WO2020135555A1 - 二价双特异性抗体及其制备方法、编码基因、宿主细胞、组合物 - Google Patents

二价双特异性抗体及其制备方法、编码基因、宿主细胞、组合物 Download PDF

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WO2020135555A1
WO2020135555A1 PCT/CN2019/128582 CN2019128582W WO2020135555A1 WO 2020135555 A1 WO2020135555 A1 WO 2020135555A1 CN 2019128582 W CN2019128582 W CN 2019128582W WO 2020135555 A1 WO2020135555 A1 WO 2020135555A1
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antibody
heavy chain
antigen
specifically binds
nucleic acid
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French (fr)
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冯晓
金磊
王涛
郭洪瑞
刘爽
陈宇珩
韩宁
梁阳秋
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长春金赛药业股份有限公司
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Priority to US17/418,856 priority Critical patent/US20220073610A1/en
Priority to CN201980078998.8A priority patent/CN113330037B/zh
Priority to EP19904064.3A priority patent/EP3904392A4/en
Priority to JP2021538377A priority patent/JP7312834B2/ja
Publication of WO2020135555A1 publication Critical patent/WO2020135555A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the invention relates to the technical field of antibody medicine, in particular to a bivalent bispecific antibody, a preparation method thereof, a coding gene, a host cell, and a composition.
  • Bispecific antibody (bispecific monoclonal antibody, BsAb) is a kind of artificially made special antibody that can bind two different antigens at the same time.
  • Bispecific antibodies can recognize tumor target cells and immune effector cells at the same time, so they have the dual functions of antibody specificity and mediating the cytotoxic effect of effector cells.
  • Bispecific antibodies can accumulate effector cells at the tumor site and activate effector cells to play an anti-tumor effect. Its mechanism of killing tumor cells includes cell proliferation, cytokine release, cytotoxic polypeptide and enzyme regulation.
  • bispecific antibody-mediated immunotherapy can alleviate the tumors of some animals, clinically can reduce the condition of tumor patients and prolong life. Therefore, the application of bispecific antibody-mediated immunocompetent cells in tumor therapy has good prospects.
  • Bispecific antibodies do not exist in a natural state and can only be prepared manually. Bi- or multispecific antibodies in the art can bind more than two antigens, and can be produced using cell fusion, chemical conjugation, or recombinant DNA technology. Recently, a wide variety of recombinant bispecific antibody formats have been developed, such as tetravalent bispecific antibodies by fusing, for example, the IgG antibody format and a single chain domain (see, eg, Cooma, MJ, et al., Nature Biotech. 15 (1997) 159-163; WO2001077342; and Morrison, SL, Nature Biotech. 25 (2007) 1233-1234).
  • the use of cell hybridoma (quadroma) technology produced bispecific antibodies very similar to natural antibodies, the cells
  • the hybridoma technology is based on the somatic fusion of two different hybridoma cell lines expressing a murine monoclonal antibody with the desired bispecific antibody specificity. Because of the random pairing of the heavy and light chains of two different antibodies in the resulting cell hybridoma cell line, up to 10 different antibody types are generated, of which only one is the desired functional bispecific antibody.
  • the method used to circumvent the problem of mismatched by-products aims to force two different antibodies to regenerate by introducing mutations into the CH3 domain to modify the contact interface Chain pairing.
  • On one chain bulky amino acids are replaced with amino acids with short side chains to form “holes.”
  • amino acids with large side chains are introduced into another CH3 domain to form "bumps”.
  • By co-expressing these two heavy chains a high yield heterodimer form (“bump-hole”) was observed compared to the homodimer form (“hole-hole” or "bump-bump”) (Ridgway, JB, Presta, LG, Carter, P.
  • the percentage of heterodimers can be stabilized by using phage display to reconstruct the interaction surface of the two CH3 domains and introducing disulfide bonds Polymers were further increased (Merchant, AM, et al., Nature Biotech 16 (1998) 677-681; Atwell, S., Ridgway, JB, Wells, JA, Carter, P., J. Mol. Biol. 270 (1997 )26-35).
  • An important constraint of this strategy is that the light chains of the two parent antibodies must be the same to prevent mismatches and the formation of inactive molecules.
  • the Fc pairing of different half-antibodies can also be achieved through the strand-exchange engineered domain (SEED) technology of IgG and IgA CH3. ,2010,23(4):195-202.).
  • SEED strand-exchange engineered domain
  • GenMab developed the FAE (Fab-arm exchange) bifunctional antibody technology (Gramer, MJ, et al., MAbs 2013, 5(6): 962-973.). Introducing two point mutations, K409R and F405L, in the CH3 region of the heavy chain of the two target antibodies IgG1, respectively, can form a half antibody exchange rearrangement similar to the IgG4 antibody.
  • the two different IgG1 antibodies after mutation were separately expressed in two CHO cell lines and the assembly between the light and heavy chains of the half antibodies was completed. After protein A affinity purification, a mild oxidant system can be used to achieve heterologous half antibodies in vitro Precise assembly.
  • Crossmab technology can also promote the correct assembly of antibody light chains.
  • the representative product is Roche's Ang-2/VEGF CrossMab CH1-CL.
  • Crossmab technology is based on the transformation of the "mortar and pestle", and the CL and CH1 in the Fab domain of the Ang-2 antibody are exchanged, while the Fab structure of the VEGF antibody remains unchanged.
  • the modified light chain of Ang-2 antibody is not easy to mismatch with the heavy chain of VEGF antibody.
  • the "mortar” structure can promote heterodimerization of two heavy chains (Schaefer, W, et al., Proc Natl. Acad. Sci .U, A, 2011, 108(27): 11187-11192.).
  • two single-chain antibodies scFv
  • two Fabs can also be linked by peptides to form bifunctional antibody fragments.
  • the representative is the BiTE (bispecific T-cellengager) series products developed by the German Micromet company. This series of products is obtained by connecting anti-CD3 single-chain antibodies and different anti-tumor cell surface antigen single-chain antibodies through peptides (Baeuerle, PA, et al., Cancer Res., 2009, 69(12):4941-4944. ).
  • the present invention provides a bivalent bispecific antibody and its preparation method, coding gene, host cell, and composition.
  • the antibody has a high light and heavy chain correct assembly rate and a moderate molecular size.
  • the present invention provides a bivalent bispecific antibody, which includes:
  • the flexible peptide is (G4S/G4SAS) n, n is an integer greater than or equal to 0, and the partial sequence of the IgG1 hinge region connected to the flexible peptide is: EPKSCDK (SEQ ID NO: 24); where (G4S/G4SAS) n represents (G4S)n or (G4SAS)n, n is an integer greater than or equal to 0;
  • the linking peptide is L/GGGC (L/GGGC stands for LGGC or GGGC), and the first cysteine residue (C) of the heavy chain hinge region connected to the linking peptide is mutated to serine (S).
  • CL and CH1 can form a heterodimer, and the terminal cysteine residue of CL can form a disulfide with the cysteine residue on the hinge region of the heavy chain key.
  • a linking peptide L/GGGC of one heavy chain and a terminal cysteine residue of a linking peptide L/GGGC of a light chain can form a disulfide bond; and one of the heavy chains The CH3 domain of the chain and the CH3 domain of the other heavy chain were changed to promote the formation of a bivalent bispecific antibody.
  • the first heavy chain variable region and the light chain variable region are respectively connected (G4S/G4SAS) n and L/GGGC, and a disulfide bond is formed between the cysteine residues of the two L/GGGC.
  • an amino acid residue having a larger volume than the original amino acid residue is composed of arginine (R), phenylalanine (P), tyrosine (Y), and tryptophan (W).
  • Amino acid residues having a smaller volume than the original amino acid residues consist of alanine (A), serine (S), threonine (T), and valine (V).
  • the bivalent bispecific antibody provided by the present invention includes:
  • the light chain and heavy chain of the antibody that specifically binds to the second antigen that is, the amino acid sequences shown in SEQ ID NO: 6 and SEQ ID NO: 15, wherein the light chain variable region is connected to the flexible peptide and the linking peptide, heavy
  • the chain variable region is connected to the heavy chain Fc fragment through a flexible peptide and a linking peptide.
  • the invention also provides a method for preparing the bivalent bispecific antibody, which includes the following steps:
  • a first vector (including the gene encoding SEQ ID NO: 2), which includes a nucleic acid molecule encoding a single-chain variable fragment of an antibody that specifically binds to the first antigen and a heavy chain constant region CH1, and
  • a second vector (including the gene encoding SEQ ID NO: 11), which includes a nucleic acid molecule encoding a single-chain variable fragment of an antibody that specifically binds to a second antigen and a light chain constant region;
  • a third vector (including the gene encoding SEQ ID NO: 14), which includes a nucleic acid molecule encoding the light chain of the antibody that specifically binds to the first antigen;
  • a fourth vector (including the gene encoding SEQ ID NO: 5), which includes a nucleic acid molecule encoding a heavy chain of an antibody that specifically binds to the first antigen;
  • a fifth vector (including the gene encoding SEQ ID NO: 15), which includes a nucleic acid molecule encoding a light chain of an antibody that specifically binds to a second antigen, wherein the light chain variable region is linked to the linking peptide;
  • the sixth vector (including the gene encoding SEQ ID NO: 6), which includes a nucleic acid molecule encoding a heavy chain of an antibody that specifically binds to a second antigen, wherein the heavy chain variable region is connected to the heavy chain Fc fragment through a linking peptide;
  • the present invention also provides a gene encoding the bivalent bispecific antibody of the present invention, which includes:
  • the first nucleic acid molecule (gene encoding SEQ ID NO: 2), which is a nucleic acid encoding a single-chain variable fragment scFv of an antibody that specifically binds to the first antigen, a flexible peptide, a heavy chain IgG1 constant region CH1, and a partial sequence of the hinge region Molecule; and
  • a second nucleic acid molecule (a gene encoding SEQ ID NO: 11), which is a nucleic acid molecule encoding a single-chain variable fragment scFv of an antibody that specifically binds to a second antigen and a light chain constant region CL;
  • a third nucleic acid molecule (a gene encoding SEQ ID NO: 14), which is a light chain encoding an antibody that specifically binds to the first antigen;
  • a fourth nucleic acid molecule (a gene encoding SEQ ID NO: 5), which is a heavy chain encoding an antibody that specifically binds to the first antigen;
  • a fifth nucleic acid molecule (a gene encoding SEQ ID NO: 15), which is a light chain encoding an antibody that specifically binds to a second antigen; wherein the light chain variable region is connected to a flexible peptide and a linking peptide; and
  • a sixth nucleic acid molecule (a gene encoding SEQ ID NO: 6), which is a heavy chain encoding an antibody that specifically binds to a second antigen, wherein the heavy chain variable region is connected to the heavy chain Fc fragment through a flexible peptide and a linking peptide.
  • the present invention also provides a host cell, including:
  • a first vector comprising a nucleic acid molecule encoding a single-chain variable fragment of an antibody that specifically binds to the first antigen and a heavy chain constant region CH1, and
  • a second vector which includes a nucleic acid molecule encoding a single-chain variable fragment of an antibody that specifically binds to a second antigen and a light chain constant region;
  • a third vector which includes a nucleic acid molecule encoding a light chain of an antibody that specifically binds to the first antigen
  • a fourth vector which includes a nucleic acid molecule encoding a heavy chain of an antibody that specifically binds to the first antigen
  • a fifth vector which includes a nucleic acid molecule encoding a light chain of an antibody that specifically binds to a second antigen, wherein the light chain variable region is linked to the linking peptide;
  • the sixth vector includes a nucleic acid molecule encoding a heavy chain of an antibody that specifically binds to a second antigen, wherein the heavy chain variable region is connected to the heavy chain Fc fragment through a linking peptide.
  • the invention also provides a composition comprising the bivalent bispecific antibody.
  • the composition of the bivalent bispecific antibody is a pharmaceutical composition or a diagnostic composition.
  • the pharmaceutical composition further includes at least one pharmaceutical excipient.
  • the invention provides a bivalent bispecific antibody, a preparation method thereof, a coding gene, a host cell, and a composition.
  • the bivalent bispecific antibody includes: a) a single chain variable fragment scFv of an antibody that specifically binds to the first antigen, a flexible peptide, a heavy chain IgG1 constant region CH1, and a hinge region partial sequence, namely CH1-partial hinge-linker- scFv2 or scFv2-linker-CH1-partial hinge; and b) a single-chain variable fragment scFv of an antibody that specifically binds a second antigen and a light chain constant region CL, namely scFv1-CL or CL-scFv1;
  • the light chain variable region is connected to the flexible peptide and the linking peptide, heavy
  • the chain variable region is connected to the heavy chain Fc fragment through a flexible peptide and a linking peptide.
  • the bivalent bispecific antibody of the present invention has a high affinity with the first antigen molecule and the second antigen molecule, and is equivalent to the parent monoclonal antibody molecule, such as the affinity of B2 and FV1 and PD-L1 are 1.09E-10M, respectively And 2.71E-10M; the affinity of B2 and FV1 to PD-L1 is 2.58E-8M and 1.50E-8M, respectively. It can be seen that the light and heavy chains of the antibodies of the present invention have a high correct assembly rate and a moderate molecular size.
  • Figure 1 is a schematic diagram of a structure B bivalent bispecific antibody, including four structures B1, B2, B3 and B4;
  • Figure 2 is a schematic diagram of a bivalent bispecific antibody of FV structure, including four structures of FV1, FV2, FV3 and FV4;
  • Figure 3 shows the results of SDS-PAGE detection of the B and FV structure bispecific antibodies: M is Maker; lanes 1, 3, 5, 7, 9, 11, 13, 15 are B1, B2, B3, B4, Non-reducing electrophoresis of FV1, FV2, FV3, FV4; lanes 2, 4, 6, 8, 10, 12, 14, 16 are the reductive electrophoresis of B1, B2, B3, B4, FV1, FV2, FV3, FV4;
  • Figure 4 shows the detection results of the bivalent bispecific antibody Elisa of the B and FV structures, including the four structures of B1, B2, B3 and B4 (4-1) and the four structures of FV1, FV2, FV3 and FV4 (4-2) Elisa test results;
  • Figure 5 shows the SDS-PAGE detection results of the preferred B2 and FV1 structure bispecific antibodies after purification: M is Maker; lanes 1, 2, and 3 are non-reducing transient supernatant of B2 bispecific antibody, Protein L affinity chromatography Non-reducing eluent, PD-L1 affinity chromatography non-reducing eluent; lanes 4, 6, and 8 are FV1 bispecific antibody Mab SelectSure affinity chromatography non-reducing eluent, Protein L affinity chromatography non-reducing Reducing eluent, hCD47 affinity chromatography non-reducing eluent; lanes 5, 7, and 9 are FV1 bispecific antibody Mab SelectSure affinity chromatography reducing eluent, Protein L affinity chromatography reducing eluent, hCD47 affinity chromatography reduction eluent;
  • Figure 6 shows the results of Elisa detection after purification of the preferred bivalent bispecific antibodies of the B2 and FV1 structures.
  • the invention discloses a bivalent bispecific antibody, a preparation method thereof, coding genes, host cells, and compositions. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. In particular, it should be noted that all similar substitutions and modifications will be obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and it is obvious that relevant personnel can modify or appropriately modify and combine the method and application described herein without departing from the content, spirit, and scope of the present invention. Apply the technology of the present invention.
  • antibody refers to a complete, monoclonal antibody.
  • the whole antibody consists of two pairs of "light chain” (LC) and “heavy chain” (HC).
  • the light chain and heavy chain of the antibody are polypeptides composed of several domains.
  • each heavy chain includes a heavy chain variable region VH and a heavy chain constant region.
  • the heavy chain constant region includes heavy chain constant domains CH1, CH2, and CH3 (antibody types IgA, IgD, and IgG) and optionally, heavy chain constant domains CH4 (antibody types IgE and IgM).
  • Each light chain includes a light chain variable domain VL and a light chain constant domain CL.
  • a naturally occurring intact antibody namely IgG antibody.
  • variable domains VH and VL can be further subdivided into hypervariable regions, called complementarity determining regions CDR, and there are more conserved regions distributed between them, called framework regions FR.
  • Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino end to the carboxyl end: FR1, CDR1, FR2, CDR2, F3, CD3, and FR4 (Janeway, CA, Jr., etc., ( 2001) Immunobiology, 5th edition, Garland Press; and Woof, J., Burton D. Nat. Rev. Immunol. 4 (2004) 89-99).
  • Two pairs of heavy and light chains can specifically bind the same antigen. Therefore, the whole antibody is a bivalent, monospecific antibody.
  • the "antibody” includes, for example, mouse antibody, human antibody, chimeric antibody, humanized antibody, and genetically engineered antibody, provided that their unique characteristics are maintained. Particularly preferred are human or humanized antibodies, especially as recombinant human or
  • Types of mammalian antibody heavy chains represented by the Greek letters: ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ (Janeway, C.A., Jr. et al., (2001) Immunobiology, 5th edition, Garland Press).
  • Types of heavy chains present Types of antisense antibodies; these chains are present in IgA, IgD, IgE, IgG, and IgM antibodies (Rhoades, RA, Sullivaner, RG (2002). Human Physiology, 4th edition, Thomson Knowledge).
  • Different heavy chains differ in size and composition; ⁇ and ⁇ contain about 450 amino acids, while ⁇ and ⁇ have about 550 amino acids.
  • Each heavy chain has two regions, a constant region and a variable region.
  • the constant region is the same in all antibodies of the same isotype, but different in antibodies of different isotypes.
  • the heavy chains ⁇ , ⁇ , and ⁇ have a constant region composed of three constant domains CH1, CH2, and CH3 and a dumpling chain region for increased flexibility (Woof, J., Burton D. Nat. Rev. Immunol. 4 (2004 )89-99)
  • the heavy chains ⁇ and ⁇ have a constant region composed of 4 constant domains CH1, CH2, CH3 and CH4 (Janeway, CA, Jr. et al., (2001) Immunobiology, 5th edition, Garland Publishing) Agency).
  • the variable region of the heavy chain differs among antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B cell clone.
  • the variable region of each heavy chain is about 110 amino acids long and consists of a single antibody domain.
  • the light chain has two consecutive domains: 1 constant domain CL and 1 variable domain VL.
  • the approximate length of the light chain is 211-217 amino acids.
  • the light chain is a K light chain, and the constant domain CL is preferably CK.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refers to a preparation of antibody molecules of a single amino acid composition.
  • the "antibody” according to the present invention may be of any type such as (IgA, IgD, IgE, IgG and IgM, preferably IgG or IgE), or subtypes such as (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1), Wherein the two antibodies derived from the bivalent bispecific antibody according to the present invention have an Fc portion of the same subtype (eg IgG1, IgG4, etc., preferably IgG1), preferably the same allotype Fc portion.
  • Fc portion of an antibody is a term well known to the skilled person and is defined based on papain cleavage of antibodies.
  • the antibody according to the present invention comprises, for example, an Fc portion, preferably an Fc portion derived from human origin, and preferably all other portions of a human constant region.
  • the Fc portion of the antibody is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding.
  • the binding to C1q is caused by the determined binding site in the Fc portion.
  • the binding site is prior art and described in, for example, Lukas, TJ, et al., J. Immunol.
  • the binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbered according to the EU catalog of Kabat).
  • Antibodies of subtypes IgG1, IgG2 and IgG3 usually show complement activation, C1q binding and C3 activation, while IgG4 does not activate the complement system, does not bind C1q and does not activate C3.
  • the Fc portion is a human Fc portion.
  • recombinant human antibody is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant methods, such as antibodies isolated from host cells, such as NSO or CHO cells, or transgenes isolated from white immunoglobulin genes Animal antibodies, or antibodies expressed using recombinant expression vectors transfected into host cells. This recombinant human antibody has variable and constant regions in rearranged form.
  • variable domain when used herein means each pair of light and heavy chains that are directly involved in the binding of an antibody to an antigen.
  • the domains of the variable human light chain and the heavy chain have the same general structure and each domain includes 4 framework regions (FR), the sequences of which are generally conserved, and are separated by 3 hypervariable regions (CDRs). connection.
  • the framework region adopts a self-folding conformation and the CDR can form a loop connecting the self-folding structures.
  • the CDRs in each chain are maintained in their three-dimensional structure by the framework region and together with the CDRs from the other chain form an antigen binding site.
  • the CDR3 regions of the antibody heavy and light chains play a particularly important role in the binding specificity/affinity of the antibodies according to the invention.
  • hypervariable region or "antigen-binding portion of an antibody” refers to the amino acid residues of the antibody responsible for antigen binding.
  • the hypervariable region includes amino acid residues from the "complementarity determining region”.
  • Framework regions are those variable domain regions other than the hypervariable region residues defined herein. Therefore, the CDRs on each chain of the antibody light chain and heavy chain from the N-terminus to the C-terminus including the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are separated by the framework amino acids.
  • the CDR3 of the heavy chain is the region most conducive to antigen binding.
  • the “constant domains" of the heavy and light chains do not directly participate in the binding of antibodies to antigens but exhibit multiple effector functions.
  • bivalent bispecific antibody refers to an antibody as described above, wherein two single-chain variable fragments specifically bind different antigens, namely the first single-chain variable fragment and the light chain constant region Specifically binds to the first antigen, the second single-chain variable fragment and the heavy chain constant region 1 and part of the hinge region specifically bind to the second antigen; the bivalent bispecific antibody can simultaneously specifically bind two different antigens, And no more than two antigens, in contrast, on the one hand, a monospecific antibody that can only bind one antigen and, on the other hand, a tetravalent, tetraspecific antibody that can simultaneously bind four antigen molecules, for example.
  • antigen or "antigen molecule” as used herein are used interchangeably and refer to all molecules capable of being specifically bound by an antibody.
  • the bivalent bispecific antibody specifically binds the first antigen and the second different antigen.
  • antigen as used herein includes, for example, proteins, different epitopes on proteins (as different antigens within the meaning of the present invention) and polysaccharides. This mainly includes parts of bacteria, viruses and other microbes (shell, envelope, cell wall, flagella, fimbria and toxins). Lipids and nucleic acids are only antigenic when they bind to proteins and polysaccharides.
  • Non-microbial exogenous (non-self) antigens can include pollen, egg whites, and proteins on the surface of blood cells from transplanted tissues and organs or infused.
  • the antigen is selected from the group consisting of cytokines, cell surface proteins, enzymes and receptor cytokines, cell surface proteins, enzymes and receptors.
  • Tumor antigens are those antigens inside the MHC I or MHC II molecules on the surface of tumor cells. These antigens can sometimes be presented by tumor cells and never by normal cells. As such, they are called tumor-specific antigens (TSAs) and are typically produced by tumor-specific mutations. More common are antigens presented by tumor cells and normal cells, and they are called tumor-associated antigens (TAAs). Cytotoxic T lymphocytes that recognize these antigens may be able to destroy tumor cells before they proliferate or metastasize. Tumor antigens can also be present on the tumor surface in the form of, for example, mutant receptors, in which case they should be recognized by B cells.
  • TSAs tumor-specific antigens
  • TAAs tumor-associated antigens
  • At least one of the two different antigens (first and second antigens) to which the bivalent bispecific antibody specifically binds is a tumor antigen.
  • the two different antigens (first and second antigens) to which the bivalent bispecific antibody specifically binds are tumor antigens; in this case, the first and second antigens also It can be two different epitopes on the same tumor-specific protein.
  • one of the two different antigens (first and second antigens) to which the bivalent bispecific antibody specifically binds is a tumor antigen, and the other is an effector cell antigen, such as T cell receptor Body, CD3, CD16, etc.
  • one of the two different antigens (first and second antigens) to which the bivalent bispecific antibody specifically binds is a tumor antigen, and the other is an anti-cancer substance such as a toxin or kinase inhibition Agent.
  • binding affinity refers to an antibody that specifically binds an antigen.
  • the binding affinity of the antibody that specifically binds to the antigen is a KD value of 10 -9 mol/L or less, such as 10 -10 mol/L, preferably having a KD value of 10 -10 mo1/L or less, such as 10 -12 mo1/ L. Binding affinity is determined using standard binding assays, such as surface plasmon resonance technology (Biacore).
  • epitope determinants include chemically clean surface groupings of molecules, such as amino acids, sugar side chains, phosphino or sulfo groups, in certain embodiments, may have specific three-dimensional structural characteristics, And or specific charging characteristics.
  • Epitopes are regions of antigen bound by antibodies. In certain embodiments, when an antibody preferably recognizes its target antigen in a complex mixture of proteins and/or macromolecules, the antibody is said to specifically bind to the antigen.
  • nucleic acid or nucleic acid molecule is intended to include DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably, and all these names include progeny. Therefore, the words “transformants” and “transformed cells” include primary subject cells and cultures derived therefrom, regardless of the number of transfers. It is also understood that the DNA content of all offspring may not be exactly the same due to intentional or unintentional mutations. This includes mutant offspring that have the same function or biological activity selected in the initially transformed cells. When different names are meant, they will be clear due to the context.
  • transfection refers to the process of transferring a vector/nucleic acid into a host cell. If cells without an insurmountable cell wall barrier are used as host cells, transfection is performed, for example, by the calcium phosphate precipitation method as described by Graham and van Eder, Virology 52 (1978) 546ff. However, other methods of introducing DNA into cells can also be used, such as by nuclear injection or by protoplast fusion. If prokaryotic cells or cells containing substantial cell wall structures are used, for example, one method of transfection is calcium treatment using calcium oxide, as described in Cohen, F.N., et al., PNAS. 69 (1972) 7110ff.
  • expression refers to the process of transcribing a nucleic acid into mRNA and/or the process of subsequently translating the transcribed mRNA into a peptide, polypeptide or protein.
  • the transcript and the encoded polypeptide are collectively called a gene product. If the polynucleotide is derived from genomic DNA, expression in eukaryotic cells may include mRNA splicing.
  • a "vector” is a nucleic acid molecule, especially self-replicating, which transfers the enclosed nucleic acid molecule into and/or between host cells.
  • the term includes vectors whose main function is to include DNA or RNA into cells, replication vectors whose main function is to replicate DNA or RNA, and expression vectors whose functions are to transcribe and/or translate DNA or RNA. It also includes carriers that provide more than one of the above functions.
  • an "expression vector” is a polynucleotide that can be transcribed and translated into a polypeptide after introduction into a suitable host cell.
  • “Expression system” generally refers to a suitable host cell that includes an expression vector whose function is to produce the desired expression product.
  • the bivalent bispecific antibody according to the invention is preferably produced by recombinant means. Such methods are generally known in the art and include protein expression in prokaryotic and eukaryotic cells and subsequent isolation of antibody polypeptides and usually purification to pharmaceutical purity. For protein expression, nucleic acids encoding light and heavy chains or fragments thereof are included in expression vectors by standard methods. Expression is performed in suitable prokaryotic or eukaryotic host cells such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, yeast or E. coli cells, and from the cells (after lysis Antibody from the supernatant or cells). Bivalent bispecific antibodies can exist in intact cells, in cell lysates, or in partially purified or substantially pure form.
  • variable domains The cloning of variable domains is described in Orlandi, R., et al., Proc. Nat. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Norderha, L., et al., J. Immunol. Methods 204 (1997) 77-87.
  • a preferred transient expression system (HEK293) is described in Schlaeger, E.J., and Christensen, K., Cytotechnology 30 (1999) 71-83 and Schlaeger, E.J., J. Immunol. Methods 194 (1996) 191-199.
  • Control sequences suitable for prokaryotes include promoters, optional operator sequences, and ribosome binding sites. It is known that eukaryotic cells utilize promoters, enhancers and polyadenylation signals.
  • a nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence.
  • the DNA of the presequence or secretion leader sequence is operably linked to the DNA of the polypeptide, provided that it is expressed as a preprotein involved in the secretion of the polypeptide;
  • the promoter or enhancer is operably linked to the coding sequence, provided that it affects the sequence Transcription; or the ribosome binding site is operably linked to the coding sequence, provided that it is positioned to promote translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and in the case of a secretory leader sequence, contiguous and in the readable bar. However, enhancers do not have to be contiguous. Ligation is achieved by ligation at convenient restriction sites. If the site does not exist, the synthetic oligonucleotide adaptor or linker is used according to conventional practice.
  • DNA and RNA encoding monoclonal antibodies are easily isolated and sequenced using conventional procedures.
  • the hybridoma cells can serve as the source of the DNA and RNA.
  • the DNA can be included in an expression vector, which is subsequently transfected into host cells that do not otherwise produce immunoglobulins, such as HEK293 cells, CHO cells, or myeloma cells, to be obtained in the host cells Synthesis of recombinant monoclonal antibodies.
  • Amino acid sequence variants (or mutants) of bivalent bispecific antibodies are prepared by introducing appropriate nucleotide changes into antibody DNA, or by nucleotide synthesis.
  • modifications can only be carried out within a very limited range, for example as described above.
  • the modification does not change the aforementioned antibody characteristics, such as IgG isotype and antigen binding, but can increase the yield of recombinant products, protein stability, or facilitate purification.
  • bivalent bispecific antibody provided by the present invention and its preparation method, coding gene, host cell, and raw materials or reagents used in the composition can all be commercially available.
  • Anti-human CD47 humanized monoclonal antibody 059-4.16.2 H1L2 VL (SEQ ID NO: 18) and VH (SEQ ID NO: 19) sequence 059-4.16.2 H1L2 is obtained by hybridoma derived from murine Antibody (201610436519.3), and then get humanized transformation;
  • VL The sequence of VL (SEQ ID NO: 20) and VH (SEQ ID NO: 21) of the human monoclonal antibody 047 anti-human PD-L1, Ab-6 is obtained by panning the natural human source library (201810044303.1), nucleotides encoding IgG1 heavy chain constant region CH1, hinge region and Fc encoding nucleotides, and Kappa chain constant region nucleotides.
  • PGS003 was selected to construct heavy and light chain expression vectors of bivalent bispecific antibodies (8, schematic structural diagrams are shown in Figures 1 and 2).
  • the nucleotides encoding VL and VH of the humanized monoclonal antibody 059-4.16.2 of H1L2 against human CD47 the VL and VH encoding nucleus of the humanized monoclonal antibody 047 of Ab-1 to human PD-L1 Glycosides, nucleotides encoding the IgG1 heavy chain constant region CH1, hinge region and Fc encoding nucleotides, and the Kappa chain constant region nucleotide sequence and multiple cloning sites in the vector design primers.
  • plasmid was extracted using Zymo Research's endotoxin-removing large extraction kit, and finally the plasmid was dissolved in 1 mL of ultrapure water, and the plasmid concentration and OD260/280 were determined with a spectrophotometer.
  • OD260/280 is plasmid DNA with higher purity from 1.8 to 1.9.
  • Heavy chain expression vector name Heavy chain amino acid sequence
  • H1, L2, L3 and H4 express anti-h scFv of CD47
  • L1, H2, H3 and L4 express anti-h scFv of PD-L1
  • H5 and L5 expresses anti-h PD-L1 VH and VL respectively
  • H6, H7, H8 and H9 express anti-h CD47 VH
  • L6, L7 and L8 express anti-h CD47 VL.
  • the above carriers are in accordance with H1+L1 (B1 structure), H2+L2 (B2 structure), H3+L3 (B3 structure), H4+L4 (B4 structure), H5+L5+H6+L6 (FV1 structure), H5+L5
  • the combination of +H7+L7 (FV2 structure), H5+L5+H8+L8 (FV3 structure) and H5+L5+H9+L7 (FV4 structure) was used to evaluate the transient transfection expression of the 2mL 293E system, and the FV1 structure was ( G4S/G4SAS) 0 +L/GGGC, where FV2 structure is (G4S/G4SAS) 1 +L/GGGC, where FV3 structure is (G4S/G4SAS) 3 +L/GGGC, where FV4 structure light chain is (G4S/G4SAS ) 1 +L/GGGC, the heavy chain is L/GGGC +(G4S/G
  • 4-1 is the B-structure bivalent bispecific antibody Elisa test result
  • 4-2 is the FV structure Test results of bivalent bispecific antibody Elisa.
  • the B2 structure is best expressed and assembled and bound to antigen in class B bispecific antibodies
  • the FV1 structure is best expressed, assembled and bound to antigen in FV bispecific antibodies. Therefore, B2 and FV1 are preferred antibodies.
  • Amplified transient transfection expression of the two preferred antibodies of B2 and FV1 structures was performed in Freestyle medium using 293E. Twenty-four hours before transfection, 300 mL of 293E cells of 0.5 ⁇ 10 6 cells/mL were inoculated in a 1L cell culture flask, and cultured in a shaker at 120 rpm in a 37°C 5% CO 2 incubator.
  • the cell culture solution was centrifuged at 2000g and 20min, and the supernatant was collected. The supernatant was filtered with a 0.22 micron filter, and subjected to Protein L (GE) chromatography, using 20 mM citric acid-sodium citrate, pH 3.0, and eluted with 1M Tris base adjusts the pH to neutral. After protein chromatography, the sample was subjected to affinity chromatography coupled with human PDL1 protein. It was eluted with 20 mM citric acid-sodium citrate, pH 3.0, and the pH was adjusted to neutrality with 1 M Tris base. Purified samples were detected by SDS-PAGE using 4-20% gradient gel (Jinsui Biotechnology Co., Ltd.). The results are shown in Figure 5. The results show that the preferred antibody B2 is 95% pure.
  • the cell culture solution was centrifuged at 2000g and 20min, and the supernatant was collected.
  • Mabselect Sure chromatography the sample was then subjected to Protein L (GE) chromatography, eluting with 20 mM citric acid-sodium citrate, pH 3.0, and adjusted the pH to neutral with 1M Tris base.
  • Example 3 ELISA detection of preferred antibodies binding to human CD47 and human PD-L1
  • Blocking After washing the plate three times, block with 3% BSA, 250 ⁇ L per well, and incubate at 37°C for 2 hours.
  • Add candidate antibody After washing the plate 3 times, add 12 concentration gradient candidate antibody samples or a positive control or a negative control with a starting concentration of 10mg/mL twice. 50 ⁇ L per well, incubated at 25°C for 1 hour.
  • Termination Directly add 50 ⁇ L of stop solution per well to terminate the reaction.
  • Biacore T200 instrument was used to test the affinity of B2 and FV1 structural antibodies.
  • the specific method is as follows: human PD-L1-His and human CD47-His are coupled to a CM5 biosensor chip (GE), antibodies of different concentrations are flowed through the chip at a flow rate of 30 ⁇ L/min, and the antigen is combined with the candidate antibody. The combination time is 120s and the dissociation time is 300s.
  • GE BIAevalution software
  • the affinity of B2 and FV1 and PD-L1 are 1.09E-10M and 2.71E-10M, respectively; B2 and FV1 and CD47 The affinity is 2.58E-8M and 1.50E-8M, respectively.

Abstract

提供二价双特异性抗体及其制备方法、编码基因、宿主细胞、组合物。该二价双特异性抗体包括：a)特异性结合第一抗原的抗体的单链可变片段scFv、柔性肽、重链IgG1恒定区CH1以及铰链区部分序列，和b)特异性结合第二抗原的抗体的单链可变片段scFv和轻链恒定区CL，即scFv1-CL或CL-scFv1；或者包括：c)特异性结合第一抗原的抗体的轻链和重链；和d)特异性结合第二抗原的抗体的轻链和重链。

Description

二价双特异性抗体及其制备方法、编码基因、宿主细胞、组合物
本申请要求于2018年12月28日提交中国专利局、申请号为201811622069.2、发明名称为“二价双特异性抗体及其制备方法、编码基因、宿主细胞、组合物”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及抗体药物技术领域，特别涉及二价双特异性抗体及其制备方法、编码基因、宿主细胞、组合物。
背景技术
双特异性抗体(bispecific monoclonal antibody,BsAb)是一种人工制作出来的可以同时结合两种不同抗原的特殊抗体。双特异性抗体能够同时识别肿瘤靶细胞和免疫效应细胞，因此兼有抗体特异性和介导效应细胞的细胞毒作用的双重功能。双特异性抗体能将效应细胞聚集于肿瘤部位并激活效应细胞发挥抗肿瘤作用，其杀伤肿瘤细胞的作用机理包括细胞增殖、细胞因子释放、细胞毒性多肽和酶的调控。体内及临床研究证明双特异性抗体介导的免疫治疗可使部分动物的肿瘤缓解，临床上可使肿瘤患者病情减轻，延长生命。因此，双特异性抗体介导的免疫活性细胞在肿瘤治疗中的应用具有良好的前景。
双特异性抗体在自然状态下不存在，只能通过人工制备。本领域中双或多特异性抗体能够结合2种以上抗原，可以利用细胞融合、化学缀合或重组DNA技术产生。最近己经开发了广泛多样的重组双特异性抗体形式，例如通过融合例如IgG抗体形式和单链结构域的四价双特异性抗体(参见例如Co1oma,M.J.,等,Nature Biotech.15(1997)159-163；WO2001077342；和Morrison,S.L.,Nature Biotech.25(2007)1233-1234)。此外，还开发了能够结合2种以上抗原的诸多其他新型形式，其中抗体中心结构(IgA、IgD、IgE、IgG或IgM)不再保持如双抗体、三链抗体或四链抗体、minibodies和若干单链形式(scFv、Bis-scFv)(Holliger,P.,等，Nature Biotech.23(2005)1126-1136；Fischer，N.，和Léger，O.，Pathobio1ogy 74(2007)3-14；Shen,J.，等，Journa1 of Immunogica1 Methods 318(2007)65-74；Wu,C.，等.,Nature Biotech.25(2007)1290-1297)。
在一种方法中，利用细胞杂交瘤(quadroma)技术(见Mi1stein,C.和 A.C.Cue11o,Nature，305(1983)537-40)生成了与天然抗体非常类似的双特异性抗体，所述细胞杂交瘤技术基于表达具有所需的双特异性抗体特异性的鼠单克隆抗体的两种不同杂交瘤细胞系的体细胞融合。因为在产生的细胞杂交瘤细胞系中的两个不同抗体重链和轻链的随机配对，所以生成至多10种不同抗体类型，其中只有一种是所需的功能双特异性抗体。由于存在错配副产物和显著降低的产率，其意味着需要复杂的纯化程序(参见例如Morrison,S.L.,Nature Biotech 25(2007)1233-1234)。一般地，如果使用重组表达技术，则相同的错配副产物问题仍存在。
用于避开错配副产物问题的方法，称为“凸起一进入一孔洞(knobs-into-ho1es)”，目的在于通过将突变引入CH3结构域以修饰接触界面来迫使两个不同抗体重链配对。在一条链上，大体积氨基酸被具有短侧链的氨基酸替换，以形成“孔洞”。相反地，将具有大侧链的氨基酸引入到另一个CH3结构域中，以形成“凸起”。通过共表达这两条重链，观察到与同型二聚体形式(“孔洞一孔洞”或“凸起-凸起”)相比高产率的异二聚体形式(“凸起-孔洞”)(Ridgway,J.B.,Presta,L.G.,Carter,P.和WO 1996027011)，异二聚体的百分比可以通过利用噬菌体展示法重建两个CH3结构域的相互作用表面和引入二硫键来稳定该异二聚体而得到进一步增加(Merchant,A.M.，等,Nature Biotech 16(1998)677-681；Atwell，S.,Ridgway,J.B.,Wells,J.A.，Carter，P.,J.Mol.Biol.270(1997)26-35)。这种策略的一个重要制约是两个母体抗体的轻链必须相同，以防止错配和形成失活的分子。
除“杵臼”结构之外，还可通过IgG和IgA CH3的链交换(strand-exchange engineered domain，SEED)技术实现不同半抗体的Fc配对(Davis,J.H,等.,Protein Eng.Des.Sel.,2010,23(4):195-202.)。
为了解决不同轻链正确装配的问题，近年来又开发了双细胞系分别表达半抗体、体外装配的新工艺。受到人体IgG4抗体在生理条件下自然发生的半抗体随机交换过程的启示，GenMab公司开发了FAE(Fab-arm exchange)双功能抗体技术(Gramer,M.J,等.,MAbs 2013,5(6):962-973.)。在两个目标抗体IgG1重链CH3区分别引入K409R和F405L两个点突变，就能够形成类似于IgG4抗体的半抗体交换重排。将突变后的两个不同IgG1抗体在两个 CHO细胞系中分别表达并完成半抗体轻重链间的装配，经过蛋白A亲和纯化后，利用温和的氧化剂系统可在体外实现异源半抗体之间的精确装配。
除了共用序列相同的轻链或进行体外装配，通过Crossmab技术也可促进抗体轻链的正确装配。代表产品是罗氏公司的Ang-2/VEGF CrossMab CH1-CL。Crossmab技术是在“杵臼”改造的基础上，将Ang-2抗体Fab结构域中的CL与CH1互换，而VEGF抗体的Fab结构则保持不变。经过改造的Ang-2抗体轻链不易与VEGF抗体的重链发生错配，同时“杵臼”结构可促进两条重链异源二聚化(Schaefer,W,等.,Proc Natl.Acad.Sci.U S A,2011,108(27):11187-11192.)。
此外，还可以将两个单链抗体(scFv)或者两个Fab通过肽段链接，形成双功能抗体片段。具有代表性的是德国Micromet公司开发的BiTE(bispecific T-cell engager)系列产品。该系列产品是将抗CD3单链抗体与不同抗肿瘤细胞表面抗原单链抗体通过肽段进行连接获得的(Baeuerle,P.A,等.,Cancer Res.,2009,69(12):4941-4944.)。这类抗体结构的优点是分子量小、可以在原核细胞中表达、不需要考虑正确装配的问题；缺点是由于没有抗体Fc段，不能介导相应的生物学功能、半衰期短等。
公开号为US2015/0284475A1和CN101896504A的专利中均公开了二价双特异性抗体，但两件专利中的二价双特异性抗体与抗原的亲和力较低。邵长利发表的《基于柔性链连接的双特异性抗体分子的设计和结构模拟》中的柔性肽结构对前述两件专利中的二价双特异性抗体进行改造，但抗体的亲和力依然不理想。
针对上述双特异性抗体技术平台所具有的轻链错配，轻重链正确装配率不高或者分子偏大或偏小等问题，需要发明新型的二价双特异性抗体。
发明内容
有鉴于此，本发明提供了二价双特异性抗体及其制备方法、编码基因、宿主细胞、组合物。该抗体的轻重链正确装配率高，且分子大小适中。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了二价双特异性抗体，其包括：
a)特异性结合第一抗原的抗体的单链可变片段scFv、柔性肽、重链IgG1 恒定区CH1以及铰链区部分序列，即CH1-partial hinge(铰链区部分序列)-linker(柔性肽)-scFv2或scFv2-linker-CH1-partial hinge；和
b)特异性结合第二抗原的抗体的单链可变片段scFv和轻链恒定区CL，即scFv1-CL或CL-scFv1；
或者包括：
c)特异性结合第一抗原的抗体的轻链和重链；和
d)特异性结合第二抗原的抗体的轻链和重链，其中轻链可变区与柔性肽以及链接肽相连，重链可变区通过柔性肽以及链接肽与重链Fc片段相连。
作为优选，柔性肽为(G4S/G4SAS)n，n为大于或等于0的整数，且与柔性肽相连的IgG1铰链区部分序列为：EPKSCDK(SEQ ID NO:24)；其中(G4S/G4SAS)n表示(G4S)n或(G4SAS)n,n为大于或等于0的整数；
链接肽为L/GGGC(L/GGGC表示LGGC或GGGC)，且与链接肽相连的重链铰链区第一个半胱氨酸残基(C)突变为丝氨酸(S)。
作为优选，在a)和b)步骤中，CL和CH1能形成异源二聚体，CL的末端半胱氨酸残基可以与重链的铰链区上的半胱氨酸残基形成二硫键。
作为优选，在c)和d)步骤中，其中一条重链的链接肽L/GGGC与一条轻链的链接肽L/GGGC的末端半胱氨酸残基能形成二硫键；和其中一条重链的CH3结构域和另一条重链的CH3结构域被改变为促进形成二价双特异性抗体的结构。
作为优选，改变为：
a)改变第一重链的CH3结构域：在与二价双特异性抗体内的第二重链的CH3结构域的初始界面相接触的第一重链的CH3结构域的初始界面内，氨基酸残基被替换为具有大于原始氨基酸残基体积的氨基酸残基，在第一重链的CH3结构域的界面内生成凸起，凸起可以定位在第二重链的CH3结构域的界面内的凹洞中；且
b)改变第二重链的CH3结构域：在与二价双特异性抗体内的第一重链的CH3结构域的初始界面相接触的第二重链的CH3结构域的初始界面内，氨基酸残基被替换为具有小于原始氨基酸残基体积的氨基酸残基，在第二重链的CH3结构域的界面内生成凹洞，在凹洞中可以定位第一重链的CH3结构域的 界面内的凸起，
其中第一重链可变区与轻链可变区分别连接(G4S/G4SAS)n以及L/GGGC，并在两个L/GGGC的半胱氨酸残基之间形成二硫键。
作为优选，具有大于原始氨基酸残基体积的氨基酸残基氨基酸残基由精氨酸(R)、苯丙氨酸(P)、酪氨酸(Y)、色氨酸(W)组成。
具有小于原始氨基酸残基体积的氨基酸残基氨基酸残基由丙氨酸(A)、丝氨酸(S)、苏氨酸(T)、缬氨酸(V)组成。
作为优选，本发明提供的二价双特异性抗体，其包括：
a)特异性结合第一抗原的抗体的单链可变片段scFv、柔性肽、重链IgG1恒定区CH1以及铰链区部分序列，即SEQ ID NO：2所示的氨基酸序列；和
b)特异性结合第二抗原的抗体的单链可变片段scFv和轻链恒定区CL，即SEQ ID NO:11所示的氨基酸序列；
或者包括：
c)特异性结合第一抗原的抗体的轻链和重链，即SEQ ID NO：5和SEQ ID NO：14所示的氨基酸序列；和
d)特异性结合第二抗原的抗体的轻链和重链，即SEQ ID NO：6和SEQ ID NO：15所示的氨基酸序列，其中轻链可变区与柔性肽以及链接肽相连，重链可变区通过柔性肽以及链接肽与重链Fc片段相连。
本发明还提供了该二价双特异性抗体的制备方法，其包括下列步骤：
a)用以下载体转化宿主细胞，
第一载体(包括编码SEQ ID NO:2的基因)，其包括编码特异性结合第一抗原的抗体的单链可变片段和重链恒定区CH1的核酸分子，和
第二载体(包括编码SEQ ID NO:11的基因)，其包括编码特异性结合第二抗原的抗体的单链可变片段和轻链恒定区的核酸分子；
或者
第三载体(包括编码SEQ ID NO:14的基因)，其包括编码特异性结合第一抗原的抗体的轻链的核酸分子；
第四载体(包括编码SEQ ID NO:5的基因)，其包括编码特异性结合第一抗原的抗体的重链的核酸分子；
第五载体(包括编码SEQ ID NO:15的基因)，其包括编码特异性结合第二抗原的抗体的轻链的核酸分子，其中轻链可变区与链接肽相连；和
第六载体(包括编码SEQ ID NO:6的基因)，其包括编码特异性结合第二抗原的抗体的重链的核酸分子，其中重链可变区通过链接肽与重链Fc片段相连；
b)在容许合成抗体分子的条件下培养宿主细胞；
c)从培养物中回收抗体分子。
本发明还提供了编码本发明二价双特异性抗体的基因，其包括：
第一核酸分子(编码SEQ ID NO:2的基因)，其为编码特异性结合第一抗原的抗体的单链可变片段scFv、柔性肽、重链IgG1恒定区CH1以及铰链区部分序列的核酸分子；和
第二核酸分子(编码SEQ ID NO:11的基因)，其为编码特异性结合第二抗原的抗体的单链可变片段scFv和轻链恒定区CL的核酸分子；
或者包括：
第三核酸分子(编码SEQ ID NO:14的基因)，其为编码特异性结合第一抗原的抗体的轻链；
第四核酸分子(编码SEQ ID NO:5的基因)，其为编码特异性结合第一抗原的抗体的重链；
第五核酸分子(编码SEQ ID NO:15的基因)，其为编码特异性结合第二抗原的抗体的轻链；其中轻链可变区与柔性肽以及链接肽相连；和
第六核酸分子(编码SEQ ID NO:6的基因)，其为编码特异性结合第二抗原的抗体的重链，其中重链可变区通过柔性肽以及链接肽与重链Fc片段相连。
本发明还提供了一种宿主细胞，其包括：
第一载体，其包括编码特异性结合第一抗原的抗体的单链可变片段和重链恒定区CH1的核酸分子，和
第二载体，其包括编码特异性结合第二抗原的抗体的单链可变片段和轻链恒定区的核酸分子；
或者包括：
第三载体，其包括编码特异性结合第一抗原的抗体的轻链的核酸分子；
第四载体，其包括编码特异性结合第一抗原的抗体的重链的核酸分子；
第五载体，其包括编码特异性结合第二抗原的抗体的轻链的核酸分子，其中轻链可变区与链接肽相连；和
第六载体，其包括编码特异性结合第二抗原的抗体的重链的核酸分子，其中重链可变区通过链接肽与重链Fc片段相连。
本发明还提供了包括该二价双特异性抗体的组合物，二价双特异性抗体的组合物为药物组合物或诊断组合物。
作为优选，药物组合物还包括至少一种药用赋形剂。
本发明提供了二价双特异性抗体及其制备方法、编码基因、宿主细胞、组合物。该二价双特异性抗体包括：a)特异性结合第一抗原的抗体的单链可变片段scFv、柔性肽、重链IgG1恒定区CH1以及铰链区部分序列，即CH1-partial hinge-linker-scFv2或scFv2-linker-CH1-partial hinge；和b)特异性结合第二抗原的抗体的单链可变片段scFv和轻链恒定区CL，即scFv1-CL或CL-scFv1；
或者包括：
c)特异性结合第一抗原的抗体的轻链和重链；和d)特异性结合第二抗原的抗体的轻链和重链，其中轻链可变区与柔性肽以及链接肽相连，重链可变区通过柔性肽以及链接肽与重链Fc片段相连。
本发明具有的技术效果为：
本发明的二价双特异性抗体与第一抗原分子及第二抗原分子的亲和力均较高，均与亲本单克隆抗体分子相当，如B2和FV1与PD-L1的亲和力分别为1.09E-10M和2.71E-10M；B2和FV1与PD-L1的亲和力分别为2.58E-8M和1.50E-8M。可见，本发明抗体的轻重链正确装配率高，且分子大小适中。
附图说明
图1为B结构二价双特异性抗体示意图，包括B1、B2、B3和B4四种结构；
图2为FV结构二价双特异性抗体示意图，包括FV1、FV2、FV3和FV4四种结构；
图3为B和FV结构双特异性抗体瞬转表达SDS-PAGE检测结果：M为Maker；泳道1、3、5、7、9、11、13、15分别为B1、B2、B3、B4、FV1、FV2、 FV3、FV4的非还原电泳；泳道2、4、6、8、10、12、14、16分别为B1、B2、B3、B4、FV1、FV2、FV3、FV4的还原电泳；
图4为B和FV结构二价双特异性抗体Elisa检测结果，包括B1、B2、B3和B4四种结构(4-1)以及包括FV1、FV2、FV3和FV4四种结构(4-2)的Elisa检测结果；
图5为优选的B2和FV1结构双特异性抗体纯化后SDS-PAGE检测结果：M为Maker；泳道1、2、3为B2双特异性抗体非还原瞬转上清、Protein L亲和层析非还原洗脱液、PD-L1亲和层析非还原洗脱液；泳道4、6、8为FV1双特异性抗体Mab SelectSure亲和层析非还原洗脱液、Protein L亲和层析非还原洗脱液、hCD47亲和层析非还原洗脱液；泳道5、7、9为FV1双特异性抗体Mab SelectSure亲和层析还原洗脱液、Protein L亲和层析还原洗脱液、hCD47亲和层析还原洗脱液；
图6为优选的B2和FV1结构二价双特异性抗体纯化后Elisa检测结果。
具体实施方式
本发明公开了二价双特异性抗体及其制备方法、编码基因、宿主细胞、组合物，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
术语解释：
术语“抗体”用于本文中时，指完整的、单克隆抗体。所述完整抗体由两对“轻链”(LC)和“重链”(HC)。所述抗体的轻链和重链是由若干结构域组成的多肽。在完整抗体中，每条重链包括重链可变区VH和重链恒定区。重链恒定区包括重链恒定结构域CH1、CH2和CH3(抗体类型IgA，IgD，和IgG)和任选地，重链恒定结构域CH4(抗体类型IgE和IgM)。每条轻链包括轻链可变结构域VL和轻链恒定结构域CL。一种天然存在的完整抗体，即IgG抗体。可变结构域VH和VL可以进一步再分为高变区，称为互补性决定区CDR，它们之间分布有更加保守的区域，称为框架区FR。每个VH和 VL由三个CDR和四个FR组成，以以下顺序从氨基端向羧基端排列：FR1、CDR1、FR2、CDR2、F3、CD3和FR4(Janeway,C.A.，Jr.等.,(2001)Immunobiology，第5版，加兰出版社；和Woof,J.,Burton D.Nat.Rev.Immunol.4(2004)89-99)。两对重链和轻链能够特异性结合相同抗原。因此所述完整抗体是二价、单特异性抗体。所述“抗体”包括例如小鼠抗体、人抗体、嵌合抗体、人源化抗体和遗传改造的抗体，条件是保持它们的特有特性。特别优选人或人源化抗体，尤其作为重组人或人源化抗体。
存在5种由希腊字母表示的哺乳动物抗体重链类型：α、δ、ε、γ和μ(Janeway,C.A.，Jr.等.,(2001)Immunobiology，第5版，加兰出版社)。存在的重链的类型运义抗体的类型；这些链分别存在于IgA，IgD，IgE，IgG,和IgM抗体中(Rhoades,R.A.,Pflanzer,R.G.(2002).Human Physiology，第4版，汤姆森知识)。不同的重链在尺寸和组成上不同；α和γ含有约450个氨基酸，而μ和ε具有约550个氨基酸。
每条重链具有两种区域，即恒定区和可变区。恒定区在相同同种型的所有抗体中相同，但在不同同种型的抗体中不同。重链γ、α和δ具有由3个恒定结构域CH1、CH2和CH3组成的恒定区和用于增加柔性的饺链区(Woof,J.,Burton D.Nat.Rev.Immunol.4(2004)89-99)重链μ和ε具有由4个恒定结构域CH1、CH2，CH3和CH4组成的恒定区(Janeway,C.A.，Jr.等.,(2001)Immunobiology，第5版，加兰出版社)。重链的可变区在由不同B细胞产生的抗体中不同，但对由单个B细胞或B细胞克隆产生的所有抗体都相同。每条重链的可变区长约110个氨基酸且由单个抗体结构域组成。
在哺乳动物中，仅存在两类轻链，其称为λ和K。轻链具有两个连续的结构域：1个恒定结构域CL和1个可变结构域VL。轻链的近似长度是211-217个氨基酸。优选地，轻链是K轻链，且恒定结构域CL优选是C K。
术语“单克隆抗体”或“单克隆抗体组合物”用于本文中时，指单个氨基酸组合物的抗体分子制剂。
按照本发明的“抗体”可以是任意类型例如(IgA、IgD、IgE、IgG和IgM，优选IgG或IgE)，或亚型例如(IgG1、IgG2、IgG3、IgG4、IgA1和IgA2，优选IgG1)，其中按照本发明的二价双特异性抗体所源自的两种抗体具有相同亚 型(例如IgGl、IgG4等，优选IgG1)的Fc部分，优选相同同种异型的Fc部分。
“抗体的Fc部分”是技术人员公知的术语并基于抗体的木瓜蛋白酶裂解而定义。按照本发明的抗体包含如Fc部分，优选源自人来源的Fc部分和优选人恒定区的全部其他部分。抗体的Fc部分直接参与补体活化、C1q结合、C3活化和Fc受体结合。虽然抗体对补体系统的影响取决于某些条件，但是与C1q的结合由Fc部分中确定的结合位点导致。所述结合位点是现有技术且记述在例如Lukas，T.J.，等.,J.Immunol.127(1981)2555-2560；Brunhouse；Cebra，J.J.,Mo l.Immunol.16(1979)907-917；Burton,D.R.，等.，Nature 288(1980)338-344；Thommesen,J.E.，等.,Mol.Immunol.37(2000)995-1004；Idusogie,E.E.，等.,J.Immunol.164(2000)4178-4184；Hezareh,M.，等.,J.Virol.75(2001)12161-12168；Morgan，A.，等.,Immunology 86(1995)319-324；和EP0307434中。所述结合位点是例如L234、L235、D270、N297、E318、K320、K322、P331和P329(按照Kabat的EU目录编号)。亚型IgGl、IgG2和IgG3的抗体通常显示补体活化、C1q结合和C3活化，而IgG4不活化补体系统，不结合C1q且不活化C3。优选地，Fc部分是人Fc部分。
用于本文时，术语“重组人抗体”意欲包括通过重组方法制备、表达、产生或分离的所有人抗体，诸如分离自宿主细胞，诸如NSO或CHO细胞的抗体或分离白人免疫球蛋白基因的转基因动物的抗体，或利用转染到宿主细胞中的重组表达载体表达的抗体。这种重组人抗体具有处于重排形式的可变区和恒定区。
“可变结构域”用于本文中时，表示直接参与抗体与抗原结合的每对轻链和重链对。可变人轻链和重链的结构域具有相同的一般结构且每个结构域包括4个框架区(FR)，所述框架区的序列普遍保守，其通过3个高变区(CDRs)相连接。框架区采用自折叠构象且CDR可以形成连接自折叠结构的环。每条链中的CDR通过框架区以其三维结构保持并与来自另一条链的CDR一起形成抗原结合位点。抗体重链和轻链CDR3区在按照本发明的抗体的结合特异性/亲和性方面起特别重要的作用。
用于本文时，术语“高变区”或“抗体的抗原结合部分”指负责抗原结合 的抗体的氨基酸残基。高变区包括来自“互补性决定区”的氨基酸残基。“框架区”是除本文中定义的高变区残基之外的那些可变结构域区域。因此，抗体的轻链和重链从N端到C端包括结构域FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4各条链上的CDR通过所述构架氨基酸分开。特别地，重链的CDR3是最有助于抗原结合的区域。按照Kabat等，有免疫学意义的蛋白质序列(Sequences of Proteins of lmmunological Interest)，第5版，公众健康服务；国家健康研究所(Public Health Service,National Institutes of Health),Bethesda,MD.(1991)的标准定义确定CDR和FR区域。
重链和轻链的“恒定结构域”不直接参与抗体与抗原的结合但是表现出多种效应子功能。
术语“二价双特异性抗体”用于本文中时，指如上所述的抗体，其中两个单链可变片段分别特异性结合不同抗原，既第一单链可变片段和轻链恒定区特异性结合第一抗原，第二单链可变片段和重链恒定区1以及部分铰链区特异性结合第二抗原；所述二价双特异性抗体能够同时特异性结合两种不同的抗原，且不超过两种抗原，与其相对照的是，一方面仅能够结合一种抗原的单特异性抗体和另一方面例如能够同时结合四种抗原分子的四价、四特异性抗体。
术语“抗原”或“抗原分子”用于本文中时，可交替使用并指能够被抗体特异性结合的所有分子。二价双特异性抗体特异性结合第一抗原和第二不同抗原。术语“抗原”用于本文中时，包括例如蛋白、蛋白上的不同表位(在本发明含义内作为不同抗原)和多糖。这主要包括细菌、病毒和其他微生物的部分(外壳、被膜、细胞壁、鞭毛、菌毛和毒素)。脂质和核酸仅在与蛋白和多糖结合时具有抗原性。非微生物外源(非自身)抗原可以包括花粉、蛋清和来自被移植组织和器官的或被灌输的血细胞表面上的蛋白。优选地，抗原选自由细胞因子、细胞表面蛋白、酶和受体细胞因子、细胞表面蛋白、酶和受体组成的组。
肿瘤抗原是由肿瘤细胞表面上的MHC I或MHC II分子里边的那些抗原。这些抗原有时可以由肿瘤细胞来呈递，且从来不由正常细胞来呈递。由此，它们称为肿瘤特异性抗原(TSAs)且典型地由肿瘤特异性突变产生。更常见的是由肿瘤细胞和正常细胞呈递的抗原，且它们称为肿瘤相关抗原(TAAs)。识 别这些抗原的细胞毒性T淋巴细胞可能能够在肿瘤细胞增殖或转移前破坏它们。肿瘤抗原还可以采用例如突变受体的形式存在于肿瘤表面上，在这种情形中它们应该被B细胞识别。
在一个优选的实施方案中，二价双特异性抗体特异性结合的两种不同抗原(第一和第二抗原)中的至少一种是肿瘤抗原。
在另一个优选的实施方案中，二价双特异性抗体特异性结合的两种不同抗原(第一和第二抗原)均是肿瘤抗原；在该情形中，所述第一和第二抗原还可以是相同肿瘤特异性蛋白上的两种不同表位。
在另一个优选的实施方案中，二价双特异性抗体特异性结合的两种不同抗原(第一和第二抗原)之一是肿瘤抗原，且另一种是效应细胞抗原，例如T细胞受体、CD3、CD16等。
在另一个优选的实施方案中，二价双特异性抗体特异性结合的两种不同抗原(第一和第二抗原)之一是肿瘤抗原，且另一种是抗癌物质诸如毒素或激酶抑制剂。
用于本文中时，“特异性结合”或“与……特异性结合”指特异性结合抗原的抗体。优选地，特异性结合该抗原的抗体的结合亲和性是KD值10 -9mol/L以下例如10 -10mol/L，优选具有KD值10 -10mo1/L以下例如10 -12mo1/L。结合亲和性使用标准结合测定，诸如表面等离振子共振技术(Biacore)来确定。
术语“表位”包括能够特异性结合抗体的任何多肽决定簇。在某些实施方案中，表位决定簇包括分子的化学洁性表面分组，诸如氨基酸、糖侧链、磷眈基或磺眈基，在某些实施方案中，可以具有特定的三维结构特征，且或特定的带电特性。表位是被抗体结合的抗原区域。在某些实施方案中，当抗体在蛋白和/或大分子的复杂混合物中优选识别其靶抗原时，将该抗体称为与抗原特异性结合。
术语“核酸或核酸分子”，用于本文中时，意欲包括DNA分子和RNA分子。核酸分子可以是单链或双链，但优选是双链DNA。
用于本文中时，表述“细胞”、“细胞系”和“细胞培养物”可交替使用，且全部这些名称都包括后代。因此，词语“转化体”和“转化的细胞”包括原代受试者细胞和由其来源的培养物，而不考虑转移数。还理解所有的后代的 DNA含量可能不精确一致，这归因于有意或无意的突变。包括在最初转化的细胞中筛选的具有相同功能或生物学活性的变异后代。在意指不同名称时，其将由于上下文而清楚。
术语“转化”用于本文中时，指将载体/核酸转移到宿主细胞中的过程。如果无难以克服的细胞壁屏障的细胞用作宿主细胞，则转染例如通过如Graham和van der Eh,Virology 52(1978)546ff所述的磷酸钙沉淀法来进行。然而，还可以使用其他将DNA引入细胞的方法，诸如通过核注射或通过原生质体融合。如果使用原核细胞或包含实质细胞壁结构的细胞，例如一种转染方法是利用氧化钙的钙处理，如Cohen，F.N.，等，PNAS.69(1972)7110ff所述。
利用转化重组生成抗体是现有技术公知的且记述在，例如，综述文章Makrides，S.C.,Protein Expr.Purif.17(1999)183-202；Geisse，S.，等.,Protein Expr.Purif.8(1996)271-282；Kaufman,R.J.,Mol.Biotechnol.16(2000)151-161；Werner,R.G.,等.,Arzneimittel Forschung 48(1998)870-880中以及US6,331,415和US4,816,567中。
用于本文中时，“表达”指将核酸转录为mRNA的过程和/或将转录的mRNA随后翻译为肽、多肽或蛋白质的过程。转录物和被编码的多肽共称为基因产物。如果多核苷酸源自基因组DNA，则真核细胞中的表达可以包括mRNA的剪接。
“载体”是核酸分子，特别是自体复制的，其将括入的核酸分子转移到宿主细胞之中和/或之间。该术语包括主要功能为将DNA或RNA括入细胞的载体，主要功能是复制DNA或RNA的复制载体，和功能是转录和/或翻译DNA或RNA的表达载体。还包括提供多于一个上述功能的载体。
“表达载体”是多核苷酸，其在引入合适的宿主细胞后能够被转录和翻译为多肽。"表达系统"通常指包括表达载体的适当宿主细胞，所述表达载体的功能是产生所需的表达产物。
按照本发明的二价双特异性抗体优选通过重组手段生成。所述方法是本领域中普遍已知的，且包括在原核和真核细胞中的蛋白质表达及随后分离抗体多肽和通常纯化到药用纯度。为了蛋白质表达，通过标准方法将编码轻链和重链的核酸或其片段括入表达载体。表达在合适的原核或真核宿主细胞如CHO细 胞、NSO细胞、SP2/0细胞、HEK293细胞、COS细胞、酵母或大肠杆菌(E.coli)细胞中进行，且从所述细胞(溶胞后的上清液或细胞)中回收抗体。二价双特异性抗体可以以完整细胞、以细胞裂解物或以部分纯化或基本纯形式存在。通过标准技术，包括碱/SDS处理，柱层析法和本领域中其他公知技术进行纯化，从而消除其他细胞成分或其他污染物，例如其他细胞核酸或蛋白。参见Ausube l，F.，等.,Current Protocols in Molecular Biology,Greene Publishing and Wileylnter science，纽约(1987)在NSO细胞中的表达记述在，例如，Barnes,L.M.，等.,Cytotechnology 32(2000)109-123；和Barnes，L.M.，等.,Biotech.Bioeng.73(2001)261-270中。瞬时表达记述在，例如，Durocher，Y.，等.,Nucl.Acids Res.30(2002)四中。可变结构域的克隆记述在Orlandi,R.，等.,Proc.Nat l.Acad.Sci.USA86(1989)3833-3837；Carter,P.，等.,Proc.Natl.Acad.Sci.USA 89(1992)4285-4289；和Norderha，L.，等.，J.Immunol.Methods 204(1997)77-87中。优选的瞬时表达系统(HEK 293)记述在Schlaeger，E.J.，和Christensen,K.,Cytotechnology 30(1999)71-83中和Schlaeger，E.J.,J.Immunol.Methods 194(1996)191-199中。
适合于原核生物的控制序列，例如，包括启动子，任选操纵子序列，和核糖体结合位点。已知真核细胞利用启动子、增强子和聚腺苷酸化信号。
核酸在被置于与另一个核酸序列的功能关系中时，是“可操作地连接的”。例如，前序列或分泌前导序列的DNA与多肽的DNA可操作地连接，条件是其表达为参与多肽分泌的前蛋白；启动子或增强子与编码序列可操作地连接，条件是其影响序列的转录；或核糖体结合位点与编码序列可操作地连接，条件是其被定位为促进翻译。一般地，“可操作地连接的”意指被连接的DNA序列是连续的，且在分泌前导序列的情形中，是连续的且在可读杠中。然而，增强子不必须是连续的。连接通过在方便的限制性位点处的连接来实现。如果所述位点不存在，则合成的寡核苷酸接合体或连接体根据常规实践使用。
通过常规免疫球蛋白纯化程序，诸如例如，蛋白A一琼脂糖、连磷灰石层析法、凝胶电泳、透析、或亲合层析法，从培养基中适当分离二价双特异性抗体。编码单克隆抗体的DNA和RNA容易利用常规程序分离和测序。杂交瘤细胞可以起所述DNA和RNA来源的作用。一旦分离后，可以将DNA括 入到表达载体中，所述表达载体随后转染到否则不产生免疫球蛋白的宿主细胞诸如HEK293细胞、CHO细胞、或骨髓瘤细胞中，以在宿主细胞中获得重组单克隆抗体的合成。
二价双特异性抗体的氨基酸序列变体(或突变体)通过将适当的核苷酸改变引入到抗体DNA中，或通过核苷酸合成来制备。然而，这样的修饰仅能在非常有限的范围内，例如如上所述的范围内进行。另外，所述修饰不改变上述抗体特征，诸如IgG同种型和抗原结合，但可以提高重组产物的产率、蛋白稳定性或促进纯化。
本发明提供的二价双特异性抗体及其制备方法、编码基因、宿主细胞、组合物中所用原料或试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1：二价双特异性抗体制备
1、二价双特异性抗体瞬时转染表达载体的构建
(1)材料
抗人CD47的人源化单克隆抗体059-4.16.2 H1L2的VL(SEQ ID NO:18)和VH(SEQ ID NO:19)的序列，059-4.16.2 H1L2是通过杂交瘤得到鼠源抗体(201610436519.3)，然后进行人源化改造得到；
抗人PD-L1的人源单克隆抗体047 Ab-6的VL(SEQ ID NO:20)和VH(SEQ ID NO:21)的序列，047 Ab-6是对天然人源库进行淘筛得到(201810044303.1)，IgG1重链恒定区CH1的编码核苷酸、铰链区和Fc的编码核苷酸，和Kappa链恒定区的核苷酸。
(2)方法
选择pGS003构建二价双特异性抗体(8个，结构示意图见图1和图2)的重链和轻链的表达载体。根据抗人CD47的人源化单克隆抗体059-4.16.2 H1L2的VL和VH的编码核苷酸，抗人PD-L1的人源化单克隆抗体047 Ab-6的VL和VH的编码核苷酸，IgG1重链恒定区CH1的编码核苷酸、铰链区和Fc的编码核苷酸，和Kappa链恒定区的核苷酸序列及载体中的多克隆位点设计引物。经PCR扩增后，使用体外重组的方法(苏州泓迅，iMulli多片段重 组克隆试剂盒)将9个重链编码序列和8个轻链编码序列克隆至pGS003，如表1。测序鉴定抗体基因正确插入后，将重组表达载体转化大肠杆菌TOP10F’，挑取单菌落接种于含100μg/mL氨苄青霉素的LB培养基中，37℃振荡培养16小时。使用Zymo Research的去内毒素大提试剂盒抽提质粒，最后将质粒溶于1mL超纯水，用分光光度计测定质粒浓度和OD260/280。OD260/280在1.8～1.9为纯度较高的质粒DNA。
表1、重链和轻链瞬时转染表达载体列表
重链表达载体名称 重链氨基酸序列 轻链表达载体名称 轻链氨基酸序列
H1 SEQ ID NO:1 L1 SEQ ID NO:10
H2 SEQ ID NO:2 L2 SEQ ID NO:11
H3 SEQ ID NO:3 L3 SEQ ID NO:12
H4 SEQ ID NO:4 L4 SEQ ID NO:13
H5 SEQ ID NO:5 L5 SEQ ID NO:14
H6 SEQ ID NO:6 L6 SEQ ID NO:15
H7 SEQ ID NO:7 L7 SEQ ID NO:16
H8 SEQ ID NO:8 L8 SEQ ID NO:17
H9 SEQ ID NO:9 / /
2、在哺乳动物细胞293E中的转染、表达和检测
将上述9个重链表达载体和8个轻链表达载体中H1、L2、L3和H4表达anti-h CD47的scFv，L1、H2、H3和L4表达anti-h PD-L1的scFv，H5和L5分别表达anti-h PD-L1的VH和VL，H6、H7、H8和H9表达anti-h CD47的VH，L6、L7和L8表达anti-h CD47的VL。
上述载体按照H1+L1(B1结构)、H2+L2(B2结构)、H3+L3(B3结构)、H4+L4(B4结构)、H5+L5+H6+L6(FV1结构)、H5+L5+H7+L7(FV2结构)、H5+L5+H8+L8(FV3结构)和H5+L5+H9+L7(FV4结构)组合后进行2mL 293E体系的瞬时转染表达评估，其中FV1结构为(G4S/G4SAS) 0+L/GGGC，其中FV2结构为(G4S/G4SAS) 1+L/GGGC，其中FV3结构为(G4S/G4SAS) 3+L/GGGC，其中FV4结构轻链为(G4S/G4SAS) 1+L/GGGC，重链为L/GGGC +(G4S/G4SAS) 1。检测表达量及抗体与人CD47和人PD-L1结合的ELISA检测值，结果见图3、图4(其中4-1为B结构二价双特异性抗体Elisa检测结果，4-2为FV结构二价双特异性抗体Elisa检测结果)。B2结构在B类结构双特异性抗体中表达、组装以及与抗原的结合都最佳；FV1结构在FV类结构双特异性抗体中表达、组装以及与抗原的结合都最佳。因此B2、FV1为优选抗体。
使用293E在Freestyle培养基中进行B2和FV1结构2个优选抗体的放大瞬时转染表达。转染前24小时，在1L细胞培养瓶中接种0.5×10 6细胞/mL的293E细胞300mL，37℃5％CO 2温箱中120rpm摇床培养。转染时先取300μL的293 fectin加入到5.7mL的OPtiMEM中，充分混匀后，室温孵育2分钟；同时将B2结构及FV1结构分子所用到的表达质粒总量各300μg使用OPtiMEM稀释至6mL。将上述稀释后的转染试剂及质粒充分混合，室温孵育15分钟，然后将混合物全部加入细胞中，混匀，37℃5％CO 2温箱中120rpm摇床培养7天。
实施例2：优选抗体的纯化及检测
B2结构蛋白纯化：
2000g、20min离心细胞培养液，收集上清，将上清用0.22微米的滤膜过滤，经过Protein L(GE)层析，利用20mM枸橼酸-枸橼酸纳，pH3.0洗脱，用1M Tris base调节pH至中性。Protein L层析后样品再经过偶联人PDL1蛋白的亲和层析，利用20mM枸橼酸-枸橼酸纳，pH3.0洗脱，用1M Tris base调节pH至中性。纯化样品利用4～20％梯度胶(金斯瑞生物科技有限公司)进行SDS-PAGE检测纯化蛋白质，结果见图5，结果显示优选抗体B2纯度为95％。
FV1结构蛋白纯化：
2000g、20min离心细胞培养液，收集上清，将上清用0.22微米的滤膜过滤，经过Mabselect Sure(GE)层析，利用20mM枸橼酸-枸橼酸纳，pH3.0洗脱，用1M Tris base调节pH至中性。Mabselect Sure层析后样品再经过Protein L(GE)层析，利用20mM枸橼酸-枸橼酸纳，pH3.0洗脱，用1M Tris base调节pH至中性。Protein L层析后样品再经过偶联人CD47蛋白的亲和层析， 利用20mM枸橼酸-枸橼酸纳，pH3.0洗脱，用1M Tris base调节pH至中性。纯化样品利用4～20％梯度胶(金斯瑞生物科技有限公司)进行SDS-PAGE检测纯化蛋白质，结果见图5，结果显示优选抗体FV1纯度为90.8％。
实施例3：优选抗体结合人CD47及人PD-L1的ELISA检测
1、第一抗原包被：人PD-L1-mFc(金赛自构，SEQ ID NO:22)用PBS稀释成1μg/mL，加至96孔酶标板中，每孔50μL，4℃下孵育过夜。
2、封闭：洗板三次后用3％BSA封闭，每孔250μL，37℃条件下孵育2小时。
3、加候选抗体：洗板3次后，加入起始浓度为10mg/mL 2倍稀释12个浓度梯度候选抗体样品或阳性对照或阴性对照。每孔50μL，25℃条件下孵育1小时。
4、加入第二抗原：人CD47-His(金赛自构，SEQ ID NO:23)用PBS稀释成10μg/mL，加至96孔酶标板中，每孔50μL，25℃条件下孵育1h。
5、加二抗：洗板3次后，加入HRP标记链霉亲和素(1:10000)，每孔50μL，25℃条件下孵育1小时。
6、显色：洗板4次后，加TMB显色液，每孔50μL，室温下避光显色10分钟。
7、终止：直接加入50μL每孔的终止液终止反应。
8、检测：终止反应后立即把酶标板放入酶标仪中，在450nm处测其OD值，保存原始数据整理。结果见图6，纯化后的优选抗体B2 EC50＝0.5636，FV1EC50＝1.662。
实施例4：优选抗体亲和力测定
使用Biacore T200仪器检测B2和FV1结构抗体的亲和力。具体方法如下：将人PD-L1-His及人CD47-His偶联在CM5生物传感芯片(GE)上，将不同浓度的抗体以30μL/min流速流过芯片，抗原与候选抗体进行结合，结合时间为120s，解离时间为300s。用BIAevalution软件(GE)进行动力学拟合，获得亲和力常数结果如下表2、表3，B2和FV1与PD-L1的亲和力分别为 1.09E-10M和2.71E-10M；B2和FV1与CD47的亲和力分别为2.58E-8M和1.50E-8M。
表2、候选抗体与PD-L1的亲和力测定结果
抗体名称 Ka(1/Ms) Kd(1/s) KD(M) Rmax(RU)
B2 3.95E+05 4.28E-05 1.09E-10 7.067
FV1 4.40E+05 1.19E-04 2.71E-10 28.06
PD-L1阳性单抗 9.08E+05 2.36E-04 2.60E-10 8 0
表3、候选抗体与CD47的亲和力测定结果
抗体名称 KD(M) Rmax(RU)
B2 2.58E-08 50.00
FV1 1.50E-08 60.00
CD47阳性单抗 2.36E-08 50.92
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (11)

  1. 二价双特异性抗体，其特征在于，其包括：
    a)特异性结合第一抗原的抗体的单链可变片段scFv、柔性肽、重链IgG1恒定区CH1以及铰链区部分序列；和
    b)特异性结合第二抗原的抗体的单链可变片段scFv和轻链恒定区CL；
    或者包括：
    c)特异性结合第一抗原的抗体的轻链和重链；和
    d)特异性结合第二抗原的抗体的轻链和重链，其中轻链可变区与柔性肽以及链接肽相连，重链可变区通过柔性肽以及链接肽与重链Fc片段相连。
  2. 根据权利要求1所述的二价双特异性抗体，其特征在于，所述柔性肽为(G4S/G4SAS)n，n为大于或等于0的整数，且与柔性肽相连的IgG1铰链区部分序列为：EPKSCDK；
    所述链接肽为L/GGGC，且与链接肽相连的重链铰链区第一个半胱氨酸残基突变为丝氨酸。
  3. 根据权利要求1或2所述的二价双特异性抗体，其特征在于，其中CL和CH1能形成异源二聚体，CL的末端半胱氨酸残基可以与重链的铰链区上的半胱氨酸残基形成二硫键。
  4. 根据权利要求1或2所述的二价双特异性抗体，其特征在于，其中一条重链的链接肽L/GGGC与一条轻链的链接肽L/GGGC的末端半胱氨酸残基能形成二硫键；和其中一条重链的CH3结构域和另一条重链的CH3结构域被改变为促进形成所述二价双特异性抗体的结构。
  5. 根据权利要求4所述的二价双特异性抗体，其特征在于，所述改变为：
    a)改变第一重链的CH3结构域：在与二价双特异性抗体内的第二重链的CH3结构域的初始界面相接触的第一重链的CH3结构域的初始界面内，氨基酸残基被替换为具有大于原始氨基酸残基体积的氨基酸残基，在第一重链的CH3结构域的界面内生成凸起，所述凸起可以定位在第二重链的CH3结构域的界面内的凹洞中；且
    b)改变第二重链的CH3结构域：在与二价双特异性抗体内的第一重链的 CH3结构域的初始界面相接触的第二重链的CH3结构域的初始界面内，氨基酸残基被替换为具有小于原始氨基酸残基体积的氨基酸残基，在第二重链的CH3结构域的界面内生成凹洞，在所述凹洞中可以定位第一重链的CH3结构域的界面内的凸起；
    其中第一重链可变区与轻链可变区分别连接(G4S/G4SAS)n以及L/GGGC，并在两个L/GGGC的半胱氨酸残基之间形成二硫键。
  6. 根据权利要求1至5中任一项所述的二价双特异性抗体，其特征在于，
    所述具有大于原始氨基酸残基体积的氨基酸残基由精氨酸、苯丙氨酸、酪氨酸、色氨酸组成；
    所述具有小于原始氨基酸残基体积的氨基酸残基由丙氨酸、丝氨酸、苏氨酸、缬氨酸组成。
  7. 根据权利要求1至6中任一项所述的二价双特异性抗体，其特征在于，所述二价双特异性抗体包括：
    a)特异性结合第一抗原的抗体的单链可变片段scFv、柔性肽、重链IgG1恒定区CH1以及铰链区部分序列，即SEQ ID NO：2所示的氨基酸序列；和
    b)特异性结合第二抗原的抗体的单链可变片段scFv和轻链恒定区CL，即SEQ ID NO:11所示的氨基酸序列；
    或者包括：
    c)特异性结合第一抗原的抗体的轻链和重链，即SEQ ID NO：5和SEQ ID NO：14所示的氨基酸序列；和
    d)特异性结合第二抗原的抗体的轻链和重链，即SEQ ID NO：6和SEQ ID NO：15所示的氨基酸序列，其中轻链可变区与柔性肽以及链接肽相连，重链可变区通过柔性肽以及链接肽与重链Fc片段相连。
  8. 权利要求1至7中任一项所述二价双特异性抗体的制备方法，其特征在于，其包括下列步骤：
    a)用以下载体转化宿主细胞，
    第一载体，其包括编码特异性结合第一抗原的抗体的单链可变片段和重链恒定区CH1的核酸分子，和
    第二载体，其包括编码特异性结合第二抗原的抗体的单链可变片段和轻链 恒定区的核酸分子；
    或者
    第三载体，其包括编码特异性结合第一抗原的抗体的轻链的核酸分子；
    第四载体，其包括编码特异性结合第一抗原的抗体的重链的核酸分子；
    第五载体，其包括编码特异性结合第二抗原的抗体的轻链的核酸分子，其中轻链可变区与链接肽相连；和
    第六载体，其包括编码特异性结合第二抗原的抗体的重链的核酸分子，其中重链可变区通过链接肽与重链Fc片段相连；
    b)在容许合成抗体分子的条件下培养所述宿主细胞；
    c)从培养物中回收所述抗体分子。
  9. 编码权利要求1至7中任一项所述二价双特异性抗体的基因，其特征在于，其包括：
    第一核酸分子，其为编码特异性结合第一抗原的抗体的单链可变片段scFv、柔性肽、重链IgG1恒定区CH1以及铰链区部分序列的核酸分子；和
    第二核酸分子，其为编码特异性结合第二抗原的抗体的单链可变片段scFv和轻链恒定区CL的核酸分子；
    或者包括：
    第三核酸分子，其为编码特异性结合第一抗原的抗体的轻链；
    第四核酸分子，其为编码特异性结合第一抗原的抗体的重链；
    第五核酸分子，其为编码特异性结合第二抗原的抗体的轻链；其中轻链可变区与柔性肽以及链接肽相连；和
    第六核酸分子，其为编码特异性结合第二抗原的抗体的重链，其中重链可变区通过柔性肽以及链接肽与重链Fc片段相连。
  10. 宿主细胞，其特征在于，其包括：
    第一载体，其包括编码特异性结合第一抗原的抗体的单链可变片段和重链恒定区CH1的核酸分子，和
    第二载体，其包括编码特异性结合第二抗原的抗体的单链可变片段和轻链恒定区的核酸分子；
    或者包括：
    第三载体，其包括编码特异性结合第一抗原的抗体的轻链的核酸分子；
    第四载体，其包括编码特异性结合第一抗原的抗体的重链的核酸分子；
    第五载体，其包括编码特异性结合第二抗原的抗体的轻链的核酸分子，其中轻链可变区与链接肽相连；和
    第六载体，其包括编码特异性结合第二抗原的抗体的重链的核酸分子，其中重链可变区通过链接肽与重链Fc片段相连。
  11. 包括权利要求1至7中任一项所述二价双特异性抗体的组合物，其特征在于，所述二价双特异性抗体的组合物为药物组合物或诊断组合物。
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ZHANG FENG, WANG KAI-QIN, WANG LAN: "Development in Bispecific Antibody", CHINESE JOURNAL OF PHARMACEUTICAL ANALYSIS, vol. 39, no. 1, 31 January 2019 (2019-01-31), pages 78 - 85, XP009522265, DOI: 10.16155/j.0254-1793.2019.01.10 *

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CN111378045A (zh) 2020-07-07
CN113330037A (zh) 2021-08-31
CN113330037B (zh) 2023-05-12
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US20220073610A1 (en) 2022-03-10
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