Bispecific polypeptides and uses thereof

Abstract

The present disclosure relates generally to a bispecific polypeptide 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, including portions and uses thereof. In a first aspect disclosed herein, there is provided a bispecific polypeptide comprising a first antigen binding protein and a second antigen binding protein, the first antigen binding protein specifically binding to CD40, and wherein the second antigen binding protein specifically binds to a FLAG tag. 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 wherein the second antigen binding protein specifically binds to a FLAG tag.

Description

Technical Field

[0001] The present disclosure generally relates to a bispecific polypeptide, including portions and uses 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.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority from Australian provisional application AU 2022902713, the entire contents of which are incorporated herein. Background Art

[0004] Adoptive cell transfer (ACT) is showing exciting potential for cancer treatment. In ACT, large numbers of autologous tumor-reactive T cells are generated in vitro and then infused into the patient. Tumor-reactive T cells can be isolated from blood or tumors and expanded in vitro using stimulation with peptides and / or cytokines.

[0005] Approaches to improve T cell responsiveness to ACT involve genetically modifying patient lymphocytes to generate tumor-reactive T cells against most malignancies, including solid cancers 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 to T cell signaling domains that redirect T cell effector functions against tumor cells. Both approaches can render T cells tumor-reactive, but the CAR approach is not restricted by MHC and can be more widely applied to more patients.

[0006] CARs can take various forms, but are typically composed of an extracellular domain consisting of a single-chain variable fragment (scFv) of an antibody specific for a tumor-associated antigen (TAA). The scFv is connected to an intracellular region consisting of one or more signal transduction moieties via a hinge and a transmembrane domain. CARs specific for a range of TAAs, including mesothelin, Her2, CEA, FBP, CD19, and BCMA, have been developed. The most advanced clinical studies have used CARs specific for CD19 to treat B-cell leukemias and lymphomas. Since 2017, the Food and Drug Administration (FDA) has approved six CAR T cell therapies. All of these therapies have been approved for the treatment of blood cancers, including lymphomas, certain forms of leukemia, and, most recently, multiple myeloma.

[0007] Attempts to optimize this therapy have combined CAR T cells with other therapeutic approaches designed to overcome tumor-induced immunosuppression, including combination therapy 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). Although some of these approaches have demonstrated success in enabling direct interactions between T cells and cancer cells, which may lead to T cell expansion in hematopoietic cancer cells, significant expansion has rarely been observed in the solid cancer setting.

[0008] The problem of optimizing therapies for delivery of CAR T cells is often exacerbated by the difficulties faced in ex vivo expansion and manufacturing of CAR T cells due to low lymphocyte counts and poor condition of cells from heavily treated patients. In addition, even if sufficient donor cells are available, large quantities of cells need to be manufactured in order to provide patients with effective doses of such treatments, and although allogeneic cells from healthy donors have been proposed to address this problem, there are still some problems, such as human leukocyte antigen (HLA) mismatch between donor and recipient, which may lead to graft-versus-host disease (GvHD). Therefore, there is still an urgent need to develop new reagents to improve the efficacy of ACT, especially CAR T therapy.

[0009] The reference to any prior art in this specification is not an acknowledgement or implication that the prior art forms part of the common general knowledge in any jurisdiction, or that the prior art could reasonably be expected to be understood, regarded as relevant and / or combined with other prior art by a person skilled in the art. Summary of the invention

[0010] 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 wherein the second antigen binding protein specifically binds to a FLAG tag.

[0011] In an embodiment of the first aspect, the first antigen binding protein binds to the external motif A of the CD40 juxtamembrane domain (D1a), optionally wherein said binding does not prevent, reduce or inhibit CD40-CD40L binding.

[0012] In an embodiment of the first aspect, the first antigen binding protein comprises an antigen binding domain comprising:

[0013] FR1–CDR1–FR2–CDR2–FR3–CDR3–FR4, and

[0014] FR1a–CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0015] in:

[0016] FR1, FR2, FR3 and FR4 are each a framework region;

[0017] CDR1, CDR2, and CDR3 are each complementarity determining regions;

[0018] FR1a, FR2a, FR3a and FR4a are each a framework region;

[0019] CDR1a, CDR2a, and CDR3a are each complementarity determining regions;

[0020] Wherein, the sequence of any one of the framework region and / or the complementarity determining region is as described herein, preferably as described in Table 1 below regarding the CD40 binding protein.

[0021] In any embodiment of the first aspect, CDR1, CDR2 and CDR3 refer to the complementary determining region from the variable heavy chain (VH) of the antibody, and CDR1a, CDR2a and CDR3a are complementary determining regions from the variable light chain (VL) of the antibody, or in the case where CDR1, CDR2 and CDR3 are complementary determining regions from VL, CDR1a, CDR2a and CDR3a are complementary determining regions from VH. In this example, CDR can refer to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 as appropriate.

[0022] Reference herein to a protein or antibody that "binds to" CD40 provides literal support for a protein or antibody that "binds specifically to or specifically binds to" CD40.

[0023] 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 of an antibody or its antigen binding fragment to CD40, and the antibody or its antigen binding fragment comprises a VH comprising the sequence shown in SEQ ID NO: 1 and a VL comprising the sequence shown in SEQ ID NO: 2.

[0024] In any embodiment of the first aspect, the invention provides a first antigen binding protein having a CDRH1, CDRH2 and / or CDRH3 of an antigen binding domain having a variable heavy chain domain as defined in SEQ ID NO:1.

[0025] In any embodiment of the first aspect, the invention provides a first antigen binding protein having CDRL1, CDRL2 and / or CDRL3 of an antigen binding domain having a variable light chain as defined in SEQ ID NO:2.

[0026] In any embodiment of the first aspect, the present invention provides a first antigen binding protein having CDR1, CDR2 and / or CDR3 of an antigen binding domain, wherein the antigen binding domain has a variable heavy chain as defined by SEQ ID NO:1 and a variable light chain as defined by SEQ ID NO:2.

[0027] In any embodiment of the first aspect, the first antigen binding protein described herein comprises:

[0028] FR1–CDR1–FR2–CDR2–FR3–CDR3–FR4–linker–FR1a–CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0029] Wherein, the sequence of any one of the framework region and / or the complementarity determining region is as described herein, preferably as described in Table 1 below.

[0030] As defined herein, a linker can be a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0031] In any embodiment of the first aspect, the first antigen binding protein comprises:

[0032] (i) and (ii):

[0033] (i) VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 3, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 6, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 10, and

[0034] (ii) 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;

[0035] or

[0036] (iii) and (iv):

[0037] (iii) a VH comprising 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:3, a CDR2 comprising or consisting of a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO:6, and a CDR3 comprising or consisting of a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO:7. NO:10 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 or consisting of the sequence shown in NO:10, and

[0038] (iv) a VL comprising 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, and 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: 12 NO:14 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 or consisting of a CDR3, wherein the CDR3 comprises or consists of 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%, at least 99% identical to or consisting of a sequence as set forth in SEQ ID NO:16;

[0039] or

[0040] (v) and (vi):

[0041] (v) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 5, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 9, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 11, and

[0042] (vi) VL comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 13, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 15, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 16;

[0043] or

[0044] (vii) and (viii):

[0045] (vii) a VH comprising 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:5, and a CDR2 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 to the sequence set forth in SEQ ID NO:5 NO:9 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 or consisting of a CDR3, wherein the CDR3 comprises 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:11, and

[0046] (viii) a VL comprising 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, and 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:13 NO:15 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 or consisting of a CDR3, wherein the CDR3 comprises 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 or consisting of a sequence as set forth in SEQ ID NO:16;

[0047] or

[0048] (ix) and (x):

[0049] (ix) VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 4, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 7, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 10, and

[0050] (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;

[0051] or

[0052] (xi) and (xii):

[0053] (xi) VH, which comprises complementarity determining region (CDR) 1, said CDR 1 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 to or consisting of the sequence shown in SEQ ID NO: 4, CDR 2, said CDR 2 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 to or consisting of the sequence shown in SEQ ID NO: 7, CDR 3, said CDR 3 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 to or consisting of the sequence shown in SEQ ID NO: 10, and

[0054] (xii) a VL comprising a CDR1 that comprises or consists 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, and a CDR2 that comprises 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. NO:14 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 or consisting of a CDR3, wherein the CDR3 comprises 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 or consisting of a sequence as set forth in SEQ ID NO:16;

[0055] or

[0056] (xiii) and (xiv):

[0057] (xiii) VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 4, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 8, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 10, and

[0058] (xiv) 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;

[0059] or

[0060] (xv) and (xvi):

[0061] (xv) a VH comprising 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, and a CDR2 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 to the sequence set forth in SEQ ID NO:4 NO:8 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 shown in SEQ ID NO:8, or consists of a CDR3, wherein the CDR3 comprises 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 shown in SEQ ID NO:10, or consists of a CDR3, and

[0062] (xvi) a VL comprising 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, and 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:12 NO:14 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 shown in NO:14, or consists of a CDR3, wherein the CDR3 comprises 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 shown in SEQ ID NO:16 or consists of a CDR3.

[0063] In any embodiment of the first aspect, the first antigen binding protein comprises a heavy chain variable domain, which comprises or consists of the amino acid sequence shown in SEQ ID NO:1, or comprises or consists 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 amino acid sequence. 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 shown in SEQ ID NO:1; optionally, the amino acid substitutions are not in the CDRs and / or the antigen-binding protein retains the ability to bind to CD40.

[0064] In any embodiment of the first aspect, the first antigen binding protein comprises a light chain variable domain, which comprises or consists of the amino acid sequence shown in SEQ ID NO:2, or comprises or consists 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 amino acid sequence. 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 shown in SEQ ID NO:2; optionally, the amino acid substitutions are not in the CDRs and / or the antigen-binding protein retains the ability to bind to CD40.

[0065] In any embodiment of the first aspect, the first antigen binding protein comprises: a heavy chain variable domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, or 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 amino acid sequence; and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 1. NO:2 or consists of the amino acid sequence shown in 2, or comprises 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 amino acid sequence. Optionally, compared to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, the heavy chain variable domain 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, respectively; optionally, the amino acid substitutions are not in the CDRs and / or the antigen binding protein retains the ability to bind to CD40.

[0066] In any of the embodiments of the first aspect, the first antigen binding protein comprises, consists essentially of, or consists of the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 (in order from N-terminus to C-terminus or C-terminus to N-terminus).

[0067] In any embodiment of the first aspect, the first antigen binding protein described herein may have, from N-terminus to C-terminus, VH then VL, or VL then VH, or any CDR1, CDR2, and CDR3 defined herein as VH then any CDR1, CDR2, and CDR3 defined herein as VL, or any CDR1, CDR2, and CDR3 defined herein as VL then any CDR1, CDR2, and CDR3 defined herein as VH.

[0068] In any embodiment of the first aspect, the first antigen binding protein may be in the form of:

[0069] (i) Single domain antibody (sdAb);

[0070] (ii) Single-chain Fv fragment (scFv);

[0071] (iii) Dimeric scFv (di-scFv); or

[0072] (iv) One of (ii) or (iii) linked to the constant region of an antibody, Fc, or the heavy chain constant domains (CH)2 and / or CH3.

[0073] Further, as described herein, in any embodiment of the first aspect, the first antigen-binding protein can be in the form of:

[0074] (i) Bispecific antibody;

[0075] (ii) Trispecific antibody;

[0076] (iii) Tetraspecific antibody;

[0077] (iv) Fab;

[0078] (v) F(ab’)2;

[0079] (vi) Fv;

[0080] (vii) Bispecific antibody or other forms of multispecific antibodies;

[0081] (viii) One of (i) to (vii) linked to the constant region of an antibody, Fc, or the heavy chain constant domains (CH)2 and / or CH3.

[0082] In any embodiment of the first aspect, the first antigen-binding protein can be in the form of an immunoglobulin G (IgG) molecule. Optionally, according to any embodiment of the first aspect of the present invention, the first antigen-binding protein can be in the form of IgG or a heterodimeric Fab-Fc, and the second antigen-binding protein can be in the form of an antigen-binding fragment of IgG (such as, scFv).

[0083] In the second aspect disclosed herein, a bispecific polypeptide is provided, which comprises a first antigen-binding protein and a second antigen-binding protein, wherein the first antigen-binding protein specifically binds to CD206, and wherein the second antigen-binding protein specifically binds to the FLAG tag.

[0084] In any embodiment of the second aspect, the first antigen binding protein binds to CD206 expressed on the surface of an immune cell, and the immune cell is 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, and the professional antigen presenting cell is selected from dendritic cells, macrophages, B cells, epithelial cells, most preferably selected from dendritic cells, and even more preferably, wherein the professional antigen presenting cell is not a tumor cell.

[0085] In an embodiment of the first aspect, the first antigen binding protein binds to human macrophage B11 antigen, optionally, wherein the B11 antigen comprises the amino acid sequence shown in SEQ ID NO:186.

[0086] In any embodiment of the second aspect, the first antigen binding protein comprises an antigen binding domain comprising:

[0087] FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4, and

[0088] FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0089] in:

[0090] FR1, FR2, FR3 and FR4 are each a framework region;

[0091] CDR1, CDR2, and CDR3 are each complementarity determining regions;

[0092] FR1a, FR2a, FR3a and FR4a are each a framework region;

[0093] CDR1a, CDR2a, and CDR3a are each complementarity determining regions;

[0094] Wherein, the sequence of any one of the framework region and / or the complementarity determining region is as described herein, preferably as described in Table 1 below for CD206 binding protein.

[0095] In this embodiment of the second aspect, CDR1, CDR2 and CDR3 refer to the complementary determining region from the variable heavy chain (VH) of the antibody, and CDR1a, CDR2a and CDR3a are complementary determining regions from the variable light chain (VL) of the antibody, or in the case where CDR1, CDR2 and CDR3 are complementary determining regions from VL, CDR1a, CDR2a and CDR3a are complementary determining regions from VH. In this example, CDR can refer to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 according to the situation.

[0096] Reference herein to a protein or antibody that "binds to" CD206 provides literal support for a protein or antibody that "binds specifically to or specifically binds to" CD206.

[0097] In any embodiment of the second aspect, the present invention provides a first antigen binding protein for binding to CD206, wherein the first antigen binding protein competitively inhibits the binding of an antibody to CD206, the antibody comprising a VH comprising the sequence shown in SEQ ID NO:33 and a VL comprising the sequence shown in SEQ ID NO:34.

[0098] In any embodiment of the second aspect, the invention provides a first antigen binding protein having a CDRH1, CDRH2 and / or CDRH3 of an antigen binding domain having a variable heavy chain domain as defined by SEQ ID NO:33.

[0099] In any embodiment of the second aspect, the invention provides a first antigen binding protein having a CDRL1, CDRL2 and / or CDRL3 antigen binding domain having a variable light chain as defined in SEQ ID NO:34.

[0100] In any embodiment of the second aspect, the present invention provides a first antigen binding protein having CDR1, CDR2 and / or CDR3 of an antigen binding domain, wherein the antigen binding domain has a variable heavy chain as defined by SEQ ID NO:33 and a variable light chain as defined by SEQ ID NO:34.

[0101] In any embodiments of the second aspect, the first antigen binding protein described herein comprises:

[0102] FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4–Linker-FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0103] Wherein, the sequence of any one of the framework region and / or the complementarity determining region is as described herein, preferably as described in Table 1 below.

[0104] As defined herein, a linker can be a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0105] In any embodiment of the second aspect, the first antigen binding protein comprises an antigen binding domain comprising:

[0106] (i) and (ii):

[0107] (i) VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 35, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 38, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 42, and

[0108] (ii) VL comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 44, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 46, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 48;

[0109] or

[0110] (iii) and (iv):

[0111] (iii) a VH comprising 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: 35, and a CDR2 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 to the sequence set forth in SEQ ID NO: 35. NO:38 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, or consisting of, a CDR3 comprising or consisting of 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%, at least 99% identical to, and

[0112] (iv) VL, which comprises CDR1, said CDR1 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 to or consisting of the sequence shown in SEQ ID NO: 44, CDR2, said CDR2 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 to or consisting of the sequence shown in SEQ ID NO: 46, CDR3, said CDR3 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 to or consisting of the sequence shown in SEQ ID NO: 48;

[0113] or

[0114] (v) and (vi):

[0115] (v) VH, which comprises complementarity determining region (CDR) 1, said complementarity determining region (CDR) 1 comprising the sequence shown in SEQ ID NO: 37 or consisting of the same, CDR2, said CDR2 comprising the sequence shown in SEQ ID NO: 41 or consisting of the same, and CDR3, said CDR3 comprising the sequence shown in SEQ ID NO: 43 or consisting of the same, and

[0116] (vi) VL, which comprises complementarity determining region (CDR) 1, said complementarity determining region (CDR) 1 comprising the sequence shown in SEQ ID NO: 45 or consisting of the same, CDR2, said CDR2 comprising the sequence shown in SEQ ID NO: 47 or consisting of the same, and CDR3, said CDR3 comprising the sequence shown in SEQ ID NO: 48 or consisting of the same;

[0117] or

[0118] (vii) and (viii):

[0119] (vii) a VH comprising 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: 37, and a CDR2 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 to the sequence set forth in SEQ ID NO: 37. NO:41 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 shown in NO:41, or consists of a CDR3, wherein the CDR3 comprises 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 shown in SEQ ID NO:43, or consists of a CDR3, and

[0120] (viii) a VL comprising 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, and 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:45. NO:47 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 or consisting of a CDR3, wherein the CDR3 comprises 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 or consisting of a sequence as set forth in SEQ ID NO:48;

[0121] or

[0122] (ix) and (x):

[0123] (ix) VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 36, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 39, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 42, and

[0124] (x) VL comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 44, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 46, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 48;

[0125] or

[0126] (xi) and (xii):

[0127] (xi) a VH comprising 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: 36, and a CDR2 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 to the sequence set forth in SEQ ID NO: 36 NO:39 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, 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%, at least 99% identical to, and

[0128] (xii) a VL comprising a CDR1 that comprises or consists 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, and a CDR2 that comprises 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. NO:46 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 or consisting of a CDR3, wherein the CDR3 comprises 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 or consisting of a sequence as set forth in SEQ ID NO:48;

[0129] or

[0130] (xiii) and (xiv):

[0131] (xiii) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 36, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 40, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 42, and

[0132] (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;

[0133] or

[0134] (xv) and (xvi):

[0135] (xv) a VH comprising 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:36, and a CDR2 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 to the sequence set forth in SEQ ID NO:36 NO:40 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, or consisting of, a CDR3 comprising or consisting of 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%, at least 99% identical to, and

[0136] (xvi) a VL comprising 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, and 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:44. NO:46 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 shown in NO:46, or consists of a CDR3, wherein the CDR3 comprises 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 shown in SEQ ID NO:48, or consists of a CDR3.

[0137] In any embodiment of the second aspect, the first antigen binding protein comprises a heavy chain variable domain, which comprises or consists of the amino acid sequence shown in SEQ ID NO:33, or comprises or consists 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 amino acid sequence. 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 shown in SEQ ID NO:33; optionally, the amino acid substitutions are not in the CDRs and / or the antigen-binding protein retains the ability to bind to CD206.

[0138] In any embodiment of the second aspect, the first antigen binding protein comprises a light chain variable domain, which comprises or consists of the amino acid sequence shown in SEQ ID NO:34, or comprises or consists 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 amino acid sequence. 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 shown in SEQ ID NO:34; optionally, the amino acid substitutions are not in the CDRs and / or the antigen binding protein retains the ability to bind to CD206.

[0139] In any embodiment of the second aspect, the first antigen binding protein comprises: a heavy chain variable domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 33, or 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 amino acid sequence; and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 34. NO:34, or 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 amino acid sequence. Optionally, the heavy chain variable domain and light chain variable domain 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 shown in SEQ ID NO:33 or SEQ ID NO:34; optionally, the amino acid substitutions are not in the CDRs and / or the antigen binding protein retains the ability to bind to CD206.

[0140] In any of the embodiments of the second aspect, the first antigen binding protein comprises, consists essentially of, or consists of the amino acid sequences of SEQ ID NO: 33 and SEQ ID NO: 34 (in order from N-terminus to C-terminus or C-terminus to N-terminus).

[0141] In any embodiment of the second aspect, the first antigen binding protein described herein may have, from N-terminus to C-terminus, VH then VL, or VL then VH, or any CDR1, CDR2, and CDR3 defined herein as VH then any CDR1, CDR2, and CDR3 defined herein as VL, or any CDR1, CDR2, and CDR3 defined herein as VL then any CDR1, CDR2, and CDR3 defined herein as VH.

[0142] In any embodiment of the second aspect, the first antigen binding protein may be in the form of:

[0143] (i) Single domain antibodies (sdAb);

[0144] (ii) single-chain Fv fragment (scFv);

[0145] (iii) dimeric scFv (di-scFv); or

[0146] (iv) one of (ii) or (iii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody.

[0147] Further, as described herein, the first antigen binding protein may be in the form of:

[0148] (i) Diabodies;

[0149] (ii) three-chain antibodies;

[0150] (iii) tetrabodies;

[0151] (iv) Fab;

[0152] (v) F(ab')2;

[0153] (vi) Fv;

[0154] (vii) bispecific antibodies or other forms of multispecific antibodies;

[0155] (viii) one of (i) to (vii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody.

[0156] 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 (IgG) molecule. Alternatively, 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 heterodimer Fab-Fc, and the second antigen binding protein may be in the form of an antigen binding fragment (e.g., scFv) of an IgG.

[0157] In any aspect (including the first aspect and the second aspect), the second antigen binding protein is used to bind to the FLAG tag, and the second antigen binding protein comprises an antigen binding domain, and the antigen binding domain comprises:

[0158] FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4, and

[0159] FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0160] in:

[0161] FR1, FR2, FR3 and FR4 are each a framework region;

[0162] CDR1, CDR2, and CDR3 are each complementarity determining regions;

[0163] FR1a, FR2a, FR3a and FR4a are each a framework region;

[0164] CDR1a, CDR2a, and CDR3a are each complementarity determining regions;

[0165] Wherein, the sequence of any one of the framework region and / or the complementarity determining region is as described herein, preferably as described in Table 1 below.

[0166] In any embodiment, CDR1, CDR2 and CDR3 refer to the complementary determining region from the variable heavy chain (VH) of an antibody, and CDR1a, CDR2a and CDR3a are complementary determining regions from the variable light chain (VL) of an antibody, or in the case where CDR1, CDR2 and CDR3 are complementary determining regions from VL, CDR1a, CDR2a and CDR3a are complementary determining regions from VH. In such examples, CDRs may refer to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, depending on the situation.

[0167] In any embodiment of any aspect of the invention, the second antigen binding protein is capable of specifically binding to a FLAG tag or a variant thereof (e.g., as defined by SEQ ID NO:80 and SEQ ID NO:99 or as otherwise defined herein). The antigen binding protein of the invention is preferably capable of specifically binding to a protein domain comprising multiple FLAG tag sequences (e.g., 2×FLAG, 3×FLAG, etc.).

[0168] In any embodiment of any aspect of the present invention, the second antigen binding protein is used to specifically bind to a peptide tag, which contains the sequence DYK, preferably the sequence DYKD (SEQ ID NO: 80), or consists of it. In addition to these sequences, other amino acids may also be present in the FLAG tag to which the second antigen binding protein binds, preferably, wherein 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).

[0169] In a preferred embodiment of any aspect of the invention, the second antigen binding protein is capable of specifically binding to a FLAG tag that contains, comprises or consists of the following 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).

[0170] As used herein, the term FLAG tag also encompasses modified FLAG tags, such as those derived from the above-mentioned FLAG tags by amino acid insertions, deletions or substitutions, in particular those derived from a tag having the sequence DYKDDDDK.

[0171] Reference herein to a protein or antibody that "binds to" a FLAG tag provides literal support for a protein or antibody that "binds specifically to or specifically binds to" a FLAG tag.

[0172] In any embodiment, the FLAG tag is present at the N-terminus, C-terminus, or within the protein to which the bispecific polypeptide of the invention is capable of binding, binds to, or specifically binds.

[0173] In any embodiment of any aspect of the invention, the second antigen binding protein competitively inhibits binding of an antibody to the FLAG tag, the antibody:

[0174] - comprising a VH comprising the sequence shown in SEQ ID NO: 70, and a VL comprising the sequence shown in SEQ ID NO: 75;

[0175] - comprising a VH comprising the sequence shown in SEQ ID NO: 71, and a VL comprising the sequence shown in SEQ ID NO: 76;

[0176] - comprising a VH comprising the sequence shown in SEQ ID NO: 71, and a VL comprising the sequence shown in SEQ ID NO: 77;

[0177] - comprising a VH comprising the sequence shown in SEQ ID NO: 71, and a VL comprising the sequence shown in SEQ ID NO: 78;

[0178] - comprising a VH comprising the sequence shown in SEQ ID NO: 71, and a VL comprising the sequence shown in SEQ ID NO: 79;

[0179] - comprising a VH comprising the sequence shown in SEQ ID NO: 72, and a VL comprising the sequence shown in SEQ ID NO: 76;

[0180] - comprising a VH comprising the sequence shown in SEQ ID NO: 72, and a VL comprising the sequence shown in SEQ ID NO: 77;

[0181] - comprising a VH comprising the sequence shown in SEQ ID NO: 72, and a VL comprising the sequence shown in SEQ ID NO: 78;

[0182] - comprising a VH comprising the sequence shown in SEQ ID NO: 72, and a VL comprising the sequence shown in SEQ ID NO: 79;

[0183] - comprising a VH comprising the sequence shown in SEQ ID NO: 73, and a VL comprising the sequence shown in SEQ ID NO: 76;

[0184] - comprising a VH comprising the sequence shown in SEQ ID NO: 73, and a VL comprising the sequence shown in SEQ ID NO: 77;

[0185] - comprising a VH comprising the sequence shown in SEQ ID NO: 73, and a VL comprising the sequence shown in SEQ ID NO: 78;

[0186] - comprising a VH comprising the sequence shown in SEQ ID NO: 73, and a VL comprising the sequence shown in SEQ ID NO: 79;

[0187] - comprising a VH comprising the sequence shown in SEQ ID NO: 74, and a VL comprising the sequence shown in SEQ ID NO: 76;

[0188] - comprising a VH comprising the sequence shown in SEQ ID NO: 74, and a VL comprising the sequence shown in SEQ ID NO: 77;

[0189] - comprises a VH comprising the sequence shown in SEQ ID NO: 74, and a VL comprising the sequence shown in SEQ ID NO: 78; or

[0190] - comprises a VH comprising the sequence shown in SEQ ID NO:74, and a VL comprising the sequence shown in SEQ ID NO:79.

[0191] In any embodiment, the second antigen binding protein comprises CDRH1, CDRH2 and / or CDRH3 of an antigen binding domain having a variable heavy chain as defined in any one of SEQ ID NO:71 to SEQ ID NO:74.

[0192] In any embodiment, the second antigen binding protein comprises CDRL1, CDRL2 and / or CDRL3 of an antigen binding domain having a variable light chain as defined in any one of SEQ ID NO:76 to SEQ ID NO:79.

[0193] In any embodiment, the second antigen binding protein comprises CDR1, CDR2 and / or CDR3 of an antigen binding domain having a variable heavy chain as defined in any one of SEQ ID NO:71 to SEQ ID NO:74 and a variable light chain as defined in any one of SEQ ID NO:76 to SEQ ID NO:79.

[0194] In any embodiment of any aspect, the second antigen binding protein described herein comprises:

[0195] FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4–Linker-FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0196] Wherein, the sequence of any one of the framework region and / or the complementarity determining region is as described herein, preferably as described in Table 1 below.

[0197] As defined herein, a linker can be a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0198] In any embodiment of any aspect of the invention, the second antigen binding domain comprises an antigen binding domain comprising:

[0199] (a) and (b):

[0200] (a) a VH comprising 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 set forth in SEQ ID NO:81, and 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 set forth in SEQ ID NO:81 NO:82, or consists 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:82, a CDR3 that comprises or consists 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

[0201] (b) a VL comprising 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:90, and 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:90 91, or consists 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:91, and a CDR3 that comprises or consists 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:92;

[0202] or

[0203] (c) and (d):

[0204] (c) VH, which comprises a complementary determining region CDR1, which comprises 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 SEQ ID NO:112, or consists of it, and CDR2, which comprises 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 SEQ ID NO:112 : 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: 113, and a CDR3 that comprises, or consists 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; and

[0205] (d) a VL comprising 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, and 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: 120 : 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:121, and a CDR3 that comprises, or consists 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:92;

[0206] or

[0207] (e) and (f):

[0208] (e) a VH comprising 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 SEQ ID NO: 112, and 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: 111 or SEQ ID NO: 112 : 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: 113, and a CDR3 that comprises, or consists 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; and

[0209] (f) a VL comprising 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, and 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: 133 : 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:121, and a CDR3 that comprises, or consists 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:92;

[0210] or

[0211] (g) and (h):

[0212] (g) a VH comprising 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 SEQ ID NO: 156, and 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: 111 or SEQ ID NO: 156 : 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: 157, and a CDR3 that comprises, or consists 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; and

[0213] (h) a VL comprising 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, and 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: 120 : 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:121, and a CDR3 that comprises, or consists 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:92;

[0214] or

[0215] (i) and (j):

[0216] (i) a VH comprising 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 SEQ ID NO: 156, and 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: 111 or SEQ ID NO: 156 : 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: 157, and a CDR3 that comprises, or consists 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; and

[0217] (j) a VL comprising 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: 133, and 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: 133 : 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:121, and a CDR3 that comprises, or consists 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:92;

[0218] or

[0219] (k) and (l):

[0220] (k) a VH comprising 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, and 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: 126 : 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: 127, and a CDR3 that comprises, or consists 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; and

[0221] (l) a VL comprising 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, and 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: 120 : 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:121, and a CDR3 that comprises, or consists 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:92;

[0222] or

[0223] (m) and (n):

[0224] (m) a VH comprising 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, and 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: 126 : 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: 127, and a CDR3 that comprises, or consists 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; and

[0225] (n) a VL comprising 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: 133, and 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: 133 : 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:121, and a CDR3 that comprises, or consists 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:92;

[0226] or

[0227] (o) and (p):

[0228] (o) a VH comprising 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, and 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: 111 : 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: 132, and a CDR3 that comprises, or consists 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; and

[0229] (p) a VL comprising 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: 133, and 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: 133 : 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:121, and a CDR3 that comprises, or consists 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:92;

[0230] or

[0231] (q) and (r):

[0232] (q) a VH comprising 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, and 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: 126 : 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: 127, and a CDR3 that comprises, or consists 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; and

[0233] (r) a VL comprising 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: 133, and 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: 133 NO:121 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:121, or consists of a CDR3, wherein the CDR3 comprises or consists 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:92.

[0234] In an embodiment of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain comprising:

[0235] (a) and (b):

[0236] (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

[0237] (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;

[0238] or

[0239] (c) and (d):

[0240] (c) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 111 or SEQ ID NO: 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

[0241] (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;

[0242] or

[0243] (e) and (f):

[0244] (e) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 111 or SEQ ID NO: 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

[0245] (f) 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;

[0246] or

[0247] (g) and (h):

[0248] (g) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 111 or SEQ ID NO: 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

[0249] (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;

[0250] or

[0251] (i) and (j):

[0252] (i) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 111 or SEQ ID NO: 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

[0253] (j) VL, which comprises CDR1, said CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 133, CDR2, said CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 121, and CDR3, said CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 92;

[0254] or

[0255] (k) and (l):

[0256] (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

[0257] (1) VL, which comprises CDR1, wherein the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 120, CDR2, wherein the CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 121, and CDR3, wherein the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 92;

[0258] or

[0259] (m) and (n):

[0260] (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

[0261] (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;

[0262] or

[0263] (o) and (p):

[0264] (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

[0265] (p) 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;

[0266] or

[0267] (q) and (r):

[0268] (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

[0269] (r) VL, which comprises CDR1, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 133, CDR2, wherein the CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 121, and CDR3, wherein the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 92.

[0270] In any embodiment of any aspect of the present invention, FR1, FR2, FR3, and FR4 may refer to framework regions from the variable heavy chain (VH) of an antibody, FR1a, FR2a, FR3a, and FR4a may refer to framework regions from the variable light chain (VL) of an antibody, or in the case where FR1, FR2, FR3, and FR4 are framework regions from VL, FR1a, FR2a, FR3a, and FR4a are framework regions from VH. In such an example, FR may refer to FR H1, FR H2, FR H3, FR H4, FR L1, FR L2, FR L3, and FR L4 as the case may be.

[0271] In any embodiment of any aspect of the present invention, the second antigen-binding protein comprises an antigen-binding domain having FR H1, FR H2, FR H3, and / or FR H4 from the human germline, wherein the human germline is IGHV1-46*01 or IGHV7-4-1*02.

[0272] In any embodiment of any aspect of the present invention, the second antigen-binding protein comprises an antigen-binding domain having FR L1, FR L2, FR L3, and / or FR L4 from the human germline, wherein the human germline is IGKV2-30*01 or IGKV4-1*01.

[0273] In any embodiment of any aspect of the present invention, the second antigen-binding protein comprises an antigen-binding domain having FR H1, FR H2, FR H3, and / or FR H4 from the human germline, wherein the human germline is IGHV1-46*01 or IGHV7-4-1*02, and having FR L1, FR L2, FR L3, and / or FRL4 from the human germline, wherein the human germline is IGKV2-30*01 or IGKV4-1*01.

[0274] In any embodiment of any aspect of the present invention, the second antigen-binding protein comprises an antigen-binding domain that comprises a VH that is more than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% identical to that of a human, preferably, the percentage identity to a human is calculated as described in Example 2, and / or a VL that is more than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% identical to that of a human, preferably, the percentage identity to a human is calculated as described in Example 2.

[0275] In an embodiment of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain having: FR H1, FR H2, FR H3 and / or FR H4 having an antigen binding domain of a variable heavy chain as defined in any one of SEQ ID NO:71 to SEQ ID NO:74.

[0276] 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 having an antigen binding domain of a variable light chain as defined in any one of SEQ ID NO: 76 to SEQ ID No: 79.

[0277] 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 as defined in any one of SEQ ID NO:71 to SEQ ID NO:74, and FR1, FR2, FR3 and / or FR4 of an antigen binding domain having a variable light chain as defined in any one of SEQ ID NO:76 to SEQ ID NO:79.

[0278] In any embodiment of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain, wherein the antigen binding domain comprises a VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (a) above, and a VL comprising a CDR1, CDR2 and CDR3 as defined in any one of (b) above, and:

[0279] A. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, and SEQ ID NO: 137, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 97, respectively;

[0280] or

[0281] B. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 87, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 97, respectively;

[0282] or

[0283] C. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 87, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, and SEQ ID NO: 97, respectively;

[0284] or

[0285] D. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 87, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97, respectively;

[0286] or

[0287] E. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 87, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 96, and SEQ ID NO: 97, respectively;

[0288] or

[0289] F. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 97, respectively;

[0290] or

[0291] G. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, and SEQ ID NO: 97, respectively;

[0292] or

[0293] H. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, and SEQ ID NO:89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:96, and SEQ ID NO:97, respectively;

[0294] or

[0295] I. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 96, and SEQ ID NO: 97, respectively;

[0296] or

[0297] J. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 149, SEQ ID NO: 85, SEQ ID NO: 151, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 97, respectively;

[0298] or

[0299] K. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 149, SEQ ID NO: 85, SEQ ID NO: 151, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, and SEQ ID NO: 97, respectively;

[0300] or

[0301] L. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 149, SEQ ID NO: 85, SEQ ID NO: 151, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97, respectively;

[0302] or

[0303] M. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 149, SEQ ID NO: 85, SEQ ID NO: 151, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 96, and SEQ ID NO: 97, respectively;

[0304] or

[0305] N. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 152, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 97, respectively;

[0306] or

[0307] O. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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, which reference sequence is set forth in SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 152, and SEQ ID NO: 89, respectively, for VH FR and SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, and SEQ ID NO: 97, respectively, for VL FR;

[0308] or

[0309] P. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 152, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97, respectively;

[0310] or

[0311] Q. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 152, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 96, and SEQ ID NO: 97, respectively.

[0312] In any embodiment, the second antigen binding protein comprises an antigen binding domain, wherein the antigen binding domain comprises a VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (c) above, and a VL comprising a CDR1, CDR2 and CDR3 as defined in any one of (d) above, and:

[0313] A. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 118, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 127, respectively;

[0314] or

[0315] B. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 118, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 127, respectively;

[0316] or

[0317] C. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 127, respectively;

[0318] or

[0319] D. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, which comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence; for VH FR, the reference sequence is set forth in SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 127, respectively.

[0320] In any embodiment of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain, wherein the antigen binding domain comprises a VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (e) above, and a VL comprising a CDR1, CDR2 and CDR3 as defined in any one of (f) above, and:

[0321] A. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 118, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 97, respectively;

[0322] or

[0323] B. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 118, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, and SEQ ID NO: 97, respectively;

[0324] or

[0325] C. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 97, respectively;

[0326] or

[0327] D. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, which comprise 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, and SEQ ID NO: 97, respectively.

[0328] In any embodiment of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain comprising a VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (g) above, and a VL comprising a CDR1, CDR2 and CDR3 as defined in any one of (h) above, and:

[0329] A. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 158, SEQ ID NO: 116, SEQ ID NO: 159, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 127, respectively;

[0330] or

[0331] B. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 158, SEQ ID NO: 116, SEQ ID NO: 159, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 127, respectively;

[0332] or

[0333] C. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 158, SEQ ID NO: 117, SEQ ID NO: 160, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 127, respectively;

[0334] or

[0335] D. VH and VL each contain framework regions (FR) 1, 2, 3, and 4, and the framework regions (FR) 1, 2, 3, and 4 contain sequences that 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%, at least 99% identical or 100% identical to a reference sequence; for VH FR, the reference sequences are shown as SEQ ID NO:158, SEQ ID NO:117, SEQ ID NO:160, and SEQ ID NO:89, respectively, and for VL FR, the reference sequences are shown as SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, and SEQ ID NO:127, respectively.

[0336] In any embodiment of any aspect of the present invention, the second antigen-binding protein comprises an antigen-binding domain, wherein the antigen-binding domain comprises a VH that comprises complementarity-determining regions (CDR) 1, CDR2, and CDR3 as defined in any one of (i) above, and a VL that comprises CDR1, CDR2, and CDR3 as defined in any one of (j) above, and:

[0337] A. VH and VL each contain framework regions (FR) 1, 2, 3, and 4, and the framework regions (FR) 1, 2, 3, and 4 contain sequences that 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%, at least 99% identical or 100% identical to a reference sequence; for VH FR, the reference sequences are shown as SEQ ID NO:158, SEQ ID NO:116, SEQ ID NO:159, and SEQ ID NO:89, respectively, and for VL FR, the reference sequences are shown as SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, and SEQ ID NO:97, respectively;

[0338] Or

[0339] B. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 158, SEQ ID NO: 116, SEQ ID NO: 159, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, and SEQ ID NO: 97, respectively;

[0340] or

[0341] C. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 158, SEQ ID NO: 117, SEQ ID NO: 160, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 97, respectively;

[0342] or

[0343] D. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, which comprise 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 158, SEQ ID NO: 117, SEQ ID NO: 160, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, and SEQ ID NO: 97, respectively.

[0344] In any embodiment of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain, wherein the antigen binding domain comprises a VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (k) above, and a VL comprising a CDR1, CDR2 and CDR3 as defined in any one of (l) above, and:

[0345] A. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 128, SEQ ID NO: 130, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 97, respectively;

[0346] or

[0347] B. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO:84, SEQ ID NO:128, SEQ ID NO:130, and SEQ ID NO:89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, and SEQ ID NO:97, respectively;

[0348] or

[0349] C. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 84, SEQ ID NO: 129, SEQ ID NO: 131, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 97, respectively;

[0350] or

[0351] D. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO:84, SEQ ID NO:129, SEQ ID NO:131, and SEQ ID NO:89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, and SEQ ID NO:97, respectively;

[0352] or

[0353] E. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 164, SEQ ID NO: 128, SEQ ID NO: 166, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 97, respectively;

[0354] or

[0355] F. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 164, SEQ ID NO: 128, SEQ ID NO: 166, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 97, respectively;

[0356] or

[0357] G. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 97, respectively;

[0358] or

[0359] H. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, which comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence; for VH FR, the reference sequence is set forth in SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 97, respectively.

[0360] In any embodiment of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain, wherein the antigen binding domain comprises a VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (m) above, and a VL comprising a CDR1, CDR2 and CDR3 as defined in any one of (n) above, and:

[0361] A. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 128, SEQ ID NO: 130, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 97, respectively;

[0362] or

[0363] B. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO:84, SEQ ID NO:128, SEQ ID NO:130, and SEQ ID NO:89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, and SEQ ID NO:97, respectively;

[0364] or

[0365] C. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO:84, SEQ ID NO:129, SEQ ID NO:131, and SEQ ID NO:89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, and SEQ ID NO:97, respectively;

[0366] or

[0367] D. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO:84, SEQ ID NO:129, SEQ ID NO:131, and SEQ ID NO:89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, and SEQ ID NO:97, respectively;

[0368] or

[0369] E. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 164, SEQ ID NO: 128, SEQ ID NO: 166, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 97, respectively;

[0370] or

[0371] F. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 164, SEQ ID NO: 128, SEQ ID NO: 166, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, and SEQ ID NO: 97, respectively;

[0372] or

[0373] G. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4, said framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 97, respectively;

[0374] or

[0375] H. VH and VL each comprise framework regions (FR) 1, 2, 3, and 4 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, and SEQ ID NO: 97, respectively.

[0376] In any embodiment of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain, wherein the antigen binding domain comprises:

[0377] A. A VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (o) above, and a VL comprising CDR1, CDR2 and CDR3 as defined in any one of (p) above, and VH and VL each comprising a framework region (FR) 1, 2, 3 and 4 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; for VH FR, the reference sequence is SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO:143 and SEQ ID NO:137, and for VLFR, the reference sequences are shown in SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146 and SEQ ID NO:97, respectively;

[0378] or

[0379] B. A VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (q) above, and a VL comprising CDR1, CDR2 and CDR3 as defined in any one of (r) above, and VH and VL each comprising a framework region (FR) 1, 2, 3 and 4 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; for VH FR, the reference sequence is SEQ ID NO: 84, SEQ ID NO: 147, SEQ ID NO: 148 and SEQ ID NO: 150, respectively. ID NO:137, and for VLFR, the reference sequences are shown in SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146 and SEQ ID NO:97, respectively.

[0380] 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 of any one of SEQ ID NO:71 to SEQ ID NO:74, 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%, at least 99% identical to said amino acid sequence. 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 shown in SEQ ID NO:71 to SEQ ID NO:74; optionally, the amino acid substitution is not in the CDR and / or the antigen-binding protein retains the ability to bind to the FLAG tag.

[0381] In embodiments of any aspect of the invention, the second antigen binding protein comprises an antigen binding domain comprising a variable light chain comprising the amino acid sequence of any one of SEQ ID NO:76 to SEQ ID NO:79, 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%, 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 shown in SEQ ID NO:76 to SEQ ID NO:79; optionally, the amino acid substitution is not in the CDR and / or the antigen-binding protein retains the ability to bind to the FLAG tag.

[0382] 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 shown in any one of SEQ ID NO:71 to SEQ ID NO:74, and a variable light chain comprising the amino acid sequence shown in any one of SEQ ID NO:76 to SEQ ID NO:79, or 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 said amino acid sequence. Optionally, compared with the amino acid sequences shown in SEQ ID NO:71 to SEQ ID NO:74 and SEQ ID NO:76 to SEQ ID NO:79, the heavy chain variable domain and light chain variable domain of the second antigen-binding protein respectively contain 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; optionally, the amino acid substitutions are not in the CDR and / or the antigen-binding protein retains the ability to bind to the FLAG tag.

[0383] As described herein, the second antigen binding protein may be in the form of:

[0384] (i) Single domain antibodies (sdAb);

[0385] (ii) single-chain Fv fragment (scFv);

[0386] (iii) dimeric scFv (di-scFv);

[0387] (iv) one of (ii) or (iii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody;

[0388] (v) one of (i) to (iv) linked to a protein that binds to immune effector cells;

[0389] (vi) one of (i) to (iv) linked to a modified immune cell receptor (eg, a modified T cell receptor).

[0390] Further, as described herein, the second antigen binding protein may be in the form of:

[0391] (i) Diabodies;

[0392] (ii) three-chain antibodies;

[0393] (iii) tetrabodies;

[0394] (iv) Fab;

[0395] (v) F(ab')2;

[0396] (vi) Fv;

[0397] (vii) bispecific antibodies or other forms of multispecific antibodies;

[0398] (viii) one of (i) to (vii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody.

[0399] In a preferred embodiment of any aspect of the invention, the second antigen binding protein is in the form of a scFv and the first antigen binding protein is in the form of an immunoglobulin G (IgG) antibody. In an alternative embodiment, the second antigen binding protein may be an antibody or antibody fragment having the same form as the first antigen binding protein. Alternatively, the first antigen binding protein and the second antigen binding protein are different forms of antigen binding proteins.

[0400] In any embodiment of the first aspect or the second aspect of the present invention, the second binding domain can be in the form of an antibody or an antigen binding fragment thereof. The antigen binding protein can be an antibody, such as a monoclonal antibody. The antigen binding protein can be in the form of a recombinant antibody or a modified antibody (e.g., a chimeric antibody, a humanized antibody, a humanized antibody, a CDR-transplanted antibody, a primatised antibody, a deimmunised antibody, a humanised antibody, a half-antibody, a bispecific antibody, a trispecific antibody or a multispecific antibody). The antibody can further include chemical modifications, such as coupling with an active agent or a radioactive marker or a reagent for improving solubility, or other modifications described herein. As used herein, the antigen binding protein can be a variable domain.

[0401] In any embodiment of any aspect of the invention, the second antigen binding junction protein comprises, consists essentially of, or consists of the following amino acid sequence (in order from N-terminus to C-terminus or from C-terminus to N-terminus):

[0402] - SEQ ID NO:71 and SEQ ID NO:79;

[0403] - SEQ ID NO:71 and SEQ ID NO:78;

[0404] - SEQ ID NO:71 and SEQ ID NO:76;

[0405] - SEQ ID NO:71 and SEQ ID NO:77;

[0406] - SEQ ID NO:72 and SEQ ID NO:76;

[0407] - SEQ ID NO:72 and SEQ ID NO:77;

[0408] - SEQ ID NO:72 and SEQ ID NO:78;

[0409] - SEQ ID NO:72 and SEQ ID NO:79;

[0410] - SEQ ID NO:73 and SEQ ID NO:76;

[0411] - SEQ ID NO:73 and SEQ ID NO:77;

[0412] - SEQ ID NO:73 and SEQ ID NO:78;

[0413] - SEQ ID NO:73 and SEQ ID NO:79;

[0414] - SEQ ID NO:74 and SEQ ID NO:76;

[0415] - SEQ ID NO:74 and SEQ ID NO:77;

[0416] - SEQ ID NO:74 and SEQ ID NO:78; or

[0417] - SEQ ID NO:74 and SEQ ID NO:79.

[0418] As used herein, the complementarity determining region (CDR) sequences of the antigen binding proteins of the invention are defined according to the IMGT, Chothia or Kabat numbering systems, or any other CDR numbering system.

[0419] In any embodiment of any aspect of the invention, the bispecific polypeptide comprises, consists essentially of, or consists of the following amino acid sequence:

[0420] - SEQ ID NO: 19 and SEQ ID NO: 20;

[0421] - SEQ ID NO:19 and SEQ ID NO:21;

[0422] - SEQ ID NO:22 and SEQ ID NO:23;

[0423] - SEQ ID NO:23 and SEQ ID NO:24;

[0424] - SEQ ID NO:19 and SEQ ID NO:25;

[0425] - SEQ ID NO:19 and SEQ ID NO:26;

[0426] - SEQ ID NO:23 and SEQ ID NO:27;

[0427] - SEQ ID NO:23 and SEQ ID NO:28;

[0428] - SEQ ID NO:19 and SEQ ID NO:29;

[0429] - SEQ ID NO:19 and SEQ ID NO:30;

[0430] - SEQ ID NO:23 and SEQ ID NO:31;

[0431] - SEQ ID NO:23 and SEQ ID NO:32;

[0432] - SEQ ID NO:171, SEQ ID NO:172 and SEQ ID NO:173;

[0433] - SEQ ID NO:172, SEQ ID NO:174 and SEQ ID NO:175;

[0434] - SEQ ID NO:171, SEQ ID NO:172 and SEQ ID NO:176;

[0435] - SEQ ID NO:172, SEQ ID NO:174 and SEQ ID NO:177;

[0436] - SEQ ID NO:50 and SEQ ID NO:51;

[0437] - SEQ ID NO:50 and SEQ ID NO:52;

[0438] - SEQ ID NO:53 and SEQ ID NO:54;

[0439] - SEQ ID NO:54 and SEQ ID NO:55;

[0440] - SEQ ID NO:50 and SEQ ID NO:56;

[0441] - SEQ ID NO:50 and SEQ ID NO:57;

[0442] - SEQ ID NO:54 and SEQ ID NO:58;

[0443] - SEQ ID NO:54 and SEQ ID NO:59;

[0444] - SEQ ID NO: 50 and SEQ ID NO: 60;

[0445] - SEQ ID NO:50 and SEQ ID NO:61;

[0446] - SEQ ID NO:54 and SEQ ID NO:62; or

[0447] - SEQ ID NO: 54 and SEQ ID NO: 63.

[0448] In any of the above embodiments, the bispecific polypeptide comprises, consists essentially of, or consists of one, two, or three polypeptide chains, wherein the one, two, or three polypeptide chains are connected by a covalent linker (e.g., any linker described herein, including (multiple) amino acids or disulfide bonds). As described herein, the bispecific polypeptide can be heterodimeric.

[0449] In a third aspect, the present invention provides a fusion protein comprising a first antigen-binding protein according to the first aspect or the second aspect of the present invention, and a second antigen-binding protein defined according to the first aspect or the second aspect of the present invention. The present invention also provides a fusion protein comprising a bispecific polypeptide defined herein according to the first aspect or the second aspect of the present invention.

[0450] In a fourth aspect, the present invention provides a nucleic acid encoding the bispecific polypeptide of the first or second aspect of the present invention or a portion thereof or the fusion protein of the third aspect. Preferably, the nucleic acid has a nucleotide sequence encoding any one or more amino acid sequences corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 17 to SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 49 to SEQ ID NO: 63, SEQ ID NO: 64 to SEQ ID NO: 67, SEQ ID NO: 71 to SEQ ID NO: 74, SEQ ID NO: 76 to SEQ ID NO: 79 and SEQ ID NO: 171 to SEQ ID NO: 177.

[0451] In any of the embodiments of the fourth aspect of the invention, the nucleic acid may be DNA (eg, cDNA) or RNA (eg, mRNA).

[0452] In a fifth aspect, the present invention provides a vector comprising the nucleic acid of the fourth aspect.

[0453] In aspects of the invention involving multiple polypeptide chains forming a bispecific polypeptide or antigen binding protein, the expression construct comprises a nucleic acid encoding a polypeptide comprising, for example, VH, operably linked to a promoter, and a nucleic acid encoding a polypeptide comprising, for example, VL, operably linked to a promoter.

[0454] In another example, the expression construct is a bicistronic expression construct, for example, comprising the following components operably linked in 5' to 3' order:

[0455] (i) a promoter;

[0456] (ii) a nucleic acid encoding a first polypeptide;

[0457] (iii) an internal ribosome entry site; and

[0458] (iv) a nucleic acid encoding a second polypeptide,

[0459] Therein, the first polypeptide comprises VH and the second polypeptide comprises VL, or vice versa.

[0460] The present invention also contemplates separate expression constructs, wherein one expression construct encodes a first polypeptide comprising VH and another expression construct encodes a second polypeptide comprising VL. For example, the present invention also provides a composition comprising:

[0461] (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising VH operably linked to a promoter; and

[0462] (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising VL operably linked to a promoter.

[0463] In a sixth aspect, the 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 of the invention, or:

[0464] (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising VH operably linked to a promoter; and

[0465] (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising VL operably linked to a promoter,

[0466] Wherein, the first polypeptide and the second polypeptide associate to form the antigen binding protein described herein.

[0467] Examples of the cells of the present invention include bacterial cells, yeast cells, insect cells or mammalian cells.

[0468] In another embodiment, an animal comprising the cell described herein or a tissue derived from the animal is provided.

[0469] In the seventh aspect, the present invention provides a pharmaceutical composition comprising the bispecific polypeptide of the first aspect or the second aspect, the fusion protein of the third aspect, the nucleic acid of the fourth aspect, the vector of the fifth aspect or the cell of the sixth aspect, and a pharmaceutically acceptable carrier, diluent or excipient.

[0470] In an eighth aspect, the present invention provides a method for producing the bispecific polypeptide of the first aspect or the second aspect or the fusion protein of the third aspect, the method comprising expressing the nucleic acid of the fourth aspect in a cell or an animal as described herein.

[0471] In the ninth aspect, the present invention provides use of the bispecific polypeptide of the first aspect or the second aspect, the fusion protein of the third aspect, the nucleic acid of the fourth aspect, the vector of the fifth aspect or the cell of the sixth aspect in the preparation of a medicament for treating or preventing cancer.

[0472] In the tenth aspect, the present invention provides a method for treating or preventing cancer, the method comprising administering the bispecific polypeptide of the first aspect or the second aspect, the fusion protein of the third aspect, the nucleic acid of the fourth aspect, the vector of the fifth aspect, or the cell of the sixth aspect to a subject, thereby treating or preventing cancer.

[0473] The antigen binding proteins described herein may be purified, substantially purified, isolated and / or recombinant.

[0474] The bispecific polypeptide of the invention may be part of a supernatant taken from a culture medium in which a hybridoma expressing a bispecific polypeptide of the invention has been grown.

[0475] As used herein, the term "comprise" and variations of the term (eg, "includes," "containing," and "comprising") are not intended to exclude other additives, components, integers or steps, unless the context requires otherwise.

[0476] 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 with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0477] Figure 1 : ELISA titer of IgG humanized FLAG tag binding monoclonal antibody.

[0478] Figure 2 : Humanized monoclonal antibodies bind to FLAG-tagged CAR to induce CAR T cells to secrete IFN-γ.

[0479] Figure 3 : Alignment of the VH and VL regions from the parent antibody and the humanized variants v4 and v7.

[0480] Figure 4 : Schematic diagram of BEAT changes in IgG domain and scFv domain.

[0481] Figure 5 : Single parameter sensorgram of CD40 binding to BEAT 1a4 (F7-G8) in the presence of 50 nM FLAG-BAP, showing CD40 injected in the presence of 50 nM FLAG-BAP after binding and saturation at 50 nM FLAG-BAP. CD40 concentrations are 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. A fit to a 1:1 binding model is shown.

[0482] Figure 6: Binding of anti-CD40 (A) and anti-CD206 (B) BEAT to monocyte-derived DCs.

[0483] Figure 7 : (A) Dose-dependent binding of BEAT to CD40 on human MoDC cells between 0.1 nM and 100 nM BEAT 1a4; (B) Dose-dependent binding of BEAT to CD40 on human B cells between 0.1 nM and 100 nM BEAT 1a4; (C) Dose-dependent binding of BEAT to CD40 on non-human primate MoDC cells between 0.1 nM and 100 nM BEAT 1a4; (D) Dose-dependent binding of BEAT to CD40 on non-human primate B cells between 0.1 nM and 100 nM BEAT 1a4.

[0484] Figure 8 : BEAT 1 (and CD40 IgG2 control) increased CD86 expression (A) and IL-12 / IL-23p40 release (B) in a dose-responsive manner between 3 nM and 30 nM (CD86) and 1 nM and 60 nM (IL-12).

[0485] Fig. 9 : (A) BEAT binds to the FLAG tag on the 7B1 anti-mesothelin CD28z CAR and increases IFN-γ. (B) MoDCs express CD40 and CD206. (C) Binding of BEAT1a4 to CAR T cells was assessed by FACS detection of the FLAG tag by GPF+ / AF647+ cells.

[0486] Fig.10 : Cytokine analysis of BEAT from one donor.

[0487] Fig.11 : Proliferation of CART cells after 120 hours of proliferation with the addition of BEAT in the presence of control compound / BEAT (30 nM) (with or without MoDc).

[0488] Fig.12 : Fig.11 Further phenotypes of CD4+, CD8+ and CD3+ as described in.

[0489] Fig.13 : Cytotoxicity of BEAT+CAR T cells in SKOV-3MSLN-positive cell line.

[0490] Fig.14: In vivo efficacy of CAR T+BEAT 1a5 in the SKOV3-mesothelin-positive model transfected with CD40 knockout cells. (A) Schematic experimental timeline. (B) Survival curve. (C) Tumor size.

[0491] Fig.15 : Schematic diagram of BEAT 3, BEAT 4, BEAT 5 and BEAT 6.

[0492] Fig.16 : ELISA binding assay of anti-CD40 anti-FLAG BEAT 3, 4, 5 and 6 to CD40 antigen.

[0493] Fig.17 : Binding of BEAT 1, BEAT 3, BEAT 4, BEAT 5, and BEAT 6 to 7B1 anti-mesothelin CD28z CAR human T cells (A) and human monocyte-derived DCs (B).

[0494] Fig.18 : CD86 (A) and IL-12 / IL-23p40 (B) were upregulated on MoDCs with the addition of BEAT 1, BEAT 3, BEAT 4, BEAT5, and BEAT 6.

[0495] Fig.19 : Proliferation of CAR T cells after 5 days of proliferation with the addition of BEAT1, BEAT 3, BEAT 4, BEAT 5 and BEAT 6 in the presence of control compound / BEAT (30 nM) (with or without MoDc) and CAR T cells.

[0496] Fig. 20 : Fig.15 Further phenotype of CD3+ T cells as described in.

[0497] Fig.21 : Fig.15 Further phenotype of CD4+ T cells as described in.

[0498] Fig. 22 : Fig.15 Further phenotype of CD8+ T cells as described in.

[0499] Fig.23 : Cytokine activities of BEAT 1, BEAT 3, BEAT 4, BEAT 5 and BEAT 6 under CAR T cells or MoDC or CAR+MoDC, including IFN-γ (A), IL12-p70 (B), IL-6 (C), IL-2 (D), MCP-1 (E), TNF-α (F).

[0500] Fig.24: In vivo efficacy of CAR T cells alone compared to CAR T + BEAT2a6 (anti-CD206 BEAT) in the SKOV3 mesothelin model with detailed description of tumor size.

[0501] Sequence Description

[0502] Table 1 Sequences of the present invention

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529] DETAILED DESCRIPTION

[0530] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned in this document or in the accompanying drawings or evident from this document or in the accompanying drawings. All these different combinations constitute various alternative aspects of the invention.

[0531] Further aspects of the invention and further embodiments of the various aspects described in the preceding paragraphs will become apparent from the following description given by way of example and with reference to the accompanying drawings.

[0532] Reference will now be made in detail to specific embodiments of the present invention. Although the present invention will be described in conjunction with the embodiments, it should be understood that it is intended not to limit the present invention to these embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications and equivalents that may be included within the scope of the present invention as defined in the claims.

[0533] The inventors have developed bispecific polypeptides, such as antibodies, which bind to CD40 on antigen presenting cells and FLAG tags present on molecules expressed on immune cells (e.g., T cells), or bind to CD206 on antigen presenting cells and FLAG tags present on molecules expressed on immune cells (e.g., T cells). These bispecific polypeptides allow immune cells (e.g., CAR T cells) expressing engineered TCR to engage with antigen presenting cells (APCs), thereby enhancing the efficacy of immune cells in vitro and in vivo, highly inhibiting tumor formation without toxicity, while reducing the effective dose required for immune cells (e.g., CAR T cells) expressing engineered TCR to achieve therapeutic effects. Importantly, immune cells (e.g., CAR T cells) expressing engineered TCR receive activation and proliferation signals in lymphoid tissues away from the immunosuppressive tumor microenvironment.

[0534] Bispecific peptides

[0535] As used herein, the term "bispecific polypeptide" means a polypeptide that can specifically bind to two different target antigens at the same time. The bispecific polypeptide described herein includes two structurally different binding proteins or domains (ie, regions), each of which specifically binds to a single target antigen. Bispecific polypeptides can be used to bind to target antigens (eg, CD40 or CD206) on APC and to different target antigens on immune cells expressing engineered TCR (eg, CARs comprising FLAG tags). Further, the bispecific polypeptides of the present invention can be used to bind to target antigens on APC and to different target antigens on engineered TCRs (eg, CARs) expressed by immune cells. Bispecific polypeptides can include polypeptide sequences (ie, domains) of one or more antibodies or antibody fragments (eg, one or more scFvs or IgG). In another embodiment, the bispecific polypeptide may include the polypeptide sequence of one or more ligands.

[0536] In any 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 can be in the form of a fusion protein comprising an immunoglobulin for binding to a first antigen and one or more scFvs fused to the immunoglobulin for binding to a second antigen. As further described herein, it should be understood that the bispecific polypeptide of the present invention can be in various forms, wherein the first antigen-binding protein and the second antigen-binding protein comprise an antigen-binding domain, and wherein the protein can be any of the various forms derived from antibodies.

[0537] The bispecific polypeptides described herein may be referred to variously as "Bispecific Engagers of APCs and T cells" or "BEATs."

[0538] "Polypeptide", "peptide", "protein" and "protein molecule" are used interchangeably herein to refer to a polymer comprising or consisting of amino acid residues, as well as variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0539] As used herein, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are the amino acids encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, such as a carbon bound to a hydrogen, carboxyl, amino, and R groups. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function similarly to naturally occurring amino acids.

[0540] Amino acids may be referred to herein by their commonly used full names (e.g., cysteine), by their well-known three letter symbols (e.g., Cys), or by the single letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission (e.g., C). Likewise, nucleotides may also be referred to by their generally accepted single-letter codes.

[0541] As used herein, the term "antigen" refers to a molecule bound by an "antibody", "antibody fragment" or "bispecific polypeptide". An antigen can 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 can be a CD40, CD206 or FLAG tag. In another embodiment, the antigen is a tag on an engineered TCR (e.g., CAR) expressed by an immune cell.

[0542] As used herein, the term "antigen presenting cell" or "APC" can be a professional antigen presenting cell (e.g., dendritic cell, macrophage, B cell, epithelial cell, etc.) or a non-professional antigen presenting cell (e.g., fibroblast, thymic epithelial cell, thyroid epithelial cell, glial cell, pancreatic β cell, vascular endothelial cell, etc.). In a preferred embodiment, the APC is an endogenous professional antigen binding cell (preferably, a dendritic cell or macrophage) and is not a tumor cell.

[0543] 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 lymphoblasts (e.g., C1R.A2, 1518B-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), wherein 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, it should be understood that in the context of using the bispecific polypeptides of the present invention, it is intended that the first antigen binding polypeptide be used to bind to CD40 or CD206 on non-tumor endogenous professional antigen presenting cells such as DCs, moDCs, etc.

[0544] The term "antigen presentation" refers to the process by which an antigen is captured by an APC and made available for recognition by a T cell, for example, as a component of an MHC-I and / or MHC-II conjugate.

[0545] "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 primarily processed for binding to MHC class II. In contrast, antigens delivered endogenously to APCs are primarily processed for binding to MHC class I.

[0546] In any embodiment, the second antigen binding protein specifically binds to an antigen on an immune cell expressing an engineered TCR (e.g., CAR) or similar receptor. In a further embodiment, the second antigen binding protein of the bispecific polypeptide of the invention can bind to an antigen on an immune cell, wherein the antigen is not part of the engineered TCR, but is an antigen present on the cell surface of the immune cell.

[0547] In a specific embodiment, immune cells are generally engineered T cells or NK cells, and generally, wherein the antigen on the engineered T cells or NK cells is a part of an engineered TCR (e.g., CAR). According to these embodiments, the second antigen-binding protein of the bispecific polypeptide of the present invention can be combined with the extracellular part of the engineered TCR. The second antigen-binding protein can be combined with the antigen-binding domain of the engineered TCR, or the second antigen-binding protein can be combined with the extracellular region of the engineered TCR that does not participate in antigen binding.

[0548] In certain embodiments, the engineered TCR (e.g., CAR) present on the immune cell may also include additional amino acids or molecules for binding to a second antigen-binding protein. As known in the art, engineered TCR (e.g., CAR) constructs can be designed to include a "tag", which is typically a short amino acid sequence specifically recognized by an antibody. In some embodiments, immune cells are engineered to express engineered TCR (e.g., CAR) comprising a tag T cell or NK cell. In the context of this embodiment, the second antigen-binding protein of the bispecific polypeptide can be combined with a tag.

[0549] In any 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 (e.g., as defined by SEQ ID NO:80 and SEQ ID NO: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., 2×FLAG, 3×FLAG, etc.).

[0550] In any embodiment, the second antigen binding protein is used to specifically bind to a peptide tag comprising or consisting of the sequence DYK, preferably the sequence DYKD (SEQ ID NO: 80). In addition to these sequences, other amino acids may also 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).

[0551] 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 comprising or consisting of the following 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).

[0552] As used herein, the term FLAG tag also refers to a modified FLAG tag, which is derived from the above FLAG tag, particularly the tag having the sequence DYKDDDDK, by amino acid insertion, deletion, or substitution.

[0553] The mention herein of a protein or antibody that "binds to" a FLAG tag provides textual support for a protein or antibody that "binds specifically to" or "specifically binds to" the FLAG tag.

[0554] In any embodiment, the FLAG tag is present at the N-terminus, C-terminus, or within the protein to which the tag is intended to bind.

[0555] 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 are familiar with the general structure and use of engineered TCRs (e.g., CARs), and more specifically, with CAR T and CAR NK cells. For example and as known in the art, a CAR is a cell surface receptor that comprises a combination of an extracellular domain, a transmembrane domain, and a cytoplasmic domain that is not naturally present in a single protein. The extracellular domain comprises an antigen-binding domain, which can be an antibody or an antibody fragment. The antibody or antibody fragment can 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, such as a Fab or an scFv. Most typically, the antigen-binding domain of a CAR is an scFv. The extracellular domain also typically comprises a spacer (or hinge region) that links the antigen-binding domain to the transmembrane domain. The spacer of a CAR can be derived from an immunoglobulin, such as IgG1 or IgG4, or the spacer can be derived from an alternative cell surface protein, including but not limited to CD4, CD8, or CD28.

[0556] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide that comprises 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 (i.e., specifically targets) an antigen (i.e., a "tumor-associated antigen") expressed on a cancer cell. Examples of tumor-associated antigens are known to those skilled in the art, and illustrative examples include mesothelin, Her2, CEA, FBP, CD19, and BCMA.

[0557] 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., when tumor-associated antigens are specific to tumor cells), tumor-associated antigens may also be referred to as "tumor-specific antigens". When tumor-associated antigens are not specific to tumor cells, tumor-associated antigens may also be expressed on non-tumor cells under conditions that fail to induce an immune tolerance state to the antigen. The expression of antigens on tumors may occur under conditions that enable 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 tumor-associated antigens may be antigens that are usually present at low levels on normal cells but expressed at much higher levels on tumor cells. Compared with corresponding non-tumor tissues, those tumor-associated antigens of greatest clinical interest are differentially expressed, and allow specific T cells or immunoglobulins to preferentially recognize tumor cells.

[0558] It will be clear from the remainder of this disclosure that the first antigen-binding protein and the second antigen-binding protein of the bispecific polypeptide of the present invention are generally in the form of antibodies or antibody fragments. As used herein, the term "antibody" refers generally to any immunoglobulin (Ig) molecule consisting of four polypeptide chains (two heavy (H) chains and two light (L) chains). Also contemplated herein are bispecific polypeptides comprising antigen-binding fragments, mutants, variants, and derivatives thereof, which retain the basic epitope binding characteristics of antibody molecules. Such mutants, variants, and derivatives are well known to those skilled in the art, and illustrative examples thereof are described elsewhere herein.

[0559] The heavy chain of an antibody generally comprises a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region generally consists of three domains: CH1, CH2 and CH3. The light chain generally comprises a light chain variable region (LCVR or VL) and a light chain constant region (CL). The VH region and the VL region can be further subdivided into hypervariable regions, also known as complementarity determining regions (CDRs), which are interlaced with the framework region (FR). Each VH and VL generally consists of three CDs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

[0560] As used herein, the term "antigen-binding fragment" or "antibody fragment" means one or more antibody fragments of an antibody that retain the ability to specifically bind to a target antigen. Illustrative examples of antigen-binding fragments include (i) Fab fragments, a monovalent fragment consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) F(ab') 2 Fragments include: (i) a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (ii) a Fd fragment consisting of a VH domain and a CH1 domain; (iii) a single-chain variable fragment (scFv) consisting of the VL domain and the VH domain of a single arm of an antibody; (iv) a dAb fragment (Ward et al., 1989, Nature, 341:544-6), which comprises a single variable domain; and (vi) isolated CDRs.

[0561] In any embodiment of the first aspect or the 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 can be an oligomeric form, wherein two polypeptide chains are connected or combined, and the second antigen-binding protein can be a single polypeptide chain. For example, the first antigen-binding protein can be an IgG form (e.g., a heavy chain and a light chain are connected or combined), and the second antigen-binding protein can be a scFv form (e.g., a single continuous polypeptide chain).

[0562] In any embodiment of the first aspect or the second aspect, the first antigen binding protein may comprise or consist of VH and VL as defined herein, and VH and VL may be arranged as VL-VH or VH-VL in order from N-terminus to C-terminus.

[0563] In any embodiment of the first aspect or the second aspect, the second antigen binding protein may comprise or consist of VH and VL as defined herein, and VH and VL may be arranged as VL-VH or VH-VL in order from N-terminus to C-terminus.

[0564] In any embodiment of the first aspect or the second aspect, the second antigen binding protein can be connected 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 can be connected to the N-terminus of the VH or VL of the first antigen binding protein. In one embodiment, the second antigen binding protein is connected 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.

[0565] In any embodiment of the first aspect or the second aspect, the second antigen binding protein can be connected 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 can be connected to the C-terminus of the VH or VL of the first antigen binding protein. In one embodiment, the second antigen binding protein is connected 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.

[0566] As used herein, the term "sequence identity" or "sequence homology" refers to the subunit sequence identity between two polymer molecules (e.g., between two nucleic acid molecules, such as two DNA molecules or two RNA molecules) or between two polypeptide molecules. When a subunit position in the two molecules is occupied by the same monomer subunit, for example, if one position in each of the two DNA molecules is occupied by adenine, the two molecules are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching positions or homologous positions, for example, if half of the positions in the two sequences (e.g., 5 positions in a polymer of 10 subunits in length) are homologous, the two sequences are 50% homologous; if 90% of the positions (i.e., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

[0567] Heterodimerization

[0568] A "CH3 domain" comprises an extension from the 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). In the bispecific polypeptides (e.g., antibodies) of the invention described herein, one corresponding CH3 domain may be arranged at the C-terminus of the VH3 domain and the VL3 domain of the third binding site. The "CH3 domain" described herein may be a variant CH3 domain, wherein the amino acid sequence of the native CH3 domain is substituted with at least one different amino acid (i.e., a modification of the amino acid sequence of the CH3 domain) to promote dimerization of two CH3 domains facing each other within the multispecific antibody.

[0569] Several methods for CH3 modification to support heterodimerization have been described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291, the disclosures of which are incorporated herein by reference in their entirety.

[0570] Typically, in heterodimerization methods known in the art, the CH3 domain of one heavy chain and the CH3 domain of another heavy chain are engineered in a complementary manner so that a heavy chain comprising an engineered CH3 domain can no longer homodimerize with another heavy chain having the same structure. Thus, a heavy chain comprising an engineered CH3 domain is forced to heterodimerize with another heavy chain comprising a CH3 domain engineered in a complementary manner.

[0571] One heterodimerization method known in the art is the so-called "knobs-into-holes" technology, and several examples are provided in the following documents to describe the technology in detail: WO 96 / 027011; Ridgway JB et al., Protein Eng., 9 (1996) 617-621; Merchant AM et al., Nat. Biotechnol., 16 (1998) 677-681; and WO98 / 050431; the disclosures of these documents are incorporated herein by reference in their entirety. In the "knobs-into-holes" technology, specific amino acids on each CH3 domain are engineered within the interface formed between the two CH3 domains in the tertiary structure of the antibody to generate a protrusion ("knob") in one CH3 domain and a cavity ("hole") in the other CH3 domain, respectively. In the tertiary structure of a multispecific antibody, a protrusion introduced in one CH3 domain can be positioned in a cavity introduced in another CH3 domain.

[0572] In any embodiment of the first aspect or the second aspect, the bispecific polypeptide may comprise a CH3 domain comprising an amino acid substitution at position T366 or Y407, such as a T366W substitution and a Y407V substitution (according to Kabat numbering).

[0573] In any embodiment of the first aspect or the second aspect, the bispecific polypeptide may comprise a CH3 domain comprising one or more amino acid substitutions at positions T366, L368, Y407, S354 and Y349. In a preferred example, the CH3 domain may comprise the following amino acid substitutions:

[0574] (a) T336W;

[0575] (b) T366S, L368A, and Y407V;

[0576] (c) Y407T; or

[0577] (d)T336Y.

[0578] In any of the above embodiments, the bispecific polypeptide may comprise two CH3 domains, wherein:

[0579] - the first CH3 domain comprises an amino acid substitution of T336W, and the second CH3 domain comprises an amino acid substitution of T366S, L368A and Y407V; or

[0580] - the first CH3 domain comprises an amino acid substitution of Y407T, and the second CH3 domain comprises an amino acid substitution of T336Y.

[0581] In addition to or as an alternative to the engineered CH3 domains described herein, the CH3 domain may comprise additional cysteine ​​substitutions to provide additional interchain disulfide bonds to stabilize the heterodimer, such as S354C substitutions and / or Y349C substitutions (according to Kabat numbering) (Atwell S. et al., J. Mol. Biol. 270 (1997) 26-35; Merchant AM et al., Nature Biotech. 16 (1998) 677 to 681).

[0582] Connectors

[0583] The bispecific polypeptides of the present invention may include a linker sequence connecting a first antigen-binding protein and a second antigen-binding protein. Alternatively or additionally, the linker sequence may connect 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. The linker may, for example, serve to connect two domains of an antigen-binding protein (e.g., VH and VL of a scFv or a double antibody), or the linker may serve to connect two antigen-binding proteins (e.g., two or more Fabs or sdAbs) together, or the linker may serve to connect an antigen-binding protein to a scaffold. In some embodiments, the bispecific polypeptide may include multiple linkers (i.e., two or more), for example, one or more scFvs connected to a scaffold may include 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 the skilled artisan based on the intended use of the linker (e.g., see Müller and Kontermann, "Bispecific Antibodies" in Handbook of Therapeutic Antibodies, Wiley-VCH Verlag GmbH, 2014).

[0584] 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), wherein n is an integer of at least 1, 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).

[0585] Other useful linkers include sequences derived from immunoglobulin hinge sequences. The linker may include all or part of a hinge sequence from any of the four IgG types, and may optionally include additional sequences. For example, the linker may include a portion of an immunoglobulin hinge sequence and a glycine-serine sequence. A non-limiting example is a linker including approximately the first 15 residues of an IgG1 hinge, followed by a GlySer linker sequence (such as a linker sequence as described above) having a length of about 10 amino acids.

[0586] The length of the linker will vary according to its application. The technician can easily select an appropriate linker length. For example, when a linker connects the VH domain and the VL domain of a scFv, the length of the linker is generally between about 5 and about 20 amino acids, for example, between about 10 and about 20 amino acids in length or between about 15 and about 20 amino acids in length. When a linker connects the VH domain and the VL domain of a diabody, the linker should be short enough to prevent the two domains from associating in the same chain. For example, the length of the linker can be between about 2 and about 12 amino acids, for example, between about 3 and about 10 amino acids in length or about 5 amino acids in length.

[0587] In some embodiments, when a linker connects two Fab fragments, a linker can be selected so that it maintains the relative spatial conformation of the complementary sites of the F(ab') fragments and is able to form a covalent bond equivalent to the disulfide bond in the IgG core hinge. In this context, suitable linkers include IgG hinge regions, such as hinge regions from IgG1, IgG2, or IgG4. Modified forms of these exemplary linkers can also be used. For example, modifications that improve IgG4 hinge stability are known in the art (see, e.g., Labrijn et al., 2009, Nature Biotechnology, 27: 767 to 771).

[0588] The linker may comprise an amino acid residue sequence connecting the first antigen-binding protein and the second antigen-binding protein. Alternatively, the first antigen-binding protein and the second antigen-binding protein may be connected via chemical coupling (e.g., forming a biaryl conjugate between the domains). Examples of suitable methods for chemically coupling the binding domains are known in the art. This method includes modifying the primary amine present on the lysine residues using succinimidyl compounds, as used in TriLink technology bioconjugators. In one embodiment, if the first antigen-binding protein and / or the second antigen-binding protein is scFv, the bispecific polypeptide comprises a linker sequence between the VH domain and the VL domain of the scFv. Suitable linker sequences are known to those skilled in the art, and illustrative examples thereof include relatively flexible and hydrophilic amino acid residues. In one embodiment, the linker sequence is selected from the group consisting of SEQ ID NO:64 or SEQ ID NO:65, or an amino acid sequence having at least 70% identity thereto, preferably an amino acid sequence having 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 to SEQ ID NO:64 or SEQ ID NO:65.

[0589] 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 epitopes or affinity tags include peptide tags (e.g., FLAG tags, HA tags, His tags, Myc tags, S tags, SBP tags, Strep tags, eXact tags) and protein tags (e.g., GST tags, MBP tags, GFP tags). In one embodiment, the epitope or affinity tag is a His tag.

[0590] Nucleic acids and vectors

[0591] In another aspect disclosed herein, a nucleic acid encoding a bispecific polypeptide described herein is provided. In this embodiment, the nucleic acid may encode one or more amino acid sequences listed in Table 1. In yet another aspect, a cell comprising a vector described herein is provided.

[0592] The terms "polynucleotide", "polynucleotide sequence", "nucleotide sequence", "nucleic acid" or "nucleic acid sequence" are used interchangeably herein to refer to mRNA, RNA, cRNA, cDNA or DNA. The term generally refers to a polymeric form of nucleotides (either ribonucleotides or deoxynucleotides) of at least 10 bases in length, or a modified form or either type of nucleotide. The term includes single-stranded and double-stranded forms of RNA and DNA.

[0593] As used herein, the term "gene" includes a nucleic acid molecule that can be used to produce mRNA, optionally with the addition of elements that aid in the process. A gene may or may not be able to be used to produce a functional protein. A gene may include coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and 5' untranslated regions and 3' untranslated regions).

[0594] The term "transgenic" is used herein to describe genetic material that has been or will be artificially introduced into the genome of a host organism and passed on to the host's offspring. In some embodiments, the transgene confers a desired characteristic to the introduced T cells, or otherwise produces a desired therapeutic outcome.

[0595] As used herein, the term "encode" or "encoding" etc. refers to the ability of a nucleic acid to provide another nucleic acid or polypeptide. For example, if a nucleic acid sequence can be transcribed and / or translated to produce a polypeptide, or if a nucleic acid sequence can be processed into a form that can be transcribed and / or translated to produce a polypeptide, the nucleic acid sequence is referred to as "encoding" a polypeptide. Such a nucleic acid sequence may include a coding sequence, or may include both a coding sequence and a non-coding sequence. Therefore, the term "encoding" etc. includes an RNA product produced by transcription of a DNA molecule, a protein produced by translation of an RNA molecule, a protein produced by transcription of a DNA molecule to form an RNA product and then translating the RNA product, or a protein produced by transcription of a DNA molecule to provide an RNA product, processing the RNA product to provide a processed RNA product (e.g., mRNA) and then translating the processed RNA product.

[0596] 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 NO: 1, SEQ ID NO: 2, SEQ ID NO: 17 to SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 49 to SEQ ID NO: 63, SEQ ID NO: 64 to SEQ ID NO: 67, SEQ ID NO: 71 to SEQ ID NO: 74, and SEQ ID NO: 76 to SEQ ID NO: 79.

[0597] In any embodiment, the nucleic acid sequence encoding the bispecific polypeptide of the invention may have one or more polynucleotide sequences selected from the group consisting of: SEQ ID NO:178 to SEQ ID NO:185.

[0598] In another aspect, a vector is provided, comprising a nucleic acid described herein operably linked to a regulatory sequence.

[0599] 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 specific cell. Regulatory sequences suitable for eukaryotic cells include promoters, polyadenylation signals, transcription enhancers, translation enhancers, leader or trailer sequences that regulate 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.

[0600] Typically, regulatory sequences include, but are not limited to, promoter sequences, 5' non-coding regions, cis-regulatory regions (e.g., functional binding sites for transcriptional regulatory proteins or translational regulatory proteins), upstream open reading frames, ribosome binding sequences, transcription initiation sites, translation initiation sites, and / or nucleotide sequences encoding leader sequences, stop codons, translation stop sites, and 3' non-translated regions. Constitutive promoters or inducible promoters known in the art are contemplated. The promoter may be a naturally occurring promoter or a hybrid promoter combining more than one promoter element.

[0601] The promoter sequence envisioned may be a native sequence of a mammalian cell or may be derived from an alternative source, wherein the region is functional in the selected organism. The choice of promoter will vary depending on the intended host cell. For example, promoters that can be used for expression in mammalian cells include metallothionein promoters (which can be induced in response to heavy metals such as cadmium), β-actin promoters, and viral promoters, such as SV40 large T antigen promoter, human cytomegalovirus (CMV) immediate early (IE) promoter, Rous sarcoma virus LTR promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus promoter, and HPV promoter, especially HPV upstream regulatory region (URR), etc. All of these promoters are well described in the art and are readily available.

[0602] Enhancer elements can also be used herein to increase the expression level of nucleic acid sequences in vector constructs. Examples include: SV40 early gene enhancers, such as Dijkema et al. (1985, EMBO Journal, 4: 761); enhancers / promoters derived from the long terminal repeats (LTR) of Rous sarcoma virus, such as Gorman et al. (1982, Proceedings of the National Academy of Science, USA, 79: 6777); and elements derived from human CMV, such as Boshart et al. (1985, Cell, 41: 521), such as elements contained in the CMV intron A sequence.

[0603] The vector construct may also include a 3' non-translated sequence. The 3' non-translated sequence refers to a gene portion comprising a DNA fragment comprising a polyadenylation signal and any other regulatory signal that can affect mRNA processing or gene expression. The polyadenylation signal is characterized by affecting the addition of polyadenylic acid tracts at the 3' end of the mRNA precursor. The polyadenylation signal is usually identified by whether there is homology with the canonical form 5'AATAAA-3', but variations are also not uncommon. The 3' non-translated regulatory DNA sequence preferably includes about 50nt to 1000nt, and in addition to the polyadenylation signal and any other regulatory signal that can affect mRNA processing or gene expression, it may also include transcription and translation termination sequences.

[0604] As used herein, the term "operably linked" or "operably linked" refers 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 means that the regulatory sequence is positioned and / or oriented relative to the coding sequence to permit expression of the coding sequence under conditions compatible with the regulatory sequence.

[0605] As used herein, the terms "open reading frame" and "ORF" are used interchangeably herein to refer to the amino acid sequence encoded between the translation start codon and the stop codon of the coding sequence. The terms "start codon" (e.g., ATG) and "stop codon" (e.g., TGA, TAA, TAG) refer to three adjacent nucleotide ("codon") units in the coding sequence that specify the start and chain termination of protein synthesis (mRNA translation), respectively.

[0606] As used herein, the term "recombinant" as applied to "nucleic acid molecule," "polynucleotide," and the like, should be 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.

[0607] The recombinant nucleic acid molecule or polynucleotide can be inserted into a vector. Non-viral vectors or viral vectors such as plasmid expression vectors can be used. The types of vectors according to the present invention and the techniques for inserting nucleic acid constructs are known in the art, and illustrative examples thereof include cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses).

[0608] In any embodiment, the vector comprises a polynucleotide sequence encoding one or more polypeptide sequences selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:17 to SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:49 to SEQ ID NO:63, SEQ ID NO:64 to SEQ ID NO:67, SEQ ID NO:71 to SEQ ID NO:74, SEQ ID NO:76 to SEQ ID NO:79.

[0609] In any embodiment, the vector comprises one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:178 to SEQ ID NO:185.

[0610] The nucleic acid sequences, polynucleotides or vector constructs described herein comprise heterologous sequences that do not occur in nature.

[0611] On the other hand, a host cell comprising a vector described herein is provided. Suitable host cells are known to those skilled in the art, and illustrative examples thereof include bacterial cells (e.g., Escherichia coli, Proteus mirabilis), fungal cells (e.g., Saccharomyces cerevisiae, Pichia pastoris, Trichoderma 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 are known to those skilled in the art, and illustrative examples thereof include CHO cells or 293T cells. These cells are widely available from commercial suppliers.

[0612] Methods for producing bispecific polypeptides

[0613] In another aspect, a method for producing a bispecific polypeptide described herein is provided, the method comprising (i) culturing a cell described herein in a culture medium and under conditions suitable for expression of the bispecific polypeptide; and (ii) isolating the bispecific polypeptide from the cell or culture medium.

[0614] The bispecific polypeptides described herein can be produced using any number of expression systems known to those skilled in the art, illustrative examples of which include production in or by production of bacteria (e.g., E. coli, Proteus mirabilis), fungi (e.g., Saccharomyces cerevisiae, Pichia pastoris, Trichoderma 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 are known to those skilled in the art, illustrative examples of which include the CHO expression system or the 293 expression system. These expression systems are widely available from commercial suppliers. In one embodiment, the bispecific polypeptides are produced using a mammalian expression system.

[0615] In another aspect, a method for producing a bispecific polypeptide described herein is provided, the method comprising combining a first antigen binding protein and a second antigen binding protein under conditions suitable for forming a chemically coupled bispecific polypeptide comprising the first antigen binding protein and the second binding protein.

[0616] In a preferred embodiment, chemically coupled bispecific polypeptides are formed following modification of primary amines present on lysine residues with succinimidyl compounds (as described elsewhere herein).

[0617] Pharmaceutical composition

[0618] In another aspect, a pharmaceutical composition is provided, comprising the bispecific polypeptide described herein and a pharmaceutically acceptable carrier.

[0619] The composition described herein can be prepared in a manner known in the art and is a composition suitable for parenteral administration to mammals, especially humans, comprising a therapeutically effective amount of the composition and one or more pharmaceutically acceptable carriers or diluents.

[0620] As used herein, the term "pharmaceutically acceptable carrier" means any suitable carrier, diluent or excipient. These include all aqueous and non-aqueous isotonic sterile injection solutions that make the composition isotonic with the blood of the intended recipient, which may contain antioxidants, buffers and solutes; aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic agents and absorbents, etc. It should be understood that the composition of the present invention may also include other supplementary physiologically active agents.

[0621] The carrier is generally pharmaceutically "acceptable", i.e., compatible with the other ingredients in the composition and harmless to the subject. The composition includes ingredients suitable for parenteral administration (including subcutaneous, intramuscular, intravenous and intradermal administration). The composition can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical art. Such methods include preparing a carrier for associating with isolated T cells. In general, the composition is prepared by uniformly and intimately associating any active ingredient with a liquid carrier.

[0622] In one embodiment, the composition is suitable for parenteral administration. In another embodiment, the composition is suitable for intravenous administration.

[0623] Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which render the compositions isotonic with the blood of the intended recipient and which may contain antioxidants, buffers, bactericides and solutes; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0624] The present disclosure also contemplates the combination of the compositions described herein with other active agents and / or with other treatment regimens or treatment modalities (such as radiotherapy or surgery). When the compositions described herein are used in combination with known active agents, the combination can be administered sequentially (continuously or interrupted by periods without treatment), in parallel, or as a mixture. Suitable anticancer agents are known to those skilled in the art. Combination therapy is also contemplated to encompass treatment with the compositions of the present invention followed by a known treatment, or treatment with a known formulation followed by treatment with the compositions of the present invention, for example as a maintenance therapy. For example, in the treatment of cancer, it is contemplated that the compositions of the invention may be administered in combination with alkylating agents (such as nitrogen mustard, cyclophosphamide, chlorambucil, ifosfamide combined with cisplatin, or platinum-containing alkylating agents such as cisplatin, carboplatin, and oxaliplatin), antimetabolites (such as purine or pyrimidine analogs, or antifolates such as azathioprine and mercaptopurine), anthracyclines (such as daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, or anthracycline analogs), implants, alkaloids (such as vinca alkaloids, or taxanes, such as vincristine, vinblastine, vinorelbine, vindesine, paclitaxel, or docetaxel), topoisomerase inhibitors (such as type I or type II topoisomerase inhibitors), podophyllotoxins (such as etoposide or teniposide), tyrosine kinase inhibitors (such as imatinib mesylate, nilotinib, or dasatinib), adenosine receptor inhibitors (such as A2aR inhibitors, SCH58261, CPI-444, SYN115, ZM241385, FSPTP, or A2 B R inhibitors, such as PSB-1115), adenosine receptor agonists (such as CCPA, IB-MECA, and CI-IB-MECA), checkpoint inhibitors (including the PDL-1:PD-1 axis, nivolumab, pembrolizumab, atezolizumab, BMS-936559, MEDI4736, MPDL33280A, or MSB0010718C), CTLA-4 pathway inhibitors (such as ipilimumab and tremelimumab), TIM-3 pathway inhibitors, or agonist monoclonal antibodies known to promote T cell function (including anti-OX40, such as MEDI6469; and anti-4-BB, such as PF-05082566).

[0625] In another aspect, the present invention provides a kit or article of manufacture comprising one or more bispecific polypeptides of the present invention, nucleic acids encoding the bispecific polypeptides, and / or pharmaceutical compositions as described above.

[0626] In other aspects, a kit for the above-mentioned therapeutic application is provided, the kit comprising:

[0627] (a) a container containing a bispecific polypeptide, nucleic acid, vector or pharmaceutical composition of the invention; and

[0628] (b) a label or package insert containing instructions for use.

[0629] The kit may also include one or more active ingredients or components for treating cancer. For example, the kit may also include immune cells expressing CAR.

[0630] 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 of a variety of materials, such as glass or plastic. The container holds a therapeutic composition effective for treating the condition and may have a sterile access port (e.g., the container may be an IV bag or a vial with a stopper pierceable by a hypodermic needle). The label or package insert indicates that the therapeutic composition is used to treat the selected condition. In one embodiment, the label or package insert includes instructions for use and indicates that the therapeutic or prophylactic composition can be used to treat cancer or other conditions described herein.

[0631] The kit may include (a) a therapeutic or prophylactic composition; and (b) a second container containing a second active ingredient or ingredient. The kit according to this embodiment of the invention may further include a drug instruction sheet indicating that the composition and other active ingredients can be used to treat cancer or disorders described herein. Alternatively or additionally, the kit may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The kit may further include other materials desired from a commercial and user perspective, which are known to those skilled in the art, and suitable examples include other buffers, diluents, filters, needles, and syringes.

[0632] Treatment

[0633] In another aspect, a method for treating cancer is provided, the method comprising co-administering to a subject in need thereof a therapeutically effective amount of: (i) immune cells expressing an engineered TCR (e.g., CAR); and (ii) a bispecific polypeptide or pharmaceutical composition as described herein, wherein the bispecific polypeptide simultaneously binds in vivo to an antigen expressed on the subject's endogenous APCs and to an antigen on the engineered TCR (e.g., CAR) expressed by the immune cells to stimulate immune cell activation and expansion to treat cancer.

[0634] In any of the methods of treatment or uses described herein, or in other aspects of the invention where a bispecific polypeptide is administered to a subject, the subject may have received or is receiving an immune cell expressing an engineered TCR (e.g., CAR).

[0635] In any of the methods of treatment, or in other aspects of the invention of administering a bispecific polypeptide to a subject, the method or other representation further comprises the step of administering an immune cell expressing an engineered TCR (e.g., CAR).

[0636] The co-administration of immune cells expressing engineered TCR (e.g., CAR) and bispecific polypeptides or pharmaceutical compositions described herein can be achieved by formulating the immune cells and bispecific polypeptides or pharmaceutical compositions in the same composition (e.g., for simultaneous co-administration), or they can be formulated into different compositions for sequential administration. The so-called "sequential" administration refers to an interval between the administration of immune cells and the administration of bispecific polypeptides or pharmaceutical compositions. The interval between sequential administration can be a few seconds, a few minutes, a few hours or a few days. In one embodiment, it may be necessary to re-administer immune cells and bispecific polypeptides or pharmaceutical compositions regularly to achieve the desired therapeutic effect. Sequential administration can be in any order.

[0637] The inventors have unexpectedly 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). Therefore, bispecific polypeptides can be referred to as "adjuvants". As used herein, the term "adjuvant" refers to a bispecific polypeptide that can increase the magnitude of the immune response caused by immune cells expressing engineered TCRs (e.g., CARs) to a magnitude that exceeds that expected from immune cells expressing engineered TCRs alone.

[0638] Immune cell activation can be, for example, by providing a primary stimulation signal through a T cell TCR / CD3 complex or via stimulating CD2 surface proteins, and secondary costimulatory signals are provided by auxiliary molecules (e.g., CD28 or 4-1BBL) to complete. In addition to the primary stimulation signal provided by TCR / CD3 complex or via CD2, inducing T cell responses also requires a second costimulatory signal. In a specific embodiment, CD28 binding agents can be used to provide costimulatory signals. Suitable costimulatory ligands include but are not limited to CD7, B7-1 (CD80), B7-2 (CD86), 4-1BBL, OX40L, inducible costimulatory ligands (ICOS-L), intercellular adhesion molecules (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, ILT3, ILT4, agonists or antibodies in conjunction with Toll-like receptors, and ligands specifically bound to B7-H3.

[0639] The terms "expanded," "expansion or expanding," "propagation," and the like are used interchangeably herein to refer to an increase in the number of immune cells before or after administration of a cell or pharmaceutical composition formulation to a subject. Immune cells can be expanded using any cell culture method known in the art. For example, immune cells can be expanded in an in vitro tissue culture system (including liquid culture, monolayer, etc.).

[0640] The treatment regimen for treating cancer can be determined by those skilled in the art, and generally depends on a variety of factors, including but not limited to the type, size, stage and receptor status of the tumor, and the age, weight and general health of the subject. Another determining factor may be the risk of developing a recurrent disease. For example, a more aggressive treatment regimen can be prescribed for a subject determined to be at a high or higher risk of developing a recurrent disease, compared to a subject deemed to be at a low or lower risk of developing a recurrent disease. Similarly, a more aggressive treatment regimen can be prescribed for a subject determined to be in the middle or late stages of cancer (e.g., stage III or stage IV disease), compared to a subject whose cancer is not at an advanced stage.

[0641] As used herein, the term "cancer" means any condition associated with abnormal cell proliferation. These conditions are known to those skilled 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 the same type of non-cancerous tissue. 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 cancer and endometrial cancer.

[0642] As used herein, the terms "treat," "treatment," or "treating" refer to any and all uses of correcting a condition or symptom in any way, or otherwise preventing, hindering, delaying, eliminating, or reversing the onset or progression of cancer or other adverse symptoms. Therefore, the terms "treating" and the like should be considered in their broadest possible context. For example, treatment does not necessarily mean that the subject is treated until full recovery or cure. In a condition exhibiting or characterized by multiple symptoms, treatment does not necessarily require correction, prevention, hindering, delaying, eliminating, or reversing all of the symptoms, but may correct, prevent, hinder, delay, eliminate, or reverse one or more of the symptoms.

[0643] The subject to be treated for cancer can be a human being, or a mammal of economic and / or social significance to humans, such as carnivores other than humans (e.g., cats and dogs), pigs (e.g., domestic pigs, barrows and wild boars), ruminants (e.g., cattle, bulls, sheep, giraffes, deer, goats, bison and camels), horses and birds, including endangered birds, birds raised in zoos and poultry, more specifically domesticated poultry (e.g., poultry such as turkeys, chickens, ducks, geese, guinea fowls), because they are of economic significance to humans. The term "subject" does not indicate a particular age. Thus, it is intended to cover adult, adolescent and neonatal subjects.

[0644] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to any subject to which the present disclosure is applicable. In one embodiment, the subject is a mammal. In another embodiment, the subject is a human.

[0645] As used herein, the term "therapeutically effective amount" means an amount of cells sufficient to treat cancer when administered to a mammal, particularly a human, in need of such treatment. 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 condition of the subject.

[0646] 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, because the modified cells replicate and proliferate after transfer, the administered cell dose will not be similar to the final steady-state cell number. In one embodiment, a pharmaceutical composition comprising the modified cells of the present invention can be administered at 10 4 Up to 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 Up to 10 6 The dosage is 10 cells / kg body weight (including all integer values ​​within these ranges).

[0647] Compositions comprising the bispecific polypeptides described herein may also be administered multiple times at these doses. One skilled in the art can readily determine the optimal dosage and treatment regimen for a particular subject by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0648] In one embodiment, the immune cells are derived from autologous cells. In another embodiment, the immune cells are derived from allogeneic cells.

[0649] The term "autologous" refers to any material derived from the same individual that is subsequently reintroduced into that individual.

[0650] The term "allogeneic" refers to any material derived from a different individual of the same species as the individual into which 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 different genetically to interact antigenically.

[0651] Cell Manufacturing

[0652] In order to obtain a sufficient therapeutic dose of immune cell composition, the method for producing immune cells generally involves one or more rounds of stimulation, activation and / or expansion. According to the methods disclosed herein, immune cells can be stimulated to activate and expand in vivo and in vitro.

[0653] Therefore, in another aspect disclosed herein, a method for stimulating activation and / or expansion of immune cells in vivo is provided, the method comprising administering to a subject an effective amount of: (i) immune cells expressing an engineered TCR (e.g., CAR); and (ii) a bispecific polypeptide or pharmaceutical composition as described herein, wherein the bispecific polypeptide simultaneously binds in vivo to an antigen expressed on endogenous APCs of the subject and to an antigen on the engineered TCR (e.g., CAR) expressed by the immune cells.

[0654] The in vivo activation and expansion of immune cells as described herein helps to reduce the number of immune cells required for each therapeutic dose of immune cells.

[0655] On the other hand, a method for stimulating in vitro activation and expansion of immune cells is provided, the method comprising culturing isolated immune cells expressing engineered TCR (e.g., CAR) in a culture medium, the culture medium comprising: (i) APC or APC mimics; and (ii) a bispecific polypeptide as described herein, wherein the bispecific polypeptide simultaneously binds to an antigen expressed on an APC or APC mimic and an engineered TCR (e.g., CAR) expressed by an immune cell. In one embodiment, the bispecific polypeptide can bind to an antigen expressed on a tumor cell.

[0656] The immune cell manufacturing method envisioned herein has simple and robust culture initiation and activation steps, which contribute to the production of immune cell compositions as high-quality therapeutic products. In one embodiment, culture initiation and activation include inoculating a cell population in a cell culture container (e.g., cell culture bag, GREX bioreactor, WAVE bioreactor, etc.), and activating immune cells by primary and costimulatory immune cell signaling pathways. Cell composition can be further cultured in the presence of one or more additional growth factors or cytokines (e.g., IL-2, IL7 and / or IL-15) or any suitable combination thereof.

[0657] In another embodiment, the activation and expansion of immune cells comprises culturing isolated immune cells using APCs or APC mimics. In one embodiment, APCs or APC mimics are selected from the group consisting of: donor-derived APCs, synthetic artificial APCs (aAPCs), microbeads functionalized with activation antibodies of CD3 and CD8 (Dynabeads), autologous monocyte-derived dendritic cells (moDCs), and scaffolds that mimic APCs.

[0658] In certain embodiments, culture initiation includes culturing cells at a desired density (e.g., 1-5×10 6 In some embodiments, a cell population comprising T cells (e.g., PBMC) is seeded in a suitable cell culture medium (e.g., 100 cells / mL) containing one or more cytokines, primary stimulatory ligands, and co-stimulatory ligands. In another embodiment, cytokines, stimulatory ligands, and co-stimulatory ligands can be subsequently added to the PBMC in the cell culture medium.

[0659] In one embodiment, the cell culture container is a cell culture bag, including but not limited to GMP cell expansion bags, GMP cell differentiation bag, EXP-PakTM Cell expansion biocontainer, VueLife TM Bag, KryoSure TM Bag, KryoVue TM bag, Bag, PermaLife TM Bag, X-Fold TM Bags, Si-Culture TM Bags and VectraCell TM Bag, as contemplated elsewhere herein.

[0660] In certain embodiments, the cells are seeded in a cell culture vessel containing a suitable cell culture medium. Illustrative examples of suitable cell culture media include, but are not limited to, TCGM, X-VIVO TM 15 (supplemented with 2mM GlutaMAX TM -I, 10 mM HEPES and 5% human AB serum), CTS TM OpTmizer TM T cell expansion SFM (Life Technologies), CTS TM AIM Culture medium (Life Technologies), RPMI 1640, Clicks, DMEM, MEM, a-MEM, F-12, X-Vivo15 (Lonza), Serum-free medium (CellGenix) and X-Vivo 20 (Lonza), in which amino acids, sodium pyruvate and vitamins are added, are serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or sufficient cytokines to allow the growth and expansion of immune cells.

[0661] The cell culture medium contemplated herein can further include one or more factors, including, but not limited to, serum (e.g., fetal bovine serum 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.

[0662] In one embodiment, the APC or APC mimetic is selected from the group consisting of donor-derived APC, synthetic artificial APC (aAPC), microbeads functionalized with activating antibodies to CD3 and CD8 (Dynabeads), autologous monocyte-derived dendritic cells (moDC), and APC-mimicking scaffolds.

[0663] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications, except those specifically described. It should 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 mentioned or indicated in this specification, either individually or in combination, and any and all combinations of any two or more of said steps or features.

[0664] Unless defined otherwise, 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.

[0665] Various embodiments implemented herein are further described by the following non-limiting examples.

[0666] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned in this document or in the accompanying drawings or evident from this document or in the accompanying drawings. All these different combinations constitute various alternative aspects of the invention.

[0667] Example

[0668] Example 1 - Generation of anti-CD40 antibodies

[0669] Obtain human anti-CD40 antibodies. 500 mL HEK293 cultures were transfected with vectors encoding anti-CD40 antibodies. Cultures were harvested by centrifugation on day 6. SDS-PAGE gel analysis was completed on day 3. Cysteine ​​mutations (C232 and C127) were performed on serines to confirm expression / aggregation. Strong expression was observed in both cysteine-mutated and non-mutated IgG2 forms.

[0670] The clarified supernatant of IgG2 protein was purified by protein A affinity chromatography and buffer (PBS) exchange. The material was dialyzed into PBS buffer at pH 7.0. The final concentration was determined by A280, and the yield, purity and endotoxin levels are detailed in the table below:

[0671]

[0672] All forms of IgG2 were determined to have high expression, low aggregation, and high purity and were suitable for BEAT design.

[0673] Example 2 - Humanization of chimeric antibodies

[0674] Obtain mouse anti-FLAG antibody.To identify the complementarity determining regions (CDRs) and analyze the closest matching germline sequences, the IMGT DomainGapAlign tool was used (Ehrenmann F., Kaas Q. and Lefranc M.-P., Nucleic Acids Res., 2010; 38: D301-D307).

[0675] 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 are available upon request for viewing in any molecular visualization software. Images were generated using PyMol (PyMol Molecular Graphics System, Version 2.0, Limited Liability Company) to generate.

[0676] The specific liability of the antibody sequence was analyzed based on the disclosed protein motifs. The analysis was performed by analyzing the following motifs, wherein X represents any amino acid except proline, as described in the following table:

[0677]

[0678] At the beginning of the humanization process, homology models of the parental VH and parental VL were constructed in single-chain Fv (scFv) format. Modeling was performed in 4 stages: collection of homologous sequences; folding library scanning; loop modeling; side chain placement. The resulting model was used to guide the selection of "donor" or "acceptor" amino acids during humanization.

[0679] The parental VH and VL sequences are aligned with a set of human germline sequences. The group has been screened to select germline sequences that do not contain unwanted sequence tendencies, especially N-linked glycosylation sites and free cysteine. The closest matching germline is selected from two different VH and VL families. The humanization algorithm is then used to select CDR and framework amino acids to be transplanted from the donor parent sequence to the human acceptor germline sequence. Four VH sequences and four VL sequences are generated, resulting in 16 possible antibodies. The IMGT DomainGapAlign tool (Ehrenmann F., Kaas Q. and Lefranc M.-P., Nucleic Acids Res., 2010; 38: D301-D307) is used to calculate the percent identity with humans. Sequence tendencies are determined based on sequence motifs.

[0680] The VH and VL sequences were run through the IMGT DomainGapAlign tool to analyze against all known antibody germline sequences. The CDR regions were assigned using the IMGT definition. As expected, the sequence was closest to the mouse, particularly the IGHV1-4*01 family of VH and the IGK1-117*01 family of VL. The closest matching germlines to the parental SEQ ID NO: 1 were IGHV1-46*01, IGHV7-4-1*02, IGKV2-30*01, and IGKV4-1*01.

[0681] The following table summarizes the original parental and humanized sequences:

[0682]

[0683] The combination of 4 different heavy and light chain framework regions resulted in 16 different V H / V L combination.

[0684] The following table summarizes the different VH / VL combinations of 16 antibodies:

[0685]

[0686] All humanized IgG1 formats were well expressed and successfully purified and characterized by SDS-PAGE and SEC-HPLC to monitor protein quality. The titer, amount of final purified antibody, and monomer content are described in the following table:

[0687]

[0688]

[0689] Example 3 - Characterization of humanized antibodies

[0690] All 16 humanized antibodies in IgG and scFv formats were evaluated for their ability to bind to the target (FLAG tag) using ELISA. Figure 1 Shown in.

[0691] Although most humanized variants retained the ability to bind to the target, three humanized IgG clones (v3, v4, and v7) had stronger affinity. These three IgG clones and the corresponding scFv formats were selected for further analysis.

[0692] Humanized antibodies can exhibit high binding affinity comparable to that of the parental mouse antibody (ie, mouse VH / VL in the context of human constant regions).

[0693] The following table summarizes the antibody affinities and stoichiometries measured by Biacore:

[0694] Antibody <![CDATA[K D (M)]]> Binding stoichiometry V4scFv 2.2E-08 2.8 V3scFv 1.9E-08 5.5 V7scFv 1.5E-08 3.7 V7IgG 1.3E-09 1.1 V3IgG 1.0E-09 1.2 Parent 9.6E-10 1.3 V4IgG 9.1E-10 1.1

[0695] Four IgG antibodies (parental mouse antibody and 3 humanized antibodies: v3, v4 and v7) were compared with the corresponding humanized scFv for binding to a single antigen by Biacore. Mouse scFv could not be expressed and therefore could not be used for affinity comparison. The IgG antibodies bound the antigen more tightly, almost 10 times more tightly, compared to the scFv. This is expected because IgG is bivalent in nature and its avidity will produce tighter binding compared to the monovalent expressed scFv.

[0696] Antibodies v3, v4, v7, and v11 (in IgG format and, in the case of v11, also 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. Antibodies induce expression of IFN-γ Figure 2 As shown. Antibody v11 was also evaluated in IgG and scFv formats. The results showed that antibody v7 and antibody v4 induced the greatest amount of IFN-γ by CAR T cells, respectively.

[0697] Based on these results, antibody variants v4 and v7 scFv were selected for future studies.

[0698] Example 4 - Design and affinity of anti-CD40-anti-FLAGBEAT (BEAT1)

[0699] Twelve anti-CD40-anti-FLAG BEATs (collectively referred to as BEAT 1) were designed and evaluated. Figure 4 The orientation of the IgG and scFv domains is outlined in . The 12 variants of BEAT 1 generated are summarized in the table below:

[0700]

[0701] The linker used between VL-VH and between scFv and IgG2 was (G4S)*3. The linker used between VH-VL was 21 AA (GGGGSGGGGSGGGGSGGGGAS).

[0702] Multi-cycle SPR experiments were used to characterize the binding of CD40 to 11 anti-CD40 BEAT antibodies, anti-CD40 IgG control, and hCD40-cMyc cBEAT. At least two sets of reliable data were obtained for each anti-CD40 antibody binding to CD40. CD40 binding to all anti-CD40 BEAT antibodies was well-fitted, with high affinity and K DThe binding of CD40 to hCD40-cMyc rBEAT showed a K D The K values ​​of the control antibody anti-CD40 IgG2 were 0.05 nM to 0.06 nM. D is 0.5 nM (Table 3).

[0703] The 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 sets of reliable data were obtained for each BEAT binding to FLAG. The binding of FLAG-BAP was well-fitted, with high affinity and a K D The binding of FLAG-BAP to the control anti-FLAG antibody showed K D The range was 0.4 nM to 35 nM (Table 4).

[0704] Table 3 Affinity of BEAT1 for CD40 antigen

[0705]

[0706] Table 4 Affinity of BEAT 1 for FLAG tag

[0707]

[0708]

[0709] Next, experiments were performed in which BEAT 1a4 (F7-G8) was simultaneously bound to CD40 and FLAG-BAP. In these experiments, after BEAT 1a4 was captured on the protein G surface, 50 nM FLAG-BAP was injected until saturation was reached, CD40 was immediately injected in the presence of 50 nM FLAG-BAP, and then the dissociation of CD40 was monitored in the case of only 50 nM FLAG-BAP injection. This was performed for different concentrations of CD40, which allowed the association and dissociation of CD40 with BEAT 1a4 to be measured, and BEAT 1a4 was always saturated with FLAG-BAP. Similarly, a reverse experiment was performed in which, after BEAT 1a4 was captured, 50 nM CD40 was injected until saturation, followed by FLAG-BAP injection in the presence of 50 nM CD40, and then the dissociation of FLAG-BAP was monitored in the case of only 50 nM CD40 injection. These experiments showed that BEAT 1a4 is able to bind CD40 in the presence of saturation with FLAG-BAP ( Figure 5). Under these conditions, CD40 binds with similar kinetic parameters, affinity, and Rmax in the presence or absence of bound FLAG-BAP. Similarly, FLAG-BAP binds to BEAT 1a4 when BEAT 1a4 is saturated with bound CD40. Binding of FLAG-BAP to BEAT 1a4 also exhibits similar kinetic parameters, affinity, and Rmax in the presence or absence of bound CD40.

[0710] Example 5 - Binding of BEAT1 to APC

[0711] BEAT 1 was evaluated for binding to APC at 10 nM, 30 nM, and 100 nM. The IgG2 isotype control showed no binding, while the anti-CD40 antibody control showed the highest binding (only at 100 nM) ( Figure 6 A). BEAT 1a7, BEAT 1a4, and BEAT 1b4 showed the highest binding.

[0712] BEAT 1a4 was expressed in human MoDC cells ( Figure 7 A) and human B cells ( Figure 7 B) and CD40 on non-human primate MoDC cells ( Figure 7 C) and non-human primate B cells ( Figure 7 D) CD40 on PD-1 displayed dose-dependent binding.

[0713] Binding of BEAT 1a4 to Human Cells / DCs: Cell binding was performed using two (2) test articles (antibody biologics), including one (1) test article of interest (Bispecific Antibody 1 (BEAT 1a4)) and one (1) human IgG2 isotype control antibody. PBMCs and MoDCs from 3 healthy human donors were evaluated. Binding of the test articles to CD3+, CD20+, CD14+, and CD11c+ cell subsets was evaluated. Binding to all suggested cell types was evaluated ( Figure 7 A and Figure 7 B), Dose responses for each test article of interest and isotype control antibody were tested in duplicate using a 200 nM top concentration, followed by a two-fold dilution series (10 points total) and untreated samples, and finally secondary antibody (5 μg / mL).

[0714] Binding of BEAT 1a4 to NHP Cells: Cell binding was performed using two (2) test articles (antibody biologics), including one (1) test article of interest (Bispecific Antibody 1 (BEAT 1a4)) and one (1) human IgG2 isotype control antibody. PBMCs and MoDCs from 3 NHP donors were evaluated. Binding of the test articles to CD3+, CD20+, CD14+, and CD11c+ cell subsets was evaluated. Binding to all suggested cell types ( Figure 7 C and Figure 7 D), the dose response of each test article of interest and isotype control antibody was tested in duplicate using a 200 nM top concentration, followed by a two-fold dilution series (10 points total) and untreated sample, and finally the secondary antibody (5 μg / mL).

[0715] Example 6 - Binding of BEAT1 to CAR T cells

[0716] Binding of BEAT 1a4 to CAR T cells expressing GFP and FLAG-tagged was evaluated. Cells were detected by FAC and were GFP+ / AF647+ cells. Binding of BEAT 1a4 was evaluated over the entire dose range of 0.1 nM to 100 nM ( Fig. 9 C).

[0717] The binding of the test article BEAT 1a4 to FLAG-tagged CAR-T target cells was evaluated with a 10-point standard curve to confirm the EC50. After staining with a viability dye and incubation with 5% FBS to block nonspecific binding, CAR-T cells were incubated with BEAT 1a4 or a human IgG2 isotype control in a dose response. In addition to the untreated control, the following concentrations were tested in duplicate: 0.01nM, 0.03nM, 0.1nM, 0.3nM, 1nM, 3nM, 10nM, 33nM, and 100nM. After incubation, cells were washed and stained with 5μg / mL of a fluorescently labeled secondary reagent (AF647-conjugated AffiniPure Fab2 fragment goat anti-human IgG Fcg specific), and then washed and collected via high-throughput flow cytometry. The geometric mean fluorescence intensity (MFI) values ​​of AF647 (viability dye-negative) in live cells were plotted on an XY graph to graph the logarithm of MFI vs. concentration and the data were fitted to a nonlinear regression curve to calculate the EC50. The percentage of AF647 in cells gated on live cells was also analyzed.

[0718] BEAT 1a4 exhibited dose-dependent binding to CAR T cells with an EC50 of 0.51 nM.

[0719] Example 7 - Functional activity of BEAT1 on APCs and CAR T cells

[0720] Lead BEAT candidates were evaluated for CD86 upregulation following addition of BEATs to monocyte-derived DCs (MoDCs). CD40 agonism is expected to upregulate CD86 expression. CD86 was upregulated in a dose-dependent manner following addition of anti-CD40 BEATs (BEAT 1a4 and BEAT 1a5) at 10 nM BEAT 1a4 ( Figure 8 A). Control antibody had no effect on the increase of CD86.

[0721] BEAT 1a4 (and anti-CD40 agonist control) increased IL-12 / IL-23p40 release in a dose-response manner between 3 nM and 30 nM (CD86) and 1 nM and 60 nM (IL-12 / IL-23p40). Figure 8 B).

[0722] With the addition of BEAT 1a4, the functional activity of CAR T cells including the FLAG tag and MoDC was evaluated. MoDC were confirmed to have CD40 expression ( Fig. 9 B). With the addition of anti-CD40 BEAT, an increase in IFN-γ was seen ( Fig. 9 A). Negative (IgG1 and IgG2 isotype) controls had no effect on IFN-γ.

[0723] Example 8 - BEAT1 cytokine analysis

[0724] Cytokine analysis was completed with one donor to test BEAT 1a4. Anti-CD40 BEAT 1 stimulated IFN-γ, TNF-α, and IL2 with some activity, while BEAT 1a4 and BEAT 1a5 stimulated IL12-p70 ( Fig.10 ). For BEAT 1a4, IL-6, IL-4, IL-10, IL-8, IP-10, MCP-1, and IL-17A had limited activity above the background of individual cells.

[0725] Example 9 - T cell proliferation after addition of BEAT1

[0726] After 120 h of proliferation, 7B128z CAR T cells were tested for proliferation in the presence of control compound / BEAT 1 (30 nM) and MoDCs.

[0727] For anti-CD40 BEAT 1a4 and 1a5, significant increases in fold changes in CD3+, CD4+, and CD8+ cells relative to controls were observed ( Fig.11 ), especially after adding anti-CD40 BEAT 1a4, the percentage of effector memory T cells, especially CD8+ T cells, increased ( Fig.12 ).

[0728] Example 10—Beat1+CAR T cell toxicity

[0729] Cytotoxicity was measured by luciferase assay using ovarian cancer cell lines SKOV-3 (parental, MSLN–ve, and MSLN expressing) + MoDC + CAR T.

[0730] BEAT had no effect on SKOV-3 cells expressing MSLN ( Fig.13 ).

[0731] Example 11 - In vivo efficacy of CAR T+BEAT

[0732] BEAT 1a5 was evaluated in the NSG mouse model using SKOV3 mesothelin-transfected CD40 knockout cells. Tumors (50 mm 2 ). A single dose of 5 million anti-mesothelin CAR / 5 million MoDC (mesothelin high expression) and BEAT was administered once every two weeks at a dose of 12.5ug BEAT (5 doses) ( Fig.14 A).

[0733] Effects on survival and tumor size ( Fig.14 B and Fig.14 C) measured that at day 21, 2 / 4 of the mice treated with CART cells + MoDC + BEAT were tumor-free, compared to only 1 / 3 of the mice treated with CART cells alone.

[0734] Example 12 - Generation of anti-CD206 antibodies

[0735] Obtain anti-CD206 antibodies. 500 mL HEK293 cultures were transfected with anti-CD206 (B11) encoding vectors. The cultures were harvested by centrifugation on day 6. SDS-PAGE gel analysis was completed on day 3. The clarified supernatant of IgG1 protein was purified by protein A affinity chromatography ProtA (agarose resin) and buffer (PBS) exchange. The material was dialyzed into PBS buffer. 15 μg samples were prepared in 4× loading buffer (reducing and non-reducing) and analyzed by SDS-PAGE. Based on size, reducing conditions gave B11 a purity of >95%.

[0736] Example 13 - Design and characterization of anti-CD206-anti-FLAGBEAT2

[0737] Anti-FLAG was designed as described in Examples 2 and 3 above. Figure 4 As outlined above, 12 anti-CD206-anti-FLAG BEATs (BEAT 2) were designed and evaluated using IgG domains and scFv domains. The 12 variants of BEAT 2 generated are summarized in the following table:

[0738]

[0739] The linker used between VL-VH and between scFv and IgG2 was (G4S)*3. The linker used between VH-VL was 21 AA (GGGGSGGGGSGGGGSGGGGAS).

[0740] 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 sets of reliable data were obtained for each anti-CD206 antibody binding to CD206. CD206 binding to all anti-CD206 BEAT antibodies was well-fitted, with high affinity and K D The binding of CD206 to the control antibody anti-CD206 IgG1 showed K D The association rate constants of all anti-CD206 BEAT antibodies with CD206 (6×10 3 -3×10 4 M -1 s -1 ) and the dissociation rate constant (2-5×10 -4 s -1 ) were similar (Table 5).

[0741] The binding of FLAG-BAP to 12 BEAT antibodies and 3 anti-FLAG IgG control antibodies was characterized using multi-cycle SPR experiments. At least two sets of reliable data were obtained for the binding of each BEAT to FLAG, except for BEAT 2a1. The binding of FLAG-BAP to BEAT antibodies was well-fitted, with high affinity and KD ranging from 1 nM to 70 nM. The binding of FLAG-BAP to the control anti-FLAG antibody showed K D The association rate constants of all BEAT antibodies with FLAG-BAP ranged from 1 × 10 5 -5×10 6 M -1 s-1 , and the dissociation rate constant range is 1×10 -3 -4×10 -2 s -1 (Table 6).

[0742] Table 5 Affinity of BEAT2 for CD206 antigen

[0743]

[0744] Table 6 Affinity of BEAT2 for FLAG tag

[0745]

[0746] Example 14—Binding of BEAT2 to APC

[0747] BEAT binding to APC was evaluated at 10 nM, 30 nM, and 100 nM. The IgG2 isotype control did not bind, while the B11 anti-CD206 Mab control had the highest binding (only at 100 nM) ( Figure 6 B). BEAT 2a6, BEAT 2a8, and BEAT 2b3 showed the highest binding.

[0748] Example 15—Functional activity of BEAT2 on APCs and CAR T cells

[0749] BEAT was used to evaluate CART and MoDC. MoDC was confirmed to express CD206 ( Fig. 9 B). Stimulation of IFN-γ response via binding of the FLAG tag. With the addition of anti-CD206 BEAT, an increase in IFN-γ was seen ( Fig. 9 A). Negative controls (IgG1 and IgG2 isotypes) had no effect on IFN-γ. Major anti-CD206 BEATs include 2b3.

[0750] Example 16 - BEAT2 cytokine analysis

[0751] Cytokine analysis was completed with one donor to test the primary BEAT 2. Anti-CD206 BEAT 2 stimulated IFN-γ, TNF-α, and IL-6 above the cell background alone ( Fig.10 ). IL-2, IL-4, IL-10, IL-8, IP-10, MCP-1, IL-12p70, and IL-17A showed limited activity.

[0752] Example 17 - T cell proliferation of 7B128z CAR+BEAT2+MoDC

[0753] After 120 h of proliferation, 7B128z CAR T cells were tested for proliferation in the presence of control compound / BEAT 2 (30 nM) and MoDCs.

[0754] For anti-CD206 BEAT 2b3 and 2b4, fold changes in CD3+, CD4+, and CD8+ cells were observed relative to controls ( Fig.11 ), especially after adding BEAT 2b4 to CD8+ T cells, the percentage of central memory T cells increased ( Fig.12 ).

[0755] Example 18 - In vivo efficacy of CAR T + anti-CD206BEAT

[0756] BEAT 2a6 was evaluated in the NSG mouse model using CD40 knockout cells transfected with SKOV3-mesothelin. Tumors (50 mm 2 ). A single dose of 5 million anti-mesothelin CAR / 5 million MoDC (mesothelin high expression) and BEAT was administered once every two weeks at a dose of 12.5ugBEAT (5 doses) ( Fig.14 A).

[0757] Tumor size of CART cells + MoDC + BEAT 2a6 was measured compared with CAR T cells alone or no treatment control ( Fig.24 ).

[0758] Example 19 - Single-arm BEAT expression and purification

[0759] Two different Knobs-in-Holes mutations were used to design single-arm anti-CD40 and anti-FLAG BEATs to form bispecific BEAT 3&4 and BEAT 5&6 ( Fig.15 ).

[0760] BEAT 3:

[0761] Single-arm anti-CD40 IgG2 (Fab+Fc) with KIH mutation set #1

[0762] One-armed anti-FLAG scFv+Fc (hM2V4 scFv in VL-(GGGGs)3-VH) with KIH mutations in KIH mutation set #1

[0763] Heavy chain 1

[0764] HC-IgG2 (anti-CD40) KIH1a

[0765] Light chain 1

[0766] LC-IgG2 (anti-CD40)

[0767] Heavy chain 2

[0768] ●HC-hM2V4_CurrusLinker-(GGGGS)3-IgG2 Fc-KIH1b

[0769] BEAT4:

[0770] Single-armed anti-CD40 IgG2 (Fab+Fc) with KIH mutation set #2

[0771] ● One-armed anti-FLAG scFv+Fc (hM2V4 scFv in VL-(GGGGs)3-VH) with KIH mutations in KIH mutation set #2

[0772] Heavy chain 1

[0773] HC-IgG2 (anti-CD40) KIH2a

[0774] Light chain 1

[0775] LC-IgG2 (anti-CD40)

[0776] Heavy chain 2

[0777] ·HC-hM2V4_CurrusLinker-(GGGGS)3-IgG2 Fc-KIH2b

[0778] BEAT 5:

[0779] One-armed bivalent anti-CD40 IgG2 with linked anti-FLAG scFv (at the C-terminus of the heavy chain) and KIH mutation set #1

[0780] Single-arm anti-CD40 IgG2 with KIH mutation set #1

[0781] Heavy chain 1

[0782] HC-IgG2 (anti-CD40) KIH1a

[0783] Light chain 1

[0784] LC-IgG2 (anti-CD40)

[0785] Heavy chain 2

[0786] ·HC-IgG2(anti-CD40)-(GGGGS)3-hM2V4_CurrusLinker-KIH1b

[0787] Light chain 2

[0788] LC-IgG2 (anti-CD40)

[0789] BEAT 6:

[0790] One-armed bivalent anti-CD40 IgG2 with linked anti-FLAG scFv (at the C-terminus of the heavy chain) and KIH mutation set #2

[0791] Single-arm anti-CD40 IgG2 with KIH mutation set #2

[0792] Heavy chain 1

[0793] HC IgG2 (anti-CD40) KIH2a

[0794] Light chain 1

[0795] LC-IgG2 (anti-CD40)

[0796] Heavy chain 2

[0797] ·HC-IgG2(anti-CD40)-(GGGGS)3-hM2V4_CurrusLinker-KIH2b

[0798] Light chain 2

[0799] LC-IgG2 (anti-CD40)

[0800] cDNAs of the variable heavy (VH) and light (VL) sequences corresponding to all four BEAT variants were synthesized and subcloned into the mammalian cell expression vector pTXs1.

[0801] 500mL XtenCHO culture was transfected with single-arm BEAT (BEAT 3, BEAT 4, BEAT 5, BEAT 6) encoding vector. 14 days after transfection, the culture was harvested by centrifugation. The clarified supernatant of the protein was purified by protein A affinity chromatography ProtA (agarose resin) and buffer (PBS). The material was dialyzed into PBS buffer. 15 μg samples were prepared in 4x loading buffer (reducing and non-reducing) and analyzed by SDS-PAGE and SEC-HPLC.

[0802] All BEAT forms were expressed with more than 10 mg of antibody, with a purity greater than 90%, as shown in the following table:

[0803]

[0804] Example 20 - Affinity of one-armed BEAT for CD40 and FLAG tags

[0805] The binding of single-arm BEATs to CD40 was evaluated by ELISA. All BEATs had similar binding to CD40 ( Fig.16 ). The affinity of the one-armed BEAT for CD40 or FLAG was confirmed by SPR using a 1:1 model. As shown in the following table, using a 1:1 model, the affinity of the one-armed BEAT was comparable to that of the anti-CD40 control Mab and BEAT 1a4:

[0806] CD40 FLAG Anti-CD40 control Mab 0.77nM not applicable BEAT1a4 0.59nM 2.59nM BEAT3 0.52nM 0.475nM BEAT4 0.92nM 0.357nM BEAT5 0.73nM 1.95nM BEAT6 0.42nM 1.44nM

[0807] Example 21 - Combination of single-arm BEAT with CAR T cells and AP cells

[0808] The binding of monovalent single-arm BEAT 3 and BEAT 4 to CAR T cells was comparable to that of the bivalent BEAT 1a4, and the binding of BEAT 5 and BEAT 6 was slightly reduced compared with BEAT 1a4, BEAT 3, and BEAT 4 ( Fig.17 A). The binding of the monovalent one-arm BEAT to MoDC is also comparable to that of the bivalent BEAT 1a4 ( Fig.17 B).

[0809] Example 22 - Functional activity of one-armed BEAT

[0810] CD86 expression and IL-12 / IL-23p40 release were assessed in the presence of BEATs and compared to BEAT 1a4 and an anti-CD40 control antibody. The activity of the single-armed BEATs was low compared to BEAT 1a4, with BEAT 3 and BEAT 4 (monovalent anti-CD40) being lower than BEAT 5 and BEAT 6 (bivalent anti-CD40) for both CD86 expression and IL-12 / IL-23p40 release ( Fig.18 ).

[0811] Example 23 - T cell proliferation of single-arm BEAT on day 5

[0812] The proliferation of single-arm BEATs was compared to BEAT 1a4 in a dose-response assay when both CAR T and MoDC were added. 7B1-CD28z CAR was incubated with MoDC and various BEATs and controls for 120 h, and the number of CAR T cells (via CD4+GFP+) was measured.

[0813] At 1 nM BEAT 1a4, a prozone effect was observed in CAR T cell proliferation. Dose responses were observed in each single-arm BEAT, with BEAT 3 being the strongest, followed by BEAT 4, BEAT 5, and BEAT 6. At the highest dose, equivalent proliferation was observed between 0.1 and 10 nM, and a prozone effect was observed in BEAT 5 above 1 nM ( Fig.19 ).

[0814] The phenotype of the cells was also assessed in this proliferation assay, where the T EM Increase( Fig. 20 A. Fig. 20 C. Fig. 20 D) and is primarily driven by increases in CD8+ cells ( Fig. 22 A. Fig. 22 B. Fig. 22 C. Fig. 22 D), CD4+ cells T EM At the highest dose of 100 nM, the increase ( Fig.21 D) Single-arm BEAT T cells ( Fig. 20 )、CD4+ cells( Fig.21 ) and CD8+ cells ( Fig. 22 ) phenotype was similar at all doses.

[0815] Example 24 - Cytokine analysis of single-arm BEAT

[0816] Cytokines were analyzed in the presence of CAR, CAR+MoDC, and MoDC alone. BEAT had no effect on IFN-γ in the presence of CAR alone or MoDC alone ( Fig.23 A). In the presence of CAR T cells + MoDC and BEAT, IFN-γ increased in a dose-response manner, with all BEATs responding comparably and the maximal response seen at 1 nM ( Fig.23 A).

[0817] In the presence of CAR alone or MoDC alone, BEAT had no effect on IL-12p70 or IL-6 ( Fig.23 B and Fig.23 C). IL-12p70 of all BEATs increased at 0.1 nM and 1 nM, and in the presence of CAR and MoDC, a prozone effect was observed above 1 nM ( Fig.23 B). IL-6 increased in a dose-response manner with the addition of BEATs to CAR and MoDC, with BEAT4 producing higher cytokine levels than other BEATs ( Fig.23 C).

[0818] IL-2 was increased in the presence of CAR alone and in a dose-responsive manner in the presence of BEAT+CAR+MoDC ( Fig.23 D). No changes in MCP-1 were observed above background ( Fig.23 E). TNF-α increased in the presence of BEAT+CAR+MoDC, but remained unchanged in the presence of CAR or MoDC alone ( Fig.23 F).

Claims

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

2. The bispecific polypeptide according to claim 1, in, The first antigen binding protein comprises an antigen binding domain, wherein the antigen binding domain comprises: FR1–CDR1–FR2–CDR2–FR3–CDR3–FR4, and FR1a–CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a, in: FR1, FR2, FR3 and FR4 are each a framework region; CDR1, CDR2, and CDR3 are each complementarity determining regions; FR1a, FR2a, FR3a and FR4a are each a framework region; CDR1a, CDR2a, and CDR3a are each complementarity determining regions; Wherein, the sequence of any one of the framework regions and / or complementarity determining regions is as described herein, preferably as described in Table 1 herein.

3. The bispecific polypeptide according to claim 2, in, The first antigen binding protein comprises: FR1–CDR1–FR2–CDR2–FR3–CDR3–FR4–linker–FR1a–CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a, Optionally, the linker is a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

4. The bispecific polypeptide according to any one of claims 1 to 3, in, The first antigen binding protein binds or specifically binds to CD40.

5. The bispecific polypeptide according to claim 4, in, The first antigen-binding protein competitively inhibits the binding of an antibody or an antigen-binding fragment thereof to CD40, wherein the antibody or the antigen-binding fragment thereof comprises a VH comprising the sequence shown in SEQ ID NO: 1 and a VL comprising the sequence shown in SEQ ID NO:

2.

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

2.

7. The bispecific polypeptide according to any one of claims 4 to 6, in, The first antigen binding protein comprises: (i) VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 3, CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 6, and CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 10, and (ii) VL comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 12, CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 14, and CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 16; or (iii) a VH comprising 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:3, a CDR2 comprising or consisting of a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO:6, and a CDR3 comprising or consisting of a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO:

7. NO:10 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 or consisting of the sequence shown in NO:10, and (iv) a VL comprising 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, and 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: 12 NO:14 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 or consisting of a CDR3, wherein the CDR3 comprises or consists of 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%, at least 99% identical to or consisting of a sequence as set forth in SEQ ID NO:16; or (v) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 5, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 9, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 11, and (vi) 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 VH comprising 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:5, and 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:5 NO:9 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 or consisting of a CDR3, wherein the CDR3 comprises 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:11, and (viii) a VL comprising 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, and 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:13 NO:15 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, or consisting of, a CDR3, wherein the CDR3 comprises 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, or consisting of, a sequence as set forth in SEQ ID NO:16; or (ix) VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 4, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 7, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 10, and (x) 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 VH comprising 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, and 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:4 NO:7 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, or consisting of, a CDR3 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 to, or consisting of, a CDR3 (xii) a VL comprising a CDR1 that comprises or consists 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, and a CDR2 that comprises 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. NO:14 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 or consisting of a CDR3, wherein the CDR3 comprises or consists of 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%, at least 99% identical to or consisting of a sequence as set forth in SEQ ID NO:16; or (ix) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 36, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 40, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 42, and (vi) 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 VH comprising 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:36, and 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:36 NO:40 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, or consisting of, a CDR3 comprising or consisting of 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%, at least 99% identical to, and (viii) a VL comprising 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, and 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:44 NO:46 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 shown in NO:46, or consists of a CDR3, wherein the CDR3 comprises 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 shown in SEQ ID NO:48, or consists of a CDR3.

8. The bispecific polypeptide according to any one of claims 4 to 7, in, The first antigen-binding protein comprises a heavy chain variable domain, which comprises or consists of the amino acid sequence shown in SEQ ID NO:1, or comprises or consists 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 amino acid sequence.

9. The bispecific polypeptide according to claim 8, in, Compared with the amino acid sequence shown in SEQ ID NO:1, 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; optionally, wherein the amino acid substitutions are not in the CDR and / or the antigen-binding protein retains the ability to bind to CD40.

10. The bispecific polypeptide according to any one of claims 4 to 9, in, The first antigen binding domain comprises a light chain variable domain, which comprises or consists of the amino acid sequence shown in SEQ ID NO:2, or comprises or consists 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 amino acid sequence.

11. The bispecific polypeptide according to claim 10, in, Compared with the amino acid sequence shown in SEQ ID NO:2, 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; optionally, wherein the amino acid substitutions are not in the CDR and / or the antigen-binding protein retains the ability to bind to CD40.

12. The bispecific polypeptide according to any one of claims 4 to 11, in, The first antigen binding protein comprises, consists essentially of, or consists of the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 (in order from N-terminus to C-terminus or from C-terminus to N-terminus).

13. The bispecific polypeptide according to any one of claims 1 to 3, in, The first antigen binding protein binds or specifically binds to CD206.

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

15. The bispecific polypeptide according to claim 14, in, The first antigen binding protein binds to CD206 expressed on the surface of the professional antigen presenting cells, wherein the professional antigen presenting cells are selected from dendritic cells, macrophages, B cells, epithelial cells, most preferably selected from dendritic cells, and even more preferably, wherein the professional antigen presenting cells are not tumor cells.

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

34.

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

34.

18. The bispecific polypeptide according to any one of claims 13 to 17, in, The first antigen binding protein comprises an antigen binding domain, wherein the antigen binding domain comprises: (i) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 35, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 38, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 42, and (ii) 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 VH comprising 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: 35, and 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: 35 NO:38 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, or consisting of, a CDR3 comprising or consisting of 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%, at least 99% identical to, and (iv) a VL comprising 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, and 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:

44. NO:46 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, or consisting of, a CDR3 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 to, or consisting of, a CDR3 or (v) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 37, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 41, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 43, and (vi) 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 VH comprising 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:37, and 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:37 NO:41 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 shown in NO:41, or consists of a CDR3, wherein the CDR3 comprises 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 shown in SEQ ID NO:43, or consists of a CDR3, and (viii) a VL comprising 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, and 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:

45. NO:47 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, or consisting of, a CDR3 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 to, or consisting of, a CDR3 or (ix) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 36, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 39, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 42, and (x) 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 VH comprising 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:36, and 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:36 NO:39 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, 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%, at least 99% identical to, and (xii) VL, which comprises CDR1, said CDR1 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 to or consisting of the sequence shown in SEQ ID NO: 44, CDR2, said CDR2 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 to or consisting of the sequence shown in SEQ ID NO: 46, CDR3, said CDR3 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 to or consisting of the sequence shown in SEQ ID NO: 48; or (ix) VH, which comprises complementarity determining region (CDR) 1, said CDR1 comprising the sequence shown in SEQ ID NO: 36 or consisting of the same, CDR2, said CDR2 comprising the sequence shown in SEQ ID NO: 40 or consisting of the same, and CDR3, said CDR3 comprising the sequence shown in SEQ ID NO: 42 or consisting of the same, and (vi) VL, which comprises complementarity determining region (CDR) 1, said CDR1 comprising the sequence shown in SEQ ID NO: 44 or consisting of the same, CDR2, said CDR2 comprising the sequence shown in SEQ ID NO: 46 or consisting of the same, and CDR3, said CDR3 comprising the sequence shown in SEQ ID NO: 48 or consisting of the same; or (vii) a VH comprising 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:36, and 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:36 NO:40 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, or consisting of, a CDR3 comprising or consisting of 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%, at least 99% identical to, and (viii) a VL comprising 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, and 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:44 NO:46 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 shown in NO:46, or consists of a CDR3, wherein the CDR3 comprises 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 shown in SEQ ID NO:48, or consists of a CDR3.

19. The bispecific polypeptide according to any one of claims 13 to 18, in, The first antigen-binding protein comprises a heavy chain variable domain, which comprises or consists of the amino acid sequence shown in SEQ ID NO:33, or comprises or consists 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 amino acid sequence.

20. The bispecific polypeptide according to claim 19, in, Compared with the amino acid sequence shown in SEQ ID NO:33, 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; optionally, wherein the amino acid substitutions are not in the CDRs and / or the antigen-binding protein retains the ability to bind to CD206.

21. The bispecific polypeptide according to any one of claims 13 to 20, in, The first antigen binding domain comprises a light chain variable domain, which comprises or consists of the amino acid sequence shown in SEQ ID NO:34, or comprises or consists 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 amino acid sequence.

22. The bispecific polypeptide according to claim 21, in, Compared with the amino acid sequence shown in SEQ ID NO:34, 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; optionally, wherein the amino acid substitutions are not in the CDRs and / or the antigen-binding protein retains the ability to bind to CD206.

23. The bispecific polypeptide according to any one of claims 13 to 22, in, The first antigen binding protein comprises, consists essentially of, or consists of the amino acid sequences of SEQ ID NO: 33 and SEQ ID NO: 34 (in order from N-terminus to C-terminus or from C-terminus to N-terminus).

24. The bispecific polypeptide according to any one of claims 1 to 23, in, The first antigen binding protein comprises, from N-terminus to C-terminus, VH and then VL, or VL and then VH, or any CDR1, CDR2 and CDR3 defined herein as VH and then any CDR1, CDR2 and CDR3 defined herein as VL, or any CDR1, CDR2 and CDR3 defined herein as VL and then any CDR1, CDR2 and CDR3 defined herein as VH.

25. The bispecific polypeptide according to any one of claims 1 to 24, in, The first antigen binding protein is in the form of: (i) Single domain antibodies (sdAb); (ii) single-chain Fv fragment (scFv); (iii) dimeric scFv (di-scFv); or (iv) one of (ii) or (iii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody.

26. The bispecific polypeptide according to any one of claims 1 to 24, in, The first antigen binding protein is in the form of: (i) Diabodies; (ii) three-chain antibodies; (iii) tetrabodies; (iv) Fab; (v) F(ab')2; (vi) Fv; (vii) bispecific antibodies or other forms of multispecific antibodies; (viii) one of (i) to (vii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody.

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

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

29. The bispecific polypeptide according to any one of claims 1 to 28, in, The second antigen binding domain is used to bind to the FLAG tag defined by SEQ ID NO:80 and SEQ ID NO:99 or a variant thereof, such as wherein the FLAG tag contains, comprises or consists of the following 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. The bispecific polypeptide according to any one of claims 1 to 29, in, The FLAG tag is present at the N-terminus, C-terminus or within the protein to which the bispecific polypeptide is capable of binding, binds or specifically binds.

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

79.

32. The bispecific polypeptide according to any one of claims 1 to 31, in, The second antigen-binding protein comprises CDRH1, CDRH2 and / or CDRH3 having an antigen-binding domain of a variable heavy chain as defined in any one of SEQ ID NO:71 to SEQ ID NO:74, and / or CDRL1, CDRL2 and / or CDRL3 having an antigen-binding domain of a variable light chain as defined in any one of SEQ ID NO:76 to SEQ ID NO:

79.

33. The bispecific polypeptide according to any one of claims 1 to 32, in, The second antigen binding domain comprises an antigen binding domain comprising: (a) a VH comprising 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 set forth in SEQ ID NO:81, and 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 set forth in SEQ ID NO:81 NO:82, or consists 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:82, a CDR3 that comprises or consists 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 VL comprising 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:90, and 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:90 91, 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:91, and a CDR3 that comprises or consists 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:92; or (c) a VH comprising 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 SEQ ID NO: 112, and 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: 111 or SEQ ID NO: 112 NO:113, or consists 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:113, a CDR3 that comprises or consists 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, and (d) a VL comprising 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, and 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: 120 : 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: 121, and a CDR3 that comprises, or consists 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: 92; or (e) a VH comprising 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 SEQ ID NO: 112, and 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: 111 or SEQ ID NO: 112 13, or consists 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: 113, and a CDR3 that comprises or consists 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; and (f) a VL comprising 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, and 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:

133. 90%, 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: 121, or consisting thereof, 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: 92; or (g) a VH comprising 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 SEQ ID NO: 156, and 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: 111 or SEQ ID NO: 156 : 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: 157, and a CDR3 that comprises, or consists 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; and (h) a VL comprising 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, and 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: 120 : 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: 121, and a CDR3 that comprises, or consists 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: 92; or (i) a VH comprising 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 SEQ ID NO: 156, and 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: 111 or SEQ ID NO: 156 : 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: 157, and a CDR3 that comprises, or consists 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; and (j) a VL comprising 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: 133, and 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: 133 : 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: 121, and a CDR3 that comprises, or consists 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: 92; or (k) a VH comprising 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, and 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: 126 NO:127 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:127, or consists of an amino acid 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%, at least 99% identical to the sequence of SEQ ID NO:114, or consists of an amino acid 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%, at least 99% identical to the sequence of SEQ ID NO:114; (l) a VL comprising 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, and 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: 120 : 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: 121, and a CDR3 that comprises, or consists 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: 92; or (m) a VH comprising 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, and 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: 126 NO:127 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:127, or consists of an amino acid 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%, at least 99% identical to the sequence of SEQ ID NO:114, or consists of an amino acid 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%, at least 99% identical to the sequence of SEQ ID NO:114; (n) a VL comprising 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: 133, and 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: 133 : 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: 121, and a CDR3 that comprises, or consists 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: 92; or (o) a VH comprising 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, and 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: 111 : 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: 132, and a CDR3 that comprises, or consists 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; and (p) a VL comprising 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: 133, and 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: 133 : 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: 121, and a CDR3 that comprises, or consists 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: 92; or (q) a VH comprising 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, and 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: 126 NO:127 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:127, or consists of an amino acid 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%, at least 99% identical to the sequence of SEQ ID NO:114, or consists of an amino acid 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%, at least 99% identical to the sequence of SEQ ID NO:114; (r) a VL comprising 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: 133, and 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: 133 NO:121 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:121, or consists 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:92, or consists 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:

34. The bispecific polypeptide according to any one of claims 1 to 33, in, The second antigen binding domain comprises an antigen binding domain comprising: (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) VL, which comprises CDR1, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 90, and CDR2, The CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 91, and the CDR3 comprises or consists 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 SEQ ID NO: 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 SEQ ID NO: 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) 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 SEQ ID NO: 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 SEQ ID NO: 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 (1) VL, which comprises CDR1, wherein the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 120, CDR2, wherein the CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 121, and CDR3, wherein the CDR3 comprises or consists 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) 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, a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 114, and (r) VL, which comprises CDR1, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 133, CDR2, wherein the CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 121, and CDR3, wherein the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

92.

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

36. The bispecific polypeptide according to any one of claims 1 to 35, in, The second antigen binding protein comprises an antigen binding domain having FR H1, FR H2, FR H3 and / or FR H4 having an antigen binding domain of a variable heavy chain as defined in any one of SEQ ID NO: 71 to SEQ ID No:

74.

37. The bispecific polypeptide according to any one of claims 1 to 36, in, The second antigen binding protein comprises an antigen binding domain having FR L1, FR L2, FR L3 and / or FR L4 having an antigen binding domain of a variable light chain as defined in any one of SEQ ID NO: 76 to SEQ ID NO:

79.

38. The bispecific polypeptide according to any one of claims 1 to 37, in, The second antigen-binding protein comprises an antigen-binding domain, wherein the antigen-binding domain comprises a VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (a) above, and a VL comprising a CDR1, CDR2 and CDR3 as defined in any one of (b) above, and: A. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136 and SEQ ID NO: 137, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 and SEQ ID NO: 97, respectively; or B. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 87 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 and SEQ ID NO: 97, respectively; or C. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 87 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155 and SEQ ID NO: 97, respectively; or D. the VH and the VL each comprise framework regions (FR) 1, 2, 3, and 4, wherein the framework regions (FR) 1, 2, 3, and 4 comprise 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 87, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97, respectively; or E. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 87 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 96 and SEQ ID NO: 97, respectively; or F. the VH and the VL each comprise framework regions (FR) 1, 2, 3, and 4, wherein the framework regions (FR) 1, 2, 3, and 4 comprise 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 97, respectively; or G. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155 and SEQ ID NO: 97, respectively; or H. the VH and the VL each comprise framework regions (FR) 1, 2, 3, and 4, wherein the framework regions (FR) 1, 2, 3, and 4 comprise 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97, respectively; or I. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequence is shown in SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is shown in SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 96 and SEQ ID NO: 97, respectively; or J. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequence is shown in SEQ ID NO: 149, SEQ ID NO: 85, SEQ ID NO: 151 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is shown in SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 and SEQ ID NO: 97, respectively; or K. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequence is shown in SEQ ID NO: 149, SEQ ID NO: 85, SEQ ID NO: 151 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is shown in SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155 and SEQ ID NO: 97, respectively; or L. the VH and the VL each comprise framework regions (FR) 1, 2, 3, and 4, wherein the framework regions (FR) 1, 2, 3, and 4 comprise 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 FR, the reference sequence is set forth in SEQ ID NO: 149, SEQ ID NO: 85, SEQ ID NO: 151, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97, respectively; or M. the VH and the VL each comprise framework regions (FR) 1, 2, 3, and 4, wherein the framework regions (FR) 1, 2, 3, and 4 comprise 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 149, SEQ ID NO: 85, SEQ ID NO: 151, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 96, and SEQ ID NO: 97, respectively; or N. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 152 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 and SEQ ID NO: 97, respectively; or O. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 152 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155 and SEQ ID NO: 97, respectively; or P. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 152 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 97, respectively; or Q. The VH and VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 152 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 96 and SEQ ID NO: 97, respectively.

39. The bispecific polypeptide according to any one of claims 1 to 37, in, The second antigen-binding protein comprises an antigen-binding domain, wherein the antigen-binding domain comprises a VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (c) above, and a VL comprising a CDR1, CDR2 and CDR3 as defined in any one of (d) above, and: A. the VH and the VL each comprise framework regions (FR) 1, 2, 3, and 4, wherein the framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence; for VH FR, the reference sequences are shown in SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 118, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 127, respectively; or B. the VH and the VL each comprise framework regions (FR) 1, 2, 3, and 4, wherein the framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence; for VH FR, the reference sequences are shown in SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 118, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 127, respectively; or C. the VH and the VL each comprise framework regions (FR) 1, 2, 3, and 4, wherein the framework regions (FR) 1, 2, 3, and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a reference sequence; for VH FR, the reference sequences are shown in SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 127, respectively; or D. The VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to a reference sequence; for VH FR, the reference sequences are shown in SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119 and SEQ ID NO: 89, respectively, and for VLFR, the reference sequences are shown in SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125 and SEQ ID NO: 127, respectively.

40. The bispecific polypeptide according to any one of claims 1 to 37, in, The second antigen-binding protein comprises an antigen-binding domain, wherein the antigen-binding domain comprises a VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (e) above, and a VL comprising a CDR1, CDR2 and CDR3 as defined in any one of (f) above, and: A. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 118 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 97, respectively; or B. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 118 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NO: 97, respectively; or C. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 97, respectively; or D. The VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119 and SEQ ID NO: 89, respectively, and for VLFR, the reference sequences are shown in SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NO: 97, respectively.

41. The bispecific polypeptide according to any one of claims 1 to 37, in, The second antigen-binding protein comprises an antigen-binding domain, comprising a VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (g) above, and a VL comprising a CDR1, CDR2 and CDR3 as defined in any one of (h) above, and: A. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 158, SEQ ID NO: 116, SEQ ID NO: 159 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 127, respectively; or B. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 158, SEQ ID NO: 116, SEQ ID NO: 159 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125 and SEQ ID NO: 127, respectively; or C. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 158, SEQ ID NO: 117, SEQ ID NO: 160 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 127, respectively; or D. The VH and VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 158, SEQ ID NO: 117, SEQ ID NO: 160 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125 and SEQ ID NO: 127, respectively.

42. The bispecific polypeptide according to any one of claims 1 to 37, in, The second antigen-binding protein comprises an antigen-binding domain, wherein the antigen-binding domain comprises a VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (i) above, and a VL comprising a CDR1, CDR2 and CDR3 as defined in any one of (j) above, and: A. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 158, SEQ ID NO: 116, SEQ ID NO: 159 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 97, respectively; or B. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 158, SEQ ID NO: 116, SEQ ID NO: 159 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NO: 97, respectively; or C. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 158, SEQ ID NO: 117, SEQ ID NO: 160 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 97, respectively; or D. The VH and VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 158, SEQ ID NO: 117, SEQ ID NO: 160 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NO: 97, respectively.

43. The bispecific polypeptide according to any one of claims 1 to 37, in, The second antigen-binding protein comprises an antigen-binding domain, wherein the antigen-binding domain comprises a VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (k) above, and a VL comprising a CDR1, CDR2 and CDR3 as defined in any one of (l) above, and: A. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 84, SEQ ID NO: 128, SEQ ID NO: 130 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 97, respectively; or B. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 84, SEQ ID NO: 128, SEQ ID NO: 130 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125 and SEQ ID NO: 97, respectively; or C. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 84, SEQ ID NO: 129, SEQ ID NO: 131 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 97, respectively; or D. the VH and the VL each comprise framework regions (FR) 1, 2, 3, and 4, wherein the framework regions (FR) 1, 2, 3, and 4 comprise 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 84, SEQ ID NO: 129, SEQ ID NO: 131, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 97, respectively; or E. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 164, SEQ ID NO: 128, SEQ ID NO: 166 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 97, respectively; or F. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 164, SEQ ID NO: 128, SEQ ID NO: 166 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125 and SEQ ID NO: 97, respectively; or G. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 97, respectively; or H. The VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to a reference sequence; for VH FR, the reference sequences are shown in SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167 and SEQ ID NO: 89, respectively, and for VLFR, the reference sequences are shown in SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125 and SEQ ID NO: 97, respectively.

44. The bispecific polypeptide according to any one of claims 1 to 37, in, The second antigen-binding protein comprises an antigen-binding domain, wherein the antigen-binding domain comprises a VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (m) above, and a VL comprising a CDR1, CDR2 and CDR3 as defined in any one of (n) above, and: A. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 84, SEQ ID NO: 128, SEQ ID NO: 130 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 97, respectively; or B. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 84, SEQ ID NO: 128, SEQ ID NO: 130 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170 and SEQ ID NO: 97, respectively; or C. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 84, SEQ ID NO: 129, SEQ ID NO: 131 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 97, respectively; or D. the VH and the VL each comprise framework regions (FR) 1, 2, 3, and 4, wherein the framework regions (FR) 1, 2, 3, and 4 comprise 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; for VH FR, the reference sequences are set forth in SEQ ID NO: 84, SEQ ID NO: 129, SEQ ID NO: 131, and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are set forth in SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, and SEQ ID NO: 97, respectively; or E. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 164, SEQ ID NO: 128, SEQ ID NO: 166 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 97, respectively; or F. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 164, SEQ ID NO: 128, SEQ ID NO: 166 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170 and SEQ ID NO: 97, respectively; or G. the VH and the VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequence is set forth in SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequence is set forth in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 97, respectively; or H. The VH and VL each comprise framework regions (FR) 1, 2, 3 and 4, wherein the framework regions (FR) 1, 2, 3 and 4 comprise 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; for VH FR, the reference sequences are shown in SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167 and SEQ ID NO: 89, respectively, and for VL FR, the reference sequences are shown in SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170 and SEQ ID NO: 97, respectively.

45. The bispecific polypeptide according to any one of claims 1 to 37, in, The second antigen binding protein comprises an antigen binding domain, wherein the antigen binding domain comprises: A. A VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (o) above, and a VL comprising CDR1, CDR2 and CDR3 as defined in any one of (p) above, and said VH and said VL each comprising a framework region (FR) 1, 2, 3 and 4, said framework region (FR) 1, 2, 3 and 4 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; for VH FR, said reference sequence is SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165 NO:143 and SEQ ID NO:137, and for VL FR, the reference sequences are shown in SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146 and SEQ ID NO:97, respectively; or B. A VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 as defined in any one of (q) above, and a VL comprising CDR1, CDR2 and CDR3 as defined in any one of (r) above, and each of the VH and VL comprises a framework region (FR) 1, 2, 3 and 4, wherein the framework region (FR) 1, 2, 3 and 4 comprises 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; for VH FR, the reference sequence is SEQ ID NO: 84, SEQ ID NO: 147, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO:148 and SEQ ID NO:137, and for VL FR, the reference sequences are shown in SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146 and SEQ ID NO:97, respectively.

46. ​​The bispecific polypeptide according to any one of claims 1 to 37, in, The second antigen binding protein comprises an antigen binding domain, which comprises a variable heavy chain, which comprises the amino acid sequence shown in any one of SEQ ID NO:71 to SEQ ID NO:74, or 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 amino acid sequence.

47. The bispecific polypeptide according to claim 46, in, Compared with the amino acid sequence shown in SEQ ID NO:71 to SEQ ID NO:74, 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; optionally, the amino acid substitutions are not in the CDR and / or the antigen-binding protein retains the ability to bind to the FLAG tag.

48. The bispecific polypeptide according to any one of claims 1 to 47, in, The second antigen binding protein comprises an antigen binding domain, which comprises a variable light chain, which comprises the amino acid sequence shown in any one of SEQ ID NO:76 to SEQ ID NO:79, or 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 amino acid sequence.

49. The bispecific polypeptide according to claim 48, in, Compared with the amino acid sequences shown in SEQ ID NO:76 to SEQ ID NO:79, 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; optionally, the amino acid substitutions are not in the CDR and / or the antigen-binding protein retains the ability to bind to the FLAG tag.

50. The bispecific polypeptide according to any one of claims 1 to 49, in, The second antigen binding protein is in the form of: (i) Single domain antibodies (sdAb); (ii) single-chain Fv fragment (scFv); (iii) dimeric scFv (di-scFv); (iv) one of (ii) or (iii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody.

51. The bispecific polypeptide according to any one of claims 1 to 49, in, The second antigen binding protein is in the form of: (i) Diabodies; (ii) three-chain antibodies; (iii) tetrabodies; (iv) Fab; (v) F(ab')2; (vi) Fv; (vii) bispecific antibodies or other forms of multispecific antibodies; (viii) one of (i) to (vii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody.

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

53. The bispecific polypeptide according to any one of claims 1 to 51, in, The second antigen binding protein and the first antigen binding protein are both antibodies or antigen binding fragments thereof; optionally, the first antigen binding protein and the second antigen binding protein have the same antibody form or fragment thereof.

54. The bispecific polypeptide according to any one of claims 1 to 53, in, The second antigen binding domain comprises, consists essentially of, or consists of the following amino acid sequence (in order from N-terminus to C-terminus or from C-terminus to N-terminus): (v) SEQ ID NO:71 and SEQ ID NO:79; (w) SEQ ID NO:71 and SEQ ID NO:78; (x) SEQ ID NO:71 and SEQ ID NO:76; (y) SEQ ID NO:71 and SEQ ID NO:77; (z) SEQ ID NO:72 and SEQ ID NO:76; (aa) SEQ ID NO:72 and SEQ ID NO:77; (bb) SEQ ID NO:72 and SEQ ID NO:78; (cc) SEQ ID NO:72 and SEQ ID NO:79; (dd) SEQ ID NO:73 and SEQ ID NO:76; (ee) SEQ ID NO:73 and SEQ ID NO:77; (ff) SEQ ID NO:73 and SEQ ID NO:78; (gg) SEQ ID NO:73 and SEQ ID NO:79; (hh) SEQ ID NO:74 and SEQ ID NO:76; (ii) SEQ ID NO:74 and SEQ ID NO:77; (jj) SEQ ID NO:74 and SEQ ID NO:78; or (kk) SEQ ID NO:74 and SEQ ID NO:

79.

55. The bispecific polypeptide according to any one of claims 1 to 54, in, The bispecific polypeptide comprises the following amino acid sequence: - SEQ ID NO: 19 and SEQ ID NO: 20; - SEQ ID NO: 19 and SEQ ID NO: 21; - SEQ ID NO: 22 and SEQ ID NO: 23; - SEQ ID NO: 23 and SEQ ID NO: 24; - SEQ ID NO: 19 and SEQ ID NO: 25; - SEQ ID NO: 19 and SEQ ID NO: 26; - SEQ ID NO: 23 and SEQ ID NO: 27; - SEQ ID NO: 23 and SEQ ID NO: 28; - SEQ ID NO: 19 and SEQ ID NO: 29; - SEQ ID NO: 19 and SEQ ID NO: 30; - SEQ ID NO: 23 and SEQ ID NO: 31; - SEQ ID NO: 23 and SEQ ID NO: 32; -SEQ ID NO:171, SEQ ID NO:172 and SEQ ID NO:173; -SEQ ID NO:172, SEQ ID NO:174 and SEQ ID NO:175; -SEQ ID NO:171, SEQ ID NO:172 and SEQ ID NO:176; -SEQ ID NO:172, SEQ ID NO:174 and SEQ ID NO:177; - SEQ ID NO:50 and SEQ ID NO:51; - SEQ ID NO:50 and SEQ ID NO:52; - SEQ ID NO:53 and SEQ ID NO:54; - SEQ ID NO:54 and SEQ ID NO:55; - SEQ ID NO:50 and SEQ ID NO:56; - SEQ ID NO:50 and SEQ ID NO:57; - SEQ ID NO:54 and SEQ ID NO:58; - SEQ ID NO:54 and SEQ ID NO:59; - SEQ ID NO: 50 and SEQ ID NO: 60; - SEQ ID NO:50 and SEQ ID NO:61; - SEQ ID NO:54 and SEQ ID NO:62; or - SEQ ID NO: 54 and SEQ ID NO:

63.

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

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

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

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

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

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

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

63. A pharmaceutical composition according to claim 61, which is used for treating or preventing cancer.

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

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