MAGE-A4 Peptide Dual T Cell Engager

Abstract

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

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 406,475, filed September 14, 2022, the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] Melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC) expression is present in many cancers, including non-small cell lung cancer (NSCLC), melanoma, bladder, head and neck, and gastroesophageal cancer (Grossman et al. N Engl J Med. 2016.375:1109-1112). Therefore, it is an attractive target for TCR-based T cell therapy. Unfortunately, TCR molecules have low binding affinity to pMHC targets. Furthermore, TCR-based T cell therapies are laborious and expensive to develop and use. In contrast, isolated monoclonal antibodies (mAbs) have extremely high binding affinity to these targets, potentially reducing off-target activity. However, generating mAbs against pMHC targets is challenging due to the small size of the binding peptide epitope in HLA.

[0003] Therefore, there is a need in the art for novel antigen binding proteins that specifically recognize the target MAGE-A4 pMHC with high affinity while retaining high specificity (i.e., low or no off-target effects on healthy tissue), possess favorable drug-like properties, and can be produced in sufficient quantities and quality at a reasonable cost. Summary of the Invention

[0004] 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 present invention have improved binding affinity to target MAGE-A4 pMHC and / or improved stability, and reduced 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 (e.g., 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, the first pMHC-binding domain binding to a first target peptide-MHC (pMHC) complex; and c) at least a second pMHC-binding domain operably linked to the light chain, the second pMHC-binding domain binding to a second pMHC complex. Bivalent targeting of pMHC using the bispecific antigen binding proteins of the invention increases cancer cell killing compared to their monovalent counterparts, and the overall specificity towards cells bearing the same HLA but not expressing the target protein is not substantially affected.

[0005] Preferably, the antigen-binding proteins of the present invention lack an Fc domain and are therefore not recognized by Fc receptors on effector cells, such as inhibitory receptors FcγRIII or FcγRIIb on macrophages and activated neutrophils, or the FcγRIIa complex on non-cytotoxic cells such as platelets and B cells. In the case of bispecific T cell engagers, Fc-mediated immune function is undesirable to avoid antigen-independent cytokine release syndrome (CRS), which results from nonspecific activation of immune cells following cross-linking of CD3 and Fcγ receptors. Rather, the Fab domain of the antigen-binding protein serves as a specific heterodimerization scaffold to which additional pMHC-binding domains are attached. The spontaneous 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 additional binding domain may be in several different formats, including, but not limited to, another Fab domain, an scFv, or an sdAb. Furthermore, in certain situations, antigen-binding proteins containing Fc may be disadvantageous due to their longer half-life. A longer half-life may lead to increased toxicity due to, among other things, excessive cytokine release from immune cells. A longer half-life may also promote T cell exhaustion. Antigen-binding proteins of the present disclosure lacking an Fc domain may have reduced cytotoxicity, in part due to their shorter half-life compared to antigen-binding proteins containing Fc.

[0006] In addition to being highly specific for their targets, the antigen binding proteins of the present invention exhibit favorable drug-like properties such as intrinsic stability and / or general physical and chemical stability.

[0007] In one aspect, the disclosure provides an antigen binding protein that specifically binds to melanoma associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), 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) 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: 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 VL 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.

[0008] In certain embodiments, the MAGE-A4 pMHC complex is a GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex. In certain embodiments, the MAGE-A4 pMHC complex is a GVYDGREHTV (SEQ ID NO: 3) HLA-A*02:01 complex.

[0009] In certain embodiments, the antigen binding protein maintains at least 94%, e.g., 94%, 95%, 96%, 97%, 98%, or 99% monomeric content at a concentration of about 1 mg / mL (e.g., 0.90-1.10 mg / mL) to about 10 mg / mL (e.g., 9.9-10.1 mg / mL) during storage in PBS at 4°C for at least two weeks, as determined by SEC-HPLC. In certain embodiments, the antigen binding protein maintains at least 80% monomeric content at a concentration of about 1 mg / mL (e.g., 0.90-1.10 mg / mL) to about 10 mg / mL (e.g., 9.9-10.1 mg / mL) during storage in PBS at 37°C for at least two weeks, as determined by SEC-HPLC. In certain embodiments, the antigen binding protein maintains at least 95% monomericity at a concentration of about 1 mg / mL (e.g., 0.90-1.10 mg / mL) to about 10 mg / mL (e.g., 9.9-10.1 mg / mL) during storage in PBS at 37°C for at least two weeks, as determined by SEC-HPLC. In certain embodiments, the antigen binding protein maintains at least 94%, e.g., 94%, 95%, 96%, 97%, 98%, or 99% monomericity at a concentration of about 1 mg / mL and about 10 mg / mL during storage in PBS at 4°C for at least two weeks, as determined by SEC-HPLC.

[0010] In certain embodiments, the antigen binding protein comprises a full-length immunoglobulin or antibody fragment, such as a Fab, Fab', F(ab')2, scFv or Fv fragment.

[0011] In a specific embodiment, a VH domain comprising an HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), an 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 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) is used in combination with a VH 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 HCDR3 amino acid sequence of ATX9X 10 X 11 SGSNFQX 12 (SEQ ID NO: 8) wherein X corresponds to the amino acid S or R, and X 10 corresponds to the amino acid D or P, and X 11 corresponds to the amino acid G, S, or F, and X 12 corresponds to amino acid L or A).

[0012] In certain embodiments, the antigen binding protein is linked to or combined with a detectable label, a therapeutic agent, or a PK-modifying moiety.

[0013] In certain embodiments, the antigen-binding protein is chemically or biologically modified, hi certain embodiments, the antigen-binding protein is glycosylated, PEGylated, PAS-ylated, XT-ylated, or HES-ylated.

[0014] In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising the antigen binding protein described above.

[0015] In another aspect, the present disclosure provides an immune cell that expresses the CAR described above. In certain embodiments, the immune cell is a T cell.

[0016] In another aspect, the present disclosure provides a multispecific antigen-binding protein comprising the antigen-binding protein described above.

[0017] In certain embodiments, the multispecific antigen binding protein is bispecific or trispecific.

[0018] In certain embodiments, the multispecific antigen-binding protein further comprises at least one additional binding domain, hi certain embodiments, the additional binding domain is an immune cell engager, in particular a CD3 binding domain or a CD16a binding domain.

[0019] 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 identical to the first MAEG-A4 pMHC antigen-binding domain defined above.

[0020] In a specific embodiment, the third antigen-binding domain comprises an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), an 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 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). 10 X 11 SGSNFQX 12(SEQ ID NO: 8) wherein X corresponds to the amino acid S or R, and X 10 corresponds to the amino acid D or P, and X 11 corresponds to the amino acid G, S, or F, and X 12 and an antibody light chain variable (VL) domain comprising:

[0021] In certain embodiments, the third antigen-binding domain comprises: i) an antibody heavy chain variable (VH) domain comprising an amino acid sequence 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 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 at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 20, and an antibody light chain variable (VL) domain comprising an amino acid sequence 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.

[0022] In one aspect, the disclosure provides a multispecific antigen binding protein comprising: a) a first antigen binding domain that specifically binds to CD3; and b) a second antigen binding domain that specifically binds to melanoma associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), wherein b1) 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. 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 heavy chain variable (VH) 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 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). 10 X 11 SGSNFQX 12 (SEQ ID NO: 8) wherein X corresponds to the amino acid S or R, and X 10 corresponds to the amino acid D or P, and X 11 corresponds to the amino acid G, S, or F, and X 12 and a second antigen-binding domain comprising an antibody light chain variable (VL) domain comprising the amino acid L(A) corresponding to amino acid L or A).

[0023] In a specific embodiment, the MAGE-A4 peptide-MHC (pMHC) is the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex.

[0024] In certain embodiments, the multispecific antigen binding protein remains at least 94%, 95%, 96%, 97%, 98%, 99% or 100% monomeric after incubation in PBS at 4°C for 14 days as determined by SEC-HPLC.

[0025] In certain embodiments, the multispecific antigen-binding protein further comprises a third antigen-binding domain, in particular, said third binding domain is identical to said second binding domain.

[0026] In certain embodiments, the multispecific antigen binding protein comprises: c) MAGE-A4 and a third antigen-binding domain that specifically binds to pMHC, the third antigen-binding domain comprising: c1) a VH domain comprising an HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), an HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6) (wherein 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) (wherein X4 corresponds to amino acid T, N, or S, X5 corresponds to amino acid D or is 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 VH 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 HCDR3 amino acid sequence of ATX9X 10 X 11 SGSNFQX 12 (SEQ ID NO: 8) wherein X corresponds to the amino acid S or R, and X 10 corresponds to the amino acid D or P, and X 11 corresponds to the amino acid G, S, or F, and X 12 and a VL domain containing the amino acid L or A).

[0027] In certain embodiments, the second and third antigen-binding domains comprise: (i) a VH comprising an HCDR1 sequence comprising the amino acid sequence of SNYAMS (SEQ ID NO: 11), an HCDR2 sequence comprising the amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 12), and an HCDR3 sequence comprising the amino acid sequence of DLYYGPNTDYSAANL (SEQ ID NO: 13), and a LCDR1 sequence comprising the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), an LCDR2 sequence comprising the amino acid sequence of RDTSRPS (SEQ ID NO: 17), and an HCDR3 sequence comprising the amino acid sequence of ATRPSSGSNFQ A (SEQ ID NO: 18), (ii) a VH comprising an HCDR1 sequence comprising the amino acid sequence of SNYAMS (SEQ ID NO: 21), an HCDR2 sequence comprising the amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 22), and an HCDR3 sequence comprising the amino acid sequence of DLYYGPSTYFVANL (SEQ ID NO: 23), and a LCDR1 sequence comprising the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 26), an LCDR2 sequence comprising the amino acid sequence of RDTSRPS (SEQ ID NO: 27), and an HCDR3 sequence comprising the amino acid sequence of ATRPSSGS (iii) a VL comprising an LCDR3 sequence comprising the amino acid sequence of NFQL (SEQ ID NO: 28); (iv) a VH comprising an HCDR1 sequence comprising the amino acid sequence of SNYAMS (SEQ ID NO: 31), an HCDR2 sequence comprising the amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 32), and an HCDR3 sequence comprising the amino acid sequence of DLYYGPTTYSAANL (SEQ ID NO: 33); and (v) a VH comprising an LCDR1 sequence comprising the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 36), an LCDR2 sequence comprising the amino acid sequence of RDTSRPS (SEQ ID NO: 37), and an ATRD (iv) a VL comprising an LCDR3 sequence comprising the amino acid sequence of FSGSNFQL (SEQ ID NO: 38), or (iv) a VH comprising an HCDR1 sequence comprising the amino acid sequence of SNYAMS (SEQ ID NO: 41), an HCDR2 sequence comprising the amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 42), and an HCDR3 sequence comprising the amino acid sequence of DLYYGPTTYSAFNL (SEQ ID NO: 43), and an LCDR1 sequence comprising the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 46), and an LCDR2 sequence comprising the amino acid sequence of RDTSRPS (SEQ ID NO: 47);and a VL comprising an LCDR3 sequence comprising the amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 48).

[0028] In certain embodiments, the second and third antigen-binding domains comprise: (i) an antibody heavy chain variable (VH) domain comprising an amino acid sequence 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 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 at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 20, and an antibody light chain variable (VL) domain comprising an amino acid sequence 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.

[0029] In certain embodiments, any one or more of the first, second, and third antigen-binding domains comprise an antibody fragment, which in certain embodiments includes a Fab fragment, a F(ab')2 fragment, a Fab' fragment, an Fv fragment, a single-chain variable fragment (scFv), and a single-domain antibody fragment.

[0030] In a specific embodiment, the immune cell or CD3 antigen binding domain is a Fab fragment, which comprises a heavy chain comprising a CH1 domain and a VH, and a light chain comprising a CL domain and a VL.

[0031] In certain embodiments, the CH1 domain comprises at least 5 amino acids of an antibody hinge region. In certain embodiments, the CH1 domain comprises the amino acid sequence EPKSC (SEQ ID NO: 88) of an antibody hinge region.

[0032] In certain embodiments, the MAGE-A4 pMHC antigen binding domain comprises an scFv.

[0033] In certain embodiments, the second antigen-binding domain is operably linked to the C-terminus of the Fab domain heavy chain or the N-terminus of the Fab domain heavy chain, hi certain embodiments, the third antigen-binding domain is operably linked to the C-terminus of the Fab domain heavy chain or the N-terminus of the Fab domain heavy chain.

[0034] In specific embodiments, a) the second antigen-binding domain comprises an scFv linked to the C-terminus of the Fab domain heavy chain, and the third antigen-binding domain comprises an scFv linked to the C-terminus of the Fab domain light chain; b) the second antigen-binding domain comprises an scFv linked to the N-terminus of the Fab domain heavy chain, and the third antigen-binding domain comprises an scFv linked to the N-terminus of the Fab domain light chain; c) the second antigen-binding domain comprises an scFv linked to the N-terminus of the Fab domain heavy chain, and the third antigen-binding domain comprises an scFv linked to the C-terminus of the Fab domain light chain; or d) the second antigen-binding domain comprises an scFv linked to the C-terminus of the Fab domain heavy chain, and the third antigen-binding domain comprises an scFv linked to the N-terminus of the Fab domain light chain.

[0035] 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 from 1 to 5. In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGS (SEQ ID NO:74), GGGGSGGGGGSGGGGGS (SEQ ID NO:75), GGGSGGGGSGGGGSGGGGS (SEQ ID NO:76), GGGSGGGGSGGGGSGGGGAS (SEQ ID NO:77), or GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:78).

[0036] In certain embodiments, the second and / or third antigen-binding domains VH and VL are connected by an amino acid linker. In certain embodiments, the amino acid linker comprises (GGGGS)n (SEQ ID NO: 73), where n is an integer from 1 to 5. In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGS (SEQ ID NO: 74), GGGGSGGGGGSGGGGS (SEQ ID NO: 75), GGGSGGGGSGGGGSGGGGS (SEQ ID NO: 76), GGGSGGGGSGGGGSGGGGAS (SEQ ID NO: 77), or GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 78).

[0037] In certain embodiments, the multispecific antigen binding protein does not comprise an Fc domain.

[0038] In certain embodiments, the multispecific antigen-binding protein comprises an (scFv)2, (scFv)3, BiTE, BIKE, Dart, diabody, tribody, Fab2, Fab3, Fab4, scFv-Fab-scFv, or minibody-scFv.

[0039] In certain embodiments, the multispecific antigen-binding protein comprises a molecular weight of about 75 kDa to about 100 kDa or about 110 kDa, hi certain embodiments, the antigen-binding protein has an increased serum half-life compared to antigen-binding proteins of molecular weight less than about 75 kDa.

[0040] In a specific embodiment, the CD3 antigen-binding domain comprises: a1) a VH comprising an HCDR1 sequence comprising the amino acid sequence of STYAMN (SEQ ID NO: 51), an HCDR2 sequence comprising the amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 52), and an HCDR3 sequence comprising the amino acid sequence of HGNFGDSYVSWFAY (SEQ ID NO: 53); and a2) a VL comprising an LCDR1 sequence comprising the amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 56), an LCDR2 sequence comprising the amino acid sequence of GTNKRAP (SEQ ID NO: 57), and an LCDR3 sequence comprising the amino acid sequence of ALWYSNHWV (SEQ ID NO: 58).

[0041] In certain embodiments, the CD3 antigen-binding domain comprises a VH comprising an amino acid sequence at least about 90% identical (e.g., 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 comprising an amino acid sequence at least about 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO:55.

[0042] In certain embodiments, the CD3 antigen-binding domain comprises a heavy chain comprising an amino acid sequence at least about 90% identical (e.g., 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 comprising an amino acid sequence at least about 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO:54.

[0043] 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 said sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to said sequence while retaining antigen specificity (i.e., while retaining specificity for the GVYDGREHTV (SEQ ID NO:3) HLA-A*02 complex and CD3).

[0044] In certain embodiments, the MAGE-A4 pMHC-binding domain, e.g. the second antigen-binding domain and / or the third antigen-binding domain described above, particularly when in scFv format, 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.

[0045] In certain embodiments, the variable heavy chain comprises a leucine (L) or serine (S) at amino acid position 11 according to Kabat numbering, a valine (V), serine (S), or threonine (T) at amino acid position 89 according to Kabat numbering, and / or a leucine (L), serine (S), or threonine (T) at amino acid position 108 according to Kabat numbering.

[0046] In certain embodiments, the polar amino acid is serine (S) and / or threonine (T).

[0047] In certain embodiments, the variable heavy chain comprises a serine (S) at amino acid position 11, a serine (S) or threonine (T) at amino acid position 89, and a serine (S) or threonine (T) at amino acid position 108, according to Kabat numbering.

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

[0049] 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 comprising an HCDR1 sequence comprising the amino acid sequence of STYAMN (SEQ ID NO: 51), an HCDR2 sequence comprising the amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 52), and an HCDR3 sequence comprising the amino acid sequence of HGNFGDSYVSWFAY (SEQ ID NO: 53), and a2) a VL comprising an LCDR1 sequence comprising the amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 56), an LCDR2 sequence comprising the amino acid sequence of GTNKRAP (SEQ ID NO: 57), and an LCDR3 sequence comprising the amino acid sequence of 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).

[0050] In certain embodiments, the second antigen-binding domain comprises b1) an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), an 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 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 heavy chain variable (VH) 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 HCDR3 amino acid sequence of ATX9X 10 X 11 SGSNFQX 12 (SEQ ID NO: 8) wherein X corresponds to the amino acid S or R, and X 10 corresponds to the amino acid D or P, and X 11 corresponds to the amino acid G, S, or F, and X 12 and an antibody light chain variable (VL) domain comprising:

[0051] In certain embodiments, the CD3 binding domain comprises a VH comprising an amino acid sequence at least about 90% identical (e.g., 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:50, and a VL comprising an amino acid sequence at least about 90% identical (e.g., 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:55.

[0052] In certain embodiments, the CD3 binding domain comprises a VH comprising an amino acid sequence at least about 90% identical (e.g., 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO: 49, and a VL comprising an amino acid sequence at least about 90% identical (e.g., 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO: 54.

[0053] In certain embodiments, the multispecific antigen-binding protein further comprises a third antigen-binding domain.

[0054] In certain embodiments, the multispecific antigen binding protein comprises: c) MAGE-A4 and a third antigen-binding domain that specifically binds to pMHC, the third antigen-binding domain comprising: c1) a VH domain comprising an HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), an HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6) (wherein 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) (wherein 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 VH 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 HCDR3 amino acid sequence of ATX9X 10 X 11 SGSNFQX 12 (SEQ ID NO: 8) wherein X corresponds to the amino acid S or R, and X 10 corresponds to the amino acid D or P, and X 11 corresponds to the amino acid G, S, or F, and X 12 and a VL domain containing the amino acid L or A).

[0055] In one aspect the present disclosure provides a multispecific antigen binding protein which binds to MAGE-A4-pMHC and CD3, the multispecific antigen binding protein comprising (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 variants of the sequences which are at least 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences while retaining antigen specificity (i.e. while retaining specificity for the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex and CD3).

[0056] 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 comprising: (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.

[0057] 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.

[0058] In certain embodiments of the multispecific antigen binding proteins described herein, the multispecific antigen binding proteins remain at least 94%, 95%, 96%, 97%, 98%, 99% or 100% monomeric after incubation at 1 mg / mL and / or 10 mg / mL in PBS at 4°C for 14 days as determined by SEC-HPLC.

[0059] In certain embodiments of the multispecific antigen binding proteins described herein, the multispecific antigen binding proteins exhibit efficacy (e.g., cytotoxicity) against target-positive tumor cells as determined in an LDH cytotoxicity assay.

[0060] In certain embodiments of the multispecific antigen binding proteins described herein, the multispecific antigen binding proteins exhibit tumor growth inhibition and tumor eradication in a cell line-derived murine non-small cell lung cancer (NSCLC) xenograft model.

[0061] In certain embodiments, the multispecific antigen-binding protein is chemically or biologically modified, hi certain embodiments, the multispecific antigen-binding protein is glycosylated, PEGylated, HESylated, PASylated, or XTENylated.

[0062] 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.

[0063] In certain embodiments, the functional entity is a toxin.

[0064] In certain embodiments of the multispecific antigen binding proteins described herein, the light and / or heavy chains comprise N-terminal and / or C-terminal truncations of 1, 2, 3, 4, or 5 amino acids.

[0065] 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.

[0066] In certain embodiments of the multispecific antigen binding proteins described herein, the multispecific antigen binding proteins comprise a pyroglutamic acid (pE) at position 1 in place of glutamine (Q) or glutamic acid (E) in the light and / or heavy chain.

[0067] In certain embodiments of the multispecific antigen binding proteins described herein, the multispecific antigen binding proteins comprise a pyroglutamic acid (pE) at position 1 in place of glutamine (Q) or glutamic acid (E) in the light chain.

[0068] In certain embodiments, the antigen binding protein is for use in diagnosis.

[0069] In certain embodiments, the multispecific antigen binding proteins described above are for use in a method for inhibiting the growth or proliferation of cancer cells.

[0070] In certain embodiments, the multispecific antigen binding proteins described above are for use in methods of redirecting T cells to MAGE-A4 expressing cancer cells.

[0071] In certain embodiments, the antigen binding protein described above or the multispecific antigen binding protein described above is for use as a medicament.

[0072] In one aspect, the present disclosure provides a nucleic acid encoding an antigen binding protein as described above or a multispecific antigen binding protein as described above.

[0073] In one aspect, the present disclosure provides a vector comprising the nucleic acid described above.

[0074] In one aspect, the present disclosure provides a population of host cells comprising the vector described above.

[0075] In one aspect, the present disclosure provides a kit comprising an antigen binding protein described above or a multispecific antigen binding protein described above.

[0076] In one aspect, the present disclosure provides a method of producing an antigen binding protein as described above or a multispecific antigen binding protein as described above, the method comprising the steps of: (i) culturing a host cell as described above under conditions that allow expression of the antigen binding protein or multispecific antigen binding protein; (ii) recovering the antigen binding protein or multispecific antigen binding protein; and optionally (iii) further purifying and / or modifying and / or formulating the antigen binding protein or multispecific antigen binding protein.

[0077] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antigen binding protein as described above or a multispecific antigen binding protein as described above and a pharmaceutically acceptable buffer.

[0078] In one aspect, the present disclosure provides the use of an antigen binding protein described above, a multispecific antigen binding protein described above, or a pharmaceutical composition described above in the manufacture of a medicament.

[0079] In one aspect, the present disclosure provides the use of an antigen binding protein described above, a CAR described above, an immune cell described above, a multispecific antigen binding protein described above, a cell described above, or a pharmaceutical composition described above in the treatment of a disease, in particular cancer.

[0080] In one aspect, the present disclosure provides a method of treating a MAGE-A4 pMHC-expressing cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an antigen binding protein as described above, a CAR as described above, an immune cell as described above, a multispecific antigen binding protein as described above, a cell as described above, or a pharmaceutical composition as described above.

[0081] 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, e.g., uterine carcinosarcoma (UCS; particularly, the UCEC subgroup), myxoid / round cell liposarcoma, gastric or gastroesophageal junction (GEJ) adenocarcinoma, epithelial ovarian cancer, e.g., high-grade serous ovarian carcinoma, synovial sarcoma, bladder urothelial carcinoma (BLCA), particularly, transitional cell carcinoma, testicular germ cell tumor (TGCT), and cervical squamous cell carcinoma (CESC).

[0082] In certain embodiments, the cancer is of squamous origin, such as head and neck squamous cell carcinoma (HNSCC) or squamous NSCLC.

[0083] In one aspect, the disclosure provides a method for selecting a patient eligible for treatment with a MAGE-A4 antagonist, the method comprising, in order: (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) selecting the patient for treatment with a MAGE-A4 antagonist if the detection antibody is bound to the sample.

[0084] In certain embodiments, the detection antibody is OTI1F9, E7O1U, or an antigen binding protein described herein.

[0085] In certain embodiments, the method further comprises the step of performing RNA sequencing for the detection of total MAGE-A4.

[0086] In certain embodiments, the MAGE-A4 antagonist is an antigen binding protein as described herein, a CAR as described herein, an immune cell as described herein, a multispecific antigen binding protein as described herein, or a pharmaceutical composition as described herein in the treatment of a disease, in particular cancer.

[0087] These and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments, taken in conjunction with the accompanying drawings. This patent or application document 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 Patent and Trademark Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0088] [Figure 1A] Figure 1 shows the binding of antibodies to HLA-A*02:01 / MAGE-A4 as determined by direct ELISA. [Figure 1B] Figure 1 shows the binding of antibodies to HLA-A*02:01 / MAGE-A4 as determined by direct ELISA. [Figure 1C] Figure 1 shows the binding of antibodies to the control complex HLA-A*02:01 / peptide mix as determined by direct ELISA. [Figure 1D] Figure 1 shows the binding of antibodies to the control complex HLA-A*02:01 / peptide mix as determined by direct ELISA. [Figure 2]Binding of antibodies M0709 (A) and M0763 (B) to T2 cells presenting MAGE-A4 or control peptides 1, 2 and 3 is shown. [Figure 3] Binding of humanized M0763 variants to HLA-A*02:01 / MAGE-A4 (A) or the control complex HLA-A*02:01 / peptide mix (B) as determined by direct ELISA is shown. [Figure 4] 1 shows a schematic diagram of one embodiment of a bispecific antibody of the invention. The embodiment shown comprises an anti-CD3 Fab fragment and two single-chain antibody fragments (scFvs) that specifically bind to a target peptide presented on an MHC complex. The pMHC-binding scFvs can be linked to the C-termini of the CH1-domain and CL-domain via a glycine-serine flexible linker. [Figure 5A] Figure 1 shows the cytotoxicity of the dual T cell engager M1048 and its monovalent engager M1041 in MAGE-A4-positive HLA-A*02:01-positive U2OS cancer cells. [Figure 5B] Figure 1 shows the cytotoxicity of the dual T cell engager M1048 and its monovalent engager M1041 in MAGE-A4 negative HLA-A*02:01 positive SK-MEL-30 cancer cells. [Figure 5C] Figure 1 shows the cytotoxicity of the dual T cell engager M1048 and its monovalent engager M1041 in MAGE-A4-negative HLA-A*02:01-positive MDA-MB-231 cancer cells. [Figure 5D] Figure 1 shows the cytotoxicity of the dual T cell engager M1048 and its monovalent engager M1041 in MAGE-A4 negative HLA-A*02:01 positive PANC-1 cancer cells. [Figure 6] Figure 1 shows the stability of dual engagers M1397 and M1403 in PBS over 0, 3, 7, and 14 days as determined by SEC-HPLC. A shows data from incubation at 4°C, and B shows data from incubation at 37°C. [Figure 7]Figure 1 shows the stability of dual engagers M1397 (A) and M1403 (B) in human serum at 37°C. [Figure 8A] Figure 1 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in MAGE-A4-positive HLA-A*02:01-positive cancer cells. Figure 2 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in U2OS. [Figure 8B] Figure 1 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in MAGE-A4-positive, HLA-A*02:01-positive cancer cells. Figure 2 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in U2OS. [Figure 8C] Figure 1 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in MAGE-A4-positive HLA-A*02:01-positive cancer cells. Figure 2 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in NCI-H1703. [Figure 8D] Figure 1 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in MAGE-A4-positive, HLA-A*02:01-positive cancer cells. Figure 2 shows the cytotoxicity of dual engagers M1403 and comparator 1 in NCI-H1703. [Figure 8E] Figure 1 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in MAGE-A4-positive HLA-A*02:01-positive cancer cells. Figure 2 shows the cytotoxicity of dual engagers M1397 and M1403 and comparator 1 in A375. [Figure 8F] Figure 1 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in MAGE-A4 negative HLA-A*02:01 positive cancer cells. Dual engager M1397 and comparator 1 in PANC-1. [Figure 8G] Figure 1 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in MAGE-A4 negative HLA-A*02:01 positive cancer cells. Figure 2 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in PANC-1 cells. [Figure 8H]Figure 1 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in MAGE-A4 negative HLA-A*02:01 positive cancer cells. Dual engager M1397 and comparator 1 in MDA-MB-231. [Figure 8I] Figure 1 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in MAGE-A4 negative HLA-A*02:01 positive cancer cells. Dual engager M1397 and comparator 1 in MDA-MB-231. [Figure 8J] Figure 1 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in MAGE-A4 negative HLA-A*02:01 positive cancer cells. Dual engager M1397 and comparator 1 in NCI-H441. [Figure 8K] Figure 1 shows the cytotoxicity of dual engagers M1397 and M1403 or comparator 1 in MAGE-A4 negative HLA-A*02:01 positive cancer cells. Dual engager M1403 and comparator 1 in NCI-H441. [Figure 9A] 1 shows cancer cell killing mediated by dual engager M1397, comparator 1, or comparator 2 against the MAGE-A4 positive HLA-A*02:01 positive cancer cell line U2OS. [Figure 9B] 1 shows cancer cell killing mediated by dual engager M1397, comparator 1, or comparator 2 against the MAGE-A4 positive HLA-A*02:01 positive cancer cell line NCI-H1703. [Figure 9C] 1 shows cancer cell killing mediated by dual engager M1397, comparator 1, or comparator 2 against the MAGE-A4 negative HLA-A*02:01 positive cancer cell line PANC-1. [Figure 9D] 1 shows cancer cell killing mediated by dual engager M1397, comparator 1, or comparator 2 against the MAGE-A4 negative HLA-A*02:01 positive cancer cell line SKMEL-30. [Figure 10A]1 shows IFN-gamma release in MAGE-A4 positive HLA-A*02:01 positive cancer cells (M1397 and Comparator 1 in U2OS) upon treatment with dual T cell engagers M1397 and M1403 or Comparator 1. [Figure 10B] 1 shows IFN-gamma release in MAGE-A4 positive HLA-A*02:01 positive cancer cells (M1403 and Comparator 1 in U2OS) upon treatment with dual T cell engagers M1397 and M1403 or Comparator 1. [Figure 10C] 1 shows IFN-gamma release in MAGE-A4 positive HLA-A*02:01 positive cancer cells (M1397 and Comparator 1 in NCI-H1703) upon treatment with dual T cell engagers M1397 and M1403 or Comparator 1. [Figure 10D] 1 shows IFN-gamma release in MAGE-A4 positive HLA-A*02:01 positive cancer cells (M1403 and Comparator 1 in NCI-H1703) upon treatment with dual T cell engagers M1397 and M1403 or Comparator 1. [Figure 10E] 1 shows IFN-gamma release in MAGE-A4 negative HLA-A*02:01 positive cancer cells (M1397 and Comparator 1 in PANC-1) upon treatment with dual T cell engagers M1397 and M1403 or Comparator 1. [Figure 10F] 1 shows IFN-gamma release in MAGE-A4 negative HLA-A*02:01 positive cancer cells (M1403 and Comparator 1 in PANC-1) upon treatment with dual T cell engagers M1397 and M1403 or Comparator 1. [Figure 10G] 1 shows IFN-gamma release in MAGE-A4 negative HLA-A*02:01 positive cancer cells (M1397 and Comparator 1 in MDA-MB-231) upon treatment with dual T cell engagers M1397 and M1403 or Comparator 1. [Figure 10H]1 shows IFN-gamma release in MAGE-A4 negative HLA-A*02:01 positive cancer cells (M1403 and Comparator 1 in MDA-MB-231) upon treatment with dual T cell engagers M1397 and M1403 or Comparator 1. [Figure 10I] 1 shows IFN-gamma release in MAGE-A4 negative HLA-A*02:01 positive cancer cells (M1397 and Comparator 1 in NCI-H441) upon treatment with dual T cell engagers M1397 and M1403 or Comparator 1. [Figure 10J] 1 shows IFN-gamma release in MAGE-A4 negative HLA-A*02:01 positive cancer cells (M1403 and Comparator 1 in NCI-H441) upon treatment with dual T cell engagers M1397 and M1403 or Comparator 1. [Figure 11A] 1 shows granzyme B release as an indicator of T cell activation in MAGE-A4-positive, HLA-A*02:01-positive cancer cells NCI-H1703 upon treatment with dual engager M1397, comparator 1, or comparator 2. [Figure 11B] 1 shows granzyme B release as an indicator of T cell activation in MAGE-A4-negative, HLA-A*02:01-positive cancer cells SKMEL-30 upon treatment with dual engager M1397, comparator 1, or comparator 2. [Figure 11C] 1 shows IFN-gamma release as an indicator of T cell activation in MAGE-A4 positive HLA-A*02:01 positive cancer cells NCI-H1703 upon treatment with dual engager M1397, comparator 1 or comparator 2. [Figure 11D] IFN-gamma release as an indicator of T cell activation in MAGE-A4-negative HLA-A*02:01-positive cancer cells SKMEL-30 is shown. [Figure 12A] Figure 1 shows cytokine release in MAGE-A4 positive HLA-A*02:01 positive U2OS cells and MAGE-A4 negative HLA-A*02:01 positive PANC-1 cells upon treatment with dual T cell engagers M1397 or M1403 and comparator 1. IL-2 was quantified. [Figure 12B] Figure 1 shows cytokine release in MAGE-A4 positive HLA-A*02:01 positive U2OS cells and MAGE-A4 negative HLA-A*02:01 positive PANC-1 cells upon treatment with dual T cell engagers M1397 or M1403 and comparator 1. IL-2 was quantified. [Figure 12C] Figure 1 shows cytokine release in MAGE-A4 positive HLA-A*02:01 positive U2OS cells and MAGE-A4 negative HLA-A*02:01 positive PANC-1 cells upon treatment with dual T cell engagers M1397 or M1403 and comparator 1. IL-2 was quantified. [Figure 12D] Figure 1 shows cytokine release in MAGE-A4 positive HLA-A*02:01 positive U2OS cells and MAGE-A4 negative HLA-A*02:01 positive PANC-1 cells upon treatment with dual T cell engagers M1397 or M1403 and comparator 1. IL-2 was quantified. [Figure 12E] 1 shows cytokine release in MAGE-A4 positive HLA-A*02:01 positive U2OS cells and MAGE-A4 negative HLA-A*02:01 positive PANC-1 cells upon treatment with dual T cell engagers M1397 or M1403 and comparator 1. [Figure 12F] 1 shows cytokine release in MAGE-A4 positive HLA-A*02:01 positive U2OS cells and MAGE-A4 negative HLA-A*02:01 positive PANC-1 cells upon treatment with dual T cell engagers M1397 or M1403 and comparator 1. [Figure 12G] Figure 1 shows cytokine release in MAGE-A4 positive HLA-A*02:01 positive U2OS cells and MAGE-A4 negative HLA-A*02:01 positive PANC-1 cells upon treatment with dual T cell engagers M1397 or M1403 and comparator 1. IL-6 was quantified. [Figure 12H]Figure 1 shows cytokine release in MAGE-A4 positive HLA-A*02:01 positive U2OS cells and MAGE-A4 negative HLA-A*02:01 positive PANC-1 cells upon treatment with dual T cell engagers M1397 or M1403 and comparator 1. IL-6 was quantified. [Figure 12I] Figure 1 shows cytokine release in MAGE-A4 positive HLA-A*02:01 positive U2OS cells and MAGE-A4 negative HLA-A*02:01 positive PANC-1 cells upon treatment with dual T cell engagers M1397 or M1403 and comparator 1. TNF-alpha was quantified. [Figure 12J] Figure 1 shows cytokine release in MAGE-A4 positive HLA-A*02:01 positive U2OS cells and MAGE-A4 negative HLA-A*02:01 positive PANC-1 cells upon treatment with dual T cell engagers M1397 or M1403 and comparator 1. TNF-alpha was quantified. [Figure 12K] Figure 1 shows cytokine release in MAGE-A4 positive HLA-A*02:01 positive U2OS cells and MAGE-A4 negative HLA-A*02:01 positive PANC-1 cells upon treatment with dual T cell engagers M1397 or M1403 and comparator 1. TNF-alpha was quantified. [Figure 12L] Figure 1 shows cytokine release in MAGE-A4 positive HLA-A*02:01 positive U2OS cells and MAGE-A4 negative HLA-A*02:01 positive PANC-1 cells upon treatment with dual T cell engagers M1397 or M1403 and comparator 1. TNF-alpha was quantified. [Figure 13A] IFN-gamma expression is shown upon incubation of TAP-deficient T2 cells stimulated with MAGE-A4 or the physiologically relevant and highly similar peptides Control 1 or Control 2 with PBMCs as effector cells and the dual engager M1397. Controls included T2 cells and PBMCs alone, and PBMCs and test compound and PBMCs alone. [Figure 13B]Figure 1 shows IFN-gamma expression upon incubation of TAP-deficient T2 cells stimulated with MAGE-A4 or the physiologically relevant and highly similar peptides Control 1 or Control 2 with PBMCs as effector cells and Comparator 1. Controls included T2 cells and PBMCs alone, and PBMCs and test compound and PBMCs alone. [Figure 13C] Figure 1 shows IFN-gamma expression upon incubation of TAP-deficient T2 cells stimulated with MAGE-A4 or the physiologically relevant and highly similar peptides Control 1 or Control 2 with PBMCs as effector cells and Comparator 2. Controls included T2 cells and PBMCs alone, and PBMCs with test compound and PBMCs alone. [Figure 14A] Figure 1 shows Granzyme B release upon treatment with dual T cell engager M1397, comparator 1 or comparator 2. The antigen-positive cell line NCI-H1703 was used as a positive control. [Figure 14B] 1 shows granzyme B release from the antigen-negative cancer cell line KLE upon treatment with the dual T cell engager M1397, comparator 1, or comparator 2. [Figure 14C] 1 shows granzyme B release from the antigen-negative cancer cell line KMRC-2 upon treatment with the dual T cell engager M1397, comparator 1, or comparator 2. [Figure 14D] 1 shows granzyme B release from the antigen-negative cancer cell line KMRC-3 upon treatment with the dual T cell engager M1397, comparator 1, or comparator 2. [Figure 14E] 1 shows granzyme B release from the antigen-negative cancer cell line LNCaP upon treatment with the dual T cell engager M1397, comparator 1, or comparator 2. [Figure 14F] 1 shows granzyme B release from antigen-negative cancer cell line 639-V upon treatment with dual T cell engager M1397, comparator 1, or comparator 2. [Figure 14G] 1 shows granzyme B release from the antigen-negative cancer cell line EKVX upon treatment with the dual T cell engager M1397, comparator 1, or comparator 2. [Figure 14H]1 shows granzyme B release from antigen-negative cancer cell line HCT116 upon treatment with dual T cell engager M1397, comparator 1, or comparator 2. [Figure 15] Safety of dual engagers M1397 and M1403 or comparator 1 in HLA-A*02:01 positive primary cells. A: HMVEC-C cells, B: NHBE cells, C: NHA cells. [Figure 16A] Shows the safety of dual engager M1397, comparator 1, and comparator 2 in various HLA-A*02:01 positive primary cells. HCM_679 cells. [Figure 16B] Shows the safety of dual engager M1397, comparator 1, and comparator 2 in various HLA-A*02:01 positive primary cells. HCM_639 cells. [Figure 16C] Shows the safety of dual engager M1397, comparator 1, and comparator 2 in various HLA-A*02:01 positive primary cells. HCM_745 cells. [Figure 16D] Shows the safety of dual engager M1397, comparator 1, and comparator 2 in various HLA-A*02:01 positive primary cells. HCM_746 cells. [Figure 16E] Shows the safety of dual engager M1397, comparator 1, and comparator 2 in various HLA-A*02:01 positive primary cells. HMVEC-L_73809 cells. [Figure 16F] Shows the safety of dual engager M1397, comparator 1, and comparator 2 in various HLA-A*02:01 positive primary cells. HCF_251 cells. [Figure 16G] Shows the safety of dual engager M1397, comparator 1, and comparator 2 in various HLA-A*02:01 positive primary cells. RPTEC_82573 cells. [Figure 16H] Shows the safety of dual engager M1397, comparator 1, and comparator 2 in various HLA-A*02:01 positive primary cells. NHLF_19232 cells. [Figure 16I]Shows the safety of dual engager M1397, comparator 1, and comparator 2 in various HLA-A*02:01 positive primary cells. NHLF_76039 cells. [Figure 16J] Shows the safety of dual engager M1397, comparator 1, and comparator 2 in various HLA-A*02:01 positive primary cells. NHA_72445 cells. [Figure 16K] Shows the safety of dual engager M1397, comparator 1, and comparator 2 in various HLA-A*02:01 positive primary cells. NHBE_35497 cells. [Figure 16L] Shows the safety of dual engager M1397, comparator 1, and comparator 2 in various HLA-A*02:01 positive primary cells. RPTEC_49985 cells. [Figure 16M] Shows the safety of dual engager M1397, comparator 1, and comparator 2 in various HLA-A*02:01 positive primary cells. HAoSMC_735 cells. [Figure 17] Figure 1 shows the in vivo efficacy of M1397 in a cell line-derived mouse NSCLC xenograft model as detailed in Example 14. Figure 1 shows the mean tumor volume over the study period at various doses of the active compound. DETAILED DESCRIPTION OF THE INVENTION

[0089] Generally, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is well known and commonly used in the art. The methods and techniques provided herein can generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the laboratory procedures and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0090] Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definitions. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and / or" unless otherwise indicated. The use of the term "including" and other forms such as "include" and "included" is not limiting.

[0091] So that the present invention may be more readily understood, certain terms are first defined.

[0092] antigen-binding proteins "MAGE-A4" stands for "melanoma-associated antigen 4," a member of the MAGE family of cancer-testis antigens (CTAs). The MAGE A family of proteins encompasses 12 highly homologous genes clustered in Xq28 and characterized by the presence of a conserved domain (MAGE homology domain, MHD). Human MAGE-A4 is listed in UniProt (www.uniprot.org) under accession number P43358 (entry version 163). "MAGE-A4p 230-239 or "203-239 peptide" refers to a peptide derived from MAGE-A4 having the amino acid sequence GVYDGREHTV (SEQ ID NO: 3) at positions 230 to 239 of the MAGE-A4 protein.

[0093] "CD3" refers to the cluster of differentiation 3 coreceptor (or coreceptor complex) of the T cell receptor, a complex consisting of four distinct chains. In mammals, this complex contains a CD3γ (gamma) chain / subunit, a CD3δ (delta) chain / subunit, and two CD3ε (epsilon) chains / subunits. Herein, CD3 is referred to 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 (epsilon) chain / subunit of CD3 (see, e.g., UniProt (www.uniprot.org) Accession No. P07766 (Version 189) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeqNP_000724.1). The CD3 molecule can be a full-length, unprocessed CD3 molecule or a fragment or variant thereof, e.g., one that has been processed intracellularly. For example, such variants may be naturally occurring variants such as splice variants or allelic variants. In certain embodiments, the antigen-binding proteins disclosed herein bind to an epitope of CD3 that is 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 are not cross-reactive with CD3 antigens from rodents (e.g., mice or rats) or minipigs.

[0094] As used herein, the term "antibody" or "antigen-binding protein" refers to an immunoglobulin molecule or immunoglobulin-derived molecule that specifically binds to or is immunologically reactive with an antigen or epitope, and includes both polyclonal and monoclonal antibodies, and also includes functional antibody fragments, including, but not limited to, fragment 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 may be a single-domain antibody, or comprise at least one variable light chain and at least one variable heavy chain. In one embodiment, at least one variable light chain and at least one variable heavy chain are presented as a single polypeptide chain. The term "antibody" or "antigen-binding protein" includes germline-derived antibodies. The term "antibody" or "antigen binding protein" includes genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFvs, tandem tri-scFvs), etc. Unless otherwise indicated, the term "antibody" or "antigen binding protein" should be understood to encompass functional antibody fragments thereof.

[0095] 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, e.g., binds to two different target molecules or two epitopes on the same target molecule. In certain embodiments, the antibody is trispecific, e.g., binds to at least three different target molecules.

[0096] Antigen-binding proteins can be monovalent or multivalent, i.e., have 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 nanobody. 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, quadroma, CrossMab, DuoBody, triabody, and tetrabody. In some embodiments, the multivalent antigen-binding protein is bivalent, 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 to two different targets or two different target sites on one target molecule. In some embodiments, the multivalent antigen-binding protein contains more than two, e.g., three or four different binding sites for three or four different antigens, respectively. Such antigen-binding proteins are multivalent and multispecific, in particular tri- or tetraspecific, respectively.

[0097] In some embodiments, the antigen-binding protein is multispecific (e.g., bispecific), including, but not limited to, a diabody, single-chain diabody, DART, BiTE, BIKE, tandem scFv, or IgG-like asymmetric heterobispecific antibody. In certain embodiments, one or more binding specificities of the multispecific antigen-binding protein are immune cell engagers (i.e., comprise 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.

[0098] As used herein, the term "monoclonal antibody" refers to a substantially homogeneous antibody, i.e., an antibody obtained from a population of antibodies that bind to the same epitope and / or have identical sequences. In contrast, a population of polyclonal antibodies contains antibodies that bind to multiple epitopes and have different sequences. Monoclonal antibody preparations may or may not contain small amounts of variant antibodies, resulting from, for example, naturally occurring mutations. Such variants can be generated, for example, through post-translational modifications such as clipping at the N- or C-terminus of the light and / or heavy chain, or pyroglutamate formation at the N-terminus of the polypeptide chain (see, e.g., Liu YD, et al. J Biol Chem. 2011 Apr 1;286(13):11211-7). Depending on the method and antibody used, the percentage of variants in the mixture will vary and may comprise substantially all of the antibodies produced or a very low percentage.

[0099] As used herein, a "single-chain variable fragment" (scFv) is an antigen-binding protein comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL). The VH and VL domains of an scFv are linked via any suitable art-recognized linker. Such linkers include, but are not limited to, repeating GGGGS (SEQ ID NO: 74) amino acid sequences or variants thereof. While scFvs generally do not contain antibody constant domain regions, the scFvs of the present disclosure may be linked or conjugated to antibody constant domain regions (e.g., antibody Fc domains) to modify various properties of the scFv, including, but not limited to, increased serum half-life or tissue half-life. ScFvs generally have a molecular weight of approximately 25 kDa and a hydrodynamic radius of approximately 2.5 nm.

[0100] As used herein, a "Fab fragment" or "Fab" or "Fab domain" is an antibody fragment containing a light chain fragment containing a variable light chain (VL) domain and a light chain constant domain (CL), and a variable heavy chain (VH) domain and the first heavy chain constant domain (CH1). F(ab')2 contains two antigen-binding regions linked at the hinge via disulfides.

[0101] As used herein, "VHH," "nanobody," "heavy chain-only antibody," "single domain antibody," or "sdAb" refers to an antigen-binding protein containing a single heavy chain variable domain derived from a species of the Camelidae family, including camel, llama, and alpaca. VHHs generally have a molecular weight of approximately 15 kDa.

[0102] The antigen binding proteins of the present disclosure may comprise one or more linkers to link domains of the antigen binding protein (e.g., linking a VH and a VL to form an scFv, or linking multiple binding domains to form a multispecific antigen binding protein).

[0103] Illustrative examples of linkers include glycine polymers (Gly) n glycine-serine polymer (Glyn Ser) n where n is an integer of at least 1, 2, 3, 4, 5, 6, 7, or 8; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art.

[0104] Glycine and glycine-serine polymers are relatively unstructured and may therefore be able to function as neutral tethers between domains of fusion proteins, such as the antigen-binding proteins described herein. Glycine has access to significantly more Φ-Ψ 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 the design of antigen-binding proteins in certain embodiments can include fully or partially flexible linkers, and thus the linker can include not only flexible linker stretches but also one or more stretches that confer reduced flexibility to provide the desired structure.

[0105] However, the linker sequence can be selected to resemble a natural linker sequence, for example, using an amino acid stretch corresponding to the first part of the human CH1 and Cκ sequences or the lower part of the hinge region of human IgG.

[0106] The peptide linker design connecting the VL and VH domains of the scFv portion is generally a flexible linker composed of small non-polar or polar residues, such as Gly, Ser, and Thr. A particular exemplary linker connecting the variable domains of the scFv portion is the (Gly4Ser)4 linker (SEQ ID NO: 76), where 4 is an exemplary number of repeats of the motif.

[0107] Linkers connecting 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 by 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 sequence GGGGSGGGGSGGGGS (SEQ ID NO: 75), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 76), GGGGSGGGGSGGGGSGGGGAS (SEQ ID NO: 77), or GGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 78).

[0108] Other exemplary linkers include 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) (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)); KESGSVGSSEQLAQFRSLD (SEQ ID NO: 83) (Bird et al., Science 242:423-426 (1988)), GGRRGGGS (SEQ ID NO: 84); LQRDGERP (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, flexible linkers can be rationally designed using computer programs capable of modeling the 3D structures of proteins and peptides, or by phage display methods.

[0109] An antibody can comprise a variable light (VL) domain and a variable heavy (VH) domain, each of which further comprises a set of three CDRs.

[0110] As used herein, the term "complementarity-determining region" or "CDR" refers to a non-contiguous sequence of amino acids in an antibody variable region that confers antigen specificity and binding affinity. Generally, each heavy chain variable domain has three CDRs (HCDR1, HCDR2, and HCDR3), and each light chain variable domain has three CDRs (LCDR1, LCDR2, and LCDR3). The term "framework region" or "FR" is known in the art to refer to the portions of heavy and light chain variable domains other than the CDRs. Generally, each heavy chain variable domain has four FRs (HFR1, HFR2, HFR3, and HFR4), and each light chain variable domain has four FRs (LFR1, LFR2, LFR3, and LFR4). Therefore, the antibody variable region amino acid sequence 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 separate amino acid sequence (or the polynucleotide sequence encoding it), and may be mutated, including one or more amino acid substitutions, deletions, and insertions. In certain embodiments, the antibody variable light chain amino acid sequence may be represented by the formula LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4. In certain embodiments, the antibody variable heavy chain amino acid sequence may be represented by the formula HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0111] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of several well-known schemes, including those described in 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 MP 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 Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 June.8;309(3):657-70 ("AHo" numbering scheme).

[0112] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering in both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, and some antibodies have insertions and deletions represented by inserted letters, e.g., "30a." The two schemes place certain insertions and deletions ("indels") at different positions, resulting in numbering differences. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0113] Table 1 below lists exemplary position boundaries for antibody LCDR1, LCDR2, LCDR3, and HCDR1, HCDR2, HCDR3, as identified by the Kabat, Chothia, and Contact schemes, respectively. For HCDR1, the residue numbering is listed using both the Kabat and Chothia numbering schemes. CDRs are located between FRs, e.g., LCDR1 is located between LFR1 and LFR2, etc. Note that the Kabat numbering scheme shown places insertions at H35A and H35B, so the end of the Chothia HCDR1 loop when numbered using the Kabat numbering convention shown varies between H32 and H34 depending on the length of the loop. [Table 1]

[0114] Thus, unless otherwise specified, the "CDR" or "complementarity determining region" of a given antibody or fragment thereof, e.g., its variable domain, or each specific CDR (e.g., HCDR1, HCDR2), should be understood to encompass the complementarity determining region (or specific complementarity determining region) defined by any of the well-known schemes. Similarly, unless otherwise specified, the "FR" or "framework region" of an antibody or fragment thereof, e.g., its variable domain, or each specific FR (e.g., "HFR1", "HFR2"), should be understood to encompass the framework region (or specific framework region) defined by any of the well-known schemes. In some cases, schemes for identifying specific CDRs or FRs are designated, such as CDRs defined by the Kabat, Chothia, or Contact methods. In other cases, the specific amino acid sequence of a CDR or FR is given.

[0115] 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" refers to an antigen-binding protein that has been modified to resemble a human antibody more closely. Non-human antigen-binding proteins, such as rabbit antigen-binding proteins, induce negative immune responses when administered to humans for therapeutic purposes. Therefore, humanizing rabbit antigen-binding proteins is advantageous for subsequent therapeutic use.

[0116] In certain embodiments, antigen binding proteins are humanized by resurfacing (i.e., remodeling solvent-exposed residues of a non-human framework to make them more human-like). Resurfacing strategies are detailed in WO2004 / 016740, WO2008 / 144757, and WO2005 / 016950, each of which is incorporated herein by reference.

[0117] 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 detailed in WO2009 / 155726, which is incorporated herein by reference.

[0118] 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 "% identical," "% identity," or similar terms are intended to refer to the percentage of nucleotides or amino acids (if applicable) that are identical, particularly after aligning the compared sequences, optionally introducing gaps to maximize identity. The percentage may be purely statistical, and the differences between the two sequences may or may not be randomly distributed over the entire length of the compared sequences. Comparison of two sequences is usually performed by comparing the sequences over a segment or "comparison window" after optimal alignment to identify local regions of corresponding sequences. For example, optimal alignment for comparison may be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or a computer program that uses an algorithm (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis., or Clustal Omega).In some embodiments, the percent homology of two sequences is determined using the BLASTN or BLASTP algorithms available at the United States National Center for Biotechnology Information (NCBI) website (e.g., 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 over which the analysis is performed.

[0119] A variant polypeptide, such as an antigen-binding protein, may contain one or more substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence. In certain embodiments, a variant polypeptide contains one, two, or three substitutions, insertions, and / or deletions relative to a reference sequence. The substitutions, insertions, or deletions may result in changes in one or more biophysical parameters, although tolerated changes are particularly preferred such that the polypeptide retains a desired activity. In some embodiments, a variant polypeptide contains one or more tolerated substitutions, such as conservative substitutions. In certain embodiments, such variant polypeptides maintain the physical, biological, chemical, and / or functional properties of the corresponding reference sequence.

[0120] "Specifically recognize" or "specifically bind" refers to the ability of an antigen-binding protein to selectively bind to an antigen, as opposed 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, the equilibrium dissociation constant (K D ) is less than about 50-fold lower than the equilibrium dissociation constant of the antigen-binding protein and its antigen, as determined, for example, by SPR.

[0121] As used herein, the term "affinity" (or "binding affinity," as 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, the affinity of an antibody or antigen-binding protein can be expressed as the equilibrium dissociation constant (K) in units of molar concentration (M). D ) can be reported as the equilibrium dissociation constant, K D is the association rate constant k a (M -1 s -1 (having units of ) and the dissociation rate constant k d (s -1 (has units of k) d / k a The antibodies of the present disclosure are calculated as follows: -8 ~10 -14 K in the M range D It may have a value.

[0122] The ability of an antibody to bind to a specific antigenic determinant (e.g., a target peptide-MHC) can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques known to those skilled in the art, such as surface plasmon resonance (SPR) techniques (e.g., performed using a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Generally, kinetic rate constants can be determined at temperatures ranging from 15°C to 37°C. This specification refers throughout to kinetic rate constants determined by SPR. Typically, in embodiments with respect to each reference to SPR throughout this specification, the association rate constant values, dissociation rate constant values, and equilibrium dissociation constant values ​​listed herein are determined by SPR at 25°C based on a monovalent antigen-binding protein. Preferably, the SPR-based system used is a BIAcore SPR system. Those skilled in the art will appreciate that binding parameters may be measured in the context of monovalent or bivalent, bi-, tri- or multispecific constructs.

[0123] In certain embodiments, the antigen-binding protein is not a T cell receptor (TCR), for example, but not limited to, a soluble TCR. As used herein, the term "T cell receptor" or "TCR" refers to a heterodimeric protein structurally composed of two distinct chains (TCRα and TCRβ) belonging 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 where stabilizing disulfide bonds form. The intracellular region forms non-covalent interactions with another transmembrane protein, CD3, which, upon correct target recognition, results in a series of conformational changes and the first T cell activation signal. Recognition and binding of peptide-MHC (pMHC) by TCRs is controlled by six hypervariable loops called complementarity-determining regions (CDRs) located in the variable domains of TCRα (CDRα1, CDRα2, CDRα3) and TCRβ (CDRβ1, CDRβ2, CDRβ3). The CDR3 loops (CDRα3 and CDRβ3) mediate recognition of processed antigens with the assistance of CDRα1 and CDRβ1, which have been shown to recognize the N- and C-terminal amino acids of presented peptides, respectively (Rudolph et al. Annu Rev Immunol. 24:419-66, 2006). MHC recognition is typically achieved through the interaction of CDRα2 and CDRβ2. The high sequence diversity of TCRs is achieved through the V(D)J rearrangement process. In this process, variable domains are generated from gene combinations: both TCRα and TCRβ are generated from V (variable) and J (joining) genes, while TCRβ is generated from an additional D (diversity) gene. The high antigen specificity of TCRs is controlled by a maturation process in the thymus, where autoreactive T cells undergo negative selection. TCR affinity and functional avidity for a particular pMHC are important factors controlling T cell activation. However, it is affinity, i.e., the strength of binding between the TCR and cell-presenting pMHC, that plays a key role in antigen recognition (Tian et al. J Immunol. 179:2952-2960, 2007).The physiological affinity of TCRs is in the range of 1 μM to 100 μM (Davidson et al. Annu Rev Immunol. 16:523-544. 1998), which is considerably lower than that of antibodies.

[0124] As used herein, the terms "peptide-MHC," or "pMHC" or "pMHC complex," as used interchangeably herein, refer to a major histocompatibility complex (MHC) molecule (MHC-I or -II) in which an antigenic peptide is bound to the peptide-binding pocket of the MHC. As known in the art, MHC molecules present peptides, particularly antigenic peptides, on the cell surface for recognition by immune cells. Therefore, as will be understood by those skilled 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 known as MHC-restricted antigen presentation. Thus, a peptide targeted by a pMHC-binding domain is an MHC-restricted peptide. Thus, a peptide can be considered a target peptide or a target antigenic peptide. Furthermore, according to the present disclosure, the terms "target pMHC-binding domain" and "pMHC-binding domain" can be used interchangeably herein, and in each case refer to at least the first and at least the second pMHC-binding domains referred to throughout the specification. The terms "target peptide / antigen presented by an MHC molecule / complex" and "MHC-restricted target peptide / antigen", or similar expressions used throughout this specification, may be used interchangeably herein.

[0125] In certain embodiments, the MHC is human. While MHC is present in all vertebrates, human MHC is known as HLA (human leukocyte antigen). HLA is highly polygenic and can be broadly divided into three classes of MHC molecules: class I, class II, and class III. Furthermore, HLA genes are the most polymorphic in the human genome. Target peptides can be presented in 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 respective subtypes) or MHC class II complexes (such as serotypes HLA-DP, HLA-DQ, HLA-DR, DM, or DO, or their respective subtypes). HLA-A proteins constitute the alpha chain of each class I MHC (major histocompatibility complex) protein and further contain a beta2 microglobulin subunit. As used interchangeably herein, "HLA-A2," "HLA-A*02," "HLA-A02," or "HLA-A*2" refer to the human leukocyte antigen serotype of HLA-A serogroup 2. Each serotype includes 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 a complex of an HLA-A*02 molecule and a MAGE-A4-derived peptide (also referred to herein as "MAGE-A4 peptide"), specifically GVYDGREHTV (SEQ ID NO: 3). "MAGE-A8 pMHC" refers to a complex of an HLA-A*02 molecule and a MAGE-A8-derived peptide (also referred to herein as a "MAGE-A8 peptide"), specifically GLYDGREHSV (SEQ ID NO: 71). "MAGE-B4 pMHC" refers to a complex of an HLA-A*02 molecule and a MAGE-B4-derived peptide (also referred to herein as a "MAGE-B4 peptide"), specifically GIYDGKRHLI (SEQ ID NO: 72).

[0126] As used herein, the term "PBS" refers to phosphate buffered saline. PBS is a pH-adjusted blend of phosphate buffer and saline. In certain embodiments, PBS contains about 100-150 mM NaCl, about 1-5 mM KCl, about 1-10 mM NaHPO, and 1-5 mM KHPO. In certain embodiments, PBS contains 130 mM NaCl, 10 mM NaHPO, and a pH of 6.0.

[0127] MAGE-A4 peptide-MHC and its antigen-binding protein The antigen binding proteins and multispecific antigen binding proteins described herein have binding specificity for MAGE-A4 peptide-MHC.

[0128] In certain embodiments, an isolated antigen binding protein is provided that binds to HLA-presented GVYDGREHTV (SEQ ID NO: 3), i.e., the isolated antigen binding protein is not associated with or bound to the surface of a cell, such as a T cell. An isolated antigen binding protein is separated from components of its natural environment. In certain embodiments, the isolated antigen binding protein is purified to greater than 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., 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.

[0129] The target peptide may be presented in an MHC class I complex (such as serotypes HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L, or each subtype thereof) or an MHC class II complex (such as serotypes HLA-DP, HLA-DQ, HLA-DR, DM, or DO, or each subtype thereof). Each serotype includes different subtypes. In one embodiment, the antigen binding protein targets peptides bound to the HLA-A2 pMHC complex, also referred to as HLA-A*02, and in particular to HLA-A*02:01.

[0130] The antigen binding proteins have surprisingly high binding affinity (i.e., low or no affinity for pMHC presenting irrelevant peptides, or beta-2-microglobulin) while retaining high specificity for their targets. As used herein, an "irrelevant peptide" corresponds to a peptide that does not contain the binding epitope of an antigen binding protein of the present disclosure. In certain embodiments, the affinity of the antigen binding protein for the irrelevant peptide MHC is less than about 50-fold lower than the binding of the antigen binding protein to HLA-presented GVYDGREHTV (SEQ ID NO: 3), as determined, for example, by SPR.

[0131] In certain embodiments, the antigen binding protein comprises specificity for the MAGE-A4 peptide amino acid sequence set forth in SEQ ID NO: 3 (GVYDGREHTV), which corresponds to amino acids 230-239 of MAGE-A4.

[0132] In certain embodiments, the antigen binding proteins bind to related HLA-presented MAGE-A4, MAGE-A8 and MAGE-B4 peptides with similar affinity ranges. Accordingly, the present disclosure provides antigen binding proteins comprising binding specificity for MAGE-A4 pMHC, MAGE-A8 pMHC and MAGE-B4 pMHC, in particular MAGE-A4-derived GVYDGREHTV (SEQ ID NO: 3), MAGE-A8-derived GLYDGREHSV (SEQ ID NO: 71) and MAGE-B4-derived GIYDGKRHLI (SEQ ID NO: 72).

[0133] In certain embodiments, the MAGE-A4 peptide, the MAGE-A8 peptide, and / or the MAGE-B4 peptide is in a complex with an HLA-A*02 polypeptide.

[0134] In certain embodiments, the HLA-A*02 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1.

[0135] In certain embodiments, the beta-2-microglobulin polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0136] Thus, in certain embodiments, the antigen binding protein comprises specificity for the peptide GVYDGREHTV (SEQ ID NO: 3) presented by HLA-A*02 / MAGE-A4, in particular HLA-A*02, more particularly the subtype HLA-A*02:01.

[0137] In one aspect, the present disclosure provides an antigen binding protein that specifically binds to melanoma associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising 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, or consists of, 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, or consists of the amino acid sequence of SEQ ID NO: 15.

[0138] In certain embodiments, such an antigen binding protein is an scFv comprising or consisting 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.

[0139] In one aspect, the present disclosure provides an antigen binding protein that specifically binds to melanoma associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising a VH domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to, or consists of, 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, or consists of the amino acid sequence of SEQ ID NO: 25.

[0140] In certain embodiments, such an antigen binding protein is an scFv comprising or consisting 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.

[0141] In one aspect, the present disclosure provides an antigen binding protein that specifically binds to melanoma associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising 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, 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%, 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.

[0142] In a specific embodiment, 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.

[0143] In certain embodiments, such an antigen binding protein is an scFv comprising or consisting 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.

[0144] In one aspect, the present disclosure provides an antigen binding protein that specifically binds to melanoma associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising 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, 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%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45.

[0145] In a specific embodiment, 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.

[0146] In certain embodiments, such an antigen binding protein is an scFv comprising or consisting 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.

[0147] Additional exemplary MAGE-A4 pMHC antigen binding proteins are described in PCT / IB2022 / 052117, filed March 9, 2022, and PCT / IB2022 / 052119, filed March 9, 2022, the contents of each of which are incorporated herein by reference.

[0148] Antigen-binding proteins can be monovalent or multivalent, e.g., bispecific or trispecific. In certain embodiments, the antigen-binding protein is monovalent. In certain aspects, monovalent antigen-binding proteins are provided that comprise the CDRs of the VL and / or VH sequences listed above. Such monovalent antigen-binding proteins include, but are not limited to, scFv, Fab, scFab, dAb, VHH, V(NAR), DARPin, affilin, and nanobody.

[0149] MAGE-A4 peptide-MHC-immune cell-engaging antigen-binding protein The multispecific antigen binding proteins described herein have at least one MAGE-A4 peptide-MHC binding domain and a binding domain with 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 with 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 a specific embodiment, the multispecific antigen binding protein comprises a MAGE-A4 peptide-MHC antigen binding protein described above.

[0150] With regard to dual pMHC engagement, targeting two MAGE-A4 peptide-MHC domains on the surface of target cells (e.g., cancer cells) increases binding avidity and improves target cell engagement. Increased binding avidity can also promote improved target cell killing compared to antigen binding proteins with only one pMHC-binding domain. The increased binding avidity provided by at least two pMHC-binding domains can be particularly useful when targeting low-copy pMHC complexes on the surface of target cells (e.g., cancer cells). Dual pMHC-engaging multispecific binding proteins are described in further detail in U.S. Patent No. 63 / 289,380, filed December 14, 2021, and U.S. Patent No. 63 / 317,256, filed March 7, 2022, the contents of each of which are incorporated herein by reference.

[0151] In certain embodiments, the multispecific antigen binding protein is bispecific or trispecific.

[0152] In certain embodiments, the multispecific antigen-binding protein is bivalent or multivalent, e.g., trivalent.

[0153] In certain embodiments, the multispecific antigen binding protein further comprises at least one additional binding domain.

[0154] In certain embodiments, the additional binding domain is an immune cell engager, in particular a CD3 binding domain or a CD16a binding domain.

[0155] In certain embodiments, the multispecific antigen-binding protein further comprises a third antigen-binding domain.

[0156] In certain embodiments, the third antigen-binding domain binds to HLA-A*02 / MAGE-A4.

[0157] In certain embodiments, the third antigen-binding domain is identical to the first HLA-A*02 / MAGE-A4 antigen-binding domain described above.

[0158] In certain embodiments, the multispecific antigen binding protein is capable of binding to two (or more) different epitopes of HLA-A2 / MAGE-A4.

[0159] In one aspect, the disclosure provides a multispecific antigen binding protein comprising: a) a first antigen binding domain that specifically binds to CD3; and b) a second antigen binding domain that specifically binds to melanoma associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), wherein b1) 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. 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 heavy chain variable (VH) 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 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). 10 X 11 SGSNFQX 12 (SEQ ID NO: 8) wherein X corresponds to the amino acid S or R, and X 10 corresponds to the amino acid D or P, and X 11 corresponds to the amino acid G, S, or F, and X 12 and a second antigen-binding domain comprising an antibody light chain variable (VL) domain comprising the amino acid L(A) corresponding to amino acid L or A).

[0160] In a specific embodiment, the MAGE-A4 peptide-MHC (pMHC) is the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex.

[0161] In certain embodiments, the multispecific antigen binding protein remains at least 94%, 95%, 96%, 97%, 98%, 99% or 100% monomeric after incubation at 1 mg / ml and / or 10 mg / ml in PBS at 4°C for 14 days as determined by SEC-HPLC.

[0162] In certain embodiments, the multispecific antigen binding protein further comprises a third antigen binding domain, in particular said third binding domain is identical to said second binding domain, i.e. the MAGE-A4 pMHC binding domain.

[0163] In certain embodiments, such a multispecific antigen binding protein comprises: c) MAGE-A4 and a third antigen-binding domain that specifically binds to pMHC, the third antigen-binding domain comprising: c1) a VH domain comprising an HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), an HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO: 6) (wherein 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) (wherein X4 corresponds to amino acid T, N, or S, X5 corresponds to amino acid D or is 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 VH 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 HCDR3 amino acid sequence of ATX9X 10 X 11 SGSNFQX 12 (SEQ ID NO: 8) wherein X corresponds to the amino acid S or R, and X 10 corresponds to the amino acid D or P, and X 11corresponds to the amino acid G, S, or F, and X 12 and a VL domain containing the amino acid L or A).

[0164] In certain embodiments, the second and third antigen-binding domains are

[0165] (i) a VH comprising an HCDR1 sequence comprising the amino acid sequence of SNYAMS (SEQ ID NO: 11), an HCDR2 sequence comprising the amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 12), and an HCDR3 sequence comprising the amino acid sequence of DLYYGPNTDYSAANL (SEQ ID NO: 13); and a VL comprising an LCDR1 sequence comprising the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), an LCDR2 sequence comprising the amino acid sequence of RDTSRPS (SEQ ID NO: 17), and an LCDR3 sequence comprising the amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 18);

[0166] (ii) VH comprising an HCDR1 sequence comprising the amino acid sequence of SNYAMS (SEQ ID NO: 21), an HCDR2 sequence comprising the amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 22), and an HCDR3 sequence comprising the amino acid sequence of DLYYGPSTYFVANL (SEQ ID NO: 23); and VL comprising an LCDR1 sequence comprising the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 26), an LCDR2 sequence comprising the amino acid sequence of RDTSRPS (SEQ ID NO: 27), and an LCDR3 sequence comprising the amino acid sequence of ATRPSSGSNFQL (SEQ ID NO: 28);

[0167] (iii) a VH comprising an HCDR1 sequence comprising the amino acid sequence of SNYAMS (SEQ ID NO: 31), an HCDR2 sequence comprising the amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 32), and an HCDR3 sequence comprising the amino acid sequence of DLYYGPTTYSAANL (SEQ ID NO: 33); and a VL comprising an LCDR1 sequence comprising the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 36), an LCDR2 sequence comprising the amino acid sequence of RDTSRPS (SEQ ID NO: 37), and an LCDR3 sequence comprising the amino acid sequence of ATRDFSGSNFQL (SEQ ID NO: 38); or

[0168] (iv) A VH comprising an HCDR1 sequence comprising the amino acid sequence of SNYAMS (SEQ ID NO: 41), an HCDR2 sequence comprising the amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 42), and an HCDR3 sequence comprising the amino acid sequence of DLYYGPTTYSAFNL (SEQ ID NO: 43), and a VL comprising an LCDR1 sequence comprising the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 46), an LCDR2 sequence comprising the amino acid sequence of RDTSRPS (SEQ ID NO: 47), and an LCDR3 sequence comprising the amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 48).

[0169] In certain embodiments, the second and third antigen-binding domains are

[0170] (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;

[0171] (ii) a VH domain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25;

[0172] (iii) a VH domain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35; or

[0173] (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.

[0174] In certain embodiments, any one or more of the first, second, and third antigen-binding domains comprise an antibody fragment, which in certain embodiments includes a Fab fragment, a F(ab')2 fragment, a Fab' fragment, an Fv fragment, a single-chain variable fragment (scFv), and a single-domain antibody fragment.

[0175] 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 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 which 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.

[0176] In a specific embodiment, the immune cell or CD3 antigen binding domain is a Fab fragment, which comprises a heavy chain comprising a CH1 domain and a VH, and a light chain comprising a CL domain and a VL.

[0177] In certain embodiments, the CH1 domain comprises at least 5 amino acids of an antibody hinge region. In certain embodiments, the CH1 domain comprises the amino acid sequence EPKSC (SEQ ID NO: 88) of an antibody hinge region.

[0178] In certain embodiments, the second antigen-binding domain is operably linked to the C-terminus of the heavy chain or the N-terminus of the heavy chain.

[0179] In certain embodiments, the third antigen-binding domain is operably linked to the C-terminus of the heavy chain or the N-terminus of the heavy chain.

[0180] In certain embodiments, the second antigen-binding domain is operably linked to the C-terminus of the light chain or the N-terminus of the light chain.

[0181] In certain embodiments, the third antigen-binding domain is operably linked to the C-terminus of the light chain or the N-terminus of the light chain.

[0182] In specific embodiments, a) the second antigen-binding domain comprises an scFv linked to the C-terminus of the Fab domain heavy chain, and the third antigen-binding domain comprises an scFv linked to the C-terminus of the Fab domain light chain; b) the second antigen-binding domain comprises an scFv linked to the N-terminus of the Fab domain heavy chain, and the third antigen-binding domain comprises an scFv linked to the N-terminus of the Fab domain light chain; c) the second antigen-binding domain comprises an scFv linked to the N-terminus of the Fab domain heavy chain, and the third antigen-binding domain comprises an scFv linked to the C-terminus of the Fab domain light chain; or d) the second antigen-binding domain comprises an scFv linked to the C-terminus of the Fab domain heavy chain, and the third antigen-binding domain comprises an scFv linked to the N-terminus of the Fab domain light chain.

[0183] In certain embodiments, the scFv is linked to the Fab domain by an amino acid linker.

[0184] In certain embodiments, the amino acid linker comprises (GGGGS)n (SEQ ID NO:73), where n is an integer from 1 to 5. In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGS (SEQ ID NO:74), GGGGSGGGGGSGGGGS (SEQ ID NO:75), GGGSGGGGSGGGGSGGGGS (SEQ ID NO:76), GGGSGGGGSGGGGSGGGGAS (SEQ ID NO:77), or GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:78).

[0185] In certain embodiments, the second and / or third antigen binding domains VH and VL are linked by an amino acid linker.

[0186] In certain embodiments, the amino acid linker comprises (GGGGS)n (SEQ ID NO:73), where n is an integer from 1 to 5. In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGS (SEQ ID NO:74), GGGGSGGGGGSGGGGS (SEQ ID NO:75), GGGSGGGGSGGGGSGGGGS (SEQ ID NO:76), GGGSGGGGSGGGGSGGGGAS (SEQ ID NO:77), or GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:78).

[0187] In certain embodiments, the multispecific antigen binding protein does not comprise an Fc domain.

[0188] In certain embodiments, the multispecific antigen-binding protein comprises an (scFv)2, (scFv)3, BiTE, BIKE, Dart, diabody, tribody, Fab2, Fab3, Fab4, scFv-Fab-scFv, or minibody-scFv.

[0189] In certain embodiments, the multispecific antigen binding protein comprises a molecular weight of about 75 kDa to about 110 kDa.

[0190] In certain embodiments, the antigen binding protein has a longer serum half-life compared to antigen binding proteins of molecular weight less than about 75 kDa.

[0191] Anti-CD3 binding domains suitable for the multispecific antigen-binding domains disclosed herein are known in the art, particularly T cell activating CD3-epsilon 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 minipig CD3 or rodent CD3, particularly rat or mouse CD3.

[0192] In a specific embodiment, the CD3 antigen-binding domain comprises: a1) a VH comprising an HCDR1 sequence comprising the amino acid sequence of STYAMN (SEQ ID NO: 51), an HCDR2 sequence comprising the amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 52), and an HCDR3 sequence comprising the amino acid sequence of HGNFGDSYVSWFAY (SEQ ID NO: 53); and a2) a VL comprising an LCDR1 sequence comprising the amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 56), an LCDR2 sequence comprising the amino acid sequence of GTNKRAP (SEQ ID NO: 57), and an LCDR3 sequence comprising the amino acid sequence of ALWYSNHWV (SEQ ID NO: 58).

[0193] In a specific embodiment, the CD3 antigen-binding domain comprises a VH comprising an amino acid sequence 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 comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:55.

[0194] In a specific embodiment, 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.

[0195] In certain embodiments, the CD3 antigen-binding domain comprises a heavy chain 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:49, and a light chain 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:54.

[0196] In a specific embodiment, 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.

[0197] 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 comprising an HCDR1 sequence comprising the amino acid sequence of STYAMN (SEQ ID NO: 51), an HCDR2 sequence comprising the amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 52), and an HCDR3 sequence comprising the amino acid sequence of HGNFGDSYVSWFAY (SEQ ID NO: 53), and a2) a VL comprising an LCDR1 sequence comprising the amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 56), an LCDR2 sequence comprising the amino acid sequence of GTNKRAP (SEQ ID NO: 57), and an LCDR3 sequence comprising the amino acid sequence of 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). Suitable embodiments of the second antigen-binding domain are described in detail above. Such multispecific antigen-binding proteins may comprise a third binding domain which may be identical to the second binding domain. Possible formats and modifications are also detailed above.

[0198] In one aspect, the disclosure provides an antigen binding protein which is multispecific, comprising: a) a Fab domain which specifically binds to CD3 on a T cell, the Fab domain comprising heavy and light chains and a CD3 antigen binding domain comprising a VH 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: 50, and a VL 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: 55; and b) a MAGE-A4 Fab domain operably linked to the C-terminus of the heavy chain, the Fab domain comprising: a VH 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 a VL comprising 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 a Fab light chain, wherein the Fab light chain comprises a VH comprising an amino acid sequence 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 comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15. In a particular embodiment, the multispecific antigen binding protein is a bispecific Fab(scFv)2, for example as shown in Figure 4.

[0199] In one aspect, the disclosure provides a multispecific antigen binding protein comprising: a) a Fab domain that specifically binds to CD3 on a T cell, the Fab domain comprising heavy and light chains and a CD3 antigen binding domain comprising a VH 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: 50, and a VL 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: 55; and b) a VH 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 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; and c) a MAGE-A4 pMHC binding domain operably linked to the C-terminus of a Fab light chain, wherein the Fab light chain comprises a VH comprising an amino acid sequence 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 comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25. In a particular embodiment, the multispecific antigen binding protein is a bispecific Fab(scFv)2, for example as shown in Figure 4.

[0200] In one aspect, the disclosure provides an antigen binding protein which is multispecific, comprising: a) a Fab domain which specifically binds to CD3 on a T cell, the Fab domain comprising heavy and light chains and a CD3 antigen binding domain comprising a VH 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: 50, and a VL 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: 55; and b) a VH 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 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; and c) a MAGE-A4 pMHC binding domain operably linked to the C-terminus of a Fab light chain, wherein the Fab light chain comprises a VH comprising an amino acid sequence 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 comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35. In a particular embodiment, the multispecific antigen binding protein is a bispecific Fab (scFv)2, for example as shown in Figure 4.

[0201] In one aspect, the disclosure provides an antigen binding protein which is multispecific, comprising: a) a Fab domain which specifically binds to CD3 on a T cell, the Fab domain comprising heavy and light chains and a CD3 antigen binding domain comprising a VH 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: 50, and a VL 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: 55; and b) a VH 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 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; and c) a MAGE-A4 pMHC binding domain operably linked to the C-terminus of a Fab light chain, wherein the Fab light chain comprises a VH comprising an amino acid sequence 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 comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45. In a particular embodiment, the multispecific antigen binding protein is a bispecific Fab(scFv)2, for example as shown in Figure 4.

[0202] In certain embodiments, the multispecific antigen binding is a Fab (scFv)2 comprising: (a) a single Fab domain targeting CD3, comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of an amino acid sequence 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 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 HLA-presented SEQ ID NO:3, each of which comprises or consists of an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% 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. In a specific embodiment, one scFv is linked to the C-terminus of the Fab heavy chain and the second scFv is linked to the C-terminus of the light chain. See exemplary embodiment in Figure 4.

[0203] In one aspect, the present disclosure provides a multispecific antigen binding protein that binds to GVYDGREHTV (SEQ ID NO: 3) HLA-A*02 complex and CD3, comprising:

[0204] (i) a first polypeptide chain comprising or consisting of 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;

[0205] (ii) a first polypeptide chain comprising or consisting of 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;

[0206] (iii) a first polypeptide chain comprising or consisting of 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

[0207] (iv) a first polypeptide chain comprising or consisting of 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;

[0208] Alternatively, multispecific antigen binding proteins are provided which comprise variants of the sequences which are at least 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences, whilst retaining antigen specificity (i.e. GVYDGREHTV (SEQ ID NO: 3) whilst retaining specificity for the HLA-A*02 complex and CD3. In certain embodiments, the pMHC binding domain, particularly when in scFv format, comprises a variable heavy chain with polar amino acids at positions 11, 89 and / or 108 according to Kabat numbering.

[0209] In certain embodiments, the Fab domain comprises a variable heavy chain having polar amino acids at positions 11, 89 and / or 108 according to the Kabat numbering system.

[0210] In certain embodiments, the variable heavy chain comprises a leucine (L) or serine (S) at amino acid position 11 according to Kabat numbering, a valine (V), serine (S), or threonine (T) at amino acid position 89 according to Kabat numbering, and / or a leucine (L), serine (S), or threonine (T) at amino acid position 108 according to Kabat numbering.

[0211] In certain embodiments, when there is a leucine (L) at amino acid position 11, there is a serine (S) or threonine (T) at amino acid position 89, and there is a serine (S) or threonine (T) at amino acid position 108, according to Kabat numbering.

[0212] In certain embodiments, when there is a valine (V) at amino acid position 89, there is a serine (S) at amino acid position 11, and there is a serine (S) or threonine (T) at amino acid position 108, according to Kabat numbering.

[0213] In certain embodiments, when there is a leucine (L) at amino acid position 108, there is a serine (S) or threonine (T) at amino acid position 11, and there is a serine (S) or threonine (T) at amino acid position 89, according to Kabat numbering.

[0214] In certain embodiments, the polar amino acid is serine (S) and / or threonine (T).

[0215] In certain embodiments, the variable heavy chain comprises a serine (S) at amino acid position 11, a serine (S) or threonine (T) at amino acid position 89, and a serine (S) or threonine (T) at amino acid position 108, according to Kabat numbering.

[0216] In certain embodiments, the variable heavy chain comprises a serine (S) at amino acid position 11, a serine (S) at amino acid position 89, and a serine (S) at amino acid position 108 according to Kabat numbering.

[0217] In certain embodiments, the Fab domain comprises a variable heavy chain in which the serine (S) at position 113 according to Kabat numbering has been deleted.

[0218] In certain embodiments, the pMHC binding domain, eg, the first and / or second pMHC binding domain, comprises a variable heavy chain in which the serine (S) at position 113 according to Kabat numbering has been deleted.

[0219] In a specific embodiment, the Fab domain comprises a variable heavy chain with a deleted serine (S) at position 112 and a deleted serine (S) at position 113 according to Kabat numbering.

[0220] In certain embodiments, the pMHC binding domain, e.g., the first and / or second pMHC binding domain, comprises a variable heavy chain with a deleted serine (S) at position 112 and a deleted serine (S) at position 113 according to Kabat numbering.

[0221] In certain embodiments, the antigen binding protein comprises an S113A, S113G, or S113T substitution according to Kabat numbering.

[0222] In certain embodiments, the antigen binding protein comprises an S113A, S113G, or S113T substitution according to Kabat numbering, and a deletion of S112.

[0223] In certain embodiments, the antigen binding protein comprises an S112A, S112G, or S112T substitution according to Kabat numbering.

[0224] In a specific embodiment, the antigen binding protein comprises an S112A, S112G, or S112T substitution according to Kabat numbering, and a deletion of S113.

[0225] In certain embodiments, the immune cell to which the multispecific antigen-binding molecule binds via the immune cell engager 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.

[0226] In certain embodiments, the Fab domain has a cytotoxicity of about 1 nM to about 100 nM (e.g., 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, , 21nM, 22nM, 23nM, 24nM, 25nM, 26nM, 27nM, 28nM, 29nM, 30nM, 31nM, 32nM, 33nM, 34nM, 35nM, 36nM, 37nM, 38nM, 39nM, 40nM, 41nM, 42nM, 43nM, 44nM, 45nM, 46nM, 47nM, 48nM, 4 9nM, 50nM, 51nM, 52nM, 53nM, 54nM, 55nM, 56nM, 57nM, 58nM, 59nM, 60nM, 61nM, 62nM, 6 3nM, 64nM, 65nM, 66nM, 67nM, 68nM, 69nM, 70nM, 71nM, 72nM, 73nM, 74nM, 75nM, 76nM, 77 In certain embodiments, the Fab domain specifically binds to CD3 with a binding affinity of about 1 nM to about 50 nM as determined by SPR. In certain embodiments, the Fab domain specifically binds to CD3 with a binding affinity of about 1 nM to about 50 nM as determined by SPR.

[0227] In certain embodiments, the Fab domain specifically binds to CD3 with a binding affinity of about 1 nM, about 10 nM, or about 50 nM, as determined by SPR.

[0228] In some embodiments, the association rate constant k of the anti-CD3 binding domain a is approximately 1 x 10 5 ~Approx. 1×10 7 M -1 s -1 , e.g., at least 1 x 10 6 M -1s -1 or at least 2×10 6 M -1 s -1 In some embodiments, the dissociation rate constant k of the anti-CD3 binding domain is d is approximately 1 x 10 -1 ~Approx. 1×10 -6 s -1 , e.g., at least 2 x 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., 2-3×10 -3 s -1 k d This value is thought to reduce overactivation of T cells and consequently reduce cytokine release.

[0229] In one embodiment, the association rate constant k a and / or the dissociation rate constant k d are equivalent or similar for both CD3 heterodimers CD3εγ (epsilon / gamma) and CD3εδ (epsilon / delta), i.e., the k of the anti-CD3 binding domain for CD3εγ (epsilon / gamma) and CD3εδ (epsilon / delta) when measured under identical conditions, in particular when determined by SPR at 25°C. a Or k d In certain embodiments thereof, there is no significant difference in either or both of the association rate constant k a and / or the dissociation rate constant k d are within 1-fold of each other, 1.5-fold of each other, 2-fold of each other, 2.5-fold of each other, or 3-fold of each other, i.e., the association rate constant k a The value is 1 x 10 5 M -1 s -1 and 3 x 10 5 M -1 s -1 is.

[0230] In certain embodiments, the pMHC binding domain binds to a target MAGE-A4-pMHC complex with a binding affinity of about 100 pM to about 5 nM (e.g., 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 a target pMHC complex with a binding affinity of about 100 pM to about 1 nM. In certain embodiments, the pMHC binding domain binds to a target pMHC complex with a binding affinity of about 100 pM to about 400 pM.

[0231] In certain embodiments, the pMHC binding domains disclosed herein bind to pMHC-presented MAGE-A8 and MAGE-B4 peptides with a binding affinity similar to that of pMHC-presented MAGE-A4 peptides. In certain embodiments, the pMHC binding domain binds to pMHC-presented MAGE-A8 peptides and / or pMHC-presented MAGE-B4 peptides with a binding affinity of about 1.5 nM to about 2.5 nM, e.g., 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, the pMHC binding domain binds to pMHC-presented MAGE-A8 peptides with a binding affinity of about 2.2 nM and / or to pMHC-presented MAGE-B4 peptides with a binding affinity of about 1.9 nM, as measured by SPR. In certain embodiments, the pMHC binding domain binds to pMHC-presented MAGE-A4, MAGE-A8, and MAGE-B4 peptides with similar binding affinities ranging from 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).

[0232] In some embodiments, the pMHC binding domain has an association rate constant, k a However, about 1 × 10 5 ~Approx. 1×10 7 M -1 s -1 , preferably about 0.5 x 10 6 M -1 s -1 ~Approx. 3×10 6 M -1 s -1 , e.g., at least 0.5 x 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 In some embodiments, the pMHC binding domain has a dissociation rate constant k for MAGE-A4 pMHC. d However, about 1 × 10 -1 ~Approx. 1×10 -6 s -1 , for example, about 1 x 10 -2 ~Approx. 1×10 -5 s -1 , e.g., at least 2 x 10 -3 s -1 , at least 4 × 10 -3 s -1 , at least 6 × 10 -3 s -1 , at least 8 x 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 This includes being.

[0233] In certain embodiments, the antigen binding protein comprises a molecular weight of about 75 kDa to about 110 kDa (e.g., 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 increased serum half-life compared to antigen binding proteins of molecular weight less than about 75 kDa.

[0234] An advantage of the antigen-binding protein scaffolds of the present disclosure is their intermediate molecular size, approximately 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 (60 kDa), blinatumomab is characterized by a short serum half-life of several hours, thus necessitating continuous infusion (see U.S. Pat. No. 7,112,324 B1). The antigen-binding proteins of the present disclosure are expected to have a significantly longer half-life than smaller bispecific antibodies, such as BiTEs, and thus do not require continuous infusion due to their favorable half-life. The intermediate size of the molecule allows it to avoid renal clearance and provides a half-life sufficient for improved tumor burden. The antigen-binding proteins of the present disclosure have an increased plasma half-life compared to other small bispecific formats while still retaining tumor penetration.

[0235] The Fab domain of the antigen binding proteins of the present disclosure can function as a specific heterodimerization scaffold to which additional pMHC binding domains are attached. The spontaneous and efficient heterodimerization properties of the heavy chain (Fd fragment) and light chain (L) of the Fab fragment make it a useful scaffold. The additional binding domain can be in several different formats, including, but not limited to, another Fab domain, an scFv, or an sdAb.

[0236] Each chain of the Fab fragment can be extended at the N- or C-terminus with an additional binding domain. The chains can be coexpressed in mammalian cells, and the host cell's binding immunoglobulin protein (BiP) chaperone induces the formation of heavy-light chain heterodimers (Fd:L). These heterodimers are stable, with each binding element retaining its specific affinity. The remaining two pMHC-binding domains can then be fused to separate Fab chains as scFvs or sdAbs, and each chain can be extended at the C-terminus with, for example, additional scFv or sdAb domains (see, e.g., Schoonjans et al. J. Immunology, 165(12):7050-7057, 2000; Schoonjans et al. Biomolecular Engineering, 17:193-202, 2001). An additional advantage of using Fab as the heterodimeric unit is that Fab molecules are abundant in serum and therefore may be non-immunogenic when administered to a subject.

[0237] In certain embodiments, the (multispecific) antigen binding proteins of the present disclosure have one or more of the following properties: (i) are capable of inhibiting tumor growth and eradicating tumors in a cell line-derived mouse NSCLC xenograft model, e.g., as exemplarily shown in Example 14, and / or (ii) exhibit efficacy against target-positive tumor cells as determined by LDH cytotoxicity, and / or (iii) are capable of inhibiting tumor growth and eradicating tumors in a cell line-derived mouse NSCLC xenograft model, e.g., as exemplarily shown in Example 14, and / or (ii) exhibit efficacy against target-positive tumor cells as determined by LDH cytotoxicity, e.g., as determined by IncuCyte exhibit efficacy against target-positive tumor cells as determined by the S3 system, and / or (iv) induce lower levels of the pro-inflammatory cytokine IFN-gamma in both antigen-positive and antigen-negative cell lines than Comparator 1 (e.g., see Example 12 for an exemplary assay), and / or (v) induce lower levels of the pro-inflammatory cytokines IL-2, IL-6, and / or TNF-alpha cytokine release than Comparator 1 (e.g., see Example 12 for an exemplary assay), (vi) possess T-cell activating properties (e.g., see Example 12 for an exemplary assay), and / or (vii) release of, for example, granzyme B. (e.g., antigen-negative cancer cell lines KLE (endometrial carcinoma), LNCaP (prostate lymph node carcinoma), KMRC-2 (clear cell renal cell carcinoma), KMRC-3 (clear cell renal cell carcinoma), 639-V (urothelial bladder carcinoma), EKVX (lung adenocarcinoma), and / or HCT116 (colorectal carcinoma), see, e.g., Example 12) and / or be safe, as measured by reactivity in healthy tissues, e.g., as determined by T cell activation assays (see Examples 12 and 13 for exemplary assays); and / or (viii) maintain at least 94% monomericity during storage at 1 mg / ml and / or 10 mg / ml in PBS at 4°C for at least 2 weeks, as determined by SEC-HPLC.

[0238] Variants of the sequences disclosed herein are also encompassed. A variant amino acid or nucleic acid sequence differs from its parent sequence by the insertion (including addition), deletion, and / or substitution of one or more amino acid residues or nucleic acid bases, but retains at least one desirable property of the parent sequence disclosed herein, e.g., specific antigen binding, efficacy against target-positive tumor cells, stability (e.g., serum stability, thermostability, and / or storage stability), productivity (e.g., expression level), safety (e.g., reactivity in healthy tissues, low or no induction of granzyme B release, pro-inflammatory cytokine IFN-gamma antigen-positive or antigen-negative cell lines, and / or pro-inflammatory cytokines IL-2, IL-6, and TNF-alpha cytokine release), efficacy (e.g., tumor growth inhibition, tumor eradication, and / or T-cell activation properties, as determined in vitro), or 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 (with a similar affinity range). In certain embodiments, the variant antigen binding protein retains the equilibrium dissociation constant K of the reference antigen binding protein (i.e., the corresponding antigen binding protein without the substitution, insertion, and / or deletion) when measured under identical conditions. D The variants retain at least 50%, e.g., 60%, 70%, 80%, 90%, or 95% of the binding to the GVYDGREHTV (SEQ ID NO: 3) HLA-A*02:01 complex. Variants can be engineered or naturally occurring, e.g., 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 an amino acid sequence disclosed herein.

[0239] Thus, in certain embodiments, the variant antigen binding protein retains specific binding to its target (i.e. MAGE-A4 pMHC, in particular GVYDGREHTV (SEQ ID NO: 3) HLA-A*02:01 complex, or CD3, respectively) and / or competes with the antigen binding protein disclosed herein for binding to its target. In certain embodiments, the antigen binding protein retains stability and remains at least 94%, 95%, 96%, 97%, 98%, 99% or 100% monomeric as determined by SEC-HPLC after 14 days of incubation at 1 mg / ml and / or 10 mg / ml in PBS at 4°C.

[0240] Decreased anti-drug antibody binding Anti-drug antibodies (ADAs) can affect the risk profile and efficacy of biologics. When neutralized, ADAs can block the ability of a drug to bind to its target. Therefore, it is a regulatory requirement to test biologics for anti-drug antibody binding and their neutralizing ability. Anti-drug antibody assays are described in detail, for example, in WO2007101661A1 (Hoffmann La Roche), WO2018178307A1 (Ablynx), WO2021046316A2 (Adverm Biotechnologies, Charles River), and US20180088140A1 (Genzyme Corporation), each of which is incorporated herein by reference.

[0241] Anti-drug antibodies that bind to the tumor-targeting domain of an antigen-binding protein can cause clustering of the antigen-binding protein when each variable domain of ADA binds to the tumor-targeting domain of one of the two antigen-binding proteins. Two or more CD3 binding domains on an antigen-binding protein cluster together, overstimulating targeted T cells in the absence of target binding, thereby causing off-target cytotoxicity. Non-specific stimulation of T cells can cause systemic cytokine release.

[0242] In general, there is a need in the art for the development of safer and more effective bispecific antibodies for cancer immunotherapy.

[0243] We have found that specific mutations in the tumor antigen-binding domain of T cell engagers simultaneously reduce ADA responses in the absence of target binding and decrease nonspecific T cell stimulation, providing a highly effective and safe approach for cancer immunotherapy.

[0244] For such purposes, the variable heavy chain amino acids at positions 11, 89, and / or 108, according to Kabat numbering, are substituted with polar amino acids, and / or the serine (S) at position 113, according to Kabat numbering, is deleted. Such substitutions are particularly preferred when the binding domain is in scFv format. In the case of Fab(scFv)2, one or both scFvs may contain such substitutions or deletions.

[0245] In certain embodiments, the polar amino acid is serine (S) and / or threonine (T).

[0246] In certain embodiments, the heavy chain amino acid has a substitution at amino acid position 11 of the heavy chain with serine (S), a substitution at amino acid position 89 of the heavy chain with serine (S) or threonine (T), and / or a substitution at amino acid position 108 of the heavy chain with serine (S) or threonine (T), according to Kabat numbering.

[0247] In certain embodiments, the heavy chain amino acid is substituted with serine (S) at amino acid position 11 of the heavy chain, with serine (S) at amino acid position 89 of the heavy chain, and with serine (S) at amino acid position 108 of the heavy chain, according to Kabat numbering.

[0248] In certain embodiments, in addition to deleting the serine(s) at position 113, the serine (S) at position 112 is deleted according to Kabat numbering.

[0249] In certain embodiments, the method further comprises adding alanine (A), glycine (G) or threonine (T), particularly alanine (A), at Kabat amino position 112 or 113.

[0250] In certain embodiments, the method further comprises adding an alanine (A) at Kabat amino position 112 or 113.

[0251] Expression of antigen-binding proteins In one aspect, polynucleotides or nucleic acids are provided that encode the antigen-binding proteins (including multispecific antigen-binding proteins) disclosed herein. Such polynucleotides or nucleic acids are typically isolated and synthesized. Methods for producing antigen-binding proteins, including expressing these polynucleotides, are also provided.

[0252] Polynucleotides encoding the antigen binding proteins disclosed herein are typically inserted into cloning or expression vectors for introduction into host cells which can be used to produce desired quantities of the antigen binding protein. Thus, in certain aspects, the present invention provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising these vectors and polynucleotides.

[0253] The term "vector" or "expression vector" is used herein to mean a vector used in accordance with the present invention as a vehicle for introducing and expressing a desired gene in a cell. As known to those skilled in the art, such vectors can be easily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, vectors compatible with the present invention contain a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0254] For purposes of the present invention, numerous expression vector systems can be employed. For example, one class of vectors utilizes 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 containing internal ribosome binding sites. Furthermore, cells into which the DNA has integrated into the chromosome can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers can confer prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by cotransformation. Additional elements may be required for optimal synthesis of mRNA. These elements can include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with heavy and light chain constant region genes (e.g., human constant region genes) synthesized as discussed above.

[0255] In other embodiments, antigen-binding proteins may be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as antibody heavy and light chains, may be produced from a single polycistronic construct. Such systems advantageously use internal ribosome entry sites (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells. Suitable IRES sequences are described 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 the present application.

[0256] 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 accomplished by a variety of techniques well known to those skilled in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See Ridgway, AAG "Mammalian Expression Vectors," Chapter 24.2, pp. 470-472, Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Introduction of the plasmid into the host can also be by electroporation. Transformed cells are grown under conditions appropriate for the production of light and heavy chains and assayed for the synthesis of heavy and / or light chain proteins. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting analysis (FACS), immunohistochemistry, and the like.

[0257] As used herein, the term "transformation" is intended to be used broadly to refer to the introduction of DNA into a recipient host cell, altering the genotype and resulting in a change in the recipient cell.

[0258] In this same sense, a "host cell" refers to a cell that has been transformed with a vector constructed using recombinant DNA technology and encoding at least one heterologous gene. In describing processes for isolating polypeptides from recombinant hosts, the terms "cells" and "cell culture" are used interchangeably to indicate the source of the antibody, unless otherwise specified. In other words, recovery of polypeptides from "cells" can mean recovery from either centrifuged whole cells or from a cell culture medium containing both medium and suspension cells.

[0259] In one embodiment, the host cell line used for antibody expression is of mammalian origin. One skilled in the art can determine the particular host cell line most suitable for the desired gene product to be expressed. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary lines, DHFR minus), HELA (human cervical carcinoma), CV-1 (monkey kidney line), COS (a derivative of CV-1 containing the SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). In one embodiment, the cell line provides for altered glycosylation (e.g., defucosylation) of antibodies expressed from the cell line (e.g., PER.C6® (Crucell) or a FUT8 knockout CHO cell line (Potelligent® cells) (Biowa, Princeton, NJ)). Host cell lines are typically available from commercial services, e.g., the American Tissue Culture Collection, or from published literature.

[0260] In vitro production allows for scale-up to obtain large amounts of the desired polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in airlift reactors or continuous stirred reactors, or immobilized or entrapped cell culture in, for example, hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatographic methods, for example, gel filtration, ion exchange chromatography, chromatography on DEAE cellulose, and / or (immuno)affinity chromatography.

[0261] Genes encoding antigen-binding proteins featured in the present invention can also be expressed in non-mammalian cells, such as bacteria or yeast, or in plant cells. In this regard, it will be understood that various unicellular non-mammalian microorganisms, such as bacteria, i.e., microorganisms that can be grown in culture or fermentation, can also be transformed. Bacteria that are susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. It will further be understood that when expressed in bacteria, the protein may become part of inclusion bodies. The protein must be isolated, purified, and then assembled into a functional molecule.

[0262] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, although other strains are commonly available. For expression in Saccharomyces, for example, the plasmid Yrp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used. This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutants lacking the ability to grow in tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). Therefore, the presence of the trp1 lesion, characteristic of the yeast host cell genome, provides an effective environment for detecting transformants by growth in the absence of tryptophan.

[0263] Engineering and Optimization of Antigen Binding Proteins Antigen-binding proteins of the present disclosure, including multispecific antigen-binding proteins, may be engineered or optimized. As used herein, "optimized" or "optimization" refers to modification of an antigen-binding protein to improve one or more functional properties. Modifications include, but are not limited to, deletion, substitution, addition, and / or modification of one or more amino acids within the antigen-binding protein.

[0264] As used herein, the term "functional property" is a property of an antigen-binding protein for which improvement (e.g., compared to conventional antigen-binding proteins such as antibodies) is desirable and / or advantageous to one of skill in the art, for example, to improve the manufacturing properties or therapeutic efficacy of the antigen-binding protein. In one embodiment, the functional property is stability (e.g., thermostability). In another embodiment, the functional property is solubility (e.g., under cellular conditions). In yet another embodiment, the functional property is aggregation behavior. In yet another embodiment, the functional property is protein expression (e.g., in prokaryotic cells). In yet another embodiment, the functional property is refolding behavior after inclusion body solubilization in a manufacturing process. In certain embodiments, the functional property is not an improvement in antigen-binding affinity. In another embodiment, an improvement in one or more functional properties does not substantially affect the binding affinity of the antigen-binding protein.

[0265] In certain embodiments, antigen binding proteins of the present disclosure comprise scFvs and are optimized by identifying amino acid residues that are preferred for substitution, deletion, and / or addition at amino acid positions of interest in the antigen binding protein (e.g., amino acid positions identified by comparing a database of scFv sequences having at least one desired property, e.g., a database of scFv sequences selected by a quality control (QC) assay, to a database of mature antibody sequences, e.g., the Kabat database). Accordingly, the present disclosure further provides an "enrichment / exclusion" method for selecting specific amino acid residues. Still further, 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" approach described herein. In certain embodiments, framework amino acid positions are mutated by replacing existing amino acid residues with residues found to be "enriched" residues using the "enrichment / exclusion" analysis method described herein. In one aspect, the disclosure provides a method for identifying amino acid positions for mutation in a single chain antibody (scFv), wherein the scFv has a VH and VL amino acid sequence, the method comprising: a) inputting the scFv VH, VL or VH and VL amino acid sequence into a database containing a large number of antibody VH, VL or VH and VL amino acid sequences to align the scFv VH, VL or VH and VL amino acid sequence 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 sequence 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 sequence are occupied by amino acid residues that are conserved at corresponding positions within the antibody VH or VL amino acid sequences of the database; and d) aligning the scFv VH, VL or VH and VL amino acid sequence with the corresponding positions within the antibody VH or VL amino acid sequences of the database. and identifying an amino acid position in the VH or VL amino acid sequence as an amino acid position for mutation if the amino acid position is occupied by an amino acid residue that is not conserved at the corresponding position in the antibody VH or VL amino acid sequences in the database.ScFv optimization is described in further detail in WO2008110348, WO2009000099, WO2009000098, and WO2009155725, all of which are incorporated herein by reference.

[0266] In certain embodiments, the antigen binding protein comprises an Fc domain that has been modified so that it does not induce a cytotoxic immune response and / or activate antibodies. For example, one or more substitutions can be introduced into the Fc domain so that ADCC / ADCP or CDC effector function is inactivated. Such antigen binding proteins have the advantage of increased half-life without mediating a cytotoxic immune response when compared to antibody fragments with a molecular weight of less than 75 kDa.

[0267] Chemical and / or biological modifications In one embodiment, 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 may be glycosylated, phosphorylated, hydroxylated, PEGylated, HES-modified, PAS-modified, XTEN-modified, sulfation, labeled with dyes and / or radioisotopes, conjugated to enzymes and / or toxins, and / or fused to albumin. Similarly, any nucleic acid sequence, plasmid or vector, and / or host cell described herein may also be modified appropriately.

[0268] Such modifications may be made, for example, to optimize its pharmacodynamics, its water solubility, or to reduce its side effects. For example, PEGylation, PASylation, XTENylation, HESylation, and / or fusion to serum albumin may be applied to delay renal clearance and thereby increase the plasma half-life of the antigen-binding protein. In one embodiment, the modification adds a different function to the antigen-binding protein, for example, a detection label for diagnosis or a toxin to more efficiently combat cancer cells.

[0269] Alternatively or additionally, in some embodiments, the antigen binding proteins and other polypeptides provided herein are subject to 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, for example, N-terminal pyroglutamate formation), removal or "clipping" of N- or C-terminal residues (e.g., C-terminal lysine residues are often removed during the manufacturing process), deamidation, isomerization, oxidation, glycation, acylation, fucosylation, peptide bond cleavage, non-reducing cross-linking, truncation, and / or incomplete processing of part or all of a signal sequence.

[0270] In certain embodiments, the CD3 antigen-binding domain comprises an N-terminal truncation of one 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 one or more amino acids (e.g., a C-terminal truncation of 1, 2, 3, 4, or 5 amino acids). In certain embodiments, the N- and / or C-terminal truncations are truncations of a 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, e.g., one or two 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, e.g., one or two 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.

[0271] In certain embodiments, the (multispecific) antigen-binding protein comprises an N-terminal glutamine or pyroglutamic acid (pE, pyrGlu, pyre, or pGlu) in place of the N-terminal glutamine. In certain embodiments, the light chain of the antigen-binding protein comprises pyroglutamic acid (pE) in place of the N-terminal glutamine. In certain embodiments, a variant sequence 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 comprises pyroglutamic acid at amino acid position 1. In certain embodiments, the light chain of the antigen-binding protein comprises pyroglutamic acid (pE) in place of the N-terminal glutamic acid. In certain embodiments, a variant sequence 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 comprises pyroglutamic acid at amino acid position 1. In certain embodiments, such pyroglutamic acid (pE) modifications do not affect the stability and / or efficacy of the (multispecific) antigen binding protein.

[0272] In one embodiment, the antigen-binding protein is glycosylated. Glycosylation refers to the process of carbohydrate attachment to proteins. In biological systems, this process is carried out enzymatically within cells as a 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 hydroxy oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side chains; utilize xylose, fucose, mannose, and N-acetylglucosamine attached to phosphoserine; and / or mannose sugars can be added to tryptophan residues found in specific recognition sequences. Glycosylation patterns can be controlled, for example, by selecting appropriate cell lines, culture media, protein engineering production modes, and process strategies (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 patterns of the antigen binding proteins described herein are modified to enhance ADCC and CDC effector function.

[0273] Antigen binding proteins can be engineered to control or modify glycosylation patterns, for example, by deleting and / or adding one or more glycosylation sites. Creation of a glycosylation site can be achieved, for example, by introducing a corresponding enzyme recognition sequence into the amino acid sequence of the antigen binding protein.

[0274] In some embodiments, the antigen-binding protein is PEGylated. PEGylation can alter the pharmacodynamic and pharmacokinetic properties of the protein. Furthermore, PEGylation can reduce immunogenicity by shielding the PEGylated antigen-binding protein from the immune system and / or modify its pharmacokinetics, for example, by increasing the antigen-binding protein's in vivo stability, protecting it from proteolysis, extending its half-life, and modifying its biodistribution. Typically, polyethylene glycol (PEG) of an appropriate molecular weight is covalently attached to the protein. A similar effect can also be achieved by using PEG mimetics, for example, by subjecting the antigen-binding protein to HESylation, XTenylation, or PASylation. HESylation utilizes a hydroxyethyl starch ("HES") derivative. In PASylation, the antigen-binding protein is linked to a non-conformal polypeptide sequence composed of the amino acids proline (P), alanine (A), and serine (S).

[0275] In certain embodiments, the antigen-binding protein (e.g., a multispecific antigen-binding protein) is linked to or combined with a detectable label, therapeutic agent, or PK-modifying moiety. For example, the antigen-binding protein may be labeled or conjugated with a second moiety that confers one or more auxiliary functions to the antigen-binding protein. For example, the second moiety may have an additional immunological effector function, be effective for drug targeting, or be useful for detection. The second moiety may 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, when detected or measured by physical or chemical means, either directly or indirectly, indicates the presence of the antigen-binding protein. For example, the label may be directly detectable by, but not limited to, absorbance, fluorescence, reflectance, light scattering, phosphorescence, or luminescence properties, a molecule or ion detectable by radioactivity, or a molecule or ion detectable by nuclear magnetic resonance or paramagnetics. Examples of indirect detection include light absorption or fluorescence, including various enzymes that convert an appropriate substrate, for example, 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 are particularly useful for in vitro and in vivo detection or diagnostic purposes. For example, antigen-binding proteins labeled with suitable radioisotopes, enzymes, fluorophores, or chromophores can be detected by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), or single-cell analysis by flow cytometry (e.g., FACS analysis), respectively. Similarly, the nucleic acids and / or vectors disclosed herein can be labeled for detection or diagnostic purposes, e.g., labeled fragments thereof can be used as probes in hybridization assays.

[0276] Non-limiting examples of second moieties include radioisotopes (35S, 32P, 14C, 18F, and / or 125I), apoenzymes, enzymes (e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and / or angiogenin), cofactors, peptide moieties (e.g., HIS tags), proteins (e.g., lectins, serum albumin), carbohydrates (e.g., mannose-6-phosphate tags), 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).

[0277] In one aspect, the invention relates to a drug conjugate (particularly an antibody-drug conjugate ADC) comprising an antigen binding protein as described herein, e.g., a monovalent or multispecific antigen binding protein as described herein, conjugated to a toxin that further enhances efficient killing of specific cells, such as, for example, MAGE-A4 positive cells. The toxin moiety is typically a low molecular weight moiety such as an anthracycline toxin, taxol, gramicidin D and / or colchicine, and may be linked to the antigen binding protein via a peptide linker.

[0278] Toxins can be conjugated to antigen-binding proteins non-site-specifically or site-specifically.Non-site-specific conjugation typically involves the use of a chemical linker, such as a maleimide functional group, which mediates conjugation to the lysine or cysteine ​​amino acid side chain or N-terminal amino acid group of antigen-binding proteins.Site-specific conjugation can be achieved using chemical, chemoenzymatic, or enzymatic conjugation known in the art, for example, by using a bifunctional linker, a linker that allows the Pictet-Spengler chemical reaction on antigen-binding proteins modified with bacterial transglutaminase or sortase enzyme, formyl-glycine generating enzyme, or a glycan-remodeled antigen-binding protein.

[0279] Methods of Administering Antigen-Binding Proteins Methods of preparing and administering to a subject an antigen-binding protein of the present disclosure (such as the multispecific antigen-binding protein described above), as well as a nucleic acid described herein, a vector described herein, a host cell described herein, or a composition described herein, are well known to or readily determined by those of skill in the art. Routes of administration of antigen-binding proteins of the present disclosure can be, for example, oral, parenteral, inhalation, or topical. The term parenteral, as used herein, includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or intravaginal administration. The term intraocular, as used herein, includes, but is not limited to, subconjunctival, intravitreal, retrobulbar, or intracameral administration. The term topical, as used herein, includes, but is not limited to, administration via liquid or solution eye drops, emulsions (e.g., oil-in-water emulsions), suspensions, and ointments.

[0280] The dosage form may be an injectable solution, although all of these dosage forms are expressly contemplated as being within the scope of the present disclosure. Typically, a suitable injectable pharmaceutical composition may include a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer (e.g., human albumin), and the like. However, in other methods consistent with the teachings herein, the modified antibody may be delivered directly to the site of the harmful cell population, thereby increasing the exposure of the affected tissue to the therapeutic agent.

[0281] The effective amount of the compositions of the present disclosure for treating the relevant conditions will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other medications being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, although non-human mammals, including transgenic mammals, can also be treated. Treatment dosages can be titrated using routine methods known to those skilled in the art to optimize safety and efficacy.

[0282] As previously mentioned, antigen binding proteins (e.g., multispecific antigen binding proteins), conjugates or recombinants thereof of the present disclosure may be administered in pharmaceutically effective amounts for the in vivo treatment of mammalian disorders. In this regard, it will be understood that the antigen binding proteins of the present disclosure will be formulated to facilitate administration and promote stability of the active agent.

[0283] Pharmaceutical compositions according to the present disclosure typically comprise a pharmaceutically acceptable, non-toxic, sterile carrier, such as physiological saline, non-toxic buffers, preservatives, and the like. For purposes of this application, a pharmaceutically effective amount of an antigen-binding protein (such as a multispecific antigen-binding protein) shall mean an amount sufficient to achieve effective binding to the antigen and provide a benefit, e.g., an amount sufficient to ameliorate the symptoms of a disease or disorder or to detect a substance or cell. In the case of tumor cells, the antigen-binding protein is typically capable of interacting with a selected immunoreactive antigen on a neoplasm or immunoreactive cells, increasing the killing of these cells. Of course, the pharmaceutical compositions of the present disclosure may be administered in a single dose or multiple doses to provide a pharmaceutically effective amount of the modified binding polypeptide.

[0284] In accordance with the scope of the present disclosure, antigen binding proteins of the present disclosure (such as the multispecific antigen binding proteins described above) may be administered to humans or other animals in amounts sufficient to provide a therapeutic or prophylactic effect according to the aforementioned methods of treatment. The antigen binding proteins of the present disclosure may be administered to such humans or other animals in conventional dosage forms prepared by combining the antigen binding proteins of the present disclosure with conventional pharmaceutically acceptable carriers or diluents in accordance with known techniques. Those skilled in the art will recognize that the form and characteristics of the pharmaceutically acceptable carrier or diluent will be determined by the amounts of active ingredients to be combined, the route of administration, and other well-known variables. Furthermore, those skilled in the art will appreciate that cocktails comprising one or more of the antigen binding proteins described in the present disclosure may prove particularly effective. Similarly, the nucleic acids described herein, the vectors described herein, the host cells described herein (particularly immune cells bearing CARs), or the compositions described herein may be administered to humans or other animals in amounts sufficient to provide a therapeutic or prophylactic effect according to the methods of treatment described above.

[0285] As used herein, "efficacy" or "in vivo efficacy" refers to a response to treatment with a pharmaceutical composition of the present disclosure, for example, using standardized efficacy criteria. Successful treatment or in vivo efficacy using a pharmaceutical composition of the present disclosure refers to the effectiveness of the composition for its intended purpose, i.e., the ability of the composition to produce the desired effect. In vivo efficacy can be monitored by standard methods established for a particular disease. In addition, various disease-specific clinical chemistry parameters and other established standard methods may be used.

[0286] 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 may 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 phototherapy, radiation therapy, or vaccine therapy.

[0287] Chimeric Antigen Receptor In one aspect, the present disclosure provides chimeric antigen receptors (CARs) comprising the antigen-binding protein sequences described herein, and immune cells engineered to express such CARs. As used herein, the term "chimeric antigen receptor" or "CAR" refers to a receptor capable of activating immune cells in response to antigen binding. CARs are recombinant transmembrane molecules that are advantageously expressed on immune cells. Their structure typically includes (i) an extracellular domain (ectodomain or antibody domain), (ii) a transmembrane domain, and (iii) a cytoplasmic domain (endodomain or intracellular signaling domain).

[0288] The ectodomain (i.e., antibody domain) typically comprises an scFv, but other formats can also be used. A spacer connects the ectodomain to the transmembrane domain and then to the endodomain. When the ectodomain binds to an antigen, a receptor cluster and an activation signal are transmitted to the cell, initiating an immune response. First-generation CARs have a simple endodomain structure, including CD3-zeta. To increase the activation signal, a costimulatory domain is added in second-generation CARs, and third-generation CARs contain two or more costimulatory domains (Maus MV et al. (2014) Blood, 123:2625-2635). The costimulatory domain can be selected from the group consisting of CD28, OX40, and / or 4-1BB. In addition to CD3-zeta, other ITAM-containing domains, including the Fc receptor of the IgE-γ domain, are also being explored.

[0289] In certain embodiments, the CAR comprises an 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 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.

[0290] 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. T cells can be CD4+ or CD8+ or a mixed population of CD4+ and CD8+ cells.

[0291] In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) that specifically recognizes peptide-MHC, comprising: i) an antigen binding protein identified from a nucleic acid library or method described herein; ii) a transmembrane domain; and iii) an intracellular signaling domain.

[0292] In certain embodiments, the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and a transmembrane domain of a type I transmembrane protein, the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0293] 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 an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0294] The antibody domain may be any of the antigen-binding proteins outlined above. Thus, in certain embodiments, the antibody domain comprises an antibody variable light chain domain (VL) comprising 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 chain domain (VH) comprising 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.

[0295] How to Treat Cancer Provided herein are methods of treating cancer with antigen binding proteins of the present disclosure, particularly multispecific antigen binding proteins (e.g., MAGE-A4 pMHC antigen binding proteins). The methods can be used to treat patients with any tumor type in which at least a portion of the 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 assessed using any method known in the art, including, but not limited to, detection of RNA expression levels or histological methods such as immunohistochemistry (IHC).

[0296] In some embodiments, the MAGE-A4 positive cancer is 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, renal cancer, synovial sarcoma, and tumors with squamous cell histology. In certain embodiments, the cancer is of squamous epithelial origin. Experimental data have shown that squamous cell carcinoma has both the highest prevalence and median expression level of MAGE-A4 mRNA. In certain embodiments, the cancer is 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, e.g., uterine carcinosarcoma (UCS; particularly, the UCEC subgroup), myxoid / round cell liposarcoma, gastric or gastroesophageal junction (GEJ) adenocarcinoma, epithelial ovarian cancer, e.g., high-grade serous ovarian carcinoma, synovial sarcoma, bladder urothelial carcinoma (BLCA), particularly, transitional cell carcinoma, testicular germ cell tumor (TGCT), and cervical squamous cell carcinoma (CESC).

[0297] The antigen binding proteins described herein also comprise binding specificity for certain relevant MAGE pMHC targets, in particular 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 methods of treating such relevant MAGE-pMHC positive cancers.

[0298] In one aspect, the present disclosure provides the use of an antigen binding protein described herein, a multispecific antigen binding protein described herein, or a pharmaceutical composition described herein in the manufacture of a medicament.

[0299] In another aspect, the present disclosure provides the use of 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 for use in the treatment of a disease, particularly cancer.

[0300] In another aspect, the present disclosure provides a method for treating a MAGE-A4 pMHC-expressing cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of 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.

[0301] In certain embodiments, patients eligible for treatment with a MAGE-A4 antagonist are selected based on RNA sequencing and / or immunohistochemistry (IHC), such as detection of total MAGE-A4.

[0302] In certain embodiments, patients eligible for treatment with a MAGE-A4 antagonist described herein (e.g., 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) are selected based on an immunohistological method comprising the following sequential steps:

[0303] (i) obtaining a tumor sample from a patient;

[0304] (ii) adding an anti-MAGEA4 detection antibody to the sample;

[0305] (iii) incubating the detection antibody and the sample;

[0306] (iv) detecting the detection antibody bound to the sample; and

[0307] (v) selecting the patient for treatment with a MAGE-A4 antagonist if the detection antibody binds to the sample.

[0308] In certain embodiments, the detection antibody is OTI1F9, E7O1U, or an antigen-binding protein described herein. The OTI1F9 detection antibody is further described in WO2019171064A1. In vitro overexpression experiments showed that E7O1U binds only to MAGE-A4, while OTI1F9 broadly cross-reacts with MAGE-A3, -A6, -A8, -A10, -A11, and -A12. Thus, E7O1U is more specific than OTI1F9. Thus, in certain embodiments, the anti-MAGE-A4 detection antibody is E7O1U. For example, E7O1U is commercially available as MAGE-A4 (E7O1U) XP rabbit mAb.

[0309] 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.

[0310] kit Also contemplated are kits comprising at least one nucleic acid library or antigen binding protein (including multispecific antigen binding proteins), or pharmaceutical composition described herein, together with a combination of reagents, typically packaged with instructions. In one embodiment, the kit comprises 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, a vial, an ampoule, a bottle, a tube, a syringe, or a blister pack. In some embodiments, the composition is a pharmaceutical composition, and the container is made of a material suitable for holding pharmaceuticals. 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 use. In one embodiment, the kit is for therapeutic purposes, and the antigen binding protein is formulated for therapeutic use.

[0311] Typically, the kit further comprises a separate sheet, pamphlet, or card with instructions for use provided in or with the container. If the kit is intended for pharmaceutical use, the kit may further comprise one or more of the following: information and administration schedule for administering the composition to a subject with the relevant disease or disorder, therapeutic drug descriptions, precautions, warnings, indications, contraindications, overdose information, and / or side effects.

[0312] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein, using appropriate equivalents, can be made without departing from the scope of the embodiments disclosed herein. While certain embodiments have been described in detail above, the embodiments will be more clearly understood by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting. [Example]

[0313] Example 1 - Production of pMHC antigens for animal immunization MHC class I heavy chain and β2m were cloned into the pET-24D(+) vector using standard molecular biology techniques (J Biol Chem. 1995 Jan 13;270(2):971-7). E. coli BL-21(DE3) was transformed with the expression vector according to the supplier's protocol. Protein expression was carried out for 16-18 hours at 37°C with shaking at 220 rpm in MagicMedium (Invitrogen) according to the supplier's instructions. Cells were harvested and lysed with BugBuster (Invitrogen), and inclusion bodies were washed twice with TBS containing 0.5% LDAO and twice with TBS. The inclusion bodies thus prepared were solubilized in denaturing buffer (8 M urea, 100 mM Tris-HCl pH 8) using 5 mL of buffer per 1 g of inclusion body pellet. Refolding and purification of MHC containing target peptides (HLA-A*02:01 extracellular domain, human β2m, and MAGE-A4 peptide 230-239) was performed essentially as described by Rodenko et al. (2006). The amino acid sequences of each element of the pMHC antigen are listed in Table 1 below.

[0314] [Table 2]

[0315] Example 2 - Rabbit Immunization To generate antibodies capable of specifically recognizing 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 complexes. Each animal received four injections of HLA-A*02:01 / MAGE-A4 complexes containing complete or incomplete Freund's adjuvant at different time points. The animals' immune responses were tested by ELISA to quantify anti-pMHC antibodies present in serum samples from immunized animals.

[0316] Example 3 - Construction of a rabbit-derived immune library A cDNA library of ScFv antibodies was constructed by PCR amplification of RNA extracted from PBMCs and splenic lymphocytes isolated from rabbits. The coding sequences for the variable light (VL) and heavy (VH) domains were amplified separately and linked by a series of overlapping PCR steps to obtain the final scFv product. The amplified DNA sequences encoding the rabbit-derived scFvs were digested with appropriate restriction enzymes and then ligated into a phagemid vector. The phagemid vector was transformed into E. coli TG1 electrocompetent cells, which are well suited for generating antibody phage display libraries. These processes yielded two antibody libraries: a kappa-based library containing 5.2 × 10 8 The lambda-based library contained 2.0 × 10 9 diversity and accuracy of 91.7%.

[0317] Example 4 - Screening of a rabbit-derived immune library A rabbit-derived immune library was screened for HLA-A*02:01 / MAGE-A4-specific binding elements. Briefly, after three rounds of phage display biopanning against the HLA-A*02:01 / MAGE-A4 antigen, the library was screened for specific hits. Screening was performed by monoclonal phage ELISA against the specific target, i.e., HLA-A*02:01 / MAGE-A4, and a nonspecific target, i.e., HLA-A*02:01 / peptide mix target. A control HLA-A*02:01 / peptide mix complex included an HLA-A*02:01 complex loaded with a mixture of 49 irrelevant peptides. The signal ratio between specific and nonspecific target binding was calculated to determine hits that specifically bound to the target. Identified hits were expressed as chimeric Fabs.

[0318] Example 5 - Antibody expression as a monovalent monospecific Fab Monovalent monospecific antibodies were expressed in Fab format. Rabbit variable domains were paired with human constant domains (heavy and kappa light chains) to generate chimeric Fabs that bind to target pMHC. The amino acid sequences of the constant domains are listed in Table 2 below.

[0319] [Table 3]

[0320] Synthetic genes encoding the different antibody chains (i.e., heavy and light chains) were constructed at Twist Bioscience Corporation and separately cloned into expression vectors for transient expression in HEK293 6E cells. Expression vector DNA was prepared using conventional plasmid DNA purification methods (e.g., Qiagen HiSpeed ​​Plasmid Maxi Kit, catalog number 12662).

[0321] Antigen-binding proteins were expressed by transient co-transfection of the respective mammalian expression vectors in suspension-cultured HEK293-6E cells using polyethyleneimine (linear PEI 40 kD). HEK293-6E cells were grown at 1.7 x 10 in Freestyle F17 medium supplemented with 2 mM L-glutamine. 6 Cells were seeded at 1000 cells / mL. DNA per mL of final production volume was prepared by adding DNA and PEI separately to 50 μL of medium without additives. Both fractions were mixed, vortexed, and left to stand for 15 minutes, resulting in a DNA:PEI ratio of 1:2.5 (1 μg DNA / mL cells). The cells and DNA / PEI mixture were combined, then transferred to a suitable container and placed in a shaker (37°C, 5% CO2, 80% RH). After 24 hours, 25 μL of Tryptone N1 was added per mL of final production volume.

[0322] After 7 days, cells were harvested by centrifugation and sterile filtered. For affinity purification of Fab-based constructs, the supernatant was loaded onto a CaptureSelect™ CH1-XL column (Thermo Fisher Scientific) equilibrated with 6 CV of PBS (pH 7.4). After a wash step with the same buffer, the antigen-binding protein was eluted from the column by stepwise elution with 100 mM citric acid (pH 3.0). Fractions containing the desired antigen-binding protein were immediately neutralized with 1 M Tris buffer (pH 9.0) at a 1:10 ratio, then pooled, dialyzed against PBS buffer, and concentrated by centrifugation. Protein purity was assessed by SDS-PAGE and size-exclusion HPLC.

[0323] Example 6 - Characterization of hits Hits were evaluated for their ability to bind to the HLA-A*02:01 / MAGE-A4 complex and control HLA-A*02:01 / peptide mix complex in a direct binding ELISA assay. Briefly, 96-well ELISA plates were coated with HLA-A*02:01 / MAGE-A4 complex or control HLA-A*02:01 complex. Serial dilutions of antigen-binding proteins in Fab format were added to the plates and detected with anti-kappa light chain-HRP (Invitrogen) followed by goat anti-rabbit IgG (H+L)-HRP (Southern Biotech). Binding entities that showed high binding to the HLA-A*02:01 / MAGE-A4 complex but no binding to the control HLA-A*02:01 / peptide mix complex were considered for further characterization. Binding of 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 is shown in Figures 1A and 1B. Binding to the negative control complex HLA-A*02:01 / peptide mix as determined by ELISA is shown in Figures 1C and 1D, respectively. All 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 mix complex.

[0324] The binding of the specific antibodies M0709 and M0763 to the HLA-A*02:01 / MAGE-A4 complex displayed on cells was determined. Briefly, TB hybrid T2 cells were incubated with serum-free RPMI 1640 medium containing MAGE-A4 or control peptides. The control peptides consisted of sequences highly identical to MAGE-A4 and were previously identified in healthy human tissues: 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 between the mean fluorescence intensity (MFI) of the HLA-A*02:01-binding antibody BB7.2 on peptide-loaded T2 cells and the MFI (>1) of unloaded T2 cells. T2 cells were incubated with each specific antibody, followed by a fluorescently labeled detection antibody (anti-kappa light chain). Cells were fixed, and fluorescence was measured by flow cytometry. Figure 2 shows the binding and specificity of M0709 and M0763 to T2 cells presenting MAGE-A4 or control peptides 1, 2, and 3. Both tested molecules demonstrated binding to HLA-A*02:01 / MAGE-A4 presented on T2 cells. Furthermore, M0763 demonstrated very high specificity for the MAGE-A4 peptide and did not demonstrate binding to any of the control peptides presented by HLA-A*02:01 on T2 cells. M0709 demonstrated lower specificity than M0763, also binding to control peptides 1 and 2.

[0325] Example 7 - Optimization of M0763 A rabbit antibody designated M0763 was humanized by CDR grafting. Briefly, human V gene germline sequences showing high sequence identity to the VH and VL of M0763, i.e., IMGT_hVH_3_23 and IMGT_hVL_3-1, respectively, were selected as CDR acceptor scaffolds. The resulting humanized antibodies, M0871-M874, retained HLA-A*02:01 / MAGE-A4 binding with EC50 values ​​of 1.18-16.02 nM as determined by direct ELISA (as described in Example 6), and showed no binding to the HLA-A*02:01 / peptide mix negative control (Figure 3).

[0326] In the next optimization step, M0873 was subjected to affinity maturation. Briefly, multiple antibody libraries were designed by randomizing three consecutive amino acids at a time across the entire length of all six CDRs. The libraries were generated using primers for site-saturation mutagenesis. Thus, each of the three amino acid positions targeted for randomization contained one of 19 possible amino acid variations. Library diversity was estimated by electroporating E. coli TG-1 cells and plating the libraries on agar plates using serial dilutions of the transfected TG-1 cells. Assuming that each E. coli colony contains a single plasmid insert, the number of colonies grown on the plate was used as an indicator of library diversity. Library quality was further assessed by sequencing a sample of 10 clones per library.

[0327] Libraries containing site-saturation mutagenesis in the light chain were combined into one library, and libraries containing site-saturation mutagenesis in the heavy chain were combined into another library. The two libraries obtained by randomizing the CDRs of the light and heavy chains were subjected to affinity selection (hereafter referred to as biopanning) against the HLA-A*02:01 / MAGE-A4 complex. The HLA-A*02:01 / MAGE-A4-specific phage library was subjected to panning (selection) with antigen adsorbed onto polystyrene tubes or plates. Briefly, after three rounds of phage display biopanning against the HLA-A*02:01 / MAGE-A4 antigen, the library was screened for hits. Screening was performed by monoclonal phage ELISA against the specific target, i.e., HLA-A*02:01 / MAGE-A4, and the nonspecific target, i.e., HLA-A*02:01 / peptide mix target.

[0328] Phage-displayed antibody clones were then classified into high, medium, and low signal ELISAs for HLA-A*02:01 in complex with the target complex protein and an unrelated peptide. Clones with high binding signals to the target complex and low binding to the HLA-A*02:01 / peptide mix complex were sequenced. Sequence analysis facilitated the identification of unique clones, which were then selected for recombinant expression in a bispecific anti-CD3 Fab x anti-MAGE-A4 scFv format. The resulting constructs were then evaluated for binding affinity to the HLA-A*02:01 / MAGE-A4 complex by SPR (Table 3).

[0329] [Table 4-1] [Table 4-2]

[0330] Affinity characterization of the anti-HLA-A*02:01 / MAGE-A4 × CD3 bispecific antibodies in Table 3 was performed by surface plasmon resonance (SPR) using a Biacore™ T200 Device (Cytiva). To determine the affinity of the bispecific antibodies for the HLA-A*02:01 / MAGE-A4 complex, a streptavidin chip (SAHC30M, XanTec) was coated with 500 RU of HLA-A*02:01 in complex with the MAGE-A4 peptide according to the manufacturer's instructions. To determine the affinity of the bispecific antibodies for CD3, a HC30M chip (XanTec) was coated with 400 RU of CD3 heterodimer (Acro Biosystems) according to the manufacturer's instructions. An uncoated channel was used as a reference. Data fitting was performed using a 1:1 Langmuir model. The affinity-matured clones exhibited binding affinities as low as double-digit picomolar, an almost 1000-fold improvement in binding affinity compared to the parental M0763 antibody.

[0331] The affinity-matured M1041 was optimized by further humanizing and stabilizing the anti-HLA-A*02:01 / MAGE-A4 binding scFv arm. The rabbit-derived residues in VH FR1 were further mutated by incorporating the amino acid substitutions S2V and V4L to obtain M1067. M1067 was further modified by introducing mutations in VH FR3, i.e., the amino acid substitutions K71R and T73N, and in CDR-L3, i.e., the amino acid substitution L97A, to obtain M1402. Each optimization step improved the human sequence identity score, thermal stability, and affinity for the target antigen, as shown in Table 4. 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). [Table 5]

[0332] At the same time, to obtain a more stable molecule with lower affinity for the HLA-A*02:01 / MAGE-A4 antigen, M1067 was further modified by incorporating a mutation in VH FR2, namely, the amino acid substitution Y47W, resulting in M1068. M1067 and M1068 were compared for stability at various concentrations and upon prolonged incubation in PBS, pH 7.4, at 4°C and 37°C. Stability was assessed by SEC-HPLC quantification of target protein monomer, and the corresponding data are shown in Table 5. M1068 exhibited superior stability to M1067, retaining more than 90% monomer content upon 2-week incubation at all tested concentrations and incubation temperatures. [Table 6]

[0333] M1068 was further subjected to affinity maturation as previously described. The affinity maturation experiments generated novel CDR-L3 and CDR-H3 sequences with distinct properties. Molecules containing these novel CDR sequences include M1302, M1382, and M1386, all of which have a K of 1.0 nM compared to M1068. D K values ​​of 0.6 nM, 0.23 nM, and 0.15 nM, respectively, compared to DM1302 was further optimized for human sequence content and stability. A different human germline VL framework, IMGT_hVL_3-19, was selected to graft the CDRs L1, L2, and L3 of M1302, resulting in M1312. While IMGT_hVL_3-19, like the previously selected IMGT_hVL_3-1, showed high sequence similarity to the M0763 VL sequence, 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, substitutions in VLFR1, i.e., amino acid substitutions S1Q and E3V, and substitutions in CDR-L3, i.e., amino acid substitution L97A, to yield M1396. As shown in Table 6, each incorporated substitution improved the human sequence identity score and / or stability of the molecule. [Table 7]

[0334] Example 8 - Generation of monovalent and bivalent pMHC-targeted T cell engagers Monovalent bispecific antigen-binding proteins were expressed by transient co-transfection in HEK293-6E cells. Cells were cultured in suspension using polyethyleneimine (linear PEI 40 kD). HEK293-6E cells were cultured at 1.7 x 10 in Freestyle F17 medium supplemented with 2 mM L-glutamine. 6Cells were seeded at 1000 cells / mL. DNA and PEI were added separately to 50 μL of medium without additives. Both fractions were mixed at a DNA:PEI ratio of 1:2.5, vortexed, and left to stand for 15 minutes. The cells and DNA / PEI mixture were combined (1 μg DNA per mL of cells) and incubated at 37°C, 5% CO2, and 80% RH. After 24 hours, tryptone N1 was added to the cells at 25 μL per mL of production volume. After 7 days, cells were harvested by centrifugation, and the supernatant was sterile filtered. Antigen-binding proteins were purified from the supernatant by affinity chromatography. The supernatant was loaded onto a CaptureSelect™ CH1-XL column (Thermo Fisher Scientific) equilibrated with 6 CV of PBS (pH 7.4). After a wash step with the same buffer, the protein was eluted from the column by stepwise elution with 100 mM citric acid (pH 3.0). Fractions containing the desired antigen-binding protein were immediately neutralized with 1 M Tris buffer (pH 9.0) at a ratio of 1:10. As an additional purification step, size exclusion chromatography was performed. The sample was run on a Superdex 200 10 / 300 GL column using PBS (pH 7.4) as the running buffer. Collected fractions were analyzed for monomer content by SE-HPLC and pooled accordingly. Final protein purity was assessed by SDS-PAGE and SE-HPLC.

[0335] Bivalent, bispecific antigen-binding proteins were produced by transient cotransfection in CHO-K1 cells. The HC and LC genes were expressed in a 2:1 vector ratio in shake flask culture for 7 days. The target protein was captured from the clarified, sterile-filtered culture supernatant by affinity chromatography using Amsphere A3 resin (JSR Life Science). The captured antigen-binding protein was further purified by strong cation exchange chromatography (CEC) on Source30S resin (Cytiva) and hydrophobic interaction chromatography (HIC) on Toyopearl PPG-600M resin (Tosoh Bioscience). An Amicon stirred cell (Merck) was applied to transfer the target protein into the final buffer (130 mM NaCl, 10 mM sodium phosphate, pH 6.5).

[0336] Example 9 - Dual pMHC-targeted T cell engager We designed antigen-binding proteins with two binding domains targeting specific pMHC and a Fab binding domain targeting CD3 as a T cell-recruiting domain. Figure 4 shows a schematic diagram of an exemplary bispecific antigen-binding protein with a Fab T cell-binding domain (e.g., an 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-targeting T cell engagers function by recruiting T cells to tumor cells expressing target pMHC molecules on their surface.

[0337] 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 an anti-CD3 Fab, with one scFv linked to the Fab heavy chain (HC) and the other to the Fab light chain (LC). The CD3 Fab has a binding affinity of 10 nM KD for CD3, and each pMHC-MAGE-A4 scFv has a binding affinity of 250 pM KD for pMHC-MAGE-A4. This multispecific antigen-binding protein was designated the "anti-MAGE-A4 dual engager" or simply "dual engager." The efficacy and safety of the dual engager M1048 and its monovalent engager M1041 were compared in an 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 PBMCs at an E:T ratio of 10:1. Cancer cell killing was measured with various concentrations of the monovalent T cell engager M1041 and the dual T cell engager M1048. Cytotoxicity was quantified by measuring the amount of LDH released into the medium from damaged cells colorimetrically after 48 hours (Figure 5). The dual T cell engager M1048 demonstrated superior cancer cell killing compared to its monovalent counterpart, M1041. At the same time, M1048 exhibited comparable low cytotoxicity to its monovalent engager, M1041, against the antigen-negative cancer cell lines SK-MEL-30, MDA-MB-231, and PANC-1; in the PANC-1 and MDA-MB-231 cell lines, the higher cytotoxicity of M1048 was observed only at 1 nM, the highest concentration of compound tested. Overall, the data demonstrated that the dual T cell engager outperformed its monovalent engager, with the dual pMHC-targeted T cell engager demonstrating approximately 10-fold increased killing of antigen-positive cancer cells, while exhibiting comparable low cytotoxicity against antigen-negative cancer cell lines.

[0338] Example 10 - Characterization of optimized anti-HLA-A*02:01 / MAGE-A4 x CD3 dual pMHC-targeted T cell engager Optimized variants of M1041, namely M1382, M1386, M1396, and M1402, were reformatted into a dual pMHC T cell engager format, resulting in molecules M1383, M1387, M1397, and M1403, respectively. Molecule production demonstrated comparable potency for both M1397 and M1403. After purification according to standardized protocols, M1397 displayed significantly lower amounts of charge variants than M1403.

[0339] The stability of M1403 and M1397 was tested at concentrations of 1 mg / mL and 10 mg / mL during long-term incubation at 4°C and 37°C in 10 mM phosphate buffer, pH 6.0, supplemented with 130 mM NaCl. Stability was assessed by SEC-HPLC quantification of target protein monomer, and the corresponding data are shown in Figure 6. Both M1397 and M1403 molecules exhibited good stability in 10 mM phosphate buffer, pH 6.0, supplemented with 130 mM NaCl, at 4°C for 14 days, retaining greater than 98% of the target protein monomer content at both concentrations tested. M1397 exhibited superior stability to M1403 when incubated at 37°C, retaining greater than 95% of the target protein monomer content at both concentrations tested.

[0340] M1397 and M1403 were subjected to detailed biophysical characterization using dynamic light scattering (DLS). Briefly, the molecular cumulant diameter (mean particle size) and polydispersity index (PDI) were determined by DLS using a Prometheus Panta instrument. Samples were mixed and filtered through 0.1 μm prior to measurement. Analysis was performed at 20°C and 100% DLS laser power. Data were analyzed using PR Panta Analysis (x64) software. The thermal stability of dual engagers M1397 and M1403 was measured using differential scanning fluorimetry (DSF). For this purpose, samples were diluted to a concentration of 1 mg / ml and heated from 20 to 95°C at a rate of 1°C / min. Data were analyzed using PR Panta Analysis (x64) software. Thermal stability, including the onset of protein unfolding (Tonset), melting temperature (TM), and aggregation temperature (Tagg), was determined. The resulting data are shown in Table 7. M1397 exhibited a highly favorable thermostability profile, with unfolding onset 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 transition occurred at 72.47° C., corresponding to Fab domain unfolding. M1403 exhibited an unfolding onset at a temperature more than 10° C. lower than M1397 and a three-step unfolding process. [Table 8]

[0341] The human serum stability of M1397 and M1403 was measured. Briefly, human serum was prepared by centrifugation of clotted whole blood at 2,000 × g. The resulting supernatant was designated as serum and directly frozen and stored at -80°C. Sterile-filtered samples were diluted with serum to a concentration of 100 μg / ml. Incubations were performed in sterile Eppendorf tubes in a humidified CO2 incubator at 8% CO2 and 37°C. A reference sample (TO) was collected before the start of incubation, and serum incubation samples were collected after 7 and 24 hours. Samples were directly quantified by SPR. SPR quantification was performed on a Biacore T200 instrument. HLA-A*02:01 / MAGE-A4 binding molecules were quantified by SPR assay. HLA-A*02:01 / MAGE-A4 was used as the ligand and diluted 1:100 in SPR running buffer before serum samples were injected. The samples were evaluated by directly comparing the signal height after injection of the serum samples. The relative reduction in binding signal compared to the TO sample was calculated. The corresponding results are shown in Figure 7.

[0342] Example 11 - Comparator molecules: Generation of a soluble TCR-anti-CD3 fusion protein (Comparator 1) and a TCR-like CD3 T cell bispecific antibody (Comparator 2) DNA sequences encoding the extracellular regions of the alpha (SEQ ID NO: 59) and beta (SEQ ID NO: 60) chains of the soluble TCR-anti-CD3 fusion (Comparative Example 1) were separately cloned into the pET-24D(+) vector using standard molecular biology techniques (J Biol Chem. 1995 Jan 13;270(2):971-7). E. 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. Cells were harvested, resuspended in TBS, and lysed via lysozyme treatment and sonication. The inclusion bodies were washed twice with TBS (50 mM Tris-HCl pH 8.1, 0.5% Triton-X100, 100 mM NaCl, 10 mM NaEDTA) supplemented with Triton-X100 and twice with TBS (50 mM Tris, 100 mM NaCl, 10 mM EDTA, pH 8.1). The inclusion bodies thus prepared were solubilized in denaturing buffer (9 M urea, 0.5 M Gua, 25 mM Tris, 1.25 mM EDTA, pH 8.1). The solubilized inclusion bodies of the alpha and beta chain-anti-CD3 scFv fusions were combined and mixed with reducing agent to a final concentration of 20 mM DTT. The solubilized and reduced inclusion bodies were gently 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. From the diluted and pH-adjusted refolding solution, the molecules were captured by anion exchange chromatography using a POROS 50HQ column. The molecules were eluted on an Äkta® purification device (Cytiva) by applying a gradient of 0 to 500 mM NaCl in 20 mM Tris pH 8.1 over 50 column volumes. An additional purification step involved size exclusion chromatography. The sample was run on a HiLoad Superdex 75, 26 / 600 column using PBS (pH 7.4) as the running buffer. Collected fractions were analyzed for monomer content by SE-HPLC and pooled accordingly.Final protein purity was assessed by SDS-PAGE and SE-HPLC.

[0343] TCR-like CD3 T cell bispecific antibodies (Comparative Example 2, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67) were expressed in HEK293-6E cells by transient co-transfection. Cells were cultured in suspension using polyethyleneimine (linear PEI 40 kD). HEK293-6E cells were cultured at 1.7 x 10 in Freestyle F17 medium supplemented with 2 mM L-glutamine. 6 Cells were seeded at 1000 cells / mL. DNA and PEI were added separately to 50 μL of medium without additives. Both fractions were mixed at a DNA:PEI ratio of 1:2.5, vortexed, and left to stand for 15 minutes. The cells and DNA / PEI mixture were combined (1 μg DNA per mL of cells) and incubated at 37°C, 5% CO2, and 80% RH. After 24 hours, tryptone N1 was added to the cells at 25 μL per mL of production volume. After 7 days, cells were harvested by centrifugation, and the supernatant was sterile filtered. Antigen-binding proteins were purified from the supernatant by affinity chromatography. The supernatant was loaded onto a CaptureSelect™ CH1-XL column (Thermo Fisher Scientific) equilibrated with 6 CV of PBS (pH 7.4). After a wash step with the same buffer, the protein was eluted from the column by stepwise elution with 100 mM citric acid (pH 3.0). Fractions containing the desired antigen-binding protein were immediately neutralized with 1 M Tris buffer (pH 9.0) at a ratio of 1:10. As an additional purification step, size exclusion chromatography was performed. The sample was run on a Superdex 200 10 / 300 GL column using PBS (pH 7.4) as the running buffer. Collected fractions were analyzed for monomer content by SE-HPLC and pooled accordingly. Final protein purity was assessed by SDS-PAGE and SE-HPLC.

[0344] Example 12 - In vitro efficacy of optimized dual pMHC-targeted T cell engagers The dual engager M1048 demonstrated improved efficacy in in vitro cytotoxicity assays compared to its monovalent engager M1041. Optimized variants of M1041, i.e., M1402 and M1396, were reformatted into a dual engager format to yield M1403 and M1397, respectively. The optimized variants of M1048, i.e., M1403 and M1397, had higher human sequence content, higher stability, and / or higher affinity. The efficacy and safety of the dual pMHC-targeted T cell engagers M1403 and M1397 were compared in an LDH assay. Additionally, the dual T cell engagers M1397 and M1403 were compared with two comparative antigen binding molecules, i.e., Comparator 1 and Comparator 2. Comparator 1 exhibited K binding to the same pMHC-MAGE-A4 antigen linked to an anti-CD3 scFv with a binding affinity of 1 nM. D Comparator 1 consists of a soluble affinity-enhanced TCR with a binding specificity of 87 pM. Comparator 1 is monovalent for target pMHC and CD3, while the dual engager is bivalent for target pMHC and monovalent for CD3. Comparator 1 is further described 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 target pMHC and monovalent for CD3 and contains an Fc domain for half-life extension. Comparator 2 is further described in US20210230278A1, which is incorporated herein by reference.

[0345] As shown in Figure 9, the percent cancer cell killing was measured in several MAGE-A4-positive, HLA-A*02:01-positive cell lines, namely, U2OS (osteosarcoma), NCI-H1703 (lung squamous cell carcinoma), and A375 (melanoma), as well as MAGE-A4-negative, HLA-A*02:01-positive cell lines, namely, PANC1 (pancreatic cancer), MDA-MB-231 (breast cancer), and NCI-H441 (lung adenocarcinoma). The cancer cell lines were incubated with dual engagers M1397, M1403, or comparator 1 and human PBMCs at an E:T ratio of 10:1. Cytotoxicity at various concentrations of the three different antigen-binding proteins was quantified for all cell lines except NCI-H1703 by colorimetrically measuring the amount of LDH released into the medium from damaged cells after 48 hours. Cytotoxicity in NCI-H1703 cells was determined by CellTiter-Glo assay (Promega) according to the instructions provided by the kit manufacturer. Data showed that cell killing mediated by dual engager M1403 was comparable to comparator 1 for all MAGE-A4-positive, HLA-A*02:01-positive cell lines tested, with M1397 demonstrating lower efficacy. In addition, lower cancer cell killing was observed with both dual T cell engagers, especially M1397, compared to comparator 1 for all MAGE-A4-negative, HLA-A*02:01-positive cancer cell lines tested, demonstrating a favorable therapeutic window for M1397 and M1403.

[0346] Additionally, cell killing mediated by M1397, Comparator 1, and Comparator 2 was analyzed in a time-resolved manner using an 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 NucLight Red fluorescent protein. Cancer cells were plated at 1.5 × 10 per well in sterile 384-well flat-bottom adherent tissue culture plates. 3Cells were seeded at a density of 1000 kJ / well and cultured overnight in an incubator at 37°C with 5% CO2. M1397, Comparator 1, and Comparator 2 were added at the indicated concentrations (ranging from 0.2 pM to 50 nM). PBMCs as effector cells were added to each well at an E:T ratio of 10:1. The plates were imaged by fluorescence microscopy, and cell growth was monitored for 72 hours. The extent of cell killing was quantified by comparing the fold growth rate of fluorescently targeted cancer cells over time with the number at time point 0. As shown in Figure 9, M1397 exhibited superior potency to Comparator 2 and inferior potency to Comparator 1 against both antigen-positive cancer cell lines tested. Furthermore, M1397 exhibited superior safety compared to both comparators against the SK-MEL-30 antigen-negative cancer cell line and a similar safety profile to both comparators against the PANC-1 antigen-negative cancer cell line.

[0347] As shown in Figure 10, the release of the pro-inflammatory cytokine IFN-gamma was measured in several MAGE-A4-positive, HLA-A*02:01-positive cell lines, namely, U2OS (osteosarcoma) and NCI-H1703 (lung squamous cell carcinoma), as well as MAGE-A4-negative, HLA-A*02:01-positive cell lines, namely, PANC1 (pancreatic ductal adenocarcinoma), MDA-MB-231 (breast adenocarcinoma), and NCI-H441 (lung adenocarcinoma). Cells were incubated with Dual Engager or Comparator 1 and human PBMCs at an E:T ratio of 10:1. After 24 hours of incubation, IFN-gamma was measured at various concentrations of the three antigen-binding proteins. The data show that the dual engager induced low levels of the pro-inflammatory cytokine IFN-gamma in both antigen-positive (Figures 10A-10D) and antigen-negative (Figures 10E-J) cell lines, indicating a low likelihood of inducing cytokine storm syndrome.

[0348] Next, M1397, Comparator 1, and Comparator 2 were further compared in granzyme B and IFN-gamma release assays as indicators of T cell activation properties. Briefly, antigen-positive (NCI-H1703) and antigen-negative (SK-MEL-30) cancer cell lines were incubated with the indicated concentrations (ranging from 7.6 pM to 50 nM) of M1397, Comparator 1, or Comparator 2 and human PBMCs at a 5:1 E:T ratio. After 24 hours of incubation, granzyme B and IFN-gamma levels were measured, and the corresponding results are shown in Figure 11, panels A-B and C-D, respectively. M1397 demonstrated a superior safety profile to both comparators when tested on the antigen-negative cell line SK-MEL-30, with no granzyme B or IFN-gamma release. Furthermore, M1397 induced higher granzyme B and IFN-gamma release than Comparator 2 against antigen-positive cell lines, and lower granzyme B and IFN-gamma release than Comparator 1 against both antigen-positive and antigen-negative cell lines.

[0349] As shown in Figure 12, the release of IL-2, IL-6, and TNF-alpha cytokines 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 dual engagers M1397 and M1403 or comparator 1 were incubated with cancer cells and human PBMCs at an E:T ratio of 10:1. After 24 hours of incubation, the cytokines IL-2, IL-6, and TNF-alpha were measured. The data show that both dual engagers M1397 and M1403 induced low levels of pro-inflammatory cytokines, indicating a low potential for cytokine storm syndrome.

[0350] Example 13 - In vitro safety of optimized dual pMHC-targeted T cell engagers Potential off-target binding of the monovalent counterparts of M1397 and M1403, i.e., M1396 and M1402, respectively, to physiologically relevant peptides similar to MAGE-A4 was investigated by SPR. The control peptides consisted of sequences highly identical to MAGE-A4 and were previously identified in healthy human tissues: 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 shown in Table 8. [Table 9]

[0351] To determine the efficacy and safety of M1397, Comparator 1, or Comparator 2, T cell activation in the presence of target HLA-A2 / MAGE-A4 or physiologically relevant control peptide antigens HLA-A2 / Control 1 and HLA-A2 / Control 2 was determined. Briefly, TAP-deficient T2 cells were incubated overnight with serum-free RPMI 1640 medium containing peptides MAGE-A4 (SEQ ID NO:3), Control 1 (SEQ ID NO:61), and Control 2 (SEQ ID NO:62) at the indicated concentrations (ranging from 0.1 nM to 1000 nM). Cells were then washed with serum-free RPMI 1640 medium and co-incubated with PBMCs (E:T 5:1) and M1397, Comparator 1, or Comparator 2 at a concentration of 1 nM for 24 hours. T cell activation was determined by quantification of IFN-gamma in the cell supernatant and is shown in Figure 13. Consistently, M1397 demonstrated a very favorable T cell activation profile with an EC50 of 5.75 nM when tested on T2 cells stimulated with the target peptide MAGE-A4, with IFN-gamma release observed only at 1 μM, the highest concentration of peptide tested, demonstrating a favorable safety window compared to the two control peptides tested. Comparator 1 exhibited increased T cell activation in the presence of off-target HLA-A2 / Control 1 antigens, demonstrating a poorer safety profile compared to M1397, while Comparator 2 exhibited poorer T cell activation properties compared to M1397, with an EC50 of 105.8 nM against T2 cells stimulated with the target peptide MAGE-A4.

[0352] To determine the safety of M1397, a panel of various antigen-negative cancer cell lines was tested for compound-induced T cell activation by measuring granzyme B release. Briefly, the antigen-negative cancer cell lines KLE (endometrial carcinoma), LNCaP (prostate lymph node carcinoma), KMRC-2 (clear cell renal cell carcinoma), KMRC-3 (clear cell renal cell carcinoma), 639-V (urothelial bladder carcinoma), EKVX (lung adenocarcinoma), and HCT116 (colorectal carcinoma) were incubated with the indicated concentrations (ranging from 7.6 pM to 50 nM) of M1397, comparator 1, or comparator 2, and human PBMCs at a 5:1 E:T ratio. The antigen-positive cell line NCI-H1703 served as a positive control. Granzyme B levels were measured after 24 h of incubation, and the corresponding results are shown in Figure 14. M1397 demonstrated a superior safety profile to Comparator 1 and Comparator 2 molecules against all antigen-negative cancer cell lines tested.

[0353] In in vitro safety experiments, various primary cell types and PBMCs derived from healthy human donors were co-cultured with compounds for 24 hours, after which the supernatants were analyzed for granzyme B released from effector cells as a function of cell-mediated cytotoxicity. Target cells included human cardiac microvascular endothelial cells (HMVEC-C), normal human bronchial epithelial cells (NHBE), and normal human astrocytes (NHAs). Target cells were prepared in assay medium (RPMI 1640 containing 10% FBS and 1% penicillin-streptomycin) and plated at 20,000 cells per well in 50 μL of assay medium. PBMC effector cells were plated at 100,000 cells per well in 50 μL of assay medium. Compounds M1397, M1403, or comparator 1 at various concentrations spanning a clinically relevant expected range were added to plated wells in 15 μL of assay medium. Final assay medium was up to 150 μL per well. Positive control wells contained ImmunoCult Human CD3 / CD28 T Cell Activator. Negative control wells contained target and effector cells alone or PBMCs alone with the highest concentration of compound. All reactions were performed in duplicate. Plates were incubated at 37°C / 5% CO2 for 24 hours. Supernatants were collected and analyzed by human granzyme B ELISA kit (MabTech) according to the manufacturer's instructions. Corresponding results are shown in Figure 15. M1397 and M1403 demonstrated superior safety profiles compared to the soluble TCR bispecific comparator single molecule.

[0354] Further safety evaluation of M1397 for Comparator 1 and Comparator 2 was performed by testing an extensive panel of primary cells derived from various essential tissues, namely human aortic smooth muscle cells (HAoSMC_735), human lung microvascular endothelial cells (HMVEC-L_73809), renal proximal tubule 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 cardiomyocytes (HCM_679, HCM_693, HCM_745, HCM_746) and human cardiac fibroblasts (HCF_251). The assay was performed essentially 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 demonstrated a favorable and superior safety profile against both comparators against the primary cells tested.

[0355] Example 14 - In vivo efficacy of cell line-derived mouse NSCLC xenograft models The antitumor activity of M1397 at doses ranging from 0.5 to 5.0 mg / kg was evaluated 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.

[0356] Immunodeficient female NCG mice were inoculated with 5 × 10 cells of the human squamous NSCLC cell line NCI-H1703, which expresses MAGE-A4 and HLA-A*02:01. 6 The cells were implanted subcutaneously (SC). The tumor size was 120 mm. 3 Once they reached an average size of 1 x 10, mice were randomized and fed 1 x 10 mice obtained from two healthy, naive donors. 7 Human peripheral blood mononuclear cells (PBMCs) were transplanted intraperitoneally. Treatment with M1397 began the following day at daily intravenous doses ranging from 0.5 to 5 mg / kg / day for up to 28 days. PBMC donors and three mice per treatment or vehicle (phosphate-buffered saline) group were evaluated twice weekly by caliper measurement and body weight.

[0357] All M1397 groups showed strong tumor growth inhibition, with the majority of mice showing complete tumor eradication at the end of the study (Figure 17). All groups, including the vehicle control group, showed minimal signs of graft-versus-host disease after day 18. M1397 was well tolerated throughout the study, and stable body weights were observed. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8] [Table 10-9] [Table 10-10] [Table 10-11] [Table 10-12] [Table 10-13]

Claims

1. 1. An antigen binding protein that specifically binds to melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising: 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; 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; The antigen-binding protein comprising:

2. 1. An antigen binding protein that specifically binds to melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising: 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; 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; The antigen-binding protein comprising:

3. 1. An antigen binding protein that specifically binds to melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising: 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; 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; The antigen-binding protein comprising:

4. 1. An antigen binding protein that specifically binds to melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising: 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; 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; The antigen-binding protein comprising:

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

6. Full length immunoglobulins or antibody fragments, e.g., Fab, Fab', F(ab') ２ , scFv, Fv fragment.

7. HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), IVSSGGTTYYAX １ X ２ X ３ The HCDR2 amino acid sequence of X １ corresponds to an amino acid or D, and X ２ corresponds to the amino acid W or S, and X ３ corresponds to amino acid A or V), and DLYYGPX ４ TX ５ YX ６ X ７ X ８ NL (SEQ ID NO: 7), ４ corresponds to the amino acid T, N, or S, and X ５ corresponds to the amino acid D or is absent, and X ６ corresponds to an amino acid or F, and X ７ corresponds to the amino acid A or V, and X ８ corresponds to amino acid F or A); The LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and ATX ９ X １０ X １１ SGSNFQX １２ The LCDR3 amino acid sequence of (SEQ ID NO: 8) ９ corresponds to an amino acid or R, and X １０ corresponds to the amino acid D or P, and X １１ corresponds to the amino acid G, S, or F, and X １２ corresponds to amino acid L or A), and 10. The antigen-binding protein of any one of the preceding claims, comprising:

8. 10. An antigen-binding protein according to any one of the preceding claims, linked to or associated with a functional entity such as a detectable label, a therapeutic agent or a PK-modifying moiety.

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

10. 10. The antigen-binding protein of claim 9, which is glycosylated, PEGylated, PASylated, XTENylated or HESylated.

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

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

13. An immune cell expressing the CAR of claim 12.

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

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

16. 16. The multispecific antigen-binding protein of claim 15, which is bispecific or trispecific.

17. 17. The multispecific antigen-binding protein of claim 15 or 16, further comprising at least one additional binding domain.

18. 18. The multispecific antigen-binding protein of claim 17, wherein said additional binding domain is an immune cell engager.

19. 19. The multispecific antigen-binding protein of claim 18, wherein said immune cell engager is a CD3 binding domain or a CD16a binding domain.

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

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

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

23. the third antigen-binding domain comprises: HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), IVSSGGTTYYAX １ X ２ X ３ The HCDR2 amino acid sequence of X １ corresponds to the amino acid S or D, and X ２ corresponds to the amino acid W or S, and X ３ corresponds to amino acid A or V), and DLYYGPX ４ TX ５ YX ６ X ７ X ８ NL (SEQ ID NO: 7), ４ corresponds to the amino acid T, N, or S, and X ５ corresponds to the amino acid D or is absent, and X ６ corresponds to the amino acid S or F, and X ７ corresponds to the amino acid A or V, and X ８ corresponds to amino acid F or A), and The LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and ATX ９ X １０ X １１ SGSNFQX １２ The LCDR3 amino acid sequence of (SEQ ID NO: 8) ９ corresponds to the amino acid S or R, and X １０ corresponds to the amino acid D or P, and X １１ corresponds to the amino acid G, S, or F, and X １２ an antibody light chain variable (VL) domain comprising:

23. The multispecific antigen-binding protein of any one of claims 20 to 22, comprising:

24. 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.

24. The multispecific antigen-binding protein of any one of claims 20 to 23, comprising:

25. 1. 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 melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), b1) HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), IVSSGGTTYYAX １ X ２ X ３ The HCDR2 amino acid sequence of X １ corresponds to the amino acid S or D, and X ２ corresponds to the amino acid W or S, and X ３ corresponds to amino acid A or V), and DLYYGPX ４ TX ５ YX ６ X ７ X ８ NL (SEQ ID NO: 7), ４ corresponds to the amino acid T, N, or S, and X ５ corresponds to the amino acid D or is absent, and X ６ corresponds to the amino acid S or F, and X ７ corresponds to the amino acid A or V, and X ８ corresponds to amino acid F or A), and b2) the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and ATX ９ X １０ X １１ SGSNFQX １２ The LCDR3 amino acid sequence of (SEQ ID NO: 8) ９ corresponds to the amino acid S or R, and X １０ corresponds to the amino acid D or P, and X １１ corresponds to the amino acid G, S, or F, and X １２ corresponding to amino acid L or A), the second antigen-binding domain comprising: c) a third antigen-binding domain that specifically binds to MAGE-A4 pMHC, c1) HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), IVSSGGTTYYAX １ X ２ X ３ The HCDR2 amino acid sequence of X １ corresponds to the amino acid S or D, and X ２ corresponds to the amino acid W or S, and X ３ corresponds to amino acid A or V), and DLYYGPX ４ TX ５ YX ６ X ７ X ８ NL (SEQ ID NO: 7), ４ corresponds to the amino acid T, N, or S, and X ５ corresponds to the amino acid D or is absent, and X ６ corresponds to the amino acid S or F, and X ７ corresponds to the amino acid A or V, and X ８ corresponds to amino acid F or A), and c2) the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and ATX ９ X １０ X １１ SGSNFQX １２ The LCDR3 amino acid sequence of (SEQ ID NO: 8) ９ corresponds to the amino acid S or R, and X １０ corresponds to the amino acid D or P, and X １１ corresponds to the amino acid G, S, or F, and X １２ corresponds to amino acid L or A), the third antigen-binding domain comprising:

2. The multispecific antigen-binding protein comprising:

26. the second and third antigen-binding domains are VH comprising an HCDR1 sequence comprising the amino acid sequence of SNYAMS (SEQ ID NO: 11), an HCDR2 sequence comprising the amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 12), and an HCDR3 sequence comprising the amino acid sequence of DLYYGPNTDYSAANL (SEQ ID NO: 13); a VL comprising an LCDR1 sequence comprising the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), an LCDR2 sequence comprising the amino acid sequence of RDTSRPS (SEQ ID NO: 17), and an LCDR3 sequence comprising the amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 18); 26. The multispecific antigen-binding protein of claim 25, comprising:

27. the second and third antigen-binding domains are 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; 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; 27. The multispecific antigen-binding protein of claim 26, comprising:

28. the second and third antigen-binding domains are VH comprising an HCDR1 sequence comprising the amino acid sequence of SNYAMS (SEQ ID NO: 21), an HCDR2 sequence comprising the amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 22), and an HCDR3 sequence comprising the amino acid sequence of DLYYGPSTYFVANL (SEQ ID NO: 23); a VL comprising an LCDR1 sequence comprising the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 26), an LCDR2 sequence comprising the amino acid sequence of RDTSRPS (SEQ ID NO: 27), and an LCDR3 sequence comprising the amino acid sequence of ATRPSSGSNFQL (SEQ ID NO: 28); 26. The multispecific antigen-binding protein of claim 25, comprising:

29. the second and third antigen-binding domains are 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; 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; 29. The multispecific antigen-binding protein of claim 28, comprising:

30. the second and third antigen-binding domains are VH comprising an HCDR1 sequence comprising the amino acid sequence of SNYAMS (SEQ ID NO: 31), an HCDR2 sequence comprising the amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 32), and an HCDR3 sequence comprising the amino acid sequence of DLYYGPTTYSAANL (SEQ ID NO: 33); a VL comprising an LCDR1 sequence comprising the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 36), an LCDR2 sequence comprising the amino acid sequence of RDTSRPS (SEQ ID NO: 37), and an LCDR3 sequence comprising the amino acid sequence of ATRDFSGSFQL (SEQ ID NO: 38); 26. The multispecific antigen-binding protein of claim 25, comprising:

31. the second and third antigen-binding domains are 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; 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; 31. The multispecific antigen-binding protein of claim 30, comprising:

32. the second and third antigen-binding domains are VH comprising an HCDR1 sequence comprising the amino acid sequence of SNYAMS (SEQ ID NO: 41), an HCDR2 sequence comprising the amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 42), and an HCDR3 sequence comprising the amino acid sequence of DLYYGPTTYSAFNL (SEQ ID NO: 43); a VL comprising an LCDR1 sequence comprising the amino acid sequence of TADTLSRSYAS (SEQ ID NO: 46), an LCDR2 sequence comprising the amino acid sequence of RDTSRPS (SEQ ID NO: 47), and an LCDR3 sequence comprising the amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 48); 26. The multispecific antigen-binding protein of claim 25, comprising:

33. the second and third antigen-binding domains are 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; 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; 26. The multispecific antigen-binding protein of claim 25, comprising:

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

35. The antibody fragment may be a Fab fragment, F(ab') ２ 35. The multispecific antigen-binding protein of claim 34, including fragments, Fab' fragments, Fv fragments, single chain variable fragments (scFv), and single domain antibody fragments.

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

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

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

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

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

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

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

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

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

45. 45. The multispecific antigen-binding protein of claim 43 or 44, wherein said amino acid linker comprises the amino acid sequence GGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGGSGGGGGSGGGGGS, GGGGSGGGGGSGGGGGSGGGGAS, or GGGGGGSGGGGGSGGGGGSGGGGGS.

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

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

48. 48. The multispecific antigen-binding protein of claim 46 or 47, wherein said amino acid linker comprises the amino acid sequence GGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGGSGGGGGSGGGGGS, GGGGSGGGGGSGGGGGSGGGGAS, or GGGGGGSGGGGGSGGGGGSGGGGGS.

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

50. (scFv) ２ , (scFv) ３ , BiTE, BIKE, Dart, diabody, tribody, Fab ２ , Fab ３ , Fab ４ 50. The multispecific antigen-binding protein of any one of claims 15 to 49, comprising a scFv-Fab-scFv or a minibody-scFv.

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

52. 52. The multispecific antigen-binding protein of claim 51 , wherein said antigen-binding protein has an increased serum half-life compared to antigen-binding proteins of molecular weight less than about 75 kDa.

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

54. the CD3 antigen-binding domain is - a VH comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 50; - a VL comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 55; 54. The multispecific antigen-binding protein of any one of claims 15 to 53, comprising:

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

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

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

58. the variable heavy chain According to Kabat numbering, leucine (L) or serine (S) at amino acid position 11; valine (V), serine (S), or threonine (T) at amino acid position 89 according to Kabat numbering, and / or According to Kabat numbering, leucine (L), serine (S), or threonine (T) is added to amino acid position 108.

58. The multispecific antigen-binding protein of claim 56 or 57, comprising:

59. 59. The multispecific antigen-binding protein of claim 57 or 58, wherein the polar amino acid is serine (S) and / or threonine (T).

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

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

62. GVYDGREHTV (SEQ ID NO: 3) A multispecific antigen binding protein that binds to the HLA-A*02 complex and CD3, comprising: (i) a first polypeptide chain comprising the amino acid sequence set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth in SEQ ID NO: 39, or a variant thereof that is 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 set forth in SEQ ID NO: 44, or a variant thereof that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

44.

2. The multispecific antigen-binding protein comprising:

63. 1. A multispecific antigen-binding protein comprising: a) a first antigen-binding domain that specifically binds to CD3, a1) a VH comprising an HCDR1 sequence comprising the amino acid sequence of STYAMN (SEQ ID NO: 51), an HCDR2 sequence comprising the amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 52), and an HCDR3 sequence comprising the amino acid sequence of HGNFGDSYVSWFAY (SEQ ID NO: 53); and a2) a VL comprising an LCDR1 sequence comprising the amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 56), an LCDR2 sequence comprising the amino acid sequence of GTNKRAP (SEQ ID NO: 57), and an LCDR3 sequence comprising the amino acid sequence of ALWYSNHWV (SEQ ID NO: 58). the first antigen-binding domain comprising: b) a second antigen-binding domain that specifically binds to melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC); 2. The multispecific antigen-binding protein comprising:

64. the second antigen-binding domain comprises: b1) HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), IVSSGGTTYYAX １ X ２ X ３ The HCDR2 amino acid sequence of X １ corresponds to the amino acid S or D, and X ２ corresponds to the amino acid W or S, and X ３ corresponds to amino acid A or V), and DLYYGPX ４ TX ５ YX ６ X ７ X ８ NL (SEQ ID NO: 7), ４ corresponds to the amino acid T, N, or S, and X ５ corresponds to the amino acid D or is absent, and X ６ corresponds to the amino acid S or F, and X ７ corresponds to the amino acid A or V, and X ８ corresponds to amino acid F or A), and b2) the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and ATX ９ X １０ X １１ SGSNFQX １２ The LCDR3 amino acid sequence of (SEQ ID NO: 8) ９ corresponds to the amino acid S or R, and X １０ corresponds to the amino acid D or P, and X １１ corresponds to the amino acid G, S, or F, and X １２ an antibody light chain variable (VL) domain comprising:

64. The multispecific antigen-binding protein of claim 63, comprising:

65. the first antigen-binding domain comprises: - a VH comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 50; - a VL comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 55; 65. The multispecific antigen-binding protein of claim 63 or 64, comprising:

66. the first antigen-binding domain comprises: - a VH comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49; - a VL comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 54; 66. The multispecific antigen-binding protein of claim 65, comprising:

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

68. c) a third antigen-binding domain that specifically binds to MAGE-A4 pMHC, c1) HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 11), IVSSGGTTYYAX １ X ２ X ３ The HCDR2 amino acid sequence of X １ corresponds to the amino acid S or D, and X ２ corresponds to the amino acid W or S, and X ３ corresponds to amino acid A or V), and DLYYGPX ４ TX ５ YX ６ X ７ X ８ NL (SEQ ID NO: 7), ４ corresponds to the amino acid T, N, or S, and X ５ corresponds to the amino acid D or is absent, and X ６ corresponds to the amino acid S or F, and X ７ corresponds to the amino acid A or V, and X ８ corresponds to amino acid F or A), c2) the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 16), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 17), and ATX ９ X １０ X １１ SGSNFQX １２ The LCDR3 amino acid sequence of (SEQ ID NO: 8) ９ corresponds to the amino acid S or R, and X １０ corresponds to the amino acid D or P, and X １１ corresponds to the amino acid G, S, or F, and X １２ corresponds to amino acid L or A), and 68. The multispecific antigen-binding protein of any one of claims 63 to 67, comprising the third antigen-binding domain comprising:

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

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

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

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

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

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

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

76. 76. The multispecific antigen-binding protein of claim 75, wherein said functional entity is a toxin.

77. 77. The multispecific antigen-binding protein of any one of claims 15 to 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. 78. The multispecific antigen-binding protein of claim 77, wherein said light chain comprises an N-terminal truncation of 1 or 2 amino acids.

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

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

81. 12. An antigen-binding protein according to any one of claims 1 to 11 for use in diagnosis.

82. 81. The multispecific antigen-binding protein of any one of claims 15 to 80 for use in a method for inhibiting the growth or proliferation of cancer cells.

83. 81. The multispecific antigen-binding protein of any one of claims 15 to 80 for use in a method for redirecting T cells towards MAGE-A4 expressing cancer cells.

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

85. 81. The antigen-binding protein of any one of claims 1 to 11, the CAR of claim 12, the immune cell of claim 13 or the multispecific antigen-binding protein of any one of claims 15 to 80 for use in the treatment of a disease, in particular cancer.

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

87. A vector comprising the nucleic acid of claim 86.

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

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

90. 80. A method for producing an antigen-binding protein according to any one of claims 1 to 11 or a multispecific antigen-binding protein according to any one of claims 15 to 80, comprising the steps of: (i) culturing the host cell of claim 87 under conditions that allow expression of said antigen binding protein or said multispecific antigen binding protein; (ii) recovering said antigen binding protein or said multispecific antigen binding protein; and optionally (iii) further purifying and / or modifying and / or formulating said antigen binding protein or said multispecific antigen binding protein; The method comprising:

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

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

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

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

95. 92. The antigen-binding protein of any one of claims 1 to 11, the CAR of claim 12, the immune cell of claim 13 or 14, the multispecific antigen-binding protein of any one of claims 15 to 80, the cell of claim 88, or the pharmaceutical composition of claim 91 for use in the treatment of a disease, in particular cancer.

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

97. 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, e.g. uterine carcinosarcoma (UCS; in particular the UCEC subgroup), myxoid / round cell liposarcoma, gastric or gastroesophageal junction (GEJ) adenocarcinoma, epithelial ovarian cancer, e.g. high-grade serous ovarian carcinoma, synovial sarcoma, bladder urothelial carcinoma (BLCA), in particular transitional cell carcinoma, testicular germ cell tumour (TGCT) and cervical squamous cell carcinoma (CESC).

98. 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. 1. A method for selecting a patient eligible for treatment with a MAGE-A4 antagonist, comprising: (i) obtaining a tumor sample from a 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) selecting the patient for treatment with a MAGE-A4 antagonist if the detection antibody binds to the sample. The method further comprises, in order:

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

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

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

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