Bispecific polypeptides and uses thereof

Abstract

The present disclosure generally relates to bispecific polypeptides and portions thereof comprising a first binding domain capable of binding to antigen-presenting cells (APCs) and a second binding domain capable of binding to T cells, and uses thereof. In a first aspect disclosed herein, a bispecific polypeptide is provided, comprising a first antigen-binding protein and a second antigen-binding protein, wherein the first antigen-binding protein specifically binds to CD40 and the second antigen-binding protein specifically binds to a FLAG tag. In a second aspect disclosed herein, a bispecific polypeptide is provided, comprising a first antigen-binding protein and a second antigen-binding protein, wherein the first antigen-binding protein specifically binds to CD206 and the second antigen-binding protein specifically binds to a FLAG tag.

Description

[Technical Field]

[0001] The present disclosure relates generally to bispecific polypeptides and portions thereof comprising a first binding domain capable of binding to an antigen-presenting cell (APC) and a second binding domain capable of binding to a T cell, and uses thereof.

[0002] Related Applications This application claims priority to Australian provisional application AU2022902713, the entire disclosure of which is incorporated herein. [Background technology]

[0003] Background of the Invention Adoptive cell transfer (ACT) has demonstrated exciting potential for cancer therapy. In ACT, large numbers of autologous tumor-reactive T cells are generated in vitro before reinfusion into the patient. Tumor-reactive T cells can be isolated from blood or tumor and expanded in vitro using stimulation with peptides and / or cytokines.

[0004] Methods for improving T cell responsiveness for ACT include genetic modification of patient lymphocytes to generate tumor-reactive T cells against most malignancies, including solid tumors and hematological tumors. The two main approaches to genetic modification involve genes encoding T cell receptors (TCRs) or chimeric antigen receptors (CARs). CARs consist of antibody-derived domains fused with T cell signaling domains and redirect the effector function of T cells against tumor cells. While both approaches can render T cells tumor-reactive, the CAR approach is not MHC-restricted and is more broadly applicable to a wider range of patients.

[0005] CARs can take various forms but typically consist of an extracellular domain consisting of a single-chain variable fragment (scFv) of an antibody specific for a tumor-associated antigen (TAA). This scFv is linked via a hinge and transmembrane domain to an intracellular region consisting of one or more signaling moieties. CARs with specificity for various TAAs, including mesothelin, Her2, CEA, FBP, CD19, and BCMA, have been developed. The most advanced clinical studies utilize CARs specific for CD19 for the treatment of B-cell leukemia and lymphoma. Since 2017, six CAR T-cell therapies have been approved by the Food and Drug Administration (FDA). All are approved for the treatment of hematological cancers, including lymphoma, some forms of leukemia, and most recently, multiple myeloma.

[0006] Attempts to optimize this type of therapy have led to the combination of CAR T cells with other therapeutic approaches designed to overcome tumor-induced immune suppression, including cotreatment with α-PD-1 monoclonal antibodies, genetic modification of signaling and cytokine pathways, and the use of adjuvants such as agonistic α-4-1BB monoclonal antibodies (mABs) and bispecific T cell engagers (BiTEs). Some of these approaches have been successful in enabling direct interactions between T cells and cancer cells, leading to T cell expansion in hematopoietic cancer cells, although significant expansion in the solid tumor setting has rarely been observed.

[0007] Optimizing therapeutic approaches for the delivery of CAR T cells is often exacerbated by the difficulties encountered in in vitro expansion and manufacturing of CAR T cells due to low lymphocyte counts and poor cell quality obtained from heavily treated patients. Furthermore, even when sufficient donor cells are available, significant numbers of cells must be manufactured to provide patients with an effective dose of these therapies. Healthy allogeneic cells from healthy donors have been suggested as a solution to this problem, but several issues, such as human leukocyte antigen (HLA) mismatches between donors and recipients, can lead to graft-versus-host disease (GvHD). Thus, there remains an urgent need for novel reagents to improve the efficacy of ACT, particularly CAR T therapy.

[0008] 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 art documents] [Patent documents]

[0009] [Patent Document 1] WO96 / 027011 [Patent Document 2] WO98 / 050431 [Patent Document 3] EP1870459 [Patent Document 4] WO2007 / 110205 [Patent Document 5] WO2007 / 147901 [Patent Document 6] WO2009 / 089004 [Patent Document 7] WO2010 / 129304 [Patent Document 8] WO2011 / 90754 [Patent Document 9] WO2011 / 143545 [Patent Document 10] WO2012 / 058768 [Patent Document 11] WO2013 / 157954 [Patent Document 12] WO2013 / 09629

Non-licensed literature

[0010]

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[0011] In a first aspect disclosed herein, there is provided a bispecific polypeptide comprising a first antigen-binding protein and a second antigen-binding protein, wherein the first antigen-binding protein specifically binds to CD40 and the second antigen-binding protein specifically binds to a FLAG tag.

[0012] In one embodiment of the first aspect, the first antigen binding protein binds to the outer A module of the membrane proximal domain (D1a) of CD40, and optionally said binding prevents, reduces or inhibits CD40-CD40L binding.

[0013] In one embodiment of the first aspect, the first antigen binding protein is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a 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 described in Table 1 below in the context of a CD40 binding protein.

[0014] In any embodiment of the first aspect, CDR1, CDR2 and CDR3 refer to complementarity determining regions from the variable heavy chain (VH) of the antibody and CDR1a, CDR2a and CDR3a are complementarity determining regions from the variable light chain (VL) of the antibody, or if CDR1, CDR2 and CDR3 are complementarity determining regions from the VL, then CDR1a, CDR2a and CDR3a are complementarity determining regions from the VH. In such examples, the CDRs may sometimes be referred to as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3.

[0015] Reference herein to a protein or antibody that "binds" to CD40 provides literal support for the protein or antibody "binds specifically to" or "specifically binds to" CD40.

[0016] In any embodiment of the first aspect, the present invention provides a first antigen binding protein for binding to CD40, wherein the first antigen binding protein competitively inhibits the binding to CD40 of an antibody or antigen-binding fragment thereof 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: 2.

[0017] In any embodiment of the first aspect, the invention provides a first antigen binding protein having antigen binding domains CDRH1, CDRH2 and / or CDRH3 which have variable heavy domains as defined in SEQ ID NO:1.

[0018] In any embodiment of the first aspect, the invention provides a first antigen binding protein having antigen binding domains CDRL1, CDRL2 and / or CDRL3 which have a light chain variable domain as defined in SEQ ID NO:2.

[0019] In any embodiment of the first aspect, the invention provides a first antigen-binding protein having CDR1, CDR2 and / or CDR3 of an antigen-binding domain having a variable heavy chain as defined in SEQ ID NO:1 and a variable light chain as defined in SEQ ID NO:2.

[0020] In embodiments of any of the first aspect, the first antigen binding protein described herein comprises: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Including, wherein the sequences of any of the framework regions and / or complementarity determining regions are as described herein, and preferably as described in Table 1 below.

[0021] As defined herein, a linker can be a chemical entity, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0022] In any embodiment of the first aspect, the first antigen binding protein is (i) and (ii): (i) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 3, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 6, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 10; and (ii) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 12, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 14, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 16; or (iii) and (iv): (iii) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:3; a VH comprising a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 10; and (iv) a CDR1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 12; a CDR2 comprising, or consisting of, a sequence that is at least about 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; a VL comprising, or consisting of, a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; or (v) and (vi): (v) a VH comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 5, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 9, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 11; and (vi) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 13, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 15, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 16; or (vii) and (viii): (vii) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:5, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 99% identical to the sequence set forth in SEQ ID NO:9; a VH comprising a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:11; (viii) a CDR1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 13; at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least a VL comprising a CDR2 comprising, or consisting of, a sequence that is at least about 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; a VL comprising, or consisting of, a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; or (ix) and (x): (ix) a VH comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 4, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 7, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 10; and (x) a VL comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 12, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 14, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 16; or (xi) and (xii): (xi) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO:4, at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89% identical to the sequence set forth in SEQ ID NO:7; a VH comprising a CDR2 comprising, or consisting of, a sequence that is 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 10; a VH comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 10; and (xii) a CDR1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 12; at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least a VL comprising a CDR2 comprising, or consisting of, a sequence that is at least about 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; a VL comprising, or consisting of, a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; or (xiii) and (xiv): (xiii) a VH comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 4, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 8, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 10; and (xiv) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 12, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 14, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 16; or (xv) and (xvi): (xv) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO:4, at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89% identical to the sequence set forth in SEQ ID NO:8; a VH comprising a CDR2 comprising, or consisting of, a sequence that is 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 10; a VH comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 10; and (xvi) a CDR1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 12; at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least a CDR2 comprising, or consisting of, a sequence that is at least about 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; and a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16. Includes.

[0023] In embodiments of any of the first aspect, the first antigen binding protein comprises a heavy chain variable domain comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO: 1 or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical thereto. Optionally, the heavy chain variable domain of the first antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to CD40.

[0024] In embodiments of any of the first aspect, the first antigen binding protein comprises a light chain variable domain comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:2, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical thereto. Optionally, the light chain variable domain of the first antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NO: 2, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to CD40.

[0025] In any embodiment of the first aspect, the first antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical thereto; or and a light chain variable domain comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:2, or a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical thereto. Optionally, the heavy and / or light chain variable domain of the first antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1 or 2, respectively, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to CD40.

[0026] In embodiments of any of the first aspect, the first antigen binding protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NOs: 1 and 2 (in N to C-terminal or C to N-terminal order).

[0027] In any embodiment of the first aspect, the first antigen binding protein described herein may have, from N to C terminus, a VH then a VL, or a VL then a VH, or any of CDR1, 2 and 3 defined herein as VH then any of CDR1, 2 and 3 defined herein as VL, or any of CDR1, 2 and 3 defined herein as VL then any of CDR1, 2 and 3 defined herein as VH.

[0028] In embodiments of any of the first aspect, the first antigen binding protein may be in the form: (i) single domain antibodies (sdAbs), (ii) single chain Fv fragment (scFv), (iii) a dimeric scFv (di-scFv), or (iv) one of (ii) or (iii) linked to the constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3.

[0029] Furthermore, as described herein, in any embodiment of the first aspect, the first antigen binding protein may be in the form: (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.

[0030] In any embodiment of the first aspect, the first antigen-binding protein may be in the form of an immunoglobulin G molecule (IgG). Optionally, according to any embodiment of the first aspect of the invention, the first antigen-binding protein may be in the form of an IgG or a heterodimeric Fab-Fc, and the second antigen-binding protein may be in the form of an antigen-binding fragment of IgG, for example an scFv.

[0031] In a second aspect disclosed herein, there is provided a bispecific polypeptide comprising a first antigen-binding protein and a second antigen-binding protein, wherein the first antigen-binding protein specifically binds to CD206 and the second antigen-binding protein specifically binds to a FLAG tag.

[0032] In embodiments of either of the second aspect, the first antigen-binding protein binds to CD206 expressed on the surface of an immune cell, preferably an antigen-presenting cell, more preferably a professional antigen-presenting cell. Preferably, the first antigen-binding protein binds to CD206 expressed on the surface of a professional antigen-presenting cell selected from a dendritic cell, a macrophage, a B cell, an epithelial cell, most preferably a dendritic cell, and even more preferably, the professional antigen-presenting cell is not a tumor cell.

[0033] In one embodiment of the first aspect, the first antigen binding protein binds to human macrophage B11 antigen, and optionally the B11 antigen comprises the amino acid sequence set forth in SEQ ID NO:186.

[0034] In embodiments of any of the second aspect, the first antigen binding protein is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a 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 described in Table 1 below in the context of a CD206 binding protein.

[0035] In this embodiment of the second aspect, 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.

[0036] Reference herein to a protein or antibody that "binds" to CD206 provides literal support for the protein or antibody "binds specifically to" or "specifically binds to" CD206.

[0037] In any embodiment of the second aspect, the invention provides a first antigen binding protein for binding to CD206, wherein the first antigen binding protein competitively inhibits the binding to CD206 of an antibody comprising a VH comprising the sequence set forth in SEQ ID NO: 33 and a VL comprising the sequence set forth in SEQ ID NO: 34.

[0038] In any embodiment of the second aspect, the invention provides a first antigen binding protein having antigen binding domains CDRH1, CDRH2 and / or CDRH3 which have variable heavy domains as defined in SEQ ID NO:33.

[0039] In any embodiment of the second aspect, the invention provides a first antigen binding protein having antigen binding domains CDRL1, CDRL2 and / or CDRL3 which have a light chain variable domain as defined in SEQ ID NO:34.

[0040] In any embodiment of the second aspect, the invention provides a first antigen-binding protein having CDR1, CDR2 and / or CDR3 of an antigen-binding domain having a variable heavy chain as defined in SEQ ID NO: 33 and a variable light chain as defined in SEQ ID NO: 34.

[0041] In embodiments of any of the second aspect, the first antigen binding protein described herein comprises: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Including, wherein the sequences of any of the framework regions and / or complementarity determining regions are as described herein, and preferably as described in Table 1 below.

[0042] As defined herein, a linker can be a chemical entity, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0043] In embodiments of any of the second aspect, the first antigen binding protein comprises: (i) and (ii): (i) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 35, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 38, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 42; and (ii) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 44, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 46, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 48; or (iii) and (iv): (iii) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 35, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the sequence set forth in SEQ ID NO: 38. , or a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO:42; (iv) a CDR1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 44; a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 48; or (v) and (vi): (v) a VH comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 37, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 41, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 43; and (vi) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 45, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 47, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 48; or (vii) and (viii): (vii) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 37, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the sequence set forth in SEQ ID NO: 41 , or a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO:43; (viii) a CDR1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 45; a CDR2 comprising, or consisting of, a sequence that is 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:48; a VL comprising, or consisting of, a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:48; or (ix) and (x): (ix) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 36, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 39, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 42; and (x) a VL comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 44, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 46, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 48; or (xi) and (xii): (xi) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 36, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the sequence set forth in SEQ ID NO: 39. , or a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO:42; (xii) a CDR1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 44; at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least a CDR2 comprising, or consisting of, a sequence that is 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:48; a VL comprising, or consisting of, a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:48; or (xiii) and (xiv): (xiii) a VH comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 36, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 40, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 42; and (xiv) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 44, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 46, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 48; or (xv) and (xvi): (xv) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 36, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the sequence set forth in SEQ ID NO: 40 , or a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO:42; (xvi) a CDR1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 44; at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least a CDR2 comprising, or consisting of, a sequence that is at least about 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:48; and a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:48. The antigen-binding domain comprises:

[0044] In embodiments of any of the second aspect, the first antigen binding protein comprises a heavy chain variable domain comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO: 33, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical thereto. Optionally, the heavy chain variable domain of the first antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NO: 33, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to CD206.

[0045] In embodiments of any of the second aspect, the first antigen binding protein comprises a light chain variable domain comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO: 34, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical thereto. Optionally, the light chain variable domain of the first antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NO: 34, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to CD206.

[0046] In any embodiment of the second aspect, the first antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 33 or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical thereto. and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 34, or a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical thereto. Optionally, the heavy and light chain variable domains of the first antigen binding protein comprise no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NO: 33 or 34, respectively, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to CD206.

[0047] In embodiments of any of the second aspect, the first antigen binding protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NOs: 33 and 34 (in N to C-terminal or C to N-terminal order).

[0048] In any embodiment of the second aspect, the first antigen binding protein described herein may have, from N to C terminus, a VH then a VL, or a VL then a VH, or any of CDR1, 2 and 3 defined herein as VH then any of CDR1, 2 and 3 defined herein as VL, or any of CDR1, 2 and 3 defined herein as VL then any of CDR1, 2 and 3 defined herein as VH.

[0049] In embodiments of either of the second aspect, the first antigen binding protein may be in the form: (i) single domain antibodies (sdAbs), (ii) single chain Fv fragment (scFv), (iii) a dimeric scFv (di-scFv), or (iv) one of (ii) or (iii) linked to the constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3.

[0050] Additionally, as described herein, the first antigen binding protein may be in the form: (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.

[0051] In any embodiment of the second aspect, the first antigen-binding protein may be in the form of an IgG. In any embodiment of the second aspect, the first antigen-binding protein may be in the form of an immunoglobulin G molecule (IgG). Optionally, according to any embodiment of the second aspect of the invention, the first antigen-binding protein may be in the form of an IgG or heterodimeric Fab-Fc, and the second antigen-binding protein may be in the form of an antigen-binding fragment of IgG, for example an scFv.

[0052] In any embodiment, including the first and second embodiment, the second antigen binding protein is for binding to a FLAG tag, wherein the second antigen binding protein is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a 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.

[0053] 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 of the variable light chain (VL) of the antibody, or where CDR1, CDR2, and CDR3 are the complementarity determining regions from the VL, 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.

[0054] In any embodiment of any aspect of the invention, the second antigen binding protein is capable of specifically binding to a FLAG tag or variants thereof (as defined in SEQ ID NOs: 80 and 99, or as defined elsewhere herein). 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.).

[0055] In any embodiment of any aspect of the invention, the second antigen binding protein is for specific binding to a peptide tag containing, or consisting of, the sequence DYK, preferably the sequence DYKD (SEQ ID NO: 80). In addition to these sequences, other amino acids may be present in the FLAG tag to which the second antigen binding protein is bound, preferably the additional amino acids in the FLAG tag are 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).

[0056] In a preferred embodiment of either aspect of the invention, the second antigen binding protein is capable of specifically binding to a FLAG tag containing, comprising, or consisting of the sequence GDYKDDDDKG (SEQ ID NO:98), DYKDDDDK (SEQ ID NO:99), MDYKDDDDK (SEQ ID NO:100), DFKDDDK (SEQ ID NO:101), DYKAFDNL (SEQ ID NO:102), DYKDHDG (SEQ ID NO:103), MDFKDDDDK (SEQ ID NO:104), MDYKAFDNL (SEQ ID NO:105), DYKDHDI (SEQ ID NO:106), DYKDH (SEQ ID NO:107), DYKDD (SEQ ID NO:108), DYKDHD (SEQ ID NO:109) and / or DYKDDD (SEQ ID NO:110). The most preferred sequence is DYKDDDDK (SEQ ID NO:99).

[0057] As used herein, the term FLAG tag also encompasses FLAG tags that have been modified by amino acid insertion, deletion or substitution, for example those derived from the FLAG tag described above, in particular tags having the sequence DYKDDDDK.

[0058] 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.

[0059] In either embodiment, the FLAG tag is present at the N-terminus, C-terminus or within the protein to which the bispecific polypeptide of the invention can bind, binds or specifically binds.

[0060] In any embodiment of any of the aspects of the invention, the second antigen binding protein is - comprising a VH comprising the sequence set forth in SEQ ID NO: 70 and a VL comprising the sequence set forth in SEQ ID NO: 75, - comprising a VH comprising the sequence set forth in SEQ ID NO: 71 and a VL comprising the sequence set forth in SEQ ID NO: 76, - comprising a VH comprising the sequence set forth in SEQ ID NO: 71 and a VL comprising the sequence set forth in SEQ ID NO: 77, - comprising a VH comprising the sequence set forth in SEQ ID NO: 71 and a VL comprising the sequence set forth in SEQ ID NO: 78, - comprising a VH comprising the sequence set forth in SEQ ID NO: 71 and a VL comprising the sequence set forth in SEQ ID NO: 79, - comprising a VH comprising the sequence set forth in SEQ ID NO: 72 and a VL comprising the sequence set forth in SEQ ID NO: 76, - comprising a VH comprising the sequence set forth in SEQ ID NO: 72 and a VL comprising the sequence set forth in SEQ ID NO: 77, - comprising a VH comprising the sequence set forth in SEQ ID NO: 72 and a VL comprising the sequence set forth in SEQ ID NO: 78, - comprising a VH comprising the sequence set forth in SEQ ID NO: 72 and a VL comprising the sequence set forth in SEQ ID NO: 79, - comprising a VH comprising the sequence set forth in SEQ ID NO: 73 and a VL comprising the sequence set forth in SEQ ID NO: 76, - comprising a VH comprising the sequence set forth in SEQ ID NO: 73 and a VL comprising the sequence set forth in SEQ ID NO: 77, - comprising a VH comprising the sequence set forth in SEQ ID NO: 73 and a VL comprising the sequence set forth in SEQ ID NO: 78, - comprising a VH comprising the sequence set forth in SEQ ID NO: 73 and a VL comprising the sequence set forth in SEQ ID NO: 79, - comprising a VH comprising the sequence set forth in SEQ ID NO: 74 and a VL comprising the sequence set forth in SEQ ID NO: 76, - comprising a VH comprising the sequence set forth in SEQ ID NO: 74 and a VL comprising the sequence set forth in SEQ ID NO: 77, - comprising a VH comprising the sequence set forth in SEQ ID NO: 74 and a VL comprising the sequence set forth in SEQ ID NO: 78, or - comprising a VH comprising the sequence set forth in SEQ ID NO: 74 and a VL comprising the sequence set forth in SEQ ID NO: 79 Competitively inhibits antibody binding to the FLAG tag.

[0061] In some embodiments, the second antigen binding protein comprises an antigen binding domain CDRH1, CDRH2 and / or CDRH3 having a variable heavy chain as defined in any one of SEQ ID NOs: 71-74.

[0062] In some embodiments, the second antigen binding protein comprises an antigen binding domain CDRL1, CDRL2 and / or CDRL3 having a variable light chain as defined in any one of SEQ ID NOs: 76-79.

[0063] In some embodiments, the second antigen binding protein comprises CDR1, CDR2 and / or CDR3 of an antigen binding domain having a variable heavy chain defined in any one of SEQ ID NOs: 71-74 and a variable light chain defined in any one of SEQ ID NOs: 76-79.

[0064] In any embodiment of any aspect, the second antigen binding protein described herein is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Including, The sequences of any framework regions and / or complementarity determining regions are as described herein, and preferably as set out in Table 1 below.

[0065] As defined herein, a linker can be a chemical entity, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0066] In any embodiment of any of the aspects of the invention, the second antigen-binding domain comprises: (a) and (b): (a) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 81; a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO: 83; and (b) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 90; 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% identical to the sequence of SEQ ID NO: 92; 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO: 111 or 112, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 99% identical to the sequence of SEQ ID NO: 113 a VH comprising a CDR2 comprising, or alternatively consisting of, an amino acid sequence that is 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%, or at least 99% identical to the sequence of SEQ ID NO: 114; a VH comprising, or alternatively consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 114; and (d) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 120; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; or (e) and (f): (e) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 111 or 112, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 99% identical to the sequence of SEQ ID NO: 113; a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 114; and (f) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 133; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; or (g) and (h): (g) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 111 or 156, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 99% identical to the sequence of SEQ ID NO: 157; a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 114; and (h) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 120; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; or (i) and (j): (i) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO: 111 or 156, at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 114; and (j) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 133; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; or (k) and (l): (k) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 126, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90% identical to the sequence of SEQ ID NO: 127 , 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% identical to the sequence of SEQ ID NO: 114; and a CDR3 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO: 114. (l) a CDR1 comprising or consisting of an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO: 120, at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least a VL comprising a CDR2 comprising, or consisting of, an amino acid sequence that is 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; a VL comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; or (m) and (n): (m) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO: 126, at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% identical to the sequence of SEQ ID NO: 127 , 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% identical to the sequence of SEQ ID NO: 114; and a CDR3 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO: 114. (n) a CDR1 comprising or consisting of an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 133; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; or (o) and (p): (o) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 111, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90% identical to the sequence of SEQ ID NO: 132 , 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% identical to the sequence of SEQ ID NO: 114; and a CDR3 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO: 114. (p) a CDR1 comprising or consisting of an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 133; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; or (q) and (r): (q) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 126, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90% identical to the sequence of SEQ ID NO: 127 , 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% identical to the sequence of SEQ ID NO: 114; and a CDR3 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO: 114. (r) a CDR1 comprising or consisting of an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 133; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; The antigen-binding domain comprises:

[0067] In one embodiment of any of the aspects of the invention, the second antigen binding protein is (a) and (b): (a) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 81, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 82, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 83; and (b) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 90, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 91, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (c) and (d): (c) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 111 or 112, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 113, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (d) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 120, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (e) and (f): (e) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 111 or 112, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 113, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (f) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 133, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (g) and (h): (g) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 111 or 156, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 157, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (h) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 120, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (i) and (j): (i) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 111 or 156, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 157, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (j) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 133, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (k) and (l): (k) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 126, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 127, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (l) VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 120, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (m) and (n): (m) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 126, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 127, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (n) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 133, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (o) and (p): (o) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 111, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 132, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (p) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 133, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (q) and (r): (q) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 126, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 127, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (r) VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 133, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92. The antigen-binding domain comprises:

[0068] In any embodiment of any aspect of the invention, 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 VL, then FR1a, FR2a, FR3a, and FR4a are framework regions from VH. In such examples, the FRs may optionally be referred to as FR H1, FR H2, FR H3, FR H4, FR L1, FR L2, FR L3, and FR L4.

[0069] In any embodiment of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain having a FR H1, a FR H2, a FR H3 and / or a FR H4 from human germline, wherein the human germline is IGHV1-46 * 01 or IGHV7-4-1 * The number is 02.

[0070]

[0013] In any embodiment of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain having FR L1, FR L2, FR L3 and / or FR L4 from human germline, wherein the human germline is selected from the group consisting of IGKV2-30 * 01 or IGKV4-1 * The number is 01.

[0071] In any embodiment of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain having a FR H1, a FR H2, a FR H3 and / or a FR H4 from human germline, wherein the human germline is IGHV1-46 * 01 or IGHV7-4-1 * 02, and comprising an antigen-binding domain 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.

[0072] In any embodiment of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain that comprises a VH with 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 2, and / or a VL with 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 2.

[0073] In one embodiment of any of the aspects of the invention, the second antigen binding protein comprises an antigen binding domain having FR H1, FR H2, FR H3 and / or FR H4 of the antigen binding domain having a variable heavy chain as defined in any one of SEQ ID NOs: 71-74.

[0074] In any embodiment of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain having FR L1, FR L2, FR L3 and / or FR L4 of an antigen binding domain having a variable light chain as defined in any one of SEQ ID NOs: 76-79.

[0075] In any embodiment of any aspect of the invention, the second antigen-binding protein comprises an antigen-binding domain 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: 71-74 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: 76-79.

[0076] In any embodiment of any of the aspects of the invention, the second antigen binding protein comprises an antigen binding domain, the antigen binding domain comprising 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 A. 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 134, 135, 136, and 137, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 138, 139, 140, and 97, respectively; or 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 87, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 138, 139, 140, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 87, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 153, 154, 155, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 87, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 95, 96, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 87, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 94, 96, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 86, 88, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 138, 139, 140, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 86, 88, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 153, 154, 155, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 86, 88, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 95, 96, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 86, 88, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 94, 96, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 85, 151, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 138, 139, 140, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 85, 151, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 153, 154, 155, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 85, 151, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 95, 96, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 85, 151, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 94, 96, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 150, 152, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 138, 139, 140, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 150, 152, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 153, 154, 155, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 150, 152, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 95, 96, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 150, 152, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 94, 96, and 97, respectively. Includes.

[0077] In any embodiment, the second antigen binding protein comprises an antigen binding domain, the antigen binding domain comprising 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 A. 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 115, 116, 118, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 127, respectively; or 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 115, 116, 118, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 127, 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: 115, 117, 119, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 127, respectively; or D. 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: 115, 117, 119, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 127, respectively. Includes.

[0078] In any embodiment of any of the aspects of the invention, the second antigen binding protein comprises an antigen binding domain, the antigen binding domain comprising 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 A. 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 115, 116, 118, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, respectively; or 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 115, 116, 118, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 161, 162, 163, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 115, 117, 119, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 115, 117, 119, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 161, 162, 163, and 97, respectively. Includes.

[0079] In any embodiment of any of the aspects of the invention, the second antigen binding protein comprises 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 A. 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 116, 159, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 127, respectively; or 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 116, 159, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 127, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 117, 160, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 127, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 117, 160, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 127, respectively. The antigen-binding domain comprises:

[0080] In any embodiment of any of the aspects of the invention, the second antigen binding protein comprises an antigen binding domain, the antigen binding domain comprising 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 A. 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 116, 159, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, respectively; or 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 116, 159, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 161, 162, 163, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 117, 160, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 117, 160, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 161, 162, 163, and 97, respectively. Includes.

[0081] In any embodiment of any of the aspects of the invention, the second antigen binding protein comprises an antigen binding domain, the antigen binding domain comprising 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 A. 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 128, 130, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 97, respectively; or 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 128, 130, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 129, 131, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 129, 131, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 164, 128, 166, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 164, 128, 166, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 164, 165, 167, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 97, 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: 164, 165, 167, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 97, respectively. Includes.

[0082] In any embodiment of any of the aspects of the invention, the second antigen binding protein comprises an antigen binding domain, the antigen binding domain comprising 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 A. 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 128, 130, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, respectively; or 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 128, 130, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 168, 169, 170, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 129, 131, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 129, 131, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 168, 169, 170, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 164, 128, 166, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 164, 128, 166, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 168, 169, 170, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 164, 165, 167, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 164, 165, 167, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 168, 169, 170, and 97, respectively. Includes.

[0083] In any embodiment of any of the aspects of the invention, the second antigen binding protein comprises 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for the VH FRs, the reference sequences are set forth in SEQ ID NOs: 141, 142, 143, and 137, respectively, and for the VL For FR, the reference sequences are shown in SEQ ID NOs: 144, 145, 146 and 97, respectively, for VH and VL, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 147, 148, and 137, respectively, and for VL For FR, the reference sequences are shown in SEQ ID NOs: 144, 145, 146 and 97, respectively. Includes.

[0084] In any embodiment of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain comprising a variable heavy chain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 71-74, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical thereto. Optionally, the heavy chain variable domain of the second antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NOs: 71-74, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to a FLAG tag.

[0085] In one embodiment of any of the aspects of the invention, the second antigen binding protein comprises an antigen binding domain comprising a variable light chain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 76-79, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical thereto. Optionally, the light chain variable domain of the second antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NOs: 76-79, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to a FLAG tag.

[0086] In any embodiment of any aspect of the invention, the second antigen binding protein comprises a variable heavy chain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 71-74 and a variable light chain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 76-79, or an antigen binding domain comprising sequences which are at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical thereto. Optionally, the heavy and light chain variable domains of the second antigen binding protein comprise no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions compared to the amino acid sequences set forth in SEQ ID NOs: 71-74 and 76-79, respectively, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to a FLAG tag.

[0087] As described herein, the second antigen binding protein may be in the form of: (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) 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.

[0088] Additionally, as described herein, the second antigen binding protein may be in the form: (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.

[0089] In a preferred embodiment of either aspect of the invention, the second antigen binding protein is in the form of an scFv and the first antigen binding protein is in the form of an immunoglobulin G (IgG) antibody. In alternative embodiments, the second antigen binding protein may be of the same antibody format as the first antigen binding protein, or a fragment thereof. Alternatively, the first and second antigen binding proteins are antigen binding proteins of different formats.

[0090] In embodiments of either the first or second aspect of the present invention, the second binding domain may be in the form of an antibody or an antigen-binding fragment thereof. The antigen-binding protein may be 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 radiolabel or an agent to improve solubility, or other modifications described herein. As used herein, an antigen-binding protein may be a variable domain.

[0091] In any embodiment of any of the aspects of the invention, the second antigen binding protein comprises, consists essentially of, or consists of (in N to C-terminal or C to N-terminal order) the following amino acid sequence: SEQ ID NOs: 71 and 79, SEQ ID NOs: 71 and 78, SEQ ID NOs: 71 and 76, SEQ ID NOs: 71 and 77, SEQ ID NOs: 72 and 76, SEQ ID NOs: 72 and 77, SEQ ID NOs: 72 and 78, SEQ ID NOs: 72 and 79, SEQ ID NOs: 73 and 76, SEQ ID NOs: 73 and 77, SEQ ID NOs: 73 and 78, SEQ ID NOs: 73 and 79, SEQ ID NOs: 74 and 76, SEQ ID NOs: 74 and 77, SEQ ID NOs: 74 and 78, or SEQ ID NOs: 74 and 79.

[0092] 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 or any other CDR numbering system.

[0093] In any embodiment of any of the aspects of the invention, the bispecific polypeptide comprises, consists essentially of, or consists of the following amino acid sequence: SEQ ID NOs: 19 and 20, SEQ ID NOs: 19 and 21, SEQ ID NOs: 22 and 23, SEQ ID NOs: 23 and 24, SEQ ID NOs: 19 and 25, SEQ ID NOs: 19 and 26, SEQ ID NOs: 23 and 27, SEQ ID NOs: 23 and 28, SEQ ID NOs: 19 and 29, SEQ ID NOs: 19 and 30, SEQ ID NOs: 23 and 31, SEQ ID NOs: 23 and 32, SEQ ID NOs: 171, 172 and 173, SEQ ID NOs: 172, 174 and 175, SEQ ID NOs: 171, 172 and 176, SEQ ID NOs: 172, 174 and 177, SEQ ID NOs: 50 and 51, SEQ ID NOs: 50 and 52, SEQ ID NOs: 53 and 54, SEQ ID NOs: 54 and 55, SEQ ID NOs: 50 and 56, SEQ ID NOs: 50 and 57, SEQ ID NOs: 54 and 58, SEQ ID NOs: 54 and 59, SEQ ID NOs: 50 and 60, SEQ ID NOs: 50 and 61, SEQ ID NOs: 54 and 62, or SEQ ID NOs: 54 and 63.

[0094] In any of the above embodiments, the bispecific polypeptide comprises, consists essentially of, or consists of one, two, or three polypeptide chains, which are linked by a covalent linker (e.g., any linker described herein, including an amino acid or disulfide bond). The bispecific polypeptide may be heterodimerized as described herein.

[0095] In a third aspect, the present invention provides a fusion protein comprising a first antigen binding protein according to either the first or second aspect of the invention and a second antigen binding protein defined according to either the first or second aspect of the invention. The present invention also provides a fusion protein comprising a bispecific polypeptide as defined herein according to either the first or second aspect of the invention.

[0096] In a fourth aspect, the present invention provides a nucleic acid encoding the bispecific polypeptide or part thereof of the first or second aspect of the present invention, or the fusion protein of the third aspect. Preferably, the nucleic acid has a nucleotide sequence encoding any one or more of the amino acid sequences corresponding to SEQ ID NOs: 1, 2, 17-32, 33, 34, 49-63, 64-67, 71-74, 76-79 and 171-177.

[0097] In any embodiment of the fourth aspect of the invention, the nucleic acid may be DNA (eg, cDNA) or RNA (eg, mRNA).

[0098] In a fifth aspect, the present invention provides a vector comprising the nucleic acid of the fourth aspect.

[0099] In aspects of the invention directed to multiple polypeptide chains forming a bispecific polypeptide or 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.

[0100] 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.

[0101] 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:

[0102] In a sixth aspect, the present invention provides a cell comprising the vector of the fifth aspect or the nucleic acid of the fourth aspect. Preferably, the cell is isolated, substantially purified, or recombinant. In one example, the cell comprises an expression construct or (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 associate to form an antigen binding protein as described herein.

[0103] Examples of cells of the invention include bacterial cells, yeast cells, insect cells or mammalian cells.

[0104] In another embodiment, an animal or tissue derived therefrom comprising the cells described herein is provided.

[0105] In a seventh aspect, the present invention provides a pharmaceutical composition comprising a bispecific polypeptide of the first or second aspect, a fusion protein of the third aspect, a nucleic acid of the fourth aspect, a vector of the fifth aspect or a cell of the sixth aspect and a pharmaceutically acceptable carrier, diluent or excipient.

[0106] In an eighth aspect, the present invention provides a method for producing a bispecific polypeptide of the first or second aspect, or a fusion protein of the third aspect, as described herein, comprising expressing a nucleic acid of the fourth aspect in a cell or animal.

[0107] In a ninth aspect, the present invention provides the use of a bispecific polypeptide of the first or second aspect, a fusion protein of the third aspect, a nucleic acid of the fourth aspect, a vector of the fifth aspect or a cell of the sixth aspect in the manufacture of a medicament for the treatment or prevention of cancer.

[0108] In a tenth aspect, the present invention provides a method of treating or preventing cancer, the method comprising the step of administering to a subject a bispecific polypeptide of the first or second aspect, a fusion protein of the third aspect, a nucleic acid of the fourth aspect, a vector of the fifth aspect or a cell of the sixth aspect, thereby treating or preventing cancer.

[0109] The antigen binding proteins described herein may be purified, substantially purified, isolated, and / or recombinant.

[0110] The bispecific polypeptide of the invention can be part of the supernatant harvested from the culture medium in which a hybridoma expressing the bispecific polypeptide of the invention has been grown.

[0111] 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.

[0112] 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]

[0113] [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. [Figure 4]FIG. 4 is a schematic diagram of BEAT fluctuations in IgG and scFv domains. [Figure 5] Figure 5 shows a single reference sensorgram of CD40 binding to BEAT1a4 (F7-G8) in the presence of 50 nM FLAG-BAP, showing 50 nM FLAG-BAP binding and saturation, followed by CD40 injection in the presence of 50 nM FLAG-BAP. CD40 concentrations: 50 nM, 16.7 nM, 5.6 nM, 1.9 nM, 0.62 nM, 0.21 nM, 0.069 nM, 0.023 nM, 0 nM. Fitting to a 1:1 binding model is shown. [Figure 6] FIG. 6 shows binding of anti-CD40 (A) and anti-CD206 (B) BEATs to monocyte-derived DCs. [Figure 7(A)] FIG. 7(A) shows the dose-dependent binding of BEAT to CD40 in human MoDC cells between 0.1 and 100 nM of BEAT1a4. [Figure 7(B)] FIG. 7(B) shows the dose-dependent binding of BEAT to CD40 in human B cells between 0.1 and 100 nM of BEAT 1a4. [Figure 7(C)] FIG. 7(C) shows the dose-dependent binding of BEAT to CD40 in non-human primate MoDC cells between 0.1 and 100 nM of BEAT1a4. [Figure 7(D)] FIG. 7(D) shows the dose-dependent binding of BEAT to CD40 in non-human primate B cells between 0.1 and 100 nM of BEAT 1a4. [Figure 8(A)] FIG. 8(A) shows that BEAT1 (and aCD40 IgG2 control) increased CD86 expression (A) in a dose-responsive manner between 3 and 30 nM (CD86) and 1 and 60 nM (IL-12). [Figure 8(B)] FIG. 8(B) shows that BEAT1 (and aCD40 IgG2 control) increased IL-12 / IL-23p40(B) release in a dose-responsive manner between 3-30 nM (CD86) and 1-60 nM (IL-12). [Figure 9]Figure 9 shows (A) BEAT binds to the FLAG tag on the 7B1 anti-mesothelin CD28z CAR and increases IFN-gamma; (B) MoDCs express CD40 and CD206; (C) BEAT1a4 binding to CAR T-cells assessed by FLAG tag detection in GPF+ / AF647+ cells by FACS. [Figure 10] FIG. 10 shows cytokine analysis using one donor with BEAT. [Figure 11] Figure 11 shows CAR T cell proliferation with BEAT added in the presence of control compound / BEAT (30 nM) after 120 hours of growth with or without MoDc. [Figure 12] FIG. 12 shows further phenotyping of the CD4+, CD8+ and CD3+ cells described in FIG. [Figure 13] Figure 13 shows the cytotoxicity of BEAT+CAR T cells in SKOV-3 MSLN-positive cell lines. [Figure 14] Figure 14 shows the in vivo efficacy of CAR T+BEAT1a5 in a SKOV3-mesothelin-positive model transfected with CD40 knockout cells. (A) Schematic experimental timeline. (B) Survival curve. (C) Tumor size. [Figure 15] FIG. 15 is a schematic diagram of BEATs 3, 4, 5 and 6. [Figure 16] FIG. 16 shows ELISA binding assay of anti-CD40 anti-FLAG BEAT3, 4, 5 and 6 to CD40 antigen. [Figure 17] FIG. 17 shows binding of BEAT1, 3, 4, 5, and 6 to 7B1 anti-mesothelin CD28z CAR human T cells (A) and human monocyte-derived DCs (B). [Figure 18(A)] FIG. 18(A) shows CD86(A) upregulation in MoDCs upon addition of BEAT1, 3, 4, 5 and 6. [Figure 18(B)]FIG. 18(B) shows the upregulation of IL-12 / IL-23p40(B) in MoDCs upon addition of BEAT1, 3, 4, 5 and 6. [Figure 19] Figure 19 shows T cell proliferation after 5 days of growth with or without MoDc and CAR T cells, with addition of BEAT1, 3, 4, 5 and 6 in the presence of control compound / BEAT (30 nM). [Figure 20] FIG. 20 shows further phenotyping of the CD3+ T cells described in FIG. [Figure 21] FIG. 21 shows further phenotyping of the CD4+ T cells described in FIG. [Figure 22] FIG. 22 shows further phenotyping of the CD8+ T cells described in FIG. [Figure 23(A)] FIG. 23(A) shows the cytokine activity of BEAT1, 3, 4, 5 and 6 with IFN-containing γ, CAR T cells or MoDCs or CAR+MoDCs. [Figure 23(B)] Figure 23(B) shows the cytokine activity of BEAT1, 3, 4, 5 and 6 with CAR T cells or MoDCs or CAR+MoDCs containing IL12-p70. [Figure 23(C)] Figure 23(C) shows the cytokine activity of BEAT1, 3, 4, 5 and 6 with CAR T cells or MoDCs or CAR+MoDCs, including IL-6. [Figure 23(D)] Figure 23(D) shows the cytokine activity of BEAT1, 3, 4, 5 and 6 with CAR T cells or MoDCs or CAR+MoDCs, including IL-2. [Figure 23(E)] Figure 23(E) shows the cytokine activity of BEAT1, 3, 4, 5 and 6 with CAR T cells or MoDCs or CAR+MoDCs, including MCP-1. [Figure 23(F)] Figure 23(F) shows the cytokine activity of BEAT1, 3, 4, 5 and 6 with CAR T cells or MoDCs or CAR+MoDCs, including TNF-α (F). [Figure 24] Figure 24 shows the in vivo efficacy of CAR T cells alone compared to CAR T+BEAT2a6 (anti-CD206 BEAT) in the SKOV3-mesothelin model detailing tumor size. DETAILED DESCRIPTION OF THE INVENTION

[0114] [Table 1A]

[0115] [Table 1B]

[0116] [Table 1C]

[0117] [Table 1D]

[0118] [Table 1E]

[0119] [Table 1F]

[0120] [Table 1G]

[0121] [Table 1H]

[0122] [Table 1I]

[0123]

Table 1J

[0124] Table 1K

[0125] Table 1L

[0126] Table 1M

[0127]

Table 1N

[0128]

Table 1O

[0129]

Table 1P

[0130]

Table 1Q

[0131]

Table 1R

[0132]

Table 1S

[0133] [Table 1T]

[0134] [Table 1U]

[0135] [Table 1V]

[0136] [Table 1W]

[0137] [Table 1X]

[0138] 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.

[0139] 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:

[0140] 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.

[0141] The present inventors have developed bispecific polypeptides, such as antibodies that bind to CD40 on antigen-presenting cells and a FLAG tag present on a molecule expressed on immune cells (e.g., T cells), or to CD206 on antigen-presenting cells and a FLAG tag present on a molecule expressed on immune cells (e.g., T cells). These bispecific polypeptides enable immune cells expressing engineered TCRs (e.g., CAR T cells) to engage with antigen-presenting cells (APCs), thereby enhancing immune cell efficacy in vitro and in vivo with high tumor inhibition without toxicity, while reducing the effective dose of immune cells expressing engineered TCRs (e.g., CAR T cells) required to achieve a therapeutic effect. Importantly, immune cells expressing engineered TCRs (e.g., CAR T cells) receive activation and proliferation signals in lymphoid tissues, away from the immunosuppressive tumor microenvironment.

[0142] Bispecific Polypeptides As used herein, the term "bispecific polypeptide" refers to a polypeptide that can specifically bind to two different target antigens simultaneously. The bispecific polypeptides described herein comprise two structurally distinct binding proteins or domains (i.e., regions), each of which specifically binds to a single target antigen. Bispecific polypeptides can be used to bind to a target antigen on an APC (e.g., CD40 or CD206) and to a different target antigen on an immune cell expressing an engineered TCR (e.g., a CAR containing a FLAG tag). Furthermore, bispecific polypeptides of the present invention can be used to bind to a target antigen on an APC and to a different target antigen on an engineered TCR (e.g., a CAR) expressed by an immune cell. Bispecific polypeptides can comprise the polypeptide sequences (i.e., domains) of one or more antibodies or antibody fragments (e.g., one or more scFvs or IgGs). In another embodiment, bispecific polypeptides can comprise the polypeptide sequences of one or more ligands.

[0143] In either embodiment, the bispecific polypeptide is a tandem single-chain variable fragment antibody (taFv) having a first scFv and a second scFv. In alternative embodiments, the bispecific polypeptide may be in the form of a fusion protein comprising an immunoglobulin protein for binding to a first antigen and one or more scFvs fused thereto for binding to a second antigen. As described further herein, it will be appreciated that the bispecific polypeptides of the invention may be in a variety of different formats, in which the first and second antigen-binding proteins comprise antigen-binding domains, and the proteins may be any one of a variety of formats derived from an antibody.

[0144] The bispecific polypeptides described herein may be variously referred to as "bispecific engagers of APCs and T cells" or "BEATs."

[0145] "Polypeptide," "peptide," "protein," and "proteinaceous molecule" are used interchangeably herein to refer to molecules comprising or consisting of polymers of amino acid residues, and to variants and synthetic analogues thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic, non-naturally occurring amino acids, e.g., chemical analogues of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0146] The term "amino acid," as used herein, refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. An amino acid analog refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, e.g., a carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones. An amino acid mimetic refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0147] Amino acids may be referred to herein by their commonly used full name (e.g., cysteine), by their commonly known three letter symbol (e.g., Cys), or by the one-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission (e.g., C). Nucleotides may likewise be referred to by their commonly accepted single-letter codes.

[0148] The term "antigen," as used herein, refers to a molecule bound by an "antibody," "antibody fragment," or "bispecific polypeptide." An antigen may be a protein recognized by an immunoglobulin, in which case the site on the protein bound by the immunoglobulin is called an "epitope." In one embodiment, the antigen may be CD40, CD206, or a FLAG tag. In another embodiment, the antigen is a tag on an engineered TCR (e.g., a CAR) expressed by an immune cell.

[0149] The term "antigen-presenting cell" or "APC," as used herein, can be a professional antigen-presenting cell (e.g., a dendritic cell, a macrophage, a B cell, an epithelial cell, etc.) or a non-professional antigen-presenting cell (e.g., a fibroblast, a thymic epithelial cell, a thyroid epithelial cell, a glial cell, a pancreatic beta cell, a vascular endothelial cell, etc.). In a preferred embodiment, the APC is an endogenous professional antigen-binding cell (preferably a dendritic cell or a macrophage), which is not a tumor cell.

[0150] Examples of APCs described herein include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes (e.g., THP-1), B lymphoblastoid cells (e.g., C1R.A2, 1518 B-LCL), and monocyte-derived dendritic cells (moDCs). In any embodiment, the first antigen-binding protein specifically binds to an antigen expressed on an APC (e.g., DC or moDC), where the antigen is selected from CD40 and CD206. In a preferred embodiment, the antigen (CD40 or CD206) specifically bound by the first antigen-binding protein is not a tumor-associated antigen. In other words, in the context of use of the bispecific polypeptides of the present invention, it will be understood that the intention is that the first antigen-binding polypeptide is for binding to CD40 or CD206 on non-tumorous, endogenous professional antigen-presenting cells, e.g., DCs, moDCs.

[0151] The term "antigen presentation" refers to the process by which APCs capture antigens and make them available for recognition by T cells, for example as components of MHC-I and / or MHC-II complexes.

[0152] "MHC molecules" include two types of molecules, MHC class I and MHC class II. MHC class I molecules present antigens to specific CD8+ T cells, and MHC class II molecules present antigens to specific CD4+ T cells. Antigens delivered exogenously to APCs are processed primarily for association with MHC class II. In contrast, antigens delivered endogenously to APCs are processed primarily for association with MHC class I.

[0153] In either embodiment, the second antigen binding protein specifically binds to an antigen on an immune cell expressing an engineered TCR (e.g., a CAR) or similar receptor. In a further embodiment, the second antigen binding protein of the bispecific polypeptide of the invention is capable of binding to an antigen on an immune cell, wherein the antigen is not part of the engineered TCR, but the antigen is an antigen present on the cell surface of the immune cell.

[0154] In certain embodiments, the immune cell is an engineered T cell or NK cell, and typically the antigen on the engineered T cell or NK cell is part of an engineered TCR (e.g., a CAR). In accordance with these embodiments, the second antigen binding protein of the bispecific polypeptide of the invention may bind to the extracellular portion of the engineered TCR. The second antigen binding protein may bind to the antigen-binding domain of the engineered TCR, or may bind to an extracellular region of the engineered TCR that is not involved in antigen binding.

[0155] In certain embodiments, the engineered TCR (e.g., CAR) present on the immune cell may also comprise additional amino acids or molecules for binding to a second antigen-binding protein. As is known in the art, engineered TCR (e.g., CAR) constructs may be designed to include a "tag," which is usually a short amino acid sequence that is specifically recognized by an antibody. In some embodiments, the immune cell is a T cell or NK cell that has been engineered to express an engineered TCR (e.g., CAR) that includes a tag. In the context of such embodiments, the second antigen-binding protein of the bispecific polypeptide may bind to the tag.

[0156] In either embodiment, the tag is a FLAG tag. Thus, the second antigen binding protein is capable of specifically binding to a FLAG tag or a variant thereof (as defined in SEQ ID NOs: 80 and 99, or as otherwise defined herein). The second antigen binding protein is preferably capable of specifically binding to a protein domain comprising multiple FLAG tag sequences (e.g., 2xFLAG, 3xFLAG, etc.).

[0157] In either embodiment, the second antigen binding protein is for specific binding to a peptide tag containing, or consisting of, the sequence DYK, preferably the sequence DYKD (SEQ ID NO: 80). 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).

[0158] In a preferred embodiment, the second antigen-binding protein of the bispecific protein of the invention is capable of specifically binding to a FLAG tag containing, or consisting of, the sequence GDYKDDDDKG (SEQ ID NO:98), DYKDDDDK (SEQ ID NO:99), MDYKDDDDK (SEQ ID NO:100), DFKDDDK (SEQ ID NO:101), DYKAFDNL (SEQ ID NO:102), DYKDHDG (SEQ ID NO:103), MDFKDDDDK (SEQ ID NO:104), MDYKAFDNL (SEQ ID NO:105), DYKDHDI (SEQ ID NO:106), DYKDH (SEQ ID NO:107), DYKDD (SEQ ID NO:108), DYKDHD (SEQ ID NO:109) and / or DYKDDD (SEQ ID NO:110). The most preferred sequence is DYKDDDDK (SEQ ID NO:99).

[0159] 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.

[0160] 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.

[0161] In either embodiment, the FLAG tag is present at the N-terminus, C-terminus, or within the protein it is intended to bind.

[0162] In certain embodiments, the immune cell is a T cell or NK cell engineered to express an engineered TCR (e.g., a CAR), and the bispecific polypeptide binds to the extracellular portion of the engineered TCR. Those skilled in the art will be familiar with the general structure and uses of engineered TCRs (e.g., CARs), and more particularly, CAR T and CAR NK cells. For example, and as known in the art, a CAR is a cell surface receptor comprising an extracellular domain, a transmembrane domain, and a cytoplasmic domain in a combination not naturally found in a single protein. The extracellular domain comprises an antigen-binding domain, which may be an antibody or antibody fragment. The antibody or antibody fragment may be a human antibody or fragment, a humanized antibody or fragment, or a non-human antibody or fragment. Typically, the antigen-binding domain of a CAR is an antibody fragment, e.g., a Fab or scFv. Most typically, the antigen-binding domain of a CAR is an scFv. The extracellular domain also typically comprises a spacer (or hinge) region linking the antigen-binding domain to the transmembrane domain. The spacer region of the CAR may be derived from an immunoglobulin, such as IgG1 or IgG4, or from alternative cell surface proteins, including but not limited to CD4, CD8, or CD28.

[0163] The term "chimeric antigen receptor" or "CAR," as used herein, refers to a recombinant polypeptide comprising an antigen-binding domain linked to at least one intracellular signaling domain. The antigen-binding domain of a CAR is the functional portion of the CAR that specifically binds to (i.e., specifically targets) an antigen expressed on a cancer cell (i.e., a "tumor-associated antigen"). Examples of tumor-associated antigens are known to those of skill in the art, and illustrative examples include mesothelin, Her2, CEA, FBP, CD19, and BCMA.

[0164] As used herein, "tumor-associated antigen" refers to an antigen expressed by cancer cells. Tumor-associated antigens may or may not be expressed by non-tumor cells. When tumor-associated antigens are not expressed by non-tumor cells (i.e., unique to tumor cells), they may be referred to as "tumor-specific antigens." When tumor-associated antigens are not unique to tumor cells, they are also expressed on non-tumor cells under conditions that do not induce a state of immune tolerance to the antigen. Expression of antigens on tumors may occur under conditions that allow the immune system to respond to the antigen. Tumor-associated antigens may be antigens expressed on non-tumor cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are normally present at low levels on normal cells but are expressed at significantly higher levels on tumor cells. Those tumor-associated antigens that are of greatest clinical interest are differentially expressed compared to corresponding non-tumor tissues, allowing preferential recognition of tumor cells by specific T cells or immunoglobulins.

[0165] As will be apparent from the remainder of this disclosure, the first and second antigen-binding proteins of the bispecific polypeptides of the invention are typically in the form of antibodies or antibody fragments. The term "antibody," as used herein, broadly refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains: two heavy (H) chains and two light (L) chains. Also contemplated herein are bispecific polypeptides including antigen-binding fragments, mutants, variants, and derivatives thereof that retain the essential epitope-binding characteristics of an antibody molecule. Such mutants, variants, and derivatives will be known to those skilled in the art, and illustrative examples thereof are described elsewhere herein.

[0166] An antibody heavy chain typically comprises a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region typically comprises three domains, CH1, CH2, and CH3. A light chain typically comprises a light chain variable region (LCVR or VL) and a light chain constant region, CL. The VH and VL regions can be further subdivided into regions of hypervariability, also known as complementarity-determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL typically comprises three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0167] The term "antigen-binding fragment" or "antibody fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen. Illustrative examples of antigen-binding fragments include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a single-chain variable fragment (scFv) consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment comprising a single variable domain (Ward et al., 1989, Nature, 341:544-6); and (vi) an isolated CDR.

[0168] In embodiments of either the first or second aspect, the first antigen-binding protein and the second antigen-binding protein are different antibodies or antibody fragments. For example, the first antigen-binding protein may be in an oligomeric format in which two polypeptide chains are linked or joined, and the second antigen-binding protein may be a single polypeptide chain. For example, the first antigen-binding protein may be in an IgG format (e.g., in which a heavy chain and a light chain are linked or joined) and the second antigen-binding protein may be in an scFv format (e.g., a single continuous polypeptide chain).

[0169] In embodiments of either the first or second aspect, the first antigen binding protein may comprise or consist of a VH and a VL as defined herein, and in N to C-terminal order the VH and VL may be arranged as VL-VH or VH-VL.

[0170] In embodiments of either the first or second aspect, the second antigen binding protein may comprise or consist of a VH and a VL as defined herein, and in N to C-terminal order the VH and VL may be arranged as VL-VH or VH-VL.

[0171] In embodiments of either the first or second aspect, the second antigen-binding protein may be linked to the N-terminus of the first antigen-binding protein or the N-terminus of the antigen-binding domain of the first antigen-binding protein. For example, the second antigen-binding protein may be linked to the N-terminus of the VH or VL of the first antigen-binding protein. In one embodiment, the second antigen-binding protein may be linked to the N-terminus of the first antigen-binding protein or the antigen-binding domain of the first antigen-binding protein via a linker, preferably a linker described herein.

[0172] In embodiments of either the first or second aspect, the second antigen-binding protein may be linked to the C-terminus of the first antigen-binding protein or the C-terminus of the antigen-binding domain of the first antigen-binding protein. For example, the second antigen-binding protein may be linked to the C-terminus of the VH or VL of the first antigen-binding protein. In one embodiment, the second antigen-binding protein is linked to the C-terminus of the first antigen-binding protein or the antigen-binding domain of the first antigen-binding protein via a linker, preferably a linker described herein.

[0173] The terms "sequence identity" or "sequence homology," as used herein, refer to the subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules, e.g., between two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit position in both of the two molecules is occupied by the same monomer subunit, e.g., if a position in each of the two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half of the positions in two sequences (e.g., five positions in a polymer 10 subunits long) are homologous, then the two sequences are 50% homologous; if 90% of the positions (i.e., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

[0174] Heterodimerization A "CH3 domain" comprises the stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to about amino acid residue 447 of IgG). Within the bispecific polypeptides (e.g., antibodies) in accordance with the invention described herein, one respective CH3 domain may be positioned C-terminal to the VH3 and VL3 domains of the third binding site. A "CH3 domain" described herein may be a variant CH3 domain, in which the amino acid sequence of a native CH3 domain has been subjected to at least one distinct amino acid substitution (i.e., modification of the amino acid sequence of the CH3 domain) in order to promote dimerization of two CH3 domains facing each other within a multispecific antibody.

[0175] Several approaches for CH3 modification to support heterodimerization are described, for example, in WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954, WO2013 / 096291, the disclosures of which are incorporated herein by reference in their entireties.

[0176] Typically, in heterodimerization approaches known in the art, the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain are both engineered in a complementary manner, such that a heavy chain containing one engineered CH3 domain can no longer homodimerize with another heavy chain of identical structure, thereby forcing the heavy chain containing one engineered CH3 domain to heterodimerize with another heavy chain containing a CH3 domain that has been engineered in a complementary manner.

[0177] One heterodimerization approach known in the art is the so-called "knobs-into-holes" technique, which provides some examples and is described in detail in, for example, WO 96 / 027011, Ridgway, JB et al., Protein Eng. 9 (1996) pp. 617-621, Merchant, AM et al., Nat. Biotechnol. 16 (1998) pp. 677-681, and WO 98 / 050431, the disclosures of which are incorporated herein by reference in their entireties. In the "knobs-into-holes" technique, within the interface formed between two CH3 domains in the tertiary structure of an antibody, specific amino acids on each CH3 domain are engineered to create a protrusion (a "knob") in one CH3 domain and a cavity (a "hole") in the other CH3 domain, respectively. In the tertiary structure of a multispecific antibody, an introduced protuberance in one CH3 domain can be positioned in an introduced cavity in the other CH3 domain.

[0178] In an embodiment of either the first or second aspect, the bispecific polypeptide may comprise a CH3 domain comprising an amino acid substitution at a T366 or a Y407 substitution, for example a T366W and a Y407V substitution (numbering according to Kabat).

[0179] In an embodiment of either the first or second aspect, the bispecific polypeptide may comprise a CH3 domain comprising one or more amino acid substitutions at T366, L368, Y407, S354 and Y349. In a preferred example, the CH3 domain may comprise the following amino acid substitutions: (a) T336W, (b) T366S, L368A and Y407V, (c) Y407T, or (d)T336Y

[0180] In any of the above embodiments, the bispecific polypeptide may comprise two CH3 domains, - the first CH3 domain comprises the amino acid substitution T336W and the second CH3 domain comprises the amino acid substitutions T366S, L368A and Y407V, or - the first CH3 domain comprises an amino acid substitution of Y407T and the second CH3 domain comprises an amino acid substitution of T336Y.

[0181] In addition to, or instead of, manipulating the CH3 domain as described herein, the CH3 domain may contain additional cysteine ​​substitutions, such as S354C and / or Y349C substitutions (numbering according to Kabat), to provide additional interchain disulfide bridges that stabilize the heterodimer (Atwell, S. et al., J. Mol. Biol. 270 (1997) pp. 26-35; Merchant, AM et al., Nature Biotech. 16 (1998) pp. 677-681).

[0182] Linker Bispecific polypeptides of the invention may comprise a linker sequence linking a first antigen-binding protein and a second antigen-binding protein. Alternatively, or in addition, the linker sequence may link one or more antigen-binding domains in the first antigen-binding protein or one or more antigen-binding domains in the second antigen-binding protein. Linkers may, for example, function to link two domains of an antigen-binding protein (e.g., the VH and VL of an scFv or diabody), or they may function to link two antigen-binding proteins together (e.g., two or more Fabs or sdAbs), or they may function to link an antigen-binding protein to a scaffold. In some embodiments, a bispecific polypeptide may comprise multiple linkers (i.e., two or more), for example, one or more scFvs linked to a scaffold may comprise a linker connecting the VH and VL of the scFv and a linker connecting the scFv to the scaffold. Suitable linkers are known in the art and can be readily selected by one of skill in the art based on the intended use of the linker (see, e.g., Muller & Kontermann, "Bispecific Antibodies," in Handbook of Therapeutic Antibodies, Wiley-VCH Verlag GmbH & Co. 2014).

[0183] Useful linkers include glycine-serine (GlySer) linkers, which are well known in the art and contain glycine and serine units combined in various orders. Examples include, but are not limited to, (GS), (GSGGS). n (SEQ ID NO: 187), (GGGS) n (SEQ ID NO: 188) and (GGGGS) n (SEQ ID NO: 189), where n is an integer of at least 1 and is typically an integer between 1 and about 10, e.g., between 1 and about 8, between 1 and about 6, or between 1 and about 5.

[0184] Other useful linkers include sequences derived from immunoglobulin hinge sequences. The linker may contain all or part of a hinge sequence derived from any one of the four IgG classes, and may optionally contain additional sequences. For example, the linker may contain a portion of an immunoglobulin hinge sequence and a glycine-serine sequence. A non-limiting example is a linker containing approximately the first 15 residues of an IgG1 hinge, followed by a GlySer linker sequence about 10 amino acids in length, such as those described above.

[0185] The length of the linker varies depending on the application. A suitable linker length can be readily selected by one skilled in the art. For example, when the linker connects the VH and VL domains of an scFv, the linker is typically between about 5 and about 20 amino acids in length, e.g., between about 10 and about 20 amino acids in length, or between about 15 and about 20 amino acids in length. When the linker is intended to connect the VH and VL domains of a diabody, the linker must be short enough to prevent association of these two domains within the same chain. For example, the linker can be between about 2 and about 12 amino acids in length, e.g., between about 3 and about 10 amino acids in length, or about 5 amino acids in length.

[0186] In some embodiments, when the linker is intended to connect two Fab fragments, the linker can be selected to maintain the relative spatial conformation of the paratopes of the F(ab') fragments and to be capable of forming a covalent bond equivalent to the disulfide bond in the core hinge of IgG. In this context, suitable linkers include those derived from IgG hinge regions, such as IgG1, IgG2, or IgG4. Modified versions of these exemplary linkers can also be used. For example, modifications to improve the stability of IgG4 hinges are known in the art (see, e.g., Labrijn et al., 2009, Nature Biotechnology, 27:767-771).

[0187] The linker may comprise a sequence of amino acid residues connecting the first and second antigen-binding proteins. Alternatively, the first and second antigen-binding proteins may be linked by chemical conjugation (e.g., to form a bis-aryl conjugate between the domains). Examples of suitable methods for chemical conjugation of binding domains are known in the art. Such methods include the use of succinimidyl compound modification of primary amines present on lysine residues, such as those used in TriLink Technologies bioconjugation reagents. In one embodiment, when the first and / or second antigen-binding proteins are scFvs, the bispecific polypeptide comprises a linker sequence between the VH and VL domains of the scFv. Suitable linker sequences are known to those of skill in the art, and illustrative examples include relatively flexible, hydrophilic amino acid residues. In one embodiment, the linker sequence is selected from the group consisting of SEQ ID NO: 64 or 65 or an amino acid sequence having at least 70% identity thereto, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or more preferably at least 99% sequence identity thereto.

[0188] The antibody or antibody fragment may contain additional amino acids or molecules for purification or identification. For example, the antibody may contain an epitope or affinity tag. Illustrative examples of such epitope or affinity tags include peptide tags (e.g., FLAG-tag, HA-tag, His-tag, Myc-tag, S-tag, SBP-tag, Strep-tag, eXact-tag) and protein tags (e.g., GST-tag, MBP-tag, GFP-tag). In one embodiment, the epitope or affinity tag is a His-tag.

[0189] Nucleic acids and vectors In another aspect disclosed herein, there is provided a nucleic acid encoding the bispecific polypeptide described herein. In this embodiment, the nucleic acid may encode one or more of the amino acid sequences listed in Table 1. In yet another aspect, there is provided a cell comprising the vector described herein.

[0190] "Polynucleotide," "polynucleotide sequence," "nucleotide sequence," "nucleic acid," or "nucleic acid sequence" are used interchangeably herein to designate mRNA, RNA, cRNA, cDNA, or DNA. The term refers to a polymeric form of nucleotides, usually at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. The term includes single- and double-stranded forms of RNA and DNA.

[0191] As used herein, the term "gene" includes a nucleic acid molecule that can be used to produce mRNA, optionally with the addition of elements to aid in this process. A gene may or may not be usable to produce a functional protein. A gene can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and 5' and 3' untranslated regions).

[0192] The term "transgene" is used herein to describe genetic material that has been artificially introduced or is being introduced into the genome of a host organism and that is transmitted to the host's progeny. In some embodiments, it confers a desired characteristic on the T cells into which it is introduced, or otherwise leads to a desired therapeutic outcome.

[0193] As used herein, the terms "encode," "encoding," and the like refer to the ability of a nucleic acid to provide another nucleic acid or a polypeptide. For example, a nucleic acid sequence is said to "encode" a polypeptide if it can be transcribed and / or translated to produce the polypeptide, or if it can be transcribed and / or translated and processed into a form that can produce a polypeptide. Such a nucleic acid sequence can include coding sequences or both coding and non-coding sequences. Thus, the terms "encode," "encoding," and the like include an RNA product that results from the transcription of a DNA molecule, a protein that results from the translation of an RNA molecule, a protein that results from the transcription of a DNA molecule to form an RNA product and the subsequent translation of the RNA product, or a protein that results from the transcription of a DNA molecule to provide an RNA product, the processing of the RNA product to provide a processed RNA product (e.g., mRNA), and the subsequent translation of the processed RNA product.

[0194] In any embodiment, the nucleic acid sequence encoding the bispecific polypeptide of the invention may have one or more polynucleotide sequences that encode, or are capable of encoding, a polypeptide selected from the group consisting of SEQ ID NOs: 1, 2, 17-32, 33, 34, 49-63, 64-67, 71-74 and 76-79.

[0195] In either embodiment, the nucleic acid sequence encoding the bispsecific polypeptide of the invention may have one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs: 178-185.

[0196] In another aspect, a vector is provided comprising a nucleic acid described herein operably linked to a regulatory sequence.

[0197] The term "regulatory element" or "regulatory sequence" refers to a nucleic acid sequence (e.g., DNA) necessary for the expression of an operably linked coding sequence in a particular cell. Regulatory sequences that are suitable for eukaryotic cells include promoters, polyadenylation signals, transcriptional enhancers, translational enhancers, leader or trailing sequences that control mRNA stability, and targeting sequences that target the product encoded by the transcribed polynucleotide to an intracellular compartment within the cell or to the extracellular environment.

[0198] Typically, regulatory sequences include, but are not limited to, promoter sequences, 5' non-coding regions, cis-regulatory regions, such as functional binding sites for transcriptional or translational regulatory proteins, upstream open reading frames, ribosomal binding sequences, transcription initiation sites, translation initiation sites and / or nucleotide sequences encoding leader sequences, stop codons, translation termination sites, and 3' non-translated regions. Constitutive or inducible promoters known in the art are also contemplated. The promoter may be either a naturally occurring promoter or a hybrid promoter that combines elements of more than one promoter.

[0199] The contemplated promoter sequences may be native to mammalian cells or may be derived from alternative sources, where the region is functional in the selected organism. The choice of promoter will depend on the intended host cell. For example, promoters that may be used for expression in mammalian cells include, among others, the metallothionein promoter, which can be induced in response to heavy metals such as cadmium, the β-actin promoter, and viral promoters, such as the SV40 large T antigen promoter, the human cytomegalovirus (CMV) immediate early (IE) promoter, the Rous sarcoma virus LTR promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus promoter, and the HPV promoter, particularly the HPV upstream regulatory region (URR). All of these promoters have been well described in the art and are readily available.

[0200] Enhancer elements can also be used herein to increase the expression level of nucleic acid sequences in vector constructs. Examples include the SV40 early gene enhancer described in Dijkema et al. (1985, EMBO Journal, 4:761), the enhancer / promoter derived from the Rous sarcoma virus long terminal repeat (LTR) described in Gorman et al. (1982, Proceedings of the National Academy of Science, USA, 79:6777), and elements derived from human CMV, such as those contained in the CMV intron A sequence, described in Boshart et al. (1985, Cell, 41:521).

[0201] The vector construct may also contain 3' non-translated sequences. The term 3' non-translated sequence refers to a portion of a gene, including a DNA segment containing a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression. A polyadenylation signal is characterized by the addition of a polyadenylic acid tract to the 3' end of a pre-mRNA. Polyadenylation signals are generally recognized by the presence of homology to the canonical form 5'AATAAA-3', although variations are not uncommon. The 3' non-translated regulatory DNA sequence preferably contains approximately 50 to 1,000 nt and may contain transcription and translation termination sequences in addition to a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression.

[0202] The terms "operably connected" or "operably linked," as used herein, refer to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence "operably linked" to a coding sequence refers to the positioning and / or orientation of the regulatory sequence relative to the coding sequence that permits expression of the coding sequence under conditions compatible with the regulatory sequence.

[0203] As used herein, the terms "open reading frame" and "ORF" are used interchangeably herein and refer to the amino acid sequence encoded between the translation initiation and termination codons of a coding sequence. The terms "start codon" (e.g., ATG) and "stop codon" (e.g., TGA, TAA, TAG) refer to the unit of three adjacent nucleotides ("codons") in a coding sequence that specify the initiation and chain termination of protein synthesis (mRNA translation), respectively.

[0204] As used herein, the term "recombinant" as applied to "nucleic acid molecule," "polynucleotide," etc., is understood to mean an artificial nucleic acid structure (i.e., a non-replicating cDNA or RNA, or a replicon, a self-replicating cDNA or RNA) that can be transcribed and / or translated in a cell as described herein.

[0205] The recombinant nucleic acid molecule or polynucleotide can be inserted into a vector. Non-viral vectors, such as plasmid expression vectors or viral vectors, can be used. The types of vectors and techniques for inserting nucleic acid constructs according to the present invention are known in the art, and illustrative examples include cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0206] In any embodiment, the vector comprises a polynucleotide sequence encoding one or more polypeptide sequences selected from the group consisting of SEQ ID NOs: 1, 2, 17-32, 33, 34, 49-63, 64-67, 71-74, and 76-79.

[0207] In any embodiment, the vector comprises one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs: 178-185.

[0208] The nucleic acid sequences, polynucleotides or vector constructs described herein include heterologous sequences that do not occur in nature.

[0209] In yet another aspect, a host cell is provided comprising a vector described herein. Suitable host cells will be known to those of skill in the art, with illustrative examples including bacterial cells (e.g., E. coli, P. mirabilis), fungal cells (e.g., S. cerevisiae, P. pastoria, T. reesei), plant cells, insect cells (e.g., SF-9, SF21, Hi-5), or mammalian cells. In one embodiment, the host cell is a mammalian cell. Suitable mammalian cells will be known to those of skill in the art, with illustrative examples including CHO or 293T cells. These cells are widely available from commercial suppliers.

[0210] Methods for producing bispecific polypeptides In another aspect, there is provided a method for producing a bispecific polypeptide described herein, the method comprising the steps of (i) culturing a cell described herein in a culture medium under conditions suitable for expression of the bispecific polypeptide, and (ii) isolating the bispecific polypeptide from the cell or from the culture medium.

[0211] The bispecific polypeptides described herein can be produced using any number of expression systems that would be known to one of skill in the art, illustrative examples of which include production in or by bacteria (e.g., E. coli, P. mirabilis), fungi (e.g., S. cerevisiae, P. pastoria, T. reesei), plants or plant cells, insects or insect cells (e.g., SF-9, SF21, Hi-5), or mammalian cells. In one embodiment, the expression system is a mammalian expression system. Suitable mammalian expression systems will be known to one of skill in the art, illustrative examples of which include CHO or 293 expression systems. These expression systems are widely available from proprietary suppliers. In one embodiment, the bispecific polypeptides are produced using a mammalian expression system.

[0212] In another aspect, there is provided a method for producing a bispecific polypeptide as described herein, the method comprising combining a first antigen binding protein and a second antigen binding protein under conditions suitable for the formation of a chemically conjugated bispecific polypeptide comprising the first antigen binding protein and the second binding protein.

[0213] In a preferred embodiment, the chemically conjugated bispecific polypeptides are formed after succinimidyl compound modification of the primary amines present on the lysine residues as described elsewhere herein.

[0214] Pharmaceutical Composition In another aspect, there is provided a pharmaceutical composition comprising a bispecific polypeptide described herein and a pharmaceutically acceptable carrier.

[0215] The compositions described herein may be prepared by methods known in the art and are suitable for parenteral administration to mammals, particularly humans, comprising a therapeutically effective amount of the composition together with one or more pharmaceutically acceptable carriers or diluents.

[0216] The term "pharmaceutically acceptable carrier," as used herein, refers to any suitable carrier, diluent, or excipient. These include all aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, and solutes which render the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic and absorption agents, etc. It will be understood that the compositions of the present invention may also include other supplementary physiologically active substances.

[0217] Carriers are generally pharmaceutically "acceptable" in the sense that they are compatible with other ingredients in the composition and are not harmful to the subject. Compositions include those suitable for parenteral administration, including subcutaneous, intramuscular, intravenous, and intradermal administration. The compositions may conveniently be presented in unit dosage form and can be prepared by any method known in the art of pharmacy. Such methods include preparing a carrier for association with isolated T cells. Generally, the compositions are prepared by uniformly and intimately associating any active ingredient with a liquid carrier.

[0218] In one embodiment, the composition is suitable for parenteral administration, hi another embodiment, the composition is suitable for intravenous administration.

[0219] Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bactericides and solutes which render the composition isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0220] The present disclosure also contemplates the compositions described herein in combination with other active agents and / or in addition to other treatment regimens or modalities, such as radiation therapy or surgery. When the compositions described herein are used in combination with known active agents, the combination can be administered either sequentially (either continuously or separated by periods of no treatment) or simultaneously or as a mixture. Suitable anti-cancer agents will be known to those skilled in the art. Combined treatment is also contemplated to include treatment with either a composition of the present invention followed by a known treatment, or treatment with a known agent followed by treatment with a composition of the present invention, for example, as maintenance therapy. For example, in the treatment of cancer, the compositions of the present invention may be used in combination with alkylating agents (e.g., mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide, cisplatin, or platinum-containing alkylating agents, e.g., cisplatin, carboplatin, and oxaliplatin) and antimetabolites (e.g., purine or pyrimidine analogs or antifolates, e.g., azathioprine and mercaptopurine), anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, or anthracycline analogs), plant alkaloids, (e.g., vinca alkaloids or taxanes, e.g., vincristine, vinblastine, vinorelbine, vindesine, paclitaxel, or doestaxel), topoisomerase inhibitors (e.g., type I or type II topoisomerase inhibitors), podophyllotoxins (e.g., etoposide or teniposide), tyrosine kinase inhibitors (e.g., imatinib mesylate, nilotinib, or dasatinib), adenosine receptor inhibitors (e.g., A2aR inhibitors, SCH58261, CPI-444, SYN115, ZM241385, FSPTP, or A2 BIt is contemplated that the agent may be administered in combination with an agonist, such as an ER inhibitor (e.g., PSB-1115), an adenosine receptor agonist (e.g., CCPA, IB-MECA, and CI-IB-MECA), one of the PDL-1:PD-1 axis, checkpoint inhibitors including nivolumab, pembrolizumab, atezolizumab, BMS-936559, MEDI4736, MPDL33280A, or MSB0010718C), an inhibitor of the CTLA-4 pathway (e.g., ipilimumab and tremelimumab), an inhibitor of the TIM-3 pathway, or an agonist monoclonal antibody known to promote T cell function (including anti-OX40, e.g., MEDI6469, and anti-4-BB, e.g., PF-05082566).

[0221] In a further aspect, the present invention provides a kit or article of manufacture comprising one or more bispecific polypeptides of the invention, a nucleic acid encoding said bispecific polypeptides and / or a pharmaceutical composition as described above.

[0222] In a further aspect, there is provided a kit for use in the above therapeutic applications, comprising: (a) a container holding a bispecific polypeptide, nucleic acid, vector or pharmaceutical composition of the invention; and (b) the label or package insert containing instructions for use; A kit is provided comprising:

[0223] The kit may also include one or more active ingredients or components for the treatment of cancer. For example, the kit may also include immune cells expressing a CAR.

[0224] A "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 to treat the condition of choice. In one embodiment, the label or package insert includes instructions for use indicating that the therapeutic or prophylactic composition can be used to treat cancer or other conditions described herein.

[0225] The kit may include (a) a therapeutic or prophylactic composition and (b) a second container containing a second active ingredient or components. The kit, according to this embodiment of the invention, may further include a package insert indicating that the composition and other active ingredients can be used to treat a cancer or condition described herein. 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. Other materials desirable from a commercial and user standpoint, as would be known to those skilled in the art, may further be included; suitable examples of which include other buffers, diluents, filters, needles, and syringes.

[0226] Treatment method In another aspect, provided is a method for the treatment of cancer, comprising co-administering to a subject in need thereof a therapeutically effective amount of (i) immune cells expressing an engineered TCR (e.g., a CAR) and (ii) a bispecific polypeptide or pharmaceutical composition described herein, wherein the bispecific polypeptide simultaneously binds to an antigen expressed on the subject's endogenous APCs in vivo and an antigen on the engineered TCR (e.g., CAR) expressed by the immune cells, thereby stimulating the activation and expansion of the immune cells for the treatment of cancer.

[0227] In any of the methods of treatment or use described herein or other aspects of the invention, wherein a bispecific polypeptide is administered to a subject, the subject may have received or have received immune cells expressing an engineered TCR (e.g., a CAR).

[0228] In any of the methods of treatment or other aspects of the invention in which a bispecific polypeptide is administered to a subject, the method or other aspects further comprise administering immune cells that express an engineered TCR (e.g., a CAR).

[0229] Co-administration of immune cells expressing an engineered TCR (e.g., a CAR) and a bispecific polypeptide or pharmaceutical composition described herein can be achieved by formulating the immune cells and the bispecific polypeptide or pharmaceutical composition in the same composition (e.g., for simultaneous co-administration), or can be formulated as different compositions for sequential administration. "Sequential" administration means that there is an interval between the administration of the immune cells and the bispecific polypeptide or pharmaceutical composition. The interval between sequential administrations can be a few seconds, minutes, hours, or days. In one embodiment, periodic re-administration of the immune cells and the bispecific polypeptide or pharmaceutical composition may be necessary to achieve the desired therapeutic effect. The sequential administration can be in any order.

[0230] The inventors have surprisingly shown that the bispecific polypeptides described herein stimulate the activation and expansion of immune cells in vivo, thereby improving the efficacy of immune cell therapy, e.g., CAR T cell therapy. Accordingly, bispecific polypeptides may be referred to as "adjuvants." The term "adjuvant," as used herein, refers to a bispecific polypeptide that can increase the magnitude of the immune response elicited by immune cells expressing an engineered TCR (e.g., a CAR) beyond that which would be expected from immune cells expressing the engineered TCR alone.

[0231] Immune cell activation can be achieved, for example, by providing a primary stimulatory signal via the T cell TCR / CD3 complex or by stimulation of the CD2 surface protein, and by providing a second, costimulatory signal via an accessory molecule, e.g., CD28 or 4-1BBL. In addition to the primary stimulatory signal provided via the TCR / CD3 complex or by CD2, a second, costimulatory signal is required for the induction of a T cell response. In certain embodiments, a CD28-binding agent can be used to provide the costimulatory signal. Suitable costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, ILT3, ILT4, agonists or antibodies that bind to Toll-like receptors, and ligands that specifically bind B7-H3.

[0232] The terms "expanded," "expansion," "expanding," "expansion," and the like are used interchangeably herein to refer to an increase in immune cell numbers either before or after administration of the cells to a subject or preparation of a pharmaceutical composition. Immune cells may be expanded using any cell culture method known in the art. For example, immune cells may be expanded in in vitro tissue culture systems, including liquid culture monolayers, and the like.

[0233] Therapeutic regimens for cancer treatment can be determined by those skilled in the art and typically vary depending on factors including, but not limited to, the subject's age, weight, and general health, as well as the type, size, stage, and receptor status of the tumor. Another determining factor may be the risk of developing recurrent disease. For example, subjects identified as being at high risk, at higher risk, or having developed recurrent disease may be prescribed a more aggressive treatment regimen compared to subjects considered to have a low or lower risk of developing recurrent disease. Similarly, subjects identified as having a more advanced stage of cancer, such as stage III or IV disease, may be prescribed a more aggressive treatment regimen compared to subjects with a less advanced stage of cancer.

[0234] The term "cancer," as used herein, refers to any condition associated with abnormal cell proliferation. Such conditions would be known to one of skill in the art. In one embodiment, the cancer is a solid cancer. In another embodiment, the cancer is a mesothelin-positive cancer. In another embodiment, the cancer does not express CD40 or CD206, or CD40 or CD206 is downregulated relative to non-cancerous tissue of the same type. In another embodiment, the cancer may express CD40. In another embodiment, the cancer is selected from the group consisting of brain cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, esophageal cancer, skin cancer, prostate cancer, pancreatic cancer, uterine cancer, gastric cancer, thymic carcinoma, and endometrial cancer.

[0235] The terms "treat," "treatment," and "treating," as used herein, refer to any and all uses of treating a condition or symptom, or otherwise preventing, hindering, delaying, inhibiting, or ameliorating in any way the onset or progression of cancer or other undesirable symptoms. Thus, terms such as "treating" should be considered in the broadest possible context. For example, treatment does not necessarily imply that a subject is treated until complete recovery or cure. In conditions exhibiting or characterized by multiple symptoms, treatment does not necessarily treat, prevent, hinder, delay, inhibit, or ameliorate all of the symptoms, but may treat, prevent, hinder, delay, inhibit, or ameliorate one or more of the symptoms.

[0236] The subject whose cancer is to be treated may be a human or a mammal that is economically and / or socially important to humans, such as non-human carnivores (e.g., cats and dogs), Sus genus animals (e.g., pigs, boars and wild boars), ruminants (e.g., cattle, cows, sheep, giraffes, deer, goats, bison and camels), horses, and birds and poultry, including endangered and zoo-kept bird species, more particularly, domesticated poultry, such as turkeys, chickens, ducks, geese, guinea fowl, etc., because they are economically important to humans. The term "subject" does not denote a specific age. Thus, adult, juvenile and newborn subjects are intended to be subjects.

[0237] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to any subject to which the present disclosure may be applicable. In one embodiment, the subject is a mammal. In another embodiment, the subject is a human.

[0238] The term "therapeutically effective amount," as used herein, means that the amount of cells, when administered to a mammal, particularly a human, in need of such treatment, is sufficient to treat cancer. The exact amount of modified cells to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the condition of the subject.

[0239] Typically, the administration of immune cell (e.g., CAR-T cell) therapy is defined by the number of cells per kilogram of body weight. However, since the modified cells replicate and expand after transfer, the administered cell dose will not resemble the final steady-state number of cells. In one embodiment, a pharmaceutical composition comprising the modified cells of the present invention is administered in a dose of 10 4 ~10 9 In another embodiment, the pharmaceutical composition comprising the modified cells of the present invention can be administered at a dose of 10 cells / kg body weight. 5 ~10 6 It may be administered at a dose of 10 cells / kg body weight, including all integer values ​​within those ranges.

[0240] Compositions comprising the bispecific polypeptides described herein can also be administered multiple times at these dosages. The optional dosage and treatment regimen for a particular subject can be readily determined by one of skill in the art by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0241] In one embodiment, the immune cells are derived from autologous cells. In another embodiment, the immune cells are derived from allogeneic cells.

[0242] The term "autologous" refers to any material originating from the same individual that is to be subsequently reintroduced into the individual.

[0243] The term "allogeneic" refers to material derived from a different individual of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently genetically distinct to interact antigenically.

[0244] cell manufacturing To achieve a sufficient therapeutic dose of an immune cell composition, methods for producing immune cells have typically involved one or more rounds of stimulation, activation, and / or expansion. According to the methods disclosed herein, immune cells can be stimulated, activated, and expanded both in vivo and in vitro.

[0245] Accordingly, in another aspect disclosed herein, there is provided a method for stimulating the activation and / or expansion of immune cells in vivo, comprising administering to a subject (i) immune cells expressing an engineered TCR (e.g., a CAR), and (ii) an effective amount of a bispecific polypeptide or pharmaceutical composition described herein, wherein the bispecific polypeptide simultaneously binds to an antigen expressed on the subject's endogenous APCs in vivo and to an antigen on the engineered TCR (e.g., CAR) expressed by the immune cells.

[0246] The in vivo activation and expansion of immune cells described herein facilitates reducing the number of immune cells required for each therapeutic dose of immune cells.

[0247] In another aspect, a method is provided for stimulating immune cell activation and expansion in vitro, comprising culturing isolated immune cells expressing an engineered TCR (e.g., a CAR) in a culture medium comprising (i) an APC or an APC mimetic, and (ii) a bispecific polypeptide described herein, wherein the bispecific polypeptide simultaneously binds to an antigen expressed on the APC or APC mimetic and to an antigen on the engineered TCR (e.g., CAR) expressed by the immune cell. In one embodiment, the bispecific polypeptide is capable of binding to an antigen expressed on a tumor cell.

[0248] The immune cell production methods contemplated herein include simple and robust culture initiation and activation steps that contribute to the resulting immune cell composition being a superior therapeutic product. In one embodiment, culture initiation and activation includes seeding a cell population into a cell culture vessel, such as a cell culture bag, a GREX bioreactor, a WAVE bioreactor, etc., and activating the immune cells via primary and costimulatory immune cell signaling pathways. The cellular composition may be further cultured in the presence of one or more additional growth factors or cytokines, such as IL-2, IL-7, and / or IL-15, or any suitable combination thereof.

[0249] In another embodiment, activating and expanding immune cells comprises culturing the isolated immune cells with APCs or APC mimetics. In one embodiment, the APCs or APC mimetics are selected from the group consisting of donor-derived APCs, synthetic artificial APCs (aAPCs), microbeads functionalized with CD3 and CD8 activating antibodies (Dynabeads), autologous monocyte-derived dendritic cells (moDCs), and scaffolds that mimic APCs.

[0250] In certain embodiments, initiation of culture involves inoculating a population of cells, including T cells, e.g., PBMCs, into a cell culture vessel at a desired density, e.g., 1-5 x 10, in a suitable cell culture medium containing one or more cytokines, a primary stimulatory ligand, and a costimulatory ligand. 6In another embodiment, cytokines, stimulatory and co-stimulatory ligands may be subsequently added to the PBMCs in cell culture medium.

[0251] In one embodiment, the cell culture vessel is a cell culture bag, including but not limited to MACS® GMP Cell Expansion Bags, MACS® GMP Cell Differentiation Bags, EXP-Pak™ Cell Expansion Bio-Containers, VueLife™ Bags, KryoSure™ Bags, KryoVue™ Bags, Lifecell® Bags, PermaLife™ Bags, X-Fold™ Bags, Si-Culture™ Bags, and VectraCell™ Bags, as contemplated elsewhere herein.

[0252] In certain embodiments, cells are seeded into cell culture vessels containing a suitable cell culture medium. Illustrative examples of suitable cell culture media include, but are not limited to, X-VIVO™ 15 supplemented with TCGM; 2 mM GlutaMAX™-I, 10 mM HEPES, and 5% human AB serum, CTS™ OpTmizer™ T Cell Expansion SFM (Life Technologies), CTS™ AIM V® Medium (Life Technologies), RPMI 1640, Clicks, DMEM, MEM, α-MEM, F-12, X-Vivo 15 (Lonza), CellGro® Serum-Free Medium (CellGenix), and X-Vivo 20 (Lonza) supplemented with amino acids, sodium pyruvate, and vitamins, either serum-free or with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in amounts sufficient for the growth and expansion of immune cells.

[0253] Cell culture media contemplated herein may further comprise one or more factors including, but not limited to, serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGF-β, and TNF-α.

[0254] In one embodiment, the APC or APC mimetic is selected from the group consisting of donor-derived APCs, synthetic artificial APCs (aAPCs), microbeads functionalized with activating antibodies to CD3 and CD8 (Dynabeads), autologous monocyte-derived dendritic cells (moDCs), and scaffolds that mimic APCs.

[0255] Those skilled in the art will recognize that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications that fall within its spirit and scope. The invention also includes all steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0256] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0257] The various embodiments enabled herein are further illustrated by the following non-limiting examples.

[0258] 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. [Example]

[0259] Example 1 Anti-CD40 antibody production A human anti-CD40 antibody was obtained. 500 mL HEK293 cultures were transfected with a vector encoding the anti-CD40 antibody. Cultures were harvested by centrifugation on day 6. SDS-PAGE gels were completed on day 3. Cysteine ​​to serine mutations (C232 and C127) were performed to confirm expression / aggregation. Strong expression was observed throughout using both the cysteine ​​mutants and the unmutated IgG2 format.

[0260] The clarified supernatant of IgG2 protein was purified by Protein A affinity chromatography & buffer exchange (PBS). The material was dialyzed into PBS buffer pH 7.0. The final concentration was determined by A280, and the yield, purity and endotoxin level are detailed in the table below:

[0261] [Table 2]

[0262] All IgG2 formats were determined to have high expression, low aggregation and high purity, and were suitable for BEAT design.

[0263] Example 2 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.

[0264] 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).

[0265] 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:

[0266] [Table 3]

[0267] 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.

[0268] 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.

[0269] 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.

[0270] The table below summarizes the original parental and humanized sequences:

[0271] [Table 4]

[0272] The combination of four different heavy and light chain framework regions resulted in the generation of 16 different VH / VL combinations.

[0273] The different VH / VL combinations of the 16 antibodies are summarized in the table below:

[0274] [Table 5]

[0275] All humanized IgG1 formats were well expressed and were successfully purified for characterization by SDS-PAGE and SEC-HPLC to monitor protein quality. The titer, quantity and monomer content of the final purified antibodies are listed in the table below:

[0276] [Table 6]

[0277] Example 3 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.

[0278] 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.

[0279] 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).

[0280] The table below summarizes the antibody affinities and stoichiometries as measured by Biacore:

[0281] [Table 7]

[0282] 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.

[0283] 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.

[0284] Based on these results, antibody variants v4 and v7 scFv were selected for further study.

[0285] Example 4 Anti-CD40-anti-FLAG BEAT (BEAT1) design and affinity Twelve anti-CD40-anti-FLAG BEATs (collectively referred to as BEAT1) were designed and evaluated. The orientation of the IgG and scFv domains is outlined in Figure 4. The 12 variations of BEAT1 that were generated are summarized in the table below:

[0286] [Table 8]

[0287] The linkers used between VL-VH and between scFv and IgG2 are (G4S) * 3. The linker used between VH and VL is 21AA (GGGGSGGGGSGGGGSGGGGAS).

[0288] CD40 binding to 11 anti-CD40 BEAT antibodies, an anti-CD40 IgG control, and hCD40-cMyc cBEAT was characterized using multi-cycle SPR experiments. At least two reliable data sets were obtained for the binding of each anti-CD40 antibody to CD40. CD40 binding to all anti-CD40 BEAT antibodies resulted in good fits, with high affinity and K values ​​ranging from 0.4 to 2 nM. D The binding of CD40 to hCD40-cMyc rBEAT had a K of 0.05–0.06 nM. D whereas the control antibody anti-CD40 IgG2 showed a K of 0.5 nM. D The results are shown in Table 3.

[0289] Binding of FLAG-BAP to anti-CD40 BEAT antibodies and three anti-FLAG IgG control antibodies was characterized using multi-cycle SPR experiments. At least two reliable data sets were obtained for each BEAT binding to FLAG. Binding of FLAG-BAP resulted in good fits, with high affinity and K values ​​ranging from 1 to 70 nM. D The binding of FLAG-BAP to the control anti-FLAG antibody had a K ranging from 0.4 to 35 nM. D The results are shown in Table 4.

[0290] [Table 9]

[0291] [Table 10]

[0292] Next, we performed BEAT 1a4 (F7-G8) simultaneous CD40 and FLAG-BAP binding experiments. In these experiments, BEAT 1a4 was captured on a protein G surface, followed by injection of 50 nM FLAG-BAP until saturation was achieved. CD40 was then immediately injected in the presence of 50 nM FLAG-BAP, and CD40 dissociation was monitored while injecting 50 nM FLAG-BAP alone. This was performed for different concentrations of CD40, allowing us to measure the association and dissociation of CD40 with BEAT 1a4, which was always saturated with FLAG-BAP. Similarly, we performed a reverse experiment in which, after capture of BEAT 1a4, 50 nM CD40 was injected until saturation, followed by injection of FLAG-BAP in the presence of 50 nM CD40, and then FLAG-BAP dissociation was monitored while injecting 50 nM CD40 alone. These experiments showed that BEAT 1a4 could bind to CD40 in a saturable manner with FLAG-BAP (Figure 5). Under these conditions, CD40 bound with similar kinetic parameters, affinity, and Rmax regardless of the presence or absence of bound FLAG-BAP. Similarly, FLAG-BAP bound to BEAT1a4 in a saturable manner with bound CD40. FLAG-BAP binding to BEAT 1a4 also showed similar kinetic parameters, affinity, and Rmax regardless of the presence or absence of bound CD40.

[0293] Example 5 BEAT1 binding to APC BEAT1 was evaluated at 10, 30, and 100 nM for binding to APCs. The IgG2 isotype control had no binding, and the anti-CD40 antibody control had the highest binding (100 nM only) (Figure 6A). BEAT1a7, 1a4, and 1b4 had the highest binding.

[0294] BEAT1a4 showed dose-dependent binding to CD40 on human MoDC cells (Figure 7A) and human B cells (Figure 7B) between 0.1 and 100 nM, and to CD40 on non-human primate MoDC cells (Figure 7C) and non-human primate B cells (Figure 7D) between 0.1 and 100 nM.

[0295] Binding of BEAT 1a4 to human cells / DCs: Cell binding using two test articles (antibody biologics), including one test article of interest (bispecific antibody 1 (BEAT 1a4)) and one human IgG2 isotype control antibody. PBMCs and moDCs obtained from three healthy human donors were evaluated. Test article binding to CD3+, CD20+, CD14+, and CD11c+ cell subsets was assessed. Dose responses of each test article of interest and isotype control antibody were examined in duplicate for binding on all proposed cell types, using a top concentration of 200 nM, followed by a two-fold dilution series (10 points total) and untreated samples, followed by secondary antibody (5 μg / mL) (Figures 7A and 7B).

[0296] Binding of BEAT 1a4 to NHP cells / DCs: Cell binding using two test articles (antibody biologics), including one test article of interest (bispecific antibody 1 (BEAT 1a4)) and one human IgG2 isotype control antibody. PBMCs and moDCs obtained from three NHP donors were evaluated. Test article binding to CD3+, CD20+, CD14+, and CD11c+ cell subsets was assessed. Dose responses of each test article of interest and isotype control antibody were examined in duplicate for binding on all proposed cell types, using a top concentration of 200 nM, followed by a two-fold dilution series (10 points total) and untreated samples, followed by secondary antibody (5 μg / mL) (Figures 7C and 7D).

[0297] Example 6 BEAT1 binding to CAR T cells BEAT1a4 was evaluated for binding to FLAG-tagged, GFP-expressing CAR T-cells. Cells were detected by FAC as GFP+ / AF647+ cells. BEAT1a4 binding was assessed over a dose range of 0.1 nM to 100 nM (Figure 9C).

[0298] Binding of the test article BEAT1a4 to FLAG-tagged CAR-T target cells was assessed in a 10-point standard curve to confirm the EC50. After viability dye staining and incubation with 5% FBS to block nonspecific binding, CAR-T cells were incubated with a dose response of either BEAT1a4 or a human IgG2 isotype control. The following concentrations were tested in duplicate: 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 33, and 100 nM, in addition to an untreated control. After incubation, cells were washed and stained with 5 μg / mL of a fluorescently labeled second reagent (AF647-conjugated AffiniPure Fab2 fragment goat anti-human IgG Fcg-specific), then washed and acquired by high-throughput flow cytometry. The geometric mean fluorescence intensity (MFI) values ​​of AF647 in live cells (viability dye negative) were plotted on an XY chart, a graph of MFI against the logarithm of concentration was generated, the data was fitted to a nonlinear regression curve, and the EC50 was calculated from it. The percentage of live-gated AF647 cells was also analyzed.

[0299] BEAT 1a4 showed dose-dependent binding to CAR T cells with an EC50 of 0.51 nM.

[0300] Example 7 Functional activity of BEAT1 in APCs and CAR T cells BEATs were added to monocyte-derived DCs (MoDCs) to evaluate CD86 upregulation for lead BEAT candidates. CD40 agonism is predicted to upregulate CD86 expression. When anti-CD40 BEATs BEAT1a4 and BEAT1a5 were added, CD86 was upregulated in a dose-dependent manner at 10 nM BEAT1a4 (Figure 8A). A control antibody had no effect on CD86 expansion.

[0301] BEAT1a4 (and the anti-CD40 agonist control) increased IL-12 / IL-23p40 release in a dose-responsive manner between 3 and 30 nM (CD86) and 1 and 60 nM (IL-12 / IL-23p40) (Figure 8B).

[0302] CAR T cells and MoDCs that contained a FLAG tag were evaluated for functional activity upon addition of BEAT1a4. MoDCs were confirmed to have CD40 expression (Figure 9B). An increase in IFN-γ was observed upon addition of anti-CD40 BEAT (Figure 9A). Negative controls (IgG1 and IgG2 isotypes) had no effect on IFN-γ.

[0303] Example 8 BEAT1 cytokine analysis Cytokine analysis was completed using one donor to test BEAT1a4. Anti-CD40 BEAT1 stimulated IFNγ, TNF-α, and IL2, and had some activity with low doses of 1a4 and 1a5 and with IL12-p70 (Figure 10). BEAT1a4 had limited activity above background with IL-6, IL-4, IL-10, IL-8, IP-10, MCP-1, and IL-17A.

[0304] Example 9 T cell proliferation with the addition of BEAT1 After 120 hours of growth, proliferation of 7B1 28z CAR T cells was examined in the presence of control compound / BEAT1 (30 nM) and MoDCs.

[0305] For anti-CD40 BEAT 1a4 and 1a5, a significant increase in the fold change over the control was observed for CD3+, CD4+ and CD8+ cells (Figure 11), and in particular, there was an increase in the percentage of effector memory T cells, especially CD8+ T cells, with the addition of anti-CD40 BEAT1a4 (Figure 12).

[0306] Example 10 Cytotoxicity of BEAT1+CAR T Cytotoxicity was measured by luciferase assay using the ovarian cancer cell line SKOV-3 (parental, MSLN-negative and MSLN-expressing) + MoDC + CAR T.

[0307] BEAT had no effect in MSLN-expressing SKOV-3 cells (FIG. 13).

[0308] Example 11 In vivo efficacy of CAR T+BEAT BEAT1a5 was evaluated in the NSG mouse model using SKOV3-mesothelin-transfected CD40 knockout cells. Tumors were first established in NSG mice (50 mm 2 ) A single dose of 5 million anti-mesothelin CAR / 5 million MoDCs (with high expression of mesothelin) and biweekly doses of 12.5 μg BEAT (5 doses) ( FIG. 14A ).

[0309] Survival and tumor size were measured (Figures 14B and C), with 2 / 4 mice tumor-free at 21 days with CAR T cells + MoDC + BEAT compared to 1 / 3 with CAR T cells alone.

[0310] Example 12 Anti-CD206 antibody production Anti-CD206 antibody was obtained. 500 mL HEK293 cultures were transfected with anti-CD206 (B11) encoding vector. Cultures were harvested by centrifugation on day 6. SDS-PAGE gels were completed on day 3. The clarified supernatant of IgG1 protein was purified by Protein A affinity chromatography ProtA (Agarose Resin) & buffer exchange (PBS). The material was dialyzed into PBS buffer. 15 μg samples were formulated in 4x loading buffer (reducing and non-reducing) and analyzed by SDS-PAGE. Based on size, >95% purity of B11 was obtained under reducing conditions.

[0311] Example 13 Anti-CD206-anti-FLAG BEAT2 design and characteristics Anti-FLAG was designed as described above in Examples 2 and 3. Twelve anti-CD206-anti-FLAG BEATs (BEAT2) were designed and evaluated using IgG and scFv domains, as outlined in Figure 4. The 12 variants of BEAT2 that were generated are summarized in the table below:

[0312] [Table 11]

[0313] The linkers used between VL-VH and between scFv and IgG2 are (G4S) * 3. The linker used between VH and VL is 21AA (GGGGSGGGGSGGGGSGGGGAS).

[0314] The binding of CD206 to 12 anti-CD206 BEAT antibodies and an anti-CD206 IgG control was characterized using multi-cycle SPR experiments. At least two reliable data sets were obtained for the binding of each anti-CD206 antibody to CD206. The binding of CD206 to all anti-CD206 BEAT antibodies resulted in good fits, with high affinity and K values ​​in the range of 10–80 nM. D Binding of CD206 to the control antibody, anti-CD206 IgG1, had a K of 11 nM. D The binding rate constants of all anti-CD206 BEAT antibodies to CD206 were similar (6 × 10 3 -3×10 4 M -1 s -1 ), and the dissociation rate constants were similar (2–5 × 10 -4 s -1 ) (Table 5).

[0315] The binding of FLAG-BAP to 12 BEAT antibodies and three anti-FLAG IgG control antibodies was characterized using multi-cycle SPR experiments. At least two reliable data sets were obtained for the binding of each BEAT antibody to FLAG, except for BEAT 2a1. The binding of FLAG-BAP to BEAT antibodies yielded good fits, with high affinity and KDs ranging from 1 to 70 nM. The binding of FLAG-BAP to the control anti-FLAG antibody showed KDs ranging from 0.4 to 35 nM. The binding rate constants of all BEAT antibodies to FLAG-BAP were 1 x 10 5 ~5×10 6 M -1 s -1 and the dissociation rate constant is in the range of 1 × 10 3 ~4×10 -2 s -1 The range was (Table 6).

[0316] [Table 12]

[0317] [Table 13]

[0318] Example 14 BEAT2 binding to APC BEATs were assessed for binding to APCs at 10, 30, and 100 nM. The IgG2 isotype control had no binding, and the B11 anti-CD206 Mab control had the highest binding (100 nM only) (Figure 6B). BEATs 2a6, 2a8, and 2b3 had the highest binding.

[0319] Example 15 Functional activity of BEAT2 in APCs and CAR T cells CART and moDCs were evaluated using BEATs. MoDCs were confirmed to have CD206 expression (Figure 9B). Binding via the FLAG tag stimulated an IFN-γ response. When anti-CD206 BEATs were added, an increase in IFN-γ was observed (Figure 9A). Negative controls (IgG1 and IgG2 isotypes) had no effect on IFN-γ. The lead anti-CD206 BEAT included 2b3.

[0320] Example 16 BEAT2 cytokine analysis Cytokine analysis was completed using one donor to test the lead BEAT2. Anti-CD206 BEAT2 stimulated IFN-γ, TNF-α, and IL-6 above cell-alone background (FIG. 10). There was limited activity with IL-2, IL-4, IL-10, IL-8, IP-10, MCP-1, IL-12p70, and IL-17A.

[0321] Example 17 T cell proliferation using 7B1 28z CAR+BEAT2+MoDC The proliferation of 7B1 28z CAR T cells in the presence of control compound / BEAT2 (30 nM) and MoDCs after 120 hours of growth was examined.

[0322] For anti-CD206 BEAT 2B3 and 2B4, an increased fold change was observed for CD3+, CD4+, and CD8+ cells relative to the control (Figure 11), and in particular, there was an increase in the percentage of central memory T cells when BEAT 2B4 was added to CD8+ T cells (Figure 12).

[0323] Example 18 In vivo efficacy of CAR T + anti-CD206 BEAT BEAT2a6 was evaluated in the NSG mouse model using SKOV3-mesothelin-transfected CD40 knockout cells. Tumors were first established in NSG mice (50 mm 2) A single dose of 5 million anti-mesothelin CAR / 5 million MoDCs (with high expression of mesothelin) and biweekly doses of 12.5 μg BEAT (5 doses) ( FIG. 14A ).

[0324] Tumor size was measured for CART cells + MoDC + BEAT 2a6 compared to CAR T cells alone or no treatment controls (Figure 24).

[0325] Example 19 One arm BEAT expression and purification Two different knob-into-hole mutations were used to design one-arm anti-CD40 and anti-FLAG BEATs, forming bispecific BEAT3&4 and BEAT5&6 (Figure 15).

[0326] BEAT3: One arm using anti-CD40 IgG2 (Fab+Fc) with KIH mutation set number 1 One arm using anti-FLAG scFv+Fc (hM2V4 scFv in VL-(GGGGs)3-VH) with KIH mutations using KIH mutation set number 1 Heavy Chain 1 ·HC-IgG2 (anti-CD40) KIH1a Light chain 1 ·LC-IgG2 (anti-CD40) Heavy Chain 2 HC-hM2V4_Currus linker-(GGGGS)3-IgG2 Fc-KIH1b

[0327] BEAT4: One arm using anti-CD40 IgG2 (Fab+Fc) with KIH mutation set number 2 One arm using anti-FLAG scFv+Fc (hM2V4 scFv in VL-(GGGGs)3-VH) with KIH mutations using KIH mutation set number 2 Heavy Chain 1 ·HC-IgG2 (anti-CD40) KIH2a Light chain 1 ·LC-IgG2 (anti-CD40) Heavy Chain 2 ·HC-hM2V4_Currus linker-(GGGGS)3-IgG2 Fc-KIH2b

[0328] BEAT5: One arm of a bivalent antibody using an anti-CD40 IgG2 coupled (at the C-terminus of the heavy chain) to an anti-FLAG scFv using KIH mutation set number 1 One arm using anti-CD40 IgG2 with KIH mutation set number 1 Heavy Chain 1 ·HC-IgG2 (anti-CD40) KIH1a Light chain 1 ·LC-IgG2 (anti-CD40) Heavy Chain 2 HC-IgG2(anti-CD40)-(GGGGS)3-hM2V4_Currus linker-KIH1b Light chain 2 ·LC-IgG2 (anti-CD40)

[0329] BEAT6: One arm of a bivalent antibody using anti-CD40 IgG2 coupled (at the C-terminus of the heavy chain) to an anti-FLAG scFv using KIH mutation set number 2 One arm using anti-CD40 IgG2 with KIH mutation set number 2 Heavy Chain 1 ·HC IgG2 (anti-CD40) KIH2a Light chain 1 ·LC-IgG2 (anti-CD40) Heavy Chain 2 HC-IgG2(anti-CD40)-(GGGGS)3-hM2V4_Currus linker-KIH2b Light chain 2 ·LC-IgG2 (anti-CD40)

[0330] cDNAs of the variable heavy (VH) and variable light (VL) sequences corresponding to all four BEAT variants were synthesized and subcloned into the mammalian cell expression vector pTXs1.

[0331] A 500 mL XtenCHO culture was transfected with a vector encoding one arm of BEAT (BEAT3, 4, 5, 6). 14 days after transfection, the culture was harvested by centrifugation. The clarified supernatant of protein was purified by Protein A affinity chromatography ProtA (Agarose Resin) and buffer exchange (PBS). The material was dialyzed into PBS buffer. 15 μg samples were formulated in 4x loading buffer (reducing and non-reducing) and analyzed by SDS-PAGE and SEC-HPLC.

[0332] As shown in the table below, greater than 10 mg of antibody was expressed for all BEAT formats with greater than 90% purity:

[0333] [Table 14]

[0334] Example 15 Affinity of one arm of BEAT for CD40 and FLAG-tag The binding of the single-arm BEATs to CD40 was evaluated by ELISA. All BEATs had similar binding to CD40 ( FIG. 16 ). The affinity of the single-arm BEATs to CD40 or FLAG was confirmed by SPR using a 1:1 model. The affinity of the single-arm BEATs was comparable to that of the anti-CD40 control Mab and BEAT1a4 using a 1:1 model, as shown in the table below:

[0335] [Table 15]

[0336] Example 16 Binding of one arm of BEAT to CAR T and AP cells Binding of monovalent one-arm BEATs 3 and 4 to CAR T cells was comparable to that of bivalent BEAT1a4, and BEATs 5 and 6 showed slightly reduced binding compared to BEATs 1a4, 3, and 4 (Figure 17A). Binding of monovalent one-arm BEATs to moDCs was also comparable to that of bivalent BEAT1a4 (Figure 17B).

[0337] Example 17 Functional activity of BEAT in one arm CD86 expression and IL-12 / IL-23p40 release were evaluated in the presence of BEATs and compared with BEAT1a4 and an anti-CD40 control antibody. One arm of BEATs had lower activity compared with BEAT1a4, and BEAT3 and BEAT4 (monovalent anti-CD40) were reduced compared with BEAT5 and BEAT6 (bivalent anti-CD40) for both CD86 expression and IL-12 / IL-23p40 release (Figure 18).

[0338] Example 18 T cell proliferation at day 5 with BEAT in one arm The proliferation of one arm of BEAT was compared to BEAT1a4 in a dose-response assay when added to both CAR T and MoDCs. 7B1-CD28z CAR was incubated with MoDCs and various BEATs and controls for 120 hours, and CAR T cell numbers were measured (by CD4+GFP+).

[0339] A prozone effect was observed with CAR T cell proliferation using 1 nM BEAT1a4. A dose response was observed with various single-arm BEATs, with BEAT3 being the most potent at the highest dose, followed by BEAT4, BEAT5, and BEAT6. Comparable proliferation was observed between 0.1 and 10 nM, and a prozone effect was observed with BEAT5 at doses higher than 1 nM (Figure 19).

[0340] This proliferation assay also assesses cell phenotype and T cell proliferation. EMincreased (Fig. 20A, C, and D), driven primarily by an expansion in CD8+ cells (Fig. 22A, B, C, D), while CD4+ cells showed a significant increase in T at the highest dose of 100 nM. EM The phenotypes of T cells (Figure 20), CD4+ cells (Figure 21), and CD8+ cells (Figure 22) with BEAT in one arm were similar across all doses.

[0341] Example 19 Cytokine analysis of one arm of BEAT Cytokines were analyzed in the presence of CAR, CAR + MoDC, and MoDC alone. BEAT had no effect on IFNγ in the presence of CAR alone or MoDC alone (Figure 23A). IFNγ increased in a dose-responsive manner in the presence of CAR T cells + MoDC and BEAT, with comparable responses seen with all BEATs, and a maximal response seen at 1 nM (Figure 23A).

[0342] BEATs had no effect on IL-12p70 or IL-6 in the presence of CARs alone or MoDCs alone (Figures 23B and 23C). IL-12p70 increased at 0.1 nM and 1 nM with all BEATs and had a prozone effect above 1 nM in the presence of CARs and MoDCs (Figure 23B). IL-6 increased in a dose-responsive manner when BEATs were added to CARs and MoDCs, with BEAT4 cytokine levels being higher than those of the other BEATs (Figure 23C).

[0343] IL-2 increased in a dose-responsive manner in the presence of CAR alone and in the presence of BEAT + CAR + MoDC (Figure 23D). No change was seen with MCP-1 above background (Figure 23E). TNF-α increased in the presence of BEAT + CAR + MoDC and was unchanged with CAR or MoDC alone (Figure 23F).

Claims

1. 1. A bispecific polypeptide comprising a first antigen-binding protein and a second antigen-binding protein, wherein the first antigen-binding protein specifically binds to CD40 or CD206, and the second antigen-binding protein specifically binds to a FLAG tag.

2. the first antigen-binding protein FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a 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; 2. The bispecific polypeptide of claim 1 , wherein the sequences of any of the framework regions and / or complementarity determining regions are as described herein, preferably as described herein in Table 1.

3. The first antigen binding protein described herein comprises: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Including, 3. The bispecific polypeptide of claim 2, wherein optionally the linker is a chemical compound, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

4. 4. The bispecific polypeptide of claim 1, wherein the first antigen-binding protein binds or specifically binds to CD40.

5. 5. The bispecific polypeptide of claim 4, wherein the first antigen-binding protein competitively inhibits the binding to CD40 of an antibody or antigen-binding fragment thereof 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:

2.

6. 6. The bispecific polypeptide of claim 4 or 5, wherein the first antigen-binding protein comprises antigen-binding domains CDRH1, CDRH2 and / or CDRH3 having a variable heavy domain as defined in SEQ ID NO: 1 and / or antigen-binding domains CDRL1, CDRL2 and / or CDRL3 having a light chain variable domain as defined in SEQ ID NO:

2.

7. the first antigen-binding protein (i) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 3, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 6, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 10; and (ii) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 12, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 14, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 16; or (iii) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:3; a VH comprising a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 10; and (iv) a CDR1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 12; a CDR2 comprising, or consisting of, a sequence that is at least about 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; a VL comprising, or consisting of, a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; or (v) a VH comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 5, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 9, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 11; and (vi) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 13, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 15, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 16; or (vii) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:5, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 99% identical to the sequence set forth in SEQ ID NO:9; a VH comprising a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:11; (viii) a CDR1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 13; at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least a VL comprising a CDR2 comprising, or consisting of, a sequence that is at least about 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; a VL comprising, or consisting of, a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; or (ix) a VH comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 4, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 7, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 10; and (x) a VL comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 12, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 14, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 16; or (xi) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO:4, at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89% identical to the sequence set forth in SEQ ID NO:7; a VH comprising a CDR2 comprising, or consisting of, a sequence that is 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 10; a VH comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 10; and (xii) a CDR1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 12; at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least a VL comprising a CDR2 comprising, or consisting of, a sequence that is at least about 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; a VL comprising, or consisting of, a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; or (ix) a VH comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 36, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 40, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 42; and (vi) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 44, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 46, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 48; or (vii) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 36, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the sequence set forth in SEQ ID NO: 40 , or a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:42; (viii) a CDR1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 44; a CDR2 comprising, or consisting of, a sequence that is 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:48; and a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:

48.

7. The bispecific polypeptide of claim 4, comprising:

8. 8. The bispecific polypeptide of any one of claims 4 to 7, wherein the first antigen-binding protein comprises a heavy chain variable domain comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO: 1 or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical thereto.

9. 9. The bispecific polypeptide of claim 8, wherein the heavy chain variable domain of the first antigen-binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1, optionally wherein the amino acid substitutions are not in the CDRs and / or wherein the antigen-binding protein retains the ability to bind to CD40.

10. 10. The bispecific polypeptide of any one of claims 4 to 9, wherein the first antigen-binding domain comprises a light chain variable domain comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO: 2, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical thereto.

11. 11. The bispecific polypeptide of claim 10, wherein the light chain variable domain of the first antigen-binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NO: 2, optionally wherein the amino acid substitutions are not in the CDRs and / or wherein the antigen-binding protein retains the ability to bind to CD40.

12. 12. The bispecific polypeptide of any one of claims 4 to 11, wherein the first antigen-binding protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NOs: 1 and 2 (in N to C-terminal or C to N-terminal order).

13. 4. The bispecific polypeptide of claim 1, wherein the first antigen-binding protein binds or specifically binds to CD206.

14. 14. The bispecific polypeptide of any one of claims 1 to 3 or 13, wherein the first antigen-binding protein binds to CD206 expressed on the surface of an immune cell, preferably an antigen-presenting cell, more preferably a professional antigen-presenting cell, in particular an endogenous professional antigen-presenting cell.

15. 15. The bispecific polypeptide of claim 14, wherein the first antigen-binding protein binds to CD206 expressed on the surface of a professional antigen-presenting cell selected from a dendritic cell, a macrophage, a B cell, an epithelial cell, most preferably a dendritic cell; even more preferably, the professional antigen-presenting cell is not a tumor cell.

16. 16. The bispecific polypeptide of any one of claims 13 to 15, wherein the first antigen-binding protein competitively inhibits the binding to CD206 of an antibody comprising a VH comprising the sequence set forth in SEQ ID NO: 33 and a VL comprising the sequence set forth in SEQ ID NO:

34.

17. 17. The bispecific polypeptide of any one of claims 13 to 16, wherein the first antigen-binding protein comprises an antigen-binding domain CDRH1, CDRH2 and / or CDRH3 having a variable heavy domain as defined in SEQ ID NO: 33 and / or an antigen-binding domain CDRL1, CDRL2 and / or CDRL3 having a variable light domain as defined in SEQ ID NO:

34.

18. the first antigen-binding protein (i) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 35, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 38, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 42; and (ii) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 44, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 46, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 48; or (iii) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 35, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the sequence set forth in SEQ ID NO:

38. , or a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO:42; (iv) a CDR1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 44; a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 48; or (v) a VH comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 37, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 41, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 43; and (vi) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 45, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 47, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 48; or (vii) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 37, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the sequence set forth in SEQ ID NO: 41 , or a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO:43; (viii) a CDR1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 45; a CDR2 comprising, or consisting of, a sequence that is 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:48; a VL comprising, or consisting of, a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:48; or (ix) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 36, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 39, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 42; and (x) a VL comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 44, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 46, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 48; or (xi) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 36, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the sequence set forth in SEQ ID NO:

39. , or a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO:42; (xii) a CDR1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 44; at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least a CDR2 comprising, or consisting of, a sequence that is 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:48; a VL comprising, or consisting of, a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:48; or (ix) a VH comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 36, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 40, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 42; and (vi) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 44, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 46, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 48; or (vii) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 36; a VH comprising a CDR2 comprising, or consisting of, a sequence that is at least about 9%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:42; a VH comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:42; (viii) a CDR1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence set forth in SEQ ID NO: 44; a CDR2 comprising, or consisting of, a sequence that is 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:48; and a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO:

48.

18. The bispecific polypeptide of any one of claims 13 to 17, comprising an antigen-binding domain comprising:

19. 19. The bispecific polypeptide of any one of claims 13 to 18, wherein the first antigen-binding protein comprises a heavy chain variable domain comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO: 33, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical thereto.

20. 20. The bispecific polypeptide of claim 19, wherein the heavy chain variable domain of the first antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NO: 33, optionally wherein the amino acid substitutions are not in the CDRs and / or wherein the antigen binding protein retains the ability to bind to CD206.

21. 21. The bispecific polypeptide of any one of claims 13 to 20, wherein the first antigen-binding domain comprises a light chain variable domain comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO: 34, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical thereto.

22. 22. The bispecific polypeptide of claim 21 , wherein the light chain variable domain of the first antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NO: 34, optionally wherein the amino acid substitutions are not in the CDRs and / or wherein the antigen binding protein retains the ability to bind to CD206.

23. 23. The bispecific polypeptide of any one of claims 13 to 22, wherein the first antigen-binding protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NOs: 33 and 34 (in N to C-terminal or C to N-terminal order).

24. 24. The bispecific polypeptide of any one of claims 1 to 23, wherein the first antigen-binding protein comprises, from N to C-terminus, a VH then a VL, or a VL then a VH, or any of CDR1, 2 and 3 defined herein as VH then any of CDR1, 2 and 3 defined herein as VL, or any of CDR1, 2 and 3 defined herein as VL then any of CDR1, 2 and 3 defined herein as VH.

25. The first antigen binding protein is in the form: (i) single domain antibodies (sdAbs), (ii) single chain Fv fragment (scFv), (iii) a dimeric scFv (di-scFv), or (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; 25. The bispecific polypeptide of any one of claims 1 to 24, wherein

26. The first antigen binding protein is in the form: (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)-(vii) linked to a constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3; 25. The bispecific polypeptide of any one of claims 1 to 24, wherein

27. 27. The bispecific polypeptide of any one of claims 1 to 26, wherein the first antigen-binding protein is in the form of an immunoglobulin G molecule (IgG).

28. 28. The bispecific polypeptide of any one of claims 1 to 27, wherein the second antigen-binding protein is capable of specifically binding to a FLAG tag or a variant or modified form thereof.

29. 29. The bispecific polypeptide of any one of claims 1 to 28, wherein the second antigen-binding domain is for binding to a FLAG tag as defined in SEQ ID NOs: 80 and 99 or a variant thereof, such as containing, comprising or consisting of the sequence GDYKDDDDKG (SEQ ID NO: 98), DYKDDDDK (SEQ ID NO: 99), MDYKDDDDK (SEQ ID NO: 100), DFKDDDK (SEQ ID NO: 101), DYKAFDNL (SEQ ID NO: 102), DYKDHDG (SEQ ID NO: 103), MDFKDDDDK (SEQ ID NO: 104), MDYKAFDNL (SEQ ID NO: 105), DYKDHDI (SEQ ID NO: 106), DYKDH (SEQ ID NO: 107), DYKDD (SEQ ID NO: 108), DYKDHD (SEQ ID NO: 109) and / or DYKDDD (SEQ ID NO: 110).

30. 30. The bispecific polypeptide of any one of Claims 1 to 29, wherein the FLAG tag is present at the N-terminus, C-terminus or within a protein to which the bispecific polypeptide can bind, binds or specifically binds.

31. the second antigen-binding protein (e) comprising a VH comprising the sequence set forth in SEQ ID NO: 70 and a VL comprising the sequence set forth in SEQ ID NO: 75; (f) a VH comprising the sequence set forth in SEQ ID NO: 71 and a VL comprising the sequence set forth in SEQ ID NO: 76; (g) comprising a VH comprising the sequence set forth in SEQ ID NO: 71 and a VL comprising the sequence set forth in SEQ ID NO: 77; (h) comprising a VH comprising the sequence set forth in SEQ ID NO: 71 and a VL comprising the sequence set forth in SEQ ID NO: 78; (i) comprising a VH comprising the sequence set forth in SEQ ID NO: 71 and a VL comprising the sequence set forth in SEQ ID NO: 79; (j) comprising a VH comprising the sequence set forth in SEQ ID NO: 72 and a VL comprising the sequence set forth in SEQ ID NO: 76; (k) comprising a VH comprising the sequence set forth in SEQ ID NO: 72 and a VL comprising the sequence set forth in SEQ ID NO: 77; (l) VH comprising the sequence shown in SEQ ID NO: 72 and VL comprising the sequence shown in SEQ ID NO: 78; (m) comprising a VH comprising the sequence set forth in SEQ ID NO: 72 and a VL comprising the sequence set forth in SEQ ID NO: 79; (n) comprising a VH comprising the sequence set forth in SEQ ID NO: 73 and a VL comprising the sequence set forth in SEQ ID NO: 76; (o) comprising a VH comprising the sequence set forth in SEQ ID NO: 73 and a VL comprising the sequence set forth in SEQ ID NO: 77; (p) comprising a VH comprising the sequence set forth in SEQ ID NO: 73 and a VL comprising the sequence set forth in SEQ ID NO: 78; (q) comprising a VH comprising the sequence set forth in SEQ ID NO: 73 and a VL comprising the sequence set forth in SEQ ID NO: 79; (r) comprising a VH comprising the sequence set forth in SEQ ID NO: 74 and a VL comprising the sequence set forth in SEQ ID NO: 76; (s) comprising a VH comprising the sequence set forth in SEQ ID NO: 74 and a VL comprising the sequence set forth in SEQ ID NO: 77; (t) a VH comprising the sequence set forth in SEQ ID NO: 74 and a VL comprising the sequence set forth in SEQ ID NO: 78; or (u) VH comprising the sequence shown in SEQ ID NO: 74 and VL comprising the sequence shown in SEQ ID NO: 79 31. The bispecific polypeptide of any one of claims 1 to 30, which competitively inhibits the binding of an antibody to the FLAG tag.

32. 32. The bispecific polypeptide of any one of claims 1 to 31, wherein the second antigen-binding protein comprises an antigen-binding domain CDRH1, CDRH2 and / or CDRH3 having a variable heavy chain as defined in any one of SEQ ID NOs: 71 to 74 and / or an antigen-binding domain CDRL1, CDRL2 and / or CDRL3 having a variable light chain as defined in any one of SEQ ID NOs: 76 to 79.

33. the second antigen-binding domain comprises: (a) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 81; a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO: 83; and (b) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 90; 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% identical to the sequence of SEQ ID NO: 92; or (c) a complementarity determining region CDR1 comprising or consisting of an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO: 111 or 112, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 99% identical to the sequence of SEQ ID NO: 113 a VH comprising a CDR2 comprising, or alternatively consisting of, an amino acid sequence that is 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%, or at least 99% identical to the sequence of SEQ ID NO: 114; a VH comprising, or alternatively consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 114; and (d) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 120; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; or (e) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 111 or 112, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 99% identical to the sequence of SEQ ID NO: 113; a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 114; and (f) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 133; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; or (g) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 111 or 156, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 99% identical to the sequence of SEQ ID NO: 157; a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 114; and (h) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 120; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; or (i) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO: 111 or 156, at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 114; and (j) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 133; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; or (k) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 126, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90% identical to the sequence of SEQ ID NO: 127 , 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% identical to the sequence of SEQ ID NO: 114; and a CDR3 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO:

114. (l) a CDR1 comprising or consisting of an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO: 120, at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least a VL comprising a CDR2 comprising, or consisting of, an amino acid sequence that is 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; a VL comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; or (m) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO: 126, at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% identical to the sequence of SEQ ID NO: 127 , 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% identical to the sequence of SEQ ID NO: 114; and a CDR3 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO:

114. (n) a CDR1 comprising or consisting of an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 133; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; or (o) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 111, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90% identical to the sequence of SEQ ID NO: 132 , 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% identical to the sequence of SEQ ID NO: 114; and a CDR3 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO:

114. (p) a CDR1 comprising or consisting of an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 133; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; or (q) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 126, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90% identical to the sequence of SEQ ID NO: 127 , 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% identical to the sequence of SEQ ID NO: 114; and a CDR3 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence of SEQ ID NO:

114. (r) a CDR1 comprising or consisting of an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 133; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the sequence of SEQ ID NO: 92; 33. The bispecific polypeptide of any one of claims 1 to 32, comprising an antigen-binding domain comprising:

34. the second antigen-binding domain comprises: (a) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 81, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 82, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 83; and (b) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 90, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 91, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (c) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 111 or 112, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 113, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (d) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 120, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (e) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 111 or 112, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 113, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (f) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 133, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (g) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 111 or 156, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 157, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (h) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 120, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (i) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 111 or 156, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 157, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (j) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 133, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (k) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 126, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 127, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (l) VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 120, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (m) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 126, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 127, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (n) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 133, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (o) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 111, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 132, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (p) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 133, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or (q) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 126, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 127, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114; and (r) VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 133, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:

92.

34. The bispecific polypeptide of any one of Claims 1 to 33, comprising an antigen-binding domain comprising:

35. the second antigen-binding protein comprises an antigen-binding domain having FR H1, FR H2, FR H3 and / or FR H4 from a human germline, the human germline being IGHV1-46 * 01 or IGHV7-4-1 * 02, and / or the second antigen binding protein comprises an antigen binding domain having FR L1, FR L2, FR L3 and / or FR L4 from human germline, wherein the human germline is selected from the group consisting of IGKV2-30 * 01 or IGKV4-1 * 35. The bispecific polypeptide of any one of claims 1 to 34, wherein said polypeptide is .Ol.

36. 36. The bispecific polypeptide of any one of claims 1 to 35, wherein the second antigen-binding protein comprises an antigen-binding domain having FR H1, FR H2, FR H3 and / or FR H4 of the antigen-binding domain having a variable heavy chain as defined in any one of SEQ ID NOs: 71 to 74.

37. 37. The bispecific polypeptide of any one of claims 1 to 36, wherein the second antigen-binding protein comprises an antigen-binding domain having FR L1, FR L2, FR L3 and / or FR L4 of the antigen-binding domain with a variable light chain as defined in any one of SEQ ID NOs: 76 to 79.

38. the second antigen binding protein comprises an antigen binding domain, the antigen binding domain comprising 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: A. 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 134, 135, 136, and 137, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 138, 139, 140, and 97, respectively; or 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 87, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 138, 139, 140, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 87, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 153, 154, 155, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 87, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 95, 96, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 85, 87, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 94, 96, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 86, 88, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 138, 139, 140, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 86, 88, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 153, 154, 155, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 86, 88, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 95, 96, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 86, 88, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 94, 96, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 85, 151, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 138, 139, 140, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 85, 151, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 153, 154, 155, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 85, 151, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 95, 96, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 85, 151, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 94, 96, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 150, 152, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 138, 139, 140, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 150, 152, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 153, 154, 155, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 150, 152, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 95, 96, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 149, 150, 152, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 93, 94, 96, and 97, respectively; 38. The bispecific polypeptide of any one of claims 1 to 37, comprising:

39. the second antigen binding protein comprises an antigen binding domain, the antigen binding domain comprising 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: A. 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 115, 116, 118, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 127, respectively; or 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 115, 116, 118, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 127, 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: 115, 117, 119, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 127, respectively; or D. 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: 115, 117, 119, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 127, respectively.

38. The bispecific polypeptide of any one of claims 1 to 37, comprising:

40. the second antigen-binding protein comprises an antigen-binding domain, wherein the antigen-binding domain comprises 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 A. 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 115, 116, 118, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, respectively; or 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 115, 116, 118, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 161, 162, 163, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 115, 117, 119, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 115, 117, 119, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 161, 162, 163, and 97, respectively.

38. The bispecific polypeptide of any one of claims 1 to 37, comprising:

41. the second antigen binding protein comprises 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: A. 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 116, 159, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 127, respectively; or 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 116, 159, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 127, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 117, 160, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 127, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 117, 160, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 127, respectively.

38. The bispecific polypeptide of any one of Claims 1 to 37, comprising an antigen-binding domain comprising:

42. the second antigen-binding protein comprises an antigen-binding domain, wherein the antigen-binding domain comprises 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 A. 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 116, 159, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, respectively; or 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 116, 159, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 161, 162, 163, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 117, 160, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 158, 117, 160, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 161, 162, 163, and 97, respectively.

38. The bispecific polypeptide of any one of claims 1 to 37, comprising:

43. the second antigen binding protein comprises an antigen binding domain, wherein the antigen binding domain comprises 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: A. 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 128, 130, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 97, respectively; or 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 128, 130, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 129, 131, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 129, 131, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 164, 128, 166, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 164, 128, 166, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 164, 165, 167, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 124, 125, and 97, 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: 164, 165, 167, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 122, 123, 125, and 97, respectively.

38. The bispecific polypeptide of any one of claims 1 to 37, comprising:

44. the second antigen-binding protein comprises an antigen-binding domain, wherein the antigen-binding domain comprises 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 A. 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 128, 130, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, respectively; or 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 128, 130, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 168, 169, 170, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 129, 131, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 129, 131, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 168, 169, 170, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 164, 128, 166, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 164, 128, 166, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 168, 169, 170, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 164, 165, 167, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 144, 145, 146, and 97, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 164, 165, 167, and 89, respectively, and for VL FRs, the reference sequences are set forth in SEQ ID NOs: 168, 169, 170, and 97, respectively.

38. The bispecific polypeptide of any one of claims 1 to 37, comprising:

45. the second antigen-binding protein comprises an antigen-binding domain, the antigen-binding domain comprising: 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for the VH FRs, the reference sequences are set forth in SEQ ID NOs: 141, 142, 143, and 137, respectively, and for the VL For FR, the reference sequences are shown in SEQ ID NOs: 144, 145, 146 and 97, respectively, for VH and VL, 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical, or 100% identical to a reference sequence, wherein for VH FRs, the reference sequences are set forth in SEQ ID NOs: 84, 147, 148, and 137, respectively, and for VL For FR, the reference sequences are shown in SEQ ID NOs: 144, 145, 146 and 97, respectively.

38. The bispecific polypeptide of any one of claims 1 to 37, comprising:

46. 38. The bispecific polypeptide of any one of claims 1 to 37, wherein the second antigen-binding protein comprises an antigen-binding domain comprising a variable heavy chain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 71 to 74, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical thereto.

47. 47. The bispecific polypeptide of claim 46, wherein the heavy chain variable domain of said second antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NOs: 71-74, optionally wherein the amino acid substitutions are not in the CDRs and / or wherein the antigen binding protein retains the ability to bind to a FLAG tag.

48. 48. The bispecific polypeptide of any one of claims 1 to 47, wherein the second antigen-binding protein comprises an antigen-binding domain comprising a variable light chain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 76-79, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical thereto.

49. 49. The bispecific polypeptide of claim 48, wherein the light chain variable domain of the second antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NOs: 76-79, optionally wherein the amino acid substitutions are not in the CDRs and / or wherein the antigen binding protein retains the ability to bind to a FLAG tag.

50. The second antigen binding protein is in the form: (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; 50. The bispecific polypeptide of any one of claims 1 to 49, wherein

51. The second antigen binding protein is in the form: (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)-(vii) linked to a constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3; 50. The bispecific polypeptide of any one of claims 1 to 49, wherein

52. 52. The bispecific polypeptide of any one of claims 1 to 51, wherein the second antigen-binding protein is in the form of an scFv and the first antigen-binding protein is in the form of an immunoglobulin G (IgG) antibody.

53. 52. The bispecific polypeptide of any one of Claims 1 to 51, wherein the second antigen-binding protein and the first antigen-binding protein are both antibodies or antigen-binding fragments thereof, and optionally the first and second antigen-binding proteins are of the same antibody type or fragment thereof.

54. the second antigen-binding domain has the following amino acid sequence (in N- to C-terminal or C- to N-terminal order): (v) SEQ ID NOs: 71 and 79; (w) SEQ ID NOs: 71 and 78; (x) SEQ ID NOs: 71 and 76; (y) SEQ ID NOs: 71 and 77; (z) SEQ ID NOs: 72 and 76; (aa) SEQ ID NOs: 72 and 77; (bb) SEQ ID NOs: 72 and 78; (cc) SEQ ID NOs: 72 and 79; (dd) SEQ ID NOs: 73 and 76; (ee) SEQ ID NOs: 73 and 77; (ff) SEQ ID NOs: 73 and 78; (gg) SEQ ID NOs: 73 and 79; (hh) SEQ ID NOs: 74 and 76; (ii) SEQ ID NOs: 74 and 77; (jj) SEQ ID NOs: 74 and 78, or (kk) SEQ ID NOs: 74 and 79 54. The bispecific polypeptide of any one of claims 1 to 53, comprising, consisting essentially of, or consisting of:

55. The bispecific polypeptide has the following amino acid sequence: SEQ ID NOs: 19 and 20, SEQ ID NOs: 19 and 21, SEQ ID NOs: 22 and 23, SEQ ID NOs: 23 and 24, SEQ ID NOs: 19 and 25, SEQ ID NOs: 19 and 26, SEQ ID NOs: 23 and 27, SEQ ID NOs: 23 and 28, SEQ ID NOs: 19 and 29, SEQ ID NOs: 19 and 30, SEQ ID NOs: 23 and 31, SEQ ID NOs: 23 and 32, SEQ ID NOs: 171, 172 and 173, SEQ ID NOs: 172, 174 and 175, SEQ ID NOs: 171, 172 and 176, SEQ ID NOs: 172, 174 and 177, SEQ ID NOs: 50 and 51, SEQ ID NOs: 50 and 52, SEQ ID NOs: 53 and 54, SEQ ID NOs: 54 and 55, SEQ ID NOs: 50 and 56, SEQ ID NOs: 50 and 57, SEQ ID NOs: 54 and 58, SEQ ID NOs: 54 and 59, SEQ ID NOs: 50 and 60, SEQ ID NOs: 50 and 61, SEQ ID NOs: 54 and 62, or SEQ ID NOs: 54 and 63 55. The bispecific polypeptide of any one of claims 1 to 54, comprising:

56. 56. The bispecific polypeptide of any one of claims 1 to 55, wherein the polypeptide is a fusion protein comprising a first and a second antigen-binding protein.

57. 57. A fusion protein comprising the bispecific polypeptide of any one of claims 1 to 56.

58. 58. A nucleic acid encoding a bispecific polypeptide according to any one of claims 1 to 56 or a fusion protein according to claim 57.

59. 59. A vector or expression construct comprising the nucleic acid of claim 58.

60. 60. A cell comprising the vector or expression construct of claim 59 or the nucleic acid of claim 58.

61. 61. A pharmaceutical composition comprising a bispecific polypeptide according to any one of claims 1 to 56, a fusion protein according to claim 57, a nucleic acid according to claim 58, a vector or expression construct according to claim 59 or a cell according to claim 60, and optionally a pharmaceutically acceptable carrier, diluent or excipient.

62. 61. Use of a bispecific polypeptide according to any one of claims 1 to 56, a fusion protein according to claim 57, a nucleic acid according to claim 58, a vector or expression construct according to claim 59 or a cell according to claim 60 in the manufacture of a medicament for the treatment or prevention of cancer.

63. 62. The pharmaceutical composition of claim 61 for the treatment or prevention of cancer.

64. 61. A method of treating or preventing cancer, said method comprising administering to a subject a bispecific polypeptide of any one of claims 1 to 56, a fusion protein of claim 57, a nucleic acid of claim 58, a vector or expression construct of claim 59, or a cell of claim 60, thereby treating or preventing cancer.

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