WO2023222027A1 - 一种抗trop-2/cd3双特异性抗体 - Google Patents

一种抗trop-2/cd3双特异性抗体 Download PDF

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WO2023222027A1
WO2023222027A1 PCT/CN2023/094746 CN2023094746W WO2023222027A1 WO 2023222027 A1 WO2023222027 A1 WO 2023222027A1 CN 2023094746 W CN2023094746 W CN 2023094746W WO 2023222027 A1 WO2023222027 A1 WO 2023222027A1
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seq
amino acid
antibody
cancer
bispecific antibody
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French (fr)
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王定和
黄浩旻
王宝丽
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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
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • 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
    • C07K2317/56—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]

Definitions

  • the present invention relates to the technical field of antibody drugs, specifically, to an anti-TROP-2/CD3 bispecific antibody.
  • 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. wait.
  • the U.S. 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 receptor
  • ITAM autoimmune tyrosine signaling motif
  • Anti-CD3 antibodies can stimulate or block T cell activation signals, eliminate effector T cells or induce the production of regulatory T cells.
  • Bispecific antibodies also known as bifunctional antibodies, are specific drugs that simultaneously target two different antigens or different epitopes of the same antigen.
  • BsAb can act on tumor cells by immune cells and viral molecules to enhance the killing effect on target cells. It can also combine with different antigens of tumor cells at the same time to enhance its binding specificity and reduce off-target effects.
  • Bispecific antibodies have broadened the application fields of antibody drugs and provided new research ideas for tumor immunity.
  • the purpose of the present invention is to provide an anti-TROP-2/CD3 bispecific antibody to solve the problems in the prior art.
  • the purpose of the present invention is to provide a new bispecific antibody against TROP-2 and CD3, which can specifically bind to TROP-2 and CD3 at the same time, thereby activating T cells to kill TROP-2 positive cells in a targeted manner. expressed in tumor cells.
  • the present invention also aims to provide a polynucleotide molecule encoding the bispecific antibody; to provide an expression vector containing the molecule; to provide a host cell containing the expression vector; to provide a method for preparing the bispecific antibody; Provide a pharmaceutical composition comprising the bispecific antibody; provide an immunoconjugate comprising the bispecific antibody; provide the bispecific antibody or the pharmaceutical composition in the preparation of drugs for treating cancer or tumors.
  • a first aspect of the invention provides a bispecific antibody, the bispecific antibody comprising a first antigen binding domain D1 and a second antigen binding domain D2, where D1 is an anti-TROP-2 antibody or its Antigen-binding fragment, the D2 is an anti-CD3 antibody or an antigen-binding fragment thereof, the anti-TROP-2 antibody or anti-CD3 antibody or their respective antigen-binding fragments comprise the heavy chain complementarity determining region HCDR1-3 and the light chain complementarity determining region LCDR1 -3.
  • the bispecific antibody comprises a monomer or a dimer or multimer formed of monomers.
  • the dimerization The monomers may be homologous or heterologous and comprise from N-terminus to C-terminus a structure selected from any of the following groups:
  • VL A represents the light chain variable region of an anti-TROP-2 antibody or its antigen-binding fragment
  • VH A represents the heavy chain variable region of an anti-TROP-2 antibody or its antigen-binding fragment
  • VL B represents the light chain variable region of an anti-CD3 antibody or its antigen-binding fragment
  • VH B represents the heavy chain variable region of an anti-CD3 antibody or its antigen-binding fragment
  • CH represents the heavy chain constant region
  • CL represents the light chain constant region
  • L1, L2, and L3 are each independently a key or connector
  • ⁇ represents disulfide bond or covalent bond
  • the bispecific antibody comprises a structure selected from any of the following groups:
  • the anti-CD3 antibody or antigen-binding fragment thereof includes a heavy chain complementarity determining region HCDR1-3 and a light chain complementarity determining region LCDR1-3, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 are respectively as SEQ ID NO. .7.
  • the anti-TROP-2 antibody or antigen-binding fragment thereof includes a heavy chain complementarity determining region HCDR1-3 and a light chain complementarity determining region LCDR1-3, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 are as SEQ ID NO.1 and SEQ ID respectively.
  • NO.2 and SEQ ID NO.3 are shown, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.
  • the complementarity determining region contains at least one amino acid mutation.
  • the mutation is used to increase the expression level of the bispecific antibody.
  • the mutation is used to reduce the affinity of the bispecific antibody to human CD3.
  • the mutation is selected from any one or more of the following:
  • X1 is not T
  • X2 is not Y
  • X3 is not I
  • X4 is not G.
  • the amino acid mutation is selected from the group consisting of X1 being G, X2 being S, H or G, X3 being D or E, and X4 being A.
  • the anti-CD3 antibody or antigen-binding fragment thereof includes a heavy chain complementarity determining region HCDR1-3 and a light chain complementarity determining region LCDR1-3, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NO. .15.
  • SEQ ID NO.8 and SEQ ID NO.9 are shown, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 respectively; and/or, The anti-TROP-2 antibody or antigen-binding fragment thereof
  • the segment includes the heavy chain complementarity determining region HCDR1-3 and the light chain complementarity determining region LCDR1-3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively as SEQ ID NO.13 or SEQ ID NO.14, SEQ ID NO.2, SEQ ID NO.3 is shown, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 respectively.
  • the HCDR1 of the anti-CD3 antibody or its antigen-binding fragment is X1X2AMN, where X1 is G, ID NO.8, SEQ ID NO.9, LCDR1, LCDR2, LCDR3 amino acid sequences are shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 respectively; and/or, the anti-TROP
  • the HCDR1 of -2 antibody or its antigen-binding fragment is X3YWLG, where X3 is D, and the amino acid sequence is shown in SEQ ID NO.13; the amino acid sequences of HCDR2 and HCDR3 are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively,
  • the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 respectively.
  • the HCDR1 of the anti-CD3 antibody or its antigen-binding fragment is X1X2AMN, where X1 is G, ID NO.8, SEQ ID NO.9, LCDR1, LCDR2, LCDR3 amino acid sequences are shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 respectively; and/or, the anti-TROP
  • the HCDR1 of -2 antibody or its antigen-binding fragment is X3YWLG, where X3 is D, and the amino acid sequence is shown in SEQ ID NO.13; the amino acid sequences of HCDR2 and HCDR3 are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively,
  • the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 respectively.
  • the HCDR1 of the anti-CD3 antibody or its antigen-binding fragment is X1X2AMN, where X1 is G, ID NO.8, SEQ ID NO.9, LCDR1, LCDR2, LCDR3 amino acid sequences are shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 respectively; and/or, the anti-TROP
  • the HCDR1 of -2 antibody or its antigen-binding fragment is X3YWLG, where X3 is D, and the amino acid sequence is shown in SEQ ID NO.13; the amino acid sequences of HCDR2 and HCDR3 are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively,
  • the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 respectively.
  • the LCDR2 of the anti-CD3 antibody or its antigen-binding fragment is X4TNKRAP, where , shown in SEQ ID NO.12, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9 respectively; and/or, the anti-TROP-2 antibody or The HCDR1 of its antigen-binding fragment is X3YWLG, where The LCDR3 amino acid sequences are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 respectively.
  • the anti-CD3 antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 22, and the light chain The amino acid sequence of the variable region is shown in SEQ ID NO. 23; and/or the anti-TROP-2 antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region The amino acid sequence is shown in SEQ ID NO.20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.21.
  • variable region contains at least one amino acid mutation.
  • variable region includes an amino acid mutation selected from any one or more of the following:
  • the SEQ ID NO.20 has an amino acid mutation at position 31;
  • the SEQ ID NO.22 has an amino acid mutation at position 30;
  • the SEQ ID NO.22 has an amino acid mutation at position 31;
  • the SEQ ID NO.22 has an amino acid mutation at position 32;
  • the SEQ ID NO. 23 has an amino acid mutation at position 50.
  • variable region of the bispecific antibody contains the mutations shown in 1), 3) and 4);
  • variable region of the bispecific antibody contains the mutations shown in 1), 2), 3) and 4);
  • variable region of the bispecific antibody contains the mutations shown in 1) and 5).
  • variable region includes an amino acid mutation selected from any one or more of the following:
  • the SEQ ID NO.20 has I31D;
  • the SEQ ID NO.20 has I31E;
  • the SEQ ID NO.22 has N30S;
  • the SEQ ID NO.22 has T31G;
  • the SEQ ID NO.22 has Y32S, Y32H, and Y32G;
  • the SEQ ID NO. 23 has G50A.
  • variable region of the bispecific antibody includes the T31G described in d), the Y32S described in e) and the I31D described in a), or the variable region of the bispecific antibody includes d).
  • the anti-CD3 antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 26, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. The sequence is shown in SEQ ID NO.
  • the anti-TROP-2 antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region amino acid sequence is as SEQ ID NO.24 and SEQ ID NO.25 are shown, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.21.
  • variable region of the bispecific antibody includes the N3OS described in c), the T31G described in d), the Y32S described in e) and the I31D mutation described in a), and the bispecific antibody has The variable region includes the N30S described in c), the T31G described in d), the Y32S described in e), and or the I31E mutation described in b);
  • variable region of the bispecific antibody comprises the T31G described in d), the Y32H described in e) and the I31D mutation described in a), or the variable region of the bispecific antibody comprises d) T31G described in, Y32H described in e) and I31E mutation described in b);
  • variable region of the bispecific antibody comprises the T31G described in d), the Y32G described in e) and the I31D mutation described in a), or the variable region of the bispecific antibody comprises d) T31G described in, Y32G described in e) and I31E mutation described in b);
  • variable region of the bispecific antibody comprises the G50A described in f) and the I31D mutation described in a), or the variable region of the bispecific antibody comprises the G50A described in f) and b)
  • the I31E mutation is described in .
  • the anti-TROP-2 or anti-CD3 antigen-binding fragment is selected from Fab, F(ab'), F(ab') 2 , Fv or single-chain Fv (scFv); the anti-TROP- 2 or the anti-CD3 antibody is an IgG antibody.
  • the anti-TROP-2 or anti-CD3 antibody is selected from chimeric antibodies (such as human-mouse chimeric antibodies), murine antibodies or humanized antibodies.
  • the anti-TROP-2 antibody is a low-endocytotic antibody, which is more conducive to sustained effects.
  • the IgG antibody includes a heavy chain constant region and a light chain constant region; more preferably, the heavy chain constant region is selected from human IgG1, human IgG2, human IgG3 or human IgG4, and the light chain The constant region is selected from human Kappa or human Lambda.
  • the IgG antibody comprises an IgG1 heavy chain constant region with an amino acid sequence shown in SEQ ID NO. 51.
  • the IgG antibody comprises a human kappa (Kappa) light chain constant region with an amino acid sequence shown in SEQ ID NO. 52.
  • the constant region of the IgG antibody contains at least one amino acid mutation.
  • the amino acid mutation is a knob-in-hole (KIH) mutation that can promote heterodimerization of two heavy chains; more preferably, the mutation is located in the CH3 region of the constant region.
  • KIH knob-in-hole
  • the D1 is an IgG antibody
  • the D2 is a scFv; more preferably, the D2 is connected to the N-terminal or C-terminal of D1, or between CH1 and CH2 of D1; furthermore Preferably, said D2 is linked to the heavy chain of D1.
  • the D2 contains one, two, three or more anti-CD3 scFvs.
  • the scFv contains a VH-L1-VL structure or a VL-L1-VH structure from the N-terminus to the C-terminus.
  • the anti-TROP-2 antibody is selected from the group consisting of anti-TROP-2-VH-I31D monoclonal antibody and anti-TROP-2-VH-I31E monoclonal antibody.
  • the anti-CD3 antigen-binding fragment is selected from CD3-scFv and CD3-scFv-VH-T31G-Y32S.
  • connection refers to direct connection through a linker or through a peptide bond.
  • amino acid sequences of the linkers L1, L2, and L3 are independently (G4S) n or 4G, and n is selected from 1, 2, 3, 4, 5, and 6.
  • amino acid sequence of the linker L1 is shown in SEQ ID NO. 27.
  • amino acid sequence of the linker L2 is shown in SEQ ID NO. 28.
  • amino acid sequence of the linker L3 is shown in SEQ ID NO. 29.
  • the bispecific antibody is a homodimer or heterodimer.
  • the bispecific antibody is a homodimer, including an anti-TROP-2 IgG antibody and two anti-CD3 scFv, wherein each scFv includes a variable region VH and The variable regions VL, VH and VL are connected through linker L1, and each anti-CD3 scFv is connected to between CH1 and CH2 of anti-TROP-2 immunoglobulin antibody IgG through linkers L2 and L3.
  • the bispecific antibody is a homodimer, including an anti-TROP-2 IgG antibody and two anti-CD3 scFv, wherein each scFv includes a variable region VH and The variable regions VL, VH and VL are connected through linker L1, and each anti-TROP-2 scFv is connected in series with the N-terminus of the anti-TROP-2 immunoglobulin antibody IgG heavy chain through linker L2.
  • the bispecific antibody is a heterodimer, comprising an anti-TROP-2 IgG antibody and an anti-CD3 scFv, wherein the scFv includes a variable region VH and a variable region VL, and VH and VL is connected through linker L1, and the anti-CD3 scFv is connected in series to the N-terminus of any anti-TROP-2 immunoglobulin antibody IgG heavy chain through linker L2.
  • the bispecific antibody comprises a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are selected from any of the following:
  • the amino acid sequence in a) to g) is formed by substituting, deleting or adding one or more amino acid residues, and has both anti-TROP-2 activity and anti-CD3 activity derived from a) to g) of peptides.
  • 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% (such as 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) of anti-TROP-2 activity and 70-100 % anti-CD3 activity.
  • the derivative of the bispecific antibody is that the bispecific antibody undergoes one or several amino acid mutations (after amino acid deletion, insertion and/or substitution) and maintains at least 85%, 86%, Polypeptides with 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.
  • the amino acid mutation is a conservative amino acid substitution.
  • the amino acid mutation is located in the framework region or constant region.
  • a second aspect of the invention provides a polynucleotide molecule encoding said bispecific antibody.
  • the polynucleotide molecule includes a polynucleotide molecule encoding the bispecific antibody heavy chain, with a nucleotide sequence such as SEQ ID NO. 43, SEQ ID NO. 44 or SEQ ID NO. As shown in 45; and, the polynucleotide molecule encoding the light chain of the bispecific antibody, the nucleotide sequence is shown in SEQ ID NO. 50.
  • the polynucleotide molecule includes a polynucleotide molecule encoding the bispecific antibody heavy chain, and the nucleotide sequence is such as SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48 or as shown in SEQ ID NO.49; and, the polynucleotide molecule encoding the light chain of the bispecific antibody, the nucleotide sequence is as shown in SEQ ID NO.50.
  • a third aspect of the invention provides an expression vector containing the polynucleotide molecule.
  • 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 fourth aspect of the present invention provides a cell containing the expression vector.
  • the cells are selected from any of the following: COS, CHO, 293F, 293E, NSO, sf9, sf21, DH5 ⁇ , BL21 (DE3) or TG1, preferably E.coli TG1, BL21 ( DE3) cells (expressing single chain antibodies or Fab antibodies) Or CHO-K1 cells (expressing full-length IgG antibodies).
  • a fifth aspect of the present invention provides a method for preparing the above-mentioned bispecific antibody, which method includes the following steps:
  • the sixth aspect of the present invention provides a pharmaceutical composition, which contains an effective amount of the above-mentioned bispecific antibody and one or more pharmaceutically acceptable carriers, diluents or excipients, etc. .
  • the pharmaceutical composition includes the above-mentioned bispecific antibody, acetate, trehalose, arginine hydrochloride or Tween, etc.
  • the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form or a parenteral dosage form.
  • the parenteral dosage form includes intravitreal injection, intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection or intracavity Injections etc.
  • the seventh aspect of the present invention provides the use of the above-mentioned bispecific antibody or pharmaceutical composition in the preparation of drugs for cancer or tumors.
  • the cancer or tumor is a TROP-2 positive cancer or tumor.
  • the cancer or tumor is selected from: lung cancer, bone cancer, gastric cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular 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 lymphoma, non-Hodgkin lymphoma, soft tissue Sarcoma, pediatric solid tumors, lymphocytic lymphoma, central nervous system tumors, primary central nervous system lymphoma, spine tumors, brainstem glioma, pitu
  • An eighth aspect of the present invention provides an immunoconjugate, the immunoconjugate comprising:
  • the conjugate part is selected from any one or more of the following: detectable markers, drugs, toxins, cytokines, radionuclides or enzymes, etc.
  • the conjugate moiety is selected from: fluorescent or luminescent markers, radioactive markers, contrast agents for magnetic resonance imaging or computerized X-ray tomography, or enzymes capable of producing detectable products, Radionuclides, biotoxins or cytokines, etc.
  • the immunoconjugate includes an antibody-drug conjugate.
  • the immunoconjugate is used to prepare a pharmaceutical composition for treating tumors or cancer.
  • a ninth aspect of the present invention provides a method for treating cancer or tumors, the method comprising administering to a subject in need the bispecific antibody of the first aspect of the present invention, the sixth aspect of the present invention.
  • the method further includes combined administration with other anti-tumor drugs.
  • the positive and progressive effects of the anti-TROP-2/CD3 bispecific antibody of the present invention are: (1) it can modulate T cells are immune active and specifically bind to tumor cells expressing TROP-2, thereby inducing immune cells to target and kill TROP-2-positive tumor cells. In particular, its tumor cell killing effect and ability to stimulate immune cells to release IL-2 cytokines are better than the control anti-CD3 monoclonal antibody; (2) high specificity and good safety; (3) high expression amount; (4) stable structure.
  • Figure 1A shows a schematic structural diagram of anti-TROP-2/CD3 double antibodies a and b.
  • Figure 1B shows a schematic structural diagram of anti-TROP-2/CD3 bis-antibody c.
  • Figure 2A shows the UPLC detection pattern of anti-TROP-2/CD3 double antibody a.
  • Figure 2B shows the UPLC detection pattern of anti-TROP-2/CD3 double antibody b.
  • Figure 2C shows the UPLC detection pattern of anti-TROP-2/CD3 double antibody c.
  • Figure 2D shows the UPLC detection pattern of anti-TROP-2/CD3 double antibody b 1 .
  • Figure 2E shows the UPLC detection pattern of anti-TROP-2/CD3 double antibody b 2 .
  • Figure 2F shows the UPLC detection pattern of anti-TROP-2/CD3 double antibody b 3 .
  • Figure 2G shows the UPLC detection pattern of anti-TROP-2/CD3 double antibody b 4 .
  • Figure 3A shows the ELISA detection of the binding of anti-TROP-2/CD3 double antibodies a and c to TROP-2.
  • Figure 3B shows the ELISA detection of the binding of anti-TROP-2/CD3 double antibodies a, b and c to CD3.
  • Figure 3C shows the ELISA detection of the binding of different mutations of anti-TROP-2/CD3 dual antibodies to CD3.
  • Figure 4 shows the kinetic characteristic parameter map of anti-TROP-2/CD3 double antibody.
  • Figure 5A shows the killing effect of hPBMC on different target cells under the action of anti-TROP-2/CD3 double antibody b.
  • Figure 5B shows the killing effect of hPBMC on Colo205 cells under the action of anti-TROP-2/CD3 double antibodies.
  • Figure 5C shows the killing effect of hPBMC on Colo205 cells under the action of different mutations of anti-TROP-2/CD3 double antibodies.
  • Figure 6A shows the IL-2 content released by hPBMC during the killing of target cells under the action of anti-TROP-2/CD3 double antibodies.
  • Figure 6B shows the IFN- ⁇ content released during the killing of target cells by hPBMC under the action of anti-TROP-2/CD3 double antibodies.
  • Figure 6C shows the content of TNF- ⁇ released during the killing of target cells by hPBMC under the action of anti-TROP-2/CD3 double antibodies.
  • Figure 6D shows the IFN- ⁇ content released by hPBMC during the killing of target cells under the action of different mutations of anti-TROP-2/CD3 double antibodies.
  • Figure 7A shows the anti-tumor effect of anti-TROP-2/CD3 dual antibody b on the NCI-H292 transplanted tumor model.
  • Figure 7B shows the toxic side effects of anti-TROP-2/CD3 double antibody b on mice in the NCI-H292 transplanted tumor model.
  • Figure 7C shows the anti-tumor effects of anti-TROP-2/CD3 dual antibodies with different mutations on the NCI-H292 transplanted tumor model.
  • Figure 7D shows the cytokine content in mouse serum under the action of anti-TROP-2/CD3 double antibodies.
  • Figure 8A shows the HPLC detection pattern of anti-TROP-2/CD3 double antibody a treated at 25°C for 28 days.
  • Figure 8B shows the HPLC detection pattern of anti-TROP-2/CD3 double antibody a treated at 37°C for 28 days.
  • the bispecific antibodies of the present invention can be used to achieve T cell-mediated immune responses, especially to effectively promote tumors expressing TROP-2. T cell-mediated killing of cells, thereby effectively suppressing tumors. Unexpectedly, its tumor cell killing effect and ability to stimulate immune cells to release IL-2 cytokines were better than the control anti-CD3 monoclonal antibody, and it was safe. Therefore, the bispecific antibody of the present invention can be developed as an anti-tumor drug with superior efficacy. On this basis, the present invention was completed.
  • the terms "Antibody (Ab)” and “Immunoglobulin G (IgG)” are heterotetrameric proteins with the same structural characteristics, which consist of two identical light chains (L) and Composed of 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).
  • 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.
  • the "immunoglobulin antibody IgG" described in the present invention is about 150kDa and consists of four peptide chains, including two identical gamma heavy chains of approximately 50kDa and two identical light chains of approximately 25kDa, thus having a tetramer structure. level 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, Fab' fragment, F(ab') 2 fragment, or a single Fv fragment, etc., which has antigen-binding activity.
  • antigen-binding fragments include: (i) Fab fragments; (ii) F(ab') 2 fragments; (iii) Fv fragments; or (iv) single chain Fv (scFv).
  • 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, dual Somatic antibodies, tribody antibodies, tetrabody antibodies, microbodies, nanobodies (such as monovalent nanobodies and bivalent nanobodies, etc.), small module immune drugs and shark variable IgNAR domains, etc.
  • the terms "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 antibody is obtained by pepsin digestion of the entire IgG antibody, removing most of the Fc region while leaving some hinge regions intact, and has two antigen-binding F( ab') part.
  • scFv single chain antibody
  • scFv single chain antibody fragment
  • VH and VL are connected through a linker containing 15-25 amino acids, wherein the fusion protein retains the same antigen specificity of the intact immunoglobulin.
  • the term "Fv fragment” or "Fv antibody” is the smallest antibody fragment that contains the heavy chain variable region and the light chain variable region of an antibody, but no constant region, and 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 held closely together by the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pp. 647-669 (1991)).
  • frame region FR refers to the amino acid sequences inserted between the CDRs, ie, those portions of the light and heavy chain variable regions of an immunoglobulin that are relatively conserved among different immunoglobulins within a single species.
  • 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%. Knowing the amino acid sequence of the CDR, those skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, and FR4-H.
  • the term "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, L2 and L3, where L1 connects the VH and VL of the single chain antibody (scFv), L2 and L3 connect the scFv to the CH1 and CH2 of the antibody heavy chain, or directly Connected to the heavy chain of the antibody through L2 (such as the N-terminus or C-terminus of the antibody heavy chain).
  • suitable linkers include single glycine (Gly) or serine (Ser) 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 the 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 10-7M, such as less than about 10-8M, 10-9M, 10-10M, 10-11M, or less.
  • 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 or a region of an antigen that is bound by an antibody.
  • the bispecific antibody of the present invention is a bispecific antibody that can specifically bind to TROP-2 and CD3, and it contains an anti-CD3 antibody part and an anti-TROP-2 antibody part.
  • the bispecific antibodies of the present invention can be dimers, trimers or multimers, preferably homo- or heterodimers.
  • the anti-CD3 or anti-TROP-2 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 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, more preferably at most 5, and most preferably at most 5.
  • the sequence of the present invention adopts the Kabat system numbering rule.
  • 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 the two antigens TROP-2 and CD3 respectively.
  • 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 polynucleotide molecule of the present invention is a conventional preparation method in the art, and preferably includes the following preparation method: through gene cloning technology such as PCR method, etc., obtain the polynucleotide molecule encoding the above-mentioned monoclonal antibody, or by The polynucleotide molecule encoding the above-mentioned monoclonal antibody is 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 homolog.
  • Polynucleotides of the present invention The system 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 polynucleotide 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 cell containing the above-mentioned expression vector.
  • the cells of the present invention are obtained by transforming the expression vector into host cells.
  • the host cell can be any conventional host cell in the art, as long as it can make the above-mentioned recombinant expression vector stably replicate itself, and the carried nucleotide 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 cell culture method and the antibody separation and purification method of the present invention are conventional methods in this field.
  • For specific operation methods please refer to the corresponding cell culture technical manual and antibody separation and purification technical manual.
  • the method for preparing anti-TROP-2/CD3 bispecific antibodies disclosed in the present invention includes: cultivating the above-mentioned cells under expression conditions to express bispecific antibodies that can specifically bind to TROP-2 and CD3; and isolating and The anti-TROP-2/CD3 bispecific antibody was purified. Using the above methods, the recombinant protein can be purified into a substantially homogeneous material.
  • the anti-TROP-2/CD3 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. Method: Elute the anti-TROP-2/CD3 bispecific antibody bound to the affinity column. The inventor of the present invention conducted detection experiments on the obtained anti-TROP-2/CD3 bispecific antibodies. The experimental results showed that the anti-TROP-2/CD3 bispecific antibodies can bind to target cells and antigens well and have high affinity.
  • compositions preferably said composition is a pharmaceutical composition.
  • 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).
  • these materials may be formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5.0-8.0, preferably at a pH of about 6.0-8.0, although the pH may vary.
  • the nature of the substance to be formulated and the disease to be treated Changes due to symptoms.
  • 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.
  • bispecific antibody preparation can be suspension, water injection, freeze-drying and other preparations commonly used in the pharmaceutical field.
  • 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 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.
  • the safe and effective dose is usually at least about 10 micrograms/kg of body weight, and in most cases does not exceed about 50 mg/kg of body weight.
  • the dose is about 10 micrograms/kg of body weight to 10 mg/kg of 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.
  • Another aspect of the present invention provides the use of the above-mentioned bispecific antibody that can specifically bind to TROP-2 and CD3, or the above-mentioned pharmaceutical composition in the preparation of medicines, and the medicines are used to treat cancer or tumors.
  • the drugs used to treat cancer or tumors 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 existing Cancer is accompanied by the reduction of symptoms and prevention of the occurrence of other symptoms, including 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, prostate cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, fallopian tube cancer, intrauterine cancer, Membrane 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 lymphoma, non-Hodgkin lymphoma , soft tissue sarcoma, childhood solid tumors, lymphocytic lymphoma, central nervous system tumors, primary central nervous system lymphoma, spine
  • 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 dose cannot exceed a certain range. Specifically, the intravenous dosage 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 acids Metabolized drugs: dihydrofolate reductase inhibitors such as methotrexate and pemetrexed (Pemetrexed), etc.; thymidine synthase inhibitors such as fluorou
  • Hormone drugs anti-estrogens such as tamoxifen, droloxifene, exemestane ( Exemestane), etc.; aromatase inhibitors such as Aminoglutethimide, Formestane, Letrozle, Anastrozole, etc.; anti-androgens: flutamide RH-LH Agonists/antagonists: Noride, Enaton, etc.; 3.
  • Biological response modifier drugs These drugs mainly achieve anti-tumor effects by regulating the body’s immune function, such as interferons (Interferon), leukocyte intermediaries Interleukin-2, Thymosins, etc.; 4.
  • Monoclonal antibody drugs Trastuzumab, Rituximab, Cetuximab , Bevacizumab, 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.
  • Competent cells Brand Shenggon, Cat. No. B528412.
  • 293F cells Brand GIBCO, Cat. No. R79007.
  • Colo205 cells Cell Bank of the Chinese Academy of Sciences, catalog number TCHu102.
  • MDA-MB-231 cells Cell Bank of Chinese Academy of Sciences, catalog number TCHu227.
  • A549 cells purchased from ATCC.
  • Human lung cancer cell NCI-H292 Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-582.
  • hPBMC purchased from Aucells, product number FPB004F-C-MLR.
  • 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.
  • Coating solution 1.59g sodium carbonate, 2.93g sodium bicarbonate, add double distilled water to make the volume to 1L.
  • ELISA blocking solution PBST+1% BSA.
  • BSA Brand Shenggong, item number A60332.
  • TROP-2-HIS protein brand Kaika, product number TRP-HM121.
  • CD3-FC protein brand Kaika, product number CD3-HM205.
  • HRP-labeled goat anti-human FC antibody Brand Boaolong, Cat. No. BF03031.
  • HRP-labeled goat anti-human Fab antibody Brand Thermo Fisher, Cat. No. A0293.
  • TMB Brand BD Biosciences, Cat. No. 555214.
  • Stop solution 2M sulfuric acid solution.
  • Trypsin-EDTA Brand GIBCO, Cat. No. 25200-072.
  • 1640 complete medium RPMI1640 medium + 10% FBS + 1% Pen Strep + 1% sodium pyruvate.
  • RPMI1640 culture medium brand GIBCO, item number 22400089.
  • FBS Brand GIBCO, item number 10099-141.
  • Pen Strep Brand GIBCO, item number 1514022.
  • DMEM complete medium DMEM high sugar medium + 10% FBS + 1% Pen Strep + 1% GLUMAX.
  • DMEM high sugar medium brand GIBCO, product number 11965-092.
  • GlutaMAX Brand GIBCO, Cat. No. 35050-061.
  • Luminescent Cell Viability Assay Purchased from Promega, Cat. No. G7572.
  • PurifiedMouse Anti-Human IL-2 Purchased from BD Pharmingen, Cat. No. 55051.
  • Biotin Mouse Anti-Human IL-2 Purchased from BD Pharmingen, Cat. No. 555040.
  • Recombinant Human IL-2 Purchased from BD Pharmingen, Cat. No. 554603.
  • Streptavidin HRP Purchased from BD Biosciences, Cat. No. 554066.
  • HiLoad 26/600Superdex 200pg column purchased from Cytiva.
  • SpectraMax i3x microplate reader purchased from Molecular Devices.
  • SpectraMaxM5 microplate reader purchased from Molecular Devices.
  • the present invention uses anti-TROP-2 monoclonal antibody IgG and anti-CD3 monoclonal antibody scFv in series to construct anti-TROP-2/CD3 bispecific antibodies a, b and c.
  • the structures of a and b are shown in Figure 1A.
  • the c structure is shown in Figure 1B.
  • the anti-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.
  • the KM4097HV3aLV0 humanized monoclonal antibody serves as the anti-TROP-2 monoclonal antibody.
  • the CDR sequence of the anti-CD3 monoclonal antibody in the anti-TROP-2/CD3 bispecific antibody is derived from SEQ ID NO.2 and SEQ ID NO.4 in US8236308B2, and has been humanized.
  • the present invention uses modified humanized SP34 as a positive control for anti-CD3 monoclonal antibodies.
  • amino acid sequences of the heavy chain and light chain variable regions of the mouse anti-human CD3 monoclonal antibody are respectively derived from SEQ ID NO.2 and SEQ ID NO.4 in US8236308B2.
  • the amino acid sequences of the heavy chain and light chain variable regions of mSP34 were analyzed, and the complementarity determining region CDR and framework region FR of mSP34 heavy chain and light chain were determined according to Kabat rules.
  • the amino acid sequences of the mSP34 heavy chain CDR are HCDR1: TYAMN (SEQ ID NO.7), HCDR2: RIRSKYNNYATYYADSVKD (SEQ ID NO.8) and HCDR3: HGNFGNSYVSWFAY (SEQ ID NO.9).
  • the amino acid sequence of the light chain CDR is LCDR1: RSSTGAVTTSNYAN (SEQ ID NO.10), LCDR2: GTNKRAP (SEQ ID NO.11) and LCDR3: ALWYSNLWV (SEQ ID NO.12).
  • the homology between the heavy chain variable region of mSP34 and the human IgG germline sequence was compared, and IGHV3-23*04 was selected as the heavy chain CDR transplantation template.
  • IGLV7-46*01 was selected as the light chain CDR transplantation template, and the light chain CDR of SP34 was transplanted into the backbone of IGLV7-46*01. region, and add FGQGTKVEIK as the fourth framework region after LCDR3 to obtain the CDR grafted light chain variable region sequence.
  • FGQGTKVEIK as the fourth framework region after LCDR3 to obtain the CDR grafted light chain variable region sequence.
  • some amino acid sites are mutated. When performing mutations, the amino acid sequence is Kabat coded, and the position of the site is indicated by the Kabat code.
  • the S at position 49 is mutated to A
  • the N at position 73 is mutated to D
  • the A at position 93 is mutated to V
  • the K at position 94 is mutated to R.
  • the above-mentioned heavy chain and light chain variable regions with backmutation sites are respectively defined as humanized SP34 heavy chain and light chain variable regions (SEQ ID NO. 18 and SEQ ID NO. 19).
  • the synthesized humanized heavy chain variable region is connected to the coding gene of the human IgG1 heavy chain constant region (SEQ ID NO. 39) to obtain the full-length humanized heavy chain gene; the humanized light chain variable region is Connected to the gene encoding the human Kappa chain constant region (SEQ ID NO. 40) to obtain the full-length humanized light chain gene.
  • the positive control monoclonal antibody was finally obtained by referring to the expression and purification method in Example 2.
  • Anti-TROP-2 heavy chain variable region VH (SEQ ID NO.20) amino acid I31D site mutation, to obtain anti-TROP-2 heavy chain variable region VH-I31D (SEQ ID NO.24), thereby obtaining anti-TROP- 2 heavy chain HC-I31D (SEQ ID NO.32), the light chain of the anti-TROP-2 monoclonal antibody (SEQ ID NO.31) remained unchanged, and the anti-TROP-2-VH-I31D monoclonal antibody was obtained.
  • Anti-TROP-2 heavy chain variable region VH (SEQ ID NO.20) amino acid I31E site mutation, to obtain anti-TROP-2 heavy chain variable region VH-I31E (SEQ ID NO.25), thereby obtaining anti-TROP- 2 heavy chain HC-I31E (SEQ ID NO.33), the light chain of the anti-TROP-2 monoclonal antibody (SEQ ID NO.31) remained unchanged, and the anti-TROP-2-VH-I31E monoclonal antibody was obtained.
  • the anti-CD3 heavy chain variable region VH-T31G-Y32S (SEQ ID NO.26) was obtained by mutating the amino acid T31G and Y32S sites of the anti-CD3 heavy chain variable region VH (SEQ ID NO.22).
  • the scFv in anti-TROP-2/CD3 bispecific antibodies a and c is VH-L1-VL, which is connected to the anti-CD3 heavy chain variable region VH (SEQ ID NO.22) and Anti-CD3 light chain variable region VL (SEQ ID NO.23) is obtained, namely CD3-scFv (SEQ ID NO.34).
  • the scFv in the anti-TROP-2/CD3 bispecific antibody b is VH-T31G-Y32S-L1-VL, which is connected to the anti-CD3 heavy chain variable region through L1 (SEQ ID NO. 27) VH-T31G-Y32S (SEQ ID NO.26) and anti-CD3 light chain variable region VL (SEQ ID NO.23), namely CD3-scFv-VH-T31G-Y32S (SEQ ID NO.35).
  • the CD3-scFv of anti-TROP-2/CD3 bispecific antibodies a and b is linked to the anti-TROP-2 heavy chain HC-I31D (SEQ ID NO. .32) Between CH1 and CH2, obtain the heavy chain of anti-TROP-2/CD3 double antibody a (SEQ ID NO.36) and the heavy chain of anti-TROP-2/CD3 double antibody b (SEQ ID NO.37), The light chain of the anti-TROP-2 monoclonal antibody (SEQ ID NO. 31) remains unchanged.
  • the CD3-scFv of anti-TROP-2/CD3 bispecific antibody c is connected to the N-terminus of anti-TROP-2 heavy chain HC-I31D (SEQ ID NO.32) through L2 (SEQ ID NO.28) to obtain anti-TROP-
  • the heavy chain of 2/CD3 double anti-c (SEQ ID NO.38) and the light chain of anti-TROP-2 monoclonal antibody (SEQ ID NO.31) remain unchanged.
  • Anti-CD3 heavy chain variable region VH (SEQ ID NO.22) amino acid N30S, T31G and Y32S site mutations, to obtain anti-CD3 heavy chain variable region VH-N30S-T31G-Y32S;
  • Anti-CD3 heavy chain variable region VH (SEQ ID NO.22) amino acid T31G and Y32H site mutations, to obtain anti-CD3 heavy chain variable region VH-T31G-Y32H;
  • Anti-CD3 heavy chain variable region VH (SEQ ID NO.22) amino acid T31G and Y32G site mutations, to obtain anti-CD3 heavy chain variable region VH-T31G-Y32G;
  • the anti-CD3 light chain variable region VL-G50A was obtained by mutating the amino acid G50A site of the anti-CD3 light chain variable region VL (SEQ ID NO. 23).
  • the anti-CD3 light chain variable region VL (SEQ ID NO.23) is combined with the anti-CD3 heavy chain variable region VH-N30S-T31G-Y32S, VH-T31G-Y32H and VH- through L1 (SEQ ID NO.27) respectively.
  • T31G-Y32G is connected to obtain VH-N30S-T31G-Y32S-L1-VL(CD3-scFv-VH-N30S-T31G-Y32S), VH-T31G-Y32H-L1-VL(CD3-scFv-VH-T31G-Y32H ) and VH-T31G-Y32G-L1-VL (CD3-scFv-VH-T31G-Y32G); combining the anti-CD3 heavy chain variable region VH (SEQ ID NO.22) with the anti-CD3 heavy chain variable region VH (SEQ ID NO.22) through L1 (SEQ ID NO.27) CD3 light chain variable region VL-G50A was connected to obtain VH-L1-VL-G50A (CD3-scFv-VL-G50A).
  • CD3-scFv-VH-N30S-T31G-Y32S, CD3-scFv-VH-T31G-Y32H, CD3-scFv-VH-T31G-Y32G through L2 (SEQ ID NO.28) and L3 (SEQ ID NO.29) and CD3-scFv-VL-G50A were sequentially connected to the anti-TROP-2 heavy chain HC-I31D (SEQ ID NO.
  • the DNA fragments of the heavy chain and light chain of the anti-TROP-2/CD3 bispecific antibody 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 then loaded into a Hitrap Mabselect Sure affinity chromatography column. The protein is eluted in one step with 100mM citric acid and a pH 3.5 eluent. The target sample was recovered and dialyzed into PBS with pH 7.4, and the purified protein was detected by UPLC-SEC.
  • the detection results of anti-TROP-2/CD3 double antibodies a, b, c, b 1 -b 4 molecules are shown in Figure 2A, Figure 2B and Figure 2C, and Figure 2D- Figure 2G.
  • the antibody molecules are in a uniform state and are monomers. The purity reaches more than 95%.
  • Remove the blocking solution (use absorbent paper to remove residual droplets), then dilute the TROP-2/CD3 bispecific antibody and anti-TROP-2 monoclonal antibody to 10 ⁇ g/ml with the blocking solution, and dilute four times to form 12 concentration gradients (the highest concentration 10 ⁇ g/ml, minimum concentration 0.002ng/ml), add 100 ⁇ l/well to the blocked enzyme plate in sequence, and leave it at room temperature for 1 hour.
  • Wash the plate three times with PBST use absorbent paper to remove residual droplets
  • dilute the HRP-labeled goat anti-human FC antibody 1:3000 with blocking solution add 100 ⁇ l/well to the enzyme plate, and leave it at room temperature for 30 minutes.
  • the test results are shown in Figure 3A.
  • the EC 50 (unit: nM) of the anti-TROP-2/CD3 bispecific antibody molecules a and c and the positive control anti-TROP-2 monoclonal antibody binding to TROP-2-HIS are 0.01649 and 0.01649, respectively.
  • TROP-2/CD3 bispecific antibody and anti-CD3 monoclonal antibody to 10 ⁇ g/ml, four-fold dilution to form 12 concentration gradients (the highest concentration is 10 ⁇ g/ml, the lowest concentration is 0.002ng/ml), and 100 ⁇ l/well is added to the blocking solution in sequence. Place in the ELISA plate at room temperature for 1 hour. Wash the plate three times with PBST (use absorbent paper to remove residual droplets), dilute the HRP-labeled goat anti-human Fab antibody 1:3000 with blocking solution, add 100 ⁇ l/well to the enzyme plate, and leave it at room temperature for 30 minutes.
  • PBST use absorbent paper to remove residual droplets
  • the test results are shown in Figure 3B.
  • the EC 50 (unit: nM) of the anti-TROP-2/CD3 bispecific antibody molecules a, b, and c and the positive control anti-CD3 monoclonal antibody binding to CD3-FC are 0.07924, 0.6071, and 0.6071, respectively. 0.04141 and 0.06182. It shows that CD3-scFv is inserted between CH1 and CH2 of the heavy chain of the anti-TROP-2 monoclonal antibody, which slightly reduces the binding ability of the bispecific antibody to CD3 antigen and is effective against the variable region of the CD3 heavy chain on the basis of the bispecific antibody a. Mutation of the amino acids of VH significantly reduces the binding ability of the bispecific antibody to the CD3 antigen.
  • Example 4 Fortibio determines the affinity of anti-TROP-2/CD3 double antibodies to CD3 antigen
  • the Fortebio Octet Molecular Interaction Instrument and Octet AR2G Biosensors probe were used to determine the kinetic parameters of anti-TROP-2/CD3 bispecific antibody and antigen CD3-Fc binding.
  • the antigen-antibody binding and dissociation rates are measured.
  • the kinetic parameters of the combination of anti-TROP-2/CD3 double antibodies and CD3-Fc are shown in Table 1, and the kinetic characteristic parameter map is shown in Figure 4.
  • the results show that the anti-TROP-2/CD3 double antibodies a, b 1 and b 4 have higher affinity to CD3-Fc, while the anti-TROP-2/CD3 double antibodies b have a weaker affinity to CD3-Fc.
  • /CD3 double antibody b 3 has the weakest affinity with CD3-Fc.
  • Example 5 Experiment on killing tumor cells by hPBMC under the action of double antibodies
  • Colo205 cells human colon cancer cells
  • MDA-MB-231 cells human breast cancer cells
  • A549 cells human lung adenocarcinoma cells
  • Colo205 cells human colon cancer cells
  • MDA-MB-231 cells human breast cancer cells
  • A549 cells human lung adenocarcinoma cells
  • hPBMC peripheral blood mononuclear cells
  • the concentration and dilution factor of adding antibodies to different tumor cells are different: dilute anti-TROP-2/CD3 double antibody b with complete culture medium to 1 ⁇ g/ml, and 6-fold gradient dilution to form 10 concentration gradients (the highest concentration is 1 ⁇ g/ml, the lowest concentration is 9.92 ⁇ 10 -8 ⁇ g/ml), add 50 ⁇ l per well to a white transparent 96-well plate containing Colo205 cells and hPBMC cells, place it in a 37°C and 5% CO 2 incubator for 48 hours; dilute with complete culture medium Anti-TROP-2/CD3 double antibody b to 20 ⁇ g/ml, diluted 4 times to form 10 concentration gradients (the highest concentration is 20 ⁇ g/ml, the lowest concentration is 7.63 ⁇ 10 -5 ⁇ g/ml), and 50 ⁇ l per well is added to the solution containing MDA.
  • tumor cells with different expression levels of TROP-2 protein were selected. Both Colo205 cells and MDA-MB-231 cells can express TROP-2 protein, and The expression of TROP-2 in Colo205 cells was higher than that in MDA-MB-231 cells, and A549 cells hardly expressed TROP-2 protein.
  • the test results are shown in Figure 5A.
  • the anti-TROP-2/CD3 double antibody b has the most obvious killing effect on Colo205 cells, and also has a certain effect on MDA-MB-231 cells. It also has a certain effect on A549 cells that do not express TROP-2 protein. There is no killing effect, indicating that anti-TROP-2/CD3 dual antibodies can be positioned on target cells to ensure that normal human tissues will not be killed by the T cell killing effect induced by anti-TROP-2/CD3 dual antibodies.
  • Colo205 cells with relatively high TROP-2 expression were selected as target cells.
  • the experimental steps and reaction conditions were the same as above.
  • the experiment The results are shown in Figure 5B.
  • the anti-TROP-2/CD3 double antibody b after T31G and Y32S site mutations has a lower killing effect on target cells than the anti-TROP-2/CD3 double antibody a, but both are higher than the anti-CD3 single antibody. anti.
  • Colo205 cells with relatively high TROP-2 expression were selected as target cells.
  • the experimental steps were the same as The reaction conditions are the same as above.
  • the test results are shown in Figure 5C.
  • the killing effect of anti-TROP-2/CD3 double antibodies b 1 , b 2 and b 4 on Colo205 cells is more obvious, and the killing effect of anti-TROP-2/CD3 double antibodies b is more obvious.
  • the effect is intermediate, and the killing effect of anti-TROP-2/CD3 double anti-b 3 is relatively weak.
  • hPBMC cells Take fresh hPBMC cells, dilute the cells with complete culture medium to 1.2 ⁇ 10 6 cells/ml, and add 50 ⁇ l per well into a white transparent 96-well plate. At the same time, set up a control well with only hPBMC cells and no tumor cells.
  • a concentration gradient (the highest concentration is 1 ⁇ g/ml, the lowest concentration is 9.92 ⁇ 10 -8 ⁇ g/ml)
  • 50 ⁇ l per well is added to a white transparent 96-well plate containing Colo205 cells and hPBMC cells, placed at 37°C and 5% CO 2 incubator for 48 hours.
  • the supernatant was collected, and the contents of IL-2, INF- ⁇ and TNF- ⁇ in the supernatant were detected by ELISA.
  • PBST containing 1% BSA blocking solution
  • anti-CD3 monoclonal antibodies with almost no killing effect will also stimulate immune cells to release small amounts of cytokines such as INF- ⁇ and TNF- ⁇ when the concentration accumulates to a certain amount, indicating that anti-TROP-2/
  • the killing effect of CD3 double antibodies on tumor cells and the impact on the autoimmune system is better than that of anti-CD3 monoclonal antibodies, and the killing effect of anti-TROP-2/CD3 double antibodies b is better than that of anti-CD3 monoclonal antibodies, but it releases relatively less Cytokines.
  • anti-TROP-2/CD3 dual antibodies cooperated with hPBMC to kill tumor cells, stimulating immune cells to release the content of cytokine INF- ⁇ .
  • the experimental results are shown in Figure 6D showed that the anti-TROP-2/CD3 double antibodies b 1 , b 2 and b 4 stimulated the highest content of cytokine INF- ⁇ , while the anti-TROP-2/CD3 double antibody b stimulated a relatively small amount of cytokine INF- ⁇ .
  • the anti-TROP-2/CD3 double antibody b 3 hardly stimulated immune cells to release cytokines.
  • the killing effect of the anti-TROP-2/CD3 double antibody on tumor cells was proportional to the content of stimulated cytokines.
  • the concentration of anti-TROP-2/CD3 double antibody b that induces the release of IL-2 cytokines is much higher than the concentration that causes tumor cell killing.
  • hPBMC Human peripheral blood mononuclear cells hPBMC were used to reconstruct the human immune system in NOG mice, and a human lung cancer NCI-H292 subcutaneous transplant tumor model was established in these mice.
  • the specific implementation steps are as follows:
  • NCI-H292 cells human non-small cell lung cancer
  • cell suspension concentration 1 ⁇ 10 8 cells/ml.
  • Matrigel in an equal ratio of 1:1.
  • Adjust the concentration of hPBMC suspension to 1 ⁇ 10 7 cells/ml.
  • 200 ⁇ l of cell mixture suspension was inoculated subcutaneously on the right upper back of NOG mice.
  • mice When the subcutaneous tumor grows to 150mm3 , the mice are randomly divided into 3 groups according to the tumor volume, with 8 mice in each group, including: blank control group, only injected with PBS as a control; anti-TROP-2/CD3 bispecific antibody
  • the diameter of the transplanted tumors was measured twice a week, and the weight of the mice was measured at the same time.
  • the growth curve of the tumors in each group over time and the weight curve of the mice were drawn.
  • hPBMC Human peripheral blood mononuclear cells hPBMC were used to reconstruct the human immune system in NOG mice, and a human lung cancer NCI-H292 subcutaneous transplant tumor model was established in these mice.
  • the specific implementation steps are as follows:
  • NCI-H292 cells cultured in vitro were collected, the cell suspension concentration was adjusted to 1 ⁇ 10 8 cells/ml, and mixed with Matrigel in an equal ratio of 1:1. Resuscitate hPBMC in vitro and resuspend hPBMC cells in PBS. Adjust the concentration of hPBMC suspension to 1 ⁇ 10 7 cells/ml. Mix the mixed tumor cell suspension and hPBMC suspension at a ratio of 1:1. The right upper back of the NOG mouse was shaved, and 200 ⁇ l of cell suspension was inoculated subcutaneously into the right upper back of the NOG mouse under sterile conditions.
  • mice When the average tumor volume of subcutaneous tumors is approximately 200mm, the mice are randomly divided into 5 groups, with 8 mice in each group, including: blank control group, only injected with PBS as a control; anti-TROP-2/CD3 bispecific antibody
  • the 1 mg/kg dose groups of a, b, b 2 and b 3 were administered by intraperitoneal injection twice a week for four consecutive weeks. During the entire experiment, the diameter of the transplanted tumors was measured twice a week.
  • the growth curve of the tumors in each group over time was drawn with the number of days of administration as the abscissa and the tumor volume as the ordinate.
  • TROP-2/CD3 dual antibodies b 2 The anti-tumor activity of TROP-2/CD3 dual antibodies b 2 is second, and the anti-tumor activity of anti-TROP-2/CD3 dual antibodies b and b 3 is slightly weaker than b 2.
  • the anti-tumor activity of each TROP-2/CD3 dual antibody is There was no significant difference in anti-tumor activity, and the anti-tumor activities were all above 75% on day 17.
  • the levels of cytokines stimulated by anti-TROP-2/CD3 double antibodies a are higher than those of anti-TROP-2/CD3 double antibodies b and b 3 ; anti-TROP-2/CD3 double antibodies Compared with anti-b and b 3 , anti-TROP-2/CD3 double anti-b stimulated higher levels of IL-1a, IL-6, G-CSF and MIP-1b cytokines, but there was no significant difference in other cytokines. , indicating that after the anti-TROP-2/CD3 double anti-a mutation reduces its affinity, it can significantly reduce the production of cytokines in animals while maintaining its anti-tumor activity.
  • This experiment can be used to evaluate protein stability in the presence of excipients, revealing important information needed to develop optimal formulations.
  • Anti-TROP-2/CD3 double antibody a is stored in a buffer with a pH of 5.0 and 20mM acetate + 6% trehalose + 1% arginine hydrochloride + 0.1% Tween 80, and placed at 25°C and 37°C respectively. After 28 days in the incubator, the HPLC-SEC results are shown in Figure 8A and Figure 8B.
  • the bispecific antibody provided by the present invention can bind to TROP-2 and CD3 at the same time, regulate the immune activity of T cells by binding to CD3, and specifically kill tumor cells expressing TROP-2 protein.
  • the results in Figures 3B and 3C show that the ability of the bispecific antibody to bind CD3 is reduced compared with its monoclonal antibody, but the results in Figures 5A and 5B show that the bispecific antibody's killing effect on tumor cells is significantly increased compared with its monoclonal antibody, and the tumor cells The higher the expression level of TROP-2, the stronger the killing effect of the bispecific antibody on tumor cells.
  • bispecific antibody b has a relatively strong killing effect, stimulates less cytokine release, and has a larger medication window.
  • the stronger the bispecific antibody's inhibitory activity on tumor cells the more TH1 and TH2 cytokines were released, which was also positively correlated.

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Abstract

涉及抗体药物技术领域，具体地，涉及一种抗TROP-2/CD3双特异性抗体。抗TROP-2/CD3双特异性抗体包含第一抗原结合结构域D1和第二抗原结合结构域D2，其中D1为抗TROP-2抗体或其抗原结合片段，D2为抗CD3抗体或其抗原结合片段。抗TROP-2/CD3双特异性抗体可用于实现T细胞介导的免疫反应，特别是有效促进针对表达TROP-2的肿瘤细胞的T细胞介导的杀伤，从而有效地抑制肿瘤。

Description

一种抗TROP-2/CD3双特异性抗体 技术领域
本发明涉及抗体药物技术领域，具体地，涉及一种抗TROP-2/CD3双特异性抗体。
背景技术
人滋养层细胞表面抗原2(human trophoblast cell surface antigen 2,TROP-2)是由TACSTD2基因编码表达的细胞表面糖蛋白。TROP-2为单次跨膜的I型膜蛋白，由323个氨基酸构成，其中信号肽26个氨基酸，胞外区248个氨基酸，跨膜区23个氨基酸，胞内区26个氨基酸。截至目前，TROP-2的配体蛋白还没有鉴定到，因此对其生理生化功能还不是十分明确。但是大量的临床研究和文献报道，TROP-2蛋白在各种人类上皮癌中高表达并且与患者的预后不良和癌细胞转移密切相关，包括乳腺癌、肺癌、胃癌、胰腺癌、前列腺癌和宫颈癌等。美国FDA已经批准TROP-2抗体偶联药物赛妥珠单抗-格卫替康(sacituzumab govitecan)用于转移性三阴性乳腺癌的治疗。
分化簇3(cluster ofdifferentiation 3,CD3)是T细胞表面重要的分化抗原，CD3分子与T细胞受体(T cell recptor,TCR)形成TCR-CD3复合体，TCRαβ亚基识别胞外信号并将胞外信号传递给CD3，CD3分子依靠自身免疫酪氨酸信号模体(immunoreceptor tyrosine-based activationmotif,ITAM)将信号向胞内传递。抗CD3抗体可激发或阻断T细胞活化信号，清除效应T细胞或诱导调节T细胞产生。
双特异性抗体(bispecific antibody,BsAb)也称为双功能抗体，是同时靶向两种不同抗原或相同抗原不同表位的特异性药物。BsAb可将免疫细胞和病毒分子等作用于肿瘤细胞，增强对靶细胞的杀伤作用，也可以同时结合肿瘤细胞的不同抗原，增强其结合特异性并降低脱靶效应。双特异性抗体拓宽了抗体药物的应用领域，为肿瘤免疫提供了新的研究思路。
然而，目前本领域尚缺乏令人满意的针对TROP-2和CD3的双特异性抗体。因此，本领域亟待开发一种新的抗TROP-2和CD3的双特异性抗体。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种抗TROP-2/CD3双特异性抗体，用于解决现有技术中的问题。
本发明的目的在于提供一种新的抗TROP-2和CD3的双特异性抗体，该双特异性抗体能同时与TROP-2及CD3特异结合，从而激活T细胞靶向性杀伤TROP-2阳性表达的肿瘤细胞。本发明的目的还在于提供编码所述双特异性抗体的多核苷酸分子；提供包含所述分子的表达载体；提供包含所述表达载体的宿主细胞；提供所述双特异性抗体的制备方法；提供包含所述双特异性抗体的药物组合物；提供包含所述双特异性抗体的免疫偶联物；提供所述双特异性抗体或所述药物组合物在制备治疗癌症或肿瘤的药物中的应用；提供所述融合蛋白、所述药物组合物或所述免疫偶联物治疗癌症或肿瘤的方法。
为了达到上述目的，本发明提供了以下技术方案：
本发明的第一个方面提供了一种双特异性抗体，所述双特异性抗体包含第一抗原结合结构域D1和第二抗原结合结构域D2，所述D1为抗TROP-2抗体或其抗原结合片段，所述D2为抗CD3抗体或其抗原结合片段，所述抗TROP-2抗体或抗CD3抗体或各自的抗原结合片段包含重链互补决定区HCDR1-3和轻链互补决定区LCDR1-3。
在另一优选例中，所述双特异性抗体包含单体或单体形成的二聚体或多聚体。所述二聚 体可以是同源的或异源的，所述单体从N端到C端包含选自以下任一组的结构：
其中，
VLA代表抗TROP-2抗体或其抗原结合片段的轻链可变区；
VHA代表抗TROP-2抗体或其抗原结合片段的重链可变区；
VLB代表抗CD3抗体或其抗原结合片段的轻链可变区；
VHB代表抗CD3抗体或其抗原结合片段的重链可变区；
CH代表重链恒定区；
CL代表轻链恒定区；
L1、L2和L3各自独立地为键或接头；
“～”代表二硫键或共价键；
“-”代表肽键。
在另一优选例中，所述双特异性抗体包含选自以下任一组的结构：
a)结构I的单体形成的同源二聚体；
b)结构II的单体形成的同源二聚体。
在另一优选例中，所述抗CD3抗体或其抗原结合片段包含重链互补决定区HCDR1-3和轻链互补决定区LCDR1-3，其中，HCDR1、HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12所示；和/或，所述抗TROP-2抗体或其抗原结合片段包含重链互补决定区HCDR1-3和轻链互补决定区LCDR1-3，其中，HCDR1、HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示。
在另一优选例中，所述互补决定区包含至少一个氨基酸突变。
在另一优选例中，所述突变用于提高双特异性抗体的表达水平。
在另一优选例中，所述突变用于降低双特异性抗体对人CD3的亲和力。
在另一优选例中，所述突变选自以下任一项或多项：
a)抗CD3抗体或其抗原结合片段的HCDR1：X1X2AMN，
b)抗TROP-2抗体或其抗原结合片段的HCDR1：X3YWLG，
c)抗CD3抗体或其抗原结合片段的LCDR2：X4TNKRAP，
其中，X1不为T、X2不为Y、X3不为I、X4不为G。
在另一优选例中，所述氨基酸突变选自X1为G、X2为S、H或G、X3为D或E、X4为A的组。
在另一优选例中，所述抗CD3抗体或其抗原结合片段包含重链互补决定区HCDR1-3和轻链互补决定区LCDR1-3，其中，HCDR1、HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.15、SEQ ID NO.8、SEQ ID NO.9所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12所示；和/或，所述抗TROP-2抗体或其抗原结合片 段包含重链互补决定区HCDR1-3和轻链互补决定区LCDR1-3，其中，HCDR1、HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.13或SEQ ID NO.14，SEQ ID NO.2，SEQ ID NO.3所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示。
在另一优选例中，所述抗CD3抗体或其抗原结合片段的HCDR1为X1X2AMN，其中X1为G，X2为S，氨基酸序列如SEQ ID NO.16所示；HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.8、SEQ ID NO.9所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12所示；和/或，所述抗TROP-2抗体或其抗原结合片段的HCDR1为X3YWLG，其中X3为D，氨基酸序列如SEQ ID NO.13所示；HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.2，SEQ ID NO.3所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示。
在另一优选例中，所述抗CD3抗体或其抗原结合片段的HCDR1为X1X2AMN，其中X1为G，X2为H，氨基酸序列如SEQ ID NO.17所示；HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.8、SEQ ID NO.9所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12所示；和/或，所述抗TROP-2抗体或其抗原结合片段的HCDR1为X3YWLG，其中X3为D，氨基酸序列如SEQ ID NO.13所示；HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.2、SEQ ID NO.3所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示。
在另一优选例中，所述抗CD3抗体或其抗原结合片段的HCDR1为X1X2AMN，其中X1为G，X2为G，氨基酸序列如SEQ ID NO.18所示；HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.8、SEQ ID NO.9所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12所示；和/或，所述抗TROP-2抗体或其抗原结合片段的HCDR1为X3YWLG，其中X3为D，氨基酸序列如SEQ ID NO.13所示；HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.2、SEQ ID NO.3所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示。
在另一优选例中，所述抗CD3抗体或其抗原结合片段的LCDR2为X4TNKRAP，其中X4为A，氨基酸序列如SEQ ID NO.19所示；LCDR1、LCDR3氨基酸序列分别如SEQ ID NO.10、SEQ ID NO.12所示，HCDR1、HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9所示；和/或，所述抗TROP-2抗体或其抗原结合片段的HCDR1为X3YWLG，其中X3为D，氨基酸序列如SEQ ID NO.13所示；HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.2、SEQ ID NO.3所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示。
在另一优选例中，所述抗CD3抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，重链可变区氨基酸序列如SEQ ID NO.22所示，轻链可变区氨基酸序列如SEQ ID NO.23所示；和/或，所述抗TROP-2抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，重链可变区氨基酸序列如SEQ ID NO.20所示，轻链可变区氨基酸序列如SEQ ID NO.21所示。
在另一优选例中，所述可变区包含至少一个氨基酸突变。
在另一优选例中，所述可变区包含选自以下任一项或多项的氨基酸突变：
1)所述SEQ ID NO.20具有第31位的氨基酸突变；
2)所述SEQ ID NO.22具有第30位的氨基酸突变；
3)所述SEQ ID NO.22具有第31位的氨基酸突变；
4)所述SEQ ID NO.22具有第32位的氨基酸突变；
5)所述SEQ ID NO.23具有第50位的氨基酸突变。
优选地，所述双特异性抗体的可变区包含1)、3)和4)所示的突变；
优选地，所述双特异性抗体的可变区包含1)、2)、3)和4)所示的突变；
优选地，所述双特异性抗体的可变区包含1)和5)所示的突变。
在另一优选例中，所述可变区包含选自以下任一项或多项的氨基酸突变：
a)所述SEQ ID NO.20具有I31D；
b)所述SEQ ID NO.20具有I31E；
c)所述SEQ ID NO.22具有N30S；
d)所述SEQ ID NO.22具有T31G；
e)所述SEQ ID NO.22具有Y32S、Y32H、Y32G；
f)所述SEQ ID NO.23具有G50A。
优选地，所述双特异性抗体的可变区包含d)中所述T31G、e)中所述Y32S和a)中所述I31D，或所述双特异性抗体的可变区包含d)中所述T31G、e)中所述Y32S和b)中所述I31E突变。相应的，所述抗CD3抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，重链可变区氨基酸序列如SEQ ID NO.26所示，轻链可变区氨基酸序列如SEQ ID NO.23所示；和/或，所述抗TROP-2抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，重链可变区氨基酸序列如SEQ ID NO.24和SEQ ID NO.25所示，轻链可变区氨基酸序列如SEQ ID NO.21所示。
优选地，所述双特异性抗体的可变区包含c)中所述N30S、d)中所述T31G、e)中所述Y32S和a)中所述I31D突变，所述双特异性抗体的可变区包含c)中所述N30S、d)中所述T31G、e)中所述Y32S和或b)中所述I31E突变；
优选地，所述双特异性抗体的可变区包含d)中所述T31G、e)中所述Y32H和a)中所述I31D突变，或所述双特异性抗体的可变区包含d)中所述T31G、e)中所述Y32H和b)中所述I31E突变；
优选地，所述双特异性抗体的可变区包含d)中所述T31G、e)中所述Y32G和a)中所述I31D突变，或所述双特异性抗体的可变区包含d)中所述T31G、e)中所述Y32G和b)中所述I31E突变；
优选地，所述双特异性抗体的可变区包含f)中所述G50A和a)中所述I31D突变，或所述双特异性抗体的可变区包含f)中所述G50A和b)中所述I31E突变。
在另一优选例中，所述抗TROP-2或抗CD3抗原结合片段选自Fab、F(ab')、F(ab')2、Fv或单链Fv(scFv)；所述抗TROP-2或抗CD3抗体为IgG抗体。
在另一优选例中，所述抗TROP-2或抗CD3抗体选自嵌合抗体(例如为人鼠嵌合抗体)、鼠源抗体或人源化抗体。
在另一优选例中，所述抗TROP-2抗体是低内吞抗体，更有利于持续发挥作用。
在另一优选例中，所述IgG抗体包含重链恒定区和轻链恒定区；更优选地，所述重链恒定区选自人IgG1、人IgG2、人IgG3或人IgG4，所述轻链恒定区选自人κ(Kappa)或人λ(Lambda)。
在另一优选例中，所述IgG抗体包含氨基酸序列如SEQ ID NO.51所示的IgG1重链恒定区。
在另一优选例中，所述IgG抗体包含氨基酸序列如SEQ ID NO.52所示的人κ(Kappa)轻链恒定区。
在另一优选例中，所述IgG抗体的恒定区包含至少一个氨基酸突变。
在另一优选例中，所述氨基酸突变是可促进两条重链异源二聚化的杵臼结构(knob-in-hole,KIH)突变；更优选地，所述突变位于恒定区CH3区。
在另一优选例中，所述D1为IgG抗体，所述D2为scFv；更优选地，所述D2连接至D1的N末端或C末端，或连接至D1的CH1和CH2之间；进一步更优选地，所述D2连接至D1的重链。
在另一优选例中，所述D2包含一个、两个、三个或多个抗CD3的scFv。
在另一优选例中，所述scFv从N端到C端包含VH-L1-VL结构或VL-L1-VH结构。
在另一优选例中，所述抗TROP-2抗体选自抗TROP-2-VH-I31D单抗和抗TROP-2-VH-I31E单抗。
在另一优选例中，所述抗CD3抗原结合片段选自CD3-scFv和CD3-scFv-VH-T31G-Y32S。
上述连接是指通过接头或通过肽键直接相连。
在另一优选例中，所述接头L1、L2、L3的氨基酸序列独立地为(G4S)n或4G，n选自1、2、3、4、5、6。
在另一优选例中，所述接头L1的氨基酸序列如SEQ ID NO.27所示。
在另一优选例中，所述接头L2的氨基酸序列如SEQ ID NO.28所示。
在另一优选例中，所述接头L3的氨基酸序列如SEQ ID NO.29所示。
在另一优选例中，所述双特异性抗体为同源二聚体或异源二聚体。
在如图1A所示的优选例中，所述双特异性抗体为同源二聚体，包含抗TROP-2的IgG抗体和两个抗CD3的scFv，其中每个scFv包含可变区VH和可变区VL，VH与VL通过接头L1连接，每个抗CD3的scFv通过接头L2和L3，连接到抗TROP-2的免疫球蛋白抗体IgG的CH1和CH2之间。
在如图1B所示的优选例中，所述双特异性抗体为同源二聚体，包含抗TROP-2的IgG抗体和两个抗CD3的scFv，其中每个scFv包含可变区VH和可变区VL，VH与VL通过接头L1连接，每个抗TROP-2的scFv通过接头L2和抗TROP-2的免疫球蛋白抗体IgG重链的N端串联。
在另一优选例中，所述双特异性抗体为异源二聚体，包含抗TROP-2的IgG抗体和一个抗CD3的scFv，其中scFv包含可变区VH和可变区VL，VH与VL通过接头L1连接，抗CD3的scFv通过接头L2与任一抗TROP-2的免疫球蛋白抗体IgG重链的N端串联。
在另一优选例中，所述双特异性抗体包含重链和轻链，所述重链和轻链的氨基酸序列选自以下任一：
a)所述双特异性抗体的重链氨基酸序列如SEQ ID NO.36所示，和所述双特异性抗体的轻链氨基酸序列如SEQ ID NO.31所示；
b)所述双特异性抗体的重链氨基酸序列如SEQ ID NO.37所示，和所述双特异性抗体的轻链氨基酸序列如SEQ ID NO.31所示；
c)所述双特异性抗体的重链氨基酸序列如SEQ ID NO.38所示，和所述双特异性抗体的轻链氨基酸序列如SEQ ID NO.31所示；
d)所述双特异性抗体的重链氨基酸序列如SEQ ID NO.39所示，和所述双特异性抗体的轻链氨基酸序列如SEQ ID NO.31所示；
e)所述双特异性抗体的重链氨基酸序列如SEQ ID NO.40所示，和所述双特异性抗体的轻链氨基酸序列如SEQ ID NO.31所示；
f)所述双特异性抗体的重链氨基酸序列如SEQ ID NO.41所示，和所述双特异性抗体的轻链氨基酸序列如SEQ ID NO.31所示；
g)所述双特异性抗体的重链氨基酸序列如SEQ ID NO.42所示，和所述双特异性抗体的轻链氨基酸序列如SEQ ID NO.31所示；
或，
h)将a)至g)中的氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有同时抗TROP-2活性和抗CD3活性的由a)至g)衍生的多肽。
在另一优选例中，所述双特异性抗体包括所述双特异性抗体的活性片段和/或衍生物，其中，所述活性片段和/或所述衍生物保留了所述双特异性抗体的70-100％(如70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、100％)的抗TROP-2活性和70-100％的抗CD3活性。
在另一优选例中，所述双特异性抗体的衍生物是所述双特异性抗体经过一个或几个氨基酸突变(氨基酸缺失、插入和/或取代后)并保持至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同一性的多肽。
在另一优选例中，所述氨基酸突变为保守性氨基酸取代。
在另一优选例中，所述氨基酸突变位于框架区或恒定区。
本发明的第二个方面提供了一种多核苷酸分子，所述多核苷酸分子编码所述的双特异性抗体。
在另一优选例中，所述多核苷酸分子包含编码所述双特异性抗体重链的多核苷酸分子，核苷酸序列如SEQ ID NO.43、SEQ ID NO.44或SEQ ID NO.45所示；和，编码所述双特异性抗体的轻链的多核苷酸分子，核苷酸序列如SEQ ID NO.50所示。
在另一优选例中，所述多核苷酸分子包含编码所述双特异性抗体重链的多核苷酸分子，核苷酸序列如SEQ ID NO.46、SEQ ID NO.47、SEQ ID NO.48或SEQ ID NO.49所示；和，编码所述双特异性抗体的轻链的多核苷酸分子，核苷酸序列如SEQ ID NO.50所示。
本发明的第三个方面提供了一种表达载体，所述表达载体含有所述的多核苷酸分子。
在另一优选例中，所述表达载体为病毒或质粒，较佳地为噬菌体或者噬菌粒。
在另一优选例中，所述表达载体选自下组：pcDNA3.4、pDR1、pcDNA3.1(+)、pcDNA3.1/ZEO(+)、pDHFR、pTT5、pDHFF、pGM-CSF或pCHO 1.0，较佳地为pcDNA3.4。
本发明的第四个方面提供了一种细胞，所述细胞含有所述的表达载体。
在另一优选例中，所述细胞选自以下任一：COS、CHO、293F、293E、NS0、sf9、sf21、DH5α、BL21(DE3)或TG1，较佳地为E.coli TG1、BL21(DE3)细胞(表达单链抗体或Fab抗体) 或者CHO-K1细胞(表达全长IgG抗体)。
本发明的第五个方面提供了上述双特异性抗体的制备方法，所述制备方法包括以下步骤：
a)在表达条件下，培养所述的细胞，从而表达双特异性抗体；
b)分离并纯化步骤a)所述的双特异性抗体。
本发明的第六个方面提供了一种药物组合物，所述药物组合物包含有效量的上述的双特异性抗体和一种或多种药学上可接受的载体、稀释剂或赋形剂等。
在另一优选例中，所述药物组合物包含上述的双特异性抗体、醋酸盐、海藻糖、盐酸精氨酸或吐温等。
在另一优选例中，所述药物组合物的剂型包括胃肠给药剂型或胃肠外给药剂型。
在另一优选例中，所述的胃肠外给药剂型包括玻璃体注射、静脉注射、静脉滴注、皮下注射、局部注射、肌肉注射、瘤内注射、腹腔内注射、颅内注射或腔内注射等。
本发明的第七个方面提供了上述的双特异性抗体或药物组合物在制备癌症或肿瘤的药物中的用途。
在另一优选例中，所述癌症或肿瘤为TROP-2阳性癌症或肿瘤。
在另一优选例中，所述癌症或肿瘤选自：肺癌、骨癌、胃癌、胰腺癌、前列腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌、睾丸癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、直肠癌、结肠癌、肛门区癌、乳腺癌、食管癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、尿道癌、阴茎癌、前列腺癌、胰腺癌、脑癌、睾丸癌、淋巴癌、移行细胞癌、膀胱癌、肾癌或输尿管癌、肾细胞癌、肾盂癌、霍奇金淋巴瘤、非霍奇金淋巴瘤、软组织肉瘤、儿童实体瘤、淋巴细胞性淋巴瘤、中枢神经系统肿瘤、原发性中枢神经系统淋巴瘤、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、黑素瘤、卡波西肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤、慢性、急性白血病或其组合。
本发明的第八个方面提供了一种免疫偶联物，所述免疫偶联物包含：
a)上述的双特异性抗体；和
b)选自以下任一种或多种的偶联物部分：可检测标记物、药物、毒素、细胞因子、放射性核素或酶等。
在另一优选例中，所述偶联物部分选自：荧光或发光标记物、放射性标记物、磁共振成像或电子计算机X射线断层扫描技术的造影剂，或能够产生可检测产物的酶、放射性核素、生物毒素或细胞因子等。
在另一优选例中，所述的免疫偶联物包括抗体-药物偶联物。
在另一优选例中，所述的免疫偶联物用于制备治疗肿瘤或癌症的药物组合物。
在本发明的第九个方面提供了一种治疗癌症或肿瘤的方法，所述方法包括向有需要的受试者施用本发明的第一方面所述的双特异性抗体、本发明的第六方面所述的药物组合物、或本发明的第八方面所述的免疫偶联物。
在另一优选例中，所述方法还包括和其他的抗肿瘤药联合给药。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
本发明的抗TROP-2/CD3双特异性抗体的积极进步效果在于：(1)可以通过结合T细胞调 节T细胞免疫活性，并特异性结合表达TROP-2的肿瘤细胞，从而诱导免疫细胞靶向杀伤TROP-2阳性的肿瘤细胞。特别地，其肿瘤细胞杀伤效果及激发免疫细胞释放IL-2细胞因子的能力均优于对照抗CD3单抗；(2)特异性高、安全性好；(3)表达量高；(4)结构稳定。
附图说明
图1A显示了抗TROP-2/CD3双抗a和b的结构示意图。
图1B显示了抗TROP-2/CD3双抗c的结构示意图。
图2A显示了抗TROP-2/CD3双抗a的UPLC检测图谱。
图2B显示了抗TROP-2/CD3双抗b的UPLC检测图谱。
图2C显示了抗TROP-2/CD3双抗c的UPLC检测图谱。
图2D显示了抗TROP-2/CD3双抗b1的UPLC检测图谱。
图2E显示了抗TROP-2/CD3双抗b2的UPLC检测图谱。
图2F显示了抗TROP-2/CD3双抗b3的UPLC检测图谱。
图2G显示了抗TROP-2/CD3双抗b4的UPLC检测图谱。
图3A显示了ELISA检测抗TROP-2/CD3双抗a和c与TROP-2的结合。
图3B显示了ELISA检测抗TROP-2/CD3双抗a、b和c与CD3的结合。
图3C显示了ELISA检测抗TROP-2/CD3双抗不同突变与CD3的结合。
图4显示了抗TROP-2/CD3双抗动力学特征参数图谱。
图5A显示了在抗TROP-2/CD3双抗b作用下，hPBMC对不同靶细胞的杀伤作用。
图5B显示了在抗TROP-2/CD3双抗作用下，hPBMC对Colo205细胞的杀伤作用。
图5C显示了在抗TROP-2/CD3双抗不同突变作用下，hPBMC对Colo205细胞的杀伤作用。
图6A显示了在抗TROP-2/CD3双抗作用下，hPBMC杀伤靶细胞过程中释放的IL-2含量。
图6B显示了在抗TROP-2/CD3双抗作用下，hPBMC杀伤靶细胞过程中释放的IFN-γ含量。
图6C显示了在抗TROP-2/CD3双抗作用下，hPBMC杀伤靶细胞过程中释放的TNF-α含量。
图6D显示了在抗TROP-2/CD3双抗不同突变作用下，hPBMC杀伤靶细胞过程中释放的IFN-γ含量。
图7A显示了抗TROP-2/CD3双抗b在NCI-H292移植瘤模型上的抗肿瘤作用。
图7B显示了抗TROP-2/CD3双抗b在NCI-H292移植瘤模型上对小鼠的毒副作用。
图7C显示了抗TROP-2/CD3双抗不同突变在NCI-H292移植瘤模型上的抗肿瘤作用。
图7D显示了抗TROP-2/CD3双抗作用下小鼠血清中的细胞因子含量。
图8A显示了抗TROP-2/CD3双抗a在25℃条件下处理28天的HPLC检测图谱。
图8B显示了抗TROP-2/CD3双抗a在37℃条件下处理28天的HPLC检测图谱。
具体实施方式
本发明人经过广泛而深入地研究，获得一种新的靶向肿瘤细胞表面分子TROP-2和CD3的双特异性抗体，其能够保持两端抗体的活性，能同时结合TROP-2和CD3抗原。本发明的双特异性抗体可用于实现T细胞介导的免疫反应，特别是有效促进针对表达TROP-2的肿瘤 细胞的T细胞介导的杀伤，从而有效地抑制肿瘤。意料不到的是，其肿瘤细胞杀伤效果及激发免疫细胞释放IL-2细胞因子的能力均优于对照抗CD3单抗，且安全性好。因此，本发明的双特异性抗体可以被开发为一种疗效优越的抗肿瘤药物。在此基础上完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
本发明中，术语“抗体(Antibody，Ab)”和“免疫球蛋白G(Immunoglobulin G，IgG)”是有相同结构特征的异四聚糖蛋白，其由两条相同的轻链(L)和两条相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型(isotype)的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是恒定区，重链恒定区由三个结构域CH1、CH2、以及CH3构成。每条轻链的一端有可变区(VL)，另一端有恒定区，轻链恒定区包括一个结构域CL；轻链的恒定区与重链恒定区的CH1结构域配对，轻链的可变区与重链的可变区配对。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体依赖的细胞介导的细胞毒性作用(antibody-dependent cell-mediatedcytotoxicity，ADCC)等。重链恒定区包括IgG1、IgG2、IgG3、IgG4亚型；轻链恒定区包括κ(Kappa)或λ(Lambda)。抗体的重链和轻链通过重链的CH1结构域和轻链的CL结构域之间的二硫键共价连接在一起，抗体的两条重链通过铰链区之间形成的多肽间二硫键共价连接在一起。
本发明所述的“免疫球蛋白抗体IgG”约150kDa，由四条肽链构成，含有两条相同的约50kDa的γ重链，和两条相同的约25kDa的轻链，从而具有四聚体四级结构。两条重链通过二硫键相互连接，并各自与一条轻链连接。所成的四聚体具有相同的两半，二者形成叉型或者类似Y的形状，叉的每一端含有一个相同的抗原结合位点。IgG抗体可以基于重链的恒定区中氨基酸序列的微小差异而分为多个亚类(例如IgG1、2、3、4)。
本发明中，术语“双特异性抗体(或双抗)”是指能同时特异性结合两种抗原(靶点)或两种表位的抗体分子。根据对称性，双特异性抗体可以分为结构对称的和不对称的分子。根据结合位点的多少，双特异性抗体可以分为二价、三价、四价和多价分子。
本发明中，术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同抗原决定簇的不同抗体的混合物)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们可以通过杂交瘤培养来合成，不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从基本均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
如本文所用，术语“抗原结合片段”，指具有抗原结合活性的Fab片段、Fab'片段、F(ab')2片段或单一Fv片段等。抗原结合片段的非限制性例子包括：(i)Fab片段；(ii)F(ab')2片段；(iii)Fv片段；或(iv)单链Fv(scFv)。如本文所用，表述“抗原结合片段”内部也涵盖其他工程化分子，如结构域特异性抗体、单结构域抗体、结构域缺失抗体、嵌合抗体、CDR移植抗体、双 体抗体、三体抗体、四体抗体、微型抗体、纳米体(例如单价纳米体和双价纳米体等)、小模块免疫药物和鲨鱼可变IgNAR域等。
本发明中，术语“Fab”和“Fc”是指木瓜蛋白酶可将抗体裂解为两个完全相同的Fab段和一个Fc段。Fab段由抗体的重链的VH和CH1以及轻链的VL和CL结构域组成。Fc段即可结晶片段(fragment crystallizable，Fc)，由抗体的CH2和CH3结构域组成。Fc段无抗原结合活性，是抗体与效应分子或细胞相互作用的部位。术语“F(ab')2”片段抗体是由胃蛋白酶消化整个IgG抗体，去除大部分Fc区同时完整保留一些铰链区后得到的，具有通过二硫键连接在一起的两个抗原结合F(ab')部分。
本发明中，术语“scFv”或“单链可变区片段scFv”为单链抗体(single chain antibody fragment，scFv)，是指包含免疫球蛋白重链VH和轻链VL可变区的融合蛋白，VH与VL通过含有15-25个氨基酸的接头(linker)相连，其中所述融合蛋白保留了完整免疫球蛋白相同的抗原特异性。
本发明中，术语“Fv片段”或“Fv抗体”含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv片段还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。
本发明中，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于重链可变区和轻链可变区中称为互补决定区(complementarity-determining region，CDR)或超变区中的三个片段中。可变区中较保守的部分称为框架区(frame region，FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷I，647-669页(1991))。
如本文所用，术语“框架区FR”指插入CDR间的氨基酸序列，即指在单一物种中不同的免疫球蛋白间相对保守的免疫球蛋白的轻链和重链可变区的那些部分。免疫球蛋白的轻链和重链各具有四个FR，分别称为FR1-L、FR2-L、FR3-L、FR4-L和FR1-H、FR2-H、FR3-H、FR4-H。相应地，轻链可变结构域可因此称作(FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L)且重链可变结构域可因此表示为(FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H)。优选地，本发明的FR是人抗体FR或其衍生物，所述人抗体FR的衍生物与天然存在的人抗体FR基本相同，即序列同一性达到85％、90％、95％、96％、97％、98％或99％。获知CDR的氨基酸序列，本领域的技术人员可轻易确定框架区FR1-L、FR2-L、FR3-L、FR4-L和/或FR1-H、FR2-H、FR3-H、FR4-H。如本文所用，术语“人框架区”是与天然存在的人抗体的框架区基本相同的(约85％或更多，具体地90％、95％、97％、99％或100％)框架区。
如本文所用，术语“接头”是指插入免疫球蛋白结构域中为轻链和重链的结构域提供足够的可动性以折叠成交换双重可变区免疫球蛋白的一个或多个氨基酸残基。在本发明中，优选的接头是指接头L1、L2和L3，其中L1连接单链抗体(scFv)的VH和VL，L2和L3将scFv连接到抗体重链的CH1和CH2之间，或直接通过L2连接到抗体的重链上(如抗体重链的N末端或C末端)。合适的接头实例包括单甘氨酸(Gly)或丝氨酸(Ser)残基，接头中氨基酸残基的标识和序列可随着接头中需要实现的次级结构要素的类型而变化。
本发明中，术语“抗”、“结合”、“特异性结合”是指两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。通常，抗体以小于大约10-7M，例如小于大约10-8M、10-9M、10-10M、10-11M或更小的平衡解离常数(KD)结合该抗原。本发明中，术语“KD”是指特定抗体-抗原相互作用的平衡解离常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。例如，使用表面等离子体共振术(surface plasmon resonance，SPR)在BIACORE仪中测定抗体与抗原的结合亲和力或使用ELISA测定抗体与抗原结合的相对亲和力。
本发明中，术语“表位”是指与抗体特异性结合的多肽决定簇或是抗原中被抗体结合的区域。
双特异性抗体
本发明的双特异性抗体为一种能与TROP-2和CD3特异结合的双特异性抗体，其包含抗CD3抗体部分和抗TROP-2抗体部分。
本发明的双特异性抗体可以为二聚体、三聚体或多聚体，优选为同源或异源二聚体。本发明的双特异性抗体中抗CD3或抗TROP-2的抗体部分可以包括一个或多个抗体或其抗原结合片段，较佳地，1、2、3、4、5或6个。
本发明的双特异性抗体还包括其保守性变异体，指与本发明的双特异性抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成的多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A.
本发明序列采用Kabat系统编号规则。
在构建本发明的双特异性抗体时，与该双特异性抗体的化学和物理稳定性相关的问题也得到了解决，诸如表达物理稳定的分子、增加热和盐依赖的稳定性、降低聚集、增加在高浓度下的溶解度以及维持分别针对两种抗原TROP-2和CD3的亲和力等。
编码核酸和表达载体
本发明另一方面提供了一种多核苷酸分子，所述多核苷酸分子编码上述所述的双特异性抗体。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
本发明所述多核苷酸分子的制备方法为本领域常规的制备方法，较佳地包括以下制备方法：通过基因克隆技术例如PCR方法等，获得编码上述单克隆抗体的多核苷酸分子，或者通过人工全序列合成的方法得到编码上述单克隆抗体的多核苷酸分子。
本领域技术人员知晓，编码上述双特异性抗体的氨基酸序列的核苷酸序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同 系物可以通过对编码该双特异性抗体基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
本发明另一方面提供了一种表达载体，所述表达载体含有上述的多核苷酸分子。
这些载体可以用于转化到适当的宿主细胞，以使其能够表达蛋白质。其中所述表达载体为本领域常规的表达载体，是指包含适当的调控序列，例如启动子序列、终止子序列、多腺苷酰化序列、增强子序列、标记基因和/或序列以及其他适当的序列的表达载体。所述表达载体可以是病毒或质粒，如适当的噬菌体或者噬菌粒，更多技术细节请参见例如Molecular Cloning：A Laboratory Manual，第二版，Cold Spring Harbor Laboratory Press，1989，Sambrook等编著。许多用于核酸操作的已知技术和方案请参见例如Current Protocols in Molecular Biology，第二版，Ausubel等编著。本发明所述表达载体较佳地为pDR1、pcDNA3.1(+)、pcDNA3.4、pcDNA3.1/ZEO(+)、pDHFR、pTT5、pDHFF、pGM-CSF或pCHO 1.0，更佳地为pcDNA3.4。
本发明另外提供了一种细胞，所述细胞含有上述的表达载体。
本发明所述的细胞为将所述表达载体转化至宿主细胞中获得。宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核苷酸可被有效表达即可。其中所述宿主细胞包括原核表达细胞和真核表达细胞，所述宿主细胞较佳地包括：COS、CHO(中国仓鼠卵巢，Chinese H amster Ovary)、NS0、sf9、sf21、DH5α、BL21(DE3)或TG1，更佳地为E.coli TG1、BL21(DE3)细胞(表达单链抗体或Fab抗体)或者CHO-K1细胞(表达全长IgG抗体)。将前述表达载体转化至宿主细胞中，可获得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
制备方法
本发明所述的细胞的培养方法、所述抗体的分离和纯化方法为本领域常规方法，具体操作方法请参考相应的细胞培养技术手册以及抗体分离纯化技术手册。本发明中公开的抗TROP-2/CD3双特异性抗体的制备方法包括：在表达条件下，培养上述的细胞，从而表达能与TROP-2和CD3特异结合的双特异性抗体；及分离和纯化所述的抗TROP-2/CD3双特异性抗体。利用上述方法，可以将重组蛋白纯化为基本均一的物质。
可以利用亲和层析的方法对本发明公开的抗TROP-2/CD3双特异性抗体进行分离纯化，根据所利用的亲和柱的特性，可以使用常规的方法例如高盐缓冲液、改变PH等方法洗脱结合在亲和柱上的抗TROP-2/CD3双特异性抗体。本发明的发明人对所得抗TROP-2/CD3双特异性抗体进行了检测实验，实验结果表明该抗TROP-2/CD3双特异性抗体能很好地与靶细胞和抗原结合，具有较高的亲和力。
药物组合物
本发明的另一方面提供了一种组合物，优选地，所述的组合物是药物组合物。
本发明中，术语“药物组合物”是指本发明的双特异性抗体可以和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的双特异性抗体的氨基酸核心序列的构象完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。
通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5.0-8.0，较佳地pH约为6.0-8.0，尽管pH值可随被配制物质的性质以及待治疗的病 症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：静脉注射、静脉滴注、皮下注射、局部注射、肌肉注射、瘤内注射、腹腔内注射(如腹膜内)、颅内注射或腔内注射。通常情况下，对于液体制剂，通常可以在2-8℃条件下至少一年保持稳定；对于冻干制剂，通常可以在30℃条件下至少六个月保持稳定。所述双特异性抗体制剂可为制药领域常用的混悬、水针、冻干等制剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％，较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的双特异性抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-50毫克/千克体重。此外，本发明的双特异性抗体还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的双特异性抗体或其免疫偶联物施用于哺乳动物。其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
应用
本发明另一方面提供了上述能与TROP-2和CD3特异结合的双特异性抗体、或上述药物组合物在制备药物中的应用，所述药物用于治疗癌症或肿瘤。
本发明所称的用于治疗癌症或肿瘤的药物，指具有抑制和/或治疗肿瘤的药物，可以包括伴随肿瘤相关症状发展的延迟和/或这些症状严重程度的降低，进一步还包括已存在的肿瘤伴随症状的减轻并防止其他症状的出现，还包括减少或防止肿瘤的转移等。
本发明所述的药物针对的肿瘤较佳地包括但不限于：肺癌、骨癌、胃癌、胰腺癌、前列腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌、睾丸癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、直肠癌、结肠癌、肛门区癌、乳腺癌、食管癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、尿道癌、阴茎癌、前列腺癌、胰腺癌、脑癌、睾丸癌、淋巴癌、移行细胞癌、膀胱癌、肾癌或输尿管癌、肾细胞癌、肾盂癌、霍奇金淋巴瘤、非霍奇金淋巴瘤、软组织肉瘤、儿童实体瘤、淋巴细胞性淋巴瘤、中枢神经系统肿瘤、原发性中枢神经系统淋巴瘤、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、黑素瘤、卡波西肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤、慢性或急性白血病和所述癌的组合。
本发明的双特异性抗体及其组合物在对包括人在内的动物给药时，给药剂量因病人的年龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况，总给药剂量不能超过一定范围。具体讲静脉注射的剂量是1-1800mg/天。
本发明的双特异性抗体及其组合物还可以和其他的抗肿瘤药联合给药以达到更加有效治疗肿瘤的目的，这些抗肿瘤药包括但不限于：1、细胞毒类药物：1)作用于核酸化学结构的药物：烷化剂如氮芥类、亚硝脲类、甲基磺酸酯类；铂类化合物如顺铂(Cisplatin)、卡铂(Carboplatin)和草酸铂(Oxaliplatin)等；抗生素类如阿霉素(Adriamycin/Doxorubicin)、放线菌素D(Dactinomycin D)、柔红霉素(Daunorubicin)、表阿霉素(Epirubicin)、光辉霉素(Mithramycin)等；2)影响核酸代谢的药物：二氢叶酸还原酶抑制剂如甲氨喋呤(Methotrexate)和培美曲塞 (Pemetrexed)等；胸腺核苷合成酶抑制剂如氟尿嘧啶类(5-氟尿嘧啶、卡培他滨)等；嘌呤核苷合成酶抑制剂如6-巯基嘌呤等；核苷酸还原酶抑制剂如羟基脲(Hydroxycarbamide)等；DNA多聚酶抑制剂如阿糖胞苷(Cytosinearabinoside)和吉西他滨(Gemcitabine)等；3)作用于微管蛋白的药物：多西他赛(Docetaxel)、长春花碱(Vincristine)、长春瑞滨(Vinorelbine)、鬼臼硷类、高三尖杉酯碱等；2、激素类药物：抗雌激素如他莫昔芬(Tamoxifen)、屈洛昔芬(Droloxifene)、依西美坦(Exemestane)等；芳香化酶抑制剂如氨鲁米特(Aminoglutethimide)、福美司坦(Formestane)、来曲唑(Letrozle)、阿那曲唑(Anastrozole)等；抗雄激素：氟它氨RH-LH激动剂/拮抗剂：诺雷德、依那通等；3、生物反应调节剂类药物：此类药物主要通过调节机体免疫功能以到抗肿瘤的效果，如干扰素类(Interferon)、白细胞介素-2(Interleukin-2)、胸腺肽类(Thymosins)等；4、单克隆抗体类药物：曲妥昔单抗(Trastuzumab)、利妥昔单抗(Rituximab)、西妥昔单抗(Cetuximab)、贝伐单抗(Bevacizumab)等。本发明公开的双特异性抗体及其组合物可以和上述的抗肿瘤药物之一或其组合联合用药。
下面结合具体实施例，进一步陈述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。
以下实施例中使用的实验材料和来源以及实验试剂的配制方法具体说明如下。
实验材料：
感受态细胞：品牌生工，货号B528412。
293F细胞：品牌GIBCO，货号R79007。
Colo205细胞：中科院细胞库，目录号TCHu102。
MDA-MB-231细胞：中科院细胞库，目录号TCHu227。
A549细胞：购自ATCC。
人肺癌细胞NCI-H292：中科院细胞库，目录号SCSP-582。
hPBMC：购自澳赛尔斯，货号FPB004F-C-MLR。
实验试剂：
无内毒素质粒大提试剂盒：品牌天根，货号DP117。
DNA纯化回收试剂盒：品牌天根，货号DP214。
ClonExpressTM II One Step Cloning Kit：品牌诺唯赞，货号C112。
HS DNA Polymerase：品牌takara，货号R010B。
包被液：碳酸钠1.59克，碳酸氢钠2.93克，加入双蒸水定容至1L。
PBST：PBS+0.05％Tween 20。
Tween 20：品牌阿拉丁，货号T104863。
ELISA封闭液：PBST+1％BSA。
BSA：品牌生工，货号A60332。
TROP-2-HIS蛋白：品牌恺佧，货号TRP-HM121。
CD3-FC蛋白：品牌恺佧，货号CD3-HM205。
HRP标记的山羊抗人FC抗体：品牌博奥龙，货号BF03031。
HRP标记的山羊抗人Fab抗体：品牌赛默飞，货号A0293。
TMB：品牌BD Biosciences，货号555214。
终止液：2M硫酸溶液。
0.25％胰酶-EDTA：品牌GIBCO，货号25200-072。
1640完全培养基：RPMI1640培养基+10％FBS+1％Pen Strep+1％丙酮酸钠。
RPMI1640培养基：品牌GIBCO，货号22400089。
FBS：品牌GIBCO，货号10099-141。
Pen Strep：品牌GIBCO，货号1514022。
丙酮酸钠：品牌GIBCO，货号11360-070。
DMEM完全培养基：DMEM高糖培养基+10％FBS+1％Pen Strep+1％GLUMAX。
DMEM高糖培养基：品牌GIBCO，货号11965-092。
GlutaMAX：品牌GIBCO，货号35050-061。
Luminescent Cell Viability Assay：购自Promega，货号G7572。
PurifiedMouse Anti-Human IL-2：购自BD Pharmingen，货号55051。
Biotin Mouse Anti-Human IL-2：购自BD Pharmingen，货号555040。
Recombinant Human IL-2：购自BD Pharmingen，货号554603。
Streptavidin HRP：购自BD Biosciences，货号554066。
实验仪器：
Mastercycler nexus PCR仪：购自Eppendorf公司。
Hitrap Mabselect Sure柱：购自Cytiva公司。
HiLoad 26/600Superdex 200pg柱：购自Cytiva公司。
SpectraMax i3x酶标仪：购自MolecularDevices公司。
SpectraMaxM5酶标仪：购自Molecular Devices公司。
本申请的序列如下表所示：
表B.





实施例1.抗TROP-2/CD3双抗分子的构建
本发明采用了抗TROP-2的单抗IgG和抗CD3单抗scFv串联的方式，构建了抗TROP-2/CD3双特异性抗体a、b和c，a和b结构如图1A所示，c结构如图1B所示。
其中，抗TROP-2/CD3双特异性抗体中的抗TROP-2单抗序列来源于US20120237518A1专利中公开的KM4097HV3aLV0人源化单克隆抗体，KM4097HV3aLV0人源化单克隆抗体作为抗TROP-2单抗阳性对照。抗TROP-2/CD3双特异性抗体中的抗CD3单抗的CDR序列来源于US8236308B2中的SEQ ID NO.2和SEQ ID NO.4，并对其进行了人源化改造。本发明使用改造后的人源化SP34作为抗CD3单抗阳性对照。
人源化SP34具体构建方法如下：
(1)鼠源抗人CD3单克隆抗体鼠源SP34(mSP34)的CDR区序列的获取
鼠源抗人CD3单克隆抗体的重链和轻链可变区氨基酸序列分别来自于US8236308B2中的SEQ ID NO.2和SEQ ID NO.4。
mSP34重链可变区氨基酸序列：
mSP34轻链可变区氨基酸序列：
对mSP34的重链和轻链可变区氨基酸序列进行分析，依据Kabat规则分别确定mSP34重链和轻链的互补决定区CDR和框架区FR。mSP34重链CDR的氨基酸序列为HCDR1：TYAMN(SEQ ID NO.7)、HCDR2：RIRSKYNNYATYYADSVKD(SEQ ID NO.8)和HCDR3：HGNFGNSYVSWFAY(SEQ ID NO.9)，轻链CDR的氨基酸序列为LCDR1：RSSTGAVTTSNYAN(SEQ ID NO.10)、LCDR2：GTNKRAP(SEQ ID NO.11)和LCDR3：ALWYSNLWV(SEQ ID NO.12)。
(2)SP34单克隆抗体的人源化构建过程
在https://www.ncbi.nlm.nih.gov/igblast/，将mSP34的重链可变区与人IgG胚系序列进行同源性比较，选择IGHV3-23*04为重链CDR移植模板，将mSP34的重链CDR移植入IGHV3-23*04骨架区，并在HCDR3之后加入WGQGTLVTVSS作为第四个框架区，获得CDR移植重链可变区序列。同样地，将mSP34的轻链可变区与人IgG胚系序列同源性比较，选择IGLV7-46*01为轻链CDR移植模板，将SP34的轻链CDR移植入IGLV7-46*01的骨架区，并在LCDR3之后加入FGQGTKVEIK作为第四个框架区，获得CDR移植轻链可变区序列。在CDR移植可变区的基础上，对一些氨基酸位点进行突变。在进行突变时，将氨基酸序列进行Kabat编码，位点的位置由Kabat码指示。
优选的，对于CDR移植重链可变区，将第49位的S突变为A，将第73位的N突变为D，将第93位的A突变为V，将第94位的K突变为R。对于CDR移植轻链可变区，将第36位的F突变为V，第46位的T突变为G，第49位的Y突变为G，第57位的W突变为G，第58位的T突变为V。上述带有回复突变位点的重链和轻链可变区分别定义为人源化SP34重链和轻链可变区(SEQ ID NO.18和SEQ ID NO.19)。
(3)全长人源化SP34单克隆抗体的构建过程：
将合成的人源化重链可变区与人IgG1重链恒定区(SEQ ID NO.39)的编码基因相连，获得全长的人源化重链基因；将人源化轻链可变区与人Kappa链恒定区(SEQ ID NO.40)的编码基因相连，获得全长的人源化轻链基因。
参照实施例2中的表达纯化方法最终获得阳性对照单克隆抗体。
在纯化产物蛋白表达量测定的过程中发现，阳性对照抗TROP-2单抗的表达量较低，约为10-20mg/l，而I31E位突变后抗TROP-2单抗蛋白表达量提高至30mg/l以上，I31D位突变后抗TROP-2单抗蛋白表达量提高至70mg/l以上，因此将I31E或I31D位突变运用于抗TROP-2/CD3双抗中，以提高双抗的表达量，TROP-2/CD3双抗的突变具体如下：
对抗TROP-2重链可变区VH(SEQ ID NO.20)的氨基酸I31D位点突变，获得抗TROP-2重链可变区VH-I31D(SEQ ID NO.24)，从而获得抗TROP-2重链HC-I31D(SEQ ID NO.32)，抗TROP-2单抗的轻链(SEQ ID NO.31)保持不变，获得抗TROP-2-VH-I31D单抗。
对抗TROP-2重链可变区VH(SEQ ID NO.20)的氨基酸I31E位点突变，获得抗TROP-2重链可变区VH-I31E(SEQ ID NO.25)，从而获得抗TROP-2重链HC-I31E(SEQ ID NO.33)，抗TROP-2单抗的轻链(SEQ ID NO.31)保持不变，获得抗TROP-2-VH-I31E单抗。
对抗CD3重链可变区VH(SEQ ID NO.22)的氨基酸T31G和Y32S位点突变，获得抗CD3重链可变区VH-T31G-Y32S(SEQ ID NO.26)。
抗TROP-2/CD3双特异性抗体a、c中的scFv为VH-L1-VL，是通过L1(SEQ ID NO.27)连接抗CD3重链可变区VH(SEQ ID NO.22)和抗CD3轻链可变区VL(SEQ ID NO.23)得到的，即CD3-scFv(SEQ ID NO.34)。
另一种实施方式中，抗TROP-2/CD3双特异性抗体b中的scFv为VH-T31G-Y32S-L1-VL，是通过L1(SEQ ID NO.27)连接抗CD3重链可变区VH-T31G-Y32S(SEQ ID NO.26)和抗CD3轻链可变区VL(SEQ ID NO.23)得到的，即CD3-scFv-VH-T31G-Y32S(SEQ ID NO.35)。
抗TROP-2/CD3双特异性抗体a和b的CD3-scFv通过L2(SEQ ID NO.28)和L3(SEQ ID NO.29)连接到抗TROP-2重链HC-I31D(SEQ ID NO.32)CH1和CH2之间，获得抗TROP-2/CD3双抗a的重链(SEQ ID NO.36)和抗TROP-2/CD3双抗b的重链(SEQ ID NO.37)，抗TROP-2单抗的轻链(SEQ ID NO.31)保持不变。
抗TROP-2/CD3双特异性抗体c的CD3-scFv通过L2(SEQ ID NO.28)连接到抗TROP-2重链HC-I31D(SEQ ID NO.32)的N末端，获得抗TROP-2/CD3双抗c的重链(SEQ ID NO.38)，抗TROP-2单抗的轻链(SEQ ID NO.31)保持不变。
对CD3可变区的氨基酸序列进行进一步的突变：
对抗CD3重链可变区VH(SEQ ID NO.22)的氨基酸N30S、T31G和Y32S位点突变，获得抗CD3重链可变区VH-N30S-T31G-Y32S；
对抗CD3重链可变区VH(SEQ ID NO.22)的氨基酸T31G和Y32H位点突变，获得抗CD3重链可变区VH-T31G-Y32H；
对抗CD3重链可变区VH(SEQ ID NO.22)的氨基酸T31G和Y32G位点突变，获得抗CD3重链可变区VH-T31G-Y32G；
对抗CD3轻链可变区VL(SEQ ID NO.23)的氨基酸G50A位点突变，获得抗CD3轻链可变区VL-G50A。
通过L1(SEQ ID NO.27)将抗CD3轻链可变区VL(SEQ ID NO.23)分别与抗CD3重链可变区VH-N30S-T31G-Y32S、VH-T31G-Y32H和VH-T31G-Y32G连接，得到VH-N30S-T31G-Y32S-L1-VL(CD3-scFv-VH-N30S-T31G-Y32S)、VH-T31G-Y32H-L1-VL(CD3-scFv-VH-T31G-Y32H)和VH-T31G-Y32G-L1-VL(CD3-scFv-VH-T31G-Y32G)；通过L1(SEQ ID NO.27)将抗CD3重链可变区VH(SEQ ID NO.22)与抗CD3轻链可变区VL-G50A连接，得到VH-L1-VL-G50A(CD3-scFv-VL-G50A)。
通过L2(SEQ ID NO.28)和L3(SEQ ID NO.29)将CD3-scFv-VH-N30S-T31G-Y32S、CD3-scFv-VH-T31G-Y32H、CD3-scFv-VH-T31G-Y32G和CD3-scFv-VL-G50A依次连接到抗TROP-2重链HC-I31D(SEQ ID NO.32)CH1和CH2之间，获得抗TROP-2/CD3双抗b1、b2、b3和b4的重链，氨基酸序列分别如SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41和SEQ ID NO.42所示，抗TROP-2单抗的轻链(SEQ ID NO.31)保持不变。
实施例2.抗TROP-2/CD3双抗的表达与纯化
将抗TROP-2/CD3双特异性抗体的重链和轻链的DNA片段分别亚克隆到载体pcDNA3.4中，提取重组质粒并共转染293F细胞和/或CHO细胞。细胞培养5-7天后，将培养液通过高速离心、0.22μm滤膜过滤后上样至Hitrap Mabselect Sure亲和层析柱中，用100mM柠檬酸，pH3.5的洗脱液一步洗脱蛋白，回收目的样品并透析换液至pH7.4的PBS中，将纯化后的蛋白用UPLC-SEC检测。
抗TROP-2/CD3双抗a、b、c、b1-b4分子的检测结果如图2A、图2B和图2C，及图2D-图2G所示，其中抗体分子状态均一，单体纯度达到95％以上。
实施例3.酶联免疫吸附法(ELISA)测定双抗对抗原的亲和力
3.1检测抗TROP-2/CD3双特异性抗体对TROP-2的亲和力
包被液稀释重组Human-TROP-2-HIS蛋白至200ng/ml，以100μl/孔加入到酶标板中，常温放置2小时或者4摄氏度过夜放置。去掉包被液(用吸水纸去掉残留液滴)，以200μl/孔加入封闭液至酶标板中，常温放置1-2小时。去掉封闭液(用吸水纸去掉残留液滴)，再用封闭液稀释TROP-2/CD3双特异性抗体和抗TROP-2单抗至10μg/ml，四倍稀释形成12个浓度梯度(最高浓度10μg/ml，最低浓度0.002ng/ml)，以100μl/孔依次加入到封闭过的酶标板中，常温放置1小时。用PBST洗板3次(用吸水纸去掉残留液滴)，用封闭液按1:3000稀释含HRP标记的山羊抗人FC抗体，以100μl/孔加入到酶标板中，常温放置30分钟。用PBST洗板3次(用吸水纸去掉残留液滴)，以100μl/孔加入TMB，室温避光放置5分钟，以80μl/孔加入终止液，终止底物显色反应，用酶标仪读取450nm处的OD值，用GraphPad对数据进行分析，作图并计算EC50。
试验结果如图3A所示，抗TROP-2/CD3双特异性抗体分子a、c和阳性对照抗TROP-2单抗与TROP-2-HIS结合的EC50(单位：nM)分别为0.01649、0.01875和0.02606，表明CD3-scFv的嵌入，无论是连接在抗TROP-2单抗重链的N末端，还是插入到抗TROP-2单抗重链的CH1和CH2之间，都不影响双特异性抗体对TROP-2抗原的结合能力。
3.2检测抗TROP-2/CD3双特异性抗体对CD3的亲和力
包被液稀释重组Human-CD3-FC蛋白至200ng/ml，以100μl/孔加入到酶标板中，常温放置2小时或者4摄氏度过夜放置。去掉包被液(用吸水纸去掉残留液滴)，以200μl/孔加入封闭液至酶标板中，常温放置1-2小时。去掉封闭液(用吸水纸去掉残留液滴)，再用封闭液稀释抗 TROP-2/CD3双特异性抗体和抗CD3单抗至10μg/ml，四倍稀释形成12个浓度梯度(最高浓度10μg/ml，最低浓度0.002ng/ml)，以100μl/孔依次加入到封闭过的酶标板中，常温放置1小时。用PBST洗板3次(用吸水纸去掉残留液滴)，用封闭液按1:3000稀释含HRP标记的山羊抗人Fab抗体，以100μl/孔加入到酶标板中，常温放置30分钟。用PBST洗板3次(用吸水纸去掉残留液滴)，以100μl/孔加入TMB，室温避光放置5分钟，以80μl/孔加入终止液，终止底物显色反应，用酶标仪读取450nm处的OD值，用GraphPad对数据进行分析，作图并计算EC50。
试验结果如图3B所示，抗TROP-2/CD3双特异性抗体分子a、b、c和阳性对照抗CD3单抗与CD3-FC结合的EC50(单位：nM)分别为0.07924、0.6071、0.04141和0.06182。表明CD3-scFv插入到抗TROP-2单抗重链的CH1和CH2之间，使双特异性抗体对CD3抗原的结合能力略有降低，在双抗a的基础上对抗CD3重链可变区VH的氨基酸进行突变后，显著降低了双特异性抗体对CD3抗原的结合能力。
同时比较抗TROP-2/CD3双抗a、b、b1、b2、b3和b4与CD3结合亲和力的差异，实验步骤与反应条件与上述相同。试验结果如图3C所示，抗TROP-2/CD3双体a、b、b1、b2、b3、b4和阳性对照抗CD3单抗与抗原CD3-FC结合的EC50(单位：nM)分别为0.0532、0.3208、0.0939、0.1431、5.4670、0.1273和0.0229。说明对抗CD3重链或轻链可变区进行突变后，抗体与CD3抗原的亲和力均有所下降，其中抗TROP-2/CD3双体b3的亲和力下降最为明显。
实施例4.Fortibio测定抗TROP-2/CD3双抗对CD3抗原的亲和力
使用Fortebio Octet分子相互作用仪和Octet AR2G Biosensors探针测定抗TROP-2/CD3双特异性抗体和抗原CD3-Fc结合的动力学参数。用1×HBS-EP缓冲液稀释TROP-2/CD3双抗浓度至10nM，2倍稀释，设置7个浓度梯度；用1×Octet Sodium Acetate缓冲液稀释抗原CD3-Fc浓度至10ug/ml，将Octet AR2G Biosensors探针活化后，测定抗原抗体结合及解离速率。
抗TROP-2/CD3双抗和CD3-Fc结合的动力学参数见表1，动力学特征参数图谱如图4所示。结果表明，抗TROP-2/CD3双抗a、b1和b4与CD3-Fc有较高的亲和力，抗TROP-2/CD3双抗b与CD3-Fc的亲和力较弱，抗TROP-2/CD3双抗b3与CD3-Fc的亲和力最弱。
表1抗TROP-2/CD3双抗与CD3-Fc抗原结合的动力学参数
KD为亲和力常数；ka为抗原抗体结合速率；kd为抗原抗体解离速率；KD＝kd/ka。
实施例5.双抗作用下hPBMC对肿瘤细胞杀伤实验
取对数期生长的Colo205细胞(人结肠癌细胞)、MDA-MB-231细胞(人乳腺癌细胞)和A549细胞(人肺腺癌细胞)，用完全培养基稀释细胞至1×105个/ml，以100μl每孔加入到白色透明96孔板中，96孔板边缘孔不加细胞，只加完全培养基进行封边处理，放置于37℃ 并5％CO2孵箱中培养过夜。
取新鲜的人外周血单个核细胞(Human peripheral blood mononuclear cell，hPBMC)，用完全培养基稀释细胞至1.2×106个/ml，以50μl每孔加入到白色透明96孔板中，同时设置只加hPBMC细胞无肿瘤细胞的对照孔。
不同肿瘤细胞加入抗体的浓度和稀释倍数不同：用完全培养基稀释抗TROP-2/CD3双抗b至1μg/ml，6倍梯度稀释形成10个浓度梯度(最高浓度1μg/ml，最低浓度9.92×10-8μg/ml)，以50μl每孔加入到含有Colo205细胞和hPBMC细胞的白色透明96孔板中，放置于37℃并5％CO2孵箱中培养48小时；用完全培养基稀释抗TROP-2/CD3双抗b至20μg/ml，4倍梯度稀释形成10个浓度梯度(最高浓度20μg/ml，最低浓度7.63×10-5μg/ml)，以50μl每孔加入到含有MDA-MB-231细胞和hPBMC细胞、含有A549细胞和hPBMC细胞的白色透明96孔板中，放置于37℃并5％CO2孵箱中培养48小时。检测前，白色透明96孔板底需用白色卡纸密封好，确保板底不透光，每孔吸出上清100μl，剩余含有细胞的培养基100μl，加入The Luminescent Cell Viability Assay中的每孔100μl，避光反应10分钟后，用SoftMax 6.3软件上机读数检测。
为了研究抗TROP-2/CD3双抗中两个靶点的协同作用，选择TROP-2蛋白表达量不同的肿瘤细胞，其中Colo205细胞和MDA-MB-231细胞都可以表达TROP-2蛋白，且Colo205细胞的TROP-2表达量高于MDA-MB-231细胞，A549细胞几乎不表达TROP-2蛋白。试验结果如图5A所示，抗TROP-2/CD3双抗b对Colo205细胞的杀伤作用最明显，对MDA-MB-231细胞的杀伤也有一定的作用，对不表达TROP-2蛋白的A549细胞无杀伤作用，说明抗TROP-2/CD3双抗可以定位于靶细胞发挥作用，确保人体正常组织不会被抗TROP-2/CD3双抗诱导的T细胞杀伤作用所杀伤。
验证抗TROP-2/CD3双抗a、b以及抗CD3单抗对肿瘤细胞杀伤的差异，选用TROP-2表达量相对较高的Colo205细胞作为靶细胞，实验步骤与反应条件与上述相同，试验结果如图5B所示，经过T31G和Y32S位点突变后的抗TROP-2/CD3双抗b对靶细胞的杀伤作用低于抗TROP-2/CD3双抗a，但均高于抗CD3单抗。
进一步验证对抗CD3重链或轻链可变区进行突变后，抗TROP-2/CD3双抗对肿瘤细胞的杀伤作用，选用TROP-2表达量相对较高的Colo205细胞作为靶细胞，实验步骤与反应条件与上述相同，试验结果如图5C所示，抗TROP-2/CD3双抗b1、b2和b4对Colo205细胞的杀伤作用较为明显，抗TROP-2/CD3双抗b的杀伤作用居中，抗TROP-2/CD3双抗b3的杀伤作用相对较弱。
实施例6.双抗作用下细胞因子释放实验
取对数期生长的Colo205细胞，用完全培养基稀释细胞至1×105个/ml，以100μl每孔加入到白色透明96孔板中，96孔板边缘孔不加细胞，只加完全培养基进行封边处理，放置于37℃并5％CO2孵箱中培养过夜。
取新鲜的hPBMC细胞,用完全培养基稀释细胞至1.2×106个/ml，以50μl每孔加入到白色透明96孔板中，同时设置只加hPBMC细胞无肿瘤细胞的对照孔。
用完全培养基稀释抗CD3单抗和抗TROP-2/CD3双抗至1μg/ml，6倍梯度稀释形成10 个浓度梯度(最高浓度1μg/ml，最低浓度9.92×10-8μg/ml)，以50μl每孔加入到含有Colo205细胞和hPBMC细胞的白色透明96孔板中，放置于37℃并5％CO2孵箱中培养48小时。
收集上清液，用ELISA检测上清液中IL-2、INF-γ和TNF-α的含量。用包被液分别稀释Purified Mouse Anti-Human IL-2、Purified Mouse Anti-Human INF-γ以及Purified Mouse Anti-Human TNF-α至4μg/ml，以50μl/孔加入到96孔酶标板中，于4℃冰箱过夜孵育。去掉包被液，用吸水纸去除残留液滴，以200μl/孔加入封闭液(含1％BSA的PBST)，室温放置2小时。去掉封闭液，用吸水纸去除残留液滴，用封闭液分别稀释Recombinant Human IL-2和Recombinant Human TNF-α至50ng ng/ml，3倍梯度稀释，形成12个浓度梯度，用于制作标准曲线；用封闭液稀释Recombinant Human INF-γ至100ng/ml，3倍梯度稀释，形成12个浓度梯度，用于制作标准曲线；用封闭液3倍稀释上清液，以100μl/孔加入到96孔酶标板中，室温放置1h。用PBST清洗酶标板3次，用封闭液按照1:1000的比例稀释Biotin Mouse Anti-Human IL-2、Biotin Mouse Anti-Human INF-γ以及Biotin Mouse Anti-Human TNF-α，以100μl/孔加入到96孔酶标板中，室温放置1h。用PBST清洗酶标板3次，用封闭液按照1:1000的比例稀释Streptavidin HRP，以100μl/孔加入到96孔酶标板中，室温放置1h。用PBST清洗酶标板3次，以100μl/孔加入TMB，室温避光放置5分钟，以70μl/孔加入终止液，终止底物显色反应后，立即用酶标仪在450nm波长处读取各孔的OD值，并使用SoftMax6.4软件绘制标准曲线，换算各孔IL-2细胞因子的浓度，用GraphPad Prism6作图分析数据。
比较抗TROP-2/CD3双抗a、b以及抗CD3单抗协同hPBMC对肿瘤细胞杀伤的过程中，激发免疫细胞释放细胞因子IL-2、INF-γ和TNF-α的含量，实验结果如图6A、6B和6C所示，在hPBMC对肿瘤细胞的杀伤过程中，抗TROP-2/CD3双抗b与双抗a相比，可激发免疫细胞释放更少的细胞因子，包括IL-2、INF-γ和TNF-α。同时与图5B比较发现，几乎没有杀伤作用的抗CD3单抗，在浓度累积到一定量时，也会激发免疫细胞释放少量的细胞因子如INF-γ和TNF-α，说明抗TROP-2/CD3双抗对肿瘤细胞的杀伤作用和对自身免疫系统的影响要优于抗CD3单抗，且抗TROP-2/CD3双抗b的杀伤作用优于抗CD3单抗，但相对释放更少的细胞因子。
比较对抗CD3重链或轻链可变区突变后，抗TROP-2/CD3双抗协同hPBMC对肿瘤细胞杀伤的过程中，激发免疫细胞释放细胞因子INF-γ的含量，实验结果如图6D所示，其中抗TROP-2/CD3双抗b1、b2和b4激发的细胞因子INF-γ含量最多，抗TROP-2/CD3双抗b激发的细胞因子INF-γ含量相对较少，抗TROP-2/CD3双抗b3几乎不激发免疫细胞释放细胞因子，同时与图5C对比发现，抗TROP-2/CD3双抗对肿瘤细胞的杀伤作用与激发的细胞因子的含量成正比。
抗TROP-2/CD3双抗b诱导IL-2细胞因子释放的浓度比引起肿瘤细胞杀伤的浓度要高的多。
实施例7.抗TROP-2/CD3双抗在NCI-H292移植瘤模型上的抗肿瘤作用
7.1抗TROP-2/CD3双抗b在NCI-H292移植瘤模型上的抗肿瘤作用
利用人外周血单核细胞hPBMC在NOG小鼠体内重建人源免疫系统，并在此小鼠上建立人肺癌NCI-H292皮下移植瘤模型。具体实施步骤如下：
收集体外培养的NCI-H292细胞(人非小细胞肺癌)，将细胞悬液浓度调整为1×108个/ml， 与基质胶以1：1等比例混合。体外复苏hPBMC并用PBS重悬hPBMC细胞，将hPBMC悬液浓度调整为1×107个/ml。将混合好的肿瘤细胞悬液和hPBMC悬液1：1混合。在无菌条件下，接种200μl细胞混合悬液于NOG小鼠右侧上背部皮下。待皮下瘤生长至150mm3时，按照肿瘤体积将小鼠随机分为3组，每组8只小鼠，包括：空白对照组，仅注射PBS作为对照；抗TROP-2/CD3双特异性抗体b的1mg/kg和5mg/kg两个剂量组，腹腔注射给药，每周给药三次，连续处理四周。整个实验过程中，每周2次测量移植瘤直径，同时称量小鼠体重，绘制的各组肿瘤随时间的生长曲线和小鼠体重曲线。
实验结果如图7A和7B所示，在NCI-H292细胞混合hPBMC移植瘤模型上，抗TROP-2/CD3双抗b对肿瘤的抑制作用明显高于对照组，且1mg/kg和5mg/kg两个剂量组间无明显差异，说明1mg/kg的剂量就可以发挥较好的抑制作用；小鼠的体重随着连续给药数周后也没有明显下降，说明TROP-2/CD3双抗b对小鼠的毒副作用较小。结果表明，在此移植瘤模型上，抗TROP-2/CD3双抗b能够显著抑制肿瘤生长，且对小鼠无明显的毒副作用。
7.2不同抗TROP-2/CD3双抗在NCI-H292移植瘤模型上的抗肿瘤作用比较
利用人外周血单核细胞hPBMC在NOG小鼠体内重建人源免疫系统，并在此小鼠上建立人肺癌NCI-H292皮下移植瘤模型。具体实施步骤如下：
收集体外培养的人非小细胞肺癌NCI-H292细胞，将细胞悬液浓度调整为1×108个/ml，与基质胶以1：1等比例混合。体外复苏hPBMC并用PBS重悬hPBMC细胞，将hPBMC悬液浓度调整为1×107个/ml，将混合好的肿瘤细胞悬液和hPBMC悬液1：1混合。将NOG小鼠右上背侧剃毛，在无菌条件下，接种200μl细胞混合悬液于NOG小鼠右侧上背部皮下。待皮下瘤平均肿瘤体积约为200mm3时，将小鼠随机分为5组，每组8只小鼠，包括：空白对照组，仅注射PBS作为对照；抗TROP-2/CD3双特异性抗体a、b、b2和b3的1mg/kg的剂量组，腹腔注射给药，每周给药两次，连续处理四周。整个实验过程中，每周2次测量移植瘤直径，以给药天数为横坐标，肿瘤体积为纵坐标绘制各组肿瘤随时间的生长曲线。
实验结果如图7C所示，在NCI-H292细胞混合hPBMC移植瘤模型上，不同的抗TROP-2/CD3双抗在1mg/kg剂量下均体现出较强的体内抗肿瘤活性，对肿瘤的抑制作用都明显高于对照组；抗TROP-2/CD3双抗间的抗肿瘤活性进行比较，在1mg/kg相同剂量下，抗TROP-2/CD3双抗a的抗肿瘤活性最强，抗TROP-2/CD3双抗b2的抗肿瘤活性次之，抗TROP-2/CD3双抗b和b3的抗肿瘤活性稍弱于b2，但总体上各TROP-2/CD3双抗的抗肿瘤活性无明显差异，第17天时抗肿瘤活性均在75％以上。
首次给药后16个小时后采集小鼠血样，分离获得血清，检测在抗TROP-2/CD3双抗a、b和b3的作用下，小鼠血清中的细胞因子含量，用Bio-plex Pro Mouse Cytokine Grp 1 Panel 23-Plex试剂盒处理血清样品，用Bio-PlexTM 200系统平台对样品进行检测，获得小鼠血清中23种细胞因子的含量，实验结果如图7D所示。除IL-1b和IL-12(P40)外，抗TROP-2/CD3双抗a激发的细胞因子水平均高于抗TROP-2/CD3双抗b和b3；抗TROP-2/CD3双抗b和b3之间相比，其中抗TROP-2/CD3双抗b激发IL-1a、IL-6、G-CSF和MIP-1b细胞因子的水平较高，对其他细胞因子无明显差异，说明对抗TROP-2/CD3双抗a突变降低其亲和力后，在保持其抗肿瘤活性的情况下，可以显著降低动物体内细胞因子的产生。
实施例8.抗TROP-2/CD3双抗a的稳定性研究
本实验可用于评估在辅料加入情况下蛋白质的稳定性，从而揭示研发最优制剂所需的重要信息。
抗TROP-2/CD3双抗a保存在20mM醋酸盐+6％海藻糖+1％盐酸精氨酸+0.1％吐温80并且pH为5.0的buffer中，分别放置于25℃和37℃的培养箱中28天，HPLC-SEC结果见图8A和图8B。
结果表明，抗TROP-2/CD3双抗a的结构比较稳定，在37℃高温条件下放置长达28天后，其纯度仍然可以保持在95％以上。
讨论
由上述实验可知，本发明提供的双特异抗体可以同时结合TROP-2和CD3，通过结合CD3调节T细胞的免疫活性功能，并特异性杀伤表达TROP-2蛋白的肿瘤细胞。图3B和3C结果显示，双特异抗体与其单抗相比结合CD3的能力有所下调，但图5A和5B结果显示，双特异抗体与其单抗相比杀伤肿瘤细胞的作用明显上升，且肿瘤细胞上TROP-2表达量越高，双特异抗体对肿瘤细胞的杀伤作用越强。比较PBMC杀伤肿瘤细胞和其细胞因子释放结果发现，双特异抗体对细胞的杀伤作用越强，释放的TH1和TH2类的细胞因子越多，成正相关，比较双特异抗体不同突变对靶细胞的杀伤作用发现，双特异抗体b的杀伤作用相对较强，同时激发的细胞因子释放较少，有较大的用药窗口。在NCI-H292移植瘤模型中，双特异抗体对肿瘤细胞抑制活性越强，释放的TH1和TH2类的细胞因子越多，也成正相关，从体内细胞免疫试验的角度再一次证明了抗TROP-2/CD3双抗b对肿瘤的杀伤作用和安全性。说明双特异抗体对CD3的亲和力下降，更有助于T细胞发挥免疫作用，且同时含有靶向TROP-2蛋白的功能，可以特异性结合并杀伤表达TROP-2的肿瘤细胞。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。

Claims (20)

  1. 一种双特异性抗体，其特征在于，所述双特异性抗体包含：
    第一抗原结合结构域D1和第二抗原结合结构域D2，
    所述第一抗原结合结构域D1为抗TROP-2抗体或其抗原结合片段，
    所述第二抗原结合结构域D2为抗CD3抗体或其抗原结合片段，
    所述抗TROP-2抗体或抗CD3抗体或各自的抗原结合片段包含重链互补决定区HCDR1-3和轻链互补决定区LCDR1-3。
  2. 如权利要求1所述的双特异性抗体，其特征在于，所述双特异性抗体包含单体或单体形成的二聚体或多聚体，所述二聚体为同源的或异源的，所述单体从N端到C端包含选自以下任一组的结构：
    其中，
    VLA代表抗TROP-2抗体或其抗原结合片段的轻链可变区；
    VHA代表抗TROP-2抗体或其抗原结合片段的重链可变区；
    VLB代表抗CD3抗体或其抗原结合片段的轻链可变区；
    VHB代表抗CD3抗体或其抗原结合片段的重链可变区；
    CH代表重链恒定区；
    CL代表轻链恒定区；
    L1、L2和L3各自独立地为键或接头；
    “～”代表二硫键或共价键；
    “-”代表肽键；
    优选地，所述双特异性抗体包含选自以下任一的结构：
    a)结构I的单体形成的同源二聚体；
    b)结构II的单体形成的同源二聚体。
  3. 如权利要求1所述的双特异性抗体，其特征在于，所述抗TROP-2抗体或其抗原结合片段的HCDR1-3和LCDR1-3氨基酸序列分别如SEQ ID NO.1-3和SEQ ID NO.4-6所示；和/或，所述抗CD3抗体或其抗原结合片段的HCDR1-3和LCDR1-3氨基酸序列分别如SEQ ID NO.7-9和SEQ ID NO.10-12所示；
    优选地，所述互补决定区包含至少一个氨基酸突变。
  4. 如权利要求3所述的双特异性抗体，其特征在于，所述突变选自以下任一项或多项：
    a)抗CD3抗体或其抗原结合片段的HCDR1：X1X2AMN，
    b)抗TROP-2抗体或其抗原结合片段的HCDR1：X3YWLG，
    c)抗CD3抗体或其抗原结合片段的LCDR2：X4TNKRAP，
    所述氨基酸突变选自X1不为T、X2不为Y、X3不为I、X4不为G的组；
    优选地，所述氨基酸突变选自X1为G，X2为S、H或G，X3为D或E，X4为A的组。
  5. 如权利要求4所述的双特异性抗体，其特征在于，所述双特异性抗体选自以下任一：
    1)所述抗CD3抗体或其抗原结合片段的HCDR1、HCDR2、HCDR3氨基酸序列分别如 SEQ ID NO.15、SEQ ID NO.8、SEQ ID NO.9所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12所示；
    和/或，所述抗TROP-2抗体或其抗原结合片段的HCDR1、HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.13或SEQ ID NO.14、SEQ ID NO.2、SEQ ID NO.3所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示；
    2)所述抗CD3抗体或其抗原结合片段的HCDR1为X1X2AMN，其中X1为G，X2为S，氨基酸序列如SEQ ID NO.16所示；HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.8、SEQ ID NO.9所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12所示；
    和/或，所述抗TROP-2抗体或其抗原结合片段的HCDR1为X3YWLG，其中X3为D，氨基酸序列如SEQ ID NO.13所示；HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.2、SEQ ID NO.3所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示；
    3)所述抗CD3抗体或其抗原结合片段的HCDR1为X1X2AMN，其中X1为G，X2为H，氨基酸序列如SEQ ID NO.17所示；HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.8、SEQ ID NO.9所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12所示；
    和/或，所述抗TROP-2抗体或其抗原结合片段的HCDR1为X3YWLG，其中X3为D，氨基酸序列如SEQ ID NO.13所示；HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.2、SEQ ID NO.3所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示；
    4)所述抗CD3抗体或其抗原结合片段的HCDR1为X1X2AMN，其中X1为G，X2为G，氨基酸序列如SEQ ID NO.18所示；HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.8、SEQ ID NO.9所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12所示；
    和/或，所述抗TROP-2抗体或其抗原结合片段的HCDR1为X3YWLG，其中X3为D，氨基酸序列如SEQ ID NO.13所示；HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.2、SEQ ID NO.3所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示；
    5)所述抗CD3抗体或其抗原结合片段的LCDR2为X4TNKRAP，其中X4为A，氨基酸序列如SEQ ID NO.19所示；LCDR1、LCDR3氨基酸序列分别如SEQ ID NO.10、SEQ ID NO.12所示，HCDR1、HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9所示；
    和/或，所述抗TROP-2抗体或其抗原结合片段的HCDR1为X3YWLG，其中X3为D，氨基酸序列如SEQ ID NO.13所示；HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.2、SEQ ID NO.3所示，LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示。
  6. 如权利要求1所述的双特异性抗体，其特征在于，所述抗CD3抗体或其抗原结合片段包 含重链可变区和轻链可变区，其中，重链可变区氨基酸序列如SEQ ID NO.22所示，轻链可变区氨基酸序列如SEQ ID NO.23所示；和/或，所述抗TROP-2抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，重链可变区氨基酸序列如SEQ ID NO.20所示，轻链可变区氨基酸序列如SEQ ID NO.21所示；
    优选地，所述可变区包含至少一个氨基酸突变；更优选地，所述氨基酸突变选自以下任一种或多种：
    1)所述SEQ ID NO.20具有第31位的氨基酸突变；
    2)所述SEQ ID NO.22具有第30位的氨基酸突变；
    3)所述SEQ ID NO.22具有第31位的氨基酸突变；
    4)所述SEQ ID NO.22具有第32位的氨基酸突变；
    5)所述SEQ ID NO.23具有第50位的氨基酸突变；
    优选地，所述双特异性抗体的可变区包含1)、3)和4)所示的突变；
    优选地，所述双特异性抗体的可变区包含1)、2)、3)和4)所示的突变；
    优选地，所述双特异性抗体的可变区包含1)和5)所示的突变。
  7. 如权利要求6所述的双特异性抗体，其特征在于，所述可变区包含选自以下任一项或多项的氨基酸突变：
    a)所述SEQ ID NO.20具有I31D的氨基酸突变；
    b)所述SEQ ID NO.20具有I31E的氨基酸突变；
    c)所述SEQ ID NO.22具有N30S的氨基酸突变；
    d)所述SEQ ID NO.22具有T31G的氨基酸突变；
    e)所述SEQ ID NO.22具有Y32S、Y32H或Y32G的氨基酸突变；
    f)所述SEQ ID NO.23具有G50A的氨基酸突变；
    优选地，所述双特异性抗体的可变区包含d)中所述T31G、e)中所述Y32S和a)中所述I31D突变，或所述双特异性抗体的可变区包含d)中所述T31G、e)中所述Y32S和b)中所述I31E突变；
    优选地，所述双特异性抗体的可变区包含c)中所述N30S、d)中所述T31G、e)中所述Y32S和a)中所述I31D突变，或所述双特异性抗体的可变区包含c)中所述N30S、d)中所述T31G、e)中所述Y32S和b)中所述I31E突变；
    优选地，所述双特异性抗体的可变区包含d)中所述T31G、e)中所述Y32H和a)中所述I31D突变，或所述双特异性抗体的可变区包含d)中所述T31G、e)中所述Y32H和b)中所述I31E突变；
    优选地，所述双特异性抗体的可变区包含d)中所述T31G、e)中所述Y32G和a)中所述I31D突变，或所述双特异性抗体的可变区包含d)中所述T31G、e)中所述Y32G和b)中所述I31E突变；
    优选地，所述双特异性抗体的可变区包含f)中所述G50A和a)中所述I31D突变，或所述双特异性抗体的可变区包含f)中所述G50A和b)中所述I31E突变。
  8. 如权利要求6所述的双特异性抗体，其特征在于，所述抗CD3抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，重链可变区氨基酸序列如SEQ ID NO.26所示，轻链可变区氨基酸序列如SEQ ID NO.23所示；和/或，所述抗TROP-2抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，重链可变区氨基酸序列如SEQ ID NO.24或SEQ  ID NO.25所示，轻链可变区氨基酸序列如SEQ ID NO.21所示。
  9. 如权利要求1所述的双特异性抗体，其特征在于，所述抗TROP-2或抗CD3抗原结合片段选自Fab、F(ab')、F(ab')2、Fv或scFv；优选地，所述抗TROP-2或抗CD3抗体为IgG抗体。
  10. 如权利要求9所述的双特异性抗体，其特征在于，所述D1为IgG抗体，所述D2为scFv；优选地，所述D2连接至D1的N端或C端，或连接至D1的CH1和CH2之间；更优选地，所述D2连接至D1的重链。
  11. 如权利要求1所述的双特异性抗体，其特征在于，所述双特异性抗体包含重链和轻链，所述重链和轻链的氨基酸序列选自以下任一：
    a)所述双特异性抗体的重链氨基酸序列如SEQ ID NO.36所示，和所述双特异性抗体的轻链氨基酸序列如SEQ ID NO.31所示；
    b)所述双特异性抗体的重链氨基酸序列如SEQ ID NO.37所示，和所述双特异性抗体的轻链氨基酸序列如SEQ ID NO.31所示；
    c)所述双特异性抗体的重链氨基酸序列如SEQ ID NO.38所示，和所述双特异性抗体的轻链氨基酸序列如SEQ ID NO.31所示；
    d)所述双特异性抗体的重链氨基酸序列如SEQ ID NO.39所示，和所述双特异性抗体的轻链氨基酸序列如SEQ ID NO.31所示；
    e)所述双特异性抗体的重链氨基酸序列如SEQ ID NO.40所示，和所述双特异性抗体的轻链氨基酸序列如SEQ ID NO.31所示；
    f)所述双特异性抗体的重链氨基酸序列如SEQ ID NO.41所示，和所述双特异性抗体的轻链氨基酸序列如SEQ ID NO.31所示；
    g)所述双特异性抗体的重链氨基酸序列如SEQ ID NO.42所示，和所述双特异性抗体的轻链氨基酸序列如SEQ ID NO.31所示；
    或，
    h)将a)至g)中的氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有同时抗TROP-2活性和抗CD3活性的由a)至g)衍生的多肽。
  12. 一种多核苷酸分子，其特征在于，所述多核苷酸分子编码如权利要求1至11中任一项所述的双特异性抗体。
  13. 一种表达载体，其特征在于，所述表达载体含有如权利要求12所述的多核苷酸分子。
  14. 一种细胞，其特征在于，所述细胞含有如权利要求13所述的表达载体。
  15. 一种如权利要求1至11中任一项所述的双特异性抗体的制备方法，其特征在于，所述制备方法包括以下步骤：
    a)在表达条件下，培养如权利要求14所述的细胞，从而表达双特异性抗体；
    b)分离并纯化步骤a)所述的双特异性抗体。
  16. 一种药物组合物，其特征在于，所述药物组合物包含有效量的如权利要求1至11中任一项所述的双特异性抗体和一种或多种药学上可接受的载体或辅料。
  17. 如权利要求1至11中任一项所述的双特异性抗体或如权利要求16所述的药物组合物在制备癌症或肿瘤的药物中的用途；优选地，所述癌症或肿瘤为TROP-2阳性癌症或肿瘤。
  18. 一种免疫偶联物，其特征在于，所述免疫偶联物包含：
    a)如权利要求1至11中任一项所述的双特异性抗体；和
    b)以下任一种或多种偶联物：可检测标记物、药物、毒素、细胞因子、放射性核素或酶。
  19. 一种治疗癌症或肿瘤的方法，其特征在于，所述方法包括向有需要的受试者施用权利要求1至11中任一项所述的双特异性抗体、权利要求16所述的药物组合物或权利要求18所述的免疫偶联物。
  20. 如权利要求19所述的方法，其特征在于，所述癌症或肿瘤选自：肺癌、骨癌、胃癌、胰腺癌、前列腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌、睾丸癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、直肠癌、结肠癌、肛门区癌、乳腺癌、食管癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、尿道癌、阴茎癌、前列腺癌、胰腺癌、脑癌、睾丸癌、淋巴癌、移行细胞癌、膀胱癌、肾癌、输尿管癌、肾细胞癌、肾盂癌、霍奇金淋巴瘤、非霍奇金淋巴瘤、软组织肉瘤、儿童实体瘤、淋巴细胞性淋巴瘤、中枢神经系统肿瘤、原发性中枢神经系统淋巴瘤、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、黑素瘤、卡波西肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤、慢性、急性白血病或其组合。
PCT/CN2023/094746 2022-05-18 2023-05-17 一种抗trop-2/cd3双特异性抗体 WO2023222027A1 (zh)

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