Binding proteins comprising h2l2 and hcab structure

Abstract

To provide bi- or multi-specific binding proteins having H2L2 and HCAb structure, and methods for preparing and using such binding proteins.SOLUTION: Disclosed is a binding protein comprising at least two protein function regions, where the binding protein comprises a protein functional region A and a protein functional region B; the protein functional region A and the protein functional region B target different antigens or antigenic epitopes, where the protein functional region A is of a Fab or scFv structure, the protein functional region B is of a VH structure, and the number of each of the protein functional region A and the protein functional region B is one or more. A multispecific binding protein of the invention has a smaller molecular weight, fewer polypeptide chains and a simpler structure. By means of different structure types, relative positions, binding valence and other parameters, the functional activities for different targets can be adjusted, and then different combinations of activities can be designed so as to meet the needs of different dose combinations of clinical combinations.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the biomedical field, and in particular to a bispecific or multispecific binding protein having H2L2 and HCAb structures and uses thereof. [Background technology]

[0002] In most species, antibodies have a tetrameric structure containing two identical heavy chains and two identical light chains, also called "H2L2". Heavy-chain antibodies (HCAb), which lack light chains, also naturally exist in the serum of camelids and sharks. The variable region VHH fragments of heavy-chain antibodies are similar to the Fab of H2L2 antibodies and have a stable structure and specific antigen-binding activity, but the molecular weight of VHH is only about 13 Kda, so they are also called nanoantibodies or single-domain antibodies. Since human antibodies have a natural structure of "H2L2", it is not possible to obtain useful human-derived heavy-chain antibodies from natural sources; however, Frank Grosveld et al. proposed a method for obtaining all-human-derived heavy-chain antibodies using transgenic animals (WO2007 / 096779), and that this method can be used to obtain stable and soluble all-human-derived VH single-domain antibodies.

[0003] Bispecific and multispecific antibodies are artificial antibodies produced by protein engineering techniques based on natural monoclonal antibodies, which are capable of binding two or more different antigens or different epitopes on the same antigen, achieving mechanisms of action and functional effects that cannot be achieved by some monospecific antibodies.

[0004] The structural design of bispecific antibodies is very important. In producing bispecific antibodies from two different H2L2 antibodies, one of the most major challenges is how to obtain bispecific antibody molecules with the functionality of the correct chain combination from more than 10 different heavy and light chain combinations, i.e., how to solve the chain mismatch problem. Scientists have already developed many strategies to solve this problem: for example, fusing VH and VL using the structure of single-chain variable region antibody fragment (scFv); or promoting heavy chain heterodimerization using the knob-into-hole (KiH) technology or other spatial structure complementary mutants; or reducing the number of different polypeptide chains using the "shared light chain" or "shared heavy chain" technology. However, various technical means have limitations, for example, the scFv structure is always unstable and prone to aggregation; the "shared light chain" technology needs to use a very complicated filtering process to obtain a VL that can pair with a different VH; the size of the bispecific antibody molecules generated by technologies such as FIT-Ig is relatively large (about 250 KDa), which may affect their ability to penetrate cells and tissues. Therefore, there remains a need for new techniques for developing bispecific antibody structures and producing bispecific antibodies.

[0005] Heavy chain antibodies and single domain antibodies derived therefrom have unique advantages in constructing bispecific or multispecific antibodies. The antigen-binding domain of a heavy chain antibody is only one-quarter the size of the Fab of a conventional antibody; and it has no light chain, which can avoid the problem of light chain mismatch. Therefore, heavy chain antibodies and single domain antibodies derived therefrom can be used to construct bispecific or even multispecific antibodies with smaller molecular weights, fewer polypeptide chains, and simpler structures. However, the potential immunogenicity risk of non-human-derived heavy chain antibodies may limit their therapeutic use. Therefore, compared with camelid nanobodies, constructing bispecific or multispecific resistance using all-human-derived heavy chain antibodies is more advantageous in terms of immunogenicity and druggability. Summary of the Invention

[0006] In order to overcome the deficiencies in the prior art in terms of lack of bispecific or multispecific binding proteins with good therapeutic effects, stability and ease of production, the present invention provides bispecific or multispecific binding proteins having H2L2 and HCAb structures and methods for making and using such binding proteins, which utilize multivalent and multispecific binding proteins constructed from fully human heavy chain antibodies and single domain antibodies derived therefrom, which have many advantages over conventional multivalent and multispecific binding proteins constructed using IgG antibodies, and are more convenient in terms of tuning specificity and valency. The multivalent and multispecific binding proteins provided herein contain at least one heavy chain variable region domain VH derived from a human heavy chain antibody, and can bind two or more antigens, or two or more epitopes of the same antigen, or two or more copies of the same epitope.

[0007] In order to solve the above technical problems, the first technical configuration of the present invention provides a binding protein containing at least two protein functional regions, the binding protein comprising a protein functional region A and a protein functional region B; the protein functional region A and the protein functional region B target different antigens or antigen epitopes, in which the protein functional region A is a Fab or scFv structure, the protein functional region B is a VH structure, and the number of the protein functional region A and the protein functional region B is one or more; preferably, the binding protein further comprises an Fc, the number of the Fc is two, and an Fc dimer is formed; preferably, the binding protein has a symmetric structure or an asymmetric structure, and / or the Fc dimer is a homodimer or a heterodimer.

[0008] In some specific embodiments, (I) the binding protein has a symmetric structure, and the symmetric structure is a bilaterally symmetric IgG-like structure, in which the number of the protein functional regions A is two, and the number of the protein functional regions B is two or four. The two protein functional regions A and the two Fc's form an IgG structure. In the bilaterally symmetric IgG-like structure, left and right are only relative spatial positions, which may be vertically symmetric or front-to-back symmetric. The bilaterally symmetric IgG-like structure means that additional protein functional regions are further connected to an antibody of an IgG structure in a bilaterally symmetric manner, and these additional protein functional regions may be connected to the C-terminus of CH3 or the N-terminus of VH or VL of the Fc functional region, or may be connected between the variable region and the Fc functional region of the IgG structure in an embedded form. The term "additional" as used herein refers to a structure or functional region other than the IgG structure.

[0009] Preferably, when the number of the protein functional region B is two, the binding protein is a tetravalent binding protein, specifically comprising the following structure: (a) the binding protein is, from the N-terminus to the C-terminus, a protein functional region A, a protein functional region B and an Fc, in which the protein functional region A and the protein functional region B are connected by a first connecting peptide (L1), and the protein functional region B and the Fc are connected by a second connecting peptide (L2); in this embodiment, the protein functional region B and the Fc of both parts form a symmetric single-chain antibody dimer form, and the protein functional region A is connected to the N-terminus of the single-chain antibody dimer, in which the protein functional region A is connected to the N-terminus of the protein functional region B at its CH1 (Figure 1 (2)) or CL (Figure 1 (1)); or (b) the binding protein is, from the N-terminus to the C-terminus, a protein functional region B, a protein functional region A and an Fc, in which the protein functional region B and the protein functional region A are connected to each other. the protein functional region A and the protein functional region B are connected by a connecting peptide, and the protein functional region A and the Fc are connected by a hinge region; in this embodiment, the protein functional region A and the Fc of both parts form a symmetric IgG structure, and the protein functional region B is connected to the N-terminus of the IgG structure, and the protein functional region B is connected to the N-terminus of the VH (Figure 1 (3)) or VL (Figure 1 (4)) of the protein functional region A; or (c) the binding protein is, from the N-terminus to the C-terminus, a protein functional region A, an Fc, and a protein functional region B, in which the protein functional region A and the Fc are connected by a hinge region, and the Fc and the protein functional region B are connected by a connecting peptide; in this embodiment, the protein functional region A and the Fc of both parts form a symmetric IgG structure, and the protein functional region B is connected to the C-terminus of the IgG structure, and the protein functional region B is connected to the C-terminus of the CH3 (Figure 1 (5)) or CL (Figure 1 (6)) of the protein functional region A;

[0010] Preferably, when the number of the protein functional regions B is four, the binding protein is a hexavalent binding protein, specifically comprising the following structure: two additional protein functional regions B are further connected to the N-terminus or C-terminus of the binding protein described in (a) or (b) or (c); preferably connected to the C-terminus of the Fc of the binding protein described in (c) or the C-terminus of the original protein functional region B, or connected to the N-terminus of the protein functional region A together with the original protein functional region B of the binding protein described in (b). The term "additional" in the present specification is merely a convenient expression to distinguish the above-mentioned protein functional region B, i.e., the "original" protein functional region, and there is no prior-subsequence distinction between the additional protein functional region and the original protein functional region when actually designing or producing an antibody. In this embodiment, the protein functional region A and the Fc of both parts form a symmetric IgG structure, and the protein functional region B is connected to the C-terminus of the IgG structure, in which two of the protein functional regions B are connected to the CH3 of the protein functional region A via a first connecting peptide, and the other two protein functional regions B are connected to the two protein functional regions B via a second connecting peptide (Figure 1(7)); or, two of the protein functional regions B are connected to the N-terminus of the VH of the protein functional region A via a first connecting peptide, and the other two protein functional regions B are connected to the N-terminus of the VL of the protein functional region A via a second connecting peptide (Figure 1(9)).

[0011] The binding protein having two different protein functional regions is also called a bispecific binding protein. Preferably, the binding protein further comprises a protein functional region C, and the binding protein is a hexavalent or octavalent trispecific binding protein, specifically comprising the following structure: the protein functional region C, the protein functional region A, and the protein functional region B target different antigens or antigen epitopes, and the protein functional region C is connected to the N-terminus or C-terminus of the antibody; preferably, the number of protein functional regions C is two, and the protein functional region C is connected to the C-terminus of the binding protein described in (c) above, or to the N-terminus of the protein functional region A, which is the same as the protein functional region B of the binding protein described in (b) above. In this embodiment, both protein functional regions C are further connected to the C-terminus of structure (5) (Figure 1 (8)) or the N-terminus of structure (3) or (4) (Figure 1 (10) shows a situation where it is connected to the N-terminus of structure (3)) by a second connecting peptide.

[0012] More preferably, the binding protein has four polypeptide chains, each divided into two identical short chains (or referred to as "polypeptide chain 1") and two identical long chains (or referred to as "polypeptide chain 2"), in which (1) the short chain comprises, from the N-terminus to the C-terminus, VH_A-CH1, and the long chain comprises, from the N-terminus to the C-terminus, VL_A-CL-L1-VH_B-L2-CH2-CH3; or (2) the short chain comprises, from the N-terminus to the C-terminus, VL_A-CL, and the long chain comprises, from the N-terminus to the C-terminus, VL_A-CL, and VH_A-CH1-L1-VH_B-L2-CH2-CH3; or (3) the short chain comprises, from N-terminus to C-terminus, VL_A-CL, and the long chain comprises, from N-terminus to C-terminus, VH_B-L-VH_A-CH1-h-CH2-CH3; or (4) the short chain comprises, from N-terminus to C-terminus, VH_B-L-VL_A-CL, and the long chain comprises, from N-terminus to C-terminus, VH_A-CH1-h-CH2-CH3; or (5) the short chain comprises, from N-terminus to C-terminus, VL_A-CL, and the long chain comprises, from N-terminus to C-terminus, VH_A-CH1-h-CH2-CH3. or (6) the short chain comprises, from the N-terminus to the C-terminus, VL_A-CL-L-VH_B, and the long chain comprises, from the N-terminus to the C-terminus, VH_A-CH1-h-CH2-CH3; or (7) the short chain comprises, from the N-terminus to the C-terminus, VL_A-CL, and the long chain comprises, from the N-terminus to the C-terminus, VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_B; or (8) the short chain comprises, from the N-terminus to the C-terminus, VL_A-CL. and the long chain comprises, from N-terminus to C-terminus, VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_C; or (9) the short chain comprises, from N-terminus to C-terminus, VH_B-L1-VL_A-CL, and the long chain comprises, from N-terminus to C-terminus, VH_B-L2-VH_A-CH1-h-CH2-CH3; or (10) the short chain comprises, from N-terminus to C-terminus, VH_B-L1-VL_A-CL, and the long chain comprises, from N-terminus to C-terminus, VH_C-L2-VH_A-CH1-h-CH2-CH3;Wherein, the L, L1 and L2 are connecting peptides, the h is a hinge region or connecting peptide, and the hinge region or connecting peptide is, for example, as shown in the amino acid sequence of "-", GS or SEQ ID NO: 495-519. The meanings of VL, VH, CL and CH in the text are all common in the art, and respectively represent light chain variable region, heavy chain variable region, light chain constant region and heavy chain constant region, in which CH includes CH1, CH2 and CH3; _A, _B and _C respectively represent that the functional region is protein functional region A or protein functional region B or protein functional region C or its composition, and "-" is a polypeptide bond for connecting different domains or for separating different domains; C-terminus is the carboxyl terminus (also written as "C'") of the peptide chain, and N-terminus is the amino terminus (also written as "N'") of the peptide chain. The different protein functional regions can be fused to the same polypeptide chain to avoid mismatch by-products. In some embodiments, L, L1 and L2 can be the same sequence. In another embodiment, L, L1, L2 may be different sequences. The protein functional regions of the present invention may be Fab, scFv or VH in some cases, F(ab)2 or full-length antibodies in other cases, and may also be referred to as antibodies or antigen-binding proteins or binding proteins in some cases. In some specific embodiments, protein functional region A is also referred to as antibody A or first antigen-binding domain for the first antigen, protein functional region B is also referred to as antibody B or second antigen-binding domain for the second antigen, protein functional region C is also referred to as antibody C or third antigen-binding domain for the third antigen, protein functional region D is also referred to as antibody D or fourth antigen-binding domain for the fourth antigen, and so on.;

[0013] In some specific embodiments, (II) the binding protein has an asymmetric structure, and the asymmetric structure exhibits a left-right asymmetric IgG-like structure, in which the left arm is a protein functional region A of a Fab or scFv structure, and the right arm is a protein functional region B of a VH structure, and the protein functional region A and the protein functional region B are each connected to one Fc; preferably, the number of the protein functional region A is one, and the number of the protein functional region B is one, two, or three. Preferably, when the number of said protein functional region B is one, said binding protein is a bivalent binding protein, comprising the following structure: said protein functional region A is (d) a conventional Fab structure, or (e) a Fab (cross VH / VL) structure, or (f) a Fab (cross Fd / LC) structure; when the number of said protein functional region B is two, said binding protein is a trivalent binding protein, comprising the following structure: said protein functional region A of the left arm is (d) or (e) or (f), and a second protein functional region B is connected to the N-terminus or C-terminus of said protein functional region A of the left arm, or to the N-terminus of the first protein functional region B of the right arm, or to the C-terminus of the Fc, preferably a second protein functional region B is connected to the N-terminus of the first protein functional region B of the right arm; when the number of said protein functional region B is three, said binding protein is a tetravalent binding protein. and specifically has the following structure: the protein functional region A of the left arm has the structure (d), (e), or (f), and a third protein functional region B is further connected to the N-terminus or C-terminus of the protein functional region A of the left arm, or the N-terminus or C-terminus of the second protein functional region B of the left arm, or the N-terminus of the first or second protein functional region B of the right arm, or the C-terminus of the Fc, preferably the second protein functional region B is connected to the N-terminus of the first protein functional region B of the right arm, and the third protein functional region B is connected to the N-terminus of the second protein functional region B.

[0014] When the number of the protein functional region B is one, the binding protein is a bivalent binding protein, specifically comprising the following structure: the protein functional region A of the left arm is (g) scFv structure, and the scFv is connected to Fc by the VH end or the VL end; when the number of the protein functional region B is two, the binding protein is a trivalent binding protein, specifically comprising the following structure: the protein functional region A of the left arm is (g), and a second protein functional region B is connected to the N-terminus of the protein functional region A of the left arm, or the N-terminus of the original protein functional region B of the right arm, or the C-terminus of the Fc, preferably the second protein functional region A of the left arm is (g). and the third protein functional region B is connected to the N-terminus of the protein functional region A of the left arm, or to the N-terminus or C-terminus of the second protein functional region B, or to the C-terminus of the Fc, preferably the second protein functional region B is connected to the N-terminus of the first protein functional region B of the right arm, and the third protein functional region B is connected to the N-terminus of the second protein functional region B of the right arm.

[0015] Preferably, said binding protein further comprises a protein functional region C, said binding protein being a trivalent or multivalent multispecific binding protein, in which said protein functional region C, said protein functional region A and said protein functional region B target different antigens or antigenic epitopes, said protein functional region C being connected to the N-terminus or C-terminus of said binding protein; preferably said protein functional region C being connected to the N-terminus of protein functional region B of the binding protein according to (d), (e), (f) or (g); Even more preferably, said binding protein further comprises a protein functional region D, said binding protein being a tetravalent or multivalent multispecific binding protein, in particular: said protein functional region D and said protein functional region A, protein functional region B and protein functional region C target different antigens or antigenic epitopes, said protein functional region D being connected to the N-terminus or C-terminus of said binding protein; preferably said protein functional region D is connected to the N-terminus of protein functional region B of the binding protein according to (d), (e), (f) or (g).

[0016] More preferably, the binding protein has three types of polypeptide chains, namely, polypeptide chain 1, polypeptide chain 2 and polypeptide chain 3, and each type of polypeptide chain is single. In the present invention, the polypeptide chain 1 is also called the first polypeptide chain, the polypeptide chain 2 is also called the second polypeptide chain, and the polypeptide chain 3 is also called the third polypeptide chain. Among them, (11) the polypeptide chain 1 comprises VL_A-CL in order from the N-terminus to the C-terminus, the polypeptide chain 2 comprises VH_A-CH1-h-CH2-CH3 in order from the N-terminus to the C-terminus, and the polypeptide chain 3 comprises VH_B-h-CH2-CH3 in order from the N-terminus to the C-terminus; or (12) the polypeptide chain 1 comprises VH_A-CL in order from the N-terminus to the C-terminus, and the polypeptide chain 2 comprises VL in order from the N-terminus to the C-terminus. _A-CH1-h-CH2-CH3, and polypeptide chain 3 comprises VH_B-h-CH2-CH3 in the order from the N-terminus to the C-terminus; or (13) said polypeptide chain 1 comprises VH_A-CH1 in the order from the N-terminus to the C-terminus, said polypeptide chain 2 comprises VL_A-CL-h-CH2-CH3 in the order from the N-terminus to the C-terminus, and said polypeptide chain 3 comprises VH_B-h-CH2-CH3 in the order from the N-terminus to the C-terminus; or (14) said Polypeptide chain 1 comprises, from the N-terminus to the C-terminus, VL_A-CL, said polypeptide chain 2 comprises, from the N-terminus to the C-terminus, VH_A-CH1-h-CH2-CH3, and said polypeptide chain 3 comprises, from the N-terminus to the C-terminus, VH_B-L-VH_B-h-CH2-CH3; or (15) said polypeptide chain 1 comprises, from the N-terminus to the C-terminus, VL_A-CL, and said polypeptide chain 2 comprises, from the N-terminus to the C-terminus, VH_A-CH (16) said polypeptide chain 1 comprises, from the N-terminus to the C-terminus, VH_A-CL, said polypeptide chain 2 comprises, from the N-terminus to the C-terminus, VL_A-CH1-h-CH2-CH3, and said polypeptide chain 3 comprises, from the N-terminus to the C-terminus, VH_B-L-VH_B-h-CH2-CH3;or (17) said polypeptide chain 1 comprises, from the N-terminus to the C-terminus, VH_A-CL, said polypeptide chain 2 comprises, from the N-terminus to the C-terminus, VL_A-CH1-h-CH2-CH3, and said polypeptide chain 3 comprises, from the N-terminus to the C-terminus, VH_C-L-VH_B-h-CH2-CH3; or (18) said polypeptide chain 1 comprises, from the N-terminus to the C-terminus, VH_A-CH1, and said polypeptide chain 2 comprises, from the N-terminus to the C-terminus, VH_C-L-VH_B-h-CH2-CH3. or (19) said polypeptide chain 1 comprises VH_A-CH1 in order from the N-terminus to the C-terminus, said polypeptide chain 2 comprises VL_A-CL-h-CH2-CH3 in order from the N-terminus to the C-terminus, and said polypeptide chain 3 comprises VH_B-L-VH_B-h-CH2-CH3 in order from the N-terminus to the C-terminus; or (20) said polypeptide chain 1 comprises VH_A-CH1 in order from the N-terminus to the C-terminus, said polypeptide chain 2 comprises VL_A-CL-h-CH2-CH3 in order from the N-terminus to the C-terminus, and said polypeptide chain 3 comprises VH_C-L- or (26) said polypeptide chain 1 comprises, from the N-terminus to the C-terminus, VL_A-CL, said polypeptide chain 2 comprises, from the N-terminus to the C-terminus, VH_A-CH1-h-CH2-CH3, and said polypeptide chain 3 comprises, from the N-terminus to the C-terminus, VH_B-L1-VH_B-L2-VH_B-h-CH2-CH3; or (27) said polypeptide chain 1 comprises, from the N-terminus to the C-terminus, V L_A-CL, said polypeptide chain 2 comprises VH_A-CH1-h-CH2-CH3 in the order from N-terminus to C-terminus, and said polypeptide chain 3 comprises VH_D-L1-VH_C-L2-VH_B-h-CH2-CH3 in the order from N-terminus to C-terminus; wherein said L, L1 and L2 are connecting peptides, and said h is a hinge region or connecting peptide, and said hinge region or connecting peptide is, for example, "-", GS or the amino acid sequence of SEQ ID NO: 495-519;

[0017] Even more preferably, the binding protein has both polypeptide chains, polypeptide chain 1 and polypeptide chain 2, each type of polypeptide chain being single, and similarly, the polypeptide chain 1 is also referred to as the first polypeptide chain, and the polypeptide chain 2 is also referred to as the second polypeptide chain. Among them, (20) the polypeptide chain 1 comprises VL_A-L-VH_A-h-CH2-CH3 in the order from the N-terminus to the C-terminus, and the polypeptide chain 2 comprises VH_B-h-CH2-CH3 in the order from the N-terminus to the C-terminus; or (21) the polypeptide chain 1 comprises VH_A-L-VL_A-h-CH2-CH3 in the order from the N-terminus to the C-terminus, and the polypeptide chain 2 comprises VH_B-h-CH2-CH3 in the order from the N-terminus to the C-terminus; or (22) the polypeptide chain 1 comprises VL_A-L1-VH_A-h-CH2-CH3 in the order from the N-terminus to the C-terminus, and the polypeptide chain 2 comprises VH_B-L2-VH_B-h-CH2-CH3 in the order from the N-terminus to the C-terminus; or (23) the polypeptide chain 1 comprises VH_A-L1-VL_A-h-CH or (24) said polypeptide chain 1 comprises, from the N-terminus to the C-terminus, VL_A-L1-VH_A-h-CH2-CH3, and said polypeptide chain 2 comprises, from the N-terminus to the C-terminus, VH_C-L2-VH_B-CH2-CH3; or (25) said polypeptide chain 1 comprises, from the N-terminus to the C-terminus, VH_A-L1-VL_A-h-CH2-CH3, and said polypeptide chain 2 comprises, from the N-terminus to the C-terminus, VH_C-L2-VH_B-h-CH2-CH3; wherein L, L1 and L2 are connecting peptides, and h is a hinge region or connecting peptide, and said hinge region or connecting peptide is, for example, "-", GS or SEQ ID NO: The amino acid sequence is shown in ID NO: 495-519.

[0018] In some specific embodiments, the binding protein comprises at least two protein functional domains, namely, protein functional domain A and protein functional domain B; the protein functional domain A and the protein functional domain B are one selected from PD-L1 antibody, HER2 antibody, B7H4 antibody, CD3 antibody, CTLA4 antibody, 4-1BB antibody, or BCMA antibody. Preferably, the protein functional domain A is PD-L1 antibody, HER2 antibody, B7H4 antibody, or CD3 antibody, and the protein functional domain B is CTLA4 antibody, 4-1BB antibody, or BCMA antibody. Preferably, the protein functional region A is a PD-L1 antibody and the protein functional region B is a CTLA4 antibody; or the protein functional region A is a PD-L1 antibody and the protein functional region B is a 4-1BB antibody; or the protein functional region A is a HER2 antibody and the protein functional region B is a CTLA4 antibody; or the protein functional region A is a B7H4 antibody and the protein functional region B is a 4-1BB antibody; or the protein functional region A is a CD3 antibody and the protein functional region B is a BCMA antibody.

[0019] In some specific embodiments, the PD-L1 antibody comprises a light chain variable region and a heavy chain variable region; wherein the light chain variable region VL comprises LCDR1, LCDR2, and LCDR3, and has the amino acid sequences set forth in SEQ ID NOs: 167, 188, and 211, respectively; wherein the heavy chain variable region VH comprises HCDR1, HCDR2, and HCDR3, and has the amino acid sequences set forth in SEQ ID NOs: 15, 62, and 122, respectively; or wherein the light chain variable region VL comprises LCDR1, LCDR2, and LCDR3, and has the amino acid sequences set forth in SEQ ID NOs: 167, 188, and 211, respectively; and wherein the heavy chain variable region VH comprises HCDR1, HCDR2, and HCDR3, and has the amino acid sequences set forth in SEQ ID NOs: 15, 69, and 122, respectively. The amino acid sequences of the listed CDRs are shown according to the Chothia definition rules. Preferably, the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO:282; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO:233; or, the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO:282; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO:240. More preferably, the PD-L1 antibody comprises two polypeptide chains; in which a first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:347 and a second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:300; or, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:353 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:308; or, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:353 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:310.

[0020] In some specific embodiments, the PD-L1 antibody has the amino acid sequence of atezolizumab known in the art.

[0021] In some specific embodiments, the HER2 antibody has the amino acid sequence of trastuzumab or pertuzumab known in the art.

[0022] In some specific embodiments, the B7H4 antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequences shown in SEQ ID NOs: 180, 191 and 225, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 32, 87 and 143, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequences shown in SEQ ID NOs: 177, 191 and 226, respectively; and the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 33, 88 and 144, respectively. The amino acid sequences of the recited CDRs are shown according to the Chothia definition rules. Preferably, the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 298; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 261; or, the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 299; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 262. More preferably, the B7H4 antibody comprises two polypeptide chains; in which a first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 360 and a second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 326; or, in which the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 361 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 327.

[0023] In some specific embodiments, the CTLA4 antibody comprises a heavy chain variable region; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which have the amino acid sequences shown in SEQ ID NOs: 17, 65 and 125, respectively. The amino acid sequences of the recited CDRs are shown according to the Chothia definition rules. Preferably, the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 236. More preferably, the CTLA4 antibody comprises one polypeptide chain, which comprises the amino acid sequence shown in SEQ ID NO: 303, which comprises the amino acid sequence shown in SEQ ID NO: 306.

[0024] In some specific embodiments, the 4-1BB antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, and has the amino acid sequences shown in SEQ ID NOs: 170, 191 and 214, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 18, 66 and 126, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, and has the amino acid sequences shown in SEQ ID NOs: 170, 191 and 214, respectively; and the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 18, 71 and 126, respectively. The amino acid sequences of the recited CDRs are shown according to the Chothia definition rules. Preferably, the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 285; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 237; or, the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 289; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 242. More preferably, the 4-1BB antibody comprises two polypeptide chains; in which a first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 350 and a second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 304; or, in which the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 355 and a second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 311.

[0025] In some specific embodiments, the 4-1BB antibody comprises a heavy chain variable region; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 27, 79 and 137, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 28, 80 and 138, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 29, 82 and 138, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 28, 89 and 145, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 28, 81 and 139; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, respectively, as shown in SEQ ID NO: 15, 83 and 140. The amino acid sequences of the recited CDRs are shown according to the Chothia definition rules. Preferably, the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 252; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 253; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 255; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 264; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 254; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 256. More preferably, the 4-1BB antibody comprises one polypeptide chain, the polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:320, the amino acid sequence shown in SEQ ID NO:321, the amino acid sequence shown in SEQ ID NO:323, the amino acid sequence shown in SEQ ID NO:322, the amino acid sequence shown in SEQ ID NO:324, or the amino acid sequence shown in SEQ ID NO:329.

[0026] In some specific embodiments, the CD3 antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequence shown in SEQ ID NO: 177, 191 and 221, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 15, 84 and 141, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequence shown in SEQ ID NO: 178, 197 and 222, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 31, 85 and 142, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequence shown in SEQ ID NO: and wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 31, 86 and 141, respectively; or wherein the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequence shown in SEQ ID NO: 179, 198 and 224, respectively; and wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 31, 85 and 142, respectively. The amino acid sequences of the recited CDRs are shown according to the Chothia definition rules. Preferably, the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 294; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 258; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 295; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 259; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 296; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 260; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 297; and the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 259.

[0027] In some specific embodiments, the CD3 antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 20, 68 and 128, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; and the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively. The amino acid sequences of the recited CDRs are shown according to the Chothia definition rules. Preferably, the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 245; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 257; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 281; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 244.

[0028] In some specific embodiments, the CD3 antibody comprises both polypeptide chains; wherein the first polypeptide chain (VL) comprises the amino acid sequence set forth in SEQ ID NO:357 and the second polypeptide chain (VH) comprises the amino acid sequence set forth in SEQ ID NO:313; or wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:325; or wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:328; or wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:346.

[0029] In some specific embodiments, the CD3 antibody comprises a polypeptide chain, the polypeptide chain comprising the amino acid sequence depicted in SEQ ID NO:489, the amino acid sequence depicted in SEQ ID NO:490, the amino acid sequence depicted in SEQ ID NO:491, the amino acid sequence depicted in SEQ ID NO:492, or the amino acid sequence depicted in SEQ ID NO:493.

[0030] In some specific embodiments, the BCMA antibody comprises a heavy chain variable region; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 15, 75 and 133, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 24, 76 and 134, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 25, 77 and 135, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 26, 78 and 136, respectively. The amino acid sequences of the recited CDRs are shown according to the Chothia definition rules. Preferably, the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 248; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 249; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 250; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 251. More preferably, the BCMA antibody comprises a single polypeptide chain, the polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 316, the amino acid sequence set forth in SEQ ID NO: 317, the amino acid sequence set forth in SEQ ID NO: 318 or the amino acid sequence set forth in SEQ ID NO: 319.

[0031] In some specific embodiments, the BCMA antibody comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence set forth in SEQ ID NOs: 26, 90 and 136, respectively; or wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence set forth in SEQ ID NOs: 34, 78 and 146, respectively; or wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence set forth in SEQ ID NOs: 35, 78 and 147, respectively; or wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence set forth in SEQ ID NOs: 35, 90 and 147, respectively; or wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence set forth in SEQ ID NOs: or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 76 and 147, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 34, 78 and 148, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 76 and 136, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 90 and 146, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 76 and 146, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 34, 76 and 146, respectively;or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 35, 90 and 148, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 35, 90 and 136, respectively. The amino acid sequences of the recited CDRs are shown according to the Chothia definition rules. Preferably, the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 265; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 266; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 267; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 268; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 269; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 270; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 271; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 272; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 273; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 274; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 275; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 276; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 277; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 278; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 279; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 280. More preferably, the BCMA antibody comprises one polypeptide chain, the polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 330; or the amino acid sequence set forth in SEQ ID NO: 331; or the amino acid sequence set forth in SEQ ID NO: 332;or the amino acid sequence set forth in SEQ ID NO:333; or the amino acid sequence set forth in SEQ ID NO:334; or the amino acid sequence set forth in SEQ ID NO:335; or the amino acid sequence set forth in SEQ ID NO:336; or the amino acid sequence set forth in SEQ ID NO:337; or the amino acid sequence set forth in SEQ ID NO:338; or the amino acid sequence set forth in SEQ ID NO:339; or the amino acid sequence set forth in SEQ ID NO:340; or the amino acid sequence set forth in SEQ ID NO:341; or the amino acid sequence set forth in SEQ ID NO:342; or the amino acid sequence set forth in SEQ ID NO:343; or the amino acid sequence set forth in SEQ ID NO:344; or the amino acid sequence set forth in SEQ ID NO:345;

[0032] In some specific embodiments, the protein functional region C is a heavy chain variable region of a BCMA heavy chain antibody, the heavy chain variable region VH of which comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 24, 76 and 134, respectively; or the heavy chain variable region VH of which comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 26, 78 and 136, respectively. Preferably, the heavy chain variable region VH of which comprises the amino acid sequence shown in SEQ ID NO: 249; or the heavy chain variable region VH of which comprises the amino acid sequence shown in SEQ ID NO: 251.

[0033] In the above specific embodiments, the binding protein further comprises a light chain constant region, which is preferably a human light chain constant region, more preferably a human light chain constant region Cκ or Cλ.

[0034] In the above specific embodiments, the binding protein further comprises a heavy chain constant region (CH1, CH2 and / or CH3), the heavy chain constant region is preferably a human heavy chain constant region, more preferably a human IgG1, IgG2, IgG3, IgG4 heavy chain constant region. In some specific embodiments, the Fc of the IgG1 heavy chain constant region has one or more mutations such as C220S, N297A, L234A, L235A, P329G, S239D, I332E, S354C, T366W, Y349C, T366S, L368A, Y407V, M252Y, S254T, T256E, and the mutation sites are represented by the EU numbering convention.

[0035] In some preferred embodiments, the binding protein comprises a protein functional region A and a protein functional region B: the protein functional region A comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, and has the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 15, 69 and 122, respectively; the protein functional region B comprises a heavy chain variable region; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 17, 65 and 125, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 62 and 122, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 17, 65 and 125, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 171, 190 and 215, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 19, 67 and 127, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 17, 65 and 125, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 176, 196 and 220, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 23, 74 and 132, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 17, 65 and 125, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 69 and 122, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 28, 80 and 138, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 69 and 122, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 27, 79 and 137, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 69 and 122, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 29, 82 and 138, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 180, 191 and 225, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 32, 87 and 143, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 28, 80 and 138, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 180, 191 and 225, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 32, 87 and 143, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 27, 79 and 137, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 180, 191 and 225, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 32, 87 and 143, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 28, 89 and 145, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 177, 191 and 226, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 33, 88 and 144, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 28, 80 and 138, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 177, 191 and 226, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 33, 88 and 144, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 28, 81 and 139, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 177, 191 and 226, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 33, 88 and 144, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 83 and 140, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 20, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 24, 76 and 134, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 20, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 20, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 75 and 133, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 75 and 133, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 26, 90 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 36, 90 and 146, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 76 and 147, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 34, 78 and 148, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 76 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 90 and 146, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 34, 76 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 76 and 146, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 34, 76 and 146, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 90 and 148, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 90 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 34, 78 and 146, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 78 and 147, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 90 and 147, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 177, 191 and 221, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 84 and 141, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 178, 197 and 222, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 31, 85 and 142, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 177, 191 and 223, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 31, 86 and 141, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 179, 198 and 224, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 31, 85 and 142, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively; or

[0036] In some preferred embodiments, the binding protein comprises a protein functional region A, a protein functional region B and a protein functional region C, wherein the protein functional region A comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, and has the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; the protein functional region B comprises a heavy chain variable region; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 15, 75 and 133, respectively; the protein functional region C comprises a heavy chain variable region; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 24, 76 and 134, respectively; or The protein functional region A comprises a light chain variable region and a heavy chain variable region, the light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; the heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; the protein functional region B comprises a heavy chain variable region, the heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and the amino acid sequences shown in SEQ ID NOs: 25, 77 and 135, respectively; the protein functional region C comprises a heavy chain variable region, the heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively. The amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.

[0037] In some more preferred embodiments, the binding protein comprises a protein functional region A and a protein functional region B: the protein functional region A comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO:282; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO:240; the protein functional region B comprises a heavy chain variable region; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO:236; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 282; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 233; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 236; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 286; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 238; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 236; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 293; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 247; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 236; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 282; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 240; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 253; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 282; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 240; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 252; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 282; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 240; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 255; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 298; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 261; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 253; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 298; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 261; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 252; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 298; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 261; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 264; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 299; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 262; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 253; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 299; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 262; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 254; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 299; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 262; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 256; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 245; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 249; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 245; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 245; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 248; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 257; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 248; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 257; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 281; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 265; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 269; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 270; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 271; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 272; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 273; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 274; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 275; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 276; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 277; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 278; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 279; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 280; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 266; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 267; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 268; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 244; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 249; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 244; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 294; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 258; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 295; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 259; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 296; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 260; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 297; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 259; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 251; or

[0038] In some more preferred embodiments, the binding protein comprises protein functional region A, protein functional region B, and protein functional region C, wherein protein functional region A comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO:291; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO:257; protein functional region B comprises a heavy chain variable region; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO:248; protein functional region C comprises a heavy chain variable region; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO:249; or said protein functional region A comprises a light chain variable region and a heavy chain variable region; said light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO:291; said heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO:257; said protein functional region B comprises a heavy chain variable region; said heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO:250; said protein functional region C comprises a heavy chain variable region; said heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO:251.

[0039] In some more preferred embodiments, the binding protein comprises both polypeptide chains, wherein a first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:371; a second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:372; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 371; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 373; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 362; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 363; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 364; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 365; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 364; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 366; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 369; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 370; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 394; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 395; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 363; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 310; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 363; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 396; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 363; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 362; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 363; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 394; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 363; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 395; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 368; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 378; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 379; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 380; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 381; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 382; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 383; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 385; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 388; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 389; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 374; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 375; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 386; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 387; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 401; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 402; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 305; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 403; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:402; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:409; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 359; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 404; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 405; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 315; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 359; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 406; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 376; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 377; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 397; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 398; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 399; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 400; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:402; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:401; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:402; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:403; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:405; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:404; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:405; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:406; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 371; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 486; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 460; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 461; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 462; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 487; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 488; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 360; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 455; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 360; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 456; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 360; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 457; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 360; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 458; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 360; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 459; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:419; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:412; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:419; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:414; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:440; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:428; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 441; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 428; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 442; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 428; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 443; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 428; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 487; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 520; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 521; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 371; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 522; or The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:360; the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:523.

[0040] In some more preferred embodiments, the binding protein comprises three polypeptide chains, in which a first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:353; a second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:407; a third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:390; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 408; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 392; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 391; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 390; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 393; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 392; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 391; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 434; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 391; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 435; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 393; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 436; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 393; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 437; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 438; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 434; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 438; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 435; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 439; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 436; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 439; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 437; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 361; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 481; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 482; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 361; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 481; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 483; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 361; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 481; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 484; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 361; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 481; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 485; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 411; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 412; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 411; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 414; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:415; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:416; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:410; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:415; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:416; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:412; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:415; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:416; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:414; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:417; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:418; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:412; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:417; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:418; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:414; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 423; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 426; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 423; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 427; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 423; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 428; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 423; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 429; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 444; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 449; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 450; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 449; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 444; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 445; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 444; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 446; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 450; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 445; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 450; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 446; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 444; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 447; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 444; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 448; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 450; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 447; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 450; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 448; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 445; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 446; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 463; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 464; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 465; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 466; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 467; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 468; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 469; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 470; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 471; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 472; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 473; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 474; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 475; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 476; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 477; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 478; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 479; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 480; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 423; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 430; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 423; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 431; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 423; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 432; or The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:423; and the third polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:433.

[0041] In order to solve the above technical problem, the second technical configuration of the present invention provides an isolated nucleic acid encoding the above-mentioned binding protein.

[0042] In order to solve the above technical problem, the third technical configuration of the present invention provides an expression vector comprising the above-mentioned isolated nucleic acid.

[0043] In order to solve the above technical problems, the fourth technical configuration of the present invention provides: a host cell, which contains the above expression vector and is a prokaryotic or eukaryotic cell.

[0044] In order to solve the above technical problem, the fifth technical configuration of the present invention provides a method for producing a binding protein as described above, the method comprising the steps of: culturing a host cell as described above, and obtaining the binding protein from the culture.

[0045] In order to solve the above technical problems, the sixth technical configuration of the present invention provides: a pharmaceutical composition comprising the above-mentioned binding protein.

[0046] In order to solve the above technical problems, the seventh technical configuration of the present invention provides a drug kit, comprising: a drug cassette 1 and a drug cassette 2, wherein the drug cassette 1 contains the binding protein or drug composition as described above, and the drug cassette 2 contains another antibody or drug composition for treating cancer.

[0047] In order to solve the above technical problems, the eighth technical configuration of the present invention provides: a use of the above-mentioned binding protein or the above-mentioned pharmaceutical composition in the preparation of a drug for treating and / or preventing cancer, wherein the cancer is preferably breast cancer, ovarian cancer, endometrial cancer, renal cancer, melanoma, lung cancer, gastric cancer, liver cancer, esophageal cancer, cervical cancer, head and neck tumor, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma, colorectal cancer, lymphoma or multiple myeloma.

[0048] In order to solve the above technical problem, the ninth technical configuration of the present invention provides a method for treating cancer, comprising: administering to a subject in need thereof the binding protein described in the first technical configuration of the present invention, or the pharmaceutical composition described in the sixth technical configuration, or the pharmaceutical kit described in the seventh technical configuration; and preferably further administering other antibodies for treating cancer, such as immune checkpoint antibodies and / or chemotherapy drugs.

[0049] The cancer is preferably breast cancer, ovarian cancer, endometrial cancer, renal cancer, melanoma, lung cancer, gastric cancer, liver cancer, esophageal cancer, cervical cancer, head and neck tumors, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma, colorectal cancer, lymphoma or multiple myeloma.

[0050] In order to solve the above technical problems, the tenth technical configuration of the present invention provides: a CD3 antibody. the CD3 antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequences shown in SEQ ID NOs: 177, 191 and 221, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 15, 84 and 141, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequences shown in SEQ ID NOs: 178, 197 and 222, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 31, 85 and 142, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequences shown in SEQ ID NOs: and wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 31, 86 and 141, respectively; or wherein the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequence shown in SEQ ID NO: 179, 198 and 224, respectively; and wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 31, 85 and 142, respectively. The amino acid sequences of the recited CDRs are shown according to the Chothia definition rules. Preferably, the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 294; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 258; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 295; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 259; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 296; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 260; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 297; and the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 259. or said CD3 antibody comprises a light chain variable region and a heavy chain variable region; wherein the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 20, 68 and 128, respectively; or wherein the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; and wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively. The amino acid sequences of the recited CDRs are shown according to the Chothia definition rules. Preferably, the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 245; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 257; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 281; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 244.

[0051] In some specific embodiments, the CD3 antibody comprises both polypeptide chains; wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:313; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:325; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:328; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:346.

[0052] In some specific embodiments, the CD3 antibody comprises a polypeptide chain, the polypeptide chain comprising the amino acid sequence depicted in SEQ ID NO:489, the amino acid sequence depicted in SEQ ID NO:490, the amino acid sequence depicted in SEQ ID NO:491, the amino acid sequence depicted in SEQ ID NO:492, or the amino acid sequence depicted in SEQ ID NO:493.

[0053] In order to solve the above technical problem, the eleventh technical configuration of the present invention provides: a BCMA antibody.

[0054] The BCMA antibody comprises a heavy chain variable region; wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence as shown in SEQ ID NOs: 15, 75 and 133, respectively; or wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence as shown in SEQ ID NOs: 24, 76 and 134, respectively; or wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence as shown in SEQ ID NOs: 25, 77 and 135, respectively; or wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence as shown in SEQ ID NOs: 26, 78 and 136, respectively. The amino acid sequences of the listed CDRs are shown according to the Chothia definition rules. Preferably, the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 248; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 249; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 250; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 251. More preferably, the BCMA antibody comprises a single polypeptide chain, the polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 316, the amino acid sequence set forth in SEQ ID NO: 317, the amino acid sequence set forth in SEQ ID NO: 318 or the amino acid sequence set forth in SEQ ID NO: 319. or the BCMA antibody comprises a heavy chain variable region, the heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 26, 90 and 136, respectively; or the heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34, 78 and 146, respectively; or the heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 35, 78 and 147, respectively; or the heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 35, 90 and 147, respectively; or the heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 76 and 147, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 34, 78 and 148, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 76 and 136, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 90 and 146, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 76 and 146, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 34, 76 and 146, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 90 and 148, respectively;or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are shown in SEQ ID NOs: 35, 90 and 136, respectively. The amino acid sequences of the recited CDRs are shown according to the Chothia definition rules;

[0055] In some specific embodiments, the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 265; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 266; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 267; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 268; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 269; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 270; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 271; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 272; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 273; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 274; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 275; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 276; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 277; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 278; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 279; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 280. In some specific embodiments, the BCMA antibody comprises a polypeptide chain, the polypeptide chain having the amino acid sequence set forth in SEQ ID NO:330; or the amino acid sequence set forth in SEQ ID NO:331; or the amino acid sequence set forth in SEQ ID NO:332; or the amino acid sequence set forth in SEQ ID NO:333; or the amino acid sequence set forth in SEQ ID NO:334; or the amino acid sequence set forth in SEQ ID NO:335; or the amino acid sequence set forth in SEQ ID NO:336; or the amino acid sequence set forth in SEQ ID NO:337; or the amino acid sequence set forth in SEQ ID NO:338; or the amino acid sequence set forth in SEQ ID NO:339; or the amino acid sequence set forth in SEQ ID NO:340; or the amino acid sequence set forth in SEQ ID NO:341; or the amino acid sequence set forth in SEQ ID NO:342; or the amino acid sequence set forth in SEQ ID NO:343; or the amino acid sequence set forth in SEQ ID NO: 344; or the amino acid sequence set forth in SEQ ID NO:345.

[0056] Positive advances of the present invention: The present invention provides bi- or multivalent bispecific or multispecific binding proteins constructed using fully human heavy chain antibodies and single domain antibodies derived therefrom, and methods for making and using such binding proteins, which have many advantages over bi- or multivalent bispecific or multispecific binding proteins constructed using conventional IgG antibodies, including more convenient adjustment of specificity and binding valency; multispecific binding proteins with smaller molecular weights, fewer polypeptide chains, and simpler structures can be constructed; and different structures can be utilized to achieve different biological functions.

[0057] In some specific embodiments, the functional activity against different targets is adjusted by parameters such as different structure types, relative positions, binding valency, etc., and different activity combinations are designed to meet the demands of different clinical combination dosage combinations. In some specific embodiments, the heavy chain antibody VH domain can be used to easily form a multivalent structure in series, which can promote cross-linking of the target through multivalent binding with the target, and the biological function induced by cross-linking of the target can be further enhanced. In some specific embodiments, the heavy chain antibody VH domain can be used to realize an asymmetric tetravalent structure such as "1+3", which cannot be achieved with a conventional IgG antibody-derived structure. [Brief description of the drawings]

[0058] Figure 1. Schematic of the molecular structure. Figure 2. Binding activity of the PD-L1×CTLA4 bibody molecule to hCTLA4-His protein. Figure 3. Binding activity of PD-L1×CTLA4 bibody molecule to CHO-K1 / hCTLA4 cells. Figure 4. Binding activity of PD-L1×CTLA4 bibody molecule to HEK293 / hCTLA4 cells. Figure 5. The activity of the PD-L1×CTLA4 dual antibody molecule in blocking the binding of human CTLA4 protein to its ligand protein B7-1. Figure 6. The activity of the PD-L1×CTLA4 dual antibody molecule to block the binding of CHO-K1 / hCTLA4 cells to its ligand protein B7-1. Figure 7. Binding activity of the PD-L1×CTLA4 dual antibody molecule to hPDL1-His protein. Figure 8. Binding activity of PD-L1×CTLA4 bibody molecule to CHO-K1 / hPDL1 cells. Figure 9. Binding activity of the PD-L1×CTLA4 bibody molecule to MDA-MB-231 cells that highly express human PD-L1. Figure 10. The activity of the PD-L1×CTLA4 dual antibody molecule to block the binding of CHO-K1 / hPDL1 cells to their ligand protein PD-1. Figure 11. Ability of PD-L1×CTLA4 bibody molecule to kill CTLA4+ target cells via ADCC effect. Figure 12. Ability of IgG-VH PD-L1×CTLA4 biantibody to activate T cells in SEB stimulation experiments. Figure 13. Ability of PD-L1×CTLA4 biantibody in Fab-HCAb format to activate T cells in SEB stimulation experiments. Figure 14. In SEB stimulation experiments, PD-L1xCTLA4 dual antibodies have comparable and potentially stronger T cell activation capacity as a 1:1 dose combination of the corresponding monoclonal antibodies when the VH is at the N-terminus of the IgG HC or LC. FIG. 15. In SEB stimulation experiments, the activity of the CTLA4 terminus is weakened in IgG_HC-VH (where VH is the C-terminus of IgG HC) and Fab-HCAb structures compared to IgG or HCAb bivalent structures, thereby reducing dose-related toxicity. Figure 16. In mixed lymphocyte reaction experiments, PD-L1×CTLA4 dual antibodies have a stronger ability to activate T cells than a 1:1 dose combination of the corresponding monoclonal antibodies when the VH is located at the N-terminus of the IgG HC or LC. Figure 17. In mixed lymphocyte reaction experiments, PD-L1×CTLA4 bibody has stronger ability to activate T cells than PD-L1 monoclonal antibody when the VH is located at the C-terminus of IgG HC or LC. Figure 18. Binding activity of the HER2×CTLA4 bibody molecule to CHO-K1 / hCTLA4 cells. Figure 19. Activity of the HER2×CTLA4 dual antibody molecule to block the binding of human CTLA4 protein to its ligand protein B7-1. Figure 20. Activity of the HER2xCTLA4 dual antibody molecule to block binding of CHO-K1 / hCTLA4 cells to their ligand protein B7-1. Figure 21. Binding activity of the HER2×CTLA4 dual antibody molecule to SK-BR-3 cells. Figure 22. Ability of HER2×CTLA4 bibody molecule to kill HER2+ target cells via ADCC effect. Figure 23. Ability of the HER2xCTLA4 dual antibody molecule to inhibit proliferation of HER2+ target cells SK-BR-3. FIG. 24. Ability of the HER2×CTLA4 bibody molecule to simultaneously bind to CTLA4+ and HER2+ cells. FIG. 25. Ability of the HER2×CTLA4 dual antibody molecule to activate T cells in SEB stimulation experiments. Figure 26. Pharmacokinetics of the HER2×CTLA4 dual antibody molecule in mice. Figure 27. Binding activity of PD-L1×4-1BB bibody molecule to CHO-K1 / hPDL1 cells. Figure 28. Binding activity of 4-1BB antibody or PD-L1×4-1BB bibody molecule to CHO-K1 / hu4-1BB cells. Figure 29. Binding activity of PD-L1×4-1BB bibody molecule to CHO-K1 / cyno 4-1BB cells. Figure 30. PD-L1x4-1BB bibody molecule mediates T cell specific activation by CHO-K1 / hPDL1 cells. Figure 31. PD-L1x4-1BB bibody molecule mediates T cell specific activation by MDA-MB-231 cells. Figure 32. In mixed lymphocyte reaction experiments, the PD-L1×4-1BB bibody molecule with an IgG-VH structure has stronger ability to activate T cells than the bibody molecule with a FIT-Ig structure. Figure 33. In mixed lymphocyte reaction experiments, the PD-L1x4-1BB bibody molecule with IgG-VH structure and Fab-HCAb structure has stronger ability to activate T cells than the bibody molecule with FIT-Ig structure. Figure 34. Pharmacokinetics of the PD-L1×4-1BB dual antibody molecule in mice. Figure 35. Binding activity of B7H4×4-1BB bibody molecule to SK-BR-3 cells. FIG. 36. Binding activity of the B7H4×4-1BB bibody molecule to CHO-K1 / hu4-1BB cells. FIG. 37. B7H4×4-1BB biantibody molecule mediates specific activation (INFγ release) by SK-BR-3 T cells. FIG. 38. B7H4×4-1BB biantibody molecule mediates specific activation (IL-2 release) by SK-BR-3 T cells. FIG. 39. T cell activation of the B7H4×4-1BB bibody molecule specifically depends on the expression of B7H4. Figure 40. Serum stability of the B7H4×4-1BB biantibody molecule PR003334. Figure 41. Serum stability of the B7H4×4-1BB biantibody molecule PR003335. Figure 42. Pharmacokinetics of the B7H4×4-1BB bibody molecule in mice. Figure 43. Antitumor effect of the B7H4×4-1BB bibody molecule in mouse tumor models. Figure 44. Binding activity of BCMA HCAb antibodies to HEK293T / hBCMA cells. Figure 45. Binding activity of BCMA HCAb antibodies to HEK293T / cynoBCMA cells. Figure 46. Binding activity of BCMA HCAb antibodies to NCI-H929 cells. Figure 47. Internalization ability of BCMA HCAb antibodies into HEK293T / hBCMA cells. Figure 48. BCMA HCAb antibody binding affinity to human BCMA (BLI method). Figure 49. Binding activity of mutant molecules derived from BCMA HCAb antibody PR001046 to NCI-H929 cells. Figure 50. Avidity of BCMAxCD3 bibody molecules to bind HEK293T / hBCMA cells. Figure 51. Binding activity of BCMAxCD3 bibody molecules to HEK293T / cynoBCMA cells. Figure 52. Avidity of BCMAxCD3 bibody molecules to bind NCI-H929 cells. Figure 53. Avidity of BCMAxCD3 bibody molecules binding to human T cells. Figure 54. Avidity of BCMAxCD3 bibody molecules binding to cynomolgus monkey T cells. Figure 55. Ability of scFv-Fc-VH(2) asymmetric BCMA×CD3 bibody molecule to kill NCI-H929 cells. Figure 56. Ability of scFv-Fc-VH(1) asymmetric BCMA×CD3 bibody molecule to kill NCI-H929 cells. Figure 57. Ability of BCMAxCD3 bibody molecules with Fab-Fc-VH(1) asymmetric structure to kill NCI-H929 cells. Figure 58. Ability of BCMAxCD3 bibody molecules with Fab-Fc-VH(2) asymmetric structure to kill NCI-H929 cells. Figure 59. BCMAxCD3 bibody molecule (PR001990, PR002309) mediates specific effector cell killing of NCI-H929 cells and detection of cytokine release. Figure 60. BCMAxCD3 bibody molecule (PR002895, PR002953, PR003178) mediates specific effector cell killing of NCI-H929 cells and detection of cytokine release. Figure 61. Effect of soluble APRIL or BAFF on the efficacy of BCMAxCD3 bibody molecules in killing target cells. Figure 62. Effect of soluble BCMA on the efficacy of BCMAxCD3 bibody molecules in killing target cells. Figure 63. Pharmacokinetics of the BCMAxCD3 bibody molecule in mice or rats. Figure 64. Antitumor effect of BCMAxCD3 bibody molecule in mouse tumor models. Figure 65. SDS-PAGE and SEC-HPLC analysis of protein samples obtained after instantaneous transfection expression and one-step affinity purification of dual antibody molecules. Figure 66. Avidity of CD3 antibodies binding to human T cells. Figure 67. Antitumor effect of the PD-L1×CTLA4 dual antibody molecule in mouse tumor models. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] Hereinafter, the present invention will be described with reference to specific embodiments, and those skilled in the art will be able to easily understand other advantages and effects of the present invention from the contents disclosed herein.

[0060] In this application, the term "antibody" or "H2L2" generally refers to a conventional four-chain antibody. Antibodies in most species of organisms exhibit a "Y"-shaped tetrameric structure containing two identical heavy chains (H chains) and two identical light chains (L chains), also called "H2L2". The heavy chain contains a heavy chain variable region (VH) near the N-terminus and a heavy chain constant region (CH) near the C-terminus; the light chain contains a light chain variable region (VL) near the N-terminus and a light chain constant region (CL) near the C-terminus. The heavy chain constant region of an IgG antibody has three domains, CH1, CH2, and CH3. There is also a hinge region between CH1 and CH2. Due to the difference between κ and λ, the light chain variable region of the light chain is further divided into Vκ and Vλ, and the corresponding light chain constant regions are divided into Cκ and Cλ, respectively. The variable region of an antibody is the main site that recognizes and binds to its antigen; the variable domains VH and VL and the constant domains CH1 and CL of an antibody together constitute the antigen-binding fragment (Fab). CH2 and CH3 constitute the fragment crystallizable (Fc) and are the main regions that exert the effector functions of the antibody, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), and affect the serum half-life of the antibody.

[0061] In this application, the term "heavy chain antibody" or "HCAb" generally refers to a class of antibodies that contain only heavy chain dimers. Heavy chain antibodies (HCAb) that lack light chains naturally occur in the serum of camelids and sharks. Heavy chain antibodies from camelids lack light chains compared to conventional H2L2 antibodies, and do not have a CH1 region between their heavy chain variable region and hinge region, but only contain one heavy chain variable region (VHH) and two heavy chain constant domains (CH2 and CH3); their basic structure is a heavy chain dimer. The VHH fragments of camelid heavy chain antibodies are also called nanobodies or single domain antibodies, because the VHH structure cloned and expressed alone has the structural stability and antigen binding activity equivalent to the original heavy chain antibody, and has a molecular weight of only about 13 Kda.

[0062] In this application, the term "binding protein" or "antigen binding protein" generally refers to a protein that includes an antigen-binding moiety, and optionally allows the antigen-binding moiety to use a conformational scaffold or framework moiety that promotes antigen binding to the antigen-binding protein. Typically, it can include an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both. The VH and VL regions can be further differentiated into highly variable regions called complementarity determining regions (CDRs) interspersed with more conservative regions called frame regions (FRs). Each VH and VL may be composed of three CDRs and four FR regions, from the amino terminus to the carboxyl terminus: FR-1, CDR1, FR-2, CDR2, FR-3, CDR3, and FR-4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The three CDRs of the VH are designated HCDR1, HCDR2, and HCDR3, respectively, and may be designated VH CDR1, VH CDR2, and VH CDR3. The three CDRs of VL are designated LCDR1, LCDR2, and LCDR3, respectively, and may be designated VL CDR1, VL CDR2, and VL CDR3. Examples of antigen-binding proteins include antibodies and antigen-binding fragments (Fab, Fab', F(ab)) that exhibit the desired antigen-binding activity. 2, Fv fragment, F(ab') 2 , scFv, di-scFv and / or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins.

[0063] In the present application, all amino acid sequences of CDRs are shown according to the Chothia definition rules. However, those skilled in the art can define antibody CDRs in various ways, such as the Kabat definition rules based on sequence variability (see Kabat et al., Immunological Protein Sequence, 5th Edition, National Institutes of Health, Bethesda, MD (1991)) and the Chothia definition rules based on structural ring region positions (see Jmol Biol 273:927-48, 1997). In embodiments of the present invention, a combined definition rule including the Kabat definition and the Chothia definition can also be used to determine amino acid residues in the variable domain sequence. Among them, the combined definition rule combines the scope of the Kabat definition and the Chothia definition, and has a broader scope based on this. For details, see the table below. Those skilled in the art should understand that unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) encompass the complementarity determining region defined by any of the above known embodiments described by the present invention. The scope of protection of the present invention is the sequence shown based on the Chothia definition rules, but the corresponding amino acid sequences according to other CDR definition rules should also be included in the scope of protection of the present invention. [Table 1]

[0064] Here, Laa-Lbb can refer to the amino acid sequence from position aa (Chothia coding rules) to position bb (Chothia coding rules) from the N-terminus of the antibody light chain; Haa-Hbb can refer to the amino acid sequence from position aa (Chothia coding rules) to position bb (Chothia coding rules) from the N-terminus of the antibody heavy chain. For example, L24-L34 can refer to the amino acid sequence from position 24 to 34 from the N-terminus of the antibody light chain according to the Chothia coding rules, and H26-H32 can refer to the amino acid sequence from position 26 to 32 from the N-terminus of the antibody heavy chain according to the Chothia coding rules. Those skilled in the art should recognize that when encoding CDRs with Chothia, some positions are inserted (http: / / bioinf.org.uk / abs / ).

[0065] In this application, the term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies in which the individual antibodies within the cluster are identical, except for minor natural mutations that may be present. Monoclonal antibodies generally have a high degree of specificity for a single antigenic site. Also, unlike conventional polyclonal antibody preparations (which usually have different antibodies against different determinant clusters), each monoclonal antibody is directed against a single determinant cluster on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma cultures without contamination by other immunoglobulins. The modifier "monoclonal" refers to the character of the antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be produced in hybridoma cells or by recombinant DNA methods.

[0066] In this application, the term "fully human antibody" generally refers to an antibody in which a human transfers all of the antibody-encoding genes into a genetically engineered antibody gene-deficient animal and expresses the antibody in the animal. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. Fully human antibodies can greatly reduce immune side reactions in the human body caused by foreign antibodies. Methods for obtaining fully human antibodies in this field include phage display technology and transgenic mouse technology.

[0067] In this application, the term "fully human heavy chain antibody" generally refers to a heavy chain antibody having a human antibody variable region VH obtained using Harbour HCAb transgenic mouse (patent application WO2007 / 096779). The endogenous antibody heavy chain locus and light chain locus of this transgenic mouse are both knocked out or inactivated to prevent mouse antibody production; then, human antibody heavy chain gene fragments (V, D, J fragments) are transferred into the mouse, and the mouse's own recombination and mutation mechanisms are used to produce an antibody having a human antibody gene sequence, whose variable region is human VH. A fully human heavy chain antibody is obtained by fusing and rearranging this human VH with a human heavy chain constant region Fc.

[0068] In this application, the term "specific binding" generally means that an antibody binds to an epitope through its antigen-binding domain, and such binding requires some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind to an antigen" if it prefers to bind to an epitope through its antigen-binding domain over random and unrelated epitopes. "Epitope" refers to a specific atomic group (e.g., sugar side chain, phosphonyl, sulfonyl) or amino acid in an antigen that binds to an antigen-binding protein (e.g., an antibody).

[0069] In this application, the term "Fab" generally refers to an antigen-binding portion of a conventional antibody (e.g., IgG), including a heavy chain variable region VH, a light chain variable region VL, and a heavy chain constant region domain CH1 and a light chain constant region CL of an antibody. In a conventional antibody, the C-terminus of VH binds to the N-terminus of CH1 to form a heavy chain Fd fragment, the C-terminus of VL binds to the N-terminus of CL to form a light chain, and the C-terminus of CH1 further binds to the hinge region of the heavy chain and other constant region domains to form a heavy chain. In some embodiments, "Fab" also refers to a mutant structure of Fab. For example, in some embodiments, the C-terminus of VH and the N-terminus of CL bind to form one polypeptide chain, and the C-terminus of VL and the N-terminus of CH1 bind to form another polypeptide chain, forming a Fab (cross VH / VL) structure; in some embodiments, the CH1 of Fab is not bound to the hinge region, and the C-terminus of CL is bound to the hinge region of the heavy chain, forming a Fab (cross Fd / LC) structure.

[0070] In the present application, the term "VH" generally refers to the VH domain of the heavy chain variable region of an antibody, i.e., the heavy chain variable region VH of a conventional antibody (H2L2 structure) of a human or other animal, the heavy chain variable region VHH of a heavy chain antibody (HCAb structure) of an animal such as a camelid, or the heavy chain variable region VH of a fully human-derived heavy chain antibody (HCAb structure) produced using a Harbour HCAb transgenic mouse.

[0071] In this application, the term "binding moieties" generally refers to any functional region of a protein capable of specifically binding to an antigen, whether it be a "Fab", a "VH", or other antigen-binding form (e.g., derivative protein structures such as lipid carrier proteins (lipocalins), neural cell adhesion molecules (NCAMs), fibronectins, overlapping fragment anchor protein proteins (DARPins), etc.).

[0072] In this application, the term "valency" generally refers to the number of "binding domains" in a binding protein, i.e., the maximum number of antigen molecules or epitopes that the binding protein can bind; for example, a conventional IgG antibody can bind to two identical antigen molecules simultaneously, and has a valency of 2; and a Fab antibody has a valency of 1.

[0073] In this application, the term "bispecific binding protein" generally refers to a binding protein having two antigen-binding specificities. The two antigen-binding specificities may bind two different antigens or may bind two different epitopes of the same antigen. Bispecific binding proteins may typically include bispecific antibodies and derivatives thereof.

[0074] In this application, the term "multispecific binding protein" generally refers to a binding protein having two or more antigen-binding specificities. Multispecific binding proteins can typically include multispecific antibodies and derivatives thereof.

[0075] In this application, the term "MFI" (Mean Fluorescent Intensity) generally refers to a fluorescence intensity signal analyzed in a flow cell FACS method and its mathematical average value, and can generally be obtained by processing and analyzing data generated by the flow cell method using specialized software, such as FlowJo (FlowJo, LLC).

[0076] In this application, the term "PD-L1" generally refers to the programmed death ligand 1 protein, functional variants thereof, and / or functional fragments thereof. PD-L1, also known as cluster of differentiation 274 (CD274) or B7 isotope 1 (B7-H1), is a protein encoded by the CD274 gene (in humans). PD-L1 sequences are known in the art. For example, an exemplary full-length human PD-L1 protein amino acid sequence can be found at NCBI Accession No. NP_054862 or UniProt Accession No. Q9NZQ7; an exemplary full-length cynomolgus monkey PD-L1 protein sequence can be found at NCBI Accession No. XP_005581836 or Uniprot Accession No. G7PSE7.

[0077] In this application, the term "PD-1" generally refers to the programmed death 1 receptor (also known as CD279), its functional variants, and / or its functional fragments. PD-1 sequences are known in the art. For example, an exemplary full-length human PD-1 protein sequence can be found at NCBI accession number NP_005009; an exemplary full-length cynomolgus monkey PD-1 protein sequence can be found at NCBI accession number NP_001271065 or Uniprot accession number B0LAJ3.

[0078] In this application, the term "CD80" generally refers to cluster of differentiation protein 80 (also called B7-1), its functional variants, and / or its functional fragments. CD80 sequences are known in the art. For example, an exemplary full-length human CD80 sequence can be found at Uniprot accession number P33681.

[0079] In this application, the term "CTLA4" generally refers to cytotoxic T-lymphocyte-associated protein-4 (also called CD152), its functional variants, and / or its functional fragments. CTLA4 sequences are known in the art. For example, an exemplary full-length human CTLA4 sequence can be found at Uniprot accession number P16410, and an exemplary full-length cynomolgus monkey CTLA4 sequence can be found at Uniprot accession number G7PL88.

[0080] In this application, the term "HER2" generally refers to the acceptor tyrosine kinase erbB-2 (also called ERBB2), its functional variants, and / or its functional fragments. HER2 sequences are known in the art. For example, an exemplary full-length human HER2 sequence can be found at Uniprot accession number P04626, and an exemplary full-length cynomolgus HER2 sequence can be found at NCBI accession number XP_005584091.

[0081] In this application, the term "B7H4" is generally meant to include V-Set domain T-cell activation inhibitor 1 (also called VTCN1), functional variants thereof, and / or functional fragments thereof. B7H4 sequences are known in the art. For example, an exemplary full-length human B7H4 sequence can be found at Uniprot accession number Q7Z7D3, an exemplary full-length cynomolgus B7H4 sequence can be found at NCBI accession number XP_005542249; an exemplary full-length mouse B7H4 sequence can be found at Uniprot accession number Q7TSP5.

[0082] In this application, the term "4-1BB" generally refers to tumor necrosis factor receptor superfamily member 9 (CD137, also known as 4-1BBL receptor), functional variants thereof, and / or functional fragments thereof. 4-1BB sequences are known in the art. For example, an exemplary full-length human 4-1BB sequence can be found at Uniprot accession number Q07011, and an exemplary full-length cynomolgus monkey 4-1BB sequence can be found at NCBI accession number XP_005544945.

[0083] In this application, the term "BCMA" generally refers to tumor necrosis factor receptor superfamily member 17 (also called CD269, B-cell maturation protein), functional variants thereof, and / or functional fragments thereof. BCMA sequences are known in the art. For example, an exemplary full-length human BCMA sequence can be found at Uniprot accession number Q02223, and an exemplary full-length cynomolgus BCMA sequence can be found at NCBI accession number XP_005591343.

[0084] In this application, the term "BAFF" generally refers to tumor necrosis factor ligand superfamily member 13B (also called CD257, B-cell activating factor), its functional variants, and / or its functional fragments. BAFF sequences are known in the art. For example, an exemplary full-length human BAFF sequence can be found at Uniprot accession number Q9Y275.

[0085] In this application, the term "APRIL" generally refers to tumor necrosis factor ligand superfamily member 13 (also called CD256, proliferation-inducing ligand), its functional variants, and / or its functional fragments. APRIL sequences are known in the art. For example, an exemplary full-length human APRIL sequence can be found in Uniprot accession number O75888.

[0086] In this application, the term "CD3" generally refers to the TCR / CD3 receptor protein complex on T cells. The specificity of T cell responses is mediated by the recognition of the TCR and CD3 molecular complex to pMHC. The TCR is a heterodimer consisting of two different transmembrane polypeptide chains, and there are four types of peptide chains, α, β, γ, and δ, and TCRs are classified as TCRαβ and TCRγδ depending on the combination of peptide chains. CD3 has different transmembrane polypeptide chains, namely γ, δ, ε, and ζ, which interact to form homodimers or heterodimers as part of the TCR-CD3 complex. Because the cytoplasmic region of the TCR peptide chain is short, it is believed that the activation signal due to the antigen recognition of the TCR is transferred into the T cell by the CD3 peptide chain.

[0087] In this application, the term "CD3E" generally refers to the epsilon peptide chain of "CD3". CD3E sequences are known in the art. For example, an exemplary full-length human CD3E sequence can be found at Uniprot accession number P07766, and an exemplary full-length cynomolgus monkey CD3E sequence can be found at Uniprot accession number Q95LI5.

[0088] Working Example The present invention will be further described below in the form of examples, but the present invention is not limited to the scope of the examples. The examples do not include detailed descriptions of conventional methods, such as methods for constructing vectors and plasmids, inserting protein-encoding genes into vectors and plasmids, or introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in many publications. Experimental methods for which specific conditions are not specified in the following examples are selected according to conventional methods and conditions or according to product instructions.

[0089] Example 1. Structural design of HCAb-based multispecific binding proteins This example shows several structures of Fc-containing, symmetric or asymmetric, multivalent and multispecific binding proteins constructed using fully human heavy chain antibodies (HCAbs) and their derived single domain antibodies (sdAbs). In some structures, the domains are linked using a connecting peptide between the domains. In some structures, amino acid mutations are introduced into the heavy chain Fc region to adjust the binding to the Fc receptor and further adjust the related effector function or other performance. In some structures, different amino acid mutations are introduced into the Fc regions of the two heavy chains to reduce the formation of heavy chain homodimerization.

[0090] Table 1-1 and Figure 1 show the molecular structures of the multispecific binding proteins included in the present application, each structure is further described below. In this embodiment and other parts of the present invention, when referring to the number of polypeptide chains included in the molecular structure, it generally refers to the number of "different polypeptide chains"; for example, a conventional IgG antibody has two different polypeptide chains, i.e., a heavy chain and a light chain, and although the IgG antibody molecule itself is a tetrapeptide chain protein molecule containing two identical heavy chains and two identical light chains, it specifically refers to two different polypeptide chains when describing its structural features. Binding valency refers to the number of antigen binding sites in the molecular structure, for example, a conventional IgG antibody can bind two identical antigen molecules simultaneously, and its binding valency is two. Table 1-2 shows connecting peptide sequences that can be used in the structural design of the present application.

[0091] [Table 2-1] TIFF2025032073000003.tif23129

[0092] [Table 2-2] TIFF2025032073000005.tif21130

[0093] Example 1.1. Fab-HCAb symmetric structure The present invention provides a method to construct a bispecific binding protein using two parent monoclonal antibodies: a conventional antibody A that binds a first antigen and a heavy chain antibody B that binds a second antigen.

[0094] As shown in Figure 1 (1-2), the Fab end is derived from conventional antibody A, and VH_A and VL_A are the heavy chain variable region and light chain variable region of antibody A, respectively. The VH end is derived from heavy chain antibody B, and VH_B is the heavy chain variable region of heavy chain antibody B. CL is the light chain constant region domain. CH1, CH2, and CH3 are the first, second, and third domains of the heavy chain constant region, respectively. L1 and L2 are the first and second connecting peptides, respectively.

[0095] Example 1.1.1. Structure (1): Fab(CL)-VH-Fc The binding protein of structure (1) comprises two different polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also called the short chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1; polypeptide chain 2 or the second polypeptide chain, also called the long chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL-L1-VH_B-L2-CH2-CH3. In structure (1), VL_A of antibody A and VH_B of heavy chain antibody B are fused to the same polypeptide chain, thereby avoiding mismatch by-products that occur upon association of VL_A and VH_B.

[0096] VH_B of polypeptide chain 2 is linked to CH2 via a connecting peptide L2; L2 may be an IgG hinge region or a connecting peptide sequence derived from a hinge region or a sequence listed in Table 1-2, preferably the sequence of human IgG1 hinge or human IgG1 hinge (C220S) or G5-LH.

[0097] In one embodiment, the CL and VH_B of polypeptide chain 2 are directly fused or linked, i.e., the length of L1 is 0. In another embodiment, the CL of polypeptide chain 2 is linked to VH_B via a connecting peptide L1; L1 is a sequence listed in Table 1-2.

[0098] Example 1.1.2. Structure (2): Fab(CH1)-VH-Fc The binding protein of structure (2) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also referred to as the short chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL; polypeptide chain 2 or the second polypeptide chain, also referred to as the long chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1-L1-VH_B-L2-CH2-CH3.

[0099] VH_B of polypeptide chain 2 is linked to CH2 via a connecting peptide L2; L2 may be an IgG hinge region or a connecting peptide sequence derived from a hinge region or a sequence listed in Table 1-2, preferably the sequence of human IgG1 hinge or human IgG1 hinge (C220S) or G5-LH.

[0100] In one embodiment, CH1 of polypeptide chain 2 and VH_B are directly fused or linked, i.e., the length of L1 is 0. In another embodiment, CH1 of polypeptide chain 2 is linked to VH_B via a connecting peptide L1; L1 is a sequence listed in Table 1-2.

[0101] Example 1.2. IgG-VH tetravalent symmetric structure The present invention provides a method to construct a bispecific binding protein using two parent monoclonal antibodies: a conventional antibody A that binds a first antigen and a heavy chain antibody B that binds a second antigen.

[0102] As shown in Figure 1 (3-6), the Fab end is derived from conventional antibody A, and VH_A and VL_A are the heavy chain variable region and light chain variable region of antibody A, respectively. The VH end is derived from heavy chain antibody B, and VH_B is the heavy chain variable region of heavy chain antibody B. CL is the light chain constant region domain. CH1, CH2, and CH3 are the first, second, and third domains of the heavy chain constant region, respectively. L is a connecting peptide, and h is the hinge region or derived sequence of an IgG antibody.

[0103] Example 1.2.1. Structure (3): VH-IgG_HC The binding protein of structure (3) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also referred to as the short chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL; polypeptide chain 2 or the second polypeptide chain, also referred to as the long chain, comprises, from the amino terminus to the carboxyl terminus, VH_B-L-VH_A-CH1-h-CH2-CH3.

[0104] In one embodiment, VH_B and VH_A of polypeptide chain 2 are directly fused or linked, i.e., the length of L is 0. In another embodiment, VH_B of polypeptide chain 2 is linked to VH_A via a connecting peptide L; L is a sequence listed in Table 1-2.

[0105] Example 1.2.2. Structure (4): VH-IgG_LC The binding protein of structure (4) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also referred to as the short chain, comprises, from the amino terminus to the carboxyl terminus, VH_B-L-VL_A-CL; polypeptide chain 2 or the second polypeptide chain, also referred to as the long chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1-h-CH2-CH3.

[0106] In one embodiment, VH_B and VL_A of polypeptide chain 1 are directly fused or linked, i.e., the length of L is 0. In another embodiment, VH_B of polypeptide chain 1 is linked to VL_A via a connecting peptide L; L is a sequence listed in Table 1-2.

[0107] Example 1.2.3. Structure (5): IgG_HC-VH The binding protein of structure (5) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also referred to as the short chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL; polypeptide chain 2 or the second polypeptide chain, also referred to as the long chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1-h-CH2-CH3-L-VH_B.

[0108] In one embodiment, CH3 of polypeptide chain 2 and VH_B are directly fused or linked, i.e., the length of L is 0. In another embodiment, CH3 of polypeptide chain 2 is linked to VH_B via a connecting peptide L; L is a sequence listed in Table 1-2.

[0109] Example 1.2.4. Structure (6): IgG_LC-VH The binding protein of structure (6) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also referred to as the short chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL-L-VH_B; polypeptide chain 2 or the second polypeptide chain, also referred to as the long chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1-h-CH2-CH3.

[0110] In one embodiment, CL and VH_B of polypeptide chain 1 are directly fused or linked, i.e., the length of L is 0. In another embodiment, CL of polypeptide chain 1 is linked to VH_B via a connecting peptide L; L is a sequence listed in Table 1-2.

[0111] Example 1.3. IgG-VH(2) Hexavalent Symmetric Structure The present invention provides a method to construct a bispecific binding protein using two parent monoclonal antibodies: a conventional antibody A that binds a first antigen and a heavy chain antibody B that binds a second antigen.

[0112] The present invention further provides a method for constructing a trispecific binding protein using three parent monoclonal antibodies: a conventional antibody A that binds a first antigen, a heavy chain antibody B that binds a second antigen, and a heavy chain antibody C that binds a third antigen.

[0113] As shown in Figure 1 (7-8), the Fab end is derived from conventional antibody A, and VH_A and VL_A are the heavy chain variable region and light chain variable region of antibody A, respectively. The VH end is derived from heavy chain antibody B or heavy chain antibody C, and VH_B is the heavy chain variable region of heavy chain antibody B, and VH_C is the heavy chain variable region of heavy chain antibody C. CL is the light chain constant region domain. CH1, CH2, and CH3 are the first, second, and third domains of the heavy chain constant region, respectively. L1 and L2 are the first and second connecting peptides, respectively, and h is the hinge region or derived sequence of an IgG antibody.

[0114] Example 1.3.1. Structure (7): IgG_HC-VH-VH Structure (7) shows a bispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also called the short chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL; polypeptide chain 2 or the second polypeptide chain, also called the long chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_B.

[0115] In one embodiment, CH3 of polypeptide chain 2 and VH_B are directly fused or linked, i.e., the length of L1 is 0. In another embodiment, CH3 of polypeptide chain 2 is linked to VH_B via a connecting peptide L1; L1 is a sequence listed in Table 1-2.

[0116] In one embodiment, the first VH_B and the second VH_B of polypeptide chain 2 are directly fused or linked, i.e., the length of L2 is 0. In another embodiment, the first VH_B of polypeptide chain 2 is linked to the second VH_B via a connecting peptide L2; L2 is a sequence listed in Table 1-2.

[0117] Example 1.3.2. Structure (8): IgG_HC-VH'-VH'' Structure (8) shows a trispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also called the short chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL; polypeptide chain 2 or the second polypeptide chain, also called the long chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_C.

[0118] In one embodiment, CH3 of polypeptide chain 2 and VH_B are directly fused or linked, i.e., the length of L1 is 0. In another embodiment, CH3 of polypeptide chain 2 is linked to VH_B via a connecting peptide L1; L1 is a sequence listed in Table 1-2.

[0119] In one embodiment, VH_B and VH_C of polypeptide chain 2 are directly fused or linked, i.e., the length of L2 is 0. In another embodiment, VH_B of polypeptide chain 2 is linked to VH_C via a connecting peptide L2; L2 is a sequence listed in Table 1-2.

[0120] Example 1.4.2xVH-IgG Hexavalent Symmetric Structure The present invention provides a method to construct a bispecific binding protein using two parent monoclonal antibodies: a conventional antibody A that binds a first antigen and a heavy chain antibody B that binds a second antigen.

[0121] The present invention further provides a method for constructing a trispecific binding protein using three parent monoclonal antibodies: a conventional antibody A that binds a first antigen, a heavy chain antibody B that binds a second antigen, and a heavy chain antibody C that binds a third antigen.

[0122] As shown in Figure 1 (9-10), the Fab end is derived from conventional antibody A, and VH_A and VL_A are the heavy chain variable region and light chain variable region of antibody A, respectively. The VH end is derived from heavy chain antibody B or heavy chain antibody C, and VH_B is the heavy chain variable region of heavy chain antibody B, and VH_C is the heavy chain variable region of heavy chain antibody C. CL is the light chain constant region domain. CH1, CH2, and CH3 are the first, second, and third domains of the heavy chain constant region, respectively. L1 and L2 are the first and second connecting peptides, respectively, and h is the hinge region or derived sequence of an IgG antibody.

[0123] Example 1.4.1. Structure (9): 2xVH-IgG(HC+LC) Structure (9) shows a bispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also called the short chain, comprises, from the amino terminus to the carboxyl terminus, VH_B-L1-VL_A-CL; polypeptide chain 2 or the second polypeptide chain, also called the long chain, comprises, from the amino terminus to the carboxyl terminus, VH_B-L2-VH_A-CH1-h-CH2-CH3.

[0124] In one embodiment, VH_B and VL_A of polypeptide chain 1 are directly fused or linked, i.e., the length of L1 is 0. In another embodiment, VH_B of polypeptide chain 1 is linked to VL_A via a connecting peptide L1; L1 is a sequence listed in Table 1-2.

[0125] In one embodiment, VH_B and VH_A of polypeptide chain 2 are directly fused or linked, i.e., the length of L2 is 0. In another embodiment, VH_B of polypeptide chain 2 is linked to VH_A via a connecting peptide L2; L2 is a sequence listed in Table 1-2.

[0126] In one embodiment, L1 and L2 are the same sequence. In another embodiment, L1 and L2 are different sequences.

[0127] Example 1.4.2. Structure (10): (VH'+VH'')-IgG(HC+LC) Structure (10) shows a trispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also called the short chain, comprises, from the amino terminus to the carboxyl terminus, VH_B-L1-VL_A-CL; polypeptide chain 2 or the second polypeptide chain, also called the long chain, comprises, from the amino terminus to the carboxyl terminus, VH_C-L2-VH_A-CH1-h-CH2-CH3.

[0128] In one embodiment, VH_B and VL_A of polypeptide chain 1 are directly fused or linked, i.e., the length of L1 is 0. In another embodiment, VH_B of polypeptide chain 1 is linked to VL_A via a connecting peptide L1; L1 is a sequence listed in Table 1-2.

[0129] In one embodiment, the VH_C and VH_A of polypeptide chain 2 are directly fused or linked, i.e., the length of L2 is 0. In another embodiment, the VH_C of polypeptide chain 2 is linked to the VH_A via a connecting peptide L2; L2 is a sequence listed in Table 1-2.

[0130] In one embodiment, L1 and L2 are the same sequence. In another embodiment, L1 and L2 are different sequences.

[0131] Example 1.5. Fab-Fc-VH bivalent asymmetric structure The present invention provides a method to construct a bispecific binding protein using two parent monoclonal antibodies: a conventional antibody A that binds a first antigen and a heavy chain antibody B that binds a second antigen.

[0132] As shown in Figure 1 (11-13), the Fab end is derived from conventional antibody A, and VH_A and VL_A are the heavy chain variable region and light chain variable region of antibody A, respectively. The VH end is derived from heavy chain antibody B, and VH_B is the heavy chain variable region of heavy chain antibody B. CL is the light chain constant region domain. CH1, CH2 and CH3 are the first, second and third domains of the heavy chain constant region, respectively. h is the hinge region or derived sequence of an IgG antibody. The Fc regions of the two heavy chains each have different amino acid mutations introduced to reduce the formation of homodimerization of the heavy chains.

[0133] In one embodiment, h in the polypeptide chain containing VH_B is the hinge region of an IgG or a connecting peptide sequence derived from the hinge region or the sequence of human IgG1 hinge (C220S) or G5-LH in Table 1-2.

[0134] Example 1.5.1. Structure (11): Fab-Fc-VH The binding protein of structure (11) comprises three distinct polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1-h-CH2-CH3; and polypeptide chain 3 comprises, from the amino terminus to the carboxyl terminus, VH_B-h-CH2-CH3.

[0135] Example 1.5.2. Structure (12): Fab(cross VH / VL)-Fc-VH The binding protein of structure (12) comprises three distinct polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CL; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CH1-h-CH2-CH3; and polypeptide chain 3 comprises, from the amino terminus to the carboxyl terminus, VH_B-h-CH2-CH3.

[0136] Example 1.5.3. Structure (13): Fab(cross Fd / LC)-Fc-VH The binding protein of structure (13) comprises three distinct polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL-h-CH2-CH3; and polypeptide chain 3, or the third polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_B-h-CH2-CH3.

[0137] In one embodiment, h in polypeptide chain 2 is a hinge region or connecting peptide, the sequence of which is the sequence of LH1 in Table 1-2.

[0138] Example 1.6. Fab-Fc-VH(2) Trivalent Asymmetric Structure The present invention provides a method to construct a bispecific binding protein using two parent monoclonal antibodies: a conventional antibody A that binds a first antigen and a heavy chain antibody B that binds a second antigen.

[0139] The present invention further provides a method for constructing a trispecific binding protein using three parent monoclonal antibodies: a conventional antibody A that binds a first antigen, a heavy chain antibody B that binds a second antigen, and a heavy chain antibody C that binds a third antigen.

[0140] As shown in Figure 1 (14-19), the Fab end is derived from conventional antibody A, and VH_A and VL_A are the heavy chain variable region and light chain variable region of antibody A, respectively. The VH end is derived from heavy chain antibody B or heavy chain antibody C, and VH_B is the heavy chain variable region of heavy chain antibody B, and VH_C is the heavy chain variable region of heavy chain antibody C. CL is the light chain constant region domain. CH1, CH2, and CH3 are the first, second, and third domains of the heavy chain constant region, respectively. L is a connecting peptide, and h is the hinge region or derived sequence of an IgG antibody. The Fc regions of the two heavy chains are each introduced with different amino acid mutations, thereby reducing the formation of homodimerization of the heavy chains.

[0141] In one embodiment, h in the polypeptide chain containing VH_B is the hinge region of an IgG or a connecting peptide sequence derived from the hinge region or the sequence of human IgG1 hinge (C220S) or G5-LH in Table 1-2.

[0142] Example 1.6.1. Structure (14): Fab-Fc-VH-VH Structure (14) shows a bispecific binding protein comprising three different polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1-h-CH2-CH3; and polypeptide chain 3, or the third polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_B-L-VH_B-h-CH2-CH3.

[0143] In one embodiment, the first VH_B and the second VH_B of polypeptide chain 3 are directly fused and linked, i.e., the length of L is 0. In another embodiment, the first VH_B of polypeptide chain 3 is linked to the second VH_B via a connecting peptide L; L is a sequence listed in Table 1-2.

[0144] Example 1.6.2. Structure (15): Fab-Fc-VH'-VH'' Structure (15) shows a trispecific binding protein comprising three different polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1-h-CH2-CH3; and polypeptide chain 3, or the third polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_C-L-VH_B-h-CH2-CH3.

[0145] In one embodiment, VH_C and VH_B of polypeptide chain 3 are directly fused or linked, i.e., the length of L is 0. In another embodiment, VH_C of polypeptide chain 3 is linked to VH_B via a connecting peptide L; L is a sequence listed in Table 1-2.

[0146] Example 1.6.3. Structure (16): Fab(cross VH / VL)-Fc-VH-VH Structure (16) shows a bispecific binding protein comprising three different polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CL; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CH1-h-CH2-CH3; and polypeptide chain 3, or the third polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_B-L-VH_B-h-CH2-CH3.

[0147] In one embodiment, the first VH_B and the second VH_B of polypeptide chain 3 are directly fused and linked, i.e., the length of L is 0. In another embodiment, the first VH_B of polypeptide chain 3 is linked to the second VH_B via a connecting peptide L; L is a sequence listed in Table 1-2.

[0148] Example 1.6.4. Structure (17): Fab(cross VH / VL)-Fc-VH'-VH'' Structure (17) shows a trispecific binding protein comprising three different polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CL; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CH1-h-CH2-CH3; and polypeptide chain 3, or the third polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_C-L-VH_B-h-CH2-CH3.

[0149] In one embodiment, VH_C and VH_B of polypeptide chain 3 are directly fused or linked, i.e., the length of L is 0. In another embodiment, VH_C of polypeptide chain 3 is linked to VH_B via a connecting peptide L; L is a sequence listed in Table 1-2.

[0150] Example 1.6.5. Structure (18): Fab(cross Fd / LC)-Fc-VH-VH Structure (18) shows a bispecific binding protein comprising three different polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL-h-CH2-CH3; and polypeptide chain 3, or the third polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_B-L-VH_B-h-CH2-CH3.

[0151] In one embodiment, the first VH_B and the second VH_B of polypeptide chain 3 are directly fused and linked, i.e., the length of L is 0. In another embodiment, the first VH_B of polypeptide chain 3 is linked to the second VH_B via a connecting peptide L; L is a sequence listed in Table 1-2.

[0152] In one embodiment, h in polypeptide chain 2 is a hinge region or connecting peptide, the sequence of which is the sequence of LH1 in Table 1-2.

[0153] Example 1.6.6. Structure (19): Fab(cross Fd / LC)-Fc-VH'-VH'' Structure (19) shows a trispecific binding protein comprising three different polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL-h-CH2-CH3; and polypeptide chain 3, or the third polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_C-L-VH_B-h-CH2-CH3.

[0154] In one embodiment, VH_C and VH_B of polypeptide chain 3 are directly fused or linked, i.e., the length of L is 0. In another embodiment, VH_C of polypeptide chain 3 is linked to VH_B via a connecting peptide L; L is a sequence listed in Table 1-2.

[0155] In one embodiment, h in polypeptide chain 2 is a hinge region or connecting peptide, the sequence of which is the sequence of LH1 in Table 1-2.

[0156] Example 1.7. scFv-Fc-VH bivalent asymmetric structure The present invention provides a method to construct a bispecific binding protein using two parent monoclonal antibodies: a conventional antibody A that binds a first antigen and a heavy chain antibody B that binds a second antigen.

[0157] As shown in Figure 1 (20-21), the scFv end is derived from conventional antibody A, and VH_A and VL_A are the heavy chain variable region and light chain variable region of antibody A, respectively. The VH end is derived from heavy chain antibody B, and VH_B is the heavy chain variable region of heavy chain antibody B. CH2 and CH3 are the second and third domains of the heavy chain constant region, respectively. L is a connecting peptide, and h is the hinge region of an IgG antibody or a derived sequence. Different amino acid mutations are introduced into the Fc regions of the two heavy chains, thereby reducing the formation of heavy chain homodimerization.

[0158] In one embodiment, the sequence of the scFv is VH-connected peptide-VL. In another embodiment, the sequence of the scFv is VL-connected peptide-VH.

[0159] In one embodiment, L in the polypeptide chain is a connecting peptide, which may be a sequence listed in Table 1-2, and is preferably a sequence of GS_15 or GS_20.

[0160] In one embodiment, h in the polypeptide chain is a hinge region or derived sequence of an IgG antibody, which may be a sequence listed in Table 1-2, preferably the sequence of human IgG1 hinge (C220S) or G5-LH.

[0161] Example 1.7.1. Structure (20): scFv(VL-VH)-Fc-VH The binding protein of structure (20) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VL_A-L-VH_A-h-CH2-CH3; polypeptide chain 2 or the second polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VH_B-h-CH2-CH3.

[0162] Example 1.7.2. Structure (21): scFv(VH-VL)-Fc-VH The binding protein of structure (21) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VH_A-L-VL_A-h-CH2-CH3; polypeptide chain 2 or the second polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VH_B-h-CH2-CH3.

[0163] Example 1.8. scFv-Fc-VH(2) Trivalent Asymmetric Structure The present invention provides a method to construct a bispecific binding protein using two parent monoclonal antibodies: a conventional antibody A that binds a first antigen and a heavy chain antibody B that binds a second antigen.

[0164] The present invention further provides a method for constructing a trispecific binding protein using three parent monoclonal antibodies: a conventional antibody A that binds a first antigen, a heavy chain antibody B that binds a second antigen, and a heavy chain antibody C that binds a third antigen.

[0165] As shown in Figure 1 (22-25), the scFv end is derived from conventional antibody A, and VH_A and VL_A are the heavy chain variable region and light chain variable region of antibody A, respectively. The VH end is derived from heavy chain antibody B or heavy chain antibody C, VH_B is the heavy chain variable region of heavy chain antibody B, and VH_C is the heavy chain variable region of heavy chain antibody C. CH2 and CH3 are the second and third domains of the heavy chain constant region, respectively. L1 and L2 are the first and second connecting peptides, respectively, and h is the hinge region or derived sequence of an IgG antibody. The Fc regions of the two heavy chains are each introduced with different amino acid mutations, thereby reducing the formation of homodimerization of the heavy chains.

[0166] In one embodiment, the sequence of the scFv is VH-connected peptide-VL. In another embodiment, the sequence of the scFv is VL-connected peptide-VH.

[0167] In one embodiment, L1 in the polypeptide chain is the connecting peptide of VH and VL in the combined scFv and may be a sequence listed in Table 1-2, preferably the sequence GS_15 or GS_20.

[0168] In one embodiment, L2 in the polypeptide chain is a connecting peptide between the heavy chain variable regions that joins the two heavy chain antibodies and is a sequence listed in Table 1-2. In another embodiment, there is a direct fusion between the heavy chain variable regions of the two heavy chain antibodies, i.e., the length of L2 is 0.

[0169] In one embodiment, h in the polypeptide chain is a hinge region or derived sequence of an IgG antibody, which may be a sequence listed in Table 1-2, preferably the sequence of human IgG1 hinge (C220S) or G5-LH.

[0170] Example 1.8.1. Structure (22): scFv(VL-VH)-Fc-VH-VH Structure (22) shows a bispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-L1-VH_A-h-CH2-CH3; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_B-L2-VH_B-h-CH2-CH3.

[0171] In one embodiment, the first VH_B and the second VH_B of polypeptide chain 2 are directly fused or linked, i.e., the length of L2 is 0. In another embodiment, the first VH_B of polypeptide chain 2 is linked to the second VH_B via a connecting peptide L2; L2 is a sequence listed in Table 1-2.

[0172] Example 1.8.2. Structure (23): scFv(VH-VL)-Fc-VH-VH Structure (23) shows a bispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-L1-VL_A-h-CH2-CH3; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_B-L2-VH_B-h-CH2-CH3.

[0173] In one embodiment, the first VH_B and the second VH_B of polypeptide chain 2 are directly fused or linked, i.e., the length of L2 is 0. In another embodiment, the first VH_B of polypeptide chain 2 is linked to the second VH_B via a connecting peptide L2; L2 is a sequence listed in Table 1-2.

[0174] Example 1.8.3. Structure (24): scFv(VL-VH)-Fc-VH'-VH'' Structure (24) shows a trispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-L1-VH_A-h-CH2-CH3; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_C-L2-VH_B-h-CH2-CH3.

[0175] In one embodiment, VH_C and VH_B of polypeptide chain 2 are directly fused or linked, i.e., the length of L2 is 0. In another embodiment, VH_C of polypeptide chain 2 is linked to VH_B via a connecting peptide L2; L2 is a sequence listed in Table 1-2.

[0176] Example 1.8.4. Structure (25): scFv(VH-VL)-Fc-VH'-VH'' Structure (25) shows a trispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-L1-VL_A-h-CH2-CH3; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_C-L2-VH_B-h-CH2-CH3.

[0177] In one embodiment, VH_C and VH_B of polypeptide chain 2 are directly fused or linked, i.e., the length of L2 is 0. In another embodiment, VH_C of polypeptide chain 2 is linked to VH_B via a connecting peptide L2; L2 is a sequence listed in Table 1-2.

[0178] Example 1.9. Fab-Fc-VH(3) tetravalent asymmetric structure The present invention provides a method to construct a bispecific binding protein using two parent monoclonal antibodies: a conventional antibody A that binds a first antigen and a heavy chain antibody B that binds a second antigen.

[0179] The present invention further provides a method for constructing a trispecific binding protein using three parent monoclonal antibodies: a conventional antibody A that binds a first antigen, a heavy chain antibody B that binds a second antigen, and a heavy chain antibody C that binds a third antigen.

[0180] The present invention further provides a method for constructing a tetraspecific binding protein using four parent monoclonal antibodies: a conventional antibody A that binds a first antigen, a heavy chain antibody B that binds a second antigen, a heavy chain antibody C that binds a third antigen, and a heavy chain antibody D that binds a fourth antigen.

[0181] As shown in Figure 1 (26-27), the Fab end is derived from conventional antibody A, and VH_A and VL_A are the heavy chain variable region and light chain variable region of antibody A, respectively. The VH end is derived from heavy chain antibody B or heavy chain antibody C or heavy chain antibody D, and VH_B is the heavy chain variable region of heavy chain antibody B, VH_C is the heavy chain variable region of heavy chain antibody C, and VH_D is the heavy chain variable region of heavy chain antibody D. CL is the light chain constant region domain. CH1, CH2, and CH3 are the first, second, and third domains of the heavy chain constant region, respectively. L1 and L2 are connecting peptides, and h is the hinge region or derived sequence of an IgG antibody. The Fc regions of the two heavy chains are each introduced with different amino acid mutations, thereby reducing the formation of homodimerization of the heavy chains.

[0182] In one embodiment, h in the polypeptide chain containing VH_B is the hinge region of an IgG or a connecting peptide sequence derived from the hinge region or the sequence of human IgG1 hinge (C220S) or G5-LH in Table 1-2.

[0183] Example 1.9.1. Structure (26): Fab-Fc-VH-VH-VH Structure (26) shows a bispecific binding protein comprising three different polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1-h-CH2-CH3; and polypeptide chain 3, or the third polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_B-L1-VH_B-L2-VH_B-h-CH2-CH3.

[0184] In one embodiment, the first VH_B and the second VH_B of polypeptide chain 3 are directly fused or linked, i.e., the length of L1 is 0. In another embodiment, the first VH_B of polypeptide chain 3 is linked to the second VH_B via a connecting peptide L1; L1 is a sequence listed in Table 1-2.

[0185] In one embodiment, the second VH_B and the third VH_B of polypeptide chain 3 are directly fused and linked, i.e., the length of L2 is 0. In another embodiment, the second VH_B of polypeptide chain 3 comprises a third VH_B linked via a connecting peptide L2; L2 is a sequence listed in Table 1-2.

[0186] Example 1.9.2. Structure (27): Fab-Fc-VH'-VH''-VH''' Structure (27) shows a tetraspecific binding protein comprising three different polypeptide chains: polypeptide chain 1, or the first polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VL_A-CL; polypeptide chain 2, or the second polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_A-CH1-h-CH2-CH3; and polypeptide chain 3, or the third polypeptide chain, comprises, from the amino terminus to the carboxyl terminus, VH_D-L1-VH_C-L2-VH_B-h-CH2-CH3.

[0187] In one embodiment, the VH_D and VH_C of polypeptide chain 3 are directly fused or linked, i.e., the length of L1 is 0. In another embodiment, the VH_D of polypeptide chain 3 is linked to the VH_C via a connecting peptide L1; L1 is a sequence listed in Table 1-2.

[0188] In one embodiment, VH_C and VH_B of polypeptide chain 3 are directly fused or linked, i.e., the length of L2 is 0. In another embodiment, VH_C of polypeptide chain 3 is linked to VH_B via a connecting peptide L2; L2 is a sequence listed in Table 1-2.

[0189] Example 2. Antibody sequence analysis, expression purification, and characterization and analysis of physicochemical properties Example 2.1. Antibody sequence analysis and optimization The heavy chain variable domain sequence of an antibody is derived from events such as genetic recombination of the germline gene V, D, and J gene fragments of the heavy chain gene group on a chromosome and high frequency somatic mutation; the light chain variable domain sequence is derived from events such as genetic recombination of the germline gene V and J gene fragments of the light chain gene group and high frequency somatic mutation. Genetic recombination and high frequency somatic mutation are the main factors that increase the diversity of antibodies. Antibodies derived from the same germline V gene fragment may also produce different sequences, but overall there is a high degree of similarity. Computational methods such as IMGT / DomainGapAlign (http: / / imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) or NCBI / IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ) can be used to estimate the possible germline gene fragments when genetic recombination occurs from the variable domain sequence of an antibody.

[0190] When a protein or polypeptide amino acid chain is translated and synthesized in a cell, chemical modifications called post-translational modifications (PTMs) may be introduced. For antibodies, some PTM sites are very conservative. For example, asparagine Asn, a conservative amino acid at position 297 (EU numbering) of the constant domain of human IgG1 antibodies, is usually modified by glycosylation to form a glycan, and this glycan structure is extremely important for antibody structure and related effect functions. However, when PTMs are present in the variable domain of an antibody, especially in the antigen-binding region such as CDR, the presence of these PTMs may have a significant effect on antigen binding and may also cause changes in the physicochemical properties of the antibody. For example, glycosylation, deamidation, isomerization, oxidation, etc. can all increase the instability or heterogeneity of the antibody molecule, thereby increasing the difficulty and risk of antibody development. Therefore, avoiding some potential PTMs is very important for the development of therapeutic antibodies. With the accumulation of experience, it has been discovered that some PTMs are highly associated with the composition of the amino acid sequence, especially the "pattern" of the adjacent amino acid composition, which allows us to predict potential PTMs from the primary amino acid sequence of a protein. For example, N-linked glycosylation sites are predicted from the sequence pattern N-x-S / T (position 1 is asparagine, position 2 is any amino acid except proline, and position 3 is serine or threonine). The amino acid sequence pattern that causes PTM may be derived from the germline gene sequence, for example, the human germline gene fragment IGHV3-33 naturally has the glycosylation pattern NST in the FR3 region; it may also be derived from somatic high frequency mutations. For example, NGS or NLT may be glycosylation sites, NS may be deamidation sites, and DG may cause isomerization of asparagine.

[0191] Amino acid mutations can disrupt the amino acid sequence pattern of PTMs, thereby reducing or eliminating the formation of certain PTMs. There are different mutation design methods depending on the antibody sequence and PTM sequence pattern. One method is to replace the "hot spot" amino acid (e.g., N or S in NS mode) with a physicochemically similar amino acid (such as mutating N to Q). If the PTM sequence pattern is derived from somatic high frequency mutations and does not exist in the germline gene sequence, another method is to replace the sequence pattern with the corresponding germline gene sequence. In practical operations, multiple mutation design methods can be adopted for the same PTM sequence pattern.

[0192] Example 2.2. Antibody Expression and Purification This example introduces a general method for producing antibodies using mammalian host cells (e.g., human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO and their derived cells), techniques such as instantaneous transfection expression and affinity capture isolation. The method applies to target antibodies that contain an Fc region; target antibodies can be composed of one or more protein polypeptide chains; and can be derived from one or more expression plasmids.

[0193] The amino acid sequence of the antibody polypeptide chain is converted into a nucleotide sequence by codon optimization; the encoded nucleotide sequence is synthesized and cloned onto an expression vector compatible with the host cell. The plasmids encoding the antibody polypeptide chain are co-transfected into mammalian host cells in a specific ratio, and recombinant antibodies with correct folding and polypeptide chain assembly can be obtained by utilizing conventional recombinant protein expression and purification techniques. Specifically, FreeStyle® 293-F cells (Thermo, #R79007) were expanded in FreeStyle® F17 Expression Medium (Thermo, #A1383504). Before starting the instantaneous transfection, the cell concentration was adjusted to 6–8 × 10 5 Adjust to 100 cells / ml and incubate at 37°C, 8% CO 2The cells were cultured in a rocker for 24 hours at a cell concentration of 1.2 × 10 6 The concentration was 100 cells / ml. 30 ml of cultured cells was prepared. Plasmids encoding antibody polypeptide chains were mixed in a specific ratio, and a total of 30 μg of plasmid (plasmid to cell ratio was 1 μg:1 ml) was dissolved in 1.5 ml of Opti-MEM serum-reduced medium (Thermo, #31985088) and sterilized by filtration using a 0.22 μm filter membrane. Further, 1.5 ml of Opti-MEM was dissolved in 120 μl of 1 mg / ml PEI (Polysciences, #23966-2) and left to stand for 5 minutes. PEI was slowly added to the plasmid and incubated at room temperature for 10 minutes. The plasmid-PEI mixed solution was slowly added dropwise while shaking the culture flask, and incubated at 37°C with 8% CO 2 The cells were cultured on a rocking bed for 5 days. After 5 days, the cell viability was observed. The culture was collected and centrifuged at 3300g for 10 minutes, and the supernatant was taken. The supernatant was then centrifuged at high speed to remove impurities. A gravity column (Bio-Rad, #7311550) containing MabSelect® (GE Healthcare, #71-5020-91) was equilibrated with PBS pH 7.4 buffer and washed with 2-5 column volumes. The supernatant sample was passed through the column; the column was washed with 5-10 column volumes of PBS buffer, and the target protein was eluted with 0.1 M glycine at pH 3.5, then adjusted to neutral with Tris-HCl at pH 8.0, and finally concentrated in an ultrafiltration tube (Millipore, #UFC901024) into a buffer containing PBS buffer or other components to obtain a purified recombinant antibody solution. Finally, the concentration is measured using a NanoDrop (Thermo NanoDrop)® One), aliquoted, and stored in advance.

[0194] Example 2.3. Protein Purity and Multimer Analysis Using SEC-HPLC In this example, analytical size exclusion chromatography (SEC) was used to analyze the purity and multimeric form of protein samples. An analytical column TSKgel G3000SWxl (Tosoh Bioscience, ##08541, 5 μm, 7.8 mm × 30 cm) was connected to a high pressure liquid chromatography HPLC (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 hour. An appropriate amount of protein sample (at least 10 μg) was filtered through a 0.22 μm filter membrane and injected into the system, and the HPLC program was set: run the sample through the column with PBS buffer at a flow rate of 1.0 ml / min, with a maximum run time of 25 minutes. The HPLC generates an analytical report, reporting the retention times of different molecular size components in the sample.

[0195] Figure 65 shows the SDS-PAGE and SEC-HPLC analysis results of protein samples obtained through instantaneous transfection expression and one-step affinity purification of several double antibody molecules with different structures in the present application. All of these double antibody molecules show good yields and purity.

[0196] Example 2.4. Measurement of thermal stability of protein molecules using DSF Differential Scanning Fluorimetry (DSF) is a common high-flux method for measuring the thermal stability of proteins. The method uses a real-time fluorescent quantitative PCR device to monitor the change in the fluorescence intensity of the dye bound to the unfolded protein molecule, thereby reflecting the process of protein denaturation and further reflecting the thermal stability of the protein molecule. In this example, the thermal denaturation temperature (Tm) of the protein molecule was measured using the DSF method. 10 μg protein was added to a 96-well PCR plate (Thermo, #AB-0700 / W), followed by adding 2 μL of 100× diluted dye SYPROTM (Invitrogen, #2008138), and adding buffer to make the final volume 40 μl per well. The PCR plate was sealed and placed in a real-time fluorescent quantitative PCR instrument (Bio-Rad CFX 96 PCR System) and incubated at 25°C for 5 min, followed by a gradual increase in temperature from 25°C to 95°C with a gradient of 0.2°C / 0.2 min, at the end of the experiment the temperature was reduced to 25°C. Data analysis was performed using Bio-Rad CFX Maestro software using the FRET scan mode, and the Tm of the samples was calculated.

[0197] Example 2.5. Measurement of protein molecular stability and molecular aggregation using Uncle Uncle (Unchained Labs) is a multifunctional one-stop protein stability analysis platform that characterizes protein stability by total fluorescence, static light scattering (SLS), and dynamic light scattering (DLS) detection methods. Samples of the same group can simultaneously obtain parameters such as melting temperature (Tm), aggregation temperature (Tagg) and particle size (diameter). In this example, the "Tm&Tagg with optional DLS" application of Uncle was selected and operated, 9 μL samples were added to Uni tubes, and the temperature was set to gradually increase from 25 °C to 95 °C with a gradient of 0.3 °C / min. Initial and final DLS measurements were taken four times for 5 seconds each. After the experiment was completed, the Uncle analysis software calculated the Tm value of each sample using the centroid mean (BCM) formula; the Tagg value was calculated according to the curve of SLS fluorescence intensity (aggregation curve) at wavelengths of 266 nm or 473 nm; the particle size and dispersity of the sample were calculated by the function related to DLS.

[0198] Example 3. PD-L1 x CTLA4 Bispecific Antibody Example 3.1. Background Programmed death receptor 1 (PD-1) is mainly expressed on immune cells such as T cells and has two ligands, namely programmed death ligand 1 (PD-L1) and PD-L2. PD-L1 is mainly expressed on antigen-presenting cells and some tumor cells. The interaction between PD-L1 and PD-1 reduces the activity of T cells, weakens the secretion of cytokines, and exerts an immunosuppressive effect. The expression of PD-L1 protein has been detected in many human tumor tissues, and the microenvironment at the tumor site can induce the expression of PD-L1 on tumor cells. The expressed PD-L1 is favorable for the development and growth of tumors, induces the apoptosis of antitumor T cells, and further protects tumor cells from immune attack. PD-1 / PD-L1 pathway inhibitors can block the binding of PD-1 and PD-L1, block the negative control signal, activate T cells, kill tumor cells, and suppress tumor growth, so immune regulation targeting PD-1 / PD-L1 is of great significance in tumor suppression.

[0199] Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) is a negative regulator expressed on T cells that binds to CD80 or CD86 on antigen-presenting cells, and then blocks the costimulatory signal of CD28, thereby reducing T cell activity and exerting an immunosuppressive effect. CTLA4-mediated suppressive mechanisms are often one of the reasons why tumor cells escape the immune system. Blocking the interaction between CTLA4 and its ligands can restore T cell activity and enhance antitumor capabilities.

[0200] At present, blocking antibodies against PD-1, PD-L1, and CTLA4 have all shown excellent antitumor effects in clinical trials, and several antibody drugs have been approved for launch. Nevertheless, these drugs and treatment methods face many problems in clinical trials, such as low response rates. Therefore, clinical trials of combination administration schemes based on blocking antibodies such as PD-1, PD-L1, and CTLA4 are being actively conducted. Because CTLA4 inhibitors show obvious toxic side effects, in the current combination administration schemes of PD-1 / PD-L1 inhibitors and CTLA4 inhibitors, the CTLA4 inhibitors generally select lower doses. For example, in clinical trials of the combination of anti-PD-1 antibody Nivolumab and anti-CTLA-4 antibody Ipilimumab to treat colorectal cancer and renal cell carcinoma, the doses of Nivolumab and Ipilimumab are 3 mg / kg and 1 mg / kg, respectively. In clinical trials treating non-small cell lung cancer using the combination of anti-PD-L1 antibody durvalumab and anti-CTLA-4 antibody tremelimummab, the doses of durvalumab and tremelimummab are 10-20 mg / kg and 1 mg / kg, respectively.

[0201] Meanwhile, the biological mechanisms of tumor immunity are also increasingly understood. It was found that blocking the CTLA4 signal pathway resulted in high expression of PD-1 on tumor-infiltrating lymphocytes (TILs), and blocking the PD-1 signal pathway enhanced the expression of CTLA4 on TILs (Rev Assoc Med Brass 2017;63(9):814-823). This suggests a mechanism of drug resistance that may be caused by blocking a single co-inhibitory signal pathway, and the combination of these antibodies to simultaneously block multiple co-inhibitory signal pathways may have a synergistic effect on T cell activation. In addition, the latest study reported a new mechanism of interaction between the PD-1 and CTLA4 signal pathways and explained the synergistic effect of PD-L1 and CTLA4 antibodies (Zhao et al., 2019, Immunity 51,1059-1073).

[0202] Based on previous knowledge, it is believed that bispecific antibodies targeting PD-L1 and CTLA4 simultaneously can enhance antitumor efficacy and safety by utilizing more than one mechanism of action. First, PD-L1×CTLA4 bispecific antibodies are activated by blocking the CTLA4 signal pathway and the PD-1 / PD-L1 signal pathway at different stages of T cells; the cis-type interaction of PD-L1 and CD80 can provide better synergy with CTLA4. Second, PD-L1 is highly expressed in tumor tissues, and PD-L1×CTLA4 bispecific antibodies can specifically release the CTLA4 inhibitory signal in the tumor microenvironment to activate T cells and reduce the toxic side effects caused by nonspecific activation of CTLA4 monoclonal antibodies in the peripheral system. Third, PD-L1×CTLA4 bispecific antibodies can selectively retain Fc effector functions (e.g., ADCC), and can specifically kill CTLA4-high expressing Treg cells, such as Treg cells, in the tumor microenvironment by CTLA4, or specifically kill PD-L1-high expressing tumor cells by PD-L1. Furthermore, bispecific antibodies, as a single drug, have advantages over the combination of two drugs in terms of economy and convenience of administration.

[0203] Example 3.2. Acquisition of anti-PD-L1 IgG antibodies and anti-CTLA4 HCAb antibodies Example 3.2.1. Obtaining fully human anti-PD-L1 IgG antibodies Harbour H2L2 mice (Harbour Antibodies BV) are transgenic mice carrying a human immunoglobulin immune library, and the antibodies they produce have fully human antibody variable domains and rat constant domains.

[0204] Harbour H2L2 mice were immunized with soluble recombinant human PD-L1 protein (NovoProtein, #CM06) for multiple rounds. After detecting that the PD-L1-specific antibody titer in the mouse serum reached a certain level, the spleen cells of the mice were taken out and fused with a myeloma cell line to obtain hybridoma cells; after multiple rounds of screening and colonization of the hybridoma cells, several monoclonal antibody molecules that specifically recognize PD-L1 were identified. These monoclonal antibodies were further identified, and several preferred candidate antibody molecules were selected based on parameters such as the binding ability to human PD-L1, the binding ability to cynomolgus monkey PD-L1, and the ability to inhibit the binding of PD-L1 to PD-1. After that, the candidate antibody molecules were sequenced and optimized to obtain several mutant sequences. The VL and VH sequences of the antibodies were fused and expressed with the corresponding human kappa light chain constant region and IgG1 heavy chain constant region sequences to obtain recombinant fully human antibody molecules. The recombinant fully human IgG antibodies against PD-L1 are shown in Tables 3-9.

[0205] Example 3.2.2. Obtaining anti-CTLA4 fully human HCAb antibodies The Harbour HCAb mouse (Harbour Antibodies BV, WO2010 / 109165A2) is a transgenic mouse carrying a human immunoglobulin immune library, capable of producing antibodies consisting only of heavy chains that are half the size of conventional IgG antibodies. The produced antibodies have only human antibody heavy chain variable domains and mouse Fc constant domains.

[0206] Harbor HCAb mice were immunized with soluble recombinant human CTLA4 protein (ACRO Biosystems, #CT4-H5229) for multiple rounds. After detecting that the CTLA4-specific antibody titer in the mouse serum reached a certain level, the mouse spleen cells were removed to separate B cells, and CD138-positive slurry cells were selected with a mouse slurry cell sorting kit (Miltenyi, #130-092-530). Human VH genes were amplified from plasma cells using conventional molecular biology methods, and the amplified human VH gene fragments were constructed into a mammalian cell expression plasmid pCAG vector encoding a human IgG1 antibody heavy chain Fc region sequence. Mammalian host cells (e.g. human embryonic kidney cells HEK293) were transfected and expressed with the plasmid to obtain total human-derived HCAb antibody supernatant. ELISA was used to test the binding of the HCAb antibody supernatant with recombinant human CTLA4 protein, and positive HCAb antibodies were identified. These HCAb antibodies were further identified, and several preferred candidate HCAb antibody molecules were selected based on parameters such as the ability to bind to human CTLA4, the ability to bind to cynomolgus monkey CTLA4, and the ability to inhibit the binding of CTLA4 to B7-1. The candidate HCAb antibody molecules were then sequenced and optimized to obtain several mutant sequences. The VH sequence of the HCAb antibody was fused with the human IgG1 heavy chain Fc sequence to obtain a fully human recombinant HCAb antibody molecule. The anti-CTLA4 recombinant fully human HCAb antibodies are shown in Table 39.

[0207] Example 3.3. Construction of bispecific antibody molecules using anti-PD-L1 IgG antibody and anti-CTLA4 HCAb antibody In this example, the antigen-binding domain Fab of the anti-PD-L1 IgG antibody PR000070 or PR000265 and the antigen-binding domain VH of the anti-CTLA4 HCAb antibody PR000184 were used to construct anti-PD-L1 x CTLA4 bispecific antibody molecules with various structures.

[0208] In this example and the following examples, the anti-PD-L1 positive control molecule is anti-PD-L1 IgG monoclonal antibody PR000070 or PR000416 or PR000265, which is the parent monoclonal antibody at the PD-L1 end of the PD-L1 x CTLA4 bibody molecule. PR000070, PR000265, and PR000416 are derived from the same anti-PD-L1 monoclonal antibody. PR000070 and PR000265 are both human IgG1 subclasses with N297A mutations in their Fc regions, and they only differ by one amino acid mutation in the heavy chain variable region VH. PR000265 and PR000416 are both human IgG1 subtypes with the same Fab structure, and only differ in the presence of N297A mutations in the Fc region of PR000265.

[0209] In this and the following examples, the anti-CTLA4 positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parent monoclonal antibody at the CTLA4 end of the PD-L1 x CTLA4 bibody molecule.

[0210] Example 3.3.1. Construction of Fab-HCAb symmetrical molecule Using anti-PD-L1 IgG antibody and anti-CTLA4 heavy chain antibody, a Fab-HCAb symmetrical PD-L1×CTLA4 bibody molecule was designed according to the structure described in Example 1.1, and the results are summarized in Table 3-1. Then, the antibody molecule samples were prepared and analyzed according to the method described in Example 2, and the results are summarized in Table 3-2.

[0211] [Table 3-1]

[0212] [Table 3-2]

[0213] Example 3.3.2. Construction of IgG-VH tetravalent symmetric molecule Using anti-PD-L1 IgG antibody and anti-CTLA4 heavy chain antibody, a PD-L1×CTLA4 bibody molecule with an IgG-VH tetravalent symmetric structure was designed according to the structure described in Example 1.2, and is summarized in Table 33. Then, antibody molecule samples were prepared and analyzed according to the method described in Example 2, and are summarized in Table 34.

[0214] [Table 3-3]

[0215] [Table 3-4]

[0216] Example 3.3.3. Construction of 2xVH-IgG hexavalent symmetric molecule Using anti-PD-L1 IgG antibody and anti-CTLA4 heavy chain antibody, a 2xVH-IgG hexavalent symmetric PD-L1xCTLA4 bibody molecule was designed according to the structure described in Example 1.4, and is summarized in Table 35. Then, antibody molecule samples were prepared and analyzed according to the methods described in Example 2, and are summarized in Table 36.

[0217] [Table 3-5]

[0218] [Table 3-6]

[0219] Example 3.3.4. Construction of Fab-Fc-VH bivalent asymmetric molecule Using anti-PD-L1 IgG antibody and anti-CTLA4 heavy chain antibody, PD-L1×CTLA4 bivalent asymmetric structured antibody molecules were designed according to the structure described in Example 1.4, and are summarized in Table 37. Then, antibody molecule samples were prepared and analyzed according to the method described in Example 2, and are summarized in Table 38.

[0220] [Table 3-7]

[0221] [Table 3-8]

[0222] Example 3.3.5. Sequence listing of PD-L1xCTLA4 dual antibody molecules and control molecules Tables 3-9, 3-10 and 3-11 provide the SEQ ID NOs corresponding to the sequences of the PD-L1 x CTLA4 bispecific antibody molecules constructed according to this example and the corresponding parent monoclonal antibody molecules and control molecules, such as PD-L1 monoclonal antibodies, CTLA4 monoclonal antibodies, etc. The structure numbers in Table 3-11 correspond to Table 1-1 and Figure 1. Table 3-12 provides the SEQ ID NOs of the CDR sequences corresponding to the first and second antigen-binding domains of the bispecific antibody molecules.

[0223] [Table 3-9]

[0224] [Table 3-10]

[0225] [Table 3-11]

[0226] [Table 3-12]

[0227] Example 3.4. Binding to CTLA4 This example was conducted to investigate the binding activity of PD-L1×CTLA4 dual antibody molecule with CTLA4.

[0228] Example 3.4.1. Binding to recombinant human CTLA4 extracellular domain protein The ability of the antibody molecules to bind to human CTLA4 recombinant protein was tested using enzyme-linked immunosorbent reaction ELISA. Specifically, 2 μg / mL human CTLA4-His protein (ACRO Biosystems, #CT 4-H5229) was first packaged in a 96-well plate at 100 μL / well and left overnight at 4 °C. After that, it was rinsed three times with PBST buffer (PBS buffer containing 0.05% Tween-20), and then a blocking solution (PBS buffer containing 5% nonfat dry milk) was added and incubated at 37 °C for 1 h. Then it was rinsed three times with PBST buffer. Then, antibody molecules diluted in a 5-fold concentration gradient with a maximum final concentration of 30 nM were added at 100 μL / well, with a total of 8 concentrations, mixed uniformly, and incubated at 37 °C for 1 h; hIgG1 iso (CrownBio, #C0001) was used as the same type control. Then it was rinsed three times with PBST buffer. Then, HRP-labeled goat anti-human IgG Fc secondary antibody (Sigma, #A0170, 1:5000 dilution) was added at 100 μL / well and incubated at 37°C for 0.5 hours. Then, rinse three times with PBST buffer. Then, TMB color development solution was added at 100 μL / well and reacted for 15 minutes. Finally, the reaction was stopped by adding a stop solution. The optical absorption value (OD value) was read at 450 nM using an Enspire® multifunction reader (Perkin Elmer, Inc.). Data processing and plot analysis were performed using the application software GraphPad Prism 8, and parameters such as binding curves and EC50 values ​​were obtained by 4-parameter nonlinear fitting.

[0229] In this example, the positive control molecule was the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parent monoclonal antibody at the CTLA4 end of the PD-L1 x CTLA4 bibody molecule.

[0230] As shown in Figure 2 and Tables 3-13, the ability of the IgG-VH tetravalent symmetrical dual antibody molecule to bind CTLA4 is related to the relative position of the VH end of anti-CTLA4 to the IgG of anti-PD-L1. When the VH is at the N-terminus of the heavy chain of IgG (PR000300) or the N-terminus of the light chain (PR000301), the EC50 value of the dual antibody molecule to bind CTLA4 is similar to that of the parent monoclonal antibody PR000184, which is slightly better. When the VH is at the C-terminus of the heavy chain of IgG (PR000303), the EC50 value of the dual antibody molecule to bind CTLA4 is 2.6-fold weaker than that of the parent monoclonal antibody PR000184; when the VH is at the C-terminus of the light chain of IgG (PR000302), the ability of the dual antibody molecule to bind CTLA4 is obviously weakened. This indicates that the ability of VH to bind the target can be adjusted by adjusting the relative position of VH on IgG.

[0231] [Table 3-13]

[0232] Example 3.4.2. Binding to CHO-K1 cells CHO-K1 / hCTLA4 that highly express human CTLA4 or HEK293 cells HEK293 / hCTLA4 that highly express human CTLA4 The ability of the antibody molecules to bind to cells such as the CHO-K1 cell line CHO-K1 / hCTLA4 (KYINNO, KC-1406) that highly expresses human CTLA4 or the HEK293T cell line HEK293 / hCTLA4 (KYINNO, KC-0209) that highly expresses human CTLA4 was tested using flow cytometry FACS. Specifically, CHO-K1 / hCTLA4 and HEK293 / hCTLA4 cells were digested and resuspended in F12K medium and DMEM medium, respectively; the cell density was 1 × 10 6The cells were then seeded at 100 μL / well in a 96-well V-plate (Corning, #3894), centrifuged at 4°C for 5 min, and the supernatant was discarded. Gradient-diluted antibody molecules were then added at 100 μL / well to the 96-well plate, mixed evenly, and diluted to a total of eight concentrations in which the antibody molecules were diluted in a 5-fold gradient from the highest final concentration of 300 nM, or in a total of eight concentrations in which the antibody molecules were diluted in a 4-fold gradient from the highest final concentration of 100 nM; hIgG1 iso (CrownBio, #C0001) was used as the homogenous control. The cells were placed at 4°C and incubated for 1 h in the dark. After that, the cells were rinsed twice by adding pre-cooled FACS buffer (PBS buffer containing 0.5% BSA) at 100 μL / well, centrifuged at 500 g for 5 min at 4°C, and the supernatant was discarded. Next, 100 μL / well of fluorescent secondary antibody (Alexa Fluor 488 anti-human IgGFc, Biolegend, #409322, diluted 1:1000) was added, and the cells were incubated at 4°C for 1 hour in the dark. Then, 200 μL / well of pre-cooled FACS buffer was added to rinse the cells twice, and the cells were then centrifuged at 500 g for 5 minutes at 4°C and the supernatant was discarded. Finally, 200 μL / well of pre-cooled FACS buffer was added to resuspend the cells. Fluorescence emission signal values ​​were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using the software FlowJov 10 (FlowJo, LLC). Data processing and plot analysis were performed using the software GraphPad Prism 8, and parameters such as binding curves and EC50 values ​​of the antibodies to the target cells were obtained by four-parameter nonlinear fitting.

[0233] In this example, the positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parent monoclonal antibody at the CTLA4 end of the PD-L1 x CTLA4 bibody molecule. An ipilimumab analog is also used as a positive control molecule. The results are shown in Figure 3, Figure 4, Tables 3-14 and 3-15.

[0234] As shown in Figure 3(A-B) and Figure 4(A), the ability of the IgG-VH tetravalent symmetrical dual antibody molecules to bind CTLA4 is related to the relative position of the VH end of anti-CTLA4 to the IgG of anti-PD-L1. When the VH is at the N-terminus of the IgG heavy chain (PR000300, PR001573) or the N-terminus of the light chain (PR000301, PR001574, PR001577), the EC50 values ​​of the dual antibody molecules to bind CTLA4 are similar or slightly weaker than that of the parent monoclonal antibody PR000184, but the maximum MFI on FACS is higher than that of the parent monoclonal antibody PR000184 or the positive control Ipilimumab. When the VH was located at the C-terminus of the heavy chain of IgG (PR000303, PR000401, PR000402), the EC50 value of the dual antibody molecule binding to CTLA4 was slightly weaker than that of the parent monoclonal antibody PR000184. When the VH was located at the C-terminus of the light chain of IgG (PR000302), the ability of the dual antibody molecule to bind to CTLA4 was clearly weakened. This indicates that by adjusting the relative position of the VH on the IgG, the ability of the VH to bind the target can be tuned.

[0235] As shown in Figure 3(C), the EC50 value of the 2xVH-IgG hexavalent symmetric structure bibody molecule binding to CTLA4 is similar to that of the parent monoclonal antibody PR000184, and the MFI maximum is lower than that of the parent monoclonal antibody PR000184. This is because the hexavalent symmetric structure of 2xVH-IgG contains four CTLA4-binding sites, which is twice as many as the CTLA4-binding sites of the parent monoclonal antibody PR000184, and fewer bibody molecules are required to bind the same number of CTLA4 molecules than the parent monoclonal antibody, indicating that the MFI value is related to the number of fluorescent secondary antibody molecules bound to the Fc region of the bibody molecule, and further indicating that the MFI maximum of the bibody molecule is lower than that of the parent monoclonal antibody.

[0236] As shown in Figure 3(D), the Fab-Fc-VH asymmetric dual antibody molecules PR001609 and PR001610 contain only one CTLA4-binding domain, and therefore their ability to bind to CTLA4 is weaker than that of the bivalent parent monoclonal antibody PR000184.

[0237] As shown in Figure 3(E) and Figure 4(B), the ability of the Fab-HCAb symmetrical dual antibody molecule to bind to CTLA4 is weaker than that of the bivalent parent monoclonal antibody PR000184, but the MFI maximum is similar to that of the positive control Ipilimumab.

[0238] [Table 3-14]

[0239] [Table 3-15]

[0240] Example 3.5. Blocking the binding of CTLA4 to its ligands This example was conducted to investigate the activity of the PD-L1×CTLA4 dual antibody molecule in inhibiting the binding of CTLA4 to its ligand B7-1 / CD80.

[0241] Example 3.5.1. Blocking the binding of human CTLA4 protein to its ligand protein The activity of the antibody molecules to inhibit the binding of human CTLA4 protein to its ligand B7-1 / CD80 was measured using enzyme-linked immunosorbent reaction ELISA. Specifically, 2 μg / mL protein human B7-1-Fc (ACRO Biosystems, #B71-H5259) was first packaged in a 96-well plate at 100 μL / well and incubated overnight at 4 °C. After that, it was rinsed three times with PBST buffer (PBS buffer containing 0.05% Tween-20), and then the blocking solution (PBS buffer containing 5% skim milk powder) was added and incubated at 37 °C for 1 h. After that, 90 μL / well of the antibody molecules diluted in a 4-fold concentration gradient with a maximum final concentration of 180 nM were added, resulting in a total of seven concentrations, which were mixed uniformly and incubated at 37 °C for 20 min; hIgG1 iso (CrownBio, #C0001) was used as the isotype control. Then, biotinylated human CTLA4-Fc protein (ACRO Biosystems, #CT4-H82F3) was added at 10 μL / well to a final concentration of 0.25 μg / ml and incubated at 37°C for 1 hour. Then rinsed three times with PBST buffer. Then, labeled Precision Protein® StrepTactin-HRP Conjugate (Bio-RAD, #1610380, diluted 1:4000) was added at 100 μL / well and incubated at 37°C for 0.5 hours. Then rinsed three times with PBST buffer. Then, TMB color development solution was added at 100 μL / well and reacted for 15 minutes. Finally, the reaction was stopped by adding a stop solution. The optical absorbance value (OD value) was read at 490 nM with an Enspire® multifunction reader (Perkin Elmer, Inc.). Data processing and graph analysis were performed using the application software GraphPad Prism 8, the OD values ​​were converted into inhibition rates, and parameters such as inhibition curves, IC50 values, and maximum inhibition rates were obtained by four-parameter nonlinear fitting.

[0242] In this example, the positive control molecule was the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parent monoclonal antibody at the CTLA4 end of the PD-L1 x CTLA4 bibody molecule. The results are shown in Figure 5 and Tables 3-16.

[0243] As shown in Figure 5, all of the IgG-VH tetravalent symmetrical double antibody molecules have the ability to block the binding of CTLA4 protein to its ligand protein (inhibitory activity), and all of them can achieve an inhibition rate of nearly 100%, but the IC50 value of the inhibitory activity is related to the relative position of the VH end of anti-CTLA4 to the IgG of anti-PD-L1. When the VH is located at the N-terminus of the heavy chain of IgG (PR000300) or the N-terminus of the light chain (PR000301), the inhibitory activity of the double antibody molecule against CTLA4 is similar to that of the parent monoclonal antibody PR000184. When the VH is located at the C-terminus of the heavy chain of IgG (PR000303), the inhibitory activity of the double antibody molecule against CTLA4 is slightly weaker than that of the parent monoclonal antibody PR000184. When the VH is located at the C-terminus of the light chain of IgG (PR000302), the IC50 value of the double antibody molecule to inhibit the activity is 4 times lower than that of the parent monoclonal antibody PR000184. This indicates that by adjusting the relative position of the VH on the IgG, it is possible to tune the target-blocking ability of the VH.

[0244] [Table 3-16]

[0245] Example 3.5.2. Blocking the binding of human CTLA4 cells to their ligand proteins The activity of the antibody molecules to inhibit the binding of cells expressing CTLA4 to its ligand B7-1 / CD80 was measured using flow cytometry (FACS). Specifically, the CHO-K1 cell line CHO-K1 / hCTLA4 (KYINNO, KC-1406), which highly expresses human CTLA4, was digested and resuspended in F12K medium. The cell density was 1 × 10 6 The cells were adjusted to 10 cells / mL and left in FACS buffer (PBS buffer containing 2% FBS) at 37°C for 15 minutes. 200 μL / well of FACS buffer was added to the 96-well plate to close it, and after incubation at 37°C for 1 hour, the closing solution in the well was discarded. Next, CHO-K1 / hCTLA4 cells were plated in a 96-well plate (2 × 10 5Cells / well) were seeded at 200 μL / well, centrifuged at 500g for 5 min at 4°C, and the supernatant was discarded. Then, the gradient diluted antibody molecules were added to the 96-well plate at 100 μL / well and mixed evenly, and the antibody molecules were diluted to a total of 8 concentrations, diluted in a 3-fold concentration gradient from the highest final concentration of 200 nM, or diluted in a 5-fold concentration gradient from the highest final concentration of 400 nM; hIgG1 iso (CrownBio, #C0001) was used as the homogenous control. Then, 1 μg / mL biotinylated ligand protein human B7-1-Fc (ACRO Biosystems, #B71-H82F2) was added to the 96-well plate at 100 μL / well and mixed evenly. The 96-well plate was placed at 4°C and incubated for 1.5 h in the dark. Then, cells were rinsed twice by adding pre-chilled FACS buffer at 200 μL / well, centrifuged at 500 g for 5 min at 4 °C, and the supernatant was discarded. Next, fluorescent label (Streptavidin, Alexa Fluor® 488 conjugate, Thermo, #S11223, diluted 1:500) was added at 100 μL / well, and incubated at 4 °C for 1 h in the dark. Then, cells were rinsed three times by adding pre-chilled FACS buffer at 200 μL / well, centrifuged at 500 g for 5 min at 4 °C, and the supernatant was discarded. Finally, cells were resuspended by adding pre-chilled FACS buffer at 200 μL / well. Fluorescence signal values ​​were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using the software FlowJov 10 (FlowJo, LLC). Data processing and graph analysis were performed using the software GraphPad Prism 8, the fluorescence signal value MFI was converted into the inhibition rate, and parameters such as the inhibition curve, IC50 value and maximum inhibition rate were obtained by four-parameter nonlinear fitting.

[0246] In this example, the positive control molecule was the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parent monoclonal antibody at the CTLA4 end of the PD-L1 x CTLA4 bibody molecule. The results are shown in Figure 6 and Tables 3-17.

[0247] As shown in Figure 6 (A-B), the inhibitory activity of the IgG-VH tetravalent symmetric dual antibody molecule against CTLA4 is related to the relative position of the VH end of anti-CTLA4 to the IgG of anti-PD-L1. When the VH is at the N-terminus of the heavy chain of IgG (PR000300, PR001573) or the N-terminus of the light chain (PR000301, PR001574, PR001577), the IC50 value of the dual antibody molecule to inhibit the activity is similar to that of the parent monoclonal antibody PR000184, both of which can achieve an inhibition rate of nearly 100%, and PR000301 is slightly stronger than the parent monoclonal antibody PR000184. When the VH is at the C-terminus of the heavy chain of IgG (PR000303), the IC50 value of the dual antibody molecule to inhibit the activity is slightly weaker than that of the parent monoclonal antibody PR000184, with the maximum inhibition rate being about 83%. When the VH is located at the C-terminus of the light chain of IgG (PR000302), the inhibitory activity of the dual antibody molecule is obviously weakened, with its maximum inhibitory rate of only 49%. This indicates that the target blocking ability of VH can be adjusted by adjusting the relative position of VH on IgG. Meanwhile, the different inhibitory activity results of the same dual antibody molecule in the blocking experiment against CTLA4 cells and against CTLA4 protein may reflect the effect of spatial disturbance of the protein on the cell membrane.

[0248] As shown in FIG. 6(C), the inhibitory activity of the 2xVH-IgG hexavalent symmetric dual antibody molecule against CTLA4 is similar to that of the parent monoclonal antibody PR000184.

[0249] As shown in Figure 6(D), the Fab-Fc-VH asymmetric dual antibody molecules PR001609 and PR001610 each contain only one CTLA4-binding domain, and therefore their inhibitory activity against CTLA4 is obviously weaker than that of the bivalent parent monoclonal antibody PR000184.

[0250] [Table 3-17]

[0251] Example 3.6. CTLA4-Mediated ADCC Effect This example is to investigate the antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1×CTLA4 dual antibody molecule against human CTLA4 highly expressing cells 293F-hCTLA4 (CHEMPARTNER). In the first step, the target cells are labeled with DELFIA BATDA (Perkin Elmer, #C136-100). The specific labeling method is as follows: 1×10 6 Target cells were labeled with 2 μL DELFIA BATDA reagent; incubated at 37 °C and CO 2 The cells were incubated in a culture chamber for 20 min; washed four times with PBS and centrifuged at 1000 rpm for 5 min. After the final wash, the pellet was resuspended in complete medium and the cell density was adjusted to 1×105 / ml. In the second step, the labeled target cells were seeded in a 96-well plate (Corning, #3599) at 100 μL / well, and then antibody molecules diluted in a 5-fold concentration gradient with a maximum final concentration of 0.8 nM were added at 50 μL / well, for a total of seven concentrations, and mixed uniformly; hIgG1 iso (CrownBio, #C0001) was used as the same type control, and incubated at 37°C, 5% CO 2 The cells were incubated in a culture box for 10 min. In the third step, NK-92 MI / CD16a cells (CHEMPARTNER) were harvested as effector cells, and the NK-92 MI / CD16a cell density was adjusted to 6 × 10 5 1 ml of 50 μL / well of the cells was then added to a 96-well plate, with effector cells:target cells=3:1. The cells were then incubated at 37° C., 5% CO 2 The plates were incubated in a culture box for 2 hours. In the fourth step, the plates were centrifuged at 500g for 5 minutes, and 25 μL of the supernatant from each well was added to a new 96-well detection plate. Then, 200 μL / well of DELFIA® Europium solution (Perkin Elmer, #C135-100) was added, and the plates were rocked at 250 rpm for 15 minutes at room temperature. Finally, the fluorescence values ​​were measured with an EnVision® 2015 multifunction reader (Perkin Elmer, Inc.).

[0252] The kill ratio is calculated based on the following formula: Kill rate (%)=(ER-ETSR) / (TMR-ETSR)*100% Here, do the following: ER = experimental well (antibody + effector cells + target cells), ETSR = spontaneous release well with mixed target and effector cells (effector cells + target cells), TMR = maximum target cell release well (target cells + lysate) Data processing and graph analysis were performed using the software GraphPad Prism 8, and the killing rate curve of the antibody against the target cells and parameters such as EC50 and maximum killing rate were obtained by four-parameter nonlinear fitting.

[0253] In this example, the positive control molecule was the anti-CTLA4 HCAb monoclonal antibody PR000184 (hIgG1 subclass), which is also the parent monoclonal antibody at the CTLA4 end of the PD-L1 x CTLA4 bibody molecule, and the negative control molecule was the anti-PD-L1 monoclonal antibody PR000416 (hIgG1 subtype).

[0254] As shown in FIG. 11 and Tables 3-18, the dual antibody molecules PR000300 and PR000301 have specific ADCC activity against CTLA4 similar to that of the parent monoclonal antibody PR000184.

[0255] [Table 3-18]

[0256] Example 3.7. Binding to PD-L1 This example was conducted to investigate the PD-L1 binding activity of the PD-L1×CTLA4 dual antibody molecule.

[0257] Example 3.7.1. Binding to recombinant human PD-L1 extracellular domain protein The ability of the antibody molecules to bind to human PD-L1 recombinant protein was tested using enzyme-linked immunosorbent ELISA. Specifically, 2 μg / mL human PD-L1-His protein (ACRO Biosystems, #PD1-H5229) was first packaged in a 96-well plate (Corning, #9018) at 100 μL / well and incubated at 4°C overnight. Then, rinsed three times with PBST buffer (PBS buffer containing 0.05% Tween-20), then added blocking solution containing 2% BSA and incubated at 37°C for 1 hour. Discard the blocking solution and rinse three times with PBST buffer. Then, antibody molecules diluted in a 5-fold concentration gradient with a maximum final concentration of 30 nM were added at 100 μL / well, with eight concentrations, mixed evenly, and incubated at 37°C for 1 hour; hIgG1 iso (CrownBio, #C0001) was used as the same type control. Then rinsed three times with PBST buffer. Then, 100 μL / well of HRP-labeled goat anti-human IgG Fc secondary antibody (Sigma, #A0170, diluted 1:5000) was added and incubated at 37°C for 0.5 hours. Then, rinse three times with PBST buffer. Then, 100 μL / well of TMB color development solution was added and reacted for 15 minutes. Finally, the reaction was stopped by adding a stop solution. The optical absorption value (OD value) was read at 450 nM using an Enspire® multifunction reader (PerkinElmer, Inc.).

[0258] Data processing and graph analysis are performed using the application software GraphPad Prism 8, and parameters such as binding curves and EC50 values ​​are obtained by four-parameter nonlinear fitting.

[0259] In this example, the positive control molecule was the anti-PD-L1 monoclonal antibody PR000070, which is also the parent monoclonal antibody at the PD-L1 end of the PD-L1 x CTLA4 bibody molecule.

[0260] As shown in Figure 7 and Tables 3-19, the ability of the IgG-VH tetravalent symmetric dual antibody molecule to bind PD-L1 is similar to that of the parent monoclonal antibody PR00070, indicating that the VH domain of anti-CTLA4 has no significant effect on the Fab domain of PD-L1.

[0261] [Table 3-19]

[0262] Example 3.7.2. Binding between CHO-K1 cells CHO-K1 / hPDL1 that highly express human PD-L1 and tumor cells MDA-MB-231 that highly express human PD-L1 The antibody molecules were tested for their binding ability to cells, such as the CHO-K1 cell line CHO-K1 / hPDL1 (GENSCRIPT, M00543) that highly expresses human PD-L1 or the tumor cell line MDA-MB-231 (ATCC, HTB-26) that highly expresses human PD-L1, using a flow cytometry cell method FACS. Specifically, cells were digested and resuspended in complete medium; the cell density was 1 × 10 6The cells were then seeded at 100 μL / well in a 96-well V-plate (Corning, #3894), centrifuged at 4°C for 5 min, and the supernatant was discarded. Gradient-diluted antibody molecules were then added at 100 μL / well to the 96-well plate, mixed evenly, and the antibody molecules were diluted to a total of 8 concentrations, either 5-fold gradient from the highest final concentration of 60 nM, or 5-fold gradient from the highest final concentration of 300 nM, or 4-fold gradient from the highest final concentration of 100 nM; hIgG1 iso (CrownBio, #C0001) was used as the homogenous control. The cells were placed at 4°C and incubated for 1 h in the dark. After that, the cells were rinsed twice by adding 100 μL / well of pre-cooled FACS buffer (PBS buffer containing 0.5% BSA), centrifuged at 500 g for 5 min at 4°C, and the supernatant was discarded. Next, 100 μL / well of fluorescent secondary antibody (Goat human lgG(H+L)Alexa Fluor 488 conjunction, Thermo, #A11013, 1:1000 dilution) was added, and the cells were incubated at 4°C for 1 hour in the dark. Then, 200 μL / well of pre-cooled FACS buffer was added to rinse the cells twice, and then the cells were centrifuged at 500g for 5 minutes at 4°C and the supernatant was discarded. Finally, 200 μL / well of pre-cooled FACS buffer was added to resuspend the cells. Fluorescence emission signal values ​​were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using the software FlowJov 10 (FlowJo, LLC). Data processing and plot analysis were performed using the software GraphPad Prism 8, and parameters such as binding curves and EC50 values ​​of antibodies to target cells were obtained by four-parameter nonlinear fitting.

[0263] In this example, the positive control molecule was the anti-PD-L1 monoclonal antibody PR000070 or PR000416 (or PR000265), which is also the parent monoclonal antibody at the PD-L1 end of the PD-L1 x CTLA4 bibody molecule. The results are displayed in Figure 8, Figure 9, Tables 3-20, and Table 3-21.

[0264] As shown in Figure 8(A-B) and Figure 9, the IgG-VH tetravalent symmetric dual antibody molecules have similar PD-L1 binding abilities to the corresponding parental monoclonal antibodies PR00070 or PR000416, with very close EC50 values ​​for PD-L1 binding. The MFI maximum is only slightly lower than the parental monoclonal antibodies when the VH is located at the N-terminus of the IgG light chain (PR000301, PR001574, PR001577).

[0265] As shown in Figure 8(C), the ability of the 2xVH-IgG hexavalent symmetric dual antibody molecule to bind PD-L1 was reduced compared to the parent monoclonal antibody PR000416; this demonstrates that when VH is simultaneously attached to the N-terminus of the IgG heavy and light chains, it has an obvious effect on the function of the Fab end of IgG.

[0266] As shown in Figure 8(D), the binding ability of the Fab-HCAb symmetrical bibody molecule to PD-L1 is similar to that of the parent monoclonal antibody PR000416, with similar EC50 and MFI maximum values, indicating that the structure at the Fab end of the anti-PD-L1 of the Fab-HCAb symmetrical bibody molecule can well retain the binding ability to PD-L1.

[0267] As shown in Figure 8(E), the PD-L1-binding domains of the Fab-Fc-VH asymmetric dual antibody molecules PR001609 and PR001610 have a monovalent Fab structure, but their PD-L1-binding abilities are similar to those of the bivalent parent monoclonal antibodies, and their MFI maximum values ​​are higher than those of the parent monoclonal antibodies.

[0268] [Table 3-20]

[0269] [Table 3-21]

[0270] Example 3.8. Blocking the binding of PD-L1 to its ligands This example was conducted to investigate the activity of the PD-L1×CTLA4 dual antibody molecule in inhibiting the binding of PD-L1 to its ligand PD-1.

[0271] Example 3.8.1. Blocking the binding of human PD-L1 cells to its ligand protein The activity of the antibody molecule to inhibit the binding of PD-L1-expressing cells to their ligand PD-1 was measured using flow cytometry (FACS). Specifically, the CHO-K1 cell line CHO-K1 / hPDL1 (GENSCRIPT, M00543), which highly expresses human PD-L1, was digested and resuspended in F12K medium at a cell density of 2 × 10 6 The cells were adjusted to 1 × 10 cells / mL and placed in FACS buffer (PBS buffer containing 1% BSA) for 15 min at 37 °C. The blocking buffer was added to the 96-well plate at 200 μL / well and incubated at 37 °C for 1 h, after which the blocking solution in the wells was discarded. CHO-K1 / hPDL1 cells were then plated in a 96-well plate (1 × 10 5Cells / well) were seeded at 50μL / well. Then, the gradient diluted antibody molecules were added to the 96-well plate at 100μL / well and mixed evenly. The antibody molecules could be diluted at a total of 8 concentrations diluted in a 5-fold concentration gradient from the highest final concentration of 100nM, and hIgG1 iso (CrownBio, #C0001) was used as the homogenous control. It was left at 4℃ and incubated for 0.5 hours in the dark. Then, PBS buffer was added at 100μL / well, centrifuged at 500g for 5 minutes at 4℃, and the supernatant was discarded. Next, biotinylated ligand protein human PD-1 protein (ACRO Biosystems, #PD 1-H82F2) at a concentration of 1μg / mL was added to the 96-well plate at 50μL / well and mixed evenly. The 96-well plate was placed at 4℃ and incubated for 0.5 hours in the dark. Afterwards, cells were rinsed twice by adding 100 μL / well pre-chilled PBS buffer, followed by centrifugation at 500 g for 5 min at 4 °C, and the supernatant was discarded. Next, fluorescent secondary antibody (PE Streptavidin, BD Biosciences, #554061, 1:200 dilution) was added at 100 μL / well, and incubated at 4 °C, protected from light, for 0.5 h. Then, cells were rinsed twice by adding 200 μL / well pre-chilled PBS buffer, followed by centrifugation at 500 g for 5 min at 4 °C, and the supernatant was discarded. Finally, cells were resuspended by adding 200 μL / well pre-chilled FACS buffer. Fluorescence signal values ​​were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using the software FlowJov 10 (FlowJo, LLC). Data processing and graph analysis were performed using the software GraphPad Prism 8, the fluorescence signal value MFI was converted into the inhibition rate, and parameters such as the inhibition curve, IC50 value and maximum inhibition rate were obtained by four-parameter nonlinear fitting.

[0272] In this example, the positive control molecule was the anti-PD-L1 monoclonal antibody PR000070 or PR000416 (or PR000265), which is also the parent monoclonal antibody at the PD-L1 end of the PD-L1 x CTLA4 bibody molecule. The results are shown in Figure 10 and Tables 3-22.

[0273] As shown in Figure 10 (A-B), the inhibitory ability of the IgG-VH tetravalent symmetric dual antibody molecules against PD-L1 is similar to that of the corresponding parent monoclonal antibodies PR000070 or PR000416, with IC50 values ​​and maximum inhibitory rates close to those of the parent monoclonal antibodies. Only when the VH is located at the N-terminus of the IgG light chain (PR001574, PR001577), the IC50 value is 2-3 times lower than that of the parent monoclonal antibodies.

[0274] As shown in Figure 10(C), the inhibitory activity of the 2xVH-IgG hexavalent symmetric dual antibody molecule against PD-L1 was weaker than that of the parent monoclonal antibody; the corresponding IC50 value was 4-6 times weaker than that of the parent monoclonal antibody, but a similar maximum inhibition rate could be achieved.

[0275] As shown in Figure 10(D), the inhibitory activity of the Fab-HCAb symmetric dual antibody molecule against PD-L1 was similar to that of the parent monoclonal antibody, with an IC50 value and maximum inhibitory rate close to those of the parent monoclonal antibody.

[0276] As shown in Figure 10(E), the PD-L1 binding domains of the Fab-Fc-VH asymmetric dual antibody molecules PR001609 and PR001610 have a monovalent Fab structure, and their inhibitory activity against PD-L1 is weaker than that of the bivalent parent monoclonal antibodies; the corresponding IC50 values ​​are 4-5 times weaker than that of the parent monoclonal antibodies, but they can achieve similar maximum inhibition rates.

[0277] [Table 3-22]

[0278] Example 3.9. Superantigen SEB stimulation experiments This example was conducted to study the activation effect of PD-L1 × CTLA4 bibody molecule on peripheral blood mononuclear cells (PBMC). In the first step, isolated human PBMC (MT-BIO) cells were cultured in 100 μL / well (1 × 10 5 The cells were added at 100μL / well (cells / well) to a 96-well plate (Corning, #3799), then different concentrations of antibody molecules were added at 100μL / well, the final concentration of the antibodies could be (100nM, 10nM, 1nM, 0.1nM, 0.01nM), or two antibodies could be mixed at a specific ratio and added to two combined wells. hIgG1 iso (CrownBio, #C0001) was used as a homogenous control. Incubated at 37℃ for 30 minutes. Then the superantigen Staphylococcal enterotoxin B (SEB) was added at 50μL / well, the final concentration was 100ng / mL or 10ng / mL. Placed at 37℃ and incubated in 5% CO 2 After incubation in the culture box for 96 or 120 hours, the supernatant was collected. In the second step, the supernatant was collected and the IL-2 concentration in the supernatant was detected using an IL-2 ELISA kit (Biolegend, #431805). Refer to the kit's instruction manual for the operation method. Data processing and graph analysis were performed using the application software GraphPad Prism 8.

[0279] In this example, the positive control molecules were the anti-CTLA4 HCAb monoclonal antibody PR000184 and the anti-PD-L1 IgG monoclonal antibody PR000416.

[0280] As shown in Figures 12 to 15, in the presence of SEB stimulation, anti-CTLA4 monoclonal antibody, anti-PD-L1 monoclonal antibody, and PD-L1 x CTLA4 bibody molecule can all promote IL-2 production in T cells to different extents, and their ability to activate T cells is related to their structure.

[0281] As shown in Figure 12, the dual antibody molecule with IgG-VH tetravalent symmetric structure can activate T cells to produce IL-2. Furthermore, the structure of anti-CTLA4 VH at the N-terminus of IgG (PR000301) has stronger T cell activation ability than the structures at the C-terminus of IgG (PR000302, PR000303).

[0282] As shown in Figure 13, the Fab-HCAb symmetric dual antibody molecule can activate T cells to produce IL-2. In addition, PR000404 has stronger T cell activation ability than PR000403. PR000404 and PR000403 have similar structures, with the difference being that PR000404 has a normal human IgG1 Fc region, while the Fc region of PR000403 contains three point mutations (L234A, L235A, P329G) to weaken the Fc-related effector function. It is speculated that PR000404 can eliminate Treg cells through CTLA4-mediated ADCC action and further enhance T cell function.

[0283] As shown in Figure 14, the dual antibody molecules PR000300 and PR000301 have comparable or stronger T cell activation capabilities compared to anti-CTLA4 monoclonal antibody, anti-PD-L1 monoclonal antibody, and their 1:1 concentration combinations (the same donor PBMCs were used in the same experiment, and the absolute values ​​of IL-2 levels can be compared).

[0284] As shown in Figure 15, the ability of the dual antibody molecules PR000303 (IgG_HC-VH structure, i.e., the heavy chain C-terminus of IgG in VH) and PR000404 (Fab-HCAb structure) to activate T is weaker than that of the CTLA4 HCAb monoclonal antibody PR000184. This indicates that the activity of the CTLA4 end is weakened in these two structures compared to conventional IgG or HCAb bivalent structures, thereby reducing the dose-dependent toxicity of CTLA4 and being suitable for the dose of combination drugs in current clinical practice.

[0285] Example 3.10. Mixed Lymphocyte Reaction (MLR) In this example, the mixed lymphocyte reaction (MLR) was used to study the activation effect of PD-L1 × CTLA4 bibody molecule on T cells. In the first step, recombinant human interleukin 4 (IL-4, R&D Systems, #204-GMP) and recombinant human GM-CSF (R&D Systems, #215-GM) were added to the first donor PBMC cells (MT-BIO), and after 6 days of induction, the immature human CD14 + Dendritic cells (iDC cells) were obtained. Then, 1 μg / ml lipopolysaccharide (LPS, Sigma, #L2630) was added and induced for 24 hours to obtain mature dendritic cells (mDC cells). In the second step, T lymphocytes were isolated from the second donor PBMC cells (MT-BIO) using a T cell isolation kit (StemCell, #17951). In the third step, the obtained T cells and mDC cells were seeded in a 96-well plate at a ratio of 10:1 (1 × 10 5 / well with T cells 1 x 10 4 Then, different concentrations of antibody molecules were added at 100μL / well, and the final concentration of the antibody could be (100nM, 10nM, 1nM, 0.1nM, 0nM), or two antibodies could be mixed in a specific ratio and added to two combined wells. hIgG1 iso (CrownBio, #C0001) was used as the same type control. 37℃, 5%CO 2 The cells were incubated in a culture box for 5 days. In the fourth step, the supernatants on the 3rd and 5th days were collected, and the IL-2 concentration in the supernatant on the 3rd day was detected with an IL-2 ELISA kit (Thermo, #88-7025-88), and the IFN-γ concentration in the supernatant on the 5th day was detected with an IFN-γ ELISA kit (Thermo, #88-7316-88).

[0286] As shown in Figure 16, in two independent MLR experiments (different donor pairs), the dual antibody molecules PR000300 and PR000301 (N-terminus of the IgG heavy or light chain of VH) both showed stronger T cell activation ability than anti-CTLA4 monoclonal antibody, anti-PD-L1 monoclonal antibody, and their 1:1 concentration combination, and could better promote the production of IL-2 and IFN-γ.

[0287] As shown in Figure 17, when the VH of anti-CTLA4 was at the C-terminus of the IgG heavy or light chain, the dual antibody molecules PR000302 and PR000303 showed stronger T cell activation ability than the anti-PD-L1 monoclonal antibody atezolizumab in MLR experiments.

[0288] Example 3.11. Antitumor efficacy of dual antibody molecules In this study, to evaluate the in vivo antitumor efficacy of the PD-L1 × CTLA4 antibody, 6-8 week old female B6-PD1 / CTLA4 transgenic mice were subcutaneously inoculated with MC38-hPDL1 cells (MC38 cells overexpressing human PD-L1), and tumors with an average size of approximately 100 mm were observed. 3 When tumor volume reached 1000, the tumor-bearing mice were grouped according to tumor volume, and antibody drugs diluted with PBS at a specific concentration were administered by intraperitoneal injection (ip) at a specific dose and frequency. The administration scheme used in this example was twice weekly, a total of 8 times (BIWx4weeks). The test used PBS as a blank control group, the double antibody administration group was 3mg / kg PR001573, and the combination administration group was 3mg / kg ipilimumab plus 3mg / kg atezolizumab. Tumor volume and mouse body weight were measured on days 6, 9, 12, 15, 19, 22, 26, 29, and 33 after tumor inoculation.

[0289] Tumor volume calculation formula: Tumor volume (mm 3 ) = 0.5 × (tumor long diameter × tumor short diameter 2 ).

[0290] As shown in Figure 67, on day 33 after tumor inoculation, the tumor volume in the control group was approximately 700 mm 3The tumors in the combination group (3mg / kg ipilimummab + 3mg / kg atezolizumab) disappeared completely, and the tumor inhibition rate (TGI) was 100%, with a significant difference of P=0.03 compared to the control group. The tumors in the dual antibody group (3mg / kg PR001573) disappeared completely, and the TGI was 100%, with a significant difference of P=0.03 compared to the control group. The mice in the treatment group showed good tolerance throughout the entire experimental process, and there was no obvious change in body weight. It was explained that the efficacy of 3mg / kg PR001573 and the efficacy of the combination of ipilimummab and atezolizumab at the same dose were equivalent.

[0291] Example 3.12. Summary In this example, the antigen-binding domain Fab of anti-PD-L1 IgG antibody and the antigen-binding domain VH of anti-CTLA4 HCAb antibody were used to construct anti-PD-L1 × CTLA4 bispecific antibody molecules with various structures, demonstrating the flexibility of constructing bispecific antibody molecular structures based on HCAb, and adjusting the functional activity of the PD-L1 end and the CTLA4 end through parameters such as different structural types, relative positions, and valency, and further designing different activity combinations to meet the needs of different clinical combination doses.

[0292] For example, when the VH of anti-CTLA4 is at the N-terminus of anti-PD-L1 IgG, the PD-L1 and CTLA4 ends of the dual antibody molecules PR000300 and PR000301 can almost completely maintain the activity of their parent monoclonal antibodies, and in in vitro functional experiments such as lymphocyte reaction and superantigen stimulation experiments, they showed comparable or stronger T cell activation ability compared to the parent monoclonal antibody 1:1 concentration combination; therefore, it can be used to achieve 1:1 dose combination of combination drugs. Mouse tumor efficacy models also confirmed that the dual antibody PR001573 with this structure has the same efficacy as the combination drug.

[0293] In addition, for example, when the VH of anti-CTLA4 is at the C-terminus of anti-PD-L1 IgG or in a Fab-HCAb structure, the PD-L1 ends of the dual antibody molecules PR000302, PR000303, and PR000404 almost completely retain the activity of their parent monoclonal antibodies, while the activity of their CTLA4 ends is weakened to different degrees, and therefore can be used to achieve clinical combination doses of 3:1 to 10:1.

[0294] Example 4. HER2xCTLA4 Bispecific Antibody Example 4.1. Background Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) is a negative regulator expressed on T cells. After binding to CD80 or CD86 on antigen-presenting cells, it blocks the costimulatory signal of CD28, while at the same time reducing the activity of T cells and exerting an immunosuppressive effect. CTLA4-mediated inhibitory mechanism is often one of the reasons why tumor cells escape from the immune system. Blocking the interaction between CTLA4 and its ligand can restore the activity of T cells and enhance their antitumor ability. Ipilimumab monoclonal antibody (trade name Yervoy®) is the first anti-CTLA4 monoclonal antibody drug to be marketed, and is also the first product to open the era of tumor immunotherapy. Ipilimumab has shown a relatively good therapeutic effect in the treatment of late-stage melanoma, but it causes relatively high immune-related side effects, and the incidence rate of its related grade 3-5 side effects reaches 50%, which has seriously affected its clinical application. Most of the toxic side effects exhibited by ipilimumab are related to the CTLA4 target, and in the current combination dosing scheme of PD-1 / PD-L1 inhibitors and CTLA4 inhibitors, the CTLA4 inhibitor is usually chosen at a lower dose, regardless of whether it is ipilimumab or tremelimummab.

[0295] In order to reduce the toxic side effects of CTLA4 inhibitors, one of the methods that should be attempted is to directional deliver CTLA4 inhibitors into tumor tissues, limit the associated T cell-mediated reaction to the tumor microenvironment, and reduce the risk of cytokine release syndrome. This directional delivery can be achieved by injecting CTLA4 inhibitors into tumors, but the method of intratumoral injection has the risk of surgical manipulation and is limited to some shallow tumor tissues that can be manipulated. This embodiment provides a directional delivery method that uses antibodies that identify tumor-associated antigens to redirect CTLA4 inhibitors to a specific tumor microenvironment, release the immunosuppressive signal of T cells in the tumor microenvironment, and restore the function of T cells.

[0296] In this embodiment, a bispecific antibody that simultaneously targets HER2 and CTLA4 was constructed, and the antitumor effect and safety were improved by one or more mechanisms of action. First, the HER2×CTLA4 bispecific antibody maintains the mechanism of action of conventional HER2 inhibitors (blocking HER2 dimerization, promoting HER2 internalization and degradation, and suppressing downstream phosphorylation signals), and activates T cells by blocking the CTLA4 signal pathway. Second, the HER2×CTLA4 bispecific antibody is enriched in tumor tissues with high HER2 expression, specifically releases the CTLA4 inhibitory signal in the tumor microenvironment, activates T cells, and reduces the toxic side effects caused by non-specific activation in the peripheral system of CTLA4 monoclonal antibodies. Third, the HER2×CTLA4 bispecific antibody can selectively maintain Fc effector functions (e.g., ADCC), and specifically kills inhibitory T cells that highly express CTLA4, such as Treg cells, by CTLA4 in the tumor microenvironment, or specifically kills tumor cells that highly express HER2 by HER2. Furthermore, as a single pharmaceutical agent, a bispecific antibody can have advantages over a combination of two pharmaceutical agents in terms of cost efficiency and ease of administration.

[0297] Example 4.2. Acquisition of anti-HER2 IgG antibodies and anti-CTLA4 HCAb antibodies This example uses the anti-HER2 IgG antibodies trastuzumab and pertuzumab, whose corresponding amino acid sequences are derived from the IMGT database and are shown in Tables 4-11.

[0298] The anti-CTLA4 fully human HCAb antibody PR000184 (Table 4-11) used in this example was derived from Harbour HCAb mice as described in Example 3.2.2.

[0299] Example 4.3. Construction of a bispecific antibody molecule using an anti-HER2 IgG antibody and an anti-CTLA4 HCAb antibody In this example, anti-HER2×CTLA4 bispecific antibody molecules with diverse structures are constructed using the antigen-binding domain Fab of the anti-HER2 IgG antibody PR000210 (trastuzumamab analog) or PR000672 (pertuzumamab analog) and the antigen-binding domain VH of the anti-CTLA4 HCAb antibody PR000184.

[0300] In this and the following examples, the positive control molecules are the anti-HER2 IgG monoclonal antibodies PR000210 (a trastuzumamab analog) or PR000672 (a pertuzumamab analog), and the parent monoclonal antibody at the HER2 end of the HER2xCTLA4 biantibody molecule.

[0301] In this and the following examples, the positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parent monoclonal antibody for the CTLA4 end of the HER2xCTLA4 bibody molecule.

[0302] Example 4.3.1. Construction of Fab-HCAb symmetric molecules Using anti-HER2 IgG antibody and anti-CTLA4 heavy chain antibody, Fab-HCAb symmetrical HER2×CTLA4 bibody molecule was designed according to the structure described in Example 1.1, and summarized in Table 4-1. Then, antibody molecule samples were prepared and analyzed according to the method described in Example 2, and summarized in Table 4-2.

[0303] [Table 4-1]

[0304] [Table 4-2]

[0305] Example 4.3.2. Construction of IgG-VH tetravalent symmetric structure molecule Using anti-HER2 IgG antibody and anti-CTLA4 heavy chain antibody, HER2×CTLA4 bibody molecules with IgG-VH tetravalent symmetric structure were designed according to the structure described in Example 1.2, and are summarized in Table 4-3. Then, antibody molecule samples were prepared and analyzed according to the method described in Example 2, and are summarized in Table 44.

[0306] [Table 4-3]

[0307] [Table 4-4]

[0308] Example 4.3.3. Construction of IgG-VH(2) Hexavalent Symmetrical Molecules Using anti-HER2 IgG antibody and anti-CTLA4 heavy chain antibody, HER2xCTLA4 bibody molecules with IgG-VH(2) hexavalent symmetric structure were designed according to the structure described in Example 1.3, and are summarized in Tables 4-5. Then, antibody molecule samples were prepared and analyzed according to the method described in Example 2, and are summarized in Tables 4-6.

[0309] [Table 4-5]

[0310] [Table 4-6]

[0311] Example 4.3.4. Construction of 2xVH-IgG hexavalent symmetric molecules Using an anti-HER2 IgG antibody and an anti-CTLA4 heavy chain antibody, a 2xVH-IgG hexavalent symmetric HER2xCTLA4 bibody molecule was designed according to the structure described in Example 1.4, and the results are summarized in Table 47. Then, antibody molecule samples were prepared and analyzed according to the method described in Example 2, and the results are summarized in Table 48.

[0312] [Table 4-7]

[0313] [Table 4-8]

[0314] Example 4.3.5. Construction of Fab-Fc-VH(n) bivalent or trivalent asymmetric molecules Using anti-HER2 IgG antibody and anti-CTLA4 heavy chain antibody, HER2×CTLA4 bibody molecules with asymmetric structure of Fab-Fc-VH(n,n={1,2}) were designed according to the structures described in Examples 1.5 and 1.6, and are summarized in Table 49. Then, antibody molecule samples were prepared and analyzed according to the method described in Example 2, and are summarized in Tables 4-10.

[0315] [Table 4-9]

[0316] [Table 4-10]

[0317] Example 4.3.6. Sequence listing of HER2xCTLA4 dual antibody molecules and control molecules Tables 4-11, 4-12 and 4-13 show the SEQ ID NOs corresponding to the sequences of the HER2xCTLA4 bispecific antibody molecules constructed according to this example and the parent monoclonal antibody molecules and control molecules such as the corresponding HER2 monoclonal antibody, CTLA4 monoclonal antibody, etc. The structure numbers in Table 4-13 correspond to Table 1-1 and Figure 1. Table 4-14 shows the SEQ ID NOs of the CDR sequences corresponding to the first and second antigen binding domains of the bispecific antibody molecules.

[0318] [Table 4-11]

[0319] [Table 4-12]

[0320] [Table 4-13] TIFF2025032073000041.tif80128

[0321] [Table 4-14]

[0322] Example 4.4. Binding to CTLA4 This example was conducted to study the binding activity of the HER2xCTLA4 dual antibody molecule to CTLA4.

[0323] Example 4.4.1. Binding to CHO-K1 cells CHO-K1 / hCTLA4 that highly express human CTLA4 The antibody molecules were tested for their ability to bind to cells, such as the CHO-K1 cell line CHO-K1 / hCTLA4 (CHEMPARTNER), which highly expresses human CTLA4, using flow cytometry (FACS). Specifically, CHO-K1 / hCTLA4 cells were digested and resuspended using F12K medium; the cell density was 2 × 10 6The concentration of antibody molecules was adjusted to 100 cells / mL. Then, CHO-K1 / hCTLA4 cells were seeded in a 96-well V-plate (Corning, #3894) at 100 μL / well, centrifuged at 4 °C for 5 min, and the supernatant was discarded. Then, the gradient-diluted antibody molecules were added to the 96-well plate at 100 μL / well, mixed evenly, and the antibody molecules were diluted to a total of 8 concentrations diluted in a 5-fold concentration gradient from the highest final concentration of 300 nM, or to a total of 12 concentrations diluted in a 4-fold concentration gradient from the highest final concentration of 300 nM; hIgG1 iso (CrownBio, #C0001) was used as the homogenous control. The cells were placed at 4 °C and incubated for 1 h in the dark. After that, the cells were rinsed twice by adding pre-cooled FACS buffer (PBS buffer containing 0.5% BSA) at 100 μL / well, centrifuged at 500 g for 5 min at 4 °C, and the supernatant was discarded. Next, 100 μL / well of fluorescent secondary antibody (Goat human lgG(H+L)Alexa Fluor 488 conjunction, Thermo, #A11013, 1:1000 dilution) was added, and the cells were incubated at 4°C for 1 hour in the dark. Then, 200 μL / well of pre-cooled FACS buffer was added to rinse the cells twice, and then the cells were centrifuged at 500g for 5 minutes at 4°C and the supernatant was discarded. Finally, 200 μL / well of pre-cooled FACS buffer was added to resuspend the cells. Fluorescence emission signal values ​​were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using the software FlowJov 10 (FlowJo, LLC). Data processing and plot analysis were performed using the software GraphPad Prism 8, and parameters such as binding curves and EC50 values ​​of antibodies to target cells were obtained by four-parameter nonlinear fitting.

[0324] In this example, the positive control molecule was the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parent monoclonal antibody for the CTLA4 end of the HER2xCTLA4 bibody molecule. The results are shown in Figure 18 and Tables 4-15.

[0325] As shown in FIG. 18(A), both of the Fab-HCAb symmetric double antibody molecules can bind to CTLA4. These molecules have a similar structure, both of which combine the Fab domain of anti-HER2 monoclonal antibody PR000210 with the VH domain of anti-CTLA4 HCAb monoclonal antibody PR000184, with the subtle differences being different first linking peptides and hinge regions of linked Fc, and different Fc types or mutations. Thus, these double antibody molecules have similar abilities to bind CTLA4. Their EC50 values ​​for binding to CTLA4 are similar to or only 1.5-3 times weaker than the parent monoclonal antibody PR000184, but the maximum binding signal (MFI max) on FACS is lower than that of the parent monoclonal antibody PR000184. This may suggest that in the Fab-HCAb structure, the Fab domain may have a "shielding" effect on the VH domain of the HCAb, making the VH domain in this structure bind fewer available CTLA4 molecules than at its free end, and for the same number of available CTLA4 molecules, its binding efficiency is comparable to that of the parent monoclonal antibody PR000184 (EC50 value). As shown in Figure 18 (B-D), the ability of IgG-VH tetravalent symmetrical bibody molecules to bind CTLA4 varies slightly depending on their specific molecular structure. Based on the relative position of the VH terminus of anti-CTLA4 to anti-HER2 IgG in the bibody structure, bibody molecules with such structure are further classified into three types: VH is at the C-terminus of the IgG heavy chain (PR000539, PR000540, PR000714, PR000715); VH is at the N-terminus of the IgG heavy chain (PR001583, PR001586, PR001588, PR001591); VH is at the N-terminus of the IgG light chain (PR001584, PR001589). When the VH is located at the C-terminus of the heavy chain of IgG, the EC50 values ​​of these bibody molecules binding to CTLA4 are similar to those of the parent monoclonal antibody PR000184, but the maximum MFI on FACS is slightly lower than that of the parent monoclonal antibody PR000184. When the VH is located at the N-terminus of either the heavy or light chain of IgG, the EC50 values ​​of these bibody molecules binding to CTLA4 are slightly weaker than that of the parent monoclonal antibody PR000184, but the maximum MFI on FACS is stronger than that of the parent monoclonal antibody PR000184. This indicates that the number of VHs binding to target molecules and the binding effect can be adjusted by adjusting the relative position of the VH on the IgG.

[0326] As shown in Figure 18 (E-F), all of the IgG-VH-VH hexavalent symmetric dual antibody molecules can bind to CTLA4. These molecules have a similar structure, and are all assembled from the VH domains of anti-HER2 monoclonal antibody PR000210 and anti-CTLA4 HCAb monoclonal antibody PR000184, with subtle differences being different first and second connecting peptides, and different Fc types or mutations. The ability of these dual antibody molecules to bind to CTLA4 is similar to that of the parent monoclonal antibody PR000184, except for PR001582 (with similar EC50 and similar MFI maximum).

[0327] As shown in Figure 18 (G-H), all of the 2xVH-IgG hexavalent symmetrical bibody molecules can bind to CTLA4. These molecules have similar structures, combining the VH domains of anti-HER2 monoclonal antibodies PR000210 or PR000672 with anti-CTLA4 HCAb monoclonal antibody PR000184. The bibody molecules (PR001585, PR001587) constructed based on the parent monoclonal antibody PR000210 have similar binding ability to CTLA4 as the parent monoclonal antibody PR000184; the bibody molecules (PR001590, PR001592) constructed based on the parent monoclonal antibody PR000672 have slightly weaker binding ability to CTLA4 than the parent monoclonal antibody PR000184.

[0328] As shown in Figure 18 (I-J), the ability of the Fab-Fc-VH (n,n=1,2) asymmetrically structured bibody molecule to bind CTLA4 is related to the number of VH domains of the parent monoclonal antibody PR000184 included in the structure. Both bibody molecules PR000916 and PR000917 contain only one CTLA4-binding domain, and therefore their ability to bind CTLA4 is obviously weaker than the bivalent parent monoclonal antibody PR000184. The bibody molecule PR002672 contains two tandemly formed CTLA4-binding domains, and its ability to bind CTLA4 is similar to that of the parent monoclonal antibody PR000184. This structure indicates that the VH domain-containing bibody molecule containing the source HCAb has the flexibility to adjust the target-binding ability by adjusting the number of tandemly connected VH domains.

[0329] [Table 4-15]

[0330] Example 4.5. Blocking the binding of CTLA4 to its ligands The present example was conducted to study the activity of the HER2×CTLA4 dual antibody molecule in inhibiting the binding of CTLA4 to its ligand B7-1 / CD80.

[0331] Example 4.5.1. Blocking the binding of human CTLA4 protein to its ligand protein The inhibitory activity of antibody molecules to the binding of human CTLA4 protein and its ligand B7-1 / CD80 protein was measured using enzyme-linked immunosorbent reaction ELISA. Specifically, 2 μg / mL protein human B7-1-Fc (ACRO Biosystems, #B71-H5259) was first packaged in a 96-well plate at 100 μL / well and incubated overnight at 4 °C. After that, it was rinsed three times with PBST buffer (PBS buffer containing 0.05% Tween-20), and then the blocking solution (PBS buffer containing 5% skim milk powder) was added and incubated at 37 °C for 1 hour. After that, 90 μL / well of antibody molecules diluted in a 3-fold concentration gradient with the highest final concentration of 200 nM were added, with 7 concentrations, mixed uniformly, and incubated at 37 °C for 20 minutes; hIgG1 iso (CrownBio, #C0001) was used as the same type control. Then, biotinylated human CTLA4-Fc protein (ACRO Biosystems, #CT4-H82F3) was added at 10 μL / well to a final concentration of 0.25 μg / ml and incubated at 37°C for 1 hour. Then rinsed three times with PBST buffer. Then, labeled Precision Protein® StrepTactin-HRP Conjugate (Bio-RAD, #1610380, diluted 1:4000) was added at 100 μL / well and incubated at 37°C for 0.5 hours. Then rinsed three times with PBST buffer. Then, TMB color development solution was added at 100 μL / well and reacted for 15 minutes. Finally, the reaction was stopped by adding a stop solution. The optical absorbance value (OD value) was read at 490 nM with an Enspire® multifunction reader (Perkin Elmer, Inc.). Data processing and graph analysis were performed using the application software GraphPad Prism 8, and inhibition curves and parameters such as IC50 values ​​were obtained by four-parameter nonlinear fitting.

[0332] In this example, the positive control molecule was the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parent monoclonal antibody for the CTLA4 end of the HER2xCTLA4 bibody molecule. The results are shown in Figure 19 and Tables 4-16.

[0333] As shown in Figure 19(A), all of the Fab-HCAb symmetric double antibody molecules had the ability to block the binding of CTLA4 protein to its ligand protein (inhibitory activity), and the inhibitory activity was comparable to or slightly weaker than that of the parent monoclonal antibody PR000184, showing a similar maximum OD difference, but the IC50 value was only 1.5 to 2.1 times lower.

[0334] As shown in Figure 19 (B-D), the inhibitory activity of the IgG-VH tetravalent symmetric dual antibody molecules varies slightly depending on their specific molecular structures. Based on the relative positions of the VH termini of anti-CTLA4 to anti-HER2 IgG in the dual antibody structure, the dual antibody molecules of such structures are further classified into three types: VH is at the C-terminus of the heavy chain of IgG (PR000539, PR000540, PR000715); VH is at the N-terminus of the heavy chain of IgG (PR001583, PR001586, PR001588, PR001591); VH is at the N-terminus of the light chain of IgG (PR001584, PR001589). When VH is at the C-terminus of the heavy chain of IgG, the inhibitory activity of these dual antibody molecules is somewhat weaker than that of the parent monoclonal antibody, showing similar maximum OD differences, but IC50 values ​​are only 1.5-2.5 times weaker. When the VH is at the N-terminus of the IgG heavy or light chain, the inhibitory activity of these bibody molecules is similar to that of the parent monoclonal antibodies.

[0335] As shown in FIG. 19 (E-F), the inhibitory activity of the IgG-VH-VH hexavalent symmetric bibody molecule is similar or slightly better than that of the parent monoclonal antibody.

[0336] As shown in FIG. 19 (GH), the inhibitory activity of the 2×VH-IgG hexavalent symmetric dual antibody molecule is similar or slightly superior to that of the parent monoclonal antibody.

[0337] [Table 4-16]

[0338] Example 4.5.2. Blocking the binding of human CTLA4 cells to their ligand proteins The activity of the antibody molecule to inhibit the binding of cells expressing CTLA4 to its ligand B7-1 / CD80 was measured using flow cytometry (FACS). Specifically, the CHO-K1 cell line CHO-K1 / hCTLA4 (CHEMPARTNER), which highly expresses human CTLA4, was digested and resuspended in FACS buffer (PBS buffer containing 2% FBS) at a cell density of 3 × 10 6 CHO-K1 / hCTLA4 cells were cultured in a 96-well plate (3 × 10 5Cells / well) were seeded at 100 μL / well, centrifuged at 500g for 5 min at 4 °C, and the supernatant was discarded. Then, gradient-diluted antibody molecules were added to a 96-well plate at 100 μL / well and mixed evenly, antibody molecules could be diluted in a total of 8 concentrations diluted in a 3-fold concentration gradient from the highest final concentration of 200 nM, and hIgG1 iso (CrownBio, #C0001) was used as an isotype control. Then, 3 μg / mL biotinylated ligand protein human B7-1-Fc-biotin (ACRO Biosystems, #B71-H82F2) was added to the 96-well plate at 100 μL / well and mixed evenly. The 96-well plate was placed at 4 °C and incubated for 1 h in the dark. After that, cells were rinsed twice by adding pre-cooled FACS buffer at 200 μL / well, centrifuged at 500g for 5 min at 4 °C, and the supernatant was discarded. Next, 100 μL / well of fluorescent label (Streptavidin, Alexa Fluor® 488 conjugate, Thermo, #S32354, diluted 1:1000) was added, and the cells were left at 4°C and incubated for 1 hour in the dark. Then, 200 μL / well of pre-cooled FACS buffer was added to rinse the cells three times, and the cells were centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded. Finally, 200 μL / well of pre-cooled FACS buffer was added to resuspend the cells. Fluorescence emission signal values ​​were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using the software FlowJov 10 (FlowJo, LLC). Data processing and plot analysis were performed using the software GraphPad Prism 8, and the fluorescence signal value MFI was converted to inhibition rate, and parameters such as inhibition curve, IC50 value and maximum inhibition rate were obtained by four-parameter nonlinear fitting.

[0339] In this example, the positive control molecule was the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parent monoclonal antibody for the CTLA4 end of the HER2xCTLA4 bibody molecule. The results are shown in Figure 20 and Table 4-17.

[0340] As shown in Figure 20 (A-B), when the VH of anti-CTLA4 is located at the C-terminus of the heavy chain of anti-HER2 IgG, the inhibitory activity of the IgG-VH tetravalent symmetric bibody molecule is significantly reduced, whereas the inhibitory activity of the IgG-VH-VH hexavalent symmetric bibody molecule (PR000541) is similar to that of the parent monoclonal antibody.

[0341] As shown in Figure 20(C), the Fab-Fc-VH(2) asymmetric double antibody molecule PR002672 contains two CTLA4-binding domains arranged in tandem, and its inhibitory activity can reach approximately 100% inhibition, although the IC50 value is approximately four times lower than that of the bivalent parent monoclonal antibody.

[0342] [Table 4-17]

[0343] Example 4.6. Binding to HER2 This example was conducted to study the binding activity of the HER2xCTLA4 dual antibody molecule to HER2.

[0344] Example 4.6.1. Binding to SK-BR-3 cells highly expressing human HER2 The binding ability of the antibodies to the tumor cell line SK-BR-3 (ATCC, HTB-30), which highly expresses human HER2, was tested using flow cytometry (FACS). Specifically, SK-BR-3 cells were digested and resuspended in DMEM complete medium at a cell density of 1 × 10 6cells / mL; then, 100μL cells / well were seeded into a 96-well V-plate (Corning, #3894), centrifuged at 4℃ for 5 min, and the supernatant was discarded. Then, 100μL / well of antibody molecules diluted in a 5-fold concentration gradient with a maximum final concentration of 100nM were added, and the eight concentrations were mixed uniformly; hIgG1 iso (CrownBio, #C0001) was used as the homogenous control. The cells were placed at 4℃ and incubated for 1 hour in the dark; then, centrifuged at 4℃ for 5 min, and the supernatant was discarded; then, 200μL / well of pre-cooled FACS buffer (PBS buffer containing 0.5% BSA) was added to rinse the cells twice, and then centrifuged at 500g for 5 min at 4℃, and the supernatant was discarded. Then, fluorescent secondary antibody (Goat human lgG (H + L) Alexa Fluor 488 conjunction, Thermo, #A11013, 1:1000 dilution) was added at 100 μL / well, left at 4 ° C, and incubated for 1 hour in the dark. Then, pre-cooled FACS buffer was added at 200 μL / well to rinse the cells twice, and then centrifuged at 500 g for 5 minutes at 4 ° C, and the supernatant was discarded. Finally, pre-cooled FACS buffer was added at 200 μL / well to resuspend the cells. Fluorescence emission signal values ​​were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using the software FlowJov 10 (FlowJo, LLC). Data processing and plot analysis were performed using the software GraphPad Prism 8, and parameters such as binding curves and EC50 values ​​of antibodies to target cells were obtained by four-parameter nonlinear fitting.

[0345] In this example, the positive control molecules were anti-HER2 monoclonal antibodies PR000210 (trastuzumab analog) or PR000672 (pertuzumab analog), which are also the parent monoclonal antibodies of the HER2xCTLA4 bibody molecule. The results are shown in Figure 21 and Table 4-18.

[0346] As shown in Figure 21(A), the binding ability of the Fab-HCAb symmetric dual antibody molecule to HER2 is similar to that of the parent monoclonal antibody PR000210, as shown by the similar EC50 value and MFI maximum value, indicating that the structure at the Fab end of the anti-HER2 dual antibody molecule with the Fab-HCAb symmetric structure can well retain the binding ability to HER2.

[0347] As shown in Figure 21 (B-E), the ability of IgG-VH tetravalent symmetrical bibody molecules to bind HER2 varies slightly depending on their specific molecular structure. Based on the relative position of the VH terminus of anti-CTLA4 to anti-HER2 IgG in the bibody structure, bibody molecules with such structure are further classified into three types: VH is at the C-terminus of the IgG heavy chain (PR000539, PR000540); VH is at the N-terminus of the IgG heavy chain (PR001583, PR0...

Claims

1. A binding protein comprising at least two protein functional regions, The binding protein comprises a protein functional region A and a protein functional region B; the protein functional region A and the protein functional region B target different antigens or antigen epitopes, wherein the protein functional region A is a Fab or scFv structure, the protein functional region B is a VH structure, and the number of the protein functional region A and the protein functional region B is one or more; preferably, the binding protein has a symmetric structure or an asymmetric structure, and / or the binding protein further comprises an Fc, the number of the Fc is two, and the Fc dimer formed thereby is preferably a homodimer or a heterodimer.

2. 2. The binding protein of claim 1 , (I) the binding protein has a symmetric structure, and the symmetric structure is a bilaterally symmetric IgG-like structure, in which the number of protein functional regions A is two and the number of protein functional regions B is two or four; Preferably, when the number of the protein functional region B is two, the binding protein is a tetravalent binding protein, and includes the following structure: (a) the binding protein is, from N-terminus to C-terminus, a protein functional region A, a protein functional region B and Fc, in which the protein functional region A and the protein functional region B are connected by a first connecting peptide (L1), and the protein functional region B and the Fc are connected by a second connecting peptide (L2); or (b) the binding protein is, from N-terminus to C-terminus, a protein functional region B, a protein functional region A and Fc, in which the protein functional region B and the protein functional region A are connected by a connecting peptide, and the protein functional region A and the Fc are connected by a hinge region; or (c) the binding protein is, from N-terminus to C-terminus, a protein functional region A, a protein functional region B and a protein functional region B, in which the protein functional region A and the Fc are connected by a hinge region, and the Fc and the protein functional region B are connected by a connecting peptide; When the number of said protein functional domains B is four, said binding protein is a hexavalent binding protein and comprises the following structure: two additional protein functional domains B are further connected to the N-terminus or C-terminus of the binding protein described in (a) or (b) or (c); preferably connected to the C-terminus of the Fc of the binding protein described in (c) or the C-terminus of the original protein functional domain B, or connected to the N-terminus of protein functional domain A together with the original protein functional domain B of the binding protein described in (b); Preferably, the binding protein further comprises a protein functional region C, wherein the protein functional region C, the protein functional region A and the protein functional region B target different antigens or antigen epitopes, and the binding protein is a hexavalent or octavalent trispecific binding protein, comprising the following structure: the protein functional region C is connected to the N-terminus or C-terminus of the binding protein; preferably, the number of the protein functional regions C is two, and the protein functional region C is connected to the C-terminus of the binding protein described in (c) or to the N-terminus of the protein functional region A, which is the same as the protein functional region B of the binding protein described in (b); or (II) the binding protein has an asymmetric structure, and the asymmetric structure exhibits an asymmetric IgG-like structure, in which the left arm is a protein functional region A having a Fab or scFv structure, and the right arm is a protein functional region B having a VH structure, and the protein functional region A and the protein functional region B are each connected to one Fc; preferably, the number of the protein functional region A is one, and the number of the protein functional region B is one, two, or three; Preferably, when the number of the protein functional region B is one, the binding protein is a bivalent binding protein, comprising the following structure: the protein functional region A is (d) a conventional Fab structure, or (e) a Fab (cross VH / VL) structure, or (f) a Fab (cross Fd / LC) structure; when the number of the protein functional region B is two, the binding protein is a trivalent binding protein, comprising the following structure: the protein functional region A of the left arm is (d), (e) or (f), and a second protein functional region B is connected to the N-terminus or C-terminus of the protein functional region A of the left arm, or to the N-terminus of the first protein functional region B of the right arm, or to the C-terminus of the Fc, preferably the second protein functional region B is connected to the N-terminus of the first protein functional region B of the right arm; when the number of the protein functional region B is three, the binding protein is a tetravalent binding protein. and specifically includes the following structure: the protein functional region A of the left arm is structure (d), (e), or (f), and a third protein functional region B is further connected to the N-terminus or C-terminus of the protein functional region A of the left arm, or to the N-terminus or C-terminus of the second protein functional region B of the left arm, or to the N-terminus of the first or second protein functional region B of the right arm, or to the C-terminus of the Fc, preferably the second protein functional region B is connected to the N-terminus of the first protein functional region B of the right arm, and the third protein functional region B is connected to the N-terminus of the second protein functional region B; or When the number of the protein functional region B is one, the binding protein is a bivalent binding protein, specifically comprising the following structure: the protein functional region A of the left arm is (g) scFv structure, and the scFv is connected to Fc by the VH end or the VL end; when the number of the protein functional region B is two, the binding protein is a trivalent binding protein, specifically comprising the following structure: the protein functional region A of the left arm is (g), and a second protein functional region B is connected to the N-terminus of the protein functional region A of the left arm, or the N-terminus of the first protein functional region B of the right arm, or the C-terminus of the Fc, preferably the second protein functional region A of the left arm is (g), and a second protein functional region B of the right arm is connected to the N-terminus of the protein functional region A of the left arm, or the N-terminus of the first protein functional region B of the right arm, or the C-terminus of the Fc; and protein functional domain B is connected to the N-terminus of the first protein functional domain B of the right arm; when the number of protein functional domains B is three, the binding protein is a tetravalent binding protein, specifically comprising the following structure: the protein functional domain A of the left arm is (g), and the third protein functional domain B is further connected to the N-terminus of the protein functional domain A of the left arm, or to the N-terminus or C-terminus of the second protein functional domain B, or to the C-terminus of the Fc, preferably the second protein functional domain B is connected to the N-terminus of the first protein functional domain B of the right arm, and the third protein functional domain B is connected to the N-terminus of the second protein functional domain B; Preferably, said binding protein further comprises a protein functional region C, said binding protein being a trivalent or multivalent multispecific binding protein, in which said protein functional region C, said protein functional region A and said protein functional region B target different antigens or antigenic epitopes, said protein functional region C being connected to the N-terminus or C-terminus of said binding protein; preferably said protein functional region C being connected to the N-terminus of protein functional region B of said binding protein according to (d), (e), (f) or (g); More preferably, said binding protein further comprises a protein functional region D, said binding protein being a tetravalent or multivalent multispecific binding protein, in particular: said protein functional region D and said protein functional region A, protein functional region B and protein functional region C target different antigens or antigenic epitopes, said protein functional region D being connected to the N-terminus or C-terminus of said binding protein; preferably said protein functional region D being connected to the N-terminus of protein functional region B of the binding protein according to (d), (e), (f) or (g).

3. 3. A binding protein according to claim 1 or 2, (A) the binding protein has four polypeptide chains, two of which are the same short chains and the other two are the same long chains, in which (1) the short chain comprises, from N-terminus to C-terminus, VH_A-CH1 and the long chain comprises, from N-terminus to C-terminus, VL_A-CL-L1-VH_B-L2-CH2-CH3; or (2) the short chain comprises, from N-terminus to C-terminus, VL_A-CL and the long chain comprises, from N-terminus to C-terminus, VH_A-CH1-L1-VH_B-L2-CH2-CH3; or (3) the short chain comprises, from N-terminus to C-terminus, VL_A-CL, and the long chain comprises, from N-terminus to C-terminus, VH_B-L-VH_A-CH1-h-CH2-CH3; or (4) the short chain comprises, from N-terminus to C-terminus, VH_B-L-VL_A-CL, and the long chain comprises, from N-terminus to C-terminus, VH_A-CH1-h-CH2-CH3; or (5) the short chain comprises, from N-terminus to C-terminus, VL_A-CL, and the long chain comprises, from N-terminus to C-terminus, VH_A-CH1-h-CH2- or (6) the short chain comprises, from N-terminus to C-terminus, VL_A-CL-L-VH_B, and the long chain comprises, from N-terminus to C-terminus, VH_A-CH1-h-CH2-CH3; or (7) the short chain comprises, from N-terminus to C-terminus, VL_A-CL, and the long chain comprises, from N-terminus to C-terminus, VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_B; or (8) the short chain comprises, from N-terminus to C-terminus, VL_A-CL, and the long chain comprises, from N-terminus to C-terminus, VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_B; or (9) the short chain comprises, from the N-terminus to the C-terminus, VH_B-L1-VL_A-CL and the long chain comprises, from the N-terminus to the C-terminus, VH_B-L2-VH_A-CH1-h-CH2-CH3; or (10) the short chain comprises, from the N-terminus to the C-terminus, VH_B-L1-VL_A-CL and the long chain comprises, from the N-terminus to the C-terminus, VH_C-L2-VH_A-CH1-h-CH2-CH3;wherein L, L1 and L2 are connecting peptides, and h is a hinge region or connecting peptide, the hinge region or connecting peptide being, for example, "-", GS or the amino acid sequence shown in SEQ ID NO: 495-519; or; (B) the binding protein has three types of polypeptide chains, polypeptide chain 1, polypeptide chain 2, and polypeptide chain 3, and each type of polypeptide chain is single, in which (11) the polypeptide chain 1 comprises VL_A-CL in order from the N-terminus to the C-terminus, the polypeptide chain 2 comprises VH_A-CH1-h-CH2-CH3 in order from the N-terminus to the C-terminus, and the polypeptide chain 3 comprises VH_B-h-CH2-CH3 in order from the N-terminus to the C-terminus; or (12) the polypeptide chain 1 comprises VH_A-CL in order from the N-terminus to the C-terminus, or (13) said polypeptide chain 1 comprises VH_A-CH1-h-CH2-CH3 from the N-terminus to the C-terminus, said polypeptide chain 2 comprises VL_A-CL-h-CH2-CH3 from the N-terminus to the C-terminus, and said polypeptide chain 3 comprises VH_B-h-CH2-CH3 from the N-terminus to the C-terminus; or (14) said polypeptide chain 1 comprises VL_A-CL from the N-terminus to the C-terminus (15) said polypeptide chain 1 comprises, from the N-terminus to the C-terminus, VL_A-CL, said polypeptide chain 2 comprises, from the N-terminus to the C-terminus, VH_A-CH1-h-CH2-CH3, and said polypeptide chain 3 comprises, from the N-terminus to the C-terminus, VH_B-L-VH_B-h-CH2-CH3; or (16) said polypeptide chain 1 comprises, from the N-terminus to the C-terminus, VL_A-CL, said polypeptide chain 2 comprises, from the N-terminus to the C-terminus, VH_A-CH1-h-CH2-CH3, and said polypeptide chain 3 comprises, from the N-terminus to the C-terminus, VH_C ... (17) said polypeptide chain 1 comprises VH_A-CL in order from the N-terminus to the C-terminus, said polypeptide chain 2 comprises VL_A-CH1-h-CH2-CH3 in order from the N-terminus to the C-terminus, and said polypeptide chain 3 comprises VH_B-L-VH_B-h-CH2-CH3 in order from the N-terminus to the C-terminus; or (17) said polypeptide chain 1 comprises VH_A-CL in order from the N-terminus to the C-terminus, said polypeptide chain 2 comprises VL_A-CH1-h-CH2-CH3 in order from the N-terminus to the C-terminus, and said polypeptide chain 3 comprises VH_C-L-VH_B-h-CH2-CH3 in order from the N-terminus to the C-terminus;or (18) said polypeptide chain 1 comprises, from the N-terminus to the C-terminus, VH_A-CH1, said polypeptide chain 2 comprises, from the N-terminus to the C-terminus, VL_A-CL-h-CH2-CH3, and said polypeptide chain 3 comprises, from the N-terminus to the C-terminus, VH_B-L-VH_B-h-CH2-CH3; or (19) said polypeptide chain 1 comprises, from the N-terminus to the C-terminus, VH_A-CH1, said polypeptide chain 2 comprises, from the N-terminus to the C-terminus, VL_A-CL-h-CH2-CH3, and said polypeptide chain 3 comprises, from the N-terminus to the C-terminus, VH_C-L-VH_B-h-CH2-CH3; or (26) said polypeptide chain 1 comprises, from the N-terminus to the C-terminus, VL_A-CL, or (27) said polypeptide chain 1 comprises, in N-terminal to C-terminal order, VL_A-CL, said polypeptide chain 2 comprises, in N-terminal to C-terminal order, VH_A-CH1-h-CH2-CH3, and said polypeptide chain 3 comprises, in N-terminal to C-terminal order, VH_D-L1-VH_C-L2-VH_B-h-CH2-CH3; wherein L, L1 and L2 are connecting peptides and said h is a hinge region or connecting peptide, the hinge region or connecting peptide being, for example, "-", GS or an amino acid sequence as set forth in SEQ ID NO: 495-519; or; (C) The binding protein has both polypeptide chains, polypeptide chain 1 and polypeptide chain 2, and each polypeptide chain is single, in which (20) the polypeptide chain 1 comprises VL_A-L-VH_A-h-CH2-CH3 from the N-terminus to the C-terminus, and the polypeptide chain 2 comprises VH_B-h-CH2-CH3 from the N-terminus to the C-terminus; or (21) the polypeptide chain 1 comprises VH_A-L-VL_A-h-CH2-CH3 from the N-terminus to the C-terminus, and the polypeptide chain 2 comprises VH_B-h-CH2-CH3 from the N-terminus to the C-terminus; or (22) the polypeptide chain 1 comprises VL_A-L1-VH_A-h-CH2-CH3 from the N-terminus to the C-terminus, and the polypeptide chain 2 comprises VH_B-L2-VH_B-h-CH2-CH3 from the N-terminus to the C-terminus; or (23) the polypeptide chain 1 comprises, from N-terminus to C-terminus, VH_A-L1-VL_A-h-CH2-CH3, and said polypeptide chain 2 comprises, from N-terminus to C-terminus, VH_B-L2-VH_B-h-CH2-CH3; or (24) said polypeptide chain 1 comprises, from N-terminus to C-terminus, VL_A-L1-VH_A-h-CH2-CH3, and said polypeptide chain 2 comprises, from N-terminus to C-terminus, VH_C-L2-VH_B-CH2-CH3. or (25) a binding protein in which polypeptide chain 1 comprises, in order from N-terminus to C-terminus, VH_A-L1-VL_A-h-CH2-CH3 and polypeptide chain 2 comprises, in order from N-terminus to C-terminus, VH_C-L2-VH_B-h-CH2-CH3; wherein L, L1 and L2 are connecting peptides and h is a hinge region or connecting peptide, e.g., the hinge region or connecting peptide is represented by "-", GS or the amino acid sequence of SEQ ID NO: 495-519.

4. A binding protein according to any one of claims 1 to 3, the protein functional region A or the protein functional region B is one selected from PD-L1 antibody, HER2 antibody, B7H4 antibody, CD3 antibody, CTLA4 antibody, 4-1BB antibody or BCMA antibody; preferably, the protein functional region A is PD-L1 antibody, HER2 antibody, B7H4 antibody or CD3 antibody, and the protein functional region B is CTLA4 antibody, 4-1BB antibody or BCMA antibody; Preferably, the protein functional region A is a PD-L1 antibody and the protein functional region B is a CTLA4 antibody; or the protein functional region A is a PD-L1 antibody and the protein functional region B is a 4-1BB antibody; or the protein functional region A is a HER2 antibody and the protein functional region B is a CTLA4 antibody; or the protein functional region A is a B7H4 antibody and the protein functional region B is a 4-1BB antibody; or the protein functional region A is a CD3 antibody and the protein functional region B is a BCMA antibody.

5. 5. The binding protein of claim 4, the PD-L1 antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises LCDR1, LCDR2, and LCDR3, and have the amino acid sequences set forth in SEQ ID NOs: 167, 188, and 211, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2, and HCDR3, and have the amino acid sequences set forth in SEQ ID NOs: 15, 62, and 122, respectively; or the light chain variable region VL comprises LCDR1, LCDR2, and LCDR3, and have the amino acid sequences set forth in SEQ ID NOs: 167, 188, and 211, respectively; and the heavy chain variable region VH comprises HCDR1, HCDR2, and HCDR3, and have the amino acid sequences set forth in SEQ ID NOs: 15, 69, and 122, respectively; preferably, the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 282; and the heavy chain variable region VH comprises SEQ ID NO: or its light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 282; its heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 240; and the PD-L1 antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises LCDR1, LCDR2, and LCDR3, and have the amino acid sequences set forth in SEQ ID NOs: 169, 190, and 213, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2, and HCDR3, and have the amino acid sequences set forth in SEQ ID NOs: 16, 64, and 124, respectively; preferably, the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 284; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 235; and The HER2 antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequence shown in SEQ ID NO: 171, 190 and 215, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 19, 67 and 127, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequence shown in SEQ ID NO: 176, 196 and 220, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 23, 74 and 132, respectively; preferably, the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 286; and the heavy chain variable region VH comprises SEQ ID NO: or its light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 293; its heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 247; and The B7H4 antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequence shown in SEQ ID NO: 180, 191 and 225, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 32, 87 and 143, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequence shown in SEQ ID NO: 177, 191 and 226, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 33, 88 and 144, respectively; preferably, the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 298; and the heavy chain variable region VH comprises SEQ ID NO: or a light chain variable region VL thereof comprising the amino acid sequence shown in SEQ ID NO: 299; a heavy chain variable region VH thereof comprising the amino acid sequence shown in SEQ ID NO: 262; and The CTLA4 antibody comprises a heavy chain variable region; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which have the amino acid sequences shown in SEQ ID NOs: 17, 65 and 125, respectively; preferably, the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 236; and The 4-1BB antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequence shown in SEQ ID NO: 170, 191 and 214, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 18, 66 and 126, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequence shown in SEQ ID NO: 170, 191 and 214, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 18, 71 and 126, respectively; preferably, the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 285; and the heavy chain variable region VH comprises SEQ ID NO: or its light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 289; its heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 242; and The 4-1BB antibody comprises a heavy chain variable region; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 27, 79 and 137, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 28, 80 and 138, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 29, 82 and 138, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 28, 89 and 145, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 28, 81 or 139; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequence shown in SEQ ID NO: 15, 83 or 140, respectively; preferably, the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 252; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 253; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 255; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 264; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 254; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 256; and the CD3 antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequences shown in SEQ ID NOs: 177, 191 and 221, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 15, 84 and 141, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequences shown in SEQ ID NOs: 178, 197 and 222, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 31, 85 and 142, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequences shown in SEQ ID NOs: the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 31, 86 and 141, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequence shown in SEQ ID NO: 179, 198 and 224, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 31, 85 and 142, respectively; preferably, the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 294; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 258; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 295; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: or its light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 296; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 260; or its light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 297; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 259; and The CD3 antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequence shown in SEQ ID NO: 172, 192 and 216, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 20, 68 and 128, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 and has the amino acid sequence shown in SEQ ID NO: 172, 192 and 216, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 30, 68 and 128, respectively; preferably, the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and the heavy chain variable region VH comprises SEQ ID NO: or its light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 291; its heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 257; or its light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 291; its heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 263; or its light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 291; its heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 281; or its light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 291; its heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 244; and The BCMA antibody comprises a heavy chain variable region; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 15, 75 and 133, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 24, 76 and 134, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 25, 77 and 135, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 26, 78 and 136, respectively; preferably, the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 248; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 249; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; and The BCMA antibody comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences set forth in SEQ ID NOs: 26, 90 and 136, respectively; or wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences set forth in SEQ ID NOs: 34, 78 and 146, respectively; or wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences set forth in SEQ ID NOs: 35, 78 and 147, respectively; or wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences set forth in SEQ ID NOs: 35, 90 and 147, respectively; or wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences set forth in SEQ ID NOs: or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 76 and 147, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 34, 78 and 148, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 76 and 136, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 90 and 146, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 76 and 146, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 34, 76 and 146, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 90 and 148, respectively;or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequence shown in SEQ ID NO: 35, 90 and 136, respectively; preferably, the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 265; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 266; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 267; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 268; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 269; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 270; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 271; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: a binding protein comprising the amino acid sequence set forth in SEQ ID NO: 272; or the heavy chain variable region VH of which comprises the amino acid sequence set forth in SEQ ID NO: 273; or the heavy chain variable region VH of which comprises the amino acid sequence set forth in SEQ ID NO: 274; or the heavy chain variable region VH of which comprises the amino acid sequence set forth in SEQ ID NO: 275; or the heavy chain variable region VH of which comprises the amino acid sequence set forth in SEQ ID NO: 276; or the heavy chain variable region VH of which comprises the amino acid sequence set forth in SEQ ID NO: 277; or the heavy chain variable region VH of which comprises the amino acid sequence set forth in SEQ ID NO: 278; or the heavy chain variable region VH of which comprises the amino acid sequence set forth in SEQ ID NO: 279; or the heavy chain variable region VH of which comprises the amino acid sequence set forth in SEQ ID NO:

280.

6. A binding protein according to any one of claims 1 to 4, (A) the binding protein comprises protein functional domain A and protein functional domain B; said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 69 and 122, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 17, 65 and 125, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 62 and 122, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 17, 65 and 125, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 171, 190 and 215, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 19, 67 and 127, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 17, 65 and 125, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 176, 196 and 220, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 23, 74 and 132, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 17, 65 and 125, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 69 and 122, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 28, 80 and 138, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 69 and 122, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 27, 79 and 137, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 69 and 122, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 29, 82 and 138, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 180, 191 and 225, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 32, 87 and 143, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 28, 80 and 138, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 180, 191 and 225, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 32, 87 and 143, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 27, 79 and 137, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 180, 191 and 225, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 32, 87 and 143, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 28, 89 and 145, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 177, 191 and 226, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 33, 88 and 144, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 28, 80 and 138, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 177, 191 and 226, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 33, 88 and 144, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 28, 81 and 139, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 177, 191 and 226, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 33, 88 and 144, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 83 and 140, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 20, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 24, 76 and 134, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 20, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 20, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 75 and 133, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 75 and 133, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 26, 90 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 36, 90 and 146, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 76 and 147, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 34, 78 and 148, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 76 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 90 and 146, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 34, 76 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 76 and 146, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 34, 76 and 146, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 90 and 148, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 90 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 34, 78 and 146, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 78 and 147, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 35, 90 and 147, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 177, 191 and 221, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 84 and 141, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 178, 197 and 222, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 31, 85 and 142, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 177, 191 and 223, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 31, 86 and 141, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 179, 198 and 224, respectively; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 31, 85 and 142, respectively; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively; or (B) the binding protein comprises protein functional domain A, protein functional domain B, and protein functional domain C; said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, and has the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; and wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 15, 75 and 133, respectively; said protein functional region C comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 24, 76 and 134, respectively; or a binding protein, wherein said protein functional region A comprises a light chain variable region and a heavy chain variable region; said light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, and have the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; said heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and have the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; said protein functional region B comprises a heavy chain variable region; said heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and have the amino acid sequences shown in SEQ ID NOs: 25, 77 and 135, respectively; and said protein functional region C comprises a heavy chain variable region; said heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and have the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively.

7. A binding protein according to any one of claims 1 to 6, (A) the binding protein comprises protein functional domain A and protein functional domain B: said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 282; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 240; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 236; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 282; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 233; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 236; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 286; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 238; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 236; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 293; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 247; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 236; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 282; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 240; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 253; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 282; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 240; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 252; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 282; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 240; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 255; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 298; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 261; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 253; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 298; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 261; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 252; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 298; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 261; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 264; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 299; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 262; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 253; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 299; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 262; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 254; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 299; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 262; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 256; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 245; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 249; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 245; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 245; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 248; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 257; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 248; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 257; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 281; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 265; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 269; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 270; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 271; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 272; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 273; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 274; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 275; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 276; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 277; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 278; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 279; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 280; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 266; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 291; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 267; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 268; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 244; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 249; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 244; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 294; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 258; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 295; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 259; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region; said light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 296; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 260; said protein functional region B comprising a heavy chain variable region; said heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 251; or said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 297; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 259; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or (B) the binding protein comprises protein functional domain A, protein functional domain B, and protein functional domain C; said protein functional region A comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; and wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 257; said protein functional region B comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 248; said protein functional region C comprising a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 249; or a binding protein, wherein said protein functional region A comprises a light chain variable region and a heavy chain variable region; said light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 291; said heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 257; said protein functional region B comprises a heavy chain variable region; said heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 250; said protein functional region C comprises a heavy chain variable region; and said heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO:

251.

8. A binding protein according to any one of claims 1 to 7, (1) The binding protein contains both types of polypeptide chains, among which: the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:371; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:372; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:371; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:373; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 362; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:363; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:364; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:365; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:364; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 366; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 369; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 370; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 394; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 395; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 363; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 310; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 363; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 396; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:363; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:362; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 363; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 394; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 363; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 395; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:368; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 378; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 379; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 380; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 381; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 382; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 383; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 385; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 388; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 389; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:374; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 375; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 386; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 387; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 401; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:402; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:305; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 403; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:402; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:409; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 359; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 404; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:405; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:315; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 359; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 406; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 376; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 377; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 397; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 398; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 399; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 400; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:402; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:401; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:402; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:403; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:405; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:404; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:405; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:406; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 371; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 486; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 460; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 461; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 462; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:487; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:488; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 360; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 455; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 360; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 456; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 360; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 457; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 360; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 458; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 360; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 459; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:419; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:412; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:419; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:414; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:440; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:428; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:441; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:428; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:442; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:428; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:443; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:428; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 487; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 520; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 353; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 521; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 371; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 522; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 360; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 523; or (2) the binding protein comprises three polypeptide chains, among which: a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 353; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 407; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 390; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 353; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 408; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 392; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:351; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:391; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:390; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:351; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:393; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:392; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 391; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 434; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 391; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 435; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 393; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 436; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 351; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 393; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 437; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 438; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 434; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 438; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 435; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 367; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 439; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 436; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 367; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 439; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 437; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:361; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:481; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:482; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:361; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:481; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:483; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:361; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:481; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:484; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 361; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 481; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 485; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 357; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 411; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 412; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 411; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 414; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:415; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:416; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:410; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:415; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:416; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:412; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:415; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:416; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:414; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:417; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:418; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:412; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:417; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:418; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:414; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:423; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:426; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 357; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 423; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 427; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:423; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:428; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:423; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:429; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:357; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:444; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:449; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:357; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:450; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:449; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:444; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:445; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:444; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:446; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 450; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 445; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:450; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:446; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:444; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:447; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:444; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:448; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 450; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 447; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:450; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:448; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:445; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:446; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:463; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:464; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:465; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:466; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:467; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:468; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:469; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:470; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:471; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:357; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:454; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:472; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 473; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:474; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:475; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:476; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:477; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:478; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:479; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:454; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:480; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 423; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 430; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 423; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 431; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 423; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 432; or A binding protein, wherein a first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:357; a second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:423; and a third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:

433.

9. A binding protein according to any one of claims 1 to 8, a binding protein further comprising a light chain constant region, said light chain constant region being preferably a human light chain constant region, more preferably a human light chain constant region Cκ or Cλ; said binding protein further comprising a heavy chain constant region CH1, CH2 and / or CH3, said heavy chain constant region being preferably a human heavy chain constant region, more preferably a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; and said IgG1 heavy chain constant region Fc further comprising one or more of the following mutations: C220S, N297A, L234A, L235A, P329G, S239D, I332E, S354C, T366W, Y349C, T366S, L368A, Y407V, M252Y, S254T, T256E, and the mutation sites are designated by the EU numbering convention.

10. 10. An isolated nucleic acid encoding a binding protein according to any one of claims 1-9.

11. 11. An expression vector comprising the isolated nucleic acid of claim 10.

12. The expression vector according to claim 11, A host cell, which may be a prokaryotic or eukaryotic cell.

13. A method for producing a binding protein according to any one of claims 1 to 9, comprising: The method for producing a binding protein includes the steps of: culturing a host cell as described above, and obtaining the binding protein from the culture.

14. A pharmaceutical composition comprising a binding protein according to any one of claims 1-9.

15. A drug kit comprising a drug cassette 1 and a drug cassette 2, wherein the drug cassette 1 contains a binding protein described in any one of claims 1-9 or a drug composition described in claim 14, and the drug cassette 2 contains another antibody or drug composition for treating cancer.

16. Use of the binding protein according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 14 in the manufacture of a drug for treating and / or preventing cancer, Preferably, the cancer is preferably breast cancer, ovarian cancer, endometrial cancer, renal cancer, melanoma, lung cancer, gastric cancer, liver cancer, esophageal cancer, cervical cancer, head and neck tumors, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma, colorectal cancer, lymphoma or multiple myeloma, etc.

17. administering to a subject in need thereof a binding protein according to any one of claims 1-9, or a pharmaceutical composition according to claim 14, or a pharmaceutical kit according to claim 15; Preferably, the method of treating cancer further comprises administering other antibodies to treat the cancer, such as immune checkpoint antibodies and / or chemotherapy drugs.

18. comprising a light chain variable region and a heavy chain variable region, its light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 177, 191 and 221, respectively; its heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 15, 84 and 141, respectively; or its light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 178, 197 and 222, respectively; its heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 31, 85 and 142, respectively; or its light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 177, 191 and 223, respectively; its heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 31, 86 and 141, respectively; or its light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 179, 198 and 224, respectively; its heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 31, 85 and 142, respectively; or its light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, and having the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; its heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and having the amino acid sequences shown in SEQ ID NOs: 20, 68 and 128, respectively; or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, and has the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; Preferably, the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 294; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 258; or its light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 295; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 259; or its light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 296; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 260; or its light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 297; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 259; or its light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 245; or its light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 257; or its light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 263; or its light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 281; or the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 291; the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 244; More preferably, the CD3 antibody comprises both polypeptide chains; wherein a first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 357 and a second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 313; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 325; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 328; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 357 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 346; or A CD3 antibody, wherein the CD3 antibody comprises one polypeptide chain, the polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 489, the amino acid sequence shown in SEQ ID NO: 490, the amino acid sequence shown in SEQ ID NO: 491, the amino acid sequence shown in SEQ ID NO: 492, or the amino acid sequence shown in SEQ ID NO:

493.

19. a heavy chain variable region, the heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3 and having the amino acid sequences shown in SEQ ID NOs: 15, 75 and 133, respectively; or the heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3 and having the amino acid sequences shown in SEQ ID NOs: 24, 76 and 134, respectively; or the heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3 and having the amino acid sequences shown in SEQ ID NOs: 25, 77 and 135, respectively; or the heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3 and having the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively; or or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 26, 90 and 136, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 34, 78 and 146, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 78 and 147, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 90 and 147, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 76 and 147, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 34, 78 and 148, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 76 and 136, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 90 and 146, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 76 and 146, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 34, 76 and 146, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequence shown in SEQ ID NO: 35, 90 and 148, respectively;or its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which have the amino acid sequences shown in SEQ ID NOs: 35, 90 and 136, respectively; Preferably, the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 248; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 249; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 250; or the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 265; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 266; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 267; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 268; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 269; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 270; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 271; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 272; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 273; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 274; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 275; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 276; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 277; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 278; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 279; or the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 280; More preferably, the BCMA antibody comprises one polypeptide chain, the polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:316, the amino acid sequence set forth in SEQ ID NO:317, the amino acid sequence set forth in SEQ ID NO:318, or the amino acid sequence set forth in SEQ ID NO:319; or The BCMA antibody comprises a single polypeptide chain, the polypeptide chain having the amino acid sequence set forth in SEQ ID NO:330; or the amino acid sequence set forth in SEQ ID NO:331; or the amino acid sequence set forth in SEQ ID NO:332; or the amino acid sequence set forth in SEQ ID NO:333; or the amino acid sequence set forth in SEQ ID NO:334; or the amino acid sequence set forth in SEQ ID NO:335; or the amino acid sequence set forth in SEQ ID NO:336; or the amino acid sequence set forth in SEQ ID NO:337; or the amino acid sequence set forth in SEQ ID NO:338; or the amino acid sequence set forth in SEQ ID NO:339; or the amino acid sequence set forth in SEQ ID NO:340; or the amino acid sequence set forth in SEQ ID NO:341; or the amino acid sequence set forth in SEQ ID NO:342; or the amino acid sequence set forth in SEQ ID NO:343; or the amino acid sequence set forth in SEQ ID NO:344; or the amino acid sequence set forth in SEQ ID NO:

345. A BCMA antibody comprising the amino acid sequence set forth in

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