WO2022117065A1 - 多特异性抗原结合蛋白 - Google Patents

多特异性抗原结合蛋白 Download PDF

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WO2022117065A1
WO2022117065A1 PCT/CN2021/135254 CN2021135254W WO2022117065A1 WO 2022117065 A1 WO2022117065 A1 WO 2022117065A1 CN 2021135254 W CN2021135254 W CN 2021135254W WO 2022117065 A1 WO2022117065 A1 WO 2022117065A1
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amino acid
seq
antigen
acid substitutions
light chain
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PCT/CN2021/135254
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English (en)
French (fr)
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张伟
姜福伟
王蕾蕾
陈思萌
廖成
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江苏恒瑞医药股份有限公司
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Priority to US18/265,217 priority Critical patent/US20240010754A1/en
Priority to CN202180079343.XA priority patent/CN116583300A/zh
Publication of WO2022117065A1 publication Critical patent/WO2022117065A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present disclosure belongs to the field of biomedicine, and in particular relates to a multispecific antigen-binding protein, a preparation method and medical use thereof.
  • bispecific antibodies Since it can recognize different antigen molecules or recognize different epitopes of the same antigen molecule, bispecific antibodies have unique biological functions that monoclonal antibodies do not have, and are gradually recognized by the market. Although the related technology about bispecific antibodies has been developed for two decades, there are still many practical technical problems that restrict the production and development of bispecific antibodies. With the advancement of technology, new molecular formats and solutions for the transformation and production of bispecific antibodies emerge in an endless stream. Taking 1+1 asymmetric (Fab A+Fab B) double antibody as an example, in order to avoid light chain mismatch (the light chain against antigen A is paired with the heavy chain against antigen B, or the light chain against antigen B is paired with Targeting on the heavy chain of antigen A), various methods have been reported so far.
  • Fab A+Fab B 1+1 asymmetric
  • Common light chain antibodies It has been reported to use in vitro display technology or to screen specific light chains from mice with a common light chain (WO2012067176; WO2013134263) to pair a heavy chain against antigen A and a heavy chain against antigen B, and maintain phase The original biological function of the corresponding antibody.
  • Two-in-one antibody It has been reported that through phage display and rational design (WO2010027981), the antibody that binds to antigen A is optimized so that it retains the original binding ability to antigen A and has the ability to bind antigen B. The ability to bind enables one antibody to bind two targets.
  • the above two methods both require a lot of engineering transformation, are technically difficult, and have yet to be proved universal. Therefore, the transformation of Fab (VH/VL or/and CH1/CL interaction interface) with orthogonal properties has attracted more and more attention in the industry in recent years.
  • IgG/TCR (WO2014014796; WO2019057122): In view of the structural similarity between the TCR constant region and antibody CH1/CL, it was reported that the potential light chain mismatch problem was avoided by replacing the CH1/CL of FabA with the constant region of TCR.
  • Crossmab (WO2012023053): Reduces the likelihood of light chain mismatches by swapping VH/VL, CH1/CL, or HC/LC for a Fab.
  • DuetMab (WO2013096291): A non-natural disulfide bond was introduced into CH1/CL of the Fab against antigen A to replace the original disulfide bond to reduce the possibility of light chain mismatch.
  • Computer-aided design Avoid light chain mismatches by computer-aided design (WO2014150973; WO2016172485).
  • bispecific antibodies As a new drug form, bispecific antibodies have special structures, and their preparation and industrialization are more difficult than monoclonal antibodies. Although various approaches have been attempted to address the mismatch between heavy and light chains, the resulting structural adjustments may alter the stability, immunogenicity or pharmacokinetic characteristics of the molecule, and new technologies still need to be developed To increase the yield of multispecific antibodies (eg bispecific antibodies).
  • the present disclosure works by removing natural disulfide bonds and introducing non-natural disulfide bonds in the CH1/CL interface, or by introducing electrostatically complementary amino acid pairs in the CH1/CL interface, or by removing natural disulfide bonds in the CH1/CL interface By introducing unnatural disulfide bonds and introducing electrostatically complementary amino acid pairs at the same time, the correct pairing ratio of the light and heavy chains of the multispecific antibody is improved compared with the wild type.
  • the present disclosure provides a dimerized polypeptide comprising a heavy chain constant region 1 (CH1) and a light chain constant region (CL), wherein: CH1 and CL are in a range selected from (i-1) to (i-6).
  • CH1 and CL are in a range selected from (i-1) to (i-6).
  • heavy chain position numbers are determined according to the EU numbering system, e.g., the positions of amino acid substitutions of CH1 are counted based on CH1 (SEQ ID NO: 88) of human IgG1; light chain position numbers are according to the Kabat numbering system The positions of amino acid substitutions, such as CL, are determined to be counted based on the human kappa light chain (IGLC, SEQ ID NO: 89).
  • IgG subtypes other than IgG1, such as IgG2, IgG3 and IgG4, contain the same type of amino acid mutation at the position corresponding to the position in IgG1 CH1 containing the amino acid mutation described in this disclosure. within the scope of protection.
  • a natural disulfide bond is or is not included between CH1 and CL.
  • CH1 retains native cysteine at position 220 and CL retains native cysteine at position 214.
  • the native cysteine at position 220 of CH1 and/or the native cysteine at position 214 of CL is substituted with an amino acid other than cysteine.
  • CH1 comprises the amino acid substitution C220A and CL comprises the amino acid substitution C214A.
  • CH1 and CL comprise the following amino acid substitutions:
  • CH1 and CL comprise the following amino acid substitutions: (a) C220A in CH1 and C214A in CL; and (b) F170C in CH1 and T164C in CL.
  • CH1 and CL comprise the following amino acid substitutions: (a) C220A in CH1 and C214A in CL; and (b) P171C in CH1 and S165C in CL.
  • CH1 and CL comprise amino acid substitutions such that an electrostatic interaction interface is formed between CH1 and CL.
  • the amino acid substitutions that result in an electrostatic interaction interface between CH1 and CL are located at position 139 of CH1 and position 114 of CL.
  • the amino acid at position 139 of CH1 is substituted with a positively charged amino acid
  • the amino acid at position 114 of CL is substituted with a negatively charged amino acid
  • the amino acid at position 139 of CH1 is substituted with a negatively charged amino acid
  • Amino acid the amino acid at position 114 of CL was substituted with a positively charged amino acid.
  • the positively charged amino acids are selected from K, R, and H; the negatively charged amino acids are selected from D and E.
  • CH1 and CL comprise amino acid substitutions selected from any of the following groups:
  • CH1 and CL comprise the following amino acid substitutions:
  • CH1 and CL comprise the following amino acid substitutions:
  • CH1 and CL comprise the following amino acid substitutions: (a) C220A in CH1 and C214A in CL; (b) F170C in CH1 and T164C in CL; and (c) T139R and CL in CH1 in the S114E.
  • CH1 and CL comprise the following amino acid substitutions: (a) C220A in CH1 and C214A in CL; (b) F170C in CH1 and T164C in CL; and (c) T139D and CL in CH1 in S114K.
  • CH1 and CL comprise the following amino acid substitutions: (a) C220A and C214A; (b) P171C in CH1 and S165C in CL; and (c) T139R in CH1 and S114E in CL.
  • CH1 and CL comprise the following amino acid substitutions: (a) C220A in CH1 and C214A in CL; (b) P171C in CH1 and S165C in CL; and (c) T139D and CL in CH1 in S114K.
  • the CL is from an antibody lambda light chain (C ⁇ ) or kappa light chain (CK).
  • the present disclosure provides an antigen-binding protein comprising the above-mentioned dimerized polypeptide.
  • the antigen binding protein comprises a first antigen binding domain comprising a Fab comprising a first heavy chain variable region VH1, a first light chain variable region VL1 and the A dimerized polypeptide, in which the CH1 is the first CH1, and the CL is the first CL; VH1 is directly connected to the first CH1 or connected through a linker, and VL1 is directly connected to the first CL or through a linker connect.
  • the C-terminus of VH1 is directly connected or connected by a linker to the N-terminus of the first CH1, and the C-terminus of VL1 is directly connected or connected by a linker to the N-terminus of the first CL.
  • the linker is a peptide linker.
  • the peptide linker is a peptide having an amino acid sequence of at least 5 amino acids in length, in one embodiment 5 to 100, and in further embodiments 10 to 50 amino acids in length.
  • the peptide linker is (G4S)4.
  • the antigen binding protein comprises a first antigen binding domain and a second antigen binding domain, wherein the second antigen binding domain comprises a second heavy chain variable region VH2 and a second light chain variable region VL2, and The first antigen binding domain and the second antigen binding domain bind different antigens or bind different epitopes on the same antigen; in some embodiments, the second antigen binding domain comprises a Fab.
  • the Fab comprises a second heavy chain variable region VH2, a second heavy chain constant region 1 (second CH1), a second light chain variable region VL2 and a second light chain constant region (second CL2).
  • the C-terminus of VH2 is directly connected or connected through a linker to the N-terminus of the second CH1, and the C-terminus of VL2 is directly connected or connected through a linker to the N-terminus of the second CL.
  • the second CH1 and the second CL do not comprise one or more groups of natural non-cysteine-to-cysteine amino acid substitutions selected from the group consisting of:
  • heavy chain position numbers are determined according to the EU numbering system, e.g., the positions of amino acid substitutions of CH1 are counted based on CH1 (SEQ ID NO: 88) of human IgG1; light chain position numbers are according to the Kabat numbering system The positions of amino acid substitutions, such as CL, are determined to be counted based on the human kappa light chain (IGLC, SEQ ID NO: 89).
  • IgG subtypes other than IgG1, such as IgG2, IgG3 and IgG4, contain the same type of amino acid mutation at the position corresponding to the position in IgG1 CH1 containing the amino acid mutation described in this disclosure. within the scope of protection.
  • the second CH1 and the second CL are free of natural non-cysteine to cysteine amino acid substitutions.
  • the native cysteines 220C and 214C are retained in the second CH1 and the second CL.
  • the second CH1 and the second CL are free of natural non-cysteine to cysteine amino acid substitutions and retain the natural cysteines 220C and 214C.
  • the first CH1 and the first CL comprise the following amino acid substitutions:
  • the first CH1 and the first CL comprise the following amino acid substitutions:
  • the first CH1 and the first CL comprise the following amino acid substitutions: (a) C220A in CH1 and C214A in CL; and (b) P171C in CH1 and S165C in CL; and the second CH1 and The second CL contains no natural non-cysteine to cysteine amino acid substitutions and retains the natural cysteines 220C and 214C.
  • the first CH1 and the first CL comprise amino acid substitutions such that an electrostatic interaction interface is formed between the first CH1 and the first CL; and/or
  • the second CH1 and the second CL comprise amino acid substitutions such that an electrostatic interaction interface is formed between the second CH1 and the second CL.
  • the charges of the amino acids used to form the electrostatic interaction interface in the first CH1 and the second CH1 are opposite, and the charges of the amino acids used to form the electrostatic interaction interface in the first CL and the second CL are opposite .
  • the amino acid substitution that results in an electrostatic interaction interface between the first CH1 and the first CL is located at position 139 of the first CH1 and position 114 of the first CL;
  • amino acid substitutions that allow for the formation of an electrostatic interaction interface between the second CH1 and the second CL are located at position 139 of the second CH1 and position 114 of the second CL.
  • position 139 of the first CH1 and position 139 of the second CH1 are substituted with oppositely charged amino acids, respectively, and positions 114 of the first CL and 114 of the second CL are respectively substituted with oppositely charged amino acids Charged amino acid substitutions.
  • the amino acid at position 139 of the first CH1 is substituted with a positively charged amino acid
  • the amino acid at position 114 of the first CL is substituted with a negatively charged amino acid
  • the amino acid at position 139 of the first CH1 is substituted with a negatively charged amino acid.
  • Substituted with a negatively charged amino acid the amino acid at position 114 of the first CL is substituted with a positively charged amino acid; and/or
  • the amino acid at position 139 of the second CH1 is substituted with a negatively charged amino acid, the amino acid at position 114 of the second CL is substituted with a positively charged amino acid; or the amino acid at position 139 of the second CH1 is substituted with a positively charged amino acid Amino acid, the amino acid at position 114 of the second CL is substituted with a negatively charged amino acid.
  • the positively charged amino acids are selected from K, R, and H; the negatively charged amino acids are selected from D and E.
  • the first CH1 and the first CL comprise amino acid substitutions selected from any of the following groups:
  • T139R in CH1 and S114E in CL T139R in CH1 and S114D in CL; T139K in CH1 and S114E in CL; T139K in CH1 and S114D in CL; T139D in CH1 and S114E in CL S114K in CH1; T139D in CH1 and S114R in CL; T139E in CH1 and S114K in CL; and T139E in CH1 and S114R in CL; and/or
  • the second CH1 and the second CL comprise amino acid substitutions selected from the group consisting of: T139R in CH1 and S114E in CL; T139R in CH1 and S114D in CL; T139K in CH1 and S114E in CL; T139K in CH1 and S114E in CL S114D in CL; T139D in CH1 and S114K in CL; T139D in CH1 and S114R in CL; T139E in CH1 and S114K in CL; and T139E in CH1 and S114R in CL.
  • the first CH1 and the first CL comprise amino acid substitutions selected from the group consisting of: T139R in CH1 and S114E in CL; T139R in CH1 and S114D in CL; T139K in CH1 and S114E in CL ; T139K in CH1 and S114D in CL; and/or
  • the second CH1 and the second CL comprise amino acid substitutions selected from the group consisting of: T139D in CH1 and S114K in CL; T139D in CH1 and S114R in CL; T139E in CH1 and S114K in CL; and T139E in CH1 and S114R in CL.
  • the first CH1 and the first CL comprise amino acid substitutions selected from the group consisting of: T139D in CH1 and S114K in CL; T139D in CH1 and S114R in CL; T139E in CH1 and S114K in CL ; and T139E in CH1 and S114R in CL; and/or
  • the second CH1 and the second CL comprise amino acid substitutions selected from the group consisting of: T139R in CH1 and S114E in CL; T139R in CH1 and S114D in CL; T139K in CH1 and S114E in CL; T139K in CH1 and S114E in CL S114D in CL.
  • the first CH1 and the first CL comprise the following amino acid substitutions:
  • the second CH1 and the second CL comprise amino acid substitutions selected from the group consisting of: T139D in CH1 and S114K in CL; T139D in CH1 and S114R in CL; T139E in CH1 and S114K in CL; and in CH1 S114R in T139E and CL.
  • the first CH1 and the first CL comprise the following amino acid substitutions:
  • the second CH1 and the second CL comprise amino acid substitutions selected from the group consisting of: T139R in CH1 and S114E in CL; T139R in CH1 and S114D in CL; T139K in CH1 and S114E in CL; and in CH1 S114D in T139K and CL.
  • the first CH1 and the first CL comprise the following amino acid substitutions:
  • the first CH1 and the first CL comprise the following amino acid substitutions:
  • the first CH1 and the first CL comprise the following amino acid substitutions:
  • the first CH1 and the first CL comprise the following amino acid substitutions:
  • the second CH1 and the second CL are free from the natural non-cysteine to cysteine Amino acid substitution of cystine, and retains 220C in CH1 and 214C in CL of native cysteines.
  • the first CL is from an antibody kappa light chain (CK); the second CL is from an antibody lambda light chain (C ⁇ ) or a kappa light chain (CK). In some embodiments, the first CL is from a kappa light chain and the second CL is from a lambda light chain.
  • the antigen binding protein further comprises an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other.
  • the Fc region is selected from the Fc of human IgGl, IgG2, IgG3, and IgG4, eg, the Fc of human IgGl.
  • Fc1 and Fc2 comprise amino acid substitutions such that Fc1 preferentially pairs with Fc2 (or such that Fc2 preferentially forms heterodimers) compared to Fc1, eg, Fc1 and Fc2 comprise such in the CH3 domain Amino acid substitution.
  • amino acid substitutions in Fc1 and Fc2 result in greater electrostatic complementarity than wild-type without the substitution.
  • the amino acid substitutions in Fc1 and Fc2 result in greater steric complementarity than the wild type without the substitution.
  • Methods for measuring electrostatic complementarity at protein/protein interfaces are known in the art and are described, for example, in Lawrence et al (1993) J Mol Biol 234, 946-950; Walls et al (1992) J Mol Biol 228, 277-297; Furman et al. (2005) Proteins 60, 187-194.
  • the term "complementarity” refers to the combination of interactions affecting heavy chain/light chain pairing, eg, at the interface of CH1 and CL (or CH3 and CH3) of the antigen binding proteins described herein.
  • spatial complementarity or “conformational complementarity” refers to, for example, the compatibility of three-dimensional structures at the interacting surfaces of CH1 and CL (or CH3 and CH3).
  • Electrical complementarity refers to the compatibility of placing negatively and/or positively charged atoms at the interacting surfaces of, for example, CH1 and CL (or CH3 and CH3).
  • one or more amino acid residues in the CH3 domain of Fc1 are replaced with one or more amino acid residues with greater side chain bulk in Fc1 and Fc2, eg, within the CH3/CH3 interface, whereby, a bump (or knob, Knob) is generated on the surface of the CH3 domain of Fc1, and one or more, preferably two or three amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1, with a small side chain Bulk amino acid residues are substituted to create a recess (or hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1.
  • the CH3 domains of Fc1 and Fc2 are altered such that within the interface, Fc2 is replaced with an equivalent number of amino acid residues with greater side chain bulk One or two amino acid residues in the CH3 domain of Fc1, thereby creating bumps (or knobs) within the interface of the CH3 domain of Fc2 that can be placed in depressions (or holes) within the surface of the CH3 domain of Fc1, altering the CH3 of Fc1 domain such that within the surface of the CH3 domain of Fc2 in interface contact with the CH3 domain of Fc2, two or three amino acid residues are substituted with an equivalent number of amino acid residues with a smaller side chain volume, thereby interfacing with the CH3 domain of Fc1 A depression within the interface is created that can place a raised depression within the CH3 domain interface of Fc2.
  • the import residue with greater side chain bulk is phenylalanine (F), tyrosine (Y), arginine (R), or tryptophan (W).
  • the bump or knob mutation comprises the substitution of tryptophan for threonine 366, amino acid numbering according to Kabat et al. (Sequences of proteins of immunological interest, 5th ed., Vol. 1 (1991; NIH, Bethesda, MD) in the EU numbering scheme on pages 688-696).
  • the import residue with a smaller side chain volume is serine (S), alanine (A), valine (V), or threonine (T).
  • the recessed CH3 domain comprises a substitution of two or more original amino acids selected from the group consisting of threonine, leucine and tyrosine. In some embodiments, the recessed CH3 domain comprises two or more import residues selected from the group consisting of alanine, serine, threonine, and valine.
  • the knob mutation modification is T366W and the hole mutation modification is at least one or at least two of T366S, L368A, and Y407V. In some embodiments, the knob mutation modification is T366W and the hole mutation modification is T366S, L368A, and Y407V.
  • positions of amino acid substitutions of Fc are determined according to the EU numbering system, eg, counted relative to the Fc of human IgGl.
  • a native non-cysteine to cysteine substitution may be included in Fc1 and Fc2, eg, CH3, eg, S354C in Fc1 and Y349C in Fc2; or Y349C in Fc1, in Fc2 Contains S354C.
  • Fc1 and/or the Fc2 comprise modifications that alter the half-life of the antigen binding protein, wherein the half-life depends on FcRn binding affinity.
  • Fc1 and/or the Fc2 comprise modifications that alter effector function, wherein the binding affinity for the Fc ⁇ receptor or C1q complement protein is increased or decreased.
  • Fc1 and Fc2, eg, within the Fc1 CH3/Fc2 CH3 interface, comprise one or more sets of amino acid substitutions selected from the group consisting of:
  • the Fc1 comprises at least one or at least two amino acid substitutions selected from T366S, L368A, and Y407V
  • the Fc2 comprises T366W
  • the Fc1 comprises T366W
  • the Fc2 comprises a selection At least one or at least two amino acid substitutions from T366S, L368A and Y407V.
  • the amino acid substitutions T366S, L368A, and Y407V are included in the Fc1 and T366W is included in the Fc2; or T366W is included in the Fc1, and the amino acid substitutions T366S, L368A, and Y407V are included in the Fc2.
  • Fc1 and Fc2 further comprise amino acid substitutions that allow for the formation of an electrostatic interaction interface between Fc1 and Fc2 (eg, CH3 and CH3).
  • the amino acid substitutions that form the electrostatic interaction interface can be one or more selected from the group consisting of:
  • domains from different antibody subtypes are included in Fc1 and/or Fc2, e.g., from different antibody subtypes CH3.
  • Fc1 and/or Fc2 e.g., from different antibody subtypes CH3.
  • Davis et al. 2010, Protein Engineering Design & Selection 23:195-202 describe a heterodimeric Fc platform using the strand-swap engineered domain (SEED) CH3 region, which is human Derivatives of IgG and IgA CH3 domains (see also WO 2007/110205).
  • Fc1 and/or Fc2, eg, CH3, comprise amino acid substitutions for altering effector function.
  • Effective functions refer to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region) and which vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (eg, B cell receptors), and B cells activation.
  • Amino acid substitutions that alter effector function are selected from one or more of the following:
  • the Fc1 and/or the Fc2 comprise amino acid substitutions L234A and L235A, or comprise amino acid substitutions L234F and L235E.
  • one or more allogeneic mutations are included in Fc1 and/or Fc2, eg, CH3.
  • the heteroallogous mutations are D356E and L358M.
  • Fc1 and Fc2, eg, CH3, comprise amino acid substitutions for altering half-life.
  • An increase in half-life may allow for a reduction in the amount of drug administered to a patient and a reduction in the frequency of dosing.
  • the antibodies herein with increased half-life can be produced by modifying (eg, substitution, deletion, or addition) amino acid residues identified as involved in the interaction between the Fc and the FcRn receptor (U.S. 7,083,784).
  • a methionine at position 252, and/or a serine at position 254, and/or a threonine at position 256 of an IgG1 isotype antibody can be changed to tyrosine, threonine, respectively acid and glutamic acid such that the resulting antibodies include tyrosine-252, threonine-254 and glutamic acid-256 (ie, M252Y, S254T, T256E).
  • This Fc region of IgGl antibodies includes a YTE modification and in IgG2, IgG3, and IgG4 antibodies, the corresponding positions can be similarly modified.
  • the half-life of the antibodies herein can be increased by conjugation to PEG or albumin by techniques known in the art.
  • Fc modifications for increasing heterodimer formation can be combined with other modifications for altering the half-life of the antibody, including but not limited to M252Y and/or S254T and/or T256E; and/or for Other known Fc modifications that alter effector function and/or alter binding to one or more Fc ligands include those described herein.
  • the antigen binding proteins provided by the present disclosure comprise a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein:
  • the sequence of the first heavy chain from the N-terminus to the C-terminus is: [VH1]-[first CH1]-[Fc1],
  • the first light chain is sequentially from N-terminal to C-terminal: [VL1]-[first CL],
  • sequence of the second heavy chain from the N-terminus to the C-terminus is: [VH2]-[the second CH1]-[Fc2],
  • the sequence of the second light chain from the N-terminus to the C-terminus is: [VL2]-[second CL].
  • the antigen binding proteins provided by the present disclosure comprise a heavy chain, a first light chain, and a second light chain, wherein:
  • the order of the heavy chain from the N-terminus to the C-terminus is: [VH1]-[first CH1]-[Fc1]-[linker]-[VH2]-[second CH1];
  • the first light chain is sequentially from N-terminal to C-terminal: [VL1]-[first CL],
  • the sequence of the second light chain from the N-terminus to the C-terminus is: [VL2]-[second CL].
  • the antigen binding proteins provided by the present disclosure comprise a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein:
  • the sequence of the first heavy chain from the N-terminus to the C-terminus is: [VH1]-[first CH1]-[Fc1]-[linker]-[VH2]-[second CH1];
  • the first light chain is sequentially from N-terminal to C-terminal: [VL1]-[first CL],
  • sequence of the second heavy chain from the N-terminus to the C-terminus is: [VH1]-[first CH1]-[Fc2]-[linker]-[VH2]-[second CH1];
  • the sequence of the second light chain from the N-terminus to the C-terminus is: [VL2]-[second CL].
  • the antigens bound by the first antigen binding domain and/or the second antigen binding domain include, but are not limited to: PD-1; PD-L1; CTLA-4; LAG-3; OX40; GTIR; A2AR; B7-H3(CD276); B7-H3; B7-H4; IDO; KIR; Tim-3; LAG-3; 4-1BB(CD137); BAFF; Folate Receptor 1; TEM1; CCR4; VISTA ; ICOS; IFN- ⁇ ; TGF-B; EGFR; Erb (ErbB1; ErbB3; ErbB4); HER2; TNF- ⁇ ; TNF- ⁇ ; TNF- ⁇ ; -B; VEGFR; ROR1; BTLA; 2B4; TIGIT; c-Met; GITR; FAP; PVRIG; BCMA; CAIX; CEA; EGP2; EGP-40; Receptor (AChR); Ganglioside G2
  • the first antigen binding domain specifically binds CTLA-4
  • the second antigen binding domain specifically binds PD-1
  • the first antigen binding domain specifically binds PD- 1.
  • the second antigen binding domain specifically binds CTLA-4.
  • the first antigen binding domain comprises a heavy chain variable region VH1 and a light chain variable region VL1
  • the second antigen binding domain comprises a heavy chain variable region VH2 and a light chain variable region VL2;
  • the VH1 Comprising: HCDR1 whose sequence is shown in SEQ ID NO: 51, HCDR2 whose sequence is shown in SEQ ID NO: 52, and HCDR3 whose sequence is shown in SEQ ID NO: 53
  • the VL1 comprises the sequence shown in SEQ ID NO: 54 LCDR1 shown, LCDR2 shown in sequence as SEQ ID NO:55, and LCDR3 shown in sequence as SEQ ID NO:56;
  • said VH2 comprises: HCDR1 shown in sequence as shown in SEQ ID NO:43, HCDR2 whose sequence is shown in SEQ ID NO: 44, and HCDR3 whose sequence is shown in SEQ ID NO: 45
  • the VL2 comprises LCDR1 whose sequence is shown in SEQ ID NO: 46, and
  • the VH1 is a heavy chain variable region with a sequence set forth in SEQ ID NO: 57
  • the VL1 is a light chain variable region with a sequence set forth in SEQ ID NO: 58
  • the Described VH2 is the heavy chain variable region whose sequence is shown in SEQ ID NO: 49
  • described VL2 is the light chain variable region whose sequence is shown in SEQ ID NO: 50.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain whose sequence is set forth in SEQ ID NO: 18, and a first light chain whose sequence is set forth in SEQ ID NO: 17, whose sequence is set forth in SEQ ID NO: The second heavy chain shown in 12, and the second light chain whose sequence is shown in SEQ ID NO: 13.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain whose sequence is shown in SEQ ID NO: 19, and a first light chain whose sequence is shown in SEQ ID NO: 20, whose sequence is shown in SEQ ID NO: The second heavy chain shown in 12, and the second light chain whose sequence is shown in SEQ ID NO: 13.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain whose sequence is shown in SEQ ID NO: 21, and a first light chain whose sequence is shown in SEQ ID NO: 22, whose sequence is shown in SEQ ID NO: The second heavy chain shown in 12, and the second light chain whose sequence is shown in SEQ ID NO: 13.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain whose sequence is shown in SEQ ID NO: 14, a first light chain whose sequence is shown in SEQ ID NO: 15, and whose sequence is shown in SEQ ID NO: The second heavy chain shown in 23, and the second light chain whose sequence is shown in SEQ ID NO:9.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain whose sequence is shown in SEQ ID NO: 14, a first light chain whose sequence is shown in SEQ ID NO: 15, and whose sequence is shown in SEQ ID NO: The second heavy chain shown in 24, and the second light chain whose sequence is shown in SEQ ID NO:9.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain whose sequence is shown in SEQ ID NO: 14, a first light chain whose sequence is shown in SEQ ID NO: 15, and whose sequence is shown in SEQ ID NO: The second heavy chain shown in 25, and the second light chain whose sequence is shown in SEQ ID NO: 10.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain whose sequence is shown in SEQ ID NO: 14, a first light chain whose sequence is shown in SEQ ID NO: 15, and whose sequence is shown in SEQ ID NO: The second heavy chain shown in 26, and the second light chain whose sequence is shown in SEQ ID NO:8.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain with a sequence shown in SEQ ID NO: 14, a first light chain with a sequence shown in SEQ ID NO: 27, and a sequence shown in SEQ ID NO: The second heavy chain shown in 12, and the second light chain whose sequence is shown in SEQ ID NO: 13.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain with a sequence shown in SEQ ID NO: 28, a first light chain with a sequence shown in SEQ ID NO: 29, and a sequence shown in SEQ ID NO: The second heavy chain shown in 12, and the second light chain whose sequence is shown in SEQ ID NO: 13.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain with a sequence shown in SEQ ID NO: 14, a first light chain with a sequence shown in SEQ ID NO: 27, and a sequence shown in SEQ ID NO: The second heavy chain shown in 25, and the second light chain whose sequence is shown in SEQ ID NO: 10.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain whose sequence is shown in SEQ ID NO: 14, a first light chain whose sequence is shown in SEQ ID NO: 15, and whose sequence is shown in SEQ ID NO: The second heavy chain shown in 31, and the second light chain whose sequence is shown in SEQ ID NO:32.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain whose sequence is shown in SEQ ID NO: 19, and a first light chain whose sequence is shown in SEQ ID NO: 20, whose sequence is shown in SEQ ID NO: The second heavy chain shown in 12, and the second light chain whose sequence is shown in SEQ ID NO:30.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain whose sequence is shown in SEQ ID NO: 35, and a first light chain whose sequence is shown in SEQ ID NO: 36, whose sequence is shown in SEQ ID NO: The second heavy chain shown in 33, and the second light chain whose sequence is shown in SEQ ID NO:34.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain whose sequence is shown in SEQ ID NO: 14, a first light chain whose sequence is shown in SEQ ID NO: 15, and whose sequence is shown in SEQ ID NO: The second heavy chain shown in 25, and the second light chain whose sequence is shown in SEQ ID NO: 10.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain whose sequence is shown in SEQ ID NO: 45, and a first light chain whose sequence is shown in SEQ ID NO: 46, whose sequence is shown in SEQ ID NO: The second heavy chain shown in 37, and the second light chain whose sequence is shown in SEQ ID NO:38.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain whose sequence is shown in SEQ ID NO: 41, and a first light chain whose sequence is shown in SEQ ID NO: 42, whose sequence is shown in SEQ ID NO: The second heavy chain shown in 39, and the second light chain whose sequence is shown in SEQ ID NO:40.
  • the first antigen binding domain specifically binds CD40, and/or the second antigen binding domain specifically binds FAP.
  • the first antigen binding domain comprises a heavy chain variable region VH1 and a light chain variable region VL1
  • the second antigen binding domain comprises a heavy chain variable region VH2 and a light chain variable region VL2;
  • the VL1 comprises LCDR1 whose sequence is shown in SEQ ID NO: 75, and the sequence LCDR2 as set forth in SEQ ID NO: 76, and LCDR3 as set forth in SEQ ID NO: 77
  • the VH2 comprises: HCDR1 as set forth in SEQ ID NO: 80, as in SEQ ID NO: HCDR2 shown in 81, and HCDR3 shown in sequence as SEQ ID NO:82, said VL2 comprising LCDR1 shown in sequence as SEQ ID NO:83, LCDR2 shown in sequence as
  • the VH1 is a heavy chain variable region with a sequence set forth in SEQ ID NO: 78
  • the VL1 is a light chain variable region with a sequence set forth in SEQ ID NO: 79
  • the Described VH2 is the heavy chain variable region whose sequence is shown in SEQ ID NO: 86
  • described VL2 is the light chain variable region whose sequence is shown in SEQ ID NO: 87.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain having a sequence as set forth in SEQ ID NO:67, a first light chain having a sequence as set forth in SEQ ID NO:68, and a first light chain having a sequence as set forth in SEQ ID NO:68 : the second light chain shown in 69.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain having a sequence as set forth in SEQ ID NO:70, a first light chain having a sequence as set forth in SEQ ID NO:71, and a first light chain having a sequence as set forth in SEQ ID NO:71 : the second light chain shown in 72.
  • the first antigen binding domain specifically binds and the second antigen binding domain specifically binds a different epitope of PSMA, respectively.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain having a sequence as set forth in SEQ ID NO: 59, a first light chain having a sequence as set forth in SEQ ID NO: 60, and a sequence as set forth in SEQ ID NO: The second heavy chain shown in 61, and the second light chain whose sequence is shown in SEQ ID NO: 62.
  • the antigen binding protein of the present disclosure comprises: a first heavy chain having a sequence as set forth in SEQ ID NO: 63, a first light chain having a sequence as set forth in SEQ ID NO: 64, and a sequence as set forth in SEQ ID NO: The second heavy chain shown in 65, and the second light chain whose sequence is shown in SEQ ID NO: 66.
  • the present disclosure provides a PD-1/CTLA-4 bispecific antibody, comprising:
  • a PD-1 antigen binding domain comprising a first light chain and a first heavy chain, wherein the following amino acid substitutions are included in CH1 of the first heavy chain and CL of the first light chain:
  • CTLA-4 antigen binding domain comprising a second light chain and a second heavy chain, wherein and in CH1 of the second heavy chain and CL of the second light chain an amino acid substitution selected from any of the following groups:
  • the present disclosure provides a PD-1/CTLA-4 bispecific antibody, comprising:
  • a PD-1 antigen binding domain comprising a first light chain and a first heavy chain, wherein CH1 of the first heavy chain and CL of the first light chain comprise amino acid substitutions selected from any of the following groups:
  • CTLA-4 antigen binding domain comprising a second light chain and a second heavy chain, wherein and in CH1 of the second heavy chain and CL of the second light chain the following amino acid substitutions are included:
  • the present disclosure provides a FAP/CD40 bispecific antibody comprising:
  • a CD40 antigen binding domain comprising a first light chain and a first heavy chain, wherein the following amino acid substitutions are included in CH1 of the first heavy chain and CL of the first light chain:
  • a FAP antigen binding domain comprising a second light chain and a second heavy chain.
  • the present disclosure provides a FAP/CD40 bispecific antibody comprising:
  • a CD40 antigen binding domain comprising a first light chain and a first heavy chain
  • a FAP antigen-binding domain comprising a second light chain and a second heavy chain; wherein the following amino acid substitutions are included in CH1 of the second heavy chain and CL of the second light chain:
  • the first heavy chain and the second heavy chain are linked by a linker.
  • the peptide linker is a peptide having an amino acid sequence of at least 5 amino acids in length, in one embodiment 5 to 100, and in further embodiments 10 to 50 amino acids in length.
  • the peptide linker is (G4S)4.
  • the present disclosure provides an antibody that binds PSMA bi-epitopes, comprising:
  • the present disclosure provides a bi-epitope antibody that binds to PSMA, comprising:
  • CH1 of the second heavy chain and CL of the second light chain comprise the following amino acid substitutions:
  • the present disclosure provides an antigen-binding protein comprising:
  • a first antigen-binding domain comprising a polypeptide H1 comprising a first CH1 linked to a first VH and a polypeptide L1 comprising a first CH1 linked to a first VH, the polypeptide L1 comprising a a CL, wherein the first CH1 and the first CL comprise natural non-cysteine to cysteine amino acid substitutions at one or more of the positions selected from (i-1) to (i-6) :
  • a second antigen binding domain comprising a polypeptide H2 comprising a second CH1 linked to a second VH and a polypeptide L2 comprising a second CH1 linked to a second VL 2 cl.
  • polypeptide H1 comprises a first CH1 linked to a first heavy chain variable region VH1; polypeptide L1 comprises a first CL linked to a first light chain variable region VL1.
  • polypeptide H1 comprises VH1 and the first CH1 in order from N-terminus to C-terminus; polypeptide L1 comprises VL1 and first CL in order from N-terminus to C-terminus.
  • polypeptide H1 comprises VH1, the first CH1 and Fc1 in order from N-terminal to C-terminal;
  • polypeptide L1 comprises VL1 and first CL in order from N-terminal to C-terminal.
  • polypeptide H1 is the first heavy chain and polypeptide L1 is the first light chain.
  • polypeptide H2 comprises a second CH1 linked to a second heavy chain variable region VH2; polypeptide L2 comprises a second CL linked to a second light chain variable region VL2.
  • polypeptide H2 comprises VH2 and a second CH1 in order from N-terminus to C-terminus;
  • polypeptide L2 comprises VL2 and a second CL in order from N-terminus to C-terminus.
  • polypeptide H2 comprises VH2, a second CH1 and Fc2 in order from N-terminus to C-terminus;
  • polypeptide L2 comprises VL2 and a second CL in order from N-terminus to C-terminus.
  • polypeptide H2 is the second heavy chain and polypeptide L2 is the second light chain.
  • polypeptide H1 and polypeptide H2 can be linked by a linker.
  • the polypeptide H1 and polypeptide H2 connected by a linker are, from N-terminus to C-terminus, [VH1]-[first CH1]-Fc1-[linker]-[VH2]-[second CH1].
  • the first CH1, the first CL, the second CH1 and the second CL are as defined above.
  • polypeptide L1 is an antibody light chain, such as a human IgG antibody light chain, which is a kappa light chain (CK); polypeptide L2 is an antibody light chain, such as a human IgG antibody light chain, which can be a lambda light chain ( C ⁇ ) or kappa light chain (CK). In some embodiments, polypeptide L1 is a kappa light chain and polypeptide L2 is a lambda light chain.
  • the polypeptide H1 comprises Fc1
  • the polypeptide H2 comprises Fc2
  • the Fc1 and/or the Fc2 is selected from the Fc of human IgG1, IgG2, IgG3 and IgG4, eg, the Fc of human IgG1.
  • Fc1 and Fc2 are engineered as defined above, or amino acid modified or substituted.
  • Fc1 and/or the Fc2 comprise modifications that alter the half-life of the antigen binding protein, wherein the half-life depends on FcRn binding affinity.
  • Fc1 and/or the Fc2 comprise modifications that alter effector function, wherein the binding affinity for the Fc ⁇ receptor or C1q complement protein is increased or decreased.
  • amino acid substitutions are included in Fc1 and Fc2 such that Fc1 preferentially pairs with Fc2 compared to Fc1.
  • the polypeptide L1 comprises amino acid substitutions: S165C and C214A
  • the polypeptide H1 comprises amino acid substitutions: P171C, C220A, L234A, L235A, D356E, L358M, Y349C, T366S, L368A, and Y407N
  • the polypeptide H2 contains amino acid substitutions: L234A, L235A, D356E, L358M, S354C and T366W;
  • polypeptide L1 contains amino acid substitutions: S165C and C214A
  • polypeptide H1 contains amino acid substitutions: P171C, C220A, L234A, L235A, D356E, L358M, S354C, and T366W
  • polypeptide H2 contains amino acid substitutions: L234A, L235A, D356E , L358M, Y349C, T366S, L368A and Y407N.
  • the polypeptide L1 comprises amino acid substitutions: T164C, C214A, and S114E
  • the polypeptide H1 comprises amino acid substitutions: T139R, F170C, C220A, L234A, L235A, D356E, L358M, Y349C, T366S, L368A, and Y407N
  • the polypeptide L2 comprises amino acid substitution S114K
  • the polypeptide H2 comprises amino acid substitutions: T139D, L234A, L235A, D356E, L358M, S354C and T366W;
  • the polypeptide L1 comprises amino acid substitutions: T164C, C214A and S114E
  • the polypeptide H1 comprises amino acid substitutions: T139R, F170C, C220A, L234A, L235A, D356E, L358M, S354C and T366W
  • the polypeptide L2 comprises amino acid substitutions S114K
  • the polypeptide H2 comprises amino acid substitutions: T139D, L234A, L235A, D356E, L358M, Y349C, T366S, L368A and Y407N.
  • the present disclosure provides a bispecific bivalent antigen binding protein comprising:
  • a first antigen-binding domain comprising a polypeptide H1 comprising a first CH1 linked to a first VH and a polypeptide L1 comprising a first CH1 linked to a first VH, the polypeptide L1 comprising a first CH1 linked to a first VL a CL, wherein: the first CH1 and the first CL each comprise a natural cysteine to non-cysteine amino acid substitution, and the first CH1 and the first CL further comprise a natural non-cysteine in a position selected from Amino acid to cysteine amino acid substitution:
  • a second antigen binding domain comprising a polypeptide H2 comprising a second CH1 linked to a second VH and a polypeptide L2 comprising a second CH1 linked to a second VL two CL;
  • polypeptide H1 includes VH, CH1 and Fc sequentially from the N-terminus to the C-terminus; the polypeptide H2 sequentially includes VH, CH1 and Fc from the N-terminus to the C-terminus.
  • the present disclosure provides a bispecific tetravalent antigen-binding protein comprising:
  • a first antigen-binding domain comprising a polypeptide H1 comprising a first CH1 linked to a first VH and a polypeptide L1 comprising a first CH1 linked to a first VH, the polypeptide L1 comprising a first CH1 linked to a first VL a CL, wherein: the first CH1 and the first CL each comprise a natural cysteine to non-cysteine amino acid substitution, and the first CH1 and the first CL further comprise a natural non-cysteine in a position selected from Amino acid to cysteine amino acid substitution:
  • a second antigen binding domain comprising a polypeptide H2 comprising a second CH1 linked to a second VH and a polypeptide L2 comprising a second CH1 linked to a second VL two CL;
  • the polypeptide H1 is composed of VH and CH1 from the N-terminus to the C-terminus
  • the polypeptide H2 is sequentially composed of VH, CH1 and Fc from the N-terminus to the C-terminus
  • the C-terminus of the polypeptide H1 is optionally connected with a peptide linker.
  • the C-terminus of the polypeptide H2 is fused; or the polypeptide H1 comprises VH, CH1 and Fc in sequence from the N-terminus to the C-terminus, the polypeptide H2 is composed of VH and CH1 from the N-terminus to the C-terminus, and the C-terminus of the polypeptide H2
  • the terminus is optionally fused to the C terminus of the polypeptide H1 via a peptide linker.
  • a peptide linker represents a peptide having an amino acid sequence.
  • the peptide linker is a peptide having an amino acid sequence of at least 5 amino acids in length, in one embodiment 5 to 100, and in further embodiments 10 to 50 amino acids in length.
  • the peptide linker is (G4S)4.
  • the polypeptide H1 consists of VH and CH1 from the N-terminus to the C-terminus
  • the polypeptide H2 comprises VH, CH1 and Fc sequentially from the N-terminus to the C-terminus
  • the C-terminus of the polypeptide H1 is optionally
  • the polypeptide H2 is fused to the C-terminus of the polypeptide H2 through a peptide linker.
  • the polypeptide H1 comprises VH, CH1 and Fc in order from N-terminal to C-terminal
  • the polypeptide H2 is composed of VH and CH1 from N-terminal to C-terminal
  • the C-terminal of the polypeptide H2 is optionally
  • the polypeptide H1 is fused to the C-terminus of the polypeptide H1 through a peptide linker.
  • the present disclosure provides a dimerized polypeptide comprising a heavy chain constant region 1 (CH1) and a light chain constant region (CL), wherein: position 139 of CH1 and position 114 of CL are included such that there is a gap between CH1 and CL Amino acid substitutions that form electrostatic interaction interfaces.
  • CH1 heavy chain constant region 1
  • CL light chain constant region
  • the amino acid at position 139 of CH1 is substituted with a positively charged amino acid
  • the amino acid at position 114 of CL is substituted with a negatively charged amino acid
  • the amino acid at position 139 of CH1 is substituted with a negatively charged amino acid
  • Amino acid the amino acid at position 114 of CL was substituted with a positively charged amino acid.
  • the positively charged amino acids are selected from K, R, and H; the negatively charged amino acids are selected from D and E.
  • CH1 and CL comprise amino acid substitutions selected from the group consisting of: T139R and S114E; T139R and S114D; T139K and S114E; T139K and S114D; T139D and S114K; T139D and S114R; T139E and S114K;
  • the present disclosure provides an antigen-binding protein comprising the above-mentioned dimerized polypeptide.
  • the antigen binding protein comprises a first antigen binding domain comprising a Fab comprising a first heavy chain variable region VH1, a first light chain variable region VL1 and the A dimerized polypeptide, in which the CH1 is the first CH1, and the CL is the first CL; VH1 is directly connected to the first CH1 or connected through a linker, and VL1 is directly connected to the first CL or through a linker connect.
  • the C-terminus of VH1 is linked directly or via a linker to the N-terminus of the first CH1 and the C-terminus of VL1 is linked directly or via a linker to the N-terminus of the first CL.
  • the antigen binding protein comprises a first antigen binding domain and a second antigen binding domain, wherein the second antigen binding domain comprises a second heavy chain variable region VH2 and a second light chain variable region VL2, and The first antigen binding domain and the second antigen binding domain bind different antigens or bind different epitopes on the same antigen; in some embodiments, the second antigen binding domain comprises a Fab.
  • the C-terminus of VH2 is directly connected or connected through a linker to the N-terminus of the second CH1
  • the C-terminus of VL2 is directly connected or connected through a linker to the N-terminus of the second CL.
  • the first CH1 and the first CL comprise amino acid substitutions such that an electrostatic interaction interface is formed between the first CH1 and the first CL; and/or
  • the second CH1 and the second CL comprise amino acid substitutions such that an electrostatic interaction interface is formed between the second CH1 and the second CL.
  • the charges of the amino acids used to form the electrostatic interaction interface in the first CH1 and the second CH1 are opposite, and the charges of the amino acids used to form the electrostatic interaction interface in the first CL and the second CL are opposite .
  • the amino acid substitution that results in an electrostatic interaction interface between the first CH1 and the first CL is located at position 139 of the first CH1 and position 114 of the first CL;
  • amino acid substitutions that allow for the formation of an electrostatic interaction interface between the second CH1 and the second CL are located at position 139 of the second CH1 and position 114 of the second CL.
  • position 139 of the first CH1 and position 139 of the second CH1 are substituted with oppositely charged amino acids, respectively, and positions 114 of the first CL and 114 of the second CL are respectively substituted with oppositely charged amino acids Charged amino acid substitutions.
  • the amino acid at position 139 of the first CH1 is substituted with a positively charged amino acid
  • the amino acid at position 114 of the first CL is substituted with a negatively charged amino acid
  • the amino acid at position 139 of the first CH1 is substituted with a negatively charged amino acid.
  • Substituted with a negatively charged amino acid the amino acid at position 114 of the first CL is substituted with a positively charged amino acid; and/or
  • the amino acid at position 139 of the second CH1 is substituted with a negatively charged amino acid, the amino acid at position 114 of the second CL is substituted with a positively charged amino acid; or the amino acid at position 139 of the second CH1 is substituted with a positively charged amino acid Amino acid, the amino acid at position 114 of the second CL is substituted with a negatively charged amino acid.
  • the positively charged amino acids are selected from K, R, and H; the negatively charged amino acids are selected from D and E.
  • the first CH1 and the first CL comprise amino acid substitutions selected from the group consisting of: T139R and S114E; T139R and S114D; T139K and S114E; T139K and S114D; T139D and S114K; T139D and S114R; T139E and S114K; and T139E and S114R; and/or
  • T139K and S114E T139K and S114D; T139D and S114K; T139D and S114R; T139E and S114K; and T139E and S114R.
  • the first CH1 and the first CL comprise amino acid substitutions selected from the group consisting of: T139R and S114E; T139R and S114D; T139K and S114E; T139K and S114D; and/or
  • the second CH1 and the second CL comprise amino acid substitutions selected from the group consisting of: T139D and S114K; T139D and S114R; T139E and S114K; and T139E and S114R.
  • the first CH1 and the first CL comprise amino acid substitutions selected from the group consisting of: T139D and S114K; T139D and S114R; T139E and S114K; and T139E and S114R; and/or
  • the second CH1 and the second CL comprise amino acid substitutions selected from the group consisting of: T139R and S114E; T139R and S114D; T139K and S114E; T139K and S114D.
  • the present disclosure provides an antigen-binding protein comprising:
  • a first antigen-binding domain comprising a polypeptide H1 comprising a first CH1 linked to a first VH and a polypeptide L1 comprising a first CH1 linked to a first VH, the polypeptide L1 comprising a first CH1 linked to a first VL a CL;
  • a second antigen binding domain comprising a polypeptide H2 comprising a second CH1 linked to a second VH and a polypeptide L2 comprising a second CH1 linked to a second VL two CL;
  • position 139 of the first CH1 and position 114 of the first CL comprise amino acid substitutions that result in an electrostatic interaction interface between the first CH1 and the first CL;
  • Position 139 of the second CH1 and position 114 of the second CL contain amino acid substitutions that allow an electrostatic interaction interface to be formed between the second CH1 and the second CL.
  • the antigen binding protein is a bispecific bivalent antigen binding protein, wherein the polypeptide H1 comprises VH, CH1 and Fc in order from N-terminus to C-terminus; and said polypeptide H2 in order from N-terminus to C-terminus Contains VH, CH1 and Fc.
  • the antigen binding protein is a bispecific tetravalent antigen binding protein, wherein the polypeptide H1 consists of VH and CH1 from N-terminus to C-terminus, and the polypeptide H2 comprises VH, CH1 and Fc, the C-terminus of the polypeptide H1 is optionally fused to the C-terminus of the polypeptide H2 through a peptide linker; or the polypeptide H1 sequentially comprises VH, CH1 and Fc from the N-terminus to the C-terminus, and the polypeptide H2 Consisting of VH and CH1 from the N-terminus to the C-terminus, the C-terminus of the polypeptide H2 is optionally fused to the C-terminus of the polypeptide H1 through a peptide linker.
  • the antigen binding proteins of the present disclosure are multispecific antibodies, eg, bispecific antibodies. In some embodiments, the antigen binding proteins of the present disclosure are chimeric, humanized or fully human antibodies, multivalent antibodies, or antibody drug conjugates.
  • the antigen binding proteins of the present disclosure comprising the above amino acid substitutions have improved polypeptide H1/L1 and polypeptide H2/L2 (eg, heavy/light chains) compared to antigen binding proteins without these amino acid substitutions Paired or improved yields are produced in single cells.
  • the antigen binding protein of the present disclosure has a correct pairing ratio of polypeptide H1/L1 and polypeptide H2/L2 (eg, heavy chain/light chain) of at least 55%, at least 60%, at least 65%, at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.
  • polypeptide H1/L1 and polypeptide H2/L2 eg, heavy chain/light chain
  • correct pairing ratio of polypeptide H1/L1 and polypeptide H2/L2 (correct first antigen-binding molecule peak intensity + correct second antigen-binding molecule peak intensity)/(correct first antigen-binding molecule peak intensity) Antigen-binding molecule peak intensity + correct second antigen-binding molecule peak intensity + other impurity peak intensity) ⁇ 100%.
  • the antigen binding protein of the present disclosure has an increased ratio of correct pairing of polypeptides H1/L1 and H2/L2 (eg, heavy/light chains) relative to wild type by at least 5%, at least 10%, at least 15% %, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49% or 50%.
  • polypeptides H1/L1 and H2/L2 eg, heavy/light chains
  • the antigen binding proteins of the present disclosure combine polypeptide H1/L1 and polypeptide H2/L2 (eg, heavy/light chain) by removing natural disulfide bonds and introducing non-natural disulfide bonds in the CH1/CL interface. at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 46%, At least 47%, at least 48%, at least 49% or 50%.
  • polypeptide H1/L1 and polypeptide H2/L2 eg, heavy/light chain
  • the antigen binding proteins of the present disclosure combine the correct pairing ratio of polypeptide H1/L1 and polypeptide H2/L2 (eg, heavy chain/light chain) with respect to the Wild type increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 46%, at least 47%, at least 48% %, at least 49% or 50%.
  • the antigen binding proteins of the present disclosure combine polypeptide H1/L1 and polypeptide H2/ by removing natural disulfide bonds and introducing non-natural disulfide bonds in the CH1/CL interface, and introducing electrostatically complementary amino acid pairs at the same time.
  • the ratio of correct pairing of L2 is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% relative to wild type , at least 45%, at least 46%, at least 47%, at least 48%, at least 49% or 50%.
  • the present disclosure also provides a nucleic acid molecule or a combination thereof encoding the aforementioned dimeric polypeptide or antigen-binding protein.
  • the present disclosure also provides a nucleic acid expression vector or a combination thereof comprising the aforementioned nucleic acid molecule or a combination thereof.
  • the present disclosure also provides a host cell comprising the aforementioned nucleic acid molecule or a combination thereof.
  • the host cell is any kind of cellular system, eg, eukaryotic or prokaryotic cells, that can be engineered to produce a dimeric polypeptide or antigen-binding protein according to the present disclosure.
  • Eukaryotic cells include, but are not limited to, for example, nucleated cells from yeast, fungi, insects, plants, animals, humans or other multicellular organisms.
  • the present disclosure also provides a method for preparing any of the aforementioned dimer polypeptides or antigen-binding proteins, comprising the following steps:
  • the aforementioned nucleic acid expression vector includes: a plasmid encoding a heavy chain and a plasmid encoding a light chain; when transforming a host cell, the plasmid encoding the light chain is in excess relative to the plasmid encoding the heavy chain, eg, encoding the heavy chain
  • the molar ratio of the plasmid to the plasmid encoding the light chain is 1:(1-10), such as 1:(1-5), such as 2:3.
  • the aforementioned nucleic acid expression vector comprises:
  • a first plasmid comprising a nucleic acid molecule encoding the polypeptide H1;
  • a second plasmid comprising a nucleic acid molecule encoding the polypeptide L1;
  • a third plasmid comprising a nucleic acid molecule encoding the polypeptide H2;
  • the fourth plasmid which comprises a nucleic acid molecule encoding the polypeptide L2.
  • the aforementioned nucleic acid expression vector comprises:
  • a first plasmid comprising a nucleic acid molecule encoding a first heavy chain
  • a second plasmid comprising a nucleic acid molecule encoding the first light chain
  • a third plasmid comprising a nucleic acid molecule encoding the second heavy chain
  • a fourth plasmid comprising a nucleic acid molecule encoding the second light chain.
  • the molar ratio of the first plasmid to the second plasmid upon transforming the host cell is 1:1 to 1:10, 1:1 to 1:9, 1:1 to 1:8, 1:1 to 1:1 1:7, 1:1 to 1:6, 1:1 to 1:5, 1:1 to 1:4, 1:1 to 1:3, 1:1 to 1:2, 1:1 to 1: 1.9, 1:1 to 1:1.8, 1:1 to 1:7, 1:1 to 1:1.6, 1:1 to 1:1.5, 1:1 to 1:1.4, 1:1 to 1:1.3, 1:1 to 1:1.2, 1:1 to 1:1.1, or 1:1 to 1:1.05.
  • the molar ratio of the first plasmid and the second plasmid when transforming the host cell is 1:1, 1:1.05, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1: 2.8, 1:2.9, 1:3.0, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4.0, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5.0, 1:5.1, 1:5.2, 1: 5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6.0, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.0,
  • the molar ratio of the third plasmid to the fourth plasmid when transforming the host cell is 1:1 to 1:10, 1:1 to 1:9, 1:1 to 1:8, 1:1 to 1:1 1:7, 1:1 to 1:6, 1:1 to 1:5, 1:1 to 1:4, 1:1 to 1:3, 1:1 to 1:2, 1:1 to 1: 1.9, 1:1 to 1:1.8, 1:1 to 1:7, 1:1 to 1:1.6, 1:1 to 1:1.5, 1:1 to 1:1.4, 1:1 to 1:1.3, 1:1 to 1:1.2, 1:1 to 1:1.1, or 1:1 to 1:1.05.
  • the molar ratio of the third plasmid and the fourth plasmid when transforming the host cell is 1:1, 1:1.05, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1: 2.8, 1:2.9, 1:3.0, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4.0, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5.0, 1:5.1, 1:5.2, 1: 5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6.0, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.0,
  • the molar ratio of the first plasmid: the second plasmid: the third plasmid: the fourth plasmid is 1:(1-10):1:(1-10), eg, 1:( 1 ⁇ 5):1:(1 ⁇ 5), for example 2:3:2:3.
  • the molar ratio of the first plasmid: the second plasmid: the third plasmid: the fourth plasmid is 1:(1-10):1:(1-10), 1:(1 ⁇ 9):1:(1 ⁇ 9), 1:(1 ⁇ 8):1:(1 ⁇ 8), 1:(1 ⁇ 7):1:(1 ⁇ 7), 1:(1 ⁇ 6 ):1:(1 ⁇ 6), 1:(1 ⁇ 5):1:(1 ⁇ 5), 1:(1 ⁇ 4):1:(1 ⁇ 4), 1:(1 ⁇ 3): 1:(1 ⁇ 3) or 1:(1 ⁇ 2):1:(1 ⁇ 2).
  • the molar ratio of the first plasmid: the second plasmid: the third plasmid: the fourth plasmid is 1:1:1:1, 1:1.05:1:1.05, 1:1.1: 1:1.1, 1:1.2:1:1.2, 1:1.3:1:1.3, 1:1.4:1:1.4, 1:1.5:1:1.5 (or 2:3:2:3), 1:1.6: 1:1.6, 1:1.7:1:1.7, 1:1.8:1:1.8, 1:1.9:1:1.9, 1:2.0:1:2.0, 1:2.1:1:2.1, 1:2.2:1: 2.2, 1:2.3:1:2.3, 1:2.4:1:2.4, 1:2.5:1:2.5, 1:2.6:1:2.6, 1:2.7:1:2.7, 1:2.8:1:2.8, 1:2.9:1:2.9, 1:3.0:1:3.0, 1:3.1:1:3.1, 1:3.2:1:3.2, 1:3.3:1:3.3, 1:3.4
  • the nucleic acid expression vector comprises:
  • a first plasmid comprising a nucleic acid molecule encoding the polypeptide H1 and a nucleic acid molecule encoding the polypeptide H2;
  • a second plasmid comprising a nucleic acid molecule encoding the polypeptide L1;
  • a third plasmid comprising a nucleic acid molecule encoding the polypeptide L2.
  • the nucleic acid expression vector comprises:
  • a first plasmid comprising a nucleic acid molecule encoding a heavy chain
  • a second plasmid comprising a nucleic acid molecule encoding the first light chain
  • a third plasmid comprising a nucleic acid molecule encoding the second light chain.
  • the molar ratio of the first plasmid: the second plasmid: the third plasmid is 1:(1-10):(1-10), preferably 1:(1-5):( 1 to 5), more preferably 2:3:3.
  • the molar ratio of the first plasmid: the second plasmid: the third plasmid is 1:(1-10):(1-10), 1:(1-9):(1 ⁇ 9), 1:(1 ⁇ 8):(1 ⁇ 8), 1:(1 ⁇ 7):(1 ⁇ 7), 1:(1 ⁇ 6):(1 ⁇ 6), 1:(1 ⁇ 5):(1 ⁇ 5), 1:(1 ⁇ 4):(1 ⁇ 4), 1:(1 ⁇ 3):(1 ⁇ 3) or 1:(1 ⁇ 2):(1 ⁇ 2 ).
  • the molar ratio of the heavy chain plasmid: the first light chain plasmid: the second light chain plasmid is 1:1:1, 1:1.05:1.05, 1:1.1:1.1, 1:1:1. 1.2:1.2, 1:1.3:1.3, 1:1.4:1.4, 1:1.5:1.5 (or 2:3:3), 1:1.6:1.6, 1:1.7:1.7, 1:1.8:1.8, 1: 1.9:1.9, 1:2.0:2.0, 1:2.1:2.1, 1:2.2:2.2, 1:2.3:2.3, 1:2.4:2.4, 1:2.5:2.5, 1:2.6:2.6, 1:2.7: 2.7, 1:2.8:2.8, 1:2.9:2.9, 1:3.0:3.0, 1:3.1:3.1, 1:3.2:3.2, 1:3.3:3.3, 1:3.4:3.4, 1:3.5:3.5, 1:3.6:3.6, 1:3.7:3.7, 1:3.8:3.8, 1:3.9:3.9, 1:4.0:4.0, 1:4.1:4.1, 1:4.2:
  • the present disclosure also provides a pharmaceutical composition comprising any one of the aforementioned antigen binding proteins and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation other than the active ingredient, which is non-toxic to the subject.
  • Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).
  • the present disclosure also provides a method of eliminating an immunosuppression-related disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an antigen binding protein as described above, or as described above
  • a pharmaceutical composition, the therapeutically effective amount of the composition in a unit dose contains 0.1-3000 mg of the aforementioned antigen-binding protein.
  • the antigen binding protein or pharmaceutical composition described herein is administered to the individual in a dose of about 10 ⁇ g/kg to about 1000 mg/kg in a single or cumulative application.
  • the present disclosure also provides the use of any of the aforementioned dimerized polypeptides or antigen-binding proteins in the preparation of medicines.
  • the present disclosure also provides use of any of the aforementioned dimerized polypeptides or antigen-binding proteins in the preparation of a medicament for treating cancer, autoimmune disease or inflammatory disease.
  • the present disclosure also provides a method of treating and/or preventing a disease, such as cancer, autoimmune disease or inflammatory disease, comprising administering to a patient in need thereof an effective amount of the aforementioned antigen binding protein or pharmaceutical composition.
  • a disease such as cancer, autoimmune disease or inflammatory disease
  • the present disclosure also provides any one of the aforementioned dimerized polypeptides, antigen binding proteins, or pharmaceutical compositions for use in the treatment of cancer, autoimmune disease, or inflammatory disease.
  • cancer includes, but is not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, and lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma , non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), T-cell/histiocyte-rich Large B-cell lymphoma
  • the autoimmune disease or inflammatory disease is selected from the group consisting of: rheumatoid arthritis, psoriasis, Crohn's disease, ankylosing spondylitis, multiple sclerosis, type I diabetes, hepatitis, myocarditis, Sjogren's syndrome , Autoimmune hemolytic anemia after transplant rejection, vesicular pemphigoid, Grave's disease, Hashimoto's thyroiditis, systemic lupus erythematosus (SLE), myasthenia gravis, pemphigus, pernicious anemia.
  • rheumatoid arthritis rheumatoid arthritis
  • psoriasis Crohn's disease
  • ankylosing spondylitis multiple sclerosis
  • type I diabetes hepatitis
  • myocarditis myocarditis
  • Sjogren's syndrome Autoimmune hemolytic anemia after transplant rejection
  • Figure 1 shows the molecular weight deconvolution mass spectrogram of the PD-1 monoclonal antibody product IdeS digested in Example 3;
  • Figure 2 shows the molecular format of Example 4: a 1+1 asymmetric bispecific antibody using native CH1/CK in one arm and CH1/CK containing a non-native disulfide bond in the other arm;
  • Figures 3A-3D show the molecular weight deconvolution mass spectrograms after papain digestion of the double antibody primary pure product in Example 4;
  • Figure 4A shows the two-step purification chromatogram of the primary product of TJ030-PR1104 protein
  • Figure 4B shows the total ion chromatogram (top) and UV spectrum (bottom) of the purified TJ030-PR1104 protein with intact molecular weight Molecular assignment information of peaks
  • Figure 4C shows the total ion chromatogram (top) and UV spectrum (bottom) of the purified TJ030-PR1104 protein, as well as the molecular assignment information of the main peaks;
  • Figure 5 shows that 1+1 asymmetric PD-1 ⁇ CTLA-4 double antibody can promote the cross-linking of PD-1 expressing cells and CTLA-4 expressing cells;
  • Figure 6 shows a schematic representation of the molecular format in Example 5: a 1+1 asymmetric bispecific antibody is shown, using native CH1/C ⁇ in one arm and CH1/C ⁇ containing a non-native disulfide bond in the other arm; or One arm uses CH1/C ⁇ containing unnatural disulfide bonds, and the other arm uses natural CH1/C ⁇ ;
  • Figure 7A shows the UV spectrum of the complete molecular weight of TJ030-PR1313 after purification and the molecular assignment information of the main peak
  • Figure 7B shows the deconvoluted mass spectrum of the Fab molecular weight of TJ030-PR1313 after Lys-C digestion
  • Figure 8 shows the molecular weight deconvolution mass spectrogram of the product of GingisKHAN protease-treated PSMA 1+1 di-epitope antibody with unnatural disulfide bond introduced into CH1/CL;
  • Figure 9A shows a schematic diagram of the molecular format of the FAP ⁇ CD40 2+2 symmetric bispecific antibody of Example 8;
  • Figure 9B shows the reduced molecular weight deconvoluted mass spectra of two FAP ⁇ CD40 antibodies;
  • Figures 9C-9D show two FAPs ⁇ The molecular weight deconvoluted mass spectrum of CD40 antibody IdeS digestion;
  • Figure 10A shows the FACS binding EC50 results of FAPxCD40 antibody to CD40
  • Figure 10B shows the FACS binding EC50 results of FAPxCD40 antibody to FAP
  • Figures 10C and 10D show the FAPxCD40 antibody in the presence and absence of FAP activating activity of CD40.
  • antigen refers to any substance that induces an immune response in the body
  • examples of antigens include, but are not limited to, peptides, proteins, glycoproteins, polysaccharides, lipids, and synthetic or naturally occurring chemical compounds or combinations thereof.
  • antigen-binding protein refers to a protein capable of binding an antigen, which includes, but is not limited to, full-length antibodies, antibody fragments, or fusion proteins of antibodies and other polypeptides.
  • binding may be, for example, specific binding.
  • antibody fragments include, but are not limited to (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, comprising linkages through disulfide bridges on the hinge region A bivalent fragment of two Fab fragments, (iii) an Fd fragment composed of VH and CH1 domains; (iv) an Fv fragment composed of the VH and VL domains of the one-armed antibody; (v) dsFv, composed of VH Antigen-binding fragments formed by interchain disulfide bonds with VL; (vi) diabodies, bispecific antibodies and multispecific antibodies comprising scFv, dsFv, Fab and other fragments.
  • scFv single-chain Fv
  • Single chain antibodies are also included within the term antibody fragment.
  • antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies.
  • Antigen binding domains can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.
  • Antibodies can be of different isotypes, for example, according to the amino acid sequence of the heavy chain constant region of the antibody, antibodies are divided into different types (for example, 5 types of IgA, IgD, IgE, IgG and IgM, and then IgG1, IgG2, IgG3). , IgG4, IgA1 and IgA2, etc. subtypes).
  • the heavy chain constant regions corresponding to the above five types are called ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , respectively.
  • the light chain of an antibody can be considered to be either Kappa ( ⁇ ) or Lamda ( ⁇ ) based on its amino acid sequence.
  • (light chain) CL region refers to the constant region of an antibody light chain, which is a region well known in the relevant art.
  • the CL region can be determined by conventional methods. For example, whether the target region is a CL region can be determined by using homology with known antibodies, etc., and the boundary of the CL region can be changed.
  • the CL region consists of 107 amino acid residues.
  • the CL region in the human ⁇ chain usually consists of 106 amino acid residues.
  • the natural cysteine in the CL region of the human kappa chain is the 214th position coded according to Kabat
  • the natural cysteine in the CL region of the human ⁇ chain is the 214th position coded according to Kabat.
  • (heavy chain) CH1 region refers to the first constant region of the heavy chain, which is a region known in the relevant art.
  • the CH1 region as defined herein may also contain a portion of the hinge region following the CH1 region (which may be included in the hinge region of the Fab region).
  • the CH1 region can be determined by a conventional method, for example, whether the target region is the CH1 region can be determined by using homology with a known antibody or the like.
  • the CH1 region is generally part of the hinge region by amino acid residue numbers 118-215 and additional hinge regions (eg amino acid residues) Residue numbers 216-224); in the heavy chain of IgM, the CH1 region as defined herein is usually composed of amino acid base numbers 118-216, but not limited thereto.
  • Fc region refers to a region corresponding to a fragment having no antigen-binding ability among two types of fragments obtained when an antibody is cleaved with papain.
  • an Fc region refers to the C-terminal region of an antibody heavy chain, which comprises a portion of the hinge region, the second constant (CH2) region and the third constant (CH3) region of the heavy chain.
  • the boundaries of the heavy chain Fc region can vary, eg, a human IgGl heavy chain Fc region consists of the amino acid residues of Thr225 to the carboxy terminus of the CH3 region.
  • ADCC antibody-dependent cytotoxicity
  • FcRs Fc receptors
  • NK cells the primary cells that mediate ADCC
  • monocytes express FcyRI, FcyRII, and FcyRIII.
  • ADCC activity on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991).
  • in vitro ADCC assays can be performed, such as those described in US Pat. Nos. 5,500,362 or 5,821,337. Effector cells used in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells.
  • the ADCC activity of the molecule of interest can be assessed in vivo, eg, in animal models such as those disclosed in Clynes et al., PNAS USA 95:652-656 (1998).
  • Fc receptor or "FcR” describes a receptor that binds the Fc region of an antibody.
  • Preferred FcRs are human FcRs.
  • preferred FcRs are those that bind IgG antibodies (gamma receptors) and include receptors of the FcyRI, FcyRII and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • FcyRII receptors include FcyRIIA ("activating receptor”) and FcyRIIB ("inhibiting receptor”), which have similar amino acid sequences that differ primarily in their cytoplasmic domains.
  • the activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • the inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see review M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)).
  • FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al, Immunomethods 4:25-34 (1994); and de Haas et al, J.Lab.Clin.Med.126: 330-41 (1995).
  • FcR herein encompasses other FcRs, including those to be identified in the future.
  • the term also includes the neonatal receptor FcRn responsible for the transfer of maternal IgG to the fetus (Guyer et al, J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)).
  • human effector cell is a leukocyte that expresses one or more FcRs and performs effector functions. Preferably, the cell expresses at least FcyRIII and performs ADCC effector function.
  • human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; PBMCs and NK cells are preferred. Effector cells can be isolated from natural sources, eg, from blood.
  • complement-dependent cytotoxicity refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to an antibody (of the appropriate subclass) that binds to its cognate antigen.
  • the CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).
  • a therapeutically effective amount refers to the amount of antibody (including multispecific antibodies), antigen-binding antibody fragments thereof, or derivatives thereof that treat a disease or disorder in an individual.
  • a therapeutically effective amount of an antibody or antibody fragment can reduce the number of cancer cells, reduce the size of the primary tumor, inhibit (ie, in a certain to some extent slow and preferably prevent) infiltration of cancer cells into peripheral organs, inhibit (i.e. slow and preferably prevent) tumor metastasis to some extent, inhibit tumor growth to some extent, and/or to some extent Relief of one or more symptoms associated with the disorder.
  • the antibody, or antibody fragment thereof, or derivative thereof can prevent growth and/or kill existing cancer cells, it can be a cytostatic and/or cytotoxic agent.
  • in vivo efficacy can be measured, for example, by assessing survival, time to disease progression (TTP), response rate (RR), duration of response, and/or quality of life.
  • natural disulfide bond refers to a cysteine-cysteine covalent bond typically present in wild-type polypeptides (antibodies, etc.).
  • non-natural disulfide bond refers to a cysteine-cysteine covalent bond formed at a position other than the above-mentioned "natural disulfide bond”.
  • multispecific antibody refers to an antibody that binds two or more different epitopes (eg, two, three, four or more different epitopes).
  • the epitopes can be on the same or different antigens.
  • a multispecific antibody is a "bispecific antibody” that binds two different epitopes.
  • valency refers to the presence of a specific number of binding sites in an antibody molecule. Natural antibodies, for example, have two binding sites and are bivalent. As such, the term “tetravalent” refers to the presence of four binding sites in the antibody molecule.
  • amino acid refers primarily to the 20 naturally occurring amino acids selected from the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), Phenylalanine (Phe or F), Glycine (Gly or G), Histidine (His or H), Isoleucine (He or I), Lysine (Lys or K) , Leucine (Leu or L), Methionine (Met or M), Asparagine (Asn or N), Proline (Pro or P), Glutamine (Gln or Q), Arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W) and tyrosine (Tyr or Y).
  • amino acid residue means that when the amino acids that make up the polypeptide are combined with each other, because some of their groups participate in the formation of peptide bonds and lose a molecule of water, the amino acid unit in a polypeptide is called an amino acid residue, that is, a peptide composed of a peptide. The remainder of the linked amino acids after dehydration.
  • amino acid and “amino acid residue” are used interchangeably herein.
  • Amino acids "positively charged” or “negatively charged” are classified according to the charge properties of amino acid side chains as measured at pH 7.4.
  • Amino acids can be grouped according to common side chain properties: (1) Hydrophobicity: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negatively charged): Asp, Glu; (4) Basic (positively charged): His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic : Trp, Tyr, Phe.
  • interface refers to a binding or contact surface derived from the interaction of one or more amino acids in the first domain of an antigen binding protein or antibody with one or more amino acids in the second domain.
  • exemplary interfaces exist, for example, between CH1/CL, between VH/VL, and/or between CH3/CH3.
  • the interface includes, for example, hydrogen bonds, electrostatic interactions, or salt bridges between amino acids that form the interface.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in the host cell into which they have been introduced (eg, bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) or may integrate into the host cell's genome after introduction into the host cell, thereby following The host genome replicates together (eg, a non-episomal mammalian vector).
  • mice can be immunized with human PD-1 or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods.
  • Antigen-binding fragments can likewise be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the present disclosure are genetically engineered to add one or more human FR regions to non-human CDRs.
  • Human FR germline sequences can be obtained by aligning the IMGT human antibody variable region germline gene database with MOE software, from the website of ImMunoGeneTics (IMGT) at http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001 ISBN012441351 get.
  • IMGT ImMunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian-like expression systems lead to glycosylation of the antibody, especially at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human PD-1, or to both PD-1 and PD-L1. Positive clones were expanded in serum-free medium in bioreactors for antibody production.
  • the antibody-secreted culture medium can be purified by conventional techniques. For example, use an A or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody was eluted by pH gradient method, and the antibody fragments were detected by SDS-PAGE and collected. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The obtained product should be frozen immediately, eg -70°C, or lyophilized.
  • first and second in the present disclosure are general identifiers only and should not be construed to identify specific or specific portions of the antigen binding proteins provided herein; in any embodiment of the present disclosure ""
  • the “first” and “second” can be reversed, eg, any amino acid substitutions described in this disclosure that are in the first CH1 and the first CL can alternatively be in the second CH1 and the second CL.
  • SEQ ID NO: 14 (CTLA-4/HC)
  • SEQ ID NO: 16 (CTLA-4/HC, F126C underlined)
  • SEQ ID NO: 18 (CTLA-4/HC, L128C underlined)
  • SEQ ID NO: 20 (CTLA-4/LC, T164C underlined)
  • SEQ ID NO: 24 (PD-1/HC, L128C underlined)
  • SEQ ID NO: 27 (CTLA-4/LC)
  • SEQ ID NO: 28 (CTLA-4/HC, P171C underlined)
  • SEQ ID NO: 36 (CTLA-4/LC, S114K underlined)
  • SEQ ID NO: 41 (CTLA-4/HC)
  • SEQ ID NO: 42 (CTLA-4/LC)
  • SEQ ID NO: 48 (PD-1/LCDR3)
  • SEQ ID NO: 51 (CTLA-4/HCDR1)
  • SEQ ID NO: 52 (CTLA-4/HCDR2)
  • SEQ ID NO: 53 (CTLA-4/HCDR3)
  • SEQ ID NO: 54 (CTLA-4/LCDR1)
  • SEQ ID NO: 56 (CTLA-4/LCDR3)
  • the expression process of the double antibody is the same as that of the monoclonal antibody PD-1, and the purification strategy is slightly more complicated than that of the monoclonal antibody: the affinity chromatography in the first step is similar to that of the monoclonal antibody, but sometimes it needs to be purified by ion exchange chromatography. According to the properties of the isoelectric point of the antibody, different anion and cation exchange chromatography methods can be selected.
  • the method of anion exchange chromatography is: load the one-step purified sample to a HiTrap Q HP column (GE, 17515601), equilibrate with A solution (20mM PB, pH 7.0), and then use 0-100% B solution ( 20 mM PB, 1 M NaCl, pH 7.0) gradient elution.
  • the method of cation exchange chromatography is: the one-step purified sample is loaded onto a Capto S ImpAct prepacked column (GE, 17-5441-22), equilibrated with solution A (50 mM NaAc, 50 mM NaCl, pH 5.0), and then equilibrated with 0- 100% B solution (50 mM NaAc, 500 mM NaCl, pH 5.0) gradient elution.
  • solution A 50 mM NaAc, 50 mM NaCl, pH 5.0
  • B solution 50 mM NaAc, 500 mM NaCl, pH 5.0
  • Protein samples were bioanalyzed in this disclosure using conventional high resolution mass spectrometry 6530B ESI-Q-TOF (Agilent) and XEVO G2-XS Q-Tof (Waters).
  • the sample After the sample is diluted, it is separated by reverse chromatography and detected by high-resolution mass spectrometry to obtain the original spectrum containing information of different mass-to-charge ratios. After processing by the deconvolution software, the complete molecular weight information of the antibody is obtained. Specifically: take 50 ⁇ g of samples and standards, dilute to 0.5 mg/mL with mobile phase A (0.1% formic acid aqueous solution), centrifuge at 12000 rpm at 4°C for 10 min, and take the supernatant to the injection bottle.
  • mobile phase A 0.1% formic acid aqueous solution
  • the column was equilibrated with 95% mobile phase A (Waters, 186008946) to be stable before injection, and gradient elution was performed with mobile phase A and mobile phase B (0.1% formic acid in acetonitrile) after injection. After the sample collection is completed, the corresponding mass spectral data is obtained at the outgoing peak of the target peak.
  • mobile phase A Waters, 186008946
  • mobile phase B 0.1% formic acid in acetonitrile
  • the sample is diluted, it is separated by reverse chromatography and detected by high-resolution mass spectrometry to obtain the original spectrum containing information of different mass-to-charge ratios. Specifically: take 100 ⁇ g of the test substance and standard substance, add 2 ⁇ L of peptide N-glycosidase F (PNGase F, BioLabs, P0704L) to each, add 50 mM ammonium bicarbonate solution to make up the volume to 100 ⁇ L, and deglycosify at 37°C for 3 hours.
  • PNGase F peptide N-glycosidase F
  • the protein concentration was diluted with mobile phase A to 0.5 ⁇ g/ ⁇ L, centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was taken into the injection bottle.
  • the column was equilibrated with 95% mobile phase A until stable, and after injection, gradient elution was performed using mobile phase A and mobile phase B (0.1% formic acid in acetonitrile). After the sample collection is completed, the corresponding mass spectral data is obtained at the outgoing peak of the target peak.
  • the sample After the sample is diluted, it is separated by reverse chromatography and detected by high-resolution mass spectrometry to obtain the original spectrum containing information of different mass-to-charge ratios.
  • the molecular weight information of antibody reduction can be obtained. Specifically: take 100 ⁇ g of the test substance and standard substance, add 50 mM ammonium bicarbonate solution to make up the volume to 90 ⁇ L, add 10 ⁇ L DTT to make the final concentration 10 mM, and incubate at 37°C for 30 min. After incubation, the protein concentration was diluted with mobile phase A to 0.5 ⁇ g/ ⁇ L, centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was taken into the injection bottle.
  • the column Before injection, the column was equilibrated with 95% mobile phase A until stable, and after injection, gradient elution was performed using mobile phase A and mobile phase B (0.1% formic acid in acetonitrile). After the sample collection is completed, the corresponding mass spectral data is obtained at the outgoing peak of the target peak.
  • the samples were digested by immunoglobulin G degrading enzyme (IdeS, Promega, v7511) to obtain Fab fragments, which were separated by reverse chromatography and entered into high-resolution mass spectrometry to obtain original spectra containing information of different mass-to-charge ratios.
  • immunoglobulin G degrading enzyme IdeS, Promega, v7511
  • Fab fragments which were separated by reverse chromatography and entered into high-resolution mass spectrometry to obtain original spectra containing information of different mass-to-charge ratios.
  • the molecular weight information of the antibody F(ab') 2 fragment was obtained, and the pairing information was obtained through the molecular weight information.
  • the sample was digested by protease (Lys-C, RHINO BIO, QIP-004-A or Papain, Solarbio, G8430) to obtain Fab fragments, which were separated by reverse chromatography and entered into high-resolution mass spectrometry detection to obtain information containing different mass-to-charge ratios.
  • protease Li-C, RHINO BIO, QIP-004-A or Papain, Solarbio, G8430
  • the molecular weight information of the Fab fragment of the antibody is obtained, and the pairing information is obtained through the molecular weight information.
  • Lys-C digestion to determine the molecular weight of Fab as an example: take 100 ⁇ g of the test and standard, add 50mM Tris-HCl (pH 7.50) solution to dilute to 0.5 ⁇ g/ ⁇ L, take 100 ⁇ L of diluted sample and add 0.25 ⁇ g Lys-C, Incubate at 37°C for 5min. After the reaction was completed, 1 ⁇ L of 10% formic acid aqueous solution was added, and the supernatant was taken into the injection bottle. Before injection, the column was equilibrated with 95% mobile phase A until stable, and after injection, gradient elution was performed using mobile phase A and mobile phase B (0.1% formic acid in acetonitrile). After the sample collection is completed, the corresponding mass spectral data is obtained at the outgoing peak of the target peak.
  • Tris-HCl pH 7.50
  • HEK293 cells were transiently transfected with human CTLA-4 plasmid, and 24 hours after transfection, HEK293 cells with high expression of CTLA-4 were labeled with Cell Trace Far red (Invitrogen, C34564), and CHO-K1/PD-1 was stably transfected
  • the strains were labeled with Cell Trace Violet (Invitrogen, C34557) and added to 96-well U-plates (Costar, 3599), 2E5 cells per well, and the antibody to be detected was diluted to 100nM, 10nM, 1nM, 0.1nM and 0.01 nM, were added to 96-well U-plate, 50 ⁇ L/well, and the total volume was 150 ⁇ L/well.
  • the cells were incubated at 4°C in the dark for 1 hour, and the percentages of double
  • CHO cells ie, CHO/FAP cells
  • HEK293 cells ie, HEK293/CD40 cells
  • Antibodies to be detected at different concentrations were added, incubated on ice for 1 h, washed with PBS, and centrifuged at 400 g for 5 min. Add goat anti-human secondary antibody Alexa Fluor 488 with a fluorophore for ice bath staining for 1 hour, wash twice with PBS, and then detect on the machine.
  • the positive cell line HEK-Blue CD40L cells with high expression of human CD40 and Flp-In CHO cells stably expressing human FAP were used.
  • the cell line was diluted to 5.5E5/mL with DMEM/F12K medium containing 10% heat-inactivated serum, and 90 ⁇ L of HEK-Blu CD40L cell suspension was added to each well of a 96-well flat-bottom cell culture plate, and 90 ⁇ L of medium or Flp was added at the same time.
  • -In CHO/FAP cell line was used.
  • the nucleic acids encoding the heavy chain (sequence shown in SEQ ID NO: 1) and light chain (sequence shown in SEQ ID NO: 2) of the PD-1-IgG1-LALA antibody were constructed into the pTT5 plasmid vector, respectively.
  • the C220A mutation encoded by EU
  • the C214A mutation encoded by Kabat
  • the simultaneous introduction of these two mutations completely removed the interchain disulfide bonds naturally present at these positions (CH1 position 220- and CL position 214).
  • S131C SEQ ID NO: 3
  • L128C SEQ ID NO: 4
  • A129C SEQ ID NO: 4
  • P119C SEQ ID NO: 8
  • S121C SEQ ID NO: 9
  • T164C SEQ ID NO: 10
  • the antibody was expressed and purified according to the methods of Examples 1.1 and 1.2, and the protein expression level of the PD-1 antibody after the introduction of non-natural disulfide bonds after one-step purification
  • the purity is comparable to that of the PD-1 antibody containing natural disulfide bonds, and there is no significant change.
  • the Fc of the PD-1 arm was mutated T366W containing the amino acid forming a knob
  • the Fc of the CTLA-4 arm was mutated T366S/L368A/Y407V containing the amino acid forming a hole
  • Fig. 2 gives a schematic diagram of the structure
  • the bispecific antibody was expressed and purified according to the methods of Examples 1.1 and 1.2.
  • TJ030-PR1104 which introduced unnatural disulfide bonds F170C-T164C
  • TJ030-PR1105 which introduced unnatural disulfide bonds S131C-P119C
  • the CTLA-4 arm peak intensity/PD-1 arm peak intensity maintained at about 1:2.
  • TJ030-PR1103 L128C-S121C
  • TJ030-PR1104 F170C-T164C
  • TJ030-PR1101 using natural disulfide bond
  • TJ030-PR1102 F126C-S121C
  • TJ030-PR1105 (S131C-P119C)
  • TJ030-PR1101 using a natural disulfide bond
  • TJ030-PR1102 F126C reported in the prior art
  • the method of 1.3.2 was used to detect the complete molecular weight of deglycosylation.
  • the results are shown in Figure 4B.
  • the deglycosylated complete molecular weight mass spectrometry found the target protein of the correctly paired 1+1 asymmetric double antibody, and at the same time found H2L1 (two heavy chains) Formation of a light chain form, CTLA-4 arm light chain deficient by-product and PD-1 light chain cysteine conjugate (LC PD-1- Cys). It may be due to the insufficient expression of the CTLA-4 arm, especially the light chain of the CTLA-4 arm, resulting in the high production of H2L1.
  • H2L1-incomplete antibody molecules require additional LC PD-1 to further stabilize the structure; even so, no light chain mismatch products were detected in purified TJ030-PR1104.
  • the method of 1.3.3 was used to detect the reduced molecular weight and the deglycosylated reduced molecular weight.
  • the results are shown in Figure 4C.
  • the reduced molecular weight and the deglycosylated reduced molecular weight were also detected by mass spectrometry.
  • Paired CTLA-4 heavy and/or light chains (HC CTLA-4- LC CTLA-4 ) further confirmed the correct formation of the F170C-T164C unnatural disulfide bond.
  • Example 1.4 the purified PD-1 ⁇ CTLA-4 bispecific antibodies TJ030-PR1102 (F126C-S121C placed in the CTLA-4 arm) and TJ030-PR1104 (F170C-T164C placed in the CTLA-4 arm) were detected by flow cytometry 4 arm), TJ030-PR1106 (F126C-S121C placed in the PD-1 arm) and TJ030-PR1108 (F170C-T164C placed in the PD-1 arm), respectively, on the co-expression of cells highly expressing human PD-1 and human CTLA-4. Binding capacity, with CTLA-4 mAb and IgG as negative controls.
  • the diabody molecules TJ030-PR1104 and TJ030-PR1108, which introduced F170C-T164C, a pair of unnatural disulfide bonds, were compared with the reported unnatural disulfide bonds TJ030-PR1102 of F126C-S121C at each concentration point of 0.03nM-100nM Significantly more double-positive cells were detected for both.
  • Example 1.1 The method of Example 1.1 was used for double antibody expression.
  • we increased the plasmid ratio of the light chain (the molar ratio of the heavy chain and light chain plasmids changed from 1:1 to 2:3), hoping to reduce the " Corresponding ratios of antibodies in two heavy chain one light chain (H2L1)" configuration.
  • H2L1 two heavy chain one light chain
  • Table 7 gives the PD-1 ⁇ CTLA-4 double antibody sequence and plasmid matching information.
  • the PD-1 ⁇ CTLA-4 double antibody was purified according to the method of Example 1, and mass spectrometry was performed. As shown in Table 8, unnatural disulfide bonds P171C-S165C (TJ030-PR1304 and TJ030-PR1309), F170C-T164C (TJ030-PR1317 and TJ030- After PR1313), whether the non-native disulfide bond was placed on the CTLA-4 arm or the PD-1 arm, the correct pairing ratio was significantly improved compared to the dual antibodies TJ030-PR1301 and TJ030-PR1306 using the natural disulfide bond.
  • Lys-C digested the double antibody purified by cation exchange indicating that the Fab is completely correctly paired, and there is no light chain mismatch.
  • Deglycosylated intact molecular weights were as expected, with no apparent light chain-deficient H2L1 by-products appearing ( Figure 7).
  • both TJ030-PR1231 and TJ030-PR1317 have the amino acid mutation F170C-T164C introduced in the PD-1 arm, and the light chain in the PD-1 arm is of ⁇ isoform.
  • the difference between the two is only in TJ030 -
  • the light chain in the CTLA-4 arm of PR1231 is of the kappa subtype
  • the light chain in the CTLA-4 arm of TJ030-PR1317 is of the lambda subtype
  • TJ030-PR1317 shows a higher proportion of correct pairing; that is, the two arms use different light chains Chain isotypes are useful for increasing the correct pairing ratio of bispecific antibodies.
  • the double antibody molecule TJ030-PR1230 (sequence and plasmid matching information are shown in Table 7) were obtained. ), the first-step purified product was free of light chain mismatches (Table 10), demonstrating that the electrostatic interaction of HC139-LC114 could further reduce light chain mismatches.
  • TJ030-PR1230 can also reduce light chain mismatch by increasing the transfection ratio of light chain (especially the light chain CTLA-4 arm with weak expression).
  • the PSMA 1+1 asymmetric bi-epitope antibody was constructed according to the antibody sequence and plasmid ratio shown in Table 11, and the method of KiH was also used to realize the 1+ heterodimerization of the heavy chain (T366W; T366S/L368A/Y407V).
  • KiH 1 Asymmetric bispecific antibody, and mass spectrometry analysis of the product after initial purification of ProteinA. No light chain mismatch products were found in the products treated with GingisKHAN protease (Fig. 8).
  • the FAP ⁇ CD40 double antibody was constructed and expressed according to the antibody sequence and plasmid ratio shown in Table 12 (for the molecular form, see FIG. 9A ).
  • SEQ ID NO: 70 CD40-Fc-FAP/HC, P171C underlined
  • the bispecific antibodies ERP2006 -BS0012 and ERP2006-BS0015 had FACS binding EC50s of 0.471 nM and 0.456 nM to CD40, respectively.
  • the FACS binding EC50 of ERP2006-BS0012 and ERP2006-BS0015 to FAP was 0.349 nM and 0.336 nM, respectively.
  • the double-antibody ERP2006-BS0012 and ERP2006-BS0015 have similar affinity to FAP as the parental FAP mAb Ab10, and the affinity to CD40 is comparable to the parental CD40 mAb 9E5-25.
  • Figures 10C and 10D show that ERP2006-BS0012 and ERP2006-BS0015 have CD40 activating activity in the absence of FAP, but their activity is weaker than that of the parental antibody 9E5-25, a feature that renders the bispecific antibody absent in the presence of FAP
  • the activation activity of CD40 in the peripheral tissues of the mice decreased, which can reduce the peripheral toxicity of CD40 mAbs.
  • the activation activity of ERP2006-BS0012 and ERP2006-BS0015 on CD40 was significantly enhanced, indicating that they have FAP-dependent CD40 activation activity, and their activation of CD40 is stronger than that of the parental CD40 mAb 9E5-25.
  • Bispecific antibodies have stronger CD40 activating activity in tumor sites with high FAP expression.

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Abstract

提供多特异性抗原结合蛋白。提供在CH1和CL中包含一个或多个氨基酸取代的多特异性抗原结合蛋白，以及包含其的组合物、其制备方法和医药用途。所述特异性抗原结合蛋白有效地减少了轻链的错配。

Description

多特异性抗原结合蛋白 技术领域
本公开属于生物医药领域，具体涉及多特异性抗原结合蛋白及其制备方法和医药用途。
背景技术
由于可以识别不同的抗原分子或者识别同一抗原分子的不同表位，双特异性抗体具有单克隆抗体不具备的独特生物学功能，并逐渐受到市场的认可。尽管关于双特异性抗体的相关技术已经发展了二十年，但仍然有很多现实的技术问题制约着双特异性抗体的生产开发。随着技术的进步，用来改造和生产双特异性抗体新型的分子形式和解决策略层出不穷。以1+1非对称(Fab A+Fab B)双抗为例，为了避免轻链错配(针对抗原A的轻链配对到针对抗原B的重链上，或者针对抗原B的轻链配对到针对抗原A的重链上)，迄今为止多种方法已被报道。
共同轻链抗体：有人报道使用体外展示技术或者从具有共同轻链的小鼠筛选特异性轻链(WO2012067176；WO2013134263)，以配对针对抗原A的重链和针对抗原B的重链，并维持相对应抗体的原有生物功能。二合一(Two-in-one)抗体：有人报道通过噬菌体展示和理性设计(WO2010027981)，将与抗原A结合的抗体进行优化，使其保留对抗原A的原有结合能力并具有与抗原B结合的能力，实现一个抗体结合两种靶点。以上两种方法均需要大量的工程化改造，技术难度大，普适性仍有待证明。因此，具有正交特性的Fab(VH/VL或/和CH1/CL相互作用界面)改造近年来越来越受到业内的关注。
IgG/TCR(WO2014014796；WO2019057122)：鉴于TCR恒定区和抗体CH1/CL中的结构相似性，有人报道通过替换FabA的CH1/CL为TCR的恒定区，以避免潜在的轻链错配问题。Crossmab(WO2012023053)：通过互换针对某Fab的VH/VL、CH1/CL、或HC/LC，降低轻链错配的可能性。DuetMab(WO2013096291)：在针对抗原A的Fab的CH1/CL引入非天然二硫键替换原有二硫键，降低轻链错配的可能性。计算机辅助设计：通过计算机辅助设计(WO2014150973；WO2016172485)，避免轻链错配。
作为新型药物形式，双特异性抗体有着特殊结构，制备与产业化也较单克隆抗体更为困难。虽已有多种方式试图解决重链和轻链之间的错配问题，但由此进行的结构调整可能会改变分子的稳定性、免疫原性或药动学特征，仍然需要开发新的技术以提高多特异性抗体(例如双特异性抗体)的产率。
发明内容
本公开通过在CH1/CL界面中去除天然二硫键并引入非天然二硫键，或者通 过在CH1/CL界面中引入静电互补的氨基酸对，或者通过在CH1/CL界面中去除天然二硫键并引入非天然二硫键，并同时引入静电互补的氨基酸对，相对于野生型提高了多特异性抗体轻重链的正确配对比例。
本公开提供了一种二聚化多肽，其包含重链恒定区1(CH1)和轻链恒定区(CL)，其中：CH1和CL在选自(i-1)至(i-6)的位置中的一组或多组包含天然非半胱氨酸至半胱氨酸的氨基酸取代：
(i-1)CH1的第170位和CL的第164位，
(i-2)CH1的第128位和CL的第121位，
(i-3)CH1的第129位和CL的第121位，
(i-4)CH1的第131位和CL的第119位，
(i-5)CH1的第141位和CL的第135位，和
(i-6)CH1的第171位和CL的第165位。
在本公开的上下文中，重链位置编号根据EU编号***确定，例如CH1的氨基酸取代的位置是以人IgG1的CH1(SEQ ID NO：88)为基准计数并；轻链位置编号根据Kabat编号***确定，例如CL的氨基酸取代的位置是以人κ轻链(IGLC，SEQ ID NO：89)为基准计数。
Figure PCTCN2021135254-appb-000001
本领域技术人员应理解，IgG1以外的其他IgG亚型如IgG2、IgG3和IgG4在与IgG1 CH1中包含本公开所述氨基酸突变的位置相对应的位置上包含相同类型的氨基酸突变也在本公开的保护范围内。
在一些实施方案中，CH1和CL之间包含或不包含天然二硫键。
在一些实施方案中，CH1保留第220位天然半胱氨酸，CL保留第214位天然半胱氨酸。
在一些实施方案中，CH1第220位的天然半胱氨酸和/或CL的第214位天然半胱氨酸被半胱氨酸以外的氨基酸取代。
在一些实施方案中，CH1包含氨基酸取代C220A，CL包含氨基酸取代C214A。
在一些实施方案中，CH1和CL包含以下氨基酸取代：
(a)CH1中的C220A和CL中的C214A；和
(b)选自以下组中的至少一组的氨基酸取代：
(b-1)CH1中的F170C和CL中的T164C；
(b-2)CH1中的L128C和CL中的S121C；
(b-3)CH1中的A129C和CL中的S121C；
(b-4)CH1中的S131C和CL中的P119C；
(b-5)CH1中的A141C和CL中的L135C；和
(b-6)CH1中的P171C和CL中的S165C。
在一些实施方案中，CH1和CL包含以下氨基酸取代:(a)CH1中的C220A和CL中的C214A；和(b)CH1中的F170C和CL中的T164C。
在一些实施方案中，CH1和CL包含以下氨基酸取代：(a)CH1中的C220A和CL中的C214A；和(b)CH1中的P171C和CL中的S165C。
在一些实施方案，CH1和CL包含使得CH1和CL之间形成静电相互作用界面的氨基酸取代。
在一些实施方案中，使得CH1和CL之间形成静电相互作用界面的氨基酸取代位于CH1的第139位和CL的第114位。
在一些实施方案中，CH1第139位的氨基酸被取代为带正电荷的氨基酸，CL第114位的氨基酸被取代为带负电荷的氨基酸；或者CH1第139位的氨基酸被取代为带负电荷的氨基酸，CL第114位的氨基酸被取代为带正电荷的氨基酸。
在一些实施方案中，带正电荷的氨基酸选自K、R和H；带负电荷的氨基酸选自D和E。
在一些实施方案中，CH1和CL包含选自以下任一组中的氨基酸取代：
(1)CH1中的T139R和CL中的S114E；
(2)CH1中的T139R和CL中的S114D；
(3)CH1中的T139K和CL中的S114E；
(4)CH1中的T139K和CL中的S114D；
(5)CH1中的T139D和CL中的S114K；
(6)CH1中的T139D和CL中的S114R；
(7)CH1中的T139E和CL中的S114K；和
(8)CH1中的T139E和CL中的S114R。
在一些实施方案中，CH1和CL包含以下氨基酸取代：
(a)CH1中的C220A和CL中的C214A；和
(b)选自以下组中的至少一组的氨基酸取代：
(b-1)CH1中的F170C和CL中的T164C；
(b-2)CH1中的L128C和CL中的S121C；
(b-3)CH1中的A129C和CL中的S121C；
(b-4)CH1中的S131C和CL中的P119C；
(b-5)CH1中的A141C和CL中的L135C；和
(b-6)CH1中的P171C和CL中的S165C；和
(c)选自以下中的任一组：
(c-1)CH1中的T139R和CL中的S114E；
(c-2)CH1中的T139R和CL中的S114D；
(c-3)CH1中的T139K和CL中的S114E；和
(c-4)CH1中的T139K和CL中的S114D。
在一些实施方案中，CH1和CL包含以下氨基酸取代：
(a)CH1中的C220A和CL中的C214A；
(b)选自以下组中的至少一组：
(b-1)CH1中的F170C和CL中的T164C；
(b-2)CH1中的L128C和CL中的S121C；
(b-3)CH1中的A129C和CL中的S121C；
(b-4)CH1中的S131C和CL中的P119C；
(b-5)CH1中的A141C和CL中的L135C；和
(b-6)CH1中的P171C和CL中的S165C；和
(c)选自以下中的任一组：
(c-1)CH1中的T139D和CL中的S114K；
(c-2)CH1中的T139D和CL中的S114R；
(c-3)CH1中的T139E和CL中的S114K；
(c-4)CH1中的T139E和CL中的S114R。
在一些实施方案中，CH1和CL包含以下氨基酸取代：(a)CH1中的C220A和CL中的C214A；(b)CH1中的F170C和CL中的T164C；和(c)CH1中的T139R和CL中的S114E。
在一些实施方案中，CH1和CL包含以下氨基酸取代：(a)CH1中的C220A和CL中的C214A；(b)CH1中的F170C和CL中的T164C；和(c)CH1中的T139D和CL中的S114K。
在一些实施方案中，CH1和CL包含以下氨基酸取代：(a)C220A和C214A；(b)CH1中的P171C和CL中的S165C；和(c)CH1中的T139R和CL中的S114E。
在一些实施方案中，CH1和CL包含以下氨基酸取代：(a)CH1中的C220A和CL中的C214A；(b)CH1中的P171C和CL中的S165C；和(c)CH1中的T139D和CL中的S114K。
在一些实施方案中，CL来自抗体λ轻链(Cλ)或κ轻链(Cκ)。
本公开提供了一种抗原结合蛋白，其包含上述二聚化多肽。
在一些实施方案中，抗原结合蛋白包含第一抗原结合域，所述第一抗原结合域包含Fab，所述Fab包含第一重链可变区VH1、第一轻链可变区VL1和所述二聚化多肽，所述二聚化多肽中所述CH1为第一CH1，所述CL为第一CL；VH1与第一CH1直接连接或通过接头连接，VL1与第一CL直接连接或通过接头连接。在一些实施方案中，VH1的C端与第一CH1的N端直接连接或通过接头连接， VL1的C端与第一CL的N端直接连接或通过接头连接。
在一些实施方案中，接头是肽接头。在一些实施方案中，肽接头是具有长度为至少5个氨基酸的氨基酸序列的肽，在一个实施方案中，长度为5至100，在进一步的实施方案中为10至50个氨基酸。在一个实施方案中，所述肽接头是(GxS)n或(GxS)nGm，其中G＝甘氨酸，S＝丝氨酸和(x＝3，n＝3，4，5或6，和m＝0，1，2或3)或(x＝4，n＝2，3，4或5和m＝0，1，2或3)。在一个实施方案中x＝4和n＝3或4。在一个实施方案中，所述肽接头是(G4S)4。
在一些实施方案中，抗原结合蛋白包含第一抗原结合域和第二抗原结合域，其中所述第二抗原结合域包含第二重链可变区VH2和第二轻链可变区VL2，并且所述第一抗原结合域和第二抗原结合域结合不同的抗原或者结合同一种抗原上的不同的表位；在一些实施方案中，所述第二抗原结合域包含Fab。所述Fab包含第二重链可变区VH2、第二重链恒定区1(第二CH1)、第二轻链可变区VL2和第二轻链恒定区(第二CL2)。在一些实施方案中，VH2的C端与第二CH1的N端直接连接或通过接头连接，VL2的C端与第二CL的N端直接连接或通过接头连接。
在一些实施方案中，第二CH1和第二CL中不包含选自以下的一组或多组天然非半胱氨酸至半胱氨酸的氨基酸取代：
(i-1)第二CH1的第170位和第二CL的第164位，
(i-2)第二CH1的第128位和第二CL的第121位，
(i-3)第二CH1的第129位和第二CL的第121位，
(i-4)第二CH1的第131位和第二CL的第119位，
(i-5)第二CH1的第141位和第二CL的第135位，和
(i-6)第二CH1的第171位和第二CL的第165位。
在本公开的上下文中，重链位置编号根据EU编号***确定，例如CH1的氨基酸取代的位置是以人IgG1的CH1(SEQ ID NO：88)为基准计数并；轻链位置编号根据Kabat编号***确定，例如CL的氨基酸取代的位置是以人κ轻链(IGLC，SEQ ID NO：89)为基准计数。
Figure PCTCN2021135254-appb-000002
本领域技术人员应理解，IgG1以外的其他IgG亚型如IgG2、IgG3和IgG4在与IgG1 CH1中包含本公开所述氨基酸突变的位置相对应的位置上包含相同类型的氨基酸突变也在本公开的保护范围内。
在一些实施方案中，第二CH1和第二CL中不含天然非半胱氨酸至半胱氨酸 的氨基酸取代。
在一些实施方案中，第二CH1和第二CL中保留天然半胱氨酸220C和214C。
在一些实施方案中，第二CH1和第二CL中不含天然非半胱氨酸至半胱氨酸的氨基酸取代，且保留天然半胱氨酸220C和214C。
在一些实施方案中，第一CH1和第一CL包含以下氨基酸取代：
(a)CH1中的C220A和CL中的C214A；和
(b)选自以下组中的至少一组：
(b-1)CH1中的F170C和CL中的T164C；
(b-2)CH1中的L128C和CL中的S121C；
(b-3)CH1中的A129C和CL中的S121C；
(b-4)CH1中的S131C和CL中的P119C；
(b-5)CH1中的A141C和CL中的L135C；和
(b-6)CH1中的P171C和CL中的S165C；
并且第二CH1和第二CL中不含天然非半胱氨酸至半胱氨酸的氨基酸取代，且保留天然半胱氨酸220C和214C。
在一些实施方案中，第一CH1和第一CL包含以下氨基酸取代：
(a)CH1中的C220A和CL中的C214A；和
(b)CH1中的F170C和CL中的T164C；
并且第二CH1和第二CL中不含天然非半胱氨酸至半胱氨酸的氨基酸取代，且保留天然半胱氨酸220C和214C。
在一些实施方案中，第一CH1和第一CL包含以下氨基酸取代：(a)CH1中的C220A和CL中的C214A；和(b)CH1中的P171C和CL中的S165C；并且第二CH1和第二CL中不含天然非半胱氨酸至半胱氨酸的氨基酸取代，且保留天然半胱氨酸220C和214C。
在一些实施方案中，第一CH1和第一CL包含使得第一CH1和第一CL之间形成静电相互作用界面的氨基酸取代；和/或
第二CH1和第二CL包含使得第二CH1和第二CL之间形成静电相互作用界面的氨基酸取代。
在一些实施方案中，第一CH1和第二CH1中用于形成静电相互作用界面的氨基酸的带电性相反，且第一CL和第二CL中用于形成静电相互作用界面的氨基酸的带电性相反。
在一些实施方案中，使得第一CH1和第一CL之间形成静电相互作用界面的氨基酸取代位于第一CH1的第139位和第一CL的第114位；和/或
使得第二CH1和第二CL之间形成静电相互作用界面的氨基酸取代位于第二CH1的第139位和第二CL的第114位。
在一些实施方案中，第一CH1的第139位和第二CH1的第139位分别被带相 反电荷的氨基酸取代，且第一CL的第114位和第二CL的第114位分别被带相反电荷的氨基酸取代。
在一些实施方案中，第一CH1第139位的氨基酸被取代为带正电荷的氨基酸，第一CL第114位的氨基酸被取代为带负电荷的氨基酸；或者第一CH1第139位的氨基酸被取代为带负电荷的氨基酸，第一CL第114位的氨基酸被取代为带正电荷的氨基酸；和/或
第二CH1第139位的氨基酸被取代为带负电荷的氨基酸，第二CL第114位的氨基酸被取代为带正电荷的氨基酸；或者第二CH1第139位的氨基酸被取代为带正电荷的氨基酸，第二CL第114位的氨基酸被取代为带负电荷的氨基酸。
在一些实施方案中，带正电荷的氨基酸选自K、R和H；带负电荷的氨基酸选自D和E。
在一些实施方案中，第一CH1和第一CL包含选自以下任一组的氨基酸取代：
(1)CH1中的T139R和CL中的S114E；CH1中的T139R和CL中的S114D；CH1中的T139K和CL中的S114E；CH1中的T139K和CL中的S114D；CH1中的T139D和CL中的S114K；CH1中的T139D和CL中的S114R；CH1中的T139E和CL中的S114K；和CH1中的T139E和CL中的S114R；和/或
第二CH1和第二CL包含选自以下的氨基酸取代：CH1中的T139R和CL中的S114E；CH1中的T139R和CL中的S114D；CH1中的T139K和CL中的S114E；CH1中的T139K和CL中的S114D；CH1中的T139D和CL中的S114K；CH1中的T139D和CL中的S114R；CH1中的T139E和CL中的S114K；和CH1中的T139E和CL中的S114R。
在一些实施方案中，第一CH1和第一CL包含选自以下的氨基酸取代：CH1中的T139R和CL中的S114E；CH1中的T139R和CL中的S114D；CH1中的T139K和CL中的S114E；CH1中的T139K和CL中的S114D；和/或
第二CH1和第二CL包含选自以下的氨基酸取代：CH1中的T139D和CL中的S114K；CH1中的T139D和CL中的S114R；CH1中的T139E和CL中的S114K；和CH1中的T139E和CL中的S114R。
在一些实施方案中，第一CH1和第一CL包含选自以下的氨基酸取代：CH1中的T139D和CL中的S114K；CH1中的T139D和CL中的S114R；CH1中的T139E和CL中的S114K；和CH1中的T139E和CL中的S114R；和/或
第二CH1和第二CL包含选自以下的氨基酸取代：CH1中的T139R和CL中的S114E；CH1中的T139R和CL中的S114D；CH1中的T139K和CL中的S114E；CH1中的T139K和CL中的S114D。
在一些实施方案中，第一CH1和第一CL包含以下氨基酸取代：
(a)CH1中的C220A和CL中的C214A；
(b)选自以下中的至少一组：CH1中的F170C和CL中的T164C；CH1中的 L128C和CL中的S121C；CH1中的A129C和CL中的S121C；CH1中的S131C和CL中的P119C；CH1中的A141C和CL中的L135C；和CH1中的P171C和CL中的S165C；和
(c)选自以下中的一组：CH1中的T139R和CL中的S114E；CH1中的T139R和CL中的S114D；CH1中的T139K和CL中的S114E；CH1中的T139K和CL中的S114D；
并且第二CH1和第二CL包含选自以下的氨基酸取代：CH1中的T139D和CL中的S114K；CH1中的T139D和CL中的S114R；CH1中的T139E和CL中的S114K；和CH1中的T139E和CL中的S114R。
在一些实施方案中，第一CH1和第一CL包含以下氨基酸取代：
(a)CH1中的C220A和CL中的C214A；
(b)选自以下中的至少一组：CH1中的F170C和CL中的T164C；CH1中的L128C和CL中的S121C；CH1中的A129C和CL中的S121C；CH1中的S131C和CL中的P119C；CH1中的A141C和CL中的L135C；和CH1中的P171C和CL中的S165C；和
(c)选自以下中的一组：CH1中的T139D和CL中的S114K；CH1中的T139D和CL中的S114R；CH1中的T139E和CL中的S114K；CH1中的T139E和CL中的S114R；
并且第二CH1和第二CL包含选自以下的氨基酸取代：CH1中的T139R和CL中的S114E；CH1中的T139R和CL中的S114D；CH1中的T139K和CL中的S114E；和CH1中的T139K和CL中的S114D。
在一些实施方案中，第一CH1和第一CL包含以下氨基酸取代：
(a)CH1中的C220A和CL中的C214A；
(b)CH1中的F170C和CL中的T164C；和
(c)CH1中的T139R和CL中的S114E；
并且第二CH1和第二CL包含以下氨基酸取代：CH1中的T139D和CL中的S114K。
在一些实施方案中，第一CH1和第一CL包含以下氨基酸取代：
(a)CH1中的C220A和CL中的C214A；
(b)CH1中的F170C和CL中的T164C；和
(c)CH1中的T139D和CL中的S114K；
并且第二CH1和第二CL包含以下氨基酸取代：CH1中的T139R和CL中的S114E。
在一些实施方案中，第一CH1和第一CL包含以下氨基酸取代：
(a)CH1中的C220A和CL中的C214A；
(b)CH1中的P171C和CL中的S165C；和
(c)CH1中的T139R和CL中的S114E；
并且第二CH1和第二CL包含以下氨基酸取代：CH1中的T139D和CL中的S114K。
在一些实施方案中，第一CH1和第一CL包含以下氨基酸取代：
(a)CH1中的C220A和CL中的C214A；
(b)CH1中的P171C和CL中的S165C；和
(c)CH1中的T139D和CL中的S114K；
并且第二CH1和第二CL包含以下氨基酸取代：CH1中的T139R和CL中的S114E。
在一些实施方案中，当第一CH1和第一CL中包含天然非半胱氨酸至半胱氨酸的氨基酸取代时，第二CH1和第二CL中不含天然非半胱氨酸至半胱氨酸的氨基酸取代，且保留天然半胱氨酸CH1中的220C和CL中的214C。
在一些实施方案中，第一CL来自抗体κ轻链(Cκ)；第二CL来自抗体λ轻链(Cλ)或κ轻链(Cκ)。在一些实施方案中，第一CL来自κ轻链且第二CL来自λ轻链。
在一些实施方案中，抗原结合蛋白还包含Fc区，所述Fc区包含能够彼此缔合的第一亚基Fc1与第二亚基Fc2。在一些实施方案中，Fc区选自人IgG1、IgG2、IgG3和IgG4的Fc，例如人IgG1的Fc。
在一些实施方案中，Fc1和Fc2中包含这样的氨基酸取代，使得与Fc1相比，Fc1优先与Fc2配对(或使得优先形成异源二聚体)，例如Fc1和Fc2在CH3域中包含这样的氨基酸取代。在一些实施方案中，Fc1和Fc2中的氨基酸取代产生比不含该取代的野生型更大的静电互补性。测量蛋白质/蛋白质界面处的静电互补性的方法为本领域已知，并描述于例如McCoy等(1997)J Mol Biol 268,570–584；Lee等,(2001)Protein Sci.10,362-377；和Chau等(1994)J Comp Mol Des8,51325中。在一些实施方案中，Fc1和Fc2中的氨基酸取代产生比不含该取代的野生型更大的空间互补性。测量蛋白质/蛋白质界面处的静电互补性的方法为本领域已知，并描述于例如Lawrence等(1993)J Mol Biol 234,946–950；Walls等(1992)J Mol Biol228,277-297；和Schueler-Furman等(2005)Proteins 60,187-194中。术语“互补性”指例如本文所述抗原结合蛋白的CH1和CL(或CH3和CH3)的界面处影响重链/轻链配对的相互作用的组合。“空间互补性”或“构象互补性”指例如CH1和CL(或CH3和CH3)的相互作用表面处的三维结构的相容性。“静电互补性”指在例如CH1和CL(或CH3和CH3)的相互作用表面处放置带负电荷和/或正电荷原子的相容性。
在一些实施方案中，在Fc1和Fc2中，例如在CH3/CH3界面内，用一个或多个具有更大侧链体积的氨基酸残基取代Fc1的CH3域中的一个或多个氨基酸残基，从而在Fc1的CH3域表面产生凸起(或杵，Knob)，Fc2的CH3域中与Fc1的CH3 域相互作用的一个或多个、优选两个或三个氨基酸残基，用具有小侧链体积的氨基酸残基取代，从而在与Fc1的CH3域相互作用的Fc2的CH3域表面产生凹陷(或臼，Hole)。在一些实施方案中，改变Fc1和Fc2(例如本文所述实施方案中任意个的Fc1和Fc2)的CH3域，使得在界面内，用等同数目的具有更大侧链体积的氨基酸残基取代Fc2的CH3域中的一个或两个氨基酸残基，从而在Fc2的CH3域的界面内产生可放置在Fc1的CH3域表面内的凹陷(或臼)的凸起(或杵)，改变Fc1的CH3域，使得在与Fc2的CH3域界面接触的Fc2的CH3域表面内，用等同数目的具有更小侧链体积的氨基酸残基取代两个或三个氨基酸残基，从而在与Fc1的CH3域界面内产生可以放置Fc2的CH3域界面内的凸起的凹陷。在一些实施方案中，具有更大侧链体积的输入残基是苯丙氨酸(F)、酪氨酸(Y)、精氨酸(R)或色氨酸(W)。在一些实施方案中，该凸起或杵突变包含用色氨酸取代366位苏氨酸，氨基酸编号按照Kabat等(Sequences of proteins ofimmunological interest,第5版,第1卷(1991；NIH,Bethesda,MD)中的688-696页)的EU编号方案。在一些实施方案中，具有更小侧链体积的输入残基是丝氨酸(S)、丙氨酸(A)、缬氨酸(V)或苏氨酸(T)。在一个实施方案中，含有凹陷的CH3域包含取代选自苏氨酸、亮氨酸和酪氨酸的两个或多个原氨基酸。在一些实施方案中，含有凹陷的CH3域包含选自丙氨酸、丝氨酸、苏氨酸和缬氨酸的两个或多个输入残基。在一些实施方案中，杵突变修饰是T366W，臼突变修饰是T366S、L368A和Y407V中的至少一个或至少两个。在一些实施方案中，杵突变修饰是T366W，臼突变修饰是T366S、L368A和Y407V。
在本公开的上下文中，Fc的氨基酸取代的位置是根据EU编号***确定的，例如以人IgG1的Fc为基准计数。
在一些实施方案中，Fc1和Fc2中，例如CH3中可以包含天然非半胱氨酸至半胱氨酸的取代，例如在Fc1中包含S354C，Fc2中包含Y349C；或Fc1中包含Y349C，Fc2中包含S354C。
在一些实施方案中，Fc1和/或所述Fc2包含改变所述抗原结合蛋白的半衰期的修饰，其中所述半衰期取决于FcRn结合亲和力。
在一些实施方案中，Fc1和/或所述Fc2包含改变效应子功能的修饰，其中对Fcγ受体或C1q补体蛋白的结合亲和力增大或减小。
在一些实施方案中，Fc1和Fc2中，例如在Fc1 CH3/Fc2CH3界面内，包含一组或更多组选自以下的氨基酸取代：
(1)T366Y/Y407T；
(2)T366W/Y407A；
(3)T366Y/Y407T；
(4)T394W/F405A；
(5)T366Y/F405AT394W/Y407T；
(6)T366W/F405WT394S/Y407A；
(7)F405W/T394S；
(8)D399C/K392C；
(9)T366W/T366S/L368A/Y407V；
(10)T366W/D399C/T366S/L368A/K392C/Y407V；
(11)T366W/K392C/T366S/D399C/L368A/Y407V；
(12)S354C/T366W/Y349C/T366S/L368A/Y407V；
(13)Y349C/T366W/S354C/T366S/L368A/Y407V；
(14)E356C/T366W/Y349C/T366S/L368A/Y407V；
(15)Y349C/T366W/E356C/T366S/L368A/Y407V；
(16)E357C/T366W/Y349C/T366S/L368A/Y407V；和
(17)Y349C/T366W/E357C/T366S/L368A/Y407V。
在一些实施方案中，所述Fc1中包含选自T366S、L368A和Y407V中的至少一个或至少两个氨基酸取代，所述Fc2中包含T366W；或者所述Fc1中包含T366W，所述Fc2中包含选自T366S、L368A和Y407V中的至少一个或至少两个氨基酸取代。
在一些实施方案中，所述Fc1中包含氨基酸取代T366S、L368A和Y407V，所述Fc2中包含T366W；或者所述Fc1中包含T366W，所述Fc2中包含氨基酸取代T366S、L368A和Y407V。
在一些实施方案中，Fc1和Fc2中还包含使得Fc1和Fc2(例如CH3和CH3)之间形成静电相互作用界面的氨基酸取代。形成静电相互作用界面的氨基酸取代可以为选自以下的一组或更多组：
(1)K370E/D399K/K439D/D356K/E357K/K409D；
(2)K409D/D399K；
(3)K409E/D399K；
(4)K409E/D399R；
(5)K409D/D399R；
(6)D339K/E356K；
(7)D399K/E356K/K409D/K392D；
(8)D399K/E356K/K409D/K439D；
(9)D399K/E357K/K409D/K370D；
(10)D399K/E356K/E357K/K409D/K392D/K370D；
(11)D399K/E357K/K409D/K392D；
(12)K392D/K409D/D399K；和
(13)K409D/K360D/D399K。
在一些实施方案中，Fc1和/或Fc2中包含来自不同抗体亚型的结构域，例如 来自不同抗体亚型CH3。例如，戴维斯等人(2010，蛋白质工程设计与选择23:195-202)描述了使用链交换工程化结构域(SEED)CH3区的一个异二聚体的Fc平台，该CH3区是人IgG和IgA CH3结构域的衍生物(还参见WO 2007/110205)。
在一些实施方案中，Fc1和/或Fc2，例如CH3中，包含用于改变效应子功能的氨基酸取代。“效应子功能”指可归因于抗体的Fc区(天然序列Fc区或氨基酸序列变体Fc区)且随抗体同种型而变的那些生物活性。抗体效应子功能的实例包括：C1q结合和依赖补体的细胞毒性、Fc受体结合、依赖抗体的细胞毒性(ADCC)、吞噬作用、细胞表面受体(例如B细胞受体)的下调和B细胞激活。改变效应子功能的氨基酸取代选自以下中的一组或更多组：
(1)S298A/E333A/K334A；
(2)S239D/I332E/A330L；
(3)S239D/I332E/G236A；
(4)G236A/S239D/A330L/I332E；
(5)F243L/R292P/Y300L/V305I/P396L；
(6)K326A/E333A；
(7)K326W/E333S；
(8)K326M/E333S；
(9)C221D/D222C；
(10)S267E/H268F/S324T；
(11)E345R；
(12)S298A/E333A/K334A/N434A；
(13)E294缺失/T307P/N434Y；
(14)T256N/A378V/S383N/N434Y；
(15)T252L/T253S/T254F；
(16)M252Y/S254T/T256E；
(17)M428L/N434S；
(18)L234A/L235A；
(19)S228P/L235E；
(20)L234A/L235A/P331S；
(21)L234A/L235A/P329G；
(22)D265A/E233P；
(23)H268Q/V309L/A330S/P331S；
(24)V234A/G237A/P238S/H268A/V309L/A300S/P331S；
(25)L234A/L235A/G237A/P238S/H268A/V309L/A300S/P331S；
(26)S228P/F234A/L235A；
(27)D270A/P329A；
(28)L234F/L235E；
(29)L234F/L235E/P331S；
(30)F241A/V264A/D265A；
(31)N297G/D265A；和
(32)L234Y/G236W/S298A。
在一些实施方案中，所述Fc1和/或所述Fc2包含氨基酸取代L234A和L235A，或者包含氨基酸取代L234F和L235E。
在一些实施方案中，Fc1和/或Fc2，例如CH3中包含一个或多个异同种异型突变。在一些实施方案中，异同种异型突变是D356E和L358M。
在一些实施方案中，Fc1和Fc2，例如CH3中，包含用于改变半衰期的氨基酸取代。半衰期的增大可以允许减少给予患者的药物的量并且降低给药频率。因此，在此的具有增大的半衰期的抗体可以通过修饰(例如，取代、缺失或添加)鉴别为Fc与FcRn受体之间的相互作用中所涉及的氨基酸残基来产生(U.S.7,083,784)。在一些方面，IgG1同型抗体的位置252处的一个甲硫氨酸、和/或位置254处的一个丝氨酸、和/或位置256处的一个苏氨酸可以分别被改变为酪氨酸、苏氨酸以及谷氨酸，这样使得所得抗体包括酪氨酸-252、苏氨酸-254以及谷氨酸-256(即，M252Y、S254T、T256E)。IgG1抗体的这种Fc区包括一个YTE修饰并且在IgG2、IgG3以及IgG4抗体中，对应位置可以进行类似地修饰。另外，在此的抗体的半衰期可以通过本领域中已知的技术通过轭合至PEG或白蛋白而增大。在一些方面，用于增加异二聚体形成的Fc修饰可以与以下相组合：用于改变抗体的半衰期的其他修饰，包括但不限于M252Y和/或S254T和/或T256E；和/或用于改变效应子功能和/或改变与一个或多个Fc配体的结合的其他已知的Fc修饰，包括在此描述的那些。
在一些实施方案中，本公开提供的抗原结合蛋白包含第一重链、第一轻链、第二重链和第二轻链，其中：
所述第一重链从N端至C端依次为：[VH1]-[第一CH1]-[Fc1]，
所述第一轻链从N端至C端依次为：[VL1]-[第一CL]，
所述第二重链从N端至C端依次为：[VH2]-[第二CH1]-[Fc2]，
所述第二轻链从N端至C端依次为：[VL2]-[第二CL]。
在一些实施方案中，本公开提供的抗原结合蛋白包含重链、第一轻链和第二轻链，其中：
所述重链从N端至C端依次为：[VH1]-[第一CH1]-[Fc1]-[接头]-[VH2]-[第二CH1]；
所述第一轻链从N端至C端依次为：[VL1]-[第一CL]，
所述第二轻链从N端至C端依次为：[VL2]-[第二CL]。
在一些实施方案中，本公开提供的抗原结合蛋白包含第一重链、第一轻链、第二重链和第二轻链，其中：
所述第一重链从N端至C端依次为：[VH1]-[第一CH1]-[Fc1]-[接头]-[VH2]-[第二CH1]；
所述第一轻链从N端至C端依次为：[VL1]-[第一CL]，
所述第二重链从N端至C端依次为：[VH1]-[第一CH1]-[Fc2]-[接头]-[VH2]-[第二CH1]；
所述第二轻链从N端至C端依次为：[VL2]-[第二CL]。
在一些实施方案中，所述第一抗原结合域和/或所述第二抗原结合域结合的抗原分别包括但不限于：PD-1；PD-L1；CTLA-4；LAG-3；OX40；GTIR；A2AR；B7-H3(CD276)；B7-H3；B7-H4；IDO；KIR；Tim-3；LAG-3；4-1BB(CD137)；BAFF；叶酸受体1；TEM1；CCR4；VISTA；ICOS；IFN-γ；TGF-B；EGFR；Erb(ErbB1；ErbB3；ErbB4)；HER2；TNF-α；TNF-β；TNF-γ；TNF-受体；BCMA；RANK；VEGF-A；VEGF-B；VEGFR；ROR1；BTLA；2B4；TIGIT；c-Met；GITR；FAP；PVRIG；BCMA；CAIX；CEA；EGP2；EGP-40；TROP-2；EpCAM；叶酸结合蛋白(FBP)；胎儿乙酰胆碱受体(AChR)；神经节苷脂G2(GD2)；神经节苷脂G3(GD3)；人端粒酶逆转录酶(hTERT)；Lewis A(CA 1.9.9)；Lewis Y(LeY)；GPC3；L1CAM；NG2D配体；癌胚抗原(h5T4)；***干细胞抗原(PSCA)；***特异性膜抗原(PSMA)；TAG-72；CLDN18.2；肾母细胞瘤蛋白(WT-1)；ROR1；黏蛋白家族成员(例如MUC1、MUC2、MUC3A、MUC3B、MUC4、MUC5AC、MUC5B、MUC6、MUC7、MUC8、MUC12、MUC13、MUC15、MUC16、MUC17、MUC19和MUC20)；白介素及其受体(例如IL-1；IL-1α；IL-1β；IL-2；IL-2R；IL-3；IL-4；IL-5；IL-4；IL-4R；IL-6；IL-6R；IL-7；IL-8；IL-9；IL-11；IL-12；IL-12β；IL-13；IL13Rα2；IL-15；IL-15R；IL-17；IL-18；IL-23；IL-23α)；白细胞分化抗原(例如CD3；CD4；CD5；CD6；CD7；CD8；CD10；CD14；CD15；CD19；CD20；CD21；CD22；CD23；CD24；CD25；CD26；CD27；CD28；CD30；CD33；CD34；CD36；CD37；CD38；CD40；CD41；CD44；CD45；CD46；CD47；CD51；CD52；CD53；CD54；CD56；CD66；CD70；CD74；CD79a/CD79b；CD80；CD92；CD103；CD122；CD123；CD126；CD133；CD138；CD147；CD148；CD150；CD152；CD171；CD261；CD262；CD317；CD362)；CA125；间皮素；干扰素A/B受体；HLA-DR；RTN4；VWF；MCP-1；EGFR；IGF-1R；TRAIL-R2；***1受体；DLL4；ILGF2；SLAMF7；TWEAKR；CD54；干扰素受体；整联蛋白Avβ3；HNGF；HGF；TYRP1；IGF-1；Cldn18.2；选择素P；SDC1；PDCD1；CFD；乙肝表面抗原；IGHE；KIR2D；TAG-72；CSF2；RON；血管生成素2；CDK4；CEACAM5/CEACAM6；CO17-1A；CO-43(血型Leb)；CO-514(血型Lea)；CTA-1；细胞角蛋白8；D1.1；D156-22；DR5；GAGE(GAGE-1；GAGE-2)；GICA 19-9；gp100；Gp37(人白细胞T细胞抗原)；gp75(黑色素瘤抗原)；gpA33；HMFG(人乳脂肪球抗原)；人***瘤病毒-E6/人***瘤病毒-E7；HMW-MAA(高分子量黑色素 瘤抗原)；I抗原；整联蛋白β6；KID3；KID31；KS1/4全抗原；L6和L20(人肺癌抗原)；LEA；LUCA-2；M18；M39；MAGE(MAGE-1；MAGE-3)；MART；Myl；N-乙酰葡糖氨基转移酶；拟糖蛋白；NS-10；OFA-1；OFA-2；致癌蛋白M；p15；p97；PEM(多态上皮黏蛋白)；PEMA(多态上皮黏蛋白抗原)；PIPA；PSA(***特异性抗原)；***酸性磷酸酶(PAP)；黑素瘤中发现的R24；阶段特异性胚胎抗原(例如SSEA-1；SSEA-3；SSEA-4)；T5A7；TAG-72；TL5(血型A)；TRA-1-85(血型H)；转铁蛋白受体；C型凝集素样分子-1(CLL-1或CLECL1)；δ样3(DLL3)；表皮生长因子受体变体III(EGFRvlll)；n抗原((Tn Ag)或(GaINAcu-Ser/Thr))；Fms样酪氨酸激酶3(FLT3)；蛋白酶丝氨酸21(Testisin或PRSS21)；PDGFR-β；神经细胞粘附分子(NCAM)；突变的延伸因子2(ELF2M)；肝配蛋白B2；蛋白酶体(Prosome，Macropain)亚基，β型，9(LMP2)；糖蛋白100(gp100)；由断点簇区(BCR)和Alelson鼠白血病病毒癌基因同源物1(AB1)组成的癌基因融合蛋白(bcr-abl)；酪氨酸酶；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；转谷氨酰胺酶5(TGS5)；STEAP1；Claudin 6；促甲状腺激素受体(TSHR)；CXORF61；ALK；聚唾液酸；PLAC1；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；LY6K；OR51E2；癌/睾丸抗原1(NY-ESO-1)；癌症/睾丸抗原2(LAGE-1A)；黑素瘤相关抗原1(MAGE-A1)；ETV6-AML；SPA17；XAGE1；Tie2；MAD-CT-1；MAD-CT-2；FOS相关抗原1；肿瘤蛋白质p53(p53)；p53突变体；PCTA-1；hTERT；细胞凋亡的黑素瘤抑制剂(ML-IAP)；PAX3；雄激素受体；细胞周期蛋白B1；MYCN；RhoC；TRP-2；CYP1B1；SART3；PAX5；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；RAGE-1；RU1；RU2；HPV E6；HPV E7；LAIR1；LILRA2；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。
在一些实施方案中，所述第一抗原结合域特异性结合CTLA-4，所述第二抗原结合域特异性结合PD-1，或者，所述所述第一抗原结合域特异性结合PD-1，所述第二抗原结合域特异性结合CTLA-4。
在一些实施方案中，第一抗原结合域包含重链可变区VH1和轻链可变区VL1，第二抗原结合域包含重链可变区VH2和轻链可变区VL2；其中所述VH1包含：序列如SEQ ID NO：51所示的HCDR1，序列如SEQ ID NO：52所示的HCDR2，和序列如SEQ ID NO：53所示的HCDR3，所述VL1包含序列如SEQ ID NO：54所示的LCDR1，序列如SEQ ID NO：55所示的LCDR2，和序列如SEQ ID NO：56所示的LCDR3；和/或所述VH2包含：序列如SEQ ID NO：43所示的HCDR1，序列如SEQ ID NO：44所示的HCDR2，和序列如SEQ ID NO：45所示的HCDR3，所述VL2包含序列如SEQ ID NO：46所示的LCDR1，序列如SEQ ID NO：47所示的LCDR2，和序列如SEQ ID NO：48所示的LCDR3。
在一些实施方案中，所述VH1为序列如SEQ ID NO：57所示的重链可变区，所述VL1为序列如SEQ ID NO：58所示的轻链可变区；和/或所述VH2为序列如SEQ ID NO：49所示的重链可变区，所述VL2为序列如SEQ ID NO：50所示的轻链可变区。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：18所示的第一重链，序列如SEQ ID NO：17所示的第一轻链，序列如SEQ ID NO：12所示的第二重链，和序列如SEQ ID NO：13所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：19所示的第一重链，序列如SEQ ID NO：20所示的第一轻链，序列如SEQ ID NO：12所示的第二重链，和序列如SEQ ID NO：13所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：21所示的第一重链，序列如SEQ ID NO：22所示的第一轻链，序列如SEQ ID NO：12所示的第二重链，和序列如SEQ ID NO：13所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：14所示的第一重链，序列如SEQ ID NO：15所示的第一轻链，序列如SEQ ID NO：23所示的第二重链，和序列如SEQ ID NO：9所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：14所示的第一重链，序列如SEQ ID NO：15所示的第一轻链，序列如SEQ ID NO：24所示的第二重链，和序列如SEQ ID NO：9所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：14所示的第一重链，序列如SEQ ID NO：15所示的第一轻链，序列如SEQ ID NO：25所示的第二重链，和序列如SEQ ID NO：10所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：14所示的第一重链，序列如SEQ ID NO：15所示的第一轻链，序列如SEQ ID NO：26所示的第二重链，和序列如SEQ ID NO：8所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：14所示的第一重链，序列如SEQ ID NO：27所示的第一轻链，序列如SEQ ID NO：12所示的第二重链，和序列如SEQ ID NO：13所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：28所示的第一重链，序列如SEQ ID NO：29所示的第一轻链，序列如SEQ ID NO：12所示的第二重链，和序列如SEQ ID NO：13所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：14所示的第一重链，序列如SEQ ID NO：27所示的第一轻链，序列如SEQ ID NO：25所示的第二重链，和序列如SEQ ID NO：10所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：14所示的第一重链，序列如SEQ ID NO：15所示的第一轻链，序列如SEQ ID NO：31 所示的第二重链，和序列如SEQ ID NO：32所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：19所示的第一重链，序列如SEQ ID NO：20所示的第一轻链，序列如SEQ ID NO：12所示的第二重链，和序列如SEQ ID NO：30所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：35所示的第一重链，序列如SEQ ID NO：36所示的第一轻链，序列如SEQ ID NO：33所示的第二重链，和序列如SEQ ID NO：34所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：14所示的第一重链，序列如SEQ ID NO：15所示的第一轻链，序列如SEQ ID NO：25所示的第二重链，和序列如SEQ ID NO：10所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：45所示的第一重链，序列如SEQ ID NO：46所示的第一轻链，序列如SEQ ID NO：37所示的第二重链，和序列如SEQ ID NO：38所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：41所示的第一重链，序列如SEQ ID NO：42所示的第一轻链，序列如SEQ ID NO：39所示的第二重链，和序列如SEQ ID NO：40所示的第二轻链。
在一些实施方案中，所述第一抗原结合域特异性结合CD40，和/或所述第二抗原结合域特异性结合FAP。
在一些实施方案中，第一抗原结合域包含重链可变区VH1和轻链可变区VL1，第二抗原结合域包含重链可变区VH2和轻链可变区VL2；其中所述VH1包含：序列如SEQ ID NO：73所示的HCDR1，序列如SEQ ID NO：74所示的HCDR2，和序列为RDY的HCDR3，所述VL1包含序列如SEQ ID NO：75所示的LCDR1，序列如SEQ ID NO：76所示的LCDR2，和序列如SEQ ID NO：77所示的LCDR3；和/或所述VH2包含：序列如SEQ ID NO：80所示的HCDR1，序列如SEQ ID NO：81所示的HCDR2，和序列如SEQ ID NO：82所示的HCDR3，所述VL2包含序列如SEQ ID NO：83所示的LCDR1，序列如SEQ ID NO：84所示的LCDR2，和序列如SEQ ID NO：85所示的LCDR3。
在一些实施方案中，所述VH1为序列如SEQ ID NO：78所示的重链可变区，所述VL1为序列如SEQ ID NO：79所示的轻链可变区；和/或所述VH2为序列如SEQ ID NO：86所示的重链可变区，所述VL2为序列如SEQ ID NO：87所示的轻链可变区。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：67所示的第一重链，序列如SEQ ID NO：68所示的第一轻链，和序列如SEQ ID NO：69所示的第二轻链。
在一个实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：70所示的第一重链，序列如SEQ ID NO：71所示的第一轻链，和序列如SEQ ID NO： 72所示的第二轻链。
在一些实施方案中，所述第一抗原结合域特异性结合和所述第二抗原结合域特异性分别结合PSMA的不同表位。
在一些实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：59所示的第一重链，序列如SEQ ID NO：60所示的第一轻链，序列如SEQ ID NO：61所示的第二重链，和序列如SEQ ID NO：62所示的第二轻链。
在一些实施方案中，本公开的抗原结合蛋白包含：序列如SEQ ID NO：63所示的第一重链，序列如SEQ ID NO：64所示的第一轻链，序列如SEQ ID NO：65所示的第二重链，和序列如SEQ ID NO：66所示的第二轻链。
本公开提供了一种PD-1/CTLA-4双特异性抗体，其包含：
(i)包含第一轻链和第一重链的PD-1抗原结合域，其中所述第一重链的CH1和第一轻链的CL中包含以下氨基酸取代：
(a)CH1中的C220A和CL中的C214A；
(b)选自以下组中的至少一组：
(b-1)CH1中的F170C和CL中的T164C；
(b-2)CH1中的L128C和CL中的S121C；
(b-3)CH1中的A129C和CL中的S121C；
(b-4)CH1中的S131C和CL中的P119C；
(b-5)CH1中的A141C和CL中的L135C；和
(b-6)CH1中的P171C和CL中的S165C；和
(c)选自以下组中的任一组：
(c-1)CH1中的T139R和CL中的S114E；
(c-2)CH1中的T139R和CL中的S114D；
(c-3)CH1中的T139K和CL中的S114E；
(c-4)CH1中的T139K和CL中的S114D；
和
(ii)包含第二轻链和第二重链的CTLA-4抗原结合域，其中并且第二重链的CH1和第二轻链的CL中包含选自以下任一组的氨基酸取代：
(1)CH1中的T139D和CL中的S114K；
(2)CH1中的T139D和CL中的S114R；
(3)CH1中的T139E和CL中的S114K；和
(4)CH1中的T139E和CL中的S114R。
本公开提供了一种PD-1/CTLA-4双特异性抗体，其包含：
(i)包含第一轻链和第一重链的PD-1抗原结合域，其中第一重链的CH1和第一轻链的CL中包含选自以下任一组的氨基酸取代：
(1)CH1中的T139D和CL中的S114K；
(2)CH1中的T139D和CL中的S114R；
(3)CH1中的T139E和CL中的S114K；和
(4)CH1中的T139E和CL中的S114R；
和
(ii)包含第二轻链和第二重链的CTLA-4抗原结合域，其中并且第二重链的CH1和第二轻链的CL中包含以下的氨基酸取代：
(a)CH1中的C220A和CL中的C214A；
(b)选自以下组中的至少一组氨基酸取代：
(b-1)CH1中的S131C和CL中的P119C；
(b-2)CH1中的L128C和CL中的S121C；
(b-3)CH1中的A129C和CL中的S121C；
(b-4)CH1中的F170C和CL中的T164C；
(b-5)CH1中的A141C和CL中的L135C；和
(b-6)CH1中的P171C和CL中的S165C；和
(c)选自以下组中的任一组的氨基酸取代：
(1)CH1中的T139R和CL中的S114E；
(2)CH1中的T139R和CL中的S114D；
(3)CH1中的T139K和CL中的S114E；和
(4)CH1中的T139K和CL中的S114D。
本公开提供了一种FAP/CD40双特异性抗体，其包含：
(i)包含第一轻链和第一重链的CD40抗原结合域，其中第一重链的CH1和第一轻链的CL中包含以下氨基酸取代：
(a)CH1中的C220A和CL中的C214A；
(b)选自以下组中的至少一组的氨基酸取代：
(b-1)CH1中的F170C和CL中的T164C；
(b-2)CH1中的L128C和CL中的S121C；
(b-3)CH1中的A129C和CL中的S121C；
(b-4)CH1中的S131C和CL中的P119C；
(b-5)CH1中的A141C和CL中的L135C；和
(b-6)CH1中的P171C和CL中的S165C；
和
(ii)包含第二轻链和第二重链的FAP抗原结合域。
本公开提供了一种FAP/CD40双特异性抗体，其包含：
(i)包含第一轻链和第一重链的CD40抗原结合域；
和
(ii)包含第二轻链和第二重链的FAP抗原结合域；其中第二重链的CH1和 第二轻链的CL中包含以下氨基酸取代：
(a)CH1中的C220A和CL中的C214A；
(b)选自以下组中的至少一组的氨基酸取代：
(b-1)CH1中的F170C和CL中的T164C；
(b-2)CH1中的L128C和CL中的S121C；
(b-3)CH1中的A129C和CL中的S121C；
(b-4)CH1中的S131C和CL中的P119C；
(b-5)CH1中的A141C和CL中的L135C；和
(b-6)CH1中的P171C和CL中的S165C。
在一些实施方案中，第一重链与第二重链通过接头连接。在一些实施方案中，肽接头是具有长度为至少5个氨基酸的氨基酸序列的肽，在一个实施方案中，长度为5至100，在进一步的实施方案中为10至50个氨基酸。在一个实施方案中，所述肽接头是(GxS)n或(GxS)nGm，其中G＝甘氨酸，S＝丝氨酸和(x＝3，n＝3，4，5或6，和m＝0，1，2或3)或(x＝4，n＝2，3，4或5和m＝0，1，2或3)。在一个实施方案中x＝4和n＝3或4。在一个实施方案中，所述肽接头是(G4S)4。
本公开提供了一种结合PSMA双表位的抗体，其包含：
(i)结合第一表位的第一轻链和第一重链，其中第二重链的CH1和第二轻链的CL中包含以下的氨基酸取代：
(a)CH1中的C220A和CL中的C214A；
(b)选自以下组中的至少一组的氨基酸取代：
(b-1)CH1中的S131C和CL中的P119C；
(b-2)CH1中的L128C和CL中的S121C；
(b-3)CH1中的A129C和CL中的S121C；
(b-4)CH1中的F170C和CL中的T164C；
(b-5)CH1中的A141C和CL中的L135C；和
(b-6)CH1中的P171C和CL中的S165C；
和
(ii)结合第二表位的第二轻链和第二重链。
本公开提供了一种结合PSMA的双表位抗体，其包含：
(i)结合第一表位的第一轻链和第一重链；和
(ii)结合第二表位的第二轻链和第二重链，其中第二重链的CH1和第二轻链的CL中包含以下的氨基酸取代：
(a)CH1中的C220A和CL中的C214A；
(b)选自以下组中的至少一组的氨基酸取代：
(b-1)CH1中的S131C和CL中的P119C；
(b-2)CH1中的L128C和CL中的S121C；
(b-3)CH1中的A129C和CL中的S121C；
(b-4)CH1中的F170C和CL中的T164C；
(b-5)CH1中的A141C和CL中的L135C；和
(b-6)CH1中的P171C和CL中的S165C。
本公开提供了一种抗原结合蛋白，其包含：
(i)第一抗原结合域，所述第一抗原结合域包含多肽H1和多肽L1，所述多肽H1包含与第一VH连接的第一CH1，所述多肽L1包含与第一VL连接的第一CL，其中，第一CH1和第一CL在选自(i-1)至(i-6)的位置中的一组或多组包含天然非半胱氨酸至半胱氨酸的氨基酸取代：
(i-1)第一CH1的第170位和第一CL的第164位，
(i-2)第一CH1的第128位和第一CL的第121位，
(i-3)第一CH1的第129位和第一CL的第121位，
(i-4)第一CH1的第131位和第一CL的第119位，
(i-5)第一CH1的第141位和第一CL的第135位，和
(i-6)第一CH1的第171位和第一CL的第165位；
和
(ii)第二抗原结合域，所述第二抗原结合域包含多肽H2和多肽L2，所述多肽H2包含与第二VH连接的第二CH1，所述多肽L2包含与第二VL连接的第二CL。
在一些实施方案中，多肽H1包含与第一重链可变区VH1连接的第一CH1；多肽L1包含与第一轻链可变区VL1连接的第一CL。
在一些实施方案中，多肽H1从N端至C端依次包含VH1和第一CH1；多肽L1从N端至C端依次包含VL1和第一CL。
在一些实施方案中，多肽H1从N端至C端依次包含VH1、第一CH1和Fc1；多肽L1从N端至C端依次包含VL1和第一CL。
在一些实施方案中，多肽H1是第一重链，多肽L1是第一轻链。
在一些实施方案中，多肽H2包含与第二重链可变区VH2连接的第二CH1；多肽L2包含与第二轻链可变区VL2连接的第二CL。
在一些实施方案中，多肽H2从N端至C端依次包含VH2和第二CH1；多肽L2从N端至C端依次包含VL2和第二CL。
在一些实施方案中，多肽H2从N端至C端依次包含VH2、第二CH1和Fc2；多肽L2从N端至C端依次包含VL2和第二CL。
在一些实施方案中，多肽H2是第二重链，多肽L2是第二轻链。
在一些实施方案中，多肽H1和多肽H2可以通过接头连接。在一些实施方案中，通过接头连接的多肽H1和多肽H2从N端至C端依次为[VH1]-[第一CH1]-Fc1-[接头]-[VH2]-[第二CH1]。
在一些实施方案中，第一CH1、第一CL、第二CH1和第二CL如上所定义。
在一些实施方案中，多肽L1是抗体轻链，例如人IgG抗体轻链，其是κ轻链(Cκ)；多肽L2是抗体轻链，例如人IgG抗体轻链，其可以是λ轻链(Cλ)或κ轻链(Cκ)。在一些实施方案中，多肽L1是κ轻链且多肽L2是λ轻链。
在一些实施方案中，所述多肽H1包含Fc1，所述多肽H2包含Fc2，所述Fc1和/或所述Fc2选自人IgG1、IgG2、IgG3和IgG4的Fc，例如人IgG1的Fc。
在一些实施方案中，Fc1和Fc2是如上所定义的经改造的、或经氨基酸修饰或取代的。
在一些实施方案中，Fc1和/或所述Fc2包含改变所述抗原结合蛋白的半衰期的修饰，其中所述半衰期取决于FcRn结合亲和力。
在一些实施方案中，Fc1和/或所述Fc2包含改变效应子功能的修饰，其中对Fcγ受体或C1q补体蛋白的结合亲和力增大或减小。
在一些实施方案中，Fc1和Fc2中包含这样的氨基酸取代，使得与Fc1相比，Fc1优先与Fc2配对。
在一些实施方案中，所述多肽L1包含氨基酸置换：S165C和C214A，所述多肽H1包含氨基酸置换：P171C、C220A、L234A、L235A、D356E、L358M、Y349C、T366S、L368A和Y407N；并且所述多肽H2包含氨基酸置换：L234A、L235A、D356E、L358M、S354C和T366W；
或者
所述多肽L1包含氨基酸置换：S165C和C214A，所述多肽H1包含氨基酸置换：P171C、C220A、L234A、L235A、D356E、L358M、S354C和T366W；并且所述多肽H2包含氨基酸置换：L234A、L235A、D356E、L358M、Y349C、T366S、L368A和Y407N。
在一些实施方案中，所述多肽L1包含氨基酸置换：T164C、C214A和S114E，所述多肽H1包含氨基酸置换：T139R、F170C、C220A、L234A、L235A、D356E、L358M、Y349C、T366S、L368A和Y407N；并且所述多肽L2包含氨基酸置换S114K，所述多肽H2包含氨基酸置换：T139D、L234A、L235A、D356E、L358M、S354C和T366W；
或者
所述多肽L1包含氨基酸置换：T164C、C214A和S114E，所述多肽H1包含氨基酸置换：T139R、F170C、C220A、L234A、L235A、D356E、L358M、S354C和T366W；并且所述多肽L2包含氨基酸置换S114K，所述多肽H2包含氨基酸置换：T139D、L234A、L235A、D356E、L358M、Y349C、T366S、L368A和Y407N。
本公开提供了一种双特异性二价抗原结合蛋白，其包含：
(i)第一抗原结合域，所述第一抗原结合域包含多肽H1和多肽L1，所述多肽H1包含与第一VH连接的第一CH1，所述多肽L1包含与第一VL连接的第一 CL，其中：第一CH1和第一CL各自包含天然半胱氨酸至非半胱氨酸的氨基酸取代，并且第一CH1和第一CL在选自以下的位置中还包含天然非半胱氨酸至半胱氨酸的氨基酸取代：
(i-1)第一CH1的第170位和第一CL的第164位，
(i-2)第一CH1的第128位和第一CL的第121位，
(i-3)第一CH1的第129位和第一CL的第121位，
(i-4)第一CH1的第131位和第一CL的第119位，
(i-5)第一CH1的第141位和第一CL的第135位，和
(i-6)第一CH1的第171位和第一CL的第165位；
和
(ii)第二抗原结合域，所述第二抗原结合域包含多肽H2和多肽L2，所述多肽H2包含与第二VH连接的第二CH1，所述多肽L2包含与第二VL连接的第二CL；
其中所述多肽H1自N端至C端依次包含VH、CH1和Fc；所述多肽H2自N端至C端依次包含VH、CH1和Fc。
本公开提供了一种双特异性四价抗原结合蛋白，其包含：
(i)第一抗原结合域，所述第一抗原结合域包含多肽H1和多肽L1，所述多肽H1包含与第一VH连接的第一CH1，所述多肽L1包含与第一VL连接的第一CL，其中：第一CH1和第一CL各自包含天然半胱氨酸至非半胱氨酸的氨基酸取代，并且第一CH1和第一CL在选自以下的位置中还包含天然非半胱氨酸至半胱氨酸的氨基酸取代：
(i-1)第一CH1的第170位和第一CL的第164位，
(i-2)第一CH1的第128位和第一CL的第121位，
(i-3)第一CH1的第129位和第一CL的第121位，
(i-4)第一CH1的第131位和第一CL的第119位，
(i-5)第一CH1的第141位和第一CL的第135位，和
(i-6)第一CH1的第171位和第一CL的第165位；
和
(ii)第二抗原结合域，所述第二抗原结合域包含多肽H2和多肽L2，所述多肽H2包含与第二VH连接的第二CH1，所述多肽L2包含与第二VL连接的第二CL；
其中：所述多肽H1自N端至C端由VH和CH1构成，所述多肽H2自N端至C端依次包含VH、CH1和Fc，所述多肽H1的C端任选地通过肽接头与所述多肽H2的C端融合；或者所述多肽H1自N端至C端依次包含VH、CH1和Fc，所述多肽H2自N端至C端由VH和CH1构成，所述多肽H2的C端任选地通过肽接头与所述多肽H1的C端融合。
肽接头表示具有氨基酸序列的肽。在一些实施方案中，肽接头是具有长度为至少5个氨基酸的氨基酸序列的肽，在一个实施方案中，长度为5至100，在进一步的实施方案中为10至50个氨基酸。在一个实施方案中，所述肽接头是(GxS)n或(GxS)nGm，其中G＝甘氨酸，S＝丝氨酸和(x＝3，n＝3，4，5或6，和m＝0，1，2或3)或(x＝4，n＝2，3，4或5和m＝0，1，2或3)。在一个实施方案中x＝4和n＝3或4。在一个实施方案中，所述肽接头是(G4S)4。
在一些实施方案中，所述多肽H1自N端至C端由VH和CH1构成，所述多肽H2自N端至C端依次包含VH、CH1和Fc，所述多肽H1的C端任选地通过肽接头与所述多肽H2的C端融合所述多肽H2。
在一些实施方案中，所述多肽H1自N端至C端依次包含VH、CH1和Fc，所述多肽H2自N端至C端由VH和CH1构成，所述多肽H2的C端任选地通过肽接头与所述多肽H1的C端融合所述多肽H1。
本公开提供了一种二聚化多肽，其包含重链恒定区1(CH1)和轻链恒定区(CL)，其中：CH1的第139位和CL的第114位包含使得CH1和CL之间形成静电相互作用界面的氨基酸取代。
在一些实施方案中，CH1第139位的氨基酸被取代为带正电荷的氨基酸，CL第114位的氨基酸被取代为带负电荷的氨基酸；或者CH1第139位的氨基酸被取代为带负电荷的氨基酸，CL第114位的氨基酸被取代为带正电荷的氨基酸。
在一些实施方案中，带正电荷的氨基酸选自K、R和H；带负电荷的氨基酸选自D和E。
在一些实施方案中，CH1和CL包含选自以下的氨基酸取代：T139R和S114E；T139R和S114D；T139K和S114E；T139K和S114D；T139D和S114K；T139D和S114R；T139E和S114K；和T139E和S114R。
本公开提供了一种抗原结合蛋白，其包含上述二聚化多肽。
在一些实施方案中，抗原结合蛋白包含第一抗原结合域，所述第一抗原结合域包含Fab，所述Fab包含第一重链可变区VH1、第一轻链可变区VL1和所述二聚化多肽，所述二聚化多肽中所述CH1为第一CH1，所述CL为第一CL；VH1与第一CH1直接连接或通过接头连接，VL1与第一CL直接连接或通过接头连接。在一些实施方案中，VH1的C端与第一CH1的N端直接连接或通过接头连接，VL1的C端与第一CL的N端直接连接或通过接头连接。
在一些实施方案中，抗原结合蛋白包含第一抗原结合域和第二抗原结合域，其中所述第二抗原结合域包含第二重链可变区VH2和第二轻链可变区VL2，并且所述第一抗原结合域和第二抗原结合域结合不同的抗原或者结合同一种抗原上的不同的表位；在一些实施方案中，所述第二抗原结合域包含Fab。在一些实施方案中，VH2的C端与第二CH1的N端直接连接或通过接头连接，VL2的C端与第二CL的N端直接连接或通过接头连接。
在一些实施方案中，第一CH1和第一CL包含使得第一CH1和第一CL之间形成静电相互作用界面的氨基酸取代；和/或
第二CH1和第二CL包含使得第二CH1和第二CL之间形成静电相互作用界面的氨基酸取代。
在一些实施方案中，第一CH1和第二CH1中用于形成静电相互作用界面的氨基酸的带电性相反，且第一CL和第二CL中用于形成静电相互作用界面的氨基酸的带电性相反。
在一些实施方案中，使得第一CH1和第一CL之间形成静电相互作用界面的氨基酸取代位于第一CH1的第139位和第一CL的第114位；和/或
使得第二CH1和第二CL之间形成静电相互作用界面的氨基酸取代位于第二CH1的第139位和第二CL的第114位。
在一些实施方案中，第一CH1的第139位和第二CH1的第139位分别被带相反电荷的氨基酸取代，且第一CL的第114位和第二CL的第114位分别被带相反电荷的氨基酸取代。
在一些实施方案中，第一CH1第139位的氨基酸被取代为带正电荷的氨基酸，第一CL第114位的氨基酸被取代为带负电荷的氨基酸；或者第一CH1第139位的氨基酸被取代为带负电荷的氨基酸，第一CL第114位的氨基酸被取代为带正电荷的氨基酸；和/或
第二CH1第139位的氨基酸被取代为带负电荷的氨基酸，第二CL第114位的氨基酸被取代为带正电荷的氨基酸；或者第二CH1第139位的氨基酸被取代为带正电荷的氨基酸，第二CL第114位的氨基酸被取代为带负电荷的氨基酸。
在一些实施方案中，带正电荷的氨基酸选自K、R和H；带负电荷的氨基酸选自D和E。
在一些实施方案中，第一CH1和第一CL包含选自以下的氨基酸取代：T139R和S114E；T139R和S114D；T139K和S114E；T139K和S114D；T139D和S114K；T139D和S114R；T139E和S114K；和T139E和S114R；和/或
第二CH1和第二CL包含选自以下的氨基酸取代：T139R和S114E；T139R和S114D；T139K和S114E；T139K和S114D；T139D和S114K；T139D和S114R；T139E和S114K；和T139E和S114R。
在一些实施方案中，第一CH1和第一CL包含选自以下的氨基酸取代：T139R和S114E；T139R和S114D；T139K和S114E；T139K和S114D；和/或
第二CH1和第二CL包含选自以下的氨基酸取代：T139D和S114K；T139D和S114R；T139E和S114K；和T139E和S114R。
在一些实施方案中，第一CH1和第一CL包含选自以下的氨基酸取代：T139D和S114K；T139D和S114R；T139E和S114K；和T139E和S114R；和/或
第二CH1和第二CL包含选自以下的氨基酸取代：T139R和S114E；T139R 和S114D；T139K和S114E；T139K和S114D。
本公开提供了一种抗原结合蛋白，其包含：
(i)第一抗原结合域，所述第一抗原结合域包含多肽H1和多肽L1，所述多肽H1包含与第一VH连接的第一CH1，所述多肽L1包含与第一VL连接的第一CL；和
(ii)第二抗原结合域，所述第二抗原结合域包含多肽H2和多肽L2，所述多肽H2包含与第二VH连接的第二CH1，所述多肽L2包含与第二VL连接的第二CL；
其中：第一CH1的第139位和第一CL的第114位包含使得第一CH1和第一CL之间形成静电相互作用界面的氨基酸取代；和/或
第二CH1的第139位和第二CL的第114位包含使得第二CH1和第二CL之间形成静电相互作用界面的氨基酸取代。
在一些实施方案中，抗原结合蛋白是双特异性二价抗原结合蛋白，其中其中所述多肽H1自N端至C端依次包含VH、CH1和Fc；所述多肽H2自N端至C端依次包含VH、CH1和Fc。
在一些实施方案中，抗原结合蛋白是双特异性四价抗原结合蛋白，其中所述多肽H1自N端至C端由VH和CH1构成，所述多肽H2自N端至C端依次包含VH、CH1和Fc，所述多肽H1的C端任选地通过肽接头与所述多肽H2的C端融合；或者所述多肽H1自N端至C端依次包含VH、CH1和Fc，所述多肽H2自N端至C端由VH和CH1构成，所述多肽H2的C端任选地通过肽接头与所述多肽H1的C端融合。
在一些实施方案中，本公开的抗原结合蛋白是多特异性抗体，例如双特异性抗体。在一些实施方案中，本公开的抗原结合蛋白是嵌合抗体、人源化抗体或全人抗体、多价抗体、或抗体药物偶联物。
在一些实施方案中，本公开的包含上述氨基酸取代的抗原结合蛋白与不含这些氨基酸取代的抗原结合蛋白相比，以改善的多肽H1/L1和多肽H2/L2(例如重链/轻链)配对或改善的产率在单细胞中产生。
在一些实施方案中，本公开的抗原结合蛋白其多肽H1/L1和多肽H2/L2(例如重链/轻链)的正确配对比例为至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。计算公式为：多肽H1/L1和多肽H2/L2(例如重链/轻链)的正确配对比例＝(正确第一抗原结合分子峰强度+正确第二抗原结合分子峰强度)/(正确第一抗原结合分子峰强度+正确第二抗原结合分子峰强度+其他杂质峰强度)×100％。
在一些实施方案中，本公开的抗原结合蛋白其多肽H1/L1和多肽H2/L2(例如重链/轻链)的正确配对比例相对于野生型提高了至少5％、至少10％、至少15％、 至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少46％、至少47％、至少48％、至少49％或50％。
在一些实施方案中，本公开的抗原结合蛋白通过在CH1/CL界面中去除天然二硫键并引入非天然二硫键，将多肽H1/L1和多肽H2/L2(例如重链/轻链)的正确配对比例相对于野生型提高了至少5％、至少10％、至少15％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少46％、至少47％、至少48％、至少49％或50％。
在一些实施方案中，本公开的抗原结合蛋白通过在CH1/CL界面中引入静电互补的氨基酸对，将多肽H1/L1和多肽H2/L2(例如重链/轻链)的正确配对比例相对于野生型提高了至少5％、至少10％、至少15％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少46％、至少47％、至少48％、至少49％或50％。
在一些实施方案中，本公开的抗原结合蛋白通过在CH1/CL界面中去除天然二硫键并引入非天然二硫键，并同时引入静电互补的氨基酸对，将多肽H1/L1和多肽H2/L2(例如重链/轻链)的正确配对比例相对于野生型提高了至少5％、至少10％、至少15％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少46％、至少47％、至少48％、至少49％或50％。
本公开还提供了一种编码前述二聚体多肽或抗原结合蛋白的核酸分子或其组合。
本公开还提供了一种核酸表达载体或其组合，其包含前述核酸分子或其组合。
本公开还提供了一种宿主细胞，其包含前述核酸分子或其组合。
在一些实施方案中，宿主细胞是可被工程化以产生根据本公开的二聚体多肽或抗原结合蛋白的任何种类的细胞***，例如真核细胞或原核细胞。真核细胞包括但不限于，例如来自于酵母、真菌、昆虫、植物、动物、人或其它的多细胞生物的有核细胞。
本公开还提供了一种制备前述任一种二聚体多肽或抗原结合蛋白的方法，其包括以下步骤：
(1)用前述核酸表达载体转化宿主细胞；
(2)在容许合成所述抗原结合蛋白的条件下培养所述宿主细胞得到细胞培养物；和
(3)从所述细胞培养物中回收抗原结合蛋白。
在一些实施方案中，前述核酸表达载体包括：编码重链的质粒和编码轻链的质粒；在转化宿主细胞时，编码轻链的质粒相对于编码重链的质粒是过量的，例如编码重链的质粒与编码轻链的质粒的摩尔比为1:(1-10)，例如1:(1-5)，例如2:3。
在一些实施方案中，前述核酸表达载体包括：
第一质粒，其包含编码所述多肽H1的核酸分子；
第二质粒，其包含编码所述多肽L1的核酸分子；
第三质粒，其包含编码所述多肽H2的核酸分子；和
第四质粒，其包含编码所述多肽L2的核酸分子。
在一些实施方案中，前述核酸表达载体包括：
第一质粒，其包含编码第一重链的核酸分子；
第二质粒，其包含编码第一轻链的核酸分子；
第三质粒，其包含编码第二重链的核酸分子；和
第四质粒，其包含编码第二轻链的核酸分子。在一些实施方案中，第一质粒和第二质粒在转化宿主细胞时的摩尔比为1:1至1:10、1:1至1:9、1:1至1:8、1:1至1:7、1:1至1:6、1:1至1:5、1:1至1:4、1:1至1:3、1:1至1:2、1:1至1:1.9、1:1至1:1.8、1:1至1:7、1:1至1:1.6、1:1至1:1.5、1:1至1:1.4、1:1至1:1.3、1:1至1:1.2、1:1至1:1.1、或1:1至1:1.05。
在一个实施方案中，第一质粒和第二质粒在转化宿主细胞时的摩尔比为1:1、1:1.05、1:1.1、1:1.2、1:1.3、1:1.4、1:1.5、1:1.6、1:1.7、1:1.8、1:1.9、1:2.0、1:2.1、1:2.2、1:2.3、1:2.4、1:2.5、1:2.6、1:2.7、1:2.8、1:2.9、1:3.0、1:3.1、1:3.2、1:3.3、1:3.4、1:3.5、1:3.6、1:3.7、1:3.8、1:3.9、1:4.0、1:4.1、1:4.2、1:4.3、1:4.4、1:4.5、1:4.6、1:4.7、1:4.8、1:4.9、1:5.0、1:5.1、1:5.2、1:5.3、1:5.4、1:5.5、1:5.6、1:5.7、1:5.8、1:5.9、1:6.0、1:6.1、1:6.2、1:6.3、1:6.4、1:6.5、1:6.6、1:6.7、1:6.8、1:6.9、1:7.0、1:7.1、1:7.2、1:7.3、1:7.4、1:7.5、1:7.6、1:7.7、1:7.8、1:7.9、1:8.0、1:8.1、1:8.2、1:8.3、1:8.4、1:8.5、1:8.6、1:8.7、1:8.8、1:8.9、1:9.0、1:9.1、1:9.2、1:9.3、1:9.4、1:9.5、1:9.6、1:9.7、1:9.8、1:9.9、或1:10.0。
在一些实施方案中，第三质粒和第四质粒在转化宿主细胞时的摩尔比为1:1至1:10、1:1至1:9、1:1至1:8、1:1至1:7、1:1至1:6、1:1至1:5、1:1至1:4、1:1至1:3、1:1至1:2、1:1至1:1.9、1:1至1:1.8、1:1至1:7、1:1至1:1.6、1:1至1:1.5、1:1至1:1.4、1:1至1:1.3、1:1至1:1.2、1:1至1:1.1、或1:1至1:1.05。
在一个实施方案中，第三质粒和第四质粒在转化宿主细胞时的摩尔比为1:1、1:1.05、1:1.1、1:1.2、1:1.3、1:1.4、1:1.5、1:1.6、1:1.7、1:1.8、1:1.9、1:2.0、1:2.1、1:2.2、1:2.3、1:2.4、1:2.5、1:2.6、1:2.7、1:2.8、1:2.9、1:3.0、1:3.1、1:3.2、1:3.3、1:3.4、1:3.5、1:3.6、1:3.7、1:3.8、1:3.9、1:4.0、1:4.1、1:4.2、1:4.3、1:4.4、1:4.5、1:4.6、1:4.7、1:4.8、1:4.9、1:5.0、1:5.1、1:5.2、1:5.3、1:5.4、1:5.5、1:5.6、1:5.7、1:5.8、1:5.9、1:6.0、1:6.1、1:6.2、1:6.3、1:6.4、1:6.5、1:6.6、1:6.7、1:6.8、1:6.9、1:7.0、1:7.1、1:7.2、1:7.3、1:7.4、1:7.5、1:7.6、1:7.7、1:7.8、1:7.9、1:8.0、1:8.1、1:8.2、1:8.3、1:8.4、1:8.5、1:8.6、1:8.7、1:8.8、1:8.9、1:9.0、1:9.1、1:9.2、1:9.3、1:9.4、1:9.5、1:9.6、1:9.7、1:9.8、1:9.9、或1:10.0。
在一些实施方案中，转化宿主细胞时，第一质粒：第二质粒：第三质粒：第四质粒的摩尔比为1:(1～10):1:(1～10)，例如1:(1～5):1:(1～5)，例如2:3:2:3。
在一些实施方案中，转化宿主细胞时，第一质粒：第二质粒：第三质粒：第四质粒的摩尔比为1:(1～10):1:(1～10)、1:(1～9):1:(1～9)、1:(1～8):1:(1～8)、1:(1～7):1:(1～7)、1:(1～6):1:(1～6)、1:(1～5):1:(1～5)、1:(1～4):1:(1～4)、1:(1～3):1:(1～3)或1:(1～2):1:(1～2)。
在一个实施方案中，转化宿主细胞时，第一质粒：第二质粒：第三质粒：第四质粒的摩尔比为1:1:1:1、1:1.05:1:1.05、1:1.1:1:1.1、1:1.2:1:1.2、1:1.3:1:1.3、1:1.4:1:1.4、1:1.5:1:1.5(或2:3:2:3)、1:1.6:1:1.6、1:1.7:1:1.7、1:1.8:1:1.8、1:1.9:1:1.9、1:2.0:1:2.0、1:2.1:1:2.1、1:2.2:1:2.2、1:2.3:1:2.3、1:2.4:1:2.4、1:2.5:1:2.5、1:2.6:1:2.6、1:2.7:1:2.7、1:2.8:1:2.8、1:2.9:1:2.9、1:3.0:1:3.0、1:3.1:1:3.1、1:3.2:1:3.2、1:3.3:1:3.3、1:3.4:1:3.4、1:3.5:1:3.5、1:3.6:1:3.6、1:3.7:1:3.7、1:3.8:1:3.8、1:3.9:1:3.9、1:4.0:1:4.0、1:4.1:1:4.1、1:4.2:1:4.2、1:4.3:1:4.3、1:4.4:1:4.4、1:4.5:1:4.5、1:4.6:1:4.6、1:4.7:1:4.7、1:4.8:1:4.8、1:4.9:1:4.9、1:5.0:1:5.0、1:5.1:1:5.1、1:5.2:1:5.2、1:5.3:1:5.3、1:5.4:1:5.4、1:5.5:1:5.5、1:5.6:1:5.6、1:5.7:1:5.7、1:5.8:1:5.8、1:5.9:1:5.9、1:6.0:1:6.0、1:6.1:1:6.1、1:6.2:1:6.2、1:6.3:1:6.3、1:6.4:1:6.4、1:6.5:1:6.5、1:6.6:1:6.6、1:6.7:1:6.7、1:6.8:1:6.8、1:6.9:1:6.9、1:7.0:1:7.0、1:7.1:1:7.1、1:7.2:1:7.2、1:7.3:1:7.3、1:7.4:1:7.4、1:7.5:1:7.5、1:7.6:1:7.6、1:7.7:1:7.7、1:7.8:1:7.8、1:7.9:1:7.9、1:8.0:1:8.0、1:8.1:1:8.1、1:8.2:1:8.2、1:8.3:1:8.3、1:8.4:1:8.4、1:8.5:1:8.5、1:8.6:1:8.6、1:8.7:1:8.7、1:8.8:1:8.8、1:8.9:1:8.9、1:9.0:1:9.0、1:9.1:1:9.1、1:9.2:1:9.2、1:9.3:1:9.3、1:9.4:1:9.4、1:9.5:1:9.5、1:9.6:1:9.6、1:9.7:1:9.7、1:9.8:1:9.8、1:9.9:1:9.9、或1:10.0:1:10.0。
在一些实施方案中，所述核酸表达载体包括：
第一质粒，其包含编码所述多肽H1的核酸分子和编码多肽H2的核酸分子；
第二质粒，其包含编码所述多肽L1的核酸分子；和
第三质粒，其包含编码所述多肽L2的核酸分子。
在一些实施方案中，所述核酸表达载体包括：
第一质粒，其包含编码重链的核酸分子；
第二质粒，其包含编码第一轻链的核酸分子；和
第三质粒，其包含编码第二轻链的核酸分子。
在一些实施方案中，转化宿主细胞时，第一质粒：第二质粒：第三质粒的摩尔比为1:(1～10):(1～10)，优选1:(1～5):(1～5)，更优选2:3:3。
在一些实施方案中，转化宿主细胞时，第一质粒：第二质粒：第三质粒的摩尔比为1:(1～10):(1～10)、1:(1～9):(1～9)、1:(1～8):(1～8)、1:(1～7):(1～7)、1:(1～6):(1～6)、1:(1～5):(1～5)、1:(1～4):(1～4)、1:(1～3):(1～3)或1:(1～2):(1～2)。
在一个实施方案中，转化宿主细胞时，重链质粒：第一轻链质粒：第二轻链质粒的摩尔比为1:1:1、1:1.05:1.05、1:1.1:1.1、1:1.2:1.2、1:1.3:1.3、1:1.4:1.4、1:1.5:1.5(或2:3:3)、1:1.6:1.6、1:1.7:1.7、1:1.8:1.8、1:1.9:1.9、1:2.0:2.0、1:2.1:2.1、1:2.2:2.2、1:2.3:2.3、1:2.4:2.4、1:2.5:2.5、1:2.6:2.6、1:2.7:2.7、1:2.8:2.8、1:2.9:2.9、1:3.0:3.0、 1:3.1:3.1、1:3.2:3.2、1:3.3:3.3、1:3.4:3.4、1:3.5:3.5、1:3.6:3.6、1:3.7:3.7、1:3.8:3.8、1:3.9:3.9、1:4.0:4.0、1:4.1:4.1、1:4.2:4.2、1:4.3:4.3、1:4.4:4.4、1:4.5:4.5、1:4.6:4.6、1:4.7:4.7、1:4.8:4.8、1:4.9:4.9、1:5.0:5.0、1:5.1:5.1、1:5.2:5.2、1:5.3:5.3、1:5.4:5.4、1:5.5:5.5、1:5.6:5.6、1:5.7:5.7、1:5.8:5.8、1:5.9:5.9、1:6.0:6.0、1:6.1:6.1、1:6.2:6.2、1:6.3:6.3、1:6.4:6.4、1:6.5:6.5、1:6.6:6.6、1:6.7:6.7、1:6.8:6.8、1:6.9:6.9、1:7.0:7.0、1:7.1:7.1、1:7.2:7.2、1:7.3:7.3、1:7.4:7.4、1:7.5:7.5、1:7.6:7.6、1:7.7:7.7、1:7.8:7.8、1:7.9:7.9、1:8.0:8.0、1:8.1:8.1、1:8.2:8.2、1:8.3:8.3、1:8.4:8.4、1:8.5:8.5、1:8.6:8.6、1:8.7:8.7、1:8.8:8.8、1:8.9:8.9、1:9.0:9.0、1:9.1:9.1、1:9.2:9.2、1:9.3:9.3、1:9.4:9.4、1:9.5:9.5、1:9.6:9.6、1:9.7:9.7、1:9.8:9.8、1:9.9:9.9、或1:10.0:10.0。
在一些实施方案中，本领域中已知的其他方法还可以用于本公开以平衡两个重链的表达水平，如强/弱启动子的使用。
本公开还提供了一种药物组合物，其包含前述任一种抗原结合蛋白和药学上可接受的载体。
药学上可接受的载体是指除了活性成分之外的药物制剂中的成分，其对受试者是无毒的。药学上可接受的载体包括生理上相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。在一个优选的实施方案中，载体适用于静脉内、肌肉内、皮下、肠胃外、脊髓或表皮施用(例如通过注射或输注)。
在另一些方面，本公开还提供一种消除受试者免疫抑制相关疾病的方法，所述方法包括向受试者施用治疗有效量的如前所述的抗原结合蛋白，或如前所述的药物组合物，所述治疗有效量为单位剂量的组合物中含有0.1-3000mg的如前所述的抗原结合蛋白。
在一些实施方案中，在单次或累积的施加中，向个体施用约10μg/kg至约1000mg/kg剂量的本公开所述的抗原结合蛋白或药物组合物。
本公开还提供了前述任一种二聚化多肽或抗原结合蛋白在制备药物中的用途。
本公开还提供了前述任一种二聚化多肽或抗原结合蛋白在制备用于治疗癌症、自身免疫性疾病或炎性疾病的药物中的用途。
本公开还提供了一种治疗和/或预防疾病例如癌症、自身免疫性疾病或炎性疾病的方法，其包括向有其需要的患者施用有效量的前述抗原结合蛋白或药物组合物。
本公开还提供了前述任一种二聚化多肽、抗原结合蛋白或药物组合物，其用于治疗癌症、自身免疫性疾病或炎性疾病。
在一些实施方案中，癌症包括但不限于癌瘤、淋巴瘤、胚细胞瘤(blastoma)、肉瘤、白血病和淋巴样恶性肿瘤。这种癌症的更具体的例子包括鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌(NSCLC)、头和颈鳞状细胞癌(HNSCC)、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤(DLBCL)、滤泡 性淋巴瘤、急性成淋巴细胞性白血病(ALL)、急性髓细胞样白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性髓细胞样白血病(CML)、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤(MCL)、小淋巴细胞性淋巴瘤(SLL)、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白(Mcl-1)、骨髓异常增生综合征(MDS)、胃肠(道)癌、肾癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、***癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、胃癌、骨癌、尤因氏肉瘤、子***、脑癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、肝细胞癌(HCC)、透明细胞肾细胞癌(RCC)、头和颈癌、咽喉癌、肝胆癌(hepatobiliary cancer)、中枢神经***癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。
在一些实施方案中，自身免疫性疾病或炎性疾病选自：类风湿关节炎、牛皮癣、克罗恩病、强硬性脊柱炎、多发性硬化症、I型糖尿病、肝炎、心肌炎、Sjogren综合征、移植排斥后的自体免疫性溶血性贫血、水疱性类天疱疮、格雷夫氏病、桥本甲状腺炎、***性红斑狼疮(SLE)、重症肌无力、天疱疮、恶性贫血。
附图说明
图1显示了实施例3中的PD-1单抗产物IdeS酶切后的分子量解卷积质谱图；
图2显示了实施例4中的分子形式：1+1非对称双特异性抗体，一臂使用天然CH1/Cκ，另一臂使用含有非天然二硫键的CH1/Cκ；
图3A-3D显示了实施例4中的双抗初纯产物木瓜蛋白酶酶切后的分子量解卷积质谱图；
图4A显示了TJ030-PR1104蛋白初纯产物的二步纯化色谱图；图4B显示了精纯后的TJ030-PR1104蛋白的脱糖完整分子量总离子流图(上)和紫外图谱(下)以及主要峰的分子归属信息；图4C显示了精纯后的TJ030-PR1104蛋白的脱糖还原分子量总离子流图(上)和紫外图谱(下)以及主要峰的分子归属信息；
图5显示了1+1非对称的PD-1×CTLA-4双抗能促成PD-1表达细胞和CTLA-4表达细胞的交联；
图6显示了实施例5中的分子形式示意图：显示了1+1非对称双特异性抗体，一臂使用天然CH1/Cκ，另一臂使用含有非天然二硫键的CH1/Cλ；或者一臂使用含有非天然二硫键的CH1/Cκ，另一臂使用含有天然CH1/Cλ；
图7A显示了TJ030-PR1313精纯后的脱糖完整分子量紫外图谱以及主峰的分子归属信息；图7B显示了TJ030-PR1313经Lys-C酶切后的Fab分子量解卷积质谱图；
图8显示了非天然二硫键引入CH1/CL的PSMA 1+1双表位抗体经 GingisKHAN蛋白酶处理后的产物的分子量解卷积质谱图；
图9A显示了实施例8的FAP×CD40 2+2对称双特异性抗体分子形式示意图；图9B显示了两种FAP×CD40抗体还原分子量解卷积质谱图；图9C-9D显示了两种FAP×CD40抗体IdeS酶切后的分子量解卷积质谱图；
图10A显示了FAP×CD40抗体与CD40的FACS结合EC 50结果；图10B显示了FAP×CD40抗体与FAP的FACS结合EC 50结果；图10C和10D显示了FAP×CD40抗体在FAP存在和不存在时对CD40的激活活性结果。
具体实施方式
术语“抗原”是指任何能诱导机体发生免疫应答的物质，抗原的实例包括但不限于肽、蛋白质、糖蛋白、多糖、脂质和合成或天然存在的化学化合物或其组合。
术语“抗原结合蛋白”是指能够结合抗原的蛋白，其包括但不限于全长抗体、抗体片段或抗体与其他多肽的融合蛋白。所述“结合”例如可以是特异性结合。抗体片段的实例包括但不限于(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab')2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)dsFv，由VH和VL经链间二硫键形成的抗原结合片段；(vi)包含scFv、dsFv、Fab等片段的双抗体、双特异性抗体和多特异性抗体。此外，虽然Fv片段的两个结构域VL和VH，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science 242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA 85:5879-5883)。此类单链抗体也包括在术语抗体片段中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合域。
抗体可以是不同同种型的抗体，例如，依据抗体的重链恒定区的氨基酸序列，抗体分为不同类型(例如IgA、IgD、IgE、IgG和IgM的5种类型，进而IgG1、IgG2、IgG3、IgG4、IgA1和IgA2等的亚型)。对应上述5种类型的重链恒定区分别称为α、δ、ε、γ和μ。
抗体的轻链基于其氨基酸序列可认为是Kappa(κ)或Lamda(λ)中任一种。
术语“(轻链)CL区”是指抗体轻链的恒定区，是相关技术领域公知的区。CL区可通过常规方法确定，例如可利用与已知抗体等的同源性而确定目的区域是否为CL区，CL区的边界可改变，通常在人κ链中，CL区由107个氨基酸残基组成，人λ链中的CL区通常由106个氨基酸残基组成。人κ链CL区中的天然半胱氨酸为按照Kabat编码的第214位，人λ链的CL区中的天然半胱氨酸为按照Kabat编 码的第214位。
术语“(重链)CH1区”是指重链的第一恒定区，其为相关技术领域已知的区。本文定义的CH1区也可含CH1区之后的绞链区的一部分(可包含于Fab区的绞链区)。CH1区可通过常规方法确定，例如可利用与已知抗体等的同源性而确定目的区域是否为CH1区。由于CH1区的边界可改变，因此，在人IgG1、IgG2、IgG3、IgG4的重链中，本文定义的CH1区通常是由氨基酸残基编号118-215和额外的绞链区的一部分(例如氨基酸残基编号216-224)组成；在IgM的重链中，本文定义CH1区域通常是由氨基酸基编号118-216组成，但不限于此。
术语“Fc区”是指将抗体以木瓜酶切割时所得的2种片段中，对应于不具有抗原结合能力的片段的区。通常，Fc区是指抗体重链的C端区，其包含一部分绞链区、重链的第二恒定(CH2)区和第三恒定(CH3)区。重链Fc区的边界可改变，例如人IgG1重链Fc区由Thr225的氨基酸残基至CH3区的羧基端组成。
术语“依赖抗体的细胞毒性(ADCC)”指分泌的Ig结合至存在于一些细胞毒性细胞(例如天然杀伤(NK)细胞、嗜中性粒细胞和巨噬细胞)上的Fc受体(FcR)上，使得这些细胞毒性效应细胞能够特异性结合具有抗原的靶细胞，随后用细胞毒剂杀死靶细胞。抗体“武装”细胞毒性细胞，且绝对为这种杀伤所需。介导ADCC的主要细胞NK细胞仅表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。造血细胞上的Fc表达总结在Ravetch和Kinet,Annu.Rev.Immunol.9:457-92(1991)的第464页上的表3中。为了评估目的分子的ADCC活性，可以进行体外ADCC测定，如描述于美国专利号5,500,362或5,821,337中的体外ADCC测定。用于这类测定的效应细胞包括外周血单核细胞(PBMC)和天然杀伤(NK)细胞。备选地，或此外，可以在体内，例如在诸如公开于Clynes等,PNAS USA 95:652-656(1998)中的动物模型的动物模型中评估目的分子的ADCC活性。
术语“Fc受体”或“FcR”描述结合抗体Fc区的受体。优选的FcR是人FcR。此外，优选的FcR是结合IgG抗体的FcR(γ受体)，且包括FcγRI、FcγRII和FcγRIII亚类的受体，包括这些受体的等位基因变体和选择性剪接形式。FcγRII受体包括FcγRIIA(“激活受体”)和FcγRIIB(“抑制受体”)，它们具有主要在其胞质结构域中不同的相似氨基酸序列。激活受体FcγRIIA在其胞质结构域中包含基于免疫受体酪氨酸的激活基序(ITAM)。抑制受体FcγRIIB在其胞质结构域中包含基于免疫受体酪氨酸的抑制基序(ITIM)(参见综述M.Daeron,Annu.Rev.Immunol.15:203-234(1997))。FcR综述于Ravetch和Kinet,Annu.Rev.Immunol.9:457-92(1991)；Capel等,Immunomethods 4:25-34(1994)；及de Haas等,J.Lab.Clin.Med.126:330-41(1995)中。本文的术语“FcR”涵盖了其他FcR，包括有待在将来鉴定的那些。该术语还包括负责将母体IgG转移至胎儿的新生儿受体FcRn(Guyer等,J.Immunol.117:587(1976)和Kim 等,J.Immunol.24:249(1994))。
术语“人效应细胞”是表达一种或多种FcR并执行效应子功能的白细胞。优选地，该细胞至少表达FcγRIII并执行ADCC效应子功能。介导ADCC的人白细胞的实例包括外周血单核细胞(PBMC)、天然杀伤(NK)细胞、单核细胞、细胞毒性T细胞和嗜中性粒细胞；优选PBMC和NK细胞。可以从天然来源例如从血液分离效应细胞。
术语“依赖补体的细胞毒性”或“CDC”指在补体存在下裂解靶细胞。经典补体途径的激活由补体***第一成分(C1q)与结合于其关连抗原的(适当亚类的)抗体的结合起始。为了评估补体激活，可以进行例如Gazzano-Santoro等,J.Immunol.Methods 202:163(1996)中所述的CDC测定。
术语“治疗有效量”指在个体中治疗疾病或障碍的抗体(包括多特异性抗体)、其抗原结合抗体片段、或其衍生物的量。在肿瘤(例如癌性肿瘤)的情况下，抗体或抗体片段(例如多特异性抗体或抗体片段)的治疗有效量可以减少癌细胞的数目、减小原发性肿瘤大小、抑制(即在某种程度上减慢和优选阻止)癌细胞浸润入外周器官、抑制(即在某种程度上减慢和优选阻止)肿瘤转移、在某种程度上抑制肿瘤生长、和/或在某种程度上减轻与障碍相关的一种或多种症状。在抗体或其抗体片段、或其衍生物可以阻止生长和/或杀死现有癌细胞的程度上，它可以是细胞抑制剂和/或细胞毒剂。对于癌症治疗，可以例如通过评估存活期、疾病进展时间(TTP)、应答率(RR)、应答持续时间和/或生活质量来测量体内功效。
术语“天然二硫键”是指通常存在于野生型多肽(抗体等)的半胱氨酸-半胱氨酸间的共价键。术语“非天然二硫键”是指在上述“天然二硫键”以外的位置所形成的半胱氨酸-半胱氨酸间的共价键。
术语“多特异性抗体”是指结合两个或更多个不同的表位(例如，两个、三个、四个或更多个不同的表位)的抗体。表位可以在相同或不同的抗原上。多特异性抗体的实例之一是结合两个不同表位的“双特异性抗体”。
术语“价”表示抗体分子中存在特定数目的结合位点。天然抗体例如具有两个结合位点并且是二价的。如此，术语“四价”表示抗体分子中存在四个结合位点。
术语“氨基酸”主要是指选自以下的20种天然存在的氨基酸：丙氨酸(Ala或A)、半胱氨酸(Cys或C)、天冬氨酸(Asp或D)、谷氨酸(Glu或E)、苯丙氨酸(Phe或F)、甘氨酸(Gly或G)、组氨酸(His或H)、异亮氨酸(He或I)、赖氨酸(Lys或K)、亮氨酸(Leu或L)、甲硫氨酸(Met或M)、天冬酰胺(Asn或N)、脯氨酸(Pro或P)、谷氨酰胺(Gln或Q)、精氨酸(Arg或R)、丝氨酸(Ser或S)、苏氨酸(Thr或T)、缬氨酸(Val或V)、色氨酸(Trp或W)和酪氨酸(Tyr或Y)。术语“氨基酸残基”是指组成多肽的氨基酸在相互结合时，由于其部分基团参与了肽键的形成而失去一分子水，因此把多肽中的氨基酸单位称为氨基酸残基，即由肽键链接的氨基酸失水后剩余部分。本文中术语“氨基酸”和“氨基酸残基”可互换使用。
氨基酸“带正电荷”或“带负电荷”是根据pH 7.4时所测定的氨基酸侧链的电荷属性进行的分类。氨基酸可根据常见的侧链性质分组：(1)疏水性：正亮氨酸、Met、Ala、Val、Leu、Ile；(2)中性亲水性：Cys、Ser、Thr、Asn、Gln；(3)酸性(带负电荷)：Asp、Glu；(4)碱性(带正电荷)：His、Lys、Arg；(5)影响链方向的残基：Gly、Pro；(6)芳香族：Trp、Tyr、Phe。
术语“界面”是指源自抗原结合蛋白或抗体的第一结构域中的一个或多个氨基酸与第二结构域的一个或多个氨基酸的相互作用的结合或接触表面。示例性界面存在于例如CH1/CL之间、VH/VL之间和/或CH3/CH3之间。在一些实施方案中，界面包括例如形成界面的氨基酸之间的氢键、静电相互作用或盐桥。
术语“载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人PD-1或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。本公开所述的抗体或抗原结合片段用基因工程方法在非人源的CDR加上一个或更多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达***会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人PD-1特异性结合的抗体、或者与PD-1和PD-L1两者结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的 培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
除另有说明外，本公开的术语“第一”和“第二”仅是通用标识符，不应被理解为标识本文提供的抗原结合蛋白的具体或特定部分；本公开任意实施方案中“第一”和“第二”可以颠倒，例如，本公开描述的处于第一CH1和第一CL中的任意氨基酸取代可以备选地处于第二CH1和第二CL中。
本公开涉及的序列如下：
SEQ ID NO：1(PD-1/HC)
Figure PCTCN2021135254-appb-000003
SEQ ID NO：2(PD-1/LC)
Figure PCTCN2021135254-appb-000004
SEQ ID NO：3(PD-1/HC，S131C下划线标注)
Figure PCTCN2021135254-appb-000005
SEQ ID NO：4(PD-1/HC，L128C下划线标注)
Figure PCTCN2021135254-appb-000006
Figure PCTCN2021135254-appb-000007
SEQ ID NO：5(PD-1/HC，A129C下划线标注)
Figure PCTCN2021135254-appb-000008
SEQ ID NO：6(PD-1/HC，F170C下划线标注)
Figure PCTCN2021135254-appb-000009
SEQ ID NO：7(PD-1/HC，A141C下划线标注)
Figure PCTCN2021135254-appb-000010
SEQ ID NO：8(PD-1/LC，P119C下划线标注)
Figure PCTCN2021135254-appb-000011
Figure PCTCN2021135254-appb-000012
SEQ ID NO：9(PD-1/LC，S121C下划线标注)
Figure PCTCN2021135254-appb-000013
SEQ ID NO：10(PD-1/LC，T164C下划线标注)
Figure PCTCN2021135254-appb-000014
SEQ ID NO：11(PD-1/LC，L135C下划线标注)
Figure PCTCN2021135254-appb-000015
SEQ ID NO：12(PD-1/HC)
Figure PCTCN2021135254-appb-000016
SEQ ID NO：13(PD-1/LC)
Figure PCTCN2021135254-appb-000017
SEQ ID NO：14(CTLA-4/HC)
Figure PCTCN2021135254-appb-000018
Figure PCTCN2021135254-appb-000019
SEQ ID NO：15(CTLA-4/LC)
Figure PCTCN2021135254-appb-000020
SEQ ID NO：16(CTLA-4/HC，F126C下划线标注)
Figure PCTCN2021135254-appb-000021
SEQ ID NO：17(CTLA-4/LC，S121C下划线标注)
Figure PCTCN2021135254-appb-000022
SEQ ID NO：18(CTLA-4/HC，L128C下划线标注)
Figure PCTCN2021135254-appb-000023
SEQ ID NO：19(CTLA-4/HC，F170C下划线标注)
Figure PCTCN2021135254-appb-000024
Figure PCTCN2021135254-appb-000025
SEQ ID NO：20(CTLA-4/LC，T164C下划线标注)
Figure PCTCN2021135254-appb-000026
SEQ ID NO：21(CTLA-4/HC，S131C下划线标注)
Figure PCTCN2021135254-appb-000027
SEQ ID NO：22(CTLA-4/LC，P119C下划线标注)
Figure PCTCN2021135254-appb-000028
SEQ ID NO：23(PD-1/HC，F126C下划线标注)
Figure PCTCN2021135254-appb-000029
SEQ ID NO：24(PD-1/HC，L128C下划线标注)
Figure PCTCN2021135254-appb-000030
Figure PCTCN2021135254-appb-000031
SEQ ID NO：25(PD-1/HC，F170C下划线标注)
Figure PCTCN2021135254-appb-000032
SEQ ID NO：26(PD-1/HC，S131C下划线标注)
Figure PCTCN2021135254-appb-000033
SEQ ID NO：27(CTLA-4/LC)
Figure PCTCN2021135254-appb-000034
SEQ ID NO：28(CTLA-4/HC，P171C下划线标注)
Figure PCTCN2021135254-appb-000035
Figure PCTCN2021135254-appb-000036
SEQ ID NO：29(CTLA-4/LC，S165C下划线标注)
Figure PCTCN2021135254-appb-000037
SEQ ID NO：30(PD-1/LC)
Figure PCTCN2021135254-appb-000038
SEQ ID NO：31(PD-1/HC，P171C下划线标注)
Figure PCTCN2021135254-appb-000039
SEQ ID NO：32(PD-1/LC，S165C下划线标注)
Figure PCTCN2021135254-appb-000040
SEQ ID NO：33(PD-1/HC，T139R，F170C下划线标注)
Figure PCTCN2021135254-appb-000041
SEQ ID NO：34(PD-1/LC，S114E，T164C下划线标注)
Figure PCTCN2021135254-appb-000042
SEQ ID NO：35(CTLA-4/HC，T139D下划线标注)
Figure PCTCN2021135254-appb-000043
SEQ ID NO：36(CTLA-4/LC，S114K下划线标注)
Figure PCTCN2021135254-appb-000044
SEQ ID NO：37(PD-1/HC)
Figure PCTCN2021135254-appb-000045
SEQ ID NO：38(PD-1/LC)
Figure PCTCN2021135254-appb-000046
SEQ ID NO：39(PD-1/HC)
Figure PCTCN2021135254-appb-000047
Figure PCTCN2021135254-appb-000048
SEQ ID NO：40(PD-1/LC)
Figure PCTCN2021135254-appb-000049
SEQ ID NO：41(CTLA-4/HC)
Figure PCTCN2021135254-appb-000050
SEQ ID NO：42(CTLA-4/LC)
Figure PCTCN2021135254-appb-000051
SEQ ID NO：43(PD-1/HCDR1)
Figure PCTCN2021135254-appb-000052
SEQ ID NO：44(PD-1/HCDR2)
Figure PCTCN2021135254-appb-000053
SEQ ID NO：45(PD-1/HCDR3)
Figure PCTCN2021135254-appb-000054
SEQ ID NO：46(PD-1/LCDR1)
Figure PCTCN2021135254-appb-000055
SEQ ID NO：47(PD-1/LCDR2)
Figure PCTCN2021135254-appb-000056
SEQ ID NO：48(PD-1/LCDR3)
Figure PCTCN2021135254-appb-000057
SEQ ID NO：49(PD-1/VH)
Figure PCTCN2021135254-appb-000058
SEQ ID NO：50(PD-1/VL)
Figure PCTCN2021135254-appb-000059
SEQ ID NO：51(CTLA-4/HCDR1)
Figure PCTCN2021135254-appb-000060
SEQ ID NO：52(CTLA-4/HCDR2)
Figure PCTCN2021135254-appb-000061
SEQ ID NO：53(CTLA-4/HCDR3)
Figure PCTCN2021135254-appb-000062
SEQ ID NO：54(CTLA-4/LCDR1)
Figure PCTCN2021135254-appb-000063
SEQ ID NO：55(CTLA-4/LCDR2)
Figure PCTCN2021135254-appb-000064
SEQ ID NO：56(CTLA-4/LCDR3)
Figure PCTCN2021135254-appb-000065
SEQ ID NO：57(CTLA-4/VH)
Figure PCTCN2021135254-appb-000066
SEQ ID NO：58(CTLA-4/VL)
Figure PCTCN2021135254-appb-000067
实施例
以下结合实施例进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1实验材料和方法
1.1单克隆抗体表达纯化
用生长状态良好、处于对数生长期的CHO-S细胞(Thermo，A29133)，离心并按照6×10 6个细胞/mL接种250mL。将溶液2(用培养液9.2mL稀释800μL转染试剂，混匀)加入溶液1(用培养液10mL稀释250μg质粒，混匀)中，总体 积是20mL，轻柔混匀后室温孵育1-5min，将混合转染液逐滴加入细胞培养液中，边摇边加。然后将培养瓶置于5％CO 2，32℃摇床培养，18-22个小时后加辅料Feed(Thermo，A29133)16mL，增强剂Enhancer(Thermo，A29133)0.6mL。第五天加辅料Feed(Thermo，A29133)16mL，120rpm，5％CO 2，32℃培养，待第12-14天离心收集上清，亲和层析法纯化(MabSelect SuRe column，GE，17-5438)。
1.2双抗表达和纯化
双抗表达过程与单抗PD-1相同，纯化策略较单抗略复杂：其中第一步的亲和层析初纯与单抗相似，但有时需要使用离子交换层析进行精纯。根据抗体等电点性质，可以选择不同的阴、阳离子交换层析方法。
其中，阴离子交换层析的方法是：将一步纯化的样品上样到HiTrap Q HP column(GE，17515601)柱，用A液(20mM PB，pH 7.0)平衡，然后用0-100％B液(20mM PB，1M NaCl，pH7.0)梯度洗脱。阳离子交换层析的方法是：将一步纯化的样品上样到Capto S ImpAct预装柱(GE，17-5441-22)，用A液(50mM NaAc，50mMNaCl，pH 5.0)平衡，然后用0-100％B液(50mM NaAc，500mM NaCl，pH5.0)梯度洗脱。
1.3质谱分析
本公开中使用常规的高分辨质谱6530B ESI-Q-TOF(Agilent)和XEVO G2-XS Q-Tof(Waters)对蛋白样品进行生物分析。
1.3.1完整分子量
将样品稀释后，利用反向色谱分离并进入高分辨率质谱检测，得到含有不同质荷比信息的原始谱图，通过解卷软件处理后，获得抗体完整分子量信息。具体为：取50μg样品及标准品用流动相A(0.1％甲酸水溶液)稀释至0.5mg/mL，4℃12000rpm离心10min，取上清至进样瓶。进样前用95％流动相A平衡色谱柱(Waters，186008946)至平稳，进样后使用流动相A和流动相B(0.1％甲酸乙腈溶液)进行梯度洗脱。样品采集结束后，在目标峰出峰处得到对应的质谱数据。
1.3.2脱糖完整分子量
将样品稀释后，利用反向色谱分离并进入高分辨率质谱检测，得到含有不同质荷比信息的原始谱图，通过解卷软件处理后，获得抗体脱糖后完整分子量信息。具体为：取100μg供试品及标准品，各加入2μL肽N-糖苷酶F(PNGase F，BioLabs，P0704L)，加入50mM碳酸氢铵溶液补足体积至100μL，37℃脱糖3小时。孵育完成后，用流动相A稀释蛋白浓度为0.5μg/μL，4℃12000rpm离心10min，取上清至进样瓶。进样前用95％流动相A平衡色谱柱至平稳，进样后使用流动相A和流动相B(0.1％甲酸乙腈溶液)进行梯度洗脱。样品采集结束后，在目标峰出峰处得到对应的质谱数据。
1.3.3还原分子量
将样品稀释后，利用反向色谱分离并进入高分辨率质谱检测，得到含有不同质 荷比信息的原始谱图，通过解卷软件处理后，获得抗体还原分子量信息。具体为：取100μg供试品及标准品，加入50mM碳酸氢铵溶液补足体积至90μL，加入10μL DTT使终浓度为10mM，37℃孵育30min。孵育完成后，用流动相A稀释蛋白浓度为0.5μg/μL，4℃12000rpm离心10min，取上清至进样瓶。进样前用95％流动相A平衡色谱柱至平稳，进样后使用流动相A和流动相B(0.1％甲酸乙腈溶液)进行梯度洗脱。样品采集结束后，在目标峰出峰处得到对应的质谱数据。
1.3.4 IdeS酶切后的F(ab’) 2分子量
将样品通过免疫球蛋白G降解酶(IdeS，Promega，v7511)酶解得到Fab片段，利用反向色谱分离并进入高分辨率质谱检测，得到含有不同质荷比信息的原始谱图，通过解卷软件处理后，获得抗体F(ab’) 2片段的分子量信息，并通过分子量信息获得配对信息。具体为：取100μg供试品及标准品，加入50mM Tris-HCl(pH7.50)溶液稀释至0.5μg/μL，取100μL稀释后样品加入1μL IdeS，37℃孵育30min。反应完成后，加入1μL 10％甲酸水溶液，取上清至进样瓶。进样前用95％流动相A平衡色谱柱至平稳，进样后使用流动相A和流动相B(0.1％甲酸乙腈溶液)进行梯度洗脱。样品采集结束后，在目标峰出峰处得到对应的质谱数据。
1.3.5 Lys-C或Papain酶切后的Fab分子量
将样品通过蛋白酶(Lys-C，RHINO BIO，QIP-004-A或者Papain，Solarbio，G8430)酶解得到Fab片段，利用反向色谱分离并进入高分辨率质谱检测，得到含有不同质荷比信息的原始谱图，通过解卷软件处理后，获得抗体的Fab片段分子量信息，并通过分子量信息获得配对信息。以Lys-C酶切测定Fab分子量为例：取100μg供试品及标准品，加入50mM Tris-HCl(pH 7.50)溶液稀释至0.5μg/μL，取100μL稀释后样品加入0.25μg Lys-C，37℃孵育5min。反应完成后，加入1μL10％甲酸水溶液，取上清至进样瓶。进样前用95％流动相A平衡色谱柱至平稳，进样后使用流动相A和流动相B(0.1％甲酸乙腈溶液)进行梯度洗脱。样品采集结束后，在目标峰出峰处得到对应的质谱数据。
1.3.6游离巯基分析
为获取供试品游离巯基的位点和比例信息：取用250μg供试品，加入8M盐酸胍溶液95μL，56℃孵育40min。加热完成后加入0.1M马来酰亚胺(NEM)5μL，混匀后暗处室温避光反应35min。13000rpm离心15min，加入50mM Tris-HCl 100μL继续离心，重复3次。随后加入50mM Tris-HCl 90μL和1M DTT溶液10μL反应40min；13000rpm离心15min，加入50mM Tris-HCl 100μL继续离心，重复3次。加入1M碘乙酰胺(IAM)20μL，混匀后暗处室温避光反应35min；13000rpm离心15min，加入50mM Tris-HCl 100μL继续离心，重复3次。随后加入胰蛋白酶Trypsin，使酶和供试品比例为1:25(w/w)，37℃孵育16小时。取出后，加入1.0μL甲酸终止反应，质谱检测，分析数据。
1.4细胞交联实验
采用流式方法检测精纯后的PD-1×CTLA-4双特异抗体对高表达人PD-1和人CTLA-4的细胞的共结合能力。首先，HEK293细胞瞬时转染人CTLA-4质粒，转染后24小时，将高表达CTLA-4的HEK293细胞用Cell Trace Far red(Invitrogen，C34564)标记，将CHO-K1/PD-1稳转株用Cell Trace Violet(Invitrogen，C34557)标记，分别加入到96孔U型板中(Costar，3599)中，2E5细胞每孔，将待检测的抗体稀释到100nM、10nM、1nM、0.1nM和0.01nM，分别加入到96孔U型板中，50μL/孔，总体积为150μL/孔。4℃避光孵育1小时，流式检测CTLA-4的HEK293/CTLA-4和CHO-K1/PD-1的双阳性细胞百分比。
1.5 FAP和CD40臂的细胞结合FACS实验
离心收集对数生长期的稳定表达人FAP的CHO细胞(即CHO/FAP细胞)以及瞬时转染48小时后的HEK293细胞(即HEK293/CD40细胞)，PBS清洗后离心。细胞铺板，每孔100μL 2E5个细胞，400g离心5min。加入不同浓度的待检测抗体，冰上孵育1h，PBS清洗，400g离心5min。加入带有荧光基团的羊抗人的二抗Alexa Fluor 488冰浴染色1小时，PBS清洗两遍后上机检测。
1.6 FAPxCD40双抗对于CD40信号通路激活的影响
为了验证FAPxCD40双特异性抗体对CD40信号通路激活的影响以及FAP存在下对CD40信号通路激活的影响，利用人CD40高表达的阳性细胞株HEK-Blue CD40L细胞以及稳定表达人FAP的Flp-In CHO细胞株，用含10％热灭活血清的DMEM/F12K培养基稀释到5.5E5/mL，向96孔平底细胞培养板每孔加入90μL HEK-Blu CD40L细胞悬液,同时加入90μL培养基或者Flp-In CHO/FAP细胞株。每孔加入20μL梯度稀释后的抗体，无抗体组作为阴性对照，37℃5％CO2培养箱过夜培养。取另外96孔平底细胞培养板加入180μL Quanti-Blue检测试剂，加入20μL细胞培养上清，室温孵育30min后用酶标仪读OD655数值。
实施例2在CH1/CL相互作用界面中引入氨基酸取代
我们设计了如表1所示的引入非天然二硫键的位置。
表1向CH1/CL引入非天然二硫键的位置信息
Figure PCTCN2021135254-appb-000068
Figure PCTCN2021135254-appb-000069
1EU编码
2Kabat编码
我们还设计了如表2所示的引入静电效应的氨基酸突变的位置。
表2向CH1/CL引入静电效应的位置信息
Figure PCTCN2021135254-appb-000070
1EU编码
2Kabat编码
实施例3 PD-1单克隆抗体表达载体的构建和蛋白表达纯化
将编码PD-1-IgG1-LALA抗体的重链(序列如SEQ ID NO：1所示)和轻链(序列如SEQ ID NO：2所示)的核酸分别构建到pTT5质粒载体上。在其基础上，在重链中引入进行C220A突变(EU编码)；在轻链中引入C214A突变(Kabat编码)。同时引入这两个突变，彻底去除了天然存在于这些位置(CH1第220位-和CL第214位)的链间二硫键。
为表达由非天然二硫键连接的PD-1单抗，我们在天然二硫键去除的重链中进一步引入S131C(SEQ ID NO：3)，L128C(SEQ ID NO：4)，A129C(SEQ ID NO：5)，或F170C(SEQ ID NO：6)；类似地，在天然二硫键去除的轻链中进一步引入P119C(SEQ ID NO：8)，S121C(SEQ ID NO：9)，或T164C(SEQ ID NO：10)，如表3所示。
表3含非天然二硫键的PD-1单抗轻重链序列及质粒配比信息
Figure PCTCN2021135254-appb-000071
Figure PCTCN2021135254-appb-000072
根据表3所示的PD-1单抗序列和质粒配比信息，按照实施例1.1和1.2的方法表达和纯化抗体，引入非天然二硫键后的PD-1抗体一步纯化后的蛋白表达量和纯度与含天然二硫键的PD-1抗体相当，无明显变化。为了确认非天然二硫键是否形成，我们使用IdeS酶酶切处理对应的PD-1抗体，得到了F(ab’) 2的分子片段(图1)，证明在上述特定位置S131C-P119C、L128C-S121C、A129C-S121C、F170C-T164C引入的半胱氨酸能够形成CH1/CL链间二硫键。
为了进一步确认非天然二硫键的形成，特别是在抗体分子中是否存在未配对的游离半胱氨酸残基，我们按照实施例1.3的方法进一步定量表征了单抗分子的游离巯基。我们设定，游离巯基比例(％)＝NEM修饰肽段质谱信号强度/肽段质谱总信号强度×100％。结果表明，由非天然二硫键S131C-P119C、L128C-S121C、A129C-S121C、F170C-T164C组成的PD-1单抗中，整体游离巯基的比例<3％，结果表明这些引入的非天然半胱氨酸残基能够配对形成二硫键。
实施例4非天然二硫键引入CH1/Cκ的PD-1×CTLA-4双抗
4.1分子形式
鉴于非天然二硫键运用于PD-1单抗上的出色表现，我们进一步设计了基于KIH(S354C/T366W；Y349C/T366S/L368A/Y407V)的1+1非对称双特异性抗体。理论上，可以在PD-1臂或者CTLA-4臂之一上使用非天然二硫键；PD-1臂的Fc中包含凹陷(hole)，CTLA-4臂的Fc中包含凸起(knob)，反之亦然；由此可以形成四种组合形式。在本实施例中，使PD-1臂的Fc在包含形成凸起(knob)的氨基酸突变T366W，使CTLA-4臂的Fc中包含形成凹陷(hole)的氨基酸突变T366S/L368A/Y407V(图2给出了结构示意图)。
根据表4所示的PD-1×CTLA-4双抗序列和质粒配比信息，按照实施例1.1和1.2的方法表达和纯化双特异性抗体。
表4含非天然二硫键的PD-1×CTLA-4双抗序列和质粒配比信息
Figure PCTCN2021135254-appb-000073
Figure PCTCN2021135254-appb-000074
4.2初纯产物的错配质谱分析
4.2.1非天然二硫键对对应Fab的表达影响
以PD-1×CTLA-4双抗为例，对于在CTLA-4臂引入非天然二硫键L128C-S121C并且在PD-1臂中保留天然二硫键的TJ030-PR1103，在编码4条链的质粒共转表达时，在PD-1臂的竞争下，CTLA-4臂的表达量显著下降。双抗初纯产物经木瓜蛋白酶酶切后的分子量解卷积质谱结果(图3)显示，TJ030-PR1101 CTLA-4臂峰强度/PD-1臂峰强度＝1:2，TJ030-PR1103CTLA-4臂峰强度/PD-1峰强度＝1:46。相比之下，引入非天然二硫键F170C-T164C的TJ030-PR1104和引入非天然二硫键S131C-P119C的TJ030-PR1105，在天然二硫键Fab臂的竞争下，并不会降低表达量，其CTLA-4臂峰强度/PD-1臂峰强度维持在1:2左右。
4.2.2轻链错配比例
为进一步定量分析轻链错配，根据图3的分子量解卷积质谱结果计算正确配对比例，计算公式为：(正确PD-1臂峰强度+正确CTLA-4臂峰强度)/(正确PD-1臂峰强度+正确CTLA-4臂峰强度+其他杂质峰强度)，结果参见表5。可以看出，TJ030-PR1103(L128C-S121C)和TJ030-PR1104(F170C-T164C)无论非天然二硫键至于CTLA-4臂还是PD-1臂，相对于TJ030-PR1101(采用天然二硫键)和现有技术报道的TJ030-PR1102(F126C-S121C)，正确配对比例均有较大提高。对于TJ030-PR1105(S131C-P119C)，当非天然二硫键置于PD-1臂时，正确配对比例相对于TJ030-PR1101(采用天然二硫键)和现有技术报道的TJ030-PR1102(F126C-S121C)，正确配对比例均有较大提高。
表5向CH1/Cκ引入非天然二硫键后PD-1×CTLA-4双抗的正确配对比例
Figure PCTCN2021135254-appb-000075
4.4精纯产物的质谱错配分析
以TJ030-PR1104为例，根据实施例1.2的方法对一步纯产物精纯后，如图4A所示收集特征峰用于后续检测。
4.4.1完整分子量和脱糖完整分子量
采用1.3.2的方法进行脱糖完整分子量检测，结果如图4B所示，脱糖完整分子量质谱发现了正确配对的1+1的非对称双抗的目的蛋白，同时发现了H2L1(两重链一轻链形式，缺CTLA-4臂轻链)的副产物以及PD-1轻链半胱氨酸缀合物(LC PD-1-Cys)的形成。可能是由于CTLA-4臂，特别是CTLA-4臂的轻链表达量不足，导致H2L1的大量产生。我们推测这类H2L1不完整的抗体分子需要额外的LC PD-1以进一步稳定结构；即便如此，经过精纯后的TJ030-PR1104仍然没有检测到轻链错配的产物。
4.4.2还原分子量检测和脱糖还原分子量
采用1.3.3的方法进行还原分子量和脱糖还原分子量检测，结果如图4C所示，除了发现双抗TJ030-PR1104对应的4条还原蛋白序列以外，还原分子量和脱糖还原分子量质谱还检测到了配对的CTLA-4重链和或轻链(HC CTLA-4-LC CTLA-4)，进一步确认了F170C-T164C非天然二硫键的正确形成。
4.5细胞交联实验
根据实施例1.4，采用流式方法检测精纯后的PD-1×CTLA-4双特异抗体TJ030-PR1102(F126C-S121C置于CTLA-4臂)、TJ030-PR1104(F170C-T164C置于CTLA-4臂)、TJ030-PR1106(F126C-S121C置于PD-1臂)和TJ030-PR1108(F170C-T164C置于PD-1臂)分别对高表达人PD-1和人CTLA-4的细胞的共结合能力，其中CTLA-4单抗和IgG作为阴性对照。交联实验的结果表明，1+1非对称的PD-1×CTLA-4双抗能够将表达PD-1的细胞和表达CTLA-4的细胞交联到一起(表6和图5)，并且双抗浓度在0.03nM到10nM的浓度范围里，随着浓度的逐渐提高，由细胞交联产生的双阳性细胞比例也逐渐升高。这种细胞交联现象仅 在双抗分子孵育的情况下出现，单抗或IgG1孵育不会出现细胞交联。另外，引入F170C-T164C这对非天然二硫键的双抗分子TJ030-PR1104和TJ030-PR1108相对于已报道的F126C-S121C的非天然二硫键TJ030-PR1102在0.03nM-100nM的各浓度点均检测到显著更多的双阳性细胞。
表6细胞交联实验中双阳性细胞比例随浓度变化的结果
Figure PCTCN2021135254-appb-000076
实施例5 PD-1×CTLA-4双抗表达中质粒配比的优化
采用实施例1.1的方法进行双抗表达，在瞬转表达过程中，我们提高了轻链的质粒配比(重链和轻链质粒摩尔比从1:1变为2:3)，希望降低“两重链一轻链(H2L1)”构型抗体的对应比例。另外，我们在1+1非对称双抗的一个臂引入CH1/Cκ或其非天然二硫键突变体，另一臂引入CH1/Cλ或其非天然二硫键突变体，以便于后续使用Kappa Select或者Lambda Select进行纯化，并且研究轻链亚型的选择对降低轻链错配的影响。表7给出了PD-1×CTLA-4双抗序列和质粒配比信息。
表7 PD-1×CTLA-4双抗序列和质粒配比信息
Figure PCTCN2021135254-appb-000077
Figure PCTCN2021135254-appb-000078
根据实施例1的方法精纯PD-1×CTLA-4双抗，并进行质谱分析。如表8所示，在PD-1×CTLA-4双抗的CH1/CL界面引入非天然二硫键P171C-S165C(TJ030-PR1304和TJ030-PR1309)、F170C-T164C(TJ030-PR1317和TJ030-PR1313)后，无论将非天然二硫键置于CTLA-4臂还是PD-1臂，相对于采用天然二硫键的双抗TJ030-PR1301和TJ030-PR1306具显著提高了正确配对比例。
表8向CH1/CL引入非天然二硫键后PD-1×CTLA-4双抗的正确配对比例
Figure PCTCN2021135254-appb-000079
*正确配对和错配配对的Fab分子量非常接近(<2Da)，比较难以判断。
以TJ030-PR1313为例，Lys-C酶切经过阳离子交换精纯后的双抗，表明Fab完全正确配对，无轻链错配现象。脱糖完整分子量符合预期，无明显缺少轻链的H2L1副产物出现(图7)。
如表9所示，TJ030-PR1231和TJ030-PR1317相比，均在PD-1臂中引入氨基酸突变F170C-T164C，并且PD-1臂中轻链为κ亚型，两者的区别仅在于TJ030-PR1231的CTLA-4臂中轻链为κ亚型，TJ030-PR1317的CTLA-4臂中轻链为λ亚型，TJ030-PR1317显示出更高的正确配对比例；即两臂采用不同的轻链亚型有利于提高双特异性抗体的正确配对比例。
表9 PD-1×CTLA-4双抗中轻链亚型的选择对正确配对比例的影响
Figure PCTCN2021135254-appb-000080
实施例6引入静电效应降低轻链错配
在CH1第139位和CL第114位引入静电效应突变后的双抗分子TJ030-PR1220、TJ030-PR1221和TJ030-PR1222的序列和质粒配比信息在表7中示出，结果显示引入静电效应能够降低轻链错配。
向TJ030-PR1231的PD-1臂和CTLA-4臂的CH1第139位和CL第114位分别引入静电效应突变后，得到双抗分子TJ030-PR1230(序列和质粒配比信息如表7所示)，一步初纯产物无轻链错配(表10)，证明了HC139-LC114的静电作用可以进一步降低轻链错配。
表10向CH1/CL引入静电效应后PD-1×CTLA-4双抗的正确配对比例
Figure PCTCN2021135254-appb-000081
另外，结合表5可以看出，TJ030-PR1230与TJ030-PR1108相比，增加轻链(特别是表达弱的轻链CTLA-4臂)的转染比例，也可以降低轻链错配。
实施例7非天然二硫键引入CH1/CL的PSMA 1+1双表位抗体
按照表11所示的抗体序列和质粒配比构建PSMA 1+1不对称双表位抗体，同样采用了KiH的方式实现重链的异源二聚(T366W；T366S/L368A/Y407V)的1+1非对称双特异性抗体，并对ProteinA初纯后的产物进行质谱分析。经过GingisKHAN蛋白酶处理后的产物，均没有发现轻链错配产物(图8)。
表11 PSMA 1+1双表位抗体序列和质粒配比信息
Figure PCTCN2021135254-appb-000082
Figure PCTCN2021135254-appb-000083
SEQ ID NO：59(J591/HC)
Figure PCTCN2021135254-appb-000084
SEQ ID NO：60(J591/LC)
Figure PCTCN2021135254-appb-000085
SEQ ID NO：61(006/HC，F170C下划线标注)
Figure PCTCN2021135254-appb-000086
SEQ ID NO：62(006/LC，T164C下划线标注)
Figure PCTCN2021135254-appb-000087
SEQ ID NO：63(J591/HC)
Figure PCTCN2021135254-appb-000088
Figure PCTCN2021135254-appb-000089
SEQ ID NO：64(J591/LC)
Figure PCTCN2021135254-appb-000090
SEQ ID NO：65(006/HC，P171C下划线标注)
Figure PCTCN2021135254-appb-000091
SEQ ID NO：66(006/LC，S165C下划线标注)
Figure PCTCN2021135254-appb-000092
实施例8 FAP×CD40双抗的表达纯化和表征
按照表12所示的抗体序列和质粒配比构建和表达FAP×CD40双抗(分子形式参见图9A)。
表12含非天然二硫键的FAP×CD40双抗质粒配比信息
Figure PCTCN2021135254-appb-000093
SEQ ID NO：67(CD40-Fc-FAP/HC，P171C下划线标注)
Figure PCTCN2021135254-appb-000094
Figure PCTCN2021135254-appb-000095
SEQ ID NO：68(CD40/LC)
Figure PCTCN2021135254-appb-000096
SEQ ID NO：69(FAP/LC，S165C下划线标注)
Figure PCTCN2021135254-appb-000097
SEQ ID NO：70(CD40-Fc-FAP/HC，P171C下划线标注)
Figure PCTCN2021135254-appb-000098
SEQ ID NO：71(CD40/LC，S165C下划线标注)
Figure PCTCN2021135254-appb-000099
Figure PCTCN2021135254-appb-000100
SEQ ID NO：72(FAP/LC)
Figure PCTCN2021135254-appb-000101
表13亲本CD40抗体9E5-25和亲本FAP抗体Ab10的CDR序列
Figure PCTCN2021135254-appb-000102
SEQ ID NO：78(CD40/VH)
Figure PCTCN2021135254-appb-000103
SEQ ID NO：79(CD40/VL)
Figure PCTCN2021135254-appb-000104
SEQ ID NO：86(FAP/VH)
Figure PCTCN2021135254-appb-000105
SEQ ID NO：87(FAP/VL)
Figure PCTCN2021135254-appb-000106
一步ProteinA纯化以后的双特异性抗体的SEC纯度>99％，脱糖完整分子量，还原分子量和IdeS酶切后的分子量均符合预期，并未发现无法归属的副产物(图9B、图9C)。
如图10A和图10B所示，双特异性抗体ERP2006-BS0012和ERP2006-BS0015与CD40的FACS结合EC 50分别为0.471nM和0.456nM。ERP2006-BS0012和ERP2006-BS0015与FAP的FACS结合EC 50分别为0.349nM和0.336nM。双抗ERP2006-BS0012和ERP2006-BS0015对FAP的亲和力与亲本FAP单抗Ab10相当，对于CD40的亲和力与亲本CD40单抗9E5-25相当。
图10C和图10D显示，ERP2006-BS0012和ERP2006-BS0015在没有FAP存在的情况下具有CD40的激活活性，但是其活性弱于亲本抗体9E5-25，这一特征使得双特异性抗体在FAP不存在的外周组织CD40激活活性下降，能降低CD40单抗的外周毒性。在FAP蛋白存在的情况下，ERP2006-BS0012和ERP2006-BS0015对CD40的激活活性明显增强，说明具有FAP依赖的CD40激活活性，其对CD40的激活要强于亲本CD40单抗9E5-25，该特征使得双特异抗体在FAP高表达的肿瘤部位具有更强的CD40激活活性。
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本公开的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚结合。

Claims (20)

  1. 一种二聚化多肽，其包含重链恒定区1(CH1)和轻链恒定区(CL)，其中：CH1和CL在选自(i-1)至(i-6)的位置中的一组或多组包含天然非半胱氨酸至半胱氨酸的氨基酸取代：
    (i-1)CH1的第170位和CL的第164位，
    (i-2)CH1的第128位和CL的第121位，
    (i-3)CH1的第129位和CL的第121位，
    (i-4)CH1的第131位和CL的第119位，
    (i-5)CH1的第141位和CL的第135位，和
    (i-6)CH1的第171位和CL的第165位。
  2. 根据权利要求1所述的二聚化多肽，其中所述CH1进一步包含天然半胱氨酸至非半胱氨酸的氨基酸取代，所述CL进一步包含天然半胱氨酸至非半胱氨酸的氨基酸取代；
    优选地，所述CH1进一步包含氨基酸取代C220A，所述CL进一步包含氨基酸取代C214A。
  3. 根据权利要求1或2所述的二聚化多肽，其中：所述CH1和CL包含以下氨基酸取代：
    (a)CH1中的C220A和CL中的C214A；和
    (b)选自以下组中的至少一组的氨基酸取代：
    (b-1)CH1中的F170C和CL中的T164C；
    (b-2)CH1中的L128C和CL中的S121C；
    (b-3)CH1中的A129C和CL中的S121C；
    (b-4)CH1中的S131C和CL中的P119C；
    (b-5)CH1中的A141C和CL中的L135C；和
    (b-6)CH1中的P171C和CL中的S165C；
    优选地，所述CH1和CL包含以下氨基酸取代:
    (a)CH1中的C220A和CL中的C214A；和
    (b)CH1中的F170C和CL中的T164C；或者
    优选地，所述CH1和CL包含以下氨基酸取代：
    (a)CH1中C220A和CL中的C214A；和
    (b)CH1中P171C和CL中的S165C。
  4. 根据权利要求1至3中任一项所述的二聚化多肽，其中：所述CH1和CL 进一步包含使得CH1和CL之间形成静电相互作用界面的氨基酸取代；
    优选地，使得CH1和CL之间形成静电相互作用界面的氨基酸取代位于CH1的第139位和CL的第114位；
    更优选地，所述CH1和CL进一步包含选自以下任一组的氨基酸取代：
    (1)CH1中的T139R和CL中的S114E；
    (2)CH1中的T139R和CL中的S114D；
    (3)CH1中的T139K和CL中的S114E；
    (4)CH1中的T139K和CL中的S114D；
    (5)CH1中的T139D和CL中的S114K；
    (6)CH1中的T139D和CL中的S114R；
    (7)CH1中的T139E和CL中的S114K；和
    (8)CH1中的T139E和CL中的S114R。
  5. 根据权利要求1至4中任一项所述的二聚化多肽，其中：所述CH1和CL包含选自(1)-(4)中任一项的氨基酸取代：
    (1)CH1中的C220A和CL中的C214A；CH1中的F170C和CL中的T164C；和CH1中的T139R和CL中的S114E；
    (2)CH1中的C220A和CL中的C214A；CH1中的F170C和CL中的T164C；和CH1中的T139D和CL中的S114K；
    (3)CH1中的C220A和CL中的C214A；CH1中的P171C和CL中的S165C；和CH1中的T139R和CL中的S114E；和
    (4)CH1中的C220A和CL中的C214A；CH1中的P171C和CL中的S165C；和CH1中的T139D和CL中的S114K。
  6. 一种抗原结合蛋白，其包含权利要求1-5中任一项所述的二聚化多肽；优选地，所述抗原结合蛋白包含或是多特异性抗体，更优选双特异性抗体。
  7. 根据权利要求7所述的抗原结合蛋白，其包含第一抗原结合域，其中所述第一抗原结合域包含Fab，所述Fab包含第一重链可变区VH1、第一轻链可变区VL1和权利要求1-6中任一项所述的二聚化多肽，所述二聚化多肽中所述CH1为第一CH1，所述CL为第一CL；
    优选地，所述抗原结合蛋白还包含第二抗原结合域，其中所述第二抗原结合域包含第二重链可变区VH2和第二轻链可变区VL2，并且所述第一抗原结合域和第二抗原结合域结合不同的抗原或者结合同一种抗原上的不同的表位；
    更优选地，所述第二抗原结合域包含Fab，所述Fab包含第二重链可变区VH2、第二CH1、第二轻链可变区VL2和第二CL2。
  8. 根据权利要求7所述的抗原结合蛋白，其中：
    所述第一CH1和第一CL包含以下氨基酸取代：
    (a)CH1中的C220A和CL中的C214A；和
    (b)选自以下组中的至少一组的氨基酸取代：
    (b-1)CH1中的F170C和CL中的T164C；
    (b-2)CH1中的L128C和CL中的S121C；
    (b-3)CH1中的A129C和CL中的S121C；
    (b-4)CH1中的S131C和CL中的P119C；
    (b-5)CH1中的A141C和CL中的L135C；和
    (b-6)CH1中的P171C和CL中的S165C；
    优选地，所述第一CH1和第一CL包含以下氨基酸取代：
    (a)CH1中的C220A和CL中的C214A；和
    (b)CH1中的F170C和CL中的T164C；
    或者
    优选地，所述第一CH1和第一CL包含以下氨基酸取代：
    (a)CH1中的C220A和CL中的C214A；和
    (b)CH1中的P171C和CL中的S165C。
  9. 根据权利要求7-8中任一项所述的抗原结合蛋白，其中：
    所述第一CH1和第一CL进一步包含使得所述第一CH1和第一CL之间形成静电相互作用界面的氨基酸取代，
    优选地，所述氨基酸取代位于第一CH1的第139位和第一CL的第114位；和/或所述第二CH1和第二CL包含使得第二CH1和第二CL之间形成静电相互作用界面的氨基酸取代，优选地，所述氨基酸取代位于第二CH1的第139位和第二CL的第114位；
    更优选地，所述第一CH1和第一CL进一步包含选自以下任一组的氨基酸取代：
    (1)CH1中的T139R和CL中的S114E；
    (2)CH1中的T139R和CL中的S114D；
    (3)CH1中的T139K和CL中的S114E；和
    (4)CH1中的T139K和CL中的S114D；和/或
    所述第二CH1和第二CL包含选自以下任一组的氨基酸取代：
    (1)CH1中的T139D和CL中的S114K；
    (2)CH1中的T139D和CL中的S114R；
    (3)CH1中的T139E和CL中的S114K；和
    (4)CH1中的T139E和CL中的S114R；或者
    更优选地，所述第一CH1和第一CL包含选自以下任一组的氨基酸取代：
    (1)CH1中的T139D和CL中的S114K；
    (2)CH1中的T139D和CL中的S114R；
    (3)CH1中的T139E和CL中的S114K；和
    (4)CH1中的T139E和CL中的S114R；和/或
    所述第二CH1和第二CL包含选自以下任一组的氨基酸取代：
    (1)CH1中的T139R和CL中的S114E；
    (2)CH1中的T139R和CL中的S114D；
    (3)CH1中的T139K和CL中的S114E；和
    (4)CH1中的T139K和CL中的S114D。
  10. 根据权利要求7-9中任一项所述的抗原结合蛋白，其中：
    所述第一CH1和第一CL包含以下氨基酸取代：
    (a)CH1中的C220A和CL中的C214A；
    (b)选自以下组中的至少一组的氨基酸取代：
    (b-1)CH1中的F170C和CL中的T164C；
    (b-2)CH1中的L128C和CL中的S121C；
    (b-3)CH1中的A129C和CL中的S121C；
    (b-4)CH1中的S131C和CL中的P119C；
    (b-5)CH1中的A141C和CL中的L135C；和
    (b-6)CH1中的P171C和CL中的S165C；和
    (c)选自以下组中的任一组的氨基酸取代：
    (c-1)CH1中的T139R和CL中的S114E；
    (c-2)CH1中的T139R和CL中的S114D；
    (c-3)CH1中的T139K和CL中的S114E；和
    (c-4)CH1中的T139K和CL中的S114D；
    并且所述第二CH1和第二CL包含选自以下任一组的氨基酸取代：
    (1)CH1中的T139D和CL中的S114K；
    (2)CH1中的T139D和CL中的S114R；
    (3)CH1中的T139E和CL中的S114K；和
    (4)CH1中的T139E和CL中的S114R；
    或者，
    所述第一CH1和第一CL包含以下氨基酸取代：
    (a)CH1中的C220A和CL中的C214A；
    (b)选自以下组中的至少一组的氨基酸取代：
    (b-1)CH1中的F170C和CL中的T164C；
    (b-2)CH1中的L128C和CL中的S121C；
    (b-3)CH1中的A129C和CL中的S121C；
    (b-4)CH1中的S131C和CL中的P119C；
    (b-5)CH1中的A141C和CL中的L135C；和
    (b-6)CH1中的P171C和CL中的S165C；和
    (c)选自以下组中的任一组的氨基酸取代：
    (c-1)CH1中的T139D和CL中的S114K；
    (c-2)CH1中的T139D和CL中的S114R；
    (c-3)CH1中的T139E和CL中的S114K；和
    (c-4)CH1中的T139E和CL中的S114R；
    并且所述第二CH1和第二CL包含选自以下组中任一组的氨基酸取代：
    (1)CH1中的T139R和CL中的S114E；
    (2)CH1中的T139R和CL中的S114D；
    (3)CH1中的T139K和CL中的S114E；和
    (4)CH1中的T139K和CL中的S114D。
  11. 根据权利要求7-10中任一项所述的抗原结合蛋白，其中：
    (1)所述第一CH1和第一CL包含以下氨基酸取代：
    (a)CH1中的C220A和CL中的C214A；
    (b)CH1中的F170C和CL中的T164C；和
    (c)CH1中的T139R和CL中的S114E；
    并且所述第二CH1和第二CL包含以下氨基酸取代：
    CH1中的T139D和CL中的S114K；
    (2)所述第一CH1和第一CL包含以下氨基酸取代：
    (a)CH1中的C220A和CL中的C214A；
    (b)CH1中的F170C和CL中的T164C；和
    (c)CH1中的T139D和CL中的S114K；
    并且所述第二CH1和第二CL包含以下氨基酸取代：
    CH1中的T139R和CL中的S114E；
    (3)所述第一CH1和第一CL包含以下氨基酸取代：
    (a)CH1中的C220A和CL中的C214A；
    (b)CH1中的P171C和CL中的S165C；和
    (c)CH1中的T139R和CL中的S114E；
    并且所述第二CH1和第二CL包含以下氨基酸取代：
    CH1中的T139D和CL中的S114K；或者
    (4)所述第一CH1和第一CL包含以下氨基酸取代：
    (a)CH1中的C220A和CL中的C214A；
    (b)CH1中的P171C和CL中的S165C；和
    (c)CH1中的T139D和CL中的S114K；
    并且所述第二CH1和第二CL包含以下氨基酸取代：
    CH1中的T139R和CL中的S114E。
  12. 根据权利要求7至11中任一项所述的抗原结合蛋白，其中：所述第一CL来自抗体κ轻链(Cκ)；所述第二CL来自抗体λ轻链(Cλ)或κ轻链(Cκ)；优选地，所述第一CL来自κ轻链且所述第二CL来自λ轻链。
  13. 根据权利要求6至12中任一项所述的抗原结合蛋白，其中：所述抗原结合蛋白还包含Fc区，所述Fc区包含能够彼此缔合的第一亚基Fc1与第二亚基Fc2，所述Fc1和/或所述Fc2选自人IgG1、IgG2、IgG3和IgG4的Fc。
  14. 根据权利要求13所述的抗原结合蛋白，其中：所述Fc1和/或所述Fc2包含改变所述抗原结合蛋白的半衰期的修饰，其中所述半衰期取决于FcRn结合亲和力；所述Fc1和/或所述Fc2包含改变效应子功能的修饰，其中对Fcγ受体或C1q补体蛋白的结合亲和力增大或减小；和/或所述Fc1和Fc2中包含这样的氨基酸取代，使得与Fc1相比，Fc1优先与Fc2配对。
  15. 根据权利要求7至14中任一项所述的抗原结合蛋白，其中所述第一抗原结合域特异性结合CTLA-4，且所述第二抗原结合域特异性结合PD-1；或者，所述第一抗原结合域特异性结合PD-1，且所述第二抗原结合域特异性结合CTLA-4；
    优选地，所述第一抗原结合域包含：序列如SEQ ID NO：51所示的HCDR1，序列如SEQ ID NO：52所示的HCDR2，序列如SEQ ID NO：53所示的HCDR3，序列如SEQ ID NO：54所示的LCDR1，序列如SEQ ID NO：55所示的LCDR2，和序列如SEQ ID NO：56所示的LCDR3；和/或所述第二抗原结合域包含：序列如SEQ ID NO：43所示的HCDR1，序列如SEQ ID NO：44所示的HCDR2，序列如SEQ ID NO：45所示的HCDR3，序列如SEQ ID NO：46所示的LCDR1，序列如SEQ ID NO：47所示的LCDR2，和序列如SEQ ID NO：48所示的LCDR3；或者
    所述第一抗原结合域包含序列如SEQ ID NO：43所示的HCDR1，序列如SEQ ID NO：44所示的HCDR2，序列如SEQ ID NO：45所示的HCDR3，序列如SEQ ID NO：46所示的LCDR1，序列如SEQ ID NO：47所示的LCDR2，和序列如SEQ ID NO：48所示的LCDR3；：和/或所述第二抗原结合域包含：序列如SEQ ID NO： 51所示的HCDR1，序列如SEQ ID NO：52所示的HCDR2，序列如SEQ ID NO：53所示的HCDR3，序列如SEQ ID NO：54所示的LCDR1，序列如SEQ ID NO：55所示的LCDR2，和序列如SEQ ID NO：56所示的LCDR3；
    更优选地，所述第一抗原结合域包含：序列如SEQ ID NO：57所示的重链可变区和序列如SEQ ID NO：58所示的轻链可变区；和/或所述第二抗原结合域包含：序列如SEQ ID NO：49所示的重链可变区和序列如SEQ ID NO：50所示的轻链可变区，或者
    所述第一抗原结合域包含：序列如SEQ ID NO：49所示的重链可变区和序列如SEQ ID NO：50所示的轻链可变区；和/或所述第二抗原结合域包含：序列如SEQ ID NO：57所示的重链可变区和序列如SEQ ID NO：58所示的轻链可变区。
  16. 核酸分子，其编码根据权利要求1至5中任一项所述的二聚化多肽或根据权利要求6至15中任一项所述的抗原结合蛋白。
  17. 宿主细胞，其包含根据权利要求16所述的核酸分子。
  18. 一种制备根据权利要求1至5中任一项所述的二聚化多肽或根据权利要求6至15中任一项所述的抗原结合蛋白的方法，其包括以下步骤：
    (1)用包含权利要求16所述的核酸分子的核酸表达载体转化宿主细胞；
    (2)在容许合成所述抗原结合蛋白的条件下培养所述宿主细胞得到细胞培养物；和
    (3)从所述细胞培养物中回收抗原结合蛋白；
    优选地，所述核酸表达载体包括编码重链的质粒和编码轻链的质粒，其中编码重链的质粒与编码轻链的质粒的摩尔比为1:(1-10)，优选1:(1-5)，更优选2:3。
  19. 一种药物组合物，其包含权利要求6至15中任一项所述的抗原结合蛋白和药学上可接受的载体。
  20. 根据权利要求1至5中任一项所述的二聚化多肽或权利要求6至15中任一项所述的抗原结合蛋白在制备药物中的用途；
    优选地，所述药物为治疗癌症、自身免疫性疾病或炎性疾病的药物。
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