WO2022002033A1 - 具有H2L2与HCAb结构的结合蛋白 - Google Patents

具有H2L2与HCAb结构的结合蛋白 Download PDF

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WO2022002033A1
WO2022002033A1 PCT/CN2021/103044 CN2021103044W WO2022002033A1 WO 2022002033 A1 WO2022002033 A1 WO 2022002033A1 CN 2021103044 W CN2021103044 W CN 2021103044W WO 2022002033 A1 WO2022002033 A1 WO 2022002033A1
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amino acid
seq
variable region
acid sequence
chain variable
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PCT/CN2021/103044
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French (fr)
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何云
石磊
罗海山
吕强
何进秋
裴增林
王永强
钟琛
黄冰
赵建勋
贾星星
张雪琨
赵楚楚
陈飞
牛磊
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和铂医药(上海)有限责任公司
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Priority to CN202180045534.4A priority Critical patent/CN115776897A/zh
Priority to CA3183565A priority patent/CA3183565A1/en
Priority to KR1020227045516A priority patent/KR20230015996A/ko
Priority to AU2021302126A priority patent/AU2021302126A1/en
Priority to JP2022578899A priority patent/JP2023531672A/ja
Priority to US18/002,655 priority patent/US20230322953A1/en
Priority to EP21833501.6A priority patent/EP4154910A1/en
Publication of WO2022002033A1 publication Critical patent/WO2022002033A1/zh

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Definitions

  • the invention relates to the field of biomedicine, in particular to a bispecific or multispecific binding protein with H2L2 and HCAb structures and applications thereof.
  • Antibodies in most species are tetrameric structures comprising two identical heavy chains and two identical light chains, also known as "H2L2".
  • H2L2 heavy-chain antibody
  • HCAb heavy-chain antibody
  • the variable region VHH fragment of heavy chain antibody is similar to the Fab of H2L2 antibody, with stable structure and specific antigen-binding activity, but the molecular weight of VHH is only about 13KDa, so it is also called nanobody or single domain antibody.
  • the natural structure of human antibodies is "H2L2", so it is impossible to obtain useful human heavy chain antibodies from natural sources; however, Frank Grosveld et al. proposed a method for obtaining fully human heavy chain antibodies using transgenic animals (WO2007/ 096779), and can use this method to obtain stable and soluble fully human VH single-domain antibodies.
  • Bispecific antibodies and multispecific antibodies (multispecific antibodies) are a class of artificial antibodies prepared by protein engineering technology on the basis of natural monoclonal antibodies, which can bind two or more different antigens or Different epitopes on the same antigen can achieve some mechanisms of action and functional effects that cannot be achieved by monospecific antibodies.
  • bispecific antibodies The structural design of bispecific antibodies is very important.
  • One of the main challenges in generating bispecific antibodies from two different H2L2 antibodies is how to obtain functional bispecific antibodies with the correct chain combination from more than 10 different combinations of heavy and light chains Molecules, i.e. how to resolve chain mismatches.
  • scientists have developed various strategies to try to solve this problem: for example, using the structure of a single-chain variable region antibody fragment (scFv) to fuse VH and VL together; alternatively, using knob-into-hole (KiH) technology or Other spatially complementary mutants to promote heterodimerization of heavy chains; alternatively, use of "consensus light chain” or “consensus heavy chain” techniques to reduce the number of distinct polypeptide chains, etc.
  • scFv single-chain variable region antibody fragment
  • KiH knob-into-hole
  • scFv structures are often unstable and prone to aggregation; "consensus light chain” technology requires the use of very complex screening processes to obtain VLs that can pair with different VHs; FIT
  • the size of double antibody molecules produced by techniques such as Ig is relatively large (about 250KDa), which may affect its ability to penetrate cells or tissues. Therefore, there is still an urgent need to develop new bispecific antibody structures and techniques for preparing bispecific antibodies.
  • Heavy chain antibodies and their derived single domain antibodies have their unique advantages in the construction of bispecific or even multispecific antibodies.
  • the antigen-binding domain of heavy chain antibodies is only one-quarter the size of the Fab of conventional antibodies; and there is no light chain, avoiding the problem of light chain mismatches. Therefore, by using heavy chain antibodies and their derived single domain antibodies, bispecific or even multispecific antibodies with smaller molecular weights, fewer polypeptide chains and simpler structures can be constructed.
  • the potential immunogenicity risk of non-human heavy chain antibodies may limit its therapeutic use. Therefore, compared with camelid nanobodies, the use of fully human heavy chain antibodies to construct bispecific or multispecific antibodies will have more advantages in immunogenicity and druggability.
  • the present invention provides a bispecific or multispecific binding protein with H2L2 and HCAb structures, and preparation and use thereof.
  • Such methods of binding proteins utilize multivalent and multispecific binding proteins constructed from fully human heavy chain antibodies and their derived single domain antibodies. Compared with multivalent and multispecific binding proteins constructed with conventional IgG antibodies, it has many advantages and is more flexible in adjusting specificity and binding valence.
  • the multivalent and multispecific binding proteins provided herein contain at least one heavy chain variable region domain VH derived from a human heavy chain antibody and are capable of binding two or more antigens, or two or more of the same antigen Multiple epitopes, or two or more copies of the same epitope.
  • one of the technical solutions of the present invention is to provide a binding protein containing at least two protein functional domains, wherein the binding protein includes a protein functional domain A and a protein functional domain B; the protein functional domain Region A and the protein functional region B target different antigens or epitopes, wherein the protein functional region A is a Fab or scFv structure, the protein functional region B is a VH structure, and the protein functional region A and the protein functional region The number of functional areas B is one or more, respectively.
  • the binding protein further includes Fc, and the number of the Fc is two to form an Fc dimer.
  • the binding protein has a symmetric structure or an asymmetric structure, and/or the Fc dimer is a homodimer or a heterodimer.
  • the binding protein has a symmetrical structure, and the symmetrical structure is a left-right symmetrical IgG-like structure, wherein the number of the protein functional regions A is two, and the protein functional regions B The number is two or four. Two of the protein functional domains A and two of the Fc form an IgG structure. In the left-right symmetrical IgG-like structure, left and right are only relative positions in space, which may be symmetrical up and down or front and rear.
  • the left-right symmetrical IgG-like structure means that additional protein functional domains are also connected symmetrically on the antibody of the IgG structure, and these additional protein functional domains can be linked to the C-terminus of CH3 of the Fc functional domain or the N of VH or VL.
  • the terminal can also be linked between the variable region of the IgG structure and the Fc functional region in an intercalated form.
  • Additional refers to structural or functional regions other than the relative IgG structure.
  • the binding protein is a tetravalent binding protein, which specifically includes the following structure: (a) The binding protein from the N-terminus to the C-terminus is the protein function in sequence; Region A, protein functional region B, and Fc, wherein the protein functional region A and the protein functional region B are linked by a first linking peptide (L1), and the protein functional domain B and the Fc are linked by a second linking peptide ( L2) ligation; in this embodiment, the two copies of the protein functional domain B and the Fc form a symmetric dimer form of a single chain antibody, and on the N-terminus of the dimer of the single chain antibody
  • the protein functional domain A is connected, and at this time, the protein functional domain A can be connected to the N-terminus of the protein functional domain B with its CH1 ((2) in Figure 1) or CL ((1) in Figure 1 ) Or, (b) the binding protein is sequentially from the N-terminus to the C-terminus of protein functional region B
  • Region B is connected by a linking peptide; in this embodiment, the two copies of the protein functional region A and the Fc form a symmetrical IgG structure, and the protein functional region B is connected to the C-terminus of the IgG structure , at this time, the protein functional region B can be connected to the C-terminal of CH3 ((5) in Figure 1 ) or CL ((6) in Figure 1 ) of the protein functional region A;
  • the binding protein is a hexavalent binding protein, which specifically includes the following structure: the two newly added protein functional regions B are further connected to the above (a) or (b) or (c) the N-terminus or C-terminus of the binding protein; preferably connected to the C-terminus of the Fc of the above-mentioned (c) binding protein or the C-terminus of the original protein functional region B, or with the above-mentioned ( b)
  • the original protein functional domain B of the binding protein is connected to the N-terminus of the protein functional domain A together.
  • the "added” mentioned in this article is only for convenience, to distinguish the protein functional region B mentioned above, that is, the "original" protein functional region.
  • the newly added protein functional region is different from the original protein functional region There is no distinction between some protein functional regions.
  • two copies of the protein functional region A and the Fc form a symmetrical IgG structure, and the protein functional region B is connected to the C-terminus of the IgG structure.
  • the functional domain B can be connected to the CH3 of the protein functional domain A through a first connecting peptide, and the other two protein functional domains B can be connected to the aforementioned two protein functional domains B through a second connecting peptide ((7) in Figure 1 ); Or wherein two protein functional regions B can be connected with the N-terminus of the VH of the protein functional region A through a first connecting peptide, and the other two protein functional regions B and the N-terminus of the VL of the protein functional region A are connected by a second linker.
  • the connecting peptide is connected ((9) in Fig. 1).
  • binding proteins with two different protein functional domains are also called bispecific binding proteins. More preferably, the binding protein also includes a protein functional region C, and the binding protein is a hexavalent or octavalent trispecific binding protein, which specifically includes the following structure: the protein functional region C and the protein functional region. A.
  • the protein functional region B targets different antigens or antigenic epitopes
  • the protein functional region C is connected to the N-terminus or C-terminus of the above-mentioned antibody; preferably the number of the protein functional region C is two, and the protein functional region Domain C is linked to the C-terminus of the binding protein described in (c) above, or, like the protein functional domain B of the binding protein of the above (b), linked to the N-terminal of the protein functional domain A.
  • the two copies of the protein functional domain C are further linked to the C-terminus of structure (5) ((8) in Figure 1), or the N-terminus of structure (3) or (4) ( (10) of Fig. 1 shows the case where it is attached to the N-terminal of the structure (3).
  • the binding protein has four polypeptide chains, which are two identical short chains (or "polypeptide chain 1") and two identical long chains (or “polypeptide chain 2"), wherein, (1) the short chain sequentially includes VH_A-CH1 from the N-terminus to the C-terminus, and the long chain sequentially includes VL_A-CL-L1-VH_B-L2-CH2-CH3 from the N-terminus to the C-terminus; or (2) the The short chain sequentially includes VL_A-CL from the N-terminus to the C-terminus, and the long chain sequentially includes VH_A-CH1-L1-VH_B-L2-CH2-CH3 from the N-terminus to the C-terminus; or (3) the short chain is composed of The N-terminus to the C-terminus includes VL_A-CL in sequence, and the long chain includes VH_B-L-VH_A-CH1-h-CH2-CH3 in sequence from the N-terminus to the C-terminus
  • the ends sequentially include VH_B-L-VL_A-CL, and the long chain sequentially includes VH_A-CH1-h-CH2-CH3 from the N-terminus to the C-terminus; or (5) the short chain sequentially includes VL_A from the N-terminus to the C-terminus -CL, the long chain sequentially includes VH_A-CH1-h-CH2-CH3-L-VH_B from the N-terminus to the C-terminus; or (6) the short chain sequentially includes VL_A-CL-L from the N-terminus to the C-terminus -VH_B, the long chain sequentially includes VH_A-CH1-h-CH2-CH3 from the N-terminus to the C-terminus; or (7) the short chain sequentially includes VL_A-CL from the N-terminus to the C-terminus, and the long chain is from the N-terminus to the C-terminus.
  • N terminus to C terminus sequentially includes VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_B; or (8) the short chain sequentially includes VL_A-CL from N terminus to C terminus, and the long chain starts from N-terminus to C-terminus sequentially includes VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_C; or (9) the short chain sequentially includes VH_B-L1-VL_A-CL from N-terminus to C-terminus, so The long chain sequentially includes VH_B-L2-VH_A-CH1-h-CH2-CH3 from the N-terminus to the C-terminus; or (10) the short chain sequentially includes VH_B-L1-VL_A-CL from the N-terminus to the C-terminus, so The long chain sequentially includes VH_C-L2-VH_A-CH1-h-CH2-CH3 from the N
  • VL, VH, CL, and CH are conventional in the art, representing light chain variable region, heavy chain variable region, light chain constant region and heavy chain constant region, respectively, wherein CH includes CH1, CH2 and CH3; _A and _B and _C respectively represent that the functional region is a protein functional region A or a protein functional region B or a protein functional region C or its composition; "-" represents a polypeptide bond that connects different structural regions or is used to separate different structural regions; The C terminus is the carboxyl terminus of the peptide chain (also written as "C'"), and the N terminus is the amino terminus of the peptide chain (also written as "N'").
  • L, L1 and L2 can be the same sequence. In other embodiments, L, L1 and L2 may be different sequences.
  • the protein domains of the present invention may be Fab, scFv or VH in some cases, F(ab)2 or full-length antibodies in other cases, also referred to as antibodies or antigen binding proteins or binding protein.
  • the protein functional region A is also referred to as the antibody A against the first antigen or the first antigen binding domain; the protein functional region B is also referred to as the antibody B against the second antigen or the first antigen binding domain.
  • the protein functional region C is also referred to as antibody C against the third antigen or the third antigen-binding domain
  • the protein functional region D is also referred to as antibody D against the fourth antigen or the fourth antigen binding domains, and so on.
  • the binding protein has an asymmetric structure, and the asymmetric structure is a left-right asymmetric IgG-like structure, wherein the left arm is the protein functional region A of the Fab or scFv structure, and the right arm is It is a protein functional region B of a VH structure, and the protein functional region A and the protein functional region B are respectively 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 Or two or three.
  • the binding protein is a bivalent binding protein, which includes the following structure: the protein functional area A is (d) a conventional Fab structure, or (e) Fab (cross VH/VL) structure, or (f) Fab (cross Fd/LC) structure; when the number of the protein functional region B is two, the binding protein is a trivalent binding protein, which includes the following structure : the protein functional region A of the left arm is the above (d) or (e) or (f), and the 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 The N-terminus of the first protein functional region B of the right arm, or the C-terminus of the Fc, preferably the second protein functional region B is connected to the N-terminus of the first protein functional region B of the right arm; when the protein When the number of functional regions B is three, the binding protein is a tetravalent binding protein, and specifically includes the following structure:
  • the N-terminus of the second protein functional region B, 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 in the right arm, and the third protein functional region B Domain B is linked to the N-terminus of domain B of the second protein.
  • the binding protein is a bivalent binding protein, which includes the following structure: the protein functional domain A of the left arm is a (g) scFv structure, and the scFv passes through the VH The end or the end of VL is connected to Fc; when the number of the protein functional region B is two, the binding protein is a trivalent binding protein, and specifically includes the following structure: the protein functional region A of the left arm is (g ), the 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 A protein functional domain B is connected to the N-terminus of the first protein functional domain B of the right arm; when the number of the protein functional domain B is three, the binding protein is a tetravalent binding protein, which specifically includes the following structure: The protein functional region A of the left arm is (g), and the third protein functional
  • the second protein functional domain B is connected to the N-terminus of the first protein functional domain B of the right arm, and the third protein functional domain B is linked to the second protein functional domain B N terminus of functional region B.
  • the binding protein also includes a protein functional region C, and the binding protein is a trivalent or multivalent multispecific binding protein, wherein the protein functional region C, the protein functional region A, the protein functional region Region B targets different antigens or antigenic epitopes, and the protein functional region C is connected to the N-terminus or C-terminus of the above-mentioned binding protein; preferably, the protein functional region C is connected to the above (d), (e), (f) ) or (g) the N-terminus of the protein functional region B of the binding protein;
  • the binding protein also includes a protein functional region D
  • the binding protein is a tetravalent or multivalent multispecific binding protein, specifically: the protein functional region D and the protein functional region A, Protein functional region B and protein functional region C target different antigens or antigenic epitopes, and the protein functional region D is connected to the N-terminus or C-terminus of the above-mentioned binding protein; preferably, the protein functional region D is connected to the above (d) , (e), (f) or (g) the N-terminus of protein domain B of the binding protein.
  • the binding protein has three polypeptide chains: polypeptide chain 1, polypeptide chain 2 and polypeptide chain 3, one for each polypeptide chain.
  • polypeptide chain 1 is also called the first polypeptide chain
  • polypeptide chain 2 is also called the second polypeptide chain
  • polypeptide chain 3 is also called the third polypeptide chain.
  • the polypeptide chain 1 sequentially includes VL_A-CL from the N-terminus to the C-terminus
  • the polypeptide chain 2 sequentially includes VH_A-CH1-h-CH2-CH3 from the N-terminus to the C-terminus
  • the polypeptide chain 3 VH_B-h-CH2-CH3 is included in sequence from the N-terminus to the C-terminus
  • the polypeptide chain 1 includes VH_A-CL in sequence from the N-terminus to the C-terminus
  • the polypeptide chain 2 sequentially includes from the N-terminus to the C-terminus.
  • the polypeptide chain 3 sequentially includes VH_B-h-CH2-CH3 from the N-terminus to the C-terminus; or (13) the polypeptide chain 1 sequentially includes VH_A- from the N-terminus to the C-terminus CH1, the polypeptide chain 2 sequentially includes VL_A-CL-h-CH2-CH3 from the N-terminus to the C-terminus, and the polypeptide chain 3 sequentially includes VH_B-h-CH2-CH3 from the N-terminus to the C-terminus; or (14) The polypeptide chain 1 sequentially includes VL_A-CL from the N-terminus to the C-terminus, the polypeptide chain 2 sequentially includes VH_A-CH1-h-CH2-CH3 from the N-terminus to the C-terminus, and the polypeptide chain 3 is from the N-terminus to C.
  • the ends include VH_B-L-VH_B-h-CH2-CH3 in sequence; or (15) the polypeptide chain 1 includes VL_A-CL sequentially from the N-terminus to the C-terminus, and the polypeptide chain 2 sequentially includes VH_A from the N-terminus to the C-terminus -CH1-h-CH2-CH3, the polypeptide chain 3 sequentially includes VH_C-L-VH_B-h-CH2-CH3 from the N-terminus to the C-terminus; or (16) the polypeptide chain 1 sequentially from the N-terminus to the C-terminus Including VH_A-CL, the polypeptide chain 2 sequentially includes VL_A-CH1-h-CH2-CH3 from the N terminus to the C terminus, and the polypeptide chain 3 sequentially includes VH_B-L-VH_B-h-CH2 from the N terminus to the C terminus -CH3; or (17) the polypeptide chain 1 includes VH_A-
  • VL_A-CL-h-CH2-CH3 is included in sequence from the end to the C terminus, and the polypeptide chain 3 includes VH_B-L-VH_B-h-CH2-CH3 in sequence from the N terminus to the C terminus; or (19) the polypeptide chain 1 From the N-terminus to the C-terminus, it includes VH_A-CH1, the polypeptide chain 2 includes VL_A-CL-h-CH2-CH3 from the N-terminus to the C-terminus, and the polypeptide chain 3 includes VH_C- L-VH_B-h-CH2-CH3; or (26) the polypeptide chain 1 includes VL_A-CL in sequence from the N-terminus to the C-terminus, and the polypeptide chain 2 is in sequence from the N-terminus to the C-terminus The second includes VH_A-CH1-h-CH2-CH3, and the polypeptide chain 3 sequentially includes VH_B-L1-VH_B-L2-VH_
  • the binding protein has two polypeptide chains: polypeptide chain 1 and polypeptide chain 2, one for each polypeptide chain.
  • polypeptide chain 1 is also called the first polypeptide chain
  • polypeptide chain 2 is also known as the second polypeptide chain.
  • the polypeptide chain 1 sequentially includes VL_A-L-VH_A-h-CH2-CH3 from the N-terminus to the C-terminus
  • the polypeptide chain 2 sequentially includes VH_B-h-CH2-CH3 from the N-terminus to the C-terminus
  • the polypeptide chain 1 sequentially includes VH_A-L-VL_A-h-CH2-CH3 from the N-terminus to the C-terminus
  • the polypeptide chain 2 sequentially includes VH_B-h-CH2-CH3 from the N-terminus to the C-terminus
  • the polypeptide chain 1 sequentially includes VL_A-L1-VH_A-h-CH2-CH3 from the N-terminus to the C-terminus
  • the polypeptide chain 2 sequentially includes VH_B-L2-VH_B-h from the N-terminus to the C-terminus -CH2-CH3
  • the polypeptide chain 1 sequentially includes VH_B-L2-VH_B
  • the terminal includes VH_C-L2-VH_B-h-CH2-CH3 in turn; wherein, the L, L1 and L2 are connecting peptides, and the h is a hinge region or a connecting peptide, such as "-", GS or as shown in the amino acid sequences of SEQ ID NOs: 495-519.
  • the binding protein contains at least two protein functional domains, namely protein functional domain A and protein functional domain B; the protein functional domain A and the protein functional domain B are PD-L1 antibody, HER2 One of antibody, B7H4 antibody, CD3 antibody, CTLA4 antibody, 4-1BB antibody or BCMA antibody.
  • the protein functional domain A is PD-L1 antibody, HER2 antibody, B7H4 antibody or CD3 antibody
  • the protein functional domain B is CTLA4 antibody, 4-1BB antibody or BCMA antibody.
  • the protein functional domain A is PD-L1 antibody, and the protein functional domain B is CTLA4 antibody; or, the protein functional domain A is PD-L1 antibody, and the protein functional domain B is 4-1BB or, the protein functional domain A is a HER2 antibody, and the protein functional domain B is a CTLA4 antibody; or, the protein functional domain A is a B7H4 antibody, and the protein functional domain B is a 4-1BB antibody; or,
  • the protein functional domain A is a CD3 antibody, and the protein functional domain B is a BCMA antibody.
  • the PD-L1 antibody comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, respectively SEQ ID NOs: 167, 188 and the amino acid sequence shown in 211; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 15, 62 and 122; or, its light chain variable region VL comprises LCDR1 , LCDR2 and LCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively shown in SEQ ID NOs: 15, 69 and 122 amino acid sequence.
  • its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 282; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 233; or, its light chain variable region Region VL includes the amino acid sequence shown in SEQ ID NO:282; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO:240.
  • the PD-L1 antibody comprises two polypeptide chains; wherein, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:347, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:300 or, the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 353, and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 308; or, the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 308 The amino acid sequence shown in SEQ ID NO:353, the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:310.
  • the PD-L1 antibody is shown in the amino acid sequence of atezolizumab as known in the art.
  • the HER2 antibody is shown as the amino acid sequence of trastuzumab or pertuzumab as known in the art.
  • the B7H4 antibody comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, respectively SEQ ID NOs: 180, 191 and 225
  • amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 298
  • its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 261
  • its light chain variable region Region VL includes the amino acid sequence shown in SEQ ID NO:299
  • its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO:262.
  • the B7H4 antibody comprises two polypeptide chains; wherein, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:360, and the second polypeptide chain comprises the amino acid shown in SEQ ID NO:326 sequence; or, the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:361, and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:327.
  • the CTLA4 antibody comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 17, 65 and 125, respectively.
  • the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO:236.
  • the CTLA4 antibody comprises a polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:303, and the amino acid sequence set forth in SEQ ID NO:306.
  • the 4-1BB antibody comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, respectively SEQ ID NOs: 170, 191 and the amino acid sequence shown in 214; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 18, 66 and 126; or, its light chain variable region VL comprises LCDR1 , LCDR2 and LCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 170, 191 and 214; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively shown in SEQ ID NOs: 18, 71 and 126 amino acid sequence.
  • amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 285
  • its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 237
  • its light chain variable region Region VL includes the amino acid sequence shown in SEQ ID NO:289
  • its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO:242.
  • the 4-1BB antibody comprises two polypeptide chains; wherein, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:350, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:304 or, the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 355, and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 311.
  • the 4-1BB antibody comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are the amino acids shown in SEQ ID NOs: 27, 79 and 137, respectively or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 28, 80 and 138, respectively; or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 , respectively the amino acid sequences shown in SEQ ID NOs: 29, 82 and 138; or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, respectively the amino acid sequences shown in SEQ ID NOs: 28, 89 and 145 Or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 28, 81 and 139; or, its heavy chain variable region VH comprises HCDR
  • its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 252; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 253; or, its heavy chain
  • the variable region VH includes the amino acid sequence shown in SEQ ID NO: 255; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 264; or, its heavy chain variable region VH includes such
  • the amino acid sequence shown in SEQ ID NO:254; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO:256.
  • the 4-1BB antibody comprises a polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:320, the amino acid sequence shown in SEQ ID NO:321, and the amino acid sequence shown in SEQ ID NO:323
  • the amino acid sequence shown is the amino acid sequence shown in SEQ ID NO: 322, the amino acid sequence shown in SEQ ID NO: 324, or the amino acid sequence shown in SEQ ID NO: 329.
  • the CD3 antibody comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, respectively SEQ ID NOs: 177, 191 and 221
  • its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 294; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 258; or, its light chain variable region Region VL includes the amino acid sequence shown in SEQ ID NO: 295; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 259; or, its light chain variable region VL includes as SEQ ID NO:
  • the amino acid sequence shown in 296; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 260; or, its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 297; its The heavy chain variable region VH includes the amino acid sequence set forth in SEQ ID NO:259.
  • the CD3 antibody comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, respectively SEQ ID NOs: 172, 192 and 216
  • its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 245; or, its light chain variable region Region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 257; or, its light chain variable region VL includes as SEQ ID NO:
  • the amino acid sequence shown in 291; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 263; or, its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its The variable region VH of the heavy chain includes the amino acid sequence shown in SEQ ID NO: 281; or, the variable region VL of the light chain includes the amino acid sequence shown in SEQ ID NO: 291; the variable region VH of the heavy chain includes the amino acid sequence shown in SEQ ID NO: 291; the variable region VH of the heavy chain includes the amino acid sequence shown
  • the CD3 antibody comprises two polypeptide chains; wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:357, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:313 the amino acid sequence shown; or, the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 357, and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 325; or, the first polypeptide The chain includes the amino acid sequence shown in SEQ ID NO:357, and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:328; or, the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357 sequence, the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:346.
  • the CD3 antibody comprises a polypeptide chain comprising the amino acid sequence as set forth in SEQ ID NO:489, the amino acid sequence as set forth in SEQ ID NO:490, as in SEQ ID NO : the amino acid sequence shown in SEQ ID NO: 491, the amino acid sequence shown in SEQ ID NO: 492, or the amino acid sequence shown in SEQ ID NO: 493.
  • the BCMA antibody comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 15, 75 and 133, respectively; Or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 24, 76 and 134; or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, respectively is the amino acid sequence shown in SEQ ID NO: 25, 77 and 135; or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are the amino acid sequences shown in SEQ ID NO: 26, 78 and 136 respectively.
  • its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 248; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 249; or, its heavy chain
  • the variable region VH includes the amino acid sequence shown in SEQ ID NO:250; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO:251.
  • the BCMA antibody comprises a polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:316, the amino acid sequence shown in SEQ ID NO:317, and the amino acid sequence shown in SEQ ID NO:318 The amino acid sequence of , or, the amino acid sequence shown in SEQ ID NO:319.
  • the BCMA antibody comprises a heavy chain variable region, and its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 26, 90 and 136, respectively; Or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 34, 78 and 146; or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, respectively is the amino acid sequence shown in SEQ ID NO: 35, 78 and 147; or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are the amino acid sequences shown in SEQ ID NO: 35, 90 and 147 respectively; or , its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 36, 90 and 146; or, its heavy chain variable region VH
  • the amino acid sequences shown in ID NOs: 35, 90 and 146; or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 34, 76 and 136, respectively; or, its The variable region VH of the heavy chain comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 35, 76 and 146; or, the variable region VH of the heavy chain comprises HCDR1, HCDR2 and HCDR3, which are respectively SEQ ID
  • the amino acid sequences shown in NO: 34, 76 and 146; or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NO: 35, 90 and 148;
  • the chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 35, 90 and 136
  • its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 265; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 266; or, its heavy chain
  • the variable region VH includes the amino acid sequence shown in SEQ ID NO: 267; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 268; or, its heavy chain variable region VH includes such
  • the amino acid sequence shown in SEQ ID NO: 269; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 270; or, its heavy chain variable region VH includes as shown in SEQ ID NO: 271.
  • its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 272; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 273; or , its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 274; Or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 275; Or, its heavy chain variable region Region VH includes the amino acid sequence shown in SEQ ID NO: 276; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 277; or, its heavy chain variable region VH includes as SEQ ID The amino acid sequence shown in NO: 278; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 279; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 280 amino acid sequence.
  • the BCMA antibody comprises a polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:330; or, the amino acid sequence shown in SEQ ID NO:331; or, as shown in SEQ ID NO : the amino acid sequence shown in 332; or, the amino acid sequence shown in SEQ ID NO: 333; or, the amino acid sequence shown in SEQ ID NO: 334; or, the amino acid sequence shown 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, as SEQ ID NO:339 or, the amino acid sequence shown in SEQ ID NO:340; or, the amino acid sequence shown in SEQ ID NO:341; or, the amino acid sequence shown 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
  • the protein functional region C is the heavy chain variable region of a BCMA heavy chain antibody; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, respectively SEQ ID NOs: 24, 76 and The amino acid sequence shown in 134; or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively.
  • its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 249; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 251.
  • the binding protein may further comprise a light chain constant region, the light chain constant region is preferably a human light chain constant region, more preferably a human light chain constant region C ⁇ or C ⁇ .
  • the binding protein may further comprise heavy chain constant regions (CH1, CH2 and/or CH3), the heavy chain constant regions are preferably human heavy chain constant regions, more preferably human IgG1, IgG2, IgG3, IgG4 heavy chain constant region.
  • the Fc of the IgG1 heavy chain constant region has C220S, N297A, L234A, L235A, P329G, S239D, I332E, S354C, T366W, Y349C, T366S, L368A, Y407V, M252Y, S254T, T256E, etc.
  • One or more of the mutations using the EU numbering scheme for the mutation sites are preferably human heavy chain constant regions, more preferably human IgG1, IgG2, IgG3, IgG4 heavy chain constant region.
  • the Fc of the IgG1 heavy chain constant region has C220S, N297A, L234A, L235A, P329G, S239
  • the binding protein contains protein functional domain A and protein functional domain B: the protein functional domain A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 are the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211, respectively; the heavy chain variable region VH includes HCDR1, HCDR2 and HCDR3, and are shown in SEQ ID NOs: 15, 69 and 122, respectively;
  • the amino acid sequence of the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 17, 65 and 125; or,
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211 respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 15, 62 and 122; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 171, 190 and 215, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 19, 67 and 127; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 176, 196 and 220, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are 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; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211 respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 15, 69 and 122; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211 respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 15, 69 and 122; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 167, 188 and 211 respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 15, 69 and 122; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 180, 191 and 225, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 32, 87 and 143; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 180, 191 and 225, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 32, 87 and 143; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 180, 191 and 225, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 32, 87 and 143; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 177, 191 and 226, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 33, 88 and 144; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 177, 191 and 226, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 33, 88 and 144; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 177, 191 and 226, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 33, 88 and 144; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 20, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 20, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 20, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 177, 191 and 221 respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 15, 84 and 141; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 178, 197 and 222, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 31, 85 and 142; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 177, 191 and 223, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are 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; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 179, 198 and 224, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 31, 85 and 142; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively; or,
  • the binding protein contains protein functional domain A, protein functional domain B and protein functional domain C: wherein, the protein functional domain A comprises a light chain variable region and a heavy chain variable region; which The light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively SEQ ID NOs: The amino acid sequences shown in 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively shown in SEQ ID NOs: 15, 75 and 133.
  • the protein functional region C comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 24, 76 and 134; or ,
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 30, 68 and 128; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 25, 77 and 135; the protein functional region C comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, 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.
  • the binding protein comprises protein functional domain A and protein functional domain B: the protein functional domain A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL Including the amino acid sequence shown in SEQ ID NO: 282; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 240; the protein functional region B includes the heavy chain variable region; its heavy chain can be The variable region VH includes the amino acid sequence shown in SEQ ID NO: 236; or,
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 282; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 233; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 236; or,
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 286; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 238; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 236; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 293; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 247; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 236; or,
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 282; its heavy chain variable region VH includes such as SEQ ID
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 282; its heavy chain variable region VH includes such as SEQ ID
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 282; its heavy chain variable region VH includes such as SEQ ID
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 298; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 261; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 253; or,
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 298; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 261; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 252; or,
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 298; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 261; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 264; or,
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 299; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 262; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 253; or,
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 299; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 262; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 254; or,
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 299; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 262; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 256; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 245; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 249; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 245; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 245; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 248; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 257; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 248; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 257; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 281; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 265; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 269; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 270; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 271; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 272; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 273; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 274; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 275; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 276; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 277; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 278; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 279; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 280; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 266; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 267; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 263; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 268; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 244; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 249; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 244; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or,
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 294; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 258; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or,
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 295; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 259; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or,
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 296; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 260; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or,
  • the protein functional region A comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 297; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 259; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 251; or,
  • the binding protein contains protein functional domain A, protein functional domain B and protein functional domain C: wherein, the protein functional domain A comprises a light chain variable region and a heavy chain variable region; Its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 257; the protein functional region B includes the heavy chain variable region B. Variable region; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO:248.
  • the protein functional region C comprises a heavy chain variable region; its heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 249; or,
  • the protein functional region A includes a light chain variable region and a heavy chain variable region; its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes such as SEQ ID The amino acid sequence shown in NO: 257; the protein functional region B comprises a heavy chain variable region; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 250.
  • the protein functional region C comprises a heavy chain variable region; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 251.
  • the binding protein comprises two polypeptide chains.
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 371; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 372; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 371; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 373; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 353; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 362; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 363; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 364; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 365; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 364; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 353; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 366; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 353; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 369; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 353; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 370; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:353; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:394; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 353; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 395; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 363; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 310; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 363; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 396; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 363; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 362; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 363; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 394; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 363; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 395; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 367; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 368; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 367; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 378; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 367; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 379; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 367; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 380; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 367; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 381; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 367; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 382; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 367; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 383; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 367; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 385; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 367; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 388; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 367; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 389; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 374; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 375; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 386; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 387; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:401; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 402; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 305; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 403; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 402; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 409; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:359; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:404; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 405; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 315; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 359; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 406; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 376; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 377; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 397; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:398; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 399; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 400; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 402; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 401; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 402; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 403; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 405; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 404; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 405; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 406; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:371; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:486; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:353; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:460; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:353; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:461; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 353; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 462; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 487; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 488; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:360; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:455; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:360; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:456; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 360; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 457; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 360; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 458; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 360; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 459; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 419; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 412; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:419; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:414; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 440; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 428; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 441; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 428; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 442; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 428; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 443; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 428; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 487; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 520; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 353; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 521; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 371; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 522; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:360; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:523.
  • the binding protein comprises three polypeptide chains.
  • the first polypeptide chain includes the amino acid sequence as shown in SEQ ID NO:353; the second polypeptide chain includes the amino acid sequence as shown in SEQ ID NO:407; the third polypeptide chain includes as SEQ ID NO:390 the amino acid sequence shown; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:353; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:408; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:392 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:391; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:390 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:393; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:392 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:391; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:434 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:391; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:435 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:393; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:436 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:351; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:393; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:437 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:367; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:438; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:434 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:367; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:438; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:435 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:367; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:439; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:436 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:367; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:439; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:437 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:361; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:481; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:482 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:361; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:481; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:483 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:361; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:481; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:484 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:361; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:481; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:485 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:411; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:412 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:411; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:414 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:415; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:416; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:410 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:415; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:416; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:412 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:415; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:416; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:414 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:417; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:418; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:412 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:417; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:418; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:414 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:423; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:426 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:423; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:427 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:423; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:428 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:423; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:429 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:444; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:449 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:450; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:449 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:444; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:445 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:444; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:446 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:450; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:445 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:450; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:446 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:444; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:447 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:444; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:448 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:450; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:447 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:450; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:448 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:445 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:446 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:463; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:464 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:465 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:466 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:467 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:468 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:469 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:470 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:471 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:472 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:473 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:474 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:475 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:476 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:477 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:478 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:479 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:454; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:480 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:423; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:430 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:423; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:431 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:423; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:432 the amino acid sequence of ; or,
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:423; the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:433 amino acid sequence.
  • the second technical solution of the present invention is to provide an isolated nucleic acid encoding the above-mentioned binding protein.
  • the third technical solution of the present invention is to provide an expression vector comprising the isolated nucleic acid as described above.
  • the fourth technical solution of the present invention is to provide a host cell, which comprises the above-mentioned expression vector, wherein the host cell is a prokaryotic cell or a eukaryotic cell.
  • the fifth technical solution of the present invention is to provide a preparation method of the binding protein as described above, characterized in that, the preparation method comprises the following steps: culturing the above-mentioned host cell, and extracting from the culture to obtain the binding protein.
  • the sixth technical solution of the present invention is to provide a pharmaceutical composition comprising the above-mentioned binding protein.
  • the seventh technical solution of the present invention is to provide a medicine kit, the medicine kit includes a medicine box 1 and a medicine box 2, and the medicine box 1 includes the above-mentioned binding protein or drug combination
  • the second drug kit includes other antibodies or pharmaceutical compositions for treating cancer.
  • the eighth technical solution of the present invention is to provide an application of the above-mentioned binding protein or the above-mentioned pharmaceutical composition in the preparation of a medicine for treating and/or preventing cancer.
  • the cancer is preferably breast cancer, ovarian cancer, endometrial cancer, kidney cancer, melanoma, lung cancer, stomach cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma, Colorectal cancer, lymphoma, or multiple myeloma.
  • the ninth technical solution of the present invention is to provide a method for treating cancer, characterized in that the binding protein according to one of the technical solutions of the present invention is administered to a subject in need, or as described in one of the technical solutions of the present invention.
  • the cancer is preferably breast cancer, ovarian cancer, endometrial cancer, kidney cancer, melanoma, lung cancer, stomach cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma, Colorectal cancer, lymphoma, or multiple myeloma.
  • the tenth technical solution of the present invention is to provide a CD3 antibody.
  • the CD3 antibody comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 177, 191 and 221;
  • the chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 15, 84 and 141; or, its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are respectively SEQ ID NOs : the amino acid sequences shown in 178, 197 and 222;
  • its heavy chain variable region VH comprises
  • its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 294; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 258; or, its light chain variable region Region VL includes the amino acid sequence shown in SEQ ID NO: 295; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 259; or, its light chain variable region VL includes as SEQ ID NO:
  • the amino acid sequence shown in 296; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 260; or, its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 297; its The heavy chain variable region VH includes the amino acid sequence set forth in SEQ ID NO:259.
  • the CD3 antibody comprises a light chain variable region and a heavy chain variable region; its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are the amino acid sequences shown in SEQ ID NOs: 172, 192 and 216, respectively; Its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 20, 68 and 128; or, its light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, which are respectively SEQ ID NOs: 20, 68 and 128.
  • amino acid sequences shown in ID NOs: 172, 192 and 216; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are 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.
  • its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 245; or, its light chain variable region Region VL includes the amino acid sequence shown in SEQ ID NO: 291; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 257; or, its light chain variable region VL includes as SEQ ID NO:
  • the amino acid sequence shown in 291; its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 263; or, its light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 291; its The variable region VH of the heavy chain includes the amino acid sequence shown in SEQ ID NO: 281; or, the variable region VL of the light chain includes the amino acid sequence shown in SEQ ID NO: 291; the variable region VH of the heavy chain includes the amino acid sequence shown in SEQ ID NO: 291; The amino acid sequence shown in SEQ ID NO:244.
  • the CD3 antibody comprises two polypeptide chains; wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:357, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:313 the amino acid sequence shown; or, the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 357, and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 325; or, the first polypeptide The chain includes the amino acid sequence shown in SEQ ID NO:357, and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:328; or, the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:357 sequence, the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:346.
  • the CD3 antibody comprises a polypeptide chain comprising the amino acid sequence as set forth in SEQ ID NO:489, the amino acid sequence as set forth in SEQ ID NO:490, as in SEQ ID NO : the amino acid sequence shown in SEQ ID NO: 491, the amino acid sequence shown in SEQ ID NO: 492, or the amino acid sequence shown in SEQ ID NO: 493.
  • the eleventh technical solution of the present invention is to provide a BCMA antibody.
  • the BCMA antibody comprises a heavy chain variable region; its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 15, 75 and 133; or, its heavy chain variable region
  • the VH comprises HCDR1, HCDR2 and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 24, 76 and 134, respectively; or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively SEQ ID NOs: 25, 77 and the amino acid sequence shown in 135; or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 26, 78 and 136, respectively.
  • its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 248; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 249; or, its heavy chain
  • the variable region VH includes the amino acid sequence shown in SEQ ID NO:250; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO:251.
  • the BCMA antibody comprises a polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:316, the amino acid sequence shown in SEQ ID NO:317, and the amino acid sequence shown in SEQ ID NO:318 The amino acid sequence of , or, the amino acid sequence shown in SEQ ID NO:319.
  • the BCMA antibody comprises a heavy chain variable region, and its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 26, 90 and 136, respectively; or, its heavy chain can be
  • the variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 34, 78 and 146; or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively SEQ ID NO:35
  • the amino acid sequences shown in , 78 and 147; or, its heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NOs: 35, 90 and 147; or, its heavy chain variable Region VH comprises HCDR1, HCDR2 and HCDR3, which are respectively the amino acid sequences shown in SEQ ID NO: 36, 90 and 146; or, its heavy chain
  • its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 265; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 266; Or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 267; Or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 268; Or, its heavy chain can
  • the variable region VH includes the amino acid sequence shown in SEQ ID NO: 269; or, its heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 270; or, its heavy chain variable region VH includes the SEQ ID NO: 270 amino acid sequence
  • the BCMA antibody comprises a polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:330; or, the amino acid sequence set forth in SEQ ID NO:331; or, The amino acid sequence set forth in SEQ ID NO:332; or, the amino acid sequence set forth in SEQ ID NO:333; or, the amino acid sequence set forth in SEQ ID NO:334; or, the amino acid sequence set forth in SEQ ID NO:335 or, the amino acid sequence shown in SEQ ID NO:336; or, the amino acid sequence shown in SEQ ID NO:337; or, the amino acid sequence shown in SEQ ID NO:338; or, as SEQ ID NO:338
  • the present invention provides bivalent to multivalent and bispecific or multispecific binding proteins constructed using fully human heavy chain antibodies and single domain antibodies derived therefrom and methods of making and using such binding proteins. Compared with bivalent to multivalent and bispecific or multispecific binding proteins constructed with conventional IgG antibodies, it has many advantages, and it is more flexible in adjusting specificity and binding valence; it can construct polypeptide chains with smaller molecular weights. Fewer multispecific binding proteins with simpler structures; and different structures can be used to achieve different biological functions.
  • the functional activities for different targets are adjusted by parameters such as different structure types, relative positions, binding valences, etc., and then different activity combinations are designed to meet the needs of different clinical combination dosage combinations.
  • the use of heavy chain antibody VH domains can easily form a multivalent structure in series, which can promote the crosslinking of the target through the multivalent binding to the target, and further enhance the biological effect induced by the crosslinking of the target.
  • asymmetric tetravalent structures such as "1+3" can be achieved using heavy chain antibody VH domains, similar structures that are difficult to achieve with conventional IgG antibody-derived structures.
  • Figure 1 Schematic diagram of the molecular structure.
  • Figure 4 Binding activity of PD-L1 x CTLA4 double antibody to HEK293/hCTLA4 cells.
  • Figure 5 The activity of PD-L1 x CTLA4 double antibody molecule to block the binding of human CTLA4 protein and its ligand protein B7-1.
  • Figure 6 The activity of PD-L1 x CTLA4 double antibody molecule to block the binding of CHO-K1/hCTLA4 cells and its ligand protein B7-1.
  • Figure 7 Binding activity of PD-L1 x CTLA4 double antibody to hPDL1-His protein.
  • Figure 8 Binding activity of PD-L1 x CTLA4 double antibody to CHO-K1/hPDL1 cells.
  • Figure 9 Binding activity of PD-L1 x CTLA4 double antibody to MDA-MB-231 cells that highly express human PD-L1.
  • Figure 10 The activity of PD-L1 x CTLA4 diabodies to block the binding of CHO-K1/hPDL1 cells to their ligand protein PD-1.
  • Figure 11 The ability of PD-L1 x CTLA4 dual antibody molecule to kill CTLA4 + target cells via ADCC effect.
  • CTLA4-terminal activity is attenuated in IgG-HC-VH (VH at the C-terminus of IgG HC) and Fab-HCAb structures compared to IgG or HCAb bivalent structures, thereby reducing its dose-related toxicity.
  • Figure 18 Binding activity of HER2 x CTLA4 double antibody to CHO-K1/hCTLA4 cells.
  • FIG. 19 Activity of HER2 x CTLA4 diabodies to block the binding of human CTLA4 protein to its ligand protein B7-1.
  • Figure 20 Activity of HER2 x CTLA4 diabodies to block the binding of CHO-K1/hCTLA4 cells to their ligand protein B7-1.
  • Figure 21 Binding activity of HER2 x CTLA4 double antibody to SK-BR-3 cells.
  • Figure 22 The ability of HER2 x CTLA4 diabodies to kill HER2 + target cells via ADCC effect.
  • Figure 23 The ability of the HER2 x CTLA4 dual antibody molecule to inhibit the proliferation of HER2 + target cells SK-BR-3.
  • Figure 24 The ability of HER2 x CTLA4 dual antibody molecules to bind CTLA4 + cells and HER2 + cells simultaneously.
  • Figure 25 The ability of HER2 x CTLA4 dual antibody molecules to activate T cells in SEB stimulation experiments.
  • Figure 26 Pharmacokinetics of HER2 x CTLA4 dual antibody molecules in mice.
  • Figure 27 Binding activity of PD-L1 x 4-1BB diabodies to CHO-K1/hPDL1 cells.
  • Figure 28 Binding activity of 4-1BB antibody or PD-L1 x 4-1BB diabody molecule to CHO-K1/hu 4-1BB cells.
  • Figure 29 Binding activity of PD-L1 x 4-1BB diabodies to CHO-K1/cyno 4-1BB cells.
  • FIG. 30 PD-L1 x 4-1BB diabodies mediate T cell-specific activation by CHO-K1/hPDL1 cells.
  • FIG. 31 PD-L1 x 4-1BB diabodies mediate T cell-specific activation by MDA-MB-231 cells.
  • Figure 33 In the mixed lymphocyte reaction experiment, the PD-L1 x 4-1BB double antibody molecules with IgG-VH structure and Fab-HCAb structure have stronger T cell activation ability than the double antibody molecules with FIT-Ig structure.
  • Figure 34 Pharmacokinetics of PD-L1 x 4-1BB double antibody molecule in mice.
  • Figure 35 Binding activity of B7H4 x 4-1BB diabodies to SK-BR-3 cells.
  • Figure 36 Binding activity of B7H4 x 4-1BB diabody molecules to CHO-K1/hu 4-1BB cells.
  • the B7H4 x 4-1BB diabody molecule specifically depends on the expression of B7H4 for T cell activation.
  • Figure 42 Pharmacokinetics of B7H4 x 4-1BB double antibody molecule in mice.
  • Figure 43 Antitumor effect of B7H4 x 4-1BB double antibody molecule in 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.
  • FIG. 47 Internalization ability of BCMA HCAb antibodies into HEK293T/hBCMA cells.
  • Figure 48 Affinity of BCMA HCAb antibodies binding to human BCMA (BLI method).
  • Figure 49 Binding activity of variant molecules derived from BCMA HCAb antibody PR001046 to NCI-H929 cells.
  • Figure 50 Binding activity of BCMA x CD3 diabodies to HEK293T/hBCMA cells.
  • Figure 51 Binding activity of BCMA x CD3 diabodies to HEK293T/cynoBCMA cells.
  • Figure 52 Binding activity of BCMA x CD3 dual antibody molecules to NCI-H929 cells.
  • Figure 53 Binding activity of BCMA x CD3 diabody molecules to human T cells.
  • Figure 54 Binding activity of BCMA x CD3 diabody molecules to cynomolgus monkey T cells.
  • Figure 55 The ability of BCMA x CD3 double antibody molecule of scFv-Fc-VH(2) asymmetric structure to kill NCI-H929 cells.
  • Figure 56 The ability of BCMA x CD3 double antibody molecule of scFv-Fc-VH(1) asymmetric structure to kill NCI-H929 cells.
  • Figure 57 The ability of Fab-Fc-VH(1) asymmetrically structured BCMA x CD3 diabodies to kill NCI-H929 cells.
  • Figure 58 The ability of Fab-Fc-VH(2) asymmetrically structured BCMA x CD3 diabodies to kill NCI-H929 cells.
  • BCMA x CD3 double antibody molecules (PR001990, PR002309) mediate specific killing of NCI-H929 cells by effector cells and detection of cytokine release.
  • BCMA x CD3 double antibody molecules (PR002895, PR002953, PR003178) mediate specific killing of NCI-H929 cells by effector cells and detection of cytokine release.
  • Figure 61 Effect of soluble APRIL or BAFF on the target cell killing effect of BCMA x CD3 dual antibody molecules.
  • Figure 62 The effect of soluble BCMA on the target cell killing effect of BCMA x CD3 diabodies.
  • Figure 63 Pharmacokinetics of BCMA x CD3 dual antibody molecules in mice or rats.
  • Figure 64 Antitumor effect of BCMA x CD3 double antibody molecule in mouse tumor model.
  • Figure 65 Analysis results of SDS-PAGE and SEC-HPLC of protein samples obtained after transient transfection expression and one-step affinity purification of double antibody molecules.
  • Figure 66 Binding activity of CD3 antibodies to human T cells.
  • the term "antibody” or “H2L2” generally refers to a conventional four-chain antibody. Antibodies in most species exhibit a "Y"-shaped tetrameric structure comprising two identical heavy chains (H chains) and two identical light chains (L chains), also referred to as "H2L2".
  • the heavy chain includes the heavy chain variable region (VH) near the N-terminus and the heavy chain constant region (CH) near the C-terminus; the light chain includes the light chain variable region (VL) near the N-terminus and the light chain constant region (CL) near the C-terminus.
  • the heavy chain constant region of IgG antibody has three domains, namely CH1, CH2 and CH3; there is also a hinge region between CH1 and CH2.
  • the light chain variable region of the light chain is further divided into V ⁇ and V ⁇ , and the corresponding light chain constant regions are C ⁇ and C ⁇ , respectively.
  • the variable region of an antibody is its main site for recognizing and binding antigen; the variable region domains VH and VL and the constant region domains CH1 and CL of the antibody together constitute an antigen-binding fragment (Fab).
  • CH2 and CH3 constitute a crystallizable fragment (Fc), which is the main factor that exerts antibody effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) and affects the serum half-life of antibodies. area.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the term "heavy chain antibody” or “HCAb” generally refers to a class of antibodies that contain only heavy chain dimers.
  • the heavy chain antibody derived from Camelidae has no CH1 region between the heavy chain variable region and the hinge region except for the lack of light chain, and only contains one heavy chain variable region (VHH) and two Heavy chain constant domains (CH2 and CH3); its basic structure is a heavy chain dimer.
  • VHH fragment of the heavy chain antibody of camelid is different from the VH characteristics of conventional antibodies.
  • the VHH structure cloned and expressed alone has the same structural stability and binding activity with the original heavy chain antibody as the original heavy chain antibody.
  • the molecular weight is only about 13KDa, Therefore, it is also called nanobody or single domain antibody.
  • binding protein or "antigen-binding protein” generally refers to a protein comprising an antigen-binding moiety, and optionally a scaffold or backbone that allows the antigen-binding moiety to adopt a conformation that facilitates the binding of the antigen-binding protein to the antigen part.
  • An antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both may typically be included.
  • VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL can consist of three CDRs and four FR regions, which can be arranged from the amino terminus to the carboxy terminus in the following order: FR-1, CDR1, FR-2, CDR2, FR-3, CDR3, and FR-4 .
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the three CDRs of VH are denoted as HCDR1, HCDR2 and HCDR3 respectively, and can also be denoted as VH CDR1, VH CDR2 and VH CDR3;
  • the three CDRs of VL are denoted as LCDR1, LCDR2 and LCDR3, respectively, and can also be denoted as VL CDR1, VL CDR2 and VL CDR3.
  • 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), immunoconjugation antibodies, multispecific antibodies (eg, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • Fab antigen binding fragments
  • Fv fragments F(ab') 2
  • scFv di-scFv and/or dAb
  • immunoconjugation antibodies eg, multispecific antibodies (eg, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • the amino acid sequences of the CDRs are shown in accordance with the Chothia definition rules.
  • the CDRs of antibodies can be defined by a variety of methods in the art, for example, based on the Kabat definition rules for sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Bethesda, MD (1991)) and Chothia definition rules based on the location of structural loop regions (see JMol Biol 273:927-48, 1997).
  • the combined definition rule including the Kabat definition and the Chothia definition can also be used to determine the amino acid residues in the variable domain sequence.
  • the Combined definition rule is the combination of the Kabat definition and the range defined by Chothia, and a larger range is taken based on this, as shown in the following table. It will be understood by those of skill in the art that, unless otherwise specified, the terms "CDRs" and “complementarity determining regions” of a given antibody or regions thereof (eg, variable regions) are to be understood to encompass the above-described already described above as described by the present invention. complementarity-determining regions defined by any of the known schemes. Although the scope of protection claimed in the present invention is based on the sequences shown in the Chothia definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the protection scope of the present invention.
  • Laa-Lbb can refer to the amino acid sequence starting from the N-terminus of the antibody light chain, from position aa (Chothia coding rule) to bb position (Chothia coding rule);
  • Haa-Hbb can refer to starting from the N-terminus of the antibody heavy chain , the amino acid sequence from position aa (Chothia coding rule) to bb position (Chothia coding rule).
  • L24-L34 may refer to the amino acid sequence from position 24 to position 34 starting from the N-terminus of the antibody light chain according to the Chothia coding rules
  • H26-H32 may refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain according to the Chothia coding rules Amino acid sequence from position 26 to position 32. It should be known to those skilled in the art that when using Chothia to encode CDRs, there will be insertion sites at some positions (see http://bioinf.org.uk/abs/).
  • the term "monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies in the population are identical except for possible minor natural mutations.
  • Monoclonal antibodies are usually highly specific for a single antigenic site.
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without contamination by other immunoglobulins.
  • monoclonal denotes a characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring the production of the antibody by any particular method.
  • monoclonal antibodies used in accordance with the present invention can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • the term "fully human antibody” generally refers to an antibody obtained by transferring all the human antibody-encoding genes into a genetically engineered antibody gene-deficient animal, so that the animal expresses the antibody. All parts of an antibody, including the variable and constant regions of the antibody, are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, and the like.
  • the term "fully human heavy chain antibody” generally refers to a heavy chain antibody with a human antibody variable region VH obtained using Harbour HCAb transgenic mice (patent application WO2007/096779).
  • the endogenous antibody heavy chain locus and light chain locus of the transgenic mouse are both knocked out or inactivated, making it unable to produce mouse antibodies; then the human antibody heavy chain gene fragments (V, D, J segment) into the mouse, using the mouse's own rearrangement and mutation mechanism to produce an antibody with a human antibody gene sequence, the variable region of which is a human VH.
  • a fully human heavy chain antibody is obtained by fusion and recombination of the human VH with the human heavy chain constant region Fc.
  • the term “specifically binds” generally refers to the binding of an antibody to an epitope through its antigen binding domain, and that binding requires some complementarity between the antigen binding domain and the epitope.
  • an antibody is said to "specifically bind" an antigen when it binds an epitope more readily through its antigen-binding domain than it would bind a random, unrelated epitope.
  • Epitope refers to a specific group of atoms (eg, sugar side chains, phosphoryl groups, sulfonyl groups) or amino acids on an antigen to which an antigen binding protein (eg, an antibody) binds.
  • Fab generally refers to the antigen-binding portion of a conventional antibody (eg, IgG), including the heavy chain variable region VH, light chain variable region VL and heavy chain constant region domain CH1 and light chain variable region of the antibody.
  • Chain constant region CL In conventional antibodies, the C-terminus of VH is linked to the N-terminus of CH1 to form a heavy chain Fd fragment, the C-terminus of VL is linked to the N-terminus of CL to form a light chain, and the C-terminus of CH1 is further linked to the hinge region of the heavy chain and other constant The domains are linked to form the heavy chain.
  • Fab also refers to variant structures of Fab.
  • the C-terminus of VH is linked to the N-terminus of CL to form a polypeptide chain
  • the C-terminus of VL is linked to the N-terminus of CH1 to form another polypeptide chain, forming a Fab (cross VH/VL) structure
  • the CH1 of the Fab is not linked to the hinge region, but the C-terminus of the CL is linked to the hinge region of the heavy chain to form a Fab (cross Fd/LC) structure.
  • VH generally refers to the heavy chain variable region VH domain of an antibody, that is, it can be the heavy chain variable region VH of a conventional antibody (H2L2 structure) of humans or other animals, or it can be Camelidae, etc.
  • the heavy chain variable region VHH of an animal heavy chain antibody (HCAb structure) can also be the heavy chain variable region VH of a fully human heavy chain antibody (HCAb structure) produced by using Harbour HCAb transgenic mice.
  • binding moieties generally refers to any protein functional region that can specifically bind to an antigen, which can be either "Fab", "VH”, or other antigen-binding forms (such as lipocalins (lipocalins), neural cell adhesion molecules (NCAM), fibronectin (fibronectin), ankyrin repeat fragment proteins (DARPins) and other derived protein structures).
  • Fab lipocalins
  • NCAM neural cell adhesion molecules
  • fibronectin fibronectin
  • DARPins ankyrin repeat fragment proteins
  • binding valency generally refers to the number of "binding domains" in a binding protein, and also refers to the maximum number of antigen molecules or epitopes that the binding protein can bind to, for example, conventional IgG antibodies can simultaneously bind two The same antigen molecule, its binding valence is two; while the binding valence of Fab antibody is one.
  • bispecific binding protein generally refers to a binding protein having two antigen binding specificities.
  • the two antigen binding specificities can be binding to two different antigens or to two different epitopes of the same antigen.
  • Bispecific binding proteins can typically include bispecific antibodies, derivatives thereof, and the like.
  • multispecific binding protein generally refers to a binding protein having two or more antigen binding specificities.
  • Multispecific binding proteins can typically include multispecific antibodies, derivatives thereof, and the like.
  • MFI Green Fluorescent Intensity
  • FACS Fluorescence Activated Cell Sorting
  • PD-L1 generally refers to programmed death ligand 1 protein, functional variants thereof and/or functional fragments thereof.
  • PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), and is a protein encoded by (in humans) the CD274 gene.
  • CD274 cluster of differentiation 274
  • B7-H1 B7 homolog 1
  • PD-L1 sequences are known in the art.
  • the amino acid sequence of an exemplary full-length human PD-L1 protein can be found under NCBI Accession No. NP_054862 or UniProt Accession No. Q9NZQ7
  • an exemplary full-length cynomolgus monkey PD-L1 protein sequence can be found under NCBI Accession No. XP_005581836 or Uniprot Accession No. Q9NZQ7; Found under accession number G7PSE7.
  • PD-1 generally refers to programmed death 1 receptor (also known as CD279), functional variants thereof and/or functional fragments thereof.
  • PD-1 sequences are known in the art.
  • an exemplary full-length human PD-1 protein sequence can be found under NCBI Accession No. NP_005009; an exemplary full-length cynomolgus monkey PD-1 protein sequence can be found under NCBI Accession No. NP_001271065 or Uniprot Accession No. BOLAJ3.
  • CD80 generally refers to Cluster of Differentiation Protein 80 (also known as B7-1), functional variants thereof and/or functional fragments thereof.
  • CD80 sequences are known in the art. For example, an exemplary full-length human CD80 sequence can be found in Uniprot Accession No. P33681.
  • CTLA4 generally refers to cytotoxic T lymphocyte-associated protein-4 (also known as CD152), functional variants thereof and/or functional fragments thereof.
  • CTLA4 sequences are known in the art. For example, an exemplary full-length human CTLA4 sequence can be found in Uniprot Accession No. P16410; an exemplary full-length cynomolgus CTLA4 sequence can be found in Uniprot Accession No. G7PL88.
  • HER2 generally refers to the receptor tyrosine kinase erbB-2 (also known as ERBB2), functional variants thereof and/or functional fragments thereof.
  • HER2 sequences are known in the art. For example, an exemplary full-length human HER2 sequence can be found in Uniprot accession number P04626; an exemplary full-length cynomolgus monkey HER2 sequence can be found in NCBI accession number XP_005584091.
  • B7H4 generally refers to V-Set domain-containing suppressor of T cell activation 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 in Uniprot Accession No. Q7Z7D3; an exemplary full-length cynomolgus monkey B7H4 sequence can be found in NCBI Accession No. XP_005542249; an exemplary full-length mouse B7H4 sequence It can be found under Uniprot accession number Q7TSP5.
  • 4-1BB generally refers to tumor necrosis factor receptor superfamily member 9 (also known as CD137, the 4-1BBL receptor), functional variants and/or functional fragments thereof.
  • the 4-1BB sequence is known in the art.
  • an exemplary full-length human 4-1BB sequence can be found in Uniprot Accession No. Q07011; an exemplary full-length cynomolgus monkey 4-1BB sequence can be found in NCBI Accession No. XP_005544945.
  • BCMA generally refers to tumor necrosis factor receptor superfamily member 17 (also known as CD269, B cell maturation protein), functional variants and/or functional fragments thereof.
  • BCMA sequences are known in the art. For example, an exemplary full-length human BCMA sequence can be found in Uniprot Accession No. Q02223; an exemplary full-length cynomolgus monkey BCMA sequence can be found in NCBI Accession No. XP_005591343.
  • BAFF generally refers to tumor necrosis factor ligand superfamily member 13B (also known as CD257, B cell activator), 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 in Uniprot Accession No. Q9Y275.
  • APRIL generally refers to tumor necrosis factor ligand superfamily member 13 (also known as CD256, proliferation-inducing ligand), functional variants thereof and/or functional fragments thereof.
  • APRIL sequences are known in the art. For example, an exemplary full-length human APRIL sequence can be found in Uniprot Accession No. 075888.
  • CD3 generally refers to the TCR/CD3 receptor protein complex on T cells.
  • the specificity of the T cell response is mediated by the recognition of pMHC by the molecular complex of TCR and CD3.
  • TCR is a heterodimer composed of two different transmembrane polypeptide chains. There are four kinds of peptide chains: ⁇ , ⁇ , ⁇ , and ⁇ . According to the different combinations of peptide chains, TCR is divided into TCR ⁇ and TCR ⁇ .
  • CD3 has different transmembrane polypeptide chains, namely ⁇ , ⁇ , ⁇ , ⁇ , which interact to form homodimers or heterodimers as part of the TCR-CD3 complex. Because the cytoplasmic region of the TCR peptide chain is very short, it is generally believed that the activation signal generated by the TCR recognizing antigen is transduced into T cells by the CD3 peptide chain.
  • 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 in Uniprot Accession No. P07766, and an exemplary full-length cynomolgus monkey CD3E sequence can be found in Uniprot Accession No. Q95LI5.
  • the present invention is further described below by way of examples, but the present invention is not limited to the scope of the described examples.
  • the examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, methods of inserting protein-encoding genes into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are useful for this study. It is well known to those of ordinary skill in the art and described in numerous publications.
  • the experimental methods that do not specify specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product description.
  • Example 1 Structural design of HCAb-based multispecific binding proteins
  • This example lists several structures of Fc-containing, symmetric or asymmetric, multivalent and multispecific binding proteins constructed by using fully human heavy chain antibodies (HCAb) and their derived single domain antibodies (sdAb) .
  • the domains are linked by linker peptides.
  • amino acid mutations are introduced in the Fc region of the heavy chain to alter its binding to Fc receptors and thereby alter the associated effector function or other properties.
  • different amino acid mutations were introduced in the Fc regions of the two heavy chains to reduce the formation of heavy chain homodimerization.
  • Table 1-1 and Figure 1 list the molecular structures of the multispecific binding proteins included in the present application, and each structure will be further described below.
  • the number of polypeptide chains contained in the molecular structure it usually refers to the number of "different polypeptide chains"; for example, conventional IgG antibodies have two different polypeptide chains, namely Heavy chain and light chain, although the IgG antibody molecule itself is a tetrapeptide protein molecule containing two identical heavy chains and two identical light chains, its two different polypeptide chains are specifically referred to when describing its structural characteristics.
  • the binding valence refers to the number of antigen-binding sites in the molecular structure. For example, a conventional IgG antibody can bind two identical antigen molecules at the same time, and its binding valence is two.
  • Tables 1-2 list the linker peptide sequences that may be used in the structural design of the present application.
  • Table 1-1 The molecular structure of the HCAb-based multispecific binding protein listed in the application of the present invention
  • the present invention provides a method for constructing bispecific binding proteins using two parental monoclonal antibodies: conventional antibody A that binds a first antigen and heavy chain antibody B that binds a second antigen.
  • the Fab end is derived from conventional antibody A
  • VH_A and VL_A are the variable region of the heavy chain and the variable region of the light chain of antibody A, respectively.
  • the VH end is derived from heavy chain antibody B
  • 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, respectively, of the heavy chain constant region.
  • L1 and L2 are the first and second linker peptides, respectively.
  • the binding protein of structure (1) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also called short chain, from the amino terminus to the carboxyl terminus, which comprises VH_A-CH1;
  • the peptide chain also called the long chain, from the amino terminus to the carboxy terminus, contains VL_A-CL-L1-VH_B-L2-CH2-CH3.
  • the VL_A of antibody A and the VH_B of heavy chain antibody B are fused on the same polypeptide chain, which can avoid mismatched by-products generated by the association of VL_A and VH_B.
  • VH_B of polypeptide chain 2 is linked to CH2 via linker peptide L2;
  • L2 can be the hinge region of IgG or a linker peptide sequence derived from the hinge region or a sequence listed in Table 1-2, preferably a human IgG1 hinge or a human IgG1 hinge (C220S) Or the sequence of G5-LH.
  • the CL of polypeptide chain 2 is directly fused to VH_B, that is, the length of L1 is 0.
  • the CL of polypeptide chain 2 is linked to VH_B via a linker peptide L1; L1 may be the sequence listed in Tables 1-2.
  • the binding protein of structure (2) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also called short chain, from the amino terminus to the carboxy terminus, which comprises VL_A-CL;
  • the peptide chain also called the long chain, from the amino terminus to the carboxy terminus, contains VH_A-CH1-L1-VH_B-L2-CH2-CH3.
  • VH_B of polypeptide chain 2 is linked to CH2 via linker peptide L2;
  • L2 can be the hinge region of IgG or a linker peptide sequence derived from the hinge region or a sequence listed in Table 1-2, preferably a human IgG1 hinge or a human IgG1 hinge (C220S) Or the sequence of G5-LH.
  • CH1 of polypeptide chain 2 is directly fused to VH_B, that is, the length of L1 is 0.
  • CH1 of polypeptide chain 2 is linked to VH_B via a linking peptide L1; L1 may be the sequence listed in Tables 1-2.
  • the present invention provides a method for constructing bispecific binding proteins using two parental monoclonal antibodies: conventional antibody A that binds a first antigen and heavy chain antibody B that binds a second antigen.
  • the Fab end is derived from conventional antibody A
  • VH_A and VL_A are the variable region of the heavy chain and the variable region of the light chain of antibody A, respectively.
  • the VH end is derived from heavy chain antibody B
  • 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, respectively, of the heavy chain constant region.
  • L is the linker peptide and h is the hinge region or derived sequence of an IgG antibody.
  • the binding protein of structure (3) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also called short chain, from the amino terminus to the carboxyl terminus, which comprises VL_A-CL;
  • the peptide chain also called long chain, from the amino terminus to the carboxy terminus, contains VH_B-L-VH_A-CH1-h-CH2-CH3.
  • VH_B of polypeptide chain 2 is directly fused to VH_A, that is, the length of L is 0.
  • VH_B of polypeptide chain 2 is linked to VH_A via a linker peptide L; L may be a sequence listed in Tables 1-2.
  • the binding protein of structure (4) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also called short chain, from the amino terminus to the carboxy terminus, which comprises VH_B-L-VL_A-CL; polypeptide chain 2 Or the second polypeptide chain, also called the long chain, from the amino terminus to the carboxy terminus, which comprises VH_A-CH1-h-CH2-CH3.
  • VH_B of polypeptide chain 1 is directly fused to VL_A, that is, the length of L is 0.
  • VH_B of polypeptide chain 1 is linked to VL_A via a linker peptide L; L may be the sequence listed in Tables 1-2.
  • the binding protein of structure (5) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also called short chain, from the amino terminus to the carboxyl terminus, which comprises VL_A-CL;
  • the peptide chain also called the long chain, from the amino terminus to the carboxy terminus, contains VH_A-CH1-h-CH2-CH3-L-VH_B.
  • CH3 of polypeptide chain 2 is directly fused to VH_B, that is, the length of L is 0.
  • CH3 of polypeptide chain 2 is linked to VH_B via a linker peptide L; L may be the sequence listed in Tables 1-2.
  • the binding protein of structure (6) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also called short chain, from the amino terminus to the carboxyl terminus, which comprises VL_A-CL-L-VH_B; polypeptide chain 2 Or the second polypeptide chain, also called the long chain, from the amino terminus to the carboxy terminus, which comprises VH_A-CH1-h-CH2-CH3.
  • CL of polypeptide chain 1 is directly fused to VH_B, that is, the length of L is 0.
  • the CL of polypeptide chain 1 is linked to VH_B via a linker peptide L; L may be a sequence listed in Tables 1-2.
  • the present invention provides a method for constructing bispecific binding proteins using two parental monoclonal antibodies: conventional antibody A that binds a first antigen and heavy chain antibody B that binds a second antigen.
  • the present invention also provides a method for constructing a trispecific binding protein using three parental monoclonal antibodies: conventional antibody A that binds to the first antigen, heavy chain antibody B that binds to the second antigen, and heavy chain antibody that binds the third antigen C.
  • the Fab end is derived from conventional antibody A
  • VH_A and VL_A are the variable region of the heavy chain and the variable region of the light chain of antibody A, respectively.
  • the VH end is derived from heavy chain antibody B or heavy chain antibody C
  • VH_B is the heavy chain variable region of heavy chain antibody B
  • VH_C is the heavy chain variable region of heavy chain antibody C.
  • CL is the light chain constant region domain.
  • CH1, CH2 and CH3 are the first, second and third domains, respectively, of the heavy chain constant region.
  • L1 and L2 are the first and second linker peptides, respectively, and h is the hinge region or derived sequence of an IgG antibody.
  • Structure (7) represents a bispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also referred to as the short chain, from the amino terminus to the carboxy terminus, which comprises VL_A-CL; Polypeptide chain 2 or the second polypeptide chain, also called the long chain, from the amino terminus to the carboxy terminus, which comprises VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_B.
  • CH3 of polypeptide chain 2 is directly fused to VH_B, that is, the length of L1 is 0.
  • CH3 of polypeptide chain 2 is linked to VH_B via a linker peptide L1; L1 may be the sequence listed in Tables 1-2.
  • first VH_B of the polypeptide chain 2 is directly fused to the second VH_B, that is, the length of L2 is 0.
  • first VH_B of polypeptide chain 2 is linked to the second VH_B via a linking peptide L2; L2 may be the sequence listed in Tables 1-2.
  • Structure (8) represents a trispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also known as the short chain, from the amino terminus to the carboxy terminus, which comprises VL_A-CL; Polypeptide chain 2 or the second polypeptide chain, also called the long chain, from the amino terminus to the carboxy terminus, which comprises VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_C.
  • CH3 of polypeptide chain 2 is directly fused to VH_B, that is, the length of L1 is 0.
  • CH3 of polypeptide chain 2 is linked to VH_B via a linker peptide L1; L1 may be the sequence listed in Tables 1-2.
  • VH_B and VH_C of polypeptide chain 2 are directly fused and linked, that is, the length of L2 is 0.
  • VH_B of polypeptide chain 2 is linked to VH_C via a linker peptide L2; L2 may be the sequence listed in Tables 1-2.
  • the present invention provides a method for constructing bispecific binding proteins using two parental monoclonal antibodies: conventional antibody A that binds a first antigen and heavy chain antibody B that binds a second antigen.
  • the present invention also provides a method for constructing a trispecific binding protein using three parental monoclonal antibodies: conventional antibody A that binds to the first antigen, heavy chain antibody B that binds to the second antigen, and heavy chain antibody that binds the third antigen C.
  • the Fab end is derived from conventional antibody A
  • VH_A and VL_A are the variable region of the heavy chain and the variable region of the light chain of antibody A, respectively.
  • the VH end is derived from heavy chain antibody B or heavy chain antibody C
  • VH_B is the heavy chain variable region of heavy chain antibody B
  • VH_C is the heavy chain variable region of heavy chain antibody C.
  • CL is the light chain constant region domain.
  • CH1, CH2 and CH3 are the first, second and third domains, respectively, of the heavy chain constant region.
  • L1 and L2 are the first and second linker peptides, respectively, and h is the hinge region or derived sequence of an IgG antibody.
  • Structure (9) represents a bispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also known as the short chain, from the amino terminus to the carboxy terminus, which comprises VH_B-L1- VL_A-CL; polypeptide chain 2 or second polypeptide chain, also called long chain, from amino terminus to carboxyl terminus, which comprises VH_B-L2-VH_A-CH1-h-CH2-CH3.
  • VH_B of polypeptide chain 1 is directly fused to VL_A, ie the length of L1 is 0.
  • VH_B of polypeptide chain 1 is linked to VL_A via a linker peptide L1; L1 may be the sequence listed in Tables 1-2.
  • VH_B of polypeptide chain 2 is directly fused to VH_A, that is, the length of L2 is 0.
  • VH_B of polypeptide chain 2 is linked to VH_A via a linker peptide L2; L2 may be the sequence listed in Tables 1-2.
  • L1 and L2 may be the same sequence. In another embodiment, L1 and L2 may be different sequences.
  • Structure (10) represents a trispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, also known as the short chain, from the amino terminus to the carboxy terminus, which comprises VH_B-L1- VL_A-CL; polypeptide chain 2 or second polypeptide chain, also called long chain, from amino terminus to carboxyl terminus, which comprises VH_C-L2-VH_A-CH1-h-CH2-CH3.
  • VH_B of polypeptide chain 1 is directly fused to VL_A, ie the length of L1 is 0.
  • VH_B of polypeptide chain 1 is linked to VL_A via a linker peptide L1; L1 may be the sequence listed in Tables 1-2.
  • VH_C of polypeptide chain 2 is directly fused to VH_A, that is, the length of L2 is 0.
  • VH_C of polypeptide chain 2 is linked to VH_A via a linker peptide L2; L2 may be the sequence listed in Tables 1-2.
  • L1 and L2 may be the same sequence. In another embodiment, L1 and L2 may be different sequences.
  • the present invention provides a method for constructing bispecific binding proteins using two parental monoclonal antibodies: conventional antibody A that binds a first antigen and heavy chain antibody B that binds a second antigen.
  • the Fab end is derived from conventional antibody A
  • VH_A and VL_A are the variable region of the heavy chain and the variable region of the light chain of antibody A, respectively.
  • the VH end is derived from heavy chain antibody B
  • 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, respectively, of the heavy chain constant region.
  • h is the hinge region or derived sequence of an IgG antibody. Different amino acid mutations were introduced in the Fc regions of the two heavy chains to reduce the formation of heavy chain homodimerization.
  • h in the VH_B-containing polypeptide chain may be the hinge region of IgG or a linker peptide sequence derived from the hinge region such as the sequence of human IgG1 hinge (C220S) or G5-LH in Tables 1-2.
  • the binding protein of structure (11) comprises three distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which contains VL_A-CL; polypeptide chain 2 or the second polypeptide chain, from the amino terminus to the carboxy terminus, which contains VH_A-CH1-h-CH2-CH3; polypeptide chain 3, from the amino terminus to the carboxy terminus, which contains VH_B-h-
  • the binding protein of structure (12) contains three distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which contains VH_A-CL; polypeptide chain 2 or the second polypeptide chain, from the amino terminus To the carboxy terminus, it contains VL_A-CH1-h-CH2-CH3; polypeptide chain 3, from the amino terminus to the carboxy terminus, it contains VH_B-h-CH2-CH3.
  • the binding protein of structure (13) comprises three distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which comprises VH_A-CH1; polypeptide chain 2 or the second polypeptide chain, from the amino terminus To the carboxy terminus, it contains VL_A-CL-h-CH2-CH3; polypeptide chain 3 or the third polypeptide chain, from the amino terminus to the carboxy terminus, it contains VH_B-h-CH2-CH3.
  • h in polypeptide chain 2 is a hinge region or a linking peptide, the sequence of which may be the sequence of LH1 in Tables 1-2.
  • the present invention provides a method for constructing bispecific binding proteins using two parental monoclonal antibodies: conventional antibody A that binds a first antigen and heavy chain antibody B that binds a second antigen.
  • the present invention also provides a method for constructing a trispecific binding protein using three parental monoclonal antibodies: conventional antibody A that binds to the first antigen, heavy chain antibody B that binds to the second antigen, and heavy chain antibody that binds the third antigen C.
  • the Fab end is derived from conventional antibody A
  • VH_A and VL_A are the variable region of the heavy chain and the variable region of the light chain of antibody A, respectively.
  • the VH end is derived from heavy chain antibody B or heavy chain antibody C
  • VH_B is the heavy chain variable region of heavy chain antibody B
  • VH_C is the heavy chain variable region of heavy chain antibody C.
  • CL is the light chain constant region domain.
  • CH1, CH2 and CH3 are the first, second and third domains, respectively, of the heavy chain constant region.
  • L is the linker peptide and h is the hinge region or derived sequence of an IgG antibody. Different amino acid mutations were introduced in the Fc regions of the two heavy chains to reduce the formation of heavy chain homodimerization.
  • h in the VH_B-containing polypeptide chain may be the hinge region of IgG or a linker peptide sequence derived from the hinge region such as the sequence of human IgG1 hinge (C220S) or G5-LH in Tables 1-2.
  • Structure (14) represents a bispecific binding protein comprising three distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which comprises VL_A-CL; polypeptide chain 2 or the second polypeptide chain Polypeptide chain, from amino terminus to carboxy terminus, VH_A-CH1-h-CH2-CH3; Polypeptide chain 3 or a third polypeptide chain, from amino terminus to carboxy terminus, which comprises VH_B-L-VH_B-h-CH2- CH3.
  • the first VH_B of polypeptide chain 3 is directly fused to the second VH_B, that is, the length of L is 0.
  • the first VH_B of polypeptide chain 3 is linked to the second VH_B via a linker peptide L; L may be a sequence listed in Tables 1-2.
  • Structure (15) represents a trispecific binding protein comprising three distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which comprises VL_A-CL; polypeptide chain 2 or the second polypeptide chain Polypeptide chain, from amino terminus to carboxy terminus, VH_A-CH1-h-CH2-CH3; Polypeptide chain 3 or a third polypeptide chain, from amino terminus to carboxy terminus, which comprises VH_C-L-VH_B-h-CH2- CH3.
  • VH_C of polypeptide chain 3 is directly fused to VH_B, that is, the length of L is 0.
  • VH_C of polypeptide chain 3 is linked to VH_B via a linker peptide L; L may be the sequence listed in Tables 1-2.
  • Structure (16) represents a bispecific binding protein comprising three distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which comprises VH_A-CL; polypeptide chain 2 or the second polypeptide chain Polypeptide chain, from amino terminus to carboxy terminus, VL_A-CH1-h-CH2-CH3; Polypeptide chain 3 or a third polypeptide chain, from amino terminus to carboxy terminus, which comprises VH_B-L-VH_B-h-CH2- CH3.
  • the first VH_B of polypeptide chain 3 is directly fused to the second VH_B, that is, the length of L is 0.
  • the first VH_B of polypeptide chain 3 is linked to the second VH_B via a linker peptide L; L may be a sequence listed in Tables 1-2.
  • Structure (17) represents a trispecific binding protein comprising three distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which comprises VH_A-CL; polypeptide chain 2 or the second polypeptide chain Polypeptide chain, from amino terminus to carboxy terminus, VL_A-CH1-h-CH2-CH3; Polypeptide chain 3 or a third polypeptide chain, from amino terminus to carboxy terminus, which comprises VH_C-L-VH_B-h-CH2- CH3.
  • VH_C of polypeptide chain 3 is directly fused to VH_B, that is, the length of L is 0.
  • VH_C of polypeptide chain 3 is linked to VH_B via a linker peptide L; L may be the sequence listed in Tables 1-2.
  • Structure (18) represents a bispecific binding protein comprising three distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which comprises VH_A-CH1; polypeptide chain 2 or the second polypeptide chain Polypeptide chain, from amino terminus to carboxy terminus, VL_A-CL-h-CH2-CH3; Polypeptide chain 3 or a third polypeptide chain, from amino terminus to carboxy terminus, which comprises VH_B-L-VH_B-h-CH2- CH3.
  • the first VH_B of polypeptide chain 3 is directly fused to the second VH_B, that is, the length of L is 0.
  • the first VH_B of polypeptide chain 3 is linked to the second VH_B via a linker peptide L; L may be a sequence listed in Tables 1-2.
  • h in polypeptide chain 2 is a hinge region or a linking peptide, the sequence of which may be the sequence of LH1 in Tables 1-2.
  • Structure (19) represents a trispecific binding protein comprising three distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which comprises VH_A-CH1; polypeptide chain 2 or the second polypeptide chain Polypeptide chain, from amino terminus to carboxy terminus, VL_A-CL-h-CH2-CH3; Polypeptide chain 3 or third polypeptide chain, from amino terminus to carboxy terminus, which comprises VH_C-L-VH_B-h-CH2- CH3.
  • VH_C of polypeptide chain 3 is directly fused to VH_B, that is, the length of L is 0.
  • VH_C of polypeptide chain 3 is linked to VH_B via a linker peptide L; L may be the sequence listed in Tables 1-2.
  • h in polypeptide chain 2 is a hinge region or a linking peptide, the sequence of which may be the sequence of LH1 in Tables 1-2.
  • the present invention provides a method for constructing bispecific binding proteins using two parental monoclonal antibodies: conventional antibody A that binds a first antigen and heavy chain antibody B that binds a second antigen.
  • the scFv end is derived from conventional antibody A
  • VH_A and VL_A are the variable region of the heavy chain and the variable region of the light chain of antibody A, respectively.
  • the VH end is derived from heavy chain antibody B
  • VH_B is the heavy chain variable region of heavy chain antibody B.
  • CH2 and CH3 are the second and third domains, respectively, of the heavy chain constant region.
  • L is the linker peptide and h is the hinge region or derived sequence of an IgG antibody. Different amino acid mutations were introduced in the Fc regions of the two heavy chains to reduce the formation of heavy chain homodimerization.
  • sequence of the scFv may be VH-linker-VL. In another embodiment, the sequence of the scFv may be VL-linker-VH.
  • L in the polypeptide chain is a linking peptide, which can be the sequence listed in Tables 1-2, preferably the sequence of GS_15 or GS_20.
  • h in the polypeptide chain is the hinge region or derived sequence of IgG antibody, which can be the sequence listed in Table 1-2, preferably the sequence of human IgG1 hinge (C220S) or G5-LH.
  • the binding protein of structure (20) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxyl terminus, which comprises VL_A-L-VH_A-h-CH2-CH3; polypeptide chain 2 or The second polypeptide chain, from the amino terminus to the carboxy terminus, contains VH_B-h-CH2-CH3.
  • the binding protein of structure (21) comprises two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxyl terminus, which comprises VH_A-L-VL_A-h-CH2-CH3; polypeptide chain 2 or The second polypeptide chain, from the amino terminus to the carboxy terminus, contains VH_B-h-CH2-CH3.
  • the present invention provides a method for constructing bispecific binding proteins using two parental monoclonal antibodies: conventional antibody A that binds a first antigen and heavy chain antibody B that binds a second antigen.
  • the present invention also provides a method for constructing a trispecific binding protein using three parental monoclonal antibodies: conventional antibody A that binds to the first antigen, heavy chain antibody B that binds to the second antigen, and heavy chain antibody that binds the third antigen C.
  • the scFv end is derived from conventional antibody A
  • VH_A and VL_A are the variable region of the heavy chain and the variable region of the light chain of antibody A, respectively.
  • the VH end is derived from heavy chain antibody B or heavy chain antibody C
  • VH_B is the heavy chain variable region of heavy chain antibody B
  • VH_C is the heavy chain variable region of heavy chain antibody C.
  • CH2 and CH3 are the second and third domains, respectively, of the heavy chain constant region.
  • L1 and L2 are the first and second linker peptides, respectively, and h is the hinge region or derived sequence of an IgG antibody. Different amino acid mutations were introduced in the Fc regions of the two heavy chains to reduce the formation of heavy chain homodimerization.
  • sequence of the scFv may be VH-linker-VL. In another embodiment, the sequence of the scFv may be VL-linker-VH.
  • L1 in the polypeptide chain is a linker peptide linking VH and VL in the scFv, which can be the sequence listed in Table 1-2, preferably the sequence of GS_15 or GS_20.
  • L2 in the polypeptide chain is a linker peptide between the heavy chain variable regions linking two heavy chain antibodies, and may be the sequence listed in Tables 1-2.
  • the heavy chain variable regions of two heavy chain antibodies are directly fused, ie, the length of L2 is zero.
  • h in the polypeptide chain is the hinge region or derived sequence of IgG antibody, which can be the sequence listed in Table 1-2, preferably the sequence of human IgG1 hinge (C220S) or G5-LH.
  • Structure (22) represents a bispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which comprises VL_A-L1-VH_A-h-CH2 -CH3; polypeptide chain 2 or second polypeptide chain, from amino terminus to carboxy terminus, which comprises VH_B-L2-VH_B-h-CH2-CH3.
  • first VH_B of the polypeptide chain 2 is directly fused to the second VH_B, that is, the length of L2 is 0.
  • first VH_B of polypeptide chain 2 is linked to the second VH_B via a linking peptide L2; L2 may be the sequence listed in Tables 1-2.
  • Structure (23) represents a bispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which comprises VH_A-L1-VL_A-h-CH2 -CH3; polypeptide chain 2 or second polypeptide chain, from amino terminus to carboxy terminus, which comprises VH_B-L2-VH_B-h-CH2-CH3.
  • first VH_B of the polypeptide chain 2 is directly fused to the second VH_B, that is, the length of L2 is 0.
  • first VH_B of polypeptide chain 2 is linked to the second VH_B via a linking peptide L2; L2 may be the sequence listed in Tables 1-2.
  • Structure (24) represents a trispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which comprises VL_A-L1-VH_A-h-CH2 -CH3; polypeptide chain 2 or second polypeptide chain, from amino terminus to carboxy terminus, which comprises VH_C-L2-VH_B-h-CH2-CH3.
  • VH_C of polypeptide chain 2 is directly fused to VH_B, that is, the length of L2 is 0.
  • VH_C of polypeptide chain 2 is linked to VH_B via a linker peptide L2; L2 may be the sequence listed in Tables 1-2.
  • Structure (25) represents a trispecific binding protein comprising two distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which comprises VH_A-L1-VL_A-h-CH2 -CH3; polypeptide chain 2 or second polypeptide chain, from amino terminus to carboxy terminus, which comprises VH_C-L2-VH_B-h-CH2-CH3.
  • VH_C of polypeptide chain 2 is directly fused to VH_B, that is, the length of L2 is 0.
  • VH_C of polypeptide chain 2 is linked to VH_B via a linker peptide L2; L2 may be the sequence listed in Tables 1-2.
  • the present invention provides a method for constructing bispecific binding proteins using two parental monoclonal antibodies: conventional antibody A that binds a first antigen and heavy chain antibody B that binds a second antigen.
  • the present invention also provides a method for constructing a trispecific binding protein using three parental monoclonal antibodies: conventional antibody A that binds to the first antigen, heavy chain antibody B that binds to the second antigen, and heavy chain antibody that binds the third antigen C.
  • the present invention also provides a method for constructing a tetraspecific binding protein using four parental monoclonal antibodies: conventional antibody A that binds to the first antigen, heavy chain antibody B that binds to the second antigen, and heavy chain antibody that binds the third antigen C and heavy chain antibody D that binds the fourth antigen.
  • the Fab end is derived from conventional antibody A
  • VH_A and VL_A are the variable region of the heavy chain and the variable region of the light chain of antibody A, respectively.
  • the VH end is derived from heavy chain antibody B or heavy chain antibody C or heavy chain antibody D
  • 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
  • VH_D is heavy chain antibody
  • CL is the light chain constant region domain.
  • CH1, CH2 and CH3 are the first, second and third domains, respectively, of the heavy chain constant region.
  • L1 and L2 are linking peptides and h is the hinge region or derived sequence of an IgG antibody. Different amino acid mutations were introduced in the Fc regions of the two heavy chains to reduce the formation of heavy chain homodimerization.
  • h in the VH_B-containing polypeptide chain may be the hinge region of IgG or a linker peptide sequence derived from the hinge region such as the sequence of human IgG1 hinge (C220S) or G5-LH in Tables 1-2.
  • Structure (26) represents a bispecific binding protein comprising three distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which comprises VL_A-CL; polypeptide chain 2 or the second polypeptide chain Polypeptide chain, from amino terminus to carboxy terminus, VH_A-CH1-h-CH2-CH3; Polypeptide chain 3 or a third polypeptide chain, from amino terminus to carboxy terminus, which comprises VH_B-L1-VH_B-L2-VH_B- h-CH2-CH3.
  • first VH_B of polypeptide chain 3 is directly fused to the second VH_B, that is, the length of L1 is 0.
  • first VH_B of polypeptide chain 3 is linked to the second VH_B via a linking peptide L1; L1 may be the sequence listed in Tables 1-2.
  • the second VH_B of polypeptide chain 3 is directly fused to the third VH_B, that is, the length of L2 is 0.
  • the second VH_B of polypeptide chain 3 is linked to the third VH_B via a linking peptide L2; L2 may be a sequence listed in Tables 1-2.
  • Structure (27) represents a tetraspecific binding protein comprising three distinct polypeptide chains: polypeptide chain 1 or the first polypeptide chain, from the amino terminus to the carboxy terminus, which comprises VL_A-CL; polypeptide chain 2 or the second polypeptide chain Polypeptide chain, from amino terminus to carboxy terminus, VH_A-CH1-h-CH2-CH3; Polypeptide chain 3 or a third polypeptide chain, from amino terminus to carboxy terminus, which comprises VH_D-L1-VH_C-L2-VH_B- h-CH2-CH3.
  • VH_D of polypeptide chain 3 is directly fused to VH_C, that is, the length of L1 is 0.
  • the VH_D of polypeptide chain 3 is linked to VH_C via a linker peptide L1; L1 may be the sequence listed in Tables 1-2.
  • VH_C of polypeptide chain 3 is directly fused to VH_B, that is, the length of L2 is 0.
  • VH_C of polypeptide chain 3 is linked to VH_B via a linking peptide L2; L2 may be the sequence listed in Tables 1-2.
  • the heavy chain variable domain sequence of the antibody is derived from the gene rearrangement and somatic hypermutation of the germline gene V, D, and J gene segments of the heavy chain gene group on the chromosome; the light chain variable domain sequence is derived from Events such as gene rearrangement and somatic hypermutation of germline genes V and J gene segments of the light chain gene group. Gene rearrangements and somatic hypermutation are the main factors that increase antibody diversity. Antibodies derived from the same germline V gene segment may also yield different sequences, but overall the similarity is high.
  • IMGT/DomainGapAlign http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi
  • NCBI/IgBLAST https://www.ncbi.nlm.nih.gov/igblast/
  • PTMs post-translational modifications
  • some PTM sites are very conserved, for example, the conserved amino acid Asparagine Asn at position 297 (EU numbering) in the constant domain of human IgG1 antibodies is usually glycosylated to form sugar chains, which are critical for antibody structure and related effector functions.
  • EU numbering conserved amino acid Asparagine Asn at position 297
  • the presence of these PTMs may have a greater impact on antigen binding, and may also affect the physicochemical properties of antibodies. to change.
  • glycosylation, deamidation, isomerization, oxidation, etc. may increase the instability or heterogeneity of antibody molecules, thereby increasing the difficulty and risk of antibody development.
  • PTMs are very important for the development of therapeutic antibodies.
  • some PTMs are highly correlated with the "pattern" of amino acid sequence composition, especially the composition of adjacent amino acids, so that potential PTMs can be predicted from the protein's primary amino acid sequence.
  • the sequence pattern of N-x-S/T (asparagine in the first position, any amino acid other than proline in the second position, and serine or threonine in the third position) predicts an N-linked glycosylation site.
  • the amino acid sequence pattern that causes PTM may be derived from germline gene sequences, such as the human germline gene fragment IGHV3-33, which naturally has a glycosylation pattern NST in the FR3 region; it may also be derived from somatic hypermutation.
  • NGS or NLT may be the glycosylation site
  • NS may be the deamidation site
  • DG may cause isomerization of aspartic acid.
  • the formation of a particular PTM can be reduced or eliminated by disrupting the amino acid sequence pattern of a PTM by amino acid mutation.
  • mutation design there are different approaches to mutation design.
  • One approach is to replace "hot spot" amino acids (eg, N or S in the NS pattern) with amino acids of similar physicochemical properties (eg, mutating N to Q). If the PTM sequence pattern is derived from somatic hypermutation and is not present in the germline gene sequence, another approach could be to replace the sequence pattern with the corresponding germline gene sequence.
  • multiple mutational design methods may be used for the same PTM sequence pattern.
  • This example describes a general method for antibody preparation using mammalian host cells (eg, human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO and its derivatives), transient transfection expression, and affinity capture isolation.
  • mammalian host cells eg, human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO and its derivatives
  • transient transfection expression e.g., transient transfection expression
  • affinity capture isolation e.g., affinity capture isolation.
  • This method is suitable for target antibody containing Fc region; the target antibody can be composed of one or more protein polypeptide chains; it can be derived from one or more expression plasmids.
  • the amino acid sequence of the antibody polypeptide chain is converted into a nucleotide sequence by a codon optimization method; the encoded nucleotide sequence is synthesized and cloned into an expression vector compatible with host cells.
  • the plasmid encoding the antibody polypeptide chain is simultaneously transfected into mammalian host cells according to a specific ratio, and the recombinant antibody with correct folding and polypeptide chain assembly can be obtained by using conventional recombinant protein expression and purification techniques. Specifically, FreeStyle TM 293-F cells (Thermo, #R79007) were expanded in FreeStyle TM F17 Expression Medium (Thermo, #A1383504).
  • the cell concentration was adjusted to 6-8 x 10 5 cells/ml and incubated for 24 hours at 37°C in an 8% CO 2 shaker at a cell concentration of 1.2 x 10 6 cells/ml.
  • a total of 30 ⁇ g of plasmids encoding antibody polypeptide chains were mixed in a certain ratio (the ratio of plasmid to cells was 1 ⁇ g: 1 ml), dissolved in 1.5 ml of Opti-MEM reduced serum medium (Thermo, #31985088), and filtered with a 0.22 ⁇ m filter. bacteria.
  • Opti-MEM 1.5 ml of Opti-MEM was dissolved in 120 ⁇ l of 1 mg/ml PEI (Polysciences, #23966-2), and it was allowed to stand for 5 minutes. Slowly add PEI to the plasmid, incubate at room temperature for 10 minutes, slowly drop the plasmid PEI mixed solution while shaking the culture flask, and culture at 37°C in an 8% CO 2 shaker for 5 days. Cell viability was observed after 5 days. The culture was collected, centrifuged at 3300g for 10 minutes, and the supernatant was taken; then the supernatant was centrifuged at high speed to remove impurities.
  • PEI Polysciences, #23966-2
  • This example uses analytical size exclusion chromatography (SEC) to analyze protein samples for purity and aggregate form.
  • An analytical column TSKgel G3000SWxl (Tosoh Bioscience, #08541, 5 ⁇ m, 7.8 mm ⁇ 30 cm) was connected to a high pressure liquid chromatograph HPLC (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer for at least 1 hour at room temperature.
  • HPLC Analogent Technologies, Agilent 1260 Infinity II
  • An appropriate amount of protein sample (at least 10 ⁇ g) was filtered with a 0.22 ⁇ m filter and injected into the system, and the HPLC program was set: the sample was flowed through the column with PBS buffer at a flow rate of 1.0 ml/min for a maximum time of 25 minutes.
  • the HPLC will generate an analytical report reporting the retention times of components of different molecular sizes within the sample.
  • Figure 65 shows the analysis results of SDS-PAGE and SEC-HPLC of protein samples obtained after transient transfection, expression and one-step affinity purification of several diabody molecules with different structures in the application. These double antibody molecules showed good yield and purity.
  • DSF Differential Scanning Fluorimetry
  • Example 2.5 Determination of molecular stability and molecular aggregation of proteins using Uncle
  • Uncle Unchained Labs is a versatile one-stop protein stability analysis platform that characterizes protein stability by 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 obtained simultaneously from the same set of samples.
  • Tm melting temperature
  • Tagg aggregation temperature
  • particle size particle size
  • Uncle's "Tm&Tagg with optional DLS” application was selected for operation, 9 ⁇ L of sample was added to the Uni tube, and the temperature was gradually increased from 25°C to 95°C with a gradient of 0.3°C/min.
  • Initial and final DLS measurements were performed with four acquisitions of 5 seconds each.
  • Hand analysis software uses the barycentric mean (BCM) formula to calculate the Tm value of each sample; the Tagg value is calculated by the SLS fluorescence intensity curve (aggregation curve) at the wavelength of 266nm or 473nm; the particle size of the sample and dispersion are calculated by DLS-related functions.
  • BCM barycentric mean
  • PD-1 Programmed death 1
  • PD-L1 is mainly expressed in immune cells such as T cells, and it has two ligands, programmed death ligand 1 (PD-L1) and PD-L2.
  • PD-L1 is mainly expressed in antigen presenting cells and various tumor cells.
  • the interaction between PD-L1 and PD-1 can down-regulate the activity of T cells, weaken the secretion of cytokines, and play an immunosuppressive effect.
  • the expression of PD-L1 protein can be detected in many human tumor tissues.
  • the microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells.
  • the expressed PD-L1 is beneficial to the occurrence and growth of tumors and induces anti-tumor effects.
  • PD-1/PD-L1 pathway inhibitors can block the combination of PD-1 and PD-L1, block negative regulatory signals, restore the activity of T cells, and play a role in killing tumor cells, thereby inhibiting tumor growth. Therefore, Immunomodulation targeting PD-1/PD-L1 has important implications for tumor suppression.
  • Cytotoxic T lymphocyte-associated antigen 4 is a negative regulator expressed on T cells. After it binds to CD80 or CD86 on antigen-presenting cells, it blocks the co-stimulatory signal of CD28 and downregulates T cells. Cell activity, play an immunosuppressive effect.
  • CTLA4-mediated inhibitory mechanism is often one of the reasons why tumor cells escape the immune system. Blocking the interaction of CTLA4 with its ligands can restore the activity of T cells and enhance the ability to fight tumors.
  • CTLA4 inhibitors have obvious toxic and side effects, in the current combination regimen of PD-1/PD-L1 inhibitors and CTLA4 inhibitors, CTLA4 inhibitors are usually selected at lower doses.
  • the doses of Nivolumab and the anti-CTLA-4 antibody Ipilimumab were 3 mg/kg and 1 mg/kg, respectively.
  • the doses of Durvalumab and Tremelimumab were 10-20 mg/kg and 1 mg/kg, respectively.
  • bispecific antibodies targeting both PD-L1 and CTLA4 can utilize one or more mechanisms of action to improve antitumor efficacy and safety.
  • the PD-L1 x CTLA4 double antibody activates T cells at different stages by blocking the CTLA4 signaling pathway and the PD-1/PD-L1 signaling pathway; the cis-interaction of PD-L1 and CD80 enables it to interact with CTLA4 There is better synergy.
  • PD-L1 is highly expressed in tumor tissues, and PD-L1 x CTLA4 double antibody can specifically release CTLA4 inhibitory signals in the tumor microenvironment to activate T cells and reduce the non-specific activation of CTLA4 monoclonal antibody in the peripheral system. toxic side effect.
  • PD-L1 x CTLA4 dual antibodies can selectively retain Fc effector functions (such as ADCC), specifically killing suppressor T cells with high CTLA4 expression such as Treg cells through CTLA4 in the tumor microenvironment, or through PD-L1 It specifically kills tumor cells that highly express PD-L1.
  • Fc effector functions such as ADCC
  • suppressor T cells with high CTLA4 expression
  • CTLA4 expression such as Treg cells through CTLA4 in the tumor microenvironment
  • PD-L1 It specifically kills tumor cells that highly express PD-L1.
  • bispecific antibody will have more advantages in terms of economy and convenience of medication than the combination of two drug products.
  • Example 3.2 Obtaining anti-PD-L1 IgG antibody and anti-CTLA4 HCAb antibody
  • the Harbour HCAb mouse (Harbour Antibodies BV, WO2010/109165A2) is a transgenic mouse carrying a human immunoglobulin immune repertoire that produces heavy chain-only antibodies that are half the size of traditional IgG antibodies. It produces antibodies with only human antibody heavy chain variable domains and mouse Fc constant domains.
  • the plasmid is transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression, and the supernatant of fully human HCAb antibody is obtained.
  • mammalian host cells such as human embryonic kidney cells HEK293
  • the binding of HCAb antibody supernatant to recombinant human CTLA4 protein was tested by ELISA, and positive HCAb antibody was identified.
  • These HCAb antibodies were further identified, and several candidate HCAb antibody molecules were selected according to parameters such as their binding ability to human CTLA4, cynomolgus monkey CTLA4 binding ability, and ability to inhibit the binding of CTLA4 to B7-1.
  • the candidate HCAb antibody molecules were then sequenced and optimized, resulting in several variant sequences.
  • VH sequence of the HCAb antibody and the human IgG1 heavy chain Fc sequence are fused and expressed to obtain a fully human recombinant HCAb antibody molecule.
  • Recombinant fully human HCAb antibodies against CTLA4 are listed in Tables 3-9.
  • Example 3.3 Construction of bispecific antibody molecules using anti-PD-L1 IgG antibody and anti-CTLA4 HCAb antibody
  • the antigen-binding domain Fab of the anti-PD-L1 IgG antibody PR000070 or PR000265 and the antigen-binding domain VH of the anti-CTLA4 HCAb antibody PR000184 were used to construct a bispecific anti-PD-L1 x CTLA4 with various structures.
  • Antibody molecules were used to construct a bispecific anti-PD-L1 x CTLA4 with various structures.
  • the positive control molecule for anti-PD-L1 is the anti-PD-L1 IgG monoclonal antibody PR000070 or PR000416 or PR000265, which is also the parental monoclonal antibody at the PD-L1 end of the PD-L1 x CTLA4 double antibody molecule anti.
  • PR000070, PR000265 and PR000416 were all derived from the same anti-PD-L1 monoclonal antibody.
  • Both PR000070 and PR000265 are human IgG1 subtypes, both have N297A mutation in their Fc region, and they only differ by one amino acid mutation in the VH of the heavy chain variable region.
  • Both PR000265 and PR000416 are of human IgG1 subtype and have the same Fab structure, the only difference between them is that the Fc region of PR000265 has the N297A mutation.
  • the anti-CTLA4 positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parent monoclonal antibody at the CTLA4 end of the PD-L1 x CTLA4 double antibody molecule.
  • a PD-L1 x CTLA4 diabody molecule with Fab-HCAb symmetrical structure was designed according to the structure described in Example 1.1, which is summarized in Table 3-1; Antibody molecule samples were prepared and analyzed by the method described in 2, which are summarized in Table 3-2.
  • Example 1.2 Using anti-PD-L1 IgG antibody and anti-CTLA4 heavy chain antibody, according to the structure described in Example 1.2, a PD-L1 x CTLA4 diabody molecule with an IgG-VH tetravalent symmetrical structure was designed, which is summarized in Table 3-3; Antibody molecule samples were prepared and analyzed by the method described in Example 2, and are summarized in Tables 3-4.
  • a PD-L1 x CTLA4 diabody molecule with a 2xVH-IgG hexavalent symmetrical structure was designed according to the structure described in Example 1.4, which is summarized in Table 3-5; Antibody molecule samples were prepared and analyzed by the method described in Example 2, and are summarized in Tables 3-6.
  • a PD-L1 x CTLA4 diabody molecule with a Fab-Fc-VH bivalent asymmetric structure was designed according to the structure described in Example 1.4, which is summarized in Table 3-7 ; and the antibody molecule samples were prepared and analyzed according to the method described in Example 2, which are summarized in Tables 3-8.
  • Table 3-9, Table 3-10 and Table 3-11 list the PD-L1 x CTLA4 double antibody molecules constructed in this example and the corresponding PD-L1 mAb, CTLA4 mAb and other parent mAb molecules and controls The sequence number corresponding to the sequence of the molecule. Structure numbers in Table 3-11 correspond to Table 1-1 and Figure 1. Tables 3-12 list the sequence numbers of the corresponding CDR sequences of the first and second antigen binding domains of the bispecific antibody molecule.
  • This example is to study the activity of PD-L1 x CTLA4 double antibody binding to CTLA4.
  • human CTLA4-His protein (ACRO Biosystems, #CT4-H5229) was first coated with 100 ⁇ L/well of 96-well plate at 4°C overnight. It was then rinsed three times with PBST buffer (PBS buffer containing 0.05% Tween-20), followed by adding blocking solution (PBS buffer containing 5% nonfat dry milk) and incubating at 37°C for 1 hour. Then rinse 3 times with PBST buffer.
  • Optical absorbance values were read at 450 nM with an Enspire TM multifunction plate reader (Perkin Elmer, Inc.).
  • the software GraphPad Prism 8 was used for data processing and graph analysis, and parameters such as binding curves and EC50 values were obtained by four-parameter nonlinear fitting.
  • the positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parental monoclonal antibody at the CTLA4 end of the PD-L1 x CTLA4 double antibody molecule.
  • the ability of IgG-VH tetravalent symmetrical diabody molecules to bind to CTLA4 is related to the relative position of the VH end of anti-CTLA4 relative to the IgG of anti-PD-L1 in the diabody structure.
  • the EC50 value of the double antibody molecule binding to CTLA4 is similar to or even slightly better than that of the parental mAb PR000184; when VH is at the C-terminus of the heavy chain of IgG When VH was at the C-terminus of IgG light chain (PR000302), the ability of the double antibody to bind CTLA4 was significantly weakened compared with that of the parental mAb PR000184 (PR000303). This suggests that the ability of VH to bind to the target can be modulated by adjusting the relative position of VH on IgG.
  • PR000302 10.6 3.55 PR000303 1.566 2.72
  • Example 3.4.2 Binding to CHO-K1 cells CHO-K1/hCTLA4 highly expressing human CTLA4 or HEK293 cells HEK293/hCTLA4 highly expressing human CTLA4
  • Flow cytometry FACS was used to test the relationship between antibody molecules and CHO-K1 cell line CHO-K1/hCTLA4 (Beijing Kangyuan Bochuang, KC-1406) or HEK293T cell line HEK293/hCTLA4 (Beijing, China) with high expression of human CTLA4. Kangyuan Borchuang, KC-0209) and other cells binding ability.
  • CHO-K1/hCTLA4 and HEK293/hCTLA4 cells were digested and resuspended with F12K medium and DMEM medium, respectively; the cell density was adjusted to 1 ⁇ 10 6 cells/mL.
  • the cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 100 ⁇ L/well, centrifuged at 4° C. for 5 minutes, and the supernatant was discarded. Then, the serially diluted antibody molecules were added to the 96-well plate at 100 ⁇ L/well and mixed evenly.
  • the antibody molecules could be diluted from the highest final concentration of 300nM according to a 5-fold concentration gradient in a total of 8 concentrations, or the highest final concentration of 100nM according to 4 A total of 8 concentrations of fold-concentration serial dilution; hIgG1iso (CrownBio, #C0001) was used as an isotype control.
  • pre-chilled FACS buffer PBS buffer containing 0.5% BSA
  • fluorescent secondary antibody Alexa Fluor 488 anti-human IgG Fc, Biolegend, #409322, 1:1000 dilution
  • the positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parental monoclonal antibody at the CTLA4 end of the PD-L1 x CTLA4 double antibody molecule.
  • Ipilimumab analogs also served as positive control molecules. The results are shown in Figure 3, Figure 4, Table 3-14 and Table 3-15.
  • the ability of the double-antibody molecule of IgG-VH tetravalent symmetrical structure to bind to CTLA4 is related to the VH terminus of anti-CTLA4 relative to the IgG of anti-PD-L1
  • the relative position in the double antibody structure is related.
  • the EC50 value of the double antibody molecule for binding to CTLA4 is similar or slightly weaker than that of the parent mAb PR000184, but its MFI maxima on FACS were higher than parental mAb PR000184 or positive control ipilimumab.
  • the VH was at the C-terminus of the heavy chain of IgG (PR000303, PR000401, PR000402), the EC50 value of the double antibody molecule for binding to CTLA4 was slightly weaker than that of the parent mAb PR000184.
  • the EC50 value of the 2 ⁇ VH-IgG hexavalent symmetrical diabody molecule for binding to CTLA4 was similar to that of the parental mAb PR000184, and the MFI maximum value was lower than that of the parental mAb PR000184.
  • the MFI value is related to the number of fluorescent secondary antibody molecules bound to the Fc region of the double-antibody molecule, which is reflected in the fact that the maximum MFI value of the double-antibody molecule is lower than that of the parental mAb.
  • the diabody molecules PR001609 and PR001610 of the Fab-Fc-VH asymmetric structure contain only one CTLA4-binding domain, and thus their ability to bind CTLA4 is weaker than that of the bivalent parent mAb PR000184.
  • the double antibody molecule with the symmetrical structure of Fab-HCAb has a weaker binding ability to CTLA4 than the parent mAb PR000184 with bivalent, but the MFI maximum value is similar to that of the positive control Ipilimumab resemblance.
  • PR000402 2.4 1764 PR000403 16.3 1408 PR000404 37.22 1615 PR000184 1.147 2261
  • This example is to study the activity of the PD-L1 x CTLA4 double antibody molecule to inhibit the binding of CTLA4 to its ligand B7-1/CD80.
  • the activity of the antibody molecule to inhibit the binding of human CTLA4 protein to its ligand B7-1/CD80 was determined by ELISA. Specifically, 2 ⁇ g/mL of protein human B7-1-Fc (ACRO Biosystems, #B71-H5259) was first coated with 100 ⁇ L/well of 96-well plate at 4°C overnight. It was then rinsed three times with PBST buffer (PBS buffer containing 0.05% Tween-20), followed by adding blocking solution (PBS buffer containing 5% nonfat dry milk) and incubating at 37°C for 1 hour.
  • PBST buffer PBS buffer containing 0.05% Tween-20
  • blocking solution PBS buffer containing 5% nonfat dry milk
  • the positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parental monoclonal antibody at the CTLA4 end of the PD-L1 x CTLA4 double antibody molecule.
  • the results are shown in Figure 5 and Tables 3-16.
  • the IgG-VH tetravalent symmetrical structure of the double antibody molecules has the ability to block the binding of CTLA4 protein to its ligand protein (inhibitory activity), and can achieve nearly 100% inhibition rate, but its inhibitory activity
  • the IC50 value is related to the relative position of the VH terminus of anti-CTLA4 relative to the IgG of anti-PD-L1 in the double antibody structure.
  • the inhibitory activity of the diabodies against CTLA4 was similar to that of the parental mAb PR000184.
  • the inhibitory activity of the double antibody against CTLA4 was slightly weaker than that of the parent mAb PR000184.
  • the IC50 value of the inhibitory activity of the double antibody molecule is 4 times weaker than that of the parent mAb PR000184. This shows that the target blocking ability of VH can be adjusted by adjusting the relative position of VH on IgG.
  • the activity of antibody molecules to inhibit the binding of CTLA4-expressing cells to its ligand B7-1/CD80 was determined by flow cytometry FACS. Specifically, the CHO-K1 cell line CHO-K1/hCTLA4 (Beijing Kangyuan Bochuang, KC-1406) with high expression of human CTLA4 was digested and resuspended in F12K medium; the cell density was adjusted to 1 ⁇ 10 6 cells/mL, and the Place in FACS buffer (2% FBS in PBS buffer) at 37°C for 15 minutes. 200 ⁇ L/well of FACS buffer was added to the 96-well plate for blocking, and after incubation at 37°C for 1 hour, the blocking solution in the well was discarded.
  • FACS buffer 2% FBS in PBS buffer
  • CHO-K1/hCTLA4 cells were seeded at 200 ⁇ L/well in a 96-well plate (2 ⁇ 10 5 cells/well), centrifuged at 500 g at 4° C. for 5 minutes, and the supernatant was discarded. Then the serially diluted antibody molecules were added to the 96-well plate at 100 ⁇ L/well and mixed evenly.
  • the antibody molecules can be diluted from a maximum final concentration of 200nM to a total of 8 concentrations in a 3-fold concentration gradient, or can be diluted from a maximum final concentration of 400nM to a total of 5 concentrations.
  • hIgG1 iso (CrownBio, #C0001) was used as an isotype control.
  • 1 ⁇ g/mL of biotinylated ligand protein human B7-1-Fc (ACRO Biosystems, #B71-H82F2) was added to the 96-well plate at 100 ⁇ L/well and mixed well.
  • the 96-well plate was placed at 4°C and incubated in the dark for 1.5 hours.
  • 200 ⁇ L/well of pre-cooled FACS buffer was added to rinse the cells twice, centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded.
  • Fluorescence signal values were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and the data were processed and analyzed with the software FlowJo v10 (FlowJo, LLC).
  • the software GraphPad Prism 8 was used for data processing and graph analysis, the fluorescence signal value MFI was converted into inhibition rate, and parameters such as inhibition curve, IC50 value and maximum inhibition rate were obtained by four-parameter nonlinear fitting.
  • the positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parental monoclonal antibody at the CTLA4 end of the PD-L1 x CTLA4 double antibody molecule.
  • the results are shown in Figure 6 and Tables 3-17.
  • the inhibitory activity of the diabody molecule with the tetravalent symmetric structure of IgG-VH against CTLA4 is related to the VH end of anti-CTLA4 relative to the IgG of anti-PD-L1 in the diabody structure. relative position.
  • the IC50 value of the inhibitory activity of the double antibody molecule is similar to that of the parental mAb PR000184, which can reach nearly 100% Inhibition rate, PR000301 was even slightly stronger than the parental mAb PR000184.
  • the IC50 value of the inhibitory activity of the double antibody molecule was slightly weaker than that of the parent mAb PR000184, and the maximum inhibition rate was about 83%.
  • the diabody molecules PR001609 and PR001610 with Fab-Fc-VH asymmetric structure both contain only one CTLA4-binding domain, so their inhibitory activity on CTLA4 is significantly weaker than that of the bivalent parental monoclonal antibody.
  • the purpose of this example is to study the antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1 x CTLA4 diabodies to cells 293F-hCTLA4 (Smart Chemical) that highly expresses human CTLA4.
  • target cells were labeled with DELFIA BATDA (Perkin Elmer, #C136-100).
  • Specific labeling method are as follows: 1x10 6 target cells labeled with 2 ⁇ L DELFIA BATDA reagent;, CO 2 incubator at 37 °C 20 min; resuspended after the last wash, the precipitate; washed 4 times with PBS, centrifuged at 1000rpm 5 minutes in complete medium and cell density was adjusted to 1x10 5 / ml.
  • the labeled target cells were seeded in 96-well plates (Corning, #3599) at 100 ⁇ L/well; then, 50 ⁇ L/well was added with a 5-fold concentration gradient dilution of antibody molecules with a maximum final concentration of 0.8nM. 7 concentrations, mixed well; hIgG1 iso (CrownBio, #C0001) as an isotype control; incubated at 37°C, 5% CO 2 incubator for 10 minutes.
  • the fourth step centrifuge at 500g for 5 minutes, then add 25 ⁇ L of supernatant from each well to a new 96-well assay plate; then add 200 ⁇ L/well Europium solution (Perkin Elmer, #C135-100) and the plate was shaken at 250 rpm for 15 minutes at room temperature. Finally, use Fluorescence values were measured with a multi-plate reader (Perkin Elmer, Inc.).
  • the software GraphPad Prism 8 was used for data processing and graph analysis, and through four-parameter nonlinear fitting, the killing rate curve, EC50 and maximum killing rate of antibodies against target cells were obtained.
  • the positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184 (hIgG1 subtype), which is also the parental monoclonal antibody at the CTLA4 end of the PD-L1 x CTLA4 double antibody molecule.
  • the negative control molecule was anti-PD-L1 monoclonal antibody PR000416 (hlgG1 subtype).
  • the double antibody molecules PR000300 and PR000301 had similar ADCC effects against CTLA4 as the parent mAb PR000184.
  • This example is to study the activity of PD-L1 x CTLA4 double antibody binding to PD-L1.
  • ELISA enzyme-linked immunosorbent assay
  • the software GraphPad Prism 8 was used for data processing and graph analysis, and parameters such as binding curves and EC50 values were obtained through four-parameter nonlinear fitting.
  • the positive control molecule is the anti-PD-L1 monoclonal antibody PR000070, which is also the parental monoclonal antibody at the PD-L1 end of the PD-L1 x CTLA4 double antibody molecule.
  • Example 3.7.2 Combination of CHO-K1 cells CHO-K1/hPDL1 with high expression of human PD-L1 and tumor cells MDA-MB-231 with high expression of human PD-L1
  • the serially diluted antibody molecules were added to the 96-well plate at 100 ⁇ L/well and mixed evenly.
  • the antibody molecules can be diluted from a maximum final concentration of 60 nM to a total of 8 concentrations in a 5-fold concentration gradient, or can be diluted from a maximum final concentration of 300 nM to a total of 5 concentrations.
  • a total of 8 concentrations were serially diluted in a 2-fold concentration, or a total of 8 concentrations that could be diluted in a 4-fold concentration gradient from a maximum final concentration of 100 nM; hIgG1 iso (CrownBio, #C0001) served as an isotype control.
  • the cells were placed at 4°C and incubated in the dark for 1 hour.
  • pre-chilled FACS buffer PBS buffer containing 0.5% BSA
  • 100 ⁇ L/well of pre-chilled FACS buffer PBS buffer containing 0.5% BSA
  • 100 ⁇ L/well of fluorescent secondary antibody Goat human IgG(H+L) Alexa Fluor 488 conjunction, Thermo, #A11013, 1:1000 dilution
  • the cells were then washed twice with 200 ⁇ L/well of pre-chilled FACS buffer, and then centrifuged at 500 g at 4° C. for 5 minutes, and the supernatant was discarded.
  • the positive control molecule is the anti-PD-L1 monoclonal antibody PR000070 or PR000416 (or PR000265), which is also the parental monoclonal antibody at the PD-L1 end of the PD-L1 x CTLA4 double-antibody molecule.
  • the results are shown in Figure 8, Figure 9, Table 3-20 and Table 3-21.
  • the ability of the IgG-VH tetravalent symmetrical diabody molecule to bind PD-L1 is similar to that of its corresponding parental mAb PR000070 or PR000416, which binds PD-L1.
  • the EC50 values for L1 are very close. Only when the VH was at the N-terminus of the light chain of IgG (PR000301, PR001574, PR001577), the MFI maximum was slightly lower than that of the parental mAb.
  • the PD-L1-binding domains of the diabody molecules PR001609 and PR001610 of the Fab-Fc-VH asymmetric structure are monovalent Fab structures, but their ability to bind to PD-L1 is similar to that of bivalent
  • the parental mAbs were similar and the MFI maxima were higher than those of the parental mAbs.
  • This example is to study the activity of PD-L1 x CTLA4 double antibody in inhibiting the binding of PD-L1 to its ligand PD-1.
  • the activity of antibody molecules to inhibit the binding of PD-L1-expressing cells to its ligand PD-1 was determined by flow cytometry FACS. Specifically, the CHO-K1 cell line CHO-K1/hPDL1 (Nanjing GenScript, M00543) highly expressing human PD-L1 was digested and resuspended in F12K medium; the cell density was adjusted to 2 x 10 6 cells/mL, and Place in FACS buffer (PBS buffer containing 1% BSA) at 37°C for 15 minutes. The blocking buffer was added to the 96-well plate at 200 ⁇ L/well, and the blocking solution in the well was discarded after incubating at 37°C for 1 hour.
  • FACS buffer PBS buffer containing 1% BSA
  • CHO-K1/hPDL1 cells were then seeded in 96-well plates at 50 ⁇ L/well (1 ⁇ 10 5 cells/well). Then the serially diluted antibody molecules were added to the 96-well plate at 100 ⁇ L/well and mixed evenly.
  • the antibody molecules can be diluted from the highest final concentration of 100nM to a total of 8 concentrations in a 5-fold concentration gradient; hIgG1 iso (CrownBio, #C0001) was used as the isotype control. Place at 4°C and incubate in the dark for 0.5 hours. Then, PBS buffer was added at 100 ⁇ L/well, centrifuged at 500 g at 4° C. for 5 minutes, and the supernatant was discarded.
  • biotinylated ligand protein human PD-1 protein (ACRO Biosystems, #PD1-H82F2) at a concentration of 1 ⁇ g/mL was added to the 96-well plate at 50 ⁇ L/well and mixed well.
  • the 96-well plate was placed at 4°C and incubated in the dark for 0.5 hours.
  • the cells were then washed twice with 100 ⁇ L/well of pre-chilled PBS buffer, then centrifuged at 500 g for 5 minutes at 4° C., and the supernatant was discarded.
  • Fluorescence signal values were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and the data were processed and analyzed with the software FlowJo v10 (FlowJo, LLC).
  • the software GraphPad Prism 8 was used for data processing and graph analysis, the fluorescence signal value MFI was converted into inhibition rate, and parameters such as inhibition curve, IC50 value and maximum inhibition rate were obtained by four-parameter nonlinear fitting.
  • the positive control molecule is the anti-PD-L1 monoclonal antibody PR000070 or PR000416 (or PR000265), which is also the parental monoclonal antibody at the PD-L1 end of the PD-L1 x CTLA4 double-antibody molecule.
  • PR000070 or PR000416 or PR000265
  • the results are shown in Figure 10 and Tables 3-22.
  • the anti-PD-L1 inhibitory ability of the IgG-VH tetravalent symmetrical structure is similar to that of the corresponding parental mAb PR000070 or PR000416, and is similar to that of the parental mAb IC50 value and maximum inhibition rate. Only when the VH is at the N-terminus of the light chain of IgG (PR001574, PR001577), the IC50 value is slightly 2-3 times weaker than that of the parental mAb.
  • the inhibitory activity of the 2xVH-IgG hexavalent symmetrical structure against PD-L1 is weaker than that of the parental mAb; although the corresponding IC50 value is 4-6 times weaker than that of the parental mAb, However, similar maximal inhibition rates were achieved.
  • the anti-PD-L1 inhibitory activity of the Fab-HCAb symmetrical diabody molecule was similar to that of the parental mAb, and had similar IC50 values and maximum inhibition rates with the parental mAb.
  • the PD-L1 binding domains of the diabody molecules PR001609 and PR001610 of the Fab-Fc-VH asymmetric structure are monovalent Fab structures, and their inhibitory activity against PD-L1 is weaker than those with bivalent
  • the corresponding IC50 value is 4-5 times weaker than that of the parental mAb, but can achieve a similar maximum inhibition rate.
  • This example is to study the activation effect of PD-L1 x CTLA4 double antibody molecule on peripheral blood mononuclear cells PBMC.
  • the isolated human PBMC cells were added to a 96-well plate (Corning, #3799) at 100 ⁇ L/well (1 ⁇ 10 5 cells/well); then different concentrations of antibody molecules were added at 100 ⁇ L/well, The final concentration of the antibody can be (100nM, 10nM, 1nM, 0.1nM, 0.01nM), or two antibodies are mixed according to a certain ratio, and two replicates are loaded; hIgG1 iso (CrownBio, #C0001) is used as an isotype control. Incubate at 37°C for 30 minutes.
  • SEB superantigen Staphylococcal enterotoxin B
  • the supernatant was collected after 96 hours or 120 hours of incubation in a 37°C, 5% CO 2 incubator.
  • the collected supernatant was detected by IL-2 ELISA kit (Biolegend, #43185) to detect the IL-2 concentration in the supernatant.
  • IL-2 ELISA kit Biolegend, #431805
  • Data processing and graph analysis were performed using the software GraphPad Prism 8.
  • the positive control molecules were anti-CTLA4 HCAb monoclonal antibody PR000184 and anti-PD-L1 IgG monoclonal antibody PR000416.
  • anti-CTLA4 monoclonal antibody, anti-PD-L1 monoclonal antibody and PD-L1 x CTLA4 double antibody molecule when stimulated by SEB, can promote T cells to produce IL-2 to varying degrees, Its ability to activate T cells is related to its structure.
  • IgG-VH tetravalent symmetric diabody molecules can activate T cells to produce IL-2.
  • the structure of anti-CTLA4 VH at the N-terminus of IgG (PR000301) has a stronger T cell activation ability than the structure of VH at the C-terminus of IgG (PR000302, PR000303).
  • the Fab-HCAb symmetrical structure of the double antibody molecule can activate T cells to produce IL-2.
  • PR000404 has stronger T cell activation ability than PR000403.
  • PR000404 and PR000403 have similar structures, the only difference being that PR000404 has the normal human IgG1 Fc region, while the Fc region of PR000403 contains three point mutations (L234A, L235A, P329G) to attenuate Fc-related effector functions. It is speculated that PR000404 can eliminate Treg cells through CTLA4-mediated ADCC, thereby further enhancing T cell function.
  • the double antibody molecules PR000300 and PR000301 have comparable or even stronger T cell activation ability than anti-CTLA4 mAb, anti-PD-L1 mAb and their 1:1 concentration combination (using the same One donor PBMC, absolute IL-2 levels can be compared).
  • the diabody molecules PR000303 IgG-HC-VH structure, ie VH is at the C-terminus of the heavy chain of IgG
  • PR000404 Fab-HCAb structure
  • MLR mixed lymphocyte reaction
  • the first step is to add recombinant human interleukin-4 (IL-4, R&D Systems, #204-GMP) and recombinant human GM-CSF (R&D Systems, #215- GM), after 6 days of induction, immature human CD14 + dendritic cells (iDC cells) were obtained.
  • IL-4 human interleukin-4
  • GM-CSF recombinant human GM-CSF
  • iDC cells immature human CD14 + dendritic cells
  • mDC cells mature dendritic cells
  • T lymphocytes were isolated from the second donor PBMC cells (Miaotong Bio) by using a T cell isolation kit (StemCell, #17951).
  • the obtained T cells and mDC cells were seeded into a 96-well plate at a ratio of 10:1 (1 ⁇ 10 5 /well of T cells and 1 ⁇ 10 4 /well of mDC cells). Then add different concentrations of antibody molecules at 100 ⁇ L/well.
  • the final concentration of the antibody can be (100nM, 10nM, 1nM, 0.1nM, 0nM), or two antibodies are mixed according to a certain ratio, and two duplicate wells are loaded; hIgG1 iso (CrownBio, #C0001) as an isotype control. Incubate for 5 days at 37 °C, 5% CO 2 incubator.
  • the fourth step is to collect the supernatant on the 3rd day and the 5th day, and use the IL-2 ELISA kit (Thermo, #88-7025-88) to detect the IL-2 concentration in the supernatant on the 3rd day.
  • the IFN- ⁇ concentration in the supernatant on day 5 was detected by a ⁇ ELISA kit (Thermo, #88-7316-88).
  • the diabody molecules PR000300 and PR000301 both showed better results than anti-CTLA4 mAb, Anti-PD-L1 monoclonal antibody and its 1:1 concentration combination have stronger T cell activation ability and can better promote the production of IL-2 and IFN- ⁇ .
  • the diabodies PR000302 and PR000303 showed stronger T cells in MLR experiments than the anti-PD-L1 mAb atezolizumab activation ability.
  • PBS was used as the blank control group
  • PR001573 at 3 mg/kg in the double-antibody administration group
  • ipilimumab at 3 mg/kg plus atezolizumab at 3 mg/kg in the combined administration group.
  • Tumor volume and mouse body weight were measured on days 6, 9, 12, 15, 19, 22, 26, 29 and 33 after tumor inoculation.
  • the tumor volume in the control group was approximately 700 mm3 .
  • the mice in the administration group tolerated well throughout the experiment, and there was no significant change in body weight. It shows that the efficacy of 3mg/kg PR001573 is equivalent to that of the combination of ipilimumab and atezolizumab at the same dose.
  • anti-PD-L1 x CTLA4 bispecific antibody molecules with various structures were constructed by using the antigen-binding domain Fab of the anti-PD-L1 IgG antibody and the antigen-binding domain VH of the anti-CTLA4 HCAb antibody. It shows the flexibility of constructing bispecific antibody molecular structures based on HCAb, and adjusts the functional activities of PD-L1 and CTLA4 ends through different structure types, relative positions, binding valences and other parameters, and then designs different activity combinations. To meet the needs of different clinical combination dosage combinations.
  • the PD-L1 and CTLA4 ends of the double-antibody molecules PR000300 and PR000301 can almost completely retain the same activity as their parental mAbs, and in mixed lymphocytes
  • in vitro functional experiments such as cell response and superantigen stimulation experiments, it has been shown that the combination of 1:1 concentration of parental monoclonal antibody has comparable or even stronger T cell activation ability; therefore, it can be used to achieve a 1:1 dose of combination therapy combination.
  • the mouse tumor efficacy model also confirmed that the double-antibody PR001573 with this structure has the same efficacy as the combination drug.
  • the PD-L1-terminus of the double-antibody molecules PR000302, PR000303 and PR000404 almost completely remains equivalent to their parental mAbs.
  • the activity of its CTLA4 terminal has been weakened to varying degrees; therefore, it can be used to achieve a clinical 3:1 or even 10:1 combination of drug doses.
  • Cytotoxic T lymphocyte-associated antigen 4 is a negative regulator expressed on T cells. After it binds to CD80 or CD86 on antigen-presenting cells, it blocks the co-stimulatory signal of CD28 and downregulates T cells. Cell activity, play an immunosuppressive effect.
  • CTLA4-mediated inhibitory mechanism is often one of the reasons why tumor cells escape the immune system. Blocking the interaction of CTLA4 with its ligands can restore the activity of T cells and enhance the ability to fight tumors.
  • Ipilimumab monoclonal antibody (trade name ) is the first approved anti-CTLA4 monoclonal antibody drug, and the first product to open the era of tumor immunotherapy.
  • Ipilimumab has shown a good therapeutic effect in the treatment of advanced melanoma, but Ipilimumab also brings high immune-related side effects, and the incidence of its related grade 3-5 adverse reactions is even as high as 50%, which seriously affect its clinical application. Most of the toxic and side effects exhibited by Ipilimumab are related to the CTLA4 target. In the current combination regimen of PD-1/PD-L1 inhibitors and CTLA4 inhibitors, CTLA4 inhibitors, whether Ipilimumab or Tremelimumab, are usually selected. low dose.
  • CTLA4 inhibitors In order to reduce the toxic and side effects of CTLA4 inhibitors, one of the methods worth trying is to deliver CTLA4 inhibitors into tumor tissues, so that the relevant T cell-mediated responses are limited to the tumor microenvironment and reduce cytokine release. Syndrome risk.
  • This targeted delivery can be achieved by intratumoral injection of CTLA4 inhibitors, but the intratumoral injection method carries the risks of surgical procedures and is limited to some superficial accessible tumor tissues.
  • This example provides another method of targeted delivery, using antibodies that recognize tumor-associated antigens to redirect CTLA4 inhibitors to a specific tumor microenvironment, so that they can deplete T cells in the tumor microenvironment The immunosuppressive signal restores T cell function.
  • HER2 x CTLA4 double antibody can block the CTLA4 signaling pathway on the basis of retaining the original mechanism of action of HER2 inhibitors (prevent HER2 dimerization, promote HER2 internalization and degradation, and inhibit downstream phosphorylation signals). to activate T cells.
  • HER2 x CTLA4 double antibody is enriched in tumor tissues with high expression of HER2, and it can specifically release CTLA4 inhibitory signal in the tumor microenvironment to activate T cells and reduce the toxic and side effects caused by non-specific activation of CTLA4 monoclonal antibody in the peripheral system. .
  • HER2 x CTLA4 dual antibodies can selectively retain Fc effector functions (such as ADCC), specifically killing suppressor T cells with high CTLA4 expression such as Treg cells through CTLA4 in the tumor microenvironment, or specifically killing through HER2 Tumor cells that highly express HER2.
  • Fc effector functions such as ADCC
  • suppressor T cells with high CTLA4 expression such as Treg cells through CTLA4 in the tumor microenvironment
  • HER2 Tumor cells that highly express HER2.
  • bispecific antibody will have more advantages in terms of economy and convenience of medication than the combination of two drug products.
  • Example 4.2 Obtaining anti-HER2 IgG antibodies and anti-CTLA4 HCAb antibodies
  • anti-HER2 IgG antibodies trastuzumab and pertuzumab were used, and their corresponding amino acid sequences were obtained from the IMGT database, as shown in Table 4-11.
  • the anti-CTLA4 fully human HCAb antibody PR000184 (Table 4-11) used in this example was derived from Harbour HCAb mice, and the discovery process was as described in Example 3.2.2.
  • Example 4.3 Construction of bispecific antibody fraction using anti-HER2 IgG antibody and anti-CTLA4 HCAb antibody
  • the antigen-binding domain Fab of the anti-HER2 IgG antibody PR000210 (trastuzumab analog) or PR000672 (pertuzumab analog), and the antigen-binding domain VH of the anti-CTLA4 HCAb antibody PR000184 were used to construct anti-HER2 antibodies with various structures.
  • the positive control molecule is an anti-HER2 IgG mAb PR000210 (trastuzumab analog) or PR000672 (pertuzumab analog), which is also the parent mAb at the HER2 end of the HER2 x CTLA4 double antibody molecule.
  • the positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parent monoclonal antibody at the CTLA4 end of the HER2 x CTLA4 double antibody molecule.
  • HER2 x CTLA4 double-antibody molecule with a Fab-HCAb symmetrical structure was designed according to the structure described in Example 1.1, which is summarized in Table 4-1; and according to the method described in Example 2 Antibody molecule samples were prepared and analyzed, summarized in Table 4-2.
  • HER2 x CTLA4 double antibody molecule with IgG-VH tetravalent symmetrical structure was designed according to the structure described in Example 1.2, which is summarized in Table 4-3; Antibody molecule samples were prepared and analyzed using the method described above, and are summarized in Tables 4-4.
  • Example 1.3 Using anti-HER2 IgG antibody and anti-CTLA4 heavy chain antibody, the IgG-VH(2) hexavalent symmetrical HER2 x CTLA4 diabody molecule was designed according to the structure described in Example 1.3, which is summarized in Table 4-5; Antibody molecule samples were prepared and analyzed as described in Example 2 and are summarized in Tables 4-6.
  • HER2 x CTLA4 diabody molecule with a 2xVH-IgG hexavalent symmetrical structure was designed according to the structure described in Example 1.4, which is summarized in Table 4-7; Antibody molecule samples were prepared and analyzed using the method described above, and are summarized in Tables 4-8.
  • Table 4-11, Table 4-12 and Table 4-13 list the sequences of the HER2 x CTLA4 double antibody molecules constructed in this example, the corresponding parent mAb molecules such as HER2 mAb, CTLA4 mAb, and control molecules. the corresponding serial number. Structure numbers in Table 4-13 correspond to Table 1-1 and Figure 1. Tables 4-14 list the sequence numbers of the corresponding CDR sequences of the first and second antigen binding domains of bispecific antibody molecules.
  • Antibody number antibody PR000210 Anti-HER2 monoclonal antibody trastuzumab analog, hIgG1 PR000672 Anti-HER2 monoclonal antibody pertuzumab analog, hIgG1 PR000184 Anti-CTLA4 heavy chain antibody CL5v3, hIgG1 PR000218 Anti-CTLA4 heavy chain antibody CL5v3, hIgG1 (S239D, I332E)
  • Table 4-13 The sequence numbering table of the HER2 x CTLA4 diabody molecule of the present embodiment
  • This example is to study the binding activity of HER2 x CTLA4 double antibody molecule to CTLA4.
  • Flow cytometry FACS was used to test the binding ability of antibody molecules to cells such as CHO-K1 cell line CHO-K1/hCTLA4 (Ruizhi Chemical) that highly expresses human CTLA4.
  • CHO-K1/hCTLA4 cells were digested and resuspended with F12K medium; the cell density was adjusted to 2 x 10 6 cells/mL.
  • CHO-K1/hCTLA4 cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 100 ⁇ L/well, centrifuged at 4° C. for 5 minutes, and the supernatant was discarded.
  • the serially diluted antibody molecules were added to the 96-well plate at 100 ⁇ L/well and mixed well.
  • the antibody molecules can be diluted from the highest final concentration of 300nM to a total of 8 concentrations in a 5-fold concentration gradient, or can be diluted from the highest final concentration of 300nM to 4 concentrations.
  • a total of 12 concentrations were serially diluted; hIgG1 iso (CrownBio, #C0001) was used as an isotype control.
  • the cells were placed at 4°C and incubated in the dark for 1 hour.
  • the positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parent monoclonal antibody at the CTLA4 end of the HER2 x CTLA4 double antibody molecule.
  • the results are shown in Figure 18 and Tables 4-15.
  • the Fab domain may have a "shadowing" effect on the VH domain of the HCAb, so that the VH domain in this structure can bind fewer CTLA4 molecules than when it is in the free end. , but the binding potency was comparable (EC50 value) to the parental mAb PR000184 for the same number of accessible CTLA4 molecules.
  • the double antibody molecules of this structure are further divided into three categories: VH is at the C-terminus of the heavy chain of IgG (PR000539, PR000540, PR000714, PR000715); VH at the N-terminus of the heavy chain of IgG (PR001583, PR001586, PR001588, PR001591); VH at the N-terminus of the light chain of IgG (PR001584, PR001589).
  • the double-antibody molecules (PR001585, PR001587) constructed based on the parental mAb PR000210 have similar binding ability to CTLA4 as the parental mAb PR000184; the double-antibody molecules (PR001590, PR001592) constructed based on the parental mAb PR000672 have the same binding ability to CTLA4 as the parental mAb PR000184 slightly weaker.
  • Both the double antibody molecules PR000916 and PR000917 contain only one CTLA4 binding domain, so their binding ability to CTLA4 is significantly weaker than that of the bivalent parent mAb PR000184.
  • the double antibody molecule PR002672 contains two CTLA4-binding domains formed in tandem, and its binding ability to CTLA4 is similar to that of the parent mAb PR000184. This structure shows that the diabody molecule containing the VH domain derived from HCAb has the flexibility to adjust the binding ability to the target by adjusting the number of VH domains in tandem.
  • This example is to study the activity of HER2 x CTLA4 double-antibody molecule in inhibiting the binding of CTLA4 to its ligand B7-1/CD80.
  • the activity of the antibody molecule to inhibit the binding of human CTLA4 protein to its ligand B7-1/CD80 protein was determined by ELISA. Specifically, 2 ⁇ g/mL of protein human B7-1-Fc (ACRO Biosystems, #B71-H5259) was first coated with 100 ⁇ L/well of 96-well plate at 4°C overnight. It was then rinsed three times with PBST buffer (PBS buffer containing 0.05% Tween-20), followed by adding blocking solution (PBS buffer containing 5% nonfat dry milk) and incubating at 37°C for 1 hour.
  • PBST buffer PBS buffer containing 0.05% Tween-20
  • blocking solution PBS buffer containing 5% nonfat dry milk
  • the positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parent monoclonal antibody at the CTLA4 end of the HER2 x CTLA4 double antibody molecule.
  • the results are shown in Figure 19 and Tables 4-16.
  • the double antibody molecules with the symmetrical structure of Fab-HCAb have the ability to block the binding of CTLA4 protein to its ligand protein (inhibitory activity), and are similar to or slightly weaker than the parent mAb PR000184, reflecting The maximum OD difference is similar but the IC50 value is slightly weaker by only 1.5-2.1 times.
  • the inhibitory activity of the diabody molecule with the tetravalent symmetric structure of IgG-VH is slightly different depending on its specific molecular structure.
  • the diabody molecules of this structure are further divided into three categories: VH at the C-terminus of the heavy chain of IgG (PR000539, PR000540, PR000715) ; VH at the N-terminus of the heavy chain of IgG (PR001583, PR001586, PR001588, PR001591); VH at the N-terminus of the light chain of IgG (PR001584, PR001589).
  • the inhibitory activity of these double antibody molecules is slightly weaker than that of the parent mAb, which is reflected in the similar maximum OD difference but the IC50 value is only 1.5-2.5 times weaker.
  • the inhibitory activity of these diabody molecules was similar to that of the parent mAb when the VH was at the N-terminal end of either the heavy or light chain of IgG.
  • the inhibitory activity of the IgG-VH-VH hexavalent symmetrical diabody molecule was similar to or slightly better than that of the parental mAb.
  • the inhibitory activity of the 2 ⁇ VH-IgG hexavalent symmetrical diabody molecule was similar to or slightly better than that of the parental mAb.
  • the activity of antibody molecules to inhibit the binding of CTLA4-expressing cells to its ligand B7-1/CD80 was determined by flow cytometry FACS. Specifically, the CHO-K1 cell line CHO-K1/hCTLA4 (Wisdom Chemical) with high expression of human CTLA4 was digested and placed in FACS buffer (PBS buffer containing 2% FBS) to resuspend, and the cell density was adjusted to 3 ⁇ 10 6 cells/mL. CHO-K1/hCTLA4 cells were seeded at 100 ⁇ L/well in a 96-well plate (3 ⁇ 10 5 cells/well), centrifuged at 500 g for 5 minutes at 4° C., and the supernatant was discarded.
  • FACS buffer PBS buffer containing 2% FBS
  • the serially diluted antibody molecules were added to the 96-well plate at 100 ⁇ L/well and mixed evenly.
  • the antibody molecules can be diluted with a 3-fold concentration gradient from the highest final concentration of 200 nM to a total of 8 concentrations; hIgG1 iso (CrownBio, #C0001) was used as the isotype control.
  • 3 ⁇ g/mL of the biotinylated ligand protein human B7-1-Fc-biotin ACRO Biosystems, #B71-H82F2
  • the 96-well plate was placed at 4°C and incubated in the dark for 1 hour.
  • Fluorescence signal values were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and the data were processed and analyzed with the software FlowJo v10 (FlowJo, LLC).
  • the software GraphPad Prism 8 was used for data processing and graph analysis, the fluorescence signal value MFI was converted into inhibition rate, and parameters such as inhibition curve, IC50 value and maximum inhibition rate were obtained by four-parameter nonlinear fitting.
  • the positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parent monoclonal antibody at the CTLA4 end of the HER2 x CTLA4 double antibody molecule.
  • the results are shown in Figure 20 and Tables 4-17.
  • the diabody molecule PR002672 of the Fab-Fc-VH(2) asymmetric structure contains two CTLA4-binding domains formed in tandem, and its inhibitory activity is compared with that of the parent mAb with bivalent , although the IC50 value is slightly weaker about 4 times, it can achieve nearly 100% inhibition.
  • This example is to study the binding activity of HER2 x CTLA4 double antibody molecule to HER2.
  • SK-BR-3 (ATCC, HTB-30) highly expressing human HER2 was tested by flow cytometry FACS. Specifically, SK-BR-3 cells were digested and resuspended in DMEM complete medium, and the cell density was adjusted to 1 x 10 6 cells/mL; then 100 ⁇ L cells/well were seeded in 96-well V-bottom plates (Corning, #3894), 4 Centrifuge at °C for 5 minutes and discard the supernatant.
  • hIgG1 iso (CrownBio, #C0001) was used as an isotype control.
  • the cells were placed at 4°C and incubated in the dark for 1 hour. After that, centrifuge at 4°C for 5 minutes, and discard the supernatant; then add pre-cooled FACS buffer (PBS buffer containing 0.5% BSA) at 200 ⁇ L/well to rinse the cells twice, and then centrifuge at 500g for 5 minutes at 4°C , discard the supernatant.
  • pre-cooled FACS buffer PBS buffer containing 0.5% BSA
  • a fluorescent secondary antibody Goat human IgG(H+L) Alexa Fluor 488 conjunction, Thermo, #A11013, 1:1000 dilution
  • a fluorescent secondary antibody Goat human IgG(H+L) Alexa Fluor 488 conjunction, Thermo, #A11013, 1:1000 dilution
  • Thermo Alexa Fluor 488 conjunction, Thermo, #A11013, 1:1000 dilution
  • Fluorescence signal values were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and the data were processed and analyzed with the software FlowJo v10 (FlowJo, LLC).
  • the software GraphPad Prism 8 was used for data processing and graph analysis, and parameters such as the binding curve of the antibody to the target cell and the EC50 value were obtained through four-parameter nonlinear fitting.
  • the positive control molecule is the anti-HER2 mAb PR000210 (trastuzumab analog) or PR000672 (pertuzumab analog), which is also the parent mAb of the HER2 x CTLA4 double-antibody molecule.
  • PR000210 to which the anti-HER2 mAb PR000210
  • PR000672 pertuzumab analog
  • the ability of the IgG-VH tetravalent symmetrical diabody molecule to bind to HER2 is slightly different depending on its specific molecular structure.
  • the diabody molecules of this structure are further divided into three categories: VH at the C-terminus of the IgG heavy chain (PR000539, PR000540); VH At the N-terminus of the heavy chain of IgG (PR001583, PR001586, PR001588, PR001591); VH at the N-terminus of the light chain of IgG (PR001584, PR001974, PR001589).
  • the 2xVH-IgG hexavalent symmetrical structure of the diabody molecule has a significantly reduced ability to bind to HER2 compared to its corresponding parental mAb; this shows that when VH is simultaneously linked to IgG After the N-terminus of the heavy chain and light chain, it has a significant impact on the function of the Fab end of IgG.
  • the HER2-binding domain of the diabody molecule PR000916 with a Fab-Fc-VH asymmetric structure is a monovalent Fab structure, but its ability to bind HER2 is very close to that of the bivalent parental mAb; It shows that the binding domain of trastuzumab has no obvious difference between monovalent binding and bivalent binding, and the double antibody molecule with asymmetric structure of Fab-Fc-VH well retains the binding ability to HER2.
  • PR000655 1.57 12201 PR001579 3.056 36444 PR000656 1.664 12358 PR001580 3.421 35754 PR000658 1.474 12964 PR001581 3.687 34744 PR000659 1.653 13672 PR001582 3.665 30517 PR000706 1.562 13355 PR001585 n.d. 26700 PR000716 1.386 13418 PR001587 n.d.
  • This example is to study the activation effect of HER2 x CTLA4 double antibody molecule on peripheral blood mononuclear cell PBMC.
  • the isolated human PBMCs (Miaotong Bio) were first adjusted to 5x10 6 cells/mL. PBMCs were then added to a 96-well plate (Corning, #3799) at 50 ⁇ L/well; then different concentrations of antibody molecules were added at 100 ⁇ L/well, and the final antibody concentration gradient could be (150nM, 30nM, 1nM) or (80nM, 8nM, 0.8 nM, 0.08 nM), loaded in duplicate; hIgG1 iso (CrownBio, #C0001) served as an isotype control.
  • the positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184 or PR000218, which is also the parental monoclonal antibody at the CTLA4 end of the HER2 x CTLA4 double antibody molecule.
  • PR000218 is an ADCC-enhanced variant constructed on PR000184 by Fc mutation.
  • both anti-CTLA4 monoclonal antibody and HER2 x CTLA4 double antibody molecule can promote T cells to produce IL-2 to varying degrees, and their ability to activate T cells is related to their structure.
  • the double antibody molecule with the symmetrical structure of Fab-HCAb has less ability to activate T cells.
  • the activity of CTLA4 terminal is weakened in Fab-HCAb structure, so that its dose-related toxicity can be reduced, and it is suitable for the current clinical combination drug dose.
  • the activating ability of IgG-VH tetravalent symmetric diabody molecules on T cells is slightly different according to their specific molecular structures.
  • the diabody molecules of this structure are further divided into three categories: VH at the C-terminus of the IgG heavy chain (PR000539, PR000715); VH At the N-terminus of the heavy chain of IgG (PR001583, PR001586); VH at the N-terminus of the light chain of IgG (PR001584, PR001974).
  • the activation ability of the double antibody molecule to T cells is significantly weakened compared with the corresponding parental mAb.
  • the activating ability of diabodies to T cells is similar to or even slightly enhanced by the corresponding parental mAbs. This shows that by adjusting the relative position of VH on IgG, the activity of anti-CTLA4 at the VH end can be adjusted to apply to different clinical combination drug dosage scenarios.
  • the IgG-VH-VH hexavalent symmetrical diabody molecule has a similar ability to activate T cells as the parental mAb, and its CTLA4-terminal activity can be regulated by using different linking peptides.
  • the 2 ⁇ VH-IgG hexavalent symmetrical diabody molecule has a similar ability to activate T cells to that of the parental monoclonal antibody, indicating that it almost completely retains the activity of the CTLA4 terminal.
  • the Fab-Fc-VH(2) asymmetric diabody molecule contains two CTLA4-binding domains formed in tandem, and its ability to activate T cells is compared with the corresponding parental mAb There is weakening. Its CTLA4 terminal can reduce its dose-related toxicity, so as to be suitable for the combination drug dose currently in clinical practice.
  • This example is to study the inhibition of the proliferation of tumor cell line SK-BR-3 (ATCC, HTB-30) highly expressing human HER2 by HER2 x CTLA4 double antibody molecule.
  • SK-BR-3 cells were digested and resuspended in DMEM complete medium. 2000 cells/50 ⁇ L were seeded in 96-well plates (Perkin Elmer, #6005225); then, incubated overnight in a 37° C., 5% CO 2 incubator.
  • hIgG1 iso (CrownBio, #C0001) was used as an isotype control; at 37°C, 5 Incubate for 7 days in a %CO 2 incubator. Afterwards, 100 ⁇ L of CellTiter-Glo (Promega, #G7573) was added per well and mixed for 10 minutes on a horizontal shaker to induce cell lysis. Finally, chemiluminescence values were determined using an Enspire TM multifunction plate reader (PerkinElmer, Inc.). The software GraphPad Prism 8 was used for data processing and graph analysis, and parameters such as the inhibitory rate of antibodies to target cells were obtained through four-parameter nonlinear fitting.
  • the positive control molecule is the anti-HER2 monoclonal antibody PR000210 (trastuzumab analog), which is also the parent monoclonal antibody of the HER2 x CTLA4 double-antibody molecule.
  • PR000210 trastuzumab analog
  • the inhibitory effects of the IgG-VH tetravalent symmetrical diabody molecule on the proliferation of SK-BR-3 cells were slightly different depending on its specific molecular structure.
  • the diabody molecules of this structure are further divided into three categories: VH is at the C-terminus of the heavy chain of IgG (PR000539, PR000540, PR000714, PR000715); VH at the N-terminus of the heavy chain of IgG (PR001583, PR001586); VH at the N-terminus of the light chain of IgG (PR001584).
  • VH When VH is at the C-terminus of the heavy chain of IgG, the structure well retains the ability to inhibit the proliferation of SK-BR-3 cells, which is reflected in the IC50 and maximum inhibition rate similar to or even slightly better than the parental mAb; indicating that VH The Fab end of the IgG has no significant effect on the Fab end.
  • VH When VH is at the N-terminus of either the heavy chain or the light chain of IgG, the inhibitory ability of the double antibody molecule on the proliferation of SK-BR-3 cells is slightly lower than that of the parental monoclonal antibody, which is reflected in a 3-4 times difference in IC50 value. , but still retains the same maximum inhibition rate.
  • This example is to study the antibody-dependent cell-mediated cytotoxicity (ADCC) of HER2 x CTLA4 double-antibody molecule to the tumor cell line BT-474 (ATCC, HTB-20) that highly expresses human HER2.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • target cells were labeled with DELFIA BATDA (Perkin Elmer, #C136-100).
  • Specific labeling method are as follows: 1x10 6 target cells labeled with 2 ⁇ L DELFIA BATDA reagent;, CO 2 incubator at 37 °C 20 min; resuspended after the last wash, the precipitate; washed 4 times with PBS, centrifuged at 1000rpm 5 minutes containing RPMI-1640 medium with 20% FBS (Thermo, # a 10491) in, and the cell density was adjusted to 1x10 5 / ml.
  • the labeled target cells were seeded at 100 ⁇ L/well in a 96-well plate (Corning, #3599); then 50 ⁇ L/well was added with a 5-fold concentration gradient dilution of antibody molecules with a maximum final concentration of 100 nM, for a total of 10 Mix well; hIgG1 iso (CrownBio, #C0001) as an isotype control; incubate at 37°C, 5% CO 2 incubator for 10 minutes.
  • the fourth step centrifuge at 500g for 5 minutes, then add 25 ⁇ L of supernatant from each well to a new 96-well assay plate; then add 200 ⁇ L/well Europium solution (Perkin Elmer, #C135-100) and the plate was shaken at 250 rpm for 15 minutes at room temperature.
  • use Fluorescence values were measured with a multi-plate reader (Perkin Elmer, Inc.).
  • the software GraphPad Prism 8 was used for data processing and graph analysis, and through four-parameter nonlinear fitting, the killing rate curve, EC50 and maximum killing rate of antibodies against target cells were obtained.
  • the positive control molecule is the anti-HER2 monoclonal antibody PR000210 (trastuzumab analog), which is also the parent monoclonal antibody of the HER2 x CTLA4 double-antibody molecule.
  • both the diabody molecule PR001582 with the IgG-VH-VH hexavalent symmetric structure and the diabody molecule PR001586 with the IgG-VH tetravalent symmetric structure can both induce strong ADCC effects.
  • Example 4.10 Determination of the ability of double antibody molecules to simultaneously bind two kinds of cells by flow cytometry
  • This example is to investigate whether the HER2 x CTLA4 double antibody molecule can simultaneously bind to human HER2-expressing cells SK-BR-3 (ATCC, HTB-30) and human CTLA4-expressing cells CHO-K1/hCTLA4 (Wisdom Chemical) .
  • CHO-K1/hCTLA4 cells and SK-BR-3 cells were fluorescently labeled with CFSE (Thermo, #C34554) and Far red (Thermo, #C34564) dyes, respectively.
  • CHO-K1/hCTLA4 cells and SK-BR-3 cells were resuspended in PBS and adjusted to 2x10 6 /ml; added to CHO-K1/hCTLA4 cells and SK-BR-3 cells, respectively 5 ⁇ M CFSE and 1 ⁇ M Far red and incubate at 37°C for 10 minutes with occasional shaking; add 5x the volume of complete medium to stop staining. After that, centrifugation was performed at 4°C for 5 minutes, the supernatant was discarded, and PBS was added for washing twice.
  • the densities of labeled CHO-K1/hCTLA4 cells and SK-BR-3 cells were adjusted to 4 x 10 6 /ml and 2 x 10 6 /ml, respectively.
  • the second step is to take 25 ⁇ l of labeled CHO-K1/hCTLA4 cells and SK-BR-3 cells, mix them with 50 ⁇ l of four-fold concentration-dilute antibody molecules, and add them to a 96-well plate.
  • the proportion of double-labeled-positive cell population (Q2) relative to all labeled SK-BR-3 cells (Q1+Q2) was calculated according to the following formula, reflecting the ability of double-labeled molecules to bind to two kinds of cells at the same time.
  • the software GraphPad Prism 8 was used for data processing and graph analysis, and through four-parameter nonlinear fitting, the curve of antibody binding to two target cells at the same time was obtained.
  • the anti-HER2 monoclonal antibody PR000210 (trastuzumab analog) and the anti-CTLA4 monoclonal antibody PR000184 are used as control molecules, which are also the parent monoclonal antibodies of the HER2 x CTLA4 double-antibody molecule.
  • IgG-VH tetravalent symmetric structure diabodies PR000539, PR000540, PR000715), IgG-VH-VH hexavalent symmetric structure diabodies (PR000541 and PR000542), Fab-Fc-
  • VH asymmetric double antibody PR000916
  • PR000210, PR000184 and hIgG1 controls.
  • Neither the control molecules HER2 mAb PR000210 nor CTLA4 mAb PR000184 could bind to both cells simultaneously.
  • the HER2 x CTLA4 diabodies shown, in either symmetric or asymmetric structures, can simultaneously bind to two cells expressing different targets.
  • This example studies the pharmacokinetic properties of antibody molecules in mice.
  • the following antibodies were tested: anti-HER2 monoclonal antibody PR000210 (trastuzumab analog); HER2 x CTLA4 double-antibody molecule PR000540 with IgG-VH tetravalent symmetric structure; HER2 with IgG-VH-VH hexavalent symmetric structure x CTLA4 double antibody PR000541.
  • the implementation method is as follows: for each test antibody molecule, 3 female C57BL/6 mice with a body weight of 18 to 22 grams were selected and administered by intravenous injection at a dose of 5 mg/kg; Whole blood was collected at 24 hours (1 day), 2 days, 4 days, 7 days, 10 days and 14 days, and the whole blood was allowed to stand for 30 minutes to clot, then centrifuged at 12,000 g at 4°C 5 min and isolated serum samples were frozen at -80°C until analysis.
  • the Fc-terminal detection ELISA method was used to quantitatively determine the drug concentration in mouse serum. That is, the fusion protein containing human Fc in mouse serum is captured by goat anti-human Fc polyclonal antibody coated on a 96-well plate, and then HRP-labeled goat anti-human Fc secondary antibody is added for detection.
  • the non-compartmental model NCA was used to analyze the plasma concentration data to evaluate its pharmacokinetics.
  • the dual antibody PR000540 and the parental mAb PR000210 had similar clearance rates, and PR000540 had a longer half-life t 1/2 value (over 20 days). Probably due to its complex structure, PR000541 showed a faster clearance rate.
  • anti-HER2 x CTLA4 bispecific antibody molecules with various structures were constructed by using the antigen-binding domain Fab of the anti-HER2 IgG antibody and the antigen-binding domain VH of the anti-CTLA4 HCAb antibody. Demonstrates the flexibility of constructing bispecific antibody molecular structures based on HCAb, and adjusts the functional activities of HER2 and CTLA4 ends through different structure types, relative positions, binding valences and other parameters, and then designs different activity combinations to achieve The need for different mechanisms of action.
  • the recommended initial dose of anti-HER2 monoclonal antibody trastuzumab in the treatment of breast cancer is 4mg/kg, and the recommended initial dose in the treatment of gastric cancer is 8mg/kg; slightly higher than the recommended dose of anti-CTLA4 monoclonal antibody Ipilimumab in the treatment of melanoma 3mg/kg.
  • both the HER2 and CTLA4 ends of the diabodies can almost completely retain activity comparable to their parental mAbs, and they are more active in tumors.
  • In vitro functional assays such as cell proliferation inhibition assays and superantigen stimulation assays show similar abilities to the corresponding parental mAbs; thus, they can be used to achieve a 1:1 dose combination of combination therapy.
  • the HER2 and CTLA4 ends of IgG-VH-VH hexavalent symmetrical diabody molecules can almost completely retain the activity equivalent to their parental mAbs, so they can be used to achieve combination. 1:1 dose combination of medication.
  • the anti-CTLA4 VH when the anti-CTLA4 VH is at the C-terminus of the anti-HER2 IgG, or when it is in the Fab-HCAb structure, the HER2-terminus activity of the double antibody molecule (such as PR000539, PR000540, PR000655) is almost equivalent to its parent mAb, but its CTLA4
  • the activity of the CTLA4 inhibitor is attenuated to varying degrees; therefore, it can be used to achieve the clinical need for moderate or low doses of CTLA4 inhibitors.
  • the HER2-binding domain of the diabody molecule is a monovalent Fab structure, but its ability to bind HER2 is very close to that of the bivalent parent monoclonal antibody; it contains two A CTLA4-binding domain formed in tandem, the ability to bind CTLA4 is similar to that of the parental mAb, but the T-cell activation ability is weakened compared with the parental mAb; thus this structure can pull tumor target cells and T cells together , to promote the formation of immune synapses, which can be used to achieve the clinical need for moderate or low doses of CTLA4 inhibitors, while also increasing the tumor-specific targeting ability.
  • PD-1 Programmed death 1
  • PD-L1 is mainly expressed in immune cells such as T cells, and it has two ligands, programmed death ligand 1 (PD-L1) and PD-L2.
  • PD-L1 is mainly expressed in antigen presenting cells and various tumor cells.
  • the interaction between PD-L1 and PD-1 can down-regulate the activity of T cells, weaken the secretion of cytokines, and play an immunosuppressive effect.
  • the expression of PD-L1 protein can be detected in many human tumor tissues.
  • the microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells.
  • the expressed PD-L1 is beneficial to the occurrence and growth of tumors and induces anti-tumor effects. apoptosis of T cells and further protect tumor cells from immune attack.
  • 4-1BB (TNFRSF9, CD137) is a transmembrane protein belonging to the TNF receptor superfamily. 4-1BB is a costimulatory molecule expressed on a variety of immune cells and is a multifunctional regulator of immune activity. It induces expression in immune cells such as activated T cells and NK cells. 4-1BB activates T cells through trimerization mediated by its ligand 4-1BBL, promoting cell proliferation and cytokine release. Agonistic antibodies against 4-1BB have the function of inhibiting tumors. The earliest 4-1BB antibodies entered clinical trials are Pfizer's Utomilumab and Bristol-Myers Squibb's (BMS) Urelumab (BMS-663513).
  • bispecific antibodies targeting both PD-L1 and 4-1BB to improve anti-tumor efficacy and safety through one or more mechanisms of action.
  • PD-L1 x 4-1BB double antibody can activate T cells by blocking the PD-1/PD-L1 signaling pathway.
  • PD-L1 molecules that are highly expressed on the surface of tumor cells can use double antibody molecules to promote the cross-linking and trimerization of 4-1BB molecules on the surface of T cells and activate downstream signaling pathways, thereby promoting the activation and proliferation of T cells.
  • the activation of T cells mediated by dual-antibody molecules is limited to the tumor microenvironment, which can avoid the toxic and side effects caused by the over-activation of T cells in normal tissues by monoclonal antibodies like Urelumab.
  • Example 5.2 Obtaining anti-PD-L1 IgG antibodies and anti-4-1BB H2L2 or HCAb antibodies
  • the anti-PD-L1 fully human IgG antibody PR000265 (Table 5-8) used in this example was derived from Harbour H2L2 mice, and the discovery process was as described in Example 3.2.1.
  • the candidate antibody molecules are then subjected to sequence analysis and optimization, resulting in several variant sequences.
  • the VL and VH sequences of the antibody are fused and expressed with the corresponding human kappa light chain constant region and IgG1 heavy chain constant region sequences to obtain recombinant fully human antibody molecules.
  • Recombinant fully human IgG antibodies PR000197 and PR000448 against 4-1BB are listed in Tables 5-8.
  • the Harbour HCAb mouse (Harbour Antibodies BV, WO2010/109165A2) is a transgenic mouse carrying a human immunoglobulin immune repertoire that produces heavy chain-only antibodies that are half the size of traditional IgG antibodies. It produces antibodies with only human antibody heavy chain variable domains and mouse Fc constant domains.
  • the human VH gene was amplified from plasma cells by conventional molecular biology methods, and the amplified human VH gene fragment was constructed into the mammalian cell expression plasmid pCAG vector encoding the Fc region sequence of the heavy chain of human IgG1 antibody.
  • the plasmid is transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression, and the supernatant of fully human HCAb antibody is obtained.
  • mammalian host cells such as human embryonic kidney cells HEK293
  • the binding of HCAb antibody supernatant to CHO-K1 cell CHO-K1/hu4-1BB highly expressing human 4-1BB was tested by FACS, and positive HCAb antibody was identified.
  • HCAb antibodies were further identified, and several candidate HCAb antibody molecules were selected according to their binding ability to human 4-1BB, cynomolgus monkey 4-1BB binding ability, T cell activation ability and other parameters.
  • the candidate HCAb antibody molecules were then sequenced and optimized, resulting in several variant sequences.
  • the VH sequence of the HCAb antibody and the human IgG1 heavy chain Fc sequence are fused and expressed to obtain a fully human recombinant HCAb antibody molecule.
  • the recombinant fully human HCAb antibodies PR001758, PR001760 and PR001836 against 4-1BB are listed in Tables 5-8.
  • Example 5.3 Using anti-PD-L1 IgG antibody and anti-4-1BB IgG antibody to construct bispecific antibody molecule with FIT-Ig structure
  • the antigen-binding domain Fab of the anti-PD-L1 IgG antibody PR000265 or PR000151 (atezolizumab analog) and the antigen-binding domain Fab of the anti-4-1BB IgG antibody PR000197 or PR000448 were used to construct FIT-Ig Structure of the anti-PD-L1 x 4-1BB bispecific antibody molecule.
  • the design of the FIT-Ig structure can refer to patent WO2015/103072A1, the structure is shown in Figure 1 (28); the constructed molecules are summarized in Table 5-1; and the antibody molecule samples were prepared and analyzed according to the method described in Example 2 , summarized in Table 5-2.
  • Table 5-3 lists the sequence numbers corresponding to the polypeptide chain sequences of the FIT-Ig structure diabody molecules.
  • Antibody number Polypeptide chain 1 Polypeptide chain 2 Polypeptide chain 3 PR000701 384 371 355 PR003052 452 451 355
  • Example 5.4 Construction of bispecific antibody molecules using anti-PD-L1 IgG antibody and anti-4-1BB HCAb antibody
  • the antigen-binding domain Fab of the anti-PD-L1 IgG antibody PR000265 and the antigen-binding domain VH of the anti-4-1BB HCAb antibodies PR001758, PR001760 or PR001836 were used to construct anti-PD-L1x with various structures 4-1BB bispecific antibody molecule.
  • the positive control molecule is the anti-PD-L1 IgG monoclonal antibody PR000265, which is also the parental monoclonal antibody at the PD-L1 end of the PD-L1 x 4-1BB double antibody molecule.
  • the positive control molecule was anti-4-1BB IgG monoclonal antibody urelumab (IgG4) or utomilumab (IgG2).
  • the positive control molecule is the PD-L1 x 4-1BB double antibody molecule PR001289, the sequence of which is derived from the anti-4-1BB and anti-PD-L1 single domain antibody sequences disclosed in patent WO2017/123650A2.
  • a PD-L1 x 4-1BB diabody molecule with Fab-HCAb symmetrical structure was designed according to the structure described in Example 1.1, which is summarized in Table 5-4; Antibody molecule samples were prepared and analyzed according to the method described in Example 2, which are summarized in Tables 5-5.
  • Example 1.2 Using anti-PD-L1 IgG antibody and anti-4-1BB heavy chain antibody, according to the structure described in Example 1.2, a PD-L1 x 4-1BB diabody molecule of IgG-VH tetravalent symmetrical structure was designed, which is summarized in Table 5- 6; and the antibody molecule samples were prepared and analyzed according to the method described in Example 2, which are summarized in Tables 5-7.
  • Table 5-8, Table 5-9 and Table 5-10 list the PD-L1 x 4-1BB double antibody molecules constructed in this example and the corresponding parental single antibody such as PD-L1 mAb and 4-1BB mAb The sequence numbers corresponding to the sequences of the anti- and control molecules. Structure numbers in Table 5-10 correspond to Table 1-1 and Figure 1. Tables 5-11 list the sequence numbers of the corresponding CDR sequences of the first and second antigen binding domains of the bispecific antibody molecule.
  • Table 5-10 The sequence numbering table of the PD-L1 x 4-1BB double antibody molecule of the present embodiment
  • This example is to study the activity of PD-L1 x 4-1BB double antibody binding to PD-L1.
  • Example 5.5.1 Binding to CHO-K1 cells CHO-K1/hPDL1 highly expressing human PD-L1
  • Flow cytometry FACS was used to test the binding ability of antibody molecules to CHO-K1 cell line CHO-K1/hPDL1 (Nanjing GenScript, M00543) that highly expresses human PD-L1. Specifically, digestion of CHO-K1 / hPDL1 cells were resuspended in complete medium; cell density was adjusted to 1x10 6 cells / mL. Next, the cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 100 ⁇ L/well, centrifuged at 4° C. for 5 minutes, and the supernatant was discarded. Then, the serially diluted antibody molecules were added to the 96-well plate at 100 ⁇ L/well and mixed evenly.
  • the antibody molecules can be diluted from the highest final concentration of 200nM to a total of 12 concentrations by 3-fold concentration; hIgG1 iso (CrownBio, #C0001) was used as the isotype control.
  • the cells were placed at 4°C and incubated in the dark for 1 hour. Then, 100 ⁇ L/well of pre-chilled FACS buffer (PBS buffer containing 0.5% BSA) was added to rinse the cells twice, centrifuged at 500 g for 5 minutes at 4° C., and the supernatant was discarded.
  • Fluorescence signal values were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and the data were processed and analyzed with the software FlowJo v10 (FlowJo, LLC).
  • the software GraphPad Prism 8 was used for data processing and graph analysis, and parameters such as the binding curve of the antibody to the target cell and the EC50 value were obtained through four-parameter nonlinear fitting.
  • the positive control molecule is the anti-PD-L1 monoclonal antibody PR000265, which is also the parent monoclonal antibody at the PD-L1 end of the PD-L1 x 4-1BB double antibody molecule.
  • the results are shown in Figure 27 and Tables 5-12.
  • the diabody molecules (PR003549, PR003550, PR003551) of IgG-VH tetravalent symmetric structure have a similar ability to bind PD-L1 to that of the parental mAb PR000265, and their binding EC50 to PD-L1
  • the value and MFI maximum value are slightly better than those of FIT-Ig structure double antibody molecules (PR000701, PR003052).
  • the double antibody molecule (PR004270) with a symmetrical structure of Fab-HCAb has a similar ability to bind to PD-L1 as the parental mAb, although the EC50 value of its binding to PD-L1 is slightly weaker than that of the parental mAb, However, the maximum MFI value for binding was higher than that of the parental mAb.

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Abstract

公开了一种含有至少两个蛋白功能区的结合蛋白，其中，所述结合蛋白包括蛋白功能区A和蛋白功能区B；所述蛋白功能区A和所述蛋白功能区B靶向不同的抗原或抗原表位，其中所述蛋白功能区A为Fab或scFv结构，所述蛋白功能区B为VH结构，所述蛋白功能区A和蛋白功能区B的数量分别为一个或多个。多特异性结合蛋白具有较小的分子量、较少的多肽链、更简单的结构，通过不同的结构类型、相对位置、结合价数等参数来调节针对不同靶点的功能活性，进而设计出不同的活性组合，以满足不同的临床联合用药剂量组合的需求。

Description

具有H2L2与HCAb结构的结合蛋白 技术领域
本发明涉及生物医药领域，尤其涉及一种具有H2L2与HCAb结构的双特异性或多特异性结合蛋白及其应用。
发明背景
大多数物种中的抗体是包含两个完全相同的重链和两个完全相同的轻链的四聚体结构，也称为“H2L2”。在骆驼科及鲨鱼科动物血清中还天然存在一种缺失轻链的重链抗体(heavy-chain antibody,HCAb)。重链抗体的可变区VHH片段与H2L2抗体的Fab类似，具有稳定的结构和特异的抗原结合活性，但是VHH的分子量只有约13KDa，因此也被称作纳米抗体或单域抗体。人的抗体天然结构为“H2L2”，因而无法从天然来源中获得有用的人源的重链抗体；但是Frank Grosveld等人提出了一种利用转基因动物获得全人源重链抗体的方法(WO2007/096779)，并可以利用该方法得到稳定和可溶的全人源VH单域抗体。
双特异性抗体(bispecific antibodies)和多特异性抗体(multispecific antibodies)是一类在天然的单克隆抗体基础上通过蛋白质工程技术制备出的人工抗体，其可以结合两种或多种不同的抗原或者同一个抗原上的不同表位，可以实现一些单特异性抗体无法实现的作用机制和功能效果。
双特异性抗体的结构设计是非常重要的。从两个不同的H2L2抗体产生双特异性抗体，其中一个最主要的挑战就是怎样从超过10种的不同的重链和轻链的组合中获得具有正确的链组合的功能性的双特异性抗体分子，即如何解决链的错配问题。科学家已经研发出多种策略来尝试解决这一问题：例如，使用单链可变区抗体片段(scFv)的结构将VH和VL融合在一起；或者，利用knob-into-hole(KiH)技术或其他空间结构互补的突变体来促进重链的异二聚化；或者，利用“共有轻链”或者“共有重链”技术来减少不同多肽链的数目，等等。但是，各种技术手段都有其局限性，例如，scFv结构经常是不稳定的且易于聚集的；“共有轻链”技术需要使用非常复杂的筛选过程来获得可以与不同VH配对的VL；FIT-Ig等技术产生的双抗分子的尺寸比较大(约250KDa)，可能会影 响其对细胞或组织的穿透能力。因此，仍然亟需开发新的双特异性抗体的结构和制备双特异性抗体的技术。
重链抗体及其衍生的单域抗体在构建双特异甚至多特异抗体方面有其独特的优势。重链抗体的抗原结合结构域只有常规抗体的Fab的四分之一大小；而且没有轻链，避免了轻链错配的问题。所以，利用重链抗体及其衍生的单域抗体，可以构建分子量较小的、多肽链较少的、结构更简单的双特异甚至多特异抗体。但是，非人源重链抗体的潜在的免疫原性风险可能会限制它的治疗性用途。因而，相较于骆驼科动物的纳米抗体，利用全人源重链抗体来构建双特异或多特异抗体会在免疫原性和成药性方面更有优势。
发明内容
为克服现有技术中缺乏疗效好、稳定且生产方便的双特异性或多特异性结合蛋白的缺陷，本发明提供了一种具有H2L2与HCAb结构的双或多特异性结合蛋白以及制备和使用此类结合蛋白的方法，其利用全人源重链抗体及其衍生的单域抗体所构建的多价和多特异性结合蛋白。其相较于利用常规IgG抗体构建的多价和多特异性结合蛋白有诸多优势，在调整特异性和结合价数方面更为灵活。本文提供的多价和多特异性结合蛋白含有至少一个来源于人源重链抗体的重链可变区结构域VH，并且能够结合两个或更多个抗原，或同一抗原的两个或更多个表位，或同一表位的两个或更多个拷贝。
为解决上述技术问题，本发明的技术方案之一为：提供一种含有至少两个蛋白功能区的结合蛋白，其中，所述结合蛋白包括蛋白功能区A和蛋白功能区B；所述蛋白功能区A和所述蛋白功能区B靶向不同的抗原或抗原表位，其中所述蛋白功能区A为Fab或scFv结构，所述蛋白功能区B为VH结构，所述蛋白功能区A和蛋白功能区B的数量分别为一个或多个。优选地，所述结合蛋白还包括Fc，所述Fc的数量为二个，形成Fc二聚体。较佳地，所述结合蛋白为对称结构或非对称结构，和/或，所述Fc二聚体为同源二聚体或异源二聚体。
在一些具体的实施例中，(I)所述结合蛋白为对称结构，所述对称结构为左右对称的类IgG结构，其中所述蛋白功能区A的数量为二个，所述蛋白功能区B的数量为二个或四个。二个所述蛋白功能区A与二个所述Fc形成IgG结构。在所述左右对称的类IgG结构中，左和右只是空间相对位置，其可以为上下对称也可以为前后对称。所述 左右对称的类IgG结构指在IgG结构的抗体上还左右对称地连接了额外的蛋白功能区，这些额外的蛋白功能区可以连接在Fc功能区的CH3的C末端或VH或VL的N末端，也可以以嵌入的形式连接在IgG结构的可变区和Fc功能区之间。本文所述“额外的”在此表示相对IgG结构以外的结构或功能区。
较佳地，当所述蛋白功能区B的数量为二个时，所述结合蛋白为四价结合蛋白，具体包括以下结构：(a)所述结合蛋白从N末端至C末端依次为蛋白功能区A、蛋白功能区B和Fc，其中所述蛋白功能区A与所述蛋白功能区B通过第一连接肽(L1)连接，所述蛋白功能区B与所述Fc通过第二连接肽(L2)连接；在该实施方式中，两份的所述蛋白功能区B与所述Fc形成对称的单链抗体的二聚体形式，并在所述单链抗体的二聚体的N末端上连接了所述蛋白功能区A，此时所述蛋白功能区A可以以其CH1(图1的(2))或CL(图1的(1))与所述蛋白功能区B的N末端连接；或，(b)所述结合蛋白从N末端至C末端依次为蛋白功能区B、蛋白功能区A和Fc，其中所述蛋白功能区B与所述蛋白功能区A通过连接肽连接，所述蛋白功能区A与所述Fc通过铰链区连接；在该实施方式中，两份的所述蛋白功能区A与所述Fc形成对称的IgG结构，并在所述IgG结构的N末端上连接了所述蛋白功能区B，此时所述蛋白功能区B可以与所述蛋白功能区A的VH(图1的(3))或VL(图1的(4))的N末端连接；或，(c)所述结合蛋白从N末端至C末端依次为蛋白功能区A、Fc和蛋白功能区B，其中所述蛋白功能区A与Fc通过铰链区连接，所述Fc与所述蛋白功能区B通过连接肽连接；在该实施方式中，两份的所述蛋白功能区A与所述Fc形成对称的IgG结构，并在所述IgG结构的C末端上连接了所述蛋白功能区B，此时所述蛋白功能区B可以与所述蛋白功能区A的CH3(图1的(5))或CL(图1的(6))的C末端连接；
较佳地，当所述蛋白功能区B的数量为四个时，所述结合蛋白为六价结合蛋白，具体包括以下结构：新增的二个蛋白功能区B进一步连接在上述(a)或(b)或(c)所述结合蛋白的N末端或C末端；优选连接在上述(c)所述结合蛋白的Fc的C末端或者原有的蛋白功能区B的C末端，或与上述(b)所述结合蛋白的原有的蛋白功能区B一同连接于蛋白功能区A的N末端。本文所述“新增的”仅为表述方便，以区分上文提到的蛋白功能区B即“原有的”蛋白功能区，在实际设计或生产抗体时，新增的蛋白功能区与原有的蛋白功能区之间并无先后之分。在该实施方式中，两份的所述蛋白功能区A与所述Fc形成对称的IgG结构，并在所述IgG结构的C末端上连接了所述蛋白功能区 B，此时其中两个蛋白功能区B可以与所述蛋白功能区A的CH3通过第一连接肽连接，另外两个蛋白功能区B与前述两个蛋白功能区B通过第二连接肽连接(图1的(7))；或其中两个蛋白功能区B可以与所述蛋白功能区A的VH的N末端通过第一连接肽连接，另外两个蛋白功能区B与所述蛋白功能区A的VL的N末端通过第二连接肽连接(图1的(9))。
上述具有二个不同的蛋白功能区的结合蛋白又称为双特异性结合蛋白。更佳地，所述结合蛋白还包括蛋白功能区C，所述结合蛋白为六价或八价的三特异性结合蛋白，其具体包括以下结构：所述蛋白功能区C与所述蛋白功能区A、蛋白功能区B靶向不同的抗原或抗原表位，所述蛋白功能区C连接在上述抗体的N末端或C末端；优选所述蛋白功能区C的数量为二个，所述蛋白功能区C连接在上述(c)所述结合蛋白的C末端，或，与上述(b)所述结合蛋白的蛋白功能区B一样，连接于所述蛋白功能区A的N末端。在该实施方式中，两份的蛋白功能区C通过第二连接肽进一步连接于结构(5)的C末端(图1的(8))，或结构(3)或(4)的N末端(图1的(10)显示了连接在结构(3)的N末端的情形)。
进一步更佳地，所述结合蛋白具有四条多肽链，分别为两条相同的短链(或称“多肽链1”)和两条相同的长链(或称“多肽链2”)，其中，(1)所述短链从N末端至C末端依次包括VH_A-CH1，所述长链从N末端至C末端依次包括VL_A-CL-L1-VH_B-L2-CH2-CH3；或(2)所述短链从N末端至C末端依次包括VL_A-CL，所述长链从N末端至C末端依次包括VH_A-CH1-L1-VH_B-L2-CH2-CH3；或(3)所述短链从N末端至C末端依次包括VL_A-CL，所述长链从N末端至C末端依次包括VH_B-L-VH_A-CH1-h-CH2-CH3；或(4)所述短链从N末端至C末端依次包括VH_B-L-VL_A-CL，所述长链从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3；或(5)所述短链从N末端至C末端依次包括VL_A-CL，所述长链从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3-L-VH_B；或(6)所述短链从N末端至C末端依次包括VL_A-CL-L-VH_B，所述长链从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3；或(7)所述短链从N末端至C末端依次包括VL_A-CL，所述长链从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_B；或(8)所述短链从N末端至C末端依次包括VL_A-CL，所述长链从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_C；或(9)所述短链从N末端至C末端依次包括VH_B-L1-VL_A-CL，所述长链从 N末端至C末端依次包括VH_B-L2-VH_A-CH1-h-CH2-CH3；或(10)所述短链从N末端至C末端依次包括VH_B-L1-VL_A-CL，所述长链从N末端至C末端依次包括VH_C-L2-VH_A-CH1-h-CH2-CH3；其中，所述L、L1和L2为连接肽，所述h为铰链区或连接肽，所述铰链区或连接肽例如“-”、GS或如SEQ ID NO:495-519的氨基酸序列所示。本文中VL、VH、CL、CH的含义均为本领域常规，分别代表轻链可变区、重链可变区、轻链恒定区和重链恒定区，其中CH包括CH1、CH2和CH3；_A与_B与_C分别代表该功能区为蛋白功能区A或蛋白功能区B或蛋白功能区C或其组成；“-”代表联结不同结构区的多肽键或用来分隔不同结构区；C末端即肽链的羧基末端(也可写成“C’”)，N末端即肽链的氨基末端(也可写成“N’”)。上述不同蛋白功能区融合在同一条多肽链上，可以避免错配副产物。在一些实施方案中，L、L1和L2可以是相同的序列。在另一些实施方案中，L、L1和L2可以是不同的序列。本发明的蛋白功能区在一些情况中可以为Fab、scFv或VH，在另一些情况中也可以为F(ab)2或全长抗体，在某些情况中也称为抗体或抗原结合蛋白或结合蛋白。在一些具体的实施例中，所述蛋白功能区A又称作针对第一抗原的抗体A或第一抗原结合结构域；所述蛋白功能区B又称作针对第二抗原的抗体B或第二抗原结合结构域；所述蛋白功能区C又称作针对第三抗原的抗体C或第三抗原结合结构域；所述蛋白功能区D又称作针对第四抗原的抗体D或第四抗原结合结构域，以此类推。
在一些具体的实施例中，(II)所述结合蛋白为非对称结构，所述非对称结构呈左右不对称的类IgG结构，其中左臂为Fab或scFv结构的蛋白功能区A，右臂为VH结构的蛋白功能区B，所述蛋白功能区A和所述蛋白功能区B分别与一个Fc连接；优选所述蛋白功能区A的数量为一个，所述蛋白功能区B的数量为一个或二个或三个。
较佳地，当所述蛋白功能区B的数量为一个时，所述结合蛋白为二价结合蛋白，其包括以下结构：所述蛋白功能区A为(d)常规Fab结构，或(e)Fab(cross VH/VL)结构，或(f)Fab(cross Fd/LC)结构；当所述蛋白功能区B的数量为二个时，所述结合蛋白为三价结合蛋白，其包括以下结构：左臂的所述蛋白功能区A为上述(d)或(e)或(f)，第二个蛋白功能区B连接在左臂的所述蛋白功能区A的N末端或C末端，或右臂的第一个蛋白功能区B的N末端，或所述Fc的C末端，优选第二个蛋白功能区B连接在右臂的第一个蛋白功能区B的N末端；当所述蛋白功能区B的数量为三个时，所述结合蛋白为四价结合蛋白，具体包括以下结构：左臂的所述蛋白功能区A为 上述(d)或(e)或(f)结构，第三个蛋白功能区B进一步连接在左臂的所述蛋白功能区A的N末端或C末端，或左臂的第二个蛋白功能区B的N末端或C末端，或右臂的第一个或第二个蛋白功能区B的N末端，或所述Fc的C末端，优选第二个蛋白功能区B连接在右臂的第一个蛋白功能区B的N末端，且第三个蛋白功能区B连接在所述第二个蛋白功能区B的N末端。
当所述蛋白功能区B的数量为一个时，所述结合蛋白为二价结合蛋白，其包括以下结构：左臂的所述蛋白功能区A为(g)scFv结构，所述scFv通过VH的末端或VL的末端与Fc连接；当所述蛋白功能区B的数量为二个时，所述结合蛋白为三价结合蛋白，具体包括以下结构：左臂的所述蛋白功能区A为(g)，第二个蛋白功能区B连接在左臂的所述蛋白功能区A的N末端，或右臂的原有的蛋白功能区B的N末端，或所述Fc的C末端，优选第二个蛋白功能区B连接在右臂的第一个蛋白功能区B的N末端；当所述蛋白功能区B的数量为三个时，所述结合蛋白为四价结合蛋白，具体包括以下结构：左臂的所述蛋白功能区A为(g)，第三个蛋白功能区B进一步连接在左臂的所述蛋白功能区A的N末端，或第二个蛋白功能区B的N末端或C末端，或所述Fc的C末端，优选第二个蛋白功能区B连接在右臂的第一个蛋白功能区B的N末端，且第三个蛋白功能区B连接在所述第二个蛋白功能区B的N末端。
更佳地，所述结合蛋白还包括蛋白功能区C，所述结合蛋白为三价或多价的多特异性结合蛋白，其中，所述蛋白功能区C与所述蛋白功能区A、蛋白功能区B靶向不同的抗原或抗原表位，所述蛋白功能区C连接在上述结合蛋白的N末端或C末端；优选所述蛋白功能区C连接在上述(d)、(e)、(f)或(g)所述结合蛋白的蛋白功能区B的N末端；
进一步更佳地，所述结合蛋白还包括蛋白功能区D，所述结合蛋白为四价或多价的多特异性结合蛋白，具体地：所述蛋白功能区D与所述蛋白功能区A、蛋白功能区B、蛋白功能区C靶向不同的抗原或抗原表位，所述蛋白功能区D连接在上述结合蛋白的N末端或C末端；优选所述蛋白功能区D连接在上述(d)、(e)、(f)或(g)所述结合蛋白的蛋白功能区B的N末端。
进一步更佳地，所述结合蛋白具有多肽链1、多肽链2和多肽链3三种多肽链，每种多肽链各一条。在本发明中，所述多肽链1又称为第一多肽链，所述多肽链2又称为第二多肽链，所述多肽链3又称为第三多肽链。其中，(11)所述多肽链1从N末端至 C末端依次包括VL_A-CL，所述多肽链2从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_B-h-CH2-CH3；或(12)所述多肽链1从N末端至C末端依次包括VH_A-CL，所述多肽链2从N末端至C末端依次包括VL_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_B-h-CH2-CH3；或(13)所述多肽链1从N末端至C末端依次包括VH_A-CH1，所述多肽链2从N末端至C末端依次包括VL_A-CL-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_B-h-CH2-CH3；或(14)所述多肽链1从N末端至C末端依次包括VL_A-CL，所述多肽链2从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_B-L-VH_B-h-CH2-CH3；或(15)所述多肽链1从N末端至C末端依次包括VL_A-CL，所述多肽链2从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_C-L-VH_B-h-CH2-CH3；或(16)所述多肽链1从N末端至C末端依次包括VH_A-CL，所述多肽链2从N末端至C末端依次包括VL_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_B-L-VH_B-h-CH2-CH3；或(17)所述多肽链1从N末端至C末端依次包括VH_A-CL，所述多肽链2从N末端至C末端依次包括VL_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_C-L-VH_B-h-CH2-CH3；或(18)所述多肽链1从N末端至C末端依次包括VH_A-CH1，所述多肽链2从N末端至C末端依次包括VL_A-CL-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_B-L-VH_B-h-CH2-CH3；或(19)所述多肽链1从N末端至C末端依次包括VH_A-CH1，所述多肽链2从N末端至C末端依次包括VL_A-CL-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_C-L-VH_B-h-CH2-CH3；或(26)所述多肽链1从N末端至C末端依次包括VL_A-CL，所述多肽链2从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_B-L1-VH_B-L2-VH_B-h-CH2-CH3；或(27)所述多肽链1从N末端至C末端依次包括VL_A-CL，所述多肽链2从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_D-L1-VH_C-L2-VH_B-h-CH2-CH3；其中，所述L、L1和L2为连接肽，所述h为铰链区或连接肽，所述铰链区或连接肽例如“-”、GS或如SEQ ID NO:495-519的氨基酸序列所示。
进一步更佳地，所述结合蛋白具有多肽链1和多肽链2两种多肽链，每种多肽链各一条，同样地，所述多肽链1又称为第一多肽链，所述多肽链2又称为第二多肽链。其 中，(20)所述多肽链1从N末端至C末端依次包括VL_A-L-VH_A-h-CH2-CH3，所述多肽链2从N末端至C末端依次包括VH_B-h-CH2-CH3；或(21)所述多肽链1从N末端至C末端依次包括VH_A-L-VL_A-h-CH2-CH3，所述多肽链2从N末端至C末端依次包括VH_B-h-CH2-CH3；或(22)所述多肽链1从N末端至C末端依次包括VL_A-L1-VH_A-h-CH2-CH3，所述多肽链2从N末端至C末端依次包括VH_B-L2-VH_B-h-CH2-CH3；或(23)所述多肽链1从N末端至C末端依次包括VH_A-L1-VL_A-h-CH2-CH3，所述多肽链2从N末端至C末端依次包括VH_B-L2-VH_B-h-CH2-CH3；或(24)所述多肽链1从N末端至C末端依次包括VL_A-L1-VH_A-h-CH2-CH3，所述多肽链2从N末端至C末端依次包括VH_C-L2-VH_B-CH2-CH3；或(25)所述多肽链1从N末端至C末端依次包括VH_A-L1-VL_A-h-CH2-CH3，所述多肽链2从N末端至C末端依次包括VH_C-L2-VH_B-h-CH2-CH3；其中，所述L、L1和L2为连接肽，所述h为铰链区或连接肽，所述铰链区或连接肽例如“-”、GS或如SEQ ID NO:495-519的氨基酸序列所示。
在一些具体的实施例中，所述结合蛋白含有至少两个蛋白功能区即蛋白功能区A和蛋白功能区B；所述蛋白功能区A和所述蛋白功能区B为PD-L1抗体、HER2抗体、B7H4抗体、CD3抗体、CTLA4抗体、4-1BB抗体或BCMA抗体中的一种。较佳地，所述蛋白功能区A为PD-L1抗体、HER2抗体、B7H4抗体或CD3抗体，所述蛋白功能区B为CTLA4抗体、4-1BB抗体或BCMA抗体。更佳地，所述蛋白功能区A为PD-L1抗体，所述蛋白功能区B为CTLA4抗体；或，所述蛋白功能区A为PD-L1抗体，所述蛋白功能区B为4-1BB抗体；或，所述蛋白功能区A为HER2抗体，所述蛋白功能区B为CTLA4抗体；或，所述蛋白功能区A为B7H4抗体，所述蛋白功能区B为4-1BB抗体；或，所述蛋白功能区A为CD3抗体，所述蛋白功能区B为BCMA抗体。
在一些具体的实施例中，所述PD-L1抗体包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:167、188和211所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、62和122所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:167、188和211所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、69和122所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。优选地，其轻链可变区VL包 括如SEQ ID NO:282所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:233所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:282所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:240所示的氨基酸序列。更优选地，所述PD-L1抗体包含两个多肽链；其中，第一多肽链包括如SEQ ID NO:347所示的氨基酸序列，第二多肽链包括如SEQ ID NO:300所示的氨基酸序列；或，第一多肽链包括如SEQ ID NO:353所示的氨基酸序列，第二多肽链包括如SEQ ID NO:308所示的氨基酸序列；或，第一多肽链包括如SEQ ID NO:353所示的氨基酸序列，第二多肽链包括如SEQ ID NO:310所示的氨基酸序列。
在一些具体的实施例中，所述PD-L1抗体的如本领域已知的atezolizumab的氨基酸序列所示。
在一些具体的实施例中，所述HER2抗体的如本领域已知的trastuzumab或pertuzumab的氨基酸序列所示。
在一些具体的实施例中，所述B7H4抗体包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:180、191和225所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:32、87和143所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和226所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:33、88和144所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。优选地，其轻链可变区VL包括如SEQ ID NO:298所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:261所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:299所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:262所示的氨基酸序列。更优选地，所述B7H4抗体包含两个多肽链；其中，第一多肽链包括如SEQ ID NO:360所示的氨基酸序列，第二多肽链包括如SEQ ID NO:326所示的氨基酸序列；或，第一多肽链包括如SEQ ID NO:361所示的氨基酸序列，第二多肽链包括如SEQ ID NO:327所示的氨基酸序列。
在一些具体的实施例中，所述CTLA4抗体包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:17、65和125所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。优选地，其重链可变区VH包括如SEQ ID NO:236所示的氨基酸序列。更优选地，所述CTLA4抗体包含一个多肽 链，所述多肽链包括如SEQ ID NO:303所示的氨基酸序列，如SEQ ID NO:306所示的氨基酸序列。
在一些具体的实施例中，所述4-1BB抗体包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:170、191和214所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:18、66和126所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:170、191和214所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:18、71和126所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。优选地，其轻链可变区VL包括如SEQ ID NO:285所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:237所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:289所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:242所示的氨基酸序列。更优选地，所述4-1BB抗体包含两个多肽链；其中，第一多肽链包括如SEQ ID NO:350所示的氨基酸序列，第二多肽链包括如SEQ ID NO:304所示的氨基酸序列；或，第一多肽链包括如SEQ ID NO:355所示的氨基酸序列，第二多肽链包括如SEQ ID NO:311所示的氨基酸序列。
在一些具体的实施例中，所述4-1BB抗体包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:27、79和137所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、80和138所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:29、82和138所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、89和145所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、81和139所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、83和140所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。优选地，其重链可变区VH包括如SEQ ID NO:252所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:253所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:255所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:264所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:254所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:256所示的氨基酸序列。 更优选地，所述4-1BB抗体包含一个多肽链，所述多肽链包括如SEQ ID NO:320所示的氨基酸序列，如SEQ ID NO:321所示的氨基酸序列，如SEQ ID NO:323所示的氨基酸序列，如SEQ ID NO:322所示的氨基酸序列，如SEQ ID NO:324所示的氨基酸序列，或如SEQ ID NO:329所示的氨基酸序列。
在一些具体的实施例中，所述CD3抗体包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和221所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、84和141所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:178、197和222所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、85和142所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和223所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、86和141所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:179、198和224所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、85和142所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。优选地，其轻链可变区VL包括如SEQ ID NO:294所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:258所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:295所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:259所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:296所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:260所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:297所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:259所示的氨基酸序列。
在一些具体的实施例中，所述CD3抗体包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:20、68和128所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。优选地，其轻链可变区VL包 括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:245所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:257所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:281所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:244所示的氨基酸序列。
在一些具体的实施例中，所述CD3抗体包含两种多肽链；其中，第一多肽链包括如SEQ ID NO:357所示的氨基酸序列，第二多肽链包括如SEQ ID NO:313所示的氨基酸序列；或，第一多肽链包括如SEQ ID NO:357所示的氨基酸序列，第二多肽链包括如SEQ ID NO:325所示的氨基酸序列；或，第一多肽链包括如SEQ ID NO:357所示的氨基酸序列，第二多肽链包括如SEQ ID NO:328所示的氨基酸序列；或，第一多肽链包括如SEQ ID NO:357所示的氨基酸序列，第二多肽链包括如SEQ ID NO:346所示的氨基酸序列。
在一些具体的实施例中，所述CD3抗体包含一个多肽链，所述多肽链包括如SEQ ID NO:489所示的氨基酸序列，如SEQ ID NO:490所示的氨基酸序列，如SEQ ID NO:491所示的氨基酸序列，如SEQ ID NO:492所示的氨基酸序列，或，如SEQ ID NO:493所示的氨基酸序列。
在一些具体的实施例中，所述BCMA抗体包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、75和133所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:24、76和134所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:25、77和135所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。优选地，其重链可变区VH包括如SEQ ID NO:248所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:249所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:250所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列。更优选地，所 述BCMA抗体包含一个多肽链，所述多肽链包含如SEQ ID NO:316所示的氨基酸序列，如SEQ ID NO:317所示的氨基酸序列，如SEQ ID NO:318所示的氨基酸序列，或，如SEQ ID NO:319所示的氨基酸序列。
在一些具体的实施例中，所述BCMA抗体包含重链可变区，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、90和136所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、78和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、78和147所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和147所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:36、90和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和147所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、78和148所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和136所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、76和136所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、76和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和148所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和136所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。优选地，其重链可变区VH包括如SEQ ID NO:265所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:266所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:267所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:268所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:269所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:270所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:271所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:272所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:273所示的氨基酸序 列；或，其重链可变区VH包括如SEQ ID NO:274所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:275所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:276所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:277所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:278所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:279所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:280所示的氨基酸序列。更优选地，所述BCMA抗体包含一个多肽链，所述多肽链包含如SEQ ID NO:330所示的氨基酸序列；或，如SEQ ID NO:331所示的氨基酸序列；或，如SEQ ID NO:332所示的氨基酸序列；或，如SEQ ID NO:333所示的氨基酸序列；或，如SEQ ID NO:334所示的氨基酸序列；或，如SEQ ID NO:335所示的氨基酸序列；或，如SEQ ID NO:336所示的氨基酸序列；或，如SEQ ID NO:337所示的氨基酸序列；或，如SEQ ID NO:338所示的氨基酸序列；或，如SEQ ID NO:339所示的氨基酸序列；或，如SEQ ID NO:340所示的氨基酸序列；或，如SEQ ID NO:341所示的氨基酸序列；或，如SEQ ID NO:342所示的氨基酸序列；或，如SEQ ID NO:343所示的氨基酸序列；或，如SEQ ID NO:344所示的氨基酸序列；或，如SEQ ID NO:345所示的氨基酸序列。
在一些具体的实施例中，所述蛋白功能区C是BCMA重链抗体的重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:24、76和134所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列。优选地，其重链可变区VH包括如SEQ ID NO:249所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列。
在以上具体实施例中，所述结合蛋白还可以包括轻链恒定区，所述轻链恒定区优选人轻链恒定区，更优选人轻链恒定区Cκ或Cλ。
在以上具体实施例中，所述结合蛋白还可以包括重链恒定区(CH1、CH2和/或CH3)，所述重链恒定区优选人重链恒定区，更优选人IgG1、IgG2、IgG3、IgG4重链恒定区。在一些具体实施例中，所述IgG1重链恒定区的Fc上具有C220S、N297A、L234A、L235A、P329G、S239D、I332E、S354C、T366W、Y349C、T366S、L368A、Y407V、M252Y、S254T、T256E等突变中的一种或多种，所述突变位点使用EU编号规则。
在一些优选的实施例中，所述结合蛋白含有蛋白功能区A和蛋白功能区B：所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:167、188和211所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、69和122所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:17、65和125所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:167、188和211所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、62和122所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:17、65和125所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:171、190和215所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:19、67和127所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:17、65和125所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:176、196和220所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:23、74和132所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:17、65和125所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:167、188和211所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、69和122所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含 HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、80和138所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:167、188和211所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、69和122所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:27、79和137所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:167、188和211所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、69和122所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:29、82和138所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:180、191和225所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:32、87和143所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、80和138所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:180、191和225所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:32、87和143所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:27、79和137所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:180、191和225所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:32、87和143 所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、89和145所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和226所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:33、88和144所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、80和138所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和226所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:33、88和144所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、81和139所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和226所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:33、88和144所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、83和140所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:20、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:24、76和134所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列； 其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:20、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:20、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、75和133所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、75和133所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、90和136所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:36、90和146所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和147所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、78和148所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和136所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含 HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和146所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、76和136所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和146所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、76和146所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和148所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128 所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和136所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、78和146所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、78和147所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和147所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和221所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、84和141所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:178、197和222所示的氨基酸序列； 其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、85和142所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和223所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、86和141所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:179、198和224所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、85和142所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列；或，
在一些优选的实施例中，所述结合蛋白含有蛋白功能区A、蛋白功能区B和蛋白功能区C：其中，所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、75和133所示的氨基酸序列；所述蛋白功能区C包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:24、76和134所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:25、77和135所示的氨基酸序列；所 述蛋白功能区C包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。
在一些更优选的实施例中，所述结合蛋白包括蛋白功能区A和蛋白功能区B：所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:282所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:240所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:236所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:282所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:233所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:236所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:286所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:238所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:236所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:293所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:247所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:236所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:282所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:240所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:253所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:282所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:240所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:252所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:282所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:240所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:255所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:298所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:261所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:253所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:298所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:261所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:252所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:298所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:261所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:264所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:299所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:262所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:253所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:299所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:262所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:254所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:299所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:262所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:256所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:245所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:249所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:245所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:245所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:248所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:257所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:248所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:257所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:281所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:265所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:269所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:270所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:271所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:272所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:273所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:274所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:275所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:276所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:277所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:278所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:279所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:280所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:266所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:267所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:268所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:244所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:249所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:244所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:294所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:258所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:295所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:259所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:296所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:260所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:297所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:259所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
在一些更优选的实施例中，所述结合蛋白含有蛋白功能区A、蛋白功能区B和蛋白功能区C：其中，所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:257所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:248所示的氨基酸序列。所述蛋白功能区C包含重链可变区；其重链可变区VH包括如SEQ ID NO:249所示的氨基酸序列；或，
所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:257所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:250所示的氨基酸序列。所述蛋白功能区C包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列。
在一些更优选的实施例中，所述结合蛋白包含两种多肽链。其中，第一多肽链包括如SEQ ID NO:371所示的氨基酸序列；第二多肽链包括如SEQ ID NO:372所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:371所示的氨基酸序列；第二多肽链包括如SEQ ID NO:373所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:362所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:363所示的氨基酸序列；第二多肽链包括如SEQ ID NO:364所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:365所示的氨基酸序列；第二多肽链包括如SEQ ID NO:364所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:366所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:369所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:370所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:394所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:395所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:363所示的氨基酸序列；第二多肽链包括如SEQ ID NO:310所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:363所示的氨基酸序列；第二多肽链包括如SEQ ID NO:396所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:363所示的氨基酸序列；第二多肽链包括如SEQ ID NO:362所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:363所示的氨基酸序列；第二多肽链包括如SEQ ID NO:394所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:363所示的氨基酸序列；第二多肽链包括如SEQ ID NO:395所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:368所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:378所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:379所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:380所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:381所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:382所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:383所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:385所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:388所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:389所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:374所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:375所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:386所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:387所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:401所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:402所示的氨基酸序列；第二多肽链包括如SEQ ID NO:305所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:403所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:402所示的氨基酸序列；第二多肽链包括如SEQ ID NO:409所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:359所示的氨基酸序列；第二多肽链包括如SEQ ID NO:404所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:405所示的氨基酸序列；第二多肽链包括如SEQ ID NO:315所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:359所示的氨基酸序列；第二多肽链包括如SEQ ID NO:406所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:376所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:377所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:397所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:398所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:399所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:400所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:402所示的氨基酸序列；第二多肽链包括如SEQ ID NO:401所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:402所示的氨基酸序列；第二多肽链包括如SEQ ID NO:403所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:405所示的氨基酸序列；第二多肽链包括如SEQ ID NO:404所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:405所示的氨基酸序列；第二多肽链包括如SEQ ID NO:406所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:371所示的氨基酸序列；第二多肽链包括如SEQ ID NO:486所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:460所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:461所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:462所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:487所示的氨基酸序列；第二多肽链包括如SEQ ID NO:488所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:360所示的氨基酸序列；第二多肽链包括如SEQ ID NO:455所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:360所示的氨基酸序列；第二多肽链包括如SEQ ID NO:456所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:360所示的氨基酸序列；第二多肽链包括如SEQ ID NO:457所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:360所示的氨基酸序列；第二多肽链包括如SEQ ID NO:458所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:360所示的氨基酸序列；第二多肽链包括如SEQ ID NO:459所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:419所示的氨基酸序列；第二多肽链包括如SEQ ID NO:412所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:419所示的氨基酸序列；第二多肽链包括如SEQ ID NO:414所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:440所示的氨基酸序列；第二多肽链包括如SEQ ID NO:428所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:441所示的氨基酸序列；第二多肽链包括如SEQ ID NO:428所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:442所示的氨基酸序列；第二多肽链包括如SEQ ID NO:428所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:443所示的氨基酸序列；第二多肽链包括如SEQ ID NO:428所示的氨基酸序列；或,，
第一多肽链包括如SEQ ID NO:487所示的氨基酸序列；第二多肽链包括如SEQ ID NO:520所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:521所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:371所示的氨基酸序列；第二多肽链包括如SEQ ID NO:522所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:360所示的氨基酸序列；第二多肽链包括如SEQ ID NO:523所示的氨基酸序列。
在一些更优选的实施例中，所述结合蛋白包含三个多肽链。其中，第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:407所示的氨基酸序列；第三多肽链包括如SEQ ID NO:390所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:408所示的氨基酸序列；第三多肽链包括如SEQ ID NO:392所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:391所示的氨基酸序列；第三多肽链包括如SEQ ID NO:390所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:393所示的氨基酸序列；第三多肽链包括如SEQ ID NO:392所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:391所示的氨基酸序列；第三多肽链包括如SEQ ID NO:434所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:391所示的氨基酸序列；第三多肽链包括如SEQ ID NO:435所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:393所示的氨基酸序列；第三多肽链包括如SEQ ID NO:436所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:393所示的氨基酸序列；第三多肽链包括如SEQ ID NO:437所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:438所示的氨基酸序列；第三多肽链包括如SEQ ID NO:434所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:438所示的氨基酸序列；第三多肽链包括如SEQ ID NO:435所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:439所示的氨基酸序列；第三多肽链包括如SEQ ID NO:436所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:439所示的氨基酸序列；第三多肽链包括如SEQ ID NO:437所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:361所示的氨基酸序列；第二多肽链包括如SEQ ID NO:481所示的氨基酸序列；第三多肽链包括如SEQ ID NO:482所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:361所示的氨基酸序列；第二多肽链包括如SEQ ID NO:481所示的氨基酸序列；第三多肽链包括如SEQ ID NO:483所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:361所示的氨基酸序列；第二多肽链包括如SEQ ID NO:481所示的氨基酸序列；第三多肽链包括如SEQ ID NO:484所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:361所示的氨基酸序列；第二多肽链包括如SEQ ID NO:481所示的氨基酸序列；第三多肽链包括如SEQ ID NO:485所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:411所示的氨基酸序列；第三多肽链包括如SEQ ID NO:412所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:411所示的氨基酸序列；第三多肽链包括如SEQ ID NO:414所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:415所示的氨基酸序列；第二多肽链包括如SEQ ID NO:416所示的氨基酸序列；第三多肽链包括如SEQ ID NO:410所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:415所示的氨基酸序列；第二多肽链包括如SEQ ID NO:416所示的氨基酸序列；第三多肽链包括如SEQ ID NO:412所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:415所示的氨基酸序列；第二多肽链包括如SEQ ID NO:416所示的氨基酸序列；第三多肽链包括如SEQ ID NO:414所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:417所示的氨基酸序列；第二多肽链包括如SEQ ID NO:418所示的氨基酸序列；第三多肽链包括如SEQ ID NO:412所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:417所示的氨基酸序列；第二多肽链包括如SEQ ID NO:418所示的氨基酸序列；第三多肽链包括如SEQ ID NO:414所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:426所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:427所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:428所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:429所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:444所示的氨基酸序列；第三多肽链包括如SEQ ID NO:449所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:450所示的氨基酸序列；第三多肽链包括如SEQ ID NO:449所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:444所示的氨基酸序列；第三多肽链包括如SEQ ID NO:445所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:444所示的氨基酸序列；第三多肽链包括如SEQ ID NO:446所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:450所示的氨基酸序列；第三多肽链包括如SEQ ID NO:445所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:450所示的氨基酸序列；第三多肽链包括如SEQ ID NO:446所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:444所示的氨基酸序列；第三多肽链包括如SEQ ID NO:447所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:444所示的氨基酸序列；第三多肽链包括如SEQ ID NO:448所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:450所示的氨基酸序列；第三多肽链包括如SEQ ID NO:447所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:450所示的氨基酸序列；第三多肽链包括如SEQ ID NO:448所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:445所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:446所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:463所示的氨基酸序列；第三多肽链包括如SEQ ID NO:464所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:465所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:466所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:467所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:468所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:469所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:470所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:471所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:472所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:473所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:474所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:475所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:476所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:477所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:478所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:479所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:480所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:430所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:431所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:432所示的氨基酸序列；或，
第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:433所示的氨基酸序列。
为解决上述技术问题，本发明的技术方案之二为：提供一种分离的核酸，其编码如上所述结合蛋白。
为解决上述技术问题，本发明的技术方案之三为：提供一种表达载体，其包含如上所述的分离的核酸。
为解决上述技术问题，本发明的技术方案之四为：提供一种宿主细胞，其包含根据以上所述的表达载体，其中所述宿主细胞是原核细胞或真核细胞。
为解决上述技术问题，本发明的技术方案之五为：提供一种如上所述结合蛋白的制备方法，其特征在于，所述制备方法包括以下步骤：培养如上所述的宿主细胞，从培养物中获得所述结合蛋白。
为解决上述技术问题，本发明的技术方案之六为：提供一种药物组合物，所述药物组合物包含如上所述结合蛋白。
为解决上述技术问题，本发明的技术方案之七为：提供一种套装药盒，所述套装药盒包括药盒一和药盒二，所述药盒一包括如上所述结合蛋白或药物组合物，所述药盒二包括治疗癌症的其它抗体或药物组合物。
为解决上述技术问题，本发明的技术方案之八为：提供一种如上所述结合蛋白或上所述的药物组合物在制备治疗和/或预防癌症的药物中的应用。所述的癌症优选乳腺癌、卵巢癌、子宫内膜癌、肾癌、黑色素瘤、肺癌、胃癌、肝癌、食管癌、宫颈癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、肉瘤、结直肠癌、淋巴瘤或者多发性骨髓瘤。
为解决上述技术问题，本发明的技术方案之九为：提供一种治疗癌症的方法，其特征在于，向有需要的受试者施用如本发明技术方案之一所述的结合蛋白，或如技术方案之六所述的药物组合物，或如技术方案之七所述的套装药盒；优选地，还包括施用治疗癌症的其它抗体例如免疫检查点抗体和/或化疗药物。
所述的癌症优选乳腺癌、卵巢癌、子宫内膜癌、肾癌、黑色素瘤、肺癌、胃癌、肝癌、食管癌、宫颈癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、肉瘤、结直肠癌、淋巴瘤或者多发性骨髓瘤。
为解决上述技术问题，本发明的技术方案之十为：提供一种CD3抗体。
所述CD3抗体包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和221所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、84和141所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:178、197和222所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、85和142所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和223所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、86和141所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:179、198和224所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、85和142所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。优选地，其轻链可变区VL包括如SEQ ID NO:294所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:258所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:295所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:259所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:296所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:260所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:297所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:259所示的氨基酸序列。
或，所述CD3抗体包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:20、68和128 所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。优选地，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:245所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:257所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:281所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:244所示的氨基酸序列。
在一些具体的实施例中，所述CD3抗体包含两种多肽链；其中，第一多肽链包括如SEQ ID NO:357所示的氨基酸序列，第二多肽链包括如SEQ ID NO:313所示的氨基酸序列；或，第一多肽链包括如SEQ ID NO:357所示的氨基酸序列，第二多肽链包括如SEQ ID NO:325所示的氨基酸序列；或，第一多肽链包括如SEQ ID NO:357所示的氨基酸序列，第二多肽链包括如SEQ ID NO:328所示的氨基酸序列；或，第一多肽链包括如SEQ ID NO:357所示的氨基酸序列，第二多肽链包括如SEQ ID NO:346所示的氨基酸序列。
在一些具体的实施例中，所述CD3抗体包含一个多肽链，所述多肽链包括如SEQ ID NO:489所示的氨基酸序列，如SEQ ID NO:490所示的氨基酸序列，如SEQ ID NO:491所示的氨基酸序列，如SEQ ID NO:492所示的氨基酸序列，或，如SEQ ID NO:493所示的氨基酸序列。
为解决上述技术问题，本发明的技术方案之十一为：提供一种BCMA抗体。
所述BCMA抗体包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、75和133所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:24、76和134所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:25、77和135所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和 HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。优选地，其重链可变区VH包括如SEQ ID NO:248所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:249所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:250所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列。更优选地，所述BCMA抗体包含一个多肽链，所述多肽链包含如SEQ ID NO:316所示的氨基酸序列，如SEQ ID NO:317所示的氨基酸序列，如SEQ ID NO:318所示的氨基酸序列，或，如SEQ ID NO:319所示的氨基酸序列。
或，所述BCMA抗体包含重链可变区，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、90和136所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、78和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、78和147所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和147所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:36、90和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和147所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、78和148所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和136所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、76和136所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、76和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和148所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和136所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。
在一些具体的实施例中，其重链可变区VH包括如SEQ ID NO:265所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:266所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:267所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:268所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:269所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:270所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:271所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:272所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:273所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:274所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:275所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:276所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:277所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:278所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:279所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:280所示的氨基酸序列。
在一些具体的实施例中，所述BCMA抗体包含一个多肽链，所述多肽链包含如SEQ ID NO:330所示的氨基酸序列；或，如SEQ ID NO:331所示的氨基酸序列；或，如SEQ ID NO:332所示的氨基酸序列；或，如SEQ ID NO:333所示的氨基酸序列；或，如SEQ ID NO:334所示的氨基酸序列；或，如SEQ ID NO:335所示的氨基酸序列；或，如SEQ ID NO:336所示的氨基酸序列；或，如SEQ ID NO:337所示的氨基酸序列；或，如SEQ ID NO:338所示的氨基酸序列；或，如SEQ ID NO:339所示的氨基酸序列；或，如SEQ ID NO:340所示的氨基酸序列；或，如SEQ ID NO:341所示的氨基酸序列；或，如SEQ ID NO:342所示的氨基酸序列；或，如SEQ ID NO:343所示的氨基酸序列；或，如SEQ ID NO:344所示的氨基酸序列；或，如SEQ ID NO:345所示的氨基酸序列。
本发明的积极进步效果：
本发明提供了利用全人源重链抗体及其衍生的单域抗体所构建的二价至多价和双特异性或多特异性结合蛋白以及制备和使用此类结合蛋白的方法。其相较于利用常规IgG抗体构建的二价至多价和双特异性或多特异性结合蛋白有诸多优势，在调整特异性和结合价数方面更为灵活；可以构建分子量较小的、多肽链较少的、结构更简单的多特异性结合蛋白；而且可以利用不同的结构来实现不同的生物学功能。
在一些具体实施例中，通过不同的结构类型、相对位置、结合价数等参数来调节针对不同靶点的功能活性，进而设计出不同的活性组合，以满足不同的临床联合用药剂量组合的需求。在一些具体实施例中，利用重链抗体VH域可以较方便地串联形成多价结构，可以通过与靶点的多价结合促使靶点的交联，进一步增强靶点交联所诱导的生物学功能。在一些具体实施例中，利用重链抗体VH域可以实现诸如“1+3”非对称四价结构，类似结构难以用常规IgG抗体衍生的结构来实现。
附图说明
图1.分子结构示意图。
图2.PD-L1 x CTLA4双抗分子结合hCTLA4-His蛋白的结合活性。
图3.PD-L1 x CTLA4双抗分子结合CHO-K1/hCTLA4细胞的结合活性。
图4.PD-L1 x CTLA4双抗分子结合HEK293/hCTLA4细胞的结合活性。
图5.PD-L1 x CTLA4双抗分子阻断人CTLA4蛋白和其配体蛋白B7-1的结合的活性。
图6.PD-L1 x CTLA4双抗分子阻断CHO-K1/hCTLA4细胞和其配体蛋白B7-1的结合的活性。
图7.PD-L1 x CTLA4双抗分子结合hPDL1-His蛋白的结合活性。
图8.PD-L1 x CTLA4双抗分子结合CHO-K1/hPDL1细胞的结合活性。
图9.PD-L1 x CTLA4双抗分子结合高表达人PD-L1的MDA-MB-231细胞的结合活性。
图10.PD-L1 x CTLA4双抗分子阻断CHO-K1/hPDL1细胞和其配体蛋白PD-1的结合的活性。
图11.PD-L1 x CTLA4双抗分子通过ADCC效应杀伤CTLA4 +靶细胞的能力。
图12.IgG-VH结构的PD-L1 x CTLA4双抗在SEB刺激实验中激活T细胞的能力。
图13.Fab-HCAb结构的PD-L1 x CTLA4双抗在SEB刺激实验中激活T细胞的能力。
图14.在SEB刺激实验中，当VH在IgG HC或LC的N端时，PD-L1 x CTLA4双抗比对应单抗的1:1剂量组合有相当甚至更强的T细胞激活能力。
图15.在SEB刺激实验中，CTLA4端的活性在IgG_HC-VH(VH在IgG HC的C端)和Fab-HCAb结构中相较于其在IgG或HCAb双价结构中会减弱，从而可以降低其剂量相关的毒性。
图16.在混合淋巴细胞反应实验中，当VH在IgG HC或LC的N端时，PD-L1 x CTLA4双抗比对应单抗的1:1剂量组合有更强的T细胞激活能力。
图17.在混合淋巴细胞反应实验中，当VH在IgG HC或LC的C端时，PD-L1 x CTLA4双抗比PD-L1单抗有更强的T细胞激活能力。
图18.HER2 x CTLA4双抗分子结合CHO-K1/hCTLA4细胞的结合活性。
图19.HER2 x CTLA4双抗分子阻断人CTLA4蛋白和其配体蛋白B7-1的结合的活性。
图20.HER2 x CTLA4双抗分子阻断CHO-K1/hCTLA4细胞和其配体蛋白B7-1的结合的活性。
图21.HER2 x CTLA4双抗分子结合SK-BR-3细胞的结合活性。
图22.HER2 x CTLA4双抗分子通过ADCC效应杀伤HER2 +靶细胞的能力。
图23.HER2 x CTLA4双抗分子抑制HER2 +靶细胞SK-BR-3增殖的能力。
图24.HER2 x CTLA4双抗分子同时结合CTLA4 +细胞和HER2 +细胞的能力。
图25.HER2 x CTLA4双抗分子在SEB刺激实验中激活T细胞的能力。
图26.HER2 x CTLA4双抗分子在小鼠体内的药代动力学。
图27.PD-L1 x 4-1BB双抗分子结合CHO-K1/hPDL1细胞的结合活性。
图28.4-1BB抗体或PD-L1 x 4-1BB双抗分子结合CHO-K1/hu 4-1BB细胞的结合活性。
图29.PD-L1 x 4-1BB双抗分子结合CHO-K1/cyno 4-1BB细胞的结合活性。
图30.PD-L1 x 4-1BB双抗分子通过CHO-K1/hPDL1细胞介导T细胞特异性激活。
图31.PD-L1 x 4-1BB双抗分子通过MDA-MB-231细胞介导T细胞特异性激活。
图32.在混合淋巴细胞反应实验中，IgG-VH结构的PD-L1 x 4-1BB双抗分子比FIT-Ig结构的双抗分子有更强的T细胞激活能力。
图33.在混合淋巴细胞反应实验中，IgG-VH结构和Fab-HCAb结构的PD-L1 x 4-1BB双抗分子比FIT-Ig结构的双抗分子有更强的T细胞激活能力。
图34.PD-L1 x 4-1BB双抗分子在小鼠体内的药代动力学.
图35.B7H4 x 4-1BB双抗分子结合SK-BR-3细胞的结合活性。
图36.B7H4 x 4-1BB双抗分子结合CHO-K1/hu 4-1BB细胞的结合活性。
图37.B7H4 x 4-1BB双抗分子通过SK-BR-3介导T细胞特异性激活(INFγ释放)。
图38.B7H4 x 4-1BB双抗分子通过SK-BR-3介导T细胞特异性激活(IL-2释放)。
图39.B7H4 x 4-1BB双抗分子对T细胞激活特异性依赖B7H4的表达。
图40.B7H4 x 4-1BB双抗分子PR003334的血清稳定性。
图41.B7H4 x 4-1BB双抗分子PR003335的血清稳定性。
图42.B7H4 x 4-1BB双抗分子在小鼠体内的药代动力学。
图43.B7H4 x 4-1BB双抗分子在小鼠肿瘤模型中的抗肿瘤效果。
图44.BCMA HCAb抗体结合HEK293T/hBCMA细胞的结合活性。
图45.BCMA HCAb抗体结合HEK293T/cynoBCMA细胞的结合活性。
图46.BCMA HCAb抗体结合NCI-H929细胞的结合活性。
图47.BCMA HCAb抗体对HEK293T/hBCMA细胞的内化能力。
图48.BCMA HCAb抗体结合人BCMA的亲和力(BLI方法)。
图49.BCMA HCAb抗体PR001046衍生的变体分子结合NCI-H929细胞的结合活性。
图50.BCMA x CD3双抗分子结合HEK293T/hBCMA细胞的结合活性。
图51.BCMA x CD3双抗分子结合HEK293T/cynoBCMA细胞的结合活性。
图52.BCMA x CD3双抗分子结合NCI-H929细胞的结合活性。
图53.BCMA x CD3双抗分子结合人T细胞的结合活性。
图54.BCMA x CD3双抗分子结合食蟹猴T细胞的结合活性。
图55.scFv-Fc-VH(2)非对称结构的BCMA x CD3双抗分子杀伤NCI-H929细胞的能力。
图56.scFv-Fc-VH(1)非对称结构的BCMA x CD3双抗分子杀伤NCI-H929细胞的能力。
图57.Fab-Fc-VH(1)非对称结构的BCMA x CD3双抗分子杀伤NCI-H929细胞的能力。
图58.Fab-Fc-VH(2)非对称结构的BCMA x CD3双抗分子杀伤NCI-H929细胞的能力。
图59.BCMA x CD3双抗分子(PR001990,PR002309)介导效应细胞对NCI-H929细胞的特异性杀伤和细胞因子释放检测。
图60.BCMA x CD3双抗分子(PR002895,PR002953,PR003178)介导效应细胞对NCI-H929细胞的特异性杀伤和细胞因子释放检测。
图61.可溶性APRIL或BAFF对BCMA x CD3双抗分子的靶细胞杀伤效果的影响。
图62.可溶性BCMA对BCMA x CD3双抗分子的靶细胞杀伤效果的影响。
图63.BCMA x CD3双抗分子在小鼠或大鼠体内的药代动力学。
图64.BCMA x CD3双抗分子在小鼠肿瘤模型中的抗肿瘤效果。
图65.双抗分子经瞬时转染表达和一步亲和纯化后得到的蛋白样品的SDS-PAGE和SEC-HPLC的分析结果。
图66.CD3抗体结合人T细胞的结合活性。
图67.PD-L1 x CTLA4双抗分子在小鼠肿瘤模型中的抗肿瘤效果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“抗体”或者“H2L2”通常是指常规的四链抗体。大多数物种中的抗体呈现“Y”型的四聚体结构，其包含两个完全相同的重链(H链)和两个完全相同的轻链(L链)，也称为“H2L2”。重链包括靠近N端的重链可变区(VH)和靠近C端的重链恒定区(CH)；轻链包括靠近N端的轻链可变区(VL)和靠近C端的轻链恒定区(CL)。IgG抗体的重链恒定区有3个结构域，分别为CH1、CH2和CH3；CH1和CH2之间还有一段铰链区(hinge)。轻链根据κ和λ的不同，其轻链可变区进一步分为Vκ和Vλ，与之对应的轻链恒定区分别为Cκ和Cλ。抗体的可变区是其识别和结合抗原的主要部位；抗体的可变区结构域VH和VL以及恒定区结构域CH1和CL共同组成了抗原结合片段(Fab)。CH2和CH3组成了可结晶片段(Fc)，是发挥抗体的效应功能如补体依赖的细胞毒作用(CDC)和抗体依赖的细胞介导的细胞毒作用(ADCC)以及影响抗体的血清半衰期的主要区域。
在本申请中，术语“重链抗体”或者“HCAb”通常是指一类只含有重链二聚体的抗体。在骆驼科及鲨鱼科动物血清中天然存在一种缺失轻链的重链抗体(heavy-chain antibody,HCAb)。来源于骆驼科的重链抗体与常规H2L2抗体相比，除了缺少轻链外，其重链可变区与铰链区之间没有CH1区，只含有一个重链可变区(VHH)和两个重链恒定结构域(CH2和CH3)；其基本结构为重链二聚体。骆驼科动物的重链抗体的VHH片段与常规抗体的VH特征不同，其单独克隆并表达出来的VHH结构具有与原重链抗体相当的结构稳定性以及与抗原的结合活性，分子量只有约13KDa，因此也被称作纳米抗体或单域抗体。
在本申请中，术语“结合蛋白”或者“抗原结合蛋白”通常是指包含结合抗原的部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述 两者。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为框架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR-1、CDR1、FR-2、CDR2、FR-3、CDR3和FR-4。重链和轻链的可变区含有与抗原相互作用的结合结构域。VH的三个CDR分别表示为HCDR1、HCDR2和HCDR3，也可表示为VH CDR1、VH CDR2和VH CDR3；VL的三个CDR分别表示为LCDR1、LCDR2和LCDR3，也可表示为VL CDR1、VL CDR2和VL CDR3。抗原结合蛋白的实例包括但不限于抗体、抗原结合片段(Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，所述CDR的氨基酸序列均是按照Chothia定义规则所示出的。但是，本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))和基于结构环区域位置的Chothia定义规则(参见JMol Biol 273:927-48,1997)。在本发明的技术方案中，还可以使用包含了Kabat定义和Chothia定义的Combined定义规则来确定可变结构域序列中的氨基酸残基。其中Combined定义规则即是将Kabat定义和Chothia定义的范围相结合，基于此取了一个更大的范围，详见下表。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应了解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。虽然本发明中请求保护的范围是基于Chothia定义规则所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当落在本发明的保护范围中。
表-I本申请抗体CDR定义方法
  Kabat Chothia Combined
LCDR1 L24--L34 L24--L34 L24-L34
LCDR2 L50--L56 L50--L56 L50-L56
LCDR3 L89--L97 L89--L97 L89-L97
HCDR1 H31--H35 H26--H32 H26-H35
HCDR2 H50--H65 H52--H56 H50-H65
HCDR3 H95--H102 H95--H102 H95-H102
其中，Laa-Lbb可以指从抗体轻链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列；Haa-Hbb可以指从抗体重链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列。例如，L24-L34可以指从抗体轻链N端开始，按照Chothia编码规则的从第24位至第34位的氨基酸序列；H26-H32可以指从抗体重链N端开始，按照Chothia编码规则的从第26位至第32位的氨基酸序列。本领域技术人员应当知晓的是，在用Chothia编码CDR时，有些位置会有插入位点的情况(可参见http://bioinf.org.uk/abs/)。
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体，即集群中的个别抗体是相同的，除了可能存在的少量的自然突变。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，根据本发明使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“全人源抗体”通常是指将人类编码抗体的基因全部转移至基因工程改造的抗体基因缺失动物中，使动物表达的抗体。抗体所有部分(包括抗体的可变区和恒定区)均由人类来源的基因所编码。全人源抗体可以大大减少异源抗体对人体造成的免疫副反应。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术等。
在本申请中，术语“全人源重链抗体”通常是指利用Harbour HCAb转基因小鼠(专利申请WO2007/096779)得到的具有人源抗体可变区VH的重链抗体。该转基因小鼠的内源的抗体重链基因座和轻链基因座都被敲除或者失活，使其无法产生小鼠的抗体；然后将人源的抗体重链基因片段(V，D，J片段)转入该小鼠，利用该小鼠自身的重排和突变机制产生具有人源抗体基因序列的抗体，其可变区为人源VH。将该人源VH与人源的重链恒定区Fc进行融合重组即得到全人源重链抗体。
在本申请中，术语“特异性结合”通常是指抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。根据该定义，当抗体相比于其将结合随机的，不相关的表位而言更容易通过其抗原结合域与表位结合时，抗体被称为“特异 性结合”该抗原。“表位”是指抗原上与抗原结合蛋白(如抗体)结合的特定的原子基团(例如，糖侧链、磷酰基、磺酰基)或氨基酸。
在本申请中，术语“Fab”通常指常规抗体(例如IgG)中与抗原结合的部分，包括抗体的重链可变区VH、轻链可变区VL和重链恒定区结构域CH1以及轻链恒定区CL。在常规抗体中，VH的C端与CH1的N端联结形成重链Fd片段，VL的C端与CL的N端联结形成轻链，CH1的C端又进一步与重链的铰链区和其他恒定区结构域联结形成重链。在一些实施例中，“Fab”也指Fab的变体结构。例如，在某些实施例中，VH的C端与CL的N端联结形成一个多肽链，VL的C端与CH1的N端联结形成另一个多肽链，形成Fab(cross VH/VL)的结构；在某些实施例中，Fab的CH1不与铰链区联结，而是CL的C端与重链的铰链区联结，形成Fab(cross Fd/LC)的结构。
在本申请中，术语“VH”通常指抗体的重链可变区VH结构域，即可以是人或者其他动物的常规抗体(H2L2结构)的重链可变区VH，也可以是骆驼科等动物的重链抗体(HCAb结构)的重链可变区VHH，还可以是利用Harbour HCAb转基因小鼠产生的全人源重链抗体(HCAb结构)的重链可变区VH。
在本申请中，术语“结合结构域”(binding moieties)通常指任何可以与抗原特异结合的蛋白功能区域，既可以是“Fab”，也可以是“VH”，还可以是其他抗原结合形式(例如脂质运载蛋白(lipocalins)、神经细胞粘附分子(NCAM)、纤维结合蛋白(fibronectin)、锚蛋白重复片段蛋白(DARPins)等衍生蛋白结构)。
在本申请中，术语“结合价数”通常指结合蛋白中的“结合结构域”的数目，也是指该结合蛋白能够结合抗原分子或者表位的最大数目，例如常规IgG抗体能同时结合两个相同的抗原分子，其结合价数为二；而Fab抗体的结合价数为一。
在本申请中，术语“双特异性结合蛋白”通常指具有两种抗原结合特异性的结合蛋白。两种抗原结合特异性可以是结合两种不同的抗原或是结合同种抗原的两个不同表位。双特异性结合蛋白可典型地包括双特异性抗体及其衍生物等。
在本申请中，术语“多特异性结合蛋白”通常指具有两种或以上抗原结合特异性的结合蛋白。多特异性结合蛋白可典型地包括多特异性抗体及其衍生物等。
在本申请中，术语“MFI”(Mean Fluorescent Intensity)通常指流式细胞术FACS中分析的荧光强度信号及其数学平均值，通常可以用专业软件如FlowJo(FlowJo,LLC)等对流式细胞仪产生的数据进行处理和分析得到。
在本申请中，术语“PD-L1”通常是指程序性死亡配体1蛋白、其功能变体和/或其功能片段。PD-L1也称为分化簇274(CD274)或B7同源物1(B7-H1)，并且是由(人类中)CD274基因编码的蛋白。PD-L1序列是本领域已知的。例如，示例性的全长人PD-L1蛋白的氨基酸序列可在NCBI登录号NP_054862或UniProt登录号Q9NZQ7下找到；示例性的全长食蟹猴PD-L1蛋白序列可在NCBI登录号XP_005581836或Uniprot登录号G7PSE7下找到。
在本申请中，术语“PD-1”通常是指程序性死亡1受体(也称为CD279)、其功能变体和/或其功能片段。PD-1序列是本领域已知的。例如，示例性的全长人PD-1蛋白序列可在NCBI登录号NP_005009下找到；示例性的全长食蟹猴的PD-1蛋白序列可在NCBI登录号NP_001271065或Uniprot登录号B0LAJ3下找到。
在本申请中，术语“CD80”通常是指分化簇蛋白80(也称为B7-1)、其功能变体和/或其功能片段。CD80序列是本领域已知的。例如，示例性的全长的人CD80序列可以在Uniprot登录号P33681中找到。
在本申请中，术语“CTLA4”通常是指细胞毒性T淋巴细胞相关蛋白-4(也称为CD152)、其功能变体和/或其功能片段。CTLA4序列是本领域已知的。例如，示例性的全长的人CTLA4序列可以在Uniprot登录号P16410中找到；示例性的全长的食蟹猴CTLA4序列可以在Uniprot登录号G7PL88中找到。
在本申请中，术语“HER2”通常是指受体酪氨酸激酶erbB-2(也称为ERBB2)、其功能变体和/或其功能片段。HER2序列是本领域已知的。例如，示例性的全长的人HER2序列可以在Uniprot登录号P04626中找到；示例性的全长的食蟹猴HER2序列可以在NCBI登录号XP_005584091中找到。
在本申请中，术语“B7H4”通常是指含V-Set域T细胞激活抑制因子1(也称为VTCN1)、其功能变体和/或其功能片段。B7H4序列是本领域已知的。例如，示例性的全长的人B7H4序列可以在Uniprot登录号Q7Z7D3中找到；示例性的全长的食蟹猴 B7H4序列可以在NCBI登录号XP_005542249中找到；示例性的全长的小鼠B7H4序列可以在Uniprot登录号Q7TSP5中找到。
在本申请中，术语“4-1BB”通常是指肿瘤坏死因子受体超家族成员9(也称为CD137，4-1BBL受体)、其功能变体和/或其功能片段。4-1BB序列是本领域已知的。例如，示例性的全长的人4-1BB序列可以在Uniprot登录号Q07011中找到；示例性的全长的食蟹猴4-1BB序列可以在NCBI登录号XP_005544945中找到。
在本申请中，术语“BCMA”通常是指肿瘤坏死因子受体超家族成员17(也称为CD269，B细胞成熟蛋白)、其功能变体和/或其功能片段。BCMA序列是本领域已知的。例如，示例性的全长的人BCMA序列可以在Uniprot登录号Q02223中找到；示例性的全长的食蟹猴BCMA序列可以在NCBI登录号XP_005591343中找到。
在本申请中，术语“BAFF”通常是指肿瘤坏死因子配体超家族成员13B(也称为CD257，B细胞激活因子)、其功能变体和/或其功能片段。BAFF序列是本领域已知的。例如，示例性的全长的人BAFF序列可以在Uniprot登录号Q9Y275中找到。
在本申请中，术语“APRIL”通常是指肿瘤坏死因子配体超家族成员13(也称为CD256，增殖诱导配体)、其功能变体和/或其功能片段。APRIL序列是本领域已知的。例如，示例性的全长的人APRIL序列可以在Uniprot登录号O75888中找到。
在本申请中，术语“CD3”通常是指T细胞上的TCR/CD3受体蛋白复合物。T细胞应答的特异性通过由TCR和CD3的分子复合物对pMHC的识别来介导。TCR是由两条不同跨膜多肽链构成的异二聚体，肽链有α、β、γ、δ四种；根据肽链的不同组合，TCR分为TCRαβ和TCRγδ。CD3有不同的跨膜多肽链即γ、δ、ε、ζ，这些肽链相互作用形成同源二聚体或异源二聚体，作为TCR-CD3复合物的一部分。由于TCR肽链的胞质区很短，一般认为TCR识别抗原所产生的活化信号由CD3肽链转导至T细胞内。
在本申请中，术语“CD3E”通常是指“CD3”的ε肽链。CD3E序列是本领域已知的。例如，示例性的全长的人CD3E序列可以在Uniprot登录号P07766中找到，示例性的全长的食蟹猴CD3E序列可以在Uniprot登录号Q95LI5中找到。
实施例
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法.这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1.基于HCAb的多特异性结合蛋白的结构设计
本实施例列举了若干种利用全人源重链抗体(HCAb)及其衍生的单域抗体(sdAb)所构建的含有Fc的、对称或者非对称的、多价和多特异性结合蛋白的结构。在有些结构中，结构域和结构域之间用连接肽进行联结。在有些结构中，重链的Fc区域引入了氨基酸突变以改变其与Fc受体的结合进而改变相关的效应功能或者其他性能。在有些结构中，两个重链的Fc区域各引入了不同的氨基酸突变以减少重链同源二聚化的形成。
表1-1和图1列出了本发明申请所包含的多特异性结合蛋白分子结构，每一个结构会在下文进一步描述。在本实施例以及本发明申请的其他部分，当提及分子结构所含多肽链数目的时候，通常是指“不同的多肽链”的数目；例如常规IgG抗体有两条不同的多肽链，即重链和轻链，尽管IgG抗体分子本身是含有两条相同的重链和两条相同的轻链的四肽链蛋白分子，但是描述其结构特征时特指其两条不同的多肽链。结合价数是指该分子结构中的抗原结合位点的数目，例如常规IgG抗体能同时结合两个相同的抗原分子，其结合价数为二。表1-2列出了本发明申请的结构设计中可能用到的连接肽序列。
Figure PCTCN2021103044-appb-000001
Figure PCTCN2021103044-appb-000002
表1-1本发明申请所列举的基于HCAb的多特异性结合蛋白分子结构
Figure PCTCN2021103044-appb-000003
Figure PCTCN2021103044-appb-000004
表1-2连接肽序列
实施例1.1.Fab-HCAb对称结构
本发明提供了一种使用两种亲本单克隆抗体构建双特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B。
如图1的(1)–(2)所示，Fab端来源于常规抗体A，VH_A和VL_A分别为抗体A的重链可变区和轻链可变区。VH端来源于重链抗体B，VH_B为重链抗体B的重链可变区。CL是轻链恒定区结构域。CH1、CH2和CH3分别是重链恒定区的第一、第二和第三结构域。L1和L2分别是第一和第二连接肽。
实施例1.1.1.结构(1):Fab(CL)-VH-Fc
结构(1)的结合蛋白包含两条不同的多肽链：多肽链1或第一多肽链，也称短链，从氨基末端到羧基末端，其包含VH_A-CH1；多肽链2或第二多肽链，也称长链，从氨基末端到羧基末端，其包含VL_A-CL-L1-VH_B-L2-CH2-CH3。在结构(1)中，抗体A的VL_A和重链抗体B的VH_B融合在同一条多肽链上，这样可以避免VL_A和VH_B的缔合产生的错配副产物。
多肽链2的VH_B经由连接肽L2联结到CH2；L2可以是IgG的铰链区或者铰链区衍生的连接肽序列或是表1-2中所列序列，优选为人IgG1铰链或者人IgG1铰链(C220S)或者G5-LH的序列。
在一个实施方案中，多肽链2的CL与VH_B直接融合联结，即L1的长度为0。在另一个实施方案中，多肽链2的CL经由连接肽L1联结到VH_B；L1可以是表1-2中所列序列。
实施例1.1.2.结构(2):Fab(CH1)-VH-Fc
结构(2)的结合蛋白包含两条不同的多肽链：多肽链1或第一多肽链，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2或第二多肽链，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-L1-VH_B-L2-CH2-CH3。
多肽链2的VH_B经由连接肽L2联结到CH2；L2可以是IgG的铰链区或者铰链区衍生的连接肽序列或是表1-2中所列序列，优选为人IgG1铰链或者人IgG1铰链(C220S)或者G5-LH的序列。
在一个实施方案中，多肽链2的CH1与VH_B直接融合联结，即L1的长度为0。在另一个实施方案中，多肽链2的CH1经由连接肽L1联结到VH_B；L1可以是表1-2中所列序列。
实施例1.2.IgG-VH四价对称结构
本发明提供了一种使用两种亲本单克隆抗体构建双特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B。
如图1的(3)–(6)所示，Fab端来源于常规抗体A，VH_A和VL_A分别为抗体A的重链可变区和轻链可变区。VH端来源于重链抗体B，VH_B为重链抗体B的重 链可变区。CL是轻链恒定区结构域。CH1、CH2和CH3分别是重链恒定区的第一、第二和第三结构域。L是连接肽，h是IgG抗体的铰链区或衍生序列。
实施例1.2.1.结构(3):VH-IgG_HC
结构(3)的结合蛋白包含两条不同的多肽链：多肽链1或第一多肽链，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2或第二多肽链，也称长链，从氨基末端到羧基末端，其包含VH_B-L-VH_A-CH1-h-CH2-CH3。
在一个实施方案中，多肽链2的VH_B与VH_A直接融合联结，即L的长度为0。在另一个实施方案中，多肽链2的VH_B经由连接肽L联结到VH_A；L可以是表1-2中所列序列。
实施例1.2.2.结构(4):VH-IgG_LC
结构(4)的结合蛋白包含两条不同的多肽链：多肽链1或第一多肽链，也称短链，从氨基末端到羧基末端，其包含VH_B-L-VL_A-CL；多肽链2或第二多肽链，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-h-CH2-CH3。
在一个实施方案中，多肽链1的VH_B与VL_A直接融合联结，即L的长度为0。在另一个实施方案中，多肽链1的VH_B经由连接肽L联结到VL_A；L可以是表1-2中所列序列。
实施例1.2.3.结构(5):IgG_HC-VH
结构(5)的结合蛋白包含两条不同的多肽链：多肽链1或第一多肽链，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2或第二多肽链，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-h-CH2-CH3-L-VH_B。
在一个实施方案中，多肽链2的CH3与VH_B直接融合联结，即L的长度为0。在另一个实施方案中，多肽链2的CH3经由连接肽L联结到VH_B；L可以是表1-2中所列序列。
实施例1.2.4.结构(6):IgG_LC-VH
结构(6)的结合蛋白包含两条不同的多肽链：多肽链1或第一多肽链，也称短链，从氨基末端到羧基末端，其包含VL_A-CL-L-VH_B；多肽链2或第二多肽链，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-h-CH2-CH3。
在一个实施方案中，多肽链1的CL与VH_B直接融合联结，即L的长度为0。在另一个实施方案中，多肽链1的CL经由连接肽L联结到VH_B；L可以是表1-2中所列序列。
实施例1.3.IgG-VH(2)六价对称结构
本发明提供了一种使用两种亲本单克隆抗体构建双特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B。
本发明还提供了一种使用三种亲本单克隆抗体构建三特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B和结合第三抗原的重链抗体C。
如图1的(7)–(8)所示，Fab端来源于常规抗体A，VH_A和VL_A分别为抗体A的重链可变区和轻链可变区。VH端来源于重链抗体B或重链抗体C，VH_B为重链抗体B的重链可变区，VH_C为重链抗体C的重链可变区。CL是轻链恒定区结构域。CH1、CH2和CH3分别是重链恒定区的第一、第二和第三结构域。L1和L2分别是第一和第二连接肽，h是IgG抗体的铰链区或衍生序列。
实施例1.3.1.结构(7):IgG_HC-VH-VH
结构(7)表示一种双特异性结合蛋白，其包含两条不同的多肽链：多肽链1或第一多肽链，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2或第二多肽链，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_B。
在一个实施方案中，多肽链2的CH3与VH_B直接融合联结，即L1的长度为0。在另一个实施方案中，多肽链2的CH3经由连接肽L1联结到VH_B；L1可以是表1-2中所列序列。
在一个实施方案中，多肽链2的第一个VH_B与第二个VH_B直接融合联结，即L2的长度为0。在另一个实施方案中，多肽链2的第一个VH_B经由连接肽L2联结到第二个VH_B；L2可以是表1-2中所列序列。
实施例1.3.2.结构(8):IgG_HC-VH’-VH”
结构(8)表示一种三特异性结合蛋白，其包含两条不同的多肽链：多肽链1或第一多肽链，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2或第二多肽 链，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_C。
在一个实施方案中，多肽链2的CH3与VH_B直接融合联结，即L1的长度为0。在另一个实施方案中，多肽链2的CH3经由连接肽L1联结到VH_B；L1可以是表1-2中所列序列。
在一个实施方案中，多肽链2的VH_B与VH_C直接融合联结，即L2的长度为0。在另一个实施方案中，多肽链2的VH_B经由连接肽L2联结到VH_C；L2可以是表1-2中所列序列。
实施例1.4. 2xVH-IgG六价对称结构
本发明提供了一种使用两种亲本单克隆抗体构建双特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B。
本发明还提供了一种使用三种亲本单克隆抗体构建三特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B和结合第三抗原的重链抗体C。
如图1的(9)–(10)所示，Fab端来源于常规抗体A，VH_A和VL_A分别为抗体A的重链可变区和轻链可变区。VH端来源于重链抗体B或重链抗体C，VH_B为重链抗体B的重链可变区，VH_C为重链抗体C的重链可变区。CL是轻链恒定区结构域。CH1、CH2和CH3分别是重链恒定区的第一、第二和第三结构域。L1和L2分别是第一和第二连接肽，h是IgG抗体的铰链区或衍生序列。
实施例1.4.1.结构(9):2xVH-IgG(HC+LC)
结构(9)表示一种双特异性结合蛋白，其包含两条不同的多肽链：多肽链1或第一多肽链，也称短链，从氨基末端到羧基末端，其包含VH_B-L1-VL_A-CL；多肽链2或第二多肽链，也称长链，从氨基末端到羧基末端，其包含VH_B-L2-VH_A-CH1-h-CH2-CH3。
在一个实施方案中，多肽链1的VH_B与VL_A直接融合联结，即L1的长度为0。在另一个实施方案中，多肽链1的VH_B经由连接肽L1联结到VL_A；L1可以是表1-2中所列序列。
在一个实施方案中，多肽链2的VH_B与VH_A直接融合联结，即L2的长度为0。在另一个实施方案中，多肽链2的VH_B经由连接肽L2联结到VH_A；L2可以是表1-2中所列序列。
在一个实施方案中，L1和L2可以是相同的序列。在另一个实施方案中，L1和L2可以是不同的序列。
实施例1.4.2.结构(10):(VH’+VH”)-IgG(HC+LC)
结构(10)表示一种三特异性结合蛋白，其包含两条不同的多肽链：多肽链1或第一多肽链，也称短链，从氨基末端到羧基末端，其包含VH_B-L1-VL_A-CL；多肽链2或第二多肽链，也称长链，从氨基末端到羧基末端，其包含VH_C-L2-VH_A-CH1-h-CH2-CH3。
在一个实施方案中，多肽链1的VH_B与VL_A直接融合联结，即L1的长度为0。在另一个实施方案中，多肽链1的VH_B经由连接肽L1联结到VL_A；L1可以是表1-2中所列序列。
在一个实施方案中，多肽链2的VH_C与VH_A直接融合联结，即L2的长度为0。在另一个实施方案中，多肽链2的VH_C经由连接肽L2联结到VH_A；L2可以是表1-2中所列序列。
在一个实施方案中，L1和L2可以是相同的序列。在另一个实施方案中，L1和L2可以是不同的序列。
实施例1.5.Fab-Fc-VH二价非对称结构
本发明提供了一种使用两种亲本单克隆抗体构建双特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B。
如图1的(11)–(13)所示，Fab端来源于常规抗体A，VH_A和VL_A分别为抗体A的重链可变区和轻链可变区。VH端来源于重链抗体B，VH_B为重链抗体B的重链可变区。CL是轻链恒定区结构域。CH1、CH2和CH3分别是重链恒定区的第一、第二和第三结构域。h是IgG抗体的铰链区或衍生序列。两个重链的Fc区域各引入了不同的氨基酸突变以减少重链同源二聚化的形成。
在一个实施方案中，含有VH_B的多肽链中的h可以是IgG的铰链区或者铰链区衍生的连接肽序列如表1-2中的人IgG1铰链(C220S)或者G5-LH的序列。
实施例1.5.1.结构(11):Fab-Fc-VH
结构(11)的结合蛋白包含三条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2或第二多肽链，从氨基末端到羧基末端，其包含VH_A-CH1-h-CH2-CH3；多肽链3，从氨基末端到羧基末端，其包含VH_B-h-
CH2-CH3。
实施例1.5.2.结构(12):Fab(cross VH/VL)-Fc-VH
结构(12)的结合蛋白包含三条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VH_A-CL；多肽链2或第二多肽链，从氨基末端到羧基末端，其包含VL_A-CH1-h-CH2-CH3；多肽链3，从氨基末端到羧基末端，其包含VH_B-h-CH2-CH3。
实施例1.5.3.结构(13):Fab(cross Fd/LC)-Fc-VH
结构(13)的结合蛋白包含三条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VH_A-CH1；多肽链2或第二多肽链，从氨基末端到羧基末端，其包含VL_A-CL-h-CH2-CH3；多肽链3或第三多肽链，从氨基末端到羧基末端，其包含VH_B-h-CH2-CH3。
在一个实施方案中，多肽链2中的h为铰链区或连接肽，其序列可以是如表1-2中的LH1的序列。
实施例1.6.Fab-Fc-VH(2)三价非对称结构
本发明提供了一种使用两种亲本单克隆抗体构建双特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B。
本发明还提供了一种使用三种亲本单克隆抗体构建三特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B和结合第三抗原的重链抗体C。
如图1的(14)–(19)所示，Fab端来源于常规抗体A，VH_A和VL_A分别为抗体A的重链可变区和轻链可变区。VH端来源于重链抗体B或重链抗体C，VH_B为重链抗体B的重链可变区，VH_C为重链抗体C的重链可变区。CL是轻链恒定区结构 域。CH1、CH2和CH3分别是重链恒定区的第一、第二和第三结构域。L是连接肽，h是IgG抗体的铰链区或衍生序列。两个重链的Fc区域各引入了不同的氨基酸突变以减少重链同源二聚化的形成。
在一个实施方案中，含有VH_B的多肽链中的h可以是IgG的铰链区或者铰链区衍生的连接肽序列如表1-2中的人IgG1铰链(C220S)或者G5-LH的序列。
实施例1.6.1.结构(14):Fab-Fc-VH-VH
结构(14)表示一种双特异性结合蛋白，其包含三条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2或第二多肽链，从氨基末端到羧基末端，VH_A-CH1-h-CH2-CH3；多肽链3或第三多肽链，从氨基末端到羧基末端，其包含VH_B-L-VH_B-h-CH2-CH3。
在一个实施方案中，多肽链3的第一个VH_B与第二个VH_B直接融合联结，即L的长度为0。在另一个实施方案中，多肽链3的第一个VH_B经由连接肽L联结到第二个VH_B；L可以是表1-2中所列序列。
实施例1.6.2.结构(15):Fab-Fc-VH’-VH”
结构(15)表示一种三特异性结合蛋白，其包含三条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2或第二多肽链，从氨基末端到羧基末端，VH_A-CH1-h-CH2-CH3；多肽链3或第三多肽链，从氨基末端到羧基末端，其包含VH_C-L-VH_B-h-CH2-CH3。
在一个实施方案中，多肽链3的VH_C与VH_B直接融合联结，即L的长度为0。在另一个实施方案中，多肽链3的VH_C经由连接肽L联结到VH_B；L可以是表1-2中所列序列。
实施例1.6.3.结构(16):Fab(cross VH/VL)-Fc-VH-VH
结构(16)表示一种双特异性结合蛋白，其包含三条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VH_A-CL；多肽链2或第二多肽链，从氨基末端到羧基末端，VL_A-CH1-h-CH2-CH3；多肽链3或第三多肽链，从氨基末端到羧基末端，其包含VH_B-L-VH_B-h-CH2-CH3。
在一个实施方案中，多肽链3的第一个VH_B与第二个VH_B直接融合联结，即L的长度为0。在另一个实施方案中，多肽链3的第一个VH_B经由连接肽L联结到第二个VH_B；L可以是表1-2中所列序列。
实施例1.6.4.结构(17):Fab(cross VH/VL)-Fc-VH’-VH”
结构(17)表示一种三特异性结合蛋白，其包含三条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VH_A-CL；多肽链2或第二多肽链，从氨基末端到羧基末端，VL_A-CH1-h-CH2-CH3；多肽链3或第三多肽链，从氨基末端到羧基末端，其包含VH_C-L-VH_B-h-CH2-CH3。
在一个实施方案中，多肽链3的VH_C与VH_B直接融合联结，即L的长度为0。在另一个实施方案中，多肽链3的VH_C经由连接肽L联结到VH_B；L可以是表1-2中所列序列。
实施例1.6.5.结构(18):Fab(cross Fd/LC)-Fc-VH-VH
结构(18)表示一种双特异性结合蛋白，其包含三条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VH_A-CH1；多肽链2或第二多肽链，从氨基末端到羧基末端，VL_A-CL-h-CH2-CH3；多肽链3或第三多肽链，从氨基末端到羧基末端，其包含VH_B-L-VH_B-h-CH2-CH3。
在一个实施方案中，多肽链3的第一个VH_B与第二个VH_B直接融合联结，即L的长度为0。在另一个实施方案中，多肽链3的第一个VH_B经由连接肽L联结到第二个VH_B；L可以是表1-2中所列序列。
在一个实施方案中，多肽链2中的h为铰链区或连接肽，其序列可以是如表1-2中的LH1的序列。
实施例1.6.6.结构(19):Fab(cross Fd/LC)-Fc-VH’-VH”
结构(19)表示一种三特异性结合蛋白，其包含三条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VH_A-CH1；多肽链2或第二多肽链，从氨基末端到羧基末端，VL_A-CL-h-CH2-CH3；多肽链3或第三多肽链，从氨基末端到羧基末端，其包含VH_C-L-VH_B-h-CH2-CH3。
在一个实施方案中，多肽链3的VH_C与VH_B直接融合联结，即L的长度为0。在另一个实施方案中，多肽链3的VH_C经由连接肽L联结到VH_B；L可以是表1-2中所列序列。
在一个实施方案中，多肽链2中的h为铰链区或连接肽，其序列可以是如表1-2中的LH1的序列。
实施例1.7.scFv-Fc-VH二价非对称结构
本发明提供了一种使用两种亲本单克隆抗体构建双特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B。
如图1的(20)–(21)所示，scFv端来源于常规抗体A，VH_A和VL_A分别为抗体A的重链可变区和轻链可变区。VH端来源于重链抗体B，VH_B为重链抗体B的重链可变区。CH2和CH3分别是重链恒定区的第二和第三结构域。L是连接肽，h是IgG抗体的铰链区或衍生序列。两个重链的Fc区域各引入了不同的氨基酸突变以减少重链同源二聚化的形成。
在一个实施方案中，scFv的序列可以是VH-连接肽-VL。在另一个实施方案中，scFv的序列可以是VL-连接肽-VH。
在一个实施方案中，多肽链中的L是连接肽，可以是表1-2中所列序列，优选为GS_15或GS_20的序列。
在一个实施方案中，多肽链中的h是IgG抗体的铰链区或衍生序列，可以是表1-2中所列序列，优选为人IgG1铰链(C220S)或者G5-LH的序列。
实施例1.7.1.结构(20):scFv(VL-VH)-Fc-VH
结构(20)的结合蛋白包含两条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VL_A-L-VH_A-h-CH2-CH3；多肽链2或第二多肽链，从氨基末端到羧基末端，其包含VH_B-h-CH2-CH3。
实施例1.7.2.结构(21):scFv(VH-VL)-Fc-VH
结构(21)的结合蛋白包含两条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VH_A-L-VL_A-h-CH2-CH3；多肽链2或第二多肽链，从氨基末端到羧基末端，其包含VH_B-h-CH2-CH3。
实施例1.8.scFv-Fc-VH(2)三价非对称结构
本发明提供了一种使用两种亲本单克隆抗体构建双特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B。
本发明还提供了一种使用三种亲本单克隆抗体构建三特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B和结合第三抗原的重链抗体C。
如图1的(22)–(25)所示，scFv端来源于常规抗体A，VH_A和VL_A分别为抗体A的重链可变区和轻链可变区。VH端来源于重链抗体B或重链抗体C，VH_B为重链抗体B的重链可变区，VH_C为重链抗体C的重链可变区。CH2和CH3分别是重链恒定区的第二和第三结构域。L1和L2分别是第一和第二连接肽，h是IgG抗体的铰链区或衍生序列。两个重链的Fc区域各引入了不同的氨基酸突变以减少重链同源二聚化的形成。
在一个实施方案中，scFv的序列可以是VH-连接肽-VL。在另一个实施方案中，scFv的序列可以是VL-连接肽-VH。
在一个实施方案中，多肽链中的L1是联结scFv中VH和VL的连接肽，可以是表1-2中所列序列，优选为GS_15或GS_20的序列。
在一个实施方案中，多肽链中的L2是联结两个重链抗体的重链可变区之间的连接肽，可以是表1-2中所列序列。在另一个实施方案中，两个重链抗体的重链可变区之间直接融合，即L2的长度为0。
在一个实施方案中，多肽链中的h是IgG抗体的铰链区或衍生序列，可以是表1-2中所列序列，优选为人IgG1铰链(C220S)或者G5-LH的序列。
实施例1.8.1.结构(22):scFv(VL-VH)-Fc-VH-VH
结构(22)表示一种双特异性结合蛋白，其包含两条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VL_A-L1-VH_A-h-CH2-CH3；多肽链2或第二多肽链，从氨基末端到羧基末端，其包含VH_B-L2-VH_B-h-CH2-CH3。
在一个实施方案中，多肽链2的第一个VH_B与第二个VH_B直接融合联结，即L2的长度为0。在另一个实施方案中，多肽链2的第一个VH_B经由连接肽L2联结到第二个VH_B；L2可以是表1-2中所列序列。
实施例1.8.2.结构(23):scFv(VH-VL)-Fc-VH-VH
结构(23)表示一种双特异性结合蛋白，其包含两条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VH_A-L1-VL_A-h-CH2-CH3；多肽链2或第二多肽链，从氨基末端到羧基末端，其包含VH_B-L2-VH_B-h-CH2-CH3。
在一个实施方案中，多肽链2的第一个VH_B与第二个VH_B直接融合联结，即L2的长度为0。在另一个实施方案中，多肽链2的第一个VH_B经由连接肽L2联结到第二个VH_B；L2可以是表1-2中所列序列。
实施例1.8.3.结构(24):scFv(VL-VH)-Fc-VH’-VH”
结构(24)表示一种三特异性结合蛋白，其包含两条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VL_A-L1-VH_A-h-CH2-CH3；多肽链2或第二多肽链，从氨基末端到羧基末端，其包含VH_C-L2-VH_B-h-CH2-CH3。
在一个实施方案中，多肽链2的VH_C与VH_B直接融合联结，即L2的长度为0。在另一个实施方案中，多肽链2的VH_C经由连接肽L2联结到VH_B；L2可以是表1-2中所列序列。
实施例1.8.4.结构(25):scFv(VH-VL)-Fc-VH’-VH”
结构(25)表示一种三特异性结合蛋白，其包含两条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VH_A-L1-VL_A-h-CH2-CH3；多肽链2或第二多肽链，从氨基末端到羧基末端，其包含VH_C-L2-VH_B-h-CH2-CH3。
在一个实施方案中，多肽链2的VH_C与VH_B直接融合联结，即L2的长度为0。在另一个实施方案中，多肽链2的VH_C经由连接肽L2联结到VH_B；L2可以是表1-2中所列序列。
实施例1.9.Fab-Fc-VH(3)四价非对称结构
本发明提供了一种使用两种亲本单克隆抗体构建双特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B。
本发明还提供了一种使用三种亲本单克隆抗体构建三特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B和结合第三抗原的重链抗体C。
本发明还提供了一种使用四种亲本单克隆抗体构建四特异性结合蛋白的方法：结合第一抗原的常规抗体A和结合第二抗原的重链抗体B和结合第三抗原的重链抗体C和结合第四抗原的重链抗体D。
如图1的(26)–(27)所示，Fab端来源于常规抗体A，VH_A和VL_A分别为抗体A的重链可变区和轻链可变区。VH端来源于重链抗体B或重链抗体C或重链抗体D，VH_B为重链抗体B的重链可变区，VH_C为重链抗体C的重链可变区，VH_D为重链抗体D的重链可变区。CL是轻链恒定区结构域。CH1、CH2和CH3分别是重链恒定区的第一、第二和第三结构域。L1和L2是连接肽，h是IgG抗体的铰链区或衍生序列。两个重链的Fc区域各引入了不同的氨基酸突变以减少重链同源二聚化的形成。
在一个实施方案中，含有VH_B的多肽链中的h可以是IgG的铰链区或者铰链区衍生的连接肽序列如表1-2中的人IgG1铰链(C220S)或者G5-LH的序列。
实施例1.9.1.结构(26):Fab-Fc-VH-VH-VH
结构(26)表示一种双特异性结合蛋白，其包含三条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2或第二多肽链，从氨基末端到羧基末端，VH_A-CH1-h-CH2-CH3；多肽链3或第三多肽链，从氨基末端到羧基末端，其包含VH_B-L1-VH_B-L2-VH_B-h-CH2-CH3。
在一个实施方案中，多肽链3的第一个VH_B与第二个VH_B直接融合联结，即L1的长度为0。在另一个实施方案中，多肽链3的第一个VH_B经由连接肽L1联结到第二个VH_B；L1可以是表1-2中所列序列。
在一个实施方案中，多肽链3的第二个VH_B与第三个VH_B直接融合联结，即L2的长度为0。在另一个实施方案中，多肽链3的第二个VH_B经由连接肽L2联结到第三个VH_B；L2可以是表1-2中所列序列。
实施例1.9.2.结构(27):Fab-Fc-VH’-VH”-VH”’
结构(27)表示一种四特异性结合蛋白，其包含三条不同的多肽链：多肽链1或第一多肽链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2或第二多肽链，从氨基末端到羧基末端，VH_A-CH1-h-CH2-CH3；多肽链3或第三多肽链，从氨基末端到羧基末端，其包含VH_D-L1-VH_C-L2-VH_B-h-CH2-CH3。
在一个实施方案中，多肽链3的VH_D与VH_C直接融合联结，即L1的长度为0。在另一个实施方案中，多肽链3的VH_D经由连接肽L1联结到VH_C；L1可以是表1-2中所列序列。
在一个实施方案中，多肽链3的VH_C与VH_B直接融合联结，即L2的长度为0。在另一个实施方案中，多肽链3的VH_C经由连接肽L2联结到VH_B；L2可以是表1-2中所列序列。
实施例2.抗体的序列分析、表达纯化、和理化性质表征分析
实施例2.1.抗体的序列分析和优化
抗体的重链可变结构域序列来源于染色体上重链基因群的胚系基因V、D、J基因片段的基因重排和体细胞高频突变等事件；轻链可变结构域序列来源于轻链基因群的胚系基因V、J基因片段的基因重排和体细胞高频突变等事件。基因重排和体细胞高频突变是增加抗体多样性的主要因素。来源于相同胚系V基因片段的抗体也可能产生不同的序列，但总体上相似性较高。利用一些算法，例如IMGT/DomainGapAlign(http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)或者NCBI/IgBLAST(https://www.ncbi.nlm.nih.gov/igblast/)可以从抗体的可变结构域序列推测出其发生基因重排时可能的胚系基因片段。
蛋白质或多肽氨基酸链在细胞中翻译合成后有时会引入化学修饰，称为翻译后修饰(PTM)。对于抗体而言，一些PTM的位点是非常保守的，例如，在人的IgG1抗体的恒定结构域的第297位(EU编号)的保守的氨基酸天冬酰胺Asn通常会发生糖基化修饰形成糖链，而该糖链结构对于抗体结构和相关的效应功能是至关重要的。但是，如果在抗体的可变结构域尤其是抗原结合区域(如CDR)中存在PTM，那么这些PTM的存在有可能会对抗原的结合有较大的影响，也可能对抗体的物理化学性质带来变化。例如，糖基化、脱酰胺、异构化、氧化等都可能增加抗体分子的不稳定性或异质性，从而增加抗体开发的难度和风险。因而避免一些潜在的PTM对于治疗性抗体的开发是非常重要的。随着经验的积累，人们发现一些PTM是和氨基酸序列的组成尤其是相邻氨基酸组成的“模式”是高度相关的，这样使得可以从蛋白质的一级氨基酸序列预测出潜在的PTM。例如，N-x-S/T(第一位是天冬酰胺，第二位是非脯氨酸以外的任意氨基酸，第 三位是丝氨酸或者苏氨酸)的序列模式预测出N-连接糖基化位点。引起PTM的氨基酸序列模式有可能来源于胚系基因序列，例如人胚系基因片段IGHV3-33天然地在FR3区域存在糖基化模式NST；也可能来源于体细胞高频突变。例如，NGS或NLT可能是糖基化位点，NS可能是脱酰胺位点，DG可能引起天冬氨酸的异构化。
可以通过氨基酸突变来破坏PTM的氨基酸序列模式，从而降低或者去除特定PTM的形成。根据抗体序列和PTM序列模式的不同，有不同的突变设计方法。一种方法是将“热点”氨基酸(如NS模式中的N或S)替换成物理化学性质相似的氨基酸(如把N突变为Q)。如果PTM序列模式来源于体细胞高频突变，而并不存在于胚系基因序列中，那么另一种方法可以是把该序列模式替换成对应的胚系基因序列。实际操作中，对同一个PTM序列模式可能采用多种突变设计方法。
实施例2.2.抗体的表达和纯化
本实施例介绍了利用哺乳动物宿主细胞(例如，人胚肾细胞HEK293或中国仓鼠卵巢细胞CHO及其衍生细胞)、瞬时转染表达和亲和捕获分离等技术来制备抗体的一般方法。本方法适用于含有Fc区的目标抗体；目标抗体可以由一条或多条蛋白质多肽链组成；可以来源于一个或多个表达质粒。
将抗体多肽链的氨基酸序列通过密码子优化方法转换成核苷酸序列；合成编码的核苷酸序列并克隆到与宿主细胞兼容的表达载体上。将编码抗体多肽链的质粒按照特定比例同时转染哺乳动物宿主细胞，利用常规的重组蛋白表达和纯化技术，可以得到具有正确折叠和多肽链组装的重组抗体。具体地，将FreeStyle TM293-F细胞(Thermo,#R79007)在FreeStyle TMF17 Expression Medium培养基(Thermo,#A1383504)中扩培。瞬时转染开始之前，调节细胞浓度至6-8x10 5细胞/ml，于37℃8％CO 2摇床中培养24小时，细胞浓度在1.2 x10 6细胞/ml。准备30ml培养的细胞。将编码抗体多肽链的质粒按照一定比例混合共计30μg质粒(质粒与细胞的比例为1μg:1ml)溶解于1.5ml Opti-MEM减血清培养基(Thermo,#31985088)，并用0.22μm滤膜过滤除菌。再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences,#23966-2)120μl，静置5分钟。把PEI缓慢加入质粒中，室温孵育10分钟，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，于37℃8％CO 2摇床中培养5天。5天后观测细胞活率。收集培养物，以3300g转速离心10分钟后取上清；然后将上清高速离心去除杂质。用PBS pH7.4缓冲液平衡含有MabSelect TM(GE Healthcare,#71-5020-91)的重力柱(Bio-Rad,#7311550)，2-5倍柱体积冲洗。将上清样品 过柱；用5-10倍柱体积的PBS缓冲液冲洗柱子，再用pH3.5的0.1M甘氨酸洗脱目的蛋白，随后用pH 8.0的Tris-HCl调节至中性，最后用超滤管(Millipore,#UFC901024)浓缩换液至PBS缓冲液或者含有其他成分的缓冲液，得到纯化的重组抗体溶液。最后用NanoDrop(Thermo,NanoDrop TMOne)测定浓度，分装、存储备用。
实施例2.3.利用SEC-HPLC分析蛋白纯度和聚体
本实施例使用分析型分子尺寸排阻层析色谱法(SEC)来分析蛋白样品的纯度和聚体形式。将分析型色谱柱TSKgel G3000SWxl(Tosoh Bioscience,#08541,5μm,7.8mm×30cm)连接到高压液相色谱仪HPLC(Agilent Technologies,Agilent 1260Infinity II)，用PBS缓冲液室温下平衡至少1小时。适量蛋白样品(至少10μg)用0.22μm滤膜过滤后注射入系统，并设定HPLC程序：用PBS缓冲液将样品以1.0ml/分钟的流速流过色谱柱，最长时间为25分钟。HPLC将生成分析报告，报告出样品内不同分子尺寸组份的滞留时间。
图65显示了本发明申请中的数个不同结构的双抗分子经过瞬时转染表达和一步亲和纯化后得到的蛋白样品的SDS-PAGE和SEC-HPLC的分析结果。这些双抗分子都体现出良好的产率和纯度。
实施例2.4.利用DSF测定蛋白分子的热稳定性
差示扫描荧光法(Differential Scanning Fluorimetry,DSF)是一种常用的高通量的测定蛋白质热稳定性的方法。它使用实时荧光定量PCR仪器通过监测与去折叠的蛋白分子结合的染料的荧光强度的变化，来反映蛋白质的变性的过程，从而反映出蛋白分子的热稳定性。本实施例利用DSF方法来测定蛋白分子热变性温度(Tm)。10μg蛋白加入96-孔PCR板(Thermo,#AB-0700/W)，接着加入2μl 100×稀释的染料SYPROTM(Invitrogen,#2008138)，然后加入缓冲液使得终体积为40μl每孔。将PCR板密封，放置于实时荧光定量PCR仪器(Bio-Rad CFX96 PCR System)，先于25℃孵育5分钟，然后以0.2℃/0.2分钟的梯度逐渐从25℃升温至95℃，在测试结束时将温度降至25℃。使用FRET扫描模式并使用Bio-Rad CFX Maestro软件进行数据分析并计算出样品的Tm。
实施例2.5.利用Uncle测定蛋白的分子稳定性和分子聚集性
Uncle(Unchained Labs)是一个多功能一站式的蛋白稳定性分析平台，它通过全荧光，静态光散射(SLS)和动态光散(DLS)检测方法来表征蛋白质的稳定性。同一组 样品可同时得到熔解温度(Tm)，聚集温度(Tagg)和粒径(diameter)等参数。在本实施例中，选择Uncle的“Tm&Tagg with optional DLS”应用程序进行操作，取9μL样品加入Uni管中，设置以0.3℃/分钟的梯度逐渐从25℃升温至95℃。进行初始和最终DLS测量四次采集，每次采集5秒。实验运行结束后，Uncle分析软件采用重心均值(BCM)公式来计算每个样品的Tm值；通过SLS在266nm或473nm波长下的荧光强度的曲线(聚集曲线)来计算Tagg值；样品的粒径和分散度则通过DLS相关的函数来计算。
实施例3.PD-L1 x CTLA4双特异性抗体
实施例3.1.背景
程序性死亡受体1(programmed death 1,PD-1)主要表达于T细胞等免疫细胞，它有两个配体，即程序性死亡配体-1(programmed death ligand 1,PD-L1)和PD-L2。PD-L1主要表达在抗原呈递细胞以及多种肿瘤细胞。PD-L1与PD-1相互作用会下调T细胞的活性，减弱细胞因子的分泌，起到免疫抑制作用。在许多人类肿瘤组织中均可检测到PD-L1蛋白的表达，肿瘤部位的微环境可诱导肿瘤细胞上的PD-L1的表达，表达的PD-L1有利于肿瘤的发生和生长，诱导抗肿瘤T细胞的凋亡，并进一步保护肿瘤细胞逃避免疫攻击。PD-1/PD-L1通路抑制剂可以阻断PD-1与PD-L1的结合，阻断负向调控信号，使T细胞恢复活性，发挥杀伤肿瘤细胞的作用，进而抑制肿瘤生长，因此，以PD-1/PD-L1为靶点的免疫调节对肿瘤抑制有重要的意义。
细胞毒性T淋巴细胞相关抗原4(CTLA4)是T细胞上表达的负调控因子，它与抗原递呈细胞上的CD80或CD86结合后，在阻断CD28的共剌激信号同时，还会下调T细胞的活性，起到免疫抑制作用。CTLA4介导的抑制机制则往往成为肿瘤细胞逃逸免疫系统的原因之一。通过阻断CTLA4与其配体的相互作用可以恢复T细胞的活性，增强抗肿瘤的能力。
目前，针对PD-1、PD-L1和CTLA4的阻断型抗体都有在临床上体现出优秀的抗肿瘤效果，且有多个抗体药物批准上市。尽管如此，这些药物和治疗手段在临床上仍面临诸多挑战，例如较低的反应率。因而，有很多基于PD-1、PD-L1和CTLA4等阻断型抗体的联合用药方案的临床试验正在积极地进行中。由于CTLA4抑制剂体现出较明显的 毒副作用，在目前的PD-1/PD-L1抑制剂和CTLA4抑制剂的联合用药方案中，CTLA4抑制剂通常选用较低剂量。例如，在联用抗PD-1抗体Nivolumab和抗CTLA-4抗体Ipilimumab治疗结直肠癌和肾细胞癌的临床试验中，Nivolumab和Ipilimumab的剂量分别是3mg/kg和1mg/kg。在联用抗PD-L1抗体Durvalumab和抗CTLA-4抗体Tremelimumab治疗非小细胞肺癌的临床试验中，Durvalumab和Tremelimumab的剂量分别是10-20mg/kg和1mg/kg。
另一方面，人们对肿瘤免疫相关的生物学机制也有越来越多的了解。有研究发现，阻断CTLA4信号通路会导致PD-1在肿瘤浸润淋巴细胞(TILs)上高表达，而且阻断PD-1信号通路会上调CTLA4在TILs上的表达(Rev Assoc Med Bras 2017；63(9):814-823)。这提示了通过阻断单一的共抑制信号通路有可能引起的耐药机制，而通过将这些抗体联用以同时阻断多个共抑制信号通路，则可能对T细胞的激活有协同作用。而且，最新的研究报道了PD-1和CTLA4信号通路相互作用的新的机制，阐释了PD-L1抗体和CTLA4抗体的协同作用(Zhao et al.,2019,Immunity 51,1059–1073)。
基于现有的知识，我们认为同时靶向PD-L1和CTLA4的双特异性抗体可以利用一个或者多个作用机制来提高抗肿瘤效果和安全性。第一，PD-L1 x CTLA4双抗通过阻断CTLA4信号通路和PD-1/PD-L1信号通路在T细胞的不同的阶段进行激活；PD-L1和CD80的顺式相互作用使其与CTLA4有更好的协同作用。第二，PD-L1高表达于肿瘤组织，PD-L1 x CTLA4双抗可以在肿瘤微环境中特异性地解除CTLA4抑制信号来激活T细胞，减少CTLA4单抗在外周系统非特异激活带来的毒副作用。第三，PD-L1 x CTLA4双抗可以选择保留Fc效应功能(如ADCC)，在肿瘤微环境中通过CTLA4特异性地杀伤高表达CTLA4的抑制性T细胞如T reg细胞，或通过PD-L1特异性地杀伤高表达PD-L1的肿瘤细胞。另外，双特异性抗体作为一个药物产品，比两个药物产品的组合，在经济性和用药的便利性等方面会更有优势。
实施例3.2.获得抗PD-L1的IgG抗体和抗CTLA4的HCAb抗体
实施例3.2.1.获得抗PD-L1的全人源IgG抗体
Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域。
用可溶的重组人PD-L1蛋白(NovoProtein,#CM06)对Harbour H2L2小鼠进行多轮免疫。当检测小鼠血清中PD-L1特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出并与骨髓瘤细胞系融合得到杂交瘤细胞；对杂交瘤细胞经过多轮筛选和克隆之后，鉴定出若干个特异识别PD-L1的单克隆抗体分子。对这些单克隆抗体进行进一步的鉴定，根据其对人PD-L1的结合能力、食蟹猴PD-L1的结合能力、抑制PD-L1与PD-1结合能力等参数，优选出数个候选抗体分子。然后对候选抗体分子进行序列分析和优化，得到数个变体序列。将抗体的VL和VH序列与相应的人的κ轻链恒定区和IgG1重链恒定区序列进行融合表达，得到重组全人源抗体分子。抗PD-L1的重组全人源IgG抗体列于表3-9。
实施例3.2.2.获得抗CTLA4的全人源HCAb抗体
Harbour HCAb小鼠(Harbour Antibodies BV，WO2010/109165A2)是一种携带人免疫球蛋白免疫库的转基因小鼠，能够产生仅有重链的抗体，该抗体的大小只有传统IgG抗体的一半。其产生的抗体仅具有人的抗体重链可变结构域和小鼠Fc恒定结构域。
用可溶的重组人CTLA4蛋白(ACRO Biosystems,#CT4-H5229)对Harbour HCAb小鼠进行多轮免疫。当检测小鼠血清中CTLA4特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出分离B细胞，用小鼠浆细胞分选试剂盒(Miltenyi,#130-092-530)分选CD138阳性的浆细胞。用常规的分子生物学手段从浆细胞中扩增人VH基因，并将扩增的人VH基因片段构建到编码人IgG1抗体重链Fc区域序列的哺乳动物细胞表达质粒pCAG载体中。质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)进行表达，得到全人源HCAb抗体上清。用ELISA测试HCAb抗体上清与重组人CTLA4蛋白的结合，鉴定出阳性HCAb抗体。对这些HCAb抗体进行进一步的鉴定，根据其对人CTLA4的结合能力、食蟹猴CTLA4的结合能力、抑制CTLA4与B7-1结合能力等参数，优选出数个候选HCAb抗体分子。然后对候选HCAb抗体分子进行序列分析和优化，得到数个变体序列。将HCAb抗体的VH序列和人的IgG1重链Fc序列进行融合表达，得到全人源重组HCAb抗体分子。抗CTLA4的重组全人源HCAb抗体列于表3-9。
实施例3.3.利用抗PD-L1的IgG抗体和抗CTLA4的HCAb抗体构建双特异性抗体分子
本实施例利用抗PD-L1的IgG抗体PR000070或PR000265的抗原结合结构域Fab，和抗CTLA4的HCAb抗体PR000184的抗原结合结构域VH，来构建多种结构的抗PD-L1 x CTLA4的双特异性抗体分子。
在本实施例及后续实施例中，抗PD-L1的阳性对照分子为抗PD-L1的IgG单抗PR000070或PR000416或PR000265，亦为PD-L1 x CTLA4双抗分子的PD-L1端的亲本单抗。PR000070、PR000265和PR000416都来源于同一个抗PD-L1的单克隆抗体。PR000070和PR000265都是人IgG1亚型，在其Fc区都有N297A突变，它们仅在重链可变区VH有一个氨基酸突变的差异。PR000265和PR000416都是人IgG1亚型且有相同的Fab结构，它们仅有差别在于PR000265的Fc区有N297A突变。
在本实施例及后续实施例中，抗CTLA4的阳性对照分子为抗CTLA4的HCAb单抗PR000184，亦为PD-L1 x CTLA4双抗分子的CTLA4端的亲本单抗。
实施例3.3.1.构建Fab-HCAb对称结构分子
利用抗PD-L1的IgG抗体和抗CTLA4的重链抗体，按照实施例1.1所述结构设计Fab-HCAb对称结构的PD-L1 x CTLA4双抗分子，总结于表3-1；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表3-2。
Figure PCTCN2021103044-appb-000005
表3-1 Fab-HCAb对称结构的PD-L1 x CTLA4双抗分子
Figure PCTCN2021103044-appb-000006
表3-2 Fab-HCAb对称结构的PD-L1 x CTLA4双抗分子蛋白的表达
实施例3.3.2.构建IgG-VH四价对称结构分子
利用抗PD-L1的IgG抗体和抗CTLA4的重链抗体，按照实施例1.2所述结构设计IgG-VH四价对称结构的PD-L1 x CTLA4双抗分子，总结于表3-3；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表3-4。
Figure PCTCN2021103044-appb-000007
表3-3 IgG-VH四价对称结构的PD-L1 x CTLA4双抗分子
Figure PCTCN2021103044-appb-000008
Figure PCTCN2021103044-appb-000009
表3-4 IgG-VH四价对称结构的PD-L1 x CTLA4双抗分子蛋白的表达
实施例3.3.3.构建2xVH-IgG六价对称结构分子
利用抗PD-L1的IgG抗体和抗CTLA4的重链抗体，按照实施例1.4所述结构设计2xVH-IgG六价对称结构的PD-L1 x CTLA4双抗分子，总结于表3-5；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表3-6。
Figure PCTCN2021103044-appb-000010
表3-5 2xVH-IgG六价对称结构的PD-L1 x CTLA4双抗分子
Figure PCTCN2021103044-appb-000011
表3-6 2xVH-IgG六价对称结构的PD-L1 x CTLA4双抗分子蛋白的表达
实施例3.3.4.构建Fab-Fc-VH二价非对称结构分子
利用抗PD-L1的IgG抗体和抗CTLA4的重链抗体，按照实施例1.4所述结构设计Fab-Fc-VH二价非对称结构的PD-L1 x CTLA4双抗分子，总结于表3-7；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表3-8。
Figure PCTCN2021103044-appb-000012
表3-7 Fab-Fc-VH二价非对称结构的PD-L1 x CTLA4双抗分子
(突变代号：Knob:S354C,T366W；Hole:Y349C,T366S,L368A,Y407V；DE:S239D,I332E.)
Figure PCTCN2021103044-appb-000013
表3-8 Fab-Fc-VH二价非对称结构的PD-L1 x CTLA4双抗分子蛋白的表达
实施例3.3.5.PD-L1 x CTLA4双抗分子及对照分子序列表
表3-9、表3-10和表3-11列出了本实施例所构建的PD-L1 x CTLA4双抗分子和对应的PD-L1单抗、CTLA4单抗等亲本单抗分子以及对照分子的序列所对应的序列号。表3-11中的结构编号对应于表1-1和图1。表3-12列出了双特异性抗体分子的第一和第二抗原结合结构域相应的CDR序列的序列编号。
抗体编号 抗体
PR000070 抗PD-L1 91G3H5H3,hIgG1(N297A)
PR000265 抗PD-L1 91G3H5H3(D54E),hIgG1(N297A)
PR000416 抗PD-L1 91G3H5H3(D54E),hIgG1
PR000184 抗CTLA4重链抗体CL5v3
PR000149 抗CTLA4单抗Ipilimumab类似物,hIgG1
PR000151 抗PD-L1单抗Atezolizumab类似物,hIgG1(N297A)
表3-9对照分子和亲本单抗
Figure PCTCN2021103044-appb-000014
表3-10对照分子和亲本单抗的序列和CDR序列的序列编号表
结构编号 抗体编号 多肽链1 多肽链2 多肽链3
3 PR000300 353 362 无
4 PR000301 363 364 无
6 PR000302 365 364 无
5 PR000303 353 366 无
5 PR000401 353 369 无
5 PR000402 353 370 无
1 PR000403 371 372 无
1 PR000404 371 373 无
9 PR001572 363 362 无
3 PR001573 353 394 无
4 PR001574 363 310 无
9 PR001575 363 394 无
3 PR001576 353 395 无
4 PR001577 363 396 无
9 PR001578 363 395 无
11 PR001609 353 407 390
11 PR001610 353 408 392
表3-11本实施例的PD-L1 x CTLA4双抗分子的序列编号表
Figure PCTCN2021103044-appb-000015
表3-12 PD-L1 x CTLA4双抗分子的抗原结合结构域的CDR的序列编号表
实施例3.4.结合CTLA4
本实施例是为了研究PD-L1 x CTLA4双抗分子结合CTLA4的活性。
实施例3.4.1.结合人CTLA4胞外区重组蛋白
利用酶联免疫吸附反应ELISA测试抗体分子结合人CTLA4重组蛋白的能力。具体地，首先将2μg/mL的人CTLA4-His蛋白(ACRO Biosystems,#CT4-H5229)以100μL/孔包被96孔板，于4℃过夜。然后用PBST缓冲液(含有0.05％Tween-20的PBS缓冲液)漂洗3次，接着加入封闭液(含有5％脱脂奶粉的PBS缓冲液)置于37℃孵育1小时。然后用PBST缓冲液漂洗3次。随后以100μL/孔加入以最高终浓度为30nM的5倍浓度梯度稀释的抗体分子，共8个浓度，混合均匀，置于37℃孵育1小时；hIgG1iso(CrownBio,#C0001)作为同型对照。然后用PBST缓冲液漂洗3次。随后以100μL/孔加入HRP标记的山羊抗人IgG Fc二抗(Sigma,#A0170,1:5000稀释)，置于37℃孵育0.5小时。然后用PBST缓冲液漂洗3次。随后以100μL/孔加入TMB显色液反应15分钟。最后加入终止液中止反应。用Enspire TM多功能读板机(Perkin Elmer,Inc.)于450nM读取光吸收值(OD值)。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及EC50值等参数。
本实施例中，阳性对照分子为抗CTLA4的HCAb单抗PR000184，亦为PD-L1 x CTLA4双抗分子的CTLA4端的亲本单抗。
图2和表3-13中所示，IgG-VH四价对称结构的双抗分子结合CTLA4的能力与抗CTLA4的VH端相对于抗PD-L1的IgG在双抗结构中的相对位置有关。当VH在IgG的重链N端(PR000300)或者轻链N端(PR000301)时，双抗分子结合CTLA4的EC50值与亲本单抗PR000184的相似甚至略优；当VH在IgG的重链C端时(PR000303)，双抗分子结合CTLA4的EC50值与亲本单抗PR000184的相比略弱2.6倍；当VH在IgG的轻链C端时(PR000302)，双抗分子结合CTLA4的能力明显减弱。这说明，通过调整VH在IgG上的相对位置可以用来调节VH结合靶点的能力。
抗体 EC50(nM) OD450最大值
PR000184 0.599 3.01
PR000300 0.567 2.95
PR000301 0.35 2.92
PR000302 10.6 3.55
PR000303 1.566 2.72
表3-13结合人CTLA4蛋白
实施例3.4.2.结合高表达人CTLA4的CHO-K1细胞CHO-K1/hCTLA4或高表达人CTLA4的HEK293细胞HEK293/hCTLA4
利用流式细胞术FACS测试抗体分子与高表达人CTLA4的CHO-K1细胞株CHO-K1/hCTLA4(北京康源博创,KC-1406)或高表达人CTLA4的HEK293T细胞株HEK293/hCTLA4(北京康源博创,KC-0209)等细胞的结合能力。具体地，消化CHO-K1/hCTLA4和HEK293/hCTLA4细胞，分别用F12K培养基和DMEM培养基重悬；将细胞密度调整为1x10 6细胞/mL。接着将细胞以100μL/孔接种于96孔V底板(Corning,#3894)，4℃下离心5分钟，弃上清。随后将梯度稀释的抗体分子以100μL/孔加入96孔板并混合均匀，抗体分子可以从最高终浓度为300nM按照5倍浓度梯度稀释的共8个浓度，或者可以从最高终浓度为100nM按照4倍浓度梯度稀释的共8个浓度；hIgG1iso(CrownBio,#C0001)作为同型对照。将细胞放置于4℃，避光孵育1小时。然后，加入100μL/孔预冷的FACS缓冲液(含有0.5％BSA的PBS缓冲液)漂洗细胞两次，4℃下500g离心5分钟，弃上清。接着，再加入100μL/孔荧光二抗(Alexa Fluor 488 anti-human IgG Fc,Biolegend,#409322,1:1000稀释)，放置于4℃，避光孵育1小时。随后以200μL/孔加入预冷的FACS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
本实施例中，阳性对照分子为抗CTLA4的HCAb单抗PR000184，亦为PD-L1 x CTLA4双抗分子的CTLA4端的亲本单抗。Ipilimumab类似物也作为阳性对照分子。结果显示于图3、图4、表3-14和表3-15。
图3的(A)-(B)和图4的(A)中所示，IgG-VH四价对称结构的双抗分子结合CTLA4的能力与抗CTLA4的VH端相对于抗PD-L1的IgG在双抗结构中的相对位置有关。当VH在IgG的重链N端(PR000300,PR001573)或轻链N端(PR000301,PR001574,PR001577)时，双抗分子结合CTLA4的EC50值与亲本单抗PR000184的相似或略弱， 但其在FACS上的MFI最大值比亲本单抗PR000184或阳性对照Ipilimumab更高。当VH在IgG的重链C端时(PR000303,PR000401,PR000402)，双抗分子结合CTLA4的EC50值与亲本单抗PR000184的相比略弱。当VH在IgG的轻链C端时(PR000302)，双抗分子结合CTLA4的能力明显减弱。这说明，通过调整VH在IgG上的相对位置可以用来调节VH结合靶点的能力。
图3的(C)中所示，2xVH-IgG六价对称结构的双抗分子结合CTLA4的EC50值与亲本单抗PR000184的相似，MFI最大值比亲本单抗PR000184的低。这可能是由于2xVH-IgG六价对称结构包含了4个结合CTLA4的结合位点，比亲本单抗PR000184的CTLA4结合位点多一倍，使得结合相同数目CTLA4分子所需的双抗分子要少于亲本单抗，而MFI值与结合到双抗分子的Fc区的荧光二抗分子的数目相关，进而体现为双抗分子的MFI最大值比亲本单抗的低。
图3的(D)中所示，Fab-Fc-VH非对称结构的双抗分子PR001609和PR001610只含有一个CTLA4结合结构域，因而其结合CTLA4的能力弱于具有二价的亲本单抗PR000184。
图3的(E)和图4的(B)中所示，Fab-HCAb对称结构的双抗分子结合CTLA4的能力弱于具有二价的亲本单抗PR000184，但是MFI最大值与阳性对照Ipilimumab的相似。
Figure PCTCN2021103044-appb-000016
表3-14结合CHO-K1/hCTLA4
抗体 EC50(nM) MFI最大值
PR000401 5.173 1911
PR000402 2.4 1764
PR000403 16.3 1408
PR000404 37.22 1615
PR000184 1.147 2261
表3-15结合HEK293/hCTLA4
实施例3.5.阻断CTLA4与其配体的结合
本实施例是为了研究PD-L1 x CTLA4双抗分子抑制CTLA4与其配体B7-1/CD80结合的活性。
实施例3.5.1.阻断人CTLA4蛋白和其配体蛋白的结合
利用酶联免疫吸附反应ELISA测定抗体分子抑制人CTLA4蛋白与其配体B7-1/CD80结合的活性。具体地，首先将2μg/mL的蛋白人B7-1-Fc(ACRO Biosystems,#B71-H5259)以100μL/孔包被96孔板，于4℃过夜。然后用PBST缓冲液(含有0.05％Tween-20的PBS缓冲液)漂洗3次，接着加入封闭液(含有5％脱脂奶粉的PBS缓冲液)置于37℃孵育1小时。随后以90μL/孔加入以最高终浓度为180nM的4倍浓度梯度稀释的抗体分子，共7个浓度，混合均匀，置于37℃孵育20分钟；hIgG1 iso(CrownBio,#C0001)作为同型对照。然后，以10μL/孔加入生物素化的人CTLA4-Fc蛋白(ACRO Biosystems,#CT4-H82F3)，使其终浓度为0.25μg/ml，置于37℃孵育1小时。然后用PBST缓冲液漂洗3次。随后以100μL/孔加入标记Precision Protein TMStrepTactin-HRP Conjugate(Bio-RAD,#1610380,1:4000稀释)，于37℃孵育0.5小时。然后用PBST缓冲液漂洗3次。随后以100μL/孔加入TMB显色液反应15分钟。最后加入终止液中止反应。用Enspire TM多功能读板机(Perkin Elmer,Inc.)于490nM读取光吸收值(OD值)。应用软件GraphPad Prism 8进行数据处理和作图分析，将OD值转换成抑制率，通过四参数非线性拟合，得到抑制曲线、IC50值和最大抑制率等参数。
本实施例中，阳性对照分子为抗CTLA4的HCAb单抗PR000184，亦为PD-L1 x CTLA4双抗分子的CTLA4端的亲本单抗。结果显示于图5和表3-16。
图5中所示，IgG-VH四价对称结构的双抗分子都有阻断CTLA4蛋白与其配体蛋白结合的能力(抑制活性)，且都能达到近100％抑制率，但其抑制活性的IC50值与抗CTLA4的VH端相对于抗PD-L1的IgG在双抗结构中的相对位置有关。当VH在IgG 的重链N端(PR000300)或轻链N端(PR000301)时，双抗分子对CTLA4的抑制活性与亲本单抗PR000184的相似。当VH在IgG的重链C端时(PR000303)，双抗分子对CTLA4的抑制活性与亲本单抗PR000184的相比略弱。当VH在IgG的轻链C端时(PR000302)，双抗分子抑制活性的IC50值比亲本单抗PR000184的弱4倍。这说明，通过调整VH在IgG上的相对位置可以用来调节VH的靶点阻断能力。
抗体 IC50(nM) 抑制率最大值(％)
PR000184 2.673 97.20
PR000300 2.091 98.17
PR000301 2.511 98.31
PR000302 11.57 98.91
PR000303 4.121 97.26
表3-16阻断人CTLA4蛋白和B7-1蛋白的结合
实施例3.5.2.阻断人CTLA4细胞和其配体蛋白的结合
利用流式细胞术FACS测定抗体分子抑制表达CTLA4的细胞与其配体B7-1/CD80结合的活性。具体地，消化高表达人CTLA4的CHO-K1细胞株CHO-K1/hCTLA4(北京康源博创,KC-1406)，并用F12K培养基重悬；将细胞密度调整为1x10 6细胞/mL，并置于FACS缓冲液(含有2％FBS的PBS缓冲液)中于37℃下15分钟。将FACS缓冲液以200μL/孔加入96孔板进行封闭，于37℃孵育1小时后，弃孔内封闭液。接着将CHO-K1/hCTLA4细胞以200μL/孔接种于96孔板(2x10 5细胞/孔)，500g转速于4℃下离心5分钟，弃上清。随后将梯度稀释的抗体分子以100μL/孔加入96孔板并混合均匀，抗体分子可以从最高终浓度为200nM按照3倍浓度梯度稀释的共8个浓度，或者可以从最高终浓度为400nM按照5倍浓度梯度稀释的共8个浓度；hIgG1 iso(CrownBio,#C0001)作为同型对照。然后，将1μg/mL的生物素化的配体蛋白人B7-1-Fc(ACRO Biosystems,#B71-H82F2)以100μL/孔加入96孔板并混合均匀。将96孔板放置于4℃，避光孵育1.5小时。然后，加入200μL/孔预冷的FACS缓冲液漂洗细胞两次，4℃下500g离心5分钟，弃上清。接着再加入100μL/孔荧光标记(Streptavidin,Alexa Fluor TM488 conjugate,Thermo,#S11223,1:500稀释)，放置于4℃，避光孵育1小时。随后以200μL/孔加入预冷的FACS缓冲液漂洗细胞三次，然后于4℃下500g离心 5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，将荧光信号值MFI转换成抑制率，通过四参数非线性拟合，得到抑制曲线、IC50值和最大抑制率等参数。
本实施例中，阳性对照分子为抗CTLA4的HCAb单抗PR000184，亦为PD-L1 x CTLA4双抗分子的CTLA4端的亲本单抗。结果显示于图6和表3-17。
图6的(A)-(B)中所示，IgG-VH四价对称结构的双抗分子对CTLA4的抑制活性与抗CTLA4的VH端相对于抗PD-L1的IgG在双抗结构中的相对位置有关。当VH在IgG的重链N端(PR000300,PR001573)或轻链N端(PR000301,PR001574,PR001577)时，双抗分子抑制活性的IC50值与亲本单抗PR000184的相似，都能达到近100％抑制率，PR000301甚至略强于亲本单抗PR000184。当VH在IgG的重链C端时(PR000303)，双抗分子抑制活性的IC50值与亲本单抗PR000184的相比略弱，且最大抑制率约83％。当VH在IgG的轻链C端时(PR000302)，双抗分子的抑制活性明显减弱，其最大抑制率只有49％。这说明，通过调整VH在IgG上的相对位置可以用来调节VH的靶点阻断能力。另一方面，同一个双抗分子在对CTLA4细胞的阻断实验和在对CTLA4蛋白的阻断实验中的不同抑制活性的结果，可能反映了细胞膜上蛋白的空间位阻的影响。
图6的(C)中所示，2xVH-IgG六价对称结构的双抗分子对CTLA4的抑制活性与亲本单抗PR000184的相似。
图6的(D)中所示，Fab-Fc-VH非对称结构的双抗分子PR001609和PR001610都只含有一个CTLA4结合结构域，因而其对CTLA4的抑制活性明显弱于具有二价的亲本单抗PR000184。
Figure PCTCN2021103044-appb-000017
Figure PCTCN2021103044-appb-000018
表3-17阻断CHO-K1/hCTLA4细胞和B7-1蛋白的结合
实施例3.6.CTLA4介导的ADCC效应
本实施例是为了研究PD-L1 x CTLA4双抗分子对高表达人CTLA4的细胞293F-hCTLA4(睿智化学)的抗体依赖的细胞介导的细胞毒性(ADCC)。第一步，用DELFIA BATDA(Perkin Elmer,#C136-100)标记靶细胞。具体标记方法如下：用2μL DELFIA BATDA试剂标记1x10 6靶细胞；于37℃，CO 2培养箱中孵育20分钟；用PBS洗涤4次，于1000rpm离心5分钟；最后一次洗涤后，将沉淀重悬于完全培养基中，并调整细胞密度至1x10 5/ml。第二步，将标记好的靶细胞以100μL/孔接种于96孔板(Corning,#3599)；随后以50μL/孔加入以最高终浓度为0.8nM的5倍浓度梯度稀释的抗体分子，共7个浓度，混合均匀；hIgG1 iso(CrownBio,#C0001)作为同型对照；于37℃，5％CO 2培养箱中孵育10分钟。第三步，收集NK-92MI/CD16a细胞(睿智化学)作为效应细胞，将NK-92MI/CD16a细胞密度调至6x10 5/ml；随后以50μL/孔加入到96孔板中，效应细胞:靶细胞＝3:1。接着，于37℃，5％CO 2培养箱中孵育2小时。第四步，以500g离心5分钟，然后从每孔取25μL上清加到一块新的96孔检测板中；随后以200μL/孔加入
Figure PCTCN2021103044-appb-000019
Europium溶液(Perkin Elmer,#C135-100)，并在室温下以250rpm摇动平板15分钟。最后，用
Figure PCTCN2021103044-appb-000020
多功能读板机(Perkin Elmer,Inc.)测量荧光值。
根据如下公式计算杀伤比率：
杀伤率(％)＝(ER–ETSR)/(TMR–ETSR)*100％
其中：
ER＝实验孔(抗体+效应细胞+靶细胞)；ETSR＝靶细胞和效应细胞混合自发释放孔(效应细胞+靶细胞)；TMR＝靶细胞最大释放孔(靶细胞+裂解液)
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的杀伤率曲线和EC50及最大杀伤率等参数。
本实施例中，阳性对照分子为抗CTLA4的HCAb单抗PR000184(hIgG1亚型)，亦为PD-L1 x CTLA4双抗分子的CTLA4端的亲本单抗。阴性对照分子为抗PD-L1的单抗PR000416(hIgG1亚型)。
图11和表3-18中所示，双抗分子PR000300和PR000301具有和亲本单抗PR000184相似的针对CTLA4特异的ADCC作用。
抗体 EC50(nM) 杀伤率最大值(％)
PR000300 0.008 23.81
PR000301 0.031 36.62
PR000184 0.015 30.20
表3-18 ADCC效应杀伤靶细胞
实施例3.7.结合PD-L1
本实施例是为了研究PD-L1 x CTLA4双抗分子结合PD-L1的活性。
实施例3.7.1.结合人PD-L1胞外区重组蛋白
利用酶联免疫吸附反应ELISA测试抗体分子结合人PD-L1重组蛋白的能力。具体地，首先将2μg/mL的人PD-L1-His蛋白(ACRO Biosystems,#PD1-H5229)以100μL/孔包被96孔板(Corning,#9018)，于4℃过夜。然后用PBST缓冲液(含有0.05％Tween-20的PBS缓冲液)漂洗3次，接着加入含2％BSA的封闭液，置于37℃孵育1小时。弃去封闭液，并用PBST缓冲液漂洗3次。随后以100μL/孔加入以最高终浓度为30nM的5倍浓度梯度稀释的抗体分子，共8个浓度，混合均匀，置于37℃孵育1小时；hIgG1 iso(CrownBio,#C0001)作为同型对照。然后用PBST缓冲液漂洗3次。随后以100μL/孔加入HRP标记的山羊抗人IgG Fc二抗(Sigma,#A0170,1:5000稀释)，置于37℃孵育0.5小时。然后用PBST缓冲液漂洗3次。随后以100μL/孔加入TMB显色液反应15分钟。最后加入终止液中止反应。用Enspire TM多功能读板机(PerkinElmer,Inc.)于450nM读取光吸收值(OD值)。
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及EC50值等参数。
本实施例中，阳性对照分子为抗PD-L1的单抗PR000070，亦为PD-L1 x CTLA4双抗分子的PD-L1端的亲本单抗。
图7和表3-19中所示，IgG-VH四价对称结构的双抗分子结合PD-L1的能力与亲本单抗PR000070的相似。这说明，抗CTLA4的VH结构域对抗PD-L1的Fab结构域没有明显的影响。
抗体 EC50(nM) OD045最大值
PR000070 0.146 3.454
PR000300 0.108 3.519
PR000301 0.131 3.528
PR000302 0.152 3.542
PR000303 0.241 3.523
表3-19结合人PDL1蛋白
实施例3.7.2.结合高表达人PD-L1的CHO-K1细胞CHO-K1/hPDL1和高表达人PD-L1的肿瘤细胞MDA-MB-231
利用流式细胞术FACS测试抗体分子与高表达人PD-L1的CHO-K1细胞株CHO-K1/hPDL1(南京金斯瑞,M00543)或高表达人PD-L1的肿瘤细胞系MDA-MB-231(ATCC,HTB-26)等细胞的结合能力。具体地，消化细胞并用完全培养基重悬；将细胞密度调整为1x10 6细胞/mL。接着将细胞以100μL/孔接种于96孔V底板(Corning,#3894)，4℃下离心5分钟，弃上清。随后将梯度稀释的抗体分子以100μL/孔加入96孔板并混合均匀，抗体分子可以从最高终浓度为60nM按照5倍浓度梯度稀释的共8个浓度，或者可以从最高终浓度为300nM按照5倍浓度梯度稀释的共8个浓度，或者可以从最高终浓度为100nM按照4倍浓度梯度稀释的共8个浓度；hIgG1 iso(CrownBio,#C0001)作为同型对照。将细胞放置于4℃，避光孵育1小时。然后，加入100μL/孔预冷的FACS缓冲液(含有0.5％BSA的PBS缓冲液)漂洗细胞两次，4℃下500g离心5分钟，弃上清。接着，再加入100μL/孔荧光二抗(Goat human lgG(H+L)Alexa Fluor 488 conjunction,Thermo,#A11013,1:1000稀释)，放置于4℃，避光孵育1小时。随后以200μL/孔加入预冷的FACS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液重悬细胞。使用BD FACS  CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
本实施例中，阳性对照分子为抗PD-L1的单抗PR000070或PR000416(或PR000265)，亦为PD-L1 x CTLA4双抗分子的PD-L1端的亲本单抗。结果显示于图8、图9、表3-20和表3-21。
图8的(A)-(B)和图9中所示，IgG-VH四价对称结构的双抗分子结合PD-L1的能力与其对应的亲本单抗PR000070或PR000416的相似，其结合PD-L1的EC50值非常接近。仅当VH在IgG的轻链N端(PR000301,PR001574,PR001577)时，MFI最大值比亲本单抗的略低。
图8的(C)中所示，2xVH-IgG六价对称结构的双抗分子结合PD-L1的能力相较于亲本单抗PR000416有降低；说明当VH同时连接到IgG的重链和轻链的N端后，对IgG的Fab端的功能带来明显的影响。
图8的(D)中所示，Fab-HCAb对称结构的双抗分子结合PD-L1的能力与亲本单抗PR000416相似，有相似的EC50值和MFI最大值。这说明，具有Fab-HCAb对称结构的双抗分子的抗PD-L1的Fab端结构可以很好地保留了对PD-L1的结合能力。
图8的(E)中所示，Fab-Fc-VH非对称结构的双抗分子PR001609和PR001610的PD-L1结合结构域是单价Fab结构，但是其结合PD-L1的能力与具有二价的亲本单抗相似，且MFI最大值比亲本单抗的更高。
Figure PCTCN2021103044-appb-000021
表3-20结合CHO-K1/hPDL1细胞
抗体 EC50(nM) MFI最大值
PR000401 0.288 994
PR000402 0.259 841
PR000265 0.163 958
表3-21结合MDA-MB-231细胞
实施例3.8.阻断PD-L1与其配体的结合
本实施例是为了研究PD-L1 x CTLA4双抗分子抑制PD-L1与其配体PD-1结合的活性。
实施例3.8.1.阻断人PD-L1细胞和其配体蛋白的结合
利用流式细胞术FACS测定抗体分子抑制表达PD-L1的细胞与其配体PD-1结合的活性。具体地，消化高表达人PD-L1的CHO-K1细胞株CHO-K1/hPDL1(南京金斯瑞,M00543)，并用F12K培养基重悬；将细胞密度调整为2 x10 6细胞/mL，并置于FACS缓冲液(含有1％BSA的PBS缓冲液)中于37℃下15分钟。将封闭缓冲液以200μL/孔加入96孔板，于37℃孵育1小时后，弃孔内封闭液。接着将CHO-K1/hPDL1细胞以50μL/孔接种于96孔板(1x10 5细胞/孔)。随后将梯度稀释的抗体分子以100μL/孔加入96孔板并混合均匀，抗体分子可以从最高终浓度为100nM按照5倍浓度梯度稀释的共8个浓度；hIgG1 iso(CrownBio,#C0001)作为同型对照。放置于4℃，避光孵育0.5小时。然后，以100μL/孔加入PBS缓冲液，并以500g转速于4℃下离心5分钟，弃上清。然后，将1μg/mL浓度的生物素化的配体蛋白人PD-1蛋白(ACRO Biosystems,#PD1-H82F2)以50μL/孔加入96孔板并混合均匀。将96孔板置于4℃，避光孵育0.5小时。随后以100μL/孔加入预冷的PBS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。接着再加入100μL/孔荧光二抗(PE Streptavidin,BD Biosciences,#554061,1:200稀释)，放置于4℃，避光孵育0.5小时。随后以200μL/孔加入预冷的PBS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，将荧光信号值MFI转换成抑制率，通过四参数非线性拟合，得到抑制曲线、IC50值和最大抑制率等参数。
本实施例中，阳性对照分子为抗PD-L1的单抗PR000070或PR000416(或PR000265)，亦为PD-L1 x CTLA4双抗分子的PD-L1端的亲本单抗。结果显示于图10和表3-22。
图10的(A)-(B)中所示，IgG-VH四价对称结构的双抗分子对PD-L1的抑制能力与其对应的亲本单抗PR000070或PR000416的相似，与亲本单抗有相近的IC50值和最大抑制率。仅当VH在IgG的轻链N端(PR001574,PR001577)时，IC50值比亲本单抗的略弱2-3倍。
图10的(C)中所示，2xVH-IgG六价对称结构的双抗分子对PD-L1的抑制活性弱于亲本单抗；相应的IC50值虽然比亲本单抗的弱4-6倍，但能达到相似的最大抑制率。
图10的(D)中所示，Fab-HCAb对称结构的双抗分子对PD-L1的抑制活性与亲本单抗的相似，与亲本单抗有相近的IC50值和最大抑制率。
图10的(E)中所示，Fab-Fc-VH非对称结构的双抗分子PR001609和PR001610的PD-L1结合结构域是单价Fab结构，其对PD-L1的抑制活性弱于具有二价的亲本单抗；相应的IC50值虽然比亲本单抗的弱4-5倍，但能达到相似的最大抑制率。
Figure PCTCN2021103044-appb-000022
表3-22阻断CHO-K1/hPDL1和PD1蛋白的结合
实施例3.9.超抗原SEB刺激实验
本实施例是为了研究PD-L1 x CTLA4双抗分子对外周血单个核细胞PBMC的激活作用。第一步，首先将分离的人PBMC(妙通生物)细胞以100μL/孔(1x10 5细胞/孔)加入96孔板(Corning,#3799)；随后以100μL/孔加入不同浓度的抗体分子，抗体终浓度 可以是(100nM,10nM,1nM,0.1nM,0.01nM)，或者是两个抗体按照一定比例的混合，两个复孔加样；hIgG1 iso(CrownBio,#C0001)作为同型对照。于37℃孵育30分钟。接着以50μL/孔加入超抗原Staphylococcal enterotoxin B(SEB)，使其终浓度为100ng/mL或10ng/mL。置于37℃，5％CO 2培养箱中孵育96小时或者120小时后收集上清。第二步，将收集上清利用IL-2ELISA试剂盒(Biolegend,#431805)检测上清中的IL-2浓度，操作方法请参考试剂盒的说明书。应用软件GraphPad Prism 8进行数据处理和作图分析。
本实施例中，阳性对照分子为抗CTLA4的HCAb单抗PR000184和抗PD-L1的IgG单抗PR000416。
如图12至图15所示，在有SEB刺激时，抗CTLA4的单抗、抗PD-L1的单抗以及PD-L1 x CTLA4双抗分子都可以不同程度地促进T细胞产生IL-2，其激活T细胞的能力与其结构有关。
图12中所示，IgG-VH四价对称结构的双抗分子可以激活T细胞产生IL-2。而且，抗CTLA4的VH在IgG的N端的结构(PR000301)相较于VH在IgG的C端的结构(PR000302,PR000303)，有更强的T细胞激活能力。
图13中所示，Fab-HCAb对称结构的双抗分子可以激活T细胞产生IL-2。而且，PR000404比PR000403有更强的T细胞激活能力。PR000404和PR000403有相似的结构，其仅有的差异在于PR000404有正常的人IgG1的Fc区，而PR000403的Fc区含有三个点突变(L234A,L235A,P329G)以减弱Fc相关的效应功能。据推测，PR000404可以通过CTLA4介导ADCC作用将Treg细胞清除掉，从而进一步增强T细胞功能。
图14中所示，双抗分子PR000300和PR000301比抗CTLA4单抗、抗PD-L1单抗及其1:1浓度组合，有相当甚至更强的T细胞激活能力(在同一次实验中使用同一个供体PBMC，可以比较IL-2水平绝对值)。
图15中所示，双抗分子PR000303(IgG_HC-VH结构，即VH在IgG的重链C端)和PR000404(Fab-HCAb结构)的激活T的能力弱于抗CTLA4的HCAb单抗PR000184。这说明，CTLA4端的活性在这两种结构中相较于其在正常的IgG或HCAb双价结构中会减弱，从而可以降低CTLA4的剂量相关的毒性，以适用于目前在临床的组合药物剂量。
实施例3.10.混合淋巴细胞反应(MLR)
本实施例是利用混合淋巴细胞反应(MLR)来研究PD-L1 x CTLA4双抗分子对T细胞的激活作用。第一步，在第一供体PBMC细胞(妙通生物)中加入重组人源白介素4(IL-4,R&D Systems,#204-GMP)和重组人源GM-CSF(R&D Systems,#215-GM)，诱导6天后，获得未成熟的人CD14 +树突状细胞(iDC细胞)。继续加入1μg/ml的脂多糖Lipopolysaccharide(LPS,Sigma,#L2630)，诱导24小时后，获得成熟的树突状细胞(mDC细胞)。第二步，用T细胞分离试剂盒(StemCell,#17951)从第二供体PBMC细胞(妙通生物)中分离得到T淋巴细胞。第三步，将获得的T细胞和mDC细胞按10:1比例接种至96孔板(1×10 5/孔的T细胞和1×10 4/孔的mDC细胞)。随后以100μL/孔加入不同浓度的抗体分子，抗体终浓度可以是(100nM,10nM,1nM,0.1nM,0nM)，或者是两个抗体按照一定比例的混合，两个复孔加样；hIgG1 iso(CrownBio,#C0001)作为同型对照。于37℃，5％CO 2培养箱孵育5天。第四步，分别收集第3天和第5天的上清液，用IL-2ELISA试剂盒(Thermo,#88-7025-88)检测第3天的上清中IL-2浓度，用IFN-γELISA试剂盒(Thermo,#88-7316-88)检测第5天的上清中IFN-γ浓度。
图16中所示，在两次独立的MLR实验中(不同的供体配对)，双抗分子PR000300和PR000301(VH在IgG重链或轻链的N端)都显示出比抗CTLA4单抗、抗PD-L1单抗及其1:1浓度组合，有更强的T细胞激活能力，能更好地促进IL-2和IFN-γ的产生。
图17中所示，当抗CTLA4的VH在IgG重链或轻链的C端时，双抗分子PR000302和PR000303在MLR实验中显示出比抗PD-L1单抗atezolizumab，有更强的T细胞激活能力。
实施例3.11.双抗分子的抗肿瘤药效
本研究为了评估PD-L1 x CTLA4抗体的体内抗肿瘤药效，采用6-8周龄雌性B6-PD1/CTLA4转基因小鼠，皮下接种MC38-hPDL1细胞(在MC38细胞上过表达人PD-L1)，待肿瘤均值达到约100mm 3时，将荷瘤小鼠按肿瘤体积分组，然后将特定浓度经PBS稀释的所述抗体药物按照特定的剂量和频次以腹腔注射(i.p.)方式给药。本实施例采用的给药方案：每周给药2次总共给药8次(BIW x 4weeks)。试验以PBS为空白对照组，双抗给药组为3mg/kg的PR001573，联合给药组为3mg/kg的ipilimumab加3 mg/kg的atezolizumab。在肿瘤接种后第6，9，12，15，19，22，26，29和33天对肿瘤体积和小鼠体重进行测量。
肿瘤体积计算公式：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。
图67中所示，在肿瘤接种后第33天，对照组肿瘤体积约700mm 3。联合给药组(3mg/kg ipilimumab+3mg/kg atezolizumab)的肿瘤完全消退，肿瘤抑制率(TGI)为100％，与对照组相比差异显著P＝0.03。双抗给药组(3mg/kg PR001573)的肿瘤完全消退，TGI为100％，与对照组相比差异显著P＝0.03。给药组小鼠在整个实验过程中耐受表现良好,体重没有明显变化。说明3mg/kg PR001573的药效和等剂量下的ipilimumab和atezolizumab的联用的药效相当。
实施例3.12.小结
本实施例利用抗PD-L1的IgG抗体的抗原结合结构域Fab和抗CTLA4的HCAb抗体的抗原结合结构域VH，构建了多种结构的抗PD-L1 x CTLA4的双特异性抗体分子。展现出了基于HCAb构建双特异性抗体分子结构的灵活性，通过不同的结构类型、相对位置、结合价数等参数来调节PD-L1端和CTLA4端的功能活性，进而设计出不同的活性组合，以满足不同的临床联合用药剂量组合的需求。
例如，当抗CTLA4的VH在抗PD-L1的IgG的N端时，双抗分子PR000300和PR000301的PD-L1端和CTLA4端都能几乎完全保持与其亲本单抗相当的活性，而且在混合淋巴细胞反应和超抗原刺激实验等体外功能实验中，显示出跟亲本单抗1:1浓度组合的有相当的甚至更强的T细胞激活能力；因而可以用来实现联合用药的1:1的剂量组合。小鼠肿瘤药效模型也证实了该结构的双抗PR001573有与联合用药相同的药效。
又例如，当抗CTLA4的VH在抗PD-L1的IgG的C端时，或者在Fab-HCAb结构时，双抗分子PR000302，PR000303和PR000404的PD-L1端几乎完全保持与其亲本单抗相当的活性，但是其CTLA4端的活性有不同程度的减弱；因而可以用来实现临床上的3:1甚至10:1的联合用药剂量组合。
实施例4.HER2 x CTLA4双特异性抗体
实施例4.1.背景
细胞毒性T淋巴细胞相关抗原4(CTLA4)是T细胞上表达的负调控因子，它与抗原递呈细胞上的CD80或CD86结合后，在阻断CD28的共剌激信号同时，还会下调T细胞的活性，起到免疫抑制作用。CTLA4介导的抑制机制则往往成为肿瘤细胞逃逸免疫系统的原因之一。通过阻断CTLA4与其配体的相互作用可以恢复T细胞的活性，增强抗肿瘤的能力。Ipilimumab单抗(商品名
Figure PCTCN2021103044-appb-000023
)是第一个获批上市的抗CTLA4单抗药物，也是开启肿瘤免疫治疗时代的第一个产品。Ipilimumab在晚期黑色素瘤的治疗上体现出较好的治疗效果，但是Ipilimumab也带来了较高的免疫相关副反应，其相关的3-5级不良反应的发生率甚至高达50％，这严重地影响了它的临床应用。Ipilimumab所表现出来的毒副作用大部分是CTLA4靶点相关的，在目前的PD-1/PD-L1抑制剂和CTLA4抑制剂的联合用药方案中，CTLA4抑制剂无论Ipilimumab或是Tremelimumab都通常选用较低剂量。
为了降低CTLA4抑制剂的毒副作用，其中一种值得尝试的方法是将CTLA4抑制剂定向输送到肿瘤组织内部，使相关的T细胞介导的反应仅局限于肿瘤微环境内，而减少细胞因子释放综合征的风险。这种定向输送可以通过瘤内注射CTLA4抑制剂来实现，但是瘤内注射的方法既有手术操作的风险，而且也仅限于一些浅表的可及的肿瘤组织。本实施例提供另一种定向输送的方法，利用识别肿瘤特异性抗原(tumor-associated antigen)的抗体将CTLA4抑制剂重定向到特定的肿瘤微环境中，使其在肿瘤微环境中解除T细胞的免疫抑制信号，恢复T细胞的功能。
在本实施例中，我们构建了同时靶向HER2和CTLA4的双特异性抗体，通过一个或者多个作用机制来提高抗肿瘤效果和安全性。第一，HER2 x CTLA4双抗在保留原有的HER2抑制剂的作用机制(阻止HER2二聚化，促进HER2的内化和降解，抑制下游磷酸化信号)的基础上，通过阻断CTLA4信号通路来激活T细胞。第二，HER2 x CTLA4双抗富集于HER2高表达的肿瘤组织，在肿瘤微环境中特异性地解除CTLA4抑制信号来激活T细胞，减少CTLA4单抗在外周系统非特异激活带来的毒副作用。第三，HER2 x CTLA4双抗可以选择保留Fc效应功能(如ADCC)，在肿瘤微环境中通过CTLA4特异性地杀伤高表达CTLA4的抑制性T细胞如T reg细胞，或通过HER2特 异性地杀伤高表达HER2的肿瘤细胞。另外，双特异性抗体作为一个药物产品，比两个药物产品的组合，在经济性和用药的便利性等方面会更有优势。
实施例4.2.获得抗HER2的IgG抗体和抗CTLA4的HCAb抗体
本实施例使用抗HER2的IgG抗体trastuzumab和pertuzumab，其相应的氨基酸序列来源于IMGT数据库，见表4-11。
本实施例使用的抗CTLA4的全人源HCAb抗体PR000184(表4-11)来源于Harbour HCAb小鼠，其发现过程如实施例3.2.2所述。
实施例4.3.利用抗HER2的IgG抗体和抗CTLA4的HCAb抗体构建双特异性抗体分
子
本实施例利用抗HER2的IgG抗体PR000210(trastuzumab类似物)或PR000672(pertuzumab类似物)的抗原结合结构域Fab，和抗CTLA4的HCAb抗体PR000184的抗原结合结构域VH，来构建多种结构的抗HER2 x CTLA4的双特异性抗体分子。
在本实施例及后续实施例中，阳性对照分子为抗HER2的IgG单抗PR000210(trastuzumab类似物)或PR000672(pertuzumab类似物)，亦为HER2 x CTLA4双抗分子的HER2端的亲本单抗。
在本实施例及后续实施例中，阳性对照分子为抗CTLA4的HCAb单抗PR000184，亦为HER2 x CTLA4双抗分子的CTLA4端的亲本单抗。
实施例4.3.1.构建Fab-HCAb对称结构分子
利用抗HER2的IgG抗体和抗CTLA4的重链抗体，按照实施例1.1所述结构设计Fab-HCAb对称结构的HER2 x CTLA4双抗分子，总结于表4-1；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表4-2。
Figure PCTCN2021103044-appb-000024
Figure PCTCN2021103044-appb-000025
表4-1 Fab-HCAb对称结构的HER2 x CTLA4双抗分子
Figure PCTCN2021103044-appb-000026
表4-2 Fab-HCAb对称结构的HER2 x CTLA4双抗分子蛋白的表达
实施例4.3.2.构建IgG-VH四价对称结构分子
利用抗HER2的IgG抗体和抗CTLA4的重链抗体，按照实施例1.2所述结构设计IgG-VH四价对称结构的HER2 x CTLA4双抗分子，总结于表4-3；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表4-4。
Figure PCTCN2021103044-appb-000027
表4-3 IgG-VH四价对称结构的HER2 x CTLA4双抗分子
Figure PCTCN2021103044-appb-000028
Figure PCTCN2021103044-appb-000029
表4-4 IgG-VH四价对称结构的HER2 x CTLA4双抗分子蛋白的表达
实施例4.3.3.构建IgG-VH(2)六价对称结构分子
利用抗HER2的IgG抗体和抗CTLA4的重链抗体，按照实施例1.3所述结构设计IgG-VH(2)六价对称结构的HER2 x CTLA4双抗分子，总结于表4-5；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表4-6。
Figure PCTCN2021103044-appb-000030
表4-5 IgG-VH(2)六价对称结构的HER2 x CTLA4双抗分子
Figure PCTCN2021103044-appb-000031
Figure PCTCN2021103044-appb-000032
表4-6 IgG-VH(2)六价对称结构的HER2 x CTLA4双抗分子蛋白的表达
实施例4.3.4.构建2xVH-IgG六价对称结构分子
利用抗HER2的IgG抗体和抗CTLA4的重链抗体，按照实施例1.4所述结构设计2xVH-IgG六价对称结构的HER2 x CTLA4双抗分子，总结于表4-7；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表4-8。
Figure PCTCN2021103044-appb-000033
表4-7 2xVH-IgG六价对称结构的HER2 x CTLA4双抗分子
Figure PCTCN2021103044-appb-000034
表4-8 2xVH-IgG六价对称结构的HER2 x CTLA4双抗分子蛋白的表达
实施例4.3.5.构建Fab-Fc-VH(n)二价或三价非对称结构分子
利用抗HER2的IgG抗体和抗CTLA4的重链抗体，按照实施例1.5和实施例1.6所述结构设计Fab-Fc-VH(n,n＝{1,2})非对称结构的HER2 x CTLA4双抗分子，总结于表4-9；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表4-10。
Figure PCTCN2021103044-appb-000035
表4-9 Fab-Fc-VH(n)非对称结构的HER2 x CTLA4双抗分子
(突变代号：Knob:S354C,T366W；Hole:Y349C,T366S,L368A,Y407V；DE:S239D,I332E.)
Figure PCTCN2021103044-appb-000036
表4-10 Fab-Fc-VH(n)非对称结构的HER2 x CTLA4双抗分子蛋白的表达
实施例4.3.6.HER2 x CTLA4双抗分子及对照分子序列表
表4-11、表4-12和表4-13列出了本实施例所构建的HER2 x CTLA4双抗分子和对应的HER2单抗、CTLA4单抗等亲本单抗分子以及对照分子的序列所对应的序列号。表4-13中的结构编号对应于表1-1和图1。表4-14列出了双特异性抗体分子的第一和第二抗原结合结构域相应的CDR序列的序列编号。
抗体编号 抗体
PR000210 抗HER2单抗trastuzumab类似物,hIgG1
PR000672 抗HER2单抗pertuzumab类似物,hIgG1
PR000184 抗CTLA4重链抗体CL5v3,hIgG1
PR000218 抗CTLA4重链抗体CL5v3,hIgG1(S239D,I332E)
表4-11对照分子和亲本单抗
Figure PCTCN2021103044-appb-000037
Figure PCTCN2021103044-appb-000038
表4-12对照分子和亲本单抗的序列和CDR序列的序列编号表
结构编号 抗体编号 多肽链1 多肽链2 多肽链3
1 PR000305 367 368 无
5 PR000539 351 374 无
5 PR000540 351 375 无
7 PR000541 351 376 无
7 PR000542 351 377 无
1 PR000653 367 378 无
1 PR000654 367 379 无
1 PR000655 367 380 无
1 PR000656 367 381 无
1 PR000658 367 382 无
1 PR000659 367 383 无
1 PR000706 367 385 无
5 PR000714 351 386 无
5 PR000715 351 387 无
1 PR000716 367 388 无
1 PR000717 367 389 无
11 PR000916 351 391 390
11 PR000917 351 393 392
7 PR001579 351 397 无
7 PR001580 351 398 无
7 PR001581 351 399 无
7 PR001582 351 400 无
3 PR001583 351 401 无
4 PR001584 402 305 无
9 PR001585 402 401 无
3 PR001586 351 403 无
9 PR001587 402 403 无
3 PR001588 359 404 无
4 PR001589 405 315 无
9 PR001590 405 404 无
3 PR001591 359 406 无
9 PR001592 405 406 无
4 PR001974 402 409 无
14 PR002666 351 391 434
14 PR002667 351 391 435
14 PR002668 351 393 436
14 PR002669 351 393 437
18 PR002670 367 438 434
18 PR002671 367 438 435
18 PR002672 367 439 436
18 PR002673 367 439 437
表4-13本实施例的HER2 x CTLA4双抗分子的序列编号表
Figure PCTCN2021103044-appb-000039
Figure PCTCN2021103044-appb-000040
表4-14 HER2 x CTLA4双抗分子的抗原结合结构域的CDR的序列编号表
实施例4.4.结合CTLA4
本实施例是为了研究HER2 x CTLA4双抗分子结合CTLA4的活性。
实施例4.4.1.结合高表达人CTLA4的CHO-K1细胞CHO-K1/hCTLA4
利用流式细胞术FACS测试抗体分子与高表达人CTLA4的CHO-K1细胞株CHO-K1/hCTLA4(睿智化学)等细胞的结合能力。具体地，消化CHO-K1/hCTLA4细胞，并用F12K培养基重悬；将细胞密度调整为2 x10 6细胞/mL。接着将CHO-K1/hCTLA4细胞以100μL/孔接种于96孔V底板(Corning,#3894)，4℃下离心5分钟，弃上清。随后将梯度稀释的抗体分子以100μL/孔加入96孔板并混合均匀，抗体分子可以从最高终浓度为300nM按照5倍浓度梯度稀释的共8个浓度，或者可以从最高终浓度为300nM按照4倍浓度梯度稀释的共12个浓度；hIgG1 iso(CrownBio,#C0001)作为同型对照。将 细胞放置于4℃，避光孵育1小时。然后，加入100μL/孔预冷FACS缓冲液(含有0.5％BSA的PBS缓冲液)漂洗细胞两次，4℃下500g离心5分钟，弃上清。接着，再加入100μL/孔荧光二抗(Goat human lgG(H+L)Alexa Fluor 488 conjunction,Thermo,#A11013,1:1000稀释)，放置于4℃，避光孵育1小时。随后以200μL/孔加入预冷的FACS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
本实施例中，阳性对照分子为抗CTLA4的HCAb单抗PR000184，亦为HER2 x CTLA4双抗分子的CTLA4端的亲本单抗。结果显示于图18和表4-15。
图18的(A)中所示，Fab-HCAb对称结构的双抗分子都可以结合CTLA4。这些分子具有相似的结构，都是由抗HER2的单抗PR000210的Fab结构域和抗CTLA4的HCAb单抗PR000184的VH结构域组合而成，其细微的差异在于不同的第一连接肽和连接Fc的铰链区以及不同的Fc类型或者突变。因而这些双抗分子有相似的结合CTLA4的能力。它们结合CTLA4的EC50值与亲本单抗PR000184的相似或者略弱仅1.5～3倍，但是在FACS上的最大结合信号(MFI最大值)比亲本单抗PR000184低。这可能暗示了，在Fab-HCAb结构中，Fab结构域可能对HCAb的VH结构域有一种“遮蔽”效应，使得在这个结构中的VH结构域能够结合的CTLA4分子比处于自由端时更少，但是在对相同数目的可及的CTLA4分子而言，其结合效力与亲本单抗PR000184相当(EC50值)。
图18的(B)-(D)中所示，IgG-VH四价对称结构的双抗分子结合CTLA4的能力根据其具体分子结构略有不同。根据抗CTLA4的VH端相对于抗HER2的IgG在双抗结构中的相对位置，将此类结构的双抗分子进一步分为三类：VH在IgG的重链C端(PR000539,PR000540,PR000714,PR000715)；VH在IgG的重链N端(PR001583,PR001586,PR001588,PR001591)；VH在IgG的轻链N端(PR001584,PR001589)。当VH在IgG的重链C端时，这些双抗分子结合CTLA4的EC50值与亲本单抗PR000184的相似，但是在FACS上的MFI最大值比亲本单抗PR000184略低。当VH在IgG的无论重链或轻链的N端时，这些双抗分子结合CTLA4的EC50值与亲本单抗 PR000184的相比略弱，但是在FACS上的MFI最大值比亲本单抗PR000184更强。这说明，通过调整VH在IgG上的相对位置可以用来调节VH结合靶点分子的数目和结合效力。
图18的(E)-(F)中所示，IgG-VH-VH六价对称结构的双抗分子都可以结合CTLA4。这些分子具有相似的结构，都是由抗HER2的单抗PR000210和抗CTLA4的HCAb单抗PR000184的VH结构域组合而成，其细微的差异在于不同的第一连接肽和第二连接肽以及不同的Fc类型或者突变。除PR001582以外，这些双抗分子结合CTLA4的能力与亲本单抗PR000184相似(相似的EC50和相似的MFI最大值)。
图18的(G)-(H)中所示，2xVH-IgG六价对称结构的双抗分子都可以结合CTLA4。这些分子具有相似的结构，都是由抗HER2的单抗PR000210或PR000672和抗CTLA4的HCAb单抗PR000184的VH结构域组合而成。基于亲本单抗PR000210构建的双抗分子(PR001585,PR001587)结合CTLA4的能力与亲本单抗PR000184相似；基于亲本单抗PR000672构建的双抗分子(PR001590,PR001592)结合CTLA4的能力与亲本单抗PR000184相比略弱。
图18的(I)-(J)中所示，Fab-Fc-VH(n,n＝1,2)非对称结构的双抗分子结合CTLA4的能力与结构中所含亲本单抗PR000184的VH结构域的数目有关。双抗分子PR000916和PR000917都只含有一个CTLA4结合结构域，因而其结合CTLA4的能力明显弱于具有二价的亲本单抗PR000184。双抗分子PR002672含有两个串联形成的CTLA4结合结构域，其结合CTLA4的能力与亲本单抗PR000184相似。这种结构显示出含有来源HCAb的VH结构域的双抗分子有这样的灵活性：通过调节串联的VH结构域的数目来调节与靶点的结合能力。
Figure PCTCN2021103044-appb-000041
Figure PCTCN2021103044-appb-000042
表4-15结合CHO-K1/hCTLA4
实施例4.5.阻断CTLA4与其配体的结合
本实施例是为了研究HER2 x CTLA4双抗分子抑制CTLA4与其配体B7-1/CD80结合的活性。
实施例4.5.1.阻断人CTLA4蛋白和其配体蛋白的结合
利用酶联免疫吸附反应ELISA测定抗体分子抑制人CTLA4蛋白与其配体B7-1/CD80蛋白结合的活性。具体地，首先将2μg/mL的蛋白人B7-1-Fc(ACRO Biosystems,#B71-H5259)以100μL/孔包被96孔板，于4℃过夜。然后用PBST缓冲液(含有0.05％Tween-20的PBS缓冲液)漂洗3次，接着加入封闭液(含有5％脱脂奶粉的PBS缓冲液)置于37℃孵育1小时。随后以90μL/孔加入以最高终浓度为200nM的3倍浓度梯度稀释的抗体分子，共7个浓度，混合均匀，置于37℃孵育20分钟；hIgG1 iso(CrownBio,#C0001)作为同型对照。然后，以10μL/孔加入生物素化的人CTLA4-Fc蛋白(ACRO Biosystems,#CT4-H82F3)，使其终浓度为0.25μg/ml，置于37℃孵育1小时。然后用PBST缓冲液漂洗3次。随后以100μL/孔加入标记Precision  Protein TMStrepTactin-HRP Conjugate(Bio-RAD,#1610380,1:4000稀释)，于37℃孵育0.5小时。然后用PBST缓冲液漂洗3次。随后以100μL/孔加入TMB显色液反应15分钟。最后加入终止液中止反应。用Enspire TM多功能读板机(Perkin Elmer,Inc.)于490nM读取光吸收值(OD值)。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抑制曲线及IC50值等参数。
本实施例中，阳性对照分子为抗CTLA4的HCAb单抗PR000184，亦为HER2 x CTLA4双抗分子的CTLA4端的亲本单抗。结果显示于图19和表4-16。
图19的(A)中所示，Fab-HCAb对称结构的双抗分子都有阻断CTLA4蛋白与其配体蛋白结合的能力(抑制活性)，且与亲本单抗PR000184的相似或者略弱，体现为相似的最大OD差值但是IC50值略弱仅1.5～2.1倍。
图19的(B)-(D)中所示，IgG-VH四价对称结构的双抗分子的抑制活性根据其具体分子结构略有不同。根据抗CTLA4的VH端相对于抗HER2的IgG在双抗结构中的相对位置，将此类结构的双抗分子进一步分为三类：VH在IgG的重链C端(PR000539,PR000540,PR000715)；VH在IgG的重链N端(PR001583,PR001586,PR001588,PR001591)；VH在IgG的轻链N端(PR001584,PR001589)。当VH在IgG的重链C端时，这些双抗分子的抑制活性与亲本单抗相比略弱，体现为相似的最大OD差值但是IC50值略弱仅1.5～2.5倍。当VH在IgG的无论重链或轻链的N端时，这些双抗分子的抑制活性与亲本单抗的相似。
图19的(E)-(F)中所示，IgG-VH-VH六价对称结构的双抗分子的抑制活性与亲本单抗的相似或略优。
图19的(G)-(H)中所示，2xVH-IgG六价对称结构的双抗分子的抑制活性与亲本单抗的相似或略优。
Figure PCTCN2021103044-appb-000043
Figure PCTCN2021103044-appb-000044
表4-16阻断人CTLA4蛋白和B7-1蛋白的结合
实施例4.5.2.阻断人CTLA4细胞和其配体蛋白的结合
利用流式细胞术FACS测定抗体分子抑制表达CTLA4的细胞与其配体B7-1/CD80结合的活性。具体地，消化高表达人CTLA4的CHO-K1细胞株CHO-K1/hCTLA4(睿智化学)，并置于FACS缓冲液(含有2％FBS的PBS缓冲液)重悬,将细胞密度调整为3x10 6细胞/mL。将CHO-K1/hCTLA4细胞以100μL/孔接种于96孔板(3x10 5细胞/孔)，500g转速于4℃下离心5分钟，弃上清。随后将梯度稀释的抗体分子以100μL/孔加入96孔板并混合均匀，抗体分子可以从最高终浓度为200nM按照3倍浓度梯度稀释的共8个浓度；hIgG1 iso(CrownBio,#C0001)作为同型对照。然后，将3μg/mL的生物素化的配体蛋白人B7-1-Fc-biotin(ACRO Biosystems,#B71-H82F2)以100μL/孔加入96孔板并混合均匀。将96孔板放置于4℃，避光孵育1小时。然后，加入200μL/孔预冷的FACS缓冲液漂洗细胞两次，4℃下500g离心5分钟，弃上清。接着再加入100μL/孔荧光标记(Streptavidin,Alexa Fluor TM488 conjugate,Thermo,#S32354,1:1000稀释)，放置于4℃，避光孵育1小时。随后以200μL/孔加入预冷的FACS缓冲液漂洗细胞三次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，将荧光信号值MFI转换成抑制率，通过四参数非线性拟合，得到抑制曲线、IC50值和最大抑制率等参数。
本实施例中，阳性对照分子为抗CTLA4的HCAb单抗PR000184，亦为HER2 x CTLA4双抗分子的CTLA4端的亲本单抗。结果显示于图20和表4-17。
图20的(A)-(B)中所示，当抗CTLA4的VH位于抗HER2的IgG的重链C端时，IgG-VH四价对称结构的双抗分子的抑制活性有较明显的降低，但是IgG-VH-VH六价对称结构的双抗分子(PR000541)的抑制活性与亲本单抗的相似。
图20的(C)中所示，Fab-Fc-VH(2)非对称结构的双抗分子PR002672含有两个串联形成的CTLA4结合结构域，其抑制活性与具有二价的亲本单抗相比，尽管IC50值略弱约4倍，但是能达到近100％抑制。
Figure PCTCN2021103044-appb-000045
表4-17阻断人CTLA4细胞和B7-1蛋白的结合
实施例4.6.结合HER2
本实施例是为了研究HER2 x CTLA4双抗分子结合HER2的活性。
实施例4.6.1.结合高表达人HER2的细胞SK-BR-3
利用流式细胞术FACS测试抗体与高表达人HER2的肿瘤细胞系SK-BR-3(ATCC,HTB-30)的结合能力。具体地，消化SK-BR-3细胞，并用DMEM完全培养基重悬，将细胞密度调整为1x10 6细胞/mL；接着以100μL细胞/孔接种于96孔V底板(Corning,#3894)，4℃下离心5分钟，弃上清。随后以100μL/孔加入以最高终浓度为100nM的5倍浓度梯度稀释的抗体分子，共8个浓度，混合均匀；hIgG1 iso(CrownBio,#C0001)作为同型对照。将细胞放置于4℃，避光孵育1小时。之后，4℃下离心5分钟，弃上清；随后以200μL/孔加入预冷的FACS缓冲液(含有0.5％BSA的PBS缓冲液)漂洗细胞两次，然后于500g，4℃下离心5分钟，弃上清。之后，以100μL/孔加入荧光二抗(Goat human lgG(H+L)Alexa Fluor 488 conjunction,Thermo,#A11013,1:1000稀释)，放置于4℃，避光孵育1小时。随后以200μL/孔加入预冷的FACS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
本实施例中，阳性对照分子为抗HER2的单抗PR000210(trastuzumab类似物)或PR000672(pertuzumab类似物)，亦为HER2 x CTLA4双抗分子的亲本单抗。结果显示于图21和表4-18。
图21的(A)中所示，Fab-HCAb对称结构的双抗分子结合HER2的能力与亲本单抗PR000210相似，体现在相似的EC50值和MFI最大值。这说明，具有Fab-HCAb对称结构的双抗分子的抗HER2的Fab端结构可以很好地保留了对HER2的结合能力。
图21的(B)-(E)中所示，IgG-VH四价对称结构的双抗分子结合HER2的能力根据其具体分子结构略有不同。根据抗CTLA4的VH端相对于抗HER2的IgG在双抗结构中的相对位置，将此类结构的双抗分子进一步分为三类：VH在IgG的重链C端(PR000539,PR000540)；VH在IgG的重链N端(PR001583,PR001586,PR001588,PR001591)；VH在IgG的轻链N端(PR001584,PR001974,PR001589)。从EC50值上看，当VH在IgG的重链C端时，该结构很好地保留了对HER2的结合能力，说明VH端没有对IgG的Fab端带来明显的影响；当VH在IgG的无论重链或轻链的N端时，双抗分子对HER2的结合能力相较于其对应的亲本单抗有1.5～2.5倍的降低。
图21的(F)-(G)中所示，IgG-VH-VH六价对称结构的双抗分子结合HER2的能力与对应的亲本单抗相似。
图21的(H)-(I)中所示，2xVH-IgG六价对称结构的双抗分子结合HER2的能力相较于其对应的亲本单抗有明显的降低；说明当VH同时连接到IgG的重链和轻链的N端后，对IgG的Fab端的功能带来明显的影响。
图21的(J)中所示，Fab-Fc-VH非对称结构的双抗分子PR000916的HER2结合结构域是单价Fab结构，但是其结合HER2的能力与具有二价的亲本单抗非常接近；说明trastuzumab的结合结构域在单价结合和二价结合上没有明显差别，而且Fab-Fc-VH非对称结构的双抗分子很好地保留了对HER2的结合能力。
抗体 EC50(nM) MFI最大值 抗体 EC50(nM) MFI最大值
PR000210 1.332 11868 PR000210 2.929 31552
PR000305 1.538 12604 PR001583 3.915 32008
PR000653 1.668 12781 PR001584 7.502 32880
PR000654 1.712 13168 PR001586 4.717 35248
PR000655 1.57 12201 PR001579 3.056 36444
PR000656 1.664 12358 PR001580 3.421 35754
PR000658 1.474 12964 PR001581 3.687 34744
PR000659 1.653 13672 PR001582 3.665 30517
PR000706 1.562 13355 PR001585 n.d. 26700
PR000716 1.386 13418 PR001587 n.d. 26800
PR000717 1.466 13684      
PR000539 1.323 9460      
PR000540 1.471 10894      
PR000541 1.282 7378      
PR000542 1.295 9019      
抗体 EC50(nM) MFI最大值 抗体 EC50(nM) MFI最大值
PR000210 1.913 7692 PR000210 1.863 10438
PR000916 3.688 9874 PR001586 2.975 9436
      PR001974 4.119 8478
抗体 EC50(nM) MFI最大值      
PR000672 3.119 33400      
PR001588 8.445 37900      
PR001589 5.52 37100      
PR001591 6.861 35900      
PR001590 未定 34000      
PR001592 未定 32800      
表4-18结合SK-BR-3
实施例4.7.超抗原SEB刺激实验
本实施例是为了研究HER2 x CTLA4双抗分子对外周血单个核细胞PBMC的激活作用。第一步，首先将分离的人PBMC(妙通生物)调整到5x10 6细胞/mL。接着将PBMC以50μL/孔加入96孔板(Corning,#3799)；随后以100μL/孔加入不同浓度的抗体分子，抗体终浓度梯度可以是(150nM,30nM,1nM)或者(80nM,8nM,0.8nM,0.08 nM)，两个复孔加样；hIgG1 iso(CrownBio,#C0001)作为同型对照。于37℃孵育30分钟。接着以50μL/孔加入400ng/mL的超抗原Staphylococcal enterotoxin B(SEB)，其终浓度为100ng/mL。置于37℃，5％CO 2培养箱中孵育72小时或者96小时后收集上清。第二步，将收集上清利用IL-2检测试剂盒(Thermo,#88-7025-77)检测上清中的IL-2浓度。应用软件GraphPad Prism 8进行数据处理和作图分析。
本实施例中，阳性对照分子为抗CTLA4的HCAb单抗PR000184或PR000218，亦为HER2 x CTLA4双抗分子的CTLA4端的亲本单抗。PR000218是在PR000184基础上通过Fc突变构建的ADCC增强的变体。
如图25所示，在有SEB刺激时，抗CTLA4的单抗以及HER2 x CTLA4双抗分子都可以不同程度地促进T细胞产生IL-2，其激活T细胞的能力与其结构有关。
图25的(F)中所示，与对应的亲本单抗相比，Fab-HCAb对称结构的双抗分子对T细胞的激活能力较弱。CTLA4端的活性在Fab-HCAb结构中相较于其在正常的IgG或HCAb双价结构中会减弱，从而可以降低其剂量相关的毒性，以适用于目前在临床的组合药物剂量。
图25的(A),(D),(G),(I)中所示，IgG-VH四价对称结构的双抗分子对T细胞的激活能力根据其具体分子结构略有不同。根据抗CTLA4的VH端相对于抗HER2的IgG在双抗结构中的相对位置，将此类结构的双抗分子进一步分为三类：VH在IgG的重链C端(PR000539,PR000715)；VH在IgG的重链N端(PR001583,PR001586)；VH在IgG的轻链N端(PR001584,PR001974)。当VH在IgG的重链C端时，双抗分子对T细胞的激活能力与对应的亲本单抗相比明显减弱。当VH在IgG的无论重链或轻链的N端时，双抗分子对T细胞的激活能力与对应的亲本单抗相似甚至略有增强。这说明，通过调整VH在IgG上的相对位置可以用来调节VH端的抗CTLA4的活性，以适用不同的临床组合药物剂量的场景。
图25的(C)中所示，IgG-VH-VH六价对称结构的双抗分子对T细胞的激活能力与亲本单抗的相似，其CTLA4端的活性可以通过使用不同的连接肽来调节。
图25的(B),(E)中所示，2xVH-IgG六价对称结构的双抗分子对T细胞的激活能力与亲本单抗的相似，说明其几乎完全保留了CTLA4端的活性。
图25(H)中所示，Fab-Fc-VH(2)非对称结构的双抗分子含有两个串联形成的CTLA4结合结构域，其对T细胞的激活能力与对应的亲本单抗相比有减弱。其CTLA4端可以降低其剂量相关的毒性，以适用于目前在临床的组合药物剂量。
实施例4.8.抑制HER2+细胞的增殖
本实施例是为了研究HER2 x CTLA4双抗分子抑制高表达人HER2的肿瘤细胞系SK-BR-3(ATCC,HTB-30)的增殖。具体地，消化SK-BR-3细胞，并用DMEM完全培养基重悬。以2000个细胞/50μL接种于96孔板中(Perkin Elmer,#6005225)；然后，于37℃，5％CO 2培养箱中孵育过夜。第二天，以50μL/孔加入以最高终浓度为100nM的5倍浓度梯度稀释的抗体分子，共6个浓度，混合均匀；hIgG1 iso(CrownBio,#C0001)作为同型对照；于37℃，5％CO 2培养箱中孵育7天。之后，每孔加入100μL CellTiter-Glo(Promega,#G7573)并在水平振动摇床上混合10分钟以诱导细胞裂解。最后，用Enspire TM多功能读板机(PerkinElmer,Inc.)测定化学发光值。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的抑制率等参数。
本实施例中，阳性对照分子为抗HER2的单抗PR000210(trastuzumab类似物)，亦为HER2 x CTLA4双抗分子的亲本单抗。结果显示于图23和表4-19。
图23的(A)中所示，Fab-HCAb对称结构的双抗分子PR000655对SK-BR-3细胞增殖的抑制能力相较于亲本单抗PR000210有较明显的降低。
图23的(B)-(C)中所示，IgG-VH四价对称结构的双抗分子对SK-BR-3细胞增殖的抑制作用根据其具体分子结构略有不同。根据抗CTLA4的VH端相对于抗HER2的IgG在双抗结构中的相对位置，将此类结构的双抗分子进一步分为三类：VH在IgG的重链C端(PR000539,PR000540,PR000714,PR000715)；VH在IgG的重链N端(PR001583,PR001586)；VH在IgG的轻链N端(PR001584)。当VH在IgG的重链C端时，该结构很好地保留了对SK-BR-3细胞增殖的抑制能力，体现在与亲本单抗有相近甚至略优的IC50和最大抑制率；说明VH端没有对IgG的Fab端带来明显的影响。当VH在IgG的无论重链或轻链的N端时，双抗分子对SK-BR-3细胞增殖的抑制能力相较于亲本单抗略有降低，体现在IC50值有3～4倍差异，但是仍能保留相同的最大抑制率。
图23的(D)-(E)中所示，除了PR000542显示出降低的最大抑制率水平以外，大多数IgG-VH-VH六价对称结构的双抗分子对SK-BR-3细胞增殖的抑制能力与亲本单抗相近。该结果与前文关于对HER2的结合能力的结果是一致的。
图23的(F)中所示，2xVH-IgG六价对称结构的双抗分子对SK-BR-3细胞增殖的抑制能力相较于亲本单抗有明显的降低。该结果与前文关于对HER2的结合能力的结果是一致的。
Figure PCTCN2021103044-appb-000046
表4-19抑制SK-BR-3的增殖
实施例4.9.HER2介导的ADCC效应
本实施例是为了研究HER2 x CTLA4双抗分子对高表达人HER2的肿瘤细胞系BT-474(ATCC,HTB-20)的抗体依赖的细胞介导的细胞毒性(ADCC)。第一步，用DELFIA BATDA(Perkin Elmer,#C136-100)标记靶细胞。具体标记方法如下：用2μL DELFIA BATDA试剂标记1x10 6靶细胞；于37℃，CO 2培养箱中孵育20分钟；用PBS洗涤4次，于1000rpm离心5分钟；最后一次洗涤后，将沉淀重悬于含20％FBS的RPMI-1640培养基(Thermo,#A 10491)中，并调整细胞密度至1x10 5/ml。第二步，将标记好的靶细胞以100μL/孔接种于96孔板(Corning,#3599)；随后以50μL/孔加入以最高终浓度为100nM的5倍浓度梯度稀释的抗体分子，共10个浓度，混合均匀；hIgG1 iso(CrownBio,#C0001)作为同型对照；于37℃，5％CO 2培养箱中孵育10分钟。第三步，收集NK-92MI/CD16a细胞(睿智化学)作为效应细胞，将NK-92MI/CD16a细胞密度调 至1.2x10 6/ml；随后以50μL/孔加入到96孔板中，效应细胞:靶细胞＝6:1。接着，于37℃，5％CO 2培养箱中孵育4小时。第四步，以500g离心5分钟，然后从每孔取25μL上清加到一块新的96孔检测板中；随后以200μL/孔加入
Figure PCTCN2021103044-appb-000047
Europium溶液(Perkin Elmer,#C135-100)，并在室温下以250rpm摇动平板15分钟。最后，用
Figure PCTCN2021103044-appb-000048
多功能读板机(Perkin Elmer,Inc.)测量荧光值。
根据如下公式计算杀伤比率：
杀伤率(％)＝(ER–ETSR)/(TMR–ETSR)*100％
其中：
ER＝实验孔(抗体+效应细胞+靶细胞)；ETSR＝靶细胞和效应细胞混合自发释放孔(效应细胞+靶细胞)；TMR＝靶细胞最大释放孔(靶细胞+裂解液)
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的杀伤率曲线和EC50及最大杀伤率等参数。
本实施例中，阳性对照分子为抗HER2的单抗PR000210(trastuzumab类似物)，亦为HER2 x CTLA4双抗分子的亲本单抗。
图22和表4-20中所示，具有IgG-VH-VH六价对称结构的双抗分子PR001582和具有IgG-VH四价对称结构的双抗分子PR001586都能引起很强的ADCC作用。
抗体 EC50(nM) 杀伤率最大值(％)
PR000210 0.183 41.95
PR001582 0.055 94.65
PR001586 0.053 94.66
表4-20 ADCC效应杀伤靶细胞BT-474
实施例4.10.利用流式细胞术测定双抗分子同时结合两种细胞的能力
本实施例是为了研究HER2 x CTLA4双抗分子是否能同时结合表达人HER2的细胞SK-BR-3(ATCC,HTB-30)和表达人CTLA4的细胞CHO-K1/hCTLA4(睿智化学)的能力。第一步，用CFSE(Thermo,#C34554)和Far red(Thermo,#C34564)染料分别对CHO-K1/hCTLA4细胞和SK-BR-3细胞进行荧光标记。具体步骤如下：将CHO- K1/hCTLA4细胞和SK-BR-3细胞分别重悬于PBS中，并调整至2x10 6/ml；分别向CHO-K1/hCTLA4细胞和SK-BR-3细胞中加入5μM CFSE和1μM Far red，并在37℃下孵育10分钟，并不时摇动；加入5倍量完全培养基以终止染色。之后，4℃下离心5分钟，弃上清，随后加入PBS洗涤两遍。将标记后的CHO-K1/hCTLA4细胞和SK-BR-3细胞的密度分别调整为4 x10 6/ml和2 x10 6/ml。第二步，将标记好的CHO-K1/hCTLA4细胞和SK-BR-3细胞各取25μl，与50μl四倍浓度梯度稀释的抗体分子混合均匀，加入到96孔板，其中抗体分子最高终浓度为31.25nM，共6个浓度，hIgG1 iso(CrownBio,#C0001)作为同型对照；避光4℃孵育1小时。之后，每孔添加100μl 4％多聚甲醛PFA固定细胞10分钟。最后，使用BD FACS CANTOII流式细胞仪读取荧光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。根据不同的荧光标记分别统计不同象限内细胞数目：Q1是Far-red标记的SK-BR-3细胞(SK-BR-3 +)；Q3是CFSE标记的CHO-K1/hCTLA4细胞(CTLA4 +)；Q2则包含了由于双抗分子同时结合两种细胞而形成的具有两种标记的细胞群(CTLA4 +&SK-BR-3 +)。根据如下公式计算双标记阳性的细胞群(Q2)相对于所有被标记的SK-BR-3细胞(Q1+Q2)的占比，反映了双抗分子同时结合两种细胞的能力。
Q2的细胞比率：(Q2/(Q1+Q2))*100％
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体同时对两种靶细胞结合的曲线。
本实施例中，抗HER2的单抗PR000210(trastuzumab类似物)和抗CTLA4的单抗PR000184作为对照分子，亦为HER2 x CTLA4双抗分子的亲本单抗。
图24的A、B和C中分别显示了IgG-VH四价对称结构双抗(PR000539,PR000540,PR000715)，IgG-VH-VH六价对称结构双抗(PR000541和PR000542)，Fab-Fc-VH非对称结构双抗(PR000916)同时结合CHOK1/CTLA4和SK-BR-3细胞的能力；其中PR000210、PR000184和hIgG1 iso为对照。对照分子HER2单抗PR000210和CTLA4单抗PR000184都不能同时结合两种细胞。所示的HER2 x CTLA4双抗分子在对称或非对称结构中，可以同时结合两种表达不同靶标的细胞。
实施例4.11.药代动力学研究
本实施例研究了抗体分子在小鼠体内的药代动力学性能。本实施例测试了如下抗体有：抗HER2的单抗PR000210(trastuzumab类似物)；具有IgG-VH四价对称结构的HER2 x CTLA4双抗分子PR000540；具有IgG-VH-VH六价对称结构的HER2 x CTLA4双抗分子PR000541。
实施方法如下：对于每一个测试抗体分子，选取体重18～22克的雌性C57BL/6小鼠3只，按5mg/kg的剂量通过静脉注射给药；于给药前以及给药后0.5小时、24小时(1天)、第2天、第4天、第7天、第10天和第14天采集全血，将全血静置30分钟使其凝固，随后在4℃下以12,000g离心5分钟并将分离的血清样品在-80℃下冻存直至分析。
本实施例采用Fc端检测ELISA方法来定量测定小鼠血清中的药物浓度。即通过包被于96孔板的山羊抗人Fc多克隆抗体来捕获小鼠血清中的含有人Fc的融合蛋白，然后加入HRP标记的山羊抗人Fc第二抗体来检测。使用Phoenix WinNonlin软件6.4版，选用非房室模型(NCA)对血药浓度数据进行分析以评价其药代动力学。
图26和表4-21中所示，双抗PR000540和亲本单抗PR000210有相似的清除速率，且PR000540有更长的半衰期t 1/2值(超过20天)。可能是由于其复杂的结构，PR000541显示出较快的清除速率。
Figure PCTCN2021103044-appb-000049
表4-21小鼠体内药代动力学
实施例4.12.小结
本实施例利用抗HER2的IgG抗体的抗原结合结构域Fab和抗CTLA4的HCAb抗体的抗原结合结构域VH，构建了多种结构的抗HER2 x CTLA4的双特异性抗体分子。展现出了基于HCAb构建双特异性抗体分子结构的灵活性，通过不同的结构类型、相对 位置、结合价数等参数来调节HER2端和CTLA4端的功能活性，进而设计出不同的活性组合，以实现不同的作用机制的需求。目前，抗HER2的单抗trastuzumab在治疗乳腺癌的推荐初始剂量是4mg/kg，在治疗胃癌的推荐初始剂量是8mg/kg,；略高于抗CTLA4的单抗Ipilimumab在治疗黑色素瘤的推荐剂量3mg/kg。
例如，当抗CTLA4的VH在抗PD-L1的IgG的N端时，双抗分子(例如PR001583和PR001584)的HER2端和CTLA4端都能几乎完全保持与其亲本单抗相当的活性，而且在肿瘤细胞增殖抑制实验和超抗原刺激实验等体外功能实验中，显示出与相应的亲本单抗的相似的能力；因而可以用来实现联合用药的1:1的剂量组合。
又例如，IgG-VH-VH六价对称结构的双抗分子(例如PR001579,PR001580,PR001581)的HER2端和CTLA4端也都能几乎完全保持与其亲本单抗相当的活性，因而可以用来实现联合用药的1:1的剂量组合。
又例如，当抗CTLA4的VH在抗HER2的IgG的C端时，或者在Fab-HCAb结构时，双抗分子(例如PR000539,PR000540,PR000655)HER2端的活性几乎与其亲本单抗相当，但是其CTLA4端的活性有不同程度的减弱；因而可以用来实现临床上对CTLA4抑制剂的中等或者低剂量的需求。
又例如，在Fab-Fc-VH(2)非对称结构中，双抗分子的HER2结合结构域是单价Fab结构，但是其结合HER2的能力与具有二价的亲本单抗非常接近；其含有两个串联形成的CTLA4结合结构域，其结合CTLA4的能力与亲本单抗相似，但是T细胞激活能力比亲本单抗相比有所减弱；因而这种结构可以把肿瘤靶细胞和T细胞拉到一起，促进免疫突触的形成，可以用来实现临床上对CTLA4抑制剂的中等或者低剂量的需求，同时还增加了肿瘤特异性地靶向能力。
实施例5.PD-L1 x 4-1BB双特异性抗体
实施例5.1.背景
程序性死亡受体1(programmed death 1,PD-1)主要表达于T细胞等免疫细胞，它有两个配体，即程序性死亡配体-1(programmed death ligand 1,PD-L1)和PD-L2。PD-L1主要表达在抗原呈递细胞以及多种肿瘤细胞。PD-L1与PD-1相互作用会下调T细胞的活性，减弱细胞因子的分泌，起到免疫抑制作用。在许多人类肿瘤组织中均可检测到 PD-L1蛋白的表达，肿瘤部位的微环境可诱导肿瘤细胞上的PD-L1的表达，表达的PD-L1有利于肿瘤的发生和生长，诱导抗肿瘤T细胞的凋亡，并进一步保护肿瘤细胞逃避免疫攻击。
4-1BB(TNFRSF9,CD137)是一种隶属于TNF受体超家族的跨膜蛋白。4-1BB是在多种免疫细胞上表达的共刺激分子，为免疫活性的多功能调节剂。其诱导表达于活化的T细胞、NK细胞等免疫细胞。4-1BB通过其配体4-1BBL介导的三聚化来激活T细胞，促进细胞增殖和细胞因子释放。抗4-1BB的激动型抗体具有抑制肿瘤的功能，最早进入临床试验的4-1BB抗体是辉瑞的Utomilumab和百时美施贵宝(BMS)公司的Urelumab(BMS-663513)。Urelumab最初的临床结果发表于2008年，尽管在部分患者上观察到令人鼓舞的疗效，但数据显示Urelumab导致肝脏毒性，且与靶标和剂量有关。并且，因为在临床试验中有两位患者因肝毒性死亡，导致相关的临床试验被终止。Utomilumab安全性更好，剂量可提高至10mg/kg，但治疗效果依然欠佳。
在本实施例中，我们构建了同时靶向PD-L1和4-1BB的双特异性抗体，通过一个或者多个作用机制来提高抗肿瘤效果和安全性。第一，PD-L1 x 4-1BB双抗可以通过阻断PD-1/PD-L1信号通路来激活T细胞。第二，高表达于肿瘤细胞表面的PD-L1分子可以利用双抗分子促进T细胞表面的4-1BB分子的交联和三聚化并激活下游信号传导通路，进而促进T细胞的活化和增殖。第三，双抗分子介导的T细胞激活仅限于在肿瘤微环境内，这样可以避免类似Urelumab的单抗在正常组织中过度激活T细胞所带来的毒副作用。
实施例5.2.获得抗PD-L1的IgG抗体和抗4-1BB的H2L2或HCAb抗体
实施例5.2.1.获得抗PD-L1的全人源IgG抗体
本实施例使用的抗PD-L1的全人源IgG抗体PR000265(表5-8)来源于Harbour H2L2小鼠，其发现过程如实施例3.2.1所述。
实施例5.2.2.获得抗4-1BB的全人源H2L2抗体
Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域。
用可溶的重组人4-1BB-Fc融合蛋白(GenScript Biotech)对Harbour H2L2小鼠进行多轮免疫。当检测小鼠血清中4-1BB特异的抗体滴度达到一定的水平后，将小鼠的脾细 胞取出并与骨髓瘤细胞系融合得到杂交瘤细胞；对杂交瘤细胞经过多轮筛选和克隆之后，鉴定出若干个特异识别4-1BB的单克隆抗体分子。对这些单克隆抗体进行进一步的鉴定，根据其对人4-1BB的结合能力、食蟹猴4-1BB的结合能力、T细胞激活能力等参数，优选出数个候选抗体分子。然后对候选抗体分子进行序列分析和优化，得到数个变体序列。将抗体的VL和VH序列与相应的人的κ轻链恒定区和IgG1重链恒定区序列进行融合表达，得到重组全人源抗体分子。抗4-1BB的重组全人源IgG抗体PR000197和PR000448列于表5-8。
利用流式细胞术FACS和实施例5.6所述方法测试4-1BB抗体PR000197、PR000448结合高表达人4-1BB的CHO-K1细胞株CHO-K1/hu4-1BB(南京金斯瑞,M00538)的结合能力，如图28的(A)–(B)所示，其结合能力与Utomilumab相似。
实施例5.2.3.获得抗4-1BB的全人源HCAb抗体
Harbour HCAb小鼠(Harbour Antibodies BV，WO2010/109165A2)是一种携带人免疫球蛋白免疫库的转基因小鼠，能够产生仅有重链的抗体，该抗体的大小只有传统IgG抗体的一半。其产生的抗体仅具有人的抗体重链可变结构域和小鼠Fc恒定结构域。
用可溶的重组人4-1BB-Fc融合蛋白(睿智化学)或者过表达了人4-1BB的NIH-3T3细胞(睿智化学)对Harbour HCAb小鼠进行多轮免疫。当检测小鼠血清中4-1BB特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出分离B细胞，用小鼠浆细胞分选试剂盒(Miltenyi,#130-092-530)分选CD138阳性的浆细胞。用常规的分子生物学手段从浆细胞中扩增人VH基因，并将扩增的人VH基因片段构建到编码人IgG1抗体重链Fc区域序列的哺乳动物细胞表达质粒pCAG载体中。质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)进行表达，得到全人源HCAb抗体上清。用FACS测试HCAb抗体上清与高表达人4-1BB的CHO-K1细胞CHO-K1/hu4-1BB的结合，鉴定出阳性HCAb抗体。对这些HCAb抗体进行进一步的鉴定，根据其对人4-1BB的结合能力、食蟹猴4-1BB的结合能力、T细胞激活能力等参数，优选出数个候选HCAb抗体分子。然后对候选HCAb抗体分子进行序列分析和优化，得到数个变体序列。将HCAb抗体的VH序列和人的IgG1重链Fc序列进行融合表达，得到全人源重组HCAb抗体分子。抗4-1BB的重组全人源HCAb抗体PR001758、PR001760和PR001836列于表5-8。
利用流式细胞术FACS和实施例5.6所述方法测试4-1BB重链抗体结合细胞CHO-K1/hu4-1BB(南京金斯瑞,M00538)的结合能力，如图28的(C)-(F)所示，其结合能力与Utomilumab相似，优于Urelumab。
实施例5.3.利用抗PD-L1的IgG抗体和抗4-1BB的IgG抗体构建具有FIT-Ig结构的双特异性抗体分子
本实施例利用抗PD-L1的IgG抗体PR000265或PR000151(atezolizumab类似物)的抗原结合结构域Fab，和抗4-1BB的IgG抗体PR000197或PR000448的抗原结合结构域Fab，来构建具有FIT-Ig结构的抗PD-L1 x 4-1BB的双特异性抗体分子。FIT-Ig结构的设计可以参考专利WO2015/103072A1，结构见图1的(28)所示；所构建的分子总结于表5-1；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表5-2。表5-3列出了所述FIT-Ig结构的双抗分子的多肽链序列对应的序列编号。
Figure PCTCN2021103044-appb-000050
表5-1具有FIT-Ig结构的PD-L1 x 4-1BB双抗分子
Figure PCTCN2021103044-appb-000051
表5-2 FIT-Ig结构的PD-L1 x 4-1BB双抗分子蛋白的表达
抗体编号 多肽链1 多肽链2 多肽链3
PR000701 384 371 355
PR003052 452 451 355
表5-3 FIT-Ig结构的PD-L1 x 4-1BB双抗分子的序列编号表
实施例5.4.利用抗PD-L1的IgG抗体和抗4-1BB的HCAb抗体构建双特异性抗体分子
本实施例利用抗PD-L1的IgG抗体PR000265的抗原结合结构域Fab，和抗4-1BB的HCAb抗体PR001758、PR001760或PR001836的抗原结合结构域VH，来构建多种结构的抗PD-L1 x 4-1BB的双特异性抗体分子。
在本实施例及后续实施例中，阳性对照分子为抗PD-L1的IgG单抗PR000265，亦为PD-L1 x 4-1BB双抗分子的PD-L1端的亲本单抗。阳性对照分子为抗4-1BB的IgG单抗urelumab(IgG4)或utomilumab(IgG2)。阳性对照分子为PD-L1 x 4-1BB双抗分子PR001289，其序列来源于专利WO2017/123650A2所公开的抗4-1BB及抗PD-L1的单域抗体序列。
实施例5.4.1.构建Fab-HCAb对称结构分子
利用抗PD-L1的IgG抗体和抗4-1BB的重链抗体，按照实施例1.1所述结构设计Fab-HCAb对称结构的PD-L1 x 4-1BB双抗分子，总结于表5-4；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表5-5。
Figure PCTCN2021103044-appb-000052
表5-4 Fab-HCAb对称结构的PD-L1 x 4-1BB双抗分子
Figure PCTCN2021103044-appb-000053
表5-5 Fab-HCAb对称结构的PD-L1 x 4-1BB双抗分子蛋白的表达
实施例5.4.2.构建IgG-VH四价对称结构分子
利用抗PD-L1的IgG抗体和抗4-1BB的重链抗体，按照实施例1.2所述结构设计IgG-VH四价对称结构的PD-L1 x 4-1BB双抗分子，总结于表5-6；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表5-7。
Figure PCTCN2021103044-appb-000054
表5-6 IgG-VH四价对称结构的PD-L1 x 4-1BB双抗分子
Figure PCTCN2021103044-appb-000055
表5-7 IgG-VH四价对称结构的PD-L1 x 4-1BB双抗分子蛋白的表达
实施例5.4.3.PD-L1 x 4-1BB双抗分子及对照分子序列表
表5-8、表5-9和表5-10列出了本实施例所构建的PD-L1 x 4-1BB双抗分子和对应的PD-L1单抗、4-1BB单抗等亲本单抗分子以及对照分子的序列所对应的序列号。表5-10中的结构编号对应于表1-1和图1。表5-11列出了双特异性抗体分子的第一和第二抗原结合结构域相应的CDR序列的序列编号。
Figure PCTCN2021103044-appb-000056
Figure PCTCN2021103044-appb-000057
表5-8对照分子和亲本单抗
Figure PCTCN2021103044-appb-000058
表5-9对照分子和亲本单抗的序列和CDR序列的序列编号表
结构编号 抗体编号 多肽链1(短链) 多肽链2(长链)
5 PR003549 353 460
5 PR003550 353 461
5 PR003551 353 462
1 PR004270 371 486
2 PR007163 353 521
1 PR007164 371 522
表5-10本实施例的PD-L1 x 4-1BB双抗分子的序列编号表
Figure PCTCN2021103044-appb-000059
表5-11 PD-L1 x 4-1BB双抗分子的抗原结合结构域的CDR的序列编号表
实施例5.5.结合PD-L1
本实施例是为了研究PD-L1 x 4-1BB双抗分子结合PD-L1的活性。
实施例5.5.1.结合高表达人PD-L1的CHO-K1细胞CHO-K1/hPDL1
利用流式细胞术FACS测试抗体分子与高表达人PD-L1的CHO-K1细胞株CHO-K1/hPDL1(南京金斯瑞,M00543)的结合能力。具体地，消化CHO-K1/hPDL1细胞并用完全培养基重悬；将细胞密度调整为1x10 6细胞/mL。接着将细胞以100μL/孔接种于96孔V底板(Corning,#3894)，4℃下离心5分钟，弃上清。随后将梯度稀释的抗体分子以100μL/孔加入96孔板并混合均匀，抗体分子可以从最高终浓度为200nM按照3倍浓度梯度稀释的共12个浓度；hIgG1 iso(CrownBio,#C0001)作为同型对照。将细胞放置于4℃，避光孵育1小时。然后，加入100μL/孔预冷的FACS缓冲液(含有0.5％BSA的 PBS缓冲液)漂洗细胞两次，4℃下500g离心5分钟，弃上清。接着，再加入100μL/孔荧光二抗(Goat human lgG(H+L)Alexa Fluor 488conjunction,Thermo,#A11013,1:1000稀释)，放置于4℃，避光孵育1小时。随后以200μL/孔加入预冷的FACS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
本实施例中，阳性对照分子为抗PD-L1的单抗PR000265，亦为PD-L1 x 4-1BB双抗分子的PD-L1端的亲本单抗。结果显示于图27和表5-12。
图27的(A)中所示，IgG-VH四价对称结构的双抗分子(PR003549,PR003550,PR003551)结合PD-L1的能力与亲本单抗PR000265的相似，而且其结合PD-L1的EC50值和MFI最大值略优于FIT-Ig结构的双抗分子(PR000701,PR003052)。
图27的(B)中所示，Fab-HCAb对称结构的双抗分子(PR004270)结合PD-L1的能力与亲本单抗相似，其结合PD-L1的EC50值虽略弱于亲本单抗，但是结合的MFI最大值比亲本单抗更高。
图27的(C)中所示，Fab-HCAb对称结构的双抗分子，无论是结构(1)的分子(PR007164)还是结构(2)的分子(PR007163)，都有几乎相同的PD-L1结合活性；说明将Fab的CL融合到VH_B所在的重链上不影响Fab端的结合活性。
Figure PCTCN2021103044-appb-000060
表5-12结合CHO-K1/hPDL1
实施例5.6.结合4-1BB
本实施例是为了研究PD-L1 x 4-1BB双抗分子结合4-1BB的活性。
利用流式细胞术FACS测试抗体分子与高表达人4-1BB的CHO-K1细胞株CHO-K1/hu4-1BB(南京金斯瑞,M00538)和高表达食蟹猴4-1BB的CHO-K1细胞株CHO-K1/cyno4-1BB(南京金斯瑞,M00569)等细胞的结合能力。具体地，消化细胞并用完全培养基重悬；将细胞密度调整为2x10 6细胞/mL。接着将细胞以100μL/孔(2x10 5细胞/孔)接种于96孔V底板(Corning,#3894)，4℃下离心5分钟，弃上清。随后将梯度稀释的抗体分子以100μL/孔加入96孔板并混合均匀，抗体分子可以从最高终浓度为200nM按照3倍浓度梯度稀释的共12个浓度；hIgG1 iso(CrownBio,#C0001)作为同型对照。将细胞放置于4℃，避光孵育1小时。然后，加入100μL/孔预冷的FACS缓冲液(含有0.5％BSA的PBS缓冲液)漂洗细胞两次，4℃下500g离心5分钟，弃上清。接着，再加入100μL/孔荧光二抗(Goat human lgG(H+L)Alexa Fluor 488 conjunction,Thermo,#A11013,1:1000稀释)，放置于4℃，避光孵育1小时。随后以200μL/孔加入预冷的FACS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
本实施例中，阳性对照分子为抗4-1BB的单抗Urelumab或Utomilumab。
实施例5.6.1.结合高表达人4-1BB的CHO-K1细胞CHO-K1/hu4-1BB
结果显示于图28和表5-13。
图28的(G)中所示，IgG-VH四价对称结构的双抗分子(PR003549,PR003550,PR003551)结合人4-1BB的能力优于FIT-Ig结构的双抗分子(PR000701,PR003052)，而且在MFI最大值上优于阳性对照Urelumab。
图28的(H)中所示，Fab-HCAb对称结构的双抗分子(PR004270)结合人4-1BB的能力在MFI最大值上优于阳性对照Urelumab和Utomilumab。
图28的(I)中所示，Fab-HCAb对称结构的双抗分子，无论是结构(1)的分子(PR007164)还是结构(2)的分子(PR007163)，都有几乎相同的4-1BB结合活性，且与亲本单抗PR001760的相当。
Figure PCTCN2021103044-appb-000061
表5-13结合CHO-K1/hu 4-1BB
实施例5.6.2.结合高表达食蟹猴4-1BB的CHO-K1细胞CHO-K1/cyno 4-1BB
图29和表5-14中所示，本实施例的双抗分子可以结合食蟹猴4-1BB，而Urelumab不能。双抗分子结合食蟹猴4-1BB(图29,(A)-(B))的能力与结合人4-1BB(图28,(G)-(H))的能力相当，有相似的结合EC50。
Figure PCTCN2021103044-appb-000062
Figure PCTCN2021103044-appb-000063
表5-14结合CHO-K1/cyno 4-1BB
实施例5.7.高表达PD-L1的靶细胞介导的T细胞的特异性激活
本实施例是为了研究PD-L1 x 4-1BB双抗分子在靶细胞的存在是通过结合4-1BB来激活T细胞的活性。靶细胞可以是不同程度表达PD-L1的细胞，例如高表达人PD-L1的CHO-K1/hPDL1(南京金斯瑞,M00543)，或者高表达人PD-L1的MDA-MB-231(ATCC,HTB-26)。效应细胞可以是分离的人PBMC或者T细胞。
具体的，首先以100μL/孔将0.3μg/mL抗CD3抗体OKT3(Thermo,#16-0037-81)包板于96孔板(Corning,#3599)。接着，将人T细胞(从人PBMC中用T细胞分选试剂盒(Miltenyi,#130-096-535)分离得到)的密度调整为2 x10 6细胞/mL，将靶细胞的密度调整为3 x10 5细胞/mL，随后把两种细胞悬液各以50μL/孔接种于96孔板，最终效靶比为20:3。然后，以100μL/孔加入不同浓度的抗体分子，抗体终浓度可以是(10nM,1nM)，或者是20nM，或者是从最高终浓度为20nM按照5倍浓度梯度稀释的共8个浓度，两个复孔加样；hIgG1 iso(CrownBio,#C0001)和hIgG4iso(CrownBio,#C0045)作为对照。将96孔板置于37℃，5％CO 2培养箱中孵育3天。分别收集培养48小时后和72小时后的上清液，用IL-2ELISA试剂盒(Thermo,#88-7025-88)检测48小时后的上清中IL-2浓度，用IFN-γELISA试剂盒(Thermo,#88-7316-77)检测72小时后的上清中IFN-γ浓度。ELISA检测方法参照相关试剂盒操作说明。应用软件GraphPad Prism 8进行数据处理和作图分析。
本实施例中，阳性对照分子为抗4-1BB的单抗Urelumab。
实施例5.7.1.CHO-K1/hPDL1介导的T细胞特异性激活
图30中所示，在靶细胞CHO-K1/hPDL1和T细胞混合的系统中，不依赖于交联的抗4-1BB单抗Urelumab可以激活T细胞释放IFN-γ，而依赖于交联的抗4-1BB单抗(PR000448,PR001758,PR001760,PR001836)则几乎不能激活T细胞。FIT-Ig结构的 双抗分子(PR003052,PR000701)和IgG-VH四价对称结构的双抗分子(PR003549,PR003550,PR003551)和Fab-HCAb结构的双抗分子(PR004270)都能够激活T细胞并释放细胞因子。这说明双抗分子对T细胞的激活是依赖于靶细胞的特异性的激活。而且IgG-VH四价对称结构的双抗分子(PR003549,PR003550)和Fab-HCAb结构的双抗分子(PR004270)比FIT-Ig结构的双抗分子(PR003052,PR000701)能够引起更高的细胞因子释放水平，显示出更强的T细胞激活能力，且优于Urelumab。而且PR003549和PR004270与对照双抗分子(PR001289)有相当甚至更强的T细胞激活能力。
实施例5.7.2.MDA-MB-231介导的T细胞特异性激活
图31的(A)中所示，在靶细胞MDA-MB-231和T细胞混合的系统中，IgG-VH四价对称结构的双抗分子(PR003549)和Fab-HCAb结构的双抗分子(PR004270)有相似的T细胞激活能力；优于FIT-Ig结构的双抗分子(PR003052,PR000701)和对照双抗分子(PR001289)。
图31的(B)-(C)和表5-15中所示，在靶细胞MDA-MB-231和T细胞混合的系统中，IgG-VH四价对称结构的双抗分子(PR003549)比Urelumab有更强T细胞激活能力，能更好地促进IFN-γ(B)和IL-2(C)的产生。
Figure PCTCN2021103044-appb-000064
表5-15细胞因子水平
实施例5.8.混合淋巴细胞反应(MLR)
本实施例是利用混合淋巴细胞反应(MLR)来研究PD-L1 x 4-1BB双抗分子对T细胞的激活作用。
第一步，利用CD14磁珠(Meltenyi,#130-050-201)从第一供体PBMC细胞(妙通生物)中分离单核细胞(monocytes)；具体操作参照相关试剂盒说明书。然后加入50ng/mL重组人源IL-4(PeproTech,#200-02-A)和100ng/mL重组人源GM-CSF(PeproTech,#300-03-A)，于37℃诱导7天后，获得未成熟的树突状细胞(iDC细胞)。 继续加入1μg/ml的脂多糖Lipopolysaccharide(LPS,Sigma,#L6529)，诱导24小时后，获得成熟的树突状细胞(mDC细胞)。第二步，利用T细胞分离试剂盒(Meltenyi,#130-096-535)从第二供体PBMC细胞(妙通生物)中分离得到T淋巴细胞。第三步，将获得的T细胞和mDC细胞按5:1比例接种至96孔板(1×10 5/孔的T细胞和2×10 4/孔的mDC细胞)。随后以50μL/孔加入不同浓度的抗体分子，抗体终浓度可以是(10nM,1nM)，或者是从最高终浓度为50nM按照3倍浓度梯度稀释的共8个浓度，两个复孔加样；hIgG1 iso(CrownBio,#C0001)或者空白孔作为对照。于37℃，5％CO 2培养箱孵育5天。第四步，分别收集第3天和第5天的上清液，用IL-2ELISA试剂盒(Thermo,#88-7025-88)检测第3天的上清中IL-2浓度，用IFN-γELISA试剂盒(Thermo,#88-7316-77)检测第5天的上清中IFN-γ浓度。ELISA检测方法参照相关试剂盒操作说明。
图32和图33中所示，在两次独立的MLR实验中(不同的供体配对)，抗4-1BB单抗对T细胞的激活作用有限，产生细胞因子的能力很弱；抗PD-L1单抗有较明显的激活作用。双抗分子可以进一步提高T细胞的功能，优于抗PD-L1单抗。
图32中所示，IgG-VH四价对称结构的双抗分子(PR003549,PR003550,PR003551)比FIT-Ig结构的双抗分子(PR003052,PR000701)能够引起更高的细胞因子释放水平，显示出更强的T细胞激活能力；而且双抗分子优于抗PD-L1单抗。
图33中所示，IgG-VH四价对称结构的双抗分子(PR003549,PR003550)和Fab-HCAb结构的双抗分子(PR004270)比FIT-Ig结构的双抗分子(PR003052,PR000701)能够引起更高的细胞因子释放水平，显示出更强的T细胞激活能力。而且相较于对照双抗分子(PR001289)，IgG-VH和和Fab-HCAb结构的双抗分子能够刺激产生更多的IFN-γ。
实施例5.9.药代动力学研究
本实施例研究了具有Fab-HCAb对称结构的PD-L1 x 4-1BB双抗分子PR004270在小鼠体内的药代动力学性能。
实施方法如下：对于每一个测试抗体分子，选取体重18～22克的雌性BALB/c小鼠6只，按5mg/kg的剂量通过静脉注射给与双特异性抗体。一组3只于给药前以及给药后15分钟、24小时(1天)、第4天、和第10天采集全血，另一组3只于只于给药前以及给药后5小时、第2天、第7天、和第14天采集全血。将全血静置30分钟使其凝 固，随后离心并将分离的血清样品在-80℃下冻存直至分析。本实施例采用两种ELISA方法来定量测定小鼠血清中的药物浓度。ELISA方法一，即Fc端检测(总体检测)方法，通过包被于96孔板的山羊抗人Fc多克隆抗体来捕获小鼠血清中的含有人Fc的抗体，然后加入HRP标记的山羊抗人Fc第二抗体来检测；ELISA方法二，即PD-L1端检测(功能结构域检测)方法，通过包被于96孔板的人PD-L1蛋白来捕获小鼠血清中的特异识别PD-L1的抗体，然后加入HRP标记的的山羊抗人Fc第二抗体来检测。使用Phoenix WinNonlin软件8.2版，选用非房室模型(NCA)分析药代动力学参数。
图34和表5-16中所示，Fab-HCAb结构的双抗分子PR004270有与常规IgG抗体相似的血清半衰期t 1/2值，功能结构域检测方法显示其t 1/2值超过10天。
Figure PCTCN2021103044-appb-000065
表5-16 PR004270在BALB/c小鼠体内的药代动力学
实施例5.10.小结
本实施例利用抗PD-L1的IgG抗体的抗原结合结构域Fab和抗4-1BB的HCAb抗体的抗原结合结构域VH，构建了多种结构的抗PD-L1 x 4-1BB的双特异性抗体分子。展现出了基于HCAb构建双特异性抗体分子结构的灵活性，通过不同的结构类型、相对位置、结合价数等参数来调节激活T细胞的功能活性。
Urelumab对T细胞的激活是没有靶点特异性，这是其临床毒副作用的原因之一。PD-L1 x 4-1BB双抗对T细胞激活作用是特异性依赖PD-L1的表达。依赖于交联的抗4-1BB的单抗不能直接激活T细胞，但是，利用这些抗4-1BB单抗构建的PD-L1 x 4-1BB双抗在高表达PD-L1的细胞存在时则可以特异地激活T细胞。
基于HCAb的双抗结构，尤其是IgG-VH四价对称结构的双抗分子和Fab-HCAb结构的双抗分子，一方面保留了PD-L1端的活性，在MLR实验中体现出比对应的抗PD-L1亲本单抗更强的T细胞激活能力；另一方面靶细胞上高表达的PD-L1分子可以介导4-1BB的交联和三聚化以传递T细胞活化信号，其激活T细胞的能力甚至优于Urelumab。而且IgG-VH四价对称结构和Fab-HCAb对称结构的双抗分子显示出比FIT-Ig结构的双抗分子拥有更强的T细胞激活能力。
综上所述，本实施例构建出了安全性好、功能活性突出、分子稳定性好的PD-L1 x4-1BB双特异性抗体分子。
实施例6.B7H4 x 4-1BB双特异性抗体
实施例6.1.背景
B7-H4(VTCN1,B7h.5,B7S1,B7x)是一种隶属于B7/CD28超家族的跨膜蛋白。B7-H4蛋白表达于一些免疫细胞如单核细胞和树突状细胞，有可能参与T细胞的负调控免疫应答。此外，B7H4还在乳腺癌、卵巢癌、子宫内膜癌、非小细胞肺癌、肾癌等的肿瘤细胞表面上高表达，而在大多数正常组织中没有表达或者表达极低。B7-H4作为这些肿瘤的一个新兴靶点，近年来受到关注。抗B7-H4的抗体可以通过多种机制作用于肿瘤细胞，但是其研发方向主要集中在单克隆抗体上，目前尚无双特异性抗体疗法。
4-1BB(TNFRSF9,CD137)是一种隶属于TNF受体超家族的跨膜蛋白。4-1BB是在多种免疫细胞上表达的共刺激分子，为免疫活性的多功能调节剂。其诱导表达于活化的T细胞、NK细胞等免疫细胞。4-1BB通过其配体4-1BBL介导的三聚化来激活T细胞，促进细胞增殖和细胞因子释放。抗4-1BB的激动型抗体具有抑制肿瘤的功能，最早进入临床试验的4-1BB抗体是辉瑞的Utomilumab和百时美施贵宝(BMS)公司的Urelumab(BMS-663513)。Urelumab最初的临床结果发表于2008年，尽管在部分患者上观察到令人鼓舞的疗效，但数据显示Urelumab导致肝脏毒性，且与靶标和剂量有关。并且，因为在临床试验中有两位患者因肝毒性死亡，导致相关的临床试验被终止。Utomilumab安全性更好，剂量可提高至10mg/kg，但治疗效果依然欠佳。
在本实施例中，我们构建了同时靶向B7H4和4-1BB的双特异性抗体，通过一个或者多个作用机制来提高抗肿瘤效果和安全性。第一，B7H4 x 4-1BB双抗可以通过解除 B7H4的负调控信号来激活T细胞。第二，B7H4 x 4-1BB双抗富集于B7H4高表达的肿瘤组织，在肿瘤微环境中，免疫细胞和肿瘤细胞通过双抗分子结合在一起，促进免疫突触的形成；同时，高表达于肿瘤细胞表面的B7H4分子可以通过双抗分子促进T细胞表面的4-1BB分子的交联，并激活下游信号传导通路，提供共刺激信号，进而促进T细胞的活化和增殖，提高抗肿瘤活性。第三，本实施例所使用的抗4-1BB的激动型抗体的功能是依赖于分子交联的，它只能在肿瘤微环境中利用靶细胞来介导T细胞的激活，以避免类似Urelumab的单抗在正常组织中过度激活T细胞所带来的毒副作用。
实施例6.2.获得抗B7H4的IgG抗体和抗4-1BB的HCAb抗体
实施例6.2.1.获得抗B7H4的全人源H2L2抗体
Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域。
用可溶的重组人B7H4-mFc融合蛋白(Sino Biological Inc.,#10738-H05H)对Harbour H2L2小鼠进行多轮免疫。当检测小鼠血清中B7H4特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出并与骨髓瘤细胞系融合得到杂交瘤细胞；对杂交瘤细胞经过多轮筛选和克隆之后，鉴定出若干个特异识别B7H4的单克隆抗体分子。对这些单克隆抗体进行进一步的鉴定，根据其对人B7H4的结合能力、食蟹猴B7H4的结合能力、靶细胞受体内化能力等参数，优选出数个候选抗体分子。然后对候选抗体分子进行序列分析和优化，得到数个变体序列。将抗体的VL和VH序列与相应的人的κ轻链恒定区和IgG1重链恒定区序列进行融合表达，得到重组全人源抗体分子。抗B7H4的重组全人源IgG抗体PR002408和PR002410列于表6-9。
实施例6.2.2.获得抗4-1BB的全人源HCAb抗体
本实施例使用的抗4-1BB的全人源HCAb抗体PR001758、PR001760、PR001771、PR001840和PR004469(表6-9)来源于Harbour HCAb小鼠，其发现过程如实施例5.2.3所述。
利用流式细胞术FACS和实施例5.6所述方法测试4-1BB重链抗体结合细胞CHO-K1/hu4-1BB(南京金斯瑞,M00538)的结合能力，如图28的(C)-(F)所示，其结合能力与Utomilumab相似，优于Urelumab。
实施例6.3.利用抗B7H4的IgG抗体和抗4-1BB的HCAb抗体构建双特异性抗体分子
本实施例利用抗B7H4的IgG抗体PR002408或PR002410的抗原结合结构域Fab，和抗4-1BB的HCAb抗体PR001758、PR001760、PR001771、PR001840或PR004469的抗原结合结构域VH，来构建多种结构的抗B7H4 x 4-1BB的双特异性抗体分子。
在本实施例及后续实施例中，阳性对照分子为抗B7H4的IgG单抗PR002408，亦为B7H4 x 4-1BB双抗分子的B7H4端的亲本单抗。
在本实施例及后续实施例中，阳性对照分子为抗4-1BB的IgG单抗urelumab。
实施例6.3.1.构建Fab-HCAb对称结构分子
利用抗B7H4的IgG抗体和抗4-1BB的重链抗体，按照实施例1.1所述结构设计Fab-HCAb对称结构的B7H4 x 4-1BB双抗分子，总结于表6-1；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表6-2。
Figure PCTCN2021103044-appb-000066
表6-1 Fab-HCAb四价对称结构的B7H4 x 4-1BB双抗分子
Figure PCTCN2021103044-appb-000067
表6-2 Fab-HCAb四价对称结构的B7H4 x 4-1BB双抗分子蛋白的表达
实施例6.3.2.构建IgG-VH四价对称结构分子
利用抗B7H4的IgG抗体和抗4-1BB的重链抗体，按照实施例1.2所述结构设计IgG-VH四价对称结构的B7H4 x 4-1BB双抗分子，总结于表6-3；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表6-4。
Figure PCTCN2021103044-appb-000068
表6-3IgG-VH四价对称结构的B7H4 x 4-1BB双抗分子
Figure PCTCN2021103044-appb-000069
表6-4 IgG-VH四价对称结构的B7H4 x 4-1BB双抗分子蛋白的表达
实施例6.3.3.构建IgG-VH(2)六价对称结构分子
利用抗B7H4的IgG抗体和抗4-1BB的重链抗体，按照实施例1.3所述结构设计IgG-VH(2)六价对称结构的B7H4 x 4-1BB双抗分子，总结于表6-5；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表6-6。
Figure PCTCN2021103044-appb-000070
Figure PCTCN2021103044-appb-000071
表6-5 IgG-VH(2)六价对称结构的B7H4 x 4-1BB双抗分子
Figure PCTCN2021103044-appb-000072
表6-6 IgG-VH(2)六价对称结构的B7H4 x 4-1BB双抗分子蛋白的表达
实施例6.3.4.构建Fab-Fc-VH(n)非对称结构分子
利用抗B7H4的IgG抗体和抗4-1BB的重链抗体，按照实施例1.6和实施例1.9所述结构设计Fab-Fc-VH(n,n＝{2,3})非对称结构的B7H4 x 4-1BB双抗分子，总结于表6-7；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表6-8。所述分子的Fc区使用“knob-into-hole”结构的突变并引入L234A和L235A双突变。
Figure PCTCN2021103044-appb-000073
表6-7 Fab-Fc-VH(n)非对称结构的B7H4 x 4-1BB双抗分子
Figure PCTCN2021103044-appb-000074
Figure PCTCN2021103044-appb-000075
表6-8 Fab-Fc-VH(n)非对称结构的B7H4 x 4-1BB双抗分子蛋白的表达
实施例6.3.5.B7H4 x 4-1BB双抗分子及对照分子序列表
表6-9、表6-10和表6-11列出了本实施例所构建的B7H4 x 4-1BB双抗分子和对应的B7H4单抗、4-1BB单抗等亲本单抗分子以及对照分子的序列所对应的序列号。表6-11中的结构编号对应于表1-1和图1。表6-12列出了双特异性抗体分子的第一和第二抗原结合结构域相应的CDR序列的序列编号。
抗体编号 抗体
PR002408 抗B7H4单抗80C8-2E9(H:G55A；L:N92Q),hIgG1
PR002410 抗B7H4单抗1025_B-1H11(L:C87Y),hIgG1
PR001758 抗4-1BB重链抗体1016P0010B11,hIgG1
PR001760 抗4-1BB重链抗体1016P0011G10,hIgG1
PR001771 抗4-1BB重链抗体1016P0042C5,hIgG1
PR001840 抗4-1BB重链抗体1016P0037D2,hIgG1
PR004469 抗4-1BB重链抗体PR001838_G53A,hIgG1
PR000628 抗4-1BB单抗urelumab类似物,hIgG4(S228P)
表6-9对照分子和亲本单抗
Figure PCTCN2021103044-appb-000076
Figure PCTCN2021103044-appb-000077
表6-10对照分子和亲本单抗的序列和CDR序列的序列编号表
结构编号 抗体编号 多肽链1(短链) 多肽链2(长链)
5 PR003334 360 455
5 PR003335 360 456
5 PR003338 360 457
7 PR003487 360 458
7 PR003488 360 459
1 PR004279 487 488
1 PR005189 487 520
2 PR007165 360 523
14 PR004160 361 481
26 PR004161 361 481
26 PR004181 361 481
26 PR004182 361 481
表6-11本实施例的B7H4 x 4-1BB双抗分子的序列编号表
Figure PCTCN2021103044-appb-000078
Figure PCTCN2021103044-appb-000079
表6-12 B7H4 x 4-1BB双抗分子的抗原结合结构域的CDR的序列编号表
实施例6.4.结合B7H4
本实施例是为了研究B7H4 x 4-1BB双抗分子结合B7H4的活性。
实施例6.4.1.结合高表达人B7H4的细胞SK-BR-3
利用流式细胞术FACS测试抗体与高表达人B7H4的肿瘤细胞系SK-BR-3(ATCC,HTB-30)的结合能力。具体地，消化SK-BR-3细胞并用完全培养基重悬，将细胞密度调整为2x10 6细胞/mL；接着以50μL细胞/孔接种于96孔V底板(Corning,#3894)。随后以50μL/孔加入5倍浓度梯度稀释的抗体分子共8个浓度，混合均匀；hIgG1 iso(CrownBio,#C0001)作为同型对照。将细胞放置于4℃，避光孵育2小时。随后以100μL/孔加入预冷的PBS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。之后，以100μL/孔加入荧光二抗(Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson ImmunoResearch,#109-605-098,1:1000稀释)，放置于4℃避光孵育1小时。随后以100μL/孔加入预冷的PBS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液(含有0.5％BSA的PBS缓冲液)重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪(ACEA Biosciences Inc.)读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处 理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
图35和表6-13中所示，双抗分子都能结合SK-BR-3细胞。图35的(A)-(C)中所示，对称结构的双抗分子(PR003334,PR003335,PR003338,PR003487,PR003488,PR004279)具有相同的二价的B7H4结合结构域(源自抗B7H4单抗PR002408)，它们结合B7H4的EC50非常接近。IgG-VH-VH六价对称结构的双抗分子PR003487和PR003488与IgG-VH四价对称结构的双抗分子PR003335结合B7H4的能力几乎相同。Fab-HCAb对称结构的双抗分子PR004279结合B7H4的活性比IgG-VH结构的PR003335的略强。图35的(D)-(E)中所示，非对称结构的双抗分子(PR004160,PR004161,PR004181,PR004182)只有一个B7H4结合结构域，故结合B7H4的能力弱于对称结构双抗分子，但是PR004182仍体现出与对称结构双抗分子近似的结合EC50。图35的(F)中所示，Fab-HCAb对称结构的双抗分子，无论是结构(1)的分子(PR005189)还是结构(2)的分子(PR007165)，都有几乎相同的结合活性；说明将Fab的CL融合到VH_B所在的重链上不影响Fab端的结合活性。
Figure PCTCN2021103044-appb-000080
表6-13结合SK-BR-3
实施例6.5.结合4-1BB
本实施例是为了研究B7H4 x 4-1BB双抗分子结合4-1BB的活性。
实施例6.5.1.结合高表达人4-1BB的CHO-K1细胞CHO-K1/hu4-1BB
利用流式细胞术FACS测试抗体分子与高表达人4-1BB的CHO-K1细胞株CHO-K1/hu4-1BB(南京金斯瑞,M00538)的结合能力。具体地，消化细胞并用完全培养基重悬；将细胞密度调整为2x10 6细胞/mL。接着将细胞以100μL/孔(2x10 5细胞/孔)接种于96孔V底板(Corning,#3894)，4℃下离心5分钟，弃上清。随后将梯度稀释的抗体分子以100μL/孔加入96孔板并混合均匀，抗体分子以5倍浓度梯度稀释的共8个浓度；hIgG1 iso(CrownBio,#C0001)作为同型对照。将细胞放置于4℃，避光孵育1小时。然后，加入100μL/孔预冷的PBS缓冲液漂洗细胞两次，4℃下500g离心5分钟，弃上清。接着，再加入100μL/孔荧光二抗(Alexa 
Figure PCTCN2021103044-appb-000081
488,Goat Anti-Human IgG,Fcγfragment specific,Jackson ImmunoResearch,#109-545-098,1:1000稀释)，放置于4℃，避光孵育1小时。随后以200μL/孔加入预冷的PBS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液(含有0.5％BSA的PBS缓冲液)重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
本实施例中，阳性对照分子为抗4-1BB的单抗Urelumab。结果显示于图36和表6-14。
图36中所示，双抗分子都能结合CHO-K1/hu4-1BB细胞，且结合曲线EC50非常接近。图36的(A)中所示，IgG-VH四价对称结构的双抗分子结合4-1BB的MFI最大值高于Urelumab；图36的(B)中所示，IgG-VH-VH六价对称结构的双抗分子结合4-1BB的活性与Urelumab相似；图36的(C)中所示，Fab-HCAb对称结构的双抗分子PR004279结合4-1BB的活性比IgG-VH结构的PR003335的略强；图36的(D)-(E)中所示，Fab-Fc-VH(n)非对称结构的双抗分子结合4-1BB的EC50与Urelumab相似，但是有更高的MFI最大值；图36的(F)中所示，Fab-HCAb对称结构的双抗分子，无论是结构(1)的分子(PR005189)还是结构(2)的分子(PR007165)，都有相似的4-1BB结合活性，且与亲本单抗PR001760的相当。
Figure PCTCN2021103044-appb-000082
Figure PCTCN2021103044-appb-000083
表6-14结合CHO-K1/hu 4-1BB
实施例6.6.高表达B7H4的靶细胞介导的T细胞的特异性激活
本实施例是为了研究B7H4 x 4-1BB双抗分子在靶细胞的存在时通过结合4-1BB来激活T细胞的活性。靶细胞可以是不同程度表达B7H4的细胞，例如高表达B7H4的SK-BR-3(ATCC,HTB-30)，或者不表达B7H4的JIMT-1(DSMZ,ACC 589)。效应细胞可以是分离的人PBMC或者T细胞。
具体的，首先将抗CD3抗体OKT3(Thermo,#16-0037-81)包板于96孔板(Corning,#3799)。接着，将人T细胞的密度调整为3 x10 6细胞/mL，将靶细胞的密度调整为3x10 5细胞/mL，随后把两种细胞悬液各以50μL/孔接种于96孔板，最终效靶比为10:1。然后，以50μL/孔加入不同浓度的抗体分子，以5倍浓度梯度稀释的共6个浓度，两个复孔加样；hIgG1 iso(CrownBio,#C0001)和hIgG4iso(CrownBio,#C0045)作为对照。将96孔板置于37℃，5％CO 2培养箱中孵育2天。孵育完成后，取100μL上清液，加入另一96孔板(Corning,#3599)，于4℃下500g离心5分钟，取出50μL上清液用于检测细胞因子的水平。用IL-2ELISA试剂盒(Thermo,#88-7025-88)检测上清中IL-2浓度，用IFN-γELISA试剂盒(Thermo,#88-7316-88)检测上清中IFN-γ浓度。ELISA检测方法参照相关试剂盒操作说明。应用软件GraphPad Prism 8进行数据处理和作图分析。
本实施例中，阳性对照分子为抗4-1BB的单抗Urelumab。
实施例6.6.1.SK-BR-3介导的T细胞特异性激活
图37和图38中所示，在高表达B7-H4的SK-BR-3细胞存在时，B7-H4 x 4-1BB双抗分子激活T细胞的能力优于阳性对照分子，表现为该体系中产生的细胞因子IFN-γ(图37)或IL-2(图38)的释放高于阳性对照分子。
图37的(A)-(B)和图38的(A)中所示，IgG-VH四价对称结构的双抗(PR003334,PR003335,PR003338)激活T细胞的能力比Urelumab略强，且PR003335和PR003338比PR003334略强。
图37的(C)和图38的(B)中所示，IgG-VH-VH六价对称结构的双抗(PR003487,PR003488)激活T细胞的能力明显强于IgG-VH四价的双抗(PR003334,PR003335)和Urelumab。
图38的(C)中所示，Fab-HCAb对称结构的双抗(PR004279)激活T细胞的能力比Urelumab略强。
图38的(D)-(F)中所示，Fab-Fc-VH(n)非对称结构的双抗(PR004160,PR004161,PR004181,PR004182)可以刺激T细胞产生IL-2。PR004160和PR004161具有相同的4-1BB结合结构域(VH)的序列，但是PR004160有两个串联的VH域(即Fab-Fc-VH(2)结构)而PR004161有三个串联的VH域(即Fab-Fc-VH(3)结构)；PR004161激活T细胞的能力明显强于PR004160和Urelumab。具有相似Fab-Fc-VH(3)结构的PR004181和PR004182也显示出优于Urelumab的T细胞激活能力。这些非对称结构双抗尽管只有一个B7H4结合结构域且结合B7H4的能力弱于对称结构双抗，但是它们仍能利用高表达B7-H4的细胞介导T细胞的激活，且激活能力优于Urelumab。
实施例6.6.2.B7H4的表达对T细胞的激活的影响
图39中所示，B7H4 x 4-1BB双抗对T细胞激活是特异性依赖B7H4的表达。(A)在高表达B7H4的细胞SK-BR-3存在时，双抗PR003334可以特异地激活T细胞释放IL-2；(B)在不表达B7H4的细胞JIMT-1存在时，PR003334不能激活T细胞。Urelumab则没有靶点特异性，即使没有B7H4的表达，也可以激活T细胞。
实施例6.7.血清稳定性
本实施例研究了具有IgG-VH对称结构的B7H4 x 4-1BB双抗分子(PR003334,PR003335)在高浓度人血清中的稳定性。具体地，双抗分子用90％人血清进行梯度稀 释，初始浓度为100nM，以3倍梯度稀释8个浓度点。每个浓度下分成6份样本，分别在37度孵育0天、1天、2天、4天、7天、14天后，液氮速冻后放置于-40度保存。然后，收集在血清中孵育不同时间后的样品，按照实施例6.4.1和实施例6.5.1所述方法分别测试双抗分子与SK-BR-3细胞和CHOK1/hu 4-1BB细胞的结合活性，以考察双抗分子在血清中放置不同时间后的结合活性的变化。
双抗分子PR003334(图40，表6-15)和PR003335(图41，表6-16)在人血清中是稳定的，即使在放置14天后，其B7H4端和4-1BB端的结合活性都没有明显的变化。
Figure PCTCN2021103044-appb-000084
表6-15 PR003334血清孵育不同时间后结合靶细胞的活性
Figure PCTCN2021103044-appb-000085
表6-16 PR003335血清孵育不同时间后结合靶细胞的活性
实施例6.8.药代动力学研究
本实施例研究了具有IgG-VH对称结构的B7H4 x 4-1BB双抗分子(PR003334,PR003335)在小鼠体内的药代动力学性能。
实施方法如下：对于每一个测试抗体分子，选取体重18～22克的雌性C57BL/6小鼠6只，按5mg/kg的剂量通过静脉注射给与双特异性抗体。一组3只于给药前以及给药后15分钟、24小时(1天)、第4天、和第10天采集全血，另一组3只于只于给药前以及给药后5小时、第2天、第7天、和第14天采集全血。将全血静置30分钟使其凝固，随后4℃下以2,000rpm离心5分钟，并将分离的血清样品在-80℃下冻存直至分析。本实施例采用两种ELISA方法来定量测定小鼠血清中的药物浓度。ELISA方法一，即Fc端检测(总体检测)，通过包被于96孔板的山羊抗人Fc多克隆抗体来捕获小鼠血清中的含有人Fc的蛋白，然后加入HRP标记的山羊抗人Fc第二抗体来检测；ELISA方法二，即4-1BB端检测(功能结构域检测)方法，通过包被于96孔板的人4-1BB蛋白(Acro biosystems,#41B-H5227)来捕获小鼠血清中的特异识别人4-1BB的抗原结合蛋白，然后加入HRP标记的的山羊抗人Fc第二抗体来检测。使用Phoenix WinNonlin软件8.2版，选用非房室模型(NCA)分析药代动力学参数。
图42和表6-17中所示，IgG-VH结构的双抗分子PR003334和PR003335有与常规IgG抗体相似的药代动力学：在总体检测方法下，PR003334和PR003335在小鼠体内的血清半衰期t 1/2值分别约为8天和14天；在功能域检测方法下，PR003334和PR003335在小鼠体内的血清半衰期t 1/2值分别约为9天和12天。
Figure PCTCN2021103044-appb-000086
表6-17 B7H4 x 4-1BB双抗分子在C57BL/6小鼠体内的药代动力学
实施例6.9.双抗分子的抗肿瘤药效
本实施例研究了B7H4 x 4-1BB双抗分子PR003334在BALB/c-hCD137/CT26-hB7H4小鼠肿瘤模型中的抗肿瘤药效。
实施方法如下：选用6-8周雌性BALB/c-hCD137小鼠(将BALB/c小鼠引入外源人4-1BB转基因，集萃药康生物科技)，然后将处于对数生长期的CT26-hB7H4肿瘤细胞(将小鼠结肠癌细胞CT26引入外源人B7H4转基因，和铂医药)以5×10 5细胞/鼠的剂量接种于实验小鼠腋右侧背部皮下。当平均肿瘤体积达到80mm 3时对小鼠进行随机分组，每组6只小鼠。分组当天，将特定浓度经PBS稀释的药物以腹腔注射(i.p.)、每周给药2次总共给药6次(BIW*3)的方式进行给药，以PBS为空白对照组。在初次给药当天及之后第4、7、11、14和18天对肿瘤体积和小鼠体重进行测量。肿瘤大小计算公式：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。实验结束后将荷瘤小鼠安乐死并剥瘤称重。计算各组小鼠的肿瘤体积、小鼠体重等实验结果以平均值±标准误差(Mean±SEM)表示。多组比较采用单因素方差分析(one way ANOVA)检验方法比较不同治疗组与对照组相比有无显著性差异。
本实施例中，抗4-1BB的单抗Urelumab为阳性对照分子。双抗分子PR003334的6mpk(mg/kg)剂量与Urelumab的5mpk剂量为同摩尔浓度(根据分子量换算)的对等剂量；双抗分子PR003334的18mpk剂量与Urelumab的15mpk剂量为同摩尔浓度的对等剂量。10mpk的抗小鼠PD-1抗体(Bio X Cell,clone RMP1-14,#BE0146)也作为对照分子。PBS为空白对照组。
图43中所示，双抗分子PR003334和Urelumab在各剂量组都有抑制肿瘤生长的药效，且优于PD-1抗体(A)。各组小鼠体重变化均在正常范围内(B)。
实施例6.10.利用UNcle测试双抗分子的稳定性和理化特性
本实施例利用实施例2.5所述方法测试具有IgG-VH对称结构的B7H4 x 4-1BB双抗分子(PR003334,PR003335,PR003338)的分子稳定性等理化表征。如表6-18中所示，这些双抗分子有良好的稳定性。
  PR003334 PR003335 PR003338
Tm(℃) 60.32 65.59 62.17
Tagg(℃)at 266nm 52.00 50.39 54.14
Tagg(℃)at 473nm / / /
Initial SLS at 266nm(counts x104) 0.26 0.22 0.20
Max SLS at 266nm(counts x104) 1.00 0.40 0.34
Initial SLS at 473nm(counts) <0 <0 <0
Max SLS at 473nm(counts) <0 <0 <0
Initial diameter(nm)(25℃) 16.22 16.38 11.74
Final diameter(nm)(95℃) 23.85 17.51 16.52
Initial PDI 0.267 0.229 0.043
Final PDI 0.089 0.194 0.185
表6-18双抗分子在UNcle中的测试参数
实施例6.11.小结
本实施例利用抗B7H4的IgG抗体的抗原结合结构域Fab和抗4-1BB的HCAb抗体的抗原结合结构域VH，构建了多种结构的抗B7H4 x 4-1BB的双特异性抗体分子。展现出了基于HCAb构建双特异性抗体分子结构的灵活性，通过不同的结构类型、相对位置、结合价数等参数来调节激活T细胞的功能活性。
Urelumab对T细胞的激活是没有靶点特异性，这是其临床毒副作用的原因之一。但是，B7H4 x 4-1BB双抗对T细胞激活作用是特异性依赖B7H4的表达。在高表达B7H4的细胞存在时，双抗可以特异地激活T细胞；而在没有B7H4表达时，双抗不能激活T细胞。因而，B7H4 x 4-1BB双抗相较于4-1BB单抗如Urelumab有更好的安全性。
另一方面，B7H4 x 4-1BB双抗在B7H4存在时，可以介导4-1BB的交联和三聚化以传递T细胞活化信号，而且其激活T细胞的能力甚至优于Urelumab。在Fab-HCAb和IgG-VH四价对称结构中，4-1BB端的VH是二价的，其激活T细胞的能力略优于Urelumab；在IgG-VH-VH六价对称结构中，4-1BB端的VH是多价的，其激活T细胞的能力得到明显的加强，显著优于Urelumab。
综上所述，本实施例构建出了安全性好、功能活性突出、分子稳定性好的B7H4 x 4-1BB双特异性抗体分子。
实施例7.BCMA x CD3双特异性抗体
实施例7.1.背景
BCMA(B-细胞成熟抗原，TNFRSF17，CD269)是一种属于TNF受体超家族的跨膜蛋白，其参与B细胞成熟、生长和存活。BCMA主要有两种配体：高亲和力配体APRIL(增殖诱导配体)以及低亲和力配体BAFF(B细胞活化因子)。BCMA是一种高度分化的浆细胞选择性蛋白，其表达限于B-细胞谱系且主要存在于浆细胞和浆母细胞上，并在一定程度上存在于记忆B-细胞上，但是不存在于外周B-细胞上。BCMA在多发性骨髓瘤(MM)患者的恶性浆细胞中表达，支持多发性骨髓瘤细胞的生长和存活。多发性骨髓瘤是继非霍奇金淋巴瘤的血液系统第二大恶性肿瘤，约占血液系统恶性肿瘤的13％。作为多发性骨髓瘤的一个新兴靶点，BCMA抗体可以通过多种机制作用于MM细胞。
目前临床阶段最快的针对BCMA的抗体是葛兰素史克(GSK)公司的抗体CA8-J6M0 hIgG1(下文又称阳性对照1，在实施例中编号为PR000274)以及在CA8-J6M0基础上制备的抗体偶联药物belantamab mafodotin(CA8-J6M0-mcMMAF,GSK2857916)。GSK2857916在多项临床试验中治疗不同类型的MM患者。来自一项小型临床研究的数据显示，在35例过度预治疗(大多数患者至少接受了5种疗法治疗失败)复发性或难治性的MM患者中，客观缓解率(ORR)达到了60％，中位无进展生存期(PFS)为12个月。安全性方面，最常见的副作用包括角膜事件、血小板减少和贫血，这些都与ADC中偶联的细胞毒性制剂有关。
目前，处于临床开发阶段的双特异性抗体有安进的AMG-420、再生元的REGN-5458、新基的CC-93269、强生JNJ-64007957、艾伯维的TNB383B。然而这些双特异性抗体还存在半衰期短、细胞因子释放综合征(CRS)等问题。TNB383B双特异性抗体于2019年进入临床一期用于MM，其具有细胞因子释放水平较低的特点。但是TNB383B既不结合食蟹猴的BCMA也不结合食蟹猴的CD3，不能在食蟹猴进行毒理学评估。
因此，急需研发更加安全有效的同时靶向人的BCMA和CD3、并能结合食蟹猴的BCMA和CD3的双特异性抗体。
实施例7.2.抗BCMA的HCAb抗体的发现
可以利用BCMA抗原对实验动物进行免疫以获得针对BCMA特异性结合的抗体分子，该实验动物可以是小鼠、大鼠、兔、羊、骆驼等。通常，其得到的抗体分子是非人源的。在获得非人源抗体后，需要对这些分子利用抗体工程技术进行人源化改造，以降低免疫原性并提高成药性。然而，抗体的人源化过程有其技术复杂性，经过人源化改造的分子往往会降低对抗原的亲和力。另一方面，转基因技术的进步使得可以培育出基因工程化小鼠，其携带人免疫球蛋白免疫库并使其内源的鼠的免疫库缺失。这种转基因小鼠产生的抗体具有全人源的序列，因而无需再进一步做人源化改造，大大提高了治疗性抗体开发的效率。Harbour HCAb小鼠(Harbour Antibodies BV,WO2010/109165A2)是一种携带人免疫球蛋白免疫库的转基因小鼠，能够产生全新的仅“重链”抗体，该抗体的大小只有常规IgG抗体的一半。其产生的抗体仅具有人的抗体“重链”可变结构域和小鼠Fc恒定结构域。由于不含轻链的这一特点，该抗体几乎解决了轻链错配和异源二聚化的问题，使得这一技术平台能够开发出常规抗体平台难以实现的产品。
实施例7.2.1.用BCMA抗原免疫小鼠
用可溶的重组人BCMA-ECD-Fc融合蛋白对Harbour HCAb小鼠进行多轮免疫。抗原蛋白与免疫佐剂混合成免疫原试剂，然后通过皮下经腹股沟注射或通过腹腔注射。在每一轮免疫中，每只小鼠接受的总注射剂量是100微升。在首轮免疫中，每只小鼠接受用50微克抗原蛋白(重组人BCMA-ECD-Fc,ACRO Biosystems,#BC7-H82F0)与完全弗氏佐剂(Sigma,#F5881)以体积比1:1混合配制的免疫原试剂的免疫。在随后的每轮增强免疫中，每只小鼠接受用25微克抗原蛋白与Sigma Adjuvant System佐剂(Sigma,#S6322)混合配制的免疫原试剂的免疫。每轮增强免疫的间隔时间至少为两周，通常不超过五轮增强免疫。免疫时间为第0、14、28、42、56、70天；并且在第49、77天，检测小鼠血清抗体滴度。在进行HCAb小鼠脾B细胞分离前5天，以每只小鼠25微克抗原蛋白的剂量进行最后一次增强免疫。
实施例7.2.2.获得HCAb单克隆和抗体序列
当检测小鼠血清中BCMA特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出分离B细胞，用BD FACSAria TMIII细胞分选仪分选CD138阳性的浆细胞和BCMA抗原阳性的B细胞群。提取RNA，反转录cDNA后PCR扩增人VH基因。扩增的VH基因片段构建到编码人IgG1抗体重链Fc结构域序列的哺乳动物细胞表达质粒pCAG载 体中，质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)进行表达，表达的HCAb的抗体上清与重组人BCMA-Fc,Avitag重组蛋白(ACRO Biosystems,#BC7-H82F0)进行Mirrorball(SPT Labtech,
Figure PCTCN2021103044-appb-000087
fluorescence cytometer)筛选，获得的阳性单克隆抗体上清用流式细胞术FACS进一步的鉴定。用FACS测试抗体上清与高表达人BCMA的HEK293T细胞株HEK293T/hBCMA(北京康源博创,KC-0233)、高表达食蟹猴BCMA的HEK293T细胞株HEK293T/cynoBCMA(北京康源博创,KC-0979)和高表达人BCMA的细胞系NCI-H929(ATCC,CRL-9068)等细胞的结合能力。通过多轮筛选，获得了四个阳性单克隆。利用常规的测序手段获得编码抗体分子可变结构域的核苷酸序列以及对应的氨基酸序列。在本实施例中，从免疫的Harbour HCAb小鼠得到的抗BCMA单克隆抗体分子可变结构域的序列是人源抗体序列。
实施例7.2.3.制备抗BCMA全人重组抗体
按照实施例2.2所述方法，用编码HCAb抗体的质粒，利用常规的重组蛋白表达和纯化技术，制备纯化的抗BCMA重组重链抗体。
表7-1列出了本实施例中抗BCMA的HCAb抗体的重链可变结构域、重链全长氨基酸序列和根据Chothia规则定义的CDR的氨基酸序列所对应的序列编号SEQ ID NO。表7-2列出了阳性对照1即CA8-J6M0(抗体编号PR000274)的轻、重链序列所对应的序列编号SEQ ID NO。
克隆号 抗体编号 重链 VH HCDR1 HCDR2 HCDR3
1005P10H8 PR000940 316 248 15 75 133
1005P16A10 PR000943 317 249 24 76 134
1005P36F3 PR001035 318 250 25 77 135
1005P63B7 PR001046 319 251 26 78 136
表7-1抗BCMA的HCAb抗体的序列编号表
抗体别名 抗体编号 重链 轻链
阳性对照1 PR000274 309 354
表7-2抗BCMA的对照抗体的序列编号表
实施例7.3.HCAb抗体结合BCMA
本实施例是为了研究抗BCMA的HCAb抗体的结合人和食蟹猴BCMA的活性。利用流式细胞术FACS测试抗BCMA重组抗体与高表达人BCMA的HEK293T细胞株HEK293T/hBCMA(北京康源博创,KC-0233)、高表达食蟹猴BCMA的HEK293T细胞株HEK293T/cynoBCMA(北京康源博创,KC-0979)和高表达人BCMA的细胞系NCI-H929(ATCC,CRL-9068)等细胞的结合能力。具体地，消化HEK293T/hBCMA细胞和HEK293T/cynoBCMA细胞，并用DMEM完全培养基重悬；另外，收集NCI-H929细胞悬液。将3种细胞密度分别调整为1x10  6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson,#109-545-06,1:500稀释)，4℃，避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
本实施例中，阳性对照1分子为抗BCMA的单抗PR000274。
实施例7.3.1.结合高表达人BCMA的细胞HEK293T/hBCMA
图44和表7-3中所示，抗人BCMA的HCAb单抗PR000943，PR001035和PR001046可以结合HEK293T/hBCMA，且结合曲线的EC50均优于阳性对照1。
抗体 EC50(nM) MFI最大值
PR000943 0.1535 69991
PR001035 0.3356 39840
PR001046 0.2129 63382
阳性对照1 4.076 86498
表7-3结合HEK293T/hBCMA
实施例7.3.2.结合高表达人BCMA的细胞NCI-H929
图46和表7-4中所示，抗人BCMA的HCAb单抗PR000940，PR000943，PR001035和PR001046可以结合NCI-H929，且结合曲线的EC50均优于阳性对照1。
抗体 EC50(nM) MFI最大值
PR000940 1.847 90962
PR000943 4.8 166453
PR001035 1.193 112747
PR001046 1.535 132791
阳性对照1 10.75 124959
表7-4结合NCI-H929
实施例7.3.3.结合高表达食蟹猴BCMA的细胞HEK293T/cynoBCMA
图45和表7-5中所示，抗人BCMA的HCAb单抗PR000943和PR001046可以结合HEK293T/cynoBCMA，且结合曲线的EC50均优于阳性对照1。PR001035结合食蟹猴BCMA的能力较弱。
抗体 EC50(nM) MFI最大值
PR000943 0.3293 63990
PR001035 n.d. 10848
PR001046 0.5641 67701
阳性对照1 4.564 116307
表7-5结合HEK293T/cynoBCMA
实施例7.4.HCAb抗体内化对靶细胞的杀伤
本实施例是为了研究抗人BCMA的HCAb抗体内化介导对表达人BCMA的细胞的杀伤。具体地，消化高表达人BCMA的HEK293T细胞株HEK293T/hBCMA(北京康源博创,KC-0233)，并用DMEM完全培养基重悬细胞，计数并接种5000或10000个/孔至96-孔黑壁透明底的平板(Perkin Elmer,#6005225)。重组抗体连续稀释至9个不同浓度并加入，最终浓度从100nM开始。加入a-hFc-MMAF(Moradec,#AH-102-AF)使最终浓 度为1μg/ml。平板在37℃、5％CO 2条件下孵育72小时，然后用CTG试剂盒(Promega,#G7573)裂解，并用Enspire TM多功能读板机(PerkinElmer,Inc.)检测发光。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值和最大杀伤率等参数。
图47和表7-6中所示，抗人BCMA的HCAb抗体PR000943和PR001046比阳性对照1分子有更优的内化能力。
抗体 EC50(nM) 最大杀伤率(％)
PR000943 0.4917 78.72
PR001046 0.5711 71.20
阳性对照1 2.180 77.19
表7-6抗体内化介导靶细胞杀伤
实施例7.5.BLI方法测定结合BCMA的亲和力
本实施例利用生物膜干涉(BLI)技术，使用Octet Red96e分子相互作用分析仪(ForteBio,Octet Red96e)，来分析抗BCMA的抗体与BCMA抗原蛋白之间的结合动力学。在本实施例中，BCMA蛋白为生物素化的人BCMA-Fc,Avitag重组蛋白(ACRO Biosystems,#BC7-H82F0)，测试缓冲液为1x动力学缓冲液(从10x动力学缓冲液(ForteBio,#18-1105)稀释)。
测定抗原和抗体(多浓度)的亲和力时，传感器转动速度为1000转/分钟。先将两列SA传感器(每列放置8个传感器；第一列称为参照SA传感器，第二列称为测试SA传感器)在测试缓冲液中平衡10分钟。然后用测试SA传感器捕获生物素化的人BCMA-Fc,Avitag重组蛋白，捕获高度0.9-1.1nm；而参照SA传感器浸入缓冲液中30秒。然后将SA传感器在测试缓冲液中平衡2分钟。两列SA传感器再与梯度稀释的抗BCMA抗体(例如，抗体浓度可以从50nM开始以两倍梯度稀释为6个浓度以及0nM)结合5分钟，然后解离10分钟。
使用Octet Data Analysis软件(Fortebio，版本11.0)进行数据分析时，选择双扣除模式(double reference)扣除参照信号，选择“1:1Global fitting”方法进行数据拟合，计算出抗原与抗体结合的动力学参数，得到kon(1/Ms)值、kdis(1/s)值和KD(M)值。
图48及表7-7中所示，抗人BCMA的HCAb抗体与人BCMA蛋白有较强的结合亲和力。
抗体编号 KD(M) kon(1/Ms) kdis(1/s) Full R^2
PR000940 1.52E-10 4.01E+05 6.09E-05 0.995
PR000943 1.62E-10 1.19E+05 1.93E-05 0.9986
PR001035 2.76E-10 3.25E+05 8.98E-05 0.9926
PR001046 2.53E-11 4.23E+05 1.07E-05 0.9983
表7-7抗BCMA的HCAb抗体结合人BCMA蛋白的亲和力
实施例7.6.优化HCAb抗体序列提高结合BCMA的亲和力
本实施例利用抗体工程的方法提高HCAb抗体PR001046结合BCMA的亲和力。
实施例7.6.1.获得PR001046的变体
通过对HCAb抗体PR001046的可变区VH的CDR区进行两轮定点突变，以获得结合BCMA的亲和力提高的突变体。
第一轮，对PR001046VH的三个CDR区域(按照Kabat CDR定义规则指定)中的35个位点进行逐点氨基酸扫描，建立35个不同位点的单点饱和突变库。对35个饱和突变库利用BCMA结合ELISA进行筛选，信号超过PR001046VH的2倍的阳性克隆被挑出来测序，并进一步对这些阳性克隆利用梯度稀释的多浓度BCMA结合ELISA来测试其结合BCMA的能力。优选出若干个突变热点。
第二轮，将第一轮中优选出来的突变热点进行随机组合，建立一个包含所有突变组合的库。然后对组合库进行筛选。对阳性克隆进行测序和序列分析，并进一步测试其结合BCMA的能力。选出若干个突变体。
PR001046VH的变体分子用对应的克隆号来表示，例如：PR001046-R1-12G7，PR001046-R2-7E7等。将变体分子的VH区构建到带有Flag和His标签的原核表达载体，然后利用常规的重组蛋白表达和纯化技术制备变体分子，其对应的序列编号列于表7-8。最后利用FACS和BLI等方法对重组突变分子进行结合能力的测定。
变体克隆号 抗体编号 重链 VH HCDR1 HCDR2 HCDR3
  PR001046 319 251 26 78 136
1046-R1-12G7 PR004559 330 265 26 90 136
1046-R2-10F1 PR004560 331 266 34 78 146
1046-R2-10H1 PR004561 332 267 35 78 147
1046-R2-10H5 PR004562 333 268 35 90 147
1046-R2-1A1 PR004563 334 269 36 90 146
1046-R2-2D4 PR004564 335 270 35 76 147
1046-R2-2G4 PR004565 336 271 34 78 148
1046-R2-4G10 PR004566 337 272 35 76 136
1046-R2-5E1 PR004567 338 273 35 90 146
1046-R2-5H1 PR004568 339 274 34 76 136
1046-R2-5H2 PR004569 340 275 35 76 146
1046-R2-6D6 PR004570 341 276 34 76 146
1046-R2-6F1 PR004571 342 277 35 90 148
1046-R2-7E7 PR004572 343 278 35 90 136
1046-R2-8B1 PR004573 344 279 35 90 136
1046-R2-9E5 PR004574 345 280 34 76 146
表7-8 PR001046衍生变体分子的序列编号表
实施例7.6.2.利用BLI方法分析变体分子结合BCMA蛋白的解离速率排序
利用实施例7.5所述方法利用BLI技术测定PR001046VH衍生出的单域结构变体分子与BCMA的结合能力。本实施例直接使用含有PR001046VH衍生变体分子的上清，用Octet Red96e分子相互作用分析仪分析变体分子和生物素化的人BCMA-Fc,Avitag重组蛋白之间的结合动力学。用结合动力学中的解离速率对变体分子进行排序，挑选出解离速率更慢(更强亲和力)的变体分子。本实施例只分析每个变体分子与BCMA结合动力学的解离速率并计算其与初始分子(PR001046VH)的相对值(Kdis Fold)；“Kdis倍数”小于1说明变体分子相较于初始分子有更慢的解离速率。
本实施例由于不使用多浓度结合动力学分析来计算抗原抗体结合亲和力，不需要对分析的抗体进行梯度稀释，因而每个抗体只需要一个SA传感器，从而在每一个分析循环中可以分析多个样品，提高了分析通量。利用这一方法可以对大量的变体分子的上清样品进行解离速率分析，筛选出解离速率更优的分子。
表7-9中所示，16个PR001046VH衍生出的变体分子相较于初始分子有更慢的解离速率(即对BCMA有更强的亲和力)。
变体克隆 抗体编号 Conc.(nM) Response Kdis(1/s) Kdis倍数
  PR001046 100 0.2606 6.96E-04 1.00
1046-R1-12G7 PR004559 100 0.2614 3.79E-04 0.54
1046-R2-10F1 PR004560 100 0.5732 3.01E-04 0.43
1046-R2-10H1 PR004561 100 0.2694 3.00E-04 0.43
1046-R2-10H5 PR004562 100 0.5352 2.17E-04 0.31
1046-R2-1A1 PR004563 100 0.4933 2.59E-04 0.37
1046-R2-2D4 PR004564 100 0.5701 2.28E-04 0.33
1046-R2-2G4 PR004565 100 0.5572 2.36E-04 0.34
1046-R2-4G10 PR004566 100 0.4245 2.86E-04 0.41
1046-R2-5E1 PR004567 100 0.5781 2.01E-04 0.29
1046-R2-5H1 PR004568 100 0.7325 2.30E-04 0.33
1046-R2-5H2 PR004569 100 0.7303 2.54E-04 0.36
1046-R2-6D6 PR004570 100 0.4373 2.34E-04 0.34
1046-R2-6F1 PR004571 100 0.7035 2.19E-04 0.31
1046-R2-7E7 PR004572 100 0.2896 2.36E-04 0.34
1046-R2-8B1 PR004573 100 0.2680 2.82E-04 0.41
1046-R2-9E5 PR004574 100 0.2901 3.14E-04 0.45
表7-9 PR001046衍生变体分子结合BCMA蛋白的解离速率Kdis
实施例7.6.3.变体分子结合高表达人BCMA的细胞NCI-H929
利用实施例7.3所述方法测定PR001046VH衍生出的单域结构变体分子与高表达人BCMA的细胞NCI-H929的结合。特别指出，其重组变体分子是带有Flag标签的VH单域抗体分子，在FACS实验中需要使用针对此标签的荧光二抗。
图49和表7-10中所示，16个PR001046VH衍生出的单域变体分子可以更好地结合细胞NCI-H929，结合力优于初始分子。
抗体编号 变体克隆 EC50(nM) MFI最大值
PR001046   未定 764
PR004559 1046-R1-12G7 22.35 1189
PR004560 1046-R2-10F1 3.75 1305
PR004561 1046-R2-10H1 4.38 1242
PR004562 1046-R2-10H5 1.24 1560
PR004563 1046-R2-1A1 7.96 1260
PR004564 1046-R2-2D4 1.34 1530
PR004565 1046-R2-2G4 2.42 1428
PR004566 1046-R2-4G10 3.17 1399
PR004567 1046-R2-5E1 1.72 1436
PR004568 1046-R2-5H1 2.85 1444
PR004569 1046-R2-5H2 5.87 1416
PR004570 1046-R2-6D6 2.15 1355
PR004571 1046-R2-6F1 2.86 1382
PR004572 1046-R2-7E7 0.54 1496
PR004573 1046-R2-8B1 1.72 1232
PR004574 1046-R2-9E5 2.90 1299
表7-10结合NCI-H929
实施例7.7.抗CD3的IgG抗体的发现
抗CD3抗体是构建T细胞桥接器双特异性抗体(T-Cell Engager Bispecific Antibody)的重要组成部分。本实施例介绍了不同方法发现和制备抗CD3的抗体。
实施例7.7.1.全人源CD3抗体的发现
Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域。用CD3抗原对Harbour H2L2小鼠进行多轮免疫；所述抗原可以是重组人CD3融合蛋白，或者 含有人CD3E的N端前27个氨基酸残基的短肽，或者人的T细胞。当检测小鼠血清中CD3特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出，并从中分离出B细胞；然后提取RNA并用RT-PCR制备cDNA。以cDNA为模板，通过PCR扩增出VH和Vκ片段，然后进行重叠PCR制备得到scFv片段，并连接到预先线性化的载体pHBM-scFv-ST。将上述所得的连接产物转化至MC1061F'电转感受态细胞(Lucigen,#60512-1)，然后制备噬菌体文库。用生物素化的抗原蛋白进行多轮生物淘选，并用高表达人CD3/TCR复合物的CHO-K1细胞CHO-K1/hCD3(和铂医药)进行筛选，鉴定出阳性克隆并测序。表7-11列出了通过噬菌体展示技术筛选得到的结合CD3的单链可变区融合蛋白(scFv-Fc结构)。
克隆号 抗体编号 多肽链 VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3
M15-C10 PR002161 490 294 258 177 191 221 15 84 141
M15-H2 PR002163 491 295 259 178 197 222 31 85 142
M23-F11 PR002337 492 296 260 177 191 223 31 86 141
M27-B11 PR002340 493 297 259 179 198 224 31 85 142
表7-11全人源的抗人CD3的抗体的序列编号表
实施例7.7.2.CD3抗体的人源化改造
SP34是来源于小鼠的抗人CD3的抗体，具有激活T细胞的功能。SP34是极少数的可以结合多种灵长类CD3(例如人和食蟹猴CD3)的抗体(参见，Salmeron,A.et al,J Immunol 147(1991)3047-3052；Conrad M.L.,et.al,Cytometry A 71(2007)925-933)。其可变区序列VH和VL来源于WO2016071004A1。
本实施例使用“CDR移植”的方法对小鼠抗体SP34的可变区的序列进行人源化，即：将SP34的VH的CDR移植到人抗体VH的框架区，将SP34的VL的CDR移植到人抗体VL的框架区。人抗体VH或VL的框架区的序列可以来源于人的胚系基因序列或者经过V(D)J重排后的抗体序列或者人抗体特定VH或VL基因家族的一致性(consensus)序列。本实施例使用人的胚系基因序列提供的框架区序列作为人源化模板序列，即：人的胚系V基因片段提供框架区FR1，FR2，FR3的序列，人的胚系J基因片段提供框架区FR4的序列。最后以(人)FR1-(鼠)CDR1-(人)FR2-(鼠)CDR2-(人)FR3-(鼠)CDR3-(人)FR4的排列方式构建人源化可变区(VH或VL)序列。
本实施例使用人胚系V基因片段IGHV3-73*01或人胚系V基因片段IGHV3-23*01结合人胚系J基因片段IGHJ1*01的序列作为人源化模板提供框架区序列。并且在第30位、第73位、第76位、第78位、第93位或第94位(按照Chothia编码规则)引入一个或者多个位点的氨基酸突变，得到多个不同的VH变体序列。本实施例还使用人胚系V基因片段IGLV7-46*02结合人胚系J基因片段IGLJ2*01的序列或者人胚系V基因片段IGKV1-39*01结合人胚系J基因片段IGKJ4*01的序列作为人源化模板提供框架区序列。并且在第2位、第36位、第46位、第49位、第66位、第69位、第71位或第87位(按照Chothia编码规则)引入零个或者多个位点的氨基酸突变，得到多个不同的VL变体序列。
将SP34衍生的VH变体序列和VL变体序列进行配对组合，并按照实施例2.2中的方法制备重组抗体。表7-12和表7-13列出了SP34及其衍生变体的重组抗体和对应的序列编号。
抗体编号 可变区变体 Fc类型(突变)
PR000260 SP34鼠抗初始序列 人IgG1(LALA)
PR000511 VH:VH3231；VL:VL7461 人IgG1(LALA)
PR001848 VH:VH3232；VL:VL7461 人IgG1(AAG)
PR003886 VH:VH3730_N30S；VL:VL7461 人IgG1(LALA)
PR004616 VH:VH3233；VL:VL7461 人IgG1(LALA)
PR000510 VH:VH3731；VL:VL7461.构建成scFv 人IgG1(LALA)
表7-12 SP34衍生的人源化重组抗体
(AAG:L234A,L235A,P329G；LALA:L234A,L235A.)
抗体编号 轻链 重链 VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3
PR000260 352 307 287 239 172 192 216 20 68 128
PR000511 357 313 291 245 172 192 216 20 68 128
PR001848 357 325 291 257 172 192 216 30 68 128
PR003886 357 328 291 263 172 192 216 30 68 128
PR004616 357 346 291 281 172 192 216 30 68 128
PR000510 无 489 291 244 172 192 216 20 68 128
表7-13 SP34衍生的人源化重组抗体的序列和CDR序列的序列编号表
实施例7.7.3.CD3抗体结合T细胞
利用流式细胞术FACS和实施例7.10所述方法测试CD3抗体结合人的T细胞的结合能力。如图66所示，所述CD3抗体都可以与人T细胞结合；特别地，全人源CD3抗体(PR002161,PR002163)结合T细胞的能力与SP34嵌合抗体(PR000260)的相似(图66的(F))；PR000510和PR000511是SP34衍生的人源化抗体，其结合T细胞的能力与PR000260的接近(图66的(A)-(B))；PR001848、PR003886和PR004616是SP34衍生的人源化抗体，通过序列优化，不同程度地减弱其与T细胞结合的能力(图66的(C)-(E))，以在后续的双抗设计中有选择性地调节T细胞活化功能。
实施例7.8.利用抗CD3的IgG抗体和抗BCMA的HCAb抗体构建双特异性抗体分子
本实施例利用抗CD3的IgG抗体的抗原结合结构域Fab，和抗BCMA的HCAb抗体的抗原结合结构域VH，来构建多种结构的抗BCMA x CD3的双特异性抗体分子。抗CD3的抗原结合结构域Fab的序列来源于表7-11和表7-13所述序列；抗BCMA的抗原结合结构域VH的序列来源于表7-1和表7-8所述序列。
在本实施例及后续实施例中，对照分子为：阳性对照1分子为抗BCMA的单抗CA8-J6M0(抗体编号PR000274，序列来源专利US9273141B2)；阳性对照2分子为BCMA x CD3双抗分子(抗体编号PR002199，序列来源专利WO2019133761A1)；阳性对照3分子为BCMA x CD3双抗分子(抗体编号PR003106，序列来源专利WO2017134134A1)；抗CD3的单抗SP34衍生的hIgG1嵌合抗体(抗体编号PR000260，序列来源专利WO2016071004A1)。对照分子的序列见表7-19。
实施例7.8.1.构建Fab-Fc-VH(n)非对称结构分子
利用抗CD3的IgG抗体和抗BCMA的重链抗体，按照实施例1.5和实施例1.6所述结构设计Fab-Fc-VH(n,n＝{1,2})非对称结构的BCMA x CD3双抗分子，总结于表7-14；并按照实施例1.6.2所述结构设计含有两个不同抗BCMA的VH结构域序列的三价非对称结构分子，总结于表7-15；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表7-16。
Figure PCTCN2021103044-appb-000088
Figure PCTCN2021103044-appb-000089
Figure PCTCN2021103044-appb-000090
Figure PCTCN2021103044-appb-000091
表7-14 Fab-Fc-VH(n)非对称结构的BCMA x CD3双抗分子
(突变代号：Knob:S354C,T366W；Hole:Y349C,T366S,L368A,Y407V；AAG:L234A,L235A,P329G；LALA:L234A,L235A；YTE:M252Y,S254T,T256E.)
Figure PCTCN2021103044-appb-000092
表7-15含有两个不同抗BCMA的VH结构域序列的Fab-Fc-VH(2)三价非对称结构的BCMA x CD3多特异性抗体分子
(突变代号：Knob:S354C,T366W；Hole:Y349C,T366S,L368A,Y407V；AAG:L234A,L235A,P329G.)
Figure PCTCN2021103044-appb-000093
Figure PCTCN2021103044-appb-000094
Figure PCTCN2021103044-appb-000095
表7-16 Fab-Fc-VH(n)非对称结构的BCMA x CD3双抗分子蛋白的表达
实施例7.8.2.构建scFv-Fc-VH(n)非对称结构分子
利用抗CD3的IgG抗体和抗BCMA的重链抗体，按照实施例1.7和实施例1.8所述结构设计scFv-Fc-VH(n,n＝{1,2})非对称结构的BCMA x CD3双抗分子，总结于表7-17；并按照实施例2所述方法制备抗体分子样品并进行分析，总结于表7-18。
Figure PCTCN2021103044-appb-000096
Figure PCTCN2021103044-appb-000097
表7-17 scFv-Fc-VH(n)非对称结构的BCMA x CD3双抗分子
(突变代号：Knob:S354C,T366W；Hole:Y349C,T366S,L368A,Y407V；AAG:L234A,L235A,P329G.)
Figure PCTCN2021103044-appb-000098
表7-18 scFv-Fc-VH(n)非对称结构的BCMA x CD3双抗分子蛋白的表达
实施例7.8.3.BCMA x CD3双抗分子及对照分子序列表
表7-20和表7-19列出了本实施例所构建的BCMA x CD3双抗分子和对照分子的序列所对应的序列编号。双抗分子中，抗CD3的抗原结合结构域Fab的序列来源于表7-11和表7-13所述序列；抗BCMA的抗原结合结构域VH的序列来源于表7-1和表7-8所述序列。表7-20中的结构编号对应于表1-1和图1。表7-21列出了BCMA x CD3双特异性或多特异性抗体分子的不同抗原结合结构域相应的CDR序列的序列编号。
Figure PCTCN2021103044-appb-000099
表7-19对照分子的序列编号表
结构编号 抗体编号 多肽链1 多肽链2 多肽链3
11 PR001987 357 411 410
11 PR001988 357 411 412
11 PR001989 357 411 413
11 PR001990 357 411 414
11 PR002929 357 444 449
11 PR002935 357 450 449
11 PR003867 357 454 465
11 PR003868 357 454 466
11 PR003869 357 454 467
11 PR003870 357 454 468
11 PR003871 357 454 469
11 PR003872 357 454 470
11 PR003873 357 454 471
11 PR003874 357 454 472
11 PR003875 357 454 473
11 PR003876 357 454 474
11 PR003877 357 454 475
11 PR003878 357 454 476
11 PR003879 357 454 477
11 PR003880 357 454 478
11 PR003881 357 454 479
11 PR003882 357 454 480
12 PR001991 415 416 410
12 PR001992 415 416 412
12 PR001993 415 416 413
12 PR001994 415 416 414
13 PR001995 417 418 410
13 PR001996 417 418 412
13 PR001997 417 418 413
13 PR001998 417 418 414
14 PR002299 357 423 424
14 PR002300 357 423 425
14 PR002301 357 423 426
14 PR002308 357 423 427
14 PR002309 357 423 428
14 PR002310 357 423 429
14 PR002892 357 444 445
14 PR002895 357 444 446
14 PR002898 357 444 447
14 PR002901 357 444 448
14 PR002950 357 450 445
14 PR002953 357 450 446
14 PR002956 357 450 447
14 PR002959 357 450 448
14 PR003177 357 454 445
14 PR003178 357 454 446
14 PR003690 357 463 464
15 PR002313 357 423 430
15 PR002326 357 423 431
15 PR002328 357 423 432
15 PR002332 357 423 433
20 PR001999 419 410 无
20 PR002000 419 412 无
20 PR002001 419 413 无
20 PR002002 419 414 无
23 PR002867 440 428 无
23 PR002868 441 428 无
23 PR002869 442 428 无
23 PR002870 443 428 无
表7-20本实施例的BCMA x CD3双抗分子的序列编号表
Figure PCTCN2021103044-appb-000100
Figure PCTCN2021103044-appb-000101
Figure PCTCN2021103044-appb-000102
Figure PCTCN2021103044-appb-000103
表7-21 BCMA x CD3双特异性或多特异性抗体分子的抗原结合结构域的CDR的序列编号表
实施例7.9.双抗分子结合BCMA
本实施例是为了研究BCMA x CD3双特异性抗体结合BCMA的能力。
按照实施例7.3所述方法用流式细胞术FACS测试双抗分子与高表达人BCMA的HEK293T细胞株HEK293T/hBCMA(北京康源博创,KC-0233)、高表达食蟹猴BCMA的HEK293T细胞株HEK293T/cynoBCMA(北京康源博创,KC-0979)和高表达人BCMA的细胞系NCI-H929(ATCC,CRL-9068)等细胞的结合能力。
本实施例中，阳性对照1分子为抗BCMA的单抗PR000274。阳性对照2分子为利用专利WO2019133761A1中序列构建的BCMA x CD3双抗分子PR002199。阳性对照3分子为利用专利WO2017134134A1中序列构建的BCMA x CD3双抗分子PR003106。
实施例7.9.1.结合高表达人BCMA的细胞HEK293T/hBCMA
图50和表7-22中所示，双抗分子都可以结合人BCMA，而且含有两个串联形成的BCMA结合结构域的Fab-Fc-VH(2)非对称结构的双抗分子比仅有一个BCMA结合结构域的Fab-Fc-VH非对称结构的双抗分子，有更强的结合BCMA的能力。
Figure PCTCN2021103044-appb-000104
表7-22结合HEK293T/hBCMA
实施例7.9.2.结合高表达人BCMA的肿瘤细胞NCI-H929
图52和表7-23中所示，双抗分子都可以结合人BCMA。总体说来，仅有一个BCMA结合结构域的双抗分子(例如在Fab-Fc-VH结构和scFv-Fc-VH结构)结合BCMA的能力比具有二价BCMA结合结构域的分子(如IgG单抗，或者Fab-Fc-VH(2)非对称结构的双抗分子)的弱。图52的(E)中所示，从PR001046衍生的亲和力提高的 HCAb变体基础上构建了一系列Fab-Fc-VH结构的双抗分子(PR003867,…,PR003882)，这些双抗分子结合BCMA的能力相对于PR002929(用PR001046 HCAb构建的Fab-Fc-VH结构)有更强的结合BCMA的能力。Fab-Fc-VH(2)非对称结构的双抗分子结合BCMA的能力优于阳性对照1和3。
Figure PCTCN2021103044-appb-000105
Figure PCTCN2021103044-appb-000106
表7-23结合NCI-H929
实施例7.9.3.结合高表达食蟹猴BCMA的细胞HEK293T/cynoBCMA
图51和表7-24中所示，双抗分子都可以结合食蟹猴BCMA，但是阳性对照2双抗分子不能结合食蟹猴BCMA。并且含有两个PR001046的VH串联结构域的Fab-Fc-VH(2)具有更强的结合食蟹猴BCMA的能力。
抗体 MFI最大值 抗体 MFI最大值 抗体 MFI最大值
PR001990 9167 PR002309 16248 PR002301 12958
PR002929 13791 PR002892 14983 PR002308 39286
PR002935 21994 PR002895 13799 PR002309 41640
    PR002950 26570 PR002310 42045
    PR002953 35655    
    PR002898 8188    
    PR002901 11033    
    PR002956 16151    
    PR002959 30644    
    阳性对照2 926    
抗体 MFI最大值 抗体 MFI最大值    
PR002313 7608 PR002309 128176    
PR002326 6444 PR002895 103462    
PR002328 7807 PR002953 109345    
    PR003178 115022    
    阳性对照2 4698    
表7-24结合HEK293T/cynoBCMA
实施例7.10.双抗分子结合T细胞
本实施例是为了研究BCMA×CD3双特异性抗体结合T细胞的能力。
利用流式细胞术FACS测试抗体分子与人或食蟹猴T细胞的结合能力。具体地，利用T细胞分离试剂盒(Meltenyi,#130-096-535)从人或食蟹猴的PBMC细胞中分离得到T细胞。将T细胞密度分别调整为1 x10 6细胞/mL。以100μL细胞/孔接种于96孔板(Corning,#3894)，随后以100μL/孔加入2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育1小时。之后，以100μL/孔加入预冷PBS漂洗细胞两次，于4℃下500g离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson,#109-545-06,1:500稀释)，于4℃下避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次，于4℃下500g离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
本实施例中，阳性对照为抗CD3的单抗SP34(抗体编号PR000260)；阳性对照2双抗分子(抗体编号PR002199)为另一个对照分子。
实施例7.10.1.结合人的T细胞
图53和表7-25中所示，双抗分子都能结合人的T细胞。阳性对照分子SP34是二价的IgG结构，其结合T细胞的能力非常强。阳性对照2双抗分子结合T细胞很弱。双抗分子的CD3端结构是单价的Fab，其Fab序列是来源于抗CD3的单抗SP34的变体序列，通过序列设计和突变，这些变体序列结合T细胞的能力有不同程度的降低，这是为了降低CD3端的T细胞激活能力并减少细胞因子的过度释放带来的副作用。不同的双抗分子的CD3端有不同的结构或者变体序列，其结合T细胞的能力也不相同。在临床上对于平衡活性和安全性的不同的需求，使得对T细胞的结合能力的要求也不同。
Figure PCTCN2021103044-appb-000107
Figure PCTCN2021103044-appb-000108
表7-25结合人的T细胞
实施例7.10.2.结合食蟹猴的T细胞
图54和表7-26中所示，由于本实施例的双抗分子的CD3端Fab序列是来源于抗CD3的单抗SP34的变体序列，因而可以结合食蟹猴的T细胞，而阳性对照2双抗分子不能结合食蟹猴的T细胞。
抗体 MFI最大值
PR001990 15557
PR002929 10128
PR002309 11664
PR003178 10725
PR002895 18601
PR002953 9708
PR003177 12107
PR002950 8827
阳性对照2 1109
hIgG1 iso 794
表7-26结合食蟹猴的T细胞
实施例7.11.BLI方法测定双抗分子结合BCMA的亲和力
本实施例按照实施例7.5所述方法使用Octet Red96e分子相互作用分析仪(ForteBio,Octet Red96e)来分析BCMA x CD3双抗分子与BCMA抗原蛋白之间的结合动力学。
本实施例的双抗分子都可以结合人和食蟹猴的BCMA(表7-27和表7-28)，但是阳性对照2双抗分子不能结合食蟹猴的BCMA。而且本实施例的双抗分子的结合人BCMA(ACRO Biosystems,#BC7-H82F0)和食蟹猴BCMA(ACRO Biosystems,#BCA-C82F4)的亲和力(KD)的比值(表7-29)在5倍以内(0.5～5)，说明有很好的相关性。
抗体 抗原 KD(M) kon(1/Ms) kdis(1/s) Full R^2
阳性对照1 huBCMA.Fc 6.55E-10 2.06E+05 1.35E-04 0.9994
PR001046 huBCMA.Fc 7.75E-11 4.59E+05 3.56E-05 0.9952
PR001990 huBCMA.Fc 1.95E-09 3.22E+05 6.29E-04 0.995
阳性对照2 huBCMA.Fc 4.52E-11 5.45E+05 2.46E-05 0.9969
PR002309 huBCMA.Fc 4.48E-11 4.22E+05 1.89E-05 0.9974
PR002895 huBCMA.Fc 1.97E-10 4.12E+05 8.11E-05 0.9974
PR002929 huBCMA.Fc 5.95E-09 2.49E+05 1.48E-03 0.9933
PR002953 huBCMA.Fc 2.25E-10 4.74E+05 1.07E-04 0.998
PR003178 huBCMA.Fc 2.36E-10 4.94E+05 1.17E-04 0.9978
表7-27 BCMA x CD3双抗分子结合人BCMA蛋白的亲和力
抗体 抗原 KD(M) kon(1/Ms) kdis(1/s) Full R^2
阳性对照1 cyBCMA.Fc 3.88E-11 2.00E+05 7.76E-06 0.9456
PR001046 cyBCMA.Fc 1.19E-10 6.33E+05 7.54E-05 0.9965
PR001990 cyBCMA.Fc 3.35E-09 7.46E+05 2.50E-03 0.9689
阳性对照2 cyBCMA.Fc \      
PR002309 cyBCMA.Fc 1.41E-10 8.23E+05 1.16E-04 0.9973
PR002895 cyBCMA.Fc 2.72E-10 8.90E+05 2.42E-04 0.9983
PR002929 cyBCMA.Fc 6.62E-09 8.79E+05 5.82E-03 0.9437
PR002953 cyBCMA.Fc 1.95E-10 3.65E+05 7.12E-05 0.9989
PR003178 cyBCMA.Fc 3.19E-10 8.22E+05 2.62E-04 0.9981
表7-28 BCMA x CD3双抗分子结合食蟹猴BCMA蛋白的亲和力
抗体 KD比值(cyBCMA/huBCMA)
阳性对照1 0.06
PR001046 1.54
PR001990 1.72
阳性对照2 N/A
PR002309 3.16
PR002895 1.38
PR002929 1.11
PR002953 0.87
PR003178 1.35
表7-29结合BCMA亲和力的比值
实施例7.12.双抗分子对靶细胞杀伤实验
本实施例研究了BCMA x CD3双抗分子介导的T细胞激活和特异性的靶细胞杀伤能力。
实施例7.12.1.对高表达BCMA的细胞NCI-H929的体外杀伤实验以及细胞因子的释放
本实施例研究了在高表达BCMA的肿瘤细胞NCI-H929(ATCC,CRL-9068)存在时，双抗分子激活T细胞，释放细胞因子并杀伤肿瘤细胞的能力。效应细胞可以是人外周血单个核细胞PBMC或者是从PBMC分离得到的T细胞。
具体地，用培养基(还有5％FBS的RPMI1640培养基)将效应细胞(PBMC或者T细胞)的密度调整为1.1 x10 6细胞/mL，NCI-H929的密度调整为0.11 x10 6细胞/mL，将两种细胞悬液各以90μL细胞/孔接种于96孔板(Corning,#3799)。随后以20μL/孔加入不同浓度的待测抗体(10倍于终浓度的梯度稀释)，抗体浓度可以是终浓度为30nM，或者从最高终浓度为1nM按照20倍浓度梯度稀释的共3个浓度，或者从最高终浓度为30nM按照4倍浓度梯度稀释的共10个浓度；hIgG1 iso(CrownBio,#C0001)作为同型对照。最终效靶比为10：1，设置两个复孔加样。加入了待测抗体的孔称为ER(实验孔，含有待测抗体样品和效应细胞及靶细胞)；同时，在板内按照下面公式中的参数组成设置不同的对照组：ESR(效应细胞自然释放孔，仅效应细胞和培养基)； TSR(靶细胞自然释放孔，仅靶细胞和培养基)；CMB(培养基参照孔，仅培养基)；TMR(靶细胞最大释放空，仅靶细胞和培养基)；VCC(体积参照孔，仅培养基)。将96孔板置于37℃二氧化碳培养箱孵育24小时。随后向TMR孔和VCC孔加入10μL裂解液，继续孵育30分钟。孵育完成后，取50μL/孔的上清液，加入96孔板(Corning,#:3599)，并以50μL/孔加入细胞毒性检测试剂(CytoTox 
Figure PCTCN2021103044-appb-000109
非放射性细胞毒性检测试剂盒，Promega,#G1780)。室温孵育30分钟后，加入50μL反应终止液来终止反应。最后用Enspire TM多功能读板机(Perkin Elmer,Inc.)于490nM读取光吸收值，并根据下面计算公式计算靶细胞杀伤率。
杀伤率＝((ER–CMB)-(ESR–CMB)-(TSR–CMB))/(TMR-VCC)×100％
其中：
ER＝实验孔，样品+效应细胞+靶细胞
ESR＝效应细胞自然释放孔，效应细胞+培养基
TSR＝靶细胞自然释放孔，靶细胞+培养基
TMR＝靶细胞最大释放空，靶细胞+培养基+裂解液
VCC＝体积参照孔，培养基+裂解液
CMB＝培养基参照孔，培养基
在检测细胞毒性的同时，还可以检测细胞上清中的细胞因子释放水平。简言之，取出50μL上清液用于检测细胞因子的水平。用TNF-αELISA试剂盒(Thermo,#88-7346-88)检测上清中TNF-α浓度；用IL-6 ELISA试剂盒(Thermo,#88-7066-88)检测上清中IL-6浓度；用IL-2ELISA试剂盒(Thermo,#88-7025-88)检测上清中IL-2浓度；用IFN-γELISA试剂盒(Thermo,#88-7316-88)检测上清中IFN-γ浓度；用IL-10 ELISA试剂盒(Thermo,#88-7106-88)检测上清中IL-10浓度。ELISA检测方法参照相关试剂盒操作说明。使用软件GraphPad Prism 8进行数据处理和作图分析。
图56中所示，scFv-Fc-VH(1)非对称结构的双抗分子(PR002000,PR002002)可以对靶细胞产生有效的杀伤。基于抗BCMA的HCAb单抗PR000940构建的双抗分子PR001999对靶细胞没有杀伤功能。这些分子结构相似，都含有相同CD3的结合结构域(scFv)的序列，差异在于BCMA的结合结构域(VH)的序列不同，不同的VH与BCMA 抗原结合的亲和力或表位的差异导致了双抗分子对靶细胞的杀伤活性的差异。抗BCMA的IgG单抗阳性对照1分子也没有杀伤功能。
图55中所示，scFv-Fc-VH(2)非对称结构的双抗分子(PR002867,PR002868,PR002869,PR002870)可以对靶细胞产生不同程度的杀伤。这些分子结构相似，都含有两个相同的串联的BCMA的结合结构域(VH)，其仅有的差异在于CD3的结合结构域scFv的序列来源于不同的抗CD3单抗。PR002867和PR002870的靶细胞杀伤能力与阳性对照2双抗分子相似，而PR002868和PR002869的靶细胞杀伤能力较弱。
图57的(A)-(B)中所示，12个Fab-Fc-VH(1)非对称结构的双抗分子(PR001987,…,PR001998)有相似的分子结构，但是BCMA的结合结构域和CD3的结合结构域的序列不同；对靶细胞的杀伤能力也是不同的。其中，PR001988、PR001990、PR001994、PR001996和PR001998有较强的靶细胞杀伤能力。图57的(C)中所示，PR001990比PR001988有更强的靶细胞杀伤能力。图57的(D)-(F)中所示，从PR001046衍生的亲和力提高的HCAb变体基础上构建的Fab-Fc-VH结构的双抗分子，对靶细胞有杀伤能力。
图58中所示，Fab-Fc-VH(2)非对称结构的双抗分子可以对靶细胞产生有效的杀伤。这些分子都含有两个串联的BCMA的结合结构域(VH)。其靶细胞杀伤能力明显优于Fab-Fc-VH(1)结构的分子PR001990，也优于阳性对照2双抗分子。图58的(A)-(B)中所示，双抗分子PR002301、PR002308、PR002309、PR002310、PR002313、PR002326和PR002328具有相同的CD3的结合结构域的序列；其中，PR002301、PR002308、PR002309和PR002310的BCMA结合结构域是由两个相同的VH序列串联而成；PR002313、PR002326和PR002328的BCMA结合结构域是由两个不同的VH序列串联而成；特别是，PR002326和PR002328的BCMA结合结构域所使用的两个不同的VH可以结合BCMA的不同表位。图58的(C)中所示，双抗分子PR002892、PR002895、PR002950、PR002953、PR003177和PR003178的BCMA结合结构域是由两个相同的来源于PR001046的VH序列通过不同长度的连接肽序列串联而成；但是它们具有不同的CD3的结合结构域的序列，有不同的结合T细胞的能力，因而也体现出不同的靶细胞杀伤效率(EC50)。图58的(D)中所示，双抗分子PR003690是在PR003178的基础上，在Fc区引入了额外的氨基酸突变以进一步提高血清半衰期，其靶点结合结构域没有变化，因而PR003690的靶细胞杀伤能力与PR003178的一致。
图59和图60中所示，更系统性地研究了双抗分子PR001990(Fab-Fc-VH(1)结构，强CD3结合)、PR002309(Fab-Fc-VH(2)结构，强CD3结合)、PR002895(Fab-Fc-VH(2)结构，强CD3结合)、PR002953(Fab-Fc-VH(2)结构，弱CD3结合)和PR003178(Fab-Fc-VH(2)结构，中等CD3结合)以及阳性对照2和阳性对照3等对靶细胞的杀伤能力和多种细胞因子释放水平(表7-30和表7-31)。这些双抗分子都能达到相似的最大杀伤率，但是杀伤效率EC50不同，诱导的细胞因子释放水平也不同。PR002309、PR002895、PR002953和PR003178的靶细胞杀伤EC50优于阳性对照2；而且，PR002895、PR002953和PR003178引起的多种细胞因子释放水平低于阳性对照3。特别是，相较于阳性对照2，PR003178的靶细胞杀伤能力优于阳性对照2(EC50强10倍)；相较于阳性对照3，PR003178有与阳性对照3相当的杀伤能力，但是有更低的细胞因子释放最大值。这说明，PR003178在保持有效的肿瘤杀伤效率的同时，能够控制细胞因子的释放水平，以减少细胞因子释放综合征的风险，可能可以在临床上更好地平衡有效性和安全性以及带来更好的药物治疗窗。
Figure PCTCN2021103044-appb-000110
表7-30 BCMA x CD3双抗分子介导效应细胞对NCI-H929细胞的体外杀伤和细胞因子释放
Figure PCTCN2021103044-appb-000111
Figure PCTCN2021103044-appb-000112
表7-31 BCMA x CD3双抗分子介导效应细胞对NCI-H929细胞的体外杀伤和细胞因子释放
实施例7.12.2.可溶性APRIL和可溶性BAFF对于双抗分子的靶细胞杀伤效果的影响
本实施例在实施例7.12.1所述方法的基础上进一步研究BCMA配体(APRIL和BAFF)对双抗分子的靶细胞杀伤效果的影响，在多发性骨髓瘤患者的血浆中APRIL和BAFF的浓度约100ng/ml(Blood 2004；103:3148–3157)。具体地，在如实施例7.12.1所述方法中，在加入不同浓度的待测抗体时，也以20μL/孔加入BCMA配体蛋白，使可溶性的配体蛋白的终浓度为100ng/ml和10ng/ml。可溶性APRIL蛋白(sAPRIL)是重组人源APRIL蛋白(Novoprotein,#CU89)；可溶性BAFF蛋白(sBAFF)是重组人源BAFF蛋白(ACRO Biosystems,#BAF-H5248)。效应细胞是人PBMC或者T细胞，靶细胞是NCI-H929。用实施例7.12.1所述方法计算靶细胞杀伤率。
图61和表7-32中所示，可溶性APRIL和BAFF对于双抗分子的靶细胞杀伤能力几乎没有影响。
表7-32不同浓度可溶性APRIL或BAFF存在时，BCMA x CD3双抗分子对NCI-H929的杀伤
Figure PCTCN2021103044-appb-000113
Figure PCTCN2021103044-appb-000114
实施例7.12.3.可溶性BCMA对于双抗分子的靶细胞杀伤效果的影响
BCMA是跨膜蛋白，但是其胞外区有可以从细胞膜上脱落下来形成可溶性BCMA(sBCMA)，在多发性骨髓瘤患者的血浆中都观察到sBCMA的水平有不同程度的升高，中位值浓度为176.0ng/ml(Leuk Res.2019Jun；81:62-66)。
本实施例在实施例7.12.1所述方法的基础上做了优化，研究可溶性BCMA(sBCMA)对双抗分子的靶细胞杀伤效果的影响。具体地，在如实施例7.12.1所述方法中，在加入不同浓度的待测抗体时，也以20μL/孔加入重组人源BCMA蛋白(ACRO Biosystems,#BCA-H522y)，使其终浓度为500ng/ml、250ng/ml、125ng/ml、和62.5ng/ml。效应细胞是人PBMC，靶细胞是NCI-H929。用实施例7.12.1所述方法计算靶细胞杀伤率。
图62和表7-33中所示，可溶性BCMA对双抗分子的靶细胞杀伤率EC50有影响，且呈剂量依赖关系，但是不影响最大杀伤率。当可溶性BCMA的浓度为176.0ng/ml 时，双抗分子PR002309和PR003178的靶细胞杀伤率EC50优于阳性对照2和阳性对照3；也就是说可溶性BCMA对双抗分子PR002309和PR003178的靶细胞杀伤率的影响小于对阳性对照2和阳性对照3的影响。
表7-33不同浓度可溶性BCMA存在时，BCMA x CD3双抗分子对NCI-H929的杀伤
Figure PCTCN2021103044-appb-000115
实施例7.13.药代动力学研究
本实施例研究了具有Fab-Fc-VH非对称结构的BCMA x CD3双抗分子(PR002929)和具有Fab-Fc-VH(2)非对称结构的BCMA x CD3双抗分子(PR003178)在小鼠或者大鼠体内的单剂量的药代动力学性能。
实施方法如下：选取体重合适的雌性BALB/c小鼠6只或者SD大鼠3只，按5mg/kg的剂量通过静脉注射给与融合蛋白药物；于给药前以及给药后0.25小时、5小时、24小时(1天)、第2天、第4天、第7天、第10天和第14天采集全血，将全血静置30分钟使其凝固，随后在4℃下以2,000rpm离心5分钟并将分离的血清样品在-80℃下冻存直至分析。本实施例采用两种方法来定量测定血清中的药物浓度。方法一，Fc端检测ELISA方法来定量测定血清中的抗体药物浓度：即通过包被于96孔板的山羊抗人Fc多克隆抗体来捕获血清中的含有人Fc的BCMA x CD3双抗分子，然后加入HRP标记的山羊抗人Fc第二抗体来检测。方法二，BCMA端检测ELISA方法来定量测定血清中的抗体药物浓度：即通过包被于96孔板的BCMA重组蛋白(Human BCMA/TNFRSF17 Protein His Tag,ACRO Biosystems,#BCA-H522y)来捕获血清中的含有抗BCMA的VH端的完整BCMA x CD3双抗分子，然后加入HRP标记的山羊抗人Fc第二抗体来检测。使用Phoenix WinNonlin软件8.2版，选用非房室模型(NCA)分析药代动力学参数。
图63的(A)-(B)中所示，双抗分子PR002929和PR003178有相似的药代动力学：在Fc端检测方法下，PR002929和PR003178在BALB/c小鼠体内的血清半衰期t 1/2值分别约为7天和9.6天；在BCMA端检测方法下，PR002929和PR003178在小鼠体内的血清半衰期t 1/2值分别约为4天和4天。
图63的(C)中所示，分别用Fc端检测方法和BCMA端检测方法，PR003178在大鼠体内的血清半衰期t 1/2值分别约为5.6天和4.6天。
表7-34总结了BCMA x CD3双抗分子在小鼠或者大鼠体内的药代动力学性能参数。
Figure PCTCN2021103044-appb-000116
Figure PCTCN2021103044-appb-000117
表7-34 BCMA x CD3双抗分子在小鼠或者大鼠体内的药代动力学参数
实施例7.14.在NCI-H929/人PBMC小鼠模型中的抗肿瘤药效评估
为了评估BCMA x CD3双特异性抗体的体内抗肿瘤药效。采用6-8周雌性NCG小鼠，瘤细胞接种前3天，所有小鼠腹腔注射(i.p.)3×10 6人PBMC，然后于肿瘤细胞接种当天每只实验小鼠皮下接种5x10 6NCI-H929细胞，细胞重悬在PBS与Matrigel(1:1)混合液中(0.1mL/只)。当肿瘤体积达到90mm 3时对小鼠进行随机分组，每组6只小鼠。分组后将特定浓度经PBS稀释的抗体药物按照特定的剂量和频次以静脉注射(i.v.)方式给药。本实施例采用了多种给药方案：1)10μg/鼠的剂量组，单次给药；2)10μg/鼠和3μg/鼠的剂量组，每周给药1次总共给药2次(QW*2)。每次试验以PBS为空白对照组。在初次给药后第3天，7天，10天和14天对肿瘤体积和小鼠体重进行测量。肿瘤大小计算公式：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。
图64的(A)-(B)中所示，PR001990，PR002309，PR002313和阳性对照2等双抗分子的10μg/鼠单次给药剂量组都显示出抑制肿瘤生长的药效，而且PR001990和
PR002309的药效显著优于同等剂量的阳性对照2分子。各组小鼠体重变化均在正常范围内。
图64的(C)-(D)中所示，所有双抗分子的不同剂量组都显示出不同程度的抑制肿瘤生长的药效。总体说来，10μg/鼠组的药效优于3μg/鼠组的药效；而且，在10μg/鼠QW*2的给药剂量下，PR003177和PR003178的药效优于同等剂量的阳性对照2分子；PR002895的3μg/鼠剂量组的药效甚至略优于阳性对照2分子的10μg/鼠剂量组。各组小鼠体重变化均在正常范围内。
实施例7.15.小结
本实施例利用抗CD3的IgG抗体的抗原结合结构域Fab(或scFv形式)和抗BCMA的HCAb抗体的抗原结合结构域VH，构建了多种非对称结构的抗BCMA x CD3的双特异性抗体分子。展现出了基于HCAb构建双特异性抗体分子结构的灵活性。本实施例的双抗分子可以同时结合人的BCMA和人的T细胞，而且可以结合食蟹猴的 BCMA和食蟹猴的T细胞，因而可以利用食蟹猴做非灵长类毒理评估，而这一点对临床开发非常重要。而阳性对照2双抗分子却没有这种结合食蟹猴靶点的性能。
本实施例构建了一系列BCMA x CD3双抗分子，通过调节CD3结合结构域的序列及细微结构来改变其结合T细胞的能力，通过调节BCMA结合结构域的序列和结构域数目来改变其结合表达BCMA的靶细胞的能力，进而调节双抗分子的T细胞激活和特异性的靶细胞杀伤能力。在体外靶细胞杀伤实验中，具有Fab-Fc-VH(2)非对称结构的双抗分子PR002309、PR002895、PR002953和PR003178的靶细胞杀伤EC50优于阳性对照2；而且，PR002895、PR002953和PR003178引起的多种细胞因子释放水平低于阳性对照3。在小鼠体内抗肿瘤药效模型中，PR002309和PR003178的药效显著优于同等剂量的阳性对照2分子；PR002895的低剂量组的药效甚至略优于阳性对照2分子的高剂量组。
特别是，PR003178的靶细胞杀伤能力明显优于阳性对照2且与阳性对照3相当；但是PR003178比阳性对照3有更低的细胞因子释放水平。这说明，PR003178在保持有效的肿瘤杀伤效率的同时，能够控制细胞因子的释放水平，以减少细胞因子释放综合征的风险，可能可以在临床上更好地平衡有效性和安全性以及带来更好的药物治疗窗。

Claims (19)

  1. 一种含有至少两个蛋白功能区的结合蛋白，其中，所述结合蛋白包括蛋白功能区A和蛋白功能区B；所述蛋白功能区A和所述蛋白功能区B靶向不同的抗原或抗原表位，其中所述蛋白功能区A为Fab或scFv结构，所述蛋白功能区B为VH结构，所述蛋白功能区A和蛋白功能区B的数量分别为一个或多个；较佳地，所述结合蛋白为对称结构或非对称结构，和/或，所述结合蛋白还包括Fc，所述Fc的数量为二个，由此形成Fc二聚体优选为同源二聚体或异源二聚体。
  2. 如权利要求1所述的结合蛋白，其中，
    (I)所述结合蛋白为对称结构，所述对称结构为左右对称的类IgG结构，其中所述蛋白功能区A的数量为二个，所述蛋白功能区B的数量为二个或四个；
    较佳地，当所述蛋白功能区B的数量为二个时，所述结合蛋白为四价结合蛋白，其包括以下结构：(a)所述结合蛋白从N末端至C末端依次为蛋白功能区A、蛋白功能区B和Fc，其中所述蛋白功能区A与所述蛋白功能区B通过第一连接肽(L1)连接，所述蛋白功能区B与所述Fc通过第二连接肽(L2)连接；或，(b)所述结合蛋白从N末端至C末端依次为蛋白功能区B、蛋白功能区A和Fc，其中所述蛋白功能区B与所述蛋白功能区A通过连接肽连接，所述蛋白功能区A与所述Fc通过铰链区连接；或，(c)所述结合蛋白从N末端至C末端依次为蛋白功能区A、Fc和蛋白功能区B，其中所述蛋白功能区A与Fc通过铰链区连接，所述Fc与所述蛋白功能区B通过连接肽连接；
    当所述蛋白功能区B的数量为四个时，所述结合蛋白为六价结合蛋白，其包括以下结构：新增的二个蛋白功能区B进一步连接在上述(a)或(b)或(c)所述结合蛋白的N末端或C末端；优选连接在上述(c)所述结合蛋白的Fc的C末端或者原有的蛋白功能区B的C末端，或与上述(b)所述结合蛋白的原有的蛋白功能区B一同连接于蛋白功能区A的N末端；
    更佳地，所述结合蛋白还包括蛋白功能区C，所述蛋白功能区C与所述蛋白功能区A、蛋白功能区B靶向不同的抗原或抗原表位，所述结合蛋白为六价或八价的三特异性结合蛋白，其包括以下结构：所述蛋白功能区C连接在上述结合蛋白的N末端或C末 端；优选所述蛋白功能区C的数量为二个，所述蛋白功能区C连接在上述(c)所述结合蛋白的C末端，或，与上述(b)所述结合蛋白的蛋白功能区B一样，连接于所述蛋白功能区A的N末端；或，
    (II)所述结合蛋白为非对称结构，所述非对称结构呈左右不对称的类IgG结构，其中左臂为Fab或scFv结构的蛋白功能区A，右臂为VH结构的蛋白功能区B，所述蛋白功能区A和所述蛋白功能区B分别与一个Fc连接；优选所述蛋白功能区A的数量为一个，所述蛋白功能区B的数量为一个或二个或三个；
    较佳地，当所述蛋白功能区B的数量为一个时，所述结合蛋白为二价结合蛋白，其包括以下结构：所述蛋白功能区A为(d)常规Fab结构，或(e)Fab(cross VH/VL)结构，或(f)Fab(cross Fd/LC)结构；当所述蛋白功能区B的数量为二个时，所述结合蛋白为三价结合蛋白，其包括以下结构：左臂的所述蛋白功能区A为上述(d)或(e)或(f)，第二个蛋白功能区B连接在左臂的所述蛋白功能区A的N末端或C末端，或右臂的第一个蛋白功能区B的N末端，或所述Fc的C末端，优选第二个蛋白功能区B连接在右臂的第一个蛋白功能区B的N末端；当所述蛋白功能区B的数量为三个时，所述结合蛋白为四价结合蛋白，具体包括以下结构：左臂的所述蛋白功能区A为上述(d)或(e)或(f)结构，第三个蛋白功能区B进一步连接在左臂的所述蛋白功能区A的N末端或C末端，或左臂的第二个蛋白功能区B的N末端或C末端，或右臂的第一个或第二个蛋白功能区B的N末端，或所述Fc的C末端，优选第二个蛋白功能区B连接在右臂的第一个蛋白功能区B的N末端，且第三个蛋白功能区B连接在所述第二个蛋白功能区B的N末端；或，
    当所述蛋白功能区B的数量为一个时，所述结合蛋白为二价结合蛋白，其包括以下结构：左臂的所述蛋白功能区A为(g)scFv结构，所述scFv通过VH的末端或VL的末端与Fc连接；当所述蛋白功能区B的数量为二个时，所述结合蛋白为三价结合蛋白，具体包括以下结构：左臂的所述蛋白功能区A为(g)，第二个蛋白功能区B连接在左臂的所述蛋白功能区A的N末端，或右臂的第一个蛋白功能区B的N末端，或所述Fc的C末端，优选第二个蛋白功能区B连接在右臂的第一个蛋白功能区B的N末端；当所述蛋白功能区B的数量为三个时，所述结合蛋白为四价结合蛋白，具体包括以下结构：左臂的所述蛋白功能区A为(g)，第三个蛋白功能区B进一步连接在左臂的所述蛋白功能区A的N末端，或第二个蛋白功能区B的N末端或C末端，或所述Fc的C末端， 优选第二个蛋白功能区B连接在右臂的第一个蛋白功能区B的N末端，且第三个蛋白功能区B连接在所述第二个蛋白功能区B的N末端；
    更佳地，所述结合蛋白还包括蛋白功能区C，所述结合蛋白为三价或多价的多特异性结合蛋白，其中，所述蛋白功能区C与所述蛋白功能区A、蛋白功能区B靶向不同的抗原或抗原表位，所述蛋白功能区C连接在上述结合蛋白的N末端或C末端；优选所述蛋白功能区C连接在上述(d)、(e)、(f)或(g)所述结合蛋白的蛋白功能区B的N末端；
    进一步更佳地，所述结合蛋白还包括蛋白功能区D，所述结合蛋白为四价或多价的多特异性结合蛋白，具体地：所述蛋白功能区D与所述蛋白功能区A、蛋白功能区B、蛋白功能区C靶向不同的抗原或抗原表位，所述蛋白功能区D连接在上述结合蛋白的N末端或C末端；优选所述蛋白功能区D连接在上述(d)、(e)、(f)或(g)所述结合蛋白的蛋白功能区B的N末端。
  3. 如权利要求1或2所述的结合蛋白，其中，(A)所述结合蛋白具有四条多肽链，其中两条为相同的短链，另两条为相同的长链，其中，(1)所述短链从N末端至C末端依次包括VH_A-CH1，所述长链从N末端至C末端依次包括VL_A-CL-L1-VH_B-L2-CH2-CH3；或(2)所述短链从N末端至C末端依次包括VL_A-CL，所述长链从N末端至C末端依次包括VH_A-CH1-L1-VH_B-L2-CH2-CH3；或(3)所述短链从N末端至C末端依次包括VL_A-CL，所述长链从N末端至C末端依次包括VH_B-L-VH_A-CH1-h-CH2-CH3；或(4)所述短链从N末端至C末端依次包括VH_B-L-VL_A-CL，所述长链从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3；或(5)所述短链从N末端至C末端依次包括VL_A-CL，所述长链从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3-L-VH_B；或(6)所述短链从N末端至C末端依次包括VL_A-CL-L-VH_B，所述长链从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3；或(7)所述短链从N末端至C末端依次包括VL_A-CL，所述长链从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_B；或(8)所述短链从N末端至C末端依次包括VL_A-CL，所述长链从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3-L1-VH_B-L2-VH_C；或(9)所述短链从N末端至C末端依次包括VH_B-L1-VL_A-CL，所述长链从N末端至C末端依次包括VH_B-L2-VH_A-CH1-h-CH2-CH3；或(10)所述短链从N末端至C末端依次包括VH_B-L1-VL_A-CL，所述长链从N末端至C末端依次包括VH_C-L2-VH_A-CH1-h-CH2- CH3；其中，所述L、L1和L2为连接肽，所述h为铰链区或连接肽，所述铰链区或连接肽例如“-”、GS或如SEQ ID NO:495-519的氨基酸序列所示；或，
    (B)所述结合蛋白具有多肽链1、多肽链2和多肽链3三种多肽链，每种多肽链各一条，其中，(11)所述多肽链1从N末端至C末端依次包括VL_A-CL，所述多肽链2从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_B-h-CH2-CH3；或(12)所述多肽链1从N末端至C末端依次包括VH_A-CL，所述多肽链2从N末端至C末端依次包括VL_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_B-h-CH2-CH3；或(13)所述多肽链1从N末端至C末端依次包括VH_A-CH1，所述多肽链2从N末端至C末端依次包括VL_A-CL-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_B-h-CH2-CH3；或(14)所述多肽链1从N末端至C末端依次包括VL_A-CL，所述多肽链2从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_B-L-VH_B-h-CH2-CH3；或(15)所述多肽链1从N末端至C末端依次包括VL_A-CL，所述多肽链2从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_C-L-VH_B-h-CH2-CH3；或(16)所述多肽链1从N末端至C末端依次包括VH_A-CL，所述多肽链2从N末端至C末端依次包括VL_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_B-L-VH_B-h-CH2-CH3；或(17)所述多肽链1从N末端至C末端依次包括VH_A-CL，所述多肽链2从N末端至C末端依次包括VL_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_C-L-VH_B-h-CH2-CH3；或(18)所述多肽链1从N末端至C末端依次包括VH_A-CH1，所述多肽链2从N末端至C末端依次包括VL_A-CL-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_B-L-VH_B-h-CH2-CH3；或(19)所述多肽链1从N末端至C末端依次包括VH_A-CH1，所述多肽链2从N末端至C末端依次包括VL_A-CL-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_C-L-VH_B-h-CH2-CH3；或(26)所述多肽链1从N末端至C末端依次包括VL_A-CL，所述多肽链2从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_B-L1-VH_B-L2-VH_B-h-CH2-CH3；或(27)所述多肽链1从N末端至C末端依次包括VL_A-CL，所述多肽链2从N末端至C末端依次包括VH_A-CH1-h-CH2-CH3，所述多肽链3从N末端至C末端依次包括VH_D-L1-VH_C-L2-VH_B-h-CH2-CH3；其中，所述L、L1和L2 为连接肽，所述h为铰链区或连接肽，所述铰链区或连接肽例如“-”、GS或如SEQ ID NO:495-519的氨基酸序列所示；或，
    (C)所述结合蛋白具有多肽链1和多肽链2两种多肽链，每种多肽链各一条，其中，(20)所述多肽链1从N末端至C末端依次包括VL_A-L-VH_A-h-CH2-CH3，所述多肽链2从N末端至C末端依次包括VH_B-h-CH2-CH3；或(21)所述多肽链1从N末端至C末端依次包括VH_A-L-VL_A-h-CH2-CH3，所述多肽链2从N末端至C末端依次包括VH_B-h-CH2-CH3；或(22)所述多肽链1从N末端至C末端依次包括VL_A-L1-VH_A-h-CH2-CH3，所述多肽链2从N末端至C末端依次包括VH_B-L2-VH_B-h-CH2-CH3；或(23)所述多肽链1从N末端至C末端依次包括VH_A-L1-VL_A-h-CH2-CH3，所述多肽链2从N末端至C末端依次包括VH_B-L2-VH_B-h-CH2-CH3；或(24)所述多肽链1从N末端至C末端依次包括VL_A-L1-VH_A-h-CH2-CH3，所述多肽链2从N末端至C末端依次包括VH_C-L2-VH_B-CH2-CH3；或(25)所述多肽链1从N末端至C末端依次包括VH_A-L1-VL_A-h-CH2-CH3，所述多肽链2从N末端至C末端依次包括VH_C-L2-VH_B-h-CH2-CH3；其中，所述L、L1和L2为连接肽，所述h为铰链区或连接肽，所述铰链区或连接肽例如“-”、GS或如SEQ ID NO:495-519的氨基酸序列所示。
  4. 如权利要求1-3任一项所述的结合蛋白，其中，所述蛋白功能区A或所述蛋白功能区B选自PD-L1抗体、HER2抗体、B7H4抗体、CD3抗体、CTLA4抗体、4-1BB抗体或BCMA抗体中的一种；较佳地，所述蛋白功能区A为PD-L1抗体、HER2抗体、B7H4抗体或CD3抗体，所述蛋白功能区B为CTLA4抗体、4-1BB抗体或BCMA抗体；
    更佳地，所述蛋白功能区A为PD-L1抗体，所述蛋白功能区B为CTLA4抗体；或，所述蛋白功能区A为PD-L1抗体，所述蛋白功能区B为4-1BB抗体；或，所述蛋白功能区A为HER2抗体，所述蛋白功能区B为CTLA4抗体；或，所述蛋白功能区A为B7H4抗体，所述蛋白功能区B为4-1BB抗体；或，所述蛋白功能区A为CD3抗体，所述蛋白功能区B为BCMA抗体。
  5. 如权利要求4所述的结合蛋白，其中，所述PD-L1抗体包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:167、188和211所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、62和122所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:167、188和211所示的氨基酸序列；其重链可 变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、69和122所示的氨基酸序列；优选地，其轻链可变区VL包括如SEQ ID NO:282所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:233所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:282所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:240所示的氨基酸序列；和，
    所述PD-L1抗体包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:169、190和213所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:16、64和124所示的氨基酸序列；优选地，其轻链可变区VL包括如SEQ ID NO:284所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:235所示的氨基酸序列；和，
    所述HER2抗体包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:171、190和215所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:19、67和127所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:176、196和220所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:23、74和132所示的氨基酸序列；优选地，其轻链可变区VL包括如SEQ ID NO:286所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:238所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:293所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:247所示的氨基酸序列；和，
    所述B7H4抗体包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:180、191和225所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:32、87和143所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和226所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:33、88和144所示的氨基酸序列；优选地，其轻链可变区VL包括如SEQ ID NO:298所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:261所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:299所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:262所示的氨基酸序列；和，
    所述CTLA4抗体包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:17、65和125所示的氨基酸序列；优选地，其重链可变区VH包括如SEQ ID NO:236所示的氨基酸序列；和，
    所述4-1BB抗体包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:170、191和214所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:18、66和126所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:170、191和214所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:18、71和126所示的氨基酸序列；优选地，其轻链可变区VL包括如SEQ ID NO:285所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:237所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:289所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:242所示的氨基酸序列；和，
    所述4-1BB抗体包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:27、79和137所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、80和138所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:29、82和138所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、89和145所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、81和139所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、83和140所示的氨基酸序列；优选地，其重链可变区VH包括如SEQ ID NO:252所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:253所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:255所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:264所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:254所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:256所示的氨基酸序列；和，
    所述CD3抗体包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和221所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、84和141所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO: 178、197和222所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、85和142所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和223所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、86和141所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:179、198和224所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、85和142所示的氨基酸序列；优选地，其轻链可变区VL包括如SEQ ID NO:294所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:258所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:295所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:259所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:296所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:260所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:297所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:259所示的氨基酸序列；和，
    所述CD3抗体包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:20、68和128所示的氨基酸序列；或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；优选地，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:245所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:257所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:281所示的氨基酸序列；或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:244所示的氨基酸序列；和，
    所述BCMA抗体包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、75和133所示的氨基酸序列；或，其重链可变区VH 包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:24、76和134所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:25、77和135所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列；优选地，其重链可变区VH包括如SEQ ID NO:248所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:249所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:250所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；和，
    所述BCMA抗体包含重链可变区，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、90和136所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、78和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、78和147所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和147所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:36、90和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和147所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、78和148所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和136所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、76和136所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、76和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和148所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和136所示的氨基酸序列；优选地，其重链可变区VH包括如SEQ ID NO:265所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:266所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:267所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:268所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:269所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID  NO:270所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:271所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:272所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:273所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:274所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:275所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:276所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:277所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:278所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:279所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:280所示的氨基酸序列。
  6. 如权利要求1-4任一项所述的结合蛋白，其中，
    (A)所述结合蛋白含有蛋白功能区A和蛋白功能区B：
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:167、188和211所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、69和122所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:17、65和125所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:167、188和211所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、62和122所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:17、65和125所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:171、190和215所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:19、67和127所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:17、65和125所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:176、196和220所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:23、74和132所示的氨 基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:17、65和125所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:167、188和211所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、69和122所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、80和138所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:167、188和211所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、69和122所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:27、79和137所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:167、188和211所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、69和122所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:29、82和138所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:180、191和225所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:32、87和143所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、80和138所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:180、191和225所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:32、87和143所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:27、79和137所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:180、191和225所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:32、87和143所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、89和145所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和226所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:33、88和144所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、80和138所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和226所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:33、88和144所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:28、81和139所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和226所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:33、88和144所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、83和140所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:20、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:24、76和134所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:20、68和128所示的氨 基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:20、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、75和133所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、75和133所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、90和136所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:36、90和146所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和147所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、78和148所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和136所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和146所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、76和136所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨 基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、76和146所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、76和146所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和148所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和136所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:34、78和146所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、78和147所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:35、90和147所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和221所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、84和141所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:178、197和222所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、85和142所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:177、191和223所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、86和141所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:179、198和224所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:31、85和142所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列；或，
    (B)所述结合蛋白含有蛋白功能区A、蛋白功能区B和蛋白功能区C：
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可 变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:15、75和133所示的氨基酸序列；所述蛋白功能区C包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:24、76和134所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:30、68和128所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:25、77和135所示的氨基酸序列；所述蛋白功能区C包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:26、78和136所示的氨基酸序列。
  7. 如权利要求1-6任一项所述的结合蛋白，其中，
    (A)所述结合蛋白包括蛋白功能区A和蛋白功能区B：
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:282所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:240所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:236所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:282所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:233所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:236所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:286所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:238所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:236所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:293所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:247所示的氨基酸序 列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:236所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:282所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:240所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:253所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:282所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:240所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:252所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:282所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:240所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:255所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:298所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:261所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:253所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:298所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:261所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:252所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:298所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:261所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:264所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:299所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:262所示的氨基酸序 列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:253所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:299所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:262所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:254所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:299所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:262所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:256所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:245所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:249所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:245所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:245所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:248所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:257所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:248所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:257所示的氨基酸序 列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:281所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:265所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:269所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:270所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:271所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序 列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:272所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:273所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:274所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:275所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:276所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:277所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:278所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序 列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:279所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:280所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:266所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:267所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:268所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:244所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:249所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:244所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:294所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:258所示的氨基酸序 列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:295所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:259所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:296所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:260所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:297所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:259所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；或，
    (B)所述结合蛋白含有蛋白功能区A、蛋白功能区B和蛋白功能区C：
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:257所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:248所示的氨基酸序列。所述蛋白功能区C包含重链可变区；其重链可变区VH包括如SEQ ID NO:249所示的氨基酸序列；或，
    所述蛋白功能区A包含轻链可变区和重链可变区；其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:257所示的氨基酸序列；所述蛋白功能区B包含重链可变区；其重链可变区VH包括如SEQ ID NO:250所示的氨基酸序列。所述蛋白功能区C包含重链可变区；其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列。
  8. 如权利要求1-7任一项所述的结合蛋白，其特征在于，
    (1)所述结合蛋白包含两种多肽链，其中，
    第一多肽链包括如SEQ ID NO:371所示的氨基酸序列；第二多肽链包括如SEQ ID NO:372所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:371所示的氨基酸序列；第二多肽链包括如SEQ ID NO:373所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:362所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:363所示的氨基酸序列；第二多肽链包括如SEQ ID NO:364所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:365所示的氨基酸序列；第二多肽链包括如SEQ ID NO:364所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:366所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:369所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:370所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:394所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:395所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:363所示的氨基酸序列；第二多肽链包括如SEQ ID NO:310所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:363所示的氨基酸序列；第二多肽链包括如SEQ ID NO:396所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:363所示的氨基酸序列；第二多肽链包括如SEQ ID NO:362所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:363所示的氨基酸序列；第二多肽链包括如SEQ ID NO:394所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:363所示的氨基酸序列；第二多肽链包括如SEQ ID NO:395所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:368所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:378所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:379所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:380所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:381所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:382所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:383所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:385所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:388所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:389所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:374所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:375所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:386所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:387所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:401所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:402所示的氨基酸序列；第二多肽链包括如SEQ ID NO:305所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:403所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:402所示的氨基酸序列；第二多肽链包括如SEQ ID NO:409所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:359所示的氨基酸序列；第二多肽链包括如SEQ ID NO:404所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:405所示的氨基酸序列；第二多肽链包括如SEQ ID NO:315所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:359所示的氨基酸序列；第二多肽链包括如SEQ ID NO:406所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:376所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:377所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:397所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:398所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:399所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:400所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:402所示的氨基酸序列；第二多肽链包括如SEQ ID NO:401所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:402所示的氨基酸序列；第二多肽链包括如SEQ ID NO:403所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:405所示的氨基酸序列；第二多肽链包括如SEQ ID NO:404所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:405所示的氨基酸序列；第二多肽链包括如SEQ ID NO:406所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:371所示的氨基酸序列；第二多肽链包括如SEQ ID NO:486所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:460所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:461所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:462所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:487所示的氨基酸序列；第二多肽链包括如SEQ ID NO:488所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:360所示的氨基酸序列；第二多肽链包括如SEQ ID NO:455所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:360所示的氨基酸序列；第二多肽链包括如SEQ ID NO:456所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:360所示的氨基酸序列；第二多肽链包括如SEQ ID NO:457所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:360所示的氨基酸序列；第二多肽链包括如SEQ ID NO:458所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:360所示的氨基酸序列；第二多肽链包括如SEQ ID NO:459所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:419所示的氨基酸序列；第二多肽链包括如SEQ ID NO:412所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:419所示的氨基酸序列；第二多肽链包括如SEQ ID NO:414所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:440所示的氨基酸序列；第二多肽链包括如SEQ ID NO:428所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:441所示的氨基酸序列；第二多肽链包括如SEQ ID NO:428所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:442所示的氨基酸序列；第二多肽链包括如SEQ ID NO:428所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:443所示的氨基酸序列；第二多肽链包括如SEQ ID NO:428所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:487所示的氨基酸序列；第二多肽链包括如SEQ ID NO:520所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:521所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:371所示的氨基酸序列；第二多肽链包括如SEQ ID NO:522所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:360所示的氨基酸序列；第二多肽链包括如SEQ ID NO:523所示的氨基酸序列；或，
    (2)所述结合蛋白包含三个多肽链，其中，
    第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:407所示的氨基酸序列；第三多肽链包括如SEQ ID NO:390所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:353所示的氨基酸序列；第二多肽链包括如SEQ ID NO:408所示的氨基酸序列；第三多肽链包括如SEQ ID NO:392所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:391所示的氨基酸序列；第三多肽链包括如SEQ ID NO:390所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:393所示的氨基酸序列；第三多肽链包括如SEQ ID NO:392所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:391所示的氨基酸序列；第三多肽链包括如SEQ ID NO:434所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:391所示的氨基酸序列；第三多肽链包括如SEQ ID NO:435所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:393所示的氨基酸序列；第三多肽链包括如SEQ ID NO:436所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:351所示的氨基酸序列；第二多肽链包括如SEQ ID NO:393所示的氨基酸序列；第三多肽链包括如SEQ ID NO:437所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:438所示的氨基酸序列；第三多肽链包括如SEQ ID NO:434所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:438所示的氨基酸序列；第三多肽链包括如SEQ ID NO:435所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:439所示的氨基酸序列；第三多肽链包括如SEQ ID NO:436所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:367所示的氨基酸序列；第二多肽链包括如SEQ ID NO:439所示的氨基酸序列；第三多肽链包括如SEQ ID NO:437所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:361所示的氨基酸序列；第二多肽链包括如SEQ ID NO:481所示的氨基酸序列；第三多肽链包括如SEQ ID NO:482所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:361所示的氨基酸序列；第二多肽链包括如SEQ ID NO:481所示的氨基酸序列；第三多肽链包括如SEQ ID NO:483所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:361所示的氨基酸序列；第二多肽链包括如SEQ ID NO:481所示的氨基酸序列；第三多肽链包括如SEQ ID NO:484所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:361所示的氨基酸序列；第二多肽链包括如SEQ ID NO:481所示的氨基酸序列；第三多肽链包括如SEQ ID NO:485所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:411所示的氨基酸序列；第三多肽链包括如SEQ ID NO:412所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:411所示的氨基酸序列；第三多肽链包括如SEQ ID NO:414所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:415所示的氨基酸序列；第二多肽链包括如SEQ ID NO:416所示的氨基酸序列；第三多肽链包括如SEQ ID NO:410所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:415所示的氨基酸序列；第二多肽链包括如SEQ ID NO:416所示的氨基酸序列；第三多肽链包括如SEQ ID NO:412所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:415所示的氨基酸序列；第二多肽链包括如SEQ ID NO:416所示的氨基酸序列；第三多肽链包括如SEQ ID NO:414所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:417所示的氨基酸序列；第二多肽链包括如SEQ ID NO:418所示的氨基酸序列；第三多肽链包括如SEQ ID NO:412所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:417所示的氨基酸序列；第二多肽链包括如SEQ ID NO:418所示的氨基酸序列；第三多肽链包括如SEQ ID NO:414所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:426所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:427所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:428所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:429所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:444所示的氨基酸序列；第三多肽链包括如SEQ ID NO:449所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:450所示的氨基酸序列；第三多肽链包括如SEQ ID NO:449所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:444所示的氨基酸序列；第三多肽链包括如SEQ ID NO:445所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:444所示的氨基酸序列；第三多肽链包括如SEQ ID NO:446所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:450所示的氨基酸序列；第三多肽链包括如SEQ ID NO:445所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:450所示的氨基酸序列；第三多肽链包括如SEQ ID NO:446所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:444所示的氨基酸序列；第三多肽链包括如SEQ ID NO:447所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:444所示的氨基酸序列；第三多肽链包括如SEQ ID NO:448所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:450所示的氨基酸序列；第三多肽链包括如SEQ ID NO:447所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:450所示的氨基酸序列；第三多肽链包括如SEQ ID NO:448所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:445所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:446所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:463所示的氨基酸序列；第三多肽链包括如SEQ ID NO:464所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:465所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:466所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:467所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:468所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:469所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:470所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:471所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:472所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:473所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:474所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:475所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:476所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:477所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:478所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:479所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:454所示的氨基酸序列；第三多肽链包括如SEQ ID NO:480所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:430所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:431所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:432所示的氨基酸序列；或，
    第一多肽链包括如SEQ ID NO:357所示的氨基酸序列；第二多肽链包括如SEQ ID NO:423所示的氨基酸序列；第三多肽链包括如SEQ ID NO:433所示的氨基酸序列。
  9. 如权利要求1-8任一项所述的结合蛋白，还可以包括轻链恒定区，所述轻链恒定区优选人轻链恒定区，更优选人轻链恒定区Cκ或Cλ；所述的结合蛋白还可以包括重链恒定区的CH1、CH2和/或CH3，所述重链恒定区优选人重链恒定区，更优选人IgG1、IgG2、IgG3、IgG4重链恒定区；所述IgG1重链恒定区的Fc上还可以具有C220S、N297A、L234A、L235A、P329G、S239D、I332E、S354C、T366W、Y349C、T366S、L368A、Y407V、M252Y、S254T、T256E等突变中的一种或多种，所述突变位点使用EU编号规则。
  10. 一种分离的核酸，其特征在于，其编码如权利要求1-9任一项所述的结合蛋白。
  11. 一种表达载体，其特征在于，其包含如权利要求10所述的分离的核酸。
  12. 一种宿主细胞，其特征在于，其包含如权利要求11所述的表达载体，其中所述宿主细胞是原核细胞或真核细胞。
  13. 一种如权利要求1-9任一项所述的结合蛋白的制备方法，其特征在于，所述制备方法包括以下步骤：培养如上所述的宿主细胞，从培养物中获得所述结合蛋白。
  14. 一种药物组合物，所述药物组合物包含如权利要求1-9任一项所述的结合蛋白。
  15. 一种套装药盒，其特征在于，所述套装药盒包括药盒一和药盒二，所述药盒一包括如权利要求1-9任一项所述的结合蛋白或如权利要求14所述的药物组合物，所述药盒二包括治疗癌症的其它抗体或药物组合物。
  16. 一种如权利要求1-9任一项所述的结合蛋白或如权利要求14所述的药物组合物在制备治疗和/或预防癌症的药物中的应用；优选地，所述的癌症优选乳腺癌、卵巢癌、子宫内膜癌、肾癌、黑色素瘤、肺癌、胃癌、肝癌、食管癌、宫颈癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、肉瘤、结直肠癌、淋巴瘤或者多发性骨髓瘤等。
  17. 一种治疗癌症的方法，其特征在于，向有需要的受试者施用如权利要求1-9任一项所述的结合蛋白，或如权利要求14所述的药物组合物，或如权利要求15所述的套装药盒；优选地，还包括施用治疗癌症的其它抗体例如免疫检查点抗体和/或化疗药物。
  18. 一种CD3抗体，其特征在于，所述CD3抗体包含轻链可变区和重链可变区；
    其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为如SEQ ID NO:177、191和221所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:15、84和141所示的氨基酸序列；
    或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为如SEQ ID NO:178、197和222所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:31、85和142所示的氨基酸序列；
    或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为如SEQ ID NO:177、191和223所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:31、86和141所示的氨基酸序列；
    或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为如SEQ ID NO:179、198和224所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:31、85和142所示的氨基酸序列；
    或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为如SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:20、68和128所示的氨基酸序列；
    或，其轻链可变区VL包含LCDR1、LCDR2和LCDR3，分别为如SEQ ID NO:172、192和216所示的氨基酸序列；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:30、68和128所示的氨基酸序列；
    优选地，其轻链可变区VL包括如SEQ ID NO:294所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:258所示的氨基酸序列；
    或，其轻链可变区VL包括如SEQ ID NO:295所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:259所示的氨基酸序列；
    或，其轻链可变区VL包括如SEQ ID NO:296所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:260所示的氨基酸序列；
    或，其轻链可变区VL包括如SEQ ID NO:297所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:259所示的氨基酸序列；
    或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:245所示的氨基酸序列；
    或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:257所示的氨基酸序列；
    或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:263所示的氨基酸序列；
    或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:281所示的氨基酸序列；
    或，其轻链可变区VL包括如SEQ ID NO:291所示的氨基酸序列；其重链可变区VH包括如SEQ ID NO:244所示的氨基酸序列；
    更优选地，所述CD3抗体包含两种多肽链；其中，第一多肽链包括如SEQ ID NO:357所示的氨基酸序列，第二多肽链包括如SEQ ID NO:313所示的氨基酸序列；
    或，第一多肽链包括如SEQ ID NO:357所示的氨基酸序列，第二多肽链包括如SEQ ID NO:325所示的氨基酸序列；
    或，第一多肽链包括如SEQ ID NO:357所示的氨基酸序列，第二多肽链包括如SEQ ID NO:328所示的氨基酸序列；
    或，第一多肽链包括如SEQ ID NO:357所示的氨基酸序列，第二多肽链包括如SEQ ID NO:346所示的氨基酸序列；
    或，所述CD3抗体包含一个多肽链，所述多肽链包括如SEQ ID NO:489所示的氨基酸序列，如SEQ ID NO:490所示的氨基酸序列，如SEQ ID NO:491所示的氨基酸序列，如SEQ ID NO:492所示的氨基酸序列，或，如SEQ ID NO:493所示的氨基酸序列。
  19. 一种BCMA抗体，其特征在于，
    所述BCMA抗体包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:15、75和133所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:24、76和134所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:25、77和135所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:26、78和136所示的氨基酸序列；
    或，所述BCMA抗体包含重链可变区，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:26、90和136所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:34、78和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:35、78和147所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:35、90和147所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:36、90和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:35、76和147所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:34、78和148所示的氨基酸序列；或，其重链可变区 VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:35、76和136所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:35、90和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:34、76和136所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:35、76和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:34、76和146所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:35、90和148所示的氨基酸序列；或，其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为如SEQ ID NO:35、90和136所示的氨基酸序列；
    优选地，其重链可变区VH包括如SEQ ID NO:248所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:249所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:250所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:251所示的氨基酸序列；
    或，其重链可变区VH包括如SEQ ID NO:265所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:266所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:267所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:268所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:269所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:270所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:271所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:272所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:273所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:274所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:275所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:276所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:277所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:278所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:279所示的氨基酸序列；或，其重链可变区VH包括如SEQ ID NO:280所示的氨基酸序列；
    更优选地，所述BCMA抗体包含一个多肽链，所述多肽链包含如SEQ ID NO:316所示的氨基酸序列，如SEQ ID NO:317所示的氨基酸序列，如SEQ ID NO:318所示的氨基酸序列，或，如SEQ ID NO:319所示的氨基酸序列；
    或，所述BCMA抗体包含一个多肽链，所述多肽链包含如SEQ ID NO:330所示的氨基酸序列；或，如SEQ ID NO:331所示的氨基酸序列；或，如SEQ ID NO:332所示的氨基酸序列；或，如SEQ ID NO:333所示的氨基酸序列；或，如SEQ ID NO:334所示的氨基酸序列；或，如SEQ ID NO:335所示的氨基酸序列；或，如SEQ ID NO:336所示的氨基酸序列；或，如SEQ ID NO:337所示的氨基酸序列；或，如SEQ ID NO:338所示的氨基酸序列；或，如SEQ ID NO:339所示的氨基酸序列；或，如SEQ ID NO:340所示的氨基酸序列；或，如SEQ ID NO:341所示的氨基酸序列；或，如SEQ ID NO:342所示的氨基酸序列；或，如SEQ ID NO:343所示的氨基酸序列；或，如SEQ ID NO:344所示的氨基酸序列；或，如SEQ ID NO:345所示的氨基酸序列。
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