Binding proteins with H2L2 and HCAb structures

Abstract

The present invention discloses a binding protein containing at least two protein functional regions, the binding protein comprising protein functional region A and 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 has a Fab or scFv structure, the protein functional region B has a VH structure, and the number of the protein functional region A and the protein functional region B can each be one or more. The multispecific binding protein of the present invention has a small molecular weight, fewer polypeptide chains, and a simpler structure, and parameters such as different structural types, relative positions, and valencies can be used to adjust the functional activity against different targets and even design different activity combinations, thereby meeting the needs for different clinical combination dosages.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine, and more particularly to a bispecific or multispecific binding protein having H2L2 and HCAb structures and its uses. [Background technology]

[0002] In most species, antibodies have a tetrameric structure containing two identical heavy chains and two identical light chains, also known as "H2L2." Heavy-chain antibodies (HCAbs), which lack light chains, also naturally occur in the serum of camelids and sharks. The variable region VHH fragments of heavy-chain antibodies resemble the Fab fragments of H2L2 antibodies and possess stable structures and specific antigen-binding activity. However, because the molecular weight of VHH is only approximately 13 kDa, these antibodies are also called nanobodies or single-domain antibodies. Because human antibodies naturally have an "H2L2" structure, it is impossible to obtain useful human-derived heavy-chain antibodies from natural sources. However, Frank Grosveld et al. proposed a method for obtaining fully human-derived heavy-chain antibodies using transgenic animals (WO 2007 / 096779), and that this method can be used to obtain stable and soluble fully human-derived VH single-domain antibodies.

[0003] Bispecific and multispecific antibodies are artificial antibodies created by protein engineering based on natural monoclonal antibodies. They can bind to two or more different antigens or different epitopes on the same antigen, achieving mechanisms of action and functional effects that cannot be achieved with some monospecific antibodies.

[0004] Structural design of bispecific antibodies is crucial. One of the biggest challenges in generating bispecific antibodies from two different H2L2 antibodies is how to obtain bispecific antibody molecules with the correct chain combination functionality from over 10 different heavy and light chain combinations, i.e., how to resolve the chain mismatch problem. Scientists have already developed many strategies to address this issue: for example, fusing VH and VL using single-chain variable fragment (scFv) structures; promoting heavy chain heterodimerization using knob-into-hole (KiH) technology or other spatially complementary variants; or reducing the number of different polypeptide chains using "shared light chain" or "shared heavy chain" technologies. However, these various technical approaches have limitations. For example, scFv structures are always unstable and prone to aggregation; "shared light chain" technologies require highly complex filtering processes to obtain VLs that can pair with different VHs; and the size of bispecific antibody molecules generated using technologies such as FIT-Ig is relatively large (approximately 250 kDa), which may affect their ability to penetrate cells and tissues. Therefore, there is still a need for new technologies for developing bispecific antibody structures and producing bispecific antibodies.

[0005] Heavy chain antibodies and single domain antibodies derived from them 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 fragment of a conventional antibody; moreover, the absence of a light chain avoids the problem of light chain mismatch. Therefore, heavy chain antibodies and single domain antibodies derived from them 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, constructing bispecific or multispecific antibodies using all-human heavy chain antibodies offers advantages in terms of immunogenicity and drug efficacy compared to camelid nanobodies. Summary of the Invention

[0006] To overcome the deficiencies of the prior art in the lack of therapeutically effective, stable, and easily produced bispecific or multispecific binding proteins, 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. This has many advantages over conventional multivalent and multispecific binding proteins constructed using IgG antibodies, including greater ease in tailoring specificity and valency. The multivalent and multispecific binding proteins provided herein contain at least one heavy chain variable region (VH) derived from a human heavy chain antibody and are capable of binding two or more antigens, two or more epitopes on the same antigen, or two or more copies of the same epitope.

[0007] In order to solve the above technical problems, a first technical configuration of the present invention provides a binding protein containing at least two protein functional regions, the binding protein comprising protein functional region A and 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 has a Fab or scFv structure, the protein functional region B has a VH structure, and the number of the protein functional regions A and B is one or more; preferably, the binding protein further comprises an Fc, the number of the Fc is two, and the 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 protein functional regions A is two and the number of protein functional regions B is two or four. Two of the protein functional regions A and two of the Fc regions form an IgG structure. In the bilaterally symmetric IgG-like structure, left and right refer only to relative spatial positions, which may be top-bottom symmetric or front-to-back symmetric. The bilaterally symmetric IgG-like structure means that additional protein functional regions are further bilaterally symmetrically connected to an antibody of IgG structure. These additional protein functional regions may be connected to the C-terminus of CH3 of the Fc functional region or the N-terminus of VH or VL, or may be connected between the variable region and the Fc functional region of the IgG structure in an embedded form. As used herein, "additional" refers to a structure or functional region other than the IgG structure.

[0009] Preferably, when the number of protein functional domains B is two, the binding protein is a tetravalent binding protein, specifically comprising the following structure: (a) the binding protein is, from N-terminus to C-terminus, composed of protein functional domain A, protein functional domain B, and Fc, in which the protein functional domain A and the protein functional domain B are connected by a first connecting peptide (L1), and the protein functional domain B and the Fc are connected by a second connecting peptide (L2); in this embodiment, the protein functional domain B and the Fc of both parts form a symmetric single-chain antibody dimer, and the protein functional domain A is connected to the N-terminus of the single-chain antibody dimer, and the protein functional domain A is connected to the N-terminus of the protein functional domain B at its CH1 (Figure 1(2)) or CL (Figure 1(1)); or (b) the binding protein is, from N-terminus to C-terminus, composed of protein functional domain B, protein functional domain A, and Fc, in which the protein functional domain B and the protein functional domain A are connected (c) the binding protein is composed of, from the N-terminus to the C-terminus, a protein functional region A, an Fc, and a protein functional region B, in that order from the N-terminus to the C-terminus, 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; (d) the binding protein is composed of, 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; (e) the binding protein is composed of, from the N-terminus to the C-terminus, a protein functional region A and the Fc, 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; (f) the binding protein is composed of, from the N-terminus to the C-terminus, a protein functional region A and the Fc, in which the protein functional region A and the Fc are connected by a hinge region, and the protein functional region B is connected to the C-terminus of the IgG structure, in which 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 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), (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 protein functional region A together with the original protein functional region B of the binding protein described in (b). The term "additional" used herein is merely a convenient expression to distinguish from the above-mentioned protein functional region B, i.e., the "original" protein functional region, and no distinction is made 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, where two of the protein functional regions B are connected to the CH3 of the protein functional region A by a first connecting peptide, and the other two protein functional regions B are connected to the two protein functional regions B by 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 by 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 by a second connecting peptide (Figure 1(9)).

[0011] A 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 they are 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 (also referred to as "polypeptide chain 1") and two identical long chains (also referred to as "polypeptide chain 2"), wherein (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, VL_A-CL. or (3) the short chain comprises, from N- to C-terminus, VL_A-CL, and the long chain comprises, from N- to C-terminus, VH_B-L-VH_A-CH1-h-CH2-CH3; or (4) the short chain comprises, from N- to C-terminus, VH_B-L-VL_A-CL, and the long chain comprises, from N- to C-terminus, VH_A-CH1-h-CH2-CH3; or (5) the short chain comprises, from N- to C-terminus, VL_A-CL, and the long chain comprises, from N- to C-terminus, VH_A-CH1-h-CH2-CH3. or (6) the short chain comprises VL_A-CL-L-VH_B in order from the N-terminus to the C-terminus, and the long chain comprises VH_A-CH1-h-CH2-CH3 in order from the N-terminus to the C-terminus; or (7) the short chain comprises VL_A-CL in order from the N-terminus to the C-terminus, and the long chain comprises VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_B in order from the N-terminus to the C-terminus; or (8) the short chain comprises VL_A-CL in order from the N-terminus to the C-terminus. (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;In the above, L, L1, and L2 are connecting peptides, and h is a hinge region or connecting peptide, which can be represented by, for example, "-", GS, or the amino acid sequence of SEQ ID NO: 495-519. The meanings of VL, VH, CL, and CH in the present specification are those commonly used in the art and represent the light chain variable region, heavy chain variable region, light chain constant region, and heavy chain constant region, respectively. CH includes CH1, CH2, and CH3; _A, _B, and _C represent that the functional region is protein functional region A, protein functional region B, or protein functional region C, or a combination thereof, respectively. "-" represents a polypeptide bond for connecting or separating different domains; the C-terminus is the carboxyl terminus (also referred to as "C'") of the peptide chain, and the N-terminus is the amino terminus (also referred to 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 have the same sequence. In another embodiment, L, L1, and L2 may be different sequences. Protein functional regions of the present invention may be Fab, scFv, or VH in some cases, or F(ab)2 or full-length antibodies in other cases, and are sometimes also referred to as antibodies or antigen-binding proteins or binding proteins. In some specific embodiments, protein functional region A is also referred to as antibody A or first antigen-binding domain against a first antigen, protein functional region B is also referred to as antibody B or second antigen-binding domain against a second antigen, protein functional region C is also referred to as antibody C or third antigen-binding domain against a third antigen, and protein functional region D is also referred to as antibody D or fourth antigen-binding domain against a fourth antigen, and so on.

[0013] In some specific embodiments, (II) the binding protein has an asymmetric structure, exhibiting 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 the protein functional region B is one, the binding protein is a bivalent binding protein, having the following structure: the protein functional region A is in (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, having the following structure: the protein functional region A of the left arm is in (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. The protein specifically has the following structure: the protein functional region A of the left arm has the structure (d), (e), or (f), and the 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 having the following structure: the protein functional region A of the left arm is a (g) scFv structure, and the scFv is connected to Fc by the end of VH or the end of VL; when the number of the protein functional region B is two, the binding protein is a trivalent binding protein, specifically having 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. 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.

[0015] Preferably, the binding protein further comprises a protein functional region C, and the binding protein is a trivalent or multivalent multispecific binding protein, in which the protein functional region C, the protein functional region A, and the protein functional region B target different antigens or antigenic epitopes, and the protein functional region C is connected to the N-terminus or C-terminus of the binding protein; preferably, the protein functional region C is connected to the N-terminus of protein functional region B of the binding protein according to (d), (e), (f) or (g); More preferably, the binding protein further comprises a protein functional region D, and the binding protein is a tetravalent or multivalent multispecific binding protein, in particular: the protein functional region D and the protein functional regions A, B and C target different antigens or antigenic epitopes, and the protein functional region D is connected to the N-terminus or C-terminus of the binding protein; preferably, the 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 polypeptide chains, polypeptide chain 1, polypeptide chain 2, and polypeptide chain 3, and each polypeptide chain is single. In the present invention, the polypeptide chain 1 is also referred to as the first polypeptide chain, the polypeptide chain 2 is also referred to as the second polypeptide chain, and the polypeptide chain 3 is also referred to as 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. (13) said polypeptide chain 1 comprises VH_A-CH1-h-CH2-CH3 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-h-CH2-CH3 in order from the N-terminus to the C-terminus; or (14) said Polypeptide chain 1 comprises VL_A-CL in order from the N-terminus to the C-terminus, said polypeptide chain 2 comprises VH_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 (15) said polypeptide chain 1 comprises VL_A-CL in order from the N-terminus to the C-terminus, and said polypeptide chain 2 comprises VH_A-CH (16) 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 VH_A-CH1 in order from the N-terminus to the C-terminus, and said polypeptide chain 2 comprises VL_A-CH1-h-CH2-CH3 in order from the N-terminus to the C-terminus. or (19) polypeptide chain 1 comprises VH_A-CH1 in order from the N-terminus to the C-terminus, polypeptide chain 2 comprises VL_A-CL-h-CH2-CH3 in order from the N-terminus to the C-terminus, and polypeptide chain 3 comprises VH_B-L-VH_B-h-CH2-CH3 in order from the N-terminus to the C-terminus; or (20) polypeptide chain 1 comprises VH_A-CH1 in order from the N-terminus to the C-terminus, polypeptide chain 2 comprises VL_A-CL-h-CH2-CH3 in order from the N-terminus to the C-terminus, and 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, wherein polypeptide chain 2 comprises, from the N-terminus to the C-terminus, VH_A-CH1-h-CH2-CH3, and polypeptide chain 3 comprises, from the N-terminus to the C-terminus, VH_D-L1-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, which is, for example, represented by "-", 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, and each type of polypeptide chain is single; similarly, polypeptide chain 1 is also referred to as the first polypeptide chain, and 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 order from the N-terminus to the C-terminus, and the polypeptide chain 2 comprises VH_B-h-CH2-CH3 in 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 order from the N-terminus to the C-terminus, and the polypeptide chain 2 comprises VH_B-h-CH2-CH3 in 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 order from the N-terminus to the C-terminus, and the polypeptide chain 2 comprises VH_B-L2-VH_B-h-CH2-CH3 in 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) 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_C-L2-VH_B-CH2-CH3 from the N-terminus to the C-terminus, in order; or (25) the polypeptide chain 1 comprises VH_A-L1-VL_A-h-CH2-CH3 from the N-terminus to the C-terminus, in order, and the polypeptide chain 2 comprises VH_C-L2-VH_B-h-CH2-CH3 from the N-terminus to the C-terminus, in order; wherein L, L1, and L2 are connecting peptides, and h is a hinge region or connecting peptide, and the hinge region or connecting peptide is, for example, "-", GS, or SEQ 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 regions, i.e., protein functional region A and protein functional region B, wherein protein functional region A and protein functional region B are selected from the group consisting of PD-L1 antibody, HER2 antibody, B7H4 antibody, CD3 antibody, CTLA4 antibody, 4-1BB antibody, and BCMA antibody. Preferably, protein functional region A is PD-L1 antibody, HER2 antibody, B7H4 antibody, or CD3 antibody, and 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.

[0019] In some specific embodiments, 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 has 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 has 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 has 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 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 a 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 a 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 set forth 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 set forth 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 set forth 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 set forth in SEQ ID NOs: 33, 88, and 144, 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: 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; wherein 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, wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 361 and a 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 set forth 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 set forth in SEQ ID NO: 236. More preferably, the CTLA4 antibody comprises one polypeptide chain, which comprises the amino acid sequence set forth in SEQ ID NO: 303 or 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 set forth 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 set forth 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 set forth 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 set forth in SEQ ID NOs: 18, 71, and 126, 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: 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, wherein 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, wherein 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth in SEQ ID NOs: or its heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 28, 81, or 139; or its heavy chain variable region VH comprises HCDR1, HCDR2, and HCDR3, and has the amino acid sequence set forth in SEQ ID NO: 15, 83, or 140, respectively. The amino acid sequences of the listed CDRs are shown according to the Chothia definition rules. Preferably, its heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 252; or its heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 253; or its heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 255; or its heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 264; or its heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 254; or its heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 256. More preferably, the 4-1BB antibody comprises one polypeptide chain, and the polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 320, the amino acid sequence set forth in SEQ ID NO: 321, the amino acid sequence set forth in SEQ ID NO: 323, the amino acid sequence set forth in SEQ ID NO: 322, the amino acid sequence set forth in SEQ ID NO: 324, or the amino acid sequence set forth 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 sequences set forth 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 set forth 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 set forth 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 set forth 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 set forth in SEQ ID NOs: the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 31, 86 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: 179, 198 and 224, respectively; and 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. 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: 294; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 258; or the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 295; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 259; or the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 296; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 260; or the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 297; and the heavy chain variable region VH comprises the amino acid sequence set forth 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 set forth 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 set forth 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 set forth 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 set forth in SEQ ID NOs: 30, 68, and 128, 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: 291; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 245; or 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; or the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 291; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 263; or the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 291; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 281; or the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 291; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 244.

[0028] In some specific embodiments, the CD3 antibody comprises both types of 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 one polypeptide chain, and the polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 489, the amino acid sequence set forth in SEQ ID NO: 490, the amino acid sequence set forth in SEQ ID NO: 491, the amino acid sequence set forth in SEQ ID NO: 492, or the amino acid sequence set forth 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 sequences set forth in SEQ ID NOs: 15, 75, and 133, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2, and HCDR3 and has the amino acid sequences set forth in SEQ ID NOs: 24, 76, and 134, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2, and HCDR3 and has the amino acid sequences set forth in SEQ ID NOs: 25, 77, and 135, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2, and HCDR3 and has the amino acid sequences set forth 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 one polypeptide chain, wherein the polypeptide chain comprises 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 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 NOs: 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 NOs: 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 NOs: 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 NOs: 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 NOs: or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 35, 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: 34, 76 and 146, respectively;Alternatively, the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which have 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, which have the amino acid sequences shown in SEQ ID NOs: 35, 90 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: 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 whose heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 274; or whose heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 275; or whose heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 276; or whose heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 277; or whose heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 278; or whose heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 279; or whose 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, wherein the polypeptide chain comprises 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, wherein the heavy chain variable region VH comprises HCDR1, HCDR2, and HCDR3, and has the amino acid sequences set forth 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 sequences set forth in SEQ ID NOs: 26, 78, and 136, respectively. Preferably, the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 249; or wherein the heavy chain variable region VH comprises the amino acid sequence set forth 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), preferably a human heavy chain constant region, more preferably a human IgG1, IgG2, IgG3, or 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, or T256E, and the mutation sites are numbered according to 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 set forth 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 set forth 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 set forth in SEQ ID NOs: 17, 65 and 125, 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 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, 62 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 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: 171, 190 and 215, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 19, 67 and 127, 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 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: 176, 196 and 220, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 23, 74 and 132, 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 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: 28, 80 and 138, 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 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: 27, 79 and 137, 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 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: 29, 82 and 138, 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 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; 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: 28, 80 and 138, 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 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; 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: 27, 79 and 137, 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 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; 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: 28, 89 and 145, 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 comprises LCDR1, LCDR2 and LCDR3, and has the amino acid sequences shown in SEQ ID NOs: 177, 191 and 226, respectively; 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 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: 28, 80 and 138, 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 comprises LCDR1, LCDR2 and LCDR3, and has the amino acid sequences shown in SEQ ID NOs: 177, 191 and 226, respectively; 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 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: 28, 81 and 139, 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 comprises LCDR1, LCDR2 and LCDR3, and has the amino acid sequences shown in SEQ ID NOs: 177, 191 and 226, respectively; 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 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, 83 and 140, 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 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; 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: 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 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; 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: 26, 78 and 136, 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 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; 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; or 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; or 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: 26, 78 and 136, 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 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: 26, 90 and 136, 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 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: 36, 90 and 146, 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 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: 35, 76 and 147, 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 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: 34, 78 and 148, 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 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: 35, 76 and 136, 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 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: 35, 90 and 146, 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 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: 34, 76 and 136, 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 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: 35, 76 and 146, 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 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: 34, 76 and 146, 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 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: 35, 90 and 148, 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 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: 35, 90 and 136, 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 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: 34, 78 and 146, 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 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: 35, 78 and 147, 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 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: 35, 90 and 147, 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 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; 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: 26, 78 and 136, 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 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; 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: 26, 78 and 136, 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 comprises LCDR1, LCDR2 and LCDR3, and has the amino acid sequences shown in SEQ ID NOs: 177, 191 and 223, respectively; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 31, 86 and 141, 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: 26, 78 and 136, 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 comprises LCDR1, LCDR2 and LCDR3, and has the amino acid sequences shown in SEQ ID NOs: 179, 198 and 224, 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; 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: 26, 78 and 136, respectively; or

[0036] In some 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 LCDR1, LCDR2, and LCDR3, and has the amino acid sequences set forth 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 set forth in SEQ ID NOs: 30, 68, and 128, respectively; 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 set forth in SEQ ID NOs: 15, 75, and 133, respectively; 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 set forth 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 has 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 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 comprising HCDR1, HCDR2 and HCDR3, and has the amino acid sequences shown in SEQ ID NOs: 25, 77 and 135, respectively; and the protein functional region C comprises a heavy chain variable region, the heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, and has 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 protein functional domain A and protein functional domain B: protein functional domain A comprises a light chain variable domain and a heavy chain variable domain; wherein the light chain variable domain VL comprises the amino acid sequence set forth in SEQ ID NO: 282; wherein the heavy chain variable domain VH comprises the amino acid sequence set forth in SEQ ID NO: 240; protein functional domain B comprises a heavy chain variable domain; wherein the heavy chain variable domain VH comprises the amino acid sequence set forth in SEQ ID NO: 236; or 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: 233; 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 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: 286; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 238; 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 the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 293; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 247; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 236; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 253; or 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: 252; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 255; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 298; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 261; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 253; or 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: 298; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 261; 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: 252; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 298; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 261; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 264; or 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: 299; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 262; 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: 253; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 299; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 262; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 254; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 299; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 262; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 256; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 245; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 249; or 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: 291; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 245; 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: 251; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 245; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 248; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 248; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 251; or 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: 291; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 281; 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: 251; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 263; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 251; or 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: 291; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 263; 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: 265; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 263; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 269; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 263; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 270; or 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: 291; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 263; 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: 271; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 263; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 272; or 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: 291; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 263; 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: 273; or 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: 291; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 263; 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: 274; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 263; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 275; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 263; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 276; or 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: 291; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 263; 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: 277; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 263; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 278; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 263; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 279; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 263; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 280; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 263; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 266; or 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: 291; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 263; 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: 267; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 263; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 268; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 244; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 249; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein 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: 244; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 251; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 294; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 258; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 251; or 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: 295; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 259; 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: 251; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 296; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 260; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 251; or the protein functional region A comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 297; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 259; the protein functional region B comprises a heavy chain variable region, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 251; or

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

[0040] In some more preferred embodiments, the binding protein comprises three polypeptide chains, wherein 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; and 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 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; and 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 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 set forth in SEQ ID NO: 357; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 423; and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 433.

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

[0042] To solve the above technical problems, the third technical aspect of the present invention provides an expression vector comprising the isolated nucleic acid as described above.

[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 problems, the fifth technical configuration of the present invention provides: a method for producing the above-mentioned binding protein, the method comprising the steps of: culturing the above-mentioned host cell, 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 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, an eighth technical configuration of the present invention provides: a use of the above-mentioned binding protein or the above-mentioned pharmaceutical composition in the production 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, a ninth technical aspect 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 aspect of the present invention, or the pharmaceutical composition described in the sixth technical aspect, or the pharmaceutical kit described in the seventh technical aspect; 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, stomach 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: the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 31, 86 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: 179, 198 and 224, respectively; and 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. 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: 294; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 258; or the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 295; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 259; or the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 296; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 260; or the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 297; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 259. Alternatively, 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 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: 291; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 245; or 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; or the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 291; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 263; or the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 291; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 281; or the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 291; and the heavy chain variable region VH comprises the amino acid sequence set forth 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 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.

[0052] In some specific embodiments, the CD3 antibody comprises one polypeptide chain, and the polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 489, the amino acid sequence set forth in SEQ ID NO: 490, the amino acid sequence set forth in SEQ ID NO: 491, the amino acid sequence set forth in SEQ ID NO: 492, or the amino acid sequence set forth 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; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences set forth in SEQ ID NOs: 15, 75 and 133, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences set forth in SEQ ID NOs: 24, 76 and 134, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences set forth in SEQ ID NOs: 25, 77 and 135, respectively; or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences set forth 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 one polypeptide chain, wherein the polypeptide chain comprises 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. Alternatively, 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 NOs: 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 NOs: 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 NOs: 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 NOs: 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 NOs: or the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 and has the amino acid sequences shown in SEQ ID NOs: 35, 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: 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;Alternatively, the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which have the amino acid sequences set forth in SEQ ID NOs: 35, 90 and 136, respectively. The amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.

[0055] In some specific examples, 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 whose heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 274; or whose heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 275; or whose heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 276; or whose heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 277; or whose heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 278; or whose heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 279; or whose 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 one polypeptide chain, and the polypeptide chain has 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 advancement effects of this invention: The present invention provides bivalent to multivalent, bispecific or multispecific binding proteins constructed using fully human heavy chain antibodies and single domain antibodies derived therefrom, as well as methods for making and using such binding proteins. These proteins have many advantages over bivalent to multivalent, bispecific or multispecific binding proteins constructed using conventional IgG antibodies, including greater ease in adjusting specificity and valency; the ability to construct multispecific binding proteins with smaller molecular weights, fewer polypeptide chains, and simpler structures; and the ability to utilize different structures to achieve different biological functions.

[0057] In some specific embodiments, functional activity against different targets can be adjusted by parameters such as different structural types, relative positions, and binding valencies, allowing for the design of different activity combinations to meet the needs of different clinical combination dosages. In some specific embodiments, heavy chain antibody VH domains can be used to easily form multivalent structures in tandem, promoting target cross-linking through multivalent binding with targets and further enhancing the biological function induced by target cross-linking. In some specific embodiments, heavy chain antibody VH domains can be used to form asymmetric tetravalent structures such as "1 + 3," which cannot be achieved with structures derived from conventional IgG antibodies. [Brief explanation 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 the PD-L1×CTLA4 bibody molecule to CHO-K1 / hCTLA4 cells. Figure 4. Binding activity of the 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 in blocking the binding of CHO-K1 / hCTLA4 cells to their 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 the 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, which highly express human PD-L1. Figure 10. The activity of the PD-L1×CTLA4 dual antibody molecule in blocking the binding of CHO-K1 / hPDL1 cells to their ligand protein PD-1. Figure 11. Ability of PD-L1×CTLA4 bibody molecules to kill CTLA4+ target cells via ADCC effect. Figure 12. Ability of IgG-VH structured PD-L1×CTLA4 biantibody to activate T cells in SEB stimulation experiments. Figure 13. Ability of PD-L1×CTLA4 biantibody in Fab-HCAb configuration to activate T cells in SEB stimulation experiments. Figure 14. In SEB stimulation experiments, when the VH is located at the N-terminus of the IgG HC or LC, the PD-L1xCTLA4 dual antibody has comparable and potentially stronger T cell activation ability than the corresponding 1:1 dose combination of the monoclonal antibodies. Figure 15. In SEB stimulation experiments, the activity of the CTLA4-terminal tail is weakened in IgG_HC-VH (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-L1xCTLA4 dual antibodies have stronger T cell activation ability 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 biantibody has stronger T cell activation ability 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 HER2xCTLA4 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. Figure 24. Ability of the HER2xCTLA4 bibody molecule to simultaneously bind to CTLA4+ and HER2+ cells. Figure 25. Ability of the HER2xCTLA4 dual antibody molecule to activate T cells in SEB stimulation experiments. Figure 26. Pharmacokinetics of the HER2×CTLA4 bibody molecule in mice. Figure 27. Binding activity of the PD-L1x4-1BB bibody molecule to CHO-K1 / hPDL1 cells. Figure 28. Binding activity of the 4-1BB antibody or PD-L1×4-1BB bibody molecule to CHO-K1 / hu4-1BB cells. Figure 29. Binding activity of the PD-L1×4-1BB bibody molecule to CHO-K1 / cyno 4-1BB cells. Figure 30. PD-L1x4-1BB biantibody molecule mediates T cell specific activation by CHO-K1 / hPDL1 cells. Figure 31. PD-L1x4-1BB biantibody molecule mediates T cell specific activation by MDA-MB-231 cells. Figure 32. In mixed lymphocyte reaction experiments, the IgG-VH structure of the PD-L1x4-1BB bibody molecule has a stronger ability to activate T cells than the FIT-Ig structure of the bibody molecule. Figure 33. In mixed lymphocyte reaction experiments, the IgG-VH and Fab-HCAb PD-L1x4-1BB bibody molecules have stronger T cell activation abilities than the FIT-Ig bibody molecule. Figure 34. Pharmacokinetics of the PD-L1×4-1BB bibody molecule in mice. Figure 35. Binding activity of B7H4×4-1BB bibody molecule to SK-BR-3 cells. Figure 36. Binding activity of the B7H4x4-1BB bibody molecule to CHO-K1 / hu4-1BB cells. FIG. 37. B7H4x4-1BB biantibody molecule mediates specific activation (IFNγ release) by SK-BR-3 T cells. FIG. 38. B7H4x4-1BB biantibody molecule mediates specific activation (IL-2 release) by SK-BR-3 T cells. FIG. 39. T cell activation of the B7H4x4-1BB bibody molecule specifically depends on B7H4 expression. 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 a mouse tumor model. 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. Binding affinity of BCMA HCAb antibodies 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 to 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 the scFv-Fc-VH(2) asymmetric BCMAxCD3 bibody molecule to kill NCI-H929 cells. Figure 56. Ability of scFv-Fc-VH(1) asymmetric BCMAxCD3 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 molecules (PR002895, PR002953, PR003178) mediate specific effector cell killing of NCI-H929 cells and detection of cytokine release. Figure 61. Effect of soluble APRIL or BAFF on the target cell killing efficacy of the BCMAxCD3 bibody molecule. Figure 62. Effect of soluble BCMA on the target cell killing efficacy of the BCMAxCD3 bibody molecule. Figure 63. Pharmacokinetics of BCMAxCD3 bibody molecules in mice or rats. Figure 64. Antitumor effect of BCMAxCD3 bibody molecules in mouse tumor models. Figure 65. SDS-PAGE and SEC-HPLC analysis of protein samples obtained from dual antibody molecules following instantaneous transfection expression and one-step affinity purification. Figure 66. Avidity of CD3 antibodies binding to human T cells. Figure 67. Antitumor effect of the PD-L1×CTLA4 bibody molecule in mouse tumor models. DETAILED DESCRIPTION OF THE INVENTION

[0059] Hereinafter, examples of the present invention will be described with reference to specific 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 exhibit a "Y"-shaped tetrameric structure containing two identical heavy chains (H chains) and two identical light chains (L chains), also referred to as "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. The light chain variable regions are further divided into Vκ and Vλ due to the difference between κ and λ, and the corresponding light chain constant regions are divided into Cκ and Cλ, respectively. The variable region of an antibody is the primary site for recognizing and binding to its antigen; the variable region domains VH and VL and the constant region domains CH1 and CL together constitute the antigen-binding fragment (Fab). CH2 and CH3 constitute the fragment crystallizable (Fc), which exerts antibody functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), and are the primary regions that affect the serum half-life of the antibody.

[0061] As used herein, the term "heavy-chain antibody" or "HCAb" generally refers to a class of antibodies containing only heavy-chain dimers. Heavy-chain antibodies (HCAb) lacking light chains naturally occur in the serum of camelids and sharks. Camelid heavy-chain antibodies lack light chains compared to conventional H2L2 antibodies, and lack a CH1 region between the heavy-chain variable region and hinge region. They contain only one heavy-chain variable region (VHH) and two heavy-chain constant domains (CH2 and CH3); their basic structure is a heavy-chain dimer. VHH fragments of camelid heavy-chain antibodies differ from the VH characteristics of conventional antibodies. When cloned and expressed, the VHH structure retains the structural stability and antigen-binding activity of the original heavy-chain antibody, but their molecular weight is only approximately 13 Kda. Therefore, they are also called nanobodies or single-domain antibodies.

[0062] As used herein, the term "binding protein" or "antigen-binding protein" generally refers to a protein comprising an antigen-binding portion, optionally utilizing a conformational scaffold or framework portion that promotes antigen binding to the antigen-binding protein. Typically, the protein may comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both. The VH and VL regions can be further distinguished into highly variable regions called complementarity-determining regions (CDRs) interspersed with more conservative regions called framework 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 heavy and light chain variable regions contain the binding domains that interact with antigen. The three CDRs of VH are designated HCDR1, HCDR2, and HCDR3, respectively, and may also be designated VH CDR1, VH CDR2, and VH CDR3. The three CDRs of VL are designated LCDR1, LCDR2, and LCDR3, respectively, and may also be designated VL CDR1, VL CDR2, and VL CDR3. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, and / or dAb), immune complexes, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, as long as they exhibit the desired antigen-binding activity.

[0063] In this application, all CDR amino acid sequences are presented according to the Chothia definition. However, those skilled in the art can define antibody CDRs by various methods known in the art, such as the Kabat definition based on sequence variability (see Kabat et al., Immunological Protein Sequences, 5th ed., National Institutes of Health, Bethesda, MD (1991)) and the Chothia definition based on structural region location (see J Mol Biol 273:927-48, 1997). In some embodiments of the present invention, a combined definition rule, including the Kabat and Chothia definitions, can also be used to determine amino acid residues in variable domain sequences. The combined definition rule combines the scope of the Kabat and Chothia definitions and provides a broader range based on this definition. 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 regions defined by any of the above-mentioned 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 convention) to position bb (Chothia coding convention) from the N-terminus of the antibody light chain; Haa-Hbb can refer to the amino acid sequence from position aa (Chothia coding convention) to position bb (Chothia coding convention) from the N-terminus of the antibody heavy chain. For example, L24-L34 can refer to the amino acid sequence from positions 24 to 34 from the N-terminus of the antibody light chain according to the Chothia coding convention, and H26-H32 can refer to the amino acid sequence from positions 26 to 32 from the N-terminus of the antibody heavy chain according to the Chothia coding convention. Those skilled in the art should recognize that when encoding CDRs using Chothia coding, some positions are inserted (http: / / bioinf.org.uk / abs / ).

[0065] As used herein, 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 possible minor natural variations. Monoclonal antibodies generally have a high degree of specificity for a single antigenic site. Furthermore, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different antigenic clusters), each monoclonal antibody is directed against a single antigenic cluster. In addition to their specificity, an advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" refers to the character of the antibody obtained from a substantially homogeneous antibody population and is not intended to require production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced in hybridoma cells or by recombinant DNA techniques.

[0066] As used herein, the term "fully human-derived antibody" generally refers to an antibody in which a human has transferred all of the antibody-encoding genes into a genetically engineered antibody gene-deficient animal and then expressed in the animal. All parts of the antibody (including the antibody variable and constant regions) are encoded by human-derived genes. Fully human-derived antibodies can significantly reduce immune side effects in the human body caused by foreign antibodies. Methods for obtaining fully human-derived 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 the Harbour HCAb transgenic mouse (patent application WO2007 / 096779). The endogenous antibody heavy chain and light chain loci of this transgenic mouse are both knocked out or inactivated, rendering the mouse unable to produce antibodies. Human antibody heavy chain gene fragments (V, D, and J fragments) are then transferred into the mouse, and the mouse's own recombination and mutation mechanisms are used to produce antibodies having a human antibody gene sequence, the variable region of which is a 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 via 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 via its antigen-binding domain over random, 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] As used herein, the term "Fab" generally refers to the antigen-binding portion of a conventional antibody (e.g., IgG), comprising an antibody heavy chain variable region (VH), a light chain variable region (VL), and heavy chain constant region domains (CH1 and CL). In conventional antibodies, 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 variant 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 this application, the term "VH" generally refers to the VH domain of the heavy chain variable region of an antibody, i.e., it may be the heavy chain variable region VH of a conventional antibody (H2L2 structure) from a human or other animal, the heavy chain variable region VHH of a heavy chain antibody (HCAb structure) from 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] As used herein, the term "binding moieties" generally refers to any functional region of a protein capable of specifically binding to an antigen, and may be a "Fab" or a "VH" or other antigen-binding form (e.g., derivative protein structures such as lipid carrier proteins (lipocalins), neural cell adhesion molecules (NCAMs), fibronectins, and overlapping fragment anchor protein proteins (DARPins)).

[0072] As used herein, 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 to; for example, a conventional IgG antibody can simultaneously bind to two identical antigen molecules and has a valency of 2; and a Fab antibody has a valency of 1.

[0073] As used herein, the term "bispecific binding protein" generally refers to a binding protein that has two antigen-binding specificities. The two antigen-binding specificities may bind two different antigens or two different epitopes that bind the same antigen. Bispecific binding proteins can typically include bispecific antibodies and derivatives thereof.

[0074] As used herein, the term "multispecific binding protein" generally refers to a binding protein that has 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 the fluorescence intensity signal analyzed in a flow cell FACS method and its mathematical mean 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] As used herein, the term "PD-L1" generally refers to the programmed death-ligand 1 protein, its functional variants, and / or functional fragments. 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 number NP_054862 or UniProt accession number Q9NZQ7; an exemplary full-length cynomolgus monkey PD-L1 protein sequence can be found at NCBI accession number XP_005581836 or Uniprot accession number G7PSE7.

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

[0078] As used herein, the term "CD80" generally refers to cluster of differentiation protein 80 (also known as B7-1), its functional variants, and / or functional fragments thereof. 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] As used herein, the term "CTLA4" generally refers to cytotoxic T-lymphocyte-associated protein-4 (also known as CD152), its functional variants, and / or 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] As used herein, the term "HER2" generally refers to the acceptor tyrosine kinase erbB-2 (also known as ERBB2), its functional variants, and / or functional fragments thereof. 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 monkey HER2 sequence can be found at NCBI accession number XP_005584091.

[0081] As used herein, the term "B7H4" is generally meant to include V-Set domain T-cell activation inhibitor 1 (also known as 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 monkey B7H4 sequence can be found at NCBI accession number XP_005542249, and an exemplary full-length mouse B7H4 sequence can be found at Uniprot accession number Q7TSP5.

[0082] As used herein, the term "4-1BB" generally refers to tumor necrosis factor receptor superfamily member 9 (CD137, also known as the 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] As used herein, the term "BCMA" generally refers to tumor necrosis factor receptor superfamily member 17 (CD269, also known as 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] As used herein, the term "BAFF" generally refers to tumor necrosis factor ligand superfamily member 13B (CD257, also known as B-cell activating factor), functional variants thereof, and / or functional fragments thereof. 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] As used herein, the term "APRIL" generally refers to tumor necrosis factor ligand superfamily member 13 (CD256, also known as proliferation-inducing ligand), its functional variants, and / or functional fragments thereof. APRIL sequences are known in the art. For example, an exemplary full-length human APRIL sequence can be found at 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 against pMHC. The TCR is a heterodimer composed of two distinct transmembrane polypeptide chains. There are four types of peptide chains: α, β, γ, and δ. Depending on the combination of peptide chains, TCRs are classified as TCRαβ and TCRγδ. CD3 has distinct 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 induced by TCR antigen recognition is transmitted into the T cell by the CD3 peptide chain.

[0087] As used herein, 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] Example The present invention will be further described in the form of examples below, but the scope of the present invention is not limited to these 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 product instructions.

[0089] Example 1. Structural design of HCAb-based multispecific binding proteins This example presents 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, domains are linked using connecting peptides. In some structures, amino acid mutations are introduced into the heavy chain Fc region to adjust binding to Fc receptors and related effector functions 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, and each structure is further described below. In this example and elsewhere in the present invention, when referring to the number of polypeptide chains included in a molecular structure, this 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. Although the IgG antibody molecule itself is a tetrapeptide chain protein molecule containing two identical heavy chains and two identical light chains, the two different polypeptide chains are specifically referred to when describing its structural features. Valency refers to the number of antigen-binding sites in the molecular structure; for example, a conventional IgG antibody can simultaneously bind to two identical antigen molecules, and its valency is two. Tables 1-2 show connecting peptide sequences that can be used in the structural design of the present application.

[0091] [Table 2-1] TIFF0007620780000003.tif23129

[0092] [Table 2-2] TIFF0007620780000005.tif21130

[0093] Example 1.1. Fab-HCAb symmetric structure The present invention provides a method for constructing bispecific binding proteins 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 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_A-CH1; polypeptide chain 2 or the second polypeptide chain, also referred to as 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 may occur during association of VL_A and VH_B.

[0096] The VH_B of polypeptide chain 2 is linked to CH2 via a connecting peptide L2; L2 may be the hinge region of an IgG 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 of polypeptide chain 2 and the VH_B 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 the 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] The VH_B of polypeptide chain 2 is linked to CH2 via a connecting peptide L2; L2 may be the hinge region of an IgG 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 for constructing bispecific binding proteins 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, the VH_B and VH_A of polypeptide chain 2 are directly fused and linked, i.e., the length of L is 0. In another embodiment, the VH_B of polypeptide chain 2 is linked to the 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, the 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, the VH_B of polypeptide chain 1 is linked to the 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, the CL and VH_B of polypeptide chain 1 are directly fused or linked, i.e., the length of L is 0. In another embodiment, the CL of polypeptide chain 1 is linked to the 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 for constructing bispecific binding proteins 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, with VH_B being the heavy chain variable region of heavy chain antibody B and VH_C being 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 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-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 and 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 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-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, the VH_B and VH_C of polypeptide chain 2 are directly fused and linked, i.e., the length of L2 is 0. In another embodiment, the VH_B of polypeptide chain 2 is linked to the 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 for constructing bispecific binding proteins 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, with VH_B being the heavy chain variable region of heavy chain antibody B and VH_C being 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 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_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, the 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, the VH_B of polypeptide chain 1 is linked to the VL_A via a connecting peptide L1; L1 is a sequence listed in Table 1-2.

[0125] In one embodiment, the VH_B and VH_A of polypeptide chain 2 are directly fused and linked, i.e., the length of L2 is 0. In another embodiment, the VH_B of polypeptide chain 2 is linked to the 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 referred to as 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 referred to as 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, the 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, the VH_B of polypeptide chain 1 is linked to the 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 and 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 for constructing bispecific binding proteins 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. Different amino acid mutations have been introduced into the Fc regions of the two heavy chains, thereby reducing the formation of heavy chain homodimerization.

[0133] In one embodiment, h in the polypeptide chain comprising 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 for constructing bispecific binding proteins 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, with VH_B being the heavy chain variable region of heavy chain antibody B, and VH_C being 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 each have different amino acid mutations introduced, thereby reducing the formation of heavy chain homodimerization.

[0141] In one embodiment, h in the polypeptide chain comprising 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, the VH_C and VH_B of polypeptide chain 3 are directly fused and linked, i.e., the length of L is 0. In another embodiment, the VH_C of polypeptide chain 3 is linked to the 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, the VH_C and VH_B of polypeptide chain 3 are directly fused and linked, i.e., the length of L is 0. In another embodiment, the VH_C of polypeptide chain 3 is linked to the 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, the VH_C and VH_B of polypeptide chain 3 are directly fused and linked, i.e., the length of L is 0. In another embodiment, the VH_C of polypeptide chain 3 is linked to the 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 for constructing bispecific binding proteins 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 have been 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, and is 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 for constructing bispecific binding proteins 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, with VH_A and VL_A representing the heavy and light chain variable regions of antibody A, respectively. The VH end is derived from heavy chain antibody B or heavy chain antibody C, with VH_B representing the heavy chain variable region of heavy chain antibody B and VH_C representing 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. Different amino acid mutations have been introduced into the Fc regions of the two heavy chains, thereby reducing the formation of heavy chain homodimerization.

[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 between 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 connects the two heavy chain antibodies and has a sequence listed in Table 1. In another embodiment, the heavy chain variable regions of the two heavy chain antibodies are directly fused together, 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, and is 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 and 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 and 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, the VH_C and VH_B of polypeptide chain 2 are directly fused and linked, i.e., the length of L2 is 0. In another embodiment, the VH_C of polypeptide chain 2 is linked to the 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, the VH_C and VH_B of polypeptide chain 2 are directly fused and linked, i.e., the length of L2 is 0. In another embodiment, the VH_C of polypeptide chain 2 is linked to the 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 for constructing bispecific binding proteins 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 and light chain variable regions of antibody A, respectively. The VH end is derived from heavy chain antibody B, heavy chain antibody C, or heavy chain antibody D, where 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 each have different amino acid mutations introduced, thereby reducing the formation of heavy chain homodimerization.

[0182] In one embodiment, h in the polypeptide chain comprising 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 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, 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 and 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 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, 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 and 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, the VH_C and VH_B of polypeptide chain 3 are directly fused and linked, i.e., the length of L2 is 0. In another embodiment, the VH_C of polypeptide chain 3 is linked to the VH_B via a connecting peptide L2; L2 is a sequence listed in Table 1-2.

[0189] Example 2. Antibody sequence analysis, expression and 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 and high-frequency somatic mutation of germline V, D, and J gene fragments of the heavy chain gene cluster on a chromosome; the light chain variable domain sequence is derived from events such as genetic recombination and high-frequency somatic mutation of germline V and J gene fragments of the light chain gene cluster. Genetic recombination and high-frequency somatic mutation are major factors that increase antibody diversity. Although antibodies derived from the same germline V gene fragment may produce different sequences, they share a high overall 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 that result from genetic recombination based on the antibody variable domain sequence.

[0190] During translation and synthesis in cells, proteins or polypeptide amino acid chains may undergo chemical modifications known as post-translational modifications (PTMs). For antibodies, some PTM sites are highly conservative. For example, the conservative amino acid asparagine Asn at position 297 (EU numbering) in the constant domain of human IgG1 antibodies is typically glycosylated to form a glycan, and this glycan structure is crucial for antibody structure and related functional effects. However, when PTMs are present in the variable domain of an antibody, particularly in the antigen-binding region such as the CDR, the presence of these PTMs can significantly affect antigen binding and can also alter the physicochemical properties of the antibody. For example, glycosylation, deamidation, isomerization, and oxidation can all increase the instability or heterogeneity of antibody molecules, thereby increasing the difficulty and risk of antibody development. Therefore, avoiding potential PTMs is crucial for the development of therapeutic antibodies. With increasing experience, it has been discovered that some PTMs are highly correlated with the amino acid sequence composition, particularly the "pattern" of adjacent amino acid composition, allowing 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 (where the first position is asparagine, the second position is any amino acid other than proline, and the third position is serine or threonine). Amino acid sequence patterns that cause PTMs may be derived from germline gene sequences. For example, the human germline gene fragment IGHV3-33 naturally has the glycosylation pattern NST in the FR3 region; they may also be derived from somatic high-frequency mutations. For example, NGS or NLT may be glycosylation sites, NS may be a deamidation site, and DG may cause asparagine isomerization.

[0191] Amino acid mutations can disrupt the amino acid sequence pattern of a PTM, thereby reducing or eliminating the formation of a specific PTM. Different mutation design methods exist depending on the antibody sequence and PTM sequence pattern. One method is to replace a "hotspot" amino acid (e.g., N or S in NS mode) with a physicochemically similar amino acid (e.g., mutating N to Q). If the PTM sequence pattern originates from a high-frequency somatic mutation and does not exist in the germline gene sequence, another method is to replace the sequence pattern with the corresponding germline gene sequence. In actual operation, multiple mutation design methods can be used 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 derivatives) and techniques such as instantaneous transfection, expression, and affinity capture isolation. This method applies to target antibodies containing 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 to a nucleotide sequence using codon optimization; the encoded nucleotide sequence is synthesized and cloned into an expression vector compatible with the host cell. Plasmids encoding the antibody polypeptide chains are co-transfected into mammalian host cells at a specific ratio, and conventional recombinant protein expression and purification techniques are used to obtain recombinant antibodies with correct folding and polypeptide chain assembly. Specifically, FreeStyle® 293-F cells (Thermo, #R79007) were expanded in FreeStyle® F17 Expression Medium (Thermo, #A1383504). Before the start of instantaneous transfection, the cell concentration was adjusted to 6–8 × 10 5 The cells were adjusted to a concentration of 1.2 × 10 cells / ml and cultured at 37°C in an 8% CO2 rocker for 24 hours.6 The cell concentration was 30 ml. Plasmids encoding antibody polypeptide chains were mixed at a specific ratio. A total of 30 μg of plasmid (1 μg:1 ml) was dissolved in 1.5 ml of Opti-MEM serum-reduced medium (Thermo, #31985088) and sterilized by filtration through a 0.22 μm filter. 1.5 ml of Opti-MEM was then dissolved in 120 μl of 1 mg / ml PEI (Polysciences, #23966-2) and allowed to stand for 5 minutes. The PEI was slowly added to the plasmid and incubated at room temperature for 10 minutes. The plasmid-PEI mixture was then slowly added dropwise while rocking the culture flask. The culture was then cultured for 5 days at 37°C on a rocking bed with 8% CO2. Cell viability was monitored after 5 days. The culture was collected and centrifuged at 3300 x g for 10 minutes, after which the supernatant was collected. 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. The target protein was eluted with 0.1 M glycine, pH 3.5, and then neutralized with Tris-HCl, pH 8.0. Finally, the purified recombinant antibody solution was concentrated using an ultrafiltration tube (Millipore, #UFC901024) into PBS buffer or a buffer containing other components. Finally, the concentration was measured using a NanoDrop (Thermo, NanoDrop)® One, aliquots were separated, and the solution was stored in advance.

[0194] Example 2.3. Analysis of Protein Purity and Multimerization 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) system (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. The HPLC program was set to run the sample through the column in PBS buffer at a flow rate of 1.0 ml / min for a maximum of 25 minutes. The HPLC generated an analytical report reporting the retention times of different molecular size components within the sample.

[0195] Figure 65 shows the SDS-PAGE and SEC-HPLC analysis results of protein samples obtained after 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 showed 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 protein thermal stability. This method uses a real-time fluorescent quantitative PCR system to monitor changes in the fluorescence intensity of a dye bound to unfolded protein molecules, thereby reflecting the process of protein denaturation and, in turn, the thermal stability of the protein molecules. In this example, the DSF method was used to measure the thermal denaturation temperature (Tm) of protein molecules. 10 μg of protein was added to a 96-well PCR plate (Thermo, #AB-0700 / W), followed by 2 μL of 100x diluted SYPRO™ dye (Invitrogen, #2008138), and buffer was added to a final volume of 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 minutes, after which the temperature was gradually increased from 25°C to 95°C at a rate of 0.2°C / 0.2 minutes, and then decreased to 25°C at the end of the experiment. Data analysis was performed using Bio-Rad CFX Maestro software in FRET scan mode, and the Tm of the samples was calculated.

[0197] Example 2.5. Measurement of protein molecular stability and aggregation using Uncle Uncle (Unchained Labs) is a multifunctional, one-stop protein stability analysis platform that characterizes protein stability using total fluorescence, static light scattering (SLS), and dynamic light scattering (DLS) detection methods. Parameters such as melting temperature (Tm), aggregation temperature (Tagg), and particle size (diameter) can be simultaneously obtained for samples from the same group. In this example, the Uncle "Tm & Tag with optional DLS" application was selected and operated. A 9 μL sample was added to a UniTube and the temperature was gradually increased from 25°C to 95°C at a rate of 0.3°C / min. Four initial and final DLS measurements were taken, each lasting 5 seconds. After the experiment was completed, the Uncle analysis software calculated the Tm value of each sample using the centroid mean (BCM) formula; the Tag value was calculated based on the SLS fluorescence intensity curve (aggregation curve) at wavelengths of 266 nm or 473 nm; and the particle size and dispersity of the sample were calculated using DLS-related functions.

[0198] Example 3. PD-L1 x CTLA4 bispecific antibody Example 3.1. Background Programmed death receptor 1 (PD-1) is primarily expressed on immune cells such as T cells and has two ligands, programmed death ligand 1 (PD-L1) and PD-L2. PD-L1 is primarily expressed on antigen-presenting cells and several tumor cells. The interaction between PD-L1 and PD-1 reduces T cell activity, attenuates cytokine secretion, and exerts an immunosuppressive effect. PD-L1 protein expression has been detected in many human tumor tissues. The tumor microenvironment can induce PD-L1 expression on tumor cells, which favors tumor development and growth, induces apoptosis of antitumor T cells, and protects tumor cells from immune attack. PD-1 / PD-L1 pathway inhibitors block the binding of PD-1 and PD-L1, block the negative control signal, activate T cells, kill tumor cells, and suppress tumor growth. Therefore, 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 regulatory factor expressed on T cells. After binding to CD80 or CD86 on antigen-presenting cells, it blocks the costimulatory signal of CD28, thereby reducing T cell activity and exerting immunosuppressive effects. CTLA4-mediated suppression is often one of the reasons why tumor cells evade the immune system. Blocking the interaction of CTLA4 with its ligand can restore T cell activity and enhance antitumor capabilities.

[0200] Currently, blocking antibodies against PD-1, PD-L1, and CTLA4 have all demonstrated excellent antitumor effects in clinical trials, and several antibody drugs have been approved for sale. However, these drugs and treatments face numerous clinical challenges, such as low response rates. Therefore, clinical trials of combination dosing schemes based on blocking antibodies against PD-1, PD-L1, and CTLA4 are actively underway. Because CTLA4 inhibitors exhibit significant toxic side effects, current combination dosing schemes involving PD-1 / PD-L1 inhibitors and CTLA4 inhibitors generally involve lower doses of CTLA4 inhibitors. For example, in clinical trials investigating the combination of the anti-PD-1 antibody nivolumab and the anti-CTLA-4 antibody ipilimumab to treat colorectal cancer and renal cell carcinoma, the doses of nivolumab and ipilimumab were 3 mg / kg and 1 mg / kg, respectively. In clinical trials treating non-small cell lung cancer using the anti-PD-L1 antibody durvalumab in combination with the anti-CTLA-4 antibody tremelimumab, the doses of durvalumab and tremelimumab are 10-20 mg / kg and 1 mg / kg, respectively.

[0201] Meanwhile, we are increasingly understanding the biological mechanisms underlying tumor immunity. Blockade of the CTLA4 signaling pathway led to elevated PD-1 expression on tumor-infiltrating lymphocytes (TILs), and blocking the PD-1 signaling pathway enhanced CTLA4 expression on TILs (Rev Assoc Med Brass 2017;63(9):814-823). This suggests a potential mechanism for drug resistance caused by blocking a single co-inhibitory signaling pathway, and the simultaneous blocking of multiple co-inhibitory signaling pathways by combining these antibodies may have a synergistic effect on T cell activation. Furthermore, a recent study reported a novel mechanism for the interaction between the PD-1 and CTLA4 signaling pathways and explained the synergistic effect of PD-L1 and CTLA4 antibodies (Zhao et al., 2019, Immunity 51, 1059-1073).

[0202] Based on previous findings, bispecific antibodies targeting PD-L1 and CTLA4 simultaneously are believed to enhance antitumor efficacy and safety by utilizing more than one mechanism of action. First, PD-L1×CTLA4 bispecific antibodies activate T cells by blocking the CTLA4 signaling pathway and the PD-1 / PD-L1 signaling pathway at different stages; the cis-interaction between PD-L1 and CD80 results in better synergy with CTLA4. Second, PD-L1 is highly expressed in tumor tissue, and PD-L1×CTLA4 bispecific antibodies specifically neutralize CTLA4 inhibitory signals in the tumor microenvironment, activating T cells and reducing the toxic side effects of CTLA4 monoclonal antibodies due to nonspecific activation in the peripheral system. Third, PD-L1×CTLA4 bispecific antibodies can selectively retain Fc-mediated effects (e.g., ADCC) and specifically kill CTLA4-highly expressing Treg cells (e.g., Treg cells) in the tumor microenvironment through CTLA4, or specifically kill PD-L1-highly expressing tumor cells through PD-L1. Furthermore, as a single drug, bispecific antibodies offer advantages over combinations of two drugs in terms of cost efficiency and ease 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 multiple times with soluble recombinant human PD-L1 protein (NovoProtein, #CM06). After detecting a specific level of PD-L1-specific antibody titer in the mouse serum, spleen cells were removed 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 their binding ability to human PD-L1, their binding ability to cynomolgus monkey PD-L1, and their ability to inhibit the binding of PD-L1 to PD-1. Subsequently, the candidate antibody molecules were sequenced and optimized, yielding several variant sequences. The antibody VL and VH sequences were fused with the corresponding human kappa light chain constant region and IgG1 heavy chain constant region sequences to obtain recombinant fully human antibody molecules. Recombinant fully human IgG antibodies against PD-L1 are listed 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 harboring 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 contain only human antibody heavy chain variable domains and mouse Fc constant domains.

[0206] Harbour HCAb mice were immunized multiple times with soluble recombinant human CTLA4 protein (ACRO Biosystems, #CT4-H5229). After detecting a specific level of CTLA4-specific antibody titer in mouse serum, mouse spleen cells were removed, B cells were isolated, and CD138-positive slurry cells were selected using a mouse slurry cell sorting kit (Miltenyi, #130-092-530). Human VH genes were amplified from plasma cells using conventional molecular biology techniques, and the amplified human VH gene fragments were then constructed into the mammalian cell expression plasmid pCAG vector encoding the human IgG1 antibody heavy chain Fc region sequence. Mammalian host cells (e.g., human embryonic kidney cells HEK293) were transfected with the plasmid for expression, yielding whole-human HCAb antibody supernatants. Binding of the HCAb antibody supernatants to recombinant human CTLA4 protein was tested using ELISA, and positive HCAb antibodies were identified. These HCAb antibodies were further characterized, 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 a human IgG1 heavy chain Fc sequence and expressed to obtain fully human-derived recombinant HCAb antibody molecules. The anti-CTLA4 recombinant fully human-derived HCAb antibodies are listed in Table 39.

[0207] Example 3.3. Construction of a bispecific antibody molecule using an anti-PD-L1 IgG antibody and an anti-CTLA4 HCAb antibody In this example, various structural anti-PD-L1 x CTLA4 bispecific antibody molecules were constructed using 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.

[0208] In this and the following examples, the anti-PD-L1 positive control molecule is the anti-PD-L1 IgG monoclonal antibody PR000070, PR000416, or PR000265, which is the parent monoclonal antibody at the PD-L1 end of the PD-L1×CTLA4 bibody molecule. PR000070, PR000265, and PR000416 are derived from the same anti-PD-L1 monoclonal antibody. PR000070 and PR000265 are all human IgG1 subclass antibodies with the N297A mutation in their Fc regions, differing only by a single amino acid mutation in the heavy chain variable region VH. PR000265 and PR000416 are all human IgG1 subtype antibodies with the same Fab structure, differing only by the N297A mutation in the Fc region of PR000265.

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

[0210] Example 3.3.1. Construction of Fab-HCAb symmetrical molecules Using an anti-PD-L1 IgG antibody and an 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. Antibody molecule samples were then 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 symmetrical structure molecules Using anti-PD-L1 IgG antibodies and anti-CTLA4 heavy chain antibodies, PD-L1×CTLA4 bibody molecules with an IgG-VH tetravalent symmetric structure were designed according to the structure described in Example 1.2, and are summarized in Table 33. Antibody molecule samples were then 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 molecules Using an anti-PD-L1 IgG antibody and an 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 the results are summarized in Table 35. Antibody molecule samples were then prepared and analyzed according to the methods described in Example 2, and the results 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 molecules Using anti-PD-L1 IgG antibodies and anti-CTLA4 heavy chain antibodies, PD-L1×CTLA4 bivalent asymmetric antibody molecules were designed according to the structure described in Example 1.4, and are summarized in Table 37. Antibody molecule samples were then 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 Bibody 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 and CTLA4 monoclonal antibodies. 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 the PD-L1×CTLA4 dual antibody molecule to CTLA4.

[0228] Example 3.4.1. Binding to recombinant human CTLA4 extracellular domain protein The ability of antibody molecules to bind to human CTLA4 recombinant protein was tested using enzyme-linked immunosorbent assay (ELISA). Specifically, 2 μg / mL human CTLA4-His protein (ACRO Biosystems, #CT4-H5229) was first loaded into a 96-well plate at 100 μL per well and incubated overnight at 4°C. After rinsing three times with PBST buffer (PBS buffer containing 0.05% Tween-20), blocking solution (PBS buffer containing 5% nonfat dry milk) was added and incubated at 37°C for 1 hour. After rinsing three times with PBST buffer, 100 μL per well of antibody molecules diluted in a 5-fold gradient to a final concentration of 30 nM was added (for a total of eight concentrations). The antibody molecules were mixed uniformly and incubated at 37°C for 1 hour; hIgG1 isoform (CrownBio, #C0001) served as an isotype control. The plate was then rinsed three times with PBST buffer. Next, 100 μL / well of HRP-conjugated goat anti-human IgG Fc secondary antibody (Sigma, #A0170, 1:5000 dilution) was added and incubated at 37°C for 0.5 hours. The cells were then rinsed three times with PBST buffer. 100 μL / well of TMB color development solution was then added and incubated for 15 minutes. The reaction was stopped by adding stop solution. Optical absorbance (OD) readings were taken at 450 nM using an Enspire® multifunction reader (Perkin Elmer, Inc.). Data processing and plot analysis were performed using GraphPad Prism 8 application software, and binding curves and parameters such as EC50 values ​​were obtained using four-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 for the CTLA4 end of the PD-L1 x CTLA4 bibody molecule.

[0230] As shown in Figure 2 and Tables 3-13, the CTLA4-binding ability of the IgG-VH tetravalent symmetrical bibody molecule is related to the relative position of the anti-CTLA4 VH terminus relative to the anti-PD-L1 IgG. When the VH was located at the N-terminus of the IgG heavy chain (PR000300) or N-terminus of the light chain (PR000301), the CTLA4-binding EC50 value of the bibody molecule was similar to, and slightly superior to, that of the parent monoclonal antibody PR000184. When the VH was located at the C-terminus of the IgG heavy chain (PR000303), the CTLA4-binding EC50 value of the bibody molecule was 2.6-fold weaker than that of the parent monoclonal antibody PR000184; when the VH was located at the C-terminus of the IgG light chain (PR000302), the CTLA4-binding ability of the bibody molecule was significantly weakened. This indicates that the target-binding ability of the VH can be tuned by adjusting the relative position of the VH on the 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 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), which 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, at a cell density of 1 x 10 6The concentration was adjusted to 100 cells / mL. The cells were then seeded into a 96-well V-plate (Corning, #3894) at 100 μL / well, centrifuged at 4°C for 5 minutes, and the supernatant was discarded. The gradient-diluted antibody molecules were then added to the 96-well plate at 100 μL / well, mixed evenly, and diluted to a total of eight concentrations, with the antibody molecules diluted five-fold from a final concentration of 300 nM, or four-fold from a final concentration of 100 nM; hIgG1 isoform (CrownBio, #C0001) served as an identical control. The cells were placed at 4°C and incubated for 1 hour in the dark. The cells were then rinsed twice with 100 μL / well of pre-chilled FACS buffer (PBS buffer containing 0.5% BSA), centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was discarded. Next, 100 μL / well of fluorescent secondary antibody (Alexa Fluor 488 anti-human IgGFc, Biolegend, #409322, 1:1000 dilution) was added and incubated at 4°C for 1 hour in the dark. Cells were then rinsed twice with 200 μL / well of pre-chilled FACS buffer, then centrifuged at 500×g for 5 minutes at 4°C and the supernatant discarded. Finally, 200 μL / well of pre-chilled FACS buffer was added and the cells were resuspended. Fluorescence signal readings were obtained using a BD FACS CANTO II flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using FlowJov 10 software (FlowJo, LLC). Data processing and plot analysis were performed using GraphPad Prism 8 software, and four-parameter nonlinear fitting was used to obtain binding curves and parameters such as EC50 values ​​for antibody target cells.

[0233] 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 PD-L1 x CTLA4 bibody molecule. An ipilimumab analog was also used as a positive control molecule. The results are shown in Figures 3 and 4, Tables 3-14 and 3-15.

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

[0235] As shown in Figure 3(C), the EC50 value of the 2xVH-IgG hexavalent symmetric bibody molecule for CTLA4 binding was similar to that of the parent monoclonal antibody PR000184, but the maximum MFI was 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 were required to bind the same number of CTLA4 molecules than the parent monoclonal antibody. This indicates that the MFI value is related to the number of fluorescent secondary antibody molecules bound to the Fc region of the bibody molecule, and the maximum MFI of the bibody molecule was lower than that of the parent monoclonal antibody.

[0236] As shown in Figure 3(D), the Fab-Fc-VH asymmetric double 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 bibody 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 inhibitory activity of antibody molecules on the binding of human CTLA4 protein to its ligand B7-1 / CD80 was measured using enzyme-linked immunosorbent assay (ELISA). Specifically, 2 μg / mL human B7-1-Fc protein (ACRO Biosystems, #B71-H5259) was first applied to a 96-well plate at 100 μL / well and incubated overnight at 4°C. After rinsing three times with PBST buffer (PBS buffer containing 0.05% Tween-20), blocking solution (PBS buffer containing 5% nonfat dry milk) was added and incubated at 37°C for 1 hour. Subsequently, 90 μL / well of antibody molecules diluted in a 4-fold gradient to a final concentration of 180 nM were added, resulting in a total of seven concentrations. The mixture was mixed uniformly and incubated at 37°C for 20 minutes; hIgG1 isoform (CrownBio, #C0001) served as an isotype control. Biotinylated human CTLA4-Fc protein (ACRO Biosystems, #CT4-H82F3) was then added at 10 μL / well to a final concentration of 0.25 μg / ml and incubated at 37°C for 1 hour. The plate was then rinsed three times with PBST buffer. Labeled Precision Protein® StrepTactin-HRP Conjugate (Bio-RAD, #1610380, 1:4000 dilution) was then added at 100 μL / well and incubated at 37°C for 0.5 hours. The plate was then rinsed three times with PBST buffer. TMB color development solution was then added at 100 μL / well and the reaction was continued for 15 minutes. The reaction was then stopped by adding stop solution. Optical absorbance (OD) readings were taken at 490 nM using 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 for 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 bibody molecules were able to block the binding of CTLA4 protein to its ligand protein (inhibitory activity), achieving nearly 100% inhibition. However, the IC50 values ​​of their inhibitory activity were related to the relative position of the VH terminus of the anti-CTLA4 antibody to the anti-PD-L1 antibody IgG. When the VH was located at the N-terminus of the IgG heavy chain (PR000300) or N-terminus of the light chain (PR000301), the inhibitory activity of the bibody molecule against CTLA4 was similar to that of the parent monoclonal antibody PR000184. When the VH was located at the C-terminus of the IgG heavy chain (PR000303), the inhibitory activity of the bibody molecule against CTLA4 was slightly weaker than that of the parent monoclonal antibody PR000184. When the VH was located at the C-terminus of the IgG light chain (PR000302), the IC50 values ​​of the bibody molecule's inhibitory activity were slightly less than fourfold that of the parent monoclonal antibody PR000184. This indicates that the target-blocking ability of VH can be tuned by adjusting the relative position of VH on IgG.

[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 CTLA4-expressing cells to their 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 2 × 10 cells / mL and placed in FACS buffer (PBS buffer containing 2% FBS) at 37°C for 15 minutes. 200 μL of FACS buffer was added to a 96-well plate to close it, and after incubation at 37°C for 1 hour, the closing solution in the wells was discarded. Next, CHO-K1 / hCTLA4 cells were placed in a 96-well plate (2 × 10 5200 μL of 1000 cells / well were seeded into a 96-well plate and centrifuged at 500 g for 5 minutes at 4°C. The supernatant was discarded. The gradient-diluted antibody molecules were then added to a 96-well plate at 100 μL / well and mixed thoroughly. The antibody molecules were diluted to a total of eight concentrations, either three-fold starting from a final concentration of 200 nM or five-fold starting from a final concentration of 400 nM; hIgG1 isoform (CrownBio, #C0001) served as an isotype control. Next, 1 μg / mL of biotinylated ligand protein human B7-1-Fc (ACRO Biosystems, #B71-H82F2) was added to the 96-well plate at 100 μL / well and mixed thoroughly. The 96-well plate was placed at 4°C and incubated for 1.5 hours in the dark. The cells were then rinsed twice with 200 μL / well of pre-chilled FACS buffer, centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded. Next, 100 μL / well of fluorescent label (Streptavidin, Alexa Fluor® 488 conjugate, Thermo, #S11223, diluted 1:500) was added and incubated at 4°C for 1 hour, protected from light. The cells were then rinsed three times with 200 μL / well of pre-chilled FACS buffer, centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded. Finally, the cells were resuspended in 200 μL / well of pre-chilled FACS buffer. Fluorescence signal values ​​were read using a BD FACS CANTO II flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using FlowJov 10 software (FlowJo, LLC). Data processing and graph analysis were performed using the software GraphPad Prism 8, and the fluorescent signal value MFI was converted into the inhibition rate. Four-parameter nonlinear fitting was performed to obtain the inhibition curve, IC50 value, maximum inhibition rate, and other parameters.

[0246] 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 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 IgG-VH tetravalent symmetrical bibody molecules against CTLA4 is related to the relative position of the VH terminus of the anti-CTLA4 molecule relative to the anti-PD-L1 IgG. When the VH was located at the N-terminus of the IgG heavy chain (PR000300, PR001573) or the N-terminus of the light chain (PR000301, PR001574, PR001577), the IC50 values ​​of the bibody molecules for inhibition were similar to those of the parent monoclonal antibody PR000184, with all achieving nearly 100% inhibition, although PR000301 was slightly stronger than the parent monoclonal antibody PR000184. When the VH was located at the C-terminus of the IgG heavy chain (PR000303), the IC50 values ​​of the bibody molecules for inhibition were slightly weaker than those of the parent monoclonal antibody PR000184, with a maximum inhibition rate of approximately 83%. When the VH was located at the C-terminus of the IgG light chain (PR000302), the inhibitory activity of the dual antibody molecule was significantly weakened, with a maximum inhibition rate of only 49%. This indicates that the target blocking ability of the VH can be tuned by adjusting the relative position of the VH on the IgG. On the other hand, the different inhibitory activity of the same dual antibody molecule in blocking experiments against CTLA4 cells and CTLA4 protein may reflect the effect of spatial obstruction of the protein on the cell membrane.

[0248] As shown in Figure 6(C), the inhibitory activity of the 2xVH-IgG hexavalent symmetrical bibody 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 double antibody molecules PR001609 and PR001610 each contain only one CTLA4-binding domain, and therefore their inhibitory activity against CTLA4 is significantly weaker than that of the bivalent parent monoclonal antibody PR000184.

[0250] [Table 3-17]

[0251] Example 3.6. CTLA4-Mediated ADCC Effect This example investigates the antibody-dependent cell-mediated cytotoxicity (ADCC) of the PD-L1 x CTLA4 dual antibody molecule against 293F-hCTLA4 (CHEMPARTNER) cells, which highly express human CTLA4. In the first step, target cells were labeled with DELFIA BATDA (Perkin Elmer, #C136-100). The specific labeling method was as follows: 1 x 10 6 Target cells were labeled with 2 μL of DELFIA BATDA reagent and incubated at 37°C in a CO2 incubator for 20 minutes. They were then washed four times with PBS and centrifuged at 1000 rpm for 5 minutes. 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 into a 96-well plate (Corning, #3599) at 100 μL / well. Then, 50 μL / well of antibody molecules diluted in a 5-fold gradient with a final concentration of 0.8 nM was added (total of seven concentrations) and mixed evenly. hIgG1 isoform (CrownBio, #C0001) served as a matched control and incubated at 37°C in a 5% CO2 incubator for 10 minutes. 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 The cells were then added to a 96-well plate at 50 μL / well, with a 3:1 effector cell:target cell ratio. The cells were then incubated at 37°C in a 5% CO2 incubator for 2 hours. Finally, the cells were centrifuged at 500 g for 5 minutes, and 25 μL of the supernatant was removed from each well and added to a new 96-well detection plate. Next, 200 μL / well of DELFIA® Europium solution (Perkin Elmer, #C135-100) was added, and the plate was rocked at 250 rpm at room temperature for 15 minutes. Finally, the fluorescence was measured using 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 containing a mixture of 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 for 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 Figure 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 antibody molecules to bind to human PD-L1 recombinant protein was tested using enzyme-linked immunosorbent assay (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 per well and incubated overnight at 4°C. After rinsing three times with PBST buffer (PBS buffer containing 0.05% Tween-20), blocking solution containing 2% BSA was added and incubated at 37°C for 1 hour. The blocking solution was then discarded and rinsed three times with PBST buffer. Eight concentrations of antibody molecules diluted five-fold to a final concentration of 30 nM were then added at 100 μL per well. The antibody molecules were mixed uniformly and incubated at 37°C for 1 hour; hIgG1 isoform (CrownBio, #C0001) was used as the matched control. The plate was then rinsed three times with PBST buffer. Next, 100 μL / well of HRP-conjugated goat anti-human IgG Fc secondary antibody (Sigma, #A0170, 1:5000 dilution) was added and incubated at 37°C for 0.5 hours. The plate was then rinsed three times with PBST buffer. 100 μL / well of TMB color development solution was then added and the reaction was allowed to proceed for 15 minutes. Finally, a stop solution was added to terminate the reaction. Optical absorbance (OD) readings were taken 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 for the PD-L1 end of the PD-L1xCTLA4 bibody molecule.

[0260] As shown in Figure 7 and Tables 3-19, the ability of the IgG-VH tetravalent symmetrical bibody 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 of CHO-K1 cells CHO-K1 / hPDL1, which highly express human PD-L1, to tumor cells MDA-MB-231, which 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 flow cytometry (FACS). Specifically, cells were digested and resuspended in complete medium; the cell density was 1 × 10 6The concentration was adjusted to cells / mL. The cells were then seeded into a 96-well V-plate (Corning, #3894) at 100 μL / well, centrifuged at 4°C for 5 minutes, and the supernatant was discarded. The gradient-diluted antibody molecules were then added to the 96-well plate at 100 μL / well and mixed evenly. The antibody molecules were diluted to a total of eight concentrations, starting from a final concentration of 60 nM in a 5-fold gradient, or starting from a final concentration of 300 nM in a 5-fold gradient, or starting from a final concentration of 100 nM in a 4-fold gradient; hIgG1 isoform (CrownBio, #C0001) served as an identical control. The cells were placed at 4°C and incubated for 1 hour in the dark. The cells were then rinsed twice with 100 μL / well of pre-chilled FACS buffer (PBS buffer containing 0.5% BSA). The mixture was centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was discarded. Next, 100 μL / well of fluorescent secondary antibody (Goat human IgG (H+L) Alexa Fluor 488 conjunction, Thermo, #A11013, 1:1000 dilution) was added and incubated at 4°C for 1 hour in the dark. Cells were then rinsed twice with 200 μL / well of pre-chilled FACS buffer, then centrifuged at 500 g for 5 minutes at 4°C and the supernatant discarded. Finally, 200 μL / well of pre-chilled FACS buffer was added to resuspend the cells. Fluorescence signal readings were read using a BD FACS CANTO II flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using FlowJov 10 software (FlowJo, LLC). Data processing and plot analysis were performed using GraphPad Prism 8 software, and four-parameter nonlinear fitting was used to obtain binding curves and parameters such as EC50 values ​​for antibody target cells.

[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 for the PD-L1 end of the PD-L1 x CTLA4 biantibody molecule. The results are displayed in Figure 8, Figure 9, Tables 3-20, and Table 3-21.

[0264] As shown in Figures 8(A-B) and 9, the PD-L1 binding ability of the IgG-VH tetravalent symmetrical bibody molecules was similar to that of the corresponding parental monoclonal antibodies PR00070 or PR000416, with very similar PD-L1 binding EC50 values. The MFI maximum was slightly lower than that of the parental monoclonal antibodies only when the VH was located at the N-terminus of the IgG light chain (PR000301, PR001574, PR001577).

[0265] As shown in Figure 8(C), the PD-L1 binding ability of the 2xVH-IgG hexavalent symmetrical bibody molecule was reduced compared to the parent monoclonal antibody PR000416, demonstrating that the simultaneous attachment of VH to the N-terminus of the IgG heavy and light chains significantly affected the function of the IgG Fab end.

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

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

[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 inhibitory activity of the antibody molecules on 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 minutes at 37°C. 200 μL of blocking buffer was added to a 96-well plate and incubated at 37°C for 1 hour, after which the blocking solution in the wells was discarded. Next, CHO-K1 / hPDL1 cells were plated in a 96-well plate (1 × 10 5The cells (cells / well) were seeded at 50 μL / well. The gradient-diluted antibody molecules were then added to a 96-well plate at 100 μL / well and mixed thoroughly. The antibody molecules were diluted 5-fold from a final concentration of 100 nM to a total of eight concentrations. hIgG1 isoform (CrownBio, #C0001) served as an identical control. The plate was then incubated at 4°C, protected from light, for 0.5 hours. PBS buffer was then added at 100 μL / well, and the plate was centrifuged at 500 g for 5 minutes at 4°C. The supernatant was discarded. Next, 1 μg / mL biotinylated ligand protein human PD-1 protein (ACRO Biosystems, #PD1-H82F2) was added to the 96-well plate at 50 μL / well and mixed thoroughly. The 96-well plate was then placed at 4°C, protected from light, and incubated for 0.5 hours. The cells were then rinsed twice with 100 μL / well of pre-chilled PBS buffer, followed by centrifugation at 500 g for 5 minutes at 4°C and discarding the supernatant. Next, 100 μL / well of fluorescent secondary antibody (PE Streptavidin, BD Biosciences, #554061, 1:200 dilution) was added and incubated at 4°C for 0.5 hours, protected from light. The cells were then rinsed twice with 200 μL / well of pre-chilled PBS buffer, followed by centrifugation at 500 g for 5 minutes at 4°C and discarding the supernatant. Finally, the cells were resuspended in 200 μL / well of pre-chilled FACS buffer. Fluorescence signal readings were read using a BD FACS CANTO II flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using FlowJov 10 software (FlowJo, LLC). Data processing and graph analysis were performed using the software GraphPad Prism 8, and the fluorescent signal value MFI was converted into the inhibition rate. Four-parameter nonlinear fitting was performed to obtain the inhibition curve, IC50 value, maximum inhibition rate, and other parameters.

[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 for the PD-L1 end of the PD-L1 x CTLA4 biantibody molecule. The results are shown in Figure 10 and Tables 3-22.

[0273] As shown in Figure 10 (A-B), the PD-L1 inhibitory activity of the IgG-VH tetravalent symmetrical bibody molecules was similar to that of the corresponding parent monoclonal antibodies PR000070 and PR000416, with IC50 values ​​and maximum inhibitory rates close to those of the parent monoclonal antibodies. Only when the VH was located at the N-terminus of the IgG light chain (PR001574, PR001577) did the IC50 value decrease by 2-3 times compared to 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 values ​​were 4-6 times weaker than those of the parent monoclonal antibody, but a similar maximum inhibition rate was achieved.

[0275] As shown in Figure 10(D), the inhibitory activity of the Fab-HCAb symmetrical 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 antibody; the corresponding IC50 values ​​are 4-5 times weaker than those of the parent monoclonal antibody, but they can achieve similar maximum inhibitory rates.

[0277] [Table 3-22]

[0278] Example 3.9. Superantigen SEB stimulation experiments This example was designed to investigate the activating effect of the PD-L1 x CTLA4 bibody molecule on peripheral blood mononuclear cells (PBMC). First, isolated human PBMC (MT-BIO) cells were plated in 100 μL / well (1 × 10 5 The cells were added to a 96-well plate (Corning, #3799) at a concentration of 100 μL / well. The final antibody concentrations were 100 nM, 10 nM, 1 nM, 0.1 nM, or 0.01 nM. Alternatively, two antibodies could be mixed at a specific ratio and added to two combined wells. hIgG1 isoform (CrownBio, #C0001) served as an identical control. The plate was then incubated at 37°C for 30 minutes. The superantigen Staphylococcal enterotoxin B (SEB) was then added at 50 μL / well to a final concentration of 100 ng / mL or 10 ng / mL. The plate was then incubated at 37°C in a 5% CO2 incubator for 96 or 120 hours, after which the supernatant was collected. Second, the supernatant was collected and the IL-2 concentration was measured using an IL-2 ELISA kit (Biolegend, #431805). Refer to the kit's instructions for the procedure. 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, with SEB stimulation, anti-CTLA4 monoclonal antibody, anti-PD-L1 monoclonal antibody, and PD-L1 x CTLA4 bibody molecule were all able to promote IL-2 production by T cells to different extents, and their ability to activate T cells was related to their structure.

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

[0282] As shown in Figure 13, the Fab-HCAb symmetrical biantibody molecule can activate T cells to produce IL-2. Furthermore, PR000404 has stronger T cell activation ability than PR000403. PR000404 and PR000403 have similar structures, but the difference is that PR000404 has a normal human IgG1 Fc region, whereas the Fc region of PR000403 contains three point mutations (L234A, L235A, and P329G) that attenuate Fc-related effector functions. It is speculated that PR000404 can eliminate Treg cells through CTLA4-mediated ADCC, further enhancing 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 combination (the same donor PBMCs were used in the same experiment, allowing for comparison of absolute IL-2 levels).

[0284] As shown in Figure 15, the dual antibody molecules PR000303 (IgG_HC-VH structure, i.e., the C-terminus of the heavy chain in IgG VH) and PR000404 (Fab-HCAb structure) have weaker T-activating abilities than 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 making it suitable for the current clinical doses of combination drugs.

[0285] Example 3.10. Mixed Lymphocyte Reaction (MLR) This example investigates the T cell activation effect of the PD-L1 × CTLA4 dual antibody molecule using a mixed lymphocyte reaction (MLR). First, primary donor PBMC cells (MT-BIO) were treated with recombinant human interleukin-4 (IL-4, R&D Systems, #204-GMP) and recombinant human GM-CSF (R&D Systems, #215-GM) for 6 days, followed by immature human CD14 T cells. + Dendritic cells (iDC cells) were obtained. Subsequently, 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 10:1 ratio (1 × 10 5 1 x 10 / well T cells 4 (mDC cells / well). Then, 100 μL of antibody molecules were added at different concentrations to each well. The final antibody concentrations were 100 nM, 10 nM, 1 nM, 0.1 nM, or 0 nM. Alternatively, two antibodies could be mixed at a specific ratio and added to two combined wells. hIgG1 isoform (CrownBio, #C0001) served as an IgG control. The cells were incubated at 37°C in a 5% CO2 culture chamber for 5 days. In the fourth step, the supernatants on days 3 and 5 were collected, respectively. IL-2 concentrations in the supernatant on day 3 were detected using an IL-2 ELISA kit (Thermo, #88-7025-88) and IFN-γ concentrations in the supernatant on day 5 were detected using 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 exhibited stronger T cell activation ability and were better able to promote IL-2 and IFN-γ production than anti-CTLA4 monoclonal antibody, anti-PD-L1 monoclonal antibody, and their 1:1 concentration combination.

[0287] As shown in Figure 17, when the anti-CTLA4 VH was located at the C-terminus of the IgG heavy or light chain, the dual antibody molecules PR000302 and PR000303 exhibited 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), resulting in tumors with an average size of approximately 100 mm. 3 Once tumor volume reached 1000 mg / kg, mice were grouped by tumor volume and administered specific doses and frequencies of antibody drugs diluted in PBS via intraperitoneal injection (ip). The administration scheme used in this study was twice weekly for a total of eight doses (BIW x 4 weeks). PBS was used as a blank control, while the dual antibody treatment group received 3 mg / kg PR001573 and the combined treatment group received 3 mg / kg ipilimumab plus 3 mg / kg atezolizumab. Tumor volume and mouse 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 treatment group (3 mg / kg ipilimummab + 3 mg / kg atezolizumab) completely disappeared, with a tumor inhibition rate (TGI) of 100%, a significant difference compared to the control group (P = 0.03). The tumors in the dual antibody treatment group (3 mg / kg PR001573) completely disappeared, with a TGI of 100%, a significant difference compared to the control group (P = 0.03). Mice in the treatment group tolerated the treatment well throughout the entire experiment, with no significant changes in body weight. The efficacy of 3 mg / kg PR001573 was comparable to that of the combination of ipilimummab and atezolizumab at the same dose.

[0291] Example 3.12. Summary In this example, anti-PD-L1 x CTLA4 bispecific antibody molecules with various structures were constructed using the antigen-binding domain Fab of an anti-PD-L1 IgG antibody and the antigen-binding domain VH of an anti-CTLA4 HCAb antibody. This demonstrates the flexibility of constructing bispecific antibody molecular structures based on HCAbs, allowing the functional activities of the PD-L1 and CTLA4 ends to be tuned through parameters such as different structural types, relative positions, and valencies, and further enabling the design of different activity combinations to meet the needs of different clinical combination doses.

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

[0293] Furthermore, for example, when the VH of an anti-CTLA4 antibody is attached to the C-terminus of an anti-PD-L1 IgG or in a Fab-HCAb configuration, the PD-L1 ends of the bibody molecules PR000302, PR000303, and PR000404 retain almost all of 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 ranging from 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 regulatory factor expressed on T cells. After binding to CD80 or CD86 on antigen-presenting cells, it blocks the costimulatory signal of CD28, thereby reducing T cell activity and exerting immunosuppressive effects. CTLA4-mediated inhibitory mechanisms are often responsible for tumor cell evasion from the immune system. Blocking the interaction between CTLA4 and its ligands can restore T cell activity and enhance antitumor capabilities. Ipilimumab monoclonal antibody (trade name Yervoy®) was the first commercially available anti-CTLA4 monoclonal antibody drug and ushered in the era of tumor immunotherapy. Although ipilimumab has demonstrated relatively good therapeutic efficacy in the treatment of late-stage melanoma, it also causes a relatively high rate of immune-related side effects, with a 50% incidence of Grade 3-5 adverse events, which has severely impacted 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, lower doses of CTLA4 inhibitors are usually selected, regardless of whether they are ipilimumab or tremelimummab.

[0295] To reduce the toxic side effects of CTLA4 inhibitors, one approach to try is to target the CTLA4 inhibitors to tumor tissue, thereby confining the associated T cell-mediated response to the tumor microenvironment and reducing the risk of cytokine release syndrome. While this targeted delivery can be achieved by injecting the CTLA4 inhibitor into the tumor, intratumoral injection carries the risk of surgical manipulation and is limited to a small, accessible tumor tissue. This example provides a targeted delivery method using antibodies that recognize tumor-associated antigens to redirect the CTLA4 inhibitor to a specific tumor microenvironment, thereby relieving the immunosuppressive signals of T cells in the tumor microenvironment and restoring T cell function.

[0296] In this example, a bispecific antibody targeting both HER2 and CTLA4 was constructed, improving antitumor efficacy and safety through multiple 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 signaling) while activating T cells by blocking the CTLA4 signaling pathway. Second, the HER2×CTLA4 bispecific antibody enriches in tumor tissues with high HER2 expression and specifically neutralizes CTLA4 inhibitory signals in the tumor microenvironment, activating T cells and reducing the toxic side effects of CTLA4 monoclonal antibodies due to nonspecific activation in the peripheral system. Third, the HER2×CTLA4 bispecific antibody selectively retains Fc effector functions (e.g., ADCC), specifically killing CTLA4-highly expressing T cells (e.g., Treg cells) in the tumor microenvironment through CTLA4, or specifically killing HER2-highly expressing tumor cells through HER2. Furthermore, as a single pharmaceutical product, a bispecific antibody can have advantages over a combination of two pharmaceutical products in terms of economy and ease of administration.

[0297] Example 4.2. Obtaining 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 (trastuzumamab analog) or PR000672 (pertuzumamab analog), which are the parent monoclonal antibodies 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 biantibody molecule.

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

[0303] [Table 4-1]

[0304] [Table 4-2]

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

[0306] [Table 4-3]

[0307] [Table 4-4]

[0308] Example 4.3.3. Construction of IgG-VH(2) hexavalent symmetrical molecule Using an anti-HER2 IgG antibody and an anti-CTLA4 heavy chain antibody, a HER2xCTLA4 bibody molecule with an IgG-VH(2) hexavalent symmetric structure was designed according to the structure described in Example 1.3, and the results are summarized in Tables 4-5. Antibody molecule samples were then prepared and analyzed according to the method described in Example 2, and the results 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. Antibody molecule samples were then 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 an anti-HER2 IgG antibody and an anti-CTLA4 heavy chain antibody, a HER2×CTLA4 bibody molecule with an asymmetric Fab-Fc-VH (n,n={1,2}) structure was designed according to the structures described in Examples 1.5 and 1.6, and the results are summarized in Table 49. Antibody molecule samples were then prepared and analyzed according to the method described in Example 2, and the results are summarized in Tables 4-10.

[0315] [Table 4-9]

[0316] [Table 4-10]

[0317] Example 4.3.6. Sequence listing of HER2xCTLA4 biantibody 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, as well as parent monoclonal antibody molecules and control molecules, such as the corresponding HER2 monoclonal antibody and CTLA4 monoclonal antibody. 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] TIFF0007620780000041.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 in F12K medium at a cell density of 2 × 10 6The concentration of antibody molecules was adjusted to 100 cells / mL. Next, CHO-K1 / hCTLA4 cells were seeded into a 96-well V-plate (Corning, #3894) at 100 μL per well, centrifuged at 4°C for 5 minutes, and the supernatant was discarded. The gradient-diluted antibody molecules were then added to the 96-well plate at 100 μL per well and mixed evenly. The antibody molecules were diluted 5-fold from a final concentration of 300 nM to a total of 8 concentrations, or 4-fold from a final concentration of 300 nM to a total of 12 concentrations; hIgG1 isoform (CrownBio, #C0001) served as an imipenem control. The cells were then incubated at 4°C for 1 hour in the dark. The cells were then rinsed twice with 100 μL per well of pre-chilled FACS buffer (PBS buffer containing 0.5% BSA). The plate was then centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was discarded. Next, 100 μL / well of fluorescent secondary antibody (Goat human IgG (H+L) Alexa Fluor 488 conjunction, Thermo, #A11013, 1:1000 dilution) was added and incubated at 4°C for 1 hour in the dark. Cells were then rinsed twice with 200 μL / well of pre-chilled FACS buffer, then centrifuged at 500 g for 5 minutes at 4°C and the supernatant discarded. Finally, 200 μL / well of pre-chilled FACS buffer was added to resuspend the cells. Fluorescence signal readings were read using a BD FACS CANTO II flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using FlowJov 10 software (FlowJo, LLC). Data processing and plot analysis were performed using GraphPad Prism 8 software, and four-parameter nonlinear fitting was used to obtain binding curves and parameters such as EC50 values ​​for antibody target cells.

[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 Figure 18(A), both Fab-HCAb symmetrical bibody molecules can bind to CTLA4. These molecules have a similar structure, combining the Fab domain of the anti-HER2 monoclonal antibody PR000210 with the VH domain of the anti-CTLA4 HCAb monoclonal antibody PR000184. The subtle differences are the first connecting peptide and hinge region of the connecting Fc, as well as different Fc types or mutations. Therefore, these bibody molecules have similar CTLA4-binding abilities. Their CTLA4-binding EC50 values ​​are similar to or only 1.5- to 3-fold lower than those of the parent monoclonal antibody PR000184, but their maximum binding signals (MFI max) on FACS are lower than those 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 less able to bind CTLA4 molecules than in the free end, and for the same number of available CTLA4 molecules, its binding efficacy 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 symmetric bibody molecules to bind to CTLA4 varies slightly depending on their specific molecular structure. Based on the relative positions of the anti-CTLA4 VH termini relative to the anti-HER2 IgG in the bibody structure, bibody molecules with such structures can be further classified into three types: VH at the C-terminus of the IgG heavy chain (PR000539, PR000540, PR000714, PR000715), VH at the N-terminus of the IgG heavy chain (PR001583, PR001586, PR001588, PR001591), and VH at the N-terminus of the IgG light chain (PR001584, PR001589). When the VH is located at the C-terminus of the IgG heavy chain, the EC50 values ​​for CTLA4 binding by these dual antibody molecules are similar to those of the parent monoclonal antibody PR000184, but the maximum MFI values ​​on FACS are slightly lower than those 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 ​​for CTLA4 binding by these dual antibody molecules are slightly weaker than those of the parent monoclonal antibody PR000184, but the maximum MFI values ​​on FACS are stronger than those of the parent monoclonal antibody PR000184. This indicates that the number of VHs that bind to target molecules and the binding efficacy 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 symmetrical bibody molecules can bind to CTLA4. These molecules have similar structures, each combining the VH domains of the anti-HER2 monoclonal antibody PR000210 and the anti-CTLA4 HCAb monoclonal antibody PR000184. The subtle differences are the different first and second connecting peptides and different Fc types or mutations. The ability of these bibody molecules to bind to CTLA4 is similar to that of the parent monoclonal antibody PR000184, with the exception of PR001582, with similar EC50 and MFI maxima.

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

[0328] As shown in Figure 18 (I-J), the CTLA4-binding ability of the Fab-Fc-VH (n, n = 1, 2) asymmetric bibody molecules is related to the number of VH domains of the parent monoclonal antibody PR000184 included in the structure. Because the bibody molecules PR000916 and PR000917 each contain only one CTLA4-binding domain, their CTLA4-binding ability is significantly weaker than that of the bivalent parent monoclonal antibody PR000184. The bibody molecule PR002672 contains two tandemly connected CTLA4-binding domains, and its CTLA4-binding ability is similar to that of the parent monoclonal antibody PR000184. This structure demonstrates the flexibility of the VH domain-containing bibody molecules, allowing the target-binding ability to be adjusted 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 This example was conducted to investigate the activity of the HER2xCTLA4 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 on the binding of human CTLA4 protein and its ligand, B7-1 / CD80 protein, was measured using enzyme-linked immunosorbent assay (ELISA). Specifically, 2 μg / mL human B7-1-Fc protein (ACRO Biosystems, #B71-H5259) was first applied to a 96-well plate at 100 μL / well and incubated overnight at 4°C. After rinsing three times with PBST buffer (PBS buffer containing 0.05% Tween-20), blocking solution (PBS buffer containing 5% nonfat dry milk) was added and incubated at 37°C for 1 hour. Seven concentrations of antibody molecules, diluted threefold to a final concentration of 200 nM, were then added at 90 μL / well. The mixture was mixed uniformly and incubated at 37°C for 20 minutes; hIgG1 isoform (CrownBio, #C0001) served as the same type control. Biotinylated human CTLA4-Fc protein (ACRO Biosystems, #CT4-H82F3) was then added at 10 μL / well to a final concentration of 0.25 μg / ml and incubated at 37°C for 1 hour. The plate was then rinsed three times with PBST buffer. Labeled Precision Protein® StrepTactin-HRP Conjugate (Bio-RAD, #1610380, 1:4000 dilution) was then added at 100 μL / well and incubated at 37°C for 0.5 hours. The plate was then rinsed three times with PBST buffer. TMB color development solution was then added at 100 μL / well and the reaction was continued for 15 minutes. The reaction was then stopped by adding stop solution. Optical absorbance (OD) readings were taken at 490 nM using an Enspire® multifunction reader (Perkin Elmer, Inc.). Data processing and graph analysis were performed using the application software GraphPad Prism 8, and parameters such as inhibition curves and 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 activity was comparable to or slightly weaker than that of the parent monoclonal antibody PR000184, showing similar maximum OD differences, but the IC50 values ​​were only 1.5 to 2.1 times lower.

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

[0335] As shown in Figure 19 (EF), the inhibitory activity of the IgG-VH-VH hexavalent symmetrical bibody molecule is similar to or slightly better than that of the parent monoclonal antibody.

[0336] As shown in Figure 19 (GH), the inhibitory activity of the 2xVH-IgG hexavalent symmetrical bibody molecule is similar to 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 molecules to inhibit the binding of CTLA4-expressing cells to their 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 plated in a 96-well plate (3 × 10 5100 μL of 1000 nM cells / well were seeded into a 96-well plate and centrifuged at 500 g for 5 minutes at 4°C. The supernatant was discarded. The gradient-diluted antibody molecules were then added to a 96-well plate at 100 μL / well and mixed thoroughly. The antibody molecules were diluted in a 3-fold gradient starting from a final concentration of 200 nM, resulting in a total of eight concentrations. hIgG1 isoform (CrownBio, #C0001) served as an isotype control. Next, 3 μg / mL of 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 thoroughly. The 96-well plate was placed at 4°C and incubated for 1 hour, protected from light. 200 μL / well of pre-chilled FACS buffer was then added to rinse the cells twice. The plate was then centrifuged at 500 g for 5 minutes at 4°C and the supernatant was discarded. Next, 100 μL / well of fluorescent label (Streptavidin, Alexa Fluor® 488 conjugate, Thermo, #S32354, 1:1000 dilution) was added and incubated at 4°C for 1 hour in the dark. Cells were then rinsed three times with 200 μL / well of pre-chilled FACS buffer, centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded. Finally, 200 μL / well of pre-chilled FACS buffer was added to resuspend the cells. Fluorescence signal values ​​were read using a BD FACS CANTO II flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using FlowJov 10 software (FlowJo, LLC). Data processing and plotting were performed using GraphPad Prism 8 software. Fluorescence signal values ​​(MFI) were converted to percent inhibition, and four-parameter nonlinear fitting was used to obtain inhibition curves, IC50 values, and parameters such as maximum inhibition.

[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 anti-CTLA4 VH is located at the C-terminus of the anti-HER2 IgG heavy chain, 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 tandemly arranged CTLA4-binding domains, and its inhibitory activity is approximately 4-fold lower than that of the bivalent parent monoclonal antibody, but can achieve approximately 100% inhibition.

[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 antibody 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 6The solution was adjusted to 100 cells / mL; 100 μL cells / well were then seeded into a 96-well V-plate (Corning, #3894), centrifuged at 4°C for 5 minutes, and the supernatant was discarded. Subsequently, 100 μL / well of antibody molecules diluted in a 5-fold gradient to a final concentration of 100 nM were added, and the eight concentrations were uniformly mixed. hIgG1 isoform (CrownBio, #C0001) was used as an identical control. The cells were placed at 4°C and incubated for 1 hour in the dark. Then, the cells were centrifuged at 4°C for 5 minutes, and the supernatant was discarded. The cells were then rinsed twice with 200 μL / well of pre-chilled FACS buffer (PBS buffer containing 0.5% BSA), followed by centrifugation at 500 g for 5 minutes at 4°C and the supernatant was discarded. Next, 100 μL of fluorescent secondary antibody (Goat human IgG (H+L) Alexa Fluor 488 conjunction, Thermo, #A11013, 1:1000 dilution) was added per well and incubated at 4°C for 1 hour in the dark. Cells were then rinsed twice with 200 μL of pre-chilled FACS buffer per well, then centrifuged at 500 g for 5 minutes at 4°C and the supernatant discarded. Finally, 200 μL of pre-chilled FACS buffer was added per well to resuspend the cells. Fluorescence signal readings were obtained using a BD FACS CANTO II flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using FlowJov 10 software (FlowJo, LLC). Data processing and plot analysis were performed using GraphPad Prism 8 software, and four-parameter nonlinear fitting was used to obtain binding curves and parameters such as EC50 values ​​for antibody target cells.

[0345] In this example, the positive control molecules were the 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 HER2-binding ability of the Fab-HCAb symmetrical bibody molecule was similar to that of the parent monoclonal antibody PR000210, as indicated by similar EC50 values ​​and maximum MFI values, indicating that the structure at the Fab end of the anti-HER2 moiety of the Fab-HCAb symmetrical bibody molecule can effectively retain its HER2-binding ability.

[0347] As shown in Figure 21 (B-E), the ability of IgG-VH tetravalent symmetrical bibody molecules to bind to HER2 varies slightly depending on their specific molecular structure. Based on the relative positions of the anti-CTLA4 VH termini relative to the anti-HER2 IgG in the bibody structure, bibody molecules with such structures are further classified into three types: VH at the C-terminus of the IgG heavy chain (PR000539, PR000540), VH at the N-terminus of the IgG heavy chain (PR001583, PR001586, PR001588, PR001591), and VH at the N-terminus of the IgG light chain (PR001584, PR001974, PR001589). The EC50 values ​​indicate that when VH is located at the C-terminus of the IgG heavy chain, this structure retains its HER2-binding ability well, indicating that the VH terminus has no obvious effect on the Fab terminus of the IgG; when VH is located at the N-terminus of either the heavy or light IgG chain, the HER2-binding ability of the dual antibody molecule is 1.5- to 2.5-fold lower than that of the corresponding parent monoclonal antibody.

[0348] As shown in Figu...

Claims

1. A binding protein comprising at least two protein functional domains, the binding protein comprises a protein functional region A and a protein functional region B, the protein functional region A being a CD3-binding fragment of a Fab or scFv structure, the protein functional region B being a BCMA-binding fragment of a VH structure, and the number of each of the protein functional regions A and B being 1 or more, the BCMA binding fragment 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, 78 and 136, respectively; the CD3-binding fragment comprises a heavy chain variable region and a light chain variable region, in which the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 having 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 having the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128, respectively; and the binding protein is selected from one of the following (i)-(ii): (i) the binding protein has three polypeptide chains including one polypeptide chain 1, one polypeptide chain 2 and one polypeptide chain 3, in which the polypeptide chain 1 includes VL_A-CL in order from the N-terminus to the C-terminus, the polypeptide chain 2 includes VH_A-CH1-h-CH2-CH3 in order from the N-terminus to the C-terminus, and the polypeptide chain 3 includes VH_B-L-VH_B-h-CH2-CH3 in order from the N-terminus to the C-terminus; (ii) the binding protein has three polypeptide chains including one polypeptide chain 1, one polypeptide chain 2 and one polypeptide chain 3, in which the polypeptide chain 1 includes VL_A-CL from the N-terminus to the C-terminus, the polypeptide chain 2 includes VH_A-CH1-h-CH2-CH3 from the N-terminus to the C-terminus, and the polypeptide chain 3 includes VH_B-h-CH2-CH3 from the N-terminus to the C-terminus; A binding protein, wherein "L" and "h" are independently selected from "-", GS, or an amino acid sequence shown in any one of SEQ ID NOs:495-519, "VH_A" is a heavy chain variable region of protein functional region A, "VL_A" is a light chain variable region of protein functional region A, and "VH_B" is a heavy chain variable region of protein functional region B.

2. 2. The binding protein of claim 1 , A binding protein, wherein the BCMA-binding fragment of the VH structure comprises the amino acid sequence shown in SEQ ID NO:

251.

3. The binding protein of claim 1, the CD3-binding fragment 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 and the 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 shown in SEQ ID NO: 291 and its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 257; or A binding protein, the light chain variable region VL of which comprises the amino acid sequence shown in SEQ ID NO: 291 and the heavy chain variable region VH of which comprises the amino acid sequence shown in SEQ ID NO:

281.

4. 4. A binding protein according to any one of claims 1 to 3, The binding peptide is selected from one of the following: (1) the binding protein has three polypeptide chains, 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:454; and a third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:445; or (2) the binding protein has three polypeptide chains, 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:454; and a third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:446; or (3) the binding protein has three polypeptide chains, 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: 463; and a third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:

464. Binding proteins.

5. 2. The binding protein of claim 1 , A binding protein comprising a light chain constant region CL, and comprising a human heavy chain constant region CH1, CH2 and / or CH3.

6. The binding protein of claim 5, A binding protein, wherein the human light chain constant region is a Cκ or Cλ and the human heavy chain constant region is a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region.

7. The binding protein of claim 6, Binding proteins in which the IgG1 heavy chain constant region further comprises one or more of the following mutations on the Fc: C220S, N297A, L234A, L235A, P329G, S239D, I332E, S354C, T366W, Y349C, T366S, L368A, Y407V, M252Y, S254T, T256E, and the sites of mutations are numbered according to the EU numbering convention.

8. 8. An isolated nucleic acid encoding a binding protein according to any one of claims 1-7.

9. An expression vector comprising the isolated nucleic acid of claim 8.

10. The expression vector according to claim 9, A host cell, which may be a prokaryotic or eukaryotic cell.

11. A method for producing a binding protein according to any one of claims 1 to 7, comprising the steps of: The method for producing a binding protein comprises the steps of: culturing a host cell according to claim 10 and obtaining the binding protein from the culture.

12. A composition comprising a binding protein according to any one of claims 1-7.

13. A pharmaceutical composition comprising a binding protein according to any one of claims 1-7 or a composition according to claim 12 for use in treating and / or preventing cancer.

14. The pharmaceutical composition of claim 13, The pharmaceutical composition, wherein the cancer is selected from 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 and multiple myeloma.

15. A BCMA antibody comprising: The BCMA antibody comprises a heavy chain variable region; and the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively.

16. The BCMA antibody of claim 15, A BCMA antibody, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO:

251.

17. The BCMA antibody of claim 15 or 16, A BCMA antibody, wherein the BCMA antibody comprises one polypeptide chain, wherein the polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:319.

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