Anti-flag antibodies

Abstract

The present invention relates to antigen binding proteins and related fragments thereof for binding FLAG tags, production of the antigen binding proteins and fragments, and uses of the antibodies and fragments.

Description

Technical Field

[0001] The present invention relates to antigen-binding proteins and related fragments thereof for binding to FLAG tags, the production of the antigen-binding proteins and fragments thereof, and the use of the antibodies and fragments thereof, wherein the antigen-binding proteins and related fragments thereof include humanized antigen-binding proteins and related fragments thereof.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority from Australian provisional application AU 2022902712, the entire contents of which are incorporated herein by reference. Background Art

[0004] The "FLAG" tag (also known as the FLAG epitope) is an artificial antigenic peptide tag that is often incorporated into proteins to facilitate detection and / or affinity purification of the target protein. The FLAG tag is one of the most commonly used protein tags in laboratories around the world.

[0005] Antibodies that bind to the FLAG tag are known, however, these antibodies are produced in mice or other rodents and are therefore not suitable for clinical applications. Although it is generally acceptable to use mouse anti-FLAG antibodies to detect or purify recombinant proteins in in vitro systems, it is not always appropriate to use mouse antibodies to detect or bind to the FLAG tag in in vivo systems.

[0006] Therefore, there is a need for new and / or improved anti-FLAG antibodies.

[0007] Reference to any prior art in the specification is not an acknowledgement or implication that the prior art forms part of the common general knowledge in any jurisdiction, or that the prior art could reasonably be expected to be understood by a person skilled in the art, could be considered relevant and / or could be combined with other prior art. Summary of the invention

[0008] The present invention relates to an antigen binding protein comprising an antigen binding domain for binding to a FLAG tag. In particular, the present invention relates to a humanized anti-FLAG antibody and a FLAG binding fragment thereof.

[0009] The present invention provides an antigen-binding protein for binding to a FLAG tag, the antigen-binding protein comprising an antigen-binding domain, the antigen-binding domain comprising:

[0010] FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4, and

[0011] FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0012] in:

[0013] FR1, FR2, FR3 and FR4 are each a framework region;

[0014] CDR1, CDR2, and CDR3 are each complementarity determining regions;

[0015] FR1a, FR2a, FR3a and FR4a are each framework regions;

[0016] CDR1a, CDR2a, and CDR3a are each complementarity determining regions;

[0017] Wherein the sequence of any of the framework regions and / or any of the complementarity determining regions is as described herein, preferably as described in Table 1 below.

[0018] In any embodiment, CDR1, CDR2 and CDR3 refer to the complementary determining region from the variable heavy chain (VH) of an antibody, and CDR1a, CDR2a and CDR3a are complementary determining regions from the variable light chain (VL) of an antibody, or CDR1, CDR2 and CDR3 are complementary determining regions from VL, and CDR1a, CDR2a and CDR3a are complementary determining regions from VH. In these examples, the CDRs may be referred to as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, respectively, depending on the specific circumstances.

[0019] In any embodiment, the antigen binding protein of the invention is capable of specifically binding to a FLAG tag or a variant thereof (such as defined in SEQ ID NO: 11 and 29 or as otherwise defined herein). The antigen binding protein of the invention is preferably capable of specifically binding to a protein domain comprising multiple FLAG tag sequences (e.g., 2×FLAG, 3×FLAG, etc.).

[0020] In any embodiment, the antigen binding protein of the invention is used to specifically bind to a peptide tag comprising or consisting of the sequence DYK, preferably the sequence DYKD (SEQ ID NO: 11). In addition to these sequences, other amino acids may be present, preferably hydrophilic amino acids such as R (arginine), D (aspartic acid), E (glutamic acid) and K (lysine), and / or amino acids with aromatic side chains such as Y (tyrosine), F (phenylalanine), H (histidine) and W (tryptophan).

[0021] In a preferred embodiment, the antigen binding protein is capable of specifically binding to a FLAG tag comprising or consisting of the following sequence: GDYKDDDDKG (SEQ ID NO: 29), DYKDDDDK (SEQ ID NO: 30), MDYKDDDDK (SEQ ID NO: 31), DFKDDDK (SEQ ID NO: 32), DYKAFDNL (SEQ ID NO: 33), DYKDHDG (SEQ ID NO: 34), MDFKDDDDK (SEQ ID NO: 35), MDYKAFDNL (SEQ ID NO: 36), DYKDHDI (SEQ ID NO: 37), DYKDH (SEQ ID NO: 38), DYKDD (SEQ ID NO: 39), DYKDHD (SEQ ID NO: 40) and / or DYKDDD (SEQ ID NO: 41). The most preferred sequence is DYKDDDDK (SEQ ID NO: 30).

[0022] As used herein, the term FLAG tag also refers to modified FLAG tags, which are tags derived from the above-mentioned FLAG tag, especially tags having the sequence of DYKDDDDK by amino acid insertion, deletion or substitution.

[0023] The protein or antibody "binding to" the FLAG tag mentioned herein literally means supporting the statement that the protein or antibody "specifically binds to" or "specifically binds to" the FLAG tag.

[0024] The antigen binding proteins of the present invention are used to specifically bind to FLAG tags, and are particularly suitable for use in the context of recombinant proteins or therapeutic proteins (including antibodies or fragments thereof, or proteins comprising antigen binding domains for treatment) comprising FLAG tags, or cell-based therapies (including cellular immunotherapy of genetically modified cells). In some examples, the antigen binding proteins can be used to bind to the FLAG tag contained in the chimeric antigen receptor, and to bind to the relevant recombinant receptors for expression by cellular immunotherapeutics (e.g., contained in chimeric antigen receptors (CARs) expressed by cytotoxic T cells).

[0025] In any embodiment, the FLAG tag is present at the N-terminus, at the C-terminus, or within the protein to which it is intended to bind.

[0026] In any embodiment, the present invention provides an antigen binding protein for binding to a FLAG tag, wherein the antigen binding protein competitively inhibits binding to the FLAG tag of the following antibodies:

[0027] - comprising a VH comprising the sequence shown in SEQ ID NO: 1 and a VL comprising the sequence shown in SEQ ID NO: 6;

[0028] - comprising a VH comprising the sequence shown in SEQ ID NO: 2 and a VL comprising the sequence shown in SEQ ID NO: 7;

[0029] - comprising a VH comprising the sequence shown in SEQ ID NO: 2 and a VL comprising the sequence shown in SEQ ID NO: 8;

[0030] - comprising a VH comprising the sequence shown in SEQ ID NO: 2 and a VL comprising the sequence shown in SEQ ID NO: 9;

[0031] - comprising a VH comprising the sequence shown in SEQ ID NO: 2 and a VL comprising the sequence shown in SEQ ID NO: 10;

[0032] - comprising a VH comprising the sequence shown in SEQ ID NO: 3 and a VL comprising the sequence shown in SEQ ID NO: 7;

[0033] - comprising a VH comprising the sequence shown in SEQ ID NO: 3 and a VL comprising the sequence shown in SEQ ID NO: 8;

[0034] - comprising a VH comprising the sequence shown in SEQ ID NO: 3 and a VL comprising the sequence shown in SEQ ID NO: 9;

[0035] - comprising a VH comprising the sequence shown in SEQ ID NO: 3 and a VL comprising the sequence shown in SEQ ID NO: 10;

[0036] - comprising a VH comprising the sequence shown in SEQ ID NO: 4 and a VL comprising the sequence shown in SEQ ID NO: 7;

[0037] - comprising a VH comprising the sequence shown in SEQ ID NO: 4 and a VL comprising the sequence shown in SEQ ID NO: 8;

[0038] - comprising a VH comprising the sequence shown in SEQ ID NO: 4 and a VL comprising the sequence shown in SEQ ID NO: 9;

[0039] - comprising a VH comprising the sequence shown in SEQ ID NO: 4 and a VL comprising the sequence shown in SEQ ID NO: 10;

[0040] - comprising a VH comprising the sequence shown in SEQ ID NO: 5 and a VL comprising the sequence shown in SEQ ID NO: 7;

[0041] - comprising a VH comprising the sequence shown in SEQ ID NO: 5 and a VL comprising the sequence shown in SEQ ID NO: 8;

[0042] - comprising a VH comprising the sequence shown in SEQ ID NO: 5 and a VL comprising the sequence shown in SEQ ID NO: 9; or

[0043] - comprising a VH comprising the sequence shown in SEQ ID NO:5 and a VL comprising the sequence shown in SEQ ID NO:10.

[0044] In any embodiment, the invention provides an antigen binding protein of CDRH1, CDRH2 and / or CDRH3 having an antigen binding domain having a variable heavy chain as defined in any one of SEQ ID NOs: 2 to 5.

[0045] In any embodiment, the invention provides an antigen binding protein of CDRL1, CDRL2 and / or CDRL3 having an antigen binding domain having a variable light chain as defined in any one of SEQ ID NOs: 7 to 10.

[0046] In any embodiment, the present invention provides an antigen binding protein comprising CDR1, CDR2 and / or CDR3 of an antigen binding domain having a variable heavy chain as defined in any one of SEQ ID NOs: 2 to 5, and comprising CDR1, CDR2 and / or CDR3 of an antigen binding domain having a variable light chain as defined in any one of SEQ ID NOs: 7 to 10.

[0047] In one embodiment, the antigen binding domain comprises:

[0048] (a) and (b):

[0049] (a) a VH comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 12; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 13; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 14. The sequence of NO:14 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity; and

[0050] (b) a VL comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 21; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 22; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 22. The sequence of NO:23 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity;

[0051] or (c) and (d):

[0052] (c) a VH comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 42 or 43; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 44; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 45. The sequence of NO:45 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity; and

[0053] (d) a VL comprising complementarity determining regions CDR1, CDR2, and CDR3, CDR1 comprising or consisting of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:51; CDR2 comprising or consisting of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:52; and CDR3 comprising or consisting of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:52. The sequence of NO:23 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity;

[0054] or (e) and (f):

[0055] (e) a VH comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 42 or 43; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 44; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 45. The sequence of NO:45 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity; and

[0056] (f) a VL comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 64; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 52; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 52. The sequence of NO:23 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity;

[0057] or (g) and (h):

[0058] (g) a VH comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 42 or 87; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 88; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 88. The sequence of NO:45 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity; and

[0059] (h) a VL comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:51; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:52; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:52. The sequence of NO:23 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity;

[0060] Or (i) and (j):

[0061] (i) a VH comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 42 or 87; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 88; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 88. The sequence of NO:45 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity; and

[0062] (j) a VL comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 64; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 52; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 52. The sequence of NO:23 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity;

[0063] Or (k) and (l):

[0064] (k) a VH comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:57; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:58; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:58. The sequence of NO:45 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity; and

[0065] (l) a VL comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:51; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:52; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:52. The sequence of NO:23 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity;

[0066] Or (m) and (n):

[0067] (m) a VH comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:57; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:58; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:58. The sequence of NO:45 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity; and

[0068] (n) a VL comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:64; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:52; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:52. The sequence of NO:23 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity;

[0069] Or (o) and (p):

[0070] (o) a VH comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:42; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:63; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:63. NO:45 has or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical; and

[0071] (p) a VL comprising complementarity determining regions CDR1, CDR2, and CDR3, CDR1 comprising or consisting of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:64; CDR2 comprising or consisting of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:52; and CDR3 comprising or consisting of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:52. The sequence of NO:23 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity;

[0072] Or (q) and (r):

[0073] (q) a VH comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:57; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:58; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:58. The sequence of NO:45 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity; and

[0074] (r) a VL comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:64; CDR2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:52; and CDR3 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:52. The sequence of NO:23 has an amino acid sequence composition of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity;

[0075] In one embodiment, the antigen binding domain comprises:

[0076] (a) and (b):

[0077] (a) a VH comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 12; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14; and

[0078] (b) a VL comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 21; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 22; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 23;

[0079] or (c) and (d):

[0080] (c) a VH comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 42 or 43; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 44; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 45; and

[0081] (d) a VL comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 51; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 52; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 23;

[0082] or (e) and (f):

[0083] (e) a VH comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 42 or 43; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 44; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 45; and

[0084] (f) a VL comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 64; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 52; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 23;

[0085] or (g) and (h):

[0086] (g) a VH comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO:42 or 87; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO:88; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:45; and

[0087] (h) a VL comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 51; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 52; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 23;

[0088] Or (i) and (j):

[0089] (i) a VH comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO:42 or 87; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO:88; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:45; and

[0090] (j) a VL comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 64; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 52; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 23;

[0091] Or (k) and (l):

[0092] (k) a VH comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 57; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 58; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 45; and

[0093] (1) a VL comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 51; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 52; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 23;

[0094] Or (m) and (n):

[0095] (m) a VH comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 57; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 58; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 45; and

[0096] (n) a VL comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 64; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 52; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 23;

[0097] Or (o) and (p):

[0098] (o) a VH comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO:42; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO:63; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:45; and

[0099] (p) a VL comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 64; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 52; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 23;

[0100] Or (q) and (r):

[0101] (q) a VH comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 57; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 58; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 45; and

[0102] (r) a VL comprising complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO:64; CDR2 comprises or consists of the amino acid sequence of SEQ ID NO:52; and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:23.

[0103] In any embodiment, FR1, FR2, FR3 and FR4 may refer to the framework region from the variable heavy chain (VH) of an antibody, FR1a, FR2a, FR3a and FR4a may refer to the framework region from the variable light chain (VL) of an antibody, or FR1, FR2, FR3 and FR4 are the framework region from VL, and FR1a, FR2a, FR3a and FR4a are the framework region from VH. In such examples, FR can be referred to as FR H1, FR H2, FR H3, FR H4, FR L1, FR L2, FR L3 and FR L4, respectively, according to the specific circumstances.

[0104] In any embodiment, the invention provides an antigen binding protein having FR H1, FR H2, FR H3 and / or FR H4 from a human germline, wherein the human germline is IGHV1-46*01 or IGHV7-4-1*02.

[0105] In any embodiment, the invention provides an antigen binding protein having FR L1, FR L2, FR L3 and / or FR L4 from a human germline, wherein the human germline is IGKV2-30*01 or IGKV4-1*01.

[0106] In any embodiment, the invention provides an antigen binding protein having FR H1, FR H2, FR H3 and / or FR H4 from a human germline, wherein the human germline is IGHV1-46*01 or IGHV7-4-1*02, and the invention provides an antigen binding protein having FR L1, FR L2, FR L3 and / or FR L4 from a human germline, wherein the human germline is IGKV2-30*01 or IGKV4-1*01.

[0107] In any embodiment, the antigen binding protein comprises a VH that is greater than 80%, 81%, 82%, 8%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% identical to a human, preferably, the percent identity to a human is calculated as described in Example 1, and / or the antigen binding protein comprises a VL that is greater than 80%, 81%, 82%, 8%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% identical to a human, preferably, the percent identity to a human is calculated as described in Example 1.

[0108] In any embodiment, the invention provides an antigen binding protein having FR H1, FR H2, FR H3 and / or FR H4 of an antigen binding domain having a variable heavy chain region as defined in any one of SEQ ID NOs: 2 to 5.

[0109] In any embodiment, the present invention provides an antigen binding protein having FR L1, FR L2, FR L3 and / or FR L4 of an antigen binding domain having a variable light chain region as defined in any one of SEQ ID NOs: 7 to 10.

[0110] In any embodiment, the present invention provides an antigen binding protein comprising FR1, FR2, FR3 and / or FR4 of an antigen binding domain having a variable heavy chain defined by any one of SEQ ID NOs: 2 to 5, and comprising FR1, FR2, FR3 and / or FR4 of an antigen binding domain having a variable light chain defined by any one of SEQ ID NOs: 7 to 10.

[0111] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises:

[0112] A. A VH comprising a complementarity determining region (CDR) 1, CDR2, and CDR3 as defined in any of (a) above, and a VL comprising CDR1, CDR2, and CDR3 as defined in any of (b) above, and the antigen binding domain comprises any of the following B., C., D., E., F., G., H., I., J., K., L., M., N., O., P., Q., or R.:

[0113] B. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 65, 66, 67, and 68, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively,

[0114] or

[0115] C. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively,

[0116] or

[0117] D. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively,

[0118] or

[0119] E. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively,

[0120] or

[0121] F. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively,

[0122] or

[0123] G. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively,

[0124] or

[0125] H. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively,

[0126] or

[0127] I. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively,

[0128] or

[0129] J. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively,

[0130] or

[0131] K. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively,

[0132] or

[0133] L. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively,

[0134] or

[0135] M. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively,

[0136] or

[0137] N. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively,

[0138] or

[0139] O. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively,

[0140] or

[0141] P. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively,

[0142] or

[0143] Q. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively,

[0144] or

[0145] R. VH and VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively.

[0146] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises:

[0147] A. A VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any of (c) above, and a VL comprising CDR1, CDR2 and CDR3 as defined in any of (d) above, and the antigen binding domain comprises any of the following B., C., D. or E.:

[0148] B. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively,

[0149] or

[0150] C. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively,

[0151] or

[0152] D. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively,

[0153] or

[0154] E. VH and VL each comprising framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively.

[0155] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises:

[0156] A. A VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any of (e) above, and a VL comprising CDR1, CDR2 and CDR3 as defined in any of (f) above, and the antigen binding domain comprises any of the following B., C., D. or E.:

[0157] B. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively,

[0158] or

[0159] C. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively,

[0160] or

[0161] D. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively,

[0162] or

[0163] E. VH and VL each comprising framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively.

[0164] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises:

[0165] A. A VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any of (g) above, and a VL comprising CDR1, CDR2 and CDR3 as defined in any of (h) above, and the antigen binding domain comprises any of the following B., C., D. or E.:

[0166] B. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively,

[0167] or

[0168] C. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively,

[0169] or

[0170] D. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively,

[0171] or

[0172] E. VH and VL each comprising framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively.

[0173] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises:

[0174] A. A VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any of (i) above, and a VL comprising CDR1, CDR2 and CDR3 as defined in any of (j) above, and the antigen binding domain comprises any of the following B., C., D. or E.:

[0175] B. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively,

[0176] or

[0177] C. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively,

[0178] or

[0179] D. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively,

[0180] or

[0181] E. VH and VL each comprising framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively.

[0182] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises:

[0183] A. A VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any of (k) above, and a VL comprising CDR1, CDR2 and CDR3 as defined in any of (l) above, and the antigen binding domain comprises any of B., C., D., E., F., G., H. or I. below:

[0184] B. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively,

[0185] or

[0186] C. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively,

[0187] or

[0188] D. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively,

[0189] or

[0190] E. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively,

[0191] or

[0192] F. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively;

[0193] or

[0194] G. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively;

[0195] or

[0196] H. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 96, 98, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively;

[0197] or

[0198] I. VH and VL each comprising framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 96, 98 and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56 and 28, respectively.

[0199] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises:

[0200] A. A VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any of (m) above, and a VL comprising CDR1, CDR2 and CDR3 as defined in any of (n) above, and the antigen binding domain comprises any of B., C., D., E., F., G., H. or I. below:

[0201] B. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively;

[0202] or

[0203] C. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 99, 100, 101, and 28, respectively;

[0204] or

[0205] D. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively;

[0206] or

[0207] E. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 99, 100, 101, and 28, respectively;

[0208] or

[0209] F. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively;

[0210] or

[0211] G. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 99, 100, 101, and 28, respectively;

[0212] or

[0213] H. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 96, 98, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively;

[0214] or

[0215] I. VH and VL each comprising framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 96, 98 and 20, respectively, and for each VLFR, the reference sequence is set forth in SEQ ID NOs: 99, 100, 101 and 28, respectively.

[0216] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises:

[0217] A. a VH comprising complementarity determining regions (CDR) 1, CDR2 and CDR3 as defined above in (o), and a VL comprising CDR1, CDR2 and CDR3 as defined above in (p), and the VH and VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 72, 73, 74 and 68, respectively, and for each VLFR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77 and 28, respectively;

[0218] or

[0219] B. A VH comprising complementarity determining regions (CDR) 1, CDR2 and CDR3 as defined in (q) above, and a VL comprising CDR1, CDR2 and CDR3 as defined in (r) above, and the VH and VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 78, 79 and 68, respectively, and for each VLFR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77 and 28, respectively.

[0220] In any embodiment, the antigen binding domain comprises a variable heavy chain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 5, or a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence.

[0221] In any embodiment, the antigen binding domain comprises a variable light chain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 7 to 10, or a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence.

[0222] In any embodiment, the antigen binding domain comprises a variable heavy chain and a variable light chain, the variable heavy chain comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 2 to 5, or comprising a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto; the variable light chain comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 7 to 10, or comprising a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0223] As described herein, the antigen binding protein may be in the form of:

[0224] (i) Single domain antibodies (sdAb);

[0225] (ii) single-chain Fv fragment (scFv);

[0226] (iii) dimeric single-chain Fv fragment (di-scFv);

[0227] (iv) one of (ii) or (iii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody;

[0228] (v) one of (i) to (iv) linked to a protein that binds to immune effector cells;

[0229] (vi) one of (i) to (iv) linked to a modified immune cell receptor (such as a modified T cell receptor); or

[0230] (vii) one of (i) to (iv) in the context of a chimeric antigen receptor (CAR) or a variant T cell receptor.

[0231] Furthermore, as described herein, the antigen binding protein may be in the form of:

[0232] (i) Diabodies;

[0233] (ii) tri-antibody;

[0234] (iii) tetrabodies;

[0235] (iv) Fab;

[0236] (v) F(ab')2;

[0237] (vi) Fv;

[0238] (vii) bispecific antibodies or other forms of multispecific antibodies;

[0239] (viii) one of (i) to (vii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody;

[0240] (ix) one of (i) to (vii) linked to a protein that binds to immune effector cells;

[0241] (x) one of (i) to (vii) linked to a protein that binds to immune effector cells;

[0242] (xi) one of (i) to (vii) linked to a modified immune cell receptor (such as a modified T cell receptor); or

[0243] (xiii) one of (i) to (vii) in the context of a chimeric antigen receptor (CAR) or variant T cell receptor (including in the context of a universal CAR system for use with a polypeptide comprising an antigen binding domain for binding to an antigen on the surface of a target cell).

[0244] The aforementioned antigen-binding protein may also be referred to as an antibody antigen-binding domain.

[0245] Preferably, the antigen-binding proteins described herein are antibodies or antigen-binding fragments thereof. Typically, the antigen-binding proteins are antibodies, such as monoclonal antibodies. The antigen-binding proteins may be in the form of recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primate antibodies, deimmunized antibodies, synthetic humanized antibodies, half antibodies, bispecific antibodies, trispecific antibodies or multispecific antibodies). The antibody may also include chemical modifications, such as coupling with an active agent or a radioactive marker, or coupling with an agent for improving solubility, or other modifications described herein.

[0246] As used herein, an antigen binding protein may be a variable domain.

[0247] The present invention provides an antigen-binding protein, which comprises the following amino acid sequence (in order from N-terminus to C-terminus or from C-terminus to N-terminus), essentially consists of the following amino acid sequence (in order from N-terminus to C-terminus or from C-terminus to N-terminus), or consists of the following amino acid sequence (in order from N-terminus to C-terminus or from C-terminus to N-terminus):

[0248] - SEQ ID NO: 2 and 10;

[0249] - SEQ ID NO: 3 and 9;

[0250] - SEQ ID NO: 2 and 9;

[0251] - SEQ ID NO: 2 and 7;

[0252] - SEQ ID NO: 2 and 8;

[0253] - SEQ ID NO: 3 and 7;

[0254] - SEQ ID NO: 3 and 8;

[0255] - SEQ ID NO: 3 and 10;

[0256] - SEQ ID NO: 4 and 7;

[0257] - SEQ ID NO: 4 and 8;

[0258] - SEQ ID NO: 4 and 9;

[0259] - SEQ ID NO: 4 and 10;

[0260] - SEQ ID NO: 5 and 7;

[0261] - SEQ ID NO: 5 and 8;

[0262] - SEQ ID NO: 5 and 9; or

[0263] - SEQ ID NO: 5 and 10.

[0264] As used herein, the complementarity determining region (CDR) sequences of the antigen binding proteins of the invention are defined according to the IMGT, Chothia or Kabat numbering systems.

[0265] The present invention provides an antigen binding protein as described herein, wherein the amino acid sequences forming one or more of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are human sequences.

[0266] The present invention provides an anti-FLAG tag antigen binding protein, an immunoglobulin variable domain, an antibody, a dab, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule or a multispecific antibody, wherein the above-mentioned substance comprises an antigen binding protein having a sequence as described herein or comprises a CDR and / or FR sequence as described herein.

[0267] The present invention provides a diabody or triabody having an antigen binding protein having a sequence as described herein or comprising a CDR and / or FR sequence as described herein.

[0268] The present invention provides a fusion protein, which comprises an antigen binding protein, an immunoglobulin variable domain, an antibody, a dab, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule or a multispecific antibody as described herein.

[0269] In one embodiment, the antigen-binding proteins of the present invention may be included as a component of a chimeric antigen protein (CAR) or a variant T cell receptor. In such embodiments, it should be understood that the generated CAR or variant T cell receptor comprising the antigen-binding proteins of the present invention may be referred to as "indirect CAR" (sometimes also referred to as universal CAR). In an indirect CAR system, the antigen-binding domain of CAR does not directly bind to the target antigen on the target cell, but binds to an intermediate, wherein the intermediate includes an antigen-binding domain for directly binding to the target cell. The example of CAR for identifying cells by an intermediate is known in the art, for example, in European patent application EP2651442.

[0270] Therefore, in the context of indirect CAR, the antigen-binding proteins of the present invention can be used to bind to the FLAG tag present in the intermediate, and wherein the intermediate also has an antigen binding domain for binding to a target cell (e.g., a cancer cell). The intermediate provides specificity to target cells (such as cancer cells), and the genetically modified cells with CAR provide efficacy and guide the immune response to the target cell. The intermediate can be a molecule such as a probe that directly binds or interacts with a target antigen on a target cell, and the intermediate also includes a FLAG tag. Non-limiting examples of such probes include antibodies, fusion proteins, Fabs of antibodies, scFv, soluble engineered TCRs or aptamers. The term "aptamer" refers to any oligonucleotide, polynucleic acid, peptide or polypeptide that specifically binds to a target or preferentially forms a complex with a target.

[0271] The present invention also provides a conjugate, which is in the form of an antigen-binding protein, an immunoglobulin variable domain, an antibody, a dab, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule, a multispecific antibody or a fusion protein as described herein, wherein the above-mentioned substance is conjugated with a marker or a cytotoxic agent.

[0272] The present invention provides an antibody for binding to an antigen-binding protein, an immunoglobulin variable domain, an antibody, a dab, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule, a multispecific antibody, a fusion protein or a conjugate as described herein.

[0273] In aspects of the invention involving multiple polypeptide chains forming an antigen binding protein, the expression construct comprises a nucleic acid encoding a polypeptide comprising, for example, a VH operably linked to a promoter, and a nucleic acid encoding a polypeptide comprising, for example, a VL operably linked to a promoter.

[0274] In another example, the expression construct is a bicistronic expression construct, for example comprising the following operably linked components in 5' to 3' order:

[0275] (i) a promoter;

[0276] (ii) a nucleic acid encoding a first polypeptide;

[0277] (iii) an internal ribosome entry site; and

[0278] (iv) a nucleic acid encoding a second polypeptide,

[0279] wherein the first polypeptide comprises VH and the second polypeptide comprises VL, or vice versa.

[0280] The present invention also encompasses separate expression constructs, wherein one expression construct encodes a first polypeptide comprising VH and another expression construct encodes a second polypeptide comprising VL. For example, the present invention also provides a composition comprising:

[0281] (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and

[0282] (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter.

[0283] The invention provides a cell comprising a vector or nucleic acid as described herein. Preferably, the cell is isolated, substantially purified or recombinant. In one example, the cell comprises an expression construct of the invention or:

[0284] (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and

[0285] (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter,

[0286] Therein, the first polypeptide and the second polypeptide are combined to form the antigen-binding protein of the present invention.

[0287] Examples of cells of the present invention include bacterial cells, yeast cells, insect cells or mammalian cells.

[0288] The present invention provides a nucleic acid encoding an antigen binding protein, an immunoglobulin variable domain, an antibody, a dab, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule, a multispecific antibody, a fusion protein or a conjugate as described herein.

[0289] The present invention provides a vector comprising the nucleic acid described herein. Preferably, the nucleic acid has a nucleotide sequence encoding any one or more of the amino acid sequences corresponding to SEQ ID NOs: 2 to 5 and SEQ ID NOs: 7 to 10.

[0290] The present invention provides a cell comprising the vector or nucleic acid as described herein.

[0291] In another embodiment, an animal comprising a cell as described herein or a tissue derived from the animal is provided.

[0292] The present invention provides a pharmaceutical composition comprising an antigen-binding protein, or comprising a CDR and / or FR sequence as described herein, or comprising an immunoglobulin variable domain, antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single-chain antibody molecule or multispecific antibody, fusion protein or conjugate as described herein, and a pharmaceutically acceptable carrier, diluent or excipient.

[0293] The present invention provides a diagnostic composition comprising an antigen binding protein, or comprising a CDR and / or FR sequence as described herein, or comprising an immunoglobulin variable domain, an antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule or a multispecific antibody, a fusion protein or a conjugate as described herein, a diluent, and an optional marker.

[0294] The present invention provides a kit or article, which comprises an antigen binding protein, or comprises a CDR and / or FR sequence as described herein, or comprises an immunoglobulin variable domain, an antibody, a dab, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody or a multispecific antibody, a fusion protein or a conjugate as described herein.

[0295] The present invention provides use of a sequence according to one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3 and FR4 as described herein in the preparation of an antigen binding protein for binding to a FLAG tag.

[0296] The present invention provides a library of nucleic acid molecules generated by mutations of the antigen binding proteins or CDR and / or FR sequences as described herein, wherein at least one nucleic acid molecule in the library encodes an antigen binding protein for binding to a FLAG tag.

[0297] The present invention provides a method for preparing an antigen-binding protein for binding to a FLAG tag as described herein, the method comprising expressing the nucleic acid as described herein in a cell or an animal as described herein.

[0298] In any aspect of the invention, the antigen binding protein comprises an Fc region that is engineered to have an enhanced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, the enhanced ability to induce ADCC is conferred by a mutation, deletion or modification of an amino acid that interacts with an Fc receptor in the Fc region. Preferably, the mutation, deletion or modification of an amino acid is located at position 239, position 330 and / or position 332 (wherein alanine is located at position 118) according to SEQ ID NO: 60, or at an equivalent position of position 239, position 330 and / or position 332. Preferably, the amino acid mutation is S239D, A330 L and I332E. Typically, Fc comprises an amino acid sequence as shown in SEQ ID NO: 62, is substantially composed of an amino acid sequence as shown in SEQ ID NO: 62, or is composed of an amino acid sequence as shown in SEQ ID NO: 62.

[0299] In any aspect of the invention, the antigen-binding protein comprises an Fc region that is not engineered to have a reduced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, according to SEQ ID NO:60 (wherein alanine is position 118), the amino acid at position 234, position 235 and / or position 331, or the amino acid at the equivalent position to position 234, position 235 and / or position 331 is not phenylalanine (F), glutamic acid (E) and / or serine (S), respectively. In other words, the amino acid at position 234 is not F, the amino acid at position 235 is not E, and / or the amino acid at position 331 is not S.

[0300] In another embodiment, the antigen binding protein comprises an Fc region that is engineered to:

[0301] - Increase in vitro or in vivo half-life;

[0302] - having the ability to induce an increase in antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity;

[0303] - reduced effector function; or

[0304] -Increase co-engagement of antigen binding proteins.

[0305] The present invention provides a use of an antigen-binding protein, an immunoglobulin variable domain, an antibody, a dab, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule or a multispecific antibody, a fusion protein, a conjugate, or a pharmaceutical composition as described herein in the preparation of a medicament for treating cancer or a disorder or disease associated with FLAG tag expression.

[0306] Antigen binding proteins, proteins or antibodies as described herein preferably comprise a human constant region, such as an IgG constant region, such as an IgG1, IgG2, IgG3 or IgG4 constant region or a mixture thereof. In the case of an antibody or protein comprising VH and VL, VH may be linked to the heavy chain constant region, and VL may be linked to the light chain constant region.

[0307] The functional characteristics of the antigen binding proteins of the invention will be used to characterize the antibodies of the invention mutatis mutandis.

[0308] The antigen binding proteins as described herein may be purified, substantially purified, isolated and / or recombinant.

[0309] The antigen-binding protein of the present invention may be part of a supernatant taken from a culture medium in which a hybridoma expressing the antigen-binding protein of the present invention has been grown.

[0310] The present invention provides single domain antibodies comprising an antigen binding protein for binding to a FLAG tag.

[0311] As used herein, the term "comprise" and variations of the term such as "comprising", "comprises" and "comprised" are not intended to exclude additional additives, components, integers or steps, unless the context requires otherwise.

[0312] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0313] Figure 1 ELISA titration of humanized FLAG-tag binding monoclonal antibodies for IgG.

[0314] Figure 2 A humanized monoclonal antibody used to bind to FLAG-tagged CAR induces IFNγ secretion from CART cells.

[0315] Figure 3 Alignment of the VH and VL regions from the parental antibody and the humanized variants v4 and v7.

[0316] Sequence information

[0317] Table 1: Sequence information

[0318]

[0319]

[0320]

[0321]

[0322]

[0323] DETAILED DESCRIPTION

[0324] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0325] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

[0326] Reference will now be made in detail to certain embodiments of the present invention. Although the present invention will be described in conjunction with each embodiment, it should be understood that the purpose of the present invention is not to limit the present invention to those embodiments. On the contrary, the present invention is intended to cover all substitutions, modifications and equivalents that may be included within the scope of the present invention defined by the claims.

[0327] The inventors have developed antigen binding proteins, such as antibodies, that bind to FLAG tags. Antigen binding proteins as described herein are used to bind to one or more FLAG tags present in any of the following, each of which is a recombinant protein, a recombinant peptide, a genetically modified cell (including a genetically modified cell modified to express a receptor on its surface, wherein the receptor includes one or more FLAG tags). In a particularly preferred embodiment, the antigen binding protein can be used to bind to one or more FLAG tags present in a chimeric antigen receptor (CAR) expressed by a genetically modified cell such as a cytotoxic cell.

[0328] The antigen-binding proteins of the present invention are humanized antibodies, making them particularly suitable for binding to CAR or other receptors or proteins, which are expressed by genetically engineered cells for human therapeutic agents. In particular, proteins engineered to include FLAG tags (such as tags described herein) can be bound by the antigen-binding proteins of the present invention in vitro or in vivo.

[0329] General

[0330] Throughout this specification, reference to a single step, composition of matter, group of steps, or group of compositions of matter shall encompass one or more (i.e., one or more) of such steps, compositions of matter, groups of steps, or groups of compositions of matter, unless expressly stated otherwise or the context requires otherwise. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects, and vice versa unless the context clearly dictates otherwise. For example, reference to "a" includes the single as well as two or more; reference to "an" includes the single as well as two or more; and reference to "the" includes the single as well as two or more, etc.

[0331] It will be appreciated by those skilled in the art that the present invention is susceptible to variations and modifications other than those specifically described. It should be understood that the present invention includes all such variations and modifications. The present invention also includes all steps, features, compositions and compounds mentioned or indicated in this specification sheet, individually or jointly, and any steps or features of these steps or features, all combinations of these steps or features, or any two or more steps or features of these steps or features.

[0332] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used to practice the present invention. The present invention is in no way limited to such methods and materials.

[0333] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0334] The present invention is not to be limited in scope by the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions and methods are clearly within the scope of the present invention.

[0335] Unless specifically stated otherwise, any example or embodiment of the invention herein should be considered applicable to any other example or embodiment of the invention.

[0336] Unless specifically defined otherwise, all technical and scientific terms used herein should be interpreted as having the same meanings as commonly understood by one of ordinary skill in the art (eg, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0337] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and illustrated in numerous references, for example: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Humes (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989). Harbour Laboratory, (1988), and JE Coligan et al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0338] The description and definition of variable regions and components thereof, immunoglobulins, antibodies and fragments thereof herein can be further clarified by the discussion in the following references: Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196: 901-917, 1987, Chothia et al. Nature 342, 877-883, 1989 and / or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

[0339] The term "and / or", such as "X and / or Y", should be understood to mean "X and Y" or "X or Y", and the term "and / or" should be understood to provide clear support for both meanings or for either meaning.

[0340] As used herein, the term "derived from" should be taken to mean that a particular thing can be obtained from a particular source, although not necessarily directly from that source.

[0341] References to ranges of, for example, residues herein should be understood to include the endpoints. For example, reference to "a region comprising amino acids 56 to 65" should be understood to include the endpoints, i.e., the region includes the amino acid sequences numbered 56, 57, 58, 59, 60, 61, 62, 63, 64, and 65 in the particular sequence.

[0342] Selected Definitions

[0343] As used herein, FLAG tag or FLAG octapeptide or FLAG epitope is a polypeptide protein tag that can be added to a protein using recombinant DNA technology, which has a sequence motif DYKDDDDK (SEQ ID NO: 29). The FLAG tag can be fused to the C-terminus or N-terminus of the protein, or can be inserted into the protein. Therefore, the antigen-binding protein of the present invention can specifically bind to the FLAG tag, whether the tag is present at the C-terminus or N-terminus of the protein or inserted into the protein. In a specific example, the FLAG tag can be included in a chimeric antigen receptor (CAR) construct, so the antigen-binding protein of the present invention can bind to CAR through the FLAG tag.

[0344] As used herein, "chimeric antigen receptor (CAR)" refers to a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain (the transmembrane domain is derived from a polypeptide different from the polypeptide from which the extracellular domain is derived), and at least one intracellular domain. "Chimeric Antigen Receptor (CAR)" is sometimes also referred to as a "chimeric receptor", "T body" or "chimeric immune receptor (CIR)". "Extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to a specific antigen. "Intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that can transmit signals to cause activation or inhibition of intracellular biological processes.

[0345] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that, due to its source or derived source, is not associated with naturally associated components with which it is naturally associated, and is substantially free of other proteins from the same source. Proteins can be substantially free of naturally associated components or substantially purified by separation using protein purification techniques known in the art. "Substantially purified" means that the protein is substantially free of contaminating substances, for example, at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% free of contaminating substances.

[0346] The term "recombination" should be understood to refer to the product of artificial gene recombination. Therefore, in the context of recombinant proteins comprising antibody antigen binding domains, the term does not include antibodies that naturally occur in the subject and are natural recombinant products during B cell maturation. However, if this antibody is isolated, then this antibody should be considered as an isolated protein comprising antibody antigen binding domains. Similarly, if the nucleic acid encoding the protein is isolated and expressed by recombinant means, the resulting protein is a recombinant protein comprising antibody antigen binding domains. Recombinant proteins also include proteins expressed by artificial recombinant means in cells, tissues or subjects (e.g., cells, tissues or subjects in which proteins are expressed).

[0347] The term "protein" should be considered to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to each other (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0348] The term "polypeptide" or "polypeptide chain" will be understood from the preceding paragraphs to mean a series of consecutive amino acids linked by peptide bonds.

[0349] As used herein, the term "antigen binding protein" is used interchangeably with "antigen binding domain" and should be considered to refer to the region of an antibody that is capable of specific binding to an antigen, i.e., VH and VL, or an Fv comprising both VH and VL. An antigen binding domain is not necessarily present in the context of an intact antibody, for example, an antigen binding domain may be in an isolated form (e.g., a domain antibody) and may also be in other forms such as scFv as described herein.

[0350] For the purposes of this disclosure, the term "antibody" includes a protein that is able to specifically bind to one or several closely related antigens by means of an antigen binding domain contained within an Fv fragment. The term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, complementary determining region transplanted antibodies, primatized antibodies, de-immunized antibodies, synthetic humanized antibodies, half antibodies, bispecific antibodies). Antibodies generally contain constant domains, which can be arranged as constant regions, constant fragments, or crystallizable fragments (Fc). An exemplary form of an antibody is a four-chain structure as its basic unit. A full-length antibody comprises two covalently linked heavy chains (~50 kD to about 70 kD) and two light chains (~23 kDa each). A light chain generally comprises a variable region (if present) and a constant domain, and in mammals, the light chain is either a kappa light chain or a lambda light chain. A heavy chain generally comprises a variable region and one or two constant domains connected to other constant domains by a hinge region. The heavy chains of mammals belong to one of the following types: α, δ, ε, γ or μ. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy chain and light chain are bound together by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds of different types of antibodies can vary. Each chain has an N-terminal variable region (VH or VL, both of which are ~110 amino acids in length) and one or more constant domains located at the C-terminus. The constant domain of the light chain (CL, which is ~110 amino acids in length) is aligned with the first constant domain of the heavy chain (CH1, which is 330 to 440 amino acids in length) and is linked by disulfide bonds. The light chain variable region is aligned with the heavy chain variable region. The antibody heavy chain may contain two or more additional CH domains (e.g., CH2, CH3, etc.), and may contain a hinge region between the CH1 constant domain and the CH2 constant domain. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (e.g., human) antibody. In one example, the antibody heavy chain lacks a C-terminal lysine residue. In one example, the antibody is humanized, synthetically humanized, chimeric, CDR-grafted, or de-immunized.

[0351] The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, rather than an antigen-binding fragment of an antibody. Specifically, whole antibodies include those comprising heavy and light chains with an Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0352] As used herein, "variable region" refers to the part of the light chain and / or heavy chain of an antibody as defined herein that can specifically bind to an antigen, and the "variable region" includes the amino acid sequence of the complementary determining region (CDR), i.e., CDR1, CDR2 and CDR3 and a framework region (FR). For example, the variable region includes three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) and three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0353] The FLAG tag according to the present invention is an amino acid-based marker, as described, for example, in EP 0150126, U.S. Pat. No. 4,703,004, U.S. Pat. No. 4,782,137 and U.S. Pat. No. 4,8151,341, and the FLAG tag according to the present invention particularly comprises or consists of the sequence DYK, preferably comprises or consists of the sequence DYKD (SEQ ID NO: 11). In addition to these sequences, other amino acids, preferably hydrophilic amino acids, such as R (Arg), D (Asp), E (Glu) and K (Lys) and / or amino acids with aromatic side chains such as Y (Tyr), F (Phe), H (His) and W (Trp) may also be present. Examples of such FLAG tags are disclosed in the aforementioned patent specifications and can be used within the scope of the present invention.

[0354] As used herein, the term "subject" should be taken to mean any animal, including humans such as mammals. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0355] "Antibodies" or "immunoglobulins" or "Ig" are gamma-globulins found in the blood or other body fluids of vertebrates that function in the immune system to bind antigens, thereby identifying and neutralizing foreign substances.

[0356] Antibodies are typically heterotetrameric glycoproteins consisting of two identical light chains (L chains) and two identical heavy chains (H chains). Each L chain is linked to an H chain by a covalent disulfide bond. Depending on the H chain isotype, the two H chains are linked to each other by one or more disulfide bonds. Each H chain and L chain also has regularly spaced intrachain disulfide bridges.

[0357] The H chain and the L chain define a specific Ig domain. More specifically, each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the α chain and the γ chain, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus and then a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain.

[0358] Antibodies can be assigned different classes or isotypes. There are five classes of immunoglobulins, namely: IgA, IgD, IgE, IgG and IgM, with heavy chains named α, δ, ε, γ and μ, respectively. The γ and α classes are further divided into subclasses based on relatively small differences in CH sequence and function, for example humans express the following subclasses, namely: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Based on the amino acid sequence of their constant domains, L chains from any vertebrate species can be classified into one of two distinct types (called κ and λ).

[0359] The constant domains include an Fc portion comprising the carboxyl terminal portions of two H chains held together by disulfides. The effector functions of antibodies such as ADCC are determined by sequences in the Fc region, which is also the portion recognized by Fc receptors (FcR) found on certain types of cells.

[0360] The pairing of VH and VL together forms a "variable region" or "variable domain" that includes the amino terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". The V domain contains antigen binding proteins that affect antigen binding and define the specificity of a particular antibody for its particular antigen. The V region spans about 110 amino acid residues and consists of relatively invariant segments, which are called framework regions (FR) of 15-30 amino acids (usually about 4), separated by shorter extreme variable regions called "hypervariable regions" (usually about 3), each of which is 9-12 amino acids in length. The FRs primarily adopt a β-sheet conformation, while the hypervariable regions form loops that connect the β-sheet conformation and in some cases also form part of the β-sheet structure.

[0361] "Hypervariable region", "HVR" or "HV" refers to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Typically, antibodies contain six hypervariable regions, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). A variety of hypervariable region demarcation methods are in use and are all contemplated herein.

[0362] As used herein, the term "complementarity determining region" (synonym: CDR, i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues in the variable region of an antibody, the presence of which plays a major role in specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs identified as CDR1, CDR2, and CDR3. The CDRs of VH are also referred to herein as CDR H1, CDR H2, and CDR H3, respectively, wherein CDR H1 corresponds to CDR1 of VH, CDR H2 corresponds to CDR2 of VH, and CDR H3 corresponds to CDR3 of VH. Similarly, the CDRs of VL are also referred to herein as CDR L1, CDR L2, and CDR L3, respectively, wherein CDR L1 corresponds to CDR 1 of VL, CDR L2 corresponds to CDR 2 of VL, and CDR L3 corresponds to CDR3 of VL. In one example, the amino acid positions assigned to CDRs and FRs are defined according to the literature on Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system"). In another example, the amino acid positions assigned to CDRs and FRs are defined according to the enhanced Chothia numbering scheme (website: http: / / www.bioinfo.org.uk / mdex.html). The present invention is not limited to FRs and CDRs defined by the Kabat numbering system, but encompasses all numbering systems, including the canonical numbering system or the numbering system discussed in Chothia and Lesk J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:877-883, 1989; Chothia et al., Nature 342:877-883, 1989; and / or the numbering system discussed in Al-Lazikani et al., J. Mol. Biol. 273:927-948, 1997; Honnegher and Plükthun J. Mol. Biol. 309:657-670, 2001; or the numbering system discussed in Giudicelli et al., Nucleic Acids The IMGT system discussed in Res. 25:206-211 1997. In one example, the CDRs are defined according to the Kabat numbering system.Alternatively, the heavy chain CDR2 according to the Kabat numbering system does not include the 5 C-terminal amino acids listed herein, or any one or more of these amino acids are substituted by another naturally occurring amino acid. In this regard, Padlan et al., FASEB J., 9: 133-139, 1995 confirmed that the 5 C-terminal amino acids of the heavy chain CDR2 are usually not involved in antigen binding.

[0363] "Framework" or "FR" residues refer to variable domain residues other than the hypervariable region or CDR residues as defined herein. The FRs of VH are also referred to herein as FR H1, FR H2, FR H3, and FR H4, respectively, wherein FR H1 corresponds to FR 1 of VH, FRH2 corresponds to FR 2 of VH, FR H3 corresponds to FR 3 of VH, and FR H4 corresponds to FR 4 of VH. Similarly, the FRs of VL are referred to herein as FR L1, FR L2, FR L3, and FR L4, respectively, wherein FR L1 corresponds to FR 1 of VL, FR L2 corresponds to FR 2 of VL, FRL3 corresponds to FR 3 of VL, and FR L4 corresponds to FR 4 of VL.

[0364] "Peptide for forming an antigen binding protein" generally refers to a peptide that can be formed into a certain structure that confers specificity to an antibody for an antigen. Examples include complete antibodies or structures related to complete antibodies, complete antibody fragments including variable domains, variable domains and fragments thereof, light and heavy chains, or fragments of light and heavy chains including part but not all of the hypervariable or constant regions.

[0365] "Complete" or "whole" antibody refers to an antibody comprising an antigen binding protein as well as CL and at least heavy chain constant domains, i.e., CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0366] "Whole antibody-related structures" include multiple forms of whole antibodies.

[0367] "Whole antibody fragments comprising variable domains" include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0368] The Fab fragment consists of the entire L chain as well as the variable region domain of the H chain (VH) and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent in terms of antigen binding, that is, each Fab fragment has a single antigen binding protein.

[0369] The Fab' fragment differs from the Fab fragment by having a few additional residues at the carboxyl terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab' in which the cysteine ​​residues of the constant domains carry a free thiol group is designated herein as Fab'-SH.

[0370] The F(ab')2 fragment roughly corresponds to two disulfide-linked Fab fragments that have divalent antigen-binding activity and are still able to cross-link antigens.

[0371] "Fv" is an antibody fragment that contains a complete antigen recognition site and an antigen binding site. The fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association.

[0372] In single-chain Fv (scFv) species, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker so that the light chain and the heavy chain can associate in a "dimer" structure similar to that of a two-chain Fv species. From the folding of these two domains, six hypervariable loops (3 loops from the H chain and 3 loops from the L chain, respectively) are emanated, which contribute amino acid residues for antigen binding and confer antigen binding specificity to the antibody.

[0373] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising a VH antibody domain and a VL antibody domain connected to form a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain, which enables the scFv to form a desired structure for antigen binding.

[0374] A "single variable domain" is half of an Fv (comprising only three CDRs specific for the antigen) that has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0375] "Diabodies" refer to antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). Small antibody fragments are prepared by constructing sFv fragments (see the previous paragraph) with short linkers (about 5-10 residues) between the VH and VL domains, so that interchain pairing of the V domains is achieved instead of intrachain pairing, thereby generating a bivalent fragment, i.e., a fragment with two antigen-binding sites.

[0376] Diabodies can be bivalent or bispecific. Bispecific diabodies are heterodimers of two "crossover" sFv fragments, in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Triabodies and tetrabodies are also well known in the art.

[0377] An "isolated antibody" is one that has been identified, separated and / or recovered from a component of its pre-existing environment. Contaminant components are materials that would interfere with the therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0378] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human being and / or has been prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigenic challenge, but whose endogenous loci have been disabled.

[0379] The non-human (e.g., rodent) antibody of "humanization" form is a chimeric antibody containing the minimum sequence derived from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the hypervariable region of the receptor are replaced by residues from the hypervariable region of a non-human species (donor antibody) such as a mouse, rat, rabbit or non-human primate with desired antibody specificity, affinity and ability. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may be included in residues not found in the receptor antibody or the donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody will include substantially all at least one, and generally two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the FRs are those of human immunoglobulin sequences. The humanized antibody optionally also includes at least a portion of an immunoglobulin constant region (Fc), generally a constant region of a human immunoglobulin.

[0380] "Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site or determinant on an antigen. In addition to their specificity, monoclonal antibodies are advantageous because they can be synthesized without contamination by other antibodies. Monoclonal antibodies can be prepared by hybridoma methods, or can be prepared using recombinant DNA methods in bacteria, eukaryotic animals or plant cells. "Monoclonal antibodies" can also be isolated from phage antibody libraries.

[0381] The monoclonal antibodies herein include "chimeric" antibodies, in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as are fragments of such antibodies, as long as they exhibit the desired biological activity. The chimeric antibodies of interest herein include "primatized" antibodies, which comprise variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0382] The term "anti-FLAG antibody" or "antibody that binds to FLAG" refers to an antibody that is capable of binding to a FLAG tag (e.g., as defined herein in SEQ ID NO: 11 or SEQ ID NO: 29) with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent when targeting proteins or cells that express or present the FLAG tag. Preferably, the extent of binding of the anti-FLAG antibody to an unrelated tag or protein is less than about 10% of the extent of binding of the antibody to FLAG when measured by methods such as radioimmunoassay (RIA). In certain embodiments, the antibody that binds to FLAG has a dissociation constant (K D )<1 μM, <100 nM, <10 nM, <1 nM or <0.1 nM.

[0383] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In general, "binding affinity" refers to intrinsic binding affinity, which reflects the one-to-one interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can often be expressed in terms of the dissociation constant (K). D ). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind to antigens faster and tend to remain bound for longer. A variety of methods for measuring binding affinity are known in the art, and any of these methods can be used for the purposes of the present invention.

[0384] As used herein, the term "binding" with respect to the interaction of an antigen-binding protein or its antigen-binding domain with an antigen means that the interaction depends on the presence of a specific structure (e.g., antigenic determinant or epitope) on the antigen. For example, antibodies generally recognize and bind to specific protein structures rather than all proteins. If an antibody binds to an epitope "A", then in a reaction containing a labeled "A" and a protein, the presence of a molecule containing an epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" bound to the antibody.

[0385] As used herein, the term "specifically binds" or "specifically binds" should be taken to mean that an antigen binding protein of the invention reacts or associates with a specific antigen or cell expressing the same more frequently, faster, longer duration and / or with greater affinity than it would with an alternative antigen or cell.

[0386] As used herein, the term "no detected binding" is understood to mean that the level of binding of an antigen-binding protein (e.g., an antibody) to a candidate antigen is less than 10%, or 8%, or 6%, or 5% above the background level. The background level can be the level of binding signal detected in the absence of the protein and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or the level of binding detected in the presence of a negative control antigen. The binding level is detected by a biosensor analysis (e.g., Biacore), in which the antigen-binding protein is immobilized and contacted with the antigen.

[0387] As used herein, the term "no significant binding" should be understood to mean that the level of binding of the antigen-binding protein of the present invention to the polypeptide is not statistically significantly higher than the background, for example, the level of binding signal detected in the absence of the antigen-binding protein and / or in the presence of a negative control protein (e.g., an isotype control antibody) and / or the level of binding detected in the presence of a negative control polypeptide. The binding level is detected by using a biosensor analysis (e.g., Biacore), in which the antigen-binding protein is immobilized and contacted with the antigen.

[0388] An "affinity matured" antibody is one that has one or more changes in one or more hypervariable regions (HVRs). These changes improve the affinity of the antibody for the antigen compared to a parent antibody that does not have these changes. An ideal affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies can be prepared by methods known in the art.

[0389] "ADCC" refers to a process called antibody-dependent cellular toxicity, which is an immune response mediated primarily by human natural killer (NK) cells. In ADCC, FcγRIII on the surface of NK cells recognizes the Fc region of antibodies bound to antigens displayed on the surface of target cells. This activates NK cells, which release perforins and granzymes, leading to lysis and apoptosis of target cells.

[0390] "CDC" refers to a complex process called complement-dependent cytotoxicity, which can lead to cell death through a protein cascade that can act through either of two major pathways.

[0391] "ADCP" refers to a process called antibody-dependent cell-mediated phagocytosis. In this Fc receptor-mediated process, target cells that have bound antibodies are engulfed by phagocytic cells such as macrophages, monocytes, neutrophils, and dendritic cells. This process involves multiple Fc receptors.

[0392] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of an antigen to which it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0393] As used herein, an "agonist antibody" is an antibody that mimics at least one functional activity of a polypeptide of interest.

[0394] As referred to herein, an "Fc region" is a dimer consisting of two polypeptide chains connected by one or more disulfide bonds, each chain comprising part or all of a hinge region and CH2 and CH3 domains. Each of the polypeptide chains is referred to as an "Fc polypeptide chain". In order to distinguish between the two Fc polypeptide chains, one Fc polypeptide chain is referred to as the "A chain" and the other Fc polypeptide chain is referred to as the "B chain". More specifically, the Fc region contemplated for use in the present invention is an IgG Fc region, which may be an Fc region of mammalian or human IgG1, IgG2, IgG3 or IgG4. In the human IgG1 Fc region, at least two allele types are known.

[0395] As used herein, "Fc-containing protein" refers to a protein comprising an Fc region as described herein and a binding region that binds to a target molecule. The term "Fc-containing protein" includes an antibody or Fc fusion protein comprising an Fc region.

[0396] The expression "therapeutically effective amount" generally refers to an amount of an antigen-binding protein of the invention that is capable of: (i) treating a specific disease, condition or disorder; (ii) alleviating, ameliorating or eliminating one or more symptoms of a specific disease, condition or disorder; or (iii) delaying the onset of one or more symptoms of a specific disease, condition or disorder described herein.

[0397] The word "treat" or "treatment" refers to therapeutic treatment, wherein the goal is to slow down (mitigate) undesirable physiological changes or illnesses. For purposes of the present invention, for detectable or undetectable illnesses, useful or desired clinical outcomes, including but not limited to relieving symptoms, alleviating disease extent, stabilizing (i.e., not worsening) disease states, delaying or slowing disease progression, improving or alleviating disease states, and alleviating (whether in part or in whole). If treatment is not received, treatment can also mean extending survival compared to expected survival. Treatment may not necessarily result in the complete removal of a disease or disorder, but can reduce or minimize complications and side effects of infection and the progression of a disease or disorder. The success of the treatment or otherwise can be monitored by individual physical examinations, cytopathology, serology DNA or mRNA detection techniques, etc.

[0398] The words "prevent" and "prevention" generally refer to prophylactic or preventative measures taken to protect or prevent an individual who does not have a given disease or condition from developing that disease or condition.

[0399] The phrase "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith.

[0400] Protein mutation

[0401] The present invention also provides an antigen binding protein having at least 80% identity with the sequence disclosed herein or a nucleic acid encoding the antigen binding protein. In one example, the antigen binding protein or nucleic acid of the present invention comprises a sequence at least about 85% or 90% or 95% or 97% or 98% or 99% identical to the sequence disclosed herein.

[0402] Alternatively or additionally, the antigen binding protein comprises CDRs (e.g., three CDRs) that are at least about 80%, or 85%, or 90%, or 95%, or 97%, or 98% or 99% identical to the CDRs of the VH or VL described herein according to any example.

[0403] In another example, the nucleic acid of the invention comprises a sequence that is at least about 80%, or 85%, or 90%, or 95%, or 97%, or 98% or 99% identical to a sequence encoding an antigen-binding protein having a function as described herein according to any example. The invention also encompasses a nucleic acid encoding an antigen-binding protein of the invention that is different from the sequences recited herein due to the degeneracy of the genetic code.

[0404] The % identity of nucleic acids or polypeptides is determined by GAP (Needleman and Wunsch. Mol. Biol. 48, 443-453, 1970) analysis (GCG program), wherein a gap creation penalty = 5 and a gap extension penalty = 0.3. The length of the query sequence is at least 50 residues, and the GAP analysis will align the two sequences over a region of at least 50 residues. For example, the length of the query sequence is at least 100 residues, and the GAP analysis will align the two sequences over a region of at least 100 residues. For another example, the two sequences will be aligned over their entire length.

[0405] The present invention also relates to a nucleic acid that hybridizes under stringent hybridization conditions with a nucleic acid encoding an antigen-binding protein described herein. "Medium stringency" is defined herein as hybridization and / or washing in a temperature range of 45°C to 65°C in 2×SSC buffer, 0.1% (w / v) SDS, or equivalent conditions. "High stringency" is defined herein as hybridization and / or washing in a solution of 0.1×SSC buffer, 0.1% (w / v) SDS or lower salt concentration and at a temperature of at least 65°C, or equivalent conditions. The specific stringency levels mentioned herein encompass equivalent conditions known to those skilled in the art using washing / hybridization solutions other than SSC. For example, the melting temperature (also known as the melting temperature, i.e., T m ) are well known in the art. m Temperatures that are similar (e.g., within 5°C or 10°C) or equal to each other are considered high stringency. Moderate stringency is considered to be at a calculated nucleic acid T. m The value is within the range of 10℃ to 20℃ or 10℃ to 15℃.

[0406] The present invention also relates to mutant forms of the antigen-binding proteins of the present invention, which comprise one or more conservative amino acid substitutions compared to the sequences described herein. In some examples, the antigen-binding protein comprises 10 or fewer conservative amino acid substitutions, such as 9, 8, 7, 6, 5, 4, 3, 2 or 1. "Conservative amino acid substitutions" refer to replacing the original amino acid residues with amino acid residues having similar side chains and / or hydrophobicity and / or hydrophilicity.

[0407] Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The hydrophobicity index is described in Kyte and Doolittle J. Mol. Biol., 157: 105-132, 1982; the hydrophilicity index is described, for example, in U.S. Pat. No. 4,554,101.

[0408] The present invention also encompasses non-conservative amino acid changes. For example, of particular interest are charged amino acids replaced by another charged amino acid and a neutral or positively charged amino acid. In some instances, the antigen binding protein comprises 10 or less, such as 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 non-conservative amino acid substitutions.

[0409] In one example, the mutation occurs in the FR of the antigen binding domain of the antigen binding protein of the invention. In another example, the mutation occurs in the CDR of the antigen binding protein of the invention.

[0410] Exemplary methods for generating mutant forms of antigen binding proteins include:

[0411] - Mutagenesis of DNA (Thie et al., Methods Mol. Biol. 525:309-322, 2009) or RNA (Kopsidas et al., Immunol. Lett. 107:163-168, 2006; Kopsidas et al. BMC Biotechnology, 7:18, 2007; and WO 1999 / 058661);

[0412] - introducing a nucleic acid encoding a polypeptide into mutant cells, such as XL-1 Red, XL-mutS and XL-mutS-Kanr bacterial cells (Stratagene);

[0413] - DNA shuffling, for example as disclosed in Stemmer, Nature 370:389-91, 1994; and

[0414] - site-directed mutagenesis, e.g. as described in Dieffenbach (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995).

[0415] Exemplary methods for determining the biological activity of mutant antigen-binding proteins of the invention will be apparent to those skilled in the art and / or from, for example, antigen binding as described herein. For example, methods for determining antigen binding, competitive inhibition of binding, affinity, binding, dissociation, and therapeutic efficacy are described herein.

[0416] As used herein, the properties of amino acids are defined in the following table:

[0417]

[0418]

[0419]

[0420] Constant region

[0421] The present invention encompasses antigen binding proteins and / or antibodies as described herein that comprise the constant region of an antibody. This includes antigen binding fragments of an antibody fused to an Fc.

[0422] The sequence of the constant region for preparing the protein of the present invention can be obtained from a variety of different sources. In some instances, the constant region of the protein or a portion thereof is derived from a human antibody. The constant region or a portion thereof can be derived from any antibody class and any antibody isotype, including IgM, IgG, IgD, IgA and IgE, and any antibody isotype including IgG1, IgG2, IgG3 and IgG4. In one example, the constant region is a human isotype IgG4 or a stabilized IgG4 constant region.

[0423] In one example, the Fc region of the constant region has a reduced ability to induce effector functions, such as compared to native or wild-type human IgG1 or IgG3 Fc regions. In one example, the effector functions are antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Methods for assessing the level of effector functions of Fc regions containing proteins are known in the art and / or as described herein.

[0424] In one example, the Fc region is an IgG4 Fc region (ie, from an IgG4 constant region), such as a human IgG4 Fc region. The sequence of a suitable IgG4 Fc region will be apparent to those skilled in the art and / or may be obtained in publicly available databases (e.g., available from the National Center for Biotechnology Information).

[0425] In one example, constant region is a stable IgG4 constant region. The term "stable IgG4 constant region" should be understood to refer to an IgG4 constant region that has been modified to reduce Fab arm exchange or reduce the experience of Fab arm exchange or to form a half antibody or to tend to form a half antibody tendency. "Fab arm exchange" refers to the protein modification type of human IgG4, wherein the IgG4 heavy chain and the attached light chain (half molecule) are exchanged into a heavy light chain pair with another IgG4 molecule. Therefore, IgG4 molecules can obtain two different Fab arms (producing bispecific molecules) that recognize two different antigens. Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. "Half antibody" is formed when IgG4 antibody dissociates to form two molecules each containing a single heavy chain and a single light chain.

[0426] In one example, a stable IgG4 constant region comprises a proline at position 241 of the hinge region according to Kabat's system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and / or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 2001 and Edelman et al., Proc. Natl. Acad. USA, 63, 78-85, 1969). In human IgG4, this residue is usually serine. After serine replaces proline, the IgG4 hinge region comprises the sequence CPPC. In this regard, those skilled in the art will appreciate that the "hinge region" is a proline-rich portion of the constant region of the heavy chain of an antibody that is connected to the Fc region and the Fab region that confer mobility on the two Fab arms of the antibody. The hinge region includes cysteine ​​residues that participate in inter-heavy chain disulfide bonds. It is generally defined as extending from Glu226 to Pro243 of human IgG1 according to the numbering system of Kabat. The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain disulfide (SS) bonds in the same position (e.g., see WO2010 / 080538).

[0427] Another example of a stable IgG4 antibody is an antibody in which the arginine at position 409 (according to the EU numbering system) in the heavy chain constant region of human IgG4 is substituted with lysine, threonine, methionine or leucine (e.g., as described in WO2006 / 033386). The Fc region of the constant region may additionally or alternatively comprise a residue selected from alanine, valine, glycine, isoleucine and leucine at a position corresponding to position 405 (according to the EU numbering system). Optionally, the hinge region includes a proline (i.e., a CPPC sequence) at position 241 (as described above).

[0428] In another example, the Fc region is a region modified to have reduced effector function, i.e., a "non-immunostimulatory Fc region". For example, the Fc region is an IgG1 Fc region comprising a substitution at one or more positions selected from the group consisting of 268, 309, 330, and 331. In another example, the Fc region is an IgG1 Fc region comprising one or more of the following changes E233P, L234V, L235A and a deletion of G236 and / or one or more of the following changes A327G, A330S, and P331S (Armour et al., Eur J Immunol. 29: 2613-2624, 1999; Shields et al., J Biol Chem. 276 (9): 6591-604, 2001). Additional examples of non-immunostimulatory Fc regions are described, for example, in Dall'Acqua et al., J Immunol. 177: 1129-1138, 2006; and / or in Hezareh J Virol; 75: 12161-12168, 2001.

[0429] In another example, the Fc region is a chimeric Fc region, e.g., comprising at least one CH2 domain from an IgG4 antibody and at least one CH3 domain from an IgG1 antibody, wherein the Fc region comprises substitutions at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409, and 427 (EU numbering) (e.g., as described in WO20 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0430] Antibody binding domain containing protein

[0431] In another embodiment, an antigen binding protein as described above is provided, wherein the amino acid sequences forming one or more of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are derived from human sequences or are in the form of human sequences.

[0432] Antigen-binding proteins can be presented in a humanized form including non-human (e.g., murine) and human immunoglobulin sequences. Typically, all sequences except the CDR sequences of antigen-binding proteins are from non-human species such as mice, rats or rabbits. In some cases, the framework residues of antigen-binding proteins can also be non-human. When antigen-binding proteins are provided in the form of complete antibodies, typically at least a portion of immunoglobulin constant region (Fc) is human, thereby allowing various human effector functions.

[0433] Methods for humanizing non-human antigen-binding proteins are well known in the art, and examples of suitable methods include those described in Jones et al., (1986) Nature, 321:522; Riechmann et al., (1988) Nature, 332:323; Verhoeyen et al., (1988) Science, 239:1534.

[0434] Phage display methods described herein using antibody libraries derived from human immunoglobulin sequences can be used to produce human antigen binding proteins and human antibodies.

[0435] In addition, transgenic mammals that can not express functional endogenous immunoglobulin but can express human immunoglobulin genes can be used.These mice can be produced by random or targeted insertion of human heavy chain and light chain immunoglobulin genes into embryonic stem cells.Host heavy chain and light chain immunoglobulin genes can become non-functional by insertion or by some other recombination events (for example, by homozygous deletion in host JH district).Transfected embryonic stem cells are amplified and microinjected into blastocysts to produce chimeric mice, and then hybridized to produce homozygous offspring expressing human antigen-binding proteins.After immunization with FLAG tags, human monoclonal antibodies can be obtained.One benefit of transgenic animal systems is that useful isotypes can be produced for treatment, because human immunoglobulin transgenes are rearranged during B cell differentiation and subsequently undergo class switching and somatic mutations in transgenic mice.

[0436] The variable domains of the present invention, including CDRs and FRs, can be made less immunogenic by replacing surface exposed residues so that the antibody appears to the immune system as a self-component. Padlan, EA, 1991, MoI. Immunol. 28, 489 provides an exemplary method. Generally, affinity is retained because the internal packing of amino acid residues near the antigen-binding protein remains unchanged, and CDR residues or adjacent residues that usually affect binding properties are not replaced in these processes.

[0437] In another embodiment, an anti-FLAG tag binding protein, immunoglobulin variable domain, antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single-chain antibody molecule or multispecific antibody as described herein is provided, preferably having a sequence as shown in Table 1.

[0438] Lower molecular weight antibody fragments may have improved access to solid tumors and more rapid clearance compared to full antibodies, which is particularly useful in therapeutic and in vivo diagnostic applications.

[0439] In certain embodiments, the antigen binding protein is provided in the form of a single-chain Fv fragment (scFv). Fv and scFv are suitable for reducing nonspecific binding during in vivo use because they have complete combining sites without constant regions. Fusion proteins including scFv can be constructed to produce fusion of effector proteins at the amino or carboxyl terminus of the scFv.

[0440] In another embodiment, a double antibody or a three-antibody or other multispecific antibody comprising the above-mentioned antigen-binding protein is provided. The multispecific antibody can be assembled using a polypeptide domain that allows multimerization. Examples include CH2 and CH3 regions of Fc and CH1 and Cκ / λ regions. Other naturally occurring protein multimerization domains can be used, including leucine zipper domains (bZIPs), helix-loop-helix motifs, Src homology domains (SH2, SH3), EF chiral structures, phosphotyrosine binding (PTB) domains or other domains known in the art.

[0441] In another embodiment, a fusion domain or heterologous protein is provided, which fusion domain or heterologous protein comprises an antigen binding protein, an immunoglobulin variable domain, an antibody, a dab, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule or a multispecific antibody as described herein.

[0442] The heterologous polypeptide can be recombinantly fused or chemically coupled to the N-terminus or C-terminus of the antigen-binding protein of the present invention or a molecule comprising the antigen-binding protein.

[0443] The antibody or antigen-binding protein fused with a heterologous polypeptide can be used to bind to a FLAG tag present on a recombinant reagent, including but not limited to: a recombinant protein, therapeutic antibody, or other antigen-binding domain containing a protein containing a FLAG tag; a cellular immunotherapy agent that has been genetically modified to express a FLAG tag or to express a receptor or other protein containing a FLAG tag. In certain instances, the antigen-binding proteins of the present invention can be used to bind to a chimeric antigen receptor (CAR) present on a cytotoxic immune cell (e.g., a CAR T cell).

[0444] In addition, the antigen-binding proteins, immunoglobulin variable domains, antibodies, dab, scFv, Fab, Fab', F(ab')2, Fv fragments, diabodies, triabodies, linear antibodies, single-chain antibody molecules or multispecific antibodies of the present invention can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, connection to cellular ligands or other proteins, etc.

[0445] Indirect CAR and related chimeric receptors

[0446] In a further embodiment, the heterologous polypeptide to which the antibody or antigen-binding protein of the present invention is fused can be included as a component of a chimeric antigen protein (CAR) or a variant T cell receptor. In such embodiments, it should be understood that the CAR or variant T cell receptor comprising the antigen-binding protein of the present invention produced can be referred to as "indirect CAR" (sometimes also referred to as universal CAR). In an indirect CAR system, the antigen binding domain of CAR does not directly bind to the target antigen on the target cell, but binds to an intermediate, wherein the intermediate comprises an antigen binding domain for directly binding to the target cell. Examples of CARs for identifying cells by intermediates are known in the art, such as described in European patent application EP 2651442.

[0447] Those skilled in the art will be familiar with the general structure of CAR and other modified receptors in which the antigen binding proteins of the present invention may be included. Typically, a "chimeric antigen receptor" refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding portion, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain").

[0448] Chimeric antigen receptors are artificially constructed proteins that can induce antigen-specific cellular responses when expressed on the cell surface. CAR includes at least two domains, the first domain is an antigen binding domain that binds to an antigen or more specifically one or more epitope portions of an antigen, and the second domain is a signaling domain that can induce or participate in the induction of an intracellular signaling pathway.

[0449] The combination of these two domains determines the antigen specificity of the CAR and the ability of the CAR to induce the desired cellular response, which also depends on the host cell in which the CAR is located. For example, a CAR expressed in a helper T cell and having a signaling domain containing a CD3 activation domain, once activated by encountering its cognate antigen, may induce CD4+ helper T cells to secrete a series of cytokines. In a further example, the same CAR, when expressed in a CD8+ cytotoxic T cell, can induce the release of cytotoxins once activated by cells expressing the cognate antigen, ultimately leading to apoptosis of the cells expressing the antigen.

[0450] In addition to the antigen recognition domain and the signaling domain, CAR may also include other components or parts. For example, CAR may include a transmembrane domain that may constitute a part of the CAR signaling domain or be associated with the CAR signaling domain. The transmembrane domain is typically one or more hydrophobic helices that span the lipid bilayer of the cell membrane and embed the CAR into the cell membrane. When CAR binds to a cell, its transmembrane domain is a factor that determines the expression pattern of CAR. For example, the use of a transmembrane domain associated with a CD3 co-receptor enables CAR to be expressed in naive T cells, while the use of a transmembrane domain derived from a CD4 co-receptor can direct CAR to be expressed in helper T cells, but not in cytotoxic T cells.

[0451] The other components or parts of CAR can be joint domains.Joint domains (also referred to as intervals or hinge domains) can span from the extracellular side of the transmembrane domain to the antigen binding domain, thereby connecting the antigen binding domain to the transmembrane domain.Although in some cases, functional CAR does not require joint domains (i.e., the antigen recognition domain can be directly connected to the transmembrane domain), in some cases, the use of joint domains allows greater efficacy of CAR.Joint domains can have a variety of functions, including allowing the flexibility of CAR to allow the necessary orientation of the antigen binding domain of CAR to bind to the antigen.Therefore, joint domains can be any amino acid sequence that performs this function.A non-limiting example of a joint domain is a domain with an amino acid sequence homology to the hinge region of an IgG antibody (e.g., an IgG1 hinge region).Alternative examples include C and C of an antibody. H2 -C H3 The region or portion of the CD3 co-receptor complex, CD4 co-receptor or CD8 co-receptor has an amino acid sequence with sequence homology.

[0452] In some embodiments, the CAR or other modified receptor may comprise an antigen binding protein as described herein in the form of an sdAb, scFv (including multivalent scFv), Fab, or another antibody-like structure.

[0453] The signaling domain of CAR can be any suitable domain that can induce or participate in inducing an intracellular signaling cascade when the antigen recognition domain of CAR recognizes the antigen and activates CAR. The signaling domain of CAR will be specifically selected based on the desired cellular outcome after CAR activation. Although there are many possible signaling domains, in immunotherapy and cancer therapy, signaling domains can be roughly divided into two categories, i.e., activation receptors and co-stimulatory receptors, according to the receptors from which they are derived.

[0454] Thus, in some embodiments of the first aspect of the invention, the signaling domain comprises a portion derived from an activated receptor. In some embodiments, the signaling domain comprises a portion derived from a co-stimulatory receptor.

[0455] As used throughout the specification, the term "portion" when used with respect to an activating receptor or a co-stimulatory receptor, relates to any fragment of a receptor that includes sequences responsible for or involved in the initiation / induction of an intracellular signaling cascade following interaction of the receptor with its cognate antigen or ligand. Examples of intracellular signaling cascades that initiate / induce T cell receptors (TCRs) via CD3 are summarized below.

[0456] Although not wishing to be bound by theory, the extracellular portion of TCR mainly comprises heterodimers of clonal TCRα and TCRβ chains (TCRα / β receptors) or TCRγ and TCRδ chains (TCRγδ receptors). These TCR heterodimers generally lack inherent signal transduction capabilities, so they are non-covalently bound to multiple signal transduction subunits (mainly CD3-ζ, -γ, -δ and -ε) of CD3. Each of the γ, δ and ε chains of CD3 has an intracellular (cytoplasmic) portion, which includes a single immunoreceptor-tyrosine-based activation motif (ITAM), and the CD3 chain includes three tandem ITAMs. When TCR binds to the cognate antigen in the presence of MHC, and binds to necessary co-receptors such as CD4 or CD8, signal transduction is initiated, which causes a tyrosine kinase called Lck to phosphorylate two tyrosine residues in the intracellular ITAM of the CD3 chain. Subsequently, a second tyrosine kinase (ZAP-70, which itself is activated by phosphorylation by Lck) is recruited to dually phosphorylate these ITAMs. As a result, several downstream target proteins are activated, which ultimately triggers conformational changes, calcium mobilization, and rearrangement of the actin cytoskeleton within the cell. These changes work together to ultimately lead to the activation of transcription factors and the induction of T cell immune responses.

[0457] As used throughout the specification, the term "activating receptor" relates to a receptor or co-receptor that forms a component of the T cell receptor (TCR) complex or participates in the formation of the T cell receptor (TCR) complex, or a receptor that participates in the specific activation of immune cells due to recognition of antigens or other immunogenic stimuli.

[0458] Non-limiting examples of such activating receptors include components of the T cell receptor-CD3 complex (CD3-ζ, -γ, -δ and -ε), CD4 co-receptors, CD8 co-receptors, Fc receptors or natural killer (NK) cell-associated activating receptors such as LY-49 (KLRA1), natural cytotoxicity receptors (NCRs, preferably NKp46, NKp44, NKp30 or NKG2 or CD94 / NKG2 heterodimers). Thus, in some embodiments of the invention, the signaling domain includes a portion derived from any one or more of a CD3 co-receptor complex member (preferably the CD3-ζ chain or a portion thereof), a CD4 co-receptor, a CD8 co-receptor, an Fc receptor (FcR) (preferably FcεRI or FcRI) or a NK-associated receptor such as LY-49.

[0459] The specific intracellular signaling portion of each of the CD3 chains is known in the art, as is the intracellular portion of the FC receptor.

[0460] Various combinations of portions of activating receptors can be used to form the transmembrane (TM) and intracellular (IC) portions of the CAR, such as CD3ζTM and CD3ζIC (Landmeier S. et al. Cancer Res. 2007; 67:8335-43; Guest RD. et al., J Immunother. 2005, 28:203-11; Hombach AA. et al. J Immunol. 2007; 178:4650-7), CD4 TM and CD3ζIC (James SE. et al. J Immunol. 2008; 180:7028-38), CD8 TM and CD3ζIC (Patel SD. et al. Gene Ther. 1999; 6:412-9), and FcεγTM and FcεγIC (Haynes NM. et al. al. J Immunol. 2001; 166: 182-7; Annenkov AE. et al. J Immunol. 1998; 161: 6604-13).

[0461] As used throughout the specification, the term "co-stimulatory receptor" relates to a receptor or co-receptor that assists immune cell activation when antigen-specific induction activates the receptor. As will be appreciated, co-stimulatory receptors do not require the presence of antigens and are not antigen-specific, but are generally one of two signals, the other being the activation signal required for inducing immune cell responses. In the case of an immune response, co-stimulatory receptors are generally activated by the presence of their expressed ligands on the surface of antigen presenting cells (APCs) such as dendritic cells or macrophages. Specifically for T cells, co-stimulation is essential for inducing cell activation, proliferation, differentiation and survival (all of which are generally classified within the scope of T cell activation). And presenting antigens to T cells in the absence of co-stimulation may result in the formation of anergy, clonal elimination and / or antigen-specific tolerance. Importantly, co-stimulatory molecules can affect the response of T cells to antigens encountered simultaneously. In general, the antigens encountered in the presence of "positive" co-stimulatory molecules can cause T cell activation and induce a cellular immune response intended to remove cells expressing the antigen.

[0462] However, antigens encountered in the context of the presence of “negative” co-receptors can result in a state of tolerance to the antigens encountered simultaneously.

[0463] Non-limiting examples of T cell co-stimulatory receptors include CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), ICOS. Specifically, CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137) and ICOS all represent "positive" co-stimulatory molecules that enhance T cell response activation. Therefore, in some embodiments of the present invention, the signaling domain includes any one or more of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137) and ICOS.

[0464] In some embodiments, the signaling domain includes a portion derived from CD28, OX40 or 4-1BB costimulatory receptors. In some embodiments, the signaling domain includes a portion of a CD28 costimulatory receptor. In some embodiments, the signaling domain includes a portion of an OX40 costimulatory receptor. Various combinations of costimulatory receptor portions can be used to form the transmembrane (TM) and intracellular (IC) portions of CAR. For example, as described in CD8 TM and DAP10 IC or CD8 TM and 4-1BB IC (Marin V. et al. Exp Hematol. 2007; 35: 1388-97), CD28 TM and CD28 IC (Wilkie S. et al. J Immunol. 2008; 180: 4901-9; Maher J. et al. Nat Biotechnol. 2002; 20: 70-5) and CD8 TM and CD28 IC (Marin V. et al. Exp Hematol. 2007; 35: 1388-97).

[0465] Further details of the antigen binding proteins of the invention

[0466] The antigen-binding proteins of the present invention can be composed of amino acids (i.e., peptide isosteres) connected to each other by peptide bonds or modified peptide bonds, and can contain amino acids other than 20 gene-encoded amino acids. The antigen-binding proteins of the present invention can be modified by natural methods such as post-translational processing or by chemical modification techniques known in the art. This modification is well described in basic texts and research literature. Modification can occur anywhere in the antigen-binding protein, including peptide backbones, amino acid side chains and amino or carboxyl termini, or on parts such as carbohydrates. It should be understood that the same type of modification can be present in several proteins in a given antigen-binding protein to the same or varying degrees. In addition, a given antigen-binding protein can contain many types of modifications. Antigen-binding proteins can be branched, for example, as a result of ubiquitination, and they can be cyclic, and have or do not have branching. Circular, branched, and branched cyclic antigen-binding proteins can be produced by natural processes after translation or can be prepared by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, adrenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination.

[0467] In another embodiment, a conjugate is provided, which is in the form of an antigen binding protein, immunoglobulin variable domain, antibody, Fab, dab, scFv, diabody, triabody or fusion protein as described above, which is conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatic toxin of bacterial, fungal, plant or animal origin or a fragment thereof) or a label such as a radioactive isotope (i.e., a radioconjugate). In another aspect, the present invention also provides a method of using the immunoconjugate. In one aspect, the immunoconjugate comprises any of the above-mentioned variable domains covalently attached to a cytotoxic agent or a detectable agent.

[0468] In another embodiment, an antibody for binding to an antigen binding protein, immunoglobulin variable domain, antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single-chain antibody molecule, or multispecific antibody, fusion protein or conjugate as described above is provided.

[0469] In another embodiment, a nucleic acid is provided that encodes an antigen-binding protein, an immunoglobulin variable domain, an antibody, a dab, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule, or a multispecific antibody, a fusion protein or a conjugate as described above.

[0470] The polynucleotides encoding or consisting of the antigen-binding proteins of CDR or FR according to any one of the general formulas in the above general formula can be produced by nucleic acids from any source, such as by chemical synthesis or separation from cDNA or genomic libraries. For example, cDNA libraries can be produced by antibody-producing cells such as B cells, plasma cells or hybridoma cells and related nucleic acids, which are separated by PCR amplification using oligonucleotides for specific clones of interest. The isolated nucleic acids can be cloned into vectors using any method known in the art. Methods known in the art such as recombinant DNA technology, site-directed mutagenesis, PCR, etc. (see, for example, the technology described in the following documents, namely: Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY) can be used to mutagenesis related nucleotide sequences to produce antigen-binding proteins with different amino acid sequences, such as to produce amino acid substitutions, deletions and / or insertions.

[0471] Protein production

[0472] In another embodiment, a method for producing the anti-FLAG antigen binding protein as described above is provided, which comprises expressing the nucleic acid as described above in the cell or non-human animal as described above.

[0473] The production of antigen-binding proteins of the present invention generally requires an expression vector containing a polynucleotide encoding the antigen-binding proteins of the present invention. Polynucleotides encoding the antigen-binding proteins of the present invention can be obtained and subcloned into a vector for use in producing antigen-binding proteins by recombinant DNA technology using techniques known in the art (including those described herein). Many different expression systems are contemplated, including the use of mammalian cells including human cells to produce and secrete antigen-binding proteins. Examples of cells include 293F cell lines, CHO cell lines, and NSO cell lines.

[0474] Methods known in the art can be used to construct expression vectors comprising protein coding sequences and appropriate transcription and translation control signals. These include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. In certain embodiments, a reproducible vector is provided having a nucleic acid encoding an antigen-binding protein operably connected to a promoter.

[0475] Cells transfected with expression vectors can be cultured to produce antigen-binding proteins by conventional techniques.Therefore, in certain embodiments, host cells or cell transfection agents are provided, which contain polynucleotides encoding antigen-binding proteins operably connected to promoters of the present invention.Promoters can be heterologous.Various host-expression vector systems can be used, and in certain systems, the transcription machinery of the vector system is particularly matched with host cells.For example, mammalian cells such as Chinese hamster ovary cells (CHO) can be transfected with a vector comprising the major intermediate early gene promoter element from human cytomegalovirus.Additionally or alternatively, host cells can be used to regulate the expression of the inserted sequence or to modify and process gene products as required, and the gene products include various forms of post-translational modifications. Examples of mammalian host cells with specific post-translational modification processes include CHO cells, VERY cells, BHK cells, HeIa cells, COS cells, MDCK cells, 293 cells, 3T3 cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT2O cells and T47D cells, NSO cells, CRL7O3O cells and HsS78Bst cells.

[0476] According to the purposes of protein molecule, many bacterial expression vectors can be advantageously selected.In an example, a carrier such as Escherichia coli expression vector pUR278 causing the expression of high-level fusion protein products that are easily purified can be used to produce a large amount of antigen-binding proteins in this carrier.The expression product can be produced in the form of a fusion protein with lacZ.Other bacterial vectors include pIN vectors etc.The pGEX vector can also be used to express exogenous polypeptides as fusion proteins with glutathione-S-transferase (GST).These fusion proteins are normally soluble and can be easily purified from lysed cells by adsorption and being attached to glutathione-agarose affinity matrix, then eluted in the presence of free glutathione.Thrombin and / or factor Xa proteinase cleavage site can be provided in the expressed polypeptide so that the cloned target gene product can be partially released from the GST.

[0477] Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes in insect systems (including Spodoptera frugiperda cells). The specific promoter used can depend on the insertion position of the protein coding sequence in the viral genomic sequence. For example, the sequence can be cloned separately into the polyhedrin gene and placed under the control of the polyhedrin promoter.

[0478] Virus-based expression systems can be used together with mammalian cells such as adenovirus, and thus the coding sequence of interest can be connected to adenovirus late promoter and three-part leader sequences. In vitro or in vivo recombination can be used to insert this chimeric gene into the adenovirus genome. Insertion region E1 or E3 will produce a live recombinant virus that can express antigen-binding proteins in the host cell of infection. The specific initiation signal including the ATG start codon and adjacent sequences may be needed to effectively translate the inserted antigen-binding proteins coding sequence. Initiation and translation control signals and codons can be obtained from natural and synthetic various sources. Transcription enhancer elements and transcription terminators can be used to improve the expression efficiency of virus-based systems.

[0479] When long-term, high-yield production of recombinant protein is required, stable expression is preferred.Usually, a selective marker gene is used, whereby after transfection, cells are grown in an enriched medium for 1-2 days, then transferred to a medium containing a selective medium, wherein cells containing corresponding selective markers such as antibiotic resistance can be screened.The result is that the cells that have stably integrated plasmids into their chromosomes grow and form a focus (foci), which can then be cloned and amplified into a cell line.Herpes simplex virus thymidine kinase gene, hypoxanthine guanine phosphoribosyltransferase gene and adenine phosphoribosyltransferase gene are examples of genes that can be used for thymidine kinase defective (tk-), hypoxanthine guanine phosphoribosyltransferase defective (hgprt-) or adenine phosphoribosyltransferase defective (aprT-) cells, thereby providing a suitable screening system. The following genes: dihydrofolate reductase gene (dhfr), which confers resistance to methotrexate; guanine phosphoribosyltransferase gene (gpt), which confers resistance to mycophenolic acid; neomycin phosphotransferase gene (neo), which confers resistance to the aminoglycoside drug G-418; and hygromycin phosphotransferase gene (hygro), which confers resistance to hygromycin, are examples of genes that can be used in antimetabolite selection systems.

[0480] The antigen binding proteins of the present invention can be purified by known methods through recombinant expression systems, including ion exchange chromatography, affinity chromatography (particularly affinity for specific antigen protein A or protein G) and gel filtration column chromatography, centrifugation, differential solubility or by any other standard technique for purifying proteins. Purification can be facilitated or assisted by providing the antigen binding protein in the form of a fusion protein.

[0481] Large amounts of antigen-binding proteins of the present invention can be produced by scalable methods starting from a pilot expression system in a research laboratory that is expanded to an analytical scale bioreactor (typically from a 5L bioreactor to about a 50L bioreactor) or a production scale bioreactor (e.g., but not limited to, 75L, 100L, 150L, ​​300L, or 500L). Ideal scalable methods include those in which the aggregation level measured by HPSEC or rCGE is low to an undetectable level, typically no more than 5% aggregation by weight of protein to no more than 0.5% aggregation by weight of protein. Additionally or alternatively, undetectable levels of fragmentation measured based on the total peak area representing the complete antigen-binding protein may be required in a scalable process, such that at least 80% and up to 99.5% or more of the total peak area represent the complete antigen-binding protein. In other embodiments, the scalable methods of the present invention produce antigen-binding proteins at a production efficiency of about 10 mg / L to about 300 mg / L or more.

[0482] Various techniques have been developed for producing antibody fragments, including proteolytic digestion of intact antibodies and recombinant expression in host cells. Regarding the latter, as described below, Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E. coli, antibody fragments can be isolated from antibody phage libraries, and Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments. In another method, F(ab')2 fragments are isolated directly from recombinant host cell cultures.

[0483] In another embodiment, a vector comprising the above-mentioned nucleic acid is provided. The vector may be, for example, in the form of a plasmid, a cosmid, a viral particle or a bacteriophage. The appropriate nucleic acid sequence may be inserted into the vector by various procedures. Typically, DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art. Vector components typically include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence. The construction of a suitable vector comprising one or more of these components employs standard ligation techniques known to those skilled in the art.

[0484] Antigen binding sites can be produced not only by direct recombinant production, but also as fusion polypeptides with heterologous polypeptides, which can be signal sequences or other polypeptides with specific cleavage sites at the N-terminus of mature proteins or polypeptides. Generally, the signal sequence can be a component of the vector, or the signal sequence can be a part of the DNA encoding the antigen binding site inserted into the vector. The signal sequence can be a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, lpp or heat-stable enterotoxin II leaders. For yeast secretion, the signal sequence can be, for example, a yeast invertase leader, an alpha factor leader or an acid phosphatase leader or a Candida albicans glucoamylase leader. In mammalian cell expression, mammalian signal sequences can be used to direct the secretion of proteins such as secretory polypeptides from the same or related species and signal sequences of viral secretory leaders.

[0485] The polynucleotide sequence encoding the polypeptide component of the antigen-binding proteins of the present invention can be obtained using standard recombinant techniques as described above. Polynucleotides can be synthesized using a nucleotide synthesizer or PCR technology. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors known in the art can be used for the purposes of the present invention. Selecting a suitable vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotides or both amplification and expression) and its compatibility with the specific host cell in which it is located.

[0486] Typically, plasmid vectors containing replicons and control sequences from species compatible with host cells are used in conjunction with these hosts. Expression and cloning vectors all contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells, and contain marker sequences that can provide phenotypic selection in transformed cells. This sequence is well known for a variety of bacteria, yeasts, and viruses. The origin of replication from plasmid pBR322 (which contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance) provides a simple means for identifying transformed cells and is applicable to most gram-negative bacteria. 2 μm plasmid sources are applicable to yeast. Various viral sources (SV40, polyoma, adenovirus, VSV or BPV) can be used for cloning vectors in mammalian cells. pBR322, its derivatives or other microbial plasmids or phages can also contain or be modified to contain promoters that can be used to express endogenous proteins by microbial organisms.

[0487] In addition, phage vectors containing replicons and control sequences compatible with the host microorganism can be used as transformation vectors in conjunction with these hosts. For example, phages such as λGEM.TM.-11 can be used to prepare recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0488] The expression vector of the present invention may contain two or more promoter-cistron (a cistron is a DNA fragment containing all the information for producing a single polypeptide) pairs. A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that is used to regulate the expression of the cistron. Prokaryotic promoters are generally divided into two categories, namely: inducible and constitutive. An inducible promoter is a promoter that initiates increased transcription levels of a cistron under its control in response to changes in culture conditions (e.g., the presence or absence of nutrients or temperature changes).

[0489] A large number of promoters recognized by a variety of potential host cells are well known. Selected promoters can be removed from source DNA by restriction enzyme digestion and the promoter sequence of separation is inserted into the carrier of the present invention, thereby being operably connected with the cistron DNA encoding light chain or heavy chain. Natural promoter sequences and many heterologous promoters can all be used to instruct the amplification and / or expression of target gene. In some embodiments, heterologous promoters are used, because compared with natural target polypeptide promoters, they usually allow larger transcription and higher expression target gene yield.

[0490] Promoters recognized by various potential host cells are well known. Promoters suitable for prokaryotic hosts include PhoA promoter, β-galactosaminidase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems and hybrid promoters such as tac or trc promoters. Promoters used in bacterial systems will also contain a Shine-Dalgarno (SD) sequence operably connected to the DNA encoding the antigen-binding protein of the present invention. However, other promoters that work in bacteria (e.g., other known bacterial or phage promoters) are also suitable. Their nucleotide sequences have been published, so those skilled in the art can use joints or adapters to effectively connect them to the cistrons encoding the target light chain and heavy chain to provide any required restriction enzyme sites.

[0491] In one aspect of the invention, each cistron in the recombinant vector comprises a secretory signal sequence component that instructs the transmembrane translocation of the expressed polypeptide. Generally, the signal sequence can be a component of the vector, or the signal sequence can be a part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purpose of the present invention should be a signal sequence recognized and processed (i.e., cut by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the natural signal sequence of heterologous polypeptides, the signal sequence is replaced by the prokaryotic signal sequence selected from the group consisting of alkaline phosphatase, penicillinase, Ipp or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PeIB, OmpA and MBP. In one embodiment of the present invention, the signal sequence used in the two cistrons of the expression system is an STII signal sequence or a variant thereof.

[0492] On the other hand, the production of immunoglobulins according to the present invention can occur in the cytoplasm of the host cell, and therefore does not require the presence of a secretion signal sequence in each cistron. In this regard, the immunoglobulin light and heavy chains are expressed, folded and assembled to form functional immunoglobulins in the cytoplasm. Certain host strains (e.g., E. coli trxB strains) can provide cytoplasmic conditions that are favorable for disulfide bond formation, thereby enabling the expressed protein subunits to fold and assemble correctly.

[0493] The present invention provides an expression system in which the quantitative ratio of expressed polypeptide components can be adjusted to maximize the yield of secreted and properly assembled antigen-binding proteins of the present invention. This regulation is achieved at least in part by simultaneously adjusting the translation strength of the polypeptide components.

[0494] For expression in eukaryotic host cells, vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0495] Vectors for eukaryotic host cells may also contain signal sequences or other polypeptides having specific cleavage sites at the N-terminus of the target mature protein or polypeptide. The heterologous signal sequence selected is preferably a signal sequence that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences and viral secretion leaders such as herpes simplex gD signals are available.

[0496] The DNA for this precursor region is linked in reading frame to the DNA encoding the antibody.

[0497] Typically, mammalian expression vectors do not require a replication origin component. For example, typically only the SV40 origin can be used because the SV40 origin contains an early promoter.

[0498] Expression and cloning vectors usually contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that have the following functions: (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline); (b) complement auxotrophic deficiencies; or (c) provide key nutrients that are not present in complex culture media, such as the gene encoding D-alanine racemase in Bacillus.

[0499] One example of a selection scheme utilizes a drug to prevent the growth of host cells. Those cells that are successfully transformed with a heterologous gene produce a protein that confers drug resistance and therefore survive the selection scheme. Examples of this dominant selection use neomycin, mycophenolic acid, and hygromycin.

[0500] Examples of suitable selection markers for mammalian cells are those that can identify cells that have the ability to take up nucleic acids encoding antigen-binding proteins (such as DHFR or thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.). When wild-type DHFR is used, suitable host cells are CHO cell lines (e.g., ATCC CRL-9096) that are prepared and propagated with insufficient DHFR activity. For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx, a competitive antagonist of DHFR). Alternatively, host cells (particularly wild-type hosts containing endogenous DHFR) transformed or co-transformed with DNA sequences encoding antibodies, wild-type DHFR proteins, and another selectable marker (e.g., aminoglycoside 3'-phosphotransferase (APH)) can be selected by growing the cells in a medium containing a selection agent for the selectable marker (e.g., an aminoglycoside antibiotic such as kanamycin, neomycin, or G418).

[0501] Expression and cloning vectors usually contain a promoter operably linked to the nucleic acid sequence encoding the antigen binding protein to direct mRNA synthesis. Promoters recognized by various potential host cells are well known.

[0502] Eukaryotic genes usually have an AT-rich region located at about 25 to 30 bases upstream of the transcription start site. Another sequence of 70 to 80 bases found in the upstream of many gene transcription starts is the CNCAAT district, in which N can be any Nucleotide. At the 3' end of most eukaryotic genes is the AATAAA sequence, which can be a signal for adding a poly-A tail to the 3' end of the encoding sequence. All these sequences are suitably inserted into the eukaryotic expression vector.

[0503] Examples of suitable promoter sequences for use with yeast hosts include the promoter for 3-phosphoglycerate kinase or promoters for other glycolytic enzymes, including enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0504] Other yeast promoters are inducible promoters that have the added advantage of transcription being controlled by growth conditions and are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes involved in nitrogen metabolism, metallothioneins, glyceraldehyde-3-phosphate dehydrogenase, and the enzymes responsible for maltose and galactose utilization.

[0505] In mammalian host cells, transcription of the antigen-binding protein on the vector is controlled, for example, by promoters obtained from viral genomes, including polyoma virus, fowl pox virus, adenovirus (such as adenovirus type 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40); it can also be controlled by heterologous mammalian promoters (such as actin promoter or immunoglobulin promoter); it can also be controlled by heat shock promoters, provided that these promoters are compatible with the host cell system.

[0506] By inserting enhancer sequences into vectors, transcription of DNA encoding antigen-binding proteins can be increased in higher eukaryotes. Enhancer sequences include those sequences known from mammalian genes (erythropoietin, elastase, albumin, alpha-fetoprotein and insulin). However, usually, enhancers from eukaryotic cell viruses will be used. Examples include SV40 enhancers at the late stage of replication origin (bp100-270), cytomegalovirus early promoter enhancers, polyoma enhancers at the late stage of replication origin, and adenovirus enhancers.

[0507] Expression vectors for eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences necessary to terminate transcription and stabilize the mRNA. Such sequences are commonly available from the 5' untranslated regions and occasionally the 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antigen binding protein.

[0508] In another embodiment, a cell comprising the above-mentioned vector or nucleic acid is provided. The nucleic acid molecule or vector can be present in the genetically modified host cell or host as an independent molecule outside the genome, preferably as a molecule capable of replication, or the nucleic acid molecule or vector can be stably integrated into the genome of the host cell or host.

[0509] The host cell of the present invention may be any prokaryotic or eukaryotic cell.

[0510] Examples of prokaryotic cells are those cells commonly used for cloning such as Escherichia coli or Bacillus subtilis. In addition, eukaryotic cells include, for example, fungi or animal cells.

[0511] Examples of suitable fungal cells are yeast cells, preferably yeast cells of the genus Saccharomyces, most preferably yeast cells of the genus Saccharomyces cerevisiae.

[0512] Examples of animal cells are, for example, insect cells, vertebrate cells, preferably mammalian cells, such as HEK293, NSO, CHO, MDCK, U2-OS, Hela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, C33A. These host cells, such as CHO cells, can provide post-translational modifications for the antibody molecules of the invention, including leader peptide removal, folding and assembly of H (heavy) and L (light) chains, glycosylation of the molecules on the correct side, and secretion of functional molecules.

[0513] Other suitable cell lines known in the art are available from cell line depositories such as the American Type Culture Collection (ATCC).

[0514] In another embodiment, an animal comprising the above-mentioned cells is provided. In certain embodiments, transgenic animals and tissues thereof can be used to produce antigen-binding proteins of the present invention. Nucleic acid molecules introduced as transgenics into non-human hosts and their subsequent expression can be used to produce antigen-binding proteins, for example, the expression of such transgenics in the milk of transgenic animals provides a means for obtaining quantitative amounts of antigen-binding proteins. In this regard, useful transgenics include nucleic acid molecules of the present invention, for example, the coding sequences of antigen-binding proteins described herein, which are operably connected to promoters and / or enhancer structures from mammary gland-specific genes such as casein or beta-lactoglobulin. The animal can be a non-human mammal, most preferably a mouse, rat, sheep, calf, dog, monkey or ape.

[0515] Binding to target antigen

[0516] Methods for determining successful binding of an antigen-binding protein of the present invention to its target antigen (i.e., FLAG tag) are well known in the art. Non-limiting examples of such methods are described herein in the Examples. Methods for confirming the specificity and binding affinity of an antigen-binding protein include the use of Western blotting, ELISA, immunohistochemistry, and Biacore methods, which are all within the skill of those skilled in the art.

[0517] Reagent test kit

[0518] In another embodiment, a kit or article is provided, which includes an antigen binding protein, an immunoglobulin variable domain, an antibody, a dab, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule or a multispecific antibody, a fusion protein, a conjugate or a pharmaceutical composition as described above.

[0519] In other embodiments, a kit for the above-mentioned use is provided, the kit comprising:

[0520] - a container containing a therapeutic composition in the form of one or more of the following: an antigen binding protein, an immunoglobulin variable domain, an antibody, a dab, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule or a multispecific antibody, a fusion protein, a conjugate or a pharmaceutical composition;

[0521] - A label or leaflet with instructions for use.

[0522] The kit or "article of manufacture" may include a container and a label or instructions attached to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. These containers may be made of a variety of materials such as glass or plastic. The container holds a therapeutic composition effective for treating a condition and may have a sterile access port (e.g., the container may be an intravenous bag, or a vial with a stopper pierced by a hypodermic needle). The label or instructions indicate that the therapeutic composition is used to treat a selected condition. In one embodiment, the label or instructions include instructions for use.

[0523] The kit may include (a) a therapeutic composition; and (b) a second container containing a second active ingredient or component. The kit in this embodiment of the invention may also include instructions indicating that the active ingredient and other active ingredients can be used to treat a condition or prevent complications from cancer. Alternatively or in addition, the kit may also include a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. The kit may also include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0524] In certain embodiments, the therapeutic composition can be provided in the form of a disposable or reusable device including a container for holding the therapeutic composition. In one embodiment, the device is a syringe. The device can hold 1 mL to 2 mL of the therapeutic composition. The therapeutic composition can be provided in the device in a ready-to-use state or in a state where other components need to be mixed or added.

[0525] In other embodiments, a kit for the above-mentioned diagnostic application is provided, the kit comprising:

[0526] - a container holding a diagnostic composition in the form of one or more of the following: an antigen binding protein, an immunoglobulin variable domain, an antibody, a Fab, a dab, a scFv, a diabody, a triabody, a fusion protein or a conjugate;

[0527] - A label or leaflet with instructions for use.

[0528] The kit may include (a) a diagnostic composition; and (b) a second container containing a second diagnostic agent or a second label. The kit may also include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, etc.

[0529] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0530] The following examples are intended to illustrate but in no way limit the present invention.

[0531] Example

[0532] Example 1: Humanization of chimeric antibodies

[0533] Obtain mouse anti-FLAG antibody.To identify the complementarity determining regions (CDRs) and analyze the closest matching germline sequences, the IMGT Domain Gap Alignment Tool was used (Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 2010; 38: D301-D307).

[0534] Molecular models were constructed for the VH and VL domains based on homology to previously published antibody crystal structures using in-house software. PDB files are available upon request for viewing in any molecular visualization software. LLC) to generate images.

[0535] The antibody sequences were analyzed for specific disadvantages based on published protein motifs. The analysis was done by analyzing the following motifs, where X represents any amino acid except proline, as described in the following table:

[0536]

[0537] At the beginning of the humanization process, homology models of the parental VH and VL were constructed in a single-chain Fv (scFv) format. Modeling was performed in 4 stages: collecting homologous sequences; folding library scanning; cycle modeling; side chain placement. The resulting model was used to guide the selection of "donor" or "acceptor" amino acids during the humanization process.

[0538] Parental VH and VL sequences were aligned with a set of human germline sequences. The panel has been filtered to select germline sequences that do not contain unwanted sequence disadvantages (particularly N-linked glycosylation sites and free cysteine). The closest matching germlines from two different VH families and VL families were selected. The humanization algorithm was then used to select CDR and framework amino acids to be transplanted from the donor parental sequence to the human acceptor germline sequence. Four VH and four VL sequences were generated to obtain 16 possible antibodies. The IMGT domain gap alignment tool (Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 2010; 38: D301-D307) was used to calculate the percent identity of the human. Sequence disadvantages were determined based on sequence motifs.

[0539] The VH and VL sequences were analyzed by the IGMT gap alignment tool for all known antibody germline sequences. The CDR regions were specified using the IMGT definition. As expected, the sequences were most closely aligned with the mouse, particularly the IGHV1-4*01 family for VH and IGK1-117*01 for VL. The most matched germlines of parent SEQ ID NO: 1 were germline IGHV1-46*01, IGHV7-4-1*02, IGKV2-30*01, and IGKV4-1*01.

[0540] The following table summarizes the original parental and humanized sequences:

[0541] ID type Human germline Percentage of identity with humans Parental - 66.3％ hu-VH1 Humanized IGHV1-46*01 84.7％ hu-VH2 Humanized IGHV1-46*01 87.8％ hu-VH3 Humanized IGHV7-4-1*02 82.7％ hu-VH4 Humanized IGHV7-4-1*02 86.7％ Parental - 80.0％ hu-VL1 Humanized IGKV2-30*01 90.0％ hu-VL2 Humanized IGKV2-30*01 91.0％ hu-VL3 Humanized IGKV4-1*01 80.2％ hu-VL4 Humanized IGKV4-1*01 81.2％

[0542] The combination of 4 different heavy chain framework regions and light chain framework regions results in 16 different V H / V L combination.

[0543] Different V of 16 antibodies H / V L The combinations are summarized in the table below:

[0544]

[0545]

[0546] All humanized IgG1 forms were well expressed and successfully purified for characterization by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion high performance liquid chromatography (SEC-HPLC) to assess protein quality. The titer, amount, and monomer content of the final purified antibodies are shown in the following table:

[0547]

[0548]

[0549] Example 2: Characterization of humanized antibodies

[0550] All 16 humanized antibodies in IgG and scFv formats were evaluated for their ability to bind to the target (FLAG tag) using ELISA. The results are shown in Figure 1 .

[0551] Although most humanized variants retained the ability to bind to the target, three humanized IgG clones (v3, v4, and v7) had greater affinity. These three IgG clones and the corresponding scFV formats were selected for further analysis.

[0552] Humanized antibodies are able to exhibit high affinity binding comparable to that of the parental mouse antibody (ie, mouse VH / VL in the context of human constant regions).

[0553] The following table summarizes the antibody affinities and binding ratios measured by Biacore:

[0554] Antibody <![CDATA[K D (M)]]> Combined ratio V4scFv 2.2E-08 2.8 V3scFv 1.9E-08 5.5 V7scFv 1.5E-08 3.7 V7IgG 1.3E-09 1.1 V3IgG 1.0E-09 1.2 Parental 9.6E-10 1.3 V4IgG 9.1E-10 1.1

[0555] Four kinds of IgG antibodies (parent mouse antibody and 3 kinds of humanized antibodies: v3, v4 and v7) are compared with the single antigen binding of corresponding humanized scFv and Biacore. Mouse scFv cannot be expressed, so it cannot be used for affinity comparison. Compared with scFv, IgG antibody is more tightly bound to antigen almost 10 times. This is expected, because IgG is essentially bivalent, and avidity (avidity) will lead to tighter combination compared with the scFv expressed by monovalent.

[0556] Antibodies v3, v4, v7, and v11 (in the case of v11, in both IgG and scFv formats) were further characterized. The antibodies were evaluated for their ability to activate anti-HER2-FLAG CART cells and induce IFN-γ. Antibodies induced IFN-γ expression, such as Figure 2 As shown. Antibody v11 was also included in the evaluation in both IgG and scFv formats. The results showed that antibodies v7 and v4 induced the greatest amount of IFN-γ by CART cells, respectively.

[0557] Based on these results, antibody variant v4 and antibody variant v7 were selected for future studies.

[0558] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

Claims

1. An antigen-binding protein for binding to a FLAG tag, the protein comprising an antigen-binding domain, the antigen-binding domain comprising CDRH1, CDRH2 and / or CDRH3 having an antigen-binding domain of a variable heavy chain (VH) as defined by any one of SEQ ID NOs: 2 to 5, and CDRL1, CDRL2 and / or CDRL3 having a variable light chain (VL) as defined by any one of SEQ ID NOs: 7 to 10.

2. The antigen-binding protein according to claim 1, wherein The protein can specifically bind to a FLAG tag or a variant thereof, wherein the FLAG tag or a variant thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs: 11 or 29 to 41.

3. The antigen-binding protein according to claim 1 or 2, wherein The protein is used to specifically bind to a peptide comprising or consisting of the amino acid sequence DYKDDDDK (SEQ ID NO: 30).

4. The antigen-binding protein according to any one of claims 1 to 3, wherein The antigen-binding protein is used to specifically bind to a FLAG tag contained in or on the following: a recombinant protein or a therapeutic protein, a cellular immunotherapeutic agent, or a receptor present on a cellular immunotherapeutic agent.

5. The antigen-binding protein according to any one of claims 1 to 4, wherein The protein competitively inhibits the binding of the FLAG tag to the following antibodies: - comprising a VH comprising the sequence shown in SEQ ID NO: 1 and a VL comprising the sequence shown in SEQ ID NO: 6; - comprising a VH comprising the sequence shown in SEQ ID NO: 2 and a VL comprising the sequence shown in SEQ ID NO: 7; - comprising a VH comprising the sequence shown in SEQ ID NO: 2 and a VL comprising the sequence shown in SEQ ID NO: 8; - comprising a VH comprising the sequence shown in SEQ ID NO: 2 and a VL comprising the sequence shown in SEQ ID NO: 9; - comprising a VH comprising the sequence shown in SEQ ID NO: 2 and a VL comprising the sequence shown in SEQ ID NO: 10; - comprising a VH comprising the sequence shown in SEQ ID NO: 3 and a VL comprising the sequence shown in SEQ ID NO: 7; - comprising a VH comprising the sequence shown in SEQ ID NO: 3 and a VL comprising the sequence shown in SEQ ID NO: 8; - comprising a VH comprising the sequence shown in SEQ ID NO: 3 and a VL comprising the sequence shown in SEQ ID NO: 9; - comprising a VH comprising the sequence shown in SEQ ID NO: 3 and a VL comprising the sequence shown in SEQ ID NO: 10; - comprising a VH comprising the sequence shown in SEQ ID NO: 4 and a VL comprising the sequence shown in SEQ ID NO: 7; - comprising a VH comprising the sequence shown in SEQ ID NO: 4 and a VL comprising the sequence shown in SEQ ID NO: 8; - comprising a VH comprising the sequence shown in SEQ ID NO: 4 and a VL comprising the sequence shown in SEQ ID NO: 9; - comprising a VH comprising the sequence shown in SEQ ID NO: 4 and a VL comprising the sequence shown in SEQ ID NO: 10; - comprising a VH comprising the sequence shown in SEQ ID NO: 5 and a VL comprising the sequence shown in SEQ ID NO: 7; - comprising a VH comprising the sequence shown in SEQ ID NO: 5 and a VL comprising the sequence shown in SEQ ID NO: 8; - comprising a VH comprising the sequence shown in SEQ ID NO: 5 and a VL comprising the sequence shown in SEQ ID NO: 9; or - comprising a VH comprising the sequence shown in SEQ ID NO:5 and a VL comprising the sequence shown in SEQ ID NO:

10.

6. The antigen-binding protein according to any one of claims 1 to 5, wherein The antigen binding domain comprises: A. VH and VL, VH comprises a complementary determining region (CDR) 1 comprising or consisting of the amino acid sequence of SEQ ID NO: 12, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 13, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 14; VL comprises a complementary determining region (CDR) 1 comprising or consisting of the amino acid sequence of SEQ ID NO: 21, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 23; and any one of B., C., D., E., F., F., G., H., I., J., K., L., M., N., O., P., Q., R. below: B. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 65, 66, 67, and 68, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively, or C. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively, or D. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively, or E. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively, or F. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively, or G. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively, or H. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively, or I. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively, or J. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively, or K. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively, or L. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively, or M. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively, or N. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively, or O. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively, or P. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively, or Q. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively, or R. VH and VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively.

7. The antigen-binding protein according to any one of claims 1 to 5, wherein The antigen binding domain comprises: A. VH and VL, VH comprises a complementary determining region CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 42 or 43, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 44, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45; VL comprises a complementary determining region CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 51, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 52, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 23; AND any of B., C., D. or E. below: B. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively, or C. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively, or D. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively, or E. VH and VL each comprising framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively.

8. The antigen-binding protein according to any one of claims 1 to 5, wherein The antigen binding domain comprises: A. VH and VL, VH comprises a complementary determining region CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 42 or 43, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 44, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45; VL comprises a complementary determining region CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 64, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 52, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 23; AND any of B., C., D. or E. below: B. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively, or C. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively, or D. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively, or E. VH and VL each comprising framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively.

9. The antigen-binding protein according to any one of claims 1 to 5, wherein The antigen binding domain comprises: A. VH and VL, VH comprises a complementary determining region CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 42 or 87, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 88, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45; VL comprises a complementary determining region CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 51, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 52, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 23; AND any of B., C., D. or E. below: B. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively, or C. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively, or D. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively, or E. VH and VL each comprising framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively.

10. The antigen-binding protein according to any one of claims 1 to 5, wherein The antigen binding domain comprises: A. VH and VL, VH comprises a complementary determining region CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 42 or 87, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 88, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45; VL comprises a complementary determining region CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 64, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 52, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 23; AND any of B., C., D. or E. below: B. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively, or C. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively, or D. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively, or E. VH and VL each comprising framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively.

11. The antigen-binding protein according to any one of claims 1 to 5, wherein The antigen binding domain comprises: A. VH and VL, VH comprises a complementary determining region CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 57, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 58, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45; VL comprises a complementary determining region CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 51, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 52, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 23; AND any of B., C., D., E., F., G., H. or I. below: B. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively, or C. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively, or D. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively, or E. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively, or F. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or G. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively; or H. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 96, 98, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or I. VH and VL each comprising framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 96, 98 and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 53, 54, 56 and 28, respectively.

12. The antigen-binding protein according to any one of claims 1 to 5, wherein The antigen binding domain comprises: A. VH and VL, VH comprises a complementary determining region CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 57, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 58, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45; VL comprises a complementary determining region CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 64, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 52, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 23; AND any of B., C., D., E., F., G., H. or I. below: B. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or C. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 99, 100, 101, and 28, respectively; or D. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or E. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 99, 100, 101, and 28, respectively; or F. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or G. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 99, 100, 101, and 28, respectively; or H. a VH and a VL each comprising framework regions (FR) 1, 2, 3, and 4, wherein framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 96, 98, and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or I. VH and VL each comprising framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 95, 96, 98 and 20, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 99, 100, 101 and 28, respectively.

13. The antigen-binding protein according to any one of claims 1 to 5, wherein The antigen binding domain comprises: A. a VH comprising complementarity determining regions (CDR) 1, CDR2 and CDR3 as defined above in (o), and a VL comprising CDR1, CDR2 and CDR3 as defined above in (p), and each of the VH and VL comprises framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 72, 73, 74 and 68, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77 and 28, respectively; or B. A VH comprising complementarity determining regions (CDR) 1, CDR2 and CDR3 as defined in (q) above, and a VL comprising CDR1, CDR2 and CDR3 as defined in (r) above, and VH and VL each comprising framework regions (FR) 1, 2, 3 and 4, wherein framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to a reference sequence, wherein for each VH FR, the reference sequence is set forth in SEQ ID NOs: 15, 78, 79 and 68, respectively, and for each VL FR, the reference sequence is set forth in SEQ ID NOs: 75, 76, 77 and 28, respectively.

14. The antigen binding domain according to any one of claims 1 to 12, wherein The antigen binding domain comprises a variable heavy chain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 5, and a variable light chain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 7 to 10.

15. The antigen binding domain of any one of claims 1 to 13, wherein The antigen binding protein is in the form of: (i) Single domain antibodies (sdAb); (ii) single-chain Fv fragment (scFv); (iii) dimeric scFv (di-scFv); (iv) one of (ii) or (iii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody; (v) one of (i) to (iv) linked to a protein that binds to immune effector cells; (vi) one of (i) to (iv) linked to a modified immune cell receptor, such as a modified T cell receptor; or (vii) one of (i) to (iv) in the context of a chimeric antigen receptor (CAR) or a variant T cell receptor.

16. The antigen binding domain of any one of claims 1 to 14, wherein The antigen binding protein is in the form of: (i) Diabodies; (ii) tri-antibody; (iii) tetrabodies; (iv) Fab; (v) F(ab')2; (vi) Fv; (vii) bispecific antibodies or other forms of multispecific antibodies; (viii) one of (i) to (vii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody; (ix) one of (i) to (vii) linked to a protein that binds to immune effector cells; (x) one of (i) to (vii) linked to a protein that binds to immune effector cells; (xi) one of (i) to (vii) linked to a modified immune cell receptor, such as a modified T cell receptor; or (xiii) one of (i) to (vii) in the context of a chimeric antigen receptor (CAR) or a variant T cell receptor, wherein the variant T cell receptor is included in the context of a universal CAR system for use with a polypeptide comprising an antigen binding domain that is used to bind to an antigen on the surface of a target cell.

17. The antigen binding domain of any one of claims 1 to 15, wherein The antigen-binding protein comprises, consists essentially of, or consists of the following amino acid sequence, wherein the following amino acid sequence is in the order from N-terminus to C-terminus or from C-terminus to N-terminus: - SEQ ID NO: 2 and 10; - SEQ ID NO: 3 and 9; - SEQ ID NO: 2 and 9; - SEQ ID NO: 2 and 7; - SEQ ID NO: 2 and 8; - SEQ ID NO: 3 and 7; - SEQ ID NO: 3 and 8; - SEQ ID NO: 3 and 10; - SEQ ID NO: 4 and 7; - SEQ ID NO: 4 and 8; - SEQ ID NO: 4 and 9; - SEQ ID NO: 4 and 10; - SEQ ID NO: 5 and 7; - SEQ ID NO: 5 and 8; - SEQ ID NO: 5 and 9; or - SEQ ID NO: 5 and 10.

18. A fusion protein comprising the antigen-binding protein of any one of claims 1 to 16.

19. A chimeric antigen receptor or variant T cell receptor comprising the antigen binding protein of any one of claims 1 to 16 or the fusion protein of claim 17.

20. A conjugate comprising the antigen-binding protein of any one of claims 1 to 16.

21. A nucleic acid encoding the antigen-binding protein of any one of claims 1 to 16 or the fusion protein of claim 17.

22. A vector construct comprising the nucleic acid of claim 20.

23. A cell comprising the nucleic acid of claim 20 or the vector construct of claim 21.

24. A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1 to 16, the fusion protein of claim 17, or the conjugate of claim 19, and a pharmaceutically acceptable carrier, diluent, or excipient.

25. A diagnostic composition comprising the antigen binding protein of any one of claims 1 to 16, the fusion protein of claim 17, or the conjugate of claim 19, and a pharmaceutically acceptable carrier, diluent or excipient.

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