WO2021136227A1 - Heterodimer induced by ch3 structural domain modification, preparation method therefor and use thereof - Google Patents

Heterodimer induced by ch3 structural domain modification, preparation method therefor and use thereof Download PDF

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WO2021136227A1
WO2021136227A1 PCT/CN2020/140595 CN2020140595W WO2021136227A1 WO 2021136227 A1 WO2021136227 A1 WO 2021136227A1 CN 2020140595 W CN2020140595 W CN 2020140595W WO 2021136227 A1 WO2021136227 A1 WO 2021136227A1
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domain
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mutated
heterodimer
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周易
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周易
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C07K2317/526—CH3 domain

Definitions

  • the invention belongs to the field of antibody engineering, and specifically relates to a heterodimer induced by the transformation of a CH3 structural domain, and a preparation method and application thereof.
  • IgG type bispecific antibodies have similar structure, physicochemical properties and Fc segment functions to common antibodies.
  • an IgG bispecific antibody consists of two heavy chains with different amino acid sequences (ie, heavy chain HC_A against antigen A and heavy chain HC_B against antigen B) and two light chains with different amino acid sequences (ie light chain against antigen A).
  • IgG-type bispecific antibodies There are many ways to construct IgG-type bispecific antibodies, and one of the important methods is to modify Fc to form heterodimers. As early as the 1990s, Carter et al. used the "knob-into-hole" model to modify the Fc segment of antibodies, and successfully realized the preparation of bispecific antibodies (Ridgway, Presta et al. 1996; Carter 2001). Carter et al.
  • US2010286374A1 discloses a method for promoting the formation of heterodimers by using static electricity. Specifically, the charged amino acids in the CH3 domains of the two heavy chains are mutated to oppositely charged amino acids, so that the CH3 domain of one heavy chain is generally positively charged, and the CH3 domain of the other heavy chain is generally positively charged. It is negatively charged, and electrostatic repulsion of the same charge will inhibit the formation of homodimers. However, electrostatic effect cannot completely inhibit the formation of homodimers. The introduction of too many mutations will cause the decrease of protein expression level, reflecting the complexity of the interaction relationship between interface amino acids in the case of multiple amino acid mutations.
  • CN106883297A discloses a method. Specifically, on the basis of the "convex-concave” model of Carter et al., the F405K mutation is further introduced at one end of the “concave” to enhance the electrostatic repulsion between amino acids at the "concave-concave” interface. K409A mutation is introduced at the "convex” end to avoid electrostatic repulsion between amino acids at the "concave-concave” interface, thereby inhibiting the formation of "concave-concave” homodimers and maintaining the “convex-concave” heterodimer form. Although the method disclosed in this patent can increase the formation of heterodimers, one of the solutions can only increase the proportion of heterodimers up to 93%.
  • US20150307628A1 discloses another method that uses the "convex-concave” model and electrostatic interaction to promote the formation of heterodimers.
  • one of the schemes created a “convex” on the CH3 domain of the first heavy chain of Fc through K409W point mutations, and created a “convex” on the CH3 of the second heavy chain through F405T and D399V point mutations.
  • the "concave”; the CH3 domain of the first heavy chain was negatively charged by the K360E point mutation, and the CH3 on the second heavy chain was positively charged by the Q347R point mutation.
  • This scheme can increase the proportion of heterodimers up to 91.4 ⁇ 1.2% without introducing disulfide bonds.
  • the purpose of the present invention is to overcome the deficiencies in the prior art, and provide a heterodimer induced by the transformation of the CH3 structure domain, and a preparation method and application thereof.
  • the present invention combines the "convex-concave" model with electrostatic interaction through comprehensive consideration of intermolecular interactions, such as ionic interactions, steric interactions, and hydrophobic interactions, and selects preferred CH3 mutation sequences, which are more likely to form heterologous two.
  • Polymers, without forming homodimers, can increase the proportion of heterodimer molecules to more than 95% without introducing disulfide bonds.
  • the heterodimer in the present invention refers to an antibody molecule or fragment containing two heavy chains with different amino acid sequences, including but not limited to bispecific antibodies, monovalent antibodies, Fc fusion proteins and the like.
  • the first aspect of the present invention is to provide a heterodimer, which contains a first polypeptide chain and a second polypeptide chain, the first polypeptide chain and the second polypeptide chain each contains an antibody heavy chain constant
  • the CH3 domain of the region respectively named CH3_A domain and CH3_B domain.
  • the CH3_A and CH3_B domains Compared with the wild-type human antibody heavy chain constant region CH3 domain, the CH3_A and CH3_B domains contain the following amino acid mutations: CH3_A domain K409 and F405 are mutated, and the CH3_A domain and CH3_B domain are respectively at one or more amino acid positions selected from the group consisting of Q347, Y349, S354, E356, E357, K360, S364, L368, K370, K392, D399 and K439 With mutations.
  • the positions of the above-mentioned amino acids are determined according to the EU index of the KABAT number.
  • the CH3_A structural domain and the CH3_B structural domain have one or more selected from the following mutations:
  • the CH3_A structural domain and the CH3_B structural domain further have the following mutations:
  • the mutation is selected from one or more of the following mutations: Q347R, Y349C, S354C, E356K, E357K, E357S, K360E, S364R, S364K, L368D, K370D, K392D, D399K, K439E, F405E and K409F.
  • Q347R refers to the replacement of glutamine Gln347 with arginine (R).
  • Y349C means that Tyrosine Tyr349 is replaced with Cysteine (C).
  • S354C means that serine Ser354 is replaced with cysteine (C).
  • E356K means that glutamic acid Glu356 is replaced with lysine (K).
  • E357K means that glutamic acid Glu357 is replaced with lysine (K).
  • E357S means that glutamic acid Glu357 is replaced with serine (S).
  • K360E means that lysine Lys360 is replaced with lysine (K).
  • S364R means that serine Ser364 is replaced with arginine (R).
  • S364K means that serine Ser364 is replaced with lysine (K).
  • L368D refers to the replacement of leucine Leu368 with aspartic acid (D).
  • K370D means that lysine Lys370 is replaced with aspartic acid (D).
  • K392D means that lysine Lys392 is replaced with aspartic acid (D).
  • D399K means that Asp399 is replaced with Lysine (K).
  • K439E means that Lys439 is replaced with glutamic acid (E).
  • F405E means phenylalanine Phe405 is replaced with glutamic acid (E).
  • K409F means that lysine Lys409 is replaced with phenylalanine (F).
  • the CH3_A domain and CH3_B domain of the heterodimer contain mutations selected from the following group:
  • CH3_A domain F405E+K409F+K370D
  • CH3_B domain S364R+E357S
  • CH3_A domain F405E+K409F+K370D+S354C
  • CH3_B domain S364R+E357S+Y349C
  • CH3_A domain F405E+K409F+K370D+Y349C
  • CH3_B domain S364R+E357S+ S354C
  • CH3_A domain F405E+K409F+K392D
  • CH3_B domain D399K
  • CH3_A domain F405E+K409F+K392D+S354C
  • CH3_B domain D399K+Y349C
  • CH3_A domain F405E+K409F+K392D+Y349C
  • CH3_B domain D399K+S354C
  • CH3_A domain F405E+K409F+K439D
  • CH3_B domain E356K+E357K
  • CH3_A domain F405E+K409F+K439D+S354C
  • CH3_B domain E356K+E357K+Y349C
  • CH3_A domain F405E+K409F+K439D+Y349C
  • CH3_B domain E356K+E357K+S354C
  • CH3_A domain F405E+K409F+L368D+Y349C
  • CH3_B domain S364R+S354C
  • CH3_A domain F405E+K409F+L368D+S354C
  • CH3_B domain S364K+Y349C
  • CH3_A domain F405E+K409F+L368D+Y349C
  • CH3_B domain S364K+S354C
  • CH3_A domain F405E+K409F+K360E+S354C
  • CH3_B domain Q347R+Y349C
  • CH3_A domain F405E+K409F+K360E+Y349C
  • CH3_B domain Q347R+S354C
  • CH3_A domain F405E+K409F+K370D+K360E
  • CH3_B domain S364R+E357S+ Q347R;
  • CH3_A domain F405E+K409F+K370D+K360E+S354C
  • CH3_B domain S364R+E357S+ Q347R+Y349C;
  • CH3_A domain F405E+K409F+K370D+K360E+Y349C
  • CH3_B domain S364R+E357S+ Q347R+S354C.
  • the antibody constant region is derived from IgG (for example, IgG1, IgG2, IgG3, IgG4), IgA (for example, IgA1, IgA2), IgD, IgE or IgM.
  • IgG for example, IgG1, IgG2, IgG3, IgG4
  • IgA for example, IgA1, IgA2
  • IgD for example, IgE or IgM.
  • the second aspect of the present invention is to provide a composition
  • a composition comprising: (1) the heterodimer according to any one of claims 1-5, and (2) a pharmaceutically acceptable carrier and/ Or diluents and/or excipients.
  • the third aspect of the present invention is to provide a polynucleotide comprising: a nucleotide encoding the first polypeptide chain of the heterodimer according to any one of claims 1-5 Molecule A, and nucleotide molecule B encoding the second polypeptide chain of the heterodimer of any one of claims 1-5;
  • the fourth aspect of the present invention is to provide a vector combination comprising: a recombinant vector A containing the nucleotide molecule A and a recombinant vector B containing the nucleotide molecule B.
  • the expression vectors used in the recombinant vector A and the recombinant vector B are conventional expression vectors in the art, which means that they contain appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, and enhancers. Expression vectors for sequences, marker genes and/or sequences and other appropriate sequences.
  • the expression vector can be a virus or a plasmid, such as an appropriate phage or phagemid.
  • Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.
  • the expression vector of the present invention is preferably pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, more preferably pTT5.
  • the fifth aspect of the present invention is to provide a recombinant host cell containing the vector combination.
  • the original host cell of the recombinant host cell of the present invention can be various conventional host cells in the art, as long as it can make the above-mentioned recombinant vector stably replicate by itself, and the nucleotides carried can be effectively expressed. .
  • the original host cell may be a prokaryotic expression cell or a eukaryotic expression cell, and the host cell preferably includes: COS, CHO (Chinese Hamster Ovary, Chinese Hamster Ovary), NS0, sf9, sf21, DH5 ⁇ , BL21 (DE3) or TG1, more preferably E.coli TG1, BL21 (DE3) cells (expressing single-chain antibody or Fab antibody) or CHO-K1 cells (expressing full-length IgG antibody).
  • the aforementioned expression vector is transformed into a host cell to obtain the preferred recombinant host cell of the present invention.
  • the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electrotransformation method.
  • the original host cell is preferably a eukaryotic cell, and more preferably a CHO cell or 293E cell.
  • the sixth aspect of the present invention is to provide the heterodimer according to the first aspect of the present invention, the composition according to the second aspect of the present invention, the polynucleotide according to the third aspect of the present invention, the present invention
  • the seventh aspect of the present invention provides a method for preparing the heterodimer according to the first aspect of the present invention, using the recombinant host cell according to the fifth aspect of the present invention to express the heterodimer .
  • the recombinant host cell contains both a recombinant vector A encoding the first polypeptide chain of the heterodimer and a recombinant vector B encoding the second polypeptide chain of the heterodimer, using the recombinant host The cells are expressed and recovered to obtain heterodimer molecules.
  • the heterodimer can be purified from the recombinant host cell by standard experimental means.
  • protein A can be used for purification. Purification methods include, but are not limited to, chromatographic techniques such as size exclusion, ion exchange, affinity chromatography, and ultrafiltration, or appropriate combinations of the above methods.
  • the transfection ratio of recombinant vector A and recombinant vector B in the recombinant host cell is 1:3 to 3:1, such as 1:2 to 2:1, such as 1:1.5 to 1.5:1, for example About 1:1.
  • the first polypeptide chain and the second polypeptide chain both contain the CH3 domain of the antibody Fc fragment, and the two polypeptide chains interact through the CH3 domain or the Fc fragment containing CH3 to form a dimer.
  • the two polypeptide chains can be in different combinations.
  • the first polypeptide chain is an antibody
  • the second polypeptide chain is a fusion protein
  • both polypeptide chains are fusion proteins
  • both polypeptide chains are antibodies.
  • the fusion protein contains the Fc portion of the antibody and the extracellular region of the cell adhesion molecule, it is also called an immunoadhesin.
  • the cell adhesion molecules mainly refer to molecules that can recognize specific ligand cell surface receptors, such as cadherins, selectins, immunoglobulin superfamily, integrins, and hyaluronic acid adhesives.
  • the CH3 is derived from an antibody Fc fragment, preferably a human antibody Fc fragment.
  • the CH3 domain of a human antibody Fc fragment is derived from a wild-type human antibody Fc fragment.
  • Wild-type human antibody Fc refers to the amino acid sequence that exists in the human population. Of course, there are some subtle differences in Fc fragments among individuals.
  • the human antibody Fc fragment of the present invention also includes individual amino acid changes to the wild-type human antibody Fc sequence, for example, including certain amino acid mutations at the glycosylation site, or other nonsense mutations.
  • the CH3 and CH2 domains may also contain other mutations that do not affect the function of the antibody, especially the Fc segment.
  • the hinge region is connected as a flexible segment between the two polypeptide segments to ensure the function of each segment of the polypeptide chain;
  • the length of the hinge region can choose the length of the hinge region as required, for example, the full-length sequence or a partial sequence thereof.
  • the numbering of amino acid positions in the Fc or its CH2, CH3 domain or hinge region is based on Kabat The position of the EU number index is determined. Those skilled in the art know that even if the amino acid sequence changes due to the insertion or deletion of amino acids or other mutations in the above-mentioned regions, according to Kabat The position number of each amino acid corresponding to the standard sequence determined by the EU numbering index remains unchanged.
  • the EU index is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991).
  • the present invention combines the "convex-concave” model with electrostatic interaction by comprehensively considering various interactions between interface amino acids, such as ionic interaction, hydrophobic interaction and steric interaction, and screens out the preferred CH3 mutation sequence. It tends to form heterodimers instead of homodimers, thus greatly increasing the yield of heterodimer molecules.
  • the "convex-concave" model construction method involved in the present invention is simpler, has fewer point mutations, and can increase the purity of the heterodimer without introducing disulfide bonds. To more than 95%.
  • Figure 1 is a schematic diagram of the crystal structure of the CH3 domain. The figure shows that K409 of CH3_A is located in the cavity surrounded by F405, D399 and K370 of CH3_B, and forms an ionic interaction with the side chain of D399.
  • Figure 2 is a schematic diagram of heterologous antibodies and homologous antibodies. From left to right are CH3_A/CH3_B heterodimer, CH3_A/CH3_A homodimer and CH3_B/CH3_B homodimer.
  • the square modules represent electrostatic interactions, and the circular modules represent spatial effects. There is repulsion between modules of different shapes. There are mainly electrostatic-hydrophobic repulsion between CH3_A/CH3_A homodimers, and electrostatic repulsion and electrostatic-hydrophobic repulsion exist between CH3_B/CH3_B homodimers.
  • Figure 3 shows the electrophoresis analysis of transiently expressing scFv-Fc/Fc heterodimer. 4-12% SDS-PAGE protein gel electrophoresis. The lanes from left to right are: protein molecular weight standard, combination 1, combination 2, combination 3 and combination 4. The homodimer and heterodimer contained in each group of products have different migration distances in gel electrophoresis due to the difference in molecular weight. The positions of different homodimer or heterodimer proteins are marked in the figure.
  • the homodimer and heterodimer contained in each group of products have different migration distances in gel electrophoresis due to the difference in molecular weight. The positions of different homodimer or heterodimer proteins are marked in the figure.
  • PBS purchased from Shenggong Biological Engineering (Shanghai) Co., Ltd., catalog number B548117.
  • Citric acid purchased from Sinopharm Chemical Reagent Co., Ltd.
  • Prime star HS DNA polymerase purchased from Takara company, catalog number R010A.
  • Endotoxin-free plasmid large-scale extraction kit purchased from TIANGEN company, item number DP117.
  • HiTrap MabSelectSuRe column purchased from GE Company.
  • AKTA-FPLC fast protein liquid chromatography system purchased from GE Company.
  • Chemidoc MP gel imager purchased from Bio-Rad.
  • G1600AX capillary electrophoresis instrument purchased from Agilent.
  • MicroCal PEAQ-DSC micro calorimeter scanning calorimeter purchased from Malvern Company.
  • IgG1 is preferably used as a template to design amino acid modifications of the CH3 domain, and these amino acid modifications are also applicable to IgG4 subtypes unless otherwise specified.
  • the CH3 domain will form a homodimer.
  • the crystal structure of the CH3 domain (pdb code: 4BSW) of the Fc segment of the IgG1 antibody is shown in Figure 1.
  • K409 of CH3_A is located in the empty space surrounded by F405, D399 and K370 of CH3_B. Inside the cavity, and form an ionic interaction with the side chain of D399.
  • the above-mentioned position is highly conserved on IgG4.
  • the only difference from IgG1 is that the amino acid with EU numbering at position 370 on the heavy chain of IgG4 is Arg.
  • the gene encoding scFv-Fc fusion protein (see SEQ ID: 1 for the sequence of scFv-Fc fusion protein) was artificially synthesized, where scFv refers to an anti-CD3 single-chain antibody. Then subcloned into mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cells to express scFv-Fc fusion protein. The Fc fragment of the above gene (see SEQ ID NO: 2 for the Fc fusion protein sequence) was subcloned into the mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cells to express the Fc fusion protein.
  • Each mutation combination includes its corresponding A chain (referring to the scFV-Fc fusion protein chain) and B chain (Refers to the Fc protein chain) recombinant expression vectors are co-transfected, and the co-transfection ratio of the A chain and B chain recombinant expression vectors is 1:1.
  • transient products all contain different ratios of homodimer proteins (scFv-Fc/scFv-Fc, Fc/Fc) and heterodimer proteins (scFv-Fc/Fc). Since the molecular weights of these three proteins (scFv-Fc/scFv-Fc, Fc/Fc, and scFv-Fc/Fc) are different, the homodimerization in each group of products can be detected by SDS-PAGE electrophoresis under non-reducing conditions
  • the composition of body protein (scFv-Fc/scFv-Fc, Fc/Fc) and heterodimer protein (scFv-Fc/Fc), the electrophoresis detection result is shown in Figure 3.
  • Combination 1 and combination 2 had significantly more homodimers than combination 3 and combination 4, indicating that the D399A mutation on CH3_B is not conducive to the formation of heterodimers.
  • Combination 4 has fewer homodimers than combination 3, indicating that the F405E mutation is better than F405D.
  • Example 1 by introducing positively charged amino acids into the CH3_B chain, electrostatic repulsion was used to inhibit the formation of homodimers (scFv-Fc/scFv-Fc).
  • scFv-Fc/scFv-Fc homodimers
  • it aims to further explore ways to reduce the mutual attraction between CH3_B chains and inhibit the formation of homodimer proteins.
  • the existing K409F in the CH3_A domain Based on the combination of F405E point mutations, the following charged amino acid mutation combinations were further introduced into the CH3_A domain and CH3_B domain:
  • CH3_A domain K392D
  • CH3_B domain D399K
  • CH3_A domain K439D
  • CH3_B domain E356K+E357K
  • CH3_A domain L368D, CH3_B domain: S364R;
  • CH3_A domain L368D
  • CH3_B domain S364K
  • CH3_A domain K360E
  • CH3_B domain Q347R
  • CH3_A domain K370D+K360E
  • CH3_B domain S364R+E357S+Q347R.
  • the purified protein fraction was analyzed by non-reducing capillary gel electrophoresis, and the percentage of peak area of each product component was calculated. The results are shown in Table 3.
  • Combinations 5-10 can obtain good heterodimer purity. Among them, the purity of the heterodimer obtained by combination 7 and combination 4 is equivalent, and combination 10 is the basis of combination 4. A new charged amino acid mutation was added to the top, and a better purity of the heterodimer was obtained.
  • the cotransformation expression vector used in the better mutation combination 4 was recombined with CH3_A.
  • the combined mutations of HC_A and HC_B encoding genes were carried out by overlap PCR, wherein the mutations for the CH3_A chain were located on the HC_A chain, and the mutations for the CH3_B chain were located on the HC_B chain.
  • the mutated genes were subcloned into pTT5, and finally recombinant expression vectors for expressing the mutations in mammalian cells were obtained.
  • the purified protein fraction was analyzed by LC-MS, and the percentage of each product component was calculated.
  • MicroCal PEAQ-DSC was used to measure the Tm value of the sample. The results are shown in Table 5.
  • Combination 11 was used as a control group, and combinations 12 and 13 introduced disulfide bonds on the basis of combination 11.
  • the obtained heterodimer ratios were 99.56% and 96.89%, which were similar to combination 11;
  • the Tm values of Fc of combination 12 and combination 13 were 69.21°C and 70.23°C, respectively, which was about 3°C higher than that of combination 11.
  • This example found that even though the control group (combination 11) already has a heterodimer ratio of greater than 95%, the introduction of disulfide bonds can still further promote the formation of heterodimers and improve the thermal stability of antibody molecules. Similarly, the introduction of disulfide bonds should also improve the heterodimer ratio and thermal stability of the combination 5-10.

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Abstract

A heterodimer, a preparation method therefor and use thereof. By comprehensively considering various interactions between interfacial amino acids, such as ionic interaction, hydrophobic interaction and steric interaction, a "convex-concave" model is combined with electrostatic interaction to screen out the preferable CH3 mutation sequence, which tends to form the heterodimer rather than a homodimer, thus greatly improving the yield of heterodimer molecules. Compared with the reference documents CN106883297A and US20150307628A1, a method for constructing the "convex-concave" model is simpler, point mutations are fewer, and the purity of the heterodimer can be increased up to 95% or more without introducing disulfide bonds.

Description

CH3结构域改造诱导形成的异源二聚体及其制备方法和应用Heterodimer induced by CH3 structural domain modification and its preparation method and application 技术领域Technical field
本发明属于抗体工程领域，具体涉及一种CH3结构域改造诱导形成的异源二聚体及其制备方法和应用。The invention belongs to the field of antibody engineering, and specifically relates to a heterodimer induced by the transformation of a CH3 structural domain, and a preparation method and application thereof.
背景技术Background technique
双特异性抗体有多种构建方式，其中IgG型双特异性抗体具有和普通抗体相似的结构、理化性质和Fc段功能。通常IgG型双特异性抗体由两条氨基酸序列不同的重链（即抗抗原A的重链HC_A和抗抗原B的重链HC_B）和两条氨基酸序列不同的轻链（即抗抗原A的轻链LC_A和抗抗原B的轻链LC_B）组成。当4条多肽链组合时，两条重链之间会形成同源二聚体和异源二聚体，轻重链之间也会形成错配，因此会产生8种不同的组合方式，其中只有一种为所需要的目标抗体分子。而从8种分子中分离纯化得到目标分子效率极低且非常困难。There are many ways to construct bispecific antibodies. Among them, IgG type bispecific antibodies have similar structure, physicochemical properties and Fc segment functions to common antibodies. Generally, an IgG bispecific antibody consists of two heavy chains with different amino acid sequences (ie, heavy chain HC_A against antigen A and heavy chain HC_B against antigen B) and two light chains with different amino acid sequences (ie light chain against antigen A). Chain LC_A and anti-antigen B light chain LC_B). When 4 polypeptide chains are combined, homodimers and heterodimers will be formed between the two heavy chains, and mismatches will also be formed between the light and heavy chains. Therefore, there will be 8 different combinations, of which only One is the desired target antibody molecule. However, it is extremely inefficient and difficult to separate and purify the target molecule from 8 kinds of molecules.
构造IgG型双特异性抗体有多种方法，通过改造Fc使之形成异源二聚体是其中重要的一种方法。早在20世纪90年代，Carter等人用“凸-凹”（knob-into-hole） 模型改造抗体Fc段，较成功的实现了双特异抗体的制备（Ridgway,Presta et al.1996 ；Carter 2001）。Carter等在Fc第一条重链的CH3结构域上通过将一个侧链小的氨基酸突变成了一个侧链大的氨基酸从而创造出了一个“凸”，并将第二条重链上的CH3上的某些氨基酸突变成了侧链小的氨基酸创造出了“凹”。“凸-凹”模型的原理是“凸-凹”的相互作用支持异源二聚体的形成，而“凸-凸”模型及“凹-凹”模型阻碍同源二聚体的形成。然而在他们的研究结果中，“凹-凹”模型对阻碍同源二聚体的形成的能力仍然不够。There are many ways to construct IgG-type bispecific antibodies, and one of the important methods is to modify Fc to form heterodimers. As early as the 1990s, Carter et al. used the "knob-into-hole" model to modify the Fc segment of antibodies, and successfully realized the preparation of bispecific antibodies (Ridgway, Presta et al. 1996; Carter 2001). Carter et al. created a “convex” on the CH3 domain of the first heavy chain of Fc by mutating an amino acid with a small side chain into an amino acid with a large side chain, and changed the Certain amino acids on CH3 are mutated into amino acids with small side chains to create "concavities." The principle of the "convex-concave" model is that the "convex-concave" interaction supports the formation of heterodimers, while the "convex-convex" and "concave-concave" models hinder the formation of homodimers. However, in their research results, the "concave-concave" model is still insufficient to hinder the formation of homodimers.
US2010286374A1公开了一种利用静电作用促进异源二聚体形成的方法。具体来说，将两条重链的CH3结构域中带电荷氨基酸分别突变为相反电荷的氨基酸，使得其中一条重链的CH3结构域总体上带正电荷，而另一条重链的CH3结构域总体上带负电荷，相同电荷的静电排斥会抑制同源二聚体的形成。然而，静电作用并不能完全抑制同源二聚体的形成，引入过多的突变反而引起蛋白表达水平的下降，反映出多个氨基酸突变情况下，界面氨基酸之间作用关系的复杂性。US2010286374A1 discloses a method for promoting the formation of heterodimers by using static electricity. Specifically, the charged amino acids in the CH3 domains of the two heavy chains are mutated to oppositely charged amino acids, so that the CH3 domain of one heavy chain is generally positively charged, and the CH3 domain of the other heavy chain is generally positively charged. It is negatively charged, and electrostatic repulsion of the same charge will inhibit the formation of homodimers. However, electrostatic effect cannot completely inhibit the formation of homodimers. The introduction of too many mutations will cause the decrease of protein expression level, reflecting the complexity of the interaction relationship between interface amino acids in the case of multiple amino acid mutations.
将“凸-凹”模型和静电作用相结合是促进异源二聚体形成的有效方法。CN106883297A公开了一种方法，具体来说，在Carter等人的“凸-凹”模型基础上进一步在“凹”的一端引入F405K 突变，增强“凹-凹”界面氨基酸之间静电排斥，同时在“凸”的一端引入K409A 突变，避免“凸-凹”界面氨基酸之间产生静电排斥，从而抑制“凹-凹”同源二聚体的形成并维持“凸-凹”异源二聚体的形成。尽管该专利公开的方法能提高异源二聚体的形成，但其中的一个方案仅能将异源二聚体的比例最高提高到93%。Combining the "convex-concave" model with electrostatic interaction is an effective way to promote the formation of heterodimers. CN106883297A discloses a method. Specifically, on the basis of the "convex-concave" model of Carter et al., the F405K mutation is further introduced at one end of the "concave" to enhance the electrostatic repulsion between amino acids at the "concave-concave" interface. K409A mutation is introduced at the "convex" end to avoid electrostatic repulsion between amino acids at the "concave-concave" interface, thereby inhibiting the formation of "concave-concave" homodimers and maintaining the "convex-concave" heterodimer form. Although the method disclosed in this patent can increase the formation of heterodimers, one of the solutions can only increase the proportion of heterodimers up to 93%.
US20150307628A1公开了另一种利用“凸-凹”模型和静电作用促进异源二聚体形成的方法。具体来说，其中一个方案在Fc第一条重链的CH3结构域上通过K409W点突变创造出了一个“凸”，并将第二条重链上的CH3上通过F405T、D399V点突变造出了“凹”；在第一条重链的CH3结构域上通过K360E点突变使之带上负电荷，并将第二条重链上的CH3上通过Q347R点突变使之带上正电荷。该方案在不引入二硫键的情况下能将异源二聚体的比例最高提高到91.4±1.2%。US20150307628A1 discloses another method that uses the "convex-concave" model and electrostatic interaction to promote the formation of heterodimers. Specifically, one of the schemes created a “convex” on the CH3 domain of the first heavy chain of Fc through K409W point mutations, and created a “convex” on the CH3 of the second heavy chain through F405T and D399V point mutations. The "concave"; the CH3 domain of the first heavy chain was negatively charged by the K360E point mutation, and the CH3 on the second heavy chain was positively charged by the Q347R point mutation. This scheme can increase the proportion of heterodimers up to 91.4±1.2% without introducing disulfide bonds.
技术问题technical problem
尽管CN106883297A和US20150307628A1公开的两种方法从原理上优于单纯的“凸-凹”模型并且进一步提高了异源二聚体的比例，但要把异源二聚体的比例提高到95%以上仍需要新的研究。Although the two methods disclosed in CN106883297A and US20150307628A1 are better than the simple "convex-concave" model in principle and further increase the proportion of heterodimers, it is still necessary to increase the proportion of heterodimers to more than 95%. New research is needed.
技术解决方案Technical solutions
本发明的目的在于克服现有技术中的不足，提供一种CH3结构域改造诱导形成的异源二聚体及其制备方法和应用。本发明通过综合考虑分子间相互作用，如离子作用、空间作用、疏水相互作用，将“凸-凹”模型和静电作用相结合，筛选到优选的CH3突变序列，其更倾向于形成异源二聚体，而不形成同源二聚体，在不引入二硫键的情况下能将异源二聚体分子的比例最高提高到95%以上。The purpose of the present invention is to overcome the deficiencies in the prior art, and provide a heterodimer induced by the transformation of the CH3 structure domain, and a preparation method and application thereof. The present invention combines the "convex-concave" model with electrostatic interaction through comprehensive consideration of intermolecular interactions, such as ionic interactions, steric interactions, and hydrophobic interactions, and selects preferred CH3 mutation sequences, which are more likely to form heterologous two. Polymers, without forming homodimers, can increase the proportion of heterodimer molecules to more than 95% without introducing disulfide bonds.
本发明所述的异源二聚体指抗体分子或片段含有两条氨基酸序列不同的重链，包括而不局限于双特异性抗体、一价抗体、Fc融合蛋白等。The heterodimer in the present invention refers to an antibody molecule or fragment containing two heavy chains with different amino acid sequences, including but not limited to bispecific antibodies, monovalent antibodies, Fc fusion proteins and the like.
本发明的第一个方面是提供一种异源二聚体，其含有第一多肽链和第二多肽链，所述第一多肽链和第二多肽链分别含有抗体重链恒定区CH3结构域，分别命名为CH3_A结构域和CH3_B结构域，与野生型的人抗体重链恒定区CH3结构域相比，所述CH3_A和CH3_B结构域含有如下位置氨基酸的突变：CH3_A结构域的K409、F405发生突变，且CH3_A结构域和CH3_B结构域还分别在选自Q347、Y349、S354、E356、E357、K360、S364、L368、K370、K392、D399和K439中的一个或多个氨基酸位置具有的突变。The first aspect of the present invention is to provide a heterodimer, which contains a first polypeptide chain and a second polypeptide chain, the first polypeptide chain and the second polypeptide chain each contains an antibody heavy chain constant The CH3 domain of the region, respectively named CH3_A domain and CH3_B domain. Compared with the wild-type human antibody heavy chain constant region CH3 domain, the CH3_A and CH3_B domains contain the following amino acid mutations: CH3_A domain K409 and F405 are mutated, and the CH3_A domain and CH3_B domain are respectively at one or more amino acid positions selected from the group consisting of Q347, Y349, S354, E356, E357, K360, S364, L368, K370, K392, D399 and K439 With mutations.
以上所述的氨基酸的位置根据KABAT 编号的EU 索引确定。The positions of the above-mentioned amino acids are determined according to the EU index of the KABAT number.
优选地，所述CH3_A结构域和CH3_B结构域具有选自下列的突变中的一种或多种：Preferably, the CH3_A structural domain and the CH3_B structural domain have one or more selected from the following mutations:
1a）CH3_B结构域的 S364、E357发生突变，且CH3_A结构域的K370发生突变；1a) S364 and E357 of CH3_B domain are mutated, and K370 of CH3_A domain is mutated;
1b）CH3_B结构域的D399发生突变，且CH3_A结构域的K392发生突变；1b) D399 of the CH3_B domain is mutated, and K392 of the CH3_A domain is mutated;
1c）CH3_B结构域的E356、E357发生突变，且CH3_A结构域的K439发生突变；1c) E356 and E357 of CH3_B domain are mutated, and K439 of CH3_A domain is mutated;
1d）CH3_B结构域的S364发生突变，且CH3_A结构域的L368发生突变；1d) S364 of the CH3_B domain is mutated, and L368 of the CH3_A domain is mutated;
1e）CH3_B结构域的Q347发生突变，且CH3_A结构域的K360发生突变。1e) Q347 of the CH3_B domain is mutated, and K360 of the CH3_A domain is mutated.
进一步优选地，所述CH3_A结构域和CH3_B结构域还具有下列突变：Further preferably, the CH3_A structural domain and the CH3_B structural domain further have the following mutations:
1f）CH3_B结构域的S354发生突变，且CH3_A结构域的Y349发生突变；或1g）CH3_B结构域的Y349发生突变，且CH3_A结构域的S354发生突变。1f) S354 of the CH3_B domain is mutated and Y349 of the CH3_A domain is mutated; or 1g) Y349 of the CH3_B domain is mutated and S354 of the CH3_A domain is mutated.
优选地，所述突变选自以下突变中的一个或数个：Q347R、Y349C、S354C、E356K、E357K、E357S、K360E、S364R、S364K、L368D、K370D、K392D、D399K、K439E、F405E和K409F。Preferably, the mutation is selected from one or more of the following mutations: Q347R, Y349C, S354C, E356K, E357K, E357S, K360E, S364R, S364K, L368D, K370D, K392D, D399K, K439E, F405E and K409F.
Q347R是指谷氨酰胺Gln347被替换为精氨酸（R）。Y349C是指酪氨酸Tyr349被替换为半胱氨酸（C）。S354C是指丝氨酸Ser354被替换为半胱氨酸（C）。E356K是指谷氨酸Glu356被替换为赖氨酸（K）。E357K是指谷氨酸Glu357被替换为赖氨酸（K）。E357S是指谷氨酸Glu357被替换为丝氨酸（S）。K360E是指赖氨酸Lys360被替换为赖氨酸（K）。S364R是指丝氨酸Ser364被替换为精氨酸（R）。S364K是指丝氨酸Ser364被替换为赖氨酸（K）。L368D是指亮氨酸Leu368被替换为天冬氨酸（D）。K370D是指赖氨酸Lys370被替换为天冬氨酸（D）。K392D是指赖氨酸Lys392被替换为天冬氨酸（D）。D399K是指天冬氨酸Asp399被替换为赖氨酸（K）。K439E是指赖氨酸Lys439被替换为谷氨酸（E）。F405E是指苯丙氨酸Phe405被替换为谷氨酸（E）。K409F是指赖氨酸Lys409被替换为苯丙氨酸（F）。Q347R refers to the replacement of glutamine Gln347 with arginine (R). Y349C means that Tyrosine Tyr349 is replaced with Cysteine (C). S354C means that serine Ser354 is replaced with cysteine (C). E356K means that glutamic acid Glu356 is replaced with lysine (K). E357K means that glutamic acid Glu357 is replaced with lysine (K). E357S means that glutamic acid Glu357 is replaced with serine (S). K360E means that lysine Lys360 is replaced with lysine (K). S364R means that serine Ser364 is replaced with arginine (R). S364K means that serine Ser364 is replaced with lysine (K). L368D refers to the replacement of leucine Leu368 with aspartic acid (D). K370D means that lysine Lys370 is replaced with aspartic acid (D). K392D means that lysine Lys392 is replaced with aspartic acid (D). D399K means that Asp399 is replaced with Lysine (K). K439E means that Lys439 is replaced with glutamic acid (E). F405E means phenylalanine Phe405 is replaced with glutamic acid (E). K409F means that lysine Lys409 is replaced with phenylalanine (F).
优选地，所述异源二聚体的CH3_A结构域和CH3_B结构域含有选自以下一组的突变：Preferably, the CH3_A domain and CH3_B domain of the heterodimer contain mutations selected from the following group:
（a1）CH3_A结构域：F405E+K409F+K370D，CH3_B结构域：S364R+E357S；(A1) CH3_A domain: F405E+K409F+K370D, CH3_B domain: S364R+E357S;
（a2）CH3_A结构域：F405E+K409F+K370D+S354C，CH3_B结构域：S364R+E357S+ Y349C；(A2) CH3_A domain: F405E+K409F+K370D+S354C, CH3_B domain: S364R+E357S+Y349C;
（a3）CH3_A结构域：F405E+K409F+K370D+Y349C，CH3_B结构域：S364R+E357S+ S354C(A3) CH3_A domain: F405E+K409F+K370D+Y349C, CH3_B domain: S364R+E357S+ S354C
（b1）CH3_A结构域：F405E+K409F+K392D，CH3_B结构域：D399K；(B1) CH3_A domain: F405E+K409F+K392D, CH3_B domain: D399K;
（b2）CH3_A结构域：F405E+K409F+K392D+S354C，CH3_B结构域：D399K+Y349C；(B2) CH3_A domain: F405E+K409F+K392D+S354C, CH3_B domain: D399K+Y349C;
（b3）CH3_A结构域：F405E+K409F+K392D+Y349C，CH3_B结构域：D399K+S354C；(B3) CH3_A domain: F405E+K409F+K392D+Y349C, CH3_B domain: D399K+S354C;
（c1）CH3_A结构域：F405E+K409F+K439D，CH3_B结构域：E356K+E357K；(C1) CH3_A domain: F405E+K409F+K439D, CH3_B domain: E356K+E357K;
（c2）CH3_A结构域：F405E+K409F+K439D+S354C，CH3_B结构域：E356K+E357K+ Y349C；(C2) CH3_A domain: F405E+K409F+K439D+S354C, CH3_B domain: E356K+E357K+Y349C;
（c3）CH3_A结构域：F405E+K409F+K439D+Y349C，CH3_B结构域：E356K+E357K+S354C；(C3) CH3_A domain: F405E+K409F+K439D+Y349C, CH3_B domain: E356K+E357K+S354C;
（d1）CH3_A结构域：F405E+K409F+L368D，CH3_B结构域：S364R；(D1) CH3_A domain: F405E+K409F+L368D, CH3_B domain: S364R;
（d2）CH3_A结构域：F405E+K409F+L368D+S354C，CH3_B结构域：S364R+ Y349C；(D2) CH3_A domain: F405E+K409F+L368D+S354C, CH3_B domain: S364R+ Y349C;
（d3）CH3_A结构域：F405E+K409F+L368D+Y349C，CH3_B结构域：S364R+S354C；(D3) CH3_A domain: F405E+K409F+L368D+Y349C, CH3_B domain: S364R+S354C;
（e1）CH3_A结构域：F405E+K409F+L368D，CH3_B结构域：S364K；(E1) CH3_A domain: F405E+K409F+L368D, CH3_B domain: S364K;
（e2）CH3_A结构域：F405E+K409F+L368D+S354C，CH3_B结构域：S364K+Y349C；(E2) CH3_A domain: F405E+K409F+L368D+S354C, CH3_B domain: S364K+Y349C;
（e3）CH3_A结构域：F405E+K409F+L368D+Y349C，CH3_B结构域：S364K+S354C；(E3) CH3_A domain: F405E+K409F+L368D+Y349C, CH3_B domain: S364K+S354C;
（f1）CH3_A结构域：F405E+K409F+K360E，CH3_B结构域：Q347R；(F1) CH3_A domain: F405E+K409F+K360E, CH3_B domain: Q347R;
（f2）CH3_A结构域：F405E+K409F+K360E+S354C，CH3_B结构域：Q347R+Y349C；(F2) CH3_A domain: F405E+K409F+K360E+S354C, CH3_B domain: Q347R+Y349C;
（f3）CH3_A结构域：F405E+K409F+K360E+Y349C，CH3_B结构域：Q347R+S354C；(F3) CH3_A domain: F405E+K409F+K360E+Y349C, CH3_B domain: Q347R+S354C;
（g1）CH3_A结构域：F405E+K409F+K370D+K360E，CH3_B结构域：S364R+E357S+ Q347R；(G1) CH3_A domain: F405E+K409F+K370D+K360E, CH3_B domain: S364R+E357S+ Q347R;
（g2）CH3_A结构域：F405E+K409F+K370D+K360E+S354C，CH3_B结构域：S364R+E357S+ Q347R+Y349C；(G2) CH3_A domain: F405E+K409F+K370D+K360E+S354C, CH3_B domain: S364R+E357S+ Q347R+Y349C;
（g3）CH3_A结构域：F405E+K409F+K370D+K360E+Y349C，CH3_B结构域：S364R+E357S+ Q347R+S354C。(G3) CH3_A domain: F405E+K409F+K370D+K360E+Y349C, CH3_B domain: S364R+E357S+ Q347R+S354C.
在本发明的实施方案中，所述的抗体恒定区来源于IgG（例如IgG1、IgG2、IgG3、IgG4）、IgA（例如 IgA1、IgA2）、IgD、IgE 或 IgM。In an embodiment of the present invention, the antibody constant region is derived from IgG (for example, IgG1, IgG2, IgG3, IgG4), IgA (for example, IgA1, IgA2), IgD, IgE or IgM.
本发明的第二个方面是提供一种组合物，其含有：（1）权利要求1-5中任意一项所述的异源二聚体，以及（2）药学上可接受的载体和/或稀释剂和/或赋形剂。The second aspect of the present invention is to provide a composition comprising: (1) the heterodimer according to any one of claims 1-5, and (2) a pharmaceutically acceptable carrier and/ Or diluents and/or excipients.
本发明的第三个方面是提供一种多核苷酸，所述多核苷酸包含：编码权利要求1-5中任意一项所述的异源二聚体的第一条多肽链的核苷酸分子A，以及编码权利要求1-5中任意一项所述的异源二聚体的第二条多肽链的核苷酸分子B；The third aspect of the present invention is to provide a polynucleotide comprising: a nucleotide encoding the first polypeptide chain of the heterodimer according to any one of claims 1-5 Molecule A, and nucleotide molecule B encoding the second polypeptide chain of the heterodimer of any one of claims 1-5;
本发明的第四个方面是提供一种载体组合，所述载体组合包括：含有所述核苷酸分子A的重组载体A，以及含有所述核苷酸分子B的重组载体B。The fourth aspect of the present invention is to provide a vector combination comprising: a recombinant vector A containing the nucleotide molecule A and a recombinant vector B containing the nucleotide molecule B.
其中，所述重组载体A和重组载体B所使用的表达载体为本领域常规的表达载体，是指包含适当的调控序列，例如启动子序列、终止子序列、多腺苷酰化序列、增强子序列、标记基因和/或序列以及其他适当的序列的表达载体。所述表达载体可以是病毒或质粒，如适当的噬菌体或者噬菌粒，更多技术细节请参见例如Sambrook等，Molecular Cloning： A Laboratory Manual，第二版，Cold Spring Harbor Laboratory Press，1989。许多用于核酸操作的已知技术和方案请参见Current Protocols in Molecular Biology，第二版，Ausubel等编著。本发明所述表达载体较佳地为pDR1，pcDNA3.1（+），pcDNA3.1/ZEO（+），pDHFR，pTT5，pDHFF，pGM-CSF或pCHO 1.0，更佳地为pTT5。Wherein, the expression vectors used in the recombinant vector A and the recombinant vector B are conventional expression vectors in the art, which means that they contain appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, and enhancers. Expression vectors for sequences, marker genes and/or sequences and other appropriate sequences. The expression vector can be a virus or a plasmid, such as an appropriate phage or phagemid. For more technical details, please refer to, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. For many known techniques and protocols for nucleic acid manipulation, please refer to Current Protocols in Molecular Biology, second edition, edited by Ausubel et al. The expression vector of the present invention is preferably pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, more preferably pTT5.
本发明的第五个方面是提供一种重组宿主细胞，所述重组宿主细胞含有所述载体组合。The fifth aspect of the present invention is to provide a recombinant host cell containing the vector combination.
本发明所述的重组宿主细胞的原始宿主细胞可以为本领域常规的各种宿主细胞，只要能满足使上述重组载体稳定地自行复制，且所携带所述的核苷酸可被有效表达即可。其中所述原始宿主细胞可以原核表达细胞或真核表达细胞，所述宿主细胞较佳地包括：COS、CHO（中国仓鼠卵巢，Chinese H amster Ovary）、NS0、sf9、sf21、DH5α、BL21（DE3）或TG1，更佳地为E.coli TG1、BL21（DE3）细胞（表达单链抗体或Fab抗体）或者CHO-K1细胞（表达全长IgG抗体）。将前述表达载体转化至宿主细胞中，即可得本发明优选的重组宿主细胞。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。The original host cell of the recombinant host cell of the present invention can be various conventional host cells in the art, as long as it can make the above-mentioned recombinant vector stably replicate by itself, and the nucleotides carried can be effectively expressed. . The original host cell may be a prokaryotic expression cell or a eukaryotic expression cell, and the host cell preferably includes: COS, CHO (Chinese Hamster Ovary, Chinese Hamster Ovary), NS0, sf9, sf21, DH5α, BL21 (DE3) or TG1, more preferably E.coli TG1, BL21 (DE3) cells (expressing single-chain antibody or Fab antibody) or CHO-K1 cells (expressing full-length IgG antibody). The aforementioned expression vector is transformed into a host cell to obtain the preferred recombinant host cell of the present invention. The transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electrotransformation method.
作为优选的方案，所述原始宿主细胞优选为真核细胞，进一步优选为CHO细胞或293E细胞。As a preferred solution, the original host cell is preferably a eukaryotic cell, and more preferably a CHO cell or 293E cell.
本发明的第六个方面是提供本发明第一个方面所述的异源二聚体、本发明第二个方面所述的组合物、本发明第三个方面所述的多核苷酸、本发明第四个方面所述的载体组合、或本发明第五个方面所述的重组宿主细胞在在制备双特异性抗体、双特异性融合蛋白和抗体-融合蛋白嵌合体中的用途。The sixth aspect of the present invention is to provide the heterodimer according to the first aspect of the present invention, the composition according to the second aspect of the present invention, the polynucleotide according to the third aspect of the present invention, the present invention The use of the vector combination according to the fourth aspect of the invention or the recombinant host cell according to the fifth aspect of the invention in the preparation of bispecific antibodies, bispecific fusion proteins and antibody-fusion protein chimeras.
本发明的第七个方面是提供一种制备本发明第一个方面所述的异源二聚体的方法，使用本发明第五个方面所述的重组宿主细胞表达所述异源二聚体。The seventh aspect of the present invention provides a method for preparing the heterodimer according to the first aspect of the present invention, using the recombinant host cell according to the fifth aspect of the present invention to express the heterodimer .
在本发明中，所述重组宿主细胞同时含有编码异源二聚体中第一多肽链的重组载体A和编码异源二聚体中第二多肽链的重组载体B，利用该重组宿主细胞表达，回收，得到异源二聚体分子。In the present invention, the recombinant host cell contains both a recombinant vector A encoding the first polypeptide chain of the heterodimer and a recombinant vector B encoding the second polypeptide chain of the heterodimer, using the recombinant host The cells are expressed and recovered to obtain heterodimer molecules.
其中，所述异源二聚体可以用标准的实验手段从重组宿主细胞中纯化。例如，当异二聚体蛋白包含抗体Fc片段，可以用蛋白A来纯化。纯化方法包括但不限于色谱技术如体积排阻法、离子交换法、亲和色谱法及超滤法，或者上述各种方法的适当组合。Wherein, the heterodimer can be purified from the recombinant host cell by standard experimental means. For example, when the heterodimeric protein contains an antibody Fc fragment, protein A can be used for purification. Purification methods include, but are not limited to, chromatographic techniques such as size exclusion, ion exchange, affinity chromatography, and ultrafiltration, or appropriate combinations of the above methods.
在本发明中，所述重组宿主细胞中重组载体A和重组载体B的转染比例为1:3～3:1，例如 1:2～2:1，例如1:1.5～1.5:1，例如约 1:1。In the present invention, the transfection ratio of recombinant vector A and recombinant vector B in the recombinant host cell is 1:3 to 3:1, such as 1:2 to 2:1, such as 1:1.5 to 1.5:1, for example About 1:1.
在本发明中，第一多肽链和第二多肽链都含有抗体Fc片段的CH3结构域，两条多肽链之间通过CH3结构域或含有CH3的Fc片段发生相互作用，形成二聚体，特别是异源二聚体。两条多肽链之间可以是不同的组合，例如第一多肽链为抗体，第二多肽链为融合蛋白，或者两条多肽链均为融合蛋白，或者两条多肽链均为抗体，靶向不同的抗原或抗原表位。当融合蛋白包含抗体的Fc段与细胞粘附分子的胞膜外区时也称为免疫粘附素。所述细胞粘附分子主要指能识别特异性配基细胞表面受体的分子，例如包括钙粘素、选择素、免疫球蛋白超家族、整合素及透明质酸粘素。In the present invention, the first polypeptide chain and the second polypeptide chain both contain the CH3 domain of the antibody Fc fragment, and the two polypeptide chains interact through the CH3 domain or the Fc fragment containing CH3 to form a dimer. , Especially heterodimers. The two polypeptide chains can be in different combinations. For example, the first polypeptide chain is an antibody, the second polypeptide chain is a fusion protein, or both polypeptide chains are fusion proteins, or both polypeptide chains are antibodies. To different antigens or epitopes. When the fusion protein contains the Fc portion of the antibody and the extracellular region of the cell adhesion molecule, it is also called an immunoadhesin. The cell adhesion molecules mainly refer to molecules that can recognize specific ligand cell surface receptors, such as cadherins, selectins, immunoglobulin superfamily, integrins, and hyaluronic acid adhesives.
在本发明中，所述CH3来源于抗体Fc片段，优选来源于人的抗体Fc片段。在一般情况下，人抗体Fc片段的CH3结构域来源于野生型的人抗体Fc片段。野生型的人抗体Fc是指存在于人群中的氨基酸序列，当然Fc片段在个体中会有一些细微的差异。本发明中人抗体Fc片段也包括对于野生型人抗体Fc序列的个别氨基酸的改变，例如包括某些在糖基化位点突变的氨基酸，或者其它无义的突变。对于 CH3 以及 CH2 结构域，除了本发明中提到的突变外，还可能含有其它不影响抗体特别是 Fc 段功能的突变。In the present invention, the CH3 is derived from an antibody Fc fragment, preferably a human antibody Fc fragment. In general, the CH3 domain of a human antibody Fc fragment is derived from a wild-type human antibody Fc fragment. Wild-type human antibody Fc refers to the amino acid sequence that exists in the human population. Of course, there are some subtle differences in Fc fragments among individuals. The human antibody Fc fragment of the present invention also includes individual amino acid changes to the wild-type human antibody Fc sequence, for example, including certain amino acid mutations at the glycosylation site, or other nonsense mutations. In addition to the mutations mentioned in the present invention, the CH3 and CH2 domains may also contain other mutations that do not affect the function of the antibody, especially the Fc segment.
在本发明中，当第一多肽链和/或第二多肽链中含有绞链区时，该绞链区作为柔性片段连接在两段多肽之间，以保证各段多肽链的功能；本领域技术人员可以根据需要选择绞链区的长度，例如可选择全长序列或其中的部分序列。In the present invention, when the first polypeptide chain and/or the second polypeptide chain contains a hinge region, the hinge region is connected as a flexible segment between the two polypeptide segments to ensure the function of each segment of the polypeptide chain; Those skilled in the art can choose the length of the hinge region as required, for example, the full-length sequence or a partial sequence thereof.
在本发明中，所述Fc或其CH2、CH3结构域或绞链区中氨基酸位置的编号均根据Kabat EU编号索引的位置确定。本领域技术人员知晓，即使上述区域中由于氨基酸的***或缺失或其它突变导致氨基酸序列的改变，根据Kabat EU编号索引确定的与标准序列对应的各氨基酸的位置编号仍然不变。所述 EU 索引描述于 Kabat 等，Sequences of Proteins of Immunological Interest, 第 5 版 Public Health Service,National Institutes of Health,Bethesda,MD.（1991）。In the present invention, the numbering of amino acid positions in the Fc or its CH2, CH3 domain or hinge region is based on Kabat The position of the EU number index is determined. Those skilled in the art know that even if the amino acid sequence changes due to the insertion or deletion of amino acids or other mutations in the above-mentioned regions, according to Kabat The position number of each amino acid corresponding to the standard sequence determined by the EU numbering index remains unchanged. The EU index is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991).
有益效果Beneficial effect
本发明通过综合考虑界面氨基酸之间的各种相互作用，例如离子作用、疏水相互作用和空间作用等，将“凸-凹”模型和静电作用相结合，筛选到优选的CH3突变序列，其更倾向于形成异源二聚体，而不形成同源二聚体，因而大大提高了异源二聚体分子的产量。与参考文献CN106883297A和US20150307628A1相比，本发明涉及的“凸-凹”模型构造方法更加简单、点突变更少，而且在不引入二硫键的情况下能将异源二聚体的纯度最高提高到95%以上。The present invention combines the "convex-concave" model with electrostatic interaction by comprehensively considering various interactions between interface amino acids, such as ionic interaction, hydrophobic interaction and steric interaction, and screens out the preferred CH3 mutation sequence. It tends to form heterodimers instead of homodimers, thus greatly increasing the yield of heterodimer molecules. Compared with references CN106883297A and US20150307628A1, the "convex-concave" model construction method involved in the present invention is simpler, has fewer point mutations, and can increase the purity of the heterodimer without introducing disulfide bonds. To more than 95%.
附图说明Description of the drawings
图1为CH3结构域晶体结构示意图。图中表明CH3_A的K409位于由CH3_B的F405、D399和K370围成的空腔内，并且和D399的侧链形成离子相互作用。 Figure 1 is a schematic diagram of the crystal structure of the CH3 domain. The figure shows that K409 of CH3_A is located in the cavity surrounded by F405, D399 and K370 of CH3_B, and forms an ionic interaction with the side chain of D399.
图2为异源抗体和同源抗体示意图。从左到右分别为CH3_A/CH3_B异源二聚体、CH3_A/CH3_A同源二聚体和CH3_B/CH3_B同源二聚体。方形模块代表静电相互作用，圆形模块代表空间作用。不同形状模块之间存在排斥作用。CH3_A/CH3_A同源二聚体之间主要存在静电-疏水排斥，CH3_B/CH3_B同源二聚体之间存在在在静电排斥和静电-疏水排斥。Figure 2 is a schematic diagram of heterologous antibodies and homologous antibodies. From left to right are CH3_A/CH3_B heterodimer, CH3_A/CH3_A homodimer and CH3_B/CH3_B homodimer. The square modules represent electrostatic interactions, and the circular modules represent spatial effects. There is repulsion between modules of different shapes. There are mainly electrostatic-hydrophobic repulsion between CH3_A/CH3_A homodimers, and electrostatic repulsion and electrostatic-hydrophobic repulsion exist between CH3_B/CH3_B homodimers.
图3为瞬时表达 scFv-Fc/Fc 异源二聚体的电泳分析。4-12% SDS-PAGE蛋白凝胶电泳。泳道从左到右依次为：蛋白分子量标准、组合1、组合2、组合3和组合4。每组产物中所包含的同源二聚体和异源二聚体因为分子量差异而在凝胶电泳中的迁移距离不同。不同的同二聚体或异二聚体蛋白所处的位置在图中被标注出。Figure 3 shows the electrophoresis analysis of transiently expressing scFv-Fc/Fc heterodimer. 4-12% SDS-PAGE protein gel electrophoresis. The lanes from left to right are: protein molecular weight standard, combination 1, combination 2, combination 3 and combination 4. The homodimer and heterodimer contained in each group of products have different migration distances in gel electrophoresis due to the difference in molecular weight. The positions of different homodimer or heterodimer proteins are marked in the figure.
图4为不同转染比例对scFv-Fc/Fc 异源二聚体影响的电泳分析。4-12% SDS-PAGE蛋白凝胶电泳。泳道从左到右依次为：共转染比例为CH3_A重组载体: CH3_B重组载体=1:1、共转染比例为CH3_A重组载体: CH3_B重组载体=1.5:1和蛋白分子量标准。每组产物中所包含的同源二聚体和异源二聚体因为分子量差异而在凝胶电泳中的迁移距离不同。不同的同二聚体或异二聚体蛋白所处的位置在图中被标注出。Figure 4 shows the electrophoresis analysis of the effect of different transfection ratios on scFv-Fc/Fc heterodimer. 4-12% SDS-PAGE protein gel electrophoresis. The lanes from left to right are: the co-transfection ratio is CH3_A recombinant vector: CH3_B recombinant vector=1:1, the co-transfection ratio is CH3_A recombinant vector: CH3_B recombinant vector=1.5:1 and protein molecular weight standard. The homodimer and heterodimer contained in each group of products have different migration distances in gel electrophoresis due to the difference in molecular weight. The positions of different homodimer or heterodimer proteins are marked in the figure.
本发明的实施方式Embodiments of the present invention
以下实施例是对本发明进行进一步的说明，不应理解为是对本发明的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因***到这样的载体和质粒的方法或将质粒引入宿主细胞的方法．这样的方法对本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook, J., Fritsch,E.F. and Maniais,T. （1989 ）Molecular Cloning :A Laboratory Manual, 2 nd edition, Cold spring Harbor Laboratory Press. The following examples are to further illustrate the present invention, and should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold spring Harbor Laboratory Press.
 To
1、实验材料：1. Experimental materials:
293E细胞，来自NRC biotechnology Research Institute。293E cells, from NRC biotechnology Research Institute.
2、实验试剂： 2. Experimental reagents:
PBS：购自生工生物工程（上海）股份有限公司，货号B548117。PBS: purchased from Shenggong Biological Engineering (Shanghai) Co., Ltd., catalog number B548117.
柠檬酸：购自国药集团化学试剂有限公司。Citric acid: purchased from Sinopharm Chemical Reagent Co., Ltd.
Prime star HS DNA polymerase：购自Takara公司，货号R010A。Prime star HS DNA polymerase: purchased from Takara company, catalog number R010A.
无内毒素质粒大提试剂盒：购自 TIANGEN公司， 货号DP117。Endotoxin-free plasmid large-scale extraction kit: purchased from TIANGEN company, item number DP117.
3、实验仪器： 3. Experimental equipment:
HiTrap MabSelectSuRe 柱：购自GE公司。HiTrap MabSelectSuRe column: purchased from GE Company.
AKTA-FPLC快速蛋白液相色谱***：购自GE公司。AKTA-FPLC fast protein liquid chromatography system: purchased from GE Company.
C1000 Touch Thermal Cycler PCR仪：购自Bio-Rad公司。C1000 Touch Thermal Cycler PCR instrument: purchased from Bio-Rad.
Chemidoc MP凝胶成像仪：购自Bio-Rad公司。Chemidoc MP gel imager: purchased from Bio-Rad.
离心机：购自Eppendorf 公司。Centrifuge: purchased from Eppendorf.
G1600AX 毛细管电泳仪：购自安捷伦公司。G1600AX capillary electrophoresis instrument: purchased from Agilent.
MicroCal PEAQ-DSC微量热差式扫描量热仪：购自马尔文公司。MicroCal PEAQ-DSC micro calorimeter scanning calorimeter: purchased from Malvern Company.
 To
实施例Example 11 第一轮突变候选组合的设计、构建、表达和纯化Design, construction, expression and purification of the first round of mutation candidate combinations
1、第一轮 CH3结构域氨基酸修饰的设计1. The first round of CH3 domain amino acid modification design
已上市的绝大多数抗体都属于IgG1或IgG4亚型，并且IgG1和IgG4的Fc段氨基酸序列高度保守。本发明优选IgG1作为模板设计CH3结构域的氨基酸修饰，未做特殊说明的情况下这些氨基酸修饰同样适用于IgG4亚型。Most of the antibodies on the market belong to the IgG1 or IgG4 subtype, and the amino acid sequences of the Fc segment of IgG1 and IgG4 are highly conserved. In the present invention, IgG1 is preferably used as a template to design amino acid modifications of the CH3 domain, and these amino acid modifications are also applicable to IgG4 subtypes unless otherwise specified.
CH3结构域会形成同源二聚体，IgG1的抗体Fc段的CH3结构域晶体结构（pdb code:4BSW）如图1所示，CH3_A的K409位于由CH3_B的F405、D399和K370围成的空腔内，并且和D399的侧链形成离子相互作用。上述位点在IgG4上高度保守，仅仅和IgG1不同的是，IgG4重链上EU编号为370位的氨基酸为Arg。US20150307628A1中提到，将CH3_A的K409突变为Trp（或叫做K409W）创造出一个“凸”，需同时在CH3_B引入F405T和D399V点突变，创造出一个“凹”，否则会引起空间位阻排斥和疏水-静电排斥。本发明意外惊奇的发现，将CH3_A的K409突变为Phe（或叫做K409F）,并不会和CH3_B上的氨基酸F405引起空间位阻排斥，也不会和CH3_B上的氨基酸K370和D399发生疏水-静电排斥。当CH3_B上的氨基酸K370突变为Arg（或叫做K409R）时，也不会发生疏水-静电排斥。相反的，将CH3_B上的D399突变为非极性并且侧链较小的氨基酸如Ala（或叫做D399A）从而创造出一个“凹”时反而不利于异源二聚体的形成（见下文图3）。因此，本发明发现了一种比对照专利US20150307628A1更加简便的构造“凸-凹”模型的方法。The CH3 domain will form a homodimer. The crystal structure of the CH3 domain (pdb code: 4BSW) of the Fc segment of the IgG1 antibody is shown in Figure 1. K409 of CH3_A is located in the empty space surrounded by F405, D399 and K370 of CH3_B. Inside the cavity, and form an ionic interaction with the side chain of D399. The above-mentioned position is highly conserved on IgG4. The only difference from IgG1 is that the amino acid with EU numbering at position 370 on the heavy chain of IgG4 is Arg. It is mentioned in US20150307628A1 that the mutation of K409 of CH3_A to Trp (or K409W) creates a "convex". It is necessary to introduce the F405T and D399V point mutations in CH3_B at the same time to create a "concave", otherwise it will cause steric rejection and Hydrophobic-electrostatic repulsion. The present invention surprisingly discovered that the mutation of K409 of CH3_A to Phe (or K409F) does not cause steric repulsion with amino acid F405 on CH3_B, nor does it cause hydrophobic-electrostatic discharge with amino acids K370 and D399 on CH3_B. Repel. When the amino acid K370 on CH3_B is mutated to Arg (or K409R), hydrophobic-electrostatic repulsion will not occur. On the contrary, mutating D399 on CH3_B to a non-polar amino acid with a smaller side chain such as Ala (or D399A) to create a "concave" is not conducive to the formation of heterodimers (see Figure 3 below) ). Therefore, the present invention has discovered a more convenient method of constructing a "convex-concave" model than the control patent US20150307628A1.
为了抑制CH3_A形成同源二聚体，进一步在CH3_A上引入F405E或F405D点突变；为了抑制CH3_B形成同源二聚体，在CH3_B上引入S364R+E357S点突变，同时在CH3_A上引入K370D或K370E点突变，如图2所示。如上所述，得到表1中的异源二聚体突变组合。To inhibit CH3_A from forming a homodimer, further introduce F405E or F405D point mutations on CH3_A; to inhibit CH3_B from forming a homodimer, introduce S364R+E357S point mutations on CH3_B, and at the same time introduce K370D or K370E points on CH3_A Mutations, as shown in Figure 2. As described above, the heterodimer mutation combinations in Table 1 were obtained.
表1 异源二聚体突变组合列表-1 Table 1 List of heterodimer mutation combinations-1
Figure 541112dest_path_image001
Figure 541112dest_path_image001
2、构建带有突变的人IgG1的Fc片段以及scFv-Fc融合蛋白的重组载体2. Construction of recombinant vector with mutant human IgG1 Fc fragment and scFv-Fc fusion protein
人工合成获得编码scFv-Fc融合蛋白（scFv-Fc融合蛋白序列见SEQ ID：1）基因，其中的scFv是指抗CD3的单链抗体。然后亚克隆至哺乳动物细胞表达载体pTT5获得用于哺乳动物细胞表达scFv-Fc融合蛋白的重组表达载体。将上述基因的Fc片段（Fc融合蛋白序列见SEQ ID NO：2）亚克隆至哺乳动物细胞表达载体pTT5获得用于哺乳动物细胞表达Fc融合蛋白的重组表达载体。根据实施例1的表1，利用重叠PCR法对scFv-Fc及Fc编码基因进行组合突变，其中针对CH3_A链的突变位于Fc融合蛋白上，针对CH3_B链的突变位于scFv-Fc融合蛋白上。将突变后的基因亚克隆到pTT5，最终分别得到用于在哺乳动物细胞中表达突变的 scFv-Fc融合蛋白及突变的Fc蛋白的重组表达载体。The gene encoding scFv-Fc fusion protein (see SEQ ID: 1 for the sequence of scFv-Fc fusion protein) was artificially synthesized, where scFv refers to an anti-CD3 single-chain antibody. Then subcloned into mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cells to express scFv-Fc fusion protein. The Fc fragment of the above gene (see SEQ ID NO: 2 for the Fc fusion protein sequence) was subcloned into the mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cells to express the Fc fusion protein. According to Table 1 of Example 1, combined mutations of scFv-Fc and Fc encoding genes were performed by overlapping PCR method, wherein the mutation for the CH3_A chain was located on the Fc fusion protein, and the mutation for the CH3_B chain was located on the scFv-Fc fusion protein. The mutated gene was subcloned into pTT5, and finally a recombinant expression vector for expressing the mutated scFv-Fc fusion protein and the mutated Fc protein in mammalian cells was obtained.
3、瞬时表达scFv-Fc/Fc异源二聚体，并检测不同突变组合对异源二聚体含量的影响3. Transiently express scFv-Fc/Fc heterodimer, and detect the influence of different mutation combinations on the content of heterodimer
将步骤1的4种突变组合相应的表达载体用PEI转染至悬浮培养的293E细胞，每一组突变组合都包括了其相对应的A链（指代scFV-Fc融合蛋白链）和B链（指代Fc蛋白链）的重组表达载体共同转染，且A链和B链的重组表达载体共转比例为1:1。培养5～6天后，收集瞬时表达培养上清液，通过Protein A亲和层析法，得到初步纯化的4组突变组合。这些瞬转产物中都包含了不同比例的同源二聚体蛋白（scFv-Fc/scFv-Fc，Fc/Fc）和异源二聚体蛋白（scFv-Fc/Fc）。由于这三种蛋白（scFv-Fc/scFv-Fc，Fc/Fc，和scFv-Fc/Fc）的分子量大小有差异，可以通过非还原条件下SDS-PAGE电泳检测每组产物中同源二聚体蛋白（scFv-Fc/scFv-Fc，Fc/Fc） 和异源二聚体蛋白 （scFv-Fc/Fc） 的组成情况，电泳检测结果如图3所示。组合1和组合2的同源二聚体明显多于组合3和组合4，表明CH3_B上的D399A突变并不利于异源二聚体的形成。组合4的同源二聚体比组合3少，表明F405E突变优于F405D。Transfect the corresponding expression vectors of the 4 mutation combinations in step 1 into suspension cultured 293E cells with PEI. Each mutation combination includes its corresponding A chain (referring to the scFV-Fc fusion protein chain) and B chain (Refers to the Fc protein chain) recombinant expression vectors are co-transfected, and the co-transfection ratio of the A chain and B chain recombinant expression vectors is 1:1. After culturing for 5-6 days, collect the transient expression culture supernatant and pass it through Protein A affinity chromatography method, to get 4 sets of mutant combinations that were preliminarily purified. These transient products all contain different ratios of homodimer proteins (scFv-Fc/scFv-Fc, Fc/Fc) and heterodimer proteins (scFv-Fc/Fc). Since the molecular weights of these three proteins (scFv-Fc/scFv-Fc, Fc/Fc, and scFv-Fc/Fc) are different, the homodimerization in each group of products can be detected by SDS-PAGE electrophoresis under non-reducing conditions The composition of body protein (scFv-Fc/scFv-Fc, Fc/Fc) and heterodimer protein (scFv-Fc/Fc), the electrophoresis detection result is shown in Figure 3. Combination 1 and combination 2 had significantly more homodimers than combination 3 and combination 4, indicating that the D399A mutation on CH3_B is not conducive to the formation of heterodimers. Combination 4 has fewer homodimers than combination 3, indicating that the F405E mutation is better than F405D.
实施例Example 2. 2. 第二轮突变候选组合的设计、构建、表达和纯化Design, construction, expression and purification of the second round of mutation candidate combinations
实施例1中通过在CH3_B链上引入带正电荷的氨基酸，利用静电排斥作用抑制同源二聚体（scFv-Fc/scFv-Fc）的形成。本实施例通过引入其他带电荷氨基酸突变的组合，旨在进一步探索减少CH3_B链之间的相互吸引，抑制同源二聚体蛋白形成的方法。在CH3_A结构域已有的K409F, F405E点突变组合基础上，进一步在CH3_A结构域和CH3_B结构域中引入下述的带电荷氨基酸突变组合：In Example 1, by introducing positively charged amino acids into the CH3_B chain, electrostatic repulsion was used to inhibit the formation of homodimers (scFv-Fc/scFv-Fc). In this example, by introducing a combination of other charged amino acid mutations, it aims to further explore ways to reduce the mutual attraction between CH3_B chains and inhibit the formation of homodimer proteins. The existing K409F in the CH3_A domain, Based on the combination of F405E point mutations, the following charged amino acid mutation combinations were further introduced into the CH3_A domain and CH3_B domain:
CH3_A结构域：K392D，CH3_B结构域：D399K；CH3_A domain: K392D, CH3_B domain: D399K;
CH3_A结构域：K439D，CH3_B结构域：E356K+E357K；CH3_A domain: K439D, CH3_B domain: E356K+E357K;
CH3_A结构域：L368D，CH3_B结构域：S364R；CH3_A domain: L368D, CH3_B domain: S364R;
CH3_A结构域：L368D，CH3_B结构域：S364K；CH3_A domain: L368D, CH3_B domain: S364K;
CH3_A结构域：K360E，CH3_B结构域：Q347R；CH3_A domain: K360E, CH3_B domain: Q347R;
CH3_A结构域：K370D+K360E，CH3_B结构域：S364R+E357S+Q347R。CH3_A domain: K370D+K360E, CH3_B domain: S364R+E357S+Q347R.
从而得到表2中所示的组合5-10。利用重叠PCR法对scFv-Fc及Fc编码基因进行组合突变，其中针对CH3_A链的突变位于Fc融合蛋白上，针对CH3_B链的突变位于scFv-Fc蛋白上。将突变后的基因亚克隆到pTT5，最终分别得到用于在哺乳动物细胞中表达突变的scFv-Fc融合蛋白及突变的Fc蛋白的重组表达载体。组合4作为对照组，组合5-10作为测试组，表达纯化方法如实施例1中步骤3所示。将纯化得到的蛋白分用非还原毛细管凝胶电泳分析，并计算各产物组分峰面积的百分比例。结果如表3所示，组合5-10均能获得较好的异源二聚体纯度，其中组合7与组合4获得的异源二聚体的纯度相当，组合10是在组合4的的基础上增加了新的带电氨基酸突变，获得了更优的异源二聚体的纯度。Thus, combinations 5-10 shown in Table 2 were obtained. The overlapping PCR method was used to carry out combined mutations of scFv-Fc and Fc encoding genes, wherein the mutation for the CH3_A chain was located on the Fc fusion protein, and the mutation for the CH3_B chain was located on the scFv-Fc protein. The mutated gene was subcloned into pTT5, and finally a recombinant expression vector for expressing the mutated scFv-Fc fusion protein and the mutated Fc protein in mammalian cells was obtained. Combination 4 was used as the control group, and combinations 5-10 were used as the test group. The expression purification method was as shown in step 3 in Example 1. The purified protein fraction was analyzed by non-reducing capillary gel electrophoresis, and the percentage of peak area of each product component was calculated. The results are shown in Table 3. Combinations 5-10 can obtain good heterodimer purity. Among them, the purity of the heterodimer obtained by combination 7 and combination 4 is equivalent, and combination 10 is the basis of combination 4. A new charged amino acid mutation was added to the top, and a better purity of the heterodimer was obtained.
表2 异源二聚体突变组合列表-2Table 2 List of heterodimer mutation combinations-2
Figure 455364dest_path_image002
Figure 455364dest_path_image002
表3 不同带电荷氨基酸组合对同源二聚体和异源二聚体的比例的影响Table 3 The influence of different charged amino acid combinations on the ratio of homodimer and heterodimer
Figure 738578dest_path_image003
Figure 738578dest_path_image003
实施例Example 3 3 转染比例对Transfection ratio scFv-Fc/Fc scFv-Fc/Fc 异源二聚体形成的影响Influence of heterodimer formation
为了进一步考察A链的重组载体和B链的重组载体共转比例对于同源二聚体和异源二聚体比例的影响，将较优的突变组合4所用的共转表达载体分别用CH3_A重组载体: CH3_B重组载体=1:1及CH3_A重组载体: CH3_B重组载体=1.5:1的比例用PEI转染至悬浮培养的293E细胞，培养5-6天后，收集细胞上清。通过Protein A亲和层析法，得到各自的瞬转产物。通过非还原条件下SDS-PAGE电泳检测同源二聚体蛋白 （scFv-Fc/scFv-Fc，Fc/Fc）和异源二聚体蛋白 （scFv-Fc/Fc） 的组成情况。具体结果见图4。从结果可以看出：重组表达载体共转比例对于产物中同源二聚体和异源二聚体比例的会带来比较明显的影响。CH3_B比CH3_A相对更容易形成同源二聚体，当共转染比例为CH3_A重组载体: CH3_B重组载体=1:1时，同源二聚体蛋白 scFv-Fc/scFv-Fc明显较多；当提高CH3_A重组载体的转染比例，如共转染比例为CH3_A重组载体: CH3_B重组载体=1.5:1时，同源二聚体蛋白 scFv-Fc/scFv-Fc明显减少。In order to further investigate the effect of the cotransformation ratio of the A chain recombination vector and the B chain recombination vector on the ratio of homodimer and heterodimer, the cotransformation expression vector used in the better mutation combination 4 was recombined with CH3_A. Carrier: The ratio of CH3_B recombinant vector=1:1 and CH3_A recombinant vector: CH3_B recombinant vector=1.5:1 was transfected into suspension cultured 293E cells with PEI. After 5-6 days of culture, the cell supernatant was collected. Through Protein A affinity chromatography, the respective transient products were obtained. The composition of homodimer proteins (scFv-Fc/scFv-Fc, Fc/Fc) and heterodimer proteins (scFv-Fc/Fc) was detected by SDS-PAGE electrophoresis under non-reducing conditions. The specific results are shown in Figure 4. It can be seen from the results that the co-transformation ratio of the recombinant expression vector will have a more obvious impact on the ratio of homodimer and heterodimer in the product. CH3_B is relatively easier to form homodimers than CH3_A. When the co-transfection ratio is CH3_A recombinant vector: CH3_B recombinant vector=1:1, the homodimer protein scFv-Fc/scFv-Fc is obviously more; Increase the transfection ratio of CH3_A recombinant vector. For example, when the co-transfection ratio is CH3_A recombinant vector: CH3_B recombinant vector = 1.5:1, the homodimer protein scFv-Fc/scFv-Fc is significantly reduced.
实施例Example 4 4 二硫键对Disulfide bond pair scFv-Fc/Fc scFv-Fc/Fc 异源二聚体形成的影响Influence of heterodimer formation
在US7695936.B2和US20150307628A1中提到二硫键可以促进异源二聚体的形成，并且提高分子的热稳定性。本实施例在突变组合4基础上引入半胱氨酸突变，得到表4所示的突变组合。人工合成抗体重链HC_A（SEQ ID NO：3）、抗体重链HC_B（SEQ ID NO：4）和抗体共同轻链LC（SEQ ID NO：5）的编码基因，然后亚克隆至哺乳动物细胞表达载体pTT5上。利用重叠PCR法对HC_A及HC_B编码基因进行组合突变，其中针对CH3_A链的突变位于HC_A链上，针对CH3_B链的突变位于HC_B链上。将突变后的基因亚克隆到pTT5，最终分别得到用于在哺乳动物细胞中表达突变的重组表达载体。表达纯化方法如实施例1中步骤3所示，重组表达载体共转比例调整为HC_A链：HC_B链：共同轻链LC=1.5:1:2。将纯化得到的蛋白分用LC-MS分析，并计算各产物组分的百分比例。使用微量热差式扫描量热仪MicroCal PEAQ-DSC测量样品的Tm值。结果如表5所示，组合11作为对照组，组合12和组合13在组合11的基础上引入二硫键，获得的异源二聚体比例分别为99.56%和96.89%，与组合11相似；组合12和组合13的Fc的Tm值分别为69.21℃和70.23℃，比组合11提高约3℃。本实施例发现，尽管对照组（组合11）已经具有大于95%的异源二聚体比例，引入二硫键依然能进一步促进异源二聚体的形成，并且提高抗体分子的热稳定性。类似的，引入二硫键也应该能提高组合5-10的异源二聚体比例和热稳定性。It is mentioned in US7695936.B2 and US20150307628A1 that disulfide bonds can promote the formation of heterodimers and improve the thermal stability of molecules. In this example, cysteine mutations were introduced on the basis of mutation combination 4, and the mutation combinations shown in Table 4 were obtained. Artificially synthesized antibody heavy chain HC_A (SEQ ID NO: 3), antibody heavy chain HC_B (SEQ ID NO: 4) and antibody common light chain LC (SEQ ID NO: 5) coding genes, and then subcloned into mammalian cells for expression On the vector pTT5. The combined mutations of HC_A and HC_B encoding genes were carried out by overlap PCR, wherein the mutations for the CH3_A chain were located on the HC_A chain, and the mutations for the CH3_B chain were located on the HC_B chain. The mutated genes were subcloned into pTT5, and finally recombinant expression vectors for expressing the mutations in mammalian cells were obtained. The expression purification method is as shown in step 3 in Example 1, and the co-transformation ratio of the recombinant expression vector is adjusted to HC_A chain: HC_B chain: common light chain LC=1.5:1:2. The purified protein fraction was analyzed by LC-MS, and the percentage of each product component was calculated. MicroCal PEAQ-DSC was used to measure the Tm value of the sample. The results are shown in Table 5. Combination 11 was used as a control group, and combinations 12 and 13 introduced disulfide bonds on the basis of combination 11. The obtained heterodimer ratios were 99.56% and 96.89%, which were similar to combination 11; The Tm values of Fc of combination 12 and combination 13 were 69.21°C and 70.23°C, respectively, which was about 3°C higher than that of combination 11. This example found that even though the control group (combination 11) already has a heterodimer ratio of greater than 95%, the introduction of disulfide bonds can still further promote the formation of heterodimers and improve the thermal stability of antibody molecules. Similarly, the introduction of disulfide bonds should also improve the heterodimer ratio and thermal stability of the combination 5-10.
表4 半胱氨酸突变组合Table 4 Cysteine mutation combinations
Figure 29882dest_path_image004
Figure 29882dest_path_image004
表5 二硫键对异源二聚体的比例和热稳定性的影响Table 5 The influence of disulfide bonds on the ratio and thermal stability of heterodimers
Figure 405238dest_path_image005
。
Figure 405238dest_path_image005
 .
以上对本发明的具体实施例进行了详细描述，但其只是作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should all fall within the scope of the present invention.
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<110>  周易<110> Zhouyi
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<120>  CH3结构域改造诱导形成的异源二聚体及其制备方法和应用<120> Heterodimer induced by CH3 structural domain modification and its preparation method and application
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<170>  PatentIn version 3.3<170> PatentIn version 3.3
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<223>  scFv-Fc融合蛋白<223> scFv-Fc fusion protein
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Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
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Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
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Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
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Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
    50                  55                  60                  50 50 55 60
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Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65                  70                  75                  80  65 70 75 80
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Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
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 To
 To
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
            100                 105                 110         100 100 105 110
 To
 To
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
        115                 120                 125             115 115 120 125
 To
 To
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
    130                 135                 140                 130 130 135 140
 To
 To
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145                 150                 155                 160 145 150 155 160
 To
 To
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
                165                 170                 175     165 165 170 175
 To
 To
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
            180                 185                 190         180 180 185 190
 To
 To
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
        195                 200                 205             195 195 200 205
 To
 To
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
    210                 215                 220                 210 210 215 220
 To
 To
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225                 230                 235                 240 225 230 235 240
 To
 To
Glu Leu Lys Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Glu Leu Lys Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro
                245                 250                 255     245 245 250 255
 To
 To
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
            260                 265                 270         260 260 265 270 270
 To
 To
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
        275                 280                 285             275 275 280 285
 To
 To
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
    290                 295                 300                 290 290 295 300
 To
 To
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
305                 310                 315                 320 305 310 315 320
 To
 To
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
                325                 330                 335     325 325 330 335
 To
 To
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
            340                 345                 350         340 345 350
 To
 To
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
        355                 360                 365             355 355 360 365
 To
 To
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
    370                 375                 380                 370 370 375 380
 To
 To
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
385                 390                 395                 400 385 390 395 400
 To
 To
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
                405                 410                 415     405 405 410 415
 To
 To
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
            420                 425                 430         420 420 425 430
 To
 To
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
        435                 440                 445             435 435 440 445
 To
 To
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
    450                 455                 460                 450 450 455 460
 To
 To
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465                 470                 475 465 470 475
 To
 To
<210>  2<210> 2
<211>  227<211> 227
<212>  PRT<212> PRT
<213>  Artificial<213> Artificial
 To
<220><220>
<223>  Fc融合蛋白<223> Fc fusion protein
 To
<400>  2<400> 2
 To
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1               5                   10                  15      1 5 10 15
 To
 To
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
            20                  25                  30          20 20 25 30
 To
 To
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
        35                  40                  45              35 35 40 45 ...
 To
 To
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
    50                  55                  60                  50 50 55 60
 To
 To
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65                  70                  75                  80  65 70 75 80
 To
 To
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
                85                  90                  95      85 85 90 95
 To
 To
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
            100                 105                 110         100 100 105 110
 To
 To
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
        115                 120                 125             115 115 120 125
 To
 To
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
    130                 135                 140                 130 130 135 140
 To
 To
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145                 150                 155                 160 145 150 155 160
 To
 To
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
                165                 170                 175     165 165 170 175
 To
 To
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
            180                 185                 190         180 180 185 190
 To
 To
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
        195                 200                 205             195 195 200 205
 To
 To
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
    210                 215                 220                 210 210 215 220
 To
 To
Pro Gly Lys Pro Gly Lys
225         225
 To
 To
<210>  3<210> 3
<211>  450<211> 450
<212>  PRT<212> PRT
<213>  Artificial<213> Artificial
 To
<220><220>
<223>  抗体重链HC_A<223> Antibody heavy chain HC_A
 To
<400>  3<400> 3
 To
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1               5                   10                  15      1 5 10 15
 To
 To
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
            20                  25                  30          20 20 25 30
 To
 To
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
        35                  40                  45              35 35 40 45
 To
 To
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
    50                  55                  60                  50 50 55 60
 To
 To
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65                  70                  75                  80  65 70 75 80
 To
 To
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                85                  90                  95      85 85 90 95
 To
 To
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
            100                 105                 110         100 100 105 110
 To
 To
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
        115                 120                 125             115 115 120 125
 To
 To
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
    130                 135                 140                 130 130 135 140
 To
 To
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145                 150                 155                 160 145 150 155 160
 To
 To
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
                165                 170                 175     165 165 170 175
 To
 To
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
            180                 185                 190         180 180 185 190
 To
 To
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
        195                 200                 205             195 195 200 205
 To
 To
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
    210                 215                 220                 210 210 215 220
 To
 To
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225                 230                 235                 240 225 230 235 240
 To
 To
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
                245                 250                 255     245 245 250 255
 To
 To
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
            260                 265                 270         260 260 265 270
 To
 To
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
        275                 280                 285             275 275 280 285
 To
 To
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
    290                 295                 300                 290 290 295 300
 To
 To
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305                 310                 315                 320 305 310 315 320
 To
 To
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
                325                 330                 335     325 325 330 335
 To
 To
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
            340                 345                 350         340 345 350
 To
 To
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
        355                 360                 365             355 355 360 365
 To
 To
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
    370                 375                 380                 370 370 375 380 380
 To
 To
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385                 390                 395                 400 385 390 395 400
 To
 To
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
                405                 410                 415     405 405 410 415
 To
 To
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
            420                 425                 430         420 420 425 430
 To
 To
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
        435                 440                 445             435 435 440 445
 To
 To
Gly Lys Gly Lys
    450 450
 To
 To
<210>  4<210> 4
<211>  449<211> 449
<212>  PRT<212> PRT
<213>  Artificial<213> Artificial
 To
<220><220>
<223>  抗体重链HC_B<223> Antibody heavy chain HC_B
 To
<400>  4<400> 4
 To
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1               5                   10                  15      1 5 10 15
 To
 To
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
            20                  25                  30          20 20 25 30
 To
 To
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
        35                  40                  45              35 35 40 45
 To
 To
Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr
    50                  55                  60                  50 50 55 60
 To
 To
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65                  70                  75                  80  65 70 75 80
 To
 To
Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala
                85                  90                  95      85 85 90 95
 To
 To
Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
            100                 105                 110         100 100 105 110
 To
 To
Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
        115                 120                 125             115 115 120 125
 To
 To
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
    130                 135                 140                 130 130 135 140
 To
 To
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145                 150                 155                 160 145 150 155 160
 To
 To
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
                165                 170                 175     165 165 170 175
 To
 To
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
            180                 185                 190         180 180 185 190
 To
 To
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
        195                 200                 205             195 195 200 205
 To
 To
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
    210                 215                 220                 210 210 215 220
 To
 To
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225                 230                 235                 240 225 230 235 240
 To
 To
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
                245                 250                 255     245 245 250 255
 To
 To
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Arg Thr Pro Glu Val Thr Cys Val Val Val Val Asp Val Ser His Glu Asp
            260                 265                 270         260 260 265 270
 To
 To
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
        275                 280                 285             275 275 280 285
 To
 To
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
    290                 295                 300                 290 290 295 300
 To
 To
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305                 310                 315                 320 305 310 315 320
 To
 To
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
                325                 330                 335     325 325 330 335
 To
 To
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
            340                 345                 350         340 345 350
 To
 To
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
        355                 360                 365             355 355 360 365
 To
 To
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
    370                 375                 380                 370 370 375 380
 To
 To
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385                 390                 395                 400 385 390 395 400
 To
 To
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
                405                 410                 415     405 405 410 415
 To
 To
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
            420                 425                 430         420 420 425 430
 To
 To
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
        435                 440                 445             435 435 440 445
 To
 To
Lys Lys
    ...
 To
 To
<210>  5<210> 5
<211>  214<211> 214
<212>  PRT<212> PRT
<213>  Artificial<213> Artificial
 To
<220><220>
<223>  抗体共同轻链LC<223> Antibody Common Light Chain LC
 To
<400>  5<400> 5
 To
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1               5                   10                  15      1 5 10 15
 To
 To
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn
            20                  25                  30          20 20 25 30
 To
 To
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
        35                  40                  45              35 35 40 45
 To
 To
Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
    50                  55                  60                  50 50 55 60 ``
 To
 To
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
65                  70                  75                  80  65 70 75 80
 To
 To
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr
                85                  90                  95      85 85 90 95
 To
 To
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala
            100                 105                 110         100 100 105 110
 To
 To
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
        115                 120                 125             115 115 120 125
 To
 To
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
    130                 135                 140                 130 130 135 140
 To
 To
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145                 150                 155                 160 145 150 155 160
 To
 To
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
                165                 170                 175     165 165 170 175
 To
 To
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
            180                 185                 190         180 180 185 190
 To
 To
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
        195                 200                 205             195 195 200 205
 To
 To
Phe Asn Arg Gly Glu Cys Phe Asn Arg Gly Glu Cys
    210                 。... 210 .

Claims (10)

  1. 一种异源二聚体，其特征在于，其含有第一多肽链和第二多肽链，所述第一多肽链和第二多肽链分别含有抗体重链恒定区CH3结构域，分别命名为CH3_A结构域和CH3_B结构域，与野生型的人抗体重链恒定区CH3结构域相比，所述CH3_A和CH3_B结构域含有如下位置氨基酸的突变：A heterodimer, characterized in that it contains a first polypeptide chain and a second polypeptide chain, the first polypeptide chain and the second polypeptide chain each containing the CH3 domain of the constant region of the antibody heavy chain, Named as the CH3_A domain and CH3_B domain, respectively, compared with the wild-type human antibody heavy chain constant region CH3 domain, the CH3_A and CH3_B domains contain amino acid mutations at the following positions:
    CH3_A结构域的K409、F405发生突变，且CH3_A结构域和CH3_B结构域还分别在选自Q347、Y349、S354、E356、E357、K360、S364、L368、K370、K392、D399和K439中的一个或多个氨基酸位置具有的突变；The K409 and F405 of the CH3_A domain are mutated, and the CH3_A domain and the CH3_B domain are respectively selected from one of Q347, Y349, S354, E356, E357, K360, S364, L368, K370, K392, D399 and K439. Mutations in multiple amino acid positions;
    优选地，所述CH3_A结构域和CH3_B结构域具有选自下列的突变中的一种或多种：Preferably, the CH3_A structural domain and the CH3_B structural domain have one or more selected from the following mutations:
    1a) CH3_B结构域的 S364、E357发生突变，且CH3_A结构域的K370发生突变；1a) S364 and E357 of CH3_B domain are mutated, and K370 of CH3_A domain is mutated;
    1b) CH3_B结构域的D399发生突变，且CH3_A结构域的K392发生突变；1b) D399 of the CH3_B domain is mutated, and K392 of the CH3_A domain is mutated;
    1c) CH3_B结构域的E356、E357发生突变，且CH3_A结构域的K439发生突变；1c) E356 and E357 of CH3_B domain are mutated, and K439 of CH3_A domain is mutated;
    1d) CH3_B结构域的S364发生突变，且CH3_A结构域的L368发生突变；1d) S364 of the CH3_B domain is mutated, and L368 of the CH3_A domain is mutated;
    1e) CH3_B结构域的Q347发生突变，且CH3_A结构域的K360发生突变；1e) Q347 of the CH3_B domain is mutated, and K360 of the CH3_A domain is mutated;
    以上所述的氨基酸的位置根据KABAT 编号的EU 索引确定。The positions of the above-mentioned amino acids are determined according to the EU index of the KABAT number.
  2. 根据权利要求1所述的异源二聚体，其特征在于，所述CH3_A结构域和CH3_B结构域还具有下列突变：The heterodimer according to claim 1, wherein the CH3_A domain and CH3_B domain further have the following mutations:
    1f)CH3_B结构域的S354发生突变，且CH3_A结构域的Y349发生突变；或1f) S354 of the CH3_B domain is mutated, and Y349 of the CH3_A domain is mutated; or
    1g)CH3_B结构域的Y349发生突变，且CH3_A结构域的S354发生突变。1g) Y349 of the CH3_B domain is mutated, and S354 of the CH3_A domain is mutated.
  3. 根据权利要求1或2所述的异源二聚体，其特征在于，所述突变选自以下突变中的一个或数个：Q347R、Y349C、S354C、E356K、E357K、E357S、K360E、S364R、S364K、L368D、K370D、K392D、D399K、K439E、F405E和K409F。The heterodimer according to claim 1 or 2, wherein the mutation is selected from one or more of the following mutations: Q347R, Y349C, S354C, E356K, E357K, E357S, K360E, S364R, S364K , L368D, K370D, K392D, D399K, K439E, F405E and K409F.
  4. 根据权利要求3所述的异源二聚体，其特征在于，所述的CH3_A结构域和CH3_B结构域含有选自以下一组的突变：The heterodimer according to claim 3, wherein the CH3_A domain and CH3_B domain contain mutations selected from the following group:
    （a1）CH3_A结构域：F405E+K409F+K370D，CH3_B结构域：S364R+E357S；(A1) CH3_A domain: F405E+K409F+K370D, CH3_B domain: S364R+E357S;
    （a2）CH3_A结构域：F405E+K409F+K370D+S354C，CH3_B结构域：S364R+E357S+ Y349C；(A2) CH3_A domain: F405E+K409F+K370D+S354C, CH3_B domain: S364R+E357S+Y349C;
    （a3）CH3_A结构域：F405E+K409F+K370D+Y349C，CH3_B结构域：S364R+E357S+ S354C(A3) CH3_A domain: F405E+K409F+K370D+Y349C, CH3_B domain: S364R+E357S+ S354C
    （b1）CH3_A结构域：F405E+K409F+K392D，CH3_B结构域：D399K；(B1) CH3_A domain: F405E+K409F+K392D, CH3_B domain: D399K;
    （b2）CH3_A结构域：F405E+K409F+K392D+S354C，CH3_B结构域：D399K+Y349C；(B2) CH3_A domain: F405E+K409F+K392D+S354C, CH3_B domain: D399K+Y349C;
    （b3）CH3_A结构域：F405E+K409F+K392D+Y349C，CH3_B结构域：D399K+S354C；(B3) CH3_A domain: F405E+K409F+K392D+Y349C, CH3_B domain: D399K+S354C;
    （c1）CH3_A结构域：F405E+K409F+K439D，CH3_B结构域：E356K+E357K；(C1) CH3_A domain: F405E+K409F+K439D, CH3_B domain: E356K+E357K;
    （c2）CH3_A结构域：F405E+K409F+K439D+S354C，CH3_B结构域：E356K+E357K+ Y349C；(C2) CH3_A domain: F405E+K409F+K439D+S354C, CH3_B domain: E356K+E357K+Y349C;
    （c3）CH3_A结构域：F405E+K409F+K439D+Y349C，CH3_B结构域：E356K+E357K+S354C；(C3) CH3_A domain: F405E+K409F+K439D+Y349C, CH3_B domain: E356K+E357K+S354C;
    （d1）CH3_A结构域：F405E+K409F+L368D，CH3_B结构域：S364R；(D1) CH3_A domain: F405E+K409F+L368D, CH3_B domain: S364R;
    （d2）CH3_A结构域：F405E+K409F+L368D+S354C，CH3_B结构域：S364R+ Y349C；(D2) CH3_A domain: F405E+K409F+L368D+S354C, CH3_B domain: S364R+ Y349C;
    （d3）CH3_A结构域：F405E+K409F+L368D+Y349C，CH3_B结构域：S364R+S354C；(D3) CH3_A domain: F405E+K409F+L368D+Y349C, CH3_B domain: S364R+S354C;
    （e1）CH3_A结构域：F405E+K409F+L368D，CH3_B结构域：S364K；(E1) CH3_A domain: F405E+K409F+L368D, CH3_B domain: S364K;
    （e2）CH3_A结构域：F405E+K409F+L368D+S354C，CH3_B结构域：S364K+Y349C；(E2) CH3_A domain: F405E+K409F+L368D+S354C, CH3_B domain: S364K+Y349C;
    （e3）CH3_A结构域：F405E+K409F+L368D+Y349C，CH3_B结构域：S364K+S354C；(E3) CH3_A domain: F405E+K409F+L368D+Y349C, CH3_B domain: S364K+S354C;
    （f1）CH3_A结构域：F405E+K409F+K360E，CH3_B结构域：Q347R；(F1) CH3_A domain: F405E+K409F+K360E, CH3_B domain: Q347R;
    （f2）CH3_A结构域：F405E+K409F+K360E+S354C，CH3_B结构域：Q347R+Y349C；(F2) CH3_A domain: F405E+K409F+K360E+S354C, CH3_B domain: Q347R+Y349C;
    （f3）CH3_A结构域：F405E+K409F+K360E+Y349C，CH3_B结构域：Q347R+S354C；(F3) CH3_A domain: F405E+K409F+K360E+Y349C, CH3_B domain: Q347R+S354C;
    （g1）CH3_A结构域：F405E+K409F+K370D+K360E，CH3_B结构域：S364R+E357S+ Q347R；(G1) CH3_A domain: F405E+K409F+K370D+K360E, CH3_B domain: S364R+E357S+ Q347R;
    （g2）CH3_A结构域：F405E+K409F+K370D+K360E+S354C，CH3_B结构域：S364R+E357S+ Q347R+Y349C；(G2) CH3_A domain: F405E+K409F+K370D+K360E+S354C, CH3_B domain: S364R+E357S+ Q347R+Y349C;
    （g3）CH3_A结构域：F405E+K409F+K370D+K360E+Y349C，CH3_B结构域：S364R+E357S+ Q347R+S354C。(G3) CH3_A domain: F405E+K409F+K370D+K360E+Y349C, CH3_B domain: S364R+E357S+Q347R+S354C.
  5. 根据权利要求1或2所述的异源二聚体，其特征在于，所述的抗体恒定区来源于IgG、IgA、IgD、IgE 或 IgM，IgG优选为IgG1、IgG2、IgG3或IgG4，IgA优选为IgA1或IgA2。The heterodimer according to claim 1 or 2, wherein the antibody constant region is derived from IgG, IgA, IgD, IgE or IgM, and IgG is preferably IgG1, IgG2, IgG3 or IgG4, and IgA is preferably It is IgA1 or IgA2.
  6. 一种组合物，其特征在于，其含有：（1）权利要求1-5中任意一项所述的异源二聚体，以及（2）药学上可接受的载体和/或稀释剂和/或赋形剂。A composition, characterized in that it contains: (1) the heterodimer according to any one of claims 1 to 5, and (2) a pharmaceutically acceptable carrier and/or diluent and/ Or excipients.
  7. 一种多核苷酸、载体组合、或重组宿主细胞，其特征在于，所述多核苷酸包含：编码权利要求1-5中任意一项所述的异源二聚体的第一条多肽链的核苷酸分子A，以及编码权利要求1-5中任意一项所述的异源二聚体的第二条多肽链的核苷酸分子B；A polynucleotide, vector combination, or recombinant host cell, characterized in that the polynucleotide comprises: the first polypeptide chain encoding the heterodimer of any one of claims 1-5 Nucleotide molecule A, and nucleotide molecule B encoding the second polypeptide chain of the heterodimer according to any one of claims 1-5;
    所述载体组合包括：含有所述核苷酸分子A的重组载体A，以及含有所述核苷酸分子B的重组载体B；The vector combination includes: a recombinant vector A containing the nucleotide molecule A, and a recombinant vector B containing the nucleotide molecule B;
    所述重组宿主细胞含有所述载体组合；其中，所述重组宿主细胞的原始宿主细胞优选为真核细胞，进一步优选为CHO细胞或293E细胞。The recombinant host cell contains the vector combination; wherein, the original host cell of the recombinant host cell is preferably a eukaryotic cell, more preferably a CHO cell or a 293E cell.
  8. 权利要求1-5中任意一项所述的异源二聚体，权利要求6所述的组合物，权利要求7所述的多核苷酸、载体组合或重组宿主细胞在在制备双特异性抗体、双特异性融合蛋白和抗体-融合蛋白嵌合体中的用途。The heterodimer according to any one of claims 1-5, the composition according to claim 6, the polynucleotide, vector combination or recombinant host cell according to claim 7 are in the preparation of bispecific antibodies , Use in bispecific fusion proteins and antibody-fusion protein chimeras.
  9. 一种制备权利要求1-5中任意一项所述的异源二聚体的方法，其特征在于，使用权利要求7所述的重组宿主细胞表达所述异源二聚体。A method for preparing the heterodimer according to any one of claims 1 to 5, characterized in that the recombinant host cell according to claim 7 is used to express the heterodimer.
  10. 根据权利要求9所述的方法，其特征在于，所述重组宿主细胞中重组载体A和重组载体B的转染比例为1:3～3:1，例如 1:2～2:1，例如1:1.5～1.5:1，例如约 1:1。The method according to claim 9, wherein the transfection ratio of the recombinant vector A and the recombinant vector B in the recombinant host cell is 1:3 to 3:1, for example, 1:2 to 2:1, for example, 1 : 1.5～1.5:1, for example, about 1:1.
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