CN117751146A

CN117751146A - MAGE-A4 peptide-MHC antigen binding proteins

Info

Publication number: CN117751146A
Application number: CN202280033825.6A
Authority: CN
Inventors: 安娜·玛丽亚·索别拉伊; 法比安·贝尔特·沙伊费勒; 斯特凡妮·容米歇尔; 莱昂纳多·博拉斯; 克里斯蒂安·瓦尔德马·温格·莱斯纳
Original assignee: Cdr Biotechnology Co ltd
Current assignee: Cdr Biotechnology Co ltd
Priority date: 2021-03-09
Filing date: 2022-03-09
Publication date: 2024-03-22

Abstract

Antigen binding proteins that specifically recognize the target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC) and nucleic acids encoding the antigen binding proteins are provided. Methods of producing antigen binding proteins that specifically recognize the target MAGE-A4pMHC and nucleic acid libraries encoding the antigen binding proteins are also provided.

Description

MAGE-A4 peptide-MHC antigen binding proteins

RELATED APPLICATIONS

The present application claims the benefit of U.S. provisional application Ser. No. 63/158,691 filed on 3 months 9 of 2021 and U.S. provisional application Ser. No. 63/172,864 filed on 4 months 9 of 2021, the disclosures of which are hereby incorporated by reference in their entireties.

Technical Field

The present disclosure relates to antigen binding proteins that specifically recognize the target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC).

Background

Melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC) expression is present in many cancers, including non-small cell lung cancer (NSCLC), melanoma, bladder cancer, head and neck cancer, and gastroesophageal cancer (Grossman et al N Engl J Med. 2016.375:1109-1112). It represents an attractive target for TCR-based T cell therapies, unfortunately TCR molecules have low binding affinity for their pMHC targets. Moreover, the development and use of TCR-based T cell therapies is laborious and costly. In contrast, isolated monoclonal antibodies provide significantly higher binding affinity to their targets, and off-target activity may also be reduced. However, due to the small epitope of peptides bound in HLA, it is difficult to generate monoclonal antibodies against pMHC targets.

Thus, there is a need in the art for novel antigen binding proteins that specifically recognize the target MAGE-A4 pM HC with high binding affinity while maintaining high specificity (i.e., low off-target to no off-target).

Disclosure of Invention

In one aspect, the present disclosure provides an antigen binding protein that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), wherein the antigen binding protein comprises one or more of the following features: (i) The antigen binding protein has a binding affinity of about 10 for the target MAGE-A4 pMHC ^-9 M to about 10 ^-14 M (e.g., about 10 ^-9 M、10 ^-10 M、10 ^-11 M、10 ^-12 M、10 ^-13 M or 10 ^-14 M) is selected from the group consisting of; (ii) The antigen binding protein has a binding affinity of about 10 for non-MAGE-A4 pMHC and/or peptide-free MHC ^-6 M or less (e.g., about 10 ^-6 M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^-1 M) is selected from the group consisting of; (iii) The antigen binding protein has a binding affinity of about 10 for non-target MAGE-A4 pMHC ^-6 M or less (e.g., about 10 ^- ⁶ M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^-1 M) is selected from the group consisting of; and (iv) the antigen binding protein has a binding affinity for the target MAGE-A4 pMHC of about 10 ^-9 M to about 10 ^-14 M (e.g., about 10 ^-9 M、10 ^-10 M、10 ^-11 M、10 ^-12 M、10 ^-13 M or 10 ^-14 M), and a binding affinity of about 10 for said MAGE-A4 peptide, HLA polypeptide and beta-2-microglobulin polypeptide alone ^-6 M or less (e.g., about 10 ^-6 M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^-1 M)。

In certain embodiments, the antigen binding protein is specific for the MAGE-A4 peptide amino acid sequence as set forth in SEQ ID NO. 3 (GVYDGREHTV).

In certain embodiments, the MAGE-A4 peptide is complexed with an HLA-A2 polypeptide. In certain embodiments, the HLA-A2 polypeptide comprises the amino acid sequence shown in SEQ ID NO. 1.

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

In certain embodiments, the antigen binding protein lacks detectable binding affinity for MAGE-A4 peptides comprising one or more mutations (e.g., substitutions, deletions, and/or insertions) in the amino acid sequence set forth in SEQ ID NO. 3 (GVYDGREHTV).

In certain embodiments, the antigen binding protein lacks detectable binding affinity for MAGE-A4 peptides comprising one, two, three, four, or five mutations (e.g., substitutions, deletions, and/or insertions) in the amino acid sequence set forth in SEQ ID NO. 3 (GVYDGREHTV).

In certain embodiments, the antigen binding protein lacks detectable binding affinity for MAGE-A4 peptides comprising the amino acid sequences set forth in one or more of SEQ ID NO:394 (GLADGRTHTV), SEQ ID NO:395 (GLYDGPVHEV), and SEQ ID NO:396 (GVFDGLHTV).

In certain embodiments, the antigen binding protein lacks detectable binding affinity for MAGE-A4 peptides comprising the amino acid sequences set forth in SEQ ID NO:394 (GLADGRTHTV), SEQ ID NO:395 (GLYDGPVHEV), and SEQ ID NO:396 (GVFDGLHTV).

In certain embodiments, the antigen binding protein has a binding affinity of about 10 for a MAGE-A4 peptide comprising one or more mutations (e.g., substitutions, deletions, and/or insertions) of the amino acid sequence set forth in SEQ ID NO. 3 (GVYDGREHTV) ^-6 M or less (e.g., about 10 ^-6 M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^-1 M)。

In certain embodiments, the antigen binding protein has a binding affinity of about 10 for MAGE-A4 peptide comprising one, two, three, four, or five mutations (e.g., substitutions, deletions, and/or insertions) of the amino acid sequence set forth in SEQ ID NO 3 (GVYDGREHTV) ^-6 M or less (e.g., about 10 ^-6 M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^-1 M)。

In certain embodiments, the antigen binding protein lacks detectable binding affinity for peptides comprising the amino acid sequences set forth in one or more of SEQ ID NOS 345 to 393.

In certain embodiments, the antigen binding protein lacks detectable binding affinity for a peptide mixture comprising the amino acid sequences set forth in SEQ ID NO 345 through to SEQ ID NO 393.

In certain embodiments, the antigen binding protein has a binding affinity of about 10 for a MAGE-A4 peptide comprising the amino acid sequence set forth in one or more of SEQ ID NO:394 (GLADGRTHTV), SEQ ID NO:395 (GLYDGPVHEV), and SEQ ID NO:396 (GVFDGLHTV) ^-6 M or less (e.g., about 10 ^-6 M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^- ¹ M)。

In certain embodiments, the antigen binding protein has a binding affinity of about 10 for a MAGE-A4 peptide comprising the amino acid sequences set forth in SEQ ID NO:394 (GLADGRTHTV), SEQ ID NO:395 (GLYDGPVHEV), and SEQ ID NO:396 (GVFDGLHTV) ^-6 M or less (e.g., about 10 ^-6 M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^-1 M)。

In certain embodiments, the antigen binding protein has a binding affinity of about 10 for a peptide comprising one or more of the amino acid sequences set forth in SEQ ID NO 345 through to SEQ ID NO 393 ^-6 M or less (e.g., about 10 ^-6 M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^-1 M)。

In certain embodiments, the antigen binding protein has a binding affinity of about 10 for a peptide mixture comprising the amino acid sequences set forth in SEQ ID NO 345 through to SEQ ID NO 393 ^-6 M or less (e.g., about 10 ^-6 M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^-1 M)。

In certain embodiments, the antigen binding protein comprises a single chain variable fragment (scFv), fab fragment, fab' fragment, fv fragment, diabody, minibody mimetic, or single domain antibody, such as sdAb, sdFv, nanobody, V-Nar, or VHH.

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0848 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0848 of table 6; (b) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0849 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0849 of table 6; (c) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0850 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0850 of table 6; (d) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0851 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0851 of table 6; (e) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0852 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0852 of table 6; (f) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as recited in M0853 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as recited in M0853 of table 6; (g) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0854 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0854 of table 6; (h) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence listed in M0855 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence listed in M0855 of table 6; (i) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0856 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0856 of table 6; (j) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence listed in M0857 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence listed in M0857 of table 6; (k) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0858 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0858 of table 6; (l) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0859 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0859 of table 6; (M) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0860 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0860 of table 6; (n) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as recited in M0861 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as recited in M0861 of table 6; (o) an antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence, HCDR2 amino acid sequence, and HCDR3 amino acid sequence listed in M0862 of table 6, and an antibody light chain Variable (VL) domain comprising the LCDR1 amino acid sequence, LCDR2 amino acid sequence, and LCDR3 amino acid sequence listed in M0862 of table 6; (p) an antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence, HCDR2 amino acid sequence, and HCDR3 amino acid sequence listed in M0863 of table 6, and an antibody light chain Variable (VL) domain comprising the LCDR1 amino acid sequence, LCDR2 amino acid sequence, and LCDR3 amino acid sequence listed in M0863 of table 6; (q) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as recited in M0864 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as recited in M0864 of table 6; (r) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0865 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0865 of table 6; (s) an antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence, HCDR2 amino acid sequence, and HCDR3 amino acid sequence shown in M0866 of table 6, and an antibody light chain Variable (VL) domain comprising the LCDR1 amino acid sequence, LCDR2 amino acid sequence, and LCDR3 amino acid sequence shown in M0866 of table 6; (t) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0700 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0700 of table 6; (u) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0701 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0701 of table 6; (v) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0702 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0702 of table 6; (w) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0703 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0703 of table 6; (x) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0704 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0704 of table 6; (y) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0705 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0705 of table 6; (z) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0706 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0706 of table 6; (aa) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence listed in M0707 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence listed in M0707 of table 6; (bb) an antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence, HCDR2 amino acid sequence, and HCDR3 amino acid sequence shown in M0708 of table 6, and an antibody light chain Variable (VL) domain comprising the LCDR1 amino acid sequence, LCDR2 amino acid sequence, and LCDR3 amino acid sequence shown in M0708 of table 6; (cc) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0709 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0709 of table 6; (dd) an antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence, HCDR2 amino acid sequence, and HCDR3 amino acid sequence shown in M0710 of table 6, and an antibody light chain Variable (VL) domain comprising the LCDR1 amino acid sequence, LCDR2 amino acid sequence, and LCDR3 amino acid sequence shown in M0710 of table 6; (ee) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0762 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0762 of table 6; (ff) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0763 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0763 of table 6; (gg) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence set forth in M0764 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence set forth in M0764 of table 6; (hh) an antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence, HCDR2 amino acid sequence and HCDR3 amino acid sequence shown in M0765 of table 6, and an antibody light chain Variable (VL) domain comprising the LCDR1 amino acid sequence, LCDR2 amino acid sequence and LCDR3 amino acid sequence shown in M0765 of table 6; (ii) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0766 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0766 of table 6; (jj) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0767 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence shown in M0767 of table 6; (kk) an antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence, HCDR2 amino acid sequence, and HCDR3 amino acid sequence shown in M0768 of table 6, and an antibody light chain Variable (VL) domain comprising the LCDR1 amino acid sequence, LCDR2 amino acid sequence, and LCDR3 amino acid sequence shown in M0768 of table 6; or (ll) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0769 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0769 of table 6.

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain as shown in M0848 of table 6 and an antibody light chain Variable (VL) domain as shown in M0848 of table 6; (b) An antibody heavy chain Variable (VH) domain as shown in M0849 of table 6 and an antibody light chain Variable (VL) domain as shown in M0849 of table 6; (c) An antibody heavy chain Variable (VH) domain as shown in M0850 of table 6 and an antibody light chain Variable (VL) domain as shown in M0850 of table 6; (d) An antibody heavy chain Variable (VH) domain as shown in M0851 of table 6 and an antibody light chain Variable (VL) domain as shown in M0851 of table 6; m (e) an antibody heavy chain Variable (VH) domain as shown in M0852 of table 6 and an antibody light chain Variable (VL) domain as shown in M0852 of table 6; (f) An antibody heavy chain Variable (VH) domain as shown in M0853 of table 6 and an antibody light chain Variable (VL) domain as shown in M0853 of table 6; (g) An antibody heavy chain Variable (VH) domain as shown in M0854 of table 6 and an antibody light chain Variable (VL) domain as shown in M0854 of table 6; (h) An antibody heavy chain Variable (VH) domain as shown in M0855 of table 6 and an antibody light chain Variable (VL) domain as shown in M0855 of table 6; (i) An antibody heavy chain Variable (VH) domain as shown in M0856 of table 6 and an antibody light chain Variable (VL) domain as shown in M0856 of table 6; (j) An antibody heavy chain Variable (VH) domain as shown in M0857 of table 6 and an antibody light chain Variable (VL) domain as shown in M0857 of table 6; (k) An antibody heavy chain Variable (VH) domain as shown in M0858 of table 6 and an antibody light chain Variable (VL) domain as shown in M0858 of table 6; (l) An antibody heavy chain Variable (VH) domain as shown in M0859 of table 6 and an antibody light chain Variable (VL) domain as shown in M0859 of table 6; (M) an antibody heavy chain Variable (VH) domain as shown in M0860 of table 6 and an antibody light chain Variable (VL) domain as shown in M0860 of table 6; (n) an antibody heavy chain Variable (VH) domain as shown in M0861 of table 6 and an antibody light chain Variable (VL) domain as shown in M0861 of table 6; (o) an antibody heavy chain Variable (VH) domain as shown in M0862 of table 6 and an antibody light chain Variable (VL) domain as shown in M0862 of table 6; (p) an antibody heavy chain Variable (VH) domain as shown in M0863 of table 6 and an antibody light chain Variable (VL) domain as shown in M0863 of table 6; (q) an antibody heavy chain Variable (VH) domain as shown in M0864 of table 6 and an antibody light chain Variable (VL) domain as shown in M0864 of table 6; (r) an antibody heavy chain Variable (VH) domain as shown in M0865 of table 6 and an antibody light chain Variable (VL) domain as shown in M0865 of table 6; (s) an antibody heavy chain Variable (VH) domain as shown in M0866 of table 6 and an antibody light chain Variable (VL) domain as shown in M0866 of table 6; (t) an antibody heavy chain Variable (VH) domain as shown in M0700 of table 6 and an antibody light chain Variable (VL) domain as shown in M0700 of table 6; (u) an antibody heavy chain Variable (VH) domain as shown in M0701 of table 6 and an antibody light chain Variable (VL) domain as shown in M0701 of table 6; (v) An antibody heavy chain Variable (VH) domain as shown in M0702 of table 6 and an antibody light chain Variable (VL) domain as shown in M0702 of table 6; (w) an antibody heavy chain Variable (VH) domain as shown in M0703 of table 6 and an antibody light chain Variable (VL) domain as shown in M0703 of table 6; (x) An antibody heavy chain Variable (VH) domain as shown in M0704 of table 6 and an antibody light chain Variable (VL) domain as shown in M0704 of table 6; (y) an antibody heavy chain Variable (VH) domain as shown in M0705 of table 6 and an antibody light chain Variable (VL) domain as shown in M0705 of table 6; (z) an antibody heavy chain Variable (VH) domain as shown in M0706 of table 6 and an antibody light chain Variable (VL) domain as shown in M0706 of table 6; (aa) an antibody heavy chain Variable (VH) domain as shown in M0707 of table 6 and an antibody light chain Variable (VL) domain as shown in M0707 of table 6; (bb) an antibody heavy chain Variable (VH) domain as shown in M0708 of table 6 and an antibody light chain Variable (VL) domain as shown in M0708 of table 6; (cc) an antibody heavy chain Variable (VH) domain as set forth in M0709 of table 6 and an antibody light chain Variable (VL) domain as set forth in M0709 of table 6; (dd) an antibody heavy chain Variable (VH) domain as shown in M0710 of table 6 and an antibody light chain Variable (VL) domain as shown in M0710 of table 6; (ee) an antibody heavy chain Variable (VH) domain as shown in M0762 of table 6 and an antibody light chain Variable (VL) domain as shown in M0762 of table 6; (ff) an antibody heavy chain Variable (VH) domain as set forth in M0763 of table 6 and an antibody light chain Variable (VL) domain as set forth in M0763 of table 6; (gg) an antibody heavy chain Variable (VH) domain as shown in M0764 of table 6 and an antibody light chain Variable (VL) domain as shown in M0764 of table 6; (hh) an antibody heavy chain Variable (VH) domain as shown in M0765 of table 6 and an antibody light chain Variable (VL) domain as shown in M0765 of table 6; (ii) An antibody heavy chain Variable (VH) domain as shown in M0766 of table 6 and an antibody light chain Variable (VL) domain as shown in M0766 of table 6; (jj) an antibody heavy chain Variable (VH) domain as shown in M0767 of table 6 and an antibody light chain Variable (VL) domain as shown in M0767 of table 6; (kk) an antibody heavy chain Variable (VH) domain as shown in M0768 of table 6 and an antibody light chain Variable (VL) domain as shown in M0768 of table 6; or (ll) an antibody heavy chain Variable (VH) domain as shown in M0769 of table 6 and an antibody light chain Variable (VL) domain as shown in M0769 of table 6.

In certain embodiments, the antigen binding protein comprises: (a) an antibody heavy chain variable (V H) domain comprising: the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469); IVSSGGTTYYAX ₁ X ₂ X ₃ The HCDR2 amino acid sequence of KG (SEQ ID NO: 881), wherein X ₁ Corresponding to amino acids S or D, X ₂ Corresponds to amino acids W or S, and X ₃ Corresponding to amino acid a or V; and DLYYGPX ₄ TX ₅ YX ₆ X ₇ X ₈ HCDR3 amino acid sequence of NL (SEQ ID NO: 882), wherein X ₄ Corresponding to amino acid T, N or S, X ₅ Corresponding to amino acid D or absence, X ₆ Corresponding to amino acids S or F, X ₇ Corresponds to amino acids A or V, and X ₈ Corresponding to amino acid F or A; and (b) an antibody light chain variable (V L) domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473), and A TX ₉ X ₁₀ X ₁₁ SGSNFQX ₁₂ (SEQ ID NO883), wherein X is ₉ Corresponding to amino acids S or R, X ₁₀ Corresponding to amino acids D or P, X ₁₁ Corresponds to amino acid G, S or F, and X ₁₂ Corresponding to amino acid L or A.

In certain embodiments, the antigen binding protein does not comprise: (a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 470) and the HCDR3 amino acid sequence of DLYYGPTTYSAFNL (SEQ ID NO: 471); and (b) an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and an LCDR3 amino acid sequence of ATSDGSGSNFQL (SEQ ID NO: 474).

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 657) and the HCDR3 amino acid sequence of DLYYGPSTYFVANL (SEQ ID NO: 731); and (b) an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and an LCDR3 amino acid sequence of ATRPSSGSNFQL (SEQ ID NO: 879).

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 653) and the HCDR3 amino acid sequence of DLYYGPTTYSAANL (SEQ ID NO: 727); and (b) an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and an LCDR3 amino acid sequence of ATRDFSGSNFQL (SEQ ID NO: 875).

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 658) and the HCDR3 amino acid sequence of DLYYGPNTDYSAANL (SEQ ID NO: 732); and (b) an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and an LCDR3 amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 880).

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 624), and the HCDR3 amino acid sequence of DLYYGPTTYSAFNL (SEQ ID NO: 698); and (b) an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and an LCDR3 amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 846).

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 470) and the HCDR3 amino acid sequence of DLYYGPTTYSAFNL (SEQ ID NO: 471); and (b) an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and an LCDR3 amino acid sequence of ATSDGSGSNFQL (SEQ ID NO: 474).

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain comprising a framework region, an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the VH domain comprises an amino acid sequence shown in SEQ ID No. 575 or an amino acid sequence having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to the framework region of the amino acid sequence shown in SEQ ID No. 575 and having 100% identity to the HCDR1 region, HCDR2 region, and HCDR3 region shown in SEQ ID No. 575; and (b) an antibody light chain Variable (VL) domain comprising a framework region, an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the VL domain comprises an amino acid sequence shown in SEQ ID NO:797 or an amino acid sequence having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to the framework region of the amino acid sequence shown in SEQ ID NO:797 and having 100% identity to the LCDR1 region, LCDR2 region, and LCDR3 region.

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain comprising a framework region, an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the VH domain comprises an amino acid sequence shown in SEQ ID NO:583 or an amino acid sequence having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to the framework region of the amino acid sequence shown in SEQ ID NO:583 and having 100% identity to the HCDR1 region, HCDR2 region, and HCDR3 region shown in SEQ ID NO: 583; and (b) an antibody light chain Variable (VL) domain comprising a framework region, an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the VL domain comprises an amino acid sequence shown in SEQ ID No. 805 or has at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to the framework region of the amino acid sequence shown in SEQ ID No. 805 and has an amino acid sequence that is 100% identical to the LCDR1 region, LCDR2 region, and LCDR3 region shown in SEQ ID No. 805.

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain comprising a framework region, an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the VH domain comprises an amino acid sequence shown in SEQ ID NO:579 or an amino acid sequence having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to the framework region of the amino acid sequence shown in SEQ ID NO:579 and having 100% identity to the HCDR1 region, HCDR2 region, and HCDR3 region; and (b) an antibody light chain Variable (VL) domain comprising a framework region, an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the VL domain comprises an amino acid sequence shown in SEQ ID No. 801 or has at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to the framework region of the amino acid sequence shown in SEQ ID No. 801 and has an amino acid sequence that is 100% identical to the LCDR1 region, LCDR2 region, and LCDR3 region shown in SEQ ID No. 801.

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain comprising a framework region, an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the VH domain comprises an amino acid sequence shown in SEQ ID No. 582, or an amino acid sequence having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to the framework region of the amino acid sequence shown in SEQ ID No. 582 and having 100% identity to the HCDR1 region, HCDR2 region, and HCDR3 region shown in SEQ ID No. 582; and (b) an antibody light chain Variable (VL) domain comprising a framework region, an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the VL domain comprises an amino acid sequence shown in SEQ ID No. 804 or has at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to the framework region of the amino acid sequence shown in SEQ ID No. 804 and has an amino acid sequence that is 100% identical to the LCDR1 region, LCDR2 region, and LCDR3 region shown in SEQ ID No. 804.

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain comprising a framework region, an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the VH domain comprises an amino acid sequence shown in SEQ ID No. 584, or an amino acid sequence having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to the framework region of the amino acid sequence shown in SEQ ID No. 584 and having 100% identity to the HCDR1 region, HCDR2 region, and HCDR3 region shown in SEQ ID No. 584; and (b) an antibody light chain Variable (VL) domain comprising a framework region, an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the VL domain comprises an amino acid sequence shown in SEQ ID No. 806 or has at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to the framework region of the amino acid sequence shown in SEQ ID No. 806 and has an amino acid sequence that is 100% identical to the LCDR1 region, LCDR2 region, and LCDR3 region shown in SEQ ID No. 806.

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain comprising a framework region, an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the VH domain comprises an amino acid sequence shown in SEQ ID No. 550 or an amino acid sequence having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to the framework region of the amino acid sequence shown in SEQ ID No. 550 and having 100% identity to the HCDR1 region, HCDR2 region, and HCDR3 region shown in SEQ ID No. 550; and (b) an antibody light chain Variable (VL) domain comprising a framework region, an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the VL domain comprises an amino acid sequence shown in SEQ ID NO 772 or has at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to the framework region of the amino acid sequence shown in SEQ ID NO 772 and has an amino acid sequence that is 100% identical to the LCDR1 region, LCDR2 region, and LCDR3 region shown in SEQ ID NO 772.

In certain embodiments, one or more of the HCDR1 amino acid sequence, the HCDR2 amino acid sequence, the HCDR3 amino acid sequence, the LCDR1 amino acid sequence, the LCDR2 amino acid sequence and the LCDR3 amino acid sequence comprises one or more amino acid substitutions.

In certain embodiments, the antigen binding protein retains binding specificity for the target MAGE-A4 pMHC after the one or more amino acid substitutions.

In certain embodiments, one or more of the VH domain and the VL domain comprises one or more amino acid substitutions.

In another aspect, the present disclosure provides an antigen binding protein that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising: (a) an antibody heavy chain Variable (VH) domain comprising: the HCD R1 amino acid sequence of SNYAMS (SEQ ID NO: 469); IVSSGGTTYYAX ₁ X ₂ X ₃ hCDR2 amino acid sequence of KG (SEQ ID NO: 881), wherein X ₁ Corresponding to amino acids S or D, X ₂ Corresponds to amino acids W or S, and X ₃ Corresponding to amino acid a or V; and DLYYGPX ₄ TX ₅ YX ₆ X ₇ X ₈ HCDR3 amino acid sequence of NL (SEQ ID NO: 882), wherein X ₄ Corresponding to amino acid T, N or S, X ₅ Corresponding to amino acid D or absence, X ₆ Corresponding to amino acids S or F, X ₇ Corresponds to amino acids A or V, and X ₈ Corresponding to amino acid F or A; and (b) an antibody light chain Variable (VL) domain comprising: TADTLSRSYAS (SEQ ID NO: 472), LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) andATX ₉ X ₁₀ X ₁₁ SGSNFQX ₁₂ (SEQ ID NO: 883) LCDR3 amino acid sequence, wherein X ₉ Corresponding to amino acids S or R, X ₁₀ Corresponding to amino acids D or P, X ₁₁ Corresponds to amino acid G, S or F, and X ₁₂ Corresponding to amino acid L or A.

In another aspect, the present disclosure provides an antigen binding protein that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising: (a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 657) and the HCDR3 amino acid sequence of DLYYGPSTYFVANL (SEQ ID NO: 731); and (b) an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and an LCDR3 amino acid sequence of ATRPSSGSNFQL (SEQ ID NO: 879).

In certain embodiments, the antigen binding protein comprises: an antibody VH domain comprising the amino acid sequence shown in SEQ ID No. 583 and an antibody VL domain comprising the amino acid sequence shown in SEQ ID No. 805, or a VH domain having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID No. 583 and a VL domain having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID No. 805.

In another aspect, the present disclosure provides an antigen binding protein that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising: (a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 653) and the HCDR3 amino acid sequence of DLYYGPTTYSAANL (SEQ ID NO: 727); and (b) an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and an LCDR3 amino acid sequence of ATRDFSGSNFQL (SEQ ID NO: 875).

In certain embodiments, the antigen binding protein comprises: an antibody VH domain comprising the amino acid sequence set forth in SEQ ID No. 579 and an antibody VL domain comprising the amino acid sequence set forth in SEQ ID No. 801, or a VH domain having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID No. 579 and a VL domain having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID No. 801.

In another aspect, the present disclosure provides an antigen binding protein that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising: (a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 658) and the HCDR3 amino acid sequence of DLYYGPNTDYSAANL (SEQ ID NO: 732); and (b) an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and an LCDR3 amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 880).

In certain embodiments, the antigen binding protein comprises: an antibody VH domain comprising the amino acid sequence shown in SEQ ID No. 584 and an antibody VL domain comprising the amino acid sequence shown in SEQ ID No. 806, or a VH domain having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID No. 584 and a VL domain having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID No. 806

In another aspect, the present disclosure provides an antigen binding protein that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising: (a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR 2 amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 624), and the HCDR3 amino acid sequence of DLYYGPTTYSAFNL (SEQ ID NO: 698); and (b) an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYA S (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473), and an LCDR3 amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 846).

In certain embodiments, the antigen binding protein comprises: an antibody VH domain comprising the amino acid sequence set forth in SEQ ID No. 550 and an antibody VL domain comprising the amino acid sequence set forth in SEQ ID No. 772, or a VH domain having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID No. 550 and a VL domain having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID No. 772.

In certain embodiments, the antigen binding protein comprises one of the following featuresOne or more of the following features: (i) The antigen binding protein has a binding affinity of about 10 for the target MAGE-A4pMHC ^-9 M to about 10 ^-14 M; (ii) The antigen binding protein has a binding affinity of about 10 for non-MAGE-A4 pMHC and/or peptide-free MHC ^-6 M or weaker; (iii) The antigen binding protein has a binding affinity of about 10 for non-target MAGE-A4pMHC ^-6 M or weaker; and (iv) the antigen binding protein has a binding affinity for the target MAGE-A4pMHC of about 10 ^-9 M to about 10 ^-14 M and has a binding affinity of about 10 for MAGE-A4 peptide, HLA polypeptide and beta-2-microglobulin polypeptide alone ^-6 M or weaker.

In certain embodiments, the VH domain and VL domain are attached with an amino acid linker. In certain embodiments, the amino acid linker comprises (GGGGS) n, wherein n is an integer from 1 to 5. In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS or GGG GSGGGGSGGGGSGGGGAS.

In certain embodiments, the antigen binding protein comprises: (a) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0734 of table 8; (b) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0735 of table 8; (c) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0736 of table 8; (d) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0737 of table 8; (e) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0738 of table 8; (f) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0739 of table 8; (g) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0740 of table 8; (h) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0741 of table 8; (i) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0742 of table 8; (j) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0743 of table 8; (k) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0744 of table 8; (l) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0745 of table 8; (M) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0746 of table 8; (n) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0747 of table 8; (o) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0748 of table 8; (p) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0749 of table 8; (q) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0750 of table 8; (r) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0751 of table 8; or(s) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0752 of table 8.

In certain embodiments, the antigen binding protein comprises: (a) an antibody VHH domain as shown in M0734 of table 8; (b) an antibody VHH domain as shown in M0735 of table 8; (c) an antibody VHH domain as shown in M0736 of table 8; (d) an antibody VHH domain as shown in M0737 of table 8; (e) an antibody VHH domain as shown in M0738 of table 8; (f) an antibody VHH domain as shown in M0739 of table 8; (g) an antibody VHH domain as shown in M0740 of table 8; (h) an antibody VHH domain as shown in M0741 of table 8; (i) an antibody VHH domain as shown in M0742 of table 8; (j) an antibody VHH domain as shown in M0743 of table 8; (k) an antibody VHH domain as shown in M0744 of table 8; (l) an antibody VHH domain as shown in M0745 of table 8; (M) an antibody VHH domain as shown in M0746 of table 8; (n) an antibody VHH domain as shown in M0747 of table 8; (o) an antibody VHH domain as shown in M0748 of table 8; (p) an antibody VHH domain as shown in M0749 of table 8; (q) an antibody VHH domain as shown in M0750 of table 8; (r) an antibody VHH domain as shown in M0751 of table 8; or(s) the antibody VHH domain shown in M0752 of Table 8.

In certain embodiments, one or more of the HCDR1 amino acid sequence, the HCDR2 amino acid sequence and the HCDR3 amino acid sequence comprises one or more amino acid substitutions.

In certain embodiments, the VHH domain comprises one or more amino acid substitutions.

In certain embodiments, the antigen binding protein has a binding affinity for the MAGE-A4 pMHC of at least about 10 ^-9 M。

In certain embodiments, the antigen binding protein has a binding affinity for the MAGE-A4 pMHC of about 10 ^-9 M to about 10 ^-14 M。

In certain embodiments, the antigen binding protein has a binding affinity for the MAGE-A4 pMHC of about 10 ^-10 M to about 10 ^-12 M。

In certain embodiments, the antigen binding protein lacks detectable binding affinity for non-MAGE-A4 pMHC.

In certain embodiments, the antigen binding protein lacks detectable binding affinity for peptide-free MHC.

In certain embodiments, the antigen binding protein lacks detectable binding affinity for non-target MAGE-A4 pMHC.

In certain embodiments, the antigen binding protein lacks detectable binding affinity for the MAGE-A4 peptide alone.

In certain embodiments, the antigen binding protein lacks detectable binding affinity for an individual HLA polypeptide.

In certain embodiments, the antigen binding protein lacks detectable binding affinity for the β -2-microglobulin polypeptide alone.

In certain embodiments, the antigen binding protein specifically binds MAGE-A4pMHC on the cell surface.

In certain embodiments, the antigen binding protein lacks detectable binding affinity for non-MAGE-A4 pMHC on the cell surface.

In certain embodiments, the antigen binding protein has cytotoxic activity against cells expressing MAGE-A4 pMHC.

In certain embodiments, the antigen binding protein lacks detectable cytotoxic activity against cells expressing non-MAGE-A4 pMHC.

In certain embodiments, the antigen binding protein is a humanized antigen binding protein.

In certain embodiments, the antigen binding protein is a human antigen binding protein.

In certain embodiments, the binding affinity is measured by Surface Plasmon Resonance (SPR).

In one aspect, the present disclosure provides a bispecific antigen binding protein comprising a first antigen binding domain comprising an antigen binding protein as described above and a second antigen binding domain specific for a cell surface protein of an immune cell.

In certain embodiments, the immune cells are 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.

In certain embodiments, the cell surface protein of the immune cell is selected from the group consisting of CD3, tcra, tcrp, CD16, NKG2D, CD89, CD64, and CD 32.

In certain embodiments, the cell surface protein of the immune cell is CD3.

In certain embodiments, the first antigen binding domain comprises an scFv or VHH and the second antigen binding domain comprises a Fab.

In certain embodiments, the bispecific antigen binding protein is multivalent.

In certain embodiments, the bispecific antigen binding protein comprises three antigen binding sites.

In certain embodiments, the bispecific antigen binding protein further comprises an immune checkpoint inhibitor.

In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-BTLA antibody, an anti-VISTA antibody, and combinations thereof.

In another aspect, the present disclosure provides the use of the above antigen binding protein or the above bispecific antigen binding protein for the manufacture of a pharmaceutical composition for treating a MAGE-A4 associated cancer in a subject.

In another aspect, the present disclosure provides a pharmaceutical composition comprising the above antigen binding protein or the above bispecific antigen binding protein and a pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides a method of treating a MAGE-A4 pMHC expressing cancer in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of the above pharmaceutical composition.

In certain embodiments, the method further comprises administering an immune checkpoint inhibitor.

In another aspect, the present disclosure provides a nucleic acid encoding the antigen binding protein described above or the bispecific antigen binding protein described above.

In another aspect, the present disclosure provides an expression vector comprising the nucleic acid described above.

In another aspect, the present disclosure provides a host cell comprising the above expression vector.

In another aspect, the present disclosure provides a method of preparing the above antigen binding protein or the above bispecific antigen binding protein, comprising the steps of: (i) Incubating the above-described host cell under conditions allowing expression of the antigen binding protein or the bispecific antigen binding protein; (ii) Recovering the antigen binding protein or bispecific antigen binding protein; and optionally (iii) further purifying and/or modifying and/or formulating the antigen binding protein or bispecific antigen binding protein.

Drawings

The foregoing and other features and advantages of the invention will be more fully understood from the following detailed description of exemplary embodiments, taken in conjunction with the accompanying drawings. The patent or application document contains at least one drawing which is drawn in color. Upon request and payment of the necessary fee, the office will provide a copy of the patent or patent application publication with one or more colored drawings.

Figure 1 depicts DNA sequence alignment of rabbit kappa light chain sequences of all 68 alleles retrieved from IMGT database. Flanking regions around the codon encoding the relevant cysteine 80 (marked with asterisks) show high sequence conservation.

FIG. 2 depicts DNA sequence alignment of randomly selected control antibodies from a rabbit immune library that have been used to identify designed primer sets by identifying mismatches. Related cysteines are marked with asterisks.

Figure 3 depicts a phylogenetic tree of 62 sequences from an optimized rabbit immune library. High coverage of sequence diversity is depicted.

FIG. 4 depicts the selection of 38 unique HLA-A2/MAGE-A4 specific antibodies generated via rabbit and llama immunization followed by construction and biopanning of the corresponding phage library. The hits selected are grouped according to amino acid sequence diversity as determined by phylogenetic analysis.

FIGS. 5A-5B depict binding of selected antibodies to HLA-A2/MAGE-A4 or control complexes as determined by direct ELISA. Antibodies designated M0709, M0739, M0742, M0743, M0747 and M0763 are shown in FIG. 5A, and antibodies designated M0700-M0710 and M0762-M0766 are shown in FIG. 5B.

FIG. 6 depicts the binding of selected antibodies M0709, M0739, M0742, M0743, M0747, M0763 to T2 cells displaying MAGE-A4 or control peptides 1, 2 and 3. TAP-deficient T2 cells were pulsed with HLA-A2 restriction peptide (MAGE-A4 or control peptide) and incubated with MAGE-A4 conjugate, followed by incubation with fluorophore-labeled specific detection antibodies and analysis by flow cytometry. Peptide loading was confirmed with PE-labelled anti-HLA-A 2 antibody BB 7.2. The ratio of binding efficiency to peptide loading results are shown as relative Median Fluorescence Intensity (MFI).

FIG. 7 depicts T cell mediated cytotoxicity triggered by CDR 4-bispecific 01. Cell killing was determined by measuring released LDH after incubation of MAGE-A4 positive cell lines with PBMC at E:T ratio 10:1 and CDR4 bispecific 01 at the indicated concentrations for 48 hours.

Figure 8 depicts EC50 values for cell killing as determined by LDH assay. LDH release was measured after co-incubation of PBMC and MAGE-A4 positive cell lines for 48 hours with or without anti-PD-1 (pambrizumab) in the presence of MAGE-A4 bispecific 01 at a ratio of 10:1.

Fig. 9 depicts T cell mediated cytotoxicity triggered by CDR4 bispecific 01 as determined by in vitro live cell imaging. MAGE-A4 positive NCI-H1703 cells were co-incubated with PBMC at E:T ratio 10:1 and CDR4 bispecific 01 at the indicated concentrations. Images were recorded by the IncuCyte S3 system for up to 72 hours. The quantification of cytotoxicity was reported as the ratio of green object count per image (dead cells, cytotox Green Dye) to red area confluence (cell line, cytolight Rapid Red). MAGE-A4 negative/HLA-A 2 positive H441 cells were used as controls at the highest concentration of bispecific antibody (6.3 nM) to demonstrate specific killing.

Fig. 10 depicts T cell mediated cytotoxicity triggered by CDR4 bispecific 01 as determined by in vitro live cell imaging. MAGE-A4 positive/HLA-A 2 positive NCI-H1703 cells or MAGE-A4 negative/HLA-A 2 positive cells (H441 and MRC 5) were co-incubated with PBMC at an E:T ratio of 10:1 and a single concentration of 0.8nM CDR 4-bispecific 01. Images were recorded with the IncuCyte S3 system for up to 72 hours. The quantification of cytotoxicity was reported as the ratio of green object count per image (dead cells, cytotox Green Dye) to red area confluence (cell line, cytolight Rapid Red).

FIG. 11 depicts the in vivo efficacy of CDR4 bispecific 02 molecules. 5x10 subcutaneous injections into NSG mice ⁶ NCI-H1703 cells and received intravenously an average tumor size of 80mm ³ 5x10 of (2) ⁶ PBMCs (2 donors, 4 mice/group). Mice were treated once daily with CDR4 bispecific 02 (2.5 mg/kg day 0-9, 5mg/kg day 10-27) or PBS control.

Detailed Description

Generally, 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. Unless otherwise specified, the methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. The enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as is commonly done in the art, or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly employed in the art. Standard techniques are used for chemical synthesis, chemical analysis, preparation, formulation and delivery of drugs, and treatment of patients.

Unless defined otherwise herein, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. If any potential ambiguity exists, the definitions provided herein take precedence over any dictionary or external definitions. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and/or" unless stated otherwise. The use of the term "include" and other forms such as "include" and "include" are not limiting.

In order that the invention may be more readily understood, certain terms are first defined.

Antigen binding proteins

As used herein, the term "antibody" or "antigen binding protein" refers to an immunoglobulin molecule or immunoglobulin-derived molecule that specifically binds or is immunoreactive with an antigen or epitope, and includes polyclonal and monoclonal antibodies, as well as functional antibody fragments, including but not limited to 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 antibodies (e.g., sdAb, sdFv, nanobody, VHH) fragments. The antibody may thus be a single domain antibody or comprise at least one variable light chain and at least one variable heavy chain. In one embodiment, the at least one variable light chain and the at least one variable heavy chain are displayed 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 other modified forms of immunoglobulins, such as endosomes, peptibodies (peptabodies), chimeric antibodies, fully human antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem diavs, tandem triavs), and the like. Unless otherwise indicated, the term "antibody" or "antigen binding protein" is understood to encompass functional antibody fragments thereof.

In certain embodiments, the antigen binding protein is not a T Cell Receptor (TCR), including but not limited to a soluble TCR.

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

The antigen binding protein may 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 nanobodies. The multivalent antigen binding protein may 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, tetravalent tumor, crossMab, duoBody, triabodies, and tetrabodies. 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 against two different targets or two different target sites on one target molecule. In some embodiments, the multivalent antigen binding protein comprises more than two binding sites, e.g., three or four different binding sites for three or four different antigens, respectively. Such antigen binding proteins are multivalent and multispecific, especially trispecific or tetraspecific, respectively.

In some embodiments, the antigen binding protein is multispecific (e.g., bispecific), such as, but not limited to, diabody, single chain diabody, DART, biTE, tandem scFv, or IgG-like asymmetric heterobispecific antibody. In certain embodiments, one or the binding specificity of the multispecific antigen-binding protein is an immune cell conjugate (i.e., comprises a binding affinity for a cell surface protein of an immune cell). Examples of immune cells that may 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, tcra, tcrp, CD16, NKG2D, CD89, CD64, and CD32. Such immune cell redirecting multispecific antigen binding proteins may in some embodiments comprise an Fc domain.

In certain embodiments, the immune cell target antigen is CD3. Exemplary CD3 antigen binding domains are described in table 7 below and in WO2016086196 and WO2017201493, which are incorporated by reference herein.

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 the scFv are connected via any suitable art-recognized linker. Such linkers include, but are not limited to, repeated GGGGS amino acid sequences or variants thereof. scFv generally do not contain antibody constant domain regions, but scFv of the present disclosure can be linked or attached to antibody constant domain regions (e.g., antibody Fc domains) to alter various characteristics of scFv, including but not limited to increased serum half-life or tissue half-life. scFv typically have a molecular weight of about 25kDa and a hydrodynamic radius of about 2.5 nm.

As used herein, a "Fab fragment" or "Fab" is an antibody fragment comprising: a light chain fragment comprising a variable light chain (VL) domain and a constant domain (CL) of a light chain, and a variable heavy chain (VH) domain and a first constant domain (CH 1) of a heavy chain.

As used herein, a "VHH", "nanobody" or "heavy chain-only antibody" is an antigen binding protein comprising a single heavy chain variable domain derived from a camelidae species including camel, llama, alpaca. The molecular weight of VHH is typically about 15kDa.

In one embodiment, the antigen binding protein comprises an Fc domain. The presence of an Fc domain may be advantageous in inducing a cytotoxic immune response and/or activating complement (e.g., ADCC/ADCP or CDC effector function). Exemplary antibody formats including, but not limited to, fc domains are full length immunoglobulins, DVD-Ig, scFv-Fc and scFv-Fc, scFv fusions, igG-scFab, scFab-dsscFv, fv2-Fc, igG-scFv fusions (such as bsAb, bs1Ab, bs2Ab, bs3Ab, ts1Ab, ts2Ab, pestle and mortar (KiH)), duoBody, and/or CrossMabs. The active Fc domain may increase the likelihood that T cells and other effector cells in the tumor microenvironment release pro-inflammatory cytokines, which is considered part of the therapeutic mechanism of action. The Fc domain may be fully active or partially silent to avoid overstimulation of the immune system. In some embodiments, the Fc domain is inactive and does not stimulate pro-inflammatory cytokine release, but still improves the half-life and/or stability of the antigen binding protein. In some embodiments, the antigen binding protein comprises a constant region selected from the group consisting of human IgG1, igG2, igG3, or IgG4 isotypes. In other embodiments, the antigen binding protein comprises a constant region selected from the group consisting of murine IgG1, igG2A, igG B, or IgG3 isotype.

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

Illustrative examples of linkers include glycine polymers (Gly) _n The method comprises the steps of carrying out a first treatment on the surface of the Glycine-serine Polymer (Gly) _n Ser) _n Wherein n is an integer of at least 1, 2, 3, 4, 5, 6, 7 or 8; glycine-alanine polymer; alanine-serine polymers; and other flexible joints known in the art.

Glycine and glycine-serine polymers are relatively unstructured and thus may be able to function as a neutral tether between domains of fusion proteins such as antigen binding proteins described herein. Glycine enters a significantly larger phi-psi space than other small side chain amino acids and is much less restricted than residues with longer side chains (Scheraga, rev. Computational chem.1:1173-142 (1992)). Those skilled in the art will recognize that the design of an antigen binding protein in particular embodiments may include a linker that is wholly or partially flexible, such that the linker may include a flexible linker segment and one or more segments that impart less flexibility to provide a desired structure.

However, linker sequences similar to the native linker sequences may be selected, for example, using an amino acid segment corresponding to the beginning of the human CH1 and Cκ sequences or an amino acid segment corresponding to the lower portion of the human IgG hinge region.

The design of the peptide linker in the scFv portion that connects the VL domain and VH domain is a flexible linker, typically consisting of small non-polar or polar residues such as Gly, ser and Thr. A particular exemplary linker linking the variable domains of scFv moieties is (Gly ₄ Ser) ₄ A linker, wherein 4 is an exemplary number of repeating sequences of the motif.

Other exemplary linkers include, but are not limited to, the following amino acid sequences: GGG; DGGGS; TGEKP (Liu et al, proc. Natl. Acad. Sci.94:5525-5530 (1997)); g GRR; (GGGGS) _n Wherein n=1, 2, 3, 4 or 5 (Kim et al proc. Natl. Acad. Sci.93:1156-1160 (1996)); EGKSSGSGSESKVD (Chaudhary et al, proc. Natl. Acad. Sci.87:1066-1070 (1990)); KESGSVSSE QLAQFRSLD (Bird et al Science 242:423-426 (1988)); GGRRGG GS; LRQRDGERP; LRQKDGGGSERP; and GSTSGSGKPGSGEGST KG (Cooper et al, blood,101 (4): 1637-1644 (2003)). Alternatively, the flexible linker may be rationally designed using a computer program capable of mimicking the 3D structure of proteins and peptides or by phage display methods.

Antibodies may comprise a variable light chain (VL) domain and a variable heavy chain (VH) domain. Each VL domain and VH domain further comprises a set of three CDRs.

As used herein, the term "complementarity determining region" or "CDR" refers to a discontinuous sequence of amino acids within the variable region of an antibody that confer antigen specificity and binding affinity. Generally, there are three CDRs (CDRH 1, CDRH2, CDRH 3) in each heavy chain variable domain and three CDRs (CDRL 1, CDRL2, CDRL 3) in each light chain variable domain. "framework region" or "FR" is known in the art and refers to the non-CDR portions of the heavy and light chain variable domains. Generally, there are four FRs (HFR 1, HFR2, HFR3, and HFR 4) in each heavy chain variable domain, and four FRs (LFR 1, L FR2, LFR3, and LFR 4) in each light chain variable domain. Thus, the antibody variable region amino acid sequence can be represented by the formula FR1-CD R1-FR2-CDR2-FR3-CDR3-FR 4. Each segment in the formula, FR1, CD R1, FR2, CDR2, FR3, CDR3, and FR4, represents a discrete amino acid sequence (or a polynucleotide sequence encoding the discrete amino acid sequence) that can be mutated, which includes 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-CDRL1-LFR2-CDRL2-LFR3-CDRL3-LFR 4. In certain embodiments, the antibody variable heavy chain amino acid sequence may be represented by the formula HFR1-CDRH1-HFR2-CDRH2-HFR3-CDRH3-HFR 4.

In certain embodiments, one or more CDR amino acid sequences of the present disclosure comprise one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, or more amino acid substitutions).

In certain embodiments, one or more framework region amino acid sequences of the present disclosure comprise one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid substitutions).

The exact amino acid sequence boundaries for a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described below: kabat et Al (1991), "Sequences of Proteins of Immunological Interest," 5 th edition Publ ic Health Service, national Institutes of Health, bethesda, md. ("Ka bat" numbering scheme), al-Lazikani et Al, (1997) JMB 273,927-948 ("Chothia" numbering scheme), macCallum et Al, J.mol. Biol.262:732-745 (1996), "anti-antigen interactions: contact analysis and bindi ng site topography," J.mol. Biol.262,732-745 ("Contact" numbering scheme), lefranc M P et Al, "IMGT unique numbering for immunoglob ulin and T cell receptor variabledomains and Ig superfamily V-like domains," Dev Comp Immunol,2003, month 1; 27 (1) 55-77 ("IMGT" numbering scheme), honyger A and Pluckaphun A, "Yet another numberi ng scheme for immunoglobulin variabledomains: an automatic mod eling and analysis tool," J Mol Biol, 6/8/2001; 309 (3) 657-70, ("AHo" numbering scheme).

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. Numbering of both Kabat and Chothia protocols is based on the most common antibody region sequence length and regulates insertion by insert letters (e.g. "30 a") and deletions occur in some antibodies. Both of these schemes place certain insertions and deletions ("indels") at different positions, resulting in different numbers. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

Table 1 below lists exemplary positional boundaries of CDRL1, CDRL2, CDRL3 and CDRH1, CDRH2, CDRH3 of antibodies identified by the Kabat, chothia and Contact schemes, respectively. For CDRH1, residue numbers are listed using the Kabat and Chothia numbering schemes. The CDRs are located between the FR, e.g., CDRL1 is located between LFR1 and LFR2, etc. It should be noted that because the Kabat numbering scheme shown places insertions at H35A and H35B, the ends of the Chothia CDRH1 loop vary between H32 and H34 when numbered using the Kabat numbering convention shown, depending on the length of the loop.

TABLE 1 exemplary position boundaries of CDRs

1-Kabat et al (1991), "Sequences of Proteins of Immunological Interest," 5 th edition, public Health Service, national Institutes of Health, bethesda, MD

2-Al-Lazikani et Al (1997), J.mol.biol.273:927-948

Thus, unless otherwise indicated, a "CDR" or "complementarity determining region" or a single designated CDR (e.g., CDRH1, CDRH 2) of a given antibody or fragment thereof (such as a variable domain thereof) is to be understood as encompassing the (or specific) complementarity determining region as defined by any known scheme. Likewise, unless otherwise specified, "FR" or "framework region" or a single designated FR (e.g., "HFR1", "HFR 2") of a given antibody or fragment thereof (such as a variable domain thereof) is to be understood to encompass a (or a particular) framework region as defined by any known scheme. In some cases, a scheme for identifying a particular CDR or FR is specified, such as a CDR defined by Kabat, chothia or Contact methods. In other cases, specific amino acid sequences of CDRs or FRs are given.

In certain embodiments, the rabbit antigen binding proteins disclosed herein are humanized. As used herein, the term "humanized" or "humanized" refers to antigen binding proteins that have been altered to make them more human-like antibodies. Non-human antigen binding proteins, such as rabbit antigen binding proteins encoded in the nucleic acid libraries disclosed herein, will elicit a negative immune response if administered to a human for treatment. Therefore, it is advantageous to humanize the rabbit antigen binding protein for later therapeutic use.

In certain embodiments, the antigen binding proteins are humanized (i.e., the solvent accessible residues of the non-human framework are remodeled such that they become more human-like) by resurfacing (resurfacing). The resurfacing strategy is described in more detail in WO2004/016740, WO2008/144757 and WO2005/016950, each of which is incorporated herein by reference.

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

As used herein, the term "affinity" refers to the strength of interaction between an antigen binding site of an antibody and an epitope to which it binds. As will be readily appreciated by those skilled in the art, antibody or antigen binding protein affinity can be reported as dissociation constant (KD) at molar concentration (M). Antibodies of the disclosure may have 10 ^-8 To 10 ^-14 KD values for M range. The high affinity antibody has 10 ^-9 M (1 nanomole, nM) and moreLow KD values. For example, a high affinity antibody may have a KD value in the range of about 1nM to about 0.01 nM. The high affinity antibody may have a KD value of about 1nM, about 0.9nM, about 0.8nM, about 0.7nM, about 0.6nM, about 0.5nM, about 0.4nM, about 0.3nM, about 0.2nM, or about 0.1 nM. Very high affinity antibody having 10 ^-12 M (1 picomolar, pM) and lower KD values. Weak or low affinity antibodies may have 10 ^-1 To 10 ^-4 KD values for M range. The low affinity antibody may have 10 ^- ⁴ M and higher KD values, such as 10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^-1 M。

The ability of an antibody to bind to a particular epitope (e.g., target peptide-MHC) can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as Surface Plasmon Resonance (SPR) techniques (e.g., analysis on a BIAcore instrument) (Liljeblad et al, glyco J17,323-329 (2000)) and conventional binding assays (Heeley, endocr Res 28,217-229 (2002)).

As used herein, the term "T cell receptor" or "TCR" refers to a heterodimeric protein consisting of two distinct chains (tcra and tcrp), which structurally belong to the immunoglobulin (Ig) superfamily. The extracellular portion of each chain consists of variable ("vα" and "vβ") domains and constant ("cα" and "cβ") domains, as well as hinge regions, in which stable disulfide bond formation occurs. The intracellular region forms a non-covalent interaction with another transmembrane protein CD3, which, in the case of correct recognition of the target, generates a series of conformational changes and a first T cell activation signal. The recognition and binding of peptide-MHC (pMHC) by TCRs is governed by six hypervariable loops called Complementarity Determining Regions (CDRs) located on the variable domains of tcrα (CDR α1, CDR α2, CDR α3) and tcrβ (CDR β1, CDR β2, CDR β3). The CDR3 loops (CDR. Alpha.3 and CDR. Beta.3) lead to the recognition of processed antigens with the support of CDR. Alpha.1 and CDR. Beta.1, CDR. Alpha.1 and CDR. Beta.1 have been implicated in the recognition of N-and C-terminal amino acids, respectively, of the presented peptides (Rudolph et al Annu Rev immunol.24:419-66.2006). Recognition of MHC is typically achieved by interaction with CDR α2 and CDR β2. The high sequence diversity of TCRs is achieved by a V (D) J recombination process in which the variable domains are generated by the combination of genes: v (variable) and J (linked) genes for tcrα and tcrβ, and additional D (diversity) genes for tcrβ. The high antigen specificity of TCRs is controlled by the thymic maturation process, in which self-reactive T cells are negatively selected. The affinity and functional avidity of TCRs for specific pMHC are key factors in controlling T cell activation. However, a key role in antigen recognition is played by affinity, i.e., the binding strength between TCR and pMHC displayed by the cell (Tian et al, J Immunol. 179:2952-2960.2007). The physiological affinity of TCRs ranges from 1. Mu.M to 100. Mu.M (Davis et al Annu Rev immunol. 16:523-544.1998), which is relatively low compared to antibodies.

As used herein, the term "peptide-MHC" refers to a Major Histocompatibility Complex (MHC) molecule (MHC-I or MHC-II) whose antigen peptide is bound in the peptide-binding pocket of the MHC. In certain embodiments, the MHC is a human MHC.

MAGE-A4pMHC antigen binding proteins

Described herein are antigen binding proteins that specifically recognize the target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC). Antigen binding proteins have surprisingly high binding affinity while maintaining high specificity for the target (i.e., low to no binding affinity for other targets, including non-MAGE-A4 pMHC, HLA polypeptides alone, or beta-2-microglobulin alone).

In one aspect, the present disclosure provides an antigen binding protein that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), wherein the antigen binding protein comprises one or more of the following features:

(i) The antigen binding protein has a binding affinity of about 10 for the target MAGE-A4pMHC ^-9 M to about 10 ^-14 M (e.g., about 10 ^-9 M、10 ^-10 M、10 ^-11 M、10 ^-12 M、10 ^-13 M or 10 ^-14 M)；

(ii) The antigen binding protein has a binding affinity of about 10 for non-MAGE-A4 peptide-MHC and/or peptide-free MHC ^-6 M or less (e.g., about 10 ^-6 M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or10 ^-1 M)；

(iii) The antigen binding protein has a binding affinity of about 10 for non-target MAGE-A4pMHC ^-6 M or less (e.g., about 10 ^-6 M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^-1 M) is selected from the group consisting of; and

(iv) The antigen binding protein has a binding affinity of about 10 for the target MAGE-A4 pMHC ^-9 M to about 10 ^-14 M (e.g., about 10 ^-9 M、10 ^-10 M、10 ^-11 M、10 ^-12 M、10 ^-13 M or 10 ^-14 M) and a binding affinity of about 10 for the MAGE-A4 peptide, HLA polypeptide and beta-2-microglobulin polypeptide alone ^-6 M or less (e.g., about 10 ^-6 M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^- ¹ M)。

In certain embodiments, the non-MAGE-A4 peptide-MHC has less than about 60% sequence identity to a MAGE-A4 polypeptide.

In certain embodiments, the non-MAGE-A4 peptide-MHC shares about 80% sequence identity with a MAGE-A4 polypeptide.

In certain embodiments, the antigen binding protein is isolated (i.e., the antigen binding protein is not associated with or bound to the surface of a cell, such as a T cell). 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 specific for the MAGE-A4 peptide amino acid sequence as set forth in SEQ ID NO. 3 (GVY DGREHTV).

In certain embodiments, the MAGE-A4 peptide is complexed with an HLA-A2 polypeptide.

In certain embodiments, the HLA-A2 polypeptide comprises the amino acid sequence shown in SEQ ID NO. 1.

In certain embodiments, the antigen binding protein lacks detectable binding affinity for MAGE-A4 peptides comprising one or more mutations (e.g., substitutions, deletions, and/or insertions) in the amino acid sequence set forth in SEQ ID NO. 3 (G VYDGREHTV).

In certain embodiments, the antigen binding protein lacks detectable binding affinity for MAGE-A4 peptides comprising one, two, three, four, or five mutations (e.g., substitutions, deletions, and/or insertions) in the amino acid sequence set forth in SEQ ID NO. 3 (G VYDGREHTV).

In certain embodiments, the antigen binding protein lacks detectable binding affinity to an M AGE-A4 peptide comprising the amino acid sequence set forth in one or more of SEQ ID NO:394 (GLADGRTHTV), SEQ ID NO:395 (GLYDGPVHEV) and SEQ ID NO:396 (GVFDGLHTV).

In certain embodiments, the antigen binding protein has a binding affinity of about 10 for a MAGE-A4 peptide comprising one or more mutations (e.g., substitutions, deletions, and/or insertions) of the amino acid sequence set forth in SEQ ID NO. 3 (G VYDGREHTV) ^-6 M or less (e.g., about 10 ^-6 M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^-1 M)。

In certain embodiments, the antigen binding protein has a binding affinity of about 10 for MAGE-A4 peptide comprising one, two, three, four, or five mutations (e.g., substitutions, deletions, and/or insertions) of the amino acid sequence set forth in SEQ ID NO 3 (G VYDGREHTV) ^-6 M or less (e.g., about 10 ^-6 M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^-1 M)。

In certain embodiments, the antigen binding protein pair comprises an amino group as set forth in one or more of SEQ ID NO 394 (GLADGRTHTV), SEQ ID NO 395 (GLYDGPVHEV), and SEQ ID NO 396 (GVFDGLHTV)The binding affinity of the MAG E-A4 peptide of the acid sequence was about 10 ^-6 M or less (e.g., about 10 ^-6 M、10 ^-5 M、10 ^-4 M、10 ^-3 M、10 ^-2 M or 10 ^- ¹ M)。

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0848 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0848 of table 6; (b) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0849 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0849 of table 6; (c) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0850 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0850 of table 6; (d) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0851 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0851 of table 6; (e) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0852 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0852 of table 6; (f) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as recited in M0853 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as recited in M0853 of table 6; (g) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0854 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0854 of table 6; (h) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0855 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0855 of table 6; (i) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0856 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0856 of table 6; (j) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0857 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0857 of table 6; (k) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0858 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0858 of table 6; (l) An antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0859 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0859 of table 6; (M) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0860 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0860 of table 6; (n) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0861 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0861 of table 6; (o) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0862 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0862 of table 6; (p) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence shown in M0863 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0863 of table 6; (q) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0864 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0864 of table 6; (r) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0865 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0865 of table 6; or(s) an antibody heavy chain Variable (VH) domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0866 of table 6, and an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence, an LCDR2 amino acid sequence, and an LCDR3 amino acid sequence as shown in M0866 of table 6.

In certain embodiments, the antigen binding proteins of the present disclosure have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence similarity or identity to any of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 amino acid sequences as shown in any of M0848 through M0866 of table 6.

In certain embodiments, the antigen binding protein comprises: (a) An antibody heavy chain Variable (VH) domain as shown in M0848 of table 6 and an antibody light chain Variable (VL) domain as shown in M0848 of table 6; (b) An antibody heavy chain Variable (VH) domain as shown in M0849 of table 6 and an antibody light chain Variable (VL) domain as shown in M0849 of table 6; (c) An antibody heavy chain Variable (VH) domain as shown in M0850 of table 6 and an antibody light chain Variable (VL) domain as shown in M0850 of table 6; (d) An antibody heavy chain Variable (VH) domain as shown in M0851 of table 6 and an antibody light chain Variable (VL) domain as shown in M0851 of table 6; (e) An antibody heavy chain Variable (VH) domain as shown in M0852 of table 6 and an antibody light chain Variable (VL) domain as shown in M0852 of table 6; (f) An antibody heavy chain Variable (VH) domain as shown in M0853 of table 6 and an antibody light chain Variable (VL) domain as shown in M0853 of table 6; (g) An antibody heavy chain Variable (VH) domain as shown in M0854 of table 6 and an antibody light chain Variable (VL) domain as shown in M0854 of table 6; (h) An antibody heavy chain Variable (VH) domain as shown in M0855 of table 6 and an antibody light chain Variable (VL) domain as shown in M0855 of table 6; (i) An antibody heavy chain Variable (VH) domain as shown in M0856 of table 6 and an antibody light chain Variable (VL) domain as shown in M0856 of table 6; (j) An antibody heavy chain Variable (VH) domain as shown in M0857 of table 6 and an antibody light chain Variable (VL) domain as shown in M0857 of table 6; (k) An antibody heavy chain Variable (VH) domain as shown in M0858 of table 6 and an antibody light chain Variable (VL) domain as shown in M0858 of table 6; (l) An antibody heavy chain Variable (VH) domain as shown in M0859 of table 6 and an antibody light chain Variable (VL) domain as shown in M0859 of table 6; (M) an antibody heavy chain Variable (VH) domain as shown in M0860 of table 6 and an antibody light chain Variable (VL) domain as shown in M0860 of table 6; (n) an antibody heavy chain Variable (VH) domain as shown in M0861 of table 6 and an antibody light chain Variable (VL) domain as shown in M0861 of table 6; (o) an antibody heavy chain Variable (VH) domain as shown in M0862 of table 6 and an antibody light chain Variable (VL) domain as shown in M0862 of table 6; (p) an antibody heavy chain Variable (VH) domain as shown in M0863 of table 6 and an antibody light chain Variable (VL) domain as shown in M0863 of table 6; (q) an antibody heavy chain Variable (VH) domain as shown in M0864 of table 6 and an antibody light chain Variable (VL) domain as shown in M0864 of table 6; (r) an antibody heavy chain Variable (VH) domain as shown in M0865 of table 6 and an antibody light chain Variable (VL) domain as shown in M0865 of table 6; or(s) an antibody heavy chain Variable (VH) domain as shown in M0866 of table 6 and an antibody light chain Variable (VL) domain as shown in M0866 of table 6.

In certain embodiments, the antigen binding proteins of the present disclosure have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence similarity or identity to any of the VH or VL amino acid sequences as set forth in any one of M0848 to M0866 of table 6.

The selected antigen binding proteins of the present disclosure have an aberrant binding affinity for MAGE-A4 pMHC of about 5nM or less (e.g., about 5nM, about 4.5nM, about 4nM, about 3.5nM, about 3nM, about 2.5nM, about 2nM, about 1.5nM, about 1nM, about 0.5nM, about 0.1nM, about 0.05nM, about 0.01nM or less). In particular embodiments, the antigen binding protein has a binding affinity of 1nM or less (e.g., about 1nM, about 0.5nM, about 0.1nM, about 0.05nM, about 0.01nM or less). The antigen binding proteins comprise a set of six CDR sequences having consensus HCDR2, HCDR3 and LCDR3 amino acid sequences and identical HCDR1, LCDR1 and LCDR2 amino acid sequences.

In certain embodiments, the antigen binding protein comprises: (a) an antibody heavy chain variable (V H) domain comprising: HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469) ；IVSSGGTTYYAX ₁ X ₂ X ₃ The HCDR2 amino acid sequence of KG (SEQ ID NO: 881), wherein X ₁ Corresponding to amino acids S or D, X ₂ Corresponds to amino acids W or S, and X ₃ Corresponding to amino acid a or V; and DLYYGPX ₄ TX ₅ YX ₆ X ₇ X ₈ HCDR3 amino acid sequence of NL (SEQ ID NO: 882), wherein X ₄ Corresponding to amino acid T, N or S, X ₅ Corresponding to amino acid D or absence, X ₆ Corresponding to amino acids S or F, X ₇ Corresponds to amino acids A or V, and X ₈ Corresponding to amino acid F or A; and (b) an antibody light chain variable (V L) domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473), and A TX ₉ X ₁₀ X ₁₁ SGSNFQX ₁₂ (SEQ ID NO: 883) LCDR3 amino acid sequence, wherein X ₉ Corresponding to amino acids S or R, X ₁₀ Corresponding to amino acids D or P, X ₁₁ Corresponds to amino acid G, S or F, and X ₁₂ Corresponding to amino acid L or A.

In another aspect, the present disclosure provides an antigen binding protein that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising: (a) Antibody heavy chain Variable (VH) junctionsA domain comprising: the HCD R1 amino acid sequence of SNYAMS (SEQ ID NO: 469); IVSSGGTTYYAX ₁ X ₂ X ₃ hCDR2 amino acid sequence of KG (SEQ ID NO: 881), wherein X ₁ Corresponding to amino acids S or D, X ₂ Corresponds to amino acids W or S, and X ₃ Corresponding to amino acid a or V; and DLYYGPX ₄ TX ₅ YX ₆ X ₇ X ₈ HCDR3 amino acid sequence of NL (SEQ ID NO: 882), wherein X ₄ Corresponding to amino acid T, N or S, X ₅ Corresponding to amino acid D or absence, X ₆ Corresponding to amino acids S or F, X ₇ Corresponds to amino acids A or V, and X ₈ Corresponding to amino acid F or A; and (b) an antibody light chain Variable (VL) domain comprising: TADTLSRSYAS (SEQ ID NO: 472), LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and ATX ₉ X ₁₀ X ₁₁ SGSNFQX ₁₂ (SEQ ID NO: 883) LCDR3 amino acid sequence, wherein X ₉ Corresponding to amino acids S or R, X ₁₀ Corresponding to amino acids D or P, X ₁₁ Corresponds to amino acid G, S or F, and X ₁₂ Corresponding to amino acid L or A.

In another aspect, the present disclosure provides an antigen binding protein that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising: (a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 624), and the HCDR3 amino acid sequence of DLYYGPTTYSAFNL (SEQ ID NO: 698); and (b) an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and an LCDR3 amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 846).

In certain embodiments, the antigen binding protein comprises one or more of the following features: (i) The antigen binding protein has a binding affinity of about 10 for the target MAGE-A4pMHC ^-9 M to about 10 ^-14 M; (ii) The antigen binding protein has a binding affinity of about 10 for non-MAGE-A4 pMHC and/or peptide-free MHC ^-6 M or weaker; (iii) The antigen binding protein has a binding affinity of about 10 for non-target MAGE-A4pMHC ^-6 M or weaker; and (iv) the antigen binding protein has a binding affinity for the target MAGE-A4pMHC of about 10 ^-9 M to about 10 ^-14 M and has a binding affinity of about 10 for MAGE-A4 peptide, HLA polypeptide and beta-2-microglobulin polypeptide alone ^-6 M or weaker.

In certain embodiments, the VH domain and VL domain are attached with an amino acid linker.

In certain embodiments, the amino acid linker comprises (GGGGS) n, wherein n is an integer from 1 to 5.

In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGSGGGG SGGGGS, ggggsggggsggggsggggggs or GGGGSGGGGSGGG GSGGGGAS.

In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequence as shown in any one of M0848 to M0866 of table 6.

In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0848 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0849 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0850 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0851 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0852 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0853 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0854 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0855 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0856 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0857 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0858 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0859 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0860 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0861 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0862 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0863 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0864 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0865 from table 6. In another aspect, the present disclosure provides a human or humanized antigen binding protein comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of M0866 from table 6.

In certain embodiments, the human or humanized antigen binding protein comprises a substitution in the LCDR2 sequence of any of M0700-M0866 disclosed in table 6, particularly any of M0709, M0739, M0742, M0743, M0747 or M0763. In some embodiments thereof, the antigen binding protein further comprises a substitution in the corresponding HCDR1, HCDR2 and/or HCDR3 sequence.

In certain embodiments, the human or humanized antigen binding protein comprises a substitution in the LCDR3 sequence of any of M0700-M0866 disclosed in table 6, particularly any of M0709, M0739, M0742, M0743, M0747 or M0763. In some embodiments thereof, the antigen binding protein further comprises a substitution in the corresponding HCDR1, HCDR2 and/or HCDR3 sequence.

In certain embodiments, the human or humanized antigen binding protein comprises a substitution in the HCDR1 sequence of any of M0700-M0866 disclosed in table 6, particularly any of M0709, M0739, M0742, M0743, M0747 or M0763. In some embodiments thereof, the antigen binding protein further comprises a substitution in the corresponding LCDR1, LCDR2 and/or LCDR3 sequence.

In certain embodiments, the human or humanized antigen binding protein comprises a substitution in the HCDR2 sequence of any of M0700-M0866 disclosed in table 6, particularly any of M0709, M0739, M0742, M0743, M0747 or M0763. In some embodiments thereof, the antigen binding protein further comprises a substitution in the corresponding LCDR1, LCDR2 and/or LCDR3 sequence.

In certain embodiments, the human or humanized antigen binding protein comprises a substitution in the HCDR3 sequence of any of M0700-M0866 disclosed in table 6, particularly any of M0709, M0739, M0742, M0743, M0747 or M0763. In some embodiments thereof, the antigen binding protein further comprises a substitution in the corresponding LCDR1, LCDR2 and/or LCDR3 sequence.

In certain embodiments, the human or humanized antigen binding protein is a variant of the sequences disclosed herein and comprises a substitution in LCDR3 and/or HCDR3 of any of M0700-M0866 disclosed in table 6, particularly any of M0709, M0739, M0742, M0743, M0747 or M0763.

In certain embodiments, the human or humanized antigen binding protein comprises substitutions in the LCDR1 and LCDR3 sequences of any of M0700-M0866, particularly any of M0709, M0739, M0742, M0743, M0747 or M0763, disclosed in table 6. In some embodiments thereof, the antigen binding protein further comprises a substitution in the corresponding HCDR1, HCDR2 and/or HCDR3 sequence.

In certain embodiments, the human or humanized antigen binding protein comprises substitutions in the LCDR2 and LCDR3 sequences of any of M0700-M0866, particularly any of M0709, M0739, M0742, M0743, M0747 or M0763, disclosed in table 6. In some embodiments, the antigen binding protein further comprises a substitution in the corresponding HCDR1, HCDR2 and/or HCDR3 sequence.

In certain embodiments, the human or humanized antigen binding protein comprises substitutions in the LCDR1, LCDR2 and LCDR3 sequences of any of M0700-M0866 disclosed in table 6, particularly any of M0709, M0739, M0742, M0743, M0747 or M0763. In some embodiments, the antigen binding protein further comprises a substitution in the corresponding HCDR1, HCDR2 and/or HCDR3 sequence.

In certain embodiments, the human or humanized antigen binding protein comprises substitutions in the HCDR1 and HCDR3 sequences of any of M0700-M0866 disclosed in table 6, particularly any of M0709, M0739, M0742, M0743, M0747 or M0763. In some embodiments, the antigen binding protein further comprises a substitution in the corresponding LCDR1, LCDR2, and/or LCDR3 sequence.

In certain embodiments, the human or humanized antigen binding protein comprises substitutions in the HCDR1 and HCDR2 sequences of any of M0700-M0866 disclosed in table 6, particularly any of M0709, M0739, M0742, M0743, M0747 or M0763. In some embodiments, the antigen binding protein further comprises a substitution in the corresponding LCDR1, LCDR2, and/or LCDR3 sequence.

In certain embodiments, the human or humanized antigen binding protein comprises substitutions in the HCDR2 and HCDR3 sequences of any of M0700-M0866 disclosed in table 6, particularly any of M0709, M0739, M0742, M0743, M0747 or M0763. In some embodiments, the antigen binding protein further comprises a substitution in the corresponding LCDR1, LCDR2, and/or LCDR3 sequence.

In certain embodiments, the human or humanized antigen binding protein comprises substitutions in the HCDR1, HCDR2 and HCDR3 sequences of any of M0700-M0866 disclosed in table 6, particularly any of M0709, M0739, M0742, M0743, M0747 or M0763. In some embodiments, the antigen binding protein further comprises a substitution in the corresponding LCDR1, LCDR2, and/or LCDR3 sequence.

For the avoidance of doubt, the above combinations refer to CDRs of matched VL-VH pairs of the antigen binding proteins depicted in table 6.

In certain embodiments, such variant antigen binding proteins retain specific binding to their targets (e.g., GVYDGREHTV) and/or compete with the antigen binding proteins disclosed herein for binding to their targets. Variants (i.e., mutant sequences) may be tested for chemical, biological, biophysical, and/or biochemical properties by conventional methods. In certain embodiments, amino acid substitutions do not substantially alter the functional and/or structural characteristics of the parent sequence. Thus, the binding properties of antigen binding proteins comprising such conservative substitutions are at least substantially unchanged. In certain embodiments, the amino acid substitutions do not substantially modify or disrupt the secondary structure of the parent sequence.

In certain embodiments, the variant antigen binding protein retains a binding affinity of about 10-9M to about 10-14M for the target MAGE-A4pMHC and/or about 10-6M or less for non-MAGE-A4 pMHC and/or peptide-free MHC; and/or a binding affinity for non-target MAGE-A4pMHC of about 10-6M or less; and/or about 10-9M to about 10-14M for the target MAGE-A4pMHC and about 10-6M or less for the MAGE-A4 peptide, HLA polypeptide, and beta-2-microglobulin polypeptide alone.

In another aspect, the disclosure provides a single domain antibody (e.g., sdAb, sdFv, nanobody, V-Nar, or VHH) that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC). Single domain antibodies, such as VHH, are smaller than traditional antibodies, which may allow them to penetrate better into the tumor microenvironment. Furthermore, the smaller binding region of single domain antibodies may confer excellent binding affinity and specificity for peptide-bound MHC.

In certain embodiments, the single domain antibody has a binding affinity of about 10 for the target MAGE-A4pMHC ^-9 M to about 10 ^-14 M。

In certain embodiments, the single domain antibody has a binding affinity of about 10 for non-MAGE-A4 peptide-MHC and/or peptide-free MHC ^-6 M or weaker.

In certain embodiments, the single domain antibody has a binding affinity of about 10 for non-target MAGE-A4 pMHC ^-6 M or weaker.

In certain embodiments, the antigen binding protein (e.g., single domain antibody) comprises: (a) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0734 of table 8; (b) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0735 of table 8; (c) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0736 of table 8; (d) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0737 of table 8; (e) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0738 of table 8; (f) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0739 of table 8; (g) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0740 of table 8; (h) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0741 of table 8; (i) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0742 of table 8; (j) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0743 of table 8; (k) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0744 of table 8; (l) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0745 of table 8; (M) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0746 of table 8; (n) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0747 of table 8; (o) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0748 of table 8; (p) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0749 of table 8; (q) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0750 of table 8; (r) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0751 of table 8; or(s) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0752 of table 8.

In certain embodiments, the antigen binding proteins of the present disclosure have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence similarity or identity to any of the HCDR1, HCDR2 or HCDR3 amino acid sequences as shown in any of M0734 to M0752 of table 8.

In certain embodiments, the antigen binding proteins of the present disclosure have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence similarity or identity to any of the VHH amino acid sequences as set forth in any one of M0734 to M0752 of table 8.

In certain embodiments, the antigen binding protein has a binding affinity for MAGE-A4 pMHC of at least about 10 ^-9 M (e.g., about 10 ^-9 M, about 10 ^-10 M, about 10 ^-11 M, about 10 ^-12 M, about 10 ^-13 M, about 10 ^-14 M)。

In certain embodiments, the antigen binding protein lacks detectable binding affinity for non-MAGE-A4 pMHC (e.g., MHC complexed with a peptide not derived from a MAGE-A4 protein). An antigen binding protein lacking detectable binding affinity is about the same binding affinity as the negative control. The negative control may be a binding affinity measurement with the antigen binding protein and no other antigen.

In certain embodiments, the antigen binding protein lacks detectable binding affinity for peptide-free MHC (e.g., MHC that is not complexed with a peptide of any origin).

In certain embodiments, the antigen binding protein lacks detectable binding affinity for non-target MAGE-A4 pMHC (e.g., M HC complexed with a MAGE-A4 peptide other than the target MAGE-A4 peptide, such as the amino acid sequence of the target MAGE-A4 peptide set forth in SEQ ID NO:3 (GVYDGRE HTV)).

In certain embodiments, the antigen binding protein lacks detectable binding affinity for a MAGE-A4 peptide alone (e.g., a MAGE-A4 peptide that is not complexed to MHC).

In certain embodiments, the antigen binding protein specifically binds MAGE-A4 pMHC on the cell surface. In certain embodiments, the cell is a T2 cell that has been pulsed with a target MAGE-A4 peptide.

In certain embodiments, the antigen binding protein lacks detectable binding affinity for non-MAGE-A4 pMHC on the cell surface. In certain embodiments, the cell is a T2 cell that has been pulsed with a target MAGE-A4 peptide.

In another aspect, the present disclosure provides a bispecific antigen binding protein comprising a first antigen binding domain comprising an antigen binding protein as described above and a second antigen binding domain specific for a cell surface protein of an immune cell.

In certain embodiments, the immune cell is a T cell.

In certain embodiments, the cell surface protein of the immune cell is CD3.

In another aspect, the present disclosure provides a method of making the above antigen binding protein or the above bispecific antigen binding protein, comprising the steps of:

(i) Incubating the above-described host cell under conditions allowing expression of the antigen binding protein or the bispecific antigen binding protein;

(ii) Recovering the antigen binding protein or bispecific antigen binding protein; and optionally

(iii) Further purifying and/or modifying and/or formulating said antigen binding protein or bispecific antigen binding protein.

MAGE-A4 peptide-MHC

The antigen binding proteins described herein have binding specificity for MAGE-A4 peptide-MHC.

The target peptide may be presented on 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 their respective subtypes) or an MHC class II complex (such as serotypes HLA-DP, HLA-DQ, HLA-DR, DM or DO or their respective subtypes). Each serotype comprises a different subtype. In one embodiment, the antigen binding protein targets peptides that bind to the HLA-A2-MHC complex, also referred to as HLA-A 02, in particular HLA-A 02:01 comprising the extracellular domain of SEQ ID No. 1.

Expression of antigen binding proteins

In one aspect, polynucleotides or nucleic acids encoding the antigen binding proteins disclosed herein are provided. Also provided are methods of making antigen binding proteins comprising expressing these polynucleotides.

Polynucleotides encoding the antigen binding proteins disclosed herein are typically inserted into expression vectors for introduction into host cells that can be used to produce the desired amount of antigen binding protein. Thus, in certain aspects, the invention provides expression vectors comprising the polynucleotides disclosed herein and host cells comprising these vectors and polynucleotides.

The term "vector" or "expression vector" is used herein to mean a vector that is used according to the present invention as a vehicle for introducing and expressing a desired gene in a cell. Such vectors may be readily selected from the group consisting of plasmids, phages, viruses and retroviruses, as known to those skilled in the art. In general, vectors compatible with the present invention will contain a selectable marker, suitable restriction sites that facilitate cloning and access of the desired gene into eukaryotic or prokaryotic cells and/or the ability to replicate in eukaryotic or prokaryotic cells.

Many expression vector systems are useful for the purposes of the present invention. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (e.g., RSV, MMTV, MOMLV, etc.), or SV40 virus. Other vectors involve the use of polycistronic systems with internal ribosome binding sites. Alternatively, cells that have DNA integrated into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The markers may provide prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene may be linked directly to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include signal sequences, splicing signals, transcriptional promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into expression vectors along with the heavy and light chain constant region genes synthesized as discussed above (e.g., human constant region genes).

In other embodiments, the antigen binding protein may be expressed using a polycistronic construct. In such expression systems, multiple gene products of interest, such as heavy and light chains of antibodies, can be produced from a single polycistronic construct. These systems advantageously use Internal Ribosome Entry Sites (IRES) to provide relatively high levels of polypeptide in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. patent No. 6,193,980, which is incorporated herein by reference in its entirety for all purposes. Those of skill in the art will appreciate that such expression systems can be used to effectively produce the full range of polypeptides disclosed herein.

More generally, once the vector or DNA sequence encoding the antibody or fragment thereof is prepared, the expression vector may be introduced into an appropriate host cell. That is, the host cell may be transformed. The plasmid may be introduced into the host cell by various techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion of the envelope DNA, microinjection, and infection with whole virus. See, ridgway, a.a.g. "Mammalian Expression Vectors" chapter 24.2, pages 470-472, vectors, rodriguez and Denhardt editions (Butterworths, boston, mass.1988). The plasmid may be introduced into the host by electroporation. The transformed cells are grown under conditions suitable for the production of light and heavy chains and heavy chain and/or light chain protein synthesis is determined. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorter analysis (FACS), immunohistochemistry, and the like.

As used herein, the term "transformation" shall be used in a broad sense to refer to the introduction of DNA into a recipient host cell, altering the genotype and thus resulting in an alteration of the recipient cell.

Likewise, "host cell" refers to a cell that has been transformed with a vector constructed using recombinant DNA techniques and encoding at least one heterologous gene. The terms "cell" and "cell culture" are used interchangeably in describing a method of isolating a polypeptide from a recombinant host, unless otherwise specifically indicated, to denote the source of the antibody. In other words, recovering the polypeptide from "cells" may mean recovering from intact cells that have been centrifuged down, or recovering from cell cultures containing both medium and suspended cells.

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 that is most suitable for expressing the desired gene product therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary cell line, DHFR-), HELA (human cervical cancer), CV-1 (monkey kidney cell line), COS (CV-1 and derivatives of SV 40T antigen), R1610 (Chinese hamster fibroblasts), BALBC/3T3 (mouse fibroblasts), HAK (hamster kidney cell line), SP2/O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), 293 (human kidney), and the like. In one embodiment, the cell line provides altered glycosylation of antibodies expressed thereby, e.g., afucosylation (e.g., PER). (Crucell) or FUT8 knockoutCHO cell line [ ]Cells) (Biowa, princeton, n.j.). Host cell lines are generally available from commercial services such as the American tissue culture Collection, or published literature.

In vitro production allows scaling 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 homogeneous suspension culture, such as in an airlift reactor or a continuously stirred reactor, or immobilized or embedded cell culture, such as in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. The polypeptide solution may be purified by conventional chromatography, such as gel filtration, ion exchange chromatography, DEAE-cellulose chromatography and/or (immuno) affinity chromatography, if necessary and/or desired.

Genes encoding the antigen binding proteins of the invention of the features may also be expressed in non-mammalian cells such as bacterial or yeast or plant cells. In this regard, it should be understood that various single-cell non-mammalian microorganisms such as bacteria may also be transformed, i.e., those capable of growing in culture or fermentation. Bacteria susceptible to transformation include members of the enterobacteriaceae family (enterobacteriaceae), such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); bacillus (bacillus) such as bacillus subtilis (Bacillus subtilis); pneumococci (pneumococci); streptococcus (Streptococcus) and haemophilus influenzae (Haemophilus influenzae). It is also understood that proteins may become part of inclusion bodies when expressed in bacteria. The proteins must be isolated, purified, and then assembled into functional molecules.

In addition to prokaryotes, eukaryotic microbes may also be used. Saccharomyces cerevisiae (Sacchar omyces cerevisiae) or Saccharomyces cerevisiae are most commonly used among eukaryotic microorganisms, but many other strains are also commonly available. For expression in Saccharomyces (Saccharomyces), the plasmid YRp7 is generally used, for example (Stinchcomb et al, nature,282:39 (1979); kingsman et al, gene,7:141 (1979); tschemper et al, gene,10:157 (1980)). This plasmid already contains the TRP1 gene which provides a selection marker for a yeast mutant which lacks the ability to grow in tryptophan, for example ATCC 44076 or PEP4-1 (Jones, genetics,85:12 (1977)). The presence of trp1 lesions, which are characteristic of the yeast host cell genome, provides an effective environment for detection of transformation by growth in the absence of tryptophan.

Engineering and optimization of antigen binding proteins

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

As used herein, the term "functional property" is a property of an antigen binding protein for which improvement (e.g., relative to conventional antigen binding proteins, such as antibodies) is desirable and/or advantageous to one skilled in the art, e.g., in order to improve the manufacturing properties or therapeutic efficacy of the antigen binding protein. In one embodiment, the functional property is stability (e.g., thermal stability). In another embodiment, the functional property is solubility (e.g., under cellular conditions). In yet another embodiment, the functional characteristic is aggregate behavior. In another embodiment, the functional property is protein expression (e.g., in a prokaryotic cell). In yet another embodiment, the functional property is refolding behavior after inclusion body lysis during manufacture. In certain embodiments, the functional property is not an improvement in antigen binding affinity. In another embodiment, the improvement in one or more functional properties has no substantial effect on the binding affinity of the antigen binding protein.

In certain embodiments, the antigen binding proteins of the present disclosure are scFv and are optimized by identifying preferred amino acid residues to be substituted, deleted and/or added at amino acid positions of interest in the antigen binding protein (e.g., by comparing a database of scFv sequences having at least one desired property (e.g., as selected by Quality Control (QC) assays) with a database of mature antibody sequences (e.g., kabat database). Thus, the present disclosure also provides "enrichment/exclusion" methods for selecting particular amino acid residues. In addition, the present disclosure provides methods of engineering antigen binding proteins (e.g., scFv) by mutating specific framework amino acid positions identified using the "functional consensus" methods described herein. In certain embodiments, the framework amino acid positions are mutated by substituting the existing amino acid residues with residues found to be "enriched" residues using the "enrichment/exclusion" assay methods described herein. In one aspect, the present disclosure provides a method of identifying a mutated amino acid position in a single chain antibody (scFv), the scFv having VH and VL amino acid sequences, the method comprising: a) Inputting the scFv VH, VL, or VH and VL amino acid sequences into a database comprising a plurality of antibody VH, VL, or VH and VL amino acid sequences such that the scFv VH, VL, or VH and VL amino acid sequences are aligned with the antibody VH, VL, or VH and VL amino acid sequences of the database; b) Comparing the amino acid positions within the scFv VH or VL amino acid sequences to corresponding positions in the antibody VH or VL amino acid sequences of the database; c) Determining whether an amino acid position within an scFv VH or VL amino acid sequence is occupied by an amino acid residue conserved at a corresponding position within an antibody VH or VL amino acid sequence of the database; and d) identifying an amino acid position in the scFv VH or VL amino acid sequence as an amino acid position for mutation when 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 of the database. scFv optimisation is described in more detail in WO2008110348, WO2009000099, WO2009000098 and WO2009155725, all of which are incorporated herein by reference.

In certain embodiments, the antigen binding protein comprises an Fc domain modified such that it does not induce a cytotoxic immune response and/or does not activate complement. For example, one or more substitutions may be introduced into the Fc domain to inactivate its ADCC/ADCP or CDC effector function. Such antigen binding proteins have the advantage of increased half-life compared to antibody fragments having a molecular weight below 60kDa, without mediating a cytotoxic immune response.

Chemical and/or biological modification

In one aspect, the antigen binding protein is chemically and/or biologically modified. For example, the antigen binding protein may be glycosylated, phosphorylated, hydroxylated, pegylated, HES-glycosylated, PAS-glycosylated, sulfated, labeled with dyes and/or radioisotopes, conjugated with enzymes and/or toxins, and/or albumin fusion techniques. Likewise, any of the nucleic acid sequences, plasmids or vectors and/or host cells described herein may be modified accordingly.

Such modifications may be made, for example, to optimize pharmacodynamics, its water solubility, or reduce its side effects. For example, pegylation, PAS, HES, and/or fusion with serum albumin may be useful to slow renal clearance, thereby increasing the plasma half-life of the antigen binding protein. In one embodiment, modification to the antigen binding protein adds a different functionality, e.g., a detection marker for diagnosis or a toxin more effective against cancer cells.

In one embodiment, the antigen binding protein is glycosylated. Glycosylation refers to the process of attaching a carbohydrate to a protein. In biological systems, this process is carried out enzymatically in the cell as co-translation and/or post-translational modification. Proteins may also be chemically glycosylated. The carbohydrate may be N-linked to the nitrogen of the asparagine or arginine side chain; o is a hydroxy oxygen attached to a serine, threonine, tyrosine, hydroxylysine or hydroxyproline side chain; xylose, fucose, mannose and N-acetylglucosamine attached to phosphoserine are used; and/or mannose to tryptophan residues found in a particular recognition sequence. Glycosylation patterns can be controlled, for example, by selection of appropriate cell lines, media, protein engineering patterns, and processing strategies (see HOSSLER, P.Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 2009, volume 19, 9, pages 936-949). In some embodiments, the glycosylation pattern of the antigen binding proteins described herein is modified to enhance ADCC and CDC effector function.

The antigen binding proteins may be engineered to control or alter the glycosylation pattern, for example by deletion and/or addition of one or more glycosylation sites. The glycosylation sites can be created, for example, by introducing the corresponding enzyme recognition sequences into the amino acid sequence of the antigen binding protein.

In some embodiments, the antigen binding protein is pegylated. PEGylation can alter the pharmacodynamic and pharmacokinetic properties of proteins. In addition, pegylation may reduce immunogenicity by shielding the pegylated antigen binding protein from the immune system and/or by, for example, increasing the in vivo stability of the antigen binding protein, protecting it from proteolytic degradation, extending its half-life and altering its pharmacokinetics by altering its biodistribution. Typically, polyethylene glycol (PEG) of appropriate molecular weight is covalently attached to the protein. Similar effects can be achieved using PEG mimics, such as HES-or PAS-formed antigen binding proteins. HES conversion utilizes hydroxyethyl starch ("HES") derivatives. During PAS, an antigen binding protein is linked to a conformationally disordered polypeptide sequence consisting of the amino acids proline (P), alanine (A) and serine (S).

In certain embodiments, the antigen binding protein is labeled with or conjugated to a second moiety that imparts one or more helper functions to the antigen binding protein. For example, the second moiety may have additional immune effector functions, be effective in drug targeting or be available 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 indicates the presence of an antigen binding protein when detected or measured directly or indirectly by physical or chemical means. For example, the label may be detected directly by, but not limited to, light absorption, fluorescence, reflectance, light scattering, phosphorescence, or luminescence properties, by a molecule or ion detectable by its radioactivity, or by a molecule or ion detectable by its nuclear magnetic resonance or paramagnetic properties. Examples of indirect detection include light absorption or fluorescence; for example, various enzymes that cause the conversion of an appropriate substrate, such as from a non-light absorbing molecule to a light absorbing molecule, or from a non-fluorescent molecule to a fluorescent molecule. The labeled antigen binding proteins are particularly useful for in vitro and in vivo detection or diagnostic purposes. For example, antigen binding proteins labeled with a suitable radioisotope, enzyme, fluorophore, or chromophore can be detected by Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), or flow cytometry-based single-cell analysis (e.g., FACS analysis), respectively. Similarly, the nucleic acids and/or vectors disclosed herein may be labeled for detection or diagnostic purposes, e.g., using labeled fragments thereof as probes in hybridization assays.

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

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

The toxin may be non-site specific or site specific conjugated to the antigen binding protein. Non-site-specific conjugation typically involves the use of chemical linkers, such as chemical linkers with maleimide functionality, that mediate conjugation to lysine or cysteine amino acid side chains of the antibody. Site-specific conjugation may be achieved using chemical, chemo-enzymatic or enzymatic conjugation known in the art, e.g. using bifunctional linkers, bacterial transglutaminase or sortase, linkers of Pictet-Spengler chemistry allowing the formation of enzyme modified antigen binding proteins on formyl-glycine, or glycan remodelled antigen binding proteins.

Chimeric antigen receptor

In one aspect, the present disclosure provides Chimeric Antigen Receptors (CARs) and immune cells engineered to express such CARs, comprising an antigen binding protein described herein. As used herein, the term "chimeric antigen receptor" or "CAR" refers to a receptor capable of activating immune cells in response to antigen binding. The CAR is a recombinant transmembrane molecule and is advantageously expressed on immune cells. Their structure typically comprises (i) an extracellular domain (extracellular domain or antibody domain), (ii) a transmembrane domain and (iii) a cytoplasmic domain (intracellular domain or intracellular signaling domain).

The extracellular domain (i.e., antibody domain) typically comprises an scFv, although other antigen binding proteins may also be used. The spacer region connects the extracellular domain and the transmembrane domain, which in turn is connected to the intracellular domain. When the extracellular domain binds to an antigen, the receptor aggregates and activates signal transmission to the cell, resulting in the initiation of an immune response. The first generation of CARs had a simple structured intracellular domain comprising CD3- ζ. To increase activation signals, costimulatory domains are added in the second generation CARs; and third generation CARs include two or more co-stimulatory domains (Maus MV et al (2014) Blood, 123:2625-2635). The co-stimulatory domain may be selected from the group consisting of CD28, OX40 and/or 4-1 BB. In addition to CD3- ζ, other Fc receptors have been explored that contain domains of ITAM, including the IgE- γ domain.

Suitable immune cells 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 may be Cytotoxic T Lymphocytes (CTLs), regulatory T lymphocytes, inflammatory T lymphocytes or helper T lymphocytes or gamma-delta T cells. T cells may be cd4+ or cd8+ or a mixed population of cd4+ and cd8+ cells.

In one aspect, the present disclosure provides a Chimeric Antigen Receptor (CAR) that specifically recognizes a peptide-MHC, comprising: i) Antigen binding proteins specific for MAGE-A4 peptide-MHC; ii) a transmembrane domain; and iii) an intracellular signaling domain.

In certain embodiments, the transmembrane domain is selected from the group consisting of the artificial hydrophobic sequence and transmembrane domain of a type I transmembrane protein, the α, β or ζ chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD 154.

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 immune receptor tyrosine based activation motif (ITAM), TCR ζ, fcrγ, fcrβ, CD3 γ, CD3 δ, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66 d.

The antibody domain may be any of the antigen binding proteins described 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-CDRL1-LFR2-CDRL2-LFR3-CDRL3-LFR 4. In certain embodiments, the antibody domain comprises an antibody variable heavy chain domain (VH) comprising an amino acid sequence represented by the formula HFR1-CDRH1-HFR2-CDRH2-HFR3-CDRH3-HFR 4. In certain embodiments, the antibody domain comprises an scFv as described herein.

Methods of administering antigen binding proteins

Methods of preparing and administering the antigen binding proteins of the present disclosure, the nucleic acids described herein, the vectors described herein, the host cells described herein (particularly CAR-bearing immune cells) or the compositions described herein to a subject are well known or readily determinable by those of skill in the art. The route of administration of the antigen binding proteins of the present disclosure may be, for example, oral, parenteral, by inhalation or topical. The term parenteral as used herein includes intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The term intraocular as used herein includes, but is not limited to, subconjunctival, intravitreal, retrobulbar, or intracameral. The term topical as used herein includes, but is not limited to, administration as liquid or solution eye drops, emulsions (e.g., oil-in-water emulsions), suspensions, and ointments.

While all of these administration forms are contemplated as being within the scope of the present disclosure, the administration form will be a solution for injection. In general, suitable pharmaceutical compositions for injection may comprise buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbate), optionally stabilizers (e.g., human albumin), and the like. However, in other methods compatible with the teachings herein, the modified antibodies may be delivered directly to the site of the adverse cell population, thereby increasing exposure of the diseased tissue to the therapeutic agent.

The effective dosage of the compositions of the present disclosure for treating a related condition varies depending on a number of different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is a human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Conventional methods known to those skilled in the art can be used to titrate the therapeutic dose to optimize safety and efficacy.

As previously discussed, the antigen binding proteins of the present disclosure, conjugates or recombinants thereof may be administered in a pharmaceutically effective amount for the in vivo treatment of a mammalian disorder. In this regard, it should be understood that the disclosed antigen binding proteins will be formulated to facilitate administration and promote stability of the active agent.

Pharmaceutical compositions according to the present disclosure generally include a pharmaceutically acceptable non-toxic, sterile carrier, such as physiological saline, non-toxic buffers, preservatives, and the like. For the purposes of this application, a pharmaceutically effective amount of an antigen binding protein shall be taken to mean an amount sufficient to achieve effective binding to an antigen and to achieve a benefit (e.g., to ameliorate a symptom of a disease or disorder or to detect a substance or cell). In the case of tumor cells, the antigen binding proteins will typically be capable of interacting with selected immunoreactive antigens on neoplastic cells or immunoreactive cells and increasing the death of those cells. Of course, the pharmaceutical compositions of the present disclosure may be administered in single or multiple doses to provide a pharmaceutically effective amount of the modified binding polypeptide.

Consistent with the scope of the present disclosure, the antigen binding proteins of the present disclosure may be administered to a human or other animal in an amount sufficient to produce a therapeutic or prophylactic effect according to the foregoing 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 according to known techniques. Those skilled in the art will recognize that the form and character of a pharmaceutically acceptable carrier or diluent is determined by the amount of active ingredient in combination therewith, the route of administration, and other well known variables. Those of skill in the art will also appreciate that mixtures comprising one or more of the antigen binding proteins described in the present disclosure may prove particularly effective. Similarly, a nucleic acid described herein, a vector described herein, a host cell described herein (particularly a CAR-bearing immune cell), or a composition described herein can be administered to a human or other animal in an amount sufficient to produce a therapeutic or prophylactic effect according to the methods of treatment described above.

As used herein, "efficacy" or "in vivo efficacy" refers to a response to a pharmaceutical composition therapy of the present disclosure using, for example, standardized response criteria (such as standard ophthalmic response criteria). The success or in vivo efficacy of a therapy 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 its desired effect. In vivo efficacy can be monitored by standard methods established for specific diseases. In addition, various disease-specific clinical chemistry parameters and other established standard methods can be used.

In some embodiments, the compounds and cells described herein are administered in combination with one or more different pharmaceutical compounds. In general, the therapeutic uses of the compounds and cells described herein may be combined with one or more therapies selected from antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy, radiation therapy, or vaccine therapy.

Methods of treating MAGE-A4 mediated diseases and conditions

In one aspect, the antigen binding proteins, nucleic acids, vectors, or host cells (particularly CAR-expressing immune cells) or vectors described above are useful as medicaments. Typically, such a drug comprises a therapeutically effective amount of a molecule or cell as provided herein. Accordingly, the corresponding molecules or host cells may be used in the manufacture of a medicament useful in the treatment of one or more disorders, particularly MAGE-A4 associated disorders.

In one aspect, methods of treating MAGE-A4 associated or mediated disorders are provided. The method comprises the step of administering a pharmaceutically effective amount of a molecule or host cell as described herein, in particular an antigen binding protein or host cell, to a subject in need thereof. In one embodiment, the above pharmaceutical composition is administered to a subject, the pharmaceutical composition comprising a pharmaceutically effective amount of an antigen binding protein, nucleic acid, vector or host cell. The above-mentioned drugs are administered to a subject.

In another aspect, the present disclosure provides a method of treating a MAGE-A4 pMHC expressing cancer in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of the antigen binding protein described above or the pharmaceutical composition described above.

The subject in need of treatment may be a human or a non-human animal. Typically, the subject is a mammal, such as a mouse, rat, rabbit, hamster, dog, cat, monkey, ape, goat, sheep, horse, chicken, guinea pig or pig. In typical embodiments, the subject is diagnosed with a MAGE-A4 disorder or such disorder can be obtained. In the case of animal models, the animals may be genetically engineered to develop MAGE-A4 associated disorders. In animal models, animals may also be genetically engineered in such a way as to display characteristics of MAGE-A4 associated diseases.

In certain embodiments, the MAGE-A4 mediated disease or disorder is selected from the group consisting of melanoma, head and neck cancer, ovarian cancer, testicular cancer, T-cell leukemia/lymphoma (e.g., ATLL), bladder cancer, and esophageal cancer. The invention also relates to antigen binding proteins as disclosed herein for use in a method of treating a MAGE-A4 mediated disease or disorder, particularly cancer, in a subject. All technical features described in the present disclosure with respect to antigen binding proteins apply.

Use in diagnostic and detection assays

The antigen binding proteins as disclosed herein may be used for in vivo and/or in vitro detection or diagnostic purposes. For example, a wide range of immunoassays for detecting expression in a particular cell or tissue using antibodies are known to those skilled in the art. For such purposes, it may be advantageous to use an antigen binding protein linked to a detectable label, such as biotin.

In one embodiment, the described antigen binding proteins can be used to detect the presence of a target peptide-MHC complex, particularly MAGE-A4, in a sample. Detection may be used for quantitative or qualitative purposes. The sample is preferably of biological origin, such as blood, urine, cerebrospinal fluid, biopsies, lymphoid and/or non-blood tissue. In certain embodiments, the biological sample comprises cells or tissue from a human patient. In certain embodiments, the method comprises contacting the biological sample with an antigen binding protein under conditions that allow binding of the inhibitor to the target peptide-MHC, and then detecting the inhibitor-target complex. Such methods may be in vitro or in vivo. In some embodiments, such methods are performed to select subjects that meet the conditions of treatment with the antigen binding proteins described herein.

Kit for detecting a substance in a sample

Kits are also contemplated that comprise at least one nucleic acid library or antigen binding protein as described herein, typically in combination with the reagents in packaging with instructions. In one embodiment, the kit comprises a composition comprising an effective amount of said antigen binding protein in unit dosage form. Such kits may comprise a sterile container containing the composition; non-limiting examples of such containers include, but are not limited to, vials, ampoules, bottles, tubes, syringes, blister packs. In some embodiments, the composition is a pharmaceutical composition and the container is made of a material suitable for containing a drug. In one embodiment, the kit may comprise the antigen binding protein in lyophilized form in a first container and a diluent (e.g., sterile water) for reconstitution or dilution of 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.

Typically, the kit will also comprise separate sheets, brochures or cards in or provided with the container with instructions for use. If the kit is intended for pharmaceutical use, it may further comprise one or more of the following: information and dosage schedules for administration of the compositions, descriptions of therapeutic agents, precautions, warnings, indications, contraindications, overdosing information, and/or adverse reactions to subjects suffering from a related disease or disorder (e.g., a MAGE-A4 mediated disease or disorder).

It will be apparent to those skilled in the art that other suitable modifications and adaptations to the methods described herein can be made using the appropriate equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the embodiments will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

Examples

EXAMPLE 1 Generation of MHC complexes as antigens for immunization

MHC class I heavy chains and β2m were cloned into pET-24D (+) vectors using standard molecular biology techniques (J Biol chem.1995, 13, 1/13; 270 (2): 971-7). Coli BL-21 (DE 3) was transformed with the expression vector according to the supplier's protocol. Protein expression was performed at 37℃for 16-18 hours with shaking at 220rpm in MagicMedium (Invitrogen), as described by the supplier. Cells were harvested and lysed with BugBuster (Invitrogen), inclusion bodies were washed twice with TBS supplemented with 0.5% ldao and twice with TBS. The inclusion bodies thus prepared were dissolved in denaturation buffer (8M urea, 100mM Tris-HCl pH 8), using 5mL buffer per 1g inclusion body pellet. Refolding and purification of MHC and target peptides (HLA-A 02:01 extracellular domain, human β2M and MAGE-A4 peptide 230-239) were performed essentially as described by Rodensko et al (2006). The amino acid sequences of the pMHC antigen components are listed in table 2 below.

TABLE 2 amino acid sequence of pMHC antigen components

EXAMPLE 2 Rabbit immunization

In order to generate a number of antibodies capable of specifically recognizing the target peptide in the case of HLA complexes, 3 new zealand white rabbits were immunized with recombinantly produced MHC complexes. Each animal received 4 injections of pMHC complex with complete or incomplete freund's adjuvant at different time points. The immune response of the animals was tested in ELISA to quantify the anti-pMHC antibodies present in the serum samples of the immunized animals. Antibody titers in serum indicate good immune responses.

EXAMPLE 3 construction of Rabbit-derived immune library

scFv antibody cDNA libraries were constructed by PCR amplification of RNA extracted from rabbit isolated PBMCs and spleen lymphocytes. The coding sequences for the variable light and heavy chain domains are amplified separately and linked by a series of overlapping Polymerase Chain Reaction (PCR) steps to yield the final scFv product. Amplified DNA sequences encoding scFv from rabbits were digested with appropriate restriction enzymes and subsequently ligated into phagemid vectors. The phagemid vector was transformed into E.coli TG1 electrocompetent cells, which are well suited for the generation of antibody phage display libraries. These methods produce twoAntibody library species, kappa based library diversity of 5.2x10 ⁸ The sequence accuracy was 87.5% and the diversity of the lambda based library sequences was 2.0x10 ⁹ The accuracy was 91.7%.

Example 4 alignment of kappa light chain alleles

68 rabbit kappa light chain alleles are listed in the IMGT database. DNA sequences of all 68 alleles were output and aligned. Only 4 of the 68 alleles did not have cysteines at position 80 (numbered according to Kabat), underscores the importance of optimizing scFv immune libraries comprising the rabbit kappa light chain repertoire. The nucleotide sequences in the flanking regions of the cysteines show high sequence conservation. This allows the design of primer sets covering the complete naive rabbit kappa light chain repertoire. An alignment of the sequences is shown in figure 1.

Example 5 design of primers

Primers were designed to mutate the cysteine at position 80 of the rabbit kappa light chain to alanine. Two forward primers were designed that contained nucleotide substitutions C80A. In addition, 10 reverse primers are required to cover the complete kappa light chain repertoire. See table 3 below. Primer design was performed according to the Q5 site-directed protocol of New England Biolabs.

Table 3-primer set for removal of cysteine 80, comprising 2 forward primers and 10 reverse primers. The primer set was intended to cover the entire primary rabbit vk pool.

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To demonstrate the concept, 20 clones of the internal rabbit immune library were randomly selected. These variants have been sequenced and aligned to the naive rabbit kappa light chain repertoire (IMGT database). The sequence alignment of the mature antibodies is set forth in figure 2. Based on these antibodies, which have undergone somatic hypermutation processes, as well as sequence rearrangements in rabbit immune responses, primer sets have been used to evaluate the functionality of the designed primer sets to mutate the immune library repertoire while restoring a high degree of diversity.

Of the 20 sequences that have been selected, 1/20 shows poor sequence quality. Of the remaining 19 sequences, 11/19 (58%) was completely covered by the primer set without any mismatches. From the remaining variants, 5/19 (26%) revealed a 1 nucleotide mismatch in either the forward or reverse primer. The other 3/19 (16%) showed two or three mismatches. Assuming that PCR was still possible for those with only 1 mismatch in the primer annealing region, the library recovery was found to be 16/19 (84%).

Example 6 optimization of internal Rabbit scFv immune library

All possible primer combinations of the explained primer sets were run using DNA of the internal rabbit scFv immune library (phagemid) as template DNA (20 PCR reactions). The Q5 site-directed mutagenesis kit of New England Biolabs was used according to the protocol provided. The annealing temperature was set to 63℃and 35 cycles were performed using 1ng of the original phagemid DNA as template. After PCR, KLD reactions (part of Q5 site-directed mutagenesis protocol) were performed on each sample, incubated for 30 min at room temperature, followed by incubation for 30 min at 16 ℃. The KLD reaction was then purified using PCR purification followed by electroporation into TG1 cells. Transformed bacteria were plated on 2xYT plates containing 100 μg/ml ampicillin+1% glucose and incubated overnight at 37 ℃. After harvesting the bacteria phage amplification was started according to standard protocols. In addition, serial dilutions of bacteria were performed to determine conversion titers, which indicate that library coverage was 8.5 times that of the original library. Several clones from each reaction were sequenced for quality control.

EXAMPLE 7 optimization of library quality

Each of the 20 PCR reactions (96 total) was sequenced to check the quality of the optimized library. Successfully optimized variants were obtained for all PCR reactions. In general, 64/96 (67%) of the correct insert with the predicted substitution C80A was identified. The remaining 32 sequences exhibited different problems such as frame shifting, sequencing problems and primer mismatches. Binding to the original library diversity 8.5-fold in the bacterial transformation reading from which 67% percent of the correct insert was identified, the total library coverage was determined to be around 6-fold.

In addition, the sequenced variants were further analyzed by designing phylogenetic circles (64/96), which indicate a good distribution of the different rabbit kappa light chain subtypes, as shown in FIG. 3.

EXAMPLE 8 biopanning with optimized library

An optimized internal rabbit scFv immune library was used for biopanning against specific pMHC targets. In parallel, the original rabbit scFv immune library has been used as a direct control to optimize the quality and efficacy of the library. Three rounds of phage display were performed prior to screening the library for specific hits. Screening was performed with a monoclonal phage ELISA against specific and non-specific targets. The ratio of signals from specific target binding to non-specific binding is then calculated to find hits that specifically bind to the target. The data can be found in table 4 (original rabbit library) and table 5 (optimized library).

Specifically, tables 4 and 5 show the output of a monoclonal phage ELISA after three rounds of biopanning applied to a rabbit derived antibody library from which Cys80 was removed. The values indicate the binding signal ratio of the target peptide MAGE-A4 in the case of HLA complexes/a mixture of 49 different unrelated peptides (SEQ ID NOS: 345-393, as shown in Table 9) in the case of HLA complexes. Ratios above 2.5 are highlighted in gray, with each data point representing one phage display clone.

Whereas only one binder could be identified for the original library after three biopanning rounds, 13 binders were found in the optimized library. This clearly shows the evidence of the removal of free cysteines from rabbit immune libraries to exploit full diversity.

Additional rounds of panning have been performed by using lambda libraries and optimized kappa libraries. 19 unique and target-specific antibodies were identified. The 19 antibody scFv sequences identified in the biopanning screening are listed in table 6 below.

Table 4-output of panning of phage display rabbit antibodies with Cys 80. A raw rabbit library. Each data point A1-H12 represents a non-specific binding to a mixture of 49 different unrelated peptides (SEQ ID NOs: 345-393, as shown in Table 9) against HLA-A2/MAGE-A4 complex, hit clones after three rounds of biopanning in a monoclonal phage ELISA. Ratios above 2.5 are highlighted in bold text.

Table 5-output of panning of phage display rabbit antibodies with Cys 80. The library is optimized. Each data point A1-H11 represents a hit clone after three rounds of biopanning in a monoclonal phage ELISA for binding to the HLA-a2/MAGE-A4 complex relative to non-specific binding to a mixture of HLA complexes/49 different unrelated peptides. Ratios above 2.5 are highlighted in bold text. H12 represents positive control.

TABLE 6 amino acid sequence of rabbit antibody. CDR sequences are highlighted in underlined bold text.

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EXAMPLE 9 expression of antibodies in the form of monovalent monospecific Fab or bispecific antibodies

Monovalent monospecific antibodies are expressed in Fab format. In addition, bispecific antibodies comprising a CD3 binding moiety are expressed based on Fab formats, which are highly stable and potent heterodimeric scaffolds. The scFv or sdAb is fused to the C-terminal region of the Fab. The rabbit variable domain was paired with human constant domains (heavy and kappa light chains) to generate chimeric Fab which bound to the target pMHC. An scFv with binding specificity for CD3 was attached to the C-terminus of the Fab light chain constant region. The amino acid sequences of the constant domains, amino acid linkers and CD3 scFv are listed in table 7 below.

TABLE 7 amino acid sequence for the production of chimeric Fab

Synthetic genes encoding different antibody chains (i.e., heavy and light chains) were constructed at Twist Bioscience Corporation and cloned separately 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 No. 12662).

Expression of the expression cassette comprising CD3 binding by transient co-transfection of the respective mammalian expression vector in HEK293-6E cellsPart of a mono-and bispecific antigen binding protein, the HEK293-6E cells were cultured in suspension using polyethylenimine (PEI 40kD linear chain). HEK293-6E cells were grown at 1.7X10 ⁶ Each cell/mL was inoculated in Freestyle F17 medium supplemented with 2mM L-glutamine. DNA per mL final production volume was prepared by adding DNA and PEI separately to 50. Mu.L of non-supplemented medium. The two fractions were mixed, vortexed and allowed to stand for 15 minutes to give a DNA to PEI ratio of 1:2.5 (1. Mu.g DNA/mL cells). The cells and DNA/PEI mixture were put together and then transferred to a suitable container (37 ℃ C., 5% CO) placed in a shaking device ₂ 80% RH). After 24 hours, 25 μl tryptone N1 was added per mL final production volume.

After 7 days, the cells were harvested by centrifugation and sterile filtered. The antigen binding proteins are purified by an affinity step. For affinity purification of Fab-based constructs, the supernatant was loaded onto a protein CH column (Thermo Fisher Scientific, # 494320005) equilibrated with 6CV PBS (pH 7.4). After the washing step with the same buffer, the antigen binding protein was eluted from the column by stepwise elution with 100mM citric acid (pH 3.0). Fractions containing the desired antigen binding protein were immediately neutralized with 1M Tris buffer (pH 9.0) at a ratio of 1:10, then pooled, dialyzed and concentrated by centrifugation.

After concentration and dialysis against PBS buffer, the content and purity of the purified protein was assessed by SDS-PAGE and size exclusion HPLC. After expression in HEK293-6E cells, the proteins were purified by a single capture step and analyzed by analytical size exclusion chromatography.

Example 10 production of llama-derived antibodies

To further increase the likelihood of identifying antibodies that specifically recognize the MAGE-A4 peptide complex, 2 llamas were immunized with the HLA a 02:01/GVYDGREHTV complex. Each animal received 4 injections of pMHC complex protein described in example 1 with complete or incomplete freund's adjuvant at different time points. The immune response of the animals was tested in ELISA to quantify the anti-HLA a 02:01/GVYDGRE HTV antibodies present in the serum samples of the immunized animals. Antibody titers in serum indicate good immune responses.

Blood samples were obtained from llamas, RNA was isolated from plasma cells of immunized animals and transcribed into cDNA using a reverse transcriptase kit. The cDNA of the heavy chain fragment was amplified using primers annealed in the leader region and CH2 region. Amplified DNA sequences encoding VHH antibodies from llamas were used as a source of repertoires for antibody library construction. Briefly, the DNA sequence is digested with the appropriate restriction enzymes and subsequently ligated into a phagemid vector. The antibody library was screened as described in example 8. The amino acid sequences of the antibodies are listed in table 8 below.

TABLE 8 amino acid sequence of a llama-derived VHH. CDR sequences are highlighted in underlined bold text.

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Example 11 characterization of hits

Phylogenetic analysis of selected 38 HLA-A2/MAGE-A4 binding hits from rabbit and llama immune libraries was performed using a maximum likelihood method based on the Jones-Taylor-Thornton (JTT) model (MEGAX software). The sequence diversity of the selected binders is depicted in figure 4. The hits selected represent a collection of HLA-A2/MAGE-A4 binders with high sequence diversity and different origins.

All available hits were evaluated for their ability to bind MAG E-A4/HLA-A2 complex and control peptide/HLA-A 2 complex in a direct binding ELISA assay. The control peptide/HLA-A 2 complex in this assay contained a HLA-A2 complex loaded with a mixture of 49 unrelated peptides, as shown in Table 9 (SEQ ID NOS: 345-393). Briefly, 96-well ELI SA plates were coated with purified human MAGE-A4/HLA-A2 complex or control HLA-A2 complex. Serial dilutions of antibody molecules were added to the plates and detected with anti-kappa light chain-HRP (I nvitrogen) or purified rabbit anti-VHH (QVQ) followed by goat anti-rabbit IgG (h+l) H RP (Southern Biotech). Conjugates were considered for further characterization when high binding to the MAGE-A4/HLA-A2 complex was shown and no binding to the control peptide/HLA-A 2 complex. Binding of six selected antibodies M0709, M0739, M0742, M0743, M0747 and M0763 to the HLA-A2/MAGE-A4 complex as determined by ELISA is shown in FIG. 5A. The binding of additional antibodies designated M0700-M0710 and M0762-M0766 to the HLA-A2/MAGE-A4 complex as determined by ELISA is shown in FIG. 5B. All molecules tested showed specific binding to the HLA-A2/MAGE-A4 complex, but not to the control HLA-A2 complex. Each antibody tested contained kappa light chains except for M0709 and M0763, which contained lambda light chains.

Specific antibodies M0709, M0739, M0742, M0743, M0747 and M0763 were assayed for binding to the MAGE-A4 peptide-HLA-A 2 complex presented on the cell. Briefly, T-B hybrid T2 cells were incubated with serum-free RPMI1640 medium containing MAGE-A4 or control peptide. The control peptides constitute sequences with high identity to MAGE-A4 and have been previously identified in healthy human tissue, namely control 1 (GLADGRTHTV; SEQ ID NO: 394), control 2 (GLYDGPVHEV; SEQ ID NO: 395) and control 3 (GVFDGLHTV; SEQ ID NO: 396) (U.S. Pat. No. 20180171024, incorporated herein by reference). Peptide loading efficiency was verified by using the ratio (> 1) between the Median Fluorescence Intensity (MFI) of HLA-A2 binding antibody BB7.2 on peptide loaded T2 cells and the MFI of non-loaded T2 cells. T2 cells were incubated with each specific antibody followed by a fluorophore-labeled detection antibody (anti-kappa light chain or anti-Flag). Cells were fixed and fluorescence was measured by flow cytometry. The binding and specificity of selected antibodies M0709, M0739, M0742, M0743, M0747, M0763 to T2 cells displaying MAGE-A4 or control peptides 1, 2 and 3 are presented in FIG. 6. All molecules tested showed binding to H LA-A2/MAGE-A4 displayed on T2 cells. Moreover, M0743, M0747 and M0763 showed very high specificity for the MAGE-A4 peptide and did not show binding to any control peptide displayed by HLA-A2 on T2 cells. M0709 showed the lowest specificity for all test molecules and also bound control peptides 1 and 2. M0739 and M0742 bind not only the MAGE-A4 display peptide, but also to control peptide 2.

EXAMPLE 12 redirected T cell killing of antigen-positive and antigen-negative cell lines Using pHLA-targeted bispecific antibodies

Redirected T cell killing of tumor cell lines by bispecific antibodies targeting peptide-HLA (pHLA) was determined by end-point cytotoxicity measurement (LDH release) and real-time imaging (IncuCyte).

Lactate dehydrogenase release assay was performed. Briefly, target cells are co-cultured with effector cells (e.g., PBMC) at an E: T ratio of about 10:1. A solution of CDR4 bispecific 01 antibody M0719 was added to the relevant wells, covering a concentration range of 0.4nM to 40nM. Cytotoxicity was quantified by colorimetric absorbance measurement of the amount of LDH released from damaged cells into the medium after 48 hours. The data obtained are presented in fig. 7 the tested antibody CDR 4-bispecific 01 shows potent T cell mediated antigen positive tumor cell killing even at low concentrations.

Furthermore, CDR 4-bispecific 01 was also tested in an LDH assay in combination with the immune checkpoint inhibitor palbociclib (anti-PD-1 antibody). Briefly, LDH assays were performed as described above. Cell killing EC50 was determined by LDH release after co-incubation of PBMC with MAGE-A4 positive cell lines a375, U20S, SCaBER and NCI-H1703 at an E: T ratio of 10:1 in the presence of MAGE-A4 bispecific 01 (concentration ranging from 0.078 to 40 nM) with or without 300nM anti-PD-1 antibody (palbociclizumab) for 48 hours. EC50 values for CDR 4-bispecific 01 and pamphlet Li Zhushan against cell killing combined with CDR 4-bispecific 01 are plotted and shown in fig. 8. The CDR 4-bispecific 01 in combination with palbociclib showed synergistic killing of HLA-A2/MAGE-A4 positive cells with EC50 values 1.4-fold to 2.7-fold higher than CDR 4-bispecific 01 alone. In addition, cell killing was analyzed in a time-resolved manner using the IncuCyte S3 system. Briefly, cells were seeded with effector cells and treated with bispecific antibodies as described above. Briefly, antigen positive target cells (e.g., NCI-H1703, a 375) or antigen negative target cells (e.g., NCI-H441, panc-1) were incubated with Cytolight Rapid Red (Sartorius, # 4706). CDR4 bispecific antibody 01 solutions were prepared at final concentrations between 6.25nM and 0.1nM and added to the relevant wells. Cytotox green dye (Sartorius, # 4633) was added to the PBMC. Plates were imaged over time to monitor cell growth. The growth of cancer cells in each image was determined and recorded as red area confluence normalized to time 0. The number of apoptotic cells in each image was determined and recorded as green/red area normalized to time 0. The bispecific antibody CDR 4-bispecific 01 tested showed potent dose-dependent T cell mediated antigen-positive tumor cell killing over time, whereas no antigen-negative cell killing was observed (fig. 9).

In addition, MAGE-A4 positive/HLA-A 2 positive NCI-H1703 cells and MAGE-A4 negative/HLA-A 2 positive cells (NCI-H441 (lung adenocarcinoma) and MRC5 (normal human fibroblasts)) were co-incubated with PBMC (E: T10:1) and CDR4 bispecific 01 at a concentration of 0.8 nM. Images were recorded with the IncuCyte S3 system for up to 72 hours and the respective cytotoxicity is depicted in fig. 10. CDR4 bispecific 01 showed effective killing of MAGE-A4 positive/HLA-A 2 positive NCI-H1703 cells, whereas no killing of control MAGE-A4 negative HLA-A2 positive cancer cells NCI-H441 and normal fibroblast MRC5 was shown, thus showing good efficacy and safety.

EXAMPLE 13 efficacy of bispecific antibodies against non-small cell lung carcinoma (NSCLC) targeting pHLA in mice

Subcutaneous implantation of NSG mice 5X10 ⁶ NCI-H1703 cells. At 80mm ³ At average tumor size (expressed as day 0), animals were randomized and received intravenously 5x10 from a total of two donors ⁶ PBMCs, wherein each donor had two mice per group. Mice were treated once daily with CDR4 bispecific 02 (2.5 mg/kg day 0-9, 5mg/kg day 10-27) or PBS control. Body weight and tumor volume (measured with calipers) were measured twice weekly. The in vivo efficacy of CDR4 bispecific 02 is presented in fig. 11. CDR4 bispecific 02 showed complete regression of lung cancer tumor xenografts in mice.

TABLE 9 HLA Complex control peptides

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Example 14 affinity enhancement of selected Rabbit antibodies

Rabbit antibody designated M0763 was used to generate a number of affinity matured variants with substitutions in selected CDR regions. CDRL1 (TADTLSRSYAS, SEQ ID NO: 472), CDRL2 (RDTSRPS, SEQ ID NO: 473) and CDRH1 (SNYAMS, SEQ ID NO: 469) were unchanged, with substitutions in CDRL3, CDRH2 and CDRH3 only.

Based on the humanized version of the M0763 antibody, affinity-enhanced variants were identified from a humanized antibody library. Briefly, multiple antibody libraries were designed to span the entire length of all 6 CDRs, at which time 3 consecutive amino acids were randomized. Primers for site-saturation mutagenesis were used to generate libraries. Thus, the three amino acid positions targeted for randomization contain one of the 19 possible amino acid variations. After electroporation into E.coli TG-1 cells, library diversity was estimated by plating the library onto agar plates using serial dilutions of transfected TG-1 cells. The number of colonies grown on the plate was used as an indication of library diversity, assuming that one plasmid was inserted into each E.coli colony. In addition, library quality was assessed by sequencing samples of approximately 10 clones per library.

Libraries comprising site-saturation mutagenesis in the light chain are combined into one library and libraries comprising site-saturation mutagenesis in the heavy chain are combined into another library. Affinity selection was performed on two resulting libraries of randomized CDRs in the light and heavy chains, hereafter referred to as biopanning against human recombinantly produced MAGE-A4/HLA-A02 complex proteins. MAGE-A4/HLA-A02 specific phage libraries were panned (selected) on antigens adsorbed onto polystyrene tubes or plates. Alternatively, panning may be performed in solution using a soluble biotinylated antigen. Several rounds of selection, typically between two and five rounds, can be performed until an antibody with the desired specificity is obtained. The stringency of biopanning conditions can be adjusted, especially in later rounds of screening, e.g. by decreasing the antigen density coated onto the solid phase or increasing the amount of washing steps. To avoid non-specific binding of phage to the surface, PBS supplemented with 2% skim milk and 0.05% tween20 can be used as blocking agent.

Selected phage antibody clones were grown in 96-well plates and assayed for their ability to specifically bind MAGE-A4/HLA-A02 complex by ELISA. To evaluate the specificity of phage antibody clones, HLA-A02 complexed with unrelated peptides was counter-screened by ELISA. Phage antibody clones were then classified as high, medium and low signals in ELISA for target complex proteins and HLA-A02 complexed with unrelated peptides. Clones with high binding signal to the target complex and low binding to the unrelated peptide-HLA-A 02 complex were sequenced. Sequence analysis facilitates identification of unique clones, followed by selection of anti-CD 3 FAB x anti-MAGE-A4 scFv expressed recombinantly in bispecific format. The resulting constructs were then evaluated for binding affinity to MAGE-A4/HLA-A02 complex in SPR (Table 10). Affinity matured clones resulted in binding affinities as low as two picomolar, nearly 1000-fold improvement compared to the parent M0763 antibody.

The amino acid sequences of the variant VH and VL domains are listed below:

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table 10 binding affinity values of the variant antibodies for MAGE-A4 pMHC.

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Optimization of amino acids rarely occurring at CDR regions

In an additional step, the anti-MAGE-A4 antibody is engineered to reduce the risk of immunogenicity. For this purpose, the CDR sequences of the anti-MAGE-A4 antibody were examined for the presence of amino acid residues that rarely occur in human repertoires. Unusual amino acid sequences in CDR sequences are replaced by frequently occurring amino acid residues in the human antibody database (these residues may have a lower risk of immunogenicity as they naturally occur in human antibodies). Germline analysis and the frequency of occurrence of defined amino acids at relatively conserved positions revealed that the presence of three amino acids in CDRH2 is rarely present in the human antibody repertoire and is therefore considered to have an increased risk factor for immunogenicity.

The relevant HCDR2 sequence is IVSSGGTTYYASWAKG (SEQ ID NO: 470). The underlined SWA motif present in parent rabbit antibody M0763 is replaced with DSV, a sequence segment frequently found in the human antibody database. While the biological relevance and potential impact of this motif on immunogenicity is still unclear, two variants of the motif SWA lacking minimal occurrence were generated by replacing SWA with DSV. Variants designated M1335 and M1342 were further characterized in SPR and the effect of this substitution on binding affinity was not considered significant. The VH, VL, HCDR, HCDR3 and LCDR3 amino acid sequences of variants M1335 and M1342 are as described above.

Claims

1. An antigen binding protein that specifically recognizes a target melanomA-Associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), wherein the antigen binding protein comprises one or more of the following features:

(i) The antigen binding protein has a binding affinity of about 10 for the target MAGE-A4 pMHC ^-9 M to about 10 ^-14 M；

(ii) The antigen binding protein has a binding affinity of about 10 for non-MAGE-A4 pMHC and/or peptide-free MHC ^-6 M or weaker;

(iii) The antigen binding protein has a binding affinity of about 10 for non-target MAGE-A4 pMHC ^-6 M or weaker; and

(iv) The antigen binding protein has a binding affinity of about 10 for the target MAGE-A4 pMHC ^-9 M to about 10 ^-14 M and has a binding affinity of about 10 for said MAGE-A4 peptide, HLA polypeptide, and beta-2-microglobulin polypeptide alone ^-6 M or weaker.

2. The antigen binding protein of claim 1, which is specific for the MAGE-A4 peptide amino acid sequence shown in SEQ ID No. 3 (GVYDGREHTV).

3. The antigen binding protein of claim 1 or 2, wherein the MAGE-A4 peptide is complexed with an HLA-A2 polypeptide.

4. The antigen binding protein of claim 3, wherein said HLA-A2 polypeptide comprises the amino acid sequence set forth in SEQ ID No. 1.

5. The antigen binding protein of claim 3, wherein said β -2-microglobulin polypeptide comprises the amino acid sequence set forth in SEQ ID No. 2.

6. The antigen binding protein of any one of claims 1-5, which lacks detectable binding affinity to a MAGE-A4 peptide comprising one or more mutations (e.g., substitutions, deletions and/or insertions) in the amino acid sequence set forth in SEQ ID No. 3 (GVYDGREHTV).

7. The antigen binding protein of any one of claims 1-6, comprising a single chain variable fragment (scFv), fab fragment, fab' fragment, fv fragment, diabody, minibody mimetic, or single domain antibody, such as sdAb, sdFv, nanobody, V-Nar, or VHH.

8. The antigen binding protein of any one of claims 1-7, comprising:

(a) An antibody heavy chain Variable (VH) domain comprising: the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469); IVSSGGTTYYAX ₁ X ₂ X ₃ The HCDR2 amino acid sequence of KG (SEQ ID NO: 881), wherein X ₁ Corresponding to amino acids S or D, X ₂ Corresponds to amino acids W or S, and X ₃ Corresponding to amino acid a or V; and DLYYGPX ₄ TX ₅ YX ₆ X ₇ X ₈ HCDR3 amino acid sequence of NL (SEQ ID NO: 882), wherein X ₄ Corresponding to amino acid T,N or S, X ₅ Corresponding to amino acid D or absence, X ₆ Corresponding to amino acids S or F, X ₇ Corresponds to amino acids A or V, and X ₈ Corresponding to amino acid F or A; and

(b) An antibody light chain Variable (VL) domain comprising: TADTLSRSYAS (SEQ ID NO: 472), LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and ATX ₉ X ₁₀ X ₁₁ SGSNFQX ₁₂ (SEQ ID NO: 883) LCDR3 amino acid sequence, wherein X ₉ Corresponding to amino acids S or R, X ₁₀ Corresponding to amino acids D or P, X ₁₁ Corresponds to amino acid G, S or F, and X ₁₂ Corresponding to amino acid L or A.

9. The antigen binding protein of any one of claims 1-8, comprising:

(a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 657) and the HCDR3 amino acid sequence of DLYYGPSTYFVANL (SEQ ID NO: 731); and

(b) An antibody light chain Variable (VL) domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and the LCDR3 amino acid sequence of ATRPSSGSNFQL (SEQ ID NO: 879).

10. The antigen binding protein of any one of claims 1-8, comprising:

(a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 653) and the HCDR3 amino acid sequence of DLYYGPTTYSAANL (SEQ ID NO: 727); and

(b) An antibody light chain Variable (VL) domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and the LCDR3 amino acid sequence of ATRDFSGSNFQL (SEQ ID NO: 875).

11. The antigen binding protein of any one of claims 1-8, comprising:

(a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 658) and the HCDR3 amino acid sequence of DLYYGPNTDYSAANL (SEQ ID NO: 732); and

(b) An antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and an LCDR3 amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 880).

12. The antigen binding protein of any one of claims 1-8, comprising:

(a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 624), and the HCDR3 amino acid sequence of DLYYGPTTYSAFNL (SEQ ID NO: 698); and

(b) An antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473), and an LCDR3 amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 846).

13. The antigen binding protein of any one of claims 1-8, comprising:

(a) An antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 470) and the HCDR3 amino acid sequence of DLYYGPTTYSAFNL (SEQ ID NO: 471); and

(b) An antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and an LCDR3 amino acid sequence of ATSDGSGSNFQL (SEQ ID NO: 474).

14. The antigen binding protein of any one of claims 1-8, comprising:

(a) An antibody heavy chain Variable (VH) domain comprising a framework region, an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the VH domain comprises an amino acid sequence shown in SEQ ID No. 575 or an amino acid sequence having at least 80% identity to the framework region of the amino acid sequence shown in SEQ ID No. 575 and 100% identity to the HCDR1, HCDR2, and HCDR3 regions shown in SEQ ID No. 575; and

(b) An antibody light chain Variable (VL) domain comprising a framework region, an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the VL domain comprises an amino acid sequence shown in SEQ ID No. 797 or an amino acid sequence having at least 80% identity to the framework region of the amino acid sequence shown in SEQ ID No. 797 and 100% identity to the LCDR1, LCDR2, and LCDR3 regions shown in SEQ ID No. 797.

15. The antigen binding protein of any one of claims 1-8, comprising:

(a) An antibody heavy chain Variable (VH) domain comprising a framework region, an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the VH domain comprises an amino acid sequence shown in SEQ ID No. 583 or an amino acid sequence having at least 80% identity to the framework region of the amino acid sequence shown in SEQ ID No. 583 and 100% identity to the HCDR1, HCDR2, and HCDR3 regions shown in SEQ ID No. 583; and

(b) An antibody light chain Variable (VL) domain comprising a framework region, an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the VL domain comprises an amino acid sequence shown in SEQ ID No. 805 or an amino acid sequence having at least 80% identity to the framework region of the amino acid sequence shown in SEQ ID No. 805 and 100% identity to the LCDR1, LCDR2, and LCDR3 regions shown in SEQ ID No. 805.

16. The antigen binding protein of any one of claims 1-8, comprising:

(a) An antibody heavy chain Variable (VH) domain comprising a framework region, an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the VH domain comprises an amino acid sequence shown in SEQ ID No. 579 or an amino acid sequence having at least 80% identity to the framework region of the amino acid sequence shown in SEQ ID No. 579 and 100% identity to the HCDR1, HCDR2, and HCDR3 regions shown in SEQ ID No. 579; and

(b) An antibody light chain Variable (VL) domain comprising a framework region, an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the VL domain comprises an amino acid sequence shown in SEQ ID No. 801 or an amino acid sequence having at least 80% identity to the framework region of the amino acid sequence shown in SEQ ID No. 801 and 100% identity to the LCDR1, LCDR2, and LCDR3 regions shown in SEQ ID No. 801.

17. The antigen binding protein of any one of claims 1-8, comprising:

(a) An antibody heavy chain Variable (VH) domain comprising a framework region, an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the VH domain comprises an amino acid sequence shown in SEQ ID No. 582 or an amino acid sequence having at least 80% identity to the framework region of the amino acid sequence shown in SEQ ID No. 582 and 100% identity to the HCDR1, HCDR2, and HCDR3 regions shown in SEQ ID No. 582; and

(b) An antibody light chain Variable (VL) domain comprising a framework region, an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the VL domain comprises an amino acid sequence shown in SEQ ID No. 804 or an amino acid sequence having at least 80% identity to the framework region of the amino acid sequence shown in SEQ ID No. 804 and 100% identity to the LCDR1, LCDR2, and LCDR3 regions shown in SEQ ID No. 804.

18. The antigen binding protein of any one of claims 1-8, comprising:

(a) An antibody heavy chain Variable (VH) domain comprising a framework region, an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the VH domain comprises an amino acid sequence shown in SEQ ID No. 584, or an amino acid sequence having at least 80% identity to the framework region of the amino acid sequence shown in SEQ ID No. 584 and 100% identity to the HCDR1, HCDR2, and HCDR3 regions shown in SEQ ID No. 584; and

(b) An antibody light chain Variable (VL) domain comprising a framework region, an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the VL domain comprises an amino acid sequence shown in SEQ ID No. 806 or an amino acid sequence having at least 80% identity to the framework region of the amino acid sequence shown in SEQ ID No. 806 and 100% identity to the LCDR1, LCDR2, and LCDR3 regions shown in SEQ ID No. 806.

19. The antigen binding protein of any one of claims 1-8, comprising:

(a) An antibody heavy chain Variable (VH) domain comprising a framework region, an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the VH domain comprises an amino acid sequence shown in SEQ ID No. 550 or an amino acid sequence having at least 80% identity to the framework region of the amino acid sequence shown in SEQ ID No. 550 and 100% identity to the HCDR1, HCDR2, and HCDR3 regions shown in SEQ ID No. 550; and

(b) An antibody light chain Variable (VL) domain comprising a framework region, an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the VL domain comprises an amino acid sequence shown in SEQ ID No. 772 or an amino acid sequence having at least 80% identity to the framework region of the amino acid sequence shown in SEQ ID No. 772 and 100% identity to the LCDR1, LCDR2, and LCDR3 regions shown in SEQ ID No. 772.

20. The antigen binding protein of any one of claims 8-19, wherein one or more of the HCDR1 amino acid sequence, the HCDR2 amino acid sequence, the HCDR3 amino acid sequence, the LCDR1 amino acid sequence, the LCDR2 amino acid sequence and the LCDR3 amino acid sequence comprises one or more amino acid substitutions.

21. The antigen binding protein of claim 20, wherein the antigen binding protein retains binding specificity for the target MAGE-A4 pMHC after the one or more amino acid substitutions.

22. The antigen binding protein of any one of claims 8-21, wherein one or more of the VH domain and the VL domain comprises one or more amino acid substitutions.

23. The antigen binding protein of claim 22, wherein the antigen binding protein retains binding specificity for the target MAGE-A4 pMHC after the one or more amino acid substitutions.

24. An antigen binding protein that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising:

an antibody heavy chain Variable (VH) domain comprising: the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469); IVSSGGTTYYAX ₁ X ₂ X ₃ The HCDR2 amino acid sequence of KG (SEQ ID NO: 881), wherein X ₁ Corresponding to amino acids S or D, X ₂ Corresponds to amino acids W or S, and X ₃ Corresponding to amino acid a or V; and DLYYGPX ₄ TX ₅ YX ₆ X ₇ X ₈ HCDR3 amino acid sequence of NL (SEQ ID NO: 882), wherein X ₄ Corresponding to amino acid T, N or S, X ₅ Corresponding to amino acid D or absence, X ₆ Corresponding to amino acids S or F, X ₇ Corresponds to amino acids A or V, and X ₈ Corresponding to amino acid F or A; and

an antibody light chain Variable (VL) domain comprising: TADTLSRSYAS (SEQ ID NO: 472), LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and ATX ₉ X ₁₀ X ₁₁ SGSNFQX ₁₂ (SEQ ID NO: 883) LCDR3 amino acid sequence, wherein X ₉ Corresponding to amino acids S or R, X ₁₀ Corresponding to amino acids D or P, X ₁₁ Corresponds to amino acid G, S or F, and X ₁₂ Corresponding to amino acid L or A.

25. An antigen binding protein that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising:

an antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 657) and the HCDR3 amino acid sequence of DLYYGPSTYFVANL (SEQ ID NO: 731); and

an antibody light chain Variable (VL) domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and the LCDR3 amino acid sequence of ATRPSSGSNFQL (SEQ ID NO: 879).

26. The antigen binding protein of claim 25, comprising: an antibody VH domain comprising the amino acid sequence shown in SEQ ID No. 583 and an antibody VL domain comprising the amino acid sequence shown in SEQ ID No. 805, or a VH domain having at least 80% identity to SEQ ID No. 583 and a VL domain having at least 80% identity to SEQ ID No. 805.

27. An antigen binding protein that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising:

an antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 653) and the HCDR3 amino acid sequence of DLYYGPTTYSAANL (SEQ ID NO: 727); and

An antibody light chain Variable (VL) domain comprising the LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), the LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and the LCDR3 amino acid sequence of ATRDFSGSNFQL (SEQ ID NO: 875).

28. The antigen binding protein of claim 27, comprising: an antibody VH domain comprising the amino acid sequence shown in SEQ ID No. 579 and an antibody VL domain comprising the amino acid sequence shown in SEQ ID No. 801, or a VH domain having at least 80% identity to SEQ ID No. 579 and a VL domain having at least 80% identity to SEQ ID No. 801.

29. An antigen binding protein that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising:

an antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYADSVKG (SEQ ID NO: 658) and the HCDR3 amino acid sequence of DLYYGPNTDYSAANL (SEQ ID NO: 732); and

an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473) and an LCDR3 amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 880).

30. The antigen binding protein of claim 29, comprising: an antibody VH domain comprising the amino acid sequence shown in SEQ ID No. 584 and an antibody VL domain comprising the amino acid sequence shown in SEQ ID No. 806, or a VH domain having at least 80% identity to SEQ ID No. 584 and a VL domain having at least 80% identity to SEQ ID No. 806.

31. An antigen binding protein that specifically recognizes a target melanoma-associated antigen A4 (MAGE-A4) peptide-MHC (pMHC), comprising:

an antibody heavy chain Variable (VH) domain comprising the HCDR1 amino acid sequence of SNYAMS (SEQ ID NO: 469), the HCDR2 amino acid sequence of IVSSGGTTYYASWAKG (SEQ ID NO: 624), and the HCDR3 amino acid sequence of DLYYGPTTYSAFNL (SEQ ID NO: 698); and

an antibody light chain Variable (VL) domain comprising an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO: 472), an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO: 473), and an LCDR3 amino acid sequence of ATRPSSGSNFQA (SEQ ID NO: 846).

32. The antigen binding protein of claim 31, comprising: an antibody VH domain comprising the amino acid sequence shown in SEQ ID No. 550 and an antibody VL domain comprising the amino acid sequence shown in SEQ ID No. 772, or a VH domain having at least 80% identity to SEQ ID No. 550 and a VL domain having at least 80% identity to SEQ ID No. 772.

33. The antigen binding protein of any one of claims 24-32, wherein the antigen binding protein comprises one or more of the following features:

34. The antigen binding protein of any one of claims 24-32, which is specific for the MAGE-A4 peptide amino acid sequence set forth in SEQ ID No. 3 (GVYDGREHTV).

35. The antigen binding protein of any one of claims 8-34, wherein the VH domain and VL domain are attached with an amino acid linker.

36. The antigen binding protein of claim 35, wherein the amino acid linker comprises (GGGGS) n, wherein n is an integer from 1 to 5.

37. The antigen binding protein of claim 35 or 36, wherein the amino acid linker comprises the amino acid sequence ggggsggggsgggs, GGGGSGGGGSGGG GSGGGGS, or GGGGSGGGGSGGGGSGGGGAS.

38. The antigen binding protein of any one of claims 1-7, comprising:

(a) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0734 of table 8;

(b) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0735 of table 8;

(c) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0736 of table 8;

(d) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0737 of table 8;

(e) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0738 of table 8;

(f) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0739 of table 8;

(g) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0740 of table 8;

(h) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0741 of table 8;

(i) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0742 of table 8;

(j) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0743 of table 8;

(k) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0744 of table 8;

(l) An antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0745 of table 8;

(M) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0746 of table 8;

(n) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0747 of table 8;

(o) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0748 of table 8;

(p) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0749 of table 8;

(q) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence, and an HCDR3 amino acid sequence as shown in M0750 of table 8;

(r) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0751 of table 8; or (b)

(s) an antibody VHH domain comprising an HCDR1 amino acid sequence, an HCDR2 amino acid sequence and an HCDR3 amino acid sequence as shown in M0752 of table 8.

39. The antigen binding protein of any one of claims 1-7, comprising:

(a) Antibody VHH domains as shown in M0734 of table 8;

(b) An antibody VHH domain as shown in M0735 of table 8;

(c) Antibody VHH domains as shown in M0736 of table 8;

(d) Antibody VHH domains as shown in M0737 of table 8;

(e) Antibody VHH domains as shown in M0738 of table 8;

(f) Antibody VHH domains as shown in M0739 of table 8;

(g) Antibody VHH domains as shown in M0740 of table 8;

(h) Antibody VHH domains as shown in M0741 of table 8;

(i) Antibody VHH domains as shown in M0742 of table 8;

(j) Antibody VHH domains as shown in M0743 of table 8;

(k) Antibody VHH domains as shown in M0744 of table 8;

(l) Antibody VHH domains as shown in M0745 of table 8;

(M) an antibody VHH domain as shown in M0746 of table 8;

(n) an antibody VHH domain as shown in M0747 of table 8;

(o) an antibody VHH domain as shown in M0748 of table 8;

(p) an antibody VHH domain as shown in M0749 of table 8;

(q) an antibody VHH domain as shown in M0750 of table 8;

(r) an antibody VHH domain as shown in M0751 of table 8; or (b)

(s) antibody VHH domain as shown in M0752 of table 8.

40. The antigen binding protein of claim 38, wherein one or more of the HCDR1 amino acid sequence, the HCDR2 amino acid sequence and the HCDR3 amino acid sequence comprises one or more amino acid substitutions.

41. The antigen binding protein of claim 40, wherein said antigen binding protein retains binding specificity for said target MAGE-A4 pMHC after said one or more amino acid substitutions.

42. The antigen binding protein of claim 39, wherein said VHH domain comprises one or more amino acid substitutions.

43. The antigen binding protein of claim 42, wherein said antigen binding protein retains binding specificity for said target MAGE-A4 pMHC after said one or more amino acid substitutions.

44. The antigen binding protein of any one of claims 1-43, which binds to said MAGE-A4 pMHCA combined affinity of at least about 10 ^-9 M。

45. The antigen binding protein of any one of claims 1-43, having a binding affinity to the MAGE-A4 pMHC of about 10 ^-9 M to about 10 ^-14 M。

46. The antigen binding protein of any one of claims 1-43, having a binding affinity to the MAGE-A4 pMHC of about 10 ^-10 M to about 10 ^-12 M。

47. The antigen binding protein of any one of claims 1-46, which lacks detectable binding affinity for non-MAGE-A4 pMHC.

48. The antigen binding protein of any one of claims 1-46, which lacks detectable binding affinity for peptide-free MHC.

49. The antigen binding protein of any one of claims 1-46, which lacks detectable binding affinity for non-target MAGE-A4 pMHC.

50. The antigen binding protein of any one of claims 1-46, which lacks detectable binding affinity for MAGE-A4 peptide alone.

51. The antigen binding protein of any one of claims 1-46, which lacks detectable binding affinity for an individual HLA polypeptide.

52. The antigen binding protein of any one of claims 1-46, which lacks detectable binding affinity for a β -2-microglobulin polypeptide alone.

53. The antigen binding protein of any one of claims 1-52, wherein the antigen binding protein specifically binds to the MAGE-A4 pMHC on the cell surface.

54. The antigen binding protein of any one of claims 1-52, which lacks detectable binding affinity for non-MAGE-A4 pMHC on the cell surface.

55. The antigen binding protein of any one of claims 1-54, which has cytotoxic activity against cells expressing MAGE-A4 pMHC.

56. The antigen binding protein of any one of claims 1-55, which lacks detectable cytotoxic activity to cells expressing non-MAGE-A4 pMHC.

57. The antigen binding protein of any one of claims 1-56, wherein the antigen binding protein is a humanized antigen binding protein.

58. The antigen binding protein of any one of claims 1-56, wherein the antigen binding protein is a human antigen binding protein.

59. The antigen binding protein of any one of claims 1-58, wherein the binding affinity is measured by Surface Plasmon Resonance (SPR).

60. A bispecific antigen binding protein comprising at least a first antigen binding domain comprising the antigen binding protein of any one of claims 1-59, and at least a second antigen binding domain specific for a cell surface protein of an immune cell.

61. The bispecific antigen binding protein of claim 60, wherein the immune cells are selected from the group consisting of T cells, B cells, natural Killer (NK) cells, natural Killer T (NKT) cells, neutrophils, monocytes and macrophages.

62. The bispecific antigen binding protein of claim 60 or 61, wherein the immune cell is a T cell.

63. The bispecific antigen binding protein of any one of claims 60-62, wherein the cell surface protein of an immune cell is selected from the group consisting of CD3, tcra, tcrp, CD16, NKG2D, CD89, CD64 and CD 32.

64. The bispecific antigen binding protein of any one of claims 60-63, wherein the cell surface protein of an immune cell is CD3.

65. The bispecific antigen binding protein of any one of claims 60-64, wherein the at least first antigen binding domain comprises an scFv or VHH and the at least second antigen binding domain comprises a Fab.

66. The bispecific antigen binding protein of any one of claims 60-65, wherein the bispecific antigen binding protein is multivalent.

67. The bispecific antigen binding protein of any one of claims 60-65, wherein the bispecific antigen binding protein comprises three antigen binding sites.

68. The bispecific antigen binding protein of any one of claims 60-67, further comprising an immune checkpoint inhibitor.

69. The bispecific antigen binding protein of claim 68, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-BTLA antibody, an anti-VISTA antibody, and combinations thereof.

70. Use of the antigen binding protein of any one of claims 1-59 or the bispecific antigen binding protein of any one of claims 60-69 for the manufacture of a pharmaceutical composition for treating MAGE-A4-associated cancer in a subject.

71. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1-59, or the bispecific antigen binding protein of any one of claims 60-69, and a pharmaceutically acceptable carrier.

72. A method of treating a MAGE-A4 pMHC expressing cancer in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of the pharmaceutical composition of claim 71.

73. The method of claim 72, further comprising administering an immune checkpoint inhibitor.

74. The method of claim 73, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-BTLA antibody, an anti-VISTA antibody, and combinations thereof.

75. A nucleic acid encoding the antigen binding protein of any one of claims 1-59, or the bispecific antigen binding protein of any one of claims 60-69.

76. An expression vector comprising the nucleic acid of claim 75.

77. A host cell comprising the expression vector of claim 76.

78. A method of making the antigen binding protein of any one of claims 1-59, or the bispecific antigen binding protein of any one of claims 60-69, comprising the steps of:

(i) Incubating the host cell of claim 77 under conditions allowing expression of the antigen binding protein of any one of claims 1-59 or the bispecific antigen binding protein of any one of claims 60-69;