WO2023098770A1 - Anticorps bispécifique anti-trop-2/pd-l1 - Google Patents

Anticorps bispécifique anti-trop-2/pd-l1 Download PDF

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WO2023098770A1
WO2023098770A1 PCT/CN2022/135706 CN2022135706W WO2023098770A1 WO 2023098770 A1 WO2023098770 A1 WO 2023098770A1 CN 2022135706 W CN2022135706 W CN 2022135706W WO 2023098770 A1 WO2023098770 A1 WO 2023098770A1
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antibody
cancer
trop
bispecific antibody
seq
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PCT/CN2022/135706
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Chinese (zh)
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王定和
朱祯平
黄浩旻
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泽达生物医药有限公司
黄浩旻
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    • 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
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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    • 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
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    • 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
    • A61K47/6851—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 the antibody targeting a determinant of a tumour cell
    • 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
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • 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
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    • C07K2317/515—Complete light chain, i.e. VL + CL
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    • 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]
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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention belongs to the field of tumor treatment and biotechnology, and relates to a bispecific antibody against TROP-2 and PD-L1.
  • 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.
  • the ligand protein of TROP-2 has not been identified, so its physiological and biochemical functions are not very clear.
  • 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 US FDA has approved the TROP-2 antibody conjugate drug-sacituzumab govitecan for the treatment of metastatic triple-negative breast cancer.
  • PD-1 protein human programmed cell death receptor-1
  • PD-L1 programmed cell death-Ligand1
  • PD- L2 programmeed cell death-Ligand2
  • High expression of PD-L1 and PD-L2 has been found in various solid tumors and hematological malignancies, and there is increasing evidence that tumor cells use PD-1 and PD-L1-mediated immunosuppressive responses to evade immunity monitor.
  • Atezolizumab monoclonal antibody is the world's first approved PD-L1 monoclonal antibody drug for the treatment of locally advanced or metastatic urothelial carcinoma and non-small cell lung cancer.
  • Bispecific antibody also known as bifunctional antibody, is a specific drug that simultaneously targets two different antigens or different epitopes of the same antigen. This dual specificity opens up a wide range of applications, including redirecting T cells around tumor cells and simultaneously blocking two different signaling pathways.
  • the purpose of the present invention is to provide an anti-TROP-2 and PD-L1 bispecific antibody and its preparation method and application.
  • the purpose of the present invention is to provide a bispecific antibody capable of specifically binding to TROP-2 and PD-L1; to provide a nucleotide molecule encoding the bispecific antibody; to provide an expression vector comprising the nucleotide molecule providing a host cell comprising the expression vector; providing a preparation method of the bispecific antibody; providing a pharmaceutical composition comprising the bispecific antibody; providing an application of the bispecific antibody in the preparation of medicine.
  • a bispecific antibody comprising:
  • D1 specifically binds to the target molecule TROP-2 protein
  • D2 specifically binds to the target molecule PD-L1 protein
  • the D1 is an antibody or an antigen-binding fragment thereof that specifically binds to the TROP-2 protein; and/or
  • the D2 is an antibody or an antigen-binding fragment thereof that specifically binds to the PD-L1 protein;
  • the structure of the antigen-binding fragment is selected from the group consisting of: (i) Fab fragment; (ii) F(ab') 2 fragment; (iii) Fv fragment; or (iv) single-chain Fv (scFv).
  • the antibodies specifically binding to TROP-2 protein include: single-chain antibodies, diabodies, nanobodies, monoclonal antibodies, chimeric antibodies, murine antibodies, humanized antibodies and diabodies specific antibody.
  • the antibodies specifically binding to PD-L1 protein include: single-chain antibodies, diabodies, nanobodies, monoclonal antibodies, chimeric antibodies, murine antibodies, humanized antibodies and diabodies specific antibody.
  • the D1 and/or D2 are IgG antibodies.
  • the IgG antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.
  • the D1 and/or D2 is scFv.
  • the D1 is an anti-TROP-2 scFv.
  • the D2 is an IgG antibody against PD-L1.
  • the D1 comprises one, two, three or more anti-TROP-2 scFvs.
  • the D2 contains one, two, three or more anti-PD-L1 IgG antibodies.
  • the D1 is connected to a region of the anti-PD-L1 antibody selected from the group consisting of heavy chain variable region, heavy chain constant region, light chain variable region, light chain constant region or its combination.
  • the D1 is an anti-TROP-2 scFv; and the D2 is an anti-PD-L1 IgG antibody, wherein D1 is connected to the end of the heavy chain variable region of D2, or D1 is connected to the end of D2 The end of the heavy chain constant region.
  • the D1 is an IgG antibody against TROP-2.
  • the D2 is an anti-PD-L1 scFv.
  • the D1 comprises one, two, three or more anti-TROP-2 IgG antibodies.
  • the D2 comprises one, two, three or more anti-PD-L1 scFvs.
  • the D2 is connected to the region of the anti-TROP-2 antibody selected from the group consisting of heavy chain variable region, heavy chain constant region, light chain variable region, light chain constant region or its combination.
  • the D1 is an anti-TROP-2 IgG antibody
  • the D2 is an anti-PD-L1 scFv, wherein D2 is connected to the end of the heavy chain variable region of D1, or D2 is connected to the end of the D1 The end of the heavy chain constant region.
  • the D1 and the D2 are connected via a linker or directly.
  • sequence of the linker is (G4S)n, preferably, n is 1-4.
  • the bispecific antibody is a homodimer or a heterodimer, preferably a homodimer.
  • the bispecific antibody comprises an anti-PD-L1 IgG antibody and two anti-TROP-2 scFvs, wherein each scFv comprises a variable region VH and a variable region VL, and VH and VL pass through Linker L1 is connected, and each anti-TROP-2 scFv is connected in series with anti-PD-L1 immunoglobulin antibody IgG through linker L2.
  • the VL or VH in the anti-TROP-2 scFv is connected with the heavy chain variable region, heavy chain constant region, and light chain variable region of the anti-PD-L1 immunoglobulin antibody IgG through the linker L2. region or the light chain constant region.
  • the bispecific antibody comprises an anti-TROP-2 IgG antibody and two anti-PD-L1 scFv, wherein each scFv comprises a variable region VH and a variable region VL, and VH and VL pass through Linker L1 is connected, and each anti-PD-L1 scFv is connected in series with anti-TROP-2 immunoglobulin antibody IgG through linker L2.
  • the VL or VH in the anti-PD-L1 scFv is connected with the heavy chain variable region, heavy chain constant region, and light chain variable region of the anti-TROP-2 immunoglobulin antibody IgG through the linker L2. region or the light chain constant region.
  • the bispecific antibody is a homodimer, which has a structure represented by formula Ia, Ib, IIa or IIb from the N-terminus to the C-terminus:
  • VLA represents the light chain variable region of an anti-TROP-2 antibody
  • VHA represents the heavy chain variable region of an anti-TROP-2 antibody
  • VL B represents the light chain variable region of the anti-PD-L1 antibody
  • VH B represents the heavy chain variable region of the anti-PD-L1 antibody
  • CH represents the heavy chain constant region
  • CL represents the light chain constant region
  • L1 and L2 are each independently a bond or a joint
  • ⁇ represents a disulfide bond or a covalent bond
  • the bispecific antibody has the activity of binding TROP-2 and PD-L1 simultaneously.
  • the heavy chain constant region is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.
  • the bispecific antibody blocks the binding of PD-1 and PD-L1.
  • the bispecific antibody degrades PD-L1 protein.
  • sequence of the linker L1 is shown in SEQ ID NO.17.
  • sequence of the linker L2 is shown in SEQ ID NO.18.
  • the anti-PD-L1 antibody comprises the following three complementarity determining regions HCDR:
  • the anti-PD-L1 antibody comprises the following three complementarity determining regions LCDR:
  • the anti-PD-L1 antibody comprises the heavy chain variable region shown in SEQ ID NO.15.
  • the anti-PD-L1 antibody comprises the light chain variable region shown in SEQ ID NO.16.
  • the anti-TROP-2 antibody comprises the following three complementarity determining regions HCDR:
  • the anti-TROP-2 antibody comprises the following three complementarity determining regions LCDR:
  • the anti-TROP-2 antibody comprises the heavy chain variable region shown in SEQ ID NO.13.
  • the anti-TROP-2 antibody comprises the light chain variable region shown in SEQ ID NO.14.
  • sequence of the anti-TROP-2 scFv is shown in SEQ ID NO.19.
  • the bispecific antibody is selected from the following group:
  • the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.20, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.21;
  • amino acid sequence in (1) or (2) is formed by the substitution, deletion or addition of one or more amino acid residues, and has simultaneous anti-TROP-2 activity and anti-PD-L1 activity ( 1) or (2) derived polypeptides.
  • the bispecific antibody comprises an active fragment and/or a derivative of the bispecific antibody, wherein the active fragment and/or the derivative retains the bispecific antibody 70-100% (such as 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) anti-TROP-2 activity and 70-100% % of anti-PD-L1 activity.
  • the derivative of the antibody has at least 85% sequence identity with the antibody of the present invention.
  • the derivative of the antibody is a sequence of the antibody of the present invention that undergoes one or several amino acid deletions, insertions and/or substitutions and maintains at least 85% identity.
  • the derivative of the antibody has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% %, 97%, 98%, 99% sequence identity.
  • the substitution is a conservative substitution.
  • the bispecific antibody comprises an anti-TROP-2 scFv comprising the heavy chain variable region shown in SEQ ID NO.13 or a derivative sequence thereof, the derivative sequence comprising a sequence selected from the group consisting of Amino acid substitutions at one or more positions: 5, 31, 32, 38, 46, 48, 50, 53, 58, 60, 61, 62, 68 and 97 of the amino acid sequence shown in SEQ ID NO.13 .
  • the derivative sequence comprises amino acid substitutions at one or more positions selected from the group consisting of: Q5V, N31D, Y32Q, K38R, K46E, M48I, W50Y, Y53K, T58Y, T60A, D61E, D62E , A68V and F97Y.
  • the bispecific antibody comprises an anti-TROP-2 scFv, which comprises the light chain variable region shown in SEQ ID NO.14 or its derivative sequence, and the derivative sequence comprises Amino acid mutations at one or more positions: 4, 20, 24, 30, 31, 53, 54, 56, 60, 85, 93 and 96 of the amino acid sequence shown in SEQ ID NO.14.
  • the derivative sequence comprises amino acid substitutions at one or more positions selected from the group consisting of L4M, S20T, K24R, S30N, I31T, Y53F, R54L, T56S, D60R, V85T, I93T and L96P .
  • the anti-PD-L1 antibody comprises the heavy chain variable region shown in SEQ ID NO.15 or its derivative sequence, and the derivative sequence comprises one or more positions selected from the following group Amino acid mutation: positions 28, 31, 40, 41, 234, 235, 252, 254 and 256 of the amino acid sequence shown in SEQ ID NO.15.
  • the derivative sequence comprises amino acid substitutions at one or more positions selected from the group consisting of S28Q, S31Q, P40A, P41A, L234A, L235A, M252Y, S254T and T256E.
  • the anti-PD-L1 antibody comprises the light chain variable region shown in SEQ ID NO.16 or its derivative sequence, and the derivative sequence comprises one or more positions selected from the following group Amino acid mutation: positions 24, 28, 29, 31, 36 and 89 of the amino acid sequence shown in SEQ ID NO.16.
  • the derivative sequence comprises amino acid substitutions at one or more positions selected from the group consisting of R24K, S28V, I29V, T31S, Y36F and Q89L.
  • a polynucleotide molecule encoding the bispecific antibody according to the first aspect of the present invention is provided.
  • polynucleotide molecular sequence encoding the heavy chain of the bispecific antibody is shown in SEQ ID NO: 23 or SEQ ID NO.25.
  • the molecular sequence of the polynucleotide encoding the light chain of the bispecific antibody is shown in SEQ ID NO: 24.
  • an expression vector containing the polynucleotide molecule according to the second aspect of the present invention is provided.
  • the expression vector is a virus or a plasmid, preferably a phage or a phagemid.
  • the expression vector is selected from the group consisting of pcDNA3.4, pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0 , preferably pcDNA3.4.
  • a host cell containing the expression vector according to the third aspect of the present invention is provided.
  • the host cell is selected from the group consisting of COS, CHO, NSO, sf9, sf21, DH5 ⁇ , BL21(DE3) or TG1, more preferably E.coli TG1, BL21(DE3) cells ( expressing single-chain antibody or Fab antibody) or CHO-K1 cells (expressing full-length IgG antibody).
  • the host cells are eukaryotic cells, preferably CHO cells or 293F cells.
  • step b) isolating and purifying the bispecific antibody described in step a).
  • a pharmaceutical composition comprising an effective amount of the bispecific antibody according to the first aspect of the present invention and one or more pharmaceutically acceptable carrier, diluent or excipient.
  • the bispecific antibody according to the first aspect of the present invention or the pharmaceutical composition according to the sixth aspect of the present invention in the preparation of a drug for cancer or tumor .
  • the cancer or tumor is selected from: lung cancer, bone cancer, gastric cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer , cervical cancer, vaginal cancer, vulvar cancer, rectal cancer, colon cancer, anal region cancer, breast cancer, esophagus cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer, penile cancer , prostate cancer, pancreatic cancer, brain cancer, testicular cancer, lymphoma, transitional cell carcinoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, Hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma , childhood solid tumors, lymphocytic lymphoma, central nervous system (CNS) tumors, primary central nervous system lymphoma, tumor angiogenesis, spinal
  • an immunoconjugate comprising:
  • a conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
  • the conjugate part is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computerized X-ray tomography) contrast agents, or capable of producing visible Detect enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.) of the products.
  • the immunoconjugate includes antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the immunoconjugate is used to prepare a pharmaceutical composition for treating cancer or tumor.
  • a method of treating cancer or tumor comprising administering to a subject in need the bispecific antibody described in the first aspect of the present invention, according to the present invention
  • the pharmaceutical composition according to the sixth aspect of the invention, and the immunoconjugate according to the eighth aspect of the present invention comprising administering to a subject in need the bispecific antibody described in the first aspect of the present invention, according to the present invention.
  • the method further includes co-administering with other antineoplastic drugs.
  • the positive and progressive effect of the present invention is that the TROP-2/PD-L1 bispecific antibody can degrade the PD-L1 of TROP-2 positive tumor cells, thereby continuously blocking the combination of PD-1/PD-L1 and exerting its superiority. Effect on PD-L1 monoclonal antibody.
  • Figure 1A shows a schematic diagram of the structure of anti-TROP-2/PD-L1 double antibody a.
  • Figure 1B shows a schematic diagram of the structure of anti-TROP-2/PD-L1 double antibody b.
  • Figure 2A shows the HPLC detection spectrum of anti-TROP-2/PD-L1 double antibody a.
  • Figure 2B shows the HPLC detection spectrum of anti-TROP-2/PD-L1 double antibody b.
  • Figure 3A shows the ELISA detection of the binding of anti-TROP-2/PD-L1 double antibody a, b to TROP-2.
  • Figure 3B shows the ELISA detection of the binding of anti-TROP-2/PD-L1 double antibody a, b to PD-L1.
  • Figure 4A shows the detection results of the affinity constant between anti-TROP-2/PD-L1 double antibody a and TROP-2.
  • Figure 4B shows the detection results of the affinity constant between anti-TROP-2/PD-L1 double antibody a and PD-L1.
  • Figure 4C shows the detection results of the affinity constant between anti-TROP-2/PD-L1 double antibody b and TROP-2.
  • Figure 4D shows the detection results of the affinity constant between anti-TROP-2/PD-L1 double antibody b and PD-L1.
  • Figure 5A shows the degradation of PD-L1 protein in NCI-H292 cells by anti-TROP-2/PD-L1 double antibody a-1.
  • Figure 5B shows the degradation of PD-L1 protein in NCI-H292 cells by anti-TROP-2/PD-L1 bis-antibody a-2.
  • Figure 6A shows the cellular level activity of anti-TROP-2/PD-L1 bis-antibody a blocking PD1/PD-L1 binding-1.
  • Figure 6B shows the cellular level activity of anti-TROP-2/PD-L1 bis-antibody a to block PD1/PD-L1 binding-2.
  • Figure 6C shows the blocking of PD1/PD-L1 binding at the cellular level by anti-TROP-2/PD-L1 bis-antibody a-3.
  • Figure 7 shows the MLR results of anti-TROP-2/PD-L1 double antibody a.
  • Figure 8A shows the anti-tumor effect of anti-TROP-2/PD-L1 double antibody a on NCI-H292 xenograft tumor model.
  • Figure 8B shows the degradation of PD-L1 protein in NCI-H292 xenograft tumor by anti-TROP-2/PD-L1 biantibody a.
  • Figure 9A shows the DSC plot of anti-TROP-2/PD-L1 double antibody a.
  • Figure 9B shows the DSC plot of anti-TROP-2/PD-L1 double antibody b.
  • Figure 9C shows the SEC results of the 37°C stability of anti-TROP-2/PD-L1 double antibody a at -0.
  • Figure 9D shows the SEC results of the 37°C stability of anti-TROP-2/PD-L1 double antibody a-28 days.
  • the inventors obtained for the first time a bispecific antibody targeting tumor cell surface molecules TROP-2 and PD-L1.
  • the bispecific antibody of the present invention is obtained by linking the single-chain variable fragment scFv and the immunoglobulin antibody IgG through a peptide linker, which can maintain the activity of both ends of the antibody and can simultaneously bind to TROP-2 and PD-L1 antigens.
  • the bispecific antibody of the present invention can degrade the PD-L1 protein of TROP-2 positive tumor cells at the cellular level, thereby blocking the binding of PD-1 on the surface of T cells; in animal experiments, hPBMC immune system
  • the experimental results of the humanized mouse NCI-H292 model show that the bispecific antibody can inhibit tumor growth and exhibit a synergistic effect of the combination of the two monoclonal antibodies.
  • the present invention has been accomplished on this basis.
  • the term “comprises” or “includes (comprising)” can be open, semi-closed and closed. In other words, the term also includes “consisting essentially of”, or “consisting of”.
  • the term "antibody (Antibody, abbreviated Ab)” and “immunoglobulin G (Immunoglobulin G, abbreviated IgG)” are heterotetrameric glycoproteins with the same structural characteristics, which consist of two identical light chains (L ) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable region (VH) at one end followed by a constant region consisting of three domains CH1, CH2, and CH3.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end.
  • the constant region of the light chain includes a domain CL; the constant region of the light chain is paired with the CH1 domain of the constant region of the heavy chain.
  • the variable region is paired with the variable region of the heavy chain.
  • the constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity) and so on.
  • the heavy chain constant region includes IgG1, IgG2, IgG3, IgG4 subtypes; the light chain constant region includes kappa (Kappa) or lambda (Lambda).
  • the heavy and light chains of an antibody are covalently linked together by a disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are linked together by an interpolypeptide disulfide formed between the hinge regions. bonded together covalently.
  • the "immunoglobulin antibody IgG" of the present invention is a molecule of about 150kDa, which is composed of four peptide chains, contains two identical heavy chains of about 50kDa gamma, and two identical light chains of about 25kDa, thus having Tetrameric quaternary structure.
  • the two heavy chains are linked to each other by disulfide bonds and each to a light chain.
  • the resulting tetramer has 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 subclasses (eg, IgGl, 2, 3, 4) based on minor differences in the amino acid sequence in the constant region of the heavy chain.
  • bispecific antibody refers to an antibody molecule that can specifically bind 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 (mAb)” refers to an antibody obtained from a substantially homogeneous population, ie, the individual antibodies contained in the population are identical except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on an antigen, unlike conventional polyclonal antibody preparations, which are typically a mixture of different antibodies directed against different antigenic determinants. 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 identity of the antibody, which is obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring any particular method for producing the antibody.
  • antigen-binding fragment refers to a Fab fragment, Fab' fragment, F(ab') 2 fragment, or a single Fv fragment having antigen-binding activity.
  • antigen-binding fragments include: (i) Fab fragments; (ii) F(ab') 2 fragments; (iii) Fv fragments; or (iv) single chain Fv (scFv).
  • the expression "antigen-binding fragment” internally also encompasses other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies , tetrabodies, minibodies, nanobodies (eg, monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.
  • SMIPs small modular immunopharmaceuticals
  • 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 of the heavy chain and the VL and CL domains of the light chain of the antibody.
  • the Fc segment is a crystallizable fragment (fragment crystallizable, Fc), which is composed of the CH2 and CH3 domains of the antibody.
  • the Fc segment has no antigen-binding activity and is the site where the antibody interacts with effector molecules or cells.
  • F(ab') 2 fragment F(ab') 2 fragment antibody is obtained by digesting whole IgG antibody with pepsin, removing most of the Fc region while leaving some hinge region intact, with the The two antigen-binding F(ab) portions of .
  • scFv single chain antibody
  • scFv single chain antibody fragment
  • scFv single chain antibody fragment
  • scFv single chain antibody fragment
  • VH and VL are linked by a 15-25 amino acid linker, wherein the fusion protein retains the same antigenic specificity of the complete immunoglobulin.
  • Fv fragment or "Fv antibody” contains the antibody heavy chain variable region, light chain variable region, but no constant region, and the smallest antibody fragment with all antigen-binding sites.
  • Fv fragments also contain a polypeptide linker between the VH and VL domains and are capable of forming structures required for antigen binding.
  • variable means that certain parts of the variable regions in antibodies differ in sequence, which contribute to the binding and specificity of various specific antibodies to their specific antigens.
  • variability is not evenly distributed throughout antibody variable domains. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy-chain variable region and the light-chain variable region.
  • CDRs complementarity-determining regions
  • FR frame region
  • the variable domains of native heavy and light chains each contain four FR regions that are roughly in a ⁇ -sheet configuration connected by three CDRs that form connecting loops, in some cases forming partial ⁇ -sheet structures.
  • the CDRs in each chain are in close proximity through the FR regions 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, Vol. I, pp. 647-669 (1991)).
  • FR framework region
  • the light and heavy chains of immunoglobulins each have four FRs, referred to as FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, 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 can thus be expressed 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, and the human antibody FR derivative is substantially identical to a naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, or 96%. , 97%, 98%, or 99%.
  • human framework region is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to that of a naturally occurring human antibody .
  • linker refers to one or more amino acid residues inserted into an immunoglobulin domain to provide sufficient mobility for the domains of the light and heavy chains to fold into an exchanged dual variable region immunoglobulin base.
  • preferred linkers refer to linkers L1 and L2, wherein L1 connects the VH and VL of a single chain antibody (scFv), and L2 is used to connect the scFv with the heavy chain of another antibody.
  • linkers include monoglycine (Gly), or serine (Ser) residues, the identity and sequence of the amino acid residues in the linker can vary with the type of secondary structural elements that need to be achieved 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 its target antigen.
  • the antibody binds the antigen with an equilibrium dissociation constant (KD) of less than about 10" 7 M, eg, less than about 10 "8 M, 10 “9 M, 10 "10 M, 10" 11 M or less.
  • KD equilibrium dissociation constant
  • the term “KD” refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • SPR Surface Plasmon Resonance
  • epitope refers to a polypeptide determinant that specifically binds to an antibody.
  • An epitope of the present invention is a region of an antigen bound by an antibody.
  • the bispecific antibody of the present invention is a bispecific antibody capable of specifically binding to TROP-2 and PD-L1, which comprises an anti-PD-L1 antibody part and an anti-TROP-2 antibody part. Specifically, it comprises an immunoglobulin antibody IgG and two identical single-chain variable fragment scFvs, wherein each single-chain variable fragment scFv comprises a variable region VH and a variable region VL, and VH and VL are passed through a peptide linker L1 Linked, each single-chain variable fragment scFv is connected in series with the immunoglobulin antibody IgG through the linker peptide L2.
  • bispecific antibody refers to a bispecific antibody that has two different antigen-binding sites and can simultaneously bind TROP-2 and PD-L1, which contains two single-chain variable fragments scFv and Conjugated with immunoglobulin antibody IgG, each scFv is connected to each heavy chain of immunoglobulin antibody IgG via a peptide linker L2 to form a heavy chain fusion protein of a bispecific antibody, wherein each scFv comprises a variable region VH And the variable region VL, VH and VL are linked by a peptide linker L1.
  • the peptide linker has sufficient length and flexibility to ensure that the two proteins connected have enough degrees of freedom in space to perform their functions.
  • a flexible peptide linker eg 0-25 amino acids, is preferred.
  • Preferably it is (G4S)n, wherein n is a positive integer of 1-5.
  • the anti-PD-L1 antibody part in the bispecific antibody of the present invention is an antibody or an antigen-binding fragment thereof that specifically binds to the PD-L1 protein
  • the anti-TROP-2 antibody part is an antibody that specifically binds to the TROP-2 protein.
  • Antibody or antigen-binding fragment thereof; the structure of the antigen-binding fragment can be selected from the group consisting of: (i) Fab fragment; (ii) F(ab') 2 fragment; (iii) Fv fragment; or (iv) single chain Fv (scFv).
  • the bispecific antibody of the present invention can be a dimer, a trimer or a multimer, preferably a homodimer or a heterodimer.
  • the anti-PD-L1 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 comprises scFv of anti-TROP-2 antibody and IgG antibody of PD-L1, wherein the VH of said anti-TROP-2 antibody comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein HCDR1
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO: 1
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3;
  • the VL of the anti-TROP-2 antibody comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 As shown in SEQ ID NO: 6;
  • the VH of the IgG antibody to PD-L1 comprises complementary determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 7, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 8, and the amino acid sequence of HCDR3 The sequence is shown in SEQ ID NO: 9;
  • the VL of the IgG antibody to PD-L1 comprises complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 10, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of LCDR3 The sequence is shown in SEQ ID NO:12.
  • the binding region of an antibody usually contains a light chain variable region and a heavy chain variable region, and each variable region contains 3 CDR domains.
  • the CDR domains of the heavy and light chains of an antibody are called HCDR and LCDR, respectively.
  • a conventional antibody antigen-binding site comprises six CDRs, including sets of CDRs from the heavy and light chain V regions, respectively.
  • the bispecific antibody of the present invention also includes its conservative variant, which means that compared with the amino acid sequence of the bispecific antibody of the present invention, there are at most 10, preferably at most 8, and more preferably at most 5, preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.
  • the amino acid sequence of the VH of the scFv is shown in SEQ ID NO: 13 or on the basis of the sequence of SEQ ID NO: 13, it also has an amino acid sequence selected from 5, 31, 32, 38, 46, 48, 50, 53, 58, 60, 61, 62, 68 and 97 in one or more amino acid mutation sequence; preferably have a sequence selected from Q5V, N31D, Y32Q, K38R, K46E, M48I, W50Y, Y53K, T58Y, T60A, D61E, The sequence of one or more amino acid mutations in D62E, A68V and F97Y; the amino acid sequence of VL is shown in SEQ ID NO: 14 or on the basis of the sequence of SEQ ID NO: 14, it also has a sequence selected from 4, 20, 24, 30, 31, 53, 54, 56, 60, 85, 93 and 96 in one or more amino acid mutation sequences; preferably having a sequence selected from L4M, S20T, K24
  • amino acid sequence of the peptide linker L1 is shown in SEQ ID NO: 17.
  • amino acid sequence of the peptide linker L2 is shown in SEQ ID NO: 18.
  • the molecular structure of the single-chain variable fragment scFv is VH-L1-VL, and the N-terminal of each scFv is connected to the C-terminal of the IgG heavy chain of an immunoglobulin antibody via a peptide linker L2.
  • the molecular structure of the single-chain variable fragment scFv is VH-L1-VL, and the C-terminal of each scFv is connected to the N-terminal of the IgG heavy chain of an immunoglobulin antibody via a peptide linker L2.
  • amino acid sequence of the single-chain variable fragment scFv is shown in SEQ ID NO: 19.
  • amino acid sequence of the heavy chain of the bispecific antibody is shown in SEQ ID NO: 20
  • amino acid sequence of the light chain is shown in SEQ ID NO: 21.
  • amino acid sequence of the heavy chain of the bispecific antibody is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain is shown in SEQ ID NO: 21.
  • bispecific antibody of the present invention problems related to the chemical and physical stability of the bispecific antibody are also addressed, such as expressing a physically stable molecule, increasing heat and salt-dependent stability, reducing aggregation, Increase solubility at high concentrations and maintain affinity for two antigens, TROP-2 and PD-L1, respectively, etc.
  • a polynucleotide of the invention may be in the form of DNA or RNA.
  • Forms of DNA include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be either the coding strand or the non-coding strand.
  • nucleotide sequence of the heavy chain of the nucleotide molecule encoding the bispecific antibody that can specifically bind to TROP-2 and PD-L1 is shown in SEQ ID NO: 23, encoding the nucleotide sequence of the light chain The nucleotide sequence is shown in SEQ ID NO: 24; or the nucleotide sequence of the nucleotide molecule encoding the heavy chain of the bispecific antibody that can specifically bind to TROP-2 and PD-L1 is shown in SEQ ID NO: 25, the nucleotide sequence encoding its light chain is shown in SEQ ID NO: 24.
  • the preparation method of the nucleotide molecule of the present invention is a conventional preparation method in the art, preferably including the following preparation method: obtain the nucleotide molecule encoding the above-mentioned monoclonal antibody by gene cloning technology such as PCR method, or obtain the nucleotide molecule encoding the above-mentioned monoclonal antibody by The nucleotide molecule encoding the above-mentioned monoclonal antibody is obtained by the method of artificial complete sequence synthesis.
  • nucleotide sequence encoding the amino acid sequence of the above-mentioned bispecific antibody can be properly introduced with substitutions, deletions, changes, insertions or additions to provide a polynucleotide homologue.
  • the homologue of the polynucleotide in the present invention can be prepared by replacing, deleting or adding one or more bases in the gene encoding the bispecific antibody within the scope of maintaining the activity of the antibody.
  • Another aspect of the present invention provides an expression vector, which contains the above-mentioned nucleotide molecules.
  • the expression vector is a conventional expression vector in the art, which refers to containing 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 a suitable phage or phagemid.
  • a suitable phage or phagemid for more technical details, please refer to, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Many known techniques and protocols for nucleic acid manipulation are found in Current Protocols in Molecular Biology, 2nd Edition, edited by Ausubel et al.
  • the expression vector of the present invention is preferably pDR1, pcDNA3.1 (+), pcDNA3.4, pcDNA3.1/ZEO (+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, more preferably pcDNA3 .4.
  • the present invention further provides a host cell containing the above expression vector.
  • the host cells described in the present invention are various conventional host cells in the field, as long as the above-mentioned recombinant expression vector can stably replicate itself and the nucleotides carried can be effectively expressed.
  • said host cells include prokaryotic expression cells and eukaryotic expression cells
  • said host cells preferably include: COS, CHO (Chinese Hamster Ovary, Chinese Hamster Ovary), NSO, sf9, sf21, DH5 ⁇ , BL21 (DE3) Or TG1, more preferably E.coli TG1, BL21 (DE3) cells (expressing single-chain antibody or Fab antibody) or CHO-K1 cells (expressing full-length IgG antibody).
  • the preferred recombinant expression transformants of the present invention can be obtained by transforming the aforementioned expression vectors into host cells.
  • the conversion method is a conventional conversion method in the art, preferably a chemical conversion method, a heat shock method or an electroporation method.
  • the host cell is a eukaryotic cell.
  • CHO cells or 293F cells are preferred.
  • recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.
  • Another aspect of the present invention provides a method for preparing the above-mentioned bispecific antibody capable of specifically binding to TROP-2 and PD-L1, the preparation method comprising the following steps:
  • step b) isolating and purifying the bispecific antibody described in step a).
  • the method for culturing host cells and the method for isolating and purifying antibodies described in the present invention are conventional methods in the art.
  • For specific operation methods please refer to the corresponding cell culture technical manual and antibody separation and purification technical manual.
  • the preparation method of the anti-TROP-2/PD-L1 bispecific antibody disclosed in the present invention comprises: cultivating the above-mentioned host cells under expression conditions, so as to express the bispecific antibody that can specifically bind to TROP-2 and PD-L1 Antibody; separating and purifying the anti-TROP-2/PD-L1 bispecific antibody.
  • recombinant proteins can be purified to a substantially homogeneous substance.
  • the anti-TROP-2/PD-L1 bispecific antibody 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 and other methods elute the anti-TROP-2/PD-L1 bispecific antibody bound to the affinity column.
  • the inventors of the present invention conducted a detection experiment on the obtained anti-TROP-2/PD-L1 bispecific antibody, and the experimental results showed that the anti-TROP-2/PD-L1 bispecific antibody can well bind to target cells and antigens , with high affinity.
  • compositions comprising the aforementioned bispecific antibody capable of specifically binding to TROP-2 and PD-L1 and one or more pharmaceutically acceptable carriers , diluent or excipient.
  • said composition is a pharmaceutical composition.
  • the bispecific antibody provided by the present invention can form a pharmaceutical preparation composition together with a pharmaceutically acceptable carrier to exert a more stable therapeutic effect.
  • These preparations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody of the present invention , while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation).
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated.
  • the prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavitary injection.
  • intravenous injection intravenous infusion
  • subcutaneous injection local injection
  • intramuscular injection intratumoral injection
  • intraperitoneal injection such as intraperitoneal injection
  • intracranial injection or intracavitary injection.
  • intracranial injection or intracavitary injection.
  • liquid formulations it can be stored at 2°C-8°C for at least one year, and for lyophilized formulations, it is stable for at least six months at 30°C.
  • the bispecific antibody preparations can be suspension, aqueous injection, freeze-dried and other preparations commonly used in the pharmaceutical field.
  • the term "pharmaceutical composition” means that the bispecific antibody of the present invention can form a pharmaceutical preparation composition together with a pharmaceutically acceptable carrier so as to exert a more stable therapeutic effect. These preparations can guarantee the bispecific antibody disclosed in the present invention.
  • the conformational integrity of the amino acid core sequence of the antibody is maintained, while the multifunctional groups of the protein are protected from degradation (including but not limited to aggregation, deamination, or oxidation).
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned bispecific antibody (or its conjugate) of the present invention and pharmaceutically acceptable carrier or excipient.
  • Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the bispecific antibodies of the invention can also be used with other therapeutic agents.
  • a safe and effective amount of the bispecific antibody or immunoconjugate thereof is administered to the mammal, wherein the safe and effective amount is usually at least about 10 ⁇ g/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • Another aspect of the present invention provides the application of the above-mentioned bispecific antibody capable of specifically binding to TROP-2 and PD-L1, or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating cancer or tumor.
  • the medicaments for treating cancer or tumor referred to in the present invention refer to the medicaments for suppressing and/or treating tumors, which may include the delay of the development of accompanying tumor-related symptoms and/or the reduction of the severity of these symptoms, and further include existing Relief of symptoms associated with tumors and prevention of other symptoms, including reduction or prevention of tumor metastasis.
  • the tumors targeted by the drug of the present invention preferably include but are not limited to: lung cancer, bone cancer, gastric cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, uterine cancer, fallopian tube cancer, uterine cancer Endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, rectal cancer, colon cancer, anal region cancer, breast cancer, esophagus cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, urethral cancer , penile cancer, prostate cancer, pancreatic cancer, brain cancer, testicular cancer, lymphoma, transitional cell carcinoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, Hodgkin's disease, non-Hodgkin's lymphoma , soft tissue sarcomas, childhood solid tumors, lymphocytic lymphomas, central nervous system (CNS) tumors, primary CNS
  • the dosage varies according to the age and body weight of the patient, the nature and severity of the disease, and the route of administration. Reference can be made to animal experiments The results and various situations, the total dosage should not exceed a certain range. Specifically, the dosage for intravenous injection is 1-1800 mg/day.
  • the bispecific antibody of the present invention and its composition can also be administered in combination with other antineoplastic drugs to achieve more effective tumor treatment.
  • antineoplastic drugs include but are not limited to: 1. Cytotoxic drugs: 1) Function Drugs based on the chemical structure of nucleic acids: alkylating agents such as nitrogen mustards, nitrosoureas, and methylsulfonates; platinum compounds such as cisplatin, carboplatin, and oxaliplatin; Antibiotics such as Adriamycin/Doxorubicin, Dactinomycin D, Daunorubicin, Epirubicin, Mithramycin, etc.; 2) affect nucleic acid metabolism Drugs: dihydrofolate reductase inhibitors such as methotrexate (MTX) and pemetrexed (Pemetrexed), etc.; thymidine synthase inhibitors such as fluorouracils (5-fluorouracil, capecitabine), etc.; Purine nucleoside syntha
  • Hormone drugs antiestrogens Hormones such as Tamoxifen, Droloxifene, Exemestane, etc.; aromatase inhibitors such as Aminoglutethimide, Formestane, Letrox Letrozle, Anastrozole, etc.; anti-androgens: flutamide RH-LH agonists/antagonists: Noreide, Enatone, etc.; 3.
  • Biological response modifier drugs these drugs Mainly by regulating the immune function of the body to achieve anti-tumor effects, such as interferon (Interferon); interleukin-2 (Interleukin-2); thymosins (Thymosins), etc.; 4.
  • Monoclonal antibody drugs tratuximab Monoclonal antibody (Trastuzumab), Rituximab (Rituximab), Cetuximab (Cetuximab), Bevacizumab (Bevacizumab), etc.; Drugs for further study, etc.
  • the bispecific antibody and the composition thereof disclosed in the present invention can be administered in combination with one or a combination of the above-mentioned anti-tumor drugs.
  • the present invention provides for the first time a bispecific antibody that can simultaneously target tumor cell surface molecules TROP-2 and PD-L1 antigens.
  • the bispecific antibody provided by the present invention can degrade the PD-L1 protein of TROP-2 positive tumor cells, thereby blocking the combination of PD-1/PD-L1, releasing the inhibitory state of T cells, and exerting anti-tumor effect effect.
  • the bispecific antibody provided by the invention can enhance the immune response.
  • 293F cells brand GIBCO, product number R79007.
  • PD-L1aAPC/CHO-K1 cells Brand Promega, Cat. No. J1252.
  • PBMC purchased from Aussells, product number: FPB004F-C-MLR.
  • DC cells purchased from Aussells, Cat. No. FPB-DC002F-C.
  • 0.45 ⁇ m filter brand Millipore, product number SLHV013SL.
  • PVDF membrane brand Millipore, product number IPVH00010.
  • Anti-Human IgG Fc probe brand FORTEBIO, Cat. No. 18-5060.
  • Coating solution 1.59 grams of sodium carbonate, 2.93 grams of sodium bicarbonate, add double distilled water to make up to 1L.
  • TROP-2-HIS protein brand Kaijia, product number TRP-HM121.
  • ELISA blocking solution PBST+1%BSA.
  • HRP-labeled goat anti-human FC antibody brand Boaolong, product number BF03031.
  • TMB Brand BD, item number 555214.
  • BSA purchased from Sangon Bioengineering (Shanghai) Co., Ltd., product number A60332.
  • Stop solution 2M sulfuric acid solution.
  • HBS-EP working fluid brand GE, product number BR-1006-69.
  • Trypsin-EDTA brand GIBCO, Cat. No. 25200-072.
  • 1640 complete medium RPMI1640 medium + 10% FBS + 1% Pen Strep + 1% sodium pyruvate.
  • RPMI1640 medium brand GIBCO, product number 22400089.
  • FBS brand GIBCO, item number 10099-141.
  • Pen Strep brand Gibco, item number 1514022.
  • RIPA Lysis Solution Brand Thermo, Cat. No. 89900.
  • Phosphatase inhibitors brand Thermo, Cat. No. 78443.
  • PMSF brand solarbio, item number P0100-1.
  • Loading buffer brand bio-RAD, Cat. No. 1610747.
  • Bio-Glo available from Promega, Cat. No. G7940.
  • Streptavidin HRP purchased from BD Biosciences, catalog number: 554066.
  • Biotin Mouse Anti-Human IL-2 purchased from BD Pharmingen, product number: 555040; 0.5mg.
  • Recombinant Human IL-2 purchased from BD Pharmingen, Cat. No.: 554603; 10ug.
  • Electrophoresis buffer Tris 75g, Glycine 360g, Sodium Lauryl Sulfate 25g.
  • 1 ⁇ transfer buffer 100mL 10 ⁇ transfer buffer, 700mL double distilled water, 200mL methanol.
  • 10 ⁇ transfer buffer 60.6g tris, 288g glycine.
  • WESTREN BOLT blocking solution TBST+5% blotting-grade blocker.
  • Blotting-grade blocker brand bio-RAD, catalog number 1706404.
  • PD-L1 rabbit IgG antibody brand cell signaling, product number 13684s.
  • HRP-labeled rabbit anti-human goat IgG antibody brand Boaolong, product number BF03008X.
  • DMEM complete medium DMEM high glucose medium + 10% FBS + 1% Pen Strep + 1% GLUMAX.
  • DMEM high glucose medium brand GIBCO, product number 11965-092.
  • GlutaMAX brand GIBCO, Cat. No. 35050-061.
  • ⁇ -mercaptoethanol brand Shanghai style, product number 80076928.
  • Polybrene co-infection agent brand sigma, product number H9268.
  • Lipofectamine 3000 brand thermo, product number L3000015.
  • F-12 complete medium F-12 medium + 10% FBS + 250 ⁇ g/ml G418 + 200 ⁇ g/ml hygromycin B.
  • F-12 medium brand GIBCO, product number 11765054.
  • G418Sulfate brand GIBCO, item number 1013102.
  • AIM-V+AlbuMAX (BSA) (1X) medium brand GIBCO, product number 31035-025.
  • Hygromycin B brand merck product number 400052.
  • Streptavidin HRP purchased from BD Biosciences, Cat. No. 554066.
  • HiLoad 26/600Superdex 200pg column purchased from Cytiva Company.
  • Beckman Coulter CytoFLEX flow cytometer purchased from Beckman Company.
  • SpectraMax i3x microplate reader purchased from Molecular Devices.
  • SpectraMaxM5 microplate reader purchased from Molecular Devices.
  • Microcalorimetric Differential Scanning Calorimeter MicroCal VP-Capillary DSC.
  • anti-TROP-2/PD-L1 bispecific antibodies a and b are constructed by tandeming PD-L1 monoclonal antibody IgG and TROP-2 monoclonal antibody scFv, the structures of which are shown in Figure 1A and Figure 1B.
  • the PD-L1 monoclonal antibody is derived from the anti-PD-L1 humanized monoclonal antibody disclosed in the patent PCT/CN2021/088154, and the TROP-2 monoclonal antibody is derived from the RS7 monoclonal antibody disclosed in the PCT/GB2003/000885 patent.
  • the anti-PD-L1 monoclonal antibody and anti-TROP-2 monoclonal antibody control in the examples are respectively: monoclonal antibodies obtained according to the amino acid sequence of the monoclonal antibody disclosed in the above-mentioned corresponding patent and referring to the same expression and purification method in Example 2.
  • the scFv is connected to TROP-2 heavy chain variable region VH (SEQ ID NO: 13) and TROP-2 light chain variable region VL (SEQ ID NO: 14) through L1 (SEQ ID NO: 17) to obtain VH- L1-VL, that is, TROP-2-scFv (SEQ ID NO: 19), and then L2 (SEQ ID NO: 18) connects the scFv to the PD-L1 monoclonal antibody, and the light chain of the PD-L1 monoclonal antibody (SEQ ID NO: 18) ID NO:21) remains unchanged.
  • a molecule is the IgG of PD-L1 monoclonal antibody and the heavy chain of TROP-2 monoclonal antibody scFv is obtained through L2 connection, that is, IgG -L2-scFv (SEQ ID NO:20)
  • b molecule is the light chain of TROP-2 monoclonal antibody scFv and PD-L1 monoclonal antibody IgG through L2 connection, namely scFv-L2-IgG (SEQ ID NO:22) .
  • the DNA fragments of the heavy chain and light chain of the anti-TROP-2/PD-L1 bispecific antibody were subcloned into the vector pcDNA3.4, and the recombinant plasmid was extracted and co-transfected into 293F cells and/or CHO cells.
  • the culture solution is passed through high-speed centrifugation and filtered with a 0.22 ⁇ m filter membrane, and then loaded onto the Hitrap Mabselect Sure affinity chromatography column, and the protein is eluted in one step with 100 mM citric acid, pH 3.5 eluent,
  • the target sample was recovered and dialyzed into PBS at pH 7.4, and the anti-TROP-2/PD-L1 bispecific antibody a protein sample was detected by UPLC-SEC.
  • the anti-TROP-2/PD-L1 bispecific antibody b was further purified by HiLoad 26/600Superdex 200pg (Cytiva) molecular sieve, and the purified protein was detected by UPLC-SEC.
  • Example 3 Enzyme-linked immunosorbent assay (ELISA) to measure the affinity of the double antibody to the antigen
  • the recombinant TROP-2-HIS protein was diluted to 200ng/ml with coating solution, added to the microtiter plate at 100 ⁇ l/well, and left at room temperature for 2 hours. Remove the coating solution (remove residual droplets with absorbent paper), add blocking solution at 200 ⁇ l/well, and place at room temperature for 1 hour.
  • test results are shown in Figure 3A.
  • the EC 50 (unit: nM) of the anti-TROP-2/PD-L1 bispecific antibody molecules a and b and the positive controls TROP-2 monoclonal antibody and TROP-2-HIS were 1.689, 0.135 and 0.123.
  • the test results are shown in Figure 3B.
  • the EC 50 (unit: nM) of the anti-TROP-2/PD-L1 bispecific antibody molecules a and b and the positive controls PD-L1 monoclonal antibody and PD-L1-HIS were 0.221, 0.253 and 0.187.
  • Example 4 Fortibio assays the affinity of the anti-TROP-2/PD-L1 double antibody to the antigen
  • the kinetic parameters of the binding of anti-TROP-2/PD-L1 bispecific antibody and antigen TROP-2-HIS were determined using Fortebio Octet molecular interaction instrument and Anti-Human IgG Fc probe capture method. Soak the Anti-Human IgG Fc probe in 1 ⁇ HBS-EP working solution for 10 minutes to activate the probe. Dilute the TROP-2/PD-L1 double antibody a and b to 20 ⁇ g/ml with 1 ⁇ HBS-EP working solution, and soak the probe in it for 180 seconds to allow the probe to bind to the antibody. Then soak the probe in 1 ⁇ HBS-EP working solution for 120 seconds to block the probe.
  • the kinetic parameters of the binding of anti-TROP-2/PD-L1 bispecific antibody and antigen PD-L1-HIS were determined using Fortebio Octet molecular interaction instrument and Anti-Human IgG Fc probe capture method. Soak the Anti-Human IgG Fc probe in 1 ⁇ HBS-EP working solution for 10 minutes to activate the probe. Dilute the TROP-2/PD-L1 double antibody a and b to 40 ⁇ g/ml with 1 ⁇ HBS-EP working solution, and soak the probe in it for 180 seconds to allow the probe to bind to the antibody. Then soak the probe in 1 ⁇ HBS-EP working solution for 120 seconds to block the probe.
  • Example 5 Detecting the degradation of target cell PD-L1 protein by anti-TROP-2/PD-L1 double antibody
  • the NCI-H292 cells were digested with 0.25% trypsin-EDTA, the NCI-H292 cells were diluted with 1640 complete medium and added to a 12-well plate with 200,000 cells per well. After the cells adhere to the wall, add anti-TROP-2/PD-L1 double antibody a, anti-TROP-2 monoclonal antibody and anti-PD-L1 monoclonal antibody, and the final concentration of the antibody is 1 ⁇ g/ml or 20 ⁇ g/ml.
  • Electrophoresis Configure 12% SDS-PAGE electrophoresis gel, assemble the electrophoresis device, add 1 ⁇ electrophoresis buffer, add the sample to the spotting well, adjust the voltage to 80v, after the blue band migrates to the separation gel, turn the voltage to Adjust to 120v until the blue strip migrates to the bottom.
  • Membrane transfer Soak 0.45 ⁇ m PVDF membrane in methanol for 1 minute, assemble the wet transfer device in the order of the negative electrode of the device, electrophoresis gel, PVDF membrane, and positive electrode of the device, add 1 ⁇ transfer buffer, adjust the current to 300mA, constant current Wet blotting for 2 hours.
  • Sealing Soak the PVDF membrane in 5% skimmed milk blocking solution, and seal it for 2 hours with slight shaking on the shaker.
  • Primary antibody incubation Dilute PD-L1 rabbit IgG antibody at a ratio of 1:1000 with 1% BSA, and incubate overnight at 4°C.
  • Membrane washing wash the PVDF membrane with TBST and soak for 3 times, and wash it for 10 minutes with a slight vibration of the shaker each time.
  • Secondary antibody incubation Dilute the HRP-labeled rabbit anti-human goat antibody at a ratio of 1:10000 with 1% skimmed milk, soak the PVDF membrane with the primary antibody, and incubate with shaking for 2 hours.
  • Membrane washing wash 3 times with TBST, and shake the shaker gently for 10 minutes each time.
  • Color development Use WESTREN BOLT color development solution A and B to mix evenly at a ratio of 1:1, and expose the PVDF film.
  • the 293TF cells were digested with 0.25% trypsin-EDTA, the 293TF cells were diluted with DMEM complete medium and added to a 6-well plate with 700,000 cells per well. After the cells adhered to the wall, the packaging virus vector PLVX (the human trop2 gene has been inserted into the vector) was reacted with 293TF cells using Lipofectamine 3000 transfection reagent.
  • PLVX the human trop2 gene has been inserted into the vector
  • the trop2 gene expression efficiency of the PD-L1-Aapc/CHO-K1/HumanTrop2 cell line was detected by flow cytometry, and the trop2 gene expression efficiency of the PD-L1-Aapc/CHO-K1/HumanTrop2 cell line was the highest at 86.71% ( Results not shown).
  • the PD-L1-Aapc/CHO-K1 and PD-L1-Aapc/CHO-K1/HumanTrop2 cells cultured to the logarithmic phase were respectively digested with trypsin into single cells and counted according to 40000 cells/well, 100 ⁇ l/well , transferred to a 3903 white bottom transparent 96-well plate, placed in a 37°C, 5% CO 2 incubator and incubated overnight. Dilute the anti-TROP-2/PD-L1 double antibody a, anti-TROP-2 monoclonal antibody, and anti-PD-L1 monoclonal antibody step by step 4 times to a working solution concentration of 2X, with an initial concentration of 100 nM.
  • the PD1 effector cells cultured to the logarithmic phase were centrifuged and counted to obtain a single cell suspension with a density of 1.25*10 6 /ml and a cell viability of over 95%.
  • the experimental results are shown in Figure 6B.
  • the IC50 of anti-TROP-2/PD-L1 double antibody a and anti-PD-L1 monoclonal antibody blocking the binding of PD-L1 on the surface of PD1 and PD-L1-Aapc/CHO-K1/HumanTrop2 cells were respectively are 0.4404nM and 0.8251nM.
  • the inhibitory activity of anti-TROP-2/PD-L1 double antibody a was comparable to that of anti-PD-L1 monoclonal antibody.
  • PD-L1-Aapc/CHO-K1/HumanTrop2 cells cultured to the logarithmic phase were digested into single cells with trypsin and counted according to 40,000 cells/well, 100 ⁇ l/well, transferred to 3903 white bottom transparent 96-well plate, placed in an incubator at 37°C, 5% CO 2 and incubated overnight.
  • Dilute TROP-2/PD-L1 double antibody a, anti-TROP-2 monoclonal antibody, and anti-PD-L1 monoclonal antibody 4-fold step by step to a 2X working solution concentration, with an initial concentration of 100nm.
  • Each PD-L1-Aapc/CHO-K1HumanTrop2 cell Add an equal volume of PD1 effector cells to the wells, and incubate for 6 h at 37° C. in an incubator with 5% CO 2 . Add 80 ⁇ l of Bio-Glo detection reagent to each well. Incubate at room temperature for 5-10min, and read Luminescence with spectrummax i3. All the data are duplicate wells, and the obtained signal values are averaged and then fitted by the 4-parameter method to draw a curve.
  • the IC50 of the anti-TROP-2/PD-L1 double antibody a blocking the combination of PD1 and PD-L1 is 5.075nM, while the anti-PD-L1 monoclonal antibody basically has no blocking function. Therefore, the anti-TROP-2/PD-L1 double antibody a synergistically degrades the PD-L1 protein of the overexpressing TROP-2 cells, thereby continuously blocking the PD-1/PD-L1 pathway.
  • Pan T cell Isolation Kit to isolate T cells (10 5 cells/well) from the purchased frozen PBMC cells, mix the frozen dendritic cells (10 4 cells/well) in proportion and inoculate them into a round bottom plate In a 96-well plate (Corning:3799), the volume is 150 ⁇ l per well; Dilute TROP-2/PD-L1 double antibody a, anti-TROP-2 monoclonal antibody, anti-PD-L1 in a 96-well plate with AIM-V medium The monoclonal antibody was diluted 4 times step by step to a working solution concentration of 2X, and the initial concentration was 100nM.
  • the supernatant was diluted with 1% BSA in PBST, the highest concentration of Recombinant Human IL-2 was 100 ng/ml, and it was used after 4-fold serial dilution.
  • the supernatant prepared above or Recombinant Human IL-2 was added and incubated at room temperature for 1 h.
  • Biotin Mouse Anti-Human IL-2 was added at a ratio of 1:1000, and incubated at room temperature for 1 h.
  • Streptavidin HRP was added at a ratio of 1:1000, and incubated at room temperature for 30 minutes.
  • anti-TROP-2/PD-L1 double antibody a can enhance the immune response, which is better than anti-PD-L1 monoclonal antibody.
  • NCI-H292 cells cultured in vitro were collected, the concentration of the cell suspension was adjusted to 1 ⁇ 10 8 /mL, and mixed with matrigel in an equal ratio of 1:1.
  • the purchased PBMC Peripheral blood mononuclear cell, peripheral blood mononuclear cell
  • the PBMC cells were resuspended with PBS
  • the concentration of the PBMC suspension was adjusted to 1 ⁇ 10 7 /mL.
  • the mixed tumor cell suspension and PBMC suspension were mixed 1:1. Under sterile conditions, inoculate 200 ⁇ L of cell mixture suspension subcutaneously on the right upper back of M-NSG mice. On the same day, the mice inoculated with the mixed cells were randomly divided into groups according to body weight.
  • the doses of anti-TROP-2/PD-L1 double antibody a, anti-TROP-2 monoclonal antibody and anti-PD-L1 monoclonal antibody were all 67nmol/kg.
  • the control group was given the same volume of PBS.
  • the administration method is intraperitoneal administration, the administration volume is 0.2mL/rat (20g), and the administration is administered twice a week for four consecutive weeks.
  • T/C (%) (TVt/CVt) ⁇ 100% (TVt: treatment group TV; CVt: negative control group TV ).
  • Tumor inhibition rate TGI (%) (1-T/C) ⁇ 100%.
  • anti-TROP-2 monoclonal antibody or anti-PD-L1 monoclonal antibody alone on inhibiting tumor growth was significantly weaker than that of anti-TROP-2/PD-L1 biclonal antibody a. Therefore, the anti-TROP-2/PD-L1 double antibody a has a synergistic effect of inhibiting tumor growth.
  • Block with 5% milk (eg: 1g milk, 20ml TBST) for 2 hours.
  • the primary antibody was incubated overnight, and the PD-L1 primary antibody was configured with 1% BSA in TBST, and the primary antibody ratio was 1:1000.
  • Membrane washing wash 3 times with TBST, 10 minutes each time. Exposure, the luminescent liquid A liquid and B liquid are mixed uniformly at a ratio of 1:1, and the exposure operation is performed.
  • thermodynamic parameters related to the interaction such as protein unfolding in the presence of excipients, thereby revealing important information for the development of optimal formulations.
  • MicroCal VP-Capillary DSC was used in the experiment, and the sample and its buffer were filtered with a 0.22um filter membrane. 400 ⁇ l of the sample and its matching buffer were placed in a 96-well plate, and the sample was scanned at 25°C-100°C. The scan rate 150°C per hour.
  • Anti-TROP-2/PD-L1 double antibody a stored in 20mM acetate + 7% trehalose + 1% arginine hydrochloride + 0.02% Tween 80, pH 5.0 buffer, anti-TROP-2/PD-L1
  • the double antibody b was stored in PBS buffer at pH 7.4, and the spectra detected by DSC are shown in Figures 9A and 9B.
  • the bispecific antibody provided by the present invention can simultaneously bind TROP-2 and PD-L1, can block the PD-1/PD-L1 pathway, can restore the immune killing function of T cells, and play the role of killing tumor cells .
  • the results in Figure 5 (5A and 5B), Figure 6C and Figure 8B show that the bispecific antibody can exert a TROP-2-dependent PD-L1 degradation function, and can continuously block the PD-1/PD-L1 pathway, which is better than Anti-PD-L1 monoclonal antibody.
  • the results in Figure 8A show that the bispecific antibody can significantly inhibit tumor growth in the transplanted tumor model, and the tumor inhibitory effect is stronger than that of anti-TROP-2 monoclonal antibody + anti-PD-L1 monoclonal antibody.

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Abstract

La présente invention concerne un anticorps bispécifique anti-TROP-2/PD-L1, son procédé de préparation et son utilisation antitumorale. En particulier, un fragment variable à chaîne unique scFv est lié à un anticorps IgG d'immunoglobuline au moyen d'un lieur peptidique pour obtenir un anticorps bispécifique, et l'anticorps bispécifique peut simultanément cibler des molécules de surface de cellules tumorales, des antigènes TROP-2 et PD-L1. Les résultats expérimentaux montrent que l'anticorps bispécifique fourni peut dégrader une protéine PD-L1 de cellules tumorales positives TROP-2, ce qui permet de bloquer la liaison de PD-I/PD-L1, de libérer l'état d'inhibition des lymphocytes T, et d'exercer l'effet antitumoral.
PCT/CN2022/135706 2021-11-30 2022-11-30 Anticorps bispécifique anti-trop-2/pd-l1 WO2023098770A1 (fr)

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