding proteins (e.g., scFvs) by mutating specific framework amino acid positions identified using the “functional consensus” method described herein. In certain embodiments, framework amino acid positions are mutated by substituting existing amino acid residues with residues that are found to be “enriched” residues using the “enrichment / exclusion” analysis method described herein. In one aspect, the present disclosure provides a method of identifying amino acid positions for mutation in a single-chain antibody (scFv) having VH and VL amino acid sequences, the method comprising: a) inputting the scFv VH, VL, or VH and VL amino acid sequences into a database comprising a plurality of antibody VH, VL, or VH and VL amino acid sequences such that the scFv VH, VL, or VH and VL amino acid sequences are aligned with the antibody VH, VL, or VH and VL amino acid sequences of the database; b) comparing amino acid positions within the scFv VH or VL amino acid sequences with corresponding positions within the antibody VH or VL amino acid sequences of the database; c) determining whether the amino acid positions within the scFv VH or VL amino acid sequences are occupied by amino acid residues that are conserved at the corresponding positions within the antibody VH or VL amino acid sequences of the database; and d) when an amino acid position is occupied by a non-conserved amino acid residue at the corresponding position within the antibody VH or VL amino acid sequences of the database, identifying the amino acid position within the scFv VH or VL amino acid sequence as an amino acid position for mutation. ScFv optimization is described in further detail in WO2008110348, WO2009000099, WO2009000098, and WO2009155725, which patents are incorporated herein by reference in their entirety.

[0293] In certain embodiments, the antigen-binding protein comprises an Fc domain that has been modified to not induce a cytotoxic immune response and / or not activate complement. For example, one or more substitutions can be introduced into the Fc domain such that its ADCC / ADCP or CDC effector functions are inactivated. Such antigen-binding proteins have the advantage of an increased half-life compared to antibody fragments having a molecular weight of less than 75 kDa and do not mediate a cytotoxic immune response.

[0294] Chemical and / or biological modification

[0295] In one aspect, the antigen-binding protein (such as the multispecific antigen-binding protein described above) is chemically and / or biologically modified. For example, the antigen-binding protein can be glycosylated, phosphorylated, hydroxylated, PEGylated, HESylated, PASylated, XTENylated, sulfated, labeled with dyes and / or radioisotopes, conjugated with enzymes and / or toxins, and / or using albumin fusion technology. Similarly, any nucleic acid sequence, plasmid or vector, and / or host cell described herein can be modified accordingly.

[0296] Such modifications may be made, for example, to optimize pharmacokinetics, its water solubility, or to reduce its side effects. For example, PEGylation, PASylation, XTENylation, HESylation, and / or fusion to serum albumin can be applied to slow renal clearance, thereby increasing the plasma half-life of the antigen-binding protein. In one embodiment, the modification adds different functionality to the antigen-binding protein, such as adding a detection label for diagnosis or adding a toxin to more effectively combat cancer cells.

[0297] Alternatively or additionally, in some embodiments, the antigen-binding proteins and other polypeptides provided herein undergo co-translational and post-translational modifications known in the art. Examples of post-translational modifications include, but are not limited to, disulfide bond formation, glycosylation, cyclization (such as N-terminal pyroglutamate formation), removal or "clipping" of N-terminal or C-terminal residues (e.g., C-terminal lysine residues are typically removed during the manufacturing process), deamidation, isomerization, oxidation, glycation, acylation, trehalosylation, peptide bond cleavage, non-reducible crosslinking, truncation, and / or incomplete processing of some or all of the signal sequence.

[0298] In certain embodiments, the CD3 antigen-binding domain comprises an N-terminal truncation of 1 or more amino acids (e.g., an N-terminal truncation of 1, 2, 3, 4, or 5 amino acids). In certain embodiments, the CD3 antigen-binding domain comprises a C-terminal truncation of 1 or more amino acids (e.g., a C-terminal truncation of 1, 2, 3, 4, or 5 amino acids). In certain embodiments, the N-terminal and / or C-terminal truncation is a truncation of the CD3-targeting Fab fragment of the heavy-chain amino acid sequence of SEQ ID NO: 49 and the light-chain amino acid sequence of SEQ ID NO: 54. In certain embodiments, the light-chain amino acid sequence of SEQ ID NO: 54 comprises an N-terminal truncation of 1, 2, 3, 4, or 5 amino acids, such as 1 or 2 truncations. In certain embodiments, the heavy-chain amino acid sequence of SEQ ID NO: 49 comprises an N-terminal truncation of 1, 2, 3, 4, or 5 amino acids, such as 1 or 2 truncations. In certain embodiments, the light-chain amino acid sequence of SEQ ID NO: 54 comprises a C-terminal truncation of 1, 2, 3, 4, or 5 amino acids. In certain embodiments, the heavy-chain amino acid sequence of SEQ ID NO: 49 comprises a C-terminal truncation of 1, 2, 3, 4, or 5 amino acids.

[0299] In certain embodiments, the (multispecific) antigen-binding protein comprises pyroglutamic acid (pE, pyrGlu, pyre, or pGlu) instead of an N-terminal glutamine or N-terminal glutamine. In certain embodiments, the light chain of the antigen-binding protein comprises pyroglutamic acid (pE) instead of an N-terminal glutamine. In certain embodiments, the variant sequences of SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 61, or SEQ ID NO: 62 comprise pyroglutamic acid at amino acid position 1. In certain embodiments, the light chain of the antigen-binding protein comprises pyroglutamic acid (pE) instead of an N-terminal glutamic acid. In certain embodiments, the variant sequences of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 65 comprise pyroglutamic acid at amino acid position 1. In certain embodiments, such pyroglutamic acid (pE) modification has no effect on the safety and / or efficacy of the (multispecific) antigen-binding protein.

[0300] In one embodiment, the antigen-binding protein is glycosylated. Glycosylation refers to the process of attaching carbohydrates to a protein. In biological systems, this process occurs enzymatically within the cell in the form of co-translational and / or post-translational modification. Proteins can also be chemically glycosylated. Carbohydrates can be N-linked to the nitrogen of asparagine or arginine side chains; O-linked to the hydroxyl oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side chains; using xylose, trehalose, mannose, and N-acetylglucosamine attached to phosphoserine; and / or adding mannose to tryptophan residues found in specific recognition sequences. The glycosylation pattern can be controlled, for example, by selecting an appropriate cell line, culture medium, protein engineering manufacturing mode, and process strategy (see HOSSLER, P. Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 2009, Vol. 19, No. 9, pp. 936-949). In some embodiments, the glycosylation pattern of the antigen-binding proteins described herein is modified to enhance ADCC and CDC effector functions.

[0301] The antigen-binding protein can be engineered to control or alter the glycosylation pattern, for example, by deleting and / or adding one or more glycosylation sites. The formation of glycosylation sites can be achieved, for example, by introducing the corresponding enzyme recognition sequence into the amino acid sequence of the antigen-binding protein.

[0302] In some embodiments, the antigen-binding protein is PEGylated. PEGylation can alter the pharmacodynamic and pharmacokinetic properties of the protein. Additionally, PEGylation can reduce immunogenicity by shielding the PEGylated antigen-binding protein from the immune system and / or alter its pharmacokinetics, for example, by increasing the in vivo stability of the antigen-binding protein, preventing its proteolytic degradation, prolonging its half-life, and altering its biodistribution. Generally, polyethylene glycol (PEG) of an appropriate molecular weight is covalently linked to the protein. Similar effects can be achieved using PEG mimetics (e.g., HESylation, XTENylation, or PASylation of the antigen-binding protein). HESylation uses hydroxyethyl starch (“HES”) derivatives. In the process of PASylation, the antigen-binding protein is linked to a conformationally disordered polypeptide sequence composed of the amino acids proline (P), alanine (A), and serine (S).

[0303] In certain embodiments, an antigen-binding protein (e.g., a multispecific antigen-binding protein) is linked to, or combined with, a detectable label, a therapeutic agent, or a PK-modifying moiety. For example, the antigen-binding protein can be labeled or conjugated with a second moiety that confers one or more ancillary functions to the antigen-binding protein. For example, the second moiety can have another immune effector function, be effective in drug targeting, or be useful for detection. The second moiety can be chemically linked or genetically fused to the antigen-binding protein, for example, using methods known in the art. As used herein, the term "label" refers to any substance or ion that indicates the presence of the antigen-binding protein when directly or indirectly detected or measured by physical or chemical means. For example, a label can be directly detected by, but is not limited to, light absorption, fluorescence, reflectance, light scattering, phosphorescence, or luminescence properties, a molecule or ion detectable by radioactive properties, or a molecule or ion detectable by nuclear magnetic resonance or paramagnetic properties. Examples of indirect detection include light absorption or fluorescence; for example, various enzymes that cause an appropriate substrate to be converted, e.g., from a non-light-absorbing molecule to a light-absorbing molecule or from a non-fluorescent molecule to a fluorescent molecule. Labeled antigen-binding proteins can be particularly useful for in vitro and in vivo detection or diagnostic purposes. For example, an antigen-binding protein labeled with a suitable radioisotope, enzyme, fluorophore, or chromophore can be detected by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), or flow cytometry-based single cell analysis (e.g., FACS analysis). Similarly, the nucleic acids and / or vectors disclosed herein can be labeled for detection or diagnostic purposes, e.g., using labeled fragments thereof as probes in hybridization assays.

[0304] Non-limiting examples of the second moiety include radioisotopes (35S, 32P, 14C, 18F, and / or 125I), apozymes, enzymes (e.g., alkaline phosphatase, horseradish peroxidase, β-galactosidase, and / or angiogenin), cofactors, peptide moieties (e.g., HIS tag), proteins (e.g., lectin, serum albumin), carbohydrates (e.g., mannose-6-phosphate tag), fluorophores (e.g., fluorescein isothiocyanate (FITC)), phycoerythrin, green / blue / red or other fluorescent proteins, allophycocyanin (APC), chromophores, vitamins (e.g., biotin), chelators, antimetabolites (e.g., methotrexate), toxins (e.g., cytotoxic drugs or radiotoxins).

[0305] In one aspect, the present invention relates to a drug conjugate (particularly an antibody-drug conjugate ADC) comprising an antigen-binding protein as described herein (such as a monovalent or multispecific antigen-binding protein as described herein) conjugated to a toxin, which further enhances the effective killing of specific cells such as MAGE-A4 positive cells. The toxin moiety is generally a small molecular weight moiety such as an anthracycline toxin, paclitaxel, gramicidin D, and / or colchicine, and the small molecular weight moiety can be linked to the antigen-binding protein via a peptide linker.

[0306] The toxin can be conjugated to the antigen-binding protein either non-site-specifically or site-specifically. Non-site-specific conjugation generally involves the use of a chemical linker, such as a maleimide functional group, which mediates conjugation to the lysine or cysteine amino acid side chains of the antigen-binding protein or to the amino group at the N-terminus. Site-specific conjugation can be achieved using chemical, chemo-enzymatic, or enzymatic conjugation known in the art, such as using a bifunctional linker, bacterial transglutaminase or sortase, a linker that allows for a Pictet-Spengler chemistry on a formyl-glycine forming enzyme-modified antigen-binding protein, or a glycan-reconstructed antigen-binding protein.

[0307] Methods of administering antigen-binding protein

[0308] Methods for preparing and administering to a subject the antigen-binding proteins of the present disclosure (such as the multispecific antigen-binding proteins described above), as well as the nucleic acids, vectors, host cells, or compositions described herein, are well-known or can be readily determined by those skilled in the art. The route of administration of the antigen-binding proteins of the present disclosure can be, for example, oral, parenteral, by inhalation, or via the surface. As used herein, the term parenteral includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. As used herein, the term intraocular includes, but is not limited to, subconjunctival, intravitreal, retrobulbar, or intracameral administration. As used herein, the term via the surface includes, but is not limited to, administration with liquid or solution eye drops, emulsions (such as oil-in-water emulsions), suspensions, and ointments.

[0309] Although all of these forms of administration are clearly contemplated within the scope of the present disclosure, the form of administration will be an injectable solution. Generally, a pharmaceutical composition suitable for injection may contain a buffer (such as acetate, phosphate, or citrate buffer), a surfactant (such as polysorbate), an optional stabilizer (such as human albumin), etc. However, in other methods compatible with the teachings herein, the modified antibody can be directly delivered to the site of the harmful cell population to increase the exposure of the diseased tissue to the therapeutic agent.

[0310] The effective dose of the compositions of the present disclosure for treating related disorders varies according to many different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Generally, the patient is human, but non-human mammals, including transgenic mammals, can also be treated. Conventional methods known to those skilled in the art can be used to titrate the therapeutic dose to optimize safety and efficacy.

[0311] As previously discussed, the antigen-binding proteins (e.g., multispecific antigen-binding proteins), conjugates, or recombinants of the present disclosure can be administered in a pharmaceutically effective amount for in vivo treatment of mammalian disorders. In this regard, it should be understood that the disclosed antigen-binding proteins will be formulated to facilitate administration and promote the stability of the active agent.

[0312] The pharmaceutical compositions according to the present disclosure generally include a pharmaceutically acceptable non-toxic sterile carrier, such as physiological saline, non-toxic buffers, preservatives, etc. For the purposes of this application, a pharmaceutically effective amount of an antigen-binding protein (such as a multispecific antigen-binding protein) should be considered to be an amount sufficient to achieve effective binding to an antigen and to achieve, for example, the benefit of ameliorating the symptoms of a disease or disorder or detecting a substance or cell. In the case of tumor cells, the antigen-binding protein is generally capable of interacting with a selected immunoreactive antigen on neoplastic cells or immunoreactive cells and increasing the death of these cells. Of course, the pharmaceutical compositions of the present disclosure can be administered in a single or multiple doses to provide a pharmaceutically effective amount of the modified binding polypeptide.

[0313] Consistent with the scope of the present disclosure, the antigen-binding proteins (such as the multispecific antigen-binding proteins described above) of the present disclosure can be administered to humans or other animals in an amount sufficient to produce a therapeutic or prophylactic effect according to the treatment methods described above. The antigen-binding proteins of the present disclosure can be administered to such humans or other animals in conventional dosage forms, which are prepared by combining the antigen-binding proteins of the present disclosure with conventional pharmaceutically acceptable carriers or diluents according to known techniques. Those skilled in the art will recognize that the form and characteristics of a pharmaceutically acceptable carrier or diluent are dictated by the amount of the active ingredient with which it is combined, the route of administration, and other well-known variables. Those skilled in the art will further understand that mixtures containing one or more species of the antigen-binding proteins described in the present disclosure may prove to be particularly effective. Similarly, the nucleic acids described herein, the vectors described herein, the host cells described herein (especially immune cells bearing a CAR), or the compositions described herein can be administered to humans or other animals in an amount sufficient to produce a therapeutic or prophylactic effect according to the treatment methods described above.

[0314] As used herein, "efficacy" or "in vivo efficacy" refers to the response to treatment with a pharmaceutical composition of the present disclosure, using, for example, standardized response criteria. The success or in vivo efficacy of a therapy using a pharmaceutical composition of the present disclosure means that the composition is effective for its intended purpose, i.e., the composition is capable of eliciting its desired effect. In vivo efficacy can be monitored by established disease-specific standard methods. Additionally, various disease-specific clinical chemistry parameters and other established standard methods can be used.

[0315] In some embodiments, the compounds and cells described herein are administered in combination with one or more different pharmaceutical compounds. Generally, the therapeutic use of the compounds and cells described herein can be in combination with one or more therapies selected from the group consisting of: antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy, radiotherapy, or vaccine therapy.

[0316] Chimeric antigen receptor

[0317] In one aspect, the present disclosure provides chimeric antigen receptors (CARs) and immune cells engineered to express such CARs, the CARs comprising the antigen-binding protein sequences described herein. As used herein, the term "chimeric antigen receptor" or "CAR" refers to a receptor capable of activating an immune cell in response to antigen binding. A CAR is a recombinant transmembrane molecule and is advantageously expressed on an immune cell. Its structure generally comprises (i) an extracellular domain (ecto-domain or antibody domain), (ii) a transmembrane domain, and (iii) a cytoplasmic domain (endo-domain or intracellular signaling domain).

[0318] The extracellular domain (i.e., the antibody domain) generally comprises an scFv, but other modalities can also be used. A spacer links the extracellular domain and the transmembrane domain, which in turn is linked to the intracellular domain. After the extracellular domain binds to an antigen, the receptor aggregates and activation signals are transmitted to the cell, thereby initiating an immune response. First-generation CARs have a simple intracellular domain comprising CD3-ξ. To increase activation signals, co-stimulatory domains are added in second-generation CARs; and third-generation CARs include two or more co-stimulatory domains (Maus MV et al. (2014) Blood, 123:2625-2635). The co-stimulatory domains can be selected from the group consisting of: CD28, OX40, and / or 4-1BB. In addition to CD3-ξ, other ITAM-containing domains have been explored, including the Fc receptor of the IgE-γ domain.

[0319] In certain embodiments, the CAR comprises a scFv that comprises the amino acid sequence of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and / or SEQ ID NO: 64, or a variant thereof that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and / or SEQ ID NO: 64, respectively.

[0320] Suitable immune cells engineered to express such CARs include, but are not limited to, T cells, natural killer T (NKT) cells, natural killer (NK) cells, human embryonic stem cells, hematopoietic stem cells (HSCs), or induced pluripotent stem cells (iPS). Such T cells can be cytotoxic T lymphocytes (CTLs), regulatory T lymphocytes, inflammatory T-lymphocytes, or helper T-lymphocytes or gamma-delta T cells. The T cells can be CD4+, CD8+, or a mixed population of CD4+ and CD8+ cells.

[0321] In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) that specifically recognizes a peptide-MHC, the chimeric antigen receptor comprising: i) an antigen-binding protein identified from a nucleic acid library or by the methods described herein; ii) a transmembrane domain; and iii) an intracellular signaling domain.

[0322] In certain embodiments, the transmembrane domain is selected from the group consisting of: artificial hydrophobic sequences and transmembrane domains of type I transmembrane proteins, the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0323] In certain embodiments, the intracellular signaling domain is selected from the group consisting of: the cytoplasmic signaling domain of the human CD3 zeta chain, FcyRIII, the cytoplasmic tail of the Fc receptor, cytoplasmic receptors with immunoreceptor tyrosine-based activation motifs (ITAMs), TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0324] The antibody domain can be any of the antigen-binding proteins outlined above. Thus, in certain embodiments, the antibody domain comprises an antibody variable light domain (VL) containing an amino acid sequence represented by the formula LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4. In certain embodiments, the antibody domain comprises an antibody variable heavy domain (VH) containing an amino acid sequence represented by the formula HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. In certain embodiments, the antibody domain comprises an scFv as described herein.

[0325] Methods of treating cancer

[0326] Provided herein are methods of treating cancer with the antigen-binding proteins of the present disclosure and, in particular, multispecific antigen-binding proteins (e.g., MAGE-A4 pMHC antigen-binding proteins). The methods can be used to treat patients having any tumor type in which at least some cancer cells express the MAGE-A4 antigen presented on pMHC, such as the GVYDGREHTV (SEQ ID NO:3)-HLA-A*02:01 complex. Such MAGE-A4-positive cancers or cancer cells can be evaluated using any method known in the art, including but not limited to detecting RNA expression levels or histological methods such as immunohistochemistry (IHC).

[0327] In some embodiments, the MAGE-A4-positive cancers are selected from the group consisting of: bladder cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, lung cancer, melanoma, esophageal cancer, ovarian cancer, kidney cancer, synovial sarcoma, and tumors having squamous cell histology. In certain embodiments, the cancer is of squamous origin. Experimental data indicate that squamous cell carcinomas have the highest incidence and median expression of MAGE-A4 mRNA. In certain embodiments, the cancers are selected from the group consisting of: head and neck squamous cell carcinoma (HNSC), head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) (particularly squamous NSCLC), triple-negative breast cancer, urothelial carcinoma, high-grade endometrial cancer (including uterine carcinosarcoma (UCS; particularly the UCEC subgroup)), myxoid / round cell liposarcoma, gastric or gastroesophageal junction (GEJ) adenocarcinoma, epithelial ovarian cancer (such as high-grade serous ovarian cancer), synovial sarcoma, bladder urothelial carcinoma (BLCA) (particularly transitional cell carcinoma), testicular germ cell tumor (TGCT), and cervical squamous cell carcinoma (CESC).

[0328] The antigen-binding proteins described herein also include binding specificities for certain related MAGE pMHC targets, particularly the MAGE-A8-derived GLYDGREHSV (SEQ ID NO: 71) HLA-A*02 complex and / or the MAGE-B4-derived GIYDGKRHLI (SEQ ID NO: 72#) HLA-A*02 complex. Accordingly, the present disclosure provides a method of treating such related MAGE-pMHC positive cancers.

[0329] In one aspect, the present disclosure provides the use of the antigen-binding proteins described herein, the multispecific antigen-binding proteins described herein, or the pharmaceutical compositions described herein in the manufacture of a medicament.

[0330] In another aspect, the present disclosure provides the use of the antigen-binding proteins described herein, the CARs described herein, the immune cells described herein, the multispecific antigen-binding proteins described herein, or the pharmaceutical compositions described herein for treating a disease (particularly cancer).

[0331] In another aspect, the present disclosure provides a method of treating a cancer expressing MAGE-A4 pMHC in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the antigen-binding proteins described herein, the CARs described herein, the immune cells described herein, the multispecific antigen-binding proteins described herein, or the pharmaceutical compositions described herein.

[0332] In certain aspects, patients suitable for treatment with a MAGE-A4 antagonist are selected based on RNA sequencing and / or immunohistochemistry (IHC) (such as for detecting total MAGE-A4).

[0333] In certain aspects, patients suitable for treatment with a MAGE-A4 antagonist described herein (such as the antigen-binding proteins described herein, the CARs described herein, the immune cells described herein, the multispecific antigen-binding proteins described herein, or the pharmaceutical compositions described herein) are selected based on immunohistological methods, the immunohistological methods comprising the following steps carried out in sequence:

[0334] (i) obtaining a tumor sample from the patient;

[0335] (ii) adding an anti-MAGEA4 detection antibody to the sample;

[0336] (iii) incubating the detection antibody and the sample;

[0337] (iv) detecting the detection antibody bound to the sample; and

[0338] (v) If the detection antibody is bound by the sample, then the patient is selected for treatment with a MAGE-A4 antagonist.

[0339] In certain embodiments, the detection antibody is OTI1F9, E7O1U, or an antigen-binding protein described herein. WO2019171064A1 further describes the OTI1F9 detection antibody. In vitro overexpression experiments confirmed that E7O1U binds only to MAGE-A4, while OTI1F9 cross-reacts extensively with MAGE-A3, MAGE-A6, MAGE-A8, MAGE-A10, MAGE-A11, and MAGE-A12. Thus, E7O1U is more specific than OTI1F9. Accordingly, in certain embodiments, the anti-MAGE-A4 detection antibody is E7O1U. For example, E7O1U is commercially available as MAGE-A4 (E7O1U) XP Rabbit MAb.

[0340] In certain embodiments, the MAGE-A4 antagonist is an antigen-binding protein described herein, a CAR described herein, an immune cell described herein, a multispecific antigen-binding protein described herein, or a pharmaceutical composition described herein.

[0341] Kit

[0342] Also contemplated are kits that contain at least one nucleic acid library or antigen-binding protein, including multispecific antigen-binding proteins, or a pharmaceutical composition as described herein, typically in combination with packaged reagents and instructions. In one embodiment, the kit includes a composition containing an effective amount of the antigen-binding protein in unit dosage form. Such kits may include a sterile container containing the composition; non-limiting examples of such containers include, but are not limited to, vials, ampoules, bottles, tubes, syringes, blister packs. In some embodiments, the composition is a pharmaceutical composition and the container is made of a material suitable for containing a medicament. In one embodiment, the kit may contain the antigen-binding protein in lyophilized form in a first container and a diluent (e.g., sterile water) for reconstituting or diluting the antigen-binding protein in a second container. In some embodiments, the diluent is a pharmaceutically acceptable diluent. In one embodiment, the kit is for diagnostic purposes and the antigen-binding protein is formulated for diagnostic applications. In one embodiment, the kit is for therapeutic purposes and the antigen-binding protein is formulated for therapeutic applications.

[0343] Typically, the medicine box will also include a separate sheet, booklet, or card with instructions for use provided in or with the container. If the medicine box is intended for medicinal use, it may also contain one or more of the following: information on administering the composition to a subject suffering from a related disease or disorder and the dosing schedule, a description of the therapeutic agent, precautions, warnings, indications, contraindications, overdose information, and / or adverse reactions.

[0344] It will be apparent to those skilled in the art that other suitable modifications and variations of the methods described herein can be made using appropriate equivalents without departing from the scope of the embodiments disclosed herein. Certain embodiments have been described in detail and will be understood more clearly by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting.

[0345] Examples

[0346] Example 1 - Preparation of pMHC Antigen for Animal Immunization

[0347] The MHC class I heavy chain and β2m were cloned into the pET-24D(+) vector using standard molecular biology techniques (J Biol Chem. January 13, 1995; 270(2): 971-7). Escherichia coli BL-21(DE3) was transformed with the expression vector according to the supplier's protocol. Protein expression was carried out in MagicMedium (Invitrogen) at 37 °C with shaking at 220 rpm for 16-18 hours as described by the supplier. The cells were harvested and lysed with BugBuster (Invitrogen), and the inclusion bodies were washed twice with TBS supplemented with 0.5% LDAO and twice with TBS. The inclusion bodies thus prepared were dissolved in denaturing buffer (8 M urea, 100 mM Tris-HCl pH 8), using 5 mL of buffer per 1 g of inclusion body pellet. The refolding and purification of MHC were carried out substantially as described by Rodenko et al. (2006) with the target peptides (HLA-A*02:01 extracellular domain, human β2m, and MAGE-A4 peptide 230-239). The amino acid sequences of each component of the pMHC antigen are listed in Table 1 below.

[0348] Table 1 - Amino Acid Sequences of pMHC Antigen Components

[0349]

[0350] Example 2 - Rabbit Immunization

[0351] To generate antibodies that can specifically recognize target peptides in the context of the HLA-A*02:01 complex, three New Zealand white rabbits were immunized with recombinantly produced HLA-A*02:01 / MAGE-A4 complex. Each animal received four injections of HLA-A*02:01 / MAGE-A4 complex with complete or incomplete Freund’s adjuvant at different time points. The immune response of the animals was tested in ELISA to quantify anti-pMHC antibodies present in the serum samples of the immunized animals.

[0352] Example 3 - Construction of a rabbit-derived immune library

[0353] An scFv antibody cDNA library was constructed by PCR amplification from RNA extracted from rabbit-derived PBMCs and splenic lymphocytes. The coding sequences of the variable light chain (VL) and heavy chain (VH) domains were amplified separately and joined by a series of overlapping PCR steps to obtain the final scFv product. The amplified DNA sequences encoding scFv from rabbits were digested with appropriate restriction enzymes and then ligated into a phagemid vector. The phagemid vector was transformed into electrocompetent Escherichia coli TG1 cells, which are well-suited for creating antibody phage display libraries. These procedures generated two antibody libraries that contained a diversity of 5.2x10 8 for the K-based library with 87.5% sequence accuracy, and a diversity of 2.0x10 9 for the λ-based library with 91.7% accuracy.

[0354] Example 4 - Screening of the rabbit-derived immune library

[0355] The rabbit-derived immune library was screened for HLA-A*02:01 / MAGE-A4 specific binders. Briefly, three rounds of phage display biopanning against the HLA-A*02:01 / MAGE-A4 antigen were performed, followed by screening of the library for specific hits. Screening was performed using monoclonal phage ELISA against specific (i.e., HLA-A*02:01 / MAGE-A4) and non-specific (i.e., HLA-A*02:01 / peptide mixture) targets. The control HLA-A*02:01 / peptide mixture complex contained the HLA-A*02:01 complex loaded with a mixture of 49 unrelated peptides. The signal ratio of specific target binding to non-specific binding was calculated to identify hits that specifically bind to the target. The identified hits were expressed as chimeric Fabs.

[0356] Example 5 - Expression of antibodies as monovalent monospecific Fabs

[0357] Monovalent monospecific antibodies are expressed in Fab format. Rabbit variable domains are paired with human constant domains (heavy chain and kappa light chain) to produce chimeric Fabs that bind to target pMHC. The amino acid sequences of the constant domains are listed in Table 2 below.

[0358] Table 2 - Amino acid sequences used to generate chimeric Fab

[0359]

[0360] Synthetic genes encoding different antibody chains (i.e., heavy and light chains) were constructed at Twist Bioscience and cloned individually into expression vectors for transient expression in HEK 2936E cells. Expression vector DNA was prepared using conventional plasmid DNA purification methods (e.g., Qiagen HiSpeed ​​plasmid maxi kit, catalog number 12662).

[0361] Antigen binding proteins were expressed by transient co-transfection of the corresponding mammalian expression vectors in HEK293-6E cells cultured in suspension using polyethyleneimine (PEI 40 kD linear). 6 HEK293-6E cells were inoculated with 10 cells / mL. DNA was prepared per milliliter of final preparation volume by adding DNA and PEI separately to 50 μL of medium without supplements. The two fractions were mixed, vortexed and left to stand for 15 minutes, resulting in a DNA:PEI ratio of 1:2.5 (1 μg DNA per milliliter of cells). The cells and DNA / PEI mixture were put together and then transferred to an appropriate container, which was placed in an oscillating device (37°C, 5% CO2, 80% RH). After 24 hours, 25 μL of tryptone N1 was added per milliliter of final preparation volume.

[0362] After 7 days, cells were harvested by centrifugation and sterile filtered. For affinity purification of Fab-based constructs, supernatants were loaded onto CaptureSelect equilibrated with 6 CV PBS (pH 7.4). TM On a CH1-XL column (Thermo Fisher Scientific). After a washing step using the same buffer, the antigen binding protein was eluted from the column by a step of eluting with 100 mM citric acid (pH 3.0). The fractions with the desired antigen binding protein were immediately neutralized with 1M Tris buffer (pH 9.0) at a 1:10 ratio, then pooled, dialyzed with PBS buffer and concentrated by centrifugation. The purity of the protein was assessed by SDS-PAGE and size exclusion HPLC.

[0363] Example 6 - Characterization of Hits

[0364] The ability of hits to bind to the HLA-A*02:01 / MAGE-A4 complex and a control HLA-A*02:01 / peptide mixture complex was evaluated in a direct binding ELISA assay. Briefly, a 96-well ELISA plate was coated with the HLA-A*02:01 / MAGE-A4 complex or the control HLA-A*02:01 complex. Serial dilutions of the antigen-binding protein in Fab format were added to the plate and detected sequentially by anti-K light chain-HRP (Invitrogen) and goat anti-rabbit IgG (H+L) HRP (Southern Biotech). Further characterization of the binder was considered when high binding to the HLA-A*02:01 / MAGE-A4 complex and no binding to the control HLA-A*02:01 / peptide mixture complex was shown. Figure 1A and Figure 1B Shown in are the bindings of the selected antibodies M0700, M0701, M0703, M0704, M0705, M0706, M0707, M0708, M0709, M0710, M0762, M0763, M0764, M0765, and M0766 to the HLA-A*02:01 / MAGE-A4 complex as determined by ELISA. Figure 1C and Figure 1D Shown in are the bindings to the negative control complex HLA-A*02:01 / peptide mixture as determined by ELISA. All the molecules tested showed specific binding to the HLA-A*02:01 / MAGE-A4 complex and no binding to the control HLA-A*02:01 / peptide mixture complex.

[0365] The binding of specific antibodies M0709 and M0763 to the HLA-A*02:01 / MAGE-A4 complex present on cells was determined. Briefly, T-B hybrid T2 cells were incubated with serum-free RPMI 1640 medium containing MAGE-A4 or control peptides. The control peptides constitute sequences with high identity to MAGE-A4 and have been previously identified in healthy human tissues, namely control 1 (GLADGRTHTV; SEQ ID NO: 68), control 2 (GLYDGPVHEV; SEQ ID NO: 69), and control 3 (GVFDGLHTV; SEQ ID NO: 70) (US20180171024, incorporated herein by reference). The peptide loading efficiency was verified by using the ratio (>1) of the median fluorescence intensity (MFI) of the HLA-A*02:01 binding antibody BB7.2 on peptide-loaded T2 cells to the MFI of unloaded T2 cells. The T2 cells were sequentially incubated with each of the specific antibodies and a fluorophore-labeled detection antibody (anti-κ light chain). The cells were fixed and fluorescence was measured by flow cytometry. The binding and specificity of M0709 and M0763 to T2 cells presenting MAGE-A4 or control peptides 1, 2, and 3 are presented in Figure 2. Both of the tested molecules showed binding to HLA-A*02:01 / MAGE-A4 presented on T2 cells. In addition, M0763 showed very high specificity for the MAGE-A4 peptide and did not show binding to any of the control peptides presented by HLA-A*02:01 on T2 cells. Compared to M0763, M0709 showed lower specificity and also bound to control peptides 1 and 2.

[0366] Example 7 - Optimization of M0763

[0367] A rabbit antibody named M0763 was humanized by CDR grafting. Briefly, human V gene germlines showing high sequence identity to the VH and VL of M0763 (i.e., IMGT_hVH_3_23 and IMGT_hVL_3-1) were selected as CDR acceptor scaffolds, respectively. As determined by direct ELISA (as described in Example 6), the resulting humanized antibodies M0871 - M874 retained HLA-A*02:01 / MAGE-A4 binding, with EC50 values of 1.18 - 16.02 nM and did not show binding to the HLA-A*02:01 / peptide mixture negative control (Figure 3).

[0368] In the next optimization step, M0873 undergoes affinity maturation. Briefly, multiple antibody libraries are designed to span the entire length of all six CDRs while randomizing three consecutive amino acids. Libraries are generated using primers for site-saturation mutagenesis. Thus, the three amino acid positions targeted for randomization contain one of 19 possible amino acid variants. After electroporation into Escherichia coli TG-1 cells, the diversity of the library is determined by plating the library on agar plates using serial dilutions of the transfected TG-1 cells. The number of colonies growing on the plates is used as an indication of library diversity, assuming one inserted plasmid per E. coli colony. Additionally, the quality of the library is evaluated by sequencing samples of 10 clones from each library.

[0369] The libraries containing site-saturation mutagenesis in the light chain are pooled into one library, and the libraries containing site-saturation mutagenesis in the heavy chain are pooled into another library. Affinity selection is performed on the two resulting libraries with randomized CDRs in the light and heavy chains, hereafter referred to as biopanning against the HLA-A*02:01 / MAGE-A4 complex. The HLA-A*02:01 / MAGE-A4-specific phage library is panned (selected) on the antigen adsorbed to polystyrene tubes or plates. Briefly, three rounds of phage display biopanning are performed against the HLA-A*02:01 / MAGE-A4 antigen, and then hits of the library are screened. Screening is performed using monoclonal phage ELISA against specific (i.e., HLA-A*02:01 / MAGE-A4) and non-specific (i.e., HLA-A*02:01 / peptide mixture) targets.

[0370] Then, in ELISA, the phage-displayed antibody clones are classified as high, medium, and low signal against the target complex protein and against the complex of HLA-A*02:01 with an unrelated peptide. Clones with high binding signal to the target complex and weak binding to the HLA-A*02:01 / peptide mixture complex are sequenced. Sequence analysis facilitates the identification of unique clones, which are then selected for recombinant expression of anti-CD3 Fab x anti-MAGE-A4 scFv in a bispecific format. Then the binding affinity of the resulting construct to the HLA-A*02:01 / MAGE-A4 complex is evaluated in SPR (Table 3).

[0371] Table 3 - Binding affinity values of variant antibodies to the HLA-A2 / MAGE-A4 complex

[0372]

[0373]

[0374]

[0375] Affinity characterization of the anti-HLA-A*02:01 / MAGE-A4×CD3 bispecific antibody in Table 3 was performed using a Biacore TM T200 instrument (Cytiva) by surface plasmon resonance (SPR). To determine the affinity of the bispecific antibody for the HLA-A*02:01 / MAGE-A4 complex, a streptavidin chip (SAHC30M, XanTec) was coated with 500 RU of the complex of HLA-A*02:01 and MAGE-A4 peptide according to the manufacturer's instructions. To determine the affinity of the bispecific antibody for CD3, an HC30M chip (XanTec) was coated with 400 RU of CD3 heterodimer (Acro Biosystems) according to the manufacturer's instructions. The uncoated channel was used as a reference. Data fitting was performed using a 1:1 Langmuir model. The affinity matured clones yielded binding affinities as low as single-digit picomolar, which was almost a 1000-fold improvement in binding affinity compared to the parental M0763 antibody.

[0376] The affinity matured M1041 was optimized by further humanization and stabilization of the anti-HLA-A*02:01 / MAGE-A4 binding scFv arm. The rabbit starting residues in VH FR1 were further mutated by incorporating the amino acid substitutions S2V and V4L to generate M1067. M1067 was further modified by incorporating mutations in VH FR3 (i.e., amino acid substitutions K71R and T73N) and CDR-L3 (i.e., amino acid substitution L97A) to generate M1402. As shown in Table 4, each optimization step improved the human sequence identity score, thermal stability, and affinity for the target antigen. The thermal stability of the bispecific antibody was measured using differential scanning fluorimetry (DSF) as described in the Protein Thermal Shift manual MAN4461806B from Applied Biosystems (Thermo Fisher).

[0377] Table 4 - Characteristics of the stabilized and humanized variants of M1041.

[0378]

[0379] Meanwhile, M1067 was further modified by incorporating the mutation in VH FR2 (i.e., amino acid substitution Y47W) to obtain a more stable molecule with lower affinity for HLA-A*02:01 / MAGE-A4 antigen, resulting in M1068. The stabilities of M1067 and M1068 were compared at various concentrations and at 4 °C and 37 °C in PBS pH 7.4 during extended incubation. Stability was evaluated by SEC-HPLC quantification of the target protein monomer, and the corresponding data are presented in Table 5. M1068 showed superior stability over M1067 and maintained >90% monomer content after two-week incubation at all tested concentrations and incubation temperatures.

[0380] Table 5 - Stabilities of M1067 and M1068

[0381]

[0382] M1068 further underwent affinity maturation as previously described. The affinity maturation campaign resulted in the generation of novel CDR-L3 and CDR-H3 sequences with unique properties. Molecules containing these novel CDR sequences include M1302, M1382, and M1386, and compared to the 1.0 nM K D of M1068, they all showed improved affinity for HLA-A*02:01 / MAGE-A4 antigen with K D values of 0.6 nM, 0.23 nM, and 0.15 nM, respectively. M1302 was also optimized for human sequence content and stability. A different human germline VL framework (i.e., IMGT_hVL_3-19) was selected to graft the CDR-L1, CDR-L2, and CDR-L3 of M1302, resulting in M1312. IMGT_hVL_3-19 showed high sequence similarity to the M0763 VL sequence similar to the previously selected IMGT_hVL_3-1; however, IMGT_hVL_3-19 provided a molecule with improved biophysical properties. M1312 was further humanized by incorporating mutations in VH FR3 (i.e., amino acid substitutions K71R and T73N), resulting in M1394. The final humanization of M1394 included substitutions in CDR-H2 (i.e., amino acid substitutions S61D, W62S, and A63V), VL FR1 (i.e., amino acid substitutions S1Q and E3V), and CDR-L3 (i.e., amino acid substitution L97A), resulting in M1396. As shown in Table 6, each incorporated substitution improved the human sequence identity score and / or the stability of the molecule.

[0383] Table 6 - Characteristics of stable and humanized variants of M1068.

[0384]

[0385] Example 8 - Preparation of Monovalent and Bivalent pMHC-Targeting T Cell Engagers

[0386] Monovalent bispecific antigen-binding proteins were expressed by transient co-transfection in HEK293-6E cells. Cells were cultured in suspension using polyethylenimine (PEI 40kD linear). HEK293-6E cells were seeded at 1.7x10 6 cells / mL in Freestyle F17 medium supplemented with 2 mM L-glutamine. DNA and PEI were added separately to 50 μL of medium without supplements. The two fractions were mixed at a 1:2.5 DNA:PEI ratio, vortexed and left to stand for 15 minutes. Cells and the DNA / PEI mixture were combined (1 μg DNA / mL cells) and incubated at 37 °C, 5% CO2, 80% RH. After 24 hours, the cells were supplemented with tryptone N1 at 25 μL per mL of preparation volume. After 7 days, the cells were harvested by centrifugation and the supernatant was sterile filtered. The antigen-binding protein was purified from the supernatant by affinity chromatography. The supernatant was loaded onto a CaptureSelect TM CH1-XL column (Thermo Fisher Scientific) equilibrated with 6CV PBS (pH 7.4). After a washing step using the same buffer, the protein was eluted from the column by elution with 100 mM citric acid (pH 3.0). Fractions containing the desired antigen-binding protein were immediately neutralized at a 1:10 ratio with 1M Tris buffer (pH 9.0). Size exclusion chromatography was performed as an additional purification step. Using PBS (pH 7.4) as the operating buffer, the sample was run on a Superdex 200 10 / 300GL column. Fractions collected were analyzed by SE-HPLC for monomer content and pooled accordingly. Final protein purity was evaluated by SDS-PAGE and SE-HPLC.

[0387] A bivalent bispecific antigen-binding protein was generated by transient co-transfection in CHO-K1 cells. The genes for HC and LC were expressed in shake flask cultures for 7 days using a 2:1 vector ratio. The target protein was captured from the clarified, sterile-filtered culture supernatant by affinity chromatography and Amsphere A3 resin (JSR Life Science). The captured antigen-binding protein was further purified by strong cation exchange chromatography (CEC) on Source 30S resin (Cytiva) and hydrophobic interaction chromatography (HIC) on Toyopearl PPG-600M resin (Tosoh Bioscience). The target protein was transferred to the final buffer (130 mM NaCl, 10 mM sodium phosphate, pH 6.5) using an Amicon stirred cell (Merck).

[0388] Example 9 - Dual pMHC-Targeted T Cell Engagers

[0389] The antigen-binding protein was designed to have two binding domains targeting specific pMHCs and a Fab binding domain targeting CD3 as a T cell recruitment domain. Figure 4 Schematic illustration of an exemplary bispecific antigen-binding protein showing a Fab T cell binding domain (e.g., anti-CD3 Fab) and two pMHC binding domains in scFv format (e.g., each pMHC binding domain specifically binds to the same target pMHC molecule on the surface of tumor cells). These dual pMHC-targeted T cell engagers function by recruiting T cells to tumor cells expressing the target pMHC molecules on their surface.

[0390] To test the functionality of the dual pMHC-targeting T cell engager, two identical scFv antigen-binding proteins with binding specificity for pMHC-MAGE-A4 were linked to anti-CD3 Fab, one scFv linked to the Fab heavy chain (HC) and one scFv linked to the Fab light chain (LC). The CD3 Fab has a binding affinity for CD3 with a KD of 10 nM and each pMHC-MAGE-A4 scFv has a binding affinity for pMHC-MAGE-A4 with a KD of 250 pM. This multispecific antigen-binding protein was named "anti-MAGE-A4 dual engager" or simply "dual engager" for short. The efficacy and safety of dual engager M1048 and its monovalent counterpart M1041 were compared in the LDH assay. The MAGE-A4-positive HLA-A*02:01-positive osteosarcoma cell line U2OS and three MAGE-A4-negative HLA-A*02:01-positive cell lines SK-MEL-30, MDA-MB-231, and PANC-1 were incubated with human PBMC at an E:T ratio of 10:1. Cancer cell killing was measured at various concentrations of the monovalent T cell engager M1041 and the dual T cell engager M1048. Cytotoxicity was quantified by the colorimetric absorbance measurement of the amount of LDH released from damaged cells into the culture medium after 48 h (Figure 5). The dual T cell engager M1048 showed superior cancer cell killing compared to its monovalent counterpart M1041. At the same time, M1048 similarly showed low cytotoxicity against antigen-negative cancer cells SK-MEL-30, MDA-MB-231, and PANC-1 compared to its monovalent counterpart M1041, with higher cytotoxicity of M1048 observed only at the highest tested compound concentration of 1 nM in the PANC-1 and MDA-MB-231 cell lines. Overall, the data showed that the dual T cell engager was superior to its monovalent counterpart, with an approximately 10-fold increase in the antigen-positive cancer cell killing potency of the dual pMHC-targeting T cell engager and similar low cytotoxicity in antigen-negative cancer cell lines.

[0391] Example 10 - Characterization of an Optimized Anti-HLA-A*02:01 / MAGE-A4 x CD3 Dual pMHC-Targeting T Cell Engager

[0392] The optimized M1041 variants (i.e., M1382, M1386, M1396, and M1402) were re-engineered into the dual pMHC T cell engager format, yielding molecules M1383, M1387, M1397, and M1403, respectively. Molecular preparation showed that M1397 and M1403 had similar potencies. After purification according to the standardized protocol, M1397 showed a much lower charge variant amount than M1403.

[0393] The stability of M1403 and M1397 was tested at 4 °C and 37 °C during extended incubation in 10 mM phosphate buffer pH 6.0 supplemented with 130 mM NaCl at concentrations of 1 mg / mL and 10 mg / mL. Stability was evaluated by SEC-HPLC quantification of the target protein monomer, and the corresponding data are presented in Figure 6. At 4 °C, molecules M1397 and M1403 showed good stability in 10 mM phosphate buffer pH 6.0 supplemented with 130 mM NaCl for 14 days, maintaining >98% target protein monomer content at both tested concentrations. When incubated at 37 °C, M1397 showed superior stability to M1403, maintaining >95% target protein monomer content at both tested concentrations.

[0394] Detailed biophysical characterization of M1397 and M1403 was performed using dynamic light scattering (DLS). Briefly, the molecular cumulative radius (average size of the particles) and polydispersity index (PDI) were determined by DLS using a Prometheus Panta instrument. Samples were mixed and 0.1 μm filtered prior to measurement. Analyses were performed at 20 °C and 100% DLS laser power. Data were analyzed using PR Panta analysis (x64) software. The thermal stability of the dual adaptors M1397 and M1403 was measured using differential scanning fluorimetry (DSF). For this purpose, samples were diluted to a concentration of 1 mg / ml. The temperature was increased from 20 °C to 95 °C at 1 °C / min. Data were analyzed using PR Panta analysis (x64) software. Thermal stability was determined, including the temperature at which the protein began to unfold (T onset), melting temperature TM and aggregation temperature (T aggregation). The resulting data are presented in Table 7. M1397 showed a very favorable thermal stability profile, with onset of unfolding at 47.01 °C and a two-step unfolding process. The first transition occurred at 53.33 °C, corresponding to scFv domain unfolding, and the second at 72.47 °C, corresponding to Fab domain unfolding. M1403 showed onset of unfolding and a three-step unfolding process at a temperature >10 °C lower than M1397.

[0395] Table 7 - Thermal stability and aggregation analysis of M1397 and M1403.

[0396]

[0397] Measure the human serum stability of M1397 and M1403. Briefly, human serum was prepared by centrifuging whole clotted blood at 2,000×g. The resulting supernatant was named serum and directly frozen and stored at -80°C. The sterile filtered samples were diluted to a concentration of 100 μg / ml in serum. Incubation was carried out in a humidified CO2 incubator at 8% CO2 and 37°C in sterile Eppendorf tubes. A reference sample (T0) was taken before the start of incubation and samples of serum incubation were collected after 7 h and 24 h. The samples were directly quantified by SPR. SPR quantification was carried out on a Biacore T200 instrument. The HLA-A*02:01 / MAGE-A4 binding molecule was quantified by SPR assay, where HLA-A*02:01 / MAGE-A4 was used as a ligand and the serum sample was injected after dilution 1:100 in the SPR operating buffer. The samples were evaluated by directly comparing the signal heights after injection of the serum samples. The relative decrease in the binding signal compared to the T0 sample was calculated. The corresponding results are presented in Figure 7.

[0398] Example 11 - Preparation of comparative molecules: soluble TCR-CD3 fusion protein (comparative 1) and TCR-like CD3 T cell bispecific antibody (comparative 2)

[0399] Using standard molecular biology techniques, the DNA sequences encoding the extracellular regions of the α (SEQ ID NO: 59) and β (SEQ ID NO: 60) chains of the soluble TCR - anti - CD3 fusion (Comparator 1) were individually cloned into the pET - 24D(+) vector (J Biol Chem. January 13, 1995; 270(2): 971 - 7). Escherichia coli BL - 21(DE3) was transformed with the expression vector according to the supplier's protocol. Protein expression was carried out in Magic Medium (Invitrogen) at 37 °C with shaking at 220 rpm for 16 - 18 hours as described by the supplier. Cells were harvested, resuspended in TBS and lysed via lysozyme treatment and sonication. Inclusion bodies were washed twice with TBS supplemented with Triton - X100 (50 mM Tris - HCl pH 8.1, 0.5% Triton - X100, 100 mM NaCl, 10 mM NaEDTA) and twice with TBS (50 mM Tris, 100 mM NaCl, 10 mM EDTA, pH 8.1). The inclusion bodies thus prepared were dissolved in denaturing buffer (9 M urea, 0.5 M Gua, 25 mM Tris, 1.25 mM EDTA, pH 8.1). The dissolved inclusion bodies from the α - chain and β - chain - anti - CD3 scFv fusion were combined with a reducing agent and mixed to a final DTT concentration of 20 mM. The dissolved and reduced inclusion bodies were slowly mixed with refolding buffer (4 M urea, 400 mM L - Arg, 2 mM EDTA, 100 mM Tris, 10 mM L - cysteine, 2.5 mM L - cystine, pH 8.1) and incubated overnight at room temperature. Molecules were captured from the diluted and pH - adjusted refolding solution by anion - exchange chromatography using a POROS 50HQ column. Molecules were eluted by applying a gradient of 0 - 500 mM NaCl in 20 mM Tris pH 8.1 over 50 column volumes on a purifier device (Cytiva). Size - exclusion chromatography was performed as an additional purification step. Using PBS (pH 7.4) as the operating buffer, the sample was run on a HiLoad Superdex 75, 26 / 600 column. Fractions collected were analyzed for monomer content by SE - HPLC and pooled accordingly. Final protein purity was evaluated by SDS - PAGE and SE - HPLC.

[0400] The TCR-like CD3 T cell bispecific antibody (Comparator 2, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67) was expressed by transient co-transfection in HEK293-6E cells. The cells were cultured in suspension using polyethylenimine (PEI 40kD linear). HEK293-6E cells were seeded at 1.7x10 6 cells / mL in Freestyle F17 medium supplemented with 2 mM L-glutamine. DNA and PEI were added separately to 50 μL of medium without supplements. The two fractions were mixed at a 1:2.5 DNA:PEI ratio, vortexed and left to stand for 15 minutes. The cells and the DNA / PEI mixture were combined (1 μg DNA / mL cells) and incubated at 37 °C, 5% CO2, 80% RH. After 24 hours, the cells were supplemented with tryptone N1 at 25 μL per mL of preparation volume. After 7 days, the cells were harvested by centrifugation and the supernatant was sterile filtered. The antigen-binding protein was purified from the supernatant by affinity chromatography. The supernatant was loaded onto a CaptureSelect TM CH1-XL column (Thermo Fisher Scientific) equilibrated with 6CV PBS (pH 7.4). After a washing step using the same buffer, the protein was eluted from the column by elution with 100 mM citric acid (pH 3.0). The fractions containing the desired antigen-binding protein were immediately neutralized at a 1:10 ratio with 1M Tris buffer (pH 9.0). Size exclusion chromatography was performed as an additional purification step. Using PBS (pH 7.4) as the operating buffer, the sample was run on a Superdex 200 10 / 300GL column. The collected fractions were analyzed by SE-HPLC for monomer content and pooled accordingly. The final protein purity was evaluated by SDS-PAGE and SE-HPLC.

[0401] Example 12 - In vitro efficacy of optimized dual pMHC-targeting T cell engagers

[0402] In an in vitro cytotoxicity assay, the dual adaptor M1048 showed improved efficacy compared to its monovalent counterpart M1041. Optimized M1041 variants (i.e., M1402 and M1396) were re-engineered into the dual adaptor format, yielding M1403 and M1397, respectively. The optimized M1048 variants (i.e., M1403 and M1397) have higher human sequence content, higher stability, and / or higher affinity. The efficacy and safety of the dual pMHC-targeting T cell adaptors M1403 and M1397 were compared in an LDH assay. Additionally, the dual T cell adaptors M1397 and M1403 were compared to two comparator antigen-binding molecules (i.e., Comparator 1 and Comparator 2). Comparator 1 consists of a soluble affinity-enhanced TCR that has a binding specificity for the same pMHC-MAGE-A4 antigen with a K D value of 87 pM and is linked to an anti-CD3 scFv with a binding affinity of 1 nM. Comparator 1 is monovalent for the target pMHC and CD3, while the dual adaptor is bivalent for the target pMHC and monovalent for CD3. Comparator 1 is further described in detail in US20190092834, which is incorporated herein by reference. Comparator 2 is a bispecific TCR-like antibody specific for the same pMHC-MAGE-A4 antigen linked to an anti-CD3 Fab. Comparator 2 is bivalent for the target pMHC, monovalent for CD3, and contains an Fc domain for half-life extension. Comparator 2 is further described in detail in US20210230278A1, which is incorporated herein by reference.

[0403] As shown in Figure 9, the percentage of cancer cell killing was measured in several MAGE-A4-positive HLA-A*02:01-positive cell lines (i.e., U2OS (osteosarcoma), NCI-H1703 (lung squamous cell carcinoma), and A375 (melanoma)) and MAGE-A4-negative HLA-A*02:01-positive cell lines (i.e., PANC1 (pancreatic cancer), MDA-MB-231 (breast cancer), and NCI-H441 (lung adenocarcinoma)). The cancer cell lines were incubated with the dual adaptors M1397, M1403, or comparator 1 and human PBMCs (E:T ratio of 10:1). For all cell lines, the cytotoxicity of the three different antigen-binding proteins at various concentrations was quantified by colorimetric absorbance measurement of the amount of LDH released from damaged cells into the culture medium after 48 h, with the exception of NCI-H1703. The cytotoxicity in NCI-H1703 was determined by CellTiter-Glo assay (Promega) according to the instructions provided by the kit manufacturer. The data showed that for all the tested MAGE-A4-positive HLA-A*02:01-positive cell lines, similar cell killing mediated by the dual adaptor M1403 compared to comparator 1, while M1397 showed lower efficacy. Additionally, for all the tested MAGE-A4-negative HLA-A*02:01-positive cancer cell lines, especially for M1397, lower cancer cell killing was observed for both dual T cell adaptors compared to comparator 1, indicating a better therapeutic window for M1397 and M1403.

[0404] Additionally, the cell killing mediated by M1397, comparator 1, and comparator 2 was analyzed in a time-resolved manner using the IncuCyte S3 system. Briefly, antigen-positive target cells (NCI-H1703 and U2OS) and antigen-negative target cells (SKMEL-30 and PANC-1) were transduced with Nuclight Red lentivirus (Sartorius) to stably express the NucLight Red fluorescent protein. In a sterile 384-well flat-bottom adherent tissue culture plate in an incubator at 37 °C and 5% CO2, at 1.5×10 3Seed cancer cells at a density of

[0405] cells / well overnight. Add molecule M1397, comparator 1, and comparator 2 at the indicated concentrations (in the range of 0.2 pM to 50 nM). Add PBMC as effector cells to each well at an E:T ratio of 10:1. Image the plates by fluorescence microscopy to monitor cell growth for 72 h. Quantify the degree of cell killing by comparing the fold growth ratio of fluorescent target cancer cells over time relative to their number at time 0. As shown in Figure 9, M1397 showed excellent potency against both tested antigen-positive cancer cell lines compared to comparator 2, and the potency was lower than that of comparator 1. In addition, compared to the two comparators, M1397 showed excellent safety against the SK-MEL-30 antigen-negative cancer cell line and a safety profile similar to that of the two comparators against the PANC-1 antigen-negative cancer cell line. Figures 10A to 10D ) and antigen-negative cell lines ( Figures 10E to 10J ), the dual adaptor induced lower levels of the pro-inflammatory cytokine IFNγ, indicating a lower likelihood of inducing cytokine storm syndrome.

[0406] Next, M1397, comparator 1, and comparator 2 were further compared in granzyme B and IFNγ release assays as a measure of T cell activation properties. Briefly, antigen-positive (NCI-H1703) and antigen-negative (SK-MEL-30) cancer cell lines were incubated with M1397, comparator 1, or comparator 2 at the indicated concentrations (ranging from 7.6 pM to 50 nM) and human PBMC (E:T ratio of 5:1). Granzyme B and IFNγ levels were measured after 24 h of incubation, and the corresponding results are shown in Figures 11A to B and C to D, respectively. Compared to the two comparators, M1397 showed an excellent safety profile with no granzyme B or IFNγ release when tested against the antigen-negative cell line SK-MEL-30. In addition, compared to comparator 2, M1397 induced higher granzyme B and IFNγ release against the antigen-positive cell line, and compared to comparator 1, it induced lower granzyme B and IFNγ release against both antigen-positive and antigen-negative cell lines.

[0407] As shown in Figure 12, IL-2, IL-6, and TNFα cytokine release was measured in MAGE-A4-positive HLA-A*02:01-positive U2OS and MAGE-A4-negative HLA-A*02:01-positive PANC-1 cells. Various concentrations of the dual adaptors M1397 and M1403 or comparator 1 were incubated with cancer cells and human PBMC (E:T ratio of 10:1). The cytokines IL-2, IL-6, and TNFα were measured after 24 h of incubation. The data indicate that both dual adaptors M1397 and M1403 induced lower levels of pro-inflammatory cytokines, indicating a lower likelihood of inducing cytokine storm syndrome.

[0408] Example 13 - In Vitro Safety of Optimized Dual pMHC-Targeted T Cell Adaptors

[0409] The potential off-target binding of the monovalent counterparts of M1397 and M1403, namely M1396 and M1402, to MAGE-A4-similar physiologically relevant peptides was investigated by SPR. Control peptides consisted of sequences with high identity to MAGE-A4 and had been previously identified in healthy human tissues, namely control 1 (GLADGRTHTV; SEQ ID NO: 68), control 2 (GLYDGPVHEV; SEQ ID NO: 69), and control 3 (GVFDGLHTV; SEQ ID NO: 70) (US20180171024, incorporated herein by reference). The corresponding data are presented in Table 8.

[0410] Table 8 - Binding of M1396 and M1402 to HLA-A*02:01 in Complex with Physiologically Relevant Control Peptides with High Identity to MAGE-A4

[0411]

[0412] To determine the potency and safety of M1397, comparator 1, or comparator 2, T cell activation was determined in the presence of the target HLA-A2 / MAGE-A4 or the physiologically relevant control peptide antigens HLA-A2 / control 1 and HLA-A2 / control 2. Briefly, TAP-deficient T2 cells were incubated overnight with serum-free RPMI 1640 medium containing the indicated concentrations (in the range of 0.1 nM to 1000 nM) of the peptide MAGE-A4 (SEQ ID NO: 3), control 1 (SEQ ID NO: 61), and control 2 (SEQ ID NO: 62). The cells were then washed in serum-free RPMI 1640 medium and co-incubated with PBMC (E:T 5:1) and M1397, comparator 1, or comparator 2 at a concentration of 1 nM for 24 h. T cell activation was determined by quantifying IFN-γ in the cell supernatant and is shown in FIG. 13. Consistently, M1397 showed an excellent T cell activation profile when tested on T2 cells pulsed with the target peptide MAGE-A4, with an EC50 of 5.75 nM, and showed a favorable safety window against the two control peptides tested, with IFNγ release observed only at the highest tested peptide concentration of 1 μM. Comparator 1 showed a secondary safety profile compared to M1397, with increased T cell activation in the presence of the off-target HLA-A2 / control 1 antigen, while comparator 2 showed secondary T cell activation characteristics compared to M1397, with an EC50 of 105.8 nM on T2 cells pulsed with the target peptide MAGE-A4.

[0413] To determine the safety of M1397, compound-induced T cell activation of a panel of various antigen-negative cancer cell lines was tested by determining granzyme B release. Briefly, the antigen-negative cancer cell lines KLE (endometrial cancer), LNCaP (prostate lymph node cancer), KMRC-2 (clear cell renal cell cancer), KMRC-3 (clear cell renal cell cancer), 639-V (urothelial bladder cancer), EKVX (lung adenocarcinoma), and HCT116 (colorectal cancer) were incubated with M1397, comparator 1, or comparator 2 at the indicated concentrations (in the range of 7.6 pM to 50 nM) and human PBMC (E:T ratio of 5:1). The antigen-positive cell line NCI-H1703 served as a positive control. Granzyme B levels were measured after 24 h incubation and the corresponding results are presented in FIG. 14. M1397 showed an excellent safety profile against all the antigen-negative cancer cell lines tested compared to the comparator 1 and comparator 2 molecules.

[0414] For in vitro safety experiments, various primary cell types and PBMCs from healthy human donors were co-cultured with the compounds for 24 h, and the supernatants were then analyzed for the release of granzyme B from effector cells in relation to cell-mediated cytotoxicity. Target cells included human cardiac microvascular endothelial cells (HMVEC-C), normal human bronchial epithelial cells (NHBE), and normal human astrocytes (NHA). Target cells were prepared in assay medium (RPMI 1640 containing 10% FBS and 1% penicillin-streptomycin) and plated at 20,000 cells / well in a 50 μL assay medium volume. PBMC effector cells were plated at 100,000 cells / well in a 50 μL assay medium volume. Different concentrations of compound M1397, M1403, or comparator 1 (covering the expected clinically relevant range) were added to the plated wells in a 15 μL assay volume. The final assay medium was made up to 150 μL per well. Positive control wells included ImmunoCult human CD3 / CD28 T cell activator. Negative control wells included target cells and effector cells alone or PBMCs alone with the maximum concentration of the compound. All reactions were performed in duplicate. The plates were incubated at 37 °C / 5% CO2 for 24 h. Supernatants were collected and analyzed by a human granzyme B ELISA kit (MabTech) according to the manufacturer's instructions. The corresponding results are shown in Figure 15. Compared to the soluble TCR bispecific comparator 1 molecule, M1397 and M1403 showed an excellent safety profile.

[0415] Further safety evaluation of M1397 relative to comparator 1 and comparator 2 was performed by testing a wider group of primary cells from various important tissues, namely human aortic smooth muscle cells (HAoSMC_735), human lung microvascular endothelial cells (HMVEC-L_73809), renal proximal tubular epithelial cells (RPTEC_49985 and RPTEC_82573), bronchial epithelial cells (NHBE_35497), normal human lung fibroblasts (NHLF_19232 and NHLF_76039), normal human astrocytes (NHA_72445), human cardiac myocytes (HCM_679, HCM_693, HCM_745, HCM_746), and human cardiac fibroblasts (HCF_251). The assay was performed substantially as described above, varying the compound concentration range (maximum test concentration 50 nM), and excluding the ImmunoCult human CD3 / CD28 T cell activator positive control. The data are shown in Figure 16. M1397 showed a favorable and excellent safety profile compared to the two comparators against the primary cells tested.

[0416] Example 14 - In Vivo Efficacy in a Cell Line-Derived Mouse NSCLC Xenograft Model

[0417] The anti-tumor activity of M1397 was evaluated at doses in the range of 0.5 to 5.0 mg / kg in a mouse cell line-derived xenograft model using the human NSCLC cell line NCI-H1703, which expresses MAGE-A4 and HLA-A*02:01.

[0418] Five x 10 6 cells of the human squamous NSCLC cell line NCI-H1703, which expresses MAGE-A4 and HLA-A*02:01, were implanted subcutaneously (SC) into immunodeficient female NCG mice. After the tumors reached an average size of 120 mm 3 , the mice were randomized and 1 x 10 7 human peripheral blood mononuclear cells (PBMCs) from two healthy untreated donors were implanted intraperitoneally. Treatment with M1397 was initiated the next day at intravenous doses in the range of 0.5 to 5 mg / kg / day for up to 28 days. Each PBMC donor and three mice in each treatment or vehicle (phosphate-buffered saline) group were evaluated twice weekly using calipers and body weight measurements.

[0419] Strong tumor growth inhibition was observed in all M1397 groups, with most mice showing complete tumor eradication ( Figure 17 ) at the end of the study. Moderate signs of graft-versus-host disease were noted after day 18 in all groups, including the vehicle control group. M1397 was well tolerated throughout the study and a constant body weight was observed.

[0420] Sequence

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

Claims

1. An antigen-binding protein that specifically binds to melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), the antigen-binding protein comprising: A variable heavy (VH) domain of an antibody, the variable heavy domain of the antibody comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and A variable light (VL) domain of an antibody, the variable light domain of the antibody comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:

15.

2. An antigen-binding protein that specifically binds to melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), the antigen-binding protein comprising: A VH domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 20, and A VL domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:

25.

3. An antigen-binding protein that specifically binds to melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), the antigen-binding protein comprising: A VH domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, wherein the VH domain comprises a C amino acid at position 44 of SEQ ID NO: 30, and A VL domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 35, wherein the VL domain comprises a C amino acid at position 102 of SEQ ID NO:

35.

4. An antigen-binding protein that specifically binds to melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), the antigen-binding protein comprising: A VH domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 40, wherein the VH domain comprises a Y amino acid at position 47 of SEQ ID NO: 40, an R amino acid at position 71, and an N amino acid at position 73, and A VL domain that comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:

45.

5. The antigen-binding protein according to any one of claims 1-4, wherein the MAGE-A4 pMHC complex is the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex.

6. The antigen-binding protein according to any one of the preceding claims, wherein the antigen-binding protein is a full-length immunoglobulin or an antibody fragment, such as Fab, Fab′, F(ab’)2, scFv, Fv fragment.

7. The antigen-binding protein according to any one of the preceding claims, wherein: the VH domain comprises the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), the HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6), wherein X1 corresponds to the amino acid S or D, X2 corresponds to the amino acid W or S, and X3 corresponds to the amino acid A or V, and the HCDR3 amino acid sequence of DLYYGPX4TX5YX6X7X8NL (SEQ ID NO: 7), wherein X4 corresponds to the amino acid T, N or S, X5 corresponds to the amino acid D or absent, X6 corresponds to the amino acid S or F, X7 corresponds to the amino acid A or V, and X8 corresponds to the amino acid F or A; and The VL domain contains the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and ATX9X 10 X 11 SGSNFQX 12 (SEQ ID NO: 8) of the LCDR3 amino acid sequence, where X9 corresponds to the amino acid S or R, X 10 corresponds to the amino acid D or P, X 11 corresponds to the amino acid G, S or F, and X 12 corresponds to the amino acid L or A.

8. The antigen-binding protein according to any one of the preceding claims, wherein the antigen-binding protein is linked to or combined with a functional entity such as a detectable label, a therapeutic agent or a PK modification moiety.

9. The antigen-binding protein according to any one of the preceding claims, wherein the antigen-binding protein is chemically or biologically modified.

10. The antigen-binding protein according to claim 9, wherein the antigen-binding protein is glycosylated, PEGylated, PASylated, XTENylated or HESylated.

11. The antigen-binding protein according to any one of claims 1-10, wherein the antigen-binding protein is linked to or combined with a functional entity such as a detectable label, a therapeutic agent or a PK modification moiety.

12. A chimeric antigen receptor (CAR) comprising the antigen-binding protein according to any one of the preceding claims.

13. An immune cell expressing the CAR according to claim 12.

14. The immune cell according to claim 13, wherein the immune cell is a T cell.

15. A multispecific antigen-binding protein comprising the antigen-binding protein according to any one of claims 1-11.

16. The multispecific antigen-binding protein according to claim 15, wherein the multispecific antigen-binding protein is bispecific or trispecific.

17. The multispecific antigen-binding protein according to any one of claims 15 or 16, wherein the multispecific antigen-binding protein further comprises at least one other binding domain.

18. The multispecific antigen-binding protein according to claim 17, wherein the other binding domain is an immune cell adaptor.

19. The multispecific antigen-binding protein according to claim 18, wherein the immune cell linker is a CD3-binding domain or a CD16a-binding domain.

20. The multispecific antigen-binding protein according to any one of claims 15-19, wherein the multispecific antigen-binding protein further comprises a third antigen-binding domain.

21. The multispecific antigen-binding protein according to claim 20, wherein the third antigen-binding domain binds to HLA-A*02 / MAGE-A4, particularly the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex.

22. The multispecific antigen-binding protein according to claim 20 or 21, wherein the third antigen-binding domain is identical to the first antigen-binding domain.

23. The multispecific antigen-binding protein according to any one of claims 20-22, wherein the third antigen-binding domain comprises: a variable heavy (VH) domain of an antibody, the variable heavy domain of the antibody comprising the amino acid sequence of HCDR1 of SNYA MS (SEQ ID NO: 11), the amino acid sequence of HCDR2 of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6), wherein X1 corresponds to the amino acid S or D, X2 corresponds to the amino acid W or S, and X3 corresponds to the amino acid A or V, and the amino acid sequence of HCDR3 of DLYYGPX4TX5YX6X7X8NL (SEQ ID NO: 7), wherein X4 corresponds to the amino acid T, N or S, X5 corresponds to the amino acid D or is absent, X6 corresponds to the amino acid S or F, X7 corresponds to the amino acid A or V, and X8 corresponds to the amino acid F or A; and Antibody light chain variable (VL) domain, said antibody light chain variable domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and the LCDR3 amino acid sequence of ATX9X 10 X 11 SGSNFQX 12 (SEQ ID NO: 8), wherein X9 corresponds to amino acid S or R, X 10 corresponds to amino acid D or P, X 11 corresponds to amino acid G, S or F, and X 12 corresponds to amino acid L or A.

24. The multispecific antigen-binding protein according to any one of claims 20-23, wherein the third antigen-binding domain comprises: i) a variable heavy (VH) domain of an antibody, the variable heavy domain of the antibody comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a variable light (VL) domain of an antibody, the variable light domain of the antibody comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 15; ii) a VH domain, the VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 20, and a VL domain, the VL domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 25; iii) a VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 30, and a VL domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 35; or iv) a VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 40, and a VL domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO:

45.

25. A multispecific antigen-binding protein, the multispecific antigen-binding protein comprising: a) a first antigen-binding domain that specifically binds to CD3; b) a second antigen-binding domain that specifically binds to melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), the second antigen-binding domain comprising: b1) an antibody heavy chain variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), the HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6), wherein X1 corresponds to the amino acid S or D, X2 corresponds to the amino acid W or S, and X3 corresponds to the amino acid A or V, and the HCDR3 amino acid sequence of DLYYGPX4TX5YX6X7X8NL (SEQ ID NO: 7), wherein X4 corresponds to the amino acid T, N or S, X5 corresponds to the amino acid D or is absent, X6 corresponds to the amino acid S or F, X7 corresponds to the amino acid A or V, and X8 corresponds to the amino acid F or A; and b2) The variable (VL) domain of an antibody light chain, said variable domain of an antibody light chain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and ATX9X 10 X 11 SGSNFQX 12 (SEQ ID NO: 8) of the LCDR3 amino acid sequence, where X9 corresponds to the amino acid S or R, X 10 corresponds to the amino acid D or P, X 11 corresponds to the amino acid G, S or F, and X 12 corresponds to the amino acid L or A; and c) a third antigen-binding domain that specifically binds to MAGE-A4 pMHC, the third antigen-binding domain comprising: c1) a VH domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), the HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6), wherein X1 corresponds to the amino acid S or D, X2 corresponds to the amino acid W or S, and X3 corresponds to the amino acid A or V, and the HCDR3 amino acid sequence of DLYYGPX4TX5YX6X7X8NL (SEQ ID NO: 7), wherein X4 corresponds to the amino acid T, N or S, X5 corresponds to the amino acid D or is absent, X6 corresponds to the amino acid S or F, X7 corresponds to the amino acid A or V, and X8 corresponds to the amino acid F or A; and c2) VL domain, said VL domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and ATX9X 10 X 11 SGSNFQX 12 (SEQ ID NO: 8) of the LCDR3 amino acid sequence, wherein X9 corresponds to the amino acid S or R, X 10 corresponds to the amino acid D or P, X 11 corresponds to the amino acid G, S or F, and X 12 corresponds to the amino acid L or A.

26. The multispecific antigen-binding protein according to claim 25, wherein the second antigen-binding domain and the third antigen-binding domain comprise: VH, wherein the VH comprises an HCDR1 sequence having an amino acid sequence containing SNYAMS (SEQ ID NO: 11), an HCDR2 sequence having an amino acid sequence containing IVSSGGTTYYADSVKG (SEQ ID NO: 12), and an HCDR3 sequence having an amino acid sequence containing DLYYGPNTDYSAANL (SEQ ID NO: 13); and VL, wherein the VL comprises an LCDR1 sequence having an amino acid sequence containing TADTLSRSYAS (SEQ ID NO: 16), an LCDR2 sequence having an amino acid sequence containing RDTSRPS (SEQ ID NO: 17), and an LCDR3 sequence having an amino acid sequence containing ATRPSSGSNFQA (SEQ ID NO: 18).

27. The multispecific antigen-binding protein according to claim 26, wherein the second antigen-binding domain and the third antigen-binding domain comprise: a variable heavy (VH) domain of an antibody, the variable heavy domain of the antibody comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a variable light (VL) domain of an antibody, the variable light domain of the antibody comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO:

15.

28. The multispecific antigen-binding protein according to claim 25, wherein the second antigen-binding domain and the third antigen-binding domain comprise: VH, wherein the VH comprises an HCDR1 sequence having an amino acid sequence containing SNYAMS (SEQ ID NO: 21), an HCDR2 sequence having an amino acid sequence containing IVSSGGTTYYADSVKG (SEQ ID NO: 22), and an HCDR3 sequence having an amino acid sequence containing DLYYGPSTYFVANL (SEQ ID NO: 23); and VL, wherein the VL comprises an LCDR1 sequence having an amino acid sequence containing TADTLSRSYAS (SEQ ID NO: 26), an LCDR2 sequence having an amino acid sequence containing RDTSRPS (SEQ ID NO: 27), and an LCDR3 sequence having an amino acid sequence containing ATRPSSGSNFQL (SEQ ID NO: 28).

29. The multispecific antigen-binding protein according to claim 28, wherein the second antigen-binding domain and the third antigen-binding domain comprise: a VH domain, the VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 20, and a VL domain, said VL domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO:

25.

30. The multispecific antigen-binding protein according to claim 25, wherein said second antigen-binding domain and said third antigen-binding domain comprise: a VH, said VH comprising an HCDR1 sequence having an amino acid sequence containing SNYAMS (SEQ ID NO: 31), an HCDR2 sequence having an amino acid sequence containing IVSSGGTTYYASWAKG (SEQ ID NO: 32), and an HCDR3 sequence having an amino acid sequence containing DLYYGPTTYSAANL (SEQ ID NO: 33); and a VL, said VL comprising an LCDR1 sequence having an amino acid sequence containing TADTLSRSYAS (SEQ ID NO: 36), an LCDR2 sequence having an amino acid sequence containing RDTSRPS (SEQ ID NO: 37), and an LCDR3 sequence having an amino acid sequence containing ATRDFSGSNFQL (SEQ ID NO: 38).

31. The multispecific antigen-binding protein according to claim 30, wherein said second antigen-binding domain and said third antigen-binding domain comprise: a VH domain, said VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 30, and a VL domain, said VL domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO:

35.

32. The multispecific antigen-binding protein according to claim 25, wherein said second antigen-binding domain and said third antigen-binding domain comprise: a VH, said VH comprising an HCDR1 sequence having an amino acid sequence containing SNYAMS (SEQ ID NO: 41), an HCDR2 sequence having an amino acid sequence containing IVSSGGTTYYASWAKG (SEQ ID NO: 42), and an HCDR3 sequence having an amino acid sequence containing DLYYGPTTYSAFNL (SEQ ID NO: 43); and a VL, said VL comprising an LCDR1 sequence having an amino acid sequence containing TADTLSRSYAS (SEQ ID NO: 46), an LCDR2 sequence having an amino acid sequence containing RDTSRPS (SEQ ID NO: 47), and an LCDR3 sequence having an amino acid sequence containing ATRPSSGSNFQA (SEQ ID NO: 48).

33. The multispecific antigen-binding protein according to claim 25, wherein said second antigen-binding domain and said third antigen-binding domain comprise: A VH domain, wherein the VH domain comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 40, and A VL domain, wherein the VL domain comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO:

45.

34. The multispecific antigen-binding protein according to any one of claims 15-33, wherein any one or more of the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain comprises an antibody fragment.

35. The multispecific antigen-binding protein according to claim 34, wherein the antibody fragment comprises a Fab fragment, an F(ab′)2 fragment, a Fab′ fragment, an Fv fragment, a single-chain variable fragment (scFv), and a single-domain antibody fragment.

36. The multispecific antigen-binding protein according to any one of claims 15-35, wherein the immune cell or CD3 antigen-binding domain is a Fab fragment, and the Fab fragment comprises a heavy chain containing a CH1 domain and the VH, and a light chain containing a CL domain and the VL.

37. The multispecific antigen-binding protein according to claim 35 or 36, wherein the MAGE-A4 pMHC antigen-binding domain comprises an scFv.

38. The multispecific antigen-binding protein according to claim 35 or 36, wherein the CH1 domain comprises at least 5 amino acids of the antibody hinge region.

39. The multispecific antigen-binding protein according to claim 38, wherein the CH1 domain comprises the amino acid sequence EPKSC of the antibody hinge region.

40. The multispecific antigen-binding protein according to any one of claims 36-39, wherein the second antigen-binding domain is operably linked to the C-terminus or the N-terminus of the heavy chain of the Fab fragment.

41. The multispecific antigen-binding protein according to any one of claims 36-40, wherein the third antigen-binding domain is operably linked to the C-terminus or the N-terminus of the heavy chain of the Fab fragment.

42. The multispecific antigen-binding protein according to any one of claims 36-41, wherein: a) the second antigen-binding domain comprises an scFv linked to the C-terminus of the heavy chain of the Fab domain and the third antigen-binding domain comprises an scFv linked to the C-terminus of the light chain of the Fab domain; b) the second antigen-binding domain comprises an scFv linked to the N-terminus of the heavy chain of the Fab domain and the third antigen-binding domain comprises an scFv linked to the N-terminus of the light chain of the Fab domain; c) the second antigen-binding domain comprises an scFv linked to the N-terminus of the heavy chain of the Fab domain and the third antigen-binding domain comprises an scFv linked to the C-terminus of the light chain of the Fab domain; or d) The second antigen-binding domain comprises an scFv linked to the C-terminus of the heavy chain of the Fab domain and the third antigen-binding domain comprises an scFv linked to the N-terminus of the light chain of the Fab domain.

43. The multispecific antigen-binding protein according to claim 42, wherein the scFv is linked to the Fab domain by an amino acid linker.

44. The multispecific antigen-binding protein according to claim 43, wherein the amino acid linker comprises (GGGGS)n, where n is an integer between 1 and 5.

45. The multispecific antigen-binding protein according to claim 43 or 44, wherein the amino acid linker comprises the amino acid sequence GGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGAS or GGGGGGSGGGGSGGGGSGGGGS.

46. The multispecific antigen-binding protein according to any one of claims 25-45, wherein the VH and VL of the second antigen-binding domain and / or the VH and VL of the third antigen-binding domain are linked by an amino acid linker.

47. The multispecific antigen-binding protein according to claim 46, wherein the amino acid linker comprises (GGGGS)n, where n is an integer between 1 and 5.

48. The multispecific antigen-binding protein according to claim 46 or 47, wherein the amino acid linker comprises the amino acid sequence GGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGAS or GGGGGGSGGGGSGGGGSGGGGS.

49. The multispecific antigen-binding protein according to any one of claims 15-48, wherein the multispecific antigen-binding protein does not comprise an Fc domain.

50. The multispecific antigen-binding protein according to any one of claims 15-49, the multispecific antigen-binding protein comprises (scFv)2, (scFv)3, BiTE, BIKE, Dart, diabody, triabody, Fab2, Fab3, Fab4, scFv-Fab-scFv or minibody-scFv.

51. The multispecific antigen-binding protein according to any one of claims 15-50, the multispecific antigen-binding protein has a molecular weight of about 75 kDa to about 110 kDa.

52. The multispecific antigen-binding protein according to claim 51, wherein the antigen-binding protein has an extended serum half-life relative to an antigen-binding protein having a molecular weight of < about 75 kDa.

53. The multispecific antigen-binding protein according to any one of claims 15-52, wherein the CD3 antigen-binding domain comprises: a1) VH, wherein the VH comprises an HCDR1 sequence having an amino acid sequence containing STYAMN (SEQ ID NO: 51), an HCDR2 sequence having an amino acid sequence containing RIRSKYNNYATYYADSVKG (SEQ ID NO: 52), and an HCDR3 sequence having an amino acid sequence containing HGNFGDSYVSWFAY (SEQ ID NO: 53); and a2) VL, wherein the VL comprises an LCDR1 sequence having an amino acid sequence containing GSSTGAVTTSNYAN (SEQ ID NO: 56), an LCDR2 sequence having an amino acid sequence containing GTNKRAP (SEQ ID NO: 57), and an LCDR3 sequence having an amino acid sequence containing ALWYSNHWV (SEQ ID NO: 58).

54. The multispecific antigen-binding protein according to any one of claims 15-53, wherein the CD3 antigen-binding domain comprises: - VH, wherein the VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 50, and - VL, wherein the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:

55.

55. The multispecific antigen-binding protein according to claim 54, wherein the CD3 antigen-binding domain comprises: - VH, wherein the VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and - VL, wherein the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:

54.

56. The multispecific antigen-binding protein according to any one of claims 34-55, wherein the second antigen-binding domain and / or the third antigen-binding domain comprises a variable heavy chain having polar amino acids at positions 11, 89, and / or 108 according to Kabat numbering.

57. The multispecific antigen-binding protein according to any one of claims 34-56, wherein the Fab domain comprises a variable heavy chain having polar amino acids at positions 11, 89, and / or 108 according to Kabat numbering.

58. The multispecific antigen-binding protein according to claim 56 or 57, wherein the variable heavy chain comprises: Leucine (L) or serine (S) at amino acid position 11 according to Kabat numbering; Valine (V), serine (S), or threonine (T) at amino acid position 89 according to Kabat numbering; and / or Leucine (L), serine (S), or threonine (T) at amino acid position 108 according to Kabat numbering.

59. The multispecific antigen-binding protein according to claim 57 or 58, wherein the polar amino acids are serine (S) and / or threonine (T).

60. The multispecific antigen-binding protein according to any one of claims 57-59, wherein the variable heavy chain comprises serine (S) at amino acid position 11, serine (S) or threonine (T) at amino acid position 89, and serine (S) or threonine (T) at amino acid position 108 according to Kabat numbering.

61. The multispecific antigen-binding protein according to any one of claims 57-60, wherein the variable heavy chain comprises serine (S) at amino acid position 11, serine (S) at amino acid position 89, and serine (S) at amino acid position 108 according to Kabat numbering.

62. A multispecific antigen-binding protein that binds to the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex and CD3, the multispecific antigen-binding protein comprising: (i) a first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 9, and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14; (ii) a first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 19 or a variant thereof that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 19, and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 24 or a variant thereof that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24; (iii) a first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 29 or a variant thereof that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29, and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 34 or a variant thereof that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 34; or (iv) A first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 39 or a variant thereof, the variant being at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 39, and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 44 or a variant thereof, the variant being at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

44.

63. A multispecific antigen-binding protein, the multispecific antigen-binding protein comprising: a) A first antigen-binding domain that specifically binds to CD3, the first antigen-binding domain comprising: a1) VH, the VH comprising an HCDR1 sequence having the amino acid sequence containing STYAMN (SEQ ID NO: 51), an HCDR2 sequence having the amino acid sequence containing RIRSKYNNYATYYADSVKG (SEQ ID NO: 52), and an HCDR3 sequence having the amino acid sequence containing HGNFGDSYVSWFAY (SEQ ID NO: 53); and a2) VL, the VL comprising an LCDR1 sequence having the amino acid sequence containing GSSTGAVTTSNYAN (SEQ ID NO: 56), an LCDR2 sequence having the amino acid sequence containing GTNKRAP (SEQ ID NO: 57), and an LCDR3 sequence having the amino acid sequence containing ALWYSNHWV (SEQ ID NO: 58); b) A second antigen-binding domain that specifically binds to melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC).

64. The multispecific antigen-binding protein according to claim 63, wherein the second antigen-binding domain comprises: b1) An antibody heavy chain variable (VH) domain, the antibody heavy chain variable domain comprising an HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), an HCDR2 amino acid sequence of IVSSGGTTYYA X1X2X3KG (SEQ ID NO: 6), wherein X1 corresponds to the amino acid S or D, X2 corresponds to the amino acid W or S, and X3 corresponds to the amino acid A or V, and an HCDR3 amino acid sequence of DLYYGPX4TX5YX6X7X8NL (SEQ ID NO: 7), wherein X4 corresponds to the amino acid T, N or S, X5 corresponds to the amino acid D or is absent, X6 corresponds to the amino acid S or F, X7 corresponds to the amino acid A or V, and X8 corresponds to the amino acid F or A; and b2) An antibody light chain variable (VL) domain, said antibody light chain variable domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and an LCDR3 amino acid sequence of ATX9X 10 X 11 SGSNFQX 12 (SEQ ID NO: 8), wherein X9 corresponds to the amino acid S or R, X 10 corresponds to the amino acid D or P, X 11 corresponds to the amino acid G, S or F, and X 12 corresponds to the amino acid L or A.

65. The multispecific antigen-binding protein according to claim 63 or 64, wherein the first antigen-binding domain comprises: - VH, the VH comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 50, and -VL, wherein the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:

55.

66. The multispecific antigen-binding protein according to claim 65, wherein the first antigen-binding domain comprises: -VH, wherein the VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and -VL, wherein the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:

54.

67. The multispecific antigen-binding protein according to any one of claims 63-66, further comprising a third antigen-binding domain.

68. The multispecific antigen-binding protein according to any one of claims 63-67, comprising: c) a third antigen-binding domain that specifically binds to MAGE-A4 pMHC, the third antigen-binding domain comprising: c1) A VH domain, said VH domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), the HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6), wherein X1 corresponds to the amino acid S or D, X2 corresponds to the amino acid W or S, and X3 corresponds to the amino acid A or V, and the HCDR3 amino acid sequence of DLYYGPX4TX5YX6X7X8NL (SEQ ID NO: 7), wherein X4 corresponds to the amino acid T, N or S, X5 corresponds to the amino acid D or absent, X6 corresponds to the amino acid S or F, X7 corresponds to the amino acid A or V, and X8 corresponds to the amino acid F or A; and c2) A VL domain, said VL domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17) and 10 X 11 SGSNFQX 12 (SEQ ID NO: 8) of the LCDR3 amino acid sequence, wherein X9 corresponds to the amino acid S or R, X 10 corresponds to the amino acid D or P, X 11 corresponds to the amino acid G, S or F, and X 12 corresponds to the amino acid L or A.

69. The multispecific antigen-binding protein according to any one of claims 15-68, wherein the MAGE-A4 peptide-MHC (pMHC) is the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex.

70. The multispecific antigen-binding protein according to any one of claims 15-68, which remains at least 94%, 95%, 96%, 97%, 98%, 99% or 100% monomeric after incubation in PBS at 4°C for 14 days at 1 mg / ml and / or 10 mg / ml as determined by SEC-HPLC.

71. The multispecific antigen-binding protein according to any one of claims 15-68, which shows efficacy against target positive tumor cells as determined by LDH cytotoxicity assay.

72. The multispecific antigen-binding protein according to any one of claims 15-68, which shows tumor growth inhibition and tumor eradication in a cell line-derived murine NSCLC xenograft model.

73. The multispecific antigen-binding protein according to any one of claims 15-72, which is chemically or biologically modified.

74. The multispecific antigen-binding protein according to claim 53, which is glycosylated, PEGylated, HESylated, PASylated or XTENylated.

75. The multispecific antigen-binding protein according to any one of claims 15-74, which is linked to or combined with a functional entity such as a detectable label, a therapeutic agent or a PK modification moiety.

76. The multispecific antigen-binding protein according to claim 75, wherein the functional entity is a toxin.

77. The multispecific antigen-binding protein according to any one of claims 15-76, wherein the light chain and / or the heavy chain comprises an N-terminal and / or C-terminal truncation of 1, 2, 3, 4 or 5 amino acids.

78. The multispecific antigen-binding protein according to claim 77, wherein the light chain comprises an N-terminal truncation of 1 or 2 amino acids.

79. The multispecific antigen-binding protein according to any one of claims 15-76, wherein the multispecific antigen-binding protein comprises pyroglutamic acid (pE) instead of glutamine (Q) or glutamic acid (E) at position 1 of the light chain and / or the heavy chain.

80. The multispecific antigen-binding protein according to claim 79, wherein the multispecific antigen-binding protein comprises pyroglutamic acid (pE) instead of glutamine (Q) or glutamic acid (E) at position 1 of the light chain.

81. The antigen-binding protein according to any one of claims 1-11, wherein the antigen-binding protein is for diagnosis.

82. The multispecific antigen-binding protein according to any one of claims 15-80, wherein the multispecific antigen-binding protein is used in a method for inhibiting cancer cell growth or proliferation.

83. The multispecific antigen-binding protein according to any one of claims 15-80, wherein the multispecific antigen-binding protein is used in a method for redirecting T cells to cancer cells expressing MAGE-A4.

84. The antigen-binding protein according to any one of claims 1-11, the CAR according to claim 12, the immune cell according to claims 13-14, or the multispecific antigen-binding protein according to any one of claims 15-80, wherein the antigen-binding protein, the CAR, the immune cell, or the multispecific antigen-binding protein is used as a medicament.

85. The antigen-binding protein according to any one of claims 1-11, the CAR according to claim 12, the immune cell according to claims 13-14, or the multispecific antigen-binding protein according to any one of claims 15-80, wherein the antigen-binding protein, the CAR, the immune cell, or the multispecific antigen-binding protein is used for the treatment of diseases, particularly cancer.

86. A nucleic acid encoding the antigen-binding protein according to any one of claims 1-11, the CAR according to claim 12, or the multispecific antigen-binding protein according to any one of claims 15-80.

87. A vector comprising the nucleic acid according to claim 86.

88. A population of host cells comprising the vector according to claim 87.

89. A kit comprising the antigen-binding protein according to any one of claims 1-11 or the multispecific antigen-binding protein according to any one of claims 15-80.

90. A method for producing the antigen-binding protein according to any one of claims 1-11 or the multispecific antigen-binding protein according to any one of claims 15-80, the method comprising the steps of: (i) culturing the host cell according to claim 87 under conditions allowing expression of the antigen-binding protein or the multispecific antigen-binding protein (ii) recovering the antigen-binding protein or the multispecific antigen-binding protein; and optionally (iii) Further purify and / or modify and / or formulate the antigen-binding protein or the multispecific antigen-binding protein.

91. A pharmaceutical composition comprising the antigen-binding protein according to any one of claims 1-11, the CAR according to claim 12, the immune cell according to claim 13 or 14, or the multispecific antigen-binding protein according to any one of claims 15-80, and a pharmaceutically acceptable buffer.

92. Use of the antigen-binding protein according to any one of claims 1-11, the CAR according to claim 12, the immune cell according to claim 13 or 14, the multispecific antigen-binding protein according to any one of claims 15-80, or the pharmaceutical composition according to claim 66 in the manufacture of a medicament.

93. The antigen-binding protein according to any one of claims 1-11, the CAR according to claim 12, the immune cell according to claim 13 or 14, the multispecific antigen-binding protein according to any one of claims 15-80, or the pharmaceutical composition according to claim 66, wherein the antigen-binding protein, the CAR, the immune cell, the multispecific antigen-binding protein, or the pharmaceutical composition is used as a medicament.

94. Use of the antigen-binding protein according to any one of claims 1-11, the CAR according to claim 12, the immune cell according to claim 13 or 14, the multispecific antigen-binding protein according to any one of claims 15-80, the cell according to claim 88, or the pharmaceutical composition according to claim 91 in the treatment of a disease, particularly cancer.

95. The antigen-binding protein according to any one of claims 1-11, the CAR according to claim 12, the immune cell according to claim 13 or 14, the multispecific antigen-binding protein according to any one of claims 15-80, the cell according to claim 88, or the pharmaceutical composition according to claim 91, wherein the antigen-binding protein, the CAR, the immune cell, the multispecific antigen-binding protein, the cell, or the pharmaceutical composition is used for treating a disease, particularly cancer.

96. A method for treating cancer expressing MAGE-A4 pMHC in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the antigen-binding protein according to any one of claims 1-11, the CAR according to claim 12, the immune cell according to claim 13 or 14, the multispecific antigen-binding protein according to any one of claims 15-80, the cell according to claim 88, or the pharmaceutical composition according to claim 91.

97. The use according to claim 94, the antigen-binding protein according to claim 95 or the method according to claim 96, wherein the cancer is selected from the group consisting of: head and neck squamous cell carcinoma (HNSC), non-small cell lung cancer (NSCLC), triple-negative breast cancer, urothelial carcinoma, high-grade endometrial cancer, including uterine carcinosarcoma (UCS; particularly the UCEC subgroup), myxoid / round cell liposarcoma, gastric or gastroesophageal junction (GEJ) adenocarcinoma, epithelial ovarian cancer, such as high-grade serous ovarian cancer, synovial sarcoma, bladder urothelial carcinoma (BLCA), particularly metastatic cell carcinoma, testicular germ cell tumor (TGCT), and cervical squamous cell carcinoma (CESC).

98. The use according to claim 97, the antigen-binding protein according to claim 97 or the method according to claim 97, wherein the cancer is of squamous origin, such as head and neck squamous cell carcinoma (HNSCC) or squamous NSCLC.

99. A method of selecting a patient suitable for treatment with a MAGE-A4 antagonist, the method comprising the following steps carried out in sequence: (i) obtaining a tumor sample from the patient; (ii) adding an anti-MAGEA4 detection antibody to the sample; (iii) incubating the detection antibody and the sample; (iv) detecting the detection antibody bound to the sample; and (v) if the detection antibody is bound by the sample, then selecting the patient for treatment with a MAGE-A4 antagonist.

100. The method according to claim 99, wherein the detection antibody is OTI1F9, E7O1U or the antigen-binding protein according to any one of claims 1-11.

101. The method according to claim 99 or 100, the method further comprising the step of performing RNA sequencing to detect total MAGE-A4.

102. The method according to any one of claims 99 to 101, wherein in the treatment of the disease, particularly cancer, the MAGE-A4 antagonist is the antigen-binding protein according to any one of claims 1-11, the CAR according to claim 12, the immune cell according to claim 13 or 14, the multispecific antigen-binding protein according to any one of claims 15-80, the cell according to claim 88 or the pharmaceutical composition according to claim 91.

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