anti-FLAG antibody

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to antigen binding proteins and related fragments thereof, including humanized antigen binding proteins and related fragments thereof, for binding to a FLAG tag, to the production of said antigen binding proteins and fragments thereof, and to the use of said antibodies and fragments thereof.

[0002] Related Applications This application claims priority to Australian provisional application AU2022902712, the entire disclosure of which is incorporated herein. [Background technology]

[0003] Background of the Invention The "FLAG" tag (also known as the FLAG epitope) is an artificial antigenic peptide tag that is frequently incorporated into proteins to facilitate detection and / or affinity purification of the target protein. It is one of the most commonly used protein tags in laboratories worldwide.

[0004] Although antibodies for binding to FLAG tags are known, such antibodies have been generated in mice or other rodents and, as a result, are not suitable for clinical use. While the use of mouse anti-FLAG antibodies is often acceptable for detecting or purifying recombinant proteins in in vitro systems, the use of mouse antibodies is not always suitable for detecting or binding to FLAG tags in in vivo systems.

[0005] Consequently, there is a need for new and / or improved anti-FLAG antibodies.

[0006] The reference to any prior art herein is not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art would be understood by, considered relevant, and / or reasonably expected to be incorporated into other pieces of prior art by those skilled in the art.

Prior Technology Literature

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Non-licensed literature

[0008] [Non-licensed document 1] J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984)

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[0009] The present invention relates to antigen-binding proteins comprising an antigen-binding domain for binding to a FLAG tag. In particular, the present invention relates to humanized anti-FLAG antibodies and FLAG-binding fragments thereof.

[0010] The present invention provides an antigen binding protein for binding to a FLAG tag, comprising: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, and providing an antigen-binding protein comprising an antigen-binding domain comprising: where: FR1, FR2, FR3, and FR4 are each a framework region; CDR1, CDR2 and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a, and FR4a are each a framework region; CDR1a, CDR2a, and CDR3a are each a complementarity determining region; The sequences of any framework regions and / or complementarity determining regions are as described herein, and preferably as set out in Table 1 below.

[0011] In either embodiment, CDR1, CDR2, and CDR3 refer to the complementarity determining regions from the variable heavy chain (VH) of the antibody, and CDR1a, CDR2a, and CDR3a are the complementarity determining regions from the variable light chain (VL) of the antibody, or if CDR1, CDR2, and CDR3 are the complementarity determining regions from the VL, then CDR1a, CDR2a, and CDR3a are the complementarity determining regions from the VH. In such instances, the CDRs may sometimes be referred to as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.

[0012] In either embodiment, the antigen binding proteins of the invention are capable of specifically binding to a FLAG tag or variants thereof (as defined in SEQ ID NOs: 11 and 29, or as defined elsewhere herein). The antigen binding proteins of the invention are preferably capable of specifically binding to protein domains comprising multiple FLAG tag sequences (e.g. 2xFLAG, 3xFLAG, etc.).

[0013] In either embodiment, the antigen binding proteins of the invention are for specific binding 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 (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).

[0014] In a preferred embodiment, the antigen binding protein is capable of specifically binding to a FLAG tag containing, or consisting of, the 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).

[0015] As used herein, the term FLAG tag also refers to FLAG tags that have been modified by amino acid insertions, deletions or substitutions derived from the above-mentioned FLAG tags, in particular tags having the sequence DYKDDDDK.

[0016] Reference herein to a protein or antibody that "binds" to a FLAG tag provides literal support for the protein or antibody "binds specifically to" or "specifically binds to" the FLAG tag.

[0017] The antigen binding proteins of the present invention are intended to specifically bind to a FLAG tag, particularly in the context of recombinant or therapeutic proteins (including antibodies or fragments thereof or proteins comprising an antigen binding domain for use in therapeutics) or cell-based therapies (cellular immunotherapy including genetically modified cells) that comprise a FLAG tag. In certain instances, the antigen binding proteins are useful for binding to FLAG tags contained in chimeric antigen receptors and related recombinant receptors for expression by cellular immunotherapeutics (e.g., contained in CARs for expression by cytotoxic T cells).

[0018] In either embodiment, the FLAG tag is present at the N-terminus, C-terminus or within the protein it is intended to bind.

[0019] In any embodiment, the present invention provides an antigen binding protein for binding to a FLAG tag, the antigen binding protein comprising: - comprising a VH comprising the sequence set forth in SEQ ID NO: 1 and a VL comprising the sequence set forth in SEQ ID NO: 6, - comprising a VH comprising the sequence set forth in SEQ ID NO: 2 and a VL comprising the sequence set forth in SEQ ID NO: 7, - comprising a VH comprising the sequence set forth in SEQ ID NO: 2 and a VL comprising the sequence set forth in SEQ ID NO: 8, - comprising a VH comprising the sequence set forth in SEQ ID NO: 2 and a VL comprising the sequence set forth in SEQ ID NO: 9, - comprising a VH comprising the sequence set forth in SEQ ID NO: 2 and a VL comprising the sequence set forth in SEQ ID NO: 10, - comprising a VH comprising the sequence set forth in SEQ ID NO: 3 and a VL comprising the sequence set forth in SEQ ID NO: 7, - comprising a VH comprising the sequence set forth in SEQ ID NO: 3 and a VL comprising the sequence set forth in SEQ ID NO: 8, - comprising a VH comprising the sequence set forth in SEQ ID NO: 3 and a VL comprising the sequence set forth in SEQ ID NO: 9, - comprising a VH comprising the sequence set forth in SEQ ID NO: 3 and a VL comprising the sequence set forth in SEQ ID NO: 10, - comprising a VH comprising the sequence set forth in SEQ ID NO: 4 and a VL comprising the sequence set forth in SEQ ID NO: 7, - comprising a VH comprising the sequence set forth in SEQ ID NO: 4 and a VL comprising the sequence set forth in SEQ ID NO: 8, - comprising a VH comprising the sequence set forth in SEQ ID NO: 4 and a VL comprising the sequence set forth in SEQ ID NO: 9, - comprising a VH comprising the sequence set forth in SEQ ID NO: 4 and a VL comprising the sequence set forth in SEQ ID NO: 10, - comprising a VH comprising the sequence set forth in SEQ ID NO: 5 and a VL comprising the sequence set forth in SEQ ID NO: 7, - comprising a VH comprising the sequence set forth in SEQ ID NO: 5 and a VL comprising the sequence set forth in SEQ ID NO: 8, - comprising a VH comprising the sequence set forth in SEQ ID NO: 5 and a VL comprising the sequence set forth in SEQ ID NO: 9, or - comprising a VH comprising the sequence set forth in SEQ ID NO: 5 and a VL comprising the sequence set forth in SEQ ID NO: 10 Competitively inhibits antibody binding to the FLAG tag.

[0020] In any embodiment, the present invention provides an antigen binding protein having an antigen binding domain CDRH1, CDRH2 and / or CDRH3 having a variable heavy chain as defined in any one of SEQ ID NOs: 2 to 5.

[0021] In any embodiment, the present invention provides an antigen binding protein having an antigen binding domain CDRL1, CDRL2 and / or CDRL3 having a variable light chain as defined in any one of SEQ ID NOs: 7 to 10.

[0022] 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 defined in any one of SEQ ID NOs: 2 to 5, and CDR1, CDR2 and / or CDR3 of an antigen-binding domain having a variable light chain defined in any one of SEQ ID NOs: 7 to 10.

[0023] In one embodiment, the antigen binding domain comprises: (a) and (b): (a) a complementarity determining region 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: 12; a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 4%, 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; a VH 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: 14; (b) a complementarity determining region (CDR) 1 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: 21; a VL comprising a CDR2 comprising, or consisting of, an amino acid sequence that is 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: 23; a VL 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: 23; or (c) and (d): (c) a complementarity determining region 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: 42 or 43, 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% or at least about 95% identical to the sequence of SEQ ID NO: 44 a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is 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; a VH 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:45; (d) a complementarity determining region 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, 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 a VL comprising a CDR2 comprising, or consisting of, an amino acid sequence that is 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: 23; a VL 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: 23; or (e) and (f): (e) a complementarity determining region 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: 42 or 43, 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% or at least about 95% identical to the sequence of SEQ ID NO: 44 a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is 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; a VH 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:45; (f) a complementarity determining region 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, 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 a VL comprising a CDR2 comprising, or consisting of, an amino acid sequence that is 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: 23; a VL 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: 23; or (g) and (h): (g) a complementarity determining region 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: 42 or 87, 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% or at least about 95% identical to the sequence of SEQ ID NO: 88 a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is 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; a VH 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:45; (h) a complementarity determining region 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, 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 a VL comprising a CDR2 comprising, or consisting of, an amino acid sequence that is 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: 23; a VL 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: 23; or (i) and (j): (i) a complementarity determining region 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: 42 or 87; a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is 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; a VH 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:45; (j) a complementarity determining region 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, 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 a VL comprising a CDR2 comprising, or consisting of, an amino acid sequence that is 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: 23; a VL 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: 23; or (k) and (l): (k) a complementarity determining region 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: 57, 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% or at least about 95% identical to the sequence of SEQ ID NO: 58; a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 4%, 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; a VH 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: 45; (l) a complementarity determining region 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, 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 a VL comprising a CDR2 comprising, or consisting of, an amino acid sequence that is 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: 23; a VL 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: 23; or (m) and (n): (m) a complementarity determining region 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: 57, 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% or at least about 95% identical to the sequence of SEQ ID NO: 58; a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 4%, 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; a VH 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: 45; (n) a complementarity determining region 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, 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 a VL comprising a CDR2 comprising, or consisting of, an amino acid sequence that is 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: 23; a VL 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: 23; or (o) and (p): (o) a complementarity determining region 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: 42; a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 4%, 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; a VH 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: 45; (p) a complementarity determining region 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, 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 a VL comprising a CDR2 comprising, or consisting of, an amino acid sequence that is 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: 23; a VL 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: 23; Or (q) and (r): (q) a complementarity determining region 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: 57; a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 4%, 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; a VH 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: 45; (r) a complementarity determining region 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, 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% or at least about 95% identical to the sequence of SEQ ID NO: 52 a CDR2 comprising, or consisting of, an amino acid sequence that is 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:23; and a 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:23. Includes:

[0024] In one embodiment, the antigen binding domain comprises: (a) and (b): (a) a VH comprising a complementarity 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; and (b) a VL comprising a complementarity-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; or (c) and (d): (c) a VH comprising a complementarity 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; and (d) a VL comprising a complementarity 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; or (e) and (f): (e) a VH comprising a complementarity 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; and (f) a VL comprising a complementarity 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; or (g) and (h): (g) a VH comprising a complementarity 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; and (h) a VL comprising a complementarity 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; or (i) and (j): (i) a VH comprising a complementarity 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; and (j) a VL comprising a complementarity 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; or (k) and (l): (k) a VH comprising a complementarity 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; and (l) a VL comprising a complementarity 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; or (m) and (n): (m) a VH comprising a complementarity 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; and (n) a VL comprising a complementarity 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; or (o) and (p): (o) a VH comprising a complementarity determining region CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 42, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 63, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45; and (p) a VL comprising a complementarity 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; Or (q) and (r): (q) a VH comprising a complementarity determining region (CDR) 1 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; and (r) a VL comprising a complementarity 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 Includes.

[0025] In either embodiment, FR1, FR2, FR3, and FR4 may refer to framework regions from the variable heavy chain (VH) of an antibody, and FR1a, FR2a, FR3a, and FR4a may refer to framework regions from the variable light chain (VL) of an antibody, or if FR1, FR2, FR3, and FR4 are framework regions from a VL, then FR1a, FR2a, FR3a, and FR4a are framework regions from a VH. In such examples, the FRs may sometimes be referred to as FR H1, FR H2, FR H3, FR H4, FR L1, FR L2, FR L3, and FR L4.

[0026] In any embodiment, the present invention provides an antigen binding protein having FR H1, FR H2, FR H3 and / or FR H4 from the human germline, wherein the human germline is IGHV1-46 * 01 or IGHV7-4-1 * The number is 02.

[0027] In any embodiment, the invention provides an antigen binding protein having FR L1, FR L2, FR L3 and / or FR L4 from the human germline, wherein the human germline is IGKV2-30. * 01 or IGKV4-1 * The number is 01.

[0028] In any embodiment, the present invention provides an antigen binding protein having FR H1, FR H2, FR H3 and / or FR H4 from the human germline, wherein the human germline is IGHV1-46 * 01 or IGHV7-4-1 *02 and 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 * The number is 01.

[0029] In either embodiment the antigen binding protein comprises a VH that has greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% identity to human, preferably where % identity to human is calculated as described in Example 1, and / or a VL that has greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% identity to human, preferably where % identity to human is calculated as described in Example 1.

[0030] In any embodiment, the present invention provides an antigen-binding protein having an antigen-binding domain FR H1, FR H2, FR H3 and / or FR H4 having a variable heavy chain defined in any one of SEQ ID NOs: 2 to 5.

[0031] In any embodiment, the present invention provides an antigen-binding protein having an antigen-binding domain FR L1, FR L2, FR L3 and / or FR L4, which has a variable light chain defined in any one of SEQ ID NOs: 7 to 10.

[0032] 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 in any one of SEQ ID NOs: 2 to 5, and FR1, FR2, FR3 and / or FR4 of an antigen-binding domain having a variable light chain defined in any one of SEQ ID NOs: 7 to 10.

[0033] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises: A. A VH comprising complementarity determining regions (CDR) 1, CDR2 and CDR3 as defined in any (a) above, and a VL comprising CDR1, CDR2 and CDR3 as defined in any (b) above. and B., C., D., E., F., G., H., I., J., K., L., M., N., O., P., Q. or R.: B. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 65, 66, 67, and 68, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively; or C. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively; or D. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively; or E. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively; or F. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively; or G. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively; or H. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively; or I. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively; or J. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively; or K. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively; or L. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively; or M. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively; or N. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively; or O. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively; or P. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively; or Q. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively; or R. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively. Any one of Includes:

[0034] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises: A. A VH comprising complementarity determining regions (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 B., C., D. or E.: B. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or C. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively; or D. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or E. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively. Any one of Includes:

[0035] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises: A. A VH comprising complementarity determining regions (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 B., C., D. or E.: B. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or C. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively; or D. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or E. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively. Any one of Includes.

[0036] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises: A. A VH comprising complementarity determining regions (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 B., C., D. or E.: B. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or C. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively; or D. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or E. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively. Any one of Includes.

[0037] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises: A. A VH comprising complementarity determining regions (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 B., C., D. or E.: B. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or C. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively; or D. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or E. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively. Any one of Includes:

[0038] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises: A. A VH comprising complementarity determining regions (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 B., C., D., E., F., G., H. or I.: B. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or C. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively; or D. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or E. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively; or F. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or G. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively; or H. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 96, 98, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or I. VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 96, 98, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively. Any one of Includes:

[0039] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises: A. A VH comprising complementarity determining regions (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 B., C., D., E., F., G., H. or I.: B. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or C. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 99, 100, 101, and 28, respectively; or D. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or E. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 99, 100, 101, and 28, respectively; or F. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or G. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 99, 100, 101, and 28, respectively; or H. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 96, 98, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or I. VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 96, 98, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 99, 100, 101, and 28, respectively. Any one of Includes:

[0040] In one embodiment, the present invention provides an antigen binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises: A. A VH comprising complementarity determining regions (CDRs) 1, CDR2, and CDR3 as defined in any of (o) above, and a VL comprising CDR1, CDR2, and CDR3 as defined in any of (p) above, and framework regions (FRs) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 72, 73, 74, and 68, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively. or B. A VH comprising complementarity determining regions (CDRs) 1, CDR2, and CDR3 as defined in any of (q) above, and a VL comprising CDR1, CDR2, and CDR3 as defined in any of (r) above, and framework regions (FRs) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 78, 79, and 68, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively. Includes:

[0041] In some embodiments, the antigen-binding domain comprises a variable heavy chain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 2-5, 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 thereto.

[0042] In some embodiments, the antigen-binding domain comprises a variable light chain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 7-10, or a sequence 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 thereto.

[0043] In some embodiments, the antigen-binding domain comprises a variable heavy chain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 5 and a variable light chain comprising the amino acid sequence set forth 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 thereto.

[0044] As described herein, an antigen binding protein is (i) single domain antibodies (sdAbs), (ii) single chain Fv fragment (scFv), (iii) dimeric scFv (di-scFv), (iv) one of (ii) or (iii) linked to a 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 an immune effector cell; (vi) one of (i) to (iv) linked to a modified immune cell receptor, e.g., 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 The form may be:

[0045] Further, as described herein, the antigen binding protein may be (i) diabodies, (ii) triabodies, (iii) tetrabodies, (iv) Fab, (v) F(ab')2, (vi) Fv, (vii) other forms of bispecific or multispecific antibodies; (viii) one of (i) to (vii) linked to the constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3; or (ix) one of (i) to (vii) linked to a protein that binds to an immune effector cell; (x) one of (i) to (vii) linked to a protein that binds to an immune effector cell; (xi) one of (i) to (vii) linked to a modified immune cell receptor, e.g., a modified T cell receptor; or (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). The form may be:

[0046] The antigen-binding protein may also be referred to as the antigen-binding domain of an antibody.

[0047] Preferably, the antigen-binding protein described herein is an antibody or an antigen-binding fragment thereof. Typically, the antigen-binding protein is an antibody, for example, a monoclonal antibody. The antigen-binding protein may be in the form of a recombinant antibody or a modified antibody (e.g., a chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, a primatized antibody, a deimmunized antibody, a synhumanized antibody, a half antibody, a bispecific antibody, a trispecific antibody, or a multispecific antibody). The antibody may further comprise chemical modifications, for example, conjugation to an active substance or a radiolabel, or an agent to improve solubility, or other modifications described herein.

[0048] As used herein, an antigen binding protein may be a variable domain.

[0049] The present invention provides SEQ ID NOs: 2 and 10, SEQ ID NOs: 3 and 9, SEQ ID NOs: 2 and 9, SEQ ID NOs: 2 and 7, SEQ ID NOs: 2 and 8, SEQ ID NOs: 3 and 7, SEQ ID NOs: 3 and 8, SEQ ID NOs: 3 and 10, SEQ ID NOs: 4 and 7, SEQ ID NOs: 4 and 8, SEQ ID NOs: 4 and 9, SEQ ID NOs: 4 and 10, SEQ ID NOs: 5 and 7, SEQ ID NOs: 5 and 8, SEQ ID NOs: 5 and 9, or SEQ ID NOs: 5 and 10 The present invention provides an antigen-binding protein comprising, consisting essentially of, or consisting of (in N- to C-terminal or C- to N-terminal order) the amino acid sequence of:

[0050] As used herein, the complementarity determining region sequences (CDRs) of the antigen binding proteins of the invention are defined according to the IMGT, Chothia or Kabat numbering systems.

[0051] 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.

[0052] The present invention provides an anti-FLAG tag 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 comprising an antigen binding protein having a sequence described herein or comprising a CDR and / or FR sequence described herein.

[0053] The present invention provides diabodies or triabodies comprising antigen binding proteins having the sequences described herein or comprising the CDR and / or FR sequences described herein.

[0054] The present invention provides a fusion protein comprising 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 as described herein.

[0055] In one embodiment, the antigen binding proteins of the present invention may be included as a component of a chimeric antigen protein (CAR) or variant T cell receptor. In such embodiments, it will be appreciated that the CAR or variant T cell receptor produced comprising the antigen binding protein of the present invention may be referred to as an "indirect CAR" (sometimes also referred to as a universal CAR). In an indirect CAR system, the antigen binding domain of the CAR does not directly bind to the target antigen on the target cell, but instead binds to an intermediate, which intermediate comprises an antigen binding domain for directly binding to the target cell. Examples of CARs that recognize cells via an intermediate are known in the art, for example, European Patent Application EP2651442.

[0056] Thus, in the context of an indirect CAR, the antigen-binding protein of the present invention may be for binding to a FLAG tag present on the intermediate and may be an antigen-binding domain for binding to a target cell (e.g., a cancer cell). The intermediate provides specificity for the target cell (e.g., a cancer cell), and the genetically modified cell bearing the CAR provides efficacy and directs an immune response against the target cell. The intermediate may be a molecule, e.g., a probe, that directly binds to or interacts with a target antigen on the target cell and also includes a FLAG tag. Non-limiting examples of such probes include antibodies, fusion proteins, antibody Fabs, scFvs, soluble engineered TCRs, or aptamers. The term "aptamer" refers to any oligonucleic acid, polynucleic acid, peptide, or polypeptide that specifically binds to or preferentially forms a complex with a target.

[0057] The present invention also provides conjugates in the form of antigen binding proteins, immunoglobulin variable domains, antibodies, dabs, scFvs, Fabs, Fab's, F(ab')2s, Fv fragments, diabodies, triabodies, linear antibodies, single chain antibody molecules or multispecific antibodies or fusion proteins as described herein conjugated to a label or a cytotoxic agent.

[0058] The invention provides antibodies for binding to the antigen binding proteins, immunoglobulin variable domains, antibodies, dabs, scFvs, Fabs, Fab's, F(ab')2s, Fv fragments, diabodies, triabodies, linear antibodies, single chain antibody molecules or multispecific antibodies, fusion proteins or conjugates described herein.

[0059] In aspects of the invention directed to multiple polypeptide chains forming an antigen binding protein, the expression construct comprises a nucleic acid encoding a polypeptide comprising, e.g., a VH operably linked to a promoter and a nucleic acid encoding a polypeptide comprising, e.g., a VL operably linked to a promoter.

[0060] In another example, an expression construct may comprise, for example, the following operably linked components in 5' to 3' order: (i) Promoter (ii) a nucleic acid encoding a first polypeptide; (iii) an internal ribosome entry site, and (iv) a nucleic acid encoding a second polypeptide a bicistronic expression construct comprising: wherein the first polypeptide comprises a VH and the second polypeptide comprises a VL, or vice versa.

[0061] The present invention also contemplates separate expression constructs, one encoding a first polypeptide comprising a VH and another encoding a second polypeptide comprising a VL. For example, the present invention also contemplates separate expression constructs, one encoding a first polypeptide comprising a VH and another encoding a second polypeptide comprising a VL. (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter; Also provided is a composition comprising:

[0062] The present invention provides cells comprising the vectors or nucleic acids described herein. Preferably, the cells are isolated, substantially purified, or recombinant. In one example, the cells contain an expression construct or a nucleic acid of the invention. (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter; Including, The first and second polypeptides together form the antigen binding protein of the present invention.

[0063] Examples of cells of the invention include bacterial cells, yeast cells, insect cells or mammalian cells.

[0064] The invention provides nucleic acids encoding the antigen binding proteins, immunoglobulin variable domains, antibodies, dabs, scFvs, Fabs, Fab's, F(ab')2s, Fv fragments, diabodies, triabodies, linear antibodies, single chain antibody molecules or multispecific antibodies, fusion proteins or conjugates described herein.

[0065] 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 7 to 10.

[0066] The invention provides cells comprising the vectors or nucleic acids described herein.

[0067] In another embodiment, an animal or tissue derived therefrom comprising the cells described herein is provided.

[0068] The present invention provides a pharmaceutical composition comprising an antigen binding protein or comprising the CDR and / or FR sequences described herein, or 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 described herein and a pharmaceutically acceptable carrier, diluent or excipient.

[0069] The present invention provides a diagnostic composition comprising an antigen binding protein or comprising the CDR and / or FR sequences described herein, or an antigen binding protein described herein, an immunoglobulin variable domain, an 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, a diluent and optionally a label.

[0070] The present invention provides kits or articles of manufacture comprising an antigen binding protein or comprising the CDR and / or FR sequences described herein, or the immunoglobulin variable domains, antibodies, dabs, scFvs, Fabs, Fab's, F(ab')2s, Fv fragments, diabodies, triabodies, linear antibodies, single chain antibody molecules or multispecific antibodies, fusion proteins or conjugates described herein.

[0071] The present invention provides for the use of sequences according to one or more of the CDR1, CDR2, CDR3, FR1, FR2, FR3 and FR4 described herein to generate antigen binding proteins for binding to a FLAG tag.

[0072] The present invention provides a library of nucleic acid molecules generated from mutations of the antigen binding proteins or CDR and / or FR sequences described herein, wherein at least one nucleic acid molecule in the library encodes an antigen binding protein for binding to a FLAG tag.

[0073] The invention provides methods for producing an antigen binding protein for binding to a FLAG tag as described herein, the method comprising expressing a nucleic acid as described herein in a cell or animal as described herein.

[0074] In any aspect of the invention, the antigen binding protein comprises an Fc region that has been engineered to have an enhanced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, the enhanced ability to induce ADCC is conferred by mutation, deletion, or modification of amino acids in the Fc region that interact with Fc receptors. Preferably, the mutated, deleted, or modified amino acids are at positions 239, 330, and / or 332, or positions equivalent to 239, 330, and / or 332, such as in SEQ ID NO: 60 (alanine is at position 118). Preferably, the amino acids are mutated to S239D, A330L, and I332E. Typically, the Fc comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 62.

[0075] In any aspect of the invention, the antigen binding protein comprises an Fc region that has not been engineered to have a reduced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, the amino acids at or equivalent to positions 234, 235 and / or 331 as in SEQ ID NO: 60 (alanine is at position 118) are not F, E and / or 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.

[0076] In another embodiment, the antigen binding protein is - increasing in vitro or in vivo half-life, - have an increased ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity, - reducing effector function, or - Increased co-engagement of antigen-binding proteins The Fc region is engineered to:

[0077] The present invention provides the use of 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, conjugate or pharmaceutical composition as described herein in the manufacture of a medicament for the treatment of cancer or a condition or disease associated with expression of a FLAG tag.

[0078] The antigen-binding proteins, proteins or antibodies described herein preferably comprise a human constant region, e.g., an IgG constant region, e.g., an IgG1, IgG2, IgG3 or IgG4 constant region, or a mixture thereof. In the case of an antibody or protein comprising a VH and a VL, the VH may be linked to a heavy chain constant region, and the VL may be linked to a light chain constant region.

[0079] The functional characteristics of antigen binding proteins of the invention apply mutatis mutandis to antibodies of the invention.

[0080] The antigen binding proteins described herein may be purified, substantially purified, isolated, and / or recombinant.

[0081] The antigen binding protein of the invention may be part of the supernatant harvested from the culture medium in which a hybridoma expressing the antigen binding protein of the invention has been grown.

[0082] The present invention provides a single domain antibody comprising an antigen binding protein for binding to a FLAG tag.

[0083] As used herein, unless the context requires otherwise, the term "comprise" and variations thereof, such as "comprising," "comprises," and "comprised," does not exclude additional additives, components, integers, or steps.

[0084] Further aspects of the 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 referring to the accompanying drawings, in which: [Brief explanation of the drawings]

[0085] [Figure 1] FIG. 1 shows the ELISA titers of IgG humanized FLAG tag-binding monoclonal antibodies. [Figure 2] FIG. 2 shows that humanized monoclonal antibodies for binding to FLAG-tagged CAR induce IFN-gamma secretion from CAR T cells. [Figure 3] FIG. 3 shows the alignment of the VH and VL regions from the parent antibody with humanized variants v4 and v7. DETAILED DESCRIPTION OF THE INVENTION

[0086] [Table 1A]

[0087] [Table 1B]

[0088] [Table 1C]

[0089] [Table 1D]

[0090] [Table 1E]

[0091] [Table 1F]

[0092] [Table 1G]

[0093] [Table 1H]

[0094] [Table 1I]

[0095] [Table 1J]

[0096] [Table 1K]

[0097] Detailed Description of the Embodiments It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent in the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

[0098] Further aspects of the 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 referring to the accompanying drawings, in which:

[0099] Reference will now be made in detail to certain specific embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims.

[0100] The present inventors have developed antigen-binding proteins, e.g., antibodies, that bind to FLAG tags. The antigen-binding proteins described herein are intended to bind to one or more FLAG tags present on either recombinant proteins, recombinant peptides, or genetically modified cells (including genetically modified cells that have been modified to express a receptor on their surface, where the receptor contains one or more FLAG tags). In a particularly preferred embodiment, the antigen-binding proteins may be used to bind to one or more FLAG tags present on a chimeric antigen receptor (CAR) expressed by a genetically modified cell, e.g., a cytotoxic cell.

[0101] The antigen binding proteins of the present invention are humanized antibodies, making them particularly suitable for binding to CAR or other receptors or proteins expressed by genetically engineered cells for use in human therapeutics. In particular, proteins that have been engineered to contain a FLAG tag, such as a tag described herein, may be bound by the antigen binding proteins of the present invention in vitro or in vivo.

[0102] General Throughout this specification, unless specifically stated otherwise or unless the context requires otherwise, a reference to a single step, composition, group of steps, or group of compositions should be taken to include one and more (i.e., one or more) of those steps, compositions, group of steps, or group of compositions. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects, and vice versa, unless the content clearly dictates otherwise. For example, reference to "a" includes the singular as well as two or more, reference to "an" includes the singular as well as two or more, reference to "the" includes the singular as well as two or more, etc.

[0103] Those skilled in the art will recognize that the present invention is susceptible to variations and modifications other than those specifically described. It is to be understood that the present invention includes all such variations and modifications. The present invention also includes all of the steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations of said steps or features, or any two or more thereof.

[0104] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0105] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0106] The present invention is not to be limited in scope by the specific examples described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention.

[0107] Any example or embodiment of the invention herein shall apply mutatis mutandis to any other example or embodiment of the invention, unless expressly stated otherwise.

[0108] Unless expressly defined otherwise, all technical and scientific terms used herein should be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0109] 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 in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Vols. 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Vols. 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all revisions to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J. E. Coligan et al. (editors), Current Protocols in It is described and explained throughout the literature in sources such as Immunology, John Wiley & Sons (including all current editions).

[0110] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein can be further clarified by a discussion in 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.

[0111] The term "and / or," e.g., "X and / or Y," must be understood to mean either "X and Y" or "X or Y," and must be taken to provide clear support for both meanings or either meaning.

[0112] As used herein, the term "derived from" should be taken to indicate that the specified integer can be obtained from a particular source, although not necessarily directly from that source.

[0113] References herein to various, e.g., residues, are understood to be inclusive, e.g., reference to "the region comprising amino acids 56-65" is understood in an inclusive manner, i.e., the region includes the sequence of amino acids numbered 56, 57, 58, 59, 60, 61, 62, 63, 64, and 65 in the specified sequence.

[0114] Selected Definitions As used herein, a FLAG-tag or FLAG octapeptide or FLAG epitope is a polypeptide protein tag that can be added to a protein using recombinant DNA technology and has the sequence motif DYKDDDDK (SEQ ID NO: 29). The FLAG tag can be fused to the C-terminus or N-terminus of the protein, or inserted within the protein. As such, the antigen binding proteins of the invention are capable of specifically binding to the FLAG tag, whether present at the C-terminus or N-terminus of the protein, or inserted within the protein. In a specific example, a FLAG tag can be included in a chimeric antigen receptor (CAR) construct, and thus the antigen binding protein of the invention can bind to the CAR via the FLAG tag.

[0115] As used herein, "chimeric antigen receptor (CAR)" refers to a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain 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 transmits a signal in a cell, causing activation or inhibition of a biological process.

[0116] An "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, by virtue of its origin or source of derivation, is not associated with naturally associated components which accompany it in its natural state and is substantially free from other proteins from the same source. A protein can be rendered substantially free of naturally associated components by isolation, or substantially purified, using protein purification techniques known in the art. "Substantially purified" means that the protein is substantially free from contaminants, e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% free.

[0117] The term "recombinant" should be understood to mean the product of artificial genetic recombination. Thus, in the context of a recombinant protein containing an antibody antigen-binding domain, this term does not encompass naturally occurring antibodies within a subject's body that are the product of natural recombination that occurs during B-cell maturation. However, when such an antibody is isolated, it should be considered an isolated protein containing an antibody antigen-binding domain. Similarly, when a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein containing an antibody antigen-binding domain. Recombinant protein also encompasses proteins expressed by artificial recombinant means, for example, when it is within the cell, tissue, or subject in which it is expressed.

[0118] The term "protein" should be taken to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains (i.e., a polypeptide complex) linked to each other by covalent or non-covalent bonds. For example, a series of polypeptide chains may be covalently linked using suitable chemicals or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0119] The term "polypeptide" or "polypeptide chain" will be understood from the previous paragraph to mean a series of consecutive amino acids linked by peptide bonds.

[0120] As used herein, the term "antigen-binding protein" is used synonymously with "antigen-binding domain" and should be taken to mean the region of an antibody that is capable of specifically binding to an antigen, i.e., an Fv comprising a VH or VL or both a VH and a VL. The antigen-binding domain need not be in the context of a whole antibody, for example, it may be isolated (e.g., a domain antibody) or in another form, such as an scFv, as described herein.

[0121] For purposes of this disclosure, the term "antibody" includes proteins capable of specifically binding to one or a small number of closely related antigens via an antigen-binding domain contained within an Fv. This term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant antibodies, or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, deimmunized antibodies, synhumanized antibodies, half antibodies, and bispecific antibodies). Antibodies generally contain a constant domain and may be arranged into a constant region, constant fragment, or crystallizable fragment (Fc). An exemplary form of an antibody contains a four-chain structure as its basic unit. Full-length antibodies contain two covalently linked heavy chains (approximately 50-70 kDa) and two light chains (approximately 23 kDa each). The light chain generally contains a variable region (if present) and a constant domain and, in mammals, is either a kappa or lambda light chain. Heavy chains generally contain a variable region and one or two constant domains linked to additional constant domains by a hinge region. Mammalian heavy chains are of 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, two heavy chains and heavy and light chains are associated by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds can vary among different types of antibodies. Each chain has an N-terminal variable region (VH or VL, each approximately 110 amino acids long) and one or more C-terminal constant domains. The light chain constant domain (CL, approximately 110 amino acids long) is aligned with and disulfide-bonded to the first heavy chain constant domain (CH1, 330-440 amino acids long). The light chain variable region is aligned with the heavy chain variable region. An antibody heavy chain may comprise two or more additional CH domains (e.g., CH2, CH3, etc.) and may comprise a hinge region between the CH1 and CH2 constant domains. The antibody may 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 is missing a C-terminal lysine residue, hi one example, the antibody is humanized, synhumanized, chimeric CDR-grafted or deimmunized.

[0122] The terms "full length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen-binding fragment thereof. Specifically, whole antibodies include those having heavy and light chains, including the 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.

[0123] As used herein, "variable region" refers to the portion of the light chain and / or heavy chain of an antibody defined herein that is capable of specifically binding to an antigen, and includes the amino acid sequences of the complementarity determining regions (CDRs), i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, a variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally, FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0124] FLAG tags according to the present invention are, for example, amino acid-based markers described 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 in particular contain or consist 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 (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). Examples of such FLAG tags are discussed in the above patent specifications and can be used within the scope of the present invention.

[0125] As used herein, the term "subject" should be taken to mean any animal, e.g., a mammal, including a human. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0126] An "antibody" or "immunoglobulin" or "Ig" is a gamma globulin protein found in the blood or other body fluids of vertebrates that functions in the immune system to bind antigens, thus identifying and neutralizing foreign substances.

[0127] Antibodies are generally heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to an H chain by one covalent disulfide bond. The two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges.

[0128] The H and L chains define specific Ig domains. More specifically, each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus, followed by 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 of the heavy chain (CH1).

[0129] Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Light chains from any vertebrate species can be assigned to two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains.

[0130] The constant domain comprises the Fc portion, which comprises the carboxy-terminal portions of both 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 (FcRs) found on certain types of cells.

[0131] The pairing of VH and VL together forms a "variable region" or "variable domain" comprising the amino-terminal domain of either the heavy or light chain of an antibody. The heavy chain variable domain is sometimes referred to as "VH." The light chain variable domain is sometimes referred to as "VL." The V domain contains antigen-binding proteins that influence antigen binding and define the specificity of a particular antibody for its particular antigen. The V region spans approximately 110 amino acid residues and consists of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) (generally about 4 regions), separated by shorter, extremely variable regions (generally about 3 regions), each 9-12 amino acids long, called "hypervariable regions." The FRs largely adopt a β-sheet configuration, and the hypervariable regions form loops that connect the β-sheets and, in some cases, form part of the β-sheet structure.

[0132] "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. Generally, antibodies contain six hypervariable regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Several hypervariable region delineations are in use and are encompassed herein.

[0133] As used herein, "complementarity determining region" (synonym: CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region whose presence is primarily responsible for 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, where 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 referred to herein as CDR L1, CDR L2, and CDR L3, respectively, where CDR L1 corresponds to CDR1 of VL, CDR L2 corresponds to CDR2 of VL, and CDR L3 corresponds to CDR3 of VL. In one example, the amino acid positions assigned to the CDRs and FRs are defined according to the Kabat Sequences of Immunologically Important Proteins, National Institutes of Health, Bethesda, Maryland, 1987 and 1991 (also referred to herein as the "Kabat numbering system"). In another example, the amino acid positions assigned to the CDRs and FRs are defined according to the Chothia numbering scheme (http: / / www.bioinfo.org.uk / mdex.html). The present invention is not limited to FRs and CDRs defined by the Kabat numbering system, but includes all numbering systems, including the canonical numbering systems or those of 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, the numbering system of Honnegher and Plukthun J. Mol. Biol. 309: 657-670, 2001, or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25: 206-211, 1997. In one example, the CDRs are defined according to the Kabat numbering system.Optionally, the heavy chain CDR2 according to the Kabat numbering system does not include the five C-terminal amino acids listed herein, or any one or more of those amino acids are substituted with another naturally occurring amino acid. In this regard, Padlan et al., FASEB J., 9:133-139, 1995, established that the five C-terminal amino acids of heavy chain CDR2 are generally not involved in antigen binding.

[0134] "Framework" or "FR" residues are variable domain residues other than 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, where FR H1 corresponds to FR 1 of VH, FR H2 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, where FR L1 corresponds to FR 1 of VL, FR L2 corresponds to FR 2 of VL, FR L3 corresponds to FR 3 of VL, and FR L4 corresponds to FR 4 of VL.

[0135] "Peptides for forming antigen-binding proteins" generally refer to peptides that can form a conformation that confers specificity of the antibody for an antigen. Examples include whole antibodies or whole antibody-related structures, whole antibody fragments including the variable domain, variable domains and fragments thereof including light and heavy chains, or fragments of the light and heavy chains that include some but not all of the hypervariable or constant regions.

[0136] An "intact" or "whole" antibody is one which comprises an antigen-binding protein as well as a CL and at least heavy chain constant domains, 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.

[0137] "Whole antibody related structures" include multimerized forms of whole antibodies.

[0138] "Whole antibody fragments comprising the variable domain" include Fab, Fab', F(ab')2 and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0139] Fab fragments consist of an entire L chain along with the variable region domain of the H chain (VH) and the first constant domain of the heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding domain.

[0140] Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group.

[0141] The F(ab')2 fragment roughly corresponds to two disulfide-linked Fab fragments that have bivalent antigen-binding activity and are still capable of cross-linking antigen.

[0142] "Fv" is an antibody fragment containing a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.

[0143] In single-chain Fv (scFv) species, one heavy-chain and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure similar to that in a two-chain Fv species. Folding of these two domains gives rise to six hypervariable loops (three loops each from the H and L chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody.

[0144] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the VH and VL antibody domains connected to form a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0145] A "single variable domain" is one 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.

[0146] "Diabody" refers to an antibody fragment having two antigen-binding sites, which fragment comprises 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 previous paragraph) using a short linker (about 5-10 residues) between the VH and VL domains, such that interchain, but not intrachain, pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites.

[0147] 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 commonly known in the art.

[0148] An "isolated antibody" is one that has been identified and separated and / or recovered from components of its existing environment. Contaminating components are materials that would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0149] A "human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies, which comprise non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Human antibodies can be prepared by administering antigen to transgenic animals that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled.

[0150] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Most frequently, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. Generally, humanized antibodies will comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of a globulin constant region (Fc), typically that of a human immunoglobulin.

[0151] A "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 possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. Monoclonal antibodies can be prepared by the hybridoma method or can be made using recombinant DNA methodology in bacterial, eukaryotic animal, or plant cells. "Monoclonal antibodies" can also be isolated from phage antibody libraries.

[0152] As used herein, monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity. Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0153] 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 29) with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting proteins or cells that express and display the FLAG tag. Preferably, the extent of binding of an anti-FLAG antibody to an unrelated tag or protein is less than about 10% of the binding of the antibody to FLAG, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to FLAG has a dissociation constant (K) of <1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM. D )

[0154] "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). Generally, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by 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 antigens slowly and tend to dissociate quickly, whereas high affinity antibodies generally bind antigens more quickly and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

[0155] As used herein, the term "bind" in reference to the interaction of an antigen-binding protein or its antigen-binding domain with an antigen means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, antibodies recognize and bind to specific protein structures rather than proteins in general. If an antibody binds to epitope "A," the presence of a molecule containing epitope "A" (or free, unlabeled "A") in a reaction containing labeled "A" and protein will reduce the amount of labeled "A" bound to the antibody.

[0156] As used herein, the terms "binds specifically" or "specifically binds" should be taken to mean that an antigen binding protein of the invention reacts with or associates with a particular antigen or cell expressing it more frequently, more rapidly, for a longer period of time and / or with greater affinity than does an alternative antigen or cell.

[0157] As used herein, the term "does not detectably bind" should be understood to mean that the antigen-binding protein, e.g., antibody, binds to the candidate antigen at a level less than 10%, or 8%, or 6%, or 5% above background. Background can be the level of binding signal detected in the absence of 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 level of binding is detected using a biosensor assay (e.g., Biacore) in which the antigen-binding protein is immobilized and contacted with the antigen.

[0158] As used herein, the term "does not significantly bind" should be understood to mean that the level of binding of an antigen-binding protein of the invention to a polypeptide is not statistically significantly higher than the background, e.g., 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 level of binding is detected using a biosensor assay (e.g., Biacore) in which the antigen-binding protein is immobilized and contacted with the antigen.

[0159] An "affinity matured" antibody is one that possesses one or more alterations in one or more HVRs thereof that result in an improvement in the affinity of the antibody for antigen compared to a parent antibody that does not possess those alterations. Preferred affinity matured antibodies have nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art.

[0160] "ADCC" refers to a process called antibody-dependent cellular cytotoxicity, which is an immune response mediated primarily by natural killer (NK) cells in humans. In ADCC, FcγRIII on the surface of NK cells recognizes the Fc region of an antibody bound to an antigen displayed on the surface of a target cell. This activates the NK cell, releasing perforin and granzymes, which leads to lysis and apoptosis of the target cell.

[0161] "CDC" refers to a complex process called complement-dependent cytotoxicity, which can lead to cell death through the action of a cascade of proteins that can act via one of two main pathways.

[0162] "ADCP" refers to a process called antibody-dependent cell-mediated phagocytosis, an Fc receptor-mediated process in which antibody-bound target cells are engulfed by phagocytes, such as macrophages, monocytes, neutrophils, and dendritic cells. Multiple Fc receptors are involved in this process.

[0163] A "blocking" or "antagonist" antibody is one that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0164] An "agonist antibody," as used herein, is an antibody that mimics at least one of the functional activities of a polypeptide of interest.

[0165] As used herein, an "Fc region" is a dimer consisting of two polypeptide chains connected by one or more disulfide bonds, each chain including part or all of the hinge domain and CH2 and CH3 domains. Each polypeptide chain is referred to as an "Fc polypeptide chain." To distinguish between the two Fc polypeptide chains, one is referred to herein as the "A chain" and the other as the "B chain." More specifically, the Fc region contemplated for use with the present invention is an IgG Fc region, which may be a mammalian or human IgG1, IgG2, IgG3, or IgG4 Fc region. At least two allelic varieties of the human IgG1 Fc region are known.

[0166] An "Fc-containing protein," as meant herein, is a protein that comprises an Fc region as described herein and a binding region that binds to a target molecule. The term "Fc-containing protein" encompasses antibodies or Fc fusion proteins that contain an Fc region.

[0167] The phrase "therapeutically effective amount" generally refers to an amount of an antigen binding protein of the invention that (i) treats a particular disease, condition, or disorder, (ii) reduces, alleviates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein.

[0168] The word "treat" or "treatment" refers to therapeutic treatment whose goal is to slow (alleviate) an undesirable physiological change or disorder. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, relief or palliation, and remission (whether partial or complete), whether detectable or undetectable. Treatment can also mean prolonging survival compared to expected survival in the absence of treatment. Treatment may not necessarily result in complete clearance of the disease or disorder, but may reduce or minimize complications and side effects of infection and progression of the disease or disorder. The success or otherwise of treatment can be monitored by, among other things, physical examination of the individual, cytopathology, serology, DNA, or mRNA detection techniques.

[0169] The words "prevent" and "prevention" generally refer to prophylactic or preventative measures to protect or prevent an individual who does not have a given disease or disorder from developing that disease or disorder.

[0170] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients that make up the formulation and / or with the mammal being treated therewith.

[0171] Protein mutations The present invention also provides antigen binding proteins or nucleic acids encoding same which have at least 80% identity to the sequences disclosed herein, hi one example, an antigen binding protein or nucleic acid of the invention comprises a sequence which is at least about 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence shown herein.

[0172] Alternatively, or in addition, 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 a VH or VL described herein according to any of the examples.

[0173] In another example, a 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 that encodes an antigen binding protein having a function as described herein according to any of the examples. The invention also encompasses nucleic acids encoding antigen binding proteins of the invention that differ from the sequences exemplified herein as a result of the degeneracy of the genetic code.

[0174] Percent identity of nucleic acids or polypeptides is determined by GAP (Needleman and Wunsch. Mol. Biol. 48, 443-453, 1970) analysis (GCG program) using a gap creation penalty of 5 and a gap extension penalty of 0.3. The query sequence is at least 50 residues in length, and GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues in length, and GAP analysis aligns the two sequences over a region of at least 100 residues. For example, the two sequences are aligned over their entire lengths.

[0175] The present invention also contemplates nucleic acids that hybridize to nucleic acids encoding the antigen-binding proteins described herein under stringent hybridization conditions. "Moderate stringency" is defined herein as hybridization and / or washing carried out at a temperature ranging from 45°C to 65°C in 2xSSC buffer, 0.1% (w / v) SDS, or equivalent conditions. "High stringency" is defined herein as hybridization and / or washing carried out at a temperature of at least 65°C or equivalent conditions in 0.1xSSC buffer, 0.1% (w / v) SDS, or lower salt concentrations. References herein to particular levels of stringency encompass equivalent conditions using wash / hybridization solutions other than SSC known to those skilled in the art. For example, the temperature at which the strands of a double-stranded nucleic acid dissociate (melting temperature or T m Methods for calculating the T of a nucleic acid (also known as the T m A temperature similar to (e.g., within 5°C or within 10°C) or equal to the calculated T of the nucleic acid is considered high stringency. m It is considered to be within 10℃ to 20℃ or 10℃ to 15℃.

[0176] The present invention also contemplates mutant forms of the antigen binding proteins of the present invention which contain one or more conservative amino acid substitutions compared to the sequences set forth herein. In some examples, the antigen binding protein contains no more than 10 conservative amino acid substitutions, for example 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain and / or hydropathic index and / or hydrophilicity.

[0177] 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Hydropathic indexes are described, for example, in Kyte and Doolittle J. Mol. Biol., 157:105-132, 1982, and hydrophilic indices are described, for example, in US4,554,101.

[0178] The present invention also contemplates non-conservative amino acid changes. For example, of particular interest are substitutions of a charged amino acid with another charged amino acid, and with an amino acid that is neutral or positively charged. In some examples, the antigen binding protein contains 10 or fewer non-conservative amino acid substitutions, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1.

[0179] In one example, the mutations occur within the FRs of the antigen binding domain of the antigen binding protein of the invention, hi another example, the mutations occur within the CDRs of the antigen binding protein of the invention.

[0180] Exemplary methods for generating mutant forms of antigen binding proteins include the following: 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 WO1999 / 058661), - introducing the nucleic acid encoding the polypeptide into a mutagenized cell, such as XL-1Red, XL-mutS and XL-mutS-Kanr bacterial cells (Stratagene); DNA shuffling, as disclosed, for example, in Stemmer, Nature 370: 389-91, 1994; and - site-directed mutagenesis, as described, for example, in Dieffenbach (eds.) and Dveksler (eds.) (PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995) Examples include:

[0181] Exemplary methods for determining the biological activity of mutant antigen binding proteins of the invention will be apparent to those of skill in the art and / or are described herein, e.g., antigen binding. For example, methods for determining antigen binding, competitive inhibition of binding, affinity, association, dissociation, and therapeutic efficacy are described herein.

[0182] As used herein, the properties of amino acids are defined in the following table:

[0183] [Table 2A]

[0184] [Table 2B]

[0185] constant region The present invention encompasses antigen binding proteins and / or antibodies described herein comprising an antibody constant region, including an antigen-binding fragment of an antibody fused to Fc.

[0186] The sequences of constant regions useful for generating the proteins of the invention can be obtained from several different sources. In some examples, 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, 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 stabilized IgG4 constant region.

[0187] In one example, the Fc region of the constant region has a reduced ability to induce effector function, for example, compared to a native or wild-type human IgG1 or IgG3 Fc region. In one example, the effector function is 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 function of an Fc region-containing protein are known in the art and / or described herein.

[0188] In one example, the Fc region is an IgG4 Fc region (i.e., derived from an IgG4 constant region), e.g., a human IgG4 Fc region. The sequence of a suitable IgG4 Fc region will be apparent to one of skill in the art and / or may be available from publicly available databases (e.g., available from the National Center for Biotechnology Information).

[0189] In one example, the constant region is a stabilized IgG4 constant region. The term "stabilized IgG4 constant region" will be understood to mean an IgG4 constant region that has been modified to reduce the tendency to undergo Fab arm exchange or the formation of half antibodies. "Fab arm exchange" refers to a type of protein modification of human IgG4 in which the IgG4 heavy chain and attached light chain (half molecule) are exchanged with a heavy chain-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two separate Fab arms that recognize two distinct antigens (resulting in a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or a reducing agent such as reduced glutathione. "Half antibodies" are formed when an IgG4 antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.

[0190] In one example, the stabilized IgG4 constant region contains a proline at position 241 of the hinge region according to the Kabat 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 generally serine. After substitution of serine with proline, the IgG4 hinge region contains the sequence CPPC. In this context, those skilled in the art will recognize that a "hinge region" is the proline-rich part of an antibody heavy chain constant region, which connects the Fc and Fab regions and confers mobility to the two Fab arms of the antibody. The hinge region contains the cysteine ​​residues involved in the inter-heavy chain disulfide bond. It is generally defined as spanning from Glu226 to Pro243 of human IgG1 according to the Kabat numbering system. Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by locating the first and last cysteine ​​residues that form the inter-heavy chain disulfide (SS) bond in the same positions (see, for example, WO2010 / 080538).

[0191] Further examples of stabilized IgG4 antibodies include antibodies in which arginine at position 409 (according to the EU numbering system) in the heavy chain constant region of human IgG4 has been 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 the group consisting of alanine, valine, glycine, isoleucine, and leucine at the position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region comprises a proline at position 241 (i.e., the CPPC sequence) (as described above).

[0192] In another example, the Fc region is a region that has been modified to have reduced effector function, i.e., a "non-immunostimulatory Fc region." For example, the Fc region is an IgG1 Fc region that includes substitutions 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 that includes one or more of the following modifications: E233P, L234V, L235A, and a deletion of G236 and / or one or more of the following modifications: 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). Further examples of non-immunostimulatory Fc regions are described, for example, in Dall'Acqua et al., J Immunol. 177: 1129-1138, 2006 and / or Hezareh J Virol; 75: 12161-12168, 2001).

[0193] In another example, the Fc region is a chimeric Fc region comprising, for example, at least one CH2 domain from an IgG4 antibody and at least one CH3 domain from an IgG1 antibody, and the Fc region comprises a substitution 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 WO2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0194] Antibody-binding domain-containing proteins In another embodiment, there is provided an antigen binding protein as described above, wherein the amino acid sequences forming one or more of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are derived from or in the form of human sequences.

[0195] Antigen-binding proteins can be presented in humanized forms, including non-human (e.g., murine) and human immunoglobulin sequences. Typically, all but the CDR sequences of the antigen-binding protein are derived from a non-human species, such as mouse, rat, or rabbit. In some cases, the framework residues of the antigen-binding protein may also be non-human. When the antigen-binding protein is provided in the form of a whole antibody, typically at least a portion of the immunoglobulin constant region (Fc) is human, thereby enabling various human effector functions.

[0196] Methods for humanizing non-human antigen-binding proteins are well known in the art, and examples of suitable processes include those in Jones et al. (1986) Nature 321:522; Riechmann et al. (1988) Nature 332:323; Verhoeyen et al. (1988) Science 239:1534.

[0197] The phage display methods described herein using antibody libraries derived from human immunoglobulin sequences are useful for making human antigen-binding proteins and human antibodies.

[0198] Alternatively, transgenic mammals can be used that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. These mice can be generated by random or targeted insertion of human heavy and light chain immunoglobulin genes into embryonic stem cells. The host heavy and light chain immunoglobulin genes can be rendered nonfunctional by insertion or some other recombination event, such as homozygous deletion of the host JH region. The transfected embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice, which are then bred to produce homozygous offspring expressing human antigen-binding proteins. Human monoclonal antibodies can be obtained after immunization using FLAG tags. One advantage of transgenic animal systems is that human immunoglobulin transgenes in transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation, allowing the generation of therapeutically useful isotypes.

[0199] The variable domains comprising the CDRs and FRs of the present invention may be made less immunogenic by replacing surface-exposed residues to make the antibody appear self to the immune system. Exemplary methods are provided in Padlan, EA, 1991, MoI. Immunol. 28, 489. The internal packing of adjacent amino acid residues in the antigen-binding protein remains unchanged, and generally, affinity is maintained because CDR residues or adjacent residues that affect binding characteristics should not be replaced in the process.

[0200] In another embodiment, there is provided 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 having a sequence as described herein, preferably as set forth in Table 1.

[0201] Antibodies with lower molecular weights compared to whole antibodies may have improved access to solid tumors and more rapid clearance, and may be particularly useful in therapeutic and in vivo diagnostic applications.

[0202] In certain embodiments, the antigen-binding protein is provided in the form of a single-chain Fv fragment (scFv). Fv and scFv have intact combining sites without constant regions, making them suitable for reduced non-specific binding during in vivo use. Fusion proteins containing scFv can be constructed to allow fusion of an effector protein at either the amino or carboxy terminus of the scFv.

[0203] In another embodiment, a diabody or triabody or other multispecific antibody is provided that comprises the antigen-binding protein described above. Multispecific antibodies can be constructed using polypeptide domains that allow for multimerization. Examples include the CH2 and CH3 regions of Fc and the CH1 and C kappa / lambda regions. Other naturally occurring protein multimerization domains can be used, including leucine zipper domains (bZIP), helix-loop-helix motifs, Src homology domains (SH2, SH3), EF hands, phosphotyrosine-binding (PTB) domains, or other domains known in the art.

[0204] In another embodiment, there is provided a fusion domain or heterologous protein comprising 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 as described herein.

[0205] The heterologous polypeptide may be recombinantly fused or chemically conjugated to the N-terminus or C-terminus of the antigen binding protein of the invention or molecule containing it.

[0206] The heterologous polypeptide to which the antibody or antigen binding protein is fused may be useful for binding to a FLAG tag present on a recombinant material including (but not limited to) a recombinant protein comprising a FLAG tag, a therapeutic antibody or other antigen binding domain comprising a protein comprising a FLAG tag, a cellular immunotherapeutic that has been genetically engineered to express a FLAG tag or to express a receptor or other protein comprising a FLAG tag. In one particular example, the antigen binding protein of the invention may be useful for binding to a chimeric antigen receptor (CAR) present on a cytotoxic immune cell (e.g., on a CAR T cell).

[0207] Furthermore, the antigen binding proteins, immunoglobulin variable domains, antibodies, dabs, scFvs, Fabs, Fab's, F(ab')2s, Fv fragments, diabodies, triabodies, linear antibodies, single chain antibody molecules or multispecific antibodies of the invention may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc.

[0208] Indirect CAR and related chimeric receptors In still further embodiments, the heterologous polypeptide to which the antibody or antigen-binding protein of the invention is fused may be included as a component of a chimeric antigen protein (CAR) or variant T cell receptor. In such embodiments, it will be appreciated that the CAR or variant T cell receptor produced comprising the antigen-binding protein of the invention may be referred to as an "indirect CAR" (sometimes referred to as a universal CAR). In an indirect CAR system, the antigen-binding domain of the CAR does not directly bind to the target antigen on the target cell, but instead binds to an intermediate, which contains the antigen-binding domain for directly binding to the target cell. Examples of CARs that recognize cells via an intermediate are known in the art, for example, European Patent Application EP2651442.

[0209] Those skilled in the art will be familiar with the general structure of CARs and other modified receptors that may comprise the antigen binding proteins of the present invention. Generally, a "chimeric antigen receptor" refers to a recombinant polypeptide construct that includes at least an extracellular antigen-binding portion, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain").

[0210] Chimeric antigen receptors are artificially constructed proteins that can induce antigen-specific cellular responses when expressed on the surface of a cell. CARs contain at least two domains: a first domain that is an antigen-binding domain that binds to an antigen, more specifically, an epitope portion(s) of the antigen, and a second domain that is a signaling domain that can induce or participate in the induction of an intracellular signaling pathway.

[0211] The combination of these two domains determines the antigen specificity of the CAR and its ability to induce the desired cellular response, which varies depending on the host cell of the CAR. For example, the activation of a CAR expressed in a helper T cell and having a signaling domain containing a CD3 activation domain can induce CD4+ helper T cells to secrete various cytokines when activated by encountering its cognate antigen. In another example, the same CAR expressed in a CD8+ cytotoxic T cell can induce the release of cytotoxins, which ultimately leads to the induction of apoptosis of the antigen-expressing cell, once activated by a cell expressing the cognate antigen.

[0212] In addition to the antigen recognition domain and the signal transduction domain, the CAR may further comprise additional components or moieties. For example, the CAR may comprise a transmembrane domain that may comprise or be associated with a portion of the signal transduction domain of the CAR. The transmembrane domain is usually one or more hydrophobic helices that span the lipid bilayer of the cell, burying the CAR within the cell membrane. The transmembrane domain of the CAR may be a determining factor in the expression pattern of the CAR when associated with a cell. For example, a transmembrane domain associated with the CD3 co-receptor may be used to enable CAR expression in naive T cells, while the use of a transmembrane domain derived from the CD4 co-receptor may direct CAR expression in helper T cells but not in cytotoxic T cells.

[0213] A further component or portion of a CAR may be a linker domain. The linker domain (also known as a spacer or hinge domain) may extend from the extracellular side of the transmembrane domain to the antigen-binding domain, thereby linking the antigen-binding domain to the transmembrane domain. In some cases, a linker domain is not required for a functional CAR (i.e., the antigen recognition domain may be directly connected to the transmembrane domain), but in some situations, the use of a linker domain allows for greater efficacy of the CAR. The linker domain may have various functions, including allowing flexibility of the CAR, which allows for the necessary orientation of the antigen-binding domain of the CAR for binding to the antigen. As a result, the linker domain may be any amino acid sequence that serves this function. One non-limiting example of a linker domain is a domain having amino acid sequence homology to the hinge region of an IgG antibody, e.g., an IgG1 hinge region. Alternatively, the C of an antibody may be a domain having amino acid sequence homology to the hinge region of an IgG1 antibody. H2 -C H3 Included are amino acid sequences that have sequence homology to regions or portions of the CD3 co-receptor complex, the CD4 co-receptor, or the CD8 co-receptor.

[0214] In some embodiments, a CAR or other modified receptor may comprise an antigen binding protein described herein in the form of an sdAb, scFv (including multivalent scFv), Fab, or another antibody-like structure.

[0215] The signaling domain of a CAR can be any suitable domain capable of inducing or participating in the induction of an intracellular signaling cascade upon activation of the CAR as a result of antigen recognition by the antigen recognition domain of the CAR. The signaling domain of a CAR is specifically selected depending on the cellular outcome desired after CAR activation. Although there are many possible signaling domains, when used in immunotherapy and cancer therapy, signaling domains can be grouped into two general categories based on the receptors from which they are derived: activating receptors and costimulatory receptors.

[0216] Thus, in some embodiments of the first aspect of the invention, the signaling domain comprises a portion derived from an activating receptor, hi some embodiments, the signaling domain comprises a portion derived from a costimulatory receptor.

[0217] As used throughout this specification, the term "portion," when used in reference to an activating or costimulatory receptor, relates to any segment of the receptor that includes sequences that are responsible for or involved in the initiation / induction of an intracellular signaling cascade following interaction of the receptor with its cognate antigen or ligand. An example of the initiation / induction of an intracellular signaling cascade of the T cell receptor (TCR) via CD3 is outlined below.

[0218] Without wishing to be bound by theory, the extracellular portion of the TCR mostly comprises a heterodimer of either clonoplasmic TCRα and TCRβ chains (TCRα / β receptor) or TCRγ and TCRδ chains (TCRγδ receptor). These TCR heterodimers generally lack intrinsic signal transduction capabilities; therefore, they noncovalently associate with multiple signaling subunits of CD3 (primarily CD3-ζ, -γ, -δ, and -ε). The gamma, delta, and epsilon chains of CD3 each have an intracellular (cytoplasmic) portion containing a single immunoreceptor tyrosine-based activation motif (ITAM), while the CD3-ζ chain contains three tandem ITAMs. When the TCR engages with its cognate antigen in the presence of MHC and associates with the required coreceptor, e.g., CD4 or CD8, signaling is initiated, resulting in tyrosine kinase (i.e., Lck) phosphorylating two tyrosine residues within the intracellular ITAMs of the CD3 chains. A second tyrosine kinase (ZAP-70, itself activated by Lck phosphorylation) is then recruited to diphosphorylate ITAMs, resulting in the activation of several downstream target proteins, ultimately leading to intracellular conformational changes, calcium fluxes, and actin cytoskeletal rearrangements, which, in combination, ultimately lead to the activation of transcription factors and the induction of T cell immune responses.

[0219] As used throughout this specification, the term "activating receptor" relates to a receptor or co-receptor that forms or is involved in the formation of a component of the T cell receptor (TCR) complex, or a receptor that is involved in the specific activation of an immune cell as a result of recognition of an antigenic or other immunogenic stimulus.

[0220] Non-limiting examples of such activating receptors include natural killer (NK) cell-associated activating receptors, such as components of the T cell receptor-CD3 complex (CD3-ζ, -γ, -δ, and -ε), CD4 co-receptors, CD8 co-receptors, Fc receptors, or LY-49 (KLRA1), natural cytotoxicity receptors (NCRs, preferably NKp46, NKp44, NKp30, or NKG2 or CD94 / NKG2 heterodimers). Consequently, in some embodiments of the invention, the signaling domain comprises a portion derived from any one or more of a member of the CD3 co-receptor complex (preferably, the CD3-ζ chain or a portion thereof), a CD4 co-receptor, a CD8 co-receptor, an Fc receptor (FcR) (preferably, FcεRI or FcγRI), or an NK-associated receptor, such as LY-49.

[0221] 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.

[0222] Various combinations of portions of the activating receptor are utilized to transmembrane (TM) and intracellular (IC) portions of the CAR, For example, CD3ζ TM and CD3ζ IC (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 CD3ζ IC (James SE. et al. J Immunol. 2008; 180:7028-38), CD8 TM and CD3ζ IC (Patel SD. et al. Gene Ther. 1999; 6:412-9), and FcεRIγ TM and FcεRIγ IC (Haynes NM. et al. J Immunol. 2001; 166:182-7; Annenkov AE. et al. J Immunol. 1998; 161: 6604-13).

[0223] As used throughout this specification, the term "costimulatory receptor" refers to a receptor or coreceptor that assists in immune cell activation upon antigen-specific induction of an activating receptor. As will be understood, costimulatory receptors do not require the presence of an antigen and are not antigen-specific, but are typically one of two signals, the other being an activating signal, required for the induction of an immune cellular response. In the context of an immune response, costimulatory receptors are typically activated by the presence of their expressed ligand on the surface of antigen-presenting cells (APCs), such as dendritic cells or macrophages. With particular reference to T cells, costimulation is necessary to lead to cellular activation, proliferation, differentiation, and survival (all of which are generally referred to under the umbrella of T cell activation), but presentation of antigen to T cells in the absence of costimulation can lead to the development of anergy, clonal deletion, and / or antigen-specific tolerance. Importantly, costimulatory molecules can inform T cell responses to simultaneously encountered antigens. Generally, an antigen encountered in the context of a "positive" costimulatory molecule leads to T cell activation and a cellular immune response aimed at eliminating cells expressing that antigen.

[0224] Antigens encountered in the context of a "negative" co-receptor lead to the induction of a state of tolerance to simultaneously encountered antigens.

[0225] Non-limiting examples of T cell costimulatory receptors include CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS. Specifically, CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS all represent "positive" costimulatory molecules that enhance activation of T cell responses. Thus, in some embodiments of the invention, the signaling domain comprises a portion derived from any one or more of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0226] In some embodiments, the signaling domain comprises a portion derived from a CD28, OX40, or 4-1BB costimulatory receptor. In some embodiments, the signaling domain comprises a portion of a CD28 costimulatory receptor. In some embodiments, the signaling domain comprises a portion of an OX40 costimulatory receptor. Various combinations of portions of costimulatory receptors can be used to form the transmembrane (TM) and intracellular (IC) portions of the CAR. For example, 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).

[0227] Further details of the antigen binding proteins of the invention The antigen-binding proteins of the present invention may be composed of amino acids joined together by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The antigen-binding proteins of the present invention may be modified by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic textbooks as well as in the research literature. Modifications can occur anywhere in the antigen-binding protein, including the peptide backbone, the amino acid side-chains, and the amino- or carboxyl-terminus, or on moieties such as carbohydrates. It will be recognized that the same type of modification may be present to the same or varying degrees in several antigen-binding proteins in a given antigen-binding protein. A given antigen-binding protein may also contain many types of modifications. Antigen-binding proteins may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic antigen-binding proteins may result from post-translational natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of a 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 phosphotidylinositol, 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, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation and ubiquitination.

[0228] In another embodiment, there is provided a conjugate in the form of an antigen-binding protein, immunoglobulin variable domain, antibody, Fab, dab, scFv, diabody, triabody or fusion protein as described above, conjugated to a cytotoxic agent, e.g., a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin or fragment thereof), or a label, e.g., a radioactive isotope (i.e., a radioconjugate). In another aspect, the invention further provides methods of using the immunoconjugates. In one aspect, an immunoconjugate comprises any of the variable domains described above covalently attached to a cytotoxic agent or a detectable agent.

[0229] In another embodiment there is provided 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.

[0230] In another embodiment, there is provided a nucleic acid encoding 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.

[0231] Polynucleotides encoding CDRs or FRs according to any one of the above general formulas, or antigen-binding proteins composed thereof, can be produced from nucleic acid from any source, for example, by chemical synthesis or isolation from a cDNA or genomic library. For example, a cDNA library can be produced from antibody-producing cells, such as B cells, plasma cells, or hybridoma cells, and the relevant nucleic acid isolated by PCR amplification using oligonucleotides directed to the specific clone of interest. The isolated nucleic acid may then be cloned into a vector using any method known in the art. The relevant nucleotide sequence can then be mutagenized using methods known in the art, for example, recombinant DNA technology, site-directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY) to generate antigen-binding proteins with different amino acid sequences, for example, to generate amino acid substitutions, deletions, and / or insertions.

[0232] Protein production In another embodiment, a method is provided for producing an anti-FLAG antigen binding protein as described above, comprising expressing a nucleic acid as described above in a cell or non-human animal as described above.

[0233] The production of antigen-binding proteins of the present invention generally requires an expression vector containing a polynucleotide encoding the antigen-binding protein of the present invention. Polynucleotides encoding antigen-binding proteins of the present invention may be obtained and subcloned into vectors for production of the antigen-binding protein by recombinant DNA technology using techniques well known in the art, including those described herein. A number of different expression systems are contemplated, including the use of mammalian cells, including human cells, for the production and secretion of antigen-binding proteins. Exemplary cells include 293F, CHO, and NSO cell lines.

[0234] Expression vectors containing the protein coding sequence and appropriate transcriptional and translational control signals can be constructed using methods known in the art. These include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. In certain embodiments, replicable vectors are provided that have a nucleic acid encoding the antigen binding protein operably linked to a promoter.

[0235] Cells transfected with the expression vector can be cultured by conventional techniques to produce the antigen binding protein. Accordingly, in certain embodiments, host cells or cell transfectants are provided that contain a polynucleotide encoding the antigen binding protein operably linked to a promoter. The promoter can be heterologous. A variety of host-expression vector systems are available, and in certain systems, the transcription machinery of the vector system is specifically adapted to the host cell. For example, mammalian cells, such as Chinese hamster ovary cells (CHO), can be transfected with a vector containing the major intermediate-early gene promoter element from human cytomegalovirus. Additionally, or alternatively, host cells can be used that modulate the expression of inserted sequences or modify and process the gene product as needed, including various forms of post-translational modifications. Examples of mammalian host cells with specific post-translational modifications include CHO, VERY, BHK, HeIa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO, CRL7O3O and HsS78Bst cells.

[0236] Depending on the intended use of the protein molecule, several bacterial expression vectors can be advantageously selected. In one example, if large quantities of an antigen-binding protein are to be produced, a vector that results in the expression of a high-level, easily purified fusion protein product, such as the E. coli expression vector pUR278, can be used. The expression product can be produced in the form of a fusion protein with lacZ. Other bacterial vectors include the pIN vector. The pGEX vector can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). These fusion proteins are generally soluble and can be easily purified from lysed cells by adsorption and binding to a glutathione-agarose affinity matrix, followed by elution in the presence of free glutathione. Thrombin and / or factor Xa protease cleavage sites can be engineered into the expressed polypeptide so that the cloned target gene product can be released from the GST moiety.

[0237] Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes in insect systems, including Spodoptera frugiperda cells. The particular promoter used can vary depending on where the protein coding sequence is inserted into the sequence. For example, sequences can be cloned individually into the polyhedrin gene and placed under the control of the polyhedrin promoter.

[0238] Viral-based expression systems can be used with mammalian cells, such as adenovirus, whereby a coding sequence of interest can be ligated to the adenovirus late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome using in vitro or in vivo recombination. Insertion into regions E1 or E3 results in a viable recombinant virus capable of expressing the antigen-binding protein in infected host cells. Specific initiation signals, including the ATG initiation codon and adjacent sequences, may be required for efficient translation of the inserted antigen-binding protein coding sequence. Initiation and translational control signals and codons can be obtained from a variety of sources, both natural and synthetic. Transcriptional enhancer elements and transcriptional terminators can be used to enhance the efficiency of expression in viral-based systems.

[0239] Stable expression is preferred when long-term, high-yield production of recombinant proteins is required. Typically, a selectable marker gene is used, whereby after transfection, cells are grown in enriched medium for 1–2 days and then transferred to a medium containing selective medium, where cells containing the corresponding selectable marker, e.g., antibiotic resistance, can be screened. The result is that cells with the plasmid stably integrated into their chromosomes grow and form foci, which can then be cloned and expanded into cell lines. The herpes simplex virus thymidine kinase, hypoxanthine guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes are examples of genes that can be used in tk-, hgprt-, or aprT- cells, respectively, thereby providing suitable selection systems. The following genes are examples of genes that can be used in antimetabolite selection systems: dhfr, which confers resistance to methotrexate; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G-418; and hygro, which confers resistance to hygromycin.

[0240] The antigen binding proteins of the present invention can be purified by recombinant expression systems, by known methods including ion exchange chromatography, affinity chromatography (particularly affinity for the specific antigens Protein A or Protein G and gel filtration column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. Purification may be facilitated or aided by providing the antigen binding protein in the form of a fusion protein.

[0241] Large quantities of the antigen-binding proteins of the invention can be produced by a scalable process that starts with a pilot expression system in a research laboratory and scales up to analytical-scale bioreactors (typically 5 L to about 50 L bioreactors) or production-scale bioreactors (e.g., but not limited to, 75 L, 100 L, 150 L, 300 L, or 500 L). Desirable scalable processes include those with low to undetectable levels of aggregation as measured by HPSEC or rCGE, typically 5% or less aggregation by mass of protein, and in some cases, aggregation of 0.5% or less by mass of protein. Additionally, or alternatively, undetectable levels of fragmentation as measured by total peak area representing intact antigen-binding protein may be desirable, such that at least 80% and as much as 99.5% or more of the total peak area represents intact antigen-binding protein. In other embodiments, the scalable process of the invention produces antigen-binding protein at a production efficiency of 10 mg / L to about 300 mg / L or higher.

[0242] Various techniques have been developed for the production of 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 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 approach, F(ab')2 fragments are directly isolated from recombinant host cell culture.

[0243] In another embodiment, a vector is provided that contains the above-described nucleic acid. The vector may be, for example, in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector by a variety of procedures. Generally, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. 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. Construction of suitable vectors containing one or more of these components uses standard ligation techniques known to those skilled in the art.

[0244] Antigen-binding sites can be produced recombinantly not only directly but also as fusion polypeptides with heterologous polypeptides, which can be signal sequences, or other polypeptides that have a specific cleavage site at the N-terminus of the mature protein or polypeptide. Generally, the signal sequence can be a component of the vector, or it can be part of the DNA encoding the antigen-binding site that is inserted into the vector. The signal sequence can be a prokaryotic signal sequence selected from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion, the signal sequence can be, for example, the yeast invertase leader, alpha-factor leader, acid phosphatase leader, or the C. albicans glucoamylase leader. In mammalian cell expression, mammalian signal sequences can be used to direct protein secretion, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.

[0245] Polynucleotide sequences encoding polypeptide components of the antigen-binding proteins of the present invention can be obtained using standard recombinant techniques, as described above. Polynucleotides can be synthesized using nucleotide synthesizers or PCR techniques. Once obtained, the polypeptide-encoding sequence is inserted into a recombinant vector that is replicable in a prokaryotic host and expresses the heterologous polynucleotide. For the purposes of the present invention, numerous vectors that are available and known in the art can be used. The selection of an appropriate vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification or expression of the heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides.

[0246] Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are generally used in connection with these hosts. Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells, as well as marking sequences capable of providing phenotypic selection in transformed cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322, which contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, provides an easy means of identifying transformed cells and is suitable for most Gram-negative bacteria. The 2 μm plasmid origin is suitable for yeast. Various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that can be used by the microorganism for expression of endogenous proteins.

[0247] Additionally, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as λGEM.TM.-11 can be used to generate recombinant vectors, which can be used to transform susceptible host cells, such as E. coli LE392.

[0248] The expression vector of the present invention may contain two or more promoter-cistron pairs (a cistron is a segment of DNA that contains all the information for the production of a single polypeptide). A promoter is a non-translated regulatory sequence located upstream (5') of a cistron and modulates its expression. Prokaryotic promoters are usually divided into two classes: inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription of the cistron under their control in response to a change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature.

[0249] Numerous promoters recognized by a variety of potential host cells are well known. The selected promoter can be operably linked to the cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention. Both the native promoter sequence and many heterologous promoters can be used to direct amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because they allow for greater transcription and higher yields of expressed target gene compared to the native target polypeptide promoter.

[0250] Promoters recognized by a variety of potential host cells are well known. Suitable promoters for use with prokaryotic hosts include the PhoA promoter, β-galactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and hybrid promoters, such as the tac or trc promoter. Promoters for use in bacterial systems also contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the antigen-binding protein of the present invention. However, other promoters functional in bacteria (e.g., other known bacterial or phage promoters) are similarly suitable. Their nucleotide sequences have been published, enabling one of skill in the art to operably ligate them to cistrons encoding the target light and heavy chains using linkers or adapters providing any necessary restriction sites.

[0251] In one aspect of the present invention, each cistron in a recombinant vector contains a secretory signal sequence component that directs translocation of an expressed polypeptide across a membrane. Generally, the signal sequence may be a component of the vector or may be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for purposes of the present invention must be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequence of a heterologous polypeptide, the signal sequence is substituted with a prokaryotic signal sequence selected from the group consisting of, for example, 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 both cistrons of the expression system is an STII signal sequence or a variant thereof.

[0252] In another embodiment, production of immunoglobulins according to the invention can occur in the cytoplasm of the host cell and therefore does not require the presence of a secretory signal sequence within each cistron. Accordingly, 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) provide cytoplasmic conditions that favor disulfide bond formation, thereby allowing proper folding and assembly of the expressed protein subunits.

[0253] The present invention provides expression systems in which the quantitative ratios of the expressed polypeptide components can be modulated to maximize the yield of secreted, properly assembled antigen binding proteins of the invention, achieved at least in part by simultaneously modulating the translational potential of the polypeptide components.

[0254] For expression in eukaryotic host cells, the 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.

[0255] Vectors for use in eukaryotic host cells may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, such as the herpes simplex gD signal, are available.

[0256] The DNA for such precursor region is ligated in reading frame to DNA encoding the antibody.

[0257] Generally, the origin of replication component is not needed for mammalian expression vectors For example, the SV40 origin may typically be used simply because it contains the early promoter.

[0258] Expression and cloning vectors usually contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement an auxotrophic deficiency, or (c) supply critical nutrients not available from complex media. For example, the gene encoding Bacillus D-alanine racemase.

[0259] One example of a selection scheme utilizes drugs to arrest growth of host cells. Cells successfully transformed with a heterologous gene produce a protein that confers drug resistance and therefore survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0260] Examples of suitable selectable markers for mammalian cells include those that enable the identification of cells competent to incorporate nucleic acid encoding an antigen-binding protein, such as DHFR or thymidine kinase, metallothionein-I and -II, preferably a primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, etc. When wild-type DHFR is used, a suitable host cell is a prepared and grown CHO cell line deficient in DHFR activity (e.g., ATCC CRL-9096). For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Alternatively, host cells transformed or co-transformed with a DNA sequence encoding an antibody, a wild-type DHFR protein, and another selectable marker, such as an aminoglycoside 3'-phosphotransferase (APH), (particularly wild-type hosts containing endogenous DHFR), can be selected by cell growth in medium containing the selection agent for the selectable marker, for example, an aminoglycoside antibiotic, such as kanamycin, neomycin, or G418.

[0261] 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 a variety of potential host cells are well known.

[0262] Eukaryotic genes generally have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence, which may be a signal for addition of a poly(A) tail to the 3' end of the coding sequence. All of these sequences are appropriately inserted into eukaryotic expression vectors.

[0263] Examples of suitable promoter sequences for use with yeast hosts include promoters of 3-phosphoglycerate kinase or 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.

[0264] Other yeast promoters that are inducible promoters with the added advantage of transcription controlled by growth conditions include the promoter regions of alcohol dehydrogenase 2, isocytochrome c, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.

[0265] Antigen binding protein transcription from the vector in mammalian host cells is controlled, for example, by promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or immunoglobulin promoters, and from heat shock promoters, provided such promoters are compatible with the host cell system.

[0266] Transcription of DNA encoding an antigen-binding protein by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancer sequences include those known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, enhancers from eukaryotic viruses are usually used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0267] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5', and occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antigen-binding protein.

[0268] In another embodiment, there is provided a cell comprising the above-described vector or nucleic acid. The nucleic acid molecule or vector may be present in the genetically modified host cell or host as an independent molecule outside the genome, preferably as a molecule that is capable of replicating, or may be stably integrated into the genome of the host cell or host.

[0269] The host cells of the present invention can be any prokaryotic or eukaryotic organism.

[0270] Examples of prokaryotic cells are those commonly used for cloning, such as E. coli or Bacillus subtilis. Furthermore, eukaryotic cells include, for example, fungal or animal cells.

[0271] Examples of suitable fungal cells are yeast cells, preferably of the genus Saccharomyces, most preferably of the species Saccharomyces cerevisiae.

[0272] Examples of animal cells include, 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, etc. These host cells, for example, CHO cells, can provide post-translational modifications to the antibody molecules of the present invention, including leader peptide removal, folding and assembly of H (heavy) and L (light) chains, glycosylation of the molecule at the correct side, and secretion of the functional molecule.

[0273] Further suitable cell lines known in the art can be obtained from cell line repositories such as the American Type Culture Collection (ATCC).

[0274] In another embodiment, an animal comprising the above-described cells is provided. In certain embodiments, animals and tissues thereof containing the transgene are useful in the production of antigen-binding proteins of the invention. Introduction of a nucleic acid molecule as a transgene into a non-human host and subsequent expression can be used to produce the antigen-binding protein; for example, expression of such a transgene in the milk of a transgenic animal provides a means of obtaining quantitative amounts of the antigen-binding protein. A transgene useful in this regard comprises the coding sequence of a nucleic acid molecule of the invention, e.g., an antigen-binding protein described herein, operably linked to a promoter and / or enhancer structure derived from a mammary gland-specific gene such as casein or β-lactoglobulin. The animal may be a non-human mammal, most preferably a mouse, rat, sheep, calf, dog, monkey, or ape.

[0275] Binding to target antigen Methods for determining successful binding of an antigen-binding protein of the 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 technology, all of which are within the skill set of one of ordinary skill in the art.

[0276] kit In another embodiment there is provided a kit or article of manufacture comprising 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, conjugate or pharmaceutical composition as described above.

[0277] In another embodiment, a kit for use in the above uses, comprising: - a container holding a therapeutic composition in the form of one or more of an antigen binding protein, an immunoglobulin variable domain, an antibody, a dab, an 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; - Label or package insert containing instructions for use A kit is provided comprising:

[0278] The kit or "article of manufacture" may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a therapeutic composition that is effective for treating a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the therapeutic composition is used for treating the condition of choice. In one embodiment, the label or package insert comprises instructions for use.

[0279] 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 further include a package insert indicating that the active ingredient and the other active ingredients can be used to treat a disorder or prevent complications resulting from cancer. Alternatively, or in addition, the kit may further 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 dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0280] In certain embodiments, the therapeutic composition may be provided in the form of a disposable or reusable device that includes a container for holding the therapeutic composition. In one embodiment, the device is a syringe. The device can hold 1-2 mL of the therapeutic composition. The therapeutic composition may be provided in the device in a ready-to-use state or one that requires mixing or addition of additional components.

[0281] In other embodiments, there is provided a kit for use in the above diagnostic applications, comprising: - a container holding a diagnostic composition in the form of one or more of 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; - Label or package insert containing instructions for use A kit is provided comprising:

[0282] The kit can include a second container containing (a) the diagnostic composition and (b) a second diagnostic agent or a second label. The kit can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, etc.

[0283] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features described or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

[0284] The following examples are illustrative and do not limit the invention in any way. [Example]

[0285] Example 1 Humanization of chimeric antibodies Mouse anti-FLAG antibody was obtained. The IMGT Domain Gap Align tool (Ehrenmann F., Kaas Q., and Lefranc M.-P. Nucleic Acids Res. 2010; 38:D301-D307) was used to identify complementarity-determining regions (CDRs) and analyze the closest matching germline sequences.

[0286] Molecular models of the VH and VL domains were constructed using in-house software based on homology to previously published antibody crystal structures. PDB files can be provided upon request for viewing in any molecular visualization software. Images were generated using PyMol (The PyMOL Molecular Graphics System, version 2.0 Schrodinger, LLC).

[0287] The antibody sequences were analyzed for certain trends based on published protein motifs. The analysis was performed by analyzing the following motifs where X represents any amino acid apart from proline as set forth in the table below:

[0288] [Table 3]

[0289] At the start of the humanization process, homology models of the parental VH and VL are constructed in single-chain Fv (scFv) format. Modeling is performed in four stages: collection of homologous sequences; fold library scanning; loop modeling; and side chain placement. The resulting models are used to guide the selection of "donor" or "acceptor" amino acids during the humanization process.

[0290] The parent VH and VL sequences are aligned with a panel of human germline sequences. This panel is filtered to select germline sequences that lack unwanted sequence preferences, particularly N-linked glycosylation sites and free cysteines. The closest germline matches from two different VH and VL families are then selected. A humanization algorithm is then used to select CDR and framework amino acids to graft from the donor parent sequence to the human acceptor germline sequence. Four VH and four VL sequences are generated, resulting in 16 potential antibodies. The IMGT Domain Gap Align tool (Ehrenmann F., Kaas Q., and Lefranc M.-P. Nucleic Acids Res., 2010; 38:D301-D307) is used to calculate the percentage identity to humans. Sequence preferences are determined based on sequence motifs.

[0291] The VH and VL sequences were analyzed against all known antibody germline sequences using the IGMT Gap Align tool. CDR regions were assigned using IMGT determination. As expected, the sequences were consistent with mouse, specifically IGHV1-4 for VH. * IGK1-117 for the 01 family and VL * The closest match to parental SEQ ID NO: 1 is germline IGHV1-46 * 01, IGHV7-4-1 * 02, IGKV2-30 * 01 and IGKV4-1 * It was 01.

[0292] The table below summarizes the original parental and humanized sequences:

[0293] [Table 4]

[0294] The combination of four different heavy and light chain framework regions resulted in the generation of 16 different VH / VL combinations.

[0295] The different VH / VL combinations of the 16 antibodies are summarized in the table below:

[0296] [Table 5]

[0297] All humanized IgG1 formats were well expressed and were successfully purified for characterization by SDS-PAGE and SEC-HPLC to assess protein quality. The titer, quantity and monomer content of the final purified antibodies are listed in the table below:

[0298] [Table 6]

[0299] Example 2 Characterization of humanized antibodies All 16 humanized antibodies in IgG and scFv formats were evaluated for their ability to bind to the target (FLAG tag) using ELISA, and the results are shown in Figure 1.

[0300] 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.

[0301] The humanized antibody was able to exhibit high affinity binding comparable to that of the parental murine antibody (ie, murine VH / VL in the context of human constant regions).

[0302] The table below summarizes the antibody affinities and stoichiometries as measured by Biacore:

[0303] [Table 7]

[0304] Four IgG antibodies (parental murine antibody and three humanized antibodies: v3, v4, and v7) were compared to the corresponding humanized scFvs for single antigen binding by Biacore. The murine scFvs could not be expressed and therefore could not be used for affinity comparison. The IgG antibodies bound antigen approximately 10-fold more tightly compared to the scFvs. This is expected due to the bivalent nature of IgG, whose avidity results in tighter binding compared to the monovalently expressed scFvs.

[0305] Antibodies v3, v4, v7, and v11 (in IgG format and, in the case of v11, in scFv format) were further characterized. The antibodies were evaluated for their ability to activate anti-HER2-FLAG CAR T cells and induce IFN-γ. As shown in Figure 2, the antibodies induced IFN-γ expression. Antibody v11 was also included in the evaluation, in both IgG and scFv format. The results show that antibodies v7 and v4, respectively, induced the greatest amount of IFN-γ by CAR T cells.

[0306] Based on these results, antibody variants v4 and v7 were selected for further study.

[0307] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features described or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

Claims

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

2. 2. The antigen-binding protein of claim 1, which is capable of specifically binding to a FLAG tag or a variant thereof comprising the amino acid sequence shown in any one of SEQ ID NOs: 11 or 29 to 41.

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

4. 4. The antigen binding protein of any one of claims 1 to 3, which is for specifically binding to a FLAG tag contained in or on a receptor present on a recombinant or therapeutic protein, cellular immunotherapeutic, or cellular immunotherapeutic.

5. - comprising a VH comprising the sequence set forth in SEQ ID NO: 1 and a VL comprising the sequence set forth in SEQ ID NO: 6, - comprising a VH comprising the sequence set forth in SEQ ID NO: 2 and a VL comprising the sequence set forth in SEQ ID NO: 7, - comprising a VH comprising the sequence set forth in SEQ ID NO: 2 and a VL comprising the sequence set forth in SEQ ID NO: 8, - comprising a VH comprising the sequence set forth in SEQ ID NO: 2 and a VL comprising the sequence set forth in SEQ ID NO: 9, - comprising a VH comprising the sequence set forth in SEQ ID NO: 2 and a VL comprising the sequence set forth in SEQ ID NO: 10, - comprising a VH comprising the sequence set forth in SEQ ID NO: 3 and a VL comprising the sequence set forth in SEQ ID NO: 7, - comprising a VH comprising the sequence set forth in SEQ ID NO: 3 and a VL comprising the sequence set forth in SEQ ID NO: 8, - comprising a VH comprising the sequence set forth in SEQ ID NO: 3 and a VL comprising the sequence set forth in SEQ ID NO: 9, - comprising a VH comprising the sequence set forth in SEQ ID NO: 3 and a VL comprising the sequence set forth in SEQ ID NO: 10, - comprising a VH comprising the sequence set forth in SEQ ID NO: 4 and a VL comprising the sequence set forth in SEQ ID NO: 7, - comprising a VH comprising the sequence set forth in SEQ ID NO: 4 and a VL comprising the sequence set forth in SEQ ID NO: 8, - comprising a VH comprising the sequence set forth in SEQ ID NO: 4 and a VL comprising the sequence set forth in SEQ ID NO: 9, - comprising a VH comprising the sequence set forth in SEQ ID NO: 4 and a VL comprising the sequence set forth in SEQ ID NO: 10, - comprising a VH comprising the sequence set forth in SEQ ID NO: 5 and a VL comprising the sequence set forth in SEQ ID NO: 7, - comprising a VH comprising the sequence set forth in SEQ ID NO: 5 and a VL comprising the sequence set forth in SEQ ID NO: 8, - comprising a VH comprising the sequence set forth in SEQ ID NO: 5 and a VL comprising the sequence set forth in SEQ ID NO: 9, or - comprising a VH comprising the sequence set forth in SEQ ID NO: 5 and a VL comprising the sequence set forth in SEQ ID NO: 10 5. The antigen-binding protein of claim 1, which competitively inhibits the binding of an antibody to a FLAG tag.

6. The antigen-binding domain is A. A VH comprising a complementarity 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, and a VL comprising a complementarity 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 B., C., D., E., F., F., G., H., I., J., K., L., M., N., O., P., Q., R.: B. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 65, 66, 67, and 68, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively; or C. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively; or D. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively; or E. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively; or F. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 16, 18, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively; or G. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively; or H. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively; or I. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively; or J. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 17, 19, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively; or K. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively; or L. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively; or M. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively; or N. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 16, 82, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively; or O. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 69, 70, 71, and 28, respectively; or P. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 86, and 28, respectively; or Q. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 26, 27, and 28, respectively; or R. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 80, 81, 83, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 24, 25, 27, and 28, respectively. Any one of 6. The antigen-binding protein of any one of claims 1 to 5, comprising:

7. The antigen-binding domain is A. A VH comprising a complementarity 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; and a VL comprising a complementarity 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 one of B., C., D. or E.: B. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or C. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively; or D. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or E. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively.

6. The antigen-binding protein of any one of claims 1 to 5, comprising:

8. The antigen-binding domain is A. A VH comprising a complementarity 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; and a VL comprising a complementarity 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 one of B., C., D. or E.: B. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or C. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 47, 49, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively; or D. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or E. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 46, 48, 50, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively.

6. The antigen-binding protein of any one of claims 1 to 5, comprising:

9. The antigen-binding domain is A. A VH comprising a complementarity 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; and a VL comprising a complementarity 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 one of B., C., D. or E.: B. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or C. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively; or D. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or E. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively.

6. The antigen-binding protein of any one of claims 1 to 5, comprising:

10. The antigen-binding domain is A. A VH comprising a complementarity 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, and a VL comprising a complementarity 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 one of B., C., D. or E.: B. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or C. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 47, 90, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively; or D. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or E. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 89, 48, 91, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 92, 93, 94, and 28, respectively.

6. The antigen-binding protein of any one of claims 1 to 5, comprising:

11. The antigen-binding domain is A. A VH comprising a complementarity 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; and a VL comprising a complementarity 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 B., C., D., E., F., G., H. or I.: B. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or C. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively; or D. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or E. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively; or F. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or G. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively; or H. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 96, 98, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 55, 56, and 28, respectively; or I. VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 96, 98, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 53, 54, 56, and 28, respectively. Any one of 6. The antigen-binding protein of any one of claims 1 to 5, comprising:

12. The antigen-binding domain is A. A VH comprising a complementarity 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, and a VL comprising a complementarity 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 B., C., D., E., F., G., H. or I.: B. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or C. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 59, 61, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 99, 100, 101, and 28, respectively; or D. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or E. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 60, 62, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 99, 100, 101, and 28, respectively; or F. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or G. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 59, 97, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 99, 100, 101, and 28, respectively; or H. A VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 96, 98, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or I. VH and VL comprising framework regions (FR) 1, 2, 3, and 4, each comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 95, 96, 98, and 20, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 99, 100, 101, and 28, respectively. Any one of 6. The antigen-binding protein of any one of claims 1 to 5, comprising:

13. The antigen-binding domain is A. A VH comprising complementarity determining regions (CDR) 1, CDR2, and CDR3 as defined in any of (o) above, and a VL comprising CDR1, CDR2, and CDR3 as defined in any of (p) above, and framework regions (FR) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 72, 73, 74, and 68, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively; or B. A VH comprising complementarity determining regions (CDRs) 1, CDR2, and CDR3 as defined in any of (q) above, and a VL comprising CDR1, CDR2, and CDR3 as defined in any of (r) above, and framework regions (FRs) 1, 2, 3, and 4, each comprising 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 VH FRs, the reference sequences are set forth in SEQ ID NOs: 15, 78, 79, and 68, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 75, 76, 77, and 28, respectively.

6. The antigen-binding protein of any one of claims 1 to 5, comprising:

14. 14. The antigen-binding domain of any one of claims 1 to 13, comprising a variable heavy chain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 5 and a variable light chain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 7 to 10.

15. The antigen-binding protein (i) single domain antibodies (sdAbs), (ii) single chain Fv fragment (scFv), (iii) dimeric scFv (di-scFv), (iv) one of (ii) or (iii) linked to a constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3; (v) one of (i)-(iv) linked to a protein that binds to an immune effector cell; (vi) one of (i) to (iv) linked to a modified immune cell receptor, e.g., 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 15. The antigen-binding domain of any one of claims 1 to 14, in the form:

16. The antigen-binding protein (i) diabodies, (ii) triabodies, (iii) tetrabodies, (iv) Fab, (v) F(ab')2, (vi) Fv, (vii) other forms of bispecific or 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; or (ix) one of (i) to (vii) linked to a protein that binds to an immune effector cell; (x) one of (i) to (vii) linked to a protein that binds to an immune effector cell; (xi) one of (i) to (vii) linked to a modified immune cell receptor, e.g., a modified T cell receptor; or (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).

16. The antigen-binding domain of any one of claims 1 to 15, in the form:

17. The antigen-binding protein SEQ ID NOs: 2 and 10, SEQ ID NOs: 3 and 9, SEQ ID NOs: 2 and 9, SEQ ID NOs: 2 and 7, SEQ ID NOs: 2 and 8, SEQ ID NOs: 3 and 7, SEQ ID NOs: 3 and 8, SEQ ID NOs: 3 and 10, SEQ ID NOs: 4 and 7, SEQ ID NOs: 4 and 8, SEQ ID NOs: 4 and 9, SEQ ID NOs: 4 and 10, SEQ ID NOs: 5 and 7, SEQ ID NOs: 5 and 8, SEQ ID NOs: 5 and 9, or SEQ ID NOs: 5 and 10 17. The antigen-binding domain of any one of claims 1 to 16, comprising, consisting essentially of, or consisting of (in N- to C-terminal or C- to N-terminal order) the amino acid sequence:

18. 18. A fusion protein comprising the antigen-binding protein of any one of claims 1 to 17.

19. 19. A chimeric antigen receptor or variant T cell receptor comprising an antigen binding protein according to any one of claims 1 to 17 or a fusion protein according to claim 18.

20. 18. A conjugate comprising the antigen-binding protein of any one of claims 1 to 17.

21. 19. A nucleic acid encoding an antigen-binding protein according to any one of claims 1 to 17 or a fusion protein according to claim 18.

22. 22. A vector construct comprising the nucleic acid of claim 21.

23. 23. A cell comprising the nucleic acid of claim 21 or the vector construct of claim 22.

24. 21. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1 to 17, a fusion protein according to claim 18 or a conjugate according to claim 20 and a pharmaceutically acceptable carrier, diluent or excipient.

25. 21. A diagnostic composition comprising an antigen-binding protein according to any one of claims 1 to 17, a fusion protein according to claim 18 or a conjugate according to claim 20 and a pharmaceutically acceptable carrier, diluent or excipient.

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