WO2024032617A1 - Protéine hétéromultimère et son procédé de préparation - Google Patents

Protéine hétéromultimère et son procédé de préparation Download PDF

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WO2024032617A1
WO2024032617A1 PCT/CN2023/111768 CN2023111768W WO2024032617A1 WO 2024032617 A1 WO2024032617 A1 WO 2024032617A1 CN 2023111768 W CN2023111768 W CN 2023111768W WO 2024032617 A1 WO2024032617 A1 WO 2024032617A1
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domain
amino acid
antibody
heavy chain
seq
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PCT/CN2023/111768
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Chinese (zh)
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王定和
黄浩旻
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三生国健药业（上海）股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • 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
    • 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
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/62—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
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention belongs to the field of biological antibodies. Specifically, it relates to heteromultimeric proteins and methods for their preparation.
  • Heteromultimeric protein is a protein product obtained by dimerization or multimerization of at least two heterologous polypeptide chains.
  • Bispecific antibodies (BsAb) with asymmetric structure belong to heteromultimeric proteins.
  • different antibodies can be combined together through genetic engineering technology. For example, by mutating the amino acid position of the CH3 or hinge region of the wild-type antibody constant region, so that the CH3-CH3 interface The steric hindrance or non-covalent interaction (electrostatic or hydrophobic interaction) changes, making the heterologous heavy chain more likely to dimerize than the homologous heavy chain.
  • the currently mature Fc segment heterodimerization technology is Knobs-into-holes (KIH).
  • the smaller threonine (T) at position 366 of the CH3 region of one antibody heavy chain is replaced with a larger tryptophan (W) to form a Knobs structure (T366W), and the larger tyrosine at position 407 of the CH3 region of another antibody is replaced.
  • the amino acid (Y) is mutated into a smaller threonine (T) to form a holes structure (Y407T), which relies on the reduction of steric hindrance after mutation and the covalent disulfide bond formed in the hinge region to promote heterologous heavy chain disulfide.
  • Polymerization which is the KIH structure.
  • T366 and L368 of the CH3 region of the heavy chain hole also had a steric hindrance effect on the T366W mutation of the heavy chain Knob.
  • the Y407T single point mutation of the heavy chain hole was finally improved to a 3-point mutation (T366S , L368A and Y407V), a more stable "1+3" pestle and mortar structure is obtained.
  • the S354C site mutation was added to the heavy chain Knob, and the S349C site mutation was added to the heavy chain hole, forming a pestle and mortar structure that is currently commonly used for specific binding of the Fc segment.
  • some Hole-Hole homodimers will be produced, which increases the difficulty of the subsequent purification process.
  • the object of the present invention is to provide a heteromultimeric protein and a preparation method thereof. Specifically, a heteromultimeric protein containing the Fc segment of an antibody is provided, and the preparation method and application of the heteromultimeric protein are developed based on the research on bispecific antibodies against HER-2 and CD47.
  • a heteromultimeric protein comprising a first polypeptide containing a first heavy chain constant domain 3 (CH3) domain and a second heavy chain constant domain 3 (CH3) domain. ) domain, wherein the first CH3 domain includes a substitution with a positively charged amino acid at amino acid position 394 relative to the wild-type CH3 domain, and/or the second CH3 domain Included was a substitution with a negatively charged amino acid at amino acid position 397 relative to the wild-type CH3 domain, the position of which was determined according to the EU index of KABAT numbering.
  • the positively charged amino acid at amino acid position 394 forms ionic and/or charge interactions with the amino acid residues in the second CH3 domain.
  • the negatively charged amino acid at amino acid position 397 forms ionic and/or charge interactions with the amino acid residues in the first CH3 domain.
  • the first CH3 domain and the second CH3 domain include residues of a pestle and mortar structure.
  • first polypeptide and the second polypeptide form a pestle and mortar structure pair.
  • the first CH3 domain contains a lysine (K) substitution at amino acid position 394.
  • the second CH3 domain contains an aspartic acid (D) substitution at amino acid position 397.
  • first CH3 domain and the second CH3 domain further include one or more amino acid substitutions, which substitutions promote the formation of the first polypeptide and the second polypeptide.
  • a pestle-mortar structure is formed between them.
  • the first CH3 domain also includes an amino acid substitution selected from the following group: S354C, T366W; and the second CH3 domain further includes an amino acid substitution selected from the following group: Y349C, T366S, L368A, Y407V, or combination thereof.
  • the residues of the pestle structure include amino acid substitutions selected from the following group: S354C, T366W; in the second CH3 domain, the residues of the pestle structure Included are amino acid substitutions selected from the group consisting of: Y349C, T366S, L368A, Y407V, or combinations thereof.
  • the first CH3 domain includes a substitution selected from the group consisting of: T394K, S354C and T366W, or a combination thereof.
  • the second CH3 domain includes a substitution selected from the group consisting of: V397D, Y349C, T366S, L368A, Y407V, or a combination thereof.
  • first CH3 domain and the second CH3 domain are human CH3 domains.
  • the heteromultimeric protein comprises an IgG Fc region.
  • the IgG Fc region is selected from IgG1, IgG2, IgG3 or IgG4.
  • the IgG is IgG1.
  • the IgG Fc is an Fc derived from human IgG1.
  • the first polypeptide and/or the second polypeptide are derived from IgG molecules.
  • the first polypeptide and/or the second polypeptide comprises heavy chain constant domain 2 (CH2 domain).
  • the first polypeptide is an antibody heavy chain
  • the second polypeptide is an antibody heavy chain
  • the heteromultimeric protein includes one or more antibody light chains.
  • heteromultimeric protein comprises
  • the heterodimerized Fc element includes a first polypeptide containing a first heavy chain constant domain 3 (CH3) domain and a second heavy chain constant domain 3 (CH3) structure.
  • a second polypeptide of a domain wherein the first CH3 domain includes an amino acid substitution selected from the group consisting of T394K, S354C and T366W, or a combination thereof, relative to a wild-type CH3 domain; and the second CH3 domain Includes amino acid substitutions selected from the group consisting of: V397D, Y349C, T366S, L368A, Y407V, or combinations thereof, the position of the amino acid being determined according to the EU index of KABAT numbering.
  • the heterodimerized Fc element further includes heavy chain constant domain 2 (CH2 domain).
  • the heteromultimeric protein is a multispecific antibody.
  • the multispecific antibody includes a bispecific antibody, a trispecific antibody or a tetraspecific antibody.
  • the multispecific antibody includes one or more antigen-binding fragments.
  • the antigen-binding fragments target the same or different antigens.
  • the antigen-binding fragments respectively target different epitopes of the same antigen.
  • the antigen-binding fragment is selected from the following group: (i) Fab fragment; (ii) F(ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single chain Fv (scFv) molecules; or (vi) single domain antibody dAb fragments.
  • the antigen is selected from the following group: EGFR, HER-2, EpCAM, CD20, CD30, CD33, CD47, TROP-2, CD52, CD133, CD3, CEA, TAG-72, CIX, PSMA , GD2, GD3, GM2, VEGF, VEGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL and FAP.
  • the antigen-binding fragment targets different antigens, and the different antigens are selected from the following antigen pairs: CD3 and Trop-2, CD20 and CD3, CD3 and CD19, CD3 and Fc- ⁇ - RIIIA, CD3 and TPBG, CD3 and Epha10, CD3 and IL-5R ⁇ , CD3 and TASCTD-2, CD3 and CLEC12A, CD3 and Prominin-1, CD3 and IL-23R, CD3 and ROR1, CD3 and IL-3R ⁇ , CD3 and PSA, CD3 and CD8, CD3 and Glypican3, CD3 and FAP, CD3 and EphA2, CD3 and ENPP3, CD3 and CD33, CD3 and CD133, CD3 and EpCAM, CD3 and CD19, CD3 and Her2, CD3 and CEA, CD3 and GD2, CD3 and PSMA, CD3 and BCMA, CD3 and A33, CD3 and B7-H3, CD3 and EGFR, CD3 and P-cadherin
  • CD3 and SSTR CD3 and FRA
  • CD16 and CD30 CD64 and Her2
  • CD137 and EGFR CD137 and Her-2
  • CD137 and PD-1 CD137 and PDL-1
  • PD-1 and PD-L1 VEGF and PD-L1
  • Lag-3 and TIM-3 OX40 and PD- 1.
  • the heteromultimeric protein includes a first heavy chain, a second heavy chain, a first light chain, a second light chain and a third light chain; wherein,
  • the first heavy chain includes from the N end to the C end: an antibody heavy chain variable domain Z1 directed against the first target, an antibody heavy chain constant region Z4, an antibody heavy chain variable domain Z2 directed against the second target, - CH1, first CH2 and first CH3 domains; the first light chain and the second light chain cooperate with the first heavy chain respectively, so that the heteromultimeric protein specifically binds to the first target and secondary target;
  • the second heavy chain includes from the N-terminus to the C-terminus: the antigen-binding fragment Z3 for the third target, the antibody heavy chain variable domain Z2, the second CH1, the second CH2 and the second CH3 structure for the second target. domain, the third light chain Cooperate with the second heavy chain respectively, so that the heteromultimeric protein specifically binds to the second target; and the antigen-binding fragment Z3 for the third target specifically binds to the third target.
  • the first light chain includes the tandem antibody light chain variable domain Y1 and the first CL directed against the first target from the N-terminus to the C-terminus.
  • the second light chain includes a tandem antibody light chain variable domain Y2 directed against the second target and an antibody second CL from the N-terminus to the C-terminus.
  • the third light chain includes the tandem antibody light chain variable domain Y2 and the third CL directed against the second target from the N-terminus to the C-terminus.
  • the antigen-binding fragment Z3 directed against the third target is selected from the group consisting of scFv molecules, VHH or Fab molecules directed against the third target.
  • the first target and the second target are the same or different epitopes.
  • the first target and the second target are different antigens.
  • the first target and the second target are different epitopes of the same antigen.
  • the third target is an epitope different from the first target and the second target.
  • the first target and the second target are HER-2, and the third target is CD47.
  • the antigen-binding fragment directed against the third target is an scFv molecule directed against CD47.
  • the antibody heavy chain variable domain Z1 directed against the first target and the antibody heavy chain variable domain Z2 directed against the second target have the same or different sequences.
  • sequences of the antibody heavy chain constant region Z4, the first CH1 and the second CH1 are the same or different.
  • first CL, the second CL and the third CL are the same or different.
  • sequences of the first light chain, the second light chain and the third light chain are the same or different.
  • the antibody heavy chain variable domain Z1 directed against the first target the antibody heavy chain constant region Z4, and the antibody heavy chain variable structure directed against the second target
  • Two adjacent elements in domain Z2 and first CH1 are directly connected or connected through a joint.
  • the antigen-binding fragment directed against the third target is directly connected to the variable domain Z2 of the antibody heavy chain directed toward the second target or connected through a linker.
  • the joints include flexible joints and rigid joints.
  • the linker is a peptide linker with a length of 1-35 amino acids, preferably a peptide linker with a length of 6-30 amino acids.
  • the first CH3 domain includes a substitution selected from the group consisting of: T394K, S354C and T366W, or a combination thereof.
  • the second CH3 domain includes a substitution selected from the following group: V397D, Y349C, T366S, L368A, Y407V, or a combination thereof.
  • the multispecific antibody is a bispecific antibody.
  • the heteromultimeric protein has two heavy chains and three light chains, wherein
  • the first heavy chain contains from N-terminus to C-terminus: Fab-VH-CH1-hinge region-Fc1;
  • the second heavy chain contains from N-terminus to C-terminus: scFv-VH-CH1-hinge region-Fc2;
  • the light chain includes from N-terminus to C-terminus: VL-CL, wherein the antigen-binding domain of the Fab of the first heavy chain binds the first target; VH-CH1 of the first heavy chain and the second heavy chain Each independently forms a junction with the VL-CL of the light chain.
  • a second antigen binding domain of the second heavy chain scFv that binds a third target.
  • the first target and the second target are the same or different.
  • the CH3 domain in Fc1 includes substitutions selected from the group consisting of: T394K, S354C and T366W; the CH3 domain in Fc2 includes substitutions selected from the group consisting of: V397D , Y349C, T366S, L368A and Y407V.
  • the first target and the second target are HER-2, and the third target is CD47.
  • the first target and the second target are TROP-2, and the third target is CD3.
  • the Fab of the first heavy chain is a Fab fragment of an anti-HER-2 IgG antibody.
  • the scFv of the second heavy chain is an anti-CD47 antibody scFv.
  • the scFv fragment of the anti-CD47 antibody includes a variable region VH and a variable region VL, and VH and VL are connected through a peptide linker L1.
  • amino acid sequence of the heavy chain variable region VH of the anti-HER-2 in the first heavy chain and the second heavy chain is shown in SEQ ID NO. 13
  • the anti-HER-2 in the light chain is shown in SEQ ID NO.
  • the sequence of the VL of the light chain variable region of 2 is shown in SEQ ID NO.14.
  • amino acid sequence of the scFv fragment of the anti-CD47 antibody in the second heavy chain is shown in SEQ ID NO. 17.
  • the heavy chain variable region of the anti-HER-2 IgG antibody is as shown in SEQ ID NO. 13; the light chain variable region of the anti-HER-2 IgG antibody is as shown in SEQ ID NO. .14; and/or the heavy chain variable region of the anti-CD47 antibody is shown in SEQ ID NO.15; the light chain variable region of the anti-CD47 antibody is shown in SEQ ID NO.16.
  • the heteromultimeric protein includes a first heavy chain, a second heavy chain, a second light chain and a third light chain; wherein,
  • the first heavy chain includes from N-terminus to C-terminus: the antigen-binding fragment Z3 for the third target, the antibody heavy chain variable domain Z2 for the second target, the first CH1, the first CH2 and the first CH3 structure. domain; the second light chain cooperates with the first heavy chain, so that the heteromultimeric protein specifically binds to the second target; the antigen-binding fragment Z3 for the third target specifically binds to the third target;
  • the second heavy chain includes from the N-terminus to the C-terminus: the antibody heavy chain variable domain Z2, the second CH1, the second CH2 and the second CH3 domain directed against the second target, and the third light chain respectively Cooperating with the second heavy chain, the heteromultimeric protein specifically binds to the second target.
  • the second target is TROP-2
  • the third target is CD3.
  • the antigen-binding fragment Z3 directed against the third target is an scFv of an anti-CD3 antibody.
  • the heteromultimeric protein has two heavy chains and two light chains, wherein
  • the first heavy chain contains from N-terminus to C-terminus: scFv-VH-CH1-hinge region-Fc1;
  • the second heavy chain contains from N-terminus to C-terminus: VH-CH1-hinge region-Fc2;
  • the light chain includes from the N end to the C end: VL-CL, wherein the VH-CH1 of the first heavy chain and the second heavy chain and the VL-CL of the light chain each independently form a binding to the second target.
  • the second antigen binding domain of the first heavy chain scFv binds the third target.
  • the second target is TROP-2
  • the third target is CD3.
  • the scFv of the first heavy chain is an anti-CD3 antibody scFv.
  • the heteromultimeric protein includes a first polypeptide containing an antibody heavy chain constant region CH3 domain and a second polypeptide containing an antibody heavy chain constant region CH3 domain.
  • the first polypeptide The constant region CH3 domain includes substitutions selected from the group consisting of: S354C, T366W, and T394K
  • the second polypeptide constant region CH3 domain includes substitutions selected from the group consisting of: Y349C, T366S, L368A, Y407V, and V397D.
  • the CH3 domain of the first polypeptide constant region includes substitutions selected from the group consisting of: S354C and T366W
  • the CH3 domain of the second polypeptide constant region includes substitutions selected from the group consisting of: Y349C, T366S, L368A, and Y407V.
  • the heteromultimeric protein is a bispecific antibody or Fc fusion protein.
  • the bispecific antibody includes:
  • D1 specifically binds to the target molecule HER-2 protein
  • D2 specifically binds to the target molecule CD47 protein
  • the D1 is an antibody that specifically binds to the HER-2 protein or an antigen-binding fragment thereof; and/or
  • the D2 is an antibody that specifically binds to CD47 protein or an antigen-binding fragment thereof;
  • the structure of the antigen-binding fragment is selected from the following group: (i) Fab fragment; (ii) F(ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single-chain Fv (scFv) molecule; or (vi) single domain antibody dAb fragment.
  • the antibodies that specifically bind to the HER-2 protein include: single-chain antibodies, diabodies, nanobodies, monoclonal antibodies, chimeric antibodies, murine antibodies, humanized antibodies and diabodies. specific antibodies.
  • the antibodies that specifically bind to CD47 protein include: single-chain antibodies, diabodies, nanobodies, monoclonal antibodies, chimeric antibodies, murine antibodies, humanized antibodies and bispecific antibodies. Antibody.
  • D1 and/or D2 are IgG antibodies.
  • the IgG antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.
  • D1 and/or D2 are scFv.
  • D1 and/or D2 are Fab fragments.
  • the D1 is an anti-HER-2 IgG antibody.
  • the D1 is an anti-HER-2 Fab fragment.
  • the D1 includes one, two, three or more anti-HER-2 IgG antibodies.
  • the D1 contains one, two, three or more anti-HER-2 Fab fragments.
  • the D1 is an anti-HER-2 Fab fragment; and the D1 is an anti-HER-2 IgG antibody, wherein the anti-HER-2 Fab fragment of D1 is connected to the anti-HER-2 antibody of D1 The N-terminus of the variable region of the IgG antibody heavy chain.
  • the Fab of D1 and the IgG of D1 are connected through a linker or directly.
  • sequence of the linker is (G4S)n, preferably, n is 1-4.
  • the D1 is an anti-HER-2 IgG antibody.
  • the D2 is an anti-CD47 scFv.
  • the D1 includes one, two, three or more anti-HER-2 IgG antibodies.
  • the D2 contains one, two, three or more anti-CD47 scFvs.
  • the D2 is connected to a region of the anti-HER-2 antibody selected from the group consisting of: heavy chain variable region, heavy chain constant region, light chain variable region, light chain constant region or its combination.
  • the D2 is an anti-CD47 scFv; and the D1 is an anti-HER-2 IgG antibody, wherein D2 is linked to the N-terminus of the heavy chain variable region of D1.
  • D1 and D2 are connected through a joint or directly.
  • sequence of the linker is (G4S)n, preferably, n is 1-4.
  • the bispecific antibody is a homodimer or a heterodimer, preferably a heterodimer.
  • the bispecific antibody comprises an anti-HER-2 IgG antibody, an anti-HER-2 Fab fragment and an anti-CD47 scFv, wherein each Fab fragment interacts with anti-HER-2 through L2 Immunoglobulin antibodies IgG are connected in series; each scFv contains a variable region VH and a variable region VL, VH and VL are connected through the linker L1, and each anti-CD47 scFv is connected to the anti-HER-2 immunoglobulin antibody through the linker L2 IgG tandem.
  • the bispecific antibody includes:
  • D1 specifically binds to the target molecule TROP-2 protein
  • D2 specifically binds to the target molecule CD3 protein
  • the D1 is an antibody that specifically binds to TROP-2 protein or an antigen-binding fragment thereof; and/or
  • the D2 is an antibody that specifically binds to CD3 protein or an antigen-binding fragment thereof;
  • the structure of the antigen-binding fragment is selected from the following group: (i) Fab fragment; (ii) F(ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single-chain Fv (scFv) molecule; or (vi) single domain antibody dAb fragment.
  • the bispecific antibody includes an anti-TROP-2 IgG antibody and an anti-CD3 scFv, and the anti-CD3 scFv is connected in series with an anti-TROP-2 immunoglobulin antibody IgG through a linker.
  • the heavy chain variable region of the anti-TROP-2 IgG antibody includes complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO. 32, and the amino acid sequence of HCDR2 As shown in SEQ ID NO.33, the amino acid sequence of HCDR3 is as shown in SEQ ID NO.34; and
  • the light chain variable region of the anti-TROP-2 IgG antibody includes the complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is as shown in SEQ ID NO.35, and the amino acid sequence of LCDR2 is as shown in SEQ ID NO.36 shown, the amino acid sequence of LCDR3 is shown in SEQ ID NO.37.
  • the heavy chain variable region of the anti-CD3 scFv includes the complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO.38, and the amino acid sequence of HCDR2 is as shown in SEQ ID As shown in NO.39, the amino acid sequence of HCDR3 is as shown in SEQ ID NO.40; and
  • the anti-CD3 scFv light chain variable region includes the complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO.41, the amino acid sequence of LCDR2 is shown in SEQ ID NO.42, and the amino acid sequence of LCDR3 The amino acid sequence is shown in SEQ ID NO.43.
  • the bispecific antibody is selected from the following group:
  • the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.48 and SEQ ID NO.49, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.50;
  • a polypeptide derived from (1) that is formed by substituting, deleting or adding one or more amino acid residues to the amino acid sequence in (1) and having both anti-TROP-2 activity and anti-CD3 activity.
  • the bispecific antibody is a heterodimer, which includes a first polypeptide and a second polypeptide, having the structures shown in formulas Ia and Ib from the N-terminus to the C-terminus respectively:
  • VLA represents the light chain variable region of the anti-HER-2 antibody
  • VH A represents the heavy chain variable region of the anti-HER-2 antibody
  • VL B represents the light chain variable region of the anti-CD47 antibody
  • VH B represents the heavy chain variable region of the anti-CD47 antibody
  • CH represents the heavy chain constant region
  • CL represents the light chain constant region
  • L1 and L2 are independently keys or joints
  • ⁇ represents disulfide bond or covalent bond
  • the bispecific antibody has the activity of binding to HER-2 and CD47 simultaneously.
  • the heavy chain constant region is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.
  • sequence of the linker L1 is shown in SEQ ID NO. 24.
  • sequence of the linker L2 is shown in SEQ ID NO. 25.
  • the heavy chain variable region of the anti-HER-2 IgG antibody includes complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO.7, and the amino acid sequence of HCDR2 As shown in SEQ ID NO.8, the amino acid sequence of HCDR3 is as shown in SEQ ID NO.9; and
  • the light chain variable region of the anti-HER-2 IgG antibody includes the complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is as shown in SEQ ID NO.10, and the amino acid sequence of LCDR2 is as shown in SEQ ID NO.11 Shown, the amino acid sequence of LCDR3 is shown in SEQ ID NO.12.
  • the anti-HER-2 antibody comprises the heavy chain variable region shown in SEQ ID NO. 13.
  • the anti-HER-2 antibody comprises the light chain variable region shown in SEQ ID NO. 14.
  • sequence of the heavy chain region of the anti-HER-2 Fab fragment is shown in SEQ ID NO. 22.
  • the heavy chain variable region of the anti-CD47 antibody includes complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO.1, and the amino acid sequence of HCDR2 is as shown in SEQ ID NO. .2, the amino acid sequence of HCDR3 is shown in SEQ ID NO.3; and
  • the light chain variable region of the anti-CD47 antibody includes the complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO.4, the amino acid sequence of LCDR2 is shown in SEQ ID NO.5, and the amino acid sequence of LCDR3 The amino acid sequence is shown in SEQ ID NO.6.
  • the anti-CD47 antibody comprises the heavy chain variable region shown in SEQ ID NO. 15.
  • the anti-CD47 antibody comprises the light chain variable region shown in SEQ ID NO. 16.
  • sequence of the anti-CD47 scFv is shown in SEQ ID NO. 17.
  • the bispecific antibody is selected from the following group:
  • the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.30 and SEQ ID NO.31, and The light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 27;
  • a polypeptide derived from (1) that is formed by substituting, deleting or adding one or more amino acid residues to the amino acid sequence in (1) and having both anti-HER-2 activity and anti-CD47 activity.
  • the bispecific antibody includes active fragments and/or derivatives of the bispecific antibody, wherein the active fragments and/or derivatives retain the bispecific antibody 70-100% anti-HER-2 activity and 70-100% anti-CD47 activity.
  • the derivative of the antibody has at least 85% sequence identity with the antibody of the present invention.
  • the derivative of the antibody is a sequence in which the antibody of the present invention undergoes one or several amino acid deletions, insertions and/or substitutions and maintains at least 85% identity.
  • the derivative of the antibody has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% sequence identity.
  • the substitution is a conservative substitution.
  • the bispecific antibody includes an anti-HER-2 IgG, which includes the heavy chain constant region shown in SEQ ID NO. 26 or a derivative sequence thereof, and the derivative sequence includes one or more positions. Amino acid substitution of points: positions 354, 366 and 394 of the amino acid sequence shown in SEQ ID NO.26.
  • the derived sequence contains amino acid substitutions at one or more positions: S354C, T366W and T394K.
  • the bispecific antibody includes an anti-HER-2 IgG, which includes the heavy chain constant region shown in SEQ ID NO. 26 or a derivative sequence thereof, and the derivative sequence includes one or more positions. Amino acid substitution of points: positions 349, 366, 368, 407 and 397 of the amino acid sequence shown in SEQ ID NO.26.
  • the derived sequence contains amino acid substitutions at one or more positions: Y349C, T366S, L368A, Y407V and V397D.
  • the bispecific antibody includes an anti-HER-2 IgG, which includes the heavy chain constant region shown in SEQ ID NO. 26 or a derivative sequence thereof, and the derivative sequence includes one or more positions. Amino acid substitution of point: positions 354 and 366 of the amino acid sequence shown in SEQ ID NO.26.
  • the derived sequence contains amino acid substitutions at one or more positions: S354C and T366W.
  • the bispecific antibody includes an anti-HER-2 IgG, which includes the heavy chain constant region shown in SEQ ID NO. 26 or a derivative sequence thereof, and the derivative sequence includes one or more positions. Amino acid substitution of points: positions 349, 366, 368 and 407 of the amino acid sequence shown in SEQ ID NO.26.
  • the derived sequence contains amino acid substitutions at one or more positions: Y349C, T366S, L368A and Y407V.
  • a polynucleotide molecule encoding a heteromultimeric protein according to the first aspect of the invention.
  • an expression vector containing the polynucleotide molecule according to the second aspect of the invention is provided.
  • the expression vector is a virus or plasmid, preferably a phage or a phagemid.
  • the expression vector is selected from the following group: pcDNA3.4, pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0 , preferably pcDNA3.4.
  • a host cell containing the expression vector according to the third aspect of the invention In a fourth aspect of the invention, there is provided a host cell containing the expression vector according to the third aspect of the invention.
  • the host cell is selected from the following group: COS, CHO, NSO, sf9, sf21, DH5 ⁇ , BL21 (DE3) or TG1, more preferably E.coli TG1, BL21 (DE3) cells ( Expressing single-chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full-length IgG antibodies).
  • the host cell is a eukaryotic cell, preferably CHO cells or 293F cells.
  • the preparation method comprising the following steps:
  • a pharmaceutical composition comprising an effective amount of the heteromultimeric protein according to the first aspect of the present invention and one or more pharmaceutically acceptable carrier, diluent or excipient.
  • the heteromultimeric protein according to the first aspect of the present invention or the pharmaceutical composition according to the sixth aspect of the present invention in the preparation of drugs for cancer or tumors. .
  • the cancer or tumor is selected from: lung cancer, bone cancer, gastric cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer , cervical cancer, vaginal cancer, vulva cancer, rectal cancer, colon cancer, anal area cancer, breast cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethra cancer, penile cancer , prostate cancer, pancreatic cancer, brain cancer, testicular cancer, lymphoma, transitional cell cancer, bladder cancer, kidney or ureter cancer, renal cell cancer, renal pelvis cancer, Hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma , pediatric solid tumors, lymphocytic lymphoma, central nervous system (CNS) tumors, primary central nervous system lymphoma, tumor angiogenesis,
  • CNS
  • an immunoconjugate is provided, and the immunoconjugate includes:
  • a conjugation moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
  • the conjugate moiety is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computerized X-ray tomography) contrast agents, or can produce Detect product enzymes, radionuclides, biotoxins, and cytokines (such as IL-2, etc.).
  • the immunoconjugate includes an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the immunoconjugate is used to prepare a pharmaceutical composition for treating cancer or tumors.
  • a method for producing a heteromultimeric protein that specifically binds a first target, a second target and a third target comprising:
  • the first CH3 domain and the second CH3 domain have the following substitutions relative to the wild-type CH3 domain: the first CH3 domain: T394K, S354C, T366W; the second CH3 domain: V397D, Y349C, T366S, L368A, Y407V.
  • the position of the amino acid is determined according to the EU index of KABAT numbering.
  • the first target and the second target are the same or different.
  • the method further includes:
  • (c) Provide a first light chain, a second light chain and a third light chain; wherein the first light chain and the second light chain are respectively coordinated with the first polypeptide such that the heteropolymer
  • the heteromeric protein specifically binds to the first target and the second target; the third light chain cooperates with the second polypeptide, so that the heteromultimeric protein specifically binds to the second target.
  • a method for producing heteromultimeric proteins that specifically bind to different targets comprising:
  • the first CH3 domain and the second CH3 domain have the following substitutions relative to the wild-type CH3 domain: the first CH3 domain: T394K, S354C, T366W; the second CH3 domain: V397D, Y349C, T366S, L368A, Y407V.
  • a method of treating cancer or tumors comprising administering to a subject in need thereof a heteromultimeric protein according to the first aspect of the invention, according to The pharmaceutical composition according to the sixth aspect of the present invention, and the immunoconjugate according to the ninth aspect of the present invention.
  • the method further includes combined administration with other anti-tumor drugs.
  • a heterodimerized Fc element comprising a first polypeptide containing a first heavy chain constant domain 3 (CH3) domain and a second heavy chain constant domain.
  • a second polypeptide of a 3(CH3) domain wherein the first CH3 domain includes an amino acid substitution selected from the group consisting of: T394K, S354C and T366W, or a combination thereof, relative to a wild-type CH3 domain; and/or
  • the second CH3 domain includes an amino acid substitution selected from the group consisting of: V397D, Y349C, T366S, L368A, Y407V, or a combination thereof, the position of the amino acid being determined according to the EU index of KABAT numbering.
  • the heterodimerized Fc element further includes heavy chain constant domain 2 (CH2).
  • the positive and progressive effect of the present invention is that it provides a pestle and mortar structural model that can be applied to heteromultimeric proteins containing the Fc segment, promotes the heterodimerization of the Fc segment of the antibody, and provides a method that can stably express Asymmetrically structured anti-HER-2 and CD47 bispecific antibodies, and derived from other bispecific antibodies that can be stably expressed Antibodies, such as anti-TROP-2 and CD3 bispecific antibodies (see Examples for details), further demonstrate the breadth of application of this model.
  • Figure 1 shows a schematic diagram of the structure of the double antibody
  • Figure 1A shows a partial view of the crystal structure of the CH3-CH3 interface of the two heavy chains in the anti-HER-2/CD47 double antibody a
  • Figure 1B shows the anti-HER-2/CD47 double antibody b
  • Figure 1C shows a schematic structural diagram of the anti-HER-2/CD47 double antibody
  • Figure 1D shows a schematic structural diagram of the anti-TROP-2/CD3 double antibody d.
  • Figure 2A shows the HPLC detection pattern of anti-HER-2/CD47 double antibody a.
  • Figure 2B shows the HPLC detection pattern of anti-HER-2/CD47 double antibody b.
  • Figure 3A shows the CE-SDS detection pattern of anti-HER-2/CD47 double antibody a.
  • Figure 3B shows the CE-SDS detection pattern of anti-HER-2/CD47 double antibody b.
  • Figure 4A shows the molecular weight detection spectrum of anti-HER-2/CD47 double antibody a.
  • Figure 4B shows the molecular weight detection pattern of anti-HER-2/CD47 double antibody b.
  • Figure 5A shows the isoelectric point detection pattern of anti-HER-2/CD47 double antibody a.
  • Figure 5B shows the isoelectric point detection pattern of anti-HER-2/CD47 double antibody b.
  • heteromultimeric protein that contains an antibody heavy chain constant domain 3 (CH3) domain with one or more amino acid substitutions that make The CH3 domain of the heteromultimeric protein has novel engineered ionic bonds and/or ionic and/or charge interactions, which are further combined with the KIH residue substitution to promote the formation of heterodimers.
  • a method for preparing the heteromultimeric protein is also provided.
  • the heteromultimeric protein preparation method of the present invention can be used to enhance the formation of heterodimers and reduce the formation of homodimers. The method is applicable to all heteromultimeric proteins containing Fc segments, such as bispecific antibodies and Fc fusion proteins. On this basis, the present invention was completed.
  • This application is based on the above-mentioned pestle and mortar structure, further mutating and screening the amino acids of CH3 of the heavy chain constant region to obtain a more stable heteromultimeric protein, and applying this structure to more heteromultimeric proteins that bind through the Fc segment.
  • multimeric proteins such as asymmetrically structured bispecific antibodies and Fc fusion proteins.
  • the present invention also provides a heteromultimeric protein, in which the CH3 domain of the Fc segment of the heteromultimeric protein contains a positively charged amino acid at amino acid position 394 relative to the wild-type CH3 domain. a substitution with an amino acid (eg, T394K); and/or the second CH3 domain includes a substitution with a negatively charged amino acid (eg, V397D) at amino acid position 397 relative to the wild-type CH3 domain.
  • a substitution with an amino acid eg, T394K
  • the second CH3 domain includes a substitution with a negatively charged amino acid (eg, V397D) at amino acid position 397 relative to the wild-type CH3 domain.
  • the first CH3 domain also includes an amino acid substitution selected from the following group: S354C and/or T366W; and the second CH3 domain also includes an amino acid substitution selected from the following group Amino acid substitutions: Y349C, T366S, L368A, Y407V.
  • the position of the amino acid is determined according to the EU index of KABAT numbering.
  • the heteromultimeric protein is an anti-HER-2 and CD47 bispecific antibody with an asymmetric structure, or an anti-TROP-2 and CD3 bispecific antibody with an asymmetric structure.
  • the term “contains” or “includes” can be open, semi-closed and closed. In other words, the term also includes “consisting essentially of,” or “consisting of.”
  • heteromultimer or “heteromultimeric protein” is a molecule comprising at least a first polypeptide and a second polypeptide, wherein the second polypeptide is different in amino acid sequence from the first polypeptide. at least one amino acid residue.
  • the heteromultimer has binding specificity for at least two different ligands or binding sites.
  • a heteromultimer may comprise a "heterodimer” formed from a first polypeptide and a second polypeptide, or may form a higher order triad in which polypeptides other than the first polypeptide and the second polypeptide are present. level structure.
  • heterodimerization generally refers to the formation or absence of a covalent bond between two different members, such as by complexing, associating, or aggregating two different members (e.g. The process of forming heterodimers between two different polypeptides).
  • knock-into-holes refers to a pair of engineered amino acid residues in the CH3 domain that results in steric modification of the contact surface of the CH3 domain.
  • the first CH3 domain is preferentially attached to the corresponding contact surface of the second CH3 domain through complementary spatial modification.
  • Such steric modifications mainly originate from different amino acid residues and side chains, for example, to generate “Knobs” or “Holes” structures, which complement each other to form “Knobs-into-Holes” dimers.
  • a “CH3 domain” (also referred to as a “C2" or “H3” domain) encompasses the C-terminal extension of residues in the Fc region to the CH2 domain (i.e., from approximately amino acid residues 341 to C of the antibody sequence terminus (usually at amino acid residue 446 or 447 of IgG)).
  • first CH3 domain “first polypeptide constant region CH3”, “first CH3”, “first CH3 domain”, “first heavy chain constant domain 3” may be used interchangeably;
  • “Second CH3 domain”, “second polypeptide constant region CH3", “second CH3", second CH3 domain” and “second heavy chain constant domain 3” may be used interchangeably.
  • Antibody (Ab) and “Immunoglobulin G (IgG)” are heterotetrameric proteins with the same structural characteristics, consisting of two identical light chains (L ) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes (isotypes) is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by a constant domain, which consists of three domains, CH1, CH2, and CH3.
  • VH variable domain
  • Each light chain has a variable region (VL) at one end and a constant region at the other end.
  • the light chain constant region includes a domain CL; the constant region of the light chain pairs with the CH1 domain of the heavy chain constant region, and the light chain can The variable region is paired with the variable region of the heavy chain.
  • Constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity), etc.
  • the heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes ⁇ (Kappa) or ⁇ (Lambda).
  • the heavy and light chains of the antibody are covalently linked together by the disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain.
  • the two heavy chains of the antibody are covalently linked together by the inter-polypeptide disulfide formed between the hinge regions. Bonds are held together covalently.
  • binding domain includes any region of a polypeptide that specifically binds a target molecule (eg, antigen, ligand, receptor, etc.).
  • exemplary binding domains include antibody variable domains, receptor binding domains, ligand binding domains, and enzymatic domains.
  • the "immunoglobulin antibody IgG" described in the present invention is a molecule of approximately 150 kDa, which is composed of four peptide chains, including two identical gamma heavy chains of approximately 50 kDa, and two identical light chains of approximately 25 kDa, thus having Tetramer quaternary structure.
  • the two heavy chains are linked to each other by disulfide bonds and to a light chain each.
  • the resulting tetramer has two identical halves that form a fork or Y-like shape, with each end of the fork containing an identical antigen-binding site.
  • IgG antibodies can be divided into multiple subclasses (eg, IgG1, 2, 3, 4) based on minor differences in amino acid sequence in the constant region of the heavy chain.
  • bispecific antibody refers to an antibody molecule that can specifically bind to two antigens (targets) or two epitopes at the same time. Based on symmetry, bispecific antibodies can be divided into structurally symmetric and asymmetric molecules. According to the number of binding sites, bispecific antibodies can be divided into bivalent, trivalent, tetravalent and multivalent molecules.
  • the term "monoclonal antibody (monoclonal antibody)” refers to an antibody obtained from a substantially homogeneous population, that is, the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may exist. Monoclonal antibodies target a single antigenic site with high specificity. Furthermore, unlike conventional polyclonal antibody preparations, which are typically mixtures of different antibodies directed against different antigenic determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefit of monoclonal antibodies is that they can be synthesized by hybridoma culture without contamination by other immunoglobulins.
  • the modifier "monoclonal" indicates the nature of the antibody as having been obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any special method to produce the antibody.
  • antigen-binding fragment refers to a Fab fragment, a Fab' fragment, an F(ab') 2 fragment, or a single Fv fragment having antigen-binding activity.
  • antigen-binding fragments include: (i) Fab fragments; (ii) F(ab') 2 fragments; (iii) Fv fragments; (iv) single chain Fv (scFv); or (vi) single domain antibodies (dAb) fragment.
  • the expression "antigen-binding fragment” also encompasses other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tribodies , tetrabody antibodies, minibodies, nanobodies (such as monovalent nanobodies, bivalent nanobodies, etc.), small module immunopharmaceuticals (SMIP) and shark variable IgNAR domains.
  • SMIP small module immunopharmaceuticals
  • Fab and Fc mean that papain can cleave an antibody into two identical Fab segments and one Fc segment.
  • the Fab segment consists of the VH and CH1 domains of the heavy chain of the antibody and the VL and CL domains of the light chain.
  • the Fc segment is a crystallizable fragment (Fc), which consists of the CH2 and CH3 domains of the antibody.
  • the Fc segment has no antigen-binding activity and is the site where the antibody interacts with effector molecules or cells.
  • F(ab') 2 fragment F(ab') 2 fragment antibody is obtained by pepsin digestion of the entire IgG antibody, removing most of the Fc region while leaving some hinge regions intact, and is linked together by disulfide bonds The two antigens bind to the F(ab) part.
  • scFv single chain antibody
  • scFv single chain antibody fragment
  • VH and VL are connected through a 15-25 amino acid linker, wherein the fusion protein retains the intact immunoglobulin The same antigen specificity.
  • the term "Fv fragment” or "Fv antibody” contains the antibody heavy chain variable region, the light chain variable region, but no constant region, and is the smallest antibody fragment that has all antigen-binding sites.
  • the Fv fragment also contains a polypeptide linker between the VH and VL domains and is capable of forming the structure required for antigen binding.
  • variable means that certain parts of the variable regions of the antibody differ in sequence, which contribute to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy chain variable region and light chain variable region. The more conservative part of the variable region is called the frame region (FR).
  • CDRs complementarity-determining regions
  • FR frame region
  • the variable regions of natural heavy and light chains each contain four FR regions, which are generally in a ⁇ -sheet configuration and are connected by three CDRs forming a connecting loop. In some cases, a partial ⁇ -sheet structure can be formed.
  • the CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (that is, forming the antigen-binding domain) (see Kabat et al., NIH Publ. No. 91- 3242, Volume I, pp. 647-669 (1991)).
  • KABAT numbered EU index generally refers to the EU index according to KABAT et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, E.A. et al. (1991) Sequences Index corresponding to the EU number of the amino acid sequence of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242.
  • FR framework region
  • the light chain and heavy chain of immunoglobulins each have four FRs, which are called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, and FR4-H respectively.
  • the light chain variable domain may thus be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain may thus be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H).
  • the FR of the present invention is a human antibody FR or a derivative thereof.
  • the derivative of the human antibody FR is essentially the same as a naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, or 96%. , 97%, 98% or 99%.
  • human framework region is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) to that of a naturally occurring human antibody. .
  • linker refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for the light and heavy chain domains to fold into an exchange dual variable region immunoglobulin. base.
  • preferred linkers refer to linkers L1 and L2, where L1 connects the VH and VL of a single chain antibody (scFv), and L2 is used to connect the scFv or Fab fragment to the heavy chain of another antibody.
  • linkers examples include single glycine (Gly), or serine (Ser) residues.
  • Gly single glycine
  • Ser serine residues.
  • the identity and sequence of the amino acid residues in the linker may vary depending on the type of secondary structural elements desired to be implemented in the linker.
  • the terms “anti”, “binding” and “specific binding” refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets.
  • the antibody binds the antigen with an equilibrium dissociation constant (KD) of less than about 10 "7 M, such as less than about 10 "8 M, 10 “9 M, 10 "10 M, 10 “11 M, or less.
  • KD equilibrium dissociation constant
  • the term “KD” refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • the smaller the equilibrium dissociation constant the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • SPR surface plasmon resonance
  • ELISA is used to determine the relative affinity of an antibody to an antigen.
  • epitope refers to a polypeptide determinant that specifically binds to an antibody.
  • An epitope of the present invention is a region of an antigen that is bound by an antibody.
  • the human epidermal growth factor receptor-2 (HER-2) gene is located on chromosome 17q21 and is a proto-oncogene.
  • the HER-2 protein encoded by it is a 185KD transmembrane protamine with tyrosine Protein kinase activity is a member of the epidermal growth factor receptor (EGFR) family.
  • EGFR epidermal growth factor receptor
  • HER-2 protein is not expressed or has low expression in normal body tissues. It is overexpressed in various tumor cells such as breast cancer, ovarian cancer, and gastric cancer. It is a typical tumor treatment target and monitoring marker.
  • the HER-2 protein forms homologous or heterodimers with other members of its family, initiating downstream signaling pathways. Currently, no ligand has been found that can directly bind to the HER-2 protein.
  • Trastuzumab (Herceptin), the world's first humanized monoclonal antibody for clinical treatment, is an antibody treatment drug specifically for HER-2-positive breast cancer. This antibody can interact with the extracellular structure of HER-2 Domain binding blocks the interaction between HER-2 and non-receptor tyrosine kinases, thereby causing down-regulation of HER-2 and has significant effects in clinical treatment.
  • CD47 Cluster of differentiation 47
  • IAP integrin-associated protein
  • CD47 belongs to the immunoglobulin superfamily.
  • natural ligands include integrin and thrombospondin 1 ( thrombospondin-1, TSP-1) and signal-regulatory protein ⁇ (Sirp ⁇ ).
  • thrombospondin-1 thrombospondin-1, TSP-1
  • signal-regulatory protein ⁇ Sirp ⁇
  • CD47 binds to Sirp ⁇ on the surface of macrophages and releases the "don't eat me” signal, thereby ensuring that normal cells are not “eaten” by macrophages.
  • some tumor cells also use the CD47/Sirp ⁇ signaling pathway. , high expression of CD47 to achieve immune evasion.
  • Anti-CD47 antibodies can target a variety of indications, mainly including hematological malignancies such as non-Hodgkin lymphoma (NHL) and acute myeloid leukemia (AML).
  • NHL non-Hodgkin lymphoma
  • AML acute myeloid leukemia
  • normal cells include red blood cells and T lymphocytes also express CD47, so the toxic and side effects of CD47-targeting drugs are a difficulty in the development of such drugs.
  • TROP-2 Human trophoblast cell surface antigen 2
  • TACSTD2 Human trophoblast cell surface antigen 2
  • TROP-2 is a single-transmembrane type I membrane protein consisting of 323 amino acids, including 26 amino acids in the signal peptide, 248 amino acids in the extracellular region, 23 amino acids in the transmembrane region, and 26 amino acids in the intracellular region.
  • TROP-2 protein is highly expressed in various human epithelial cancers and is closely related to poor prognosis and cancer cell metastasis, including breast cancer, lung cancer, Gastric cancer, pancreatic cancer, prostate cancer and cervical cancer, etc.
  • the US FDA has approved the TROP-2 antibody conjugate drug sacituzumab govitecan for the treatment of metastatic triple-negative breast cancer.
  • CD3 Cluster of differentiation 3
  • TCR T cell receptors
  • ITAM autoimmune tyrosine-based activation motif
  • Anti-CD3 antibodies can stimulate or block T cell activation signals, eliminate effector T cells or induce the production of regulatory T cells.
  • heteromultimeric proteins such as heterodimeric proteins, which comprise a first polypeptide containing an antibody heavy chain constant region CH3 and a second polypeptide containing an antibody heavy chain constant region CH3, wherein the first polypeptide
  • the constant region CH3 and the second polypeptide constant region CH3 contain engineered ionic and/or charge interactions and electrostatic interactions.
  • the heteromultimeric protein comprises an engineered Fc segment comprising a first polypeptide constant region CH3 and a second polypeptide constant region CH3.
  • the first polypeptide constant region CH3 and the second polypeptide constant region CH3 comprise "Knobs-into-Holes" residues.
  • Exemplary structures of heteromultimeric proteins include heterodimers (e.g., Fc fusion proteins), heterotrimers (e.g., antibody-immunoadhesin chimeras), heterotetramers (e.g., bispecific antibodies), and others Oligomeric structure.
  • the heteromultimeric protein is a bispecific antibody, such as a bispecific antibody with an asymmetric structure (comprising two different heavy chains).
  • the present invention provides a heteromultimeric protein comprising a modified Fc segment, wherein the first polypeptide constant region CH3 includes a substitution with a positively charged amino acid at amino acid position 394 relative to the wild-type constant region CH3, and /or the second polypeptide constant region CH3 comprises a substitution with a negatively charged amino acid at amino acid position 397 relative to the wild-type constant region CH3.
  • the positively charged amino acid at 394 in CH3 of the first polypeptide constant region forms ionic and/or charge interactions with the amino acid residues in CH3 of the second polypeptide constant region, and the band at 397 in the second polypeptide constant region CH3
  • the negatively charged amino acids form ionic and/or charge interactions with the amino acid residues in the CH3 constant region of the first polypeptide.
  • the heteromultimeric protein is a bispecific antibody.
  • the bispecific antibody is an anti-HER-2 and CD47 bispecific antibody, which includes an engineered Fc segment that includes an engineered Fc segment that promotes engineered ion and/or charge interactions. Amino acid substitutions and "Knobs-into-Holes" residues.
  • the engineered Fc segment includes a first polypeptide constant region CH3 and a second polypeptide constant region CH3, which includes amino acid substitutions selected from the group consisting of:
  • the first polypeptide constant region CH3 T394K, S354C, T366W; the second polypeptide constant region CH3: V397D, Y349C, T366S, L368A, Y407V.
  • the bispecific antibody of the present invention is an asymmetric bispecific antibody that can specifically bind to HER-2 and CD47, and contains an anti-HER-2 antibody part and an anti-CD47 antibody part. Specifically, it contains immunoglobulin anti- Body IgG, a Fab fragment and a variable region fragment scFv.
  • the Fab fragment contains the variable region VH and the constant region CH1, as well as the variable region VL and the constant region CL.
  • the Fab fragment is connected in series with the immunoglobulin antibody IgG through the linker peptide L2;
  • the single-chain variable fragment scFv contains variable regions VH and variable regions VL. VH and VL are connected through a peptide linker L1.
  • the single-chain variable fragment scFv is connected in series with the immunoglobulin antibody IgG through the linker peptide L2.
  • the "bispecific antibody” described in the present invention refers to a bispecific antibody that has two different antigen-binding sites and can bind to HER-2 and CD47 at the same time. It contains a Fab fragment and a variable region fragment scFv. And the immunoglobulin antibody IgG conjugated to it, the Fab fragment and scFv are connected to different IgG antibody heavy chains via the peptide linker L2, forming two different heavy chains of the bispecific antibody and obtaining a fusion protein, where scFv contains variable regions VH and VL, and VH and VL are connected through a peptide linker L1.
  • the anti-HER-2 antibody part in the bispecific antibody of the present invention is an antibody that specifically binds to the HER-2 protein or its antigen-binding fragment
  • the anti-CD47 antibody part is an antibody that specifically binds to the CD47 protein or its antigen.
  • Binding fragment; the structure of the antigen-binding fragment can be selected from the following group: (i) Fab fragment; (ii) F(ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single chain Fv (scFv) molecules; or (vi) single domain antibody dAb fragments.
  • the bispecific antibodies of the present invention can be dimers, trimers or multimers, preferably homo- or heterodimers.
  • the anti-HER-2 or anti-CD47 antibody portion of the bispecific antibody of the present invention may include one or more antibodies or antigen-binding fragments thereof, preferably 1, 2, 3, 4, 5, or 6.
  • the bispecific antibody of the present invention includes the scFv of an anti-CD47 antibody and an IgG antibody of HER-2, wherein the VH of the anti-CD47 antibody includes complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is as follows SEQ ID NO.1 is shown, the amino acid sequence of HCDR2 is shown in SEQ ID NO.2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO.3;
  • the VL of the anti-CD47 antibody includes the complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO.4, the amino acid sequence of LCDR2 is shown in SEQ ID NO.5, and the amino acid sequence of LCDR3 is shown in SEQ Shown as ID NO.6;
  • the VH of the anti-HER-2 IgG antibody includes the complementary determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO.7, the amino acid sequence of HCDR2 is shown in SEQ ID NO.8, and the amino acid sequence of HCDR3 The amino acid sequence is shown in SEQ ID NO.9;
  • the VL of the anti-HER-2 IgG antibody includes the complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO.10, the amino acid sequence of LCDR2 is shown in SEQ ID NO.11, and the amino acid sequence of LCDR3 The amino acid sequence is shown in SEQ ID NO.12.
  • the binding region of an antibody usually contains a light chain variable region and a heavy chain variable region, and each variable region contains 3 CDR domains.
  • the CDR domains of the heavy and light chains of antibodies are called HCDR and LCDR respectively. Therefore, a conventional antibody antigen-binding site contains six CDRs, including a set of CDRs from the heavy chain and light chain V regions respectively.
  • the bispecific antibody of the present invention also includes conservative variants thereof, which means that compared with the amino acid sequence of the bispecific antibody of the present invention, there are at most 10, preferably at most 8, and more preferably at most 5, preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.
  • the amino acid sequence of the first heavy chain constant region is as shown in SEQ ID NO.18, or a sequence based on the SEQ ID NO.18 sequence and also has an amino acid mutation selected from positions 354, 366 and 394. , preferably a sequence having one or more amino acid mutations selected from S354C, T366W and T394K; the amino acid sequence of the second heavy chain constant region is as shown in SEQ ID NO.18, or based on the SEQ ID NO.18 sequence
  • the sequence also has an amino acid mutation selected from positions 349, 366, 368, 407 and 397, preferably a sequence having one or more amino acid mutations selected from Y349C, T366S, L368A, Y407V and V397D.
  • the amino acid sequence of the first heavy chain constant region is as shown in SEQ ID NO.18, or a sequence based on the SEQ ID NO.18 sequence and also has an amino acid mutation selected from positions 354 and 366, preferably A sequence with one or more amino acid mutations selected from S354C and T366W;
  • the amino acid sequence of the second heavy chain constant region is as shown in SEQ ID NO.18, or based on the SEQ ID NO.18 sequence, it also has
  • the sequence selected from amino acid mutations at positions 349, 366, 368 and 407 is preferably a sequence having amino acid mutations at one or more positions selected from Y349C, T366S, L368A and Y407V.
  • amino acid sequence of the peptide linker L1 is shown in SEQ ID NO. 24.
  • amino acid sequence of the peptide linker L2 is shown in SEQ ID NO. 25.
  • the molecular structure of the single-chain variable fragment scFv is VH-L1-VL, and the C-terminus of each scFv is connected to the N-terminus of the immunoglobulin antibody IgG heavy chain via a peptide linker L2.
  • amino acid sequence of the single-chain variable fragment scFv is shown in SEQ ID NO. 17.
  • the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 30 and SEQ ID NO. 31, and the light chain amino acid sequence is shown in SEQ ID NO. 27.
  • bispecific antibodies of the invention issues related to the chemical and physical stability of the bispecific antibodies are also addressed, such as expressing physically stable molecules, increasing heat and salt-dependent stability, reducing aggregation, Increase solubility at high concentrations and maintain affinity for two antigens, HER-2 and CD47 respectively.
  • polynucleotide molecule encoding the heteromultimeric protein described above.
  • the polynucleotides of the invention may be in DNA form or RNA form.
  • Forms of DNA include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the preparation method of the nucleotide molecules of the present invention is a conventional preparation method in the art, and preferably includes the following preparation methods: obtaining the nucleotide molecules encoding the above-mentioned monoclonal antibodies through gene cloning technology such as PCR methods, or by The nucleotide molecules encoding the above-mentioned monoclonal antibodies are obtained by artificial full-sequence synthesis.
  • nucleotide sequence encoding the amino acid sequence of the above-mentioned bispecific antibody can be appropriately introduced with substitutions, deletions, changes, insertions or additions to provide a polynucleotide homologue.
  • Homologues of the polynucleotides of the present invention can be prepared by substituting, deleting or adding one or more bases encoding the bispecific antibody gene within the range of maintaining the activity of the antibody.
  • Another aspect of the present invention provides an expression vector containing the above-mentioned nucleotide molecule.
  • the expression vector is a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate The expression vector of the sequence.
  • the expression vector can be a virus or a plasmid, such as an appropriate phage or phagemid.
  • a virus or a plasmid such as an appropriate phage or phagemid.
  • the expression vector of the present invention is preferably pDR1, pcDNA3.1(+), pcDNA3.4, pcDNA3.1/ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, more preferably pcDNA3 .4.
  • the present invention further provides a host cell containing the above-mentioned expression vector.
  • the host cells described in the present invention are various conventional host cells in this field, as long as the recombinant expression vector can stably replicate itself and the nucleotides carried can be effectively expressed.
  • the host cells include prokaryotic expression cells and eukaryotic expression cells, and the host cells preferably include: COS, CHO (Chinese Hamster Ovary), NSO, sf9, sf21, DH5 ⁇ , BL21 (DE3) Or TG1, more preferably E.coli TG1, BL21 (DE3) cells (expressing single chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full-length IgG antibodies).
  • the preferred recombinant expression transformant of the present invention can be obtained by transforming the aforementioned expression vector into a host cell.
  • the transformation method is a conventional transformation method in this field, preferably a chemical transformation method, a heat shock method or an electroporation method.
  • the host cell is a eukaryotic cell.
  • CHO cells or 293F cells are preferred.
  • recombination can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.
  • the invention also relates to vectors comprising the appropriate DNA sequences as described above and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells to enable expression of the protein.
  • Another aspect of the present invention provides the above-mentioned bispecific antibody that can specifically bind to HER-2 and CD47 and a preparation method thereof.
  • the preparation method includes the following steps:
  • the host cell culture method and the antibody isolation and purification method of the present invention are conventional methods in this field.
  • the preparation method of the anti-HER-2 and CD47 bispecific antibodies disclosed in the present invention includes: cultivating the above-mentioned host cells under expression conditions to express bispecific antibodies that can specifically bind to HER-2 and CD47; isolating and The anti-HER-2 and CD47 bispecific antibodies were purified.
  • the recombinant protein can be purified into a substantially homogeneous material.
  • the anti-HER-2 and CD47 bispecific antibodies disclosed in the present invention can be separated and purified by affinity chromatography. According to the characteristics of the affinity column used, conventional methods such as high-salt buffer, changing pH, etc. can be used. Methods Elute anti-HER-2 and CD47 bispecific antibodies bound to the affinity column. The inventor of the present invention conducted detection experiments on the obtained anti-HER-2 and CD47 bispecific antibodies, and the experimental results showed that the anti-HER-2 and CD47 Bispecific antibodies can bind to target cells and antigens well and have high affinity.
  • compositions comprising the above-mentioned bispecific antibody capable of specifically binding to HER-2 and CD47 and one or more pharmaceutically acceptable carriers, diluents agents or excipients.
  • the composition is a pharmaceutical composition.
  • the bispecific antibodies provided by the present invention can be combined with pharmaceutically acceptable carriers to form pharmaceutical preparation compositions to exert therapeutic effects more stably. These preparations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibodies of the present invention. , while also protecting the multi-functional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation).
  • these materials may be formulated in a nontoxic, inert, and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary. It will vary depending on the nature of the substance formulated and the condition to be treated.
  • the prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal injection). ), intracranial injection, or intracavity injection.
  • intravenous injection intravenous drip
  • subcutaneous injection local injection
  • intramuscular injection intratumoral injection
  • intraperitoneal injection such as intraperitoneal injection).
  • intracranial injection or intracavity injection.
  • liquid preparations they can usually be stored at 2°C-8°C and are stable for at least one year, and for lyophilized preparations, they are stable at 30°C for at least six months.
  • the bispecific antibody preparation can be suspension, water injection, freeze-drying and other preparations commonly used in the pharmaceutical field.
  • the term "pharmaceutical composition” means that the bispecific antibody of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition to exert a more stable therapeutic effect. These preparations can ensure the bispecific antibodies disclosed in the present invention. It protects the conformational integrity of the amino acid core sequence of the antibody while also protecting the polyfunctional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation).
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned bispecific antibody (or conjugate thereof) of the present invention and Pharmaceutically acceptable carrier or excipient.
  • Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the bispecific antibodies of the invention can also be used with other therapeutic agents.
  • a safe and effective amount of the bispecific antibody or immunoconjugate thereof is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 mg/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.
  • the present invention provides bispecific antibodies that specifically bind to HER-2 and CD47, or the use of the above pharmaceutical composition in the preparation of medicines for treating cancer or tumors.
  • the drugs used to treat cancer or tumors as referred to in the present invention refer to drugs that inhibit and/or treat tumors, which may include delaying the development of tumor-related symptoms and/or reducing the severity of these symptoms, and further include This includes alleviating symptoms associated with existing tumors and preventing the occurrence of other symptoms, as well as reducing or preventing tumor metastasis.
  • Tumors targeted by the medicine of the present invention preferably include, but are not limited to: lung cancer, bone cancer, gastric cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, uterine cancer, fallopian tube cancer, uterine cancer Endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, rectal cancer, colon cancer, anal area cancer, breast cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, urethra cancer , penile cancer, prostate cancer, pancreatic cancer, brain cancer, testicular cancer, lymphoma, transitional cell cancer, bladder cancer, kidney or ureter cancer, renal cell cancer, renal pelvis cancer, Hodgkin's disease, non-Hodgkin's lymphoma , soft tissue sarcoma, pediatric solid tumors, lymphocytic lymphoma, central nervous system (CNS) tumors, primary
  • the dosage varies depending on the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. Animal experiments can be referred to Depending on the results and various circumstances, the total dosage cannot exceed a certain range. Specifically, the dosage for intravenous injection is 1-1800mg/day.
  • the bispecific antibodies and their compositions of the present invention can also be administered in combination with other anti-tumor drugs to achieve more effective treatment of tumors.
  • anti-tumor drugs include but are not limited to: 1.
  • Cytotoxic drugs 1) Action Drugs based on the chemical structure of nucleic acids: alkylating agents such as nitrogen mustards, nitrosoureas, and methanesulfonates; platinum compounds such as cisplatin (Cisplatin), carboplatin (Carboplatin), and oxaliplatin (Oxaliplatin), etc.; Antibiotics such as Adriamycin/Doxorubicin, Dactinomycin D, Daunorubicin, Epirubicin, Mithramycin, etc.; 2) Affect nucleic acid metabolism Drugs: dihydrofolate reductase inhibitors such as methotrexate (MTX) and pemetrexed (Pemetrexed), etc.; thymidine synthase inhibitors such as
  • Hormone drugs antiestrogens Hormones such as Tamoxifen, Droloxifene, Exemestane, etc.; aromatase inhibitors such as Aminoglutethimide, Formestane, Letrox Letrozle, Anastrozole, etc.; anti-androgens: flutamide RH-LH agonists/antagonists: Noride, enastrone, etc.; 3.
  • Biological response modifier drugs these drugs Mainly by regulating the body's immune function to achieve anti-tumor effects, such as interferons (Interferon); interleukin-2 (Interleukin-2); thymosins (Thymosins), etc.; 4.
  • Monoclonal antibody drugs Tratuximab Trastuzumab, Rituximab, Cetuximab, Bevacizumab, etc.; 5.
  • Other anti-tumor drugs including some whose mechanisms are still unclear and need to be Drugs for further research, etc.
  • the bispecific antibodies and their compositions disclosed in the present invention can be used in combination with one of the above-mentioned anti-tumor drugs or a combination thereof.
  • the heteromultimeric protein of the present invention contains an Fc segment with specific amino acid substitutions that promote ionic and/or charge interactions, and effectively promotes the formation and reduction of heterodimers by combining with the KIH mutation. Homodimer formation.
  • the present invention provides anti-HER-2 and CD47 and anti-TROP-2 and CD3 dual antibodies with engineered ion and/or charge interaction amino acid substitutions and "Knobs-into-Holes" pestle structures in the Fc region. Specific antibodies that can be stably expressed with high purity.
  • Competent cells Brand Shenggon, Cat. No. B528412.
  • 293F cells Brand GIBCO, Cat. No. R79007.
  • 0.45 ⁇ m filter Brand Millipore, Cat. No. SLHV013SL.
  • AdvancedBio SEC 300A column purchased from Agilent.
  • GXIIHT protein express labchip: purchased from PerkinElmer.
  • MassPREP Micro Desalting column purchased from Waters Company.
  • Endotoxin-free plasmid maximal extraction kit brand Tiangen, product number DP117.
  • DNA purification and recovery kit brand Tiangen, product number DP214.
  • ClonExpressTM II One Step Cloning Kit: Brand Novozymes, item number C112.
  • Protein express reagent kit Brand PerkinElmer, product number CLS960008.
  • PNGase F enzyme Brand NEB, Cat. No. P0704S.
  • Heraeus Fresco17 centrifuge purchased from Thermo Company.
  • iCE3 Capillary Isopoint Focusing Analyzer purchased from ProteinSimple Company.
  • the present invention adopts the tandem connection method of HER-2 monoclonal antibody IgG and CD47 monoclonal antibody scFv, and the tandem connection method of HER-2 monoclonal antibody IgG and HER-2 monoclonal antibody Fab fragment heavy chain region, and constructs it through the knob-into-hole mode.
  • Anti-HER-2 and CD47 bispecific antibodies a and b with asymmetric structures were developed. The structures are shown in Figure 1C.
  • the partial views of the CH3-CH3 interface crystal structures of bispecific antibodies a and b are shown in Figures 1A and 1B respectively. Show.
  • the HER-2 monoclonal antibody is derived from: the human-mouse chimeric monoclonal antibody obtained by Sansheng Guojian Pharmaceutical according to the amino acid sequence of the variable region of Herceptin, expressed using the CHO cell expression system, and independently developed by the cell culture production process. .
  • the CD47 monoclonal antibody is derived from: the humanized anti-CD47 antibody in WO2021218684A1, whose heavy chain variable region and light chain variable region are Anti-CD47B-Hu-VH and Anti-CD47B-Hu-VL respectively.
  • anti-HER-2 monoclonal antibodies and anti-CD47 monoclonal antibody controls in the examples are respectively: monoclonal antibodies obtained according to the monoclonal antibody amino acid sequences disclosed in the above-mentioned corresponding patents and with reference to the same expression and purification method in Example 2.
  • Anti-HER-2 and CD47 bispecific antibodies a and b are heterodimers and require amino acid mutations in the constant region CH3 (SEQ ID NO.21) of the heavy chain. S354C, T366W and T394K of the constant region CH3 are required. The sites were mutated to obtain the anti-HER-2 monoclonal antibody heavy chain HC-Knob (SEQ ID NO.28); the Y349C, T366S, L368A, Y407V and V397D sites of the constant region CH3 were mutated to obtain the anti-HER-2 monoclonal antibody. Anti-heavy chain HC-Hole (SEQ ID NO.29).
  • the anti-HER-2 monoclonal antibody Fab fragment heavy chain region (SEQ ID NO. 22) is composed of the anti-HER-2 monoclonal antibody heavy chain.
  • the variable region VH (SEQ ID NO. 13) is directly connected to the heavy chain constant region CH1 (SEQ ID NO. 19); the anti-CD47 monoclonal antibody variable fragment scFv is connected to the CD47 heavy chain through the peptide linker L1 (SEQ ID NO. 24).
  • the C-terminus of the heavy chain region of the anti-HER-2 monoclonal antibody Fab fragment is connected to the N-terminus of the anti-HER-2 monoclonal antibody heavy chain HC-Knob through the peptide linker L2 (SEQ ID NO: 25) to obtain the anti-HER-2 and CD47 dual Anti- ⁇ heavy chain I (SEQ ID NO:30); the C-terminus of the variable fragment scFv of the anti-CD47 monoclonal antibody is connected to the anti-HER-2 monoclonal antibody heavy chain HC-Hole through the peptide linker L2 to obtain anti-HER-2 and
  • the heavy chain II of the CD47 double antibody a SEQ ID NO.31
  • the light chain of the anti-HER-2 monoclonal antibody SEQ ID NO:27
  • anti-HER-2 and CD47 bispecific antibody a On the basis of anti-HER-2 and CD47 bispecific antibody a, the amino acid of CH3 in the heavy chain constant region was mutated to obtain anti-HER-2 and CD47 bispecific antibodies a', b, c and c', as shown in the table 2 shown.
  • the DNA fragments of the heavy chain and light chain of anti-HER-2 and CD47 bispecific antibodies were subcloned into vector pcDNA3.4 respectively, and the recombinant plasmid was extracted and co-transfected into 293F cells and/or CHO cells. After the cells have been cultured for 5-7 days, the culture medium is centrifuged at high speed, filtered with a 0.22 ⁇ m filter membrane, and loaded into a Hitrap Mabselect Sure affinity chromatography column. The protein is eluted in one step with 100mM citric acid, pH 3.5 eluent. The target sample is recovered and dialyzed into PBS at pH 7.4, and further purified by HiLoad 26/600 Superdex 200pg molecular sieve.
  • anti-TROP-2 and CD3 bispecific antibody molecule d which includes anti-TROP-2 IgG Antibody, scFv fragment of anti-CD3 antibody
  • its heavy chain I contains S354C, T366W and T394K mutations
  • heavy chain II contains Y349C, T366S, L368A, Y407V and V397D
  • substitutions in the Fc segment of molecule d are completely consistent with those of anti-HER- 2 is the same molecule as CD47 double antibody a.
  • Table 3 the monomer purity still reaches over 90%.
  • TROP-2/CD3 double antibody d is as follows (see Figure 1D for the structure).
  • the TROP-2 monoclonal antibody sequence in the anti-TROP-2/CD3 bispecific antibody is derived from the KM4097HV3aLV0 humanized monoclonal antibody disclosed in the US20120237518A1 patent, and the CD3 monoclonal antibody in the anti-TROP-2/CD3 bispecific antibody
  • the CDR sequence is derived from SEQ ID NO 2 and 4 in US8236308B2.
  • Adjust the concentration of anti-HER-2 and CD47 bispecific antibodies a and b to 1mg/mL with ultrapure water prepare reduced and non-reduced samples, follow the instructions of the protein express reagent kit, use the GXIIHT protein express labchip chip, and use The CE value of the sample was detected by the GX II capillary electrophoresis system, and the CE-SDS detection patterns of anti-HER-2 and CD47 bispecific antibodies a and b were obtained, as shown in Figure 3A and Figure 3B.
  • the experimental results show that the main peak proportion of the anti-HER-2 and CD47 double antibody a molecule is 93.2%, which is consistent with the UPLC-SEC results; the main peak proportion of the anti-HER-2 and CD47 double antibody b molecule is 94.2%, but in this antibody size There is a 5.8% impurity peak at about half of the peak, indicating that there is a certain amount of half-antibody structure in the molecule.
  • the anti-HER-2 and CD47 bispecific antibodies a and b were desaccharified with glycocleaving enzyme PNGase F, diluted the protein concentration to 1mg/mL with 50mM NH 4 HCO 3 , and centrifuged at 12000rpm for 10min. The supernatant was taken and passed through a MassPREPTM Micro Desalting Column (20 ⁇ m, 2.1 ⁇ 5 mm) desalting column for mass spectrometry analysis. The molecular weight detection patterns of the anti-HER-2 and CD47 bispecific antibodies a and b were obtained, as shown in Figure 4A and Figure 4B.
  • the experimental results show that the molecular state of the anti-HER-2 and CD47 double antibody a molecules is uniform, and the theoretical molecular weight is consistent with the tested molecular weight, accounting for 100%; the anti-HER-2 and CD47 double antibody b molecules have a theoretical molecular weight that is basically consistent with the tested molecular weight.
  • the proportion is 61.9%, which also contains 38.1% protein molecules with a size of 100KD, indicating that there is a certain amount of unknown structural impurities in this molecule.
  • the size exclusion chromatography, capillary electrophoresis, molecular weight mass spectrometry and isoelectricity of the anti-HER-2 and CD47 bispecific antibody a introduced the S354C, T366W and T394K mutations and the Y349C, T366S, L368A, Y407V and V397D mutations.
  • the results of spot detection were better than the introduction of L351K and L351D anti-HER-2 and CD47 bispecific antibodies b.
  • the anti-HER-2 and CD47 bispecific antibody a has a higher target protein content and a narrower isoelectric point range, making it relatively more stable, and this mutation is suitable for the preparation of asymmetric biantibodies for other targets.

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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

La présente invention concerne une protéine hétéromultimère et son procédé de préparation. Plus particulièrement, la protéine hétéromultimère comprend une première chaîne lourde d'anticorps avec introduction d'une mutation T394K par rapport aux mutations S354C et T366W de la chaîne unique Knob de Knob-into-Hole, et une seconde chaîne lourde d'anticorps avec addition d'une mutation V397D par rapport aux mutations Y349C, T366S, L368A et Y407V de la chaîne unique Knob de Knob-into-Hole. L'invention concerne en outre un procédé de préparation d'un anticorps bispécifique ciblant des molécules de surface de cellule tumorale HER-2 et CD47 ou un anticorps bispécifique ciblant une cellule tumorale et des molécules de surface de cellule immunitaire TROP-2 et CD3 au moyen du procédé de production de la protéine hétéromultimère, l'anticorps bispécifique comprenant un anticorps bispécifique produit par liaison d'un fragment variable scFv et d'un anticorps d'immunoglobuline IgG par l'intermédiaire d'un lieur peptidique, ou la liaison d'un fragment Fab ayant une activité antigénique et un anticorps d'immunoglobuline IgG par l'intermédiaire d'un lieur peptidique.
PCT/CN2023/111768 2022-08-09 2023-08-08 Protéine hétéromultimère et son procédé de préparation WO2024032617A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109476763A (zh) * 2016-07-19 2019-03-15 伊班绰斯有限责任公司 双特异性蛋白质及其制备方法
CN111527109A (zh) * 2017-12-26 2020-08-11 南京金斯瑞生物科技有限公司 以抗体Fc区为骨架的融合蛋白二聚体及其应用
CN112566937A (zh) * 2018-05-23 2021-03-26 辉瑞公司 对cd3特异性的抗体及其用途
WO2021213511A1 (fr) * 2020-04-24 2021-10-28 Virtuoso Binco, Inc. Anticorps bispécifiques pour le traitement de maladies associées à cd47

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109476763A (zh) * 2016-07-19 2019-03-15 伊班绰斯有限责任公司 双特异性蛋白质及其制备方法
CN111527109A (zh) * 2017-12-26 2020-08-11 南京金斯瑞生物科技有限公司 以抗体Fc区为骨架的融合蛋白二聚体及其应用
CN112566937A (zh) * 2018-05-23 2021-03-26 辉瑞公司 对cd3特异性的抗体及其用途
WO2021213511A1 (fr) * 2020-04-24 2021-10-28 Virtuoso Binco, Inc. Anticorps bispécifiques pour le traitement de maladies associées à cd47

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