WO2023087344A1 - Recombinant antibody and application thereof - Google Patents

Recombinant antibody and application thereof Download PDF

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WO2023087344A1
WO2023087344A1 PCT/CN2021/132785 CN2021132785W WO2023087344A1 WO 2023087344 A1 WO2023087344 A1 WO 2023087344A1 CN 2021132785 W CN2021132785 W CN 2021132785W WO 2023087344 A1 WO2023087344 A1 WO 2023087344A1
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antibody
cancer
nucleic acid
vegf
terminal
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Chinese (zh)
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柯航
张发明
彭飞宇
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杭州翰思生物医药有限公司
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—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 CD28 or CD152
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
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    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
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    • 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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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention belongs to the field of biomedicine, and specifically relates to recombinant antibodies and their applications, more specifically, to recombinant antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors, recombinant cells, compositions and the recombinant antibodies or antigen-binding fragments, Use of nucleic acid molecules, expression vectors, recombinant cells or compositions in the preparation of medicines.
  • PD-1 and its ligands programmed death ligand 1 and 2 are a class of inhibitory co-stimulatory molecules.
  • Human PD-1 is approximately 55kDa.
  • PD-1 is expressed in activated T and B cells, and also in monocytes, dendritic cells (DCs) and regulatory T cells (T regulatory cells, Tregs).
  • DCs dendritic cells
  • Tregs regulatory T cells
  • the two ligands of PD-1, PD-L1 (B7-H1, also known as CD274) and PD-L2 (B7-DC) are expressed in activated T cells, B cells, macrophages, dendritic cells and In addition to a variety of tumor cells, PD-L1 is also widely expressed in non-lymphoid organs, such as the heart, blood vessels, placenta, skeletal muscle, lung, liver, spleen, and thymus. PD-L2 is mainly expressed on activated macrophages, DCs and a few tumor cells. Tumor cells will use the immunosuppressive effect of PD-1/PD-L1 signaling pathway to implement immune escape.
  • PD-L1 non-small cell lung cancer
  • NSCLC Non-Small Cell Lung Cancer
  • melanoma lymphoma
  • breast cancer leukemia
  • leukemia various urological tumors
  • gastrointestinal tumors germline tumors Tumor etc.
  • Overexpression of PD-L1 interacts with the PD-1 receptor on the surface of T cells, inhibiting the transcription and translation of genes and cytokines required for T cell activation.
  • the PD-1/PD-L1 signaling pathway has become the most popular molecular target in tumor immunotherapy, and some monoclonal antibodies are used for Hodgkin's lymphoma, kidney cancer, gastric cancer, anal cancer, liver cancer, colorectal cancer and other tumors All types have achieved good clinical trial results, and several anti-PD-1 and anti-PDL1 antibodies have entered clinical trials for the treatment of various tumors.
  • PD-1/PD-L1 monoclonal antibody has been proved by a large number of clinical studies to be more effective and safe than the existing standard treatment in the treatment of various solid tumors and hematological tumors; however, PD-1/PD- L1 monoclonal antibody still has the following disadvantages: (1) The response rate for the effective population still needs to be improved, and the overall response rate for different tumor indications is different, but the average is less than 30%, and another 70% of people cannot benefit; (2) ) In order to improve the response rate of the population, PD-1/PD-L1 monoclonal antibodies need to be identified with the help of diagnostic reagents (Biomarker).
  • VEGF Vascular Endothelial Growth Factor
  • VEGFR vascular endothelial growth factor receptor
  • VEGF-A vascular endothelial growth factor receptor
  • VEGFR-2 vascular endothelial growth factor receptor
  • VEGFR-1 binds to VEGFR-1
  • VEGF-C and VEGF-D specifically bind to VEGFR-3.
  • VEGFR1 tyrosine kinase receptor 1
  • VEGFR2 tyrosine kinase receptor 2
  • VEGFR3 tyrosine kinase receptor 3
  • each ligand has different affinity and selectivity for different receptors.
  • VEGFR-2 plays the most important role, mediating almost all known cellular responses to VEGF.
  • VEGF vascular endothelial growth factor
  • PLC ⁇ phospholipase C ⁇
  • PI3K phosphatidylinositol 3-kinase
  • VEGF can increase the permeability of blood vessels, leading to the spread of tumors through the circulation and promoting the metastasis of tumor cells to other parts.
  • VEGF can also recruit endothelial precursor cells in the circulatory system, and act as a constituent of new tumor blood vessels, promote tumor angiogenesis, and further shape the typical tumor microenvironment. Therefore, VEGF has become a target of much concern in tumor targeted therapy.
  • a variety of targeted drugs against VEGF/VEGFR2 have been developed for the first-line or second-line treatment of various tumors including colorectal cancer, breast cancer, non-small cell lung cancer and thyroid cancer; the types include: monoclonal antibody (eg, anti-VEGF bevacizumab and anti-VEGFR2 ramucirumab), antibody decoys (eg, aflibercept), and small molecule inhibitors (eg, sorafenib, sunitinib )wait.
  • monoclonal antibody eg, anti-VEGF bevacizumab and anti-VEGFR2 ramucirumab
  • antibody decoys eg, aflibercept
  • small molecule inhibitors eg, sorafenib, sunitinib
  • VEGF has been shown to suppress immune responses.
  • VEGF can inhibit the maturation of dendritic cells (DC) and destroy the normal differentiation of hematopoietic precursor cells;
  • VEGF can also induce the expression of PD-L1 on DC;
  • Nirin 1 (NRP1) signaling activates antigen-specific immunoregulatory T cells, thereby downregulating tumor immune responses.
  • VEGF monoclonal antibody drugs and immunomodulatory antibodies such as anti-PD-1 and CTLA-4 monoclonal antibodies can not only restore the adverse effect of VEGF on immune response suppression, but also indirectly promote the tumor infiltration effect of T cells, Activate T cells to respond to immune antigens, and the two have anti-tumor synergistic effects. Therefore, the development of a bifunctional antibody that can simultaneously target VEGF/VEGFR and block the PD-1/PD-L1 immune checkpoint can overcome the weaknesses of each target and increase the auxiliary effect of each other, thereby exerting a greater anti-inflammatory effect. tumor effect.
  • the present invention is based on the inventor's discovery and recognition of the following facts and problems:
  • PD-1/PD-L1 or VEGF monoclonal antibodies have certain defects in the treatment of tumors, such as the response rate for effective population or different tumor indications when only PD-1/PD-L1 monoclonal antibodies are used to treat tumors
  • the overall response rate for different tumor indications is relatively low, and there are large differences in the overall response rate for different tumor indications, and drug resistance, tumor recurrence, or tumor hyperprogression may occur; only using VEGF monoclonal antibody to treat tumors will lead to immune response suppression in the body.
  • the inventor unexpectedly found that the recombinant antibody obtained by combining PD-1/PD-L1 and VEGF/VEGFR monoclonal antibody or partial fragments of monoclonal antibody in a specific way can specifically recognize PD-1 At least one of PD-L1, VEGF and VEGFR, overcome the problem of PD-1 monoclonal antibody resistance, reduce the dependence on Biomarker, and the immune response suppression of the body, thus increasing the indications of various tumors response rate.
  • the present invention proposes a recombinant antibody.
  • it includes: an anti-PD-1/PD-L1 antibody, the anti-PD-1 antibody comprising a heavy chain and a light chain; and a human anti-VEGF antibody, a human anti-VEGFR antibody or a VEGF decoy protein, the N-terminal of the human anti-VEGF antibody is connected to the C-terminal of the heavy chain of the anti-PD-1 antibody; or the N-terminal of the VEGF trap protein is connected to the heavy chain of the anti-PD-1 antibody or the C-terminus of the VEGF-trap protein is connected to the N-terminus of the heavy chain of the anti-PD-1 antibody, or the N-terminus of the human anti-VEGFR antibody is connected to the anti-PD-1 antibody The C-terminus of the heavy chain is connected.
  • the recombinant antibody is composed of an anti-PD-1 antibody and a human anti-VEGF antibody, a human anti-VEGFR antibody or a human VEGF trap protein.
  • -1 antibody combination was explored, and it was unexpectedly found that when the N-terminal of the human anti-VEGF antibody was connected to the C-terminal of the heavy chain of the anti-PD-1 antibody, the recombinant antibody and PD-1 antibody were combined.
  • VEGF has a strong binding activity, when the N-terminal of the VEGF-trap protein is connected to the C-terminal of the heavy chain of the anti-PD-1 antibody or the C-terminal of the VEGF-trap protein is connected to the anti-PD
  • the N-terminus of the heavy chain of the antibody of -1 is connected, and the binding activity of the recombinant antibody to PD-1 and/or VEGF is strong, when the N-terminus of the human anti-VEGFR antibody is connected to the anti-PD-1
  • the C-terminus of the heavy chain of the antibody is connected, and the recombinant antibody has strong binding activity to PD-1 and/or human anti-VEGFR antibody.
  • the recombinant antibody according to the embodiment of the present invention can effectively bind to PD-1, VEGF or VEGFR, overcome the problem of drug resistance of PD-1 monoclonal antibody and the problem of immune response suppression caused by VEGF monoclonal antibody, and play a greater role
  • the immunomodulatory effect of the PD-1 target has been significantly improved, and the response rate of various tumor indications has been significantly improved, and the anti-tumor effect has been significantly improved compared with monoclonal antibodies.
  • the above-mentioned recombinant antibody may further include at least one of the following additional technical features:
  • the human anti-VEGFR antibody comprises a Ramucirumab single-chain antibody.
  • Ramucirumab single-chain antibody is a single-chain antibody that can block the human vascular endothelial growth factor-vascular endothelial growth factor receptor (VEGF-VEGFR) axis, and Ramucirumab single-chain antibody can bind to VEGFR2.
  • VEGF-VEGFR human vascular endothelial growth factor-vascular endothelial growth factor receptor
  • the Ramucirumab single-chain antibody has a heavy chain variable region shown in SEQ ID NO: 18 and a light chain variable region shown in SEQ ID NO: 19.
  • Ramucirumab single-chain antibodies have the heavy chain variable region shown below:
  • Ramucirumab single-chain antibodies have the light chain variable regions shown below:
  • the human VEGF trap protein comprises VEGFR1D2 or VEGFR2D3.
  • the VEGFR1D2 and VEGFR2D3 are domains of VEGFR, which can be combined with VEGF and can block the binding of human vascular endothelial growth factor-vascular endothelial growth factor receptor (VEGF-VEGFR).
  • VEGF-VEGFR human vascular endothelial growth factor-vascular endothelial growth factor receptor
  • the VEGFR1D2, VEGFR2D3 has the amino acid sequence shown below.
  • VEGFR1D2 has the amino acid sequence shown below:
  • VEGFR2D3 has the amino acid sequence shown below:
  • the human anti-VEGF antibody has the amino acid sequence shown below:
  • the anti-PD-1 antibody is an anti-PD-1 IgG antibody.
  • it further includes a connecting peptide, the N-terminal of the connecting peptide is connected with the C-terminal of the heavy chain of the anti-PD-1 antibody, and the C-terminal of the connecting peptide is connected with the human anti-VEGF
  • the N-termini of the antibodies are linked.
  • the N-terminal of the connecting peptide is connected to the C-terminal of the heavy chain of the anti-PD-1 antibody, and the C-terminal of the connecting peptide is connected to the N-terminal of the Ramucirumab single-chain antibody or VEGFR1D2 connected.
  • the N-terminal of the connecting peptide is connected to the C-terminal of the VEGFR1D2, and the C-terminal of the connecting peptide is connected to the N-terminal of the heavy chain of the anti-PD-1 antibody.
  • the C-terminus of the connecting peptide is connected to the N-terminus of the heavy chain of the anti-PD-1 antibody, the N-terminus of the connecting peptide is connected to the C-terminus of the VEGFR2D3, and the VEGFR2D3
  • the N-terminal of VEGFR1D2 is connected with the C-terminal of VEGFR1D2.
  • the N-terminal of the connecting peptide is connected to the C-terminal of the heavy chain of the anti-PD-1 antibody
  • the C-terminal of the connecting peptide is connected to the N-terminal of the VEGFR1D2
  • the VEGFR1D2 is connected with the N-terminal of VEGFR2D3.
  • the connecting peptide has the amino acid sequence shown in SEQ ID NO:1.
  • the present invention provides a recombinant antibody.
  • the light chain of the recombinant antibody has the amino acid sequence shown in SEQ ID NO: 2
  • the heavy chain of the recombinant antibody is selected from SEQ ID NO: 3-6, SEQ ID NO: 23 and SEQ ID NO: ID NO: at least one of the amino acid sequences shown in 25.
  • the recombinant antibody according to the embodiment of the present invention can effectively bind to PD-1, VEGF or VEGFR, overcome the problem of drug resistance of PD-1 monoclonal antibody and the problem of immune response suppression caused by VEGF monoclonal antibody, and significantly improve the The response rate and anti-tumor effect of tumor indications have been significantly improved compared with monoclonal antibodies.
  • the present invention proposes a nucleic acid.
  • the nucleic acid encodes the recombinant antibody described in the first aspect.
  • the recombinant antibody encoded by the nucleic acid according to the embodiment of the present invention can effectively bind to PD-1, VEGF or VEGFR, overcome the problem of PD-1 monoclonal antibody drug resistance and the problem of immune response suppression caused by VEGF monoclonal antibody, significantly The response rate of various tumor indications has been improved, and the anti-tumor effect has been significantly improved compared with monoclonal antibodies.
  • the above-mentioned nucleic acid may further include at least one of the following additional technical features:
  • the nucleic acid has a nucleotide sequence shown in SEQ ID NO: 7, and the nucleic acid further includes SEQ ID NO: 8-11, SEQ ID NO: 27 and SEQ ID NO: 28 at least one of the nucleotide sequences.
  • the nucleotide sequence shown in SEQ ID NO: 7 can encode the light chain of the recombinant antibody
  • the nucleotide sequences shown in SEQ ID NO: 8-11, SEQ ID NO: 27 and SEQ ID NO: 28 can encode The heavy chain of the recombinant antibody.
  • the present invention provides an expression vector.
  • the expression vector includes: a first nucleic acid molecule encoding an anti-PD-1 antibody light chain; a second nucleic acid molecule encoding an anti-PD-1 the heavy chain of the antibody; a third nucleic acid molecule encoding a human anti-VEGF antibody, a human anti-VEGFR antibody, or a human VEGF trap.
  • the recombinant antibody expressed by the expression vector according to the embodiment of the present invention can effectively bind to PD-1, VEGF or VEGFR, and overcome the problem of drug resistance of PD-1 monoclonal antibody and the suppression of the immune response of the body caused by VEGF monoclonal antibody
  • the response rate of various tumor indications has been significantly improved, and the anti-tumor effect has been significantly improved compared with monoclonal antibodies.
  • the above-mentioned expression vector may further include at least one of the following additional technical features:
  • the present invention further includes: a fourth nucleic acid molecule, the fourth nucleic acid molecule is arranged between the second nucleic acid molecule and the third nucleic acid molecule, and the fourth nucleic acid molecule encodes a connecting peptide.
  • the nucleic acid molecule can be directly or indirectly linked to the control elements on the carrier, as long as these control elements can control the translation and expression of the nucleic acid molecule.
  • these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself.
  • it is sufficient that the nucleic acid molecule is operably linked to the control element.
  • the first nucleic acid molecule has a nucleotide sequence as shown below:
  • the second nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 13.
  • the third nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 14, 15, 16 or 29.
  • the fourth nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 17.
  • the expression vectors include but are not limited to recombinant lentiviruses, adenoviruses, and retroviruses. Electrical stimulation or liposome transfection of DNA vectors expressing the above-mentioned proteins into cells has therapeutic value Cell therapy products, including but not limited to CAR-T, TCR-T, TIL, CAR-NK, CAR-M or CAR-DC, etc.
  • the present invention proposes a method for preparing the recombinant antibody described in the first aspect. According to an embodiment of the present invention, it includes: introducing the expression vector described in the fourth aspect into eukaryotic cells; culturing the eukaryotic cells under conditions suitable for protein expression and secretion, so as to obtain the recombinant antibody .
  • the recombinant antibody obtained according to the method of the embodiment of the present invention can effectively bind to PD-1, VEGF or VEGFR, overcome the problem of PD-1 monoclonal antibody drug resistance and the problem of immune response suppression caused by VEGF monoclonal antibody, The response rate of various tumor indications has been significantly improved, and the anti-tumor effect has been significantly improved compared with monoclonal antibodies.
  • the above method may further include at least one of the following additional technical features:
  • the eukaryotic cells are mammalian cells.
  • the mammalian cells include at least one selected from CHO-K1, CHOS, 293F, 293T, CAR-T, TCR-T, TIL, CAR-NK, CAR-M and CAR-DC. one.
  • the mammalian cells do not include animal germ cells, fertilized eggs or embryonic stem cells.
  • the present invention provides a recombinant cell.
  • the recombinant cell carries the nucleic acid described in the third aspect, or the expression vector described in the fourth aspect.
  • the recombinant antibody secreted by recombinant cells under certain conditions can effectively bind to PD-1, VEGF or VEGFR, and overcome the problem of PD-1 monoclonal antibody resistance and the emergence of the body caused by VEGF monoclonal antibody
  • the problem of immune response suppression has significantly improved the response rate of various tumor indications, and the anti-tumor effect has been significantly improved compared with monoclonal antibodies.
  • the present invention proposes a composition for treating cancer.
  • it includes: the recombinant antibody described in the first or second aspect, the nucleic acid described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cell described in the sixth aspect.
  • the composition according to the embodiment of the present invention can effectively bind to PD-1, VEGF or VEGFR, overcome the problem of PD-1 monoclonal antibody drug resistance and the problem of immune response suppression caused by VEGF monoclonal antibody, and significantly improve each The response rate and anti-tumor effect of tumor indications have been significantly improved compared with monoclonal antibodies.
  • the above composition may further include at least one of the following additional technical features:
  • the cancer includes at least one selected from lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, leukemia, skin cancer and kidney cancer. one.
  • the composition further includes: a pharmaceutically acceptable adjuvant.
  • the pharmaceutically acceptable adjuvant includes at least one of a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent.
  • the medicine is in the form of at least one of tablet, granule, powder, capsule, solution, suspension, and freeze-dried preparation.
  • the present invention provides the recombinant antibody described in the first or second aspect, the nucleic acid described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the sixth aspect or Use of the composition described in the seventh aspect in the preparation of medicaments.
  • the medicament is used for treating or preventing cancer.
  • the recombinant antibody, nucleic acid, expression vector, recombinant cell or composition according to the embodiments of the present invention can effectively bind to PD-1, VEGF or VEGFR under certain conditions, and can effectively treat or prevent cancer.
  • the above use may further include at least one of the following additional technical features:
  • the cancer includes at least one selected from lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, leukemia, skin cancer, and kidney cancer .
  • the present invention provides a method for preventing or treating cancer. According to the embodiments of the present invention, it includes administering to the subject an effective dose of at least one of the following: the aforementioned recombinant antibody, nucleic acid, expression vector, recombinant cell and composition. Under certain conditions, the recombinant antibodies, nucleic acids, expression vectors, recombinant cells, and compositions can effectively bind to PD-1, VEGF, or VEGFR. Inhibition, on the other hand, blocks the binding of VEGF to VEGFR and inhibits tumor angiogenesis, thereby effectively treating or preventing cancer. Therefore, the method according to the embodiment of the present invention can effectively prevent or treat cancer.
  • the above method may further include at least one of the following additional technical features:
  • the cancer includes at least one selected from lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, leukemia, skin cancer, and kidney cancer .
  • Figure 1 is a graph showing the ELISA binding activity results of the recombinant antibody and the target molecule human PD-1 according to an embodiment of the present invention, wherein HX008 is an anti-PD-1 antibody, and HX016-1 is the first PD-1/VEGF recombinant antibody.
  • HX016-2 is the second structure of PD-1/VEGF recombinant antibody
  • HX016-4 is the fourth structure of PD-1/VEGF recombinant antibody
  • HX016-6 is the structure of PD-1/VEGF recombinant antibody
  • the sixth structure, 293F-HX016-4-0919AC represents the sample with batch number 0919AC expressed transiently by 293F cells
  • HX016-6-0929UF is the sample of HX016-6 with batch number 0929UF
  • R2 is the four-parameter simulation of the data
  • Abs is the absorbance at 450nm
  • Antibody Conc. is the concentration of the antibody to be tested;
  • Figure 2 is a graph showing the ELISA binding activity results of the recombinant antibody and the target molecule human VEGF165 according to an embodiment of the present invention, wherein HX006 is a VEGF antibody, Avastin is a marketed VEGF monoclonal antibody product, and HX016-2 is PD-1/VEGF
  • HX016-4 is the fourth structure of the PD-1/VEGF recombinant antibody
  • HX016-6 is the sixth structure of the PD-1/VEGF recombinant antibody
  • Abs is the absorbance at 450nm
  • Antibody Conc. is the concentration of the antibody to be tested;
  • Figure 3 is a graph showing the results of ELISA binding activity between different batches of HX016-1 and the target molecule human VEGFR2 according to an embodiment of the present invention, wherein, HX016-1 is the first structure of the PD-1/VEGF recombinant antibody, in brackets Represents different batches (Lot) of HX016-1 samples, such as HX016-1-5K-2-E2 (Lot20190419) represents the HX016-1 produced in the 20190419 batch, Abs is the absorbance at 450nm, Antibody Conc. is to be tested Antibody concentration;
  • Figure 4 is a graph showing the results of an ELISA experiment in which recombinant antibodies competitively block PD-1/PD-L1 binding according to an embodiment of the present invention, wherein HX008 is a PD-1 antibody, and HX016-1 is a PD-1/VEGF recombinant antibody
  • the first structure, HX016-2 is the second structure of PD-1/VEGF recombinant antibody
  • HX016-4 is the fourth structure of PD-1/VEGF recombinant antibody
  • HX016-6 is PD-1/VEGF recombinant antibody
  • the sixth structure of HX016 in parentheses represents the batch (Lot) of each structure of HX016, such as HX016-1 (Lot20190103) represents the HX016-1 produced in the 20190103 batch
  • Abs is the absorbance at 450nm
  • Antibody Conc. is The concentration of the antibody to be tested;
  • FIG. 5 is a diagram showing the results of an ELISA experiment in which recombinant antibodies competitively block the binding of VEGF/VEGFR2 according to an embodiment of the present invention, wherein HX006 is a VEGF antibody, Avastin is a marketed VEGF monoclonal antibody product, and HX016-2 is a PD-1/VEGFR2 antibody product.
  • the second structure of VEGF recombinant antibody HX016-4 is the fourth structure of PD-1/VEGF recombinant antibody
  • HX016-6 is the sixth structure of PD-1/VEGF recombinant antibody
  • the brackets represent the structures of HX016
  • the lot (Lot) of the sample such as HX016-1 (Lot20190103) means the HX016-1 produced in the 20190103 batch
  • Abs is the absorbance at 450nm
  • Antibody Conc. is the concentration of the antibody to be tested;
  • Figure 6 is a graph showing the experimental results of recombinant antibody HX016-2 binding to 293T cells expressing human PD-1 on the surface according to an embodiment of the present invention, wherein HX008 is the PD-1 antibody, and HX016-2 is the first PD-1/VEGF recombinant antibody Two structures, the abscissa Conc. is the concentration of the analyte, and the ordinate MFI is the average fluorescence intensity;
  • Figure 7 is a graph showing the experimental results of the recombinant antibody HX016-2 blocking the binding of PD-L1 to 293T cells expressing PD-1 on the cell membrane surface according to an embodiment of the present invention, wherein HX008 is a PD-1 antibody, and HX016-2 is a PD- 1/The second structure of VEGF recombinant antibody, the abscissa Conc. is the concentration of the analyte, and the ordinate MFI indicates the average fluorescence intensity;
  • Figure 8 is a graph showing the experimental results of blocking the expression of luciferase by CHO-scOKT3-PD-L1 cells on Jurkat-NFAT-luc cells according to an embodiment of the present invention, and restoring the expression of luciferase, wherein, PD -1 mAb is PD-1 antibody, PD-1/VEGFR2bsAb-1 is the first structure of PD-1/VEGF recombinant antibody (HX016-1), PD-1/VEGFbsAb-2 is PD-1/VEGF recombinant antibody PD-1/VEGFbsAb-4 is the fourth structure of PD-1/VEGF recombinant antibody (HX016-4), Fluorescent Intensity is the fluorescence intensity, Antibody Conc. is the antibody to be tested concentration;
  • Figure 9 is a graph showing the experimental results of the recombinant antibody blocking the activation of luciferase expressed by human VEGF165 on 293T-VEGFR2-NFAT-luc cells and inhibiting the expression of luciferase according to an embodiment of the present invention, wherein VEGF mAb is a VEGF antibody , PD-1/VEGFR2bsAb-1 is the first structure of PD-1/VEGF recombinant antibody (HX016-1), PD-1/VEGFbsAb-2 is the second structure of PD-1/VEGF recombinant antibody (HX016- 2), PD-1/VEGFbsAb-4 is the fourth structure of PD-1/VEGF recombinant antibody (HX016-4), Fluorescent Intensity is the fluorescence intensity, Antibody Conc. is the concentration of the antibody to be tested;
  • Figure 10 is a graph showing the results of recombinant antibody HX016-2 stimulating T cells to secrete IFN- ⁇ in a mixed lymphatic reaction according to an embodiment of the present invention, wherein HX008 represents the PD-1 antibody, and PD-1/VEGFR2bsAb-1 is PD-1
  • HX016-2 is the second structure of PD-1/VEGF recombinant antibody (HX016-2)
  • PD-1/VEGFbsAb-4 is PD-1/VEGF
  • Fluorescent Intensity is the fluorescence intensity
  • Conc. is the concentration of the analyte;
  • Figure 11 is a graph showing the results of recombinant antibody HX016-2 reactivating T cells to secrete IL-2 in the SEB stimulation test according to an embodiment of the present invention, wherein the abscissa Conc. is the concentration of the analyte, and the ordinate indicates the concentration of IL-2 content;
  • Figure 12 is a graph showing the results of recombinant antibodies HX016-2 and HX016-6 inhibiting human VEGF165-mediated HUVEC cell proliferation according to an embodiment of the present invention, wherein the abscissa Conc. is the concentration of the analyte, and the ordinate FLU indicates the fluorescence intensity; as well as
  • Figure 13-A is a diagram of the tumor volume change of the subcutaneous CT26.WT colon cancer model of different groups of BALB/c-hPD1 mice according to an embodiment of the present invention, wherein the abscissa Days Post Treatment represents the time of drug or antibody treatment, and the ordinate Tumor Volume indicates the tumor volume (mm2), G1 is the IgG4 isotype negative control 5mg/kg group, G2 is the anti-PD-1 monoclonal antibody Keytruda 10mg/kg group, G3 is the anti-PD-1 monoclonal antibody 10mg/kg group, G4 is the anti-PD-1 monoclonal antibody 10mg/kg group, and G4 is the anti-PD-1 monoclonal antibody 10mg/kg group.
  • G1 is the IgG4 isotype negative control 5mg/kg group
  • G2 is the anti-PD-1 monoclonal antibody Keytruda 10mg/kg group
  • G3 is the anti-PD-1 monoclonal antibody 10mg/kg
  • PD-1 monoclonal antibody 5mg/kg group G5 is anti-VEGF monoclonal antibody 5mg/kg group, G6 is VEGFR1D2 binding domain 5mg/kg group, G7 is HX016-2, the second structure of PD-1/VEGF recombinant antibody 5mg /kg group, G8 is HX016-4, the fourth structure 5mg/kg group of PD-1/VEGF recombinant antibody, G9 is HX016-6, the sixth structure 5mg/kg group of PD-1/VEGF recombinant antibody;
  • Figure 13-B is a graph showing the change in tumor volume of each mouse in different groups of G1-G9 according to an embodiment of the present invention, wherein the abscissa Days Post Treatment represents the time of drug or antibody treatment, and the ordinate Tumor Volume represents the tumor volume (mm2 );
  • Figure 13-C is a graph showing the change in tumor weight of the BALB/c-hPD1 mouse subcutaneous CT26.WT colon cancer model in different groups of G1-G9 according to an embodiment of the present invention, wherein the abscissa represents different groups, and the ordinate represents Tumor Weight Indicates tumor weight (g); and
  • Fig. 13-D is a diagram of body weight changes of subcutaneous CT26.WT colon cancer model mice of different groups of BALB/c-hPD1 mice according to an embodiment of the present invention, wherein the abscissa Days Post Treatment represents the time of drug or antibody treatment, and the ordinate Coordinate Body Weight represents the mouse body weight (g).
  • Embodiments of the present invention are described in detail below.
  • the embodiments described below are exemplary only for explaining the present invention and should not be construed as limiting the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features.
  • the features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • “Operably linked” herein refers to linking the exogenous gene to the vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended role in regulating the transcription and translation of the exogenous gene function.
  • VEGF trapping protein refers to a protein capable of binding to VEGF protein, which is not particularly limited, and any protein that can bind to VEGF is within this scope, such as the structural domain of VEGF receptor, as described in the present invention VEGFR1D2 mentioned above is domain 2 of VEGF receptor 1.
  • anti-tumor drugs are mainly monoclonal antibodies, especially the first-generation immune checkpoint blocking antibody therapy represented by anti-human PD-1/PD-L1 monoclonal antibody and anti-human CTLA-4 monoclonal antibody.
  • Significant success has been achieved in cancer therapy.
  • the immunomodulatory mechanism of PD-1/PD-L1 monoclonal antibody endows it with broad-spectrum anti-tumor activity.
  • PD-1/PD-L1 monoclonal antibody has been proved by a large number of clinical research results to show efficacy and safety superior to existing standard treatment regimens in the treatment of various solid tumors and blood tumors.
  • a single PD-1/PD-L1 monoclonal antibody still has the following disadvantages: the response rate for the effective population is still low, and the overall response rate for different tumor indications is different, but the average is less than 30%, and another 70% People cannot benefit; in order to improve the response rate of the population, PD-1/PD-L1 monoclonal antibodies need to be identified with the help of diagnostic reagents (Biomarker).
  • Biomarker diagnostic reagents
  • the 6 bispecific antibody molecules were constructed, and the 6 bispecific antibody molecules were PD-1 mAb molecules obtained by linking the peptide with a protein molecule selected from one of the following: Ramucirumab single-chain antibody , VEGF single-chain antibody, VEGFR1D2, the connection method is that the heavy chain of the PD-1 mAb molecule is connected to the above-mentioned protein molecule through a connecting peptide.
  • the connecting peptide has the amino acid sequence shown in SEQ ID NO: 01
  • the light chain of the PD-1 mAb is the light chain of the 6 recombinant protein molecules, having the sequence as SEQ ID NO:
  • the amino acid sequence shown in 02, the heavy chain of the PD-1 mAb has the amino acid sequence shown in SEQ ID NO: 21:
  • amino acid sequences of PD-1 mAb light chains are as follows:
  • amino acid sequence of the PD-1 mAb heavy chain is as follows:
  • nucleotide sequence encoding the connecting peptide is as follows:
  • the nucleotide sequences encoding the PD-1 mAb light chains (6 kinds of bispecific recombinant antibody light chains) are as follows:
  • nucleotide sequence encoding the heavy chain of the PD-1 mAb is as follows:
  • the amino acid sequence of the heavy chain of HX016-1 is shown in SEQ ID NO: 3, which is the C-terminal of the heavy chain of the humanized anti-PD-1 antibody (PD-1 mAb) and the linker is used to connect the Ramucirumab single-chain antibody (SEQ ID NO: 22) N-terminal.
  • the whole gene synthesis codes the nucleic acid sequence of PD-1 mAb light chain, heavy chain, linker-Ramucirumab single-chain antibody, and the specific experimental operation of constructing expression vectors 1, 2, and 3. Please refer to CN 105330739 to encode the light chain of PD-1 mAb
  • the nucleic acid sequence of the expression vector was first connected to the expression vector to obtain the expression vector 1; the nucleic acid sequence encoding the heavy chain of PD-1 mAb was directly connected to the expression vector 1 to obtain the expression vector 2 expressing the anti-PD1 monoclonal antibody HX008; by Over- Lap PCR fused the C-terminal of the heavy chain of PD-1 mAb to the nucleotide sequence of the N-terminal of the linker (the C-terminal of the linker is connected with Ramucirumab single-chain antibody) to obtain the nucleotide sequence encoding the heavy chain of HX016-1 acid sequence (SEQ ID NO: 8), the nucleot
  • the amino acid sequence of the heavy chain of recombinant antibody No. 1 (HX016-1) is as follows:
  • the amino acid sequence of Ramucirumab single-chain antibody is as follows:
  • amino acid sequence of the heavy chain of HX016-2 is shown in SEQ ID NO: 4, which is the C-terminal of the amino acid sequence of VEGFR1D2 (SEQ ID NO: 20), which is connected to the N-terminal of the heavy chain of PD-1 mAb with a linker;
  • nucleotide sequences encoding the light chain, heavy chain, and VEGFR1D2-linker of the humanized anti-PD-1 antibody (PD-1 mAb) were synthesized in the whole gene, and the specific procedures for constructing expression vectors 1, 2, and 4 were carried out.
  • the experimental operation refers to CN 105330739, the nucleic acid sequence of the light chain of PD-1 mAb is first connected to the expression vector to obtain expression vector 1; the heavy chain sequence of PD-1 mAb is connected to expression vector 1 to obtain a monoclonal expression of anti-PD1 Expression vector 2 of antibody HX008; the N-terminal of the heavy chain of PD-1 mAb was fused to the C-terminal nucleotide sequence of the linker (the N-terminal of the linker is connected with VEGFR1D2) by Over-lap PCR to obtain the HX016-2 encoding The nucleotide sequence of the heavy chain (SEQ ID NO: 9) was connected to the expression vector 1 to obtain the expression vector 4 expressing the bispecific antibody HX016-2.
  • the amino acid sequence of the heavy chain of recombinant antibody No. 2 (HX016-2) is as follows:
  • VEGFR1D2 has the amino acid sequence shown below:
  • the nucleotide sequence encoding the heavy chain of recombinant antibody No. 2 (HX016-2) is as follows:
  • the amino acid sequence of the HX016-3 heavy chain is shown in SEQ ID NO: 23.
  • the C-terminal of VEGFR1D2 is connected to the N-terminal of VEGFR2D3 (SEQ ID NO: 24), and then the C-terminal of VEGFR2D3 is connected to the heavy chain of PD-1 mAb with a linker N-terminal.
  • the nucleotide sequences encoding the light chain, heavy chain, and VEGFR1D2-VEGFR2D3-linker of the humanized anti-PD-1 antibody by whole gene synthesis, and the specific experimental operation of constructing expression vectors 1, 2, and 5 refer to CN 105330739
  • the light chain encoding the humanized anti-PD-1 antibody was first connected to the expression vector to obtain expression vector 1;
  • the heavy chain sequence of PD-1 mAb was connected to expression vector 1 to obtain the monoclonal antibody expressing anti-PD1 Expression vector 2 of HX008;
  • the HX016- 3 the nucleotide sequence encoding the heavy chain of HX016-3 was connected to the expression vector 1 to obtain the expression vector 5 expressing the bispecific antibody HX016-3.
  • the amino acid sequence of the heavy chain of recombinant antibody No. 3 (HX016-3) is as follows:
  • VEGFR2D3 The amino acid sequence of VEGFR2D3 is shown below:
  • VEGFR2D3 The nucleotide sequence encoding VEGFR2D3 is as follows:
  • the nucleotide sequence encoding the heavy chain of recombinant antibody No. 3 (HX016-3) is as follows:
  • the heavy chain of HX016-4 (SEQ ID NO: 5) is the C-terminal of the heavy chain of PD-1 mAb connected to the N-terminal of VEGFR1D2 with a linker;
  • the whole gene synthesis codes the light chain, heavy chain, and linker-VEGFR1D2 nucleotide sequences of PD-1 mAb, and the specific experimental operations for constructing expression vectors 1, 2, and 6 are the same as described above.
  • the C-terminal of the PD-1 mAb heavy chain was fused to the N-terminal nucleotide sequence of the linker (the C-terminal of the linker is connected with VEGFR1D2) by Over-lap PCR to obtain the nucleotide sequence (SEQ ID NO: 10) , connected to the expression vector 1 to obtain the expression vector 6 expressing the bispecific antibody HX016-4.
  • the final concentration of plasmid DNA is 4 ⁇ g/mL
  • the final concentration of transfection reagent PEI is 8 ⁇ g/mL.
  • In the cells mix well while adding dropwise.
  • After cell transfection antibodies are expressed in mammalian cells and secreted extracellularly. Then, the expressed antibody was purified through a protein A affinity chromatography column to obtain the HX016-4 protein.
  • the amino acid sequence of the heavy chain of recombinant antibody No. 4 (HX016-4) is as follows:
  • the nucleotide sequence encoding the heavy chain of recombinant antibody No. 4 (HX016-4) is as follows:
  • the amino acid sequence of the heavy chain of HX016-5 is shown in SEQ ID NO: 25.
  • the C-terminal of the heavy chain of PD-1 mAb is connected to the N-terminal of VEGFR1D2 with a linker, and then the C-terminal of VEGFR1D2 is connected to the N-terminal of VEGFR2D3;
  • the whole gene synthesis encodes the light chain, heavy chain, linker-VEGFR1D2-VEGFR2D3 nucleic acid sequence of PD-1 mAb, and the specific experimental operation of constructing expression vectors 1, 2, and 7 is the same as above.
  • the PD-1 mAb The nucleic acid sequence of the light chain was first connected to the expression vector to obtain the expression vector 1; the heavy chain sequence of PD-1 mAb was connected to the expression vector 1 to obtain the expression vector 2 expressing the anti-PD1 monoclonal antibody HX008; by Over-lap PCR After fusing the C-terminal of the PD-1 mAb heavy chain with the N-terminal nucleotide sequence of the linker (the C-terminal of the linker is connected with VEGFR1D2-VEGFR2D3), the nucleotide sequence encoding the HX016-5 heavy chain was obtained ( SEQ ID NO: 28), the nucleotide sequence encoding the heavy chain of HX016-5 was connected to the expression vector 1 to obtain the expression vector 7 expressing the bispecific antibody HX016-5.
  • the amino acid sequence of the heavy chain of recombinant antibody No. 5 (HX016-5) is as follows:
  • the heavy chain of HX016-6 (SEQ ID NO: 6) is the C-terminal of the heavy chain of PD-1 mAb, and the N-terminal of the single-chain antibody (SEQ ID NO: 26) that can bind to VEGF is connected with a linker.
  • nucleic acid sequences encoding PD-1 mAb light chain, heavy chain, linker+VEGF single-chain antibody, and the specific experimental operations for constructing vectors 1, 2, and 8 are the same as above.
  • the nucleus encoding the HX016-6 heavy chain was obtained by fusing the C-terminal of the heavy chain of PD-1 mAb with the N-terminal nucleotide sequence of the linker (the C-terminal of the linker is linked with VEGF single-chain antibody) by Over-lap PCR
  • the nucleotide sequence (SEQ ID NO: 11) was connected to the expression vector 1 to obtain the expression vector 8 expressing the bispecific antibody HX016-6. Extract the DNA of expression vectors 1 and 8, and transfect them into 293 mammalian cells respectively, with a final concentration of plasmid DNA of 4 ⁇ g/mL and a final concentration of transfection reagent PEI of 8 ⁇ g/mL for transfection. In the cells, mix well while adding dropwise. After cell transfection, antibodies are expressed in mammalian cells and secreted extracellularly. Then, the expressed antibody is purified through a protein A affinity chromatography column to obtain the HX016-6 protein.
  • the amino acid sequence of the heavy chain of recombinant antibody No. 6 (HX016-6) is as follows:
  • the amino acid sequence of the VEGF single-chain antibody is as follows:
  • HX008 group anti-PD-1 mAb
  • HX016-1 group HX016-2 group
  • HX016-4 group HX016-6 group.
  • HX008 group was used as the control group , to detect the binding activity of HX016-1 group, HX016-2 group, HX016-4 group, HX016-6 group and PD-1,
  • amino acid sequence of the light chain of HX008 (PD-1 mAb) is as follows:
  • amino acid sequence of the heavy chain of HX008 (PD-1 mAb) is as follows:
  • the EC50 was calculated according to the OD value in the ELISA results. The specific results are shown in Figure 1. Among them, the binding activities of HX016-1, HX016-4, HX016-6 and PD-1 are relatively close to 25.0ng/mL, and the activities are stable. The results were good, the binding activity of HX016-2 to PD-1 was 36.6ng/mL.
  • HX006 group anti-VEGF mAb
  • HX016-2 group HX016-4 group
  • HX016-6 group HX016-6 group.
  • amino acid sequence of the HX006 heavy chain is as follows:
  • amino acid sequence of the HX006 light chain is as follows:
  • VEGF165 into the wells of the microplate, coat at 4°C overnight, and add 200 ⁇ L PBST the next day to wash with shaking for 10 minutes, wash 3 times, then add 200 ⁇ L PBS to wash with shaking for 10 minutes, wash 3 times, to wash
  • add 1% BSA blocking solution block at 37°C for 1 hour, wash 3 times, remove the blocking solution, dilute the sample to be tested with BSA with a concentration of 1 ⁇ to 2 ⁇ g/mL, and then dilute 6 times in a 5-fold gradient Load the samples at different degrees, so that the loading concentrations are 2000, 400, 80, 16, 3.2, 0.64, and 0.128 ng/mL.
  • the EC50 was calculated according to the OD value in the ELISA results. The results are shown in Figure 2. Among them, the binding activities of HX016-2 and HX016-4 to VEGF165 are relatively close, both at about 25.0ng/mL, and the activity is better. HX016-6 and VEGF165 The binding activity was 93.5 ng/mL.
  • HX016-1 (Lot.20190103) group HX016-1-5K-2-E2 group, HX016-1-7.5K-3-F4 group, HX016-1-7.5K- 4-F6 group, HX016-1-7.5K-5-F10 group, by ELISA method, with HX016-1 (Lot.20190103) group as the control group, detect the difference between the samples of each batch of HX016-1 and VEGFR2-his Combined activity, the specific experimental operation is as follows:
  • VEGFR2-his into the wells of the microtiter plate, coat at 4°C overnight, and add 200 ⁇ L PBST to shake and wash for 10 minutes the next day, wash 3 times, then add 200 ⁇ L PBS to shake and wash for 10 minutes, wash 3 times.
  • BSA blocking solution block at 37°C for 1 hour, wash 3 times, remove the blocking solution, dilute the sample to be tested with BSA at a concentration of 1 ⁇ to 10 ⁇ g/mL, and then dilute 6 samples in a 5-fold gradient A total of 7 gradients of dilution were loaded, and the duplicate wells were set as blank.
  • the EC50 was calculated according to the OD value in the ELISA results, and the specific results are shown in Figure 3, where the binding activity of HX016-1 (Lot.20190103) to VEGFR2 was 83.6 ng/mL, and the binding activity of HX016-1-5K-2-E2 to VEGFR2 The activity is 59.7ng/mL, the binding activity of HX016-1-7.5K-3-F4 and VEGFR2 is 78.7ng/mL, the binding activity of HX016-1-7.5K-4-F6 and VEGFR2 is 84.3ng/mL, HX016-1 The binding activity of -7.5K-5-F10 to VEGFR2 was 130.4ng/mL. The binding activity of each batch of HX016-1 to VEGFR2 was relatively close, ranging from 59.7 to 130.4 ng/mL.
  • HX008 group anti-PD-1 mAb
  • HX016-1 group HX016-2 group
  • HX016-4 group HX016-6 group
  • HX008 group as the control group
  • HX008 group the specific experimental operation is as follows:
  • the IC50 is calculated according to the OD value in the ELISA result, as shown in Figure 4, where the IC50 of HX016-1 (Lot.20190103) is 97.5ng/mL, and the IC50 of HX016-2 (Lot.20190121) is 121ng/mL , the IC50 of HX016-4 (Lot.20190311) is 64.9ng/mL, the IC50 of HX016-4 (Lot.20190726) is 63.7ng/mL, the IC50 of HX016-6 (Lot.20190726) is 83.5ng/mL, HX008 (Lot.A103C20171201) has an IC50 of 53.1ng/mL. Therefore, the activity of a series of recombinant antibodies that can block the binding of PD-1/PD-L1 is more consistent with the activity of PD-1 monoclonal antibody.
  • HX006 group anti-VEGF mAb
  • HX016-1 group HX016-2 group
  • HX016-4 group HX016-6 group
  • HX006 group is used as the control group.
  • VEGF165 into the wells of the microplate, coat at 4°C overnight, add 200 ⁇ L PBST to shake and wash for 10 minutes the next day, wash 3 times, then add 200 ⁇ L PBS to shake and wash for 10 minutes, wash 3 times to wash away
  • Add 1% BSA blocking solution after excess antigen, block at 37°C for 1 hour, wash 3 times, remove the blocking solution, dilute HX016-1 with 1 ⁇ BSA to 6 ⁇ g/mL, and dilute HX016-2 with 1 ⁇ BSA to 20 ⁇ g/mL , HX006, HX016-4 and HX016-6 were diluted with 1 ⁇ BSA to 60 ⁇ g/mL, and then 3-fold serially diluted 6 dilutions to load the samples, so that the loading concentrations were 2000, 400, 80, 16, 3.2, 0.64, 0.128ng/mL, the samples to be tested at each dilution concentration were mixed with 0.6 ⁇ g/mL VEGFR2-mFc at a volume
  • the IC50 was calculated according to the OD value in the ELISA results, and the specific results are shown in Figure 5. Among them, the IC50 of HX016-1 (Lot.20190103) is 136.7ng/mL, the IC50 of HX016-2 (Lot.20190121) is 944.1ng/mL, the IC50 of HX016-4 (Lot.20190311) is 1345.4ng/mL, The IC50 of HX016-4 (Lot.20190726) is 6258.4ng/mL, and the IC50 of HX006 (Lot.20170614) is 1097.3ng/mL. Therefore, both HX016 and HX006 have the activity of blocking the binding of VEGF/VEGFR, among which HX016- 1 has the best activity.
  • Example 4 HX016-2 binds to 293T cells expressing PD-1 on the surface
  • HX016-2 is an anti-PD-1/VEGF bispecific immune antibody.
  • 293T-PD-1 stably transfected cell line cells were used as experimental cells.
  • FACS method was used to detect HX016-2 and the expression of PD-1 on the cell membrane surface. Protein cell binding, the specific operation is as follows:
  • the cells were centrifuged at 450 ⁇ g for 5 min, the supernatant was discarded, and 10 diluted samples were added in turn, 100 ⁇ L/well.
  • FACS buffer blank incubate at 4°C for 1 hour, centrifuge at 450xg for 5 minutes, discard the supernatant; wash 2 times with 250 ⁇ L/well FACS buffer, add 100 ⁇ L/well to 200X diluted Alexa 647 fluorescently-labeled anti-human secondary antibody, incubated at 4°C for 30 minutes, centrifuged at 450x g for 5 minutes, discarded the supernatant; washed the precipitate twice with 250 ⁇ L/well FACS buffer, and reconstituted with 200 ⁇ L/well FACS buffer. Hang and transfer to flow tube. Detection of fluorescence intensity with CytoFLEX flow cytometer, Alexa 647 uses APC channel.
  • the FACS method was used to detect the binding of HX016-2 to cells expressing PD-1 protein on the cell membrane surface.
  • HX008 is an anti-PD-1 mAb.
  • the specific results are shown in Figure 6.
  • the EC50 value of the binding of HX016-2 to PD-1 is 0.2111ug/mL. Compared with HX008, the binding activity is slightly weaker, but the difference between the two is not significant.
  • HX016-2 is an anti-PD-1/VEGF bispecific immune antibody.
  • 293T-PD-1 stably transfected cell line cells were used as experimental cells.
  • FACS method was used to detect whether HX016-2 could block PD-L1
  • the specific operation of binding to cells expressing PD-1 on the cell membrane surface is as follows:
  • the cells were centrifuged at 450 g for 5 min, and after the supernatant was discarded, 8 diluted samples were sequentially added, 50 ⁇ L/well.
  • Two accompanying FACS buffer blanks were incubated at 4°C for 1 hour. Dilute the sample PD-L1-mFC (Akesobio, 20 ⁇ g/mL) to 10 ⁇ g/mL with FACS buffer, add 50 ⁇ L/well to 8 diluted sample wells and one blank well, and incubate at 4 °C for 1 h. Centrifuge at 450g for 5min, discard the supernatant; wash the resulting precipitate twice with 250 ⁇ L/well FACS buffer, add 100 ⁇ L/well of 200X diluted Alexa 647 fluorescently labeled anti-mouse secondary antibody, incubated at 4°C for 30min.
  • HX008 is anti-PD-1 mAb.
  • the specific results are shown in Figure 7.
  • HX016-2 and PD-1 The binding EC50 value of 1 is 0.2735ug/mL, and the binding EC5 value of HX008 (anti-PD-1 mAb) and PD-1 is 0.0301ug/mL. Therefore, both HX016 and HX008 can block the binding of PD-1/PD-L1 activity, and its blocking activity varies.
  • Example 6 Inhibition of luciferase expression in Jurkat-NFAT-luc cells by target protein blocking CHO-scOKT3-PD-L1 cells
  • HX016 is an anti-PD-1/VEGF bispecific immune antibody.
  • CHO-scOKT3-PD-L1 and Jurkat-NFAT-luc stably transfected cell lines were used as experimental cells.
  • the luciferase reporter gene was used to detect HX016 -2 Block the inhibitory effect of CHO-scOKT3-PD-L1 cells on the expression of luciferase in Jurkat-NFAT-luc cells, restore the expression of luciferase, and detect the biological activity of PD-1, the specific operation is as follows:
  • CHO-PDL1-CD3L cells Adjust the density of CHO-PDL1-CD3L cells to 5x10 5 cells/mL. On the first day, add 100 ⁇ L of CHO-PDL1-CD3L cells with a density of 5x10 5 cells/mL into a white-bottomed 96-well plate. Incubate in an incubator at 37°C, 5% CO 2 for 16 ⁇ 2h.
  • Use assay medium for Jurkat-PD1-NFAT cells to adjust the density to 2x10 6 cells/mL take out the 96-well white plate cultured with CHO-PDL1-CD3L cells, suck out the cell supernatant medium, and add 50 ⁇ L of different concentrations of HX016 to each well solution and 50 ⁇ L of Jurkat-PD1-NFAT cell suspension, and the cell mixture was incubated in an incubator at 37°C and 5% CO 2 for 6h.
  • the luciferase assay solution was taken out in advance and pre-equilibrated to room temperature in the dark.
  • luciferase detection solution 100 ⁇ L of luciferase detection solution to each well, incubate at room temperature in the dark for 1 min, and then read the autoluminescence with a Perkin Elmer fluorescence microplate reader.
  • the detection wavelength in the Protocol is 560 nm, and the detection height is 6.5 cm.
  • the detection time is 5s, and the value is read after selecting a suitable detection hole.
  • Example 7 The target protein blocks the activation experiment of VEGF on the expression of luciferase in 293T-VEGFR2-NFAT-luc cells
  • HX01 is an anti-PD-1/VEGF bispecific immune antibody.
  • 293T-VEGFR2-CNFAT-5F8 stably transfected cell line cells were used as experimental cells.
  • the luciferase reporter gene detected HX016-2 to block the effect of VEGF on 293T- VEGFR2-CNFAT-5F8 cells express the activation of luciferase, and inhibit the expression of luciferase to detect the biological activity of VEGF terminal.
  • the specific operation is as follows:
  • the 293T-VEGFR2-CNFAT-5F8 cell density was adjusted to 2x10 6 cells/mL, and the VEGF protein was diluted to 400ng/mL with assay medium.
  • luciferase detection solution 100 ⁇ L was added to each well; after incubation at room temperature in the dark for 1 min, the autoluminescence was read with a Perkin Elmer fluorescence microplate reader.
  • the detection wavelength in the Protocol was 560 nm, and the detection height was 6.5 cm. The time is 4s, select the appropriate detection hole and read the value.
  • each antibody of luciferase reporter gene detection HX016 can block the activation of VEGF on the expression of luciferase in 293T-VEGFR2-CNFAT-5F8 cells, and inhibit the expression of luciferase.
  • HX016-2 is an anti-PD-1/VEGF bispecific immune antibody.
  • the mixed lymphocyte reaction (MLR) uses one person's DC cells to stimulate another person's CD4+T cells, and activates CD4+T cells to make their surface PD -1 is highly expressed, while PD-L1 expressed on the surface of DC cells binds to PD-1 on the surface of CD4+ T cells to inhibit its downstream PI3k signaling pathway, thereby inhibiting T cell function (cytokine secretion).
  • MLR mixed lymphocyte reaction
  • PD-L1 expressed on the surface of DC cells binds to PD-1 on the surface of CD4+ T cells to inhibit its downstream PI3k signaling pathway, thereby inhibiting T cell function (cytokine secretion).
  • This experiment mainly verifies whether HX016-2 can block the inhibition of DC on T cells to enhance immune function.
  • Use CBA to detect the secretion level of IFN- ⁇ to determine the efficacy of HX016-2, the specific operation is as follows:
  • monocytes (CD14+) were sorted out by magnetic bead sorting, and the cell density was adjusted to 1x10 with DC induction medium + 1000IU/mL GM-CSF + 1000IU/mL IL-4 6 cells/mL, inoculated in a 6-well plate at 2 mL/well, cultured at 37°C and 5% CO 2 , and added fresh DC induction medium + 1000IU/mL GM-CSF + 1000IU/mL IL-4; continue to culture at 37°C and 5% CO 2 ; culture until the 7th day, draw 1mL of supernatant from each well Discard, add 1mL of induction medium A to each well, and add 100IU/mL TNF- ⁇ , shake gently; continue to culture at 37°C, 5% CO2 ; culture until the 8th day, Day 9: Add 100IU/mL TNF- ⁇ to each well, shake gently; continue to culture at 37°C and 5% CO 2
  • HX016-2 Dilute HX016-2 to 40 ⁇ g/mL with MLR co-cultivation medium, and then carry out 10-fold serial dilution, and set 5 concentration gradients in total (the specific concentrations are 40, 4, 0.4, 0.04, 0.004 ⁇ g/mL). Blow down the DC, adjust the cell density to 2x10 5 /mL with MLR co-cultivation medium, spread 100 ⁇ L of DC and 50 ⁇ L of T cells into a flat-bottomed 96-well plate, add 50 ⁇ L of different concentrations of HX016-2 diluted samples to each well, Cultured at 37°C and 5% CO 2 for 5 days, collected the supernatant after 5 days and detected IFN- ⁇ with a CBA kit.
  • the secretion level of IFN- ⁇ in the MLR system was detected by the CBA method to determine whether HX016-2 could block the inhibition of DCs on T cells.
  • the concentration increases, and there is a dose-dependent effect; when the tested concentration of HX016-2 is 1ug/mL, the IFN- ⁇ secretion levels of HX016-2 and HX008 are similar, and at the concentration of 10ug/mL, the IFN- ⁇ secretion level of HX016-2
  • the secretion level of IFN- ⁇ was higher than that of HX008.
  • HX016-2 is an anti-PD-1/VEGF bispecific immune antibody.
  • SEB Staphylococcal enterotoxin B, staphylococcal enterotoxin B
  • PBMC Peripheral Blood Mononuclear Cell, peripheral blood mononuclear cells
  • the T cells are in a state of exhaustion, and the function of the immune system is reduced.
  • This example mainly verifies whether HX016-2 can enhance the ability of the immune system by reactivating T cells under the stimulation system of SEB.
  • Use CBA to detect the secretion level of IL-2 to determine the biological activity of the PD-1 end of HX016-2, the specific operation is as follows:
  • PBMC complete medium After isolating a human PBMC cell, resuspend it with PBMC complete medium, dilute the SEB concentration to 100ng/mL, centrifuge the PBMC cell suspension at 450xg for 10min, and resuspend the cell with 100ng/mL SEB to a density of 2x10 6 cell/ mL, add 100 ⁇ L to each well of a flat-bottomed 96-well plate.
  • the concentration of HX016-2 was diluted to 20 ⁇ g/mL with complete PBMC medium, and then 10-fold serial dilution was performed successively, and a total of 6 concentration gradients (20000, 2000, 200, 20, 2, 0.2 ng/mL) were established. Add different concentrations of HX016-2 to each well to dilute 100 ⁇ L of samples, and culture them at 37°C and 5% CO 2 for 3 days. After 3 days, collect the supernatant and use the CBA kit to detect IFN-r.
  • CBA detection of cytokines After the sample is diluted, add 50 ⁇ L and 50 ⁇ L of mixed capture microspheres to each well of the V-bottom 96-well plate, and incubate at room temperature for 1 hour. Add 50 ⁇ L/well PE-labeled detection antibody and incubate at room temperature for 2 hours. Wash the sample once with 200 ⁇ L of washing solution and resuspend it. After the instrument is debugged and verified, it is tested on the machine; after the sample is collected (FCS 2.0 format), the standard curve is drawn and data analysis is performed with the CBA professional analysis software FCAP Array v1.0.
  • the secretion level of IL-2 in PBMC stimulated by HX016-2 was detected by CBA method.
  • the specific results are shown in Figure 11. With the increase of the dose of HX016-2, the concentration of IL-2 increased, and there was a dose-dependent effect. And at the concentration of 10ug/mL, the IL-2 secretion levels of HX016-2 and HX008 were basically the same.
  • HX016 is an anti-PD-1/VEGF bispecific immune antibody.
  • Human umbilical vein endothelial cells Human Umbilical Vein Endothelial Cells, HUVEC are usually used as cell models when performing vascular endothelial cell experiments.
  • VEGF vascular Endothelial growth factor
  • HUVEC cells are used as experimental cells to detect whether HX016-2 and HX016-6 can inhibit VEGF-mediated HUVEC cell proliferation. The specific operations are as follows:
  • the cell concentration was adjusted to 5 ⁇ 10 4 cells/mL with inoculation medium for HUVEC cells, and mix well. According to the volume of 100 ⁇ L per well, the cell suspension was evenly added to the 96-well plate with a discharge gun, and cultured at 37° C. and 5% CO 2 for 18-24 hours.
  • HX016-2 and HX016-6 were mixed with 100ng/mL rhVEGF165 monoclonal antibody at a volume ratio of 1:1, followed by individual rhVEGF165 wells, and incubated in a 37°C incubator for 2h.
  • HX016-2 and HX016-6 can inhibit VEGF-mediated HUVEC cell proliferation, the specific results are shown in Figure 12, the EC50 of HX016-2 inhibiting HUVEC cell proliferation experiment is 2940ng/mL, HX016-6 inhibits HUVEC cell proliferation The EC50 of the experiment was 4620ng/mL. Compared with HX006, the activity is weaker, and HX016-2 is more active than HX016-6.
  • Mouse colon cancer CT26.WT cells were revived, and the CT26.WT cells in the logarithmic growth phase were collected, the culture medium was removed, washed twice with PBS, and then inoculated into 54 mice at an inoculation volume of 5 ⁇ 10 5 cells/ 100 ⁇ L/piece.
  • 54 mice were randomly divided into 9 groups according to the tumor volume, 6 mice in each group. The day of grouping was defined as D0 day, and the administration started on D0 day, and the administration was given twice a week according to the dose mentioned in the figure, and a total of 7 administrations were administered.

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Abstract

Provided is a recombinant antibody or an antigen-binding fragment. The recombinant antibody comprises: an anti-PD-1 antibody, and a humanized anti-VEGF antibody, a human VEGF trap protein or a humanized anti-VEGFR antibody; the anti-PD-1 antibody comprises a heavy chain and a light chain; the N-terminus of the humanized anti-VEGF antibody is linked with the C-terminus of the heavy chain of the anti-PD-1 antibody; the N-terminus of the human VEGF trap protein is linked with the C-terminus of the heavy chain of the anti-PD-1 antibody; or the C-terminus of the human VEGF trap protein is linked with the N-terminus of the heavy chain of the anti-PD-1 antibody; the N-terminus of the humanized anti-VEGFR antibody is linked with the C-terminus of the heavy chain of the anti-PD-1 antibody.

Description

重组抗体及其应用Recombinant antibody and its application 技术领域technical field
本发明属于生物医药领域，具体地，涉及重组抗体及其应用，更具体地，涉及重组抗体或抗原结合片段、核酸分子、表达载体、重组细胞、组合物以及所述重组抗体或抗原结合片段、核酸分子、表达载体、重组细胞或组合物在制备药物中的用途。The present invention belongs to the field of biomedicine, and specifically relates to recombinant antibodies and their applications, more specifically, to recombinant antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors, recombinant cells, compositions and the recombinant antibodies or antigen-binding fragments, Use of nucleic acid molecules, expression vectors, recombinant cells or compositions in the preparation of medicines.
背景技术Background technique
近年来癌症成为全球导致死亡的主要原因之一。晚期恶性肿瘤患者生存期短，生活质量差，是我国公民健康的重要挑战。单克隆抗体，以其高亲和力、高靶向性、高安全性和低异源性，满足针对肿瘤治疗的需求，逐渐成为抑制肿瘤的研发重点药物。2018年来，NMPA相继批准了抗细胞毒性T淋巴细胞相关蛋白-4(cytotoxic T-lymphocyte-associated protein-4，CTLA-4，又称CD152)和抗程序性死亡受体－1(programmed death-1，PD-1，又称CD279)单克隆抗体用于对抗对抗肿瘤疾病。其中，抗PD-1单克隆抗体在我国开始了广泛的应用。Cancer has become one of the leading causes of death worldwide in recent years. Patients with advanced malignant tumors have a short survival period and poor quality of life, which is an important challenge to the health of our citizens. Monoclonal antibody, with its high affinity, high targeting, high safety and low heterogeneity, meets the needs of tumor treatment, and has gradually become a key drug in the research and development of tumor inhibition. Since 2018, NMPA has successively approved anti-cytotoxic T-lymphocyte-associated protein-4 (CTLA-4, also known as CD152) and anti-programmed death-1 (programmed death-1). , PD-1, also known as CD279) monoclonal antibody is used to fight against tumor diseases. Among them, anti-PD-1 monoclonal antibody has been widely used in my country.
PD-1及其配体程序性死亡受体配体1和2(PD-L1和PD-L2)是一类抑制性的共刺激分子。人PD-1约为55kDa。PD-1在活化的T及B细胞表达，也在单核细胞、树突状细胞(dendritic cells，DCs)和调节性T细胞(T regulatory cells，Tregs)中表达。PD-1的两种配体，PD-L1(B7-H1，又称CD274)和PD-L2(B7-DC)除了表达在活化的T细胞、B细胞、巨噬细胞、树突状细胞和多种肿瘤细胞外，PD-L1还广泛表达在非淋巴器官，如心脏、血管、胎盘、骨骼肌、肺、肝、脾及胸腺等。PD-L2主要在活化的巨噬细胞、DCs和少数肿瘤细胞上表达。肿瘤细胞会利用PD-1/PD-L1信号通路的免疫抑制作用来实施免疫逃逸。多种肿瘤细胞会上调表达PD-L1，如非小细胞肺癌(Non-Small Cell Lung Cancer，NSCLC)、黑色素瘤、淋巴瘤、乳腺癌、白血病及各种泌尿系肿瘤、消化道肿瘤、生殖系肿瘤等。过量表达PD-L1从而与T细胞表面的PD-1受体相互作用，使T细胞活化所需的基因及细胞因子的转录和翻译受到抑制。PD-1 and its ligands programmed death ligand 1 and 2 (PD-L1 and PD-L2) are a class of inhibitory co-stimulatory molecules. Human PD-1 is approximately 55kDa. PD-1 is expressed in activated T and B cells, and also in monocytes, dendritic cells (DCs) and regulatory T cells (T regulatory cells, Tregs). The two ligands of PD-1, PD-L1 (B7-H1, also known as CD274) and PD-L2 (B7-DC) are expressed in activated T cells, B cells, macrophages, dendritic cells and In addition to a variety of tumor cells, PD-L1 is also widely expressed in non-lymphoid organs, such as the heart, blood vessels, placenta, skeletal muscle, lung, liver, spleen, and thymus. PD-L2 is mainly expressed on activated macrophages, DCs and a few tumor cells. Tumor cells will use the immunosuppressive effect of PD-1/PD-L1 signaling pathway to implement immune escape. A variety of tumor cells will up-regulate the expression of PD-L1, such as non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC), melanoma, lymphoma, breast cancer, leukemia and various urological tumors, gastrointestinal tumors, germline tumors Tumor etc. Overexpression of PD-L1 interacts with the PD-1 receptor on the surface of T cells, inhibiting the transcription and translation of genes and cytokines required for T cell activation.
因此，PD-1/PD-L1信号通路成为了肿瘤免疫治疗中最热门的分子靶标，一些单抗药物用于霍奇金淋巴瘤、肾癌、胃癌、肛门癌、肝癌、结直肠癌等肿瘤类型都已经取得了较好的临床试验结果，且还有多个抗PD-1和抗PDL1的抗体进入临床试验用于多种肿瘤的治疗。Therefore, the PD-1/PD-L1 signaling pathway has become the most popular molecular target in tumor immunotherapy, and some monoclonal antibodies are used for Hodgkin's lymphoma, kidney cancer, gastric cancer, anal cancer, liver cancer, colorectal cancer and other tumors All types have achieved good clinical trial results, and several anti-PD-1 and anti-PDL1 antibodies have entered clinical trials for the treatment of various tumors.
目前PD-1/PD-L1单抗的免疫调节作用机理赋予了其广谱抗肿瘤活性。PD-1/PD-L1单抗已经被大量的临床研究证明在治疗各类实体瘤和血液肿瘤时都显示了优于现有标准治疗方案的疗效和安全性；但是，PD-1/PD-L1单抗仍然具有以下缺点：(1)针对有效人群的响应率仍然有待提高，针对不同肿瘤适应症总体响应率不同，但是平均不到30％，另有70％的人无法获益；(2)为了提高人群响应率，PD-1/PD-L1单抗需要借助诊断试剂(Biomarker)帮助鉴别，单一Biomarker人群筛选存在弊端，多Biomarker则测 量成本较高；(3)容易产生耐药：癌症易复发，其耐药的机制可能有多种，例如肿瘤又产生了新的免疫逃逸通路，或者抗体药物因免疫原性产生了抗药抗体；(4)接受PD-1/PD-L1单抗治疗的肿瘤病人当中，会有一部分病人出现超进展的情况，即用药后肿瘤生长超过50％，在多种肿瘤中均存在此类情况，其发生率、结局和预测因素尚不清楚。因此，利用基因工程制备肿瘤抑制能力更强、肿瘤适应症的响应率更大的双抗分子是非常有必要的。The current immunomodulatory mechanism of PD-1/PD-L1 monoclonal antibody endows it with broad-spectrum anti-tumor activity. PD-1/PD-L1 monoclonal antibody has been proved by a large number of clinical studies to be more effective and safe than the existing standard treatment in the treatment of various solid tumors and hematological tumors; however, PD-1/PD- L1 monoclonal antibody still has the following disadvantages: (1) The response rate for the effective population still needs to be improved, and the overall response rate for different tumor indications is different, but the average is less than 30%, and another 70% of people cannot benefit; (2) ) In order to improve the response rate of the population, PD-1/PD-L1 monoclonal antibodies need to be identified with the help of diagnostic reagents (Biomarker). There are disadvantages in population screening with a single Biomarker, and the measurement cost of multiple Biomarkers is high; (3) It is easy to produce drug resistance: cancer It is prone to relapse, and its drug resistance may have multiple mechanisms, such as the tumor has developed a new immune escape pathway, or antibody drugs have produced anti-drug antibodies due to immunogenicity; (4) receiving PD-1/PD-L1 monoclonal antibody Among the treated tumor patients, some patients will experience hyperprogression, that is, the tumor growth exceeds 50% after treatment. This condition exists in many tumors, and its incidence, outcome and predictors are still unclear. Therefore, it is very necessary to use genetic engineering to prepare double antibody molecules with stronger tumor suppression ability and higher response rate for tumor indications.
血管内皮生长因子(Vascular Endothelial Growth Factor，VEGF)是细胞内刺激血管生成的信号蛋白，具备促进血管新生和再生的功能。细胞内VEGF通过结合并活化细胞表面的血管内皮生长因子受体(VascularEndothelial Growth Factor Receptor，VEGFR，又称酪氨酸激酶受体)，最终导致血管生成。VEGF包含5种配体，分别为VEGF-A、VEGF-B、VEGF-C、VEGF-D和PIGF，其中，VEGF-A能与VEGFR-1和VEGFR-2结合，是起最主要作用的血管内皮生长因子。PIGF和VEGF-B与VEGFR-1结合，VEGF-C和VEGF-D与VEGFR-3特异性结合。三种酪氨酸激酶受体VEGFR1、VEGFR2和VEGFR3，各配体对不同受体的亲和力和选择性存在差异。在所有受体之中，VEGFR-2扮演着最主要的作用，几乎介导所有VEGF已知的细胞应答。Vascular Endothelial Growth Factor (VEGF) is a signaling protein that stimulates angiogenesis in cells, and has the function of promoting angiogenesis and regeneration. Intracellular VEGF binds and activates the vascular endothelial growth factor receptor (VEGFR, also known as tyrosine kinase receptor) on the cell surface, eventually leading to angiogenesis. VEGF contains five kinds of ligands, namely VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF. Among them, VEGF-A can bind to VEGFR-1 and VEGFR-2 and play the most important role in vascular endothelial growth factor. PIGF and VEGF-B bind to VEGFR-1, and VEGF-C and VEGF-D specifically bind to VEGFR-3. There are three kinds of tyrosine kinase receptors VEGFR1, VEGFR2 and VEGFR3, each ligand has different affinity and selectivity for different receptors. Among all receptors, VEGFR-2 plays the most important role, mediating almost all known cellular responses to VEGF.
肿瘤微环境中低氧的特性、炎症因子以及炎症相关细胞聚集，使得VEGF表达量上调。在许多实体瘤中，肿瘤细胞往往都有着VEGFRs高表达基因突变。VEGF与VEGFR-2的结合激活一系列信号转导分子，例如磷脂酶Cγ(PLCγ)和磷脂酰肌醇3-激酶(PI3K)，进而引发细胞内一系列级联反应，最终的作用包括细胞分裂、迁移、血管渗透性改变和促进细胞存活。前期研究证实VEGF能增加血管通透性，导致肿瘤通过循环传播，促进肿瘤细胞向其他部位转移。此外，VEGF也能招募循环系统中的内皮前体细胞，并作为新生肿瘤血管的构成成分，促进肿瘤血管的生成，更进一步塑造典型的肿瘤微环境。因此，VEGF成为了肿瘤靶向治疗中备受关注的靶点。The characteristics of hypoxia in the tumor microenvironment, the accumulation of inflammatory factors and inflammation-related cells make the expression of VEGF up-regulated. In many solid tumors, tumor cells often have gene mutations with high expression of VEGFRs. The binding of VEGF to VEGFR-2 activates a series of signal transduction molecules, such as phospholipase Cγ (PLCγ) and phosphatidylinositol 3-kinase (PI3K), which in turn initiates a series of cascade reactions in the cell, and the final effects include cell division , migration, changes in vascular permeability and promotion of cell survival. Previous studies have confirmed that VEGF can increase the permeability of blood vessels, leading to the spread of tumors through the circulation and promoting the metastasis of tumor cells to other parts. In addition, VEGF can also recruit endothelial precursor cells in the circulatory system, and act as a constituent of new tumor blood vessels, promote tumor angiogenesis, and further shape the typical tumor microenvironment. Therefore, VEGF has become a target of much concern in tumor targeted therapy.
多种针对VEGF/VEGFR2的靶向药物被研制出来，用于针对包括结直肠癌、乳腺癌、非小细胞肺癌和甲状腺癌等多种肿瘤的一线治疗或者二线治疗；其类型包括：单克隆抗体(如：anti-VEGF贝伐珠单抗和anti-VEGFR2雷莫芦单抗)、抗体诱骗剂(如：阿柏西普)和小分子抑制剂(如：索拉非尼、舒尼替尼)等。A variety of targeted drugs against VEGF/VEGFR2 have been developed for the first-line or second-line treatment of various tumors including colorectal cancer, breast cancer, non-small cell lung cancer and thyroid cancer; the types include: monoclonal antibody (eg, anti-VEGF bevacizumab and anti-VEGFR2 ramucirumab), antibody decoys (eg, aflibercept), and small molecule inhibitors (eg, sorafenib, sunitinib )wait.
然而，单一的VEGF/VEGFR靶向药物仍然有其缺点。VEGF被证实会抑制免疫应答。VEGF可以抑制树突细胞(DC)的成熟并破坏造血前体细胞的正常分化；VEGF还可以诱导DC上PD-L1的表达；还可以通过免疫调节T细胞(Regulatory T cells，Treg)上的神经毡蛋白1(NRP1)信号激活抗原特异性免疫调节T细胞，从而下调肿瘤免疫应答。研究表明，VEGF单抗药物和抗PD-1以及CTLA-4单抗这类免疫调节型抗体联用，不仅能挽回VEGF对免疫应答抑制的不利影响，还能间接促进T细胞的肿瘤浸润效果，激活T细胞对免疫抗原的响应，二者具有抗肿瘤协同效应。所以，开发一款能同时靶向VEGF/VEGFR 并能阻断PD-1/PD-L1免疫检查点的双功能抗体能克服各靶点的弱点，增加彼此的辅助作用，从而发挥更大的抗肿瘤效应。However, single VEGF/VEGFR targeting drugs still have their disadvantages. VEGF has been shown to suppress immune responses. VEGF can inhibit the maturation of dendritic cells (DC) and destroy the normal differentiation of hematopoietic precursor cells; VEGF can also induce the expression of PD-L1 on DC; Nirin 1 (NRP1) signaling activates antigen-specific immunoregulatory T cells, thereby downregulating tumor immune responses. Studies have shown that the combination of VEGF monoclonal antibody drugs and immunomodulatory antibodies such as anti-PD-1 and CTLA-4 monoclonal antibodies can not only restore the adverse effect of VEGF on immune response suppression, but also indirectly promote the tumor infiltration effect of T cells, Activate T cells to respond to immune antigens, and the two have anti-tumor synergistic effects. Therefore, the development of a bifunctional antibody that can simultaneously target VEGF/VEGFR and block the PD-1/PD-L1 immune checkpoint can overcome the weaknesses of each target and increase the auxiliary effect of each other, thereby exerting a greater anti-inflammatory effect. tumor effect.
发明内容Contents of the invention
本发明是基于发明人对以下事实和问题的发现和认识作出的：The present invention is based on the inventor's discovery and recognition of the following facts and problems:
研究表明，PD-1/PD-L1或VEGF单抗在治疗肿瘤时存在一定的缺陷，如仅使用PD-1/PD-L1单抗治疗肿瘤时，针对有效人群或不同肿瘤适应症的响应率较低，且针对不同肿瘤适应症总体响应率存在较大差异，会出现耐药、肿瘤复发或肿瘤超进展等情况；仅使用VEGF单抗治疗肿瘤会导致机体出现免疫应答抑制。经过大量的研究后，发明人意外地发现，将PD-1/PD-L1和VEGF/VEGFR单抗或单抗的部分片段通过特定的方式进行组合，获得的重组抗体可特异性识别PD-1、PD-L1、VEGF和VEGFR中的至少之一，克服了PD-1单抗耐药的问题，减少了对Biomarker的依赖，以及机体出现免疫应答抑制等问题，从而增大了各肿瘤适应症的响应率。Studies have shown that PD-1/PD-L1 or VEGF monoclonal antibodies have certain defects in the treatment of tumors, such as the response rate for effective population or different tumor indications when only PD-1/PD-L1 monoclonal antibodies are used to treat tumors The overall response rate for different tumor indications is relatively low, and there are large differences in the overall response rate for different tumor indications, and drug resistance, tumor recurrence, or tumor hyperprogression may occur; only using VEGF monoclonal antibody to treat tumors will lead to immune response suppression in the body. After a lot of research, the inventor unexpectedly found that the recombinant antibody obtained by combining PD-1/PD-L1 and VEGF/VEGFR monoclonal antibody or partial fragments of monoclonal antibody in a specific way can specifically recognize PD-1 At least one of PD-L1, VEGF and VEGFR, overcome the problem of PD-1 monoclonal antibody resistance, reduce the dependence on Biomarker, and the immune response suppression of the body, thus increasing the indications of various tumors response rate.
因此，在本发明的第一方面，本发明提出了一种重组抗体。根据本发明的实施例，包括：抗PD-1/PD-L1的抗体，所述抗PD-1的抗体包含重链和轻链；以及人抗VEGF的抗体、人抗VEGFR的抗体或VEGF诱捕蛋白，所述人抗VEGF的抗体的N端与所述抗PD-1的抗体的重链的C端相连；或所述VEGF诱捕蛋白的N端与所述抗PD-1的抗体的重链的C端相连或所述VEGF诱捕蛋白的C端与所述抗PD-1的抗体的重链的N端相连，或所述人抗VEGFR的抗体的N端与所述抗PD-1的抗体的重链的C端相连。所述重组抗体由抗PD-1的抗体和人抗VEGF的抗体、人抗VEGFR的抗体或人VEGF诱捕蛋白组成，发明人通过大量实验对两种抗体及人VEGF诱捕蛋白分别与所述抗PD-1的抗体的组合方式进行探索，意外地发现，当所述人抗VEGF的抗体的N端与所述抗PD-1的抗体的重链的C端相连时，所述重组抗体与PD-1和/或VEGF的结合活性较强，当所述VEGF诱捕蛋白的N端与所述抗PD-1的抗体的重链的C端相连或所述VEGF诱捕蛋白的C端与所述抗PD-1的抗体的重链的N端相连，所述重组抗体与PD-1和/或VEGF的结合活性均较强，当所述人抗VEGFR的抗体的N端与所述抗PD-1的抗体的重链的C端相连，所述重组抗体与PD-1和/或人抗VEGFR的抗体的结合活性均较强。根据本发明实施例的重组抗体可以有效与PD-1、VEGF或VEGFR进行结合，克服了PD-1单抗耐药的问题以及VEGF单抗导致的机体出现免疫应答抑制的问题，更大的发挥了PD-1靶点的免疫调节作用，显著提高了各肿瘤适应症的响应率，抗肿瘤效果较单抗得到显著提升。Therefore, in the first aspect of the present invention, the present invention proposes a recombinant antibody. According to an embodiment of the present invention, it includes: an anti-PD-1/PD-L1 antibody, the anti-PD-1 antibody comprising a heavy chain and a light chain; and a human anti-VEGF antibody, a human anti-VEGFR antibody or a VEGF decoy protein, the N-terminal of the human anti-VEGF antibody is connected to the C-terminal of the heavy chain of the anti-PD-1 antibody; or the N-terminal of the VEGF trap protein is connected to the heavy chain of the anti-PD-1 antibody or the C-terminus of the VEGF-trap protein is connected to the N-terminus of the heavy chain of the anti-PD-1 antibody, or the N-terminus of the human anti-VEGFR antibody is connected to the anti-PD-1 antibody The C-terminus of the heavy chain is connected. The recombinant antibody is composed of an anti-PD-1 antibody and a human anti-VEGF antibody, a human anti-VEGFR antibody or a human VEGF trap protein. -1 antibody combination was explored, and it was unexpectedly found that when the N-terminal of the human anti-VEGF antibody was connected to the C-terminal of the heavy chain of the anti-PD-1 antibody, the recombinant antibody and PD-1 antibody were combined. 1 and/or VEGF has a strong binding activity, when the N-terminal of the VEGF-trap protein is connected to the C-terminal of the heavy chain of the anti-PD-1 antibody or the C-terminal of the VEGF-trap protein is connected to the anti-PD The N-terminus of the heavy chain of the antibody of -1 is connected, and the binding activity of the recombinant antibody to PD-1 and/or VEGF is strong, when the N-terminus of the human anti-VEGFR antibody is connected to the anti-PD-1 The C-terminus of the heavy chain of the antibody is connected, and the recombinant antibody has strong binding activity to PD-1 and/or human anti-VEGFR antibody. The recombinant antibody according to the embodiment of the present invention can effectively bind to PD-1, VEGF or VEGFR, overcome the problem of drug resistance of PD-1 monoclonal antibody and the problem of immune response suppression caused by VEGF monoclonal antibody, and play a greater role The immunomodulatory effect of the PD-1 target has been significantly improved, and the response rate of various tumor indications has been significantly improved, and the anti-tumor effect has been significantly improved compared with monoclonal antibodies.
根据本发明的实施例，上述重组抗体还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned recombinant antibody may further include at least one of the following additional technical features:
根据本发明的实施例，所述人抗VEGFR的抗体包含Ramucirumab单链抗体。Ramucirumab单链抗体为能阻断人血管内皮生长因子-血管内皮生长因子受体(VEGF-VEGFR)轴的单链抗体，Ramucirumab单链抗体可与VEGFR2进行结合。其中，Ramucirumab单链抗体具有如SEQ ID NO：18所示的重链可变 区和SEQ ID NO：19所示的轻链可变区。According to an embodiment of the present invention, the human anti-VEGFR antibody comprises a Ramucirumab single-chain antibody. Ramucirumab single-chain antibody is a single-chain antibody that can block the human vascular endothelial growth factor-vascular endothelial growth factor receptor (VEGF-VEGFR) axis, and Ramucirumab single-chain antibody can bind to VEGFR2. Wherein, the Ramucirumab single-chain antibody has a heavy chain variable region shown in SEQ ID NO: 18 and a light chain variable region shown in SEQ ID NO: 19.
Ramucirumab单链抗体具有如下所示的重链可变区：Ramucirumab single-chain antibodies have the heavy chain variable region shown below:
Figure PCTCN2021132785-appb-000001
Figure PCTCN2021132785-appb-000001
Ramucirumab单链抗体具有如下所示的轻链可变区：Ramucirumab single-chain antibodies have the light chain variable regions shown below:
Figure PCTCN2021132785-appb-000002
Figure PCTCN2021132785-appb-000002
根据本发明的实施例，所述人VEGF诱捕蛋白包含VEGFR1D2或VEGFR2D3。所述VEGFR1D2和VEGFR2D3为VEGFR的结构域，可以与VEGF结合，能阻断人血管内皮生长因子-血管内皮生长因子受体(VEGF-VEGFR)结合，根据本发明的具体实施例，所述VEGFR1D2、VEGFR2D3具有如下所示的氨基酸序列。According to an embodiment of the present invention, the human VEGF trap protein comprises VEGFR1D2 or VEGFR2D3. The VEGFR1D2 and VEGFR2D3 are domains of VEGFR, which can be combined with VEGF and can block the binding of human vascular endothelial growth factor-vascular endothelial growth factor receptor (VEGF-VEGFR). According to specific embodiments of the present invention, the VEGFR1D2, VEGFR2D3 has the amino acid sequence shown below.
VEGFR1D2具有如下所示的氨基酸序列：VEGFR1D2 has the amino acid sequence shown below:
Figure PCTCN2021132785-appb-000003
Figure PCTCN2021132785-appb-000003
VEGFR2D3具有如下所示的氨基酸序列：VEGFR2D3 has the amino acid sequence shown below:
Figure PCTCN2021132785-appb-000004
Figure PCTCN2021132785-appb-000004
人抗VEGF的抗体具有如下所示的氨基酸序列：The human anti-VEGF antibody has the amino acid sequence shown below:
Figure PCTCN2021132785-appb-000005
Figure PCTCN2021132785-appb-000005
根据本发明的实施例，所述抗PD-1的抗体为抗PD-1的IgG类抗体。According to an embodiment of the present invention, the anti-PD-1 antibody is an anti-PD-1 IgG antibody.
根据本发明的实施例，进一步包括连接肽，所述连接肽的N端与所述抗PD-1的抗体的重链的C端相连，所述连接肽的C端与所述人抗VEGF的抗体的N端相连。According to an embodiment of the present invention, it further includes a connecting peptide, the N-terminal of the connecting peptide is connected with the C-terminal of the heavy chain of the anti-PD-1 antibody, and the C-terminal of the connecting peptide is connected with the human anti-VEGF The N-termini of the antibodies are linked.
根据本发明的实施例，所述连接肽的N端与所述抗PD-1的抗体的重链的C端相连，所述连接肽的C端与所述Ramucirumab单链抗体或VEGFR1D2的N端相连。According to an embodiment of the present invention, the N-terminal of the connecting peptide is connected to the C-terminal of the heavy chain of the anti-PD-1 antibody, and the C-terminal of the connecting peptide is connected to the N-terminal of the Ramucirumab single-chain antibody or VEGFR1D2 connected.
根据本发明的实施例，所述连接肽的N端与所述VEGFR1D2的C端相连，所述连接肽的C端与所述抗PD-1的抗体的重链的N端相连。According to an embodiment of the present invention, the N-terminal of the connecting peptide is connected to the C-terminal of the VEGFR1D2, and the C-terminal of the connecting peptide is connected to the N-terminal of the heavy chain of the anti-PD-1 antibody.
根据本发明的实施例，所述连接肽的C端与所述抗PD-1的抗体的重链的N端相连，所述连接肽的 N端与所述VEGFR2D3的C端相连，所述VEGFR2D3的N端与所述VEGFR1D2的C端相连。According to an embodiment of the present invention, the C-terminus of the connecting peptide is connected to the N-terminus of the heavy chain of the anti-PD-1 antibody, the N-terminus of the connecting peptide is connected to the C-terminus of the VEGFR2D3, and the VEGFR2D3 The N-terminal of VEGFR1D2 is connected with the C-terminal of VEGFR1D2.
根据本发明的实施例，所述连接肽的N端与所述抗PD-1的抗体的重链的C端相连，所述连接肽的C端与所述VEGFR1D2的N端相连，所述VEGFR1D2的C端与所述VEGFR2D3的N端相连。According to an embodiment of the present invention, the N-terminal of the connecting peptide is connected to the C-terminal of the heavy chain of the anti-PD-1 antibody, the C-terminal of the connecting peptide is connected to the N-terminal of the VEGFR1D2, and the VEGFR1D2 The C-terminal of VEGFR2D3 is connected with the N-terminal of VEGFR2D3.
根据本发明的实施例，所述连接肽具有SEQ ID NO：1所示的氨基酸序列。According to an embodiment of the present invention, the connecting peptide has the amino acid sequence shown in SEQ ID NO:1.
Figure PCTCN2021132785-appb-000006
Figure PCTCN2021132785-appb-000006
在本发明的第二方面，本发明提出了一种重组抗体。根据本发明的实施例，所述重组抗体的轻链具有SEQ ID NO：2所示的氨基酸序列，所述重组抗体的重链选自SEQ ID NO：3-6、SEQ ID NO：23和SEQ ID NO：25所示的氨基酸序列的至少之一。根据本发明实施例的重组抗体可以有效与PD-1、VEGF或VEGFR进行结合，克服了PD-1单抗耐药的问题以及VEGF单抗导致的机体出现免疫应答抑制的问题，显著提高了各肿瘤适应症的响应率，抗肿瘤效果较单抗得到显著提升。In the second aspect of the present invention, the present invention provides a recombinant antibody. According to an embodiment of the present invention, the light chain of the recombinant antibody has the amino acid sequence shown in SEQ ID NO: 2, and the heavy chain of the recombinant antibody is selected from SEQ ID NO: 3-6, SEQ ID NO: 23 and SEQ ID NO: ID NO: at least one of the amino acid sequences shown in 25. The recombinant antibody according to the embodiment of the present invention can effectively bind to PD-1, VEGF or VEGFR, overcome the problem of drug resistance of PD-1 monoclonal antibody and the problem of immune response suppression caused by VEGF monoclonal antibody, and significantly improve the The response rate and anti-tumor effect of tumor indications have been significantly improved compared with monoclonal antibodies.
Figure PCTCN2021132785-appb-000007
Figure PCTCN2021132785-appb-000007
Figure PCTCN2021132785-appb-000008
Figure PCTCN2021132785-appb-000008
Figure PCTCN2021132785-appb-000009
Figure PCTCN2021132785-appb-000009
在本发明的第三方面，本发明提出了一种核酸。根据本发明的实施例，所述核酸编码第一方面所述的重组抗体。根据本发明实施例的核酸编码的重组抗体可以有效与PD-1、VEGF或VEGFR进行结合，克服了PD-1单抗耐药的问题以及VEGF单抗导致的机体出现免疫应答抑制的问题，显著提高了各肿瘤适应症的响应率，抗肿瘤效果较单抗得到显著提升。In a third aspect of the present invention, the present invention proposes a nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes the recombinant antibody described in the first aspect. The recombinant antibody encoded by the nucleic acid according to the embodiment of the present invention can effectively bind to PD-1, VEGF or VEGFR, overcome the problem of PD-1 monoclonal antibody drug resistance and the problem of immune response suppression caused by VEGF monoclonal antibody, significantly The response rate of various tumor indications has been improved, and the anti-tumor effect has been significantly improved compared with monoclonal antibodies.
根据本发明的实施例，上述核酸还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned nucleic acid may further include at least one of the following additional technical features:
根据本发明的实施例，所述核酸具有SEQ ID NO：7所示的核苷酸序列，所述核酸进一步包括SEQ ID NO：8-11、SEQ ID NO：27和SEQ ID NO：28所示的核苷酸序列的至少之一。SEQ ID NO：7所示的核苷酸序列可编码所述重组抗体的轻链，SEQ ID NO：8-11、SEQ ID NO：27和SEQ ID NO：28所示的核苷酸序列可编码所述重组抗体的重链。According to an embodiment of the present invention, the nucleic acid has a nucleotide sequence shown in SEQ ID NO: 7, and the nucleic acid further includes SEQ ID NO: 8-11, SEQ ID NO: 27 and SEQ ID NO: 28 at least one of the nucleotide sequences. The nucleotide sequence shown in SEQ ID NO: 7 can encode the light chain of the recombinant antibody, and the nucleotide sequences shown in SEQ ID NO: 8-11, SEQ ID NO: 27 and SEQ ID NO: 28 can encode The heavy chain of the recombinant antibody.
编码重组抗体轻链的核苷酸序列：Nucleotide sequence encoding recombinant antibody light chain:
Figure PCTCN2021132785-appb-000010
Figure PCTCN2021132785-appb-000010
编码重组抗体1号重链的核苷酸序列：Nucleotide sequence encoding recombinant antibody No. 1 heavy chain:
Figure PCTCN2021132785-appb-000011
Figure PCTCN2021132785-appb-000011
Figure PCTCN2021132785-appb-000012
Figure PCTCN2021132785-appb-000012
编码重组抗体2号重链的核苷酸序列：The nucleotide sequence encoding the No. 2 heavy chain of the recombinant antibody:
Figure PCTCN2021132785-appb-000013
Figure PCTCN2021132785-appb-000013
Figure PCTCN2021132785-appb-000014
Figure PCTCN2021132785-appb-000014
编码重组抗体4号重链的核苷酸序列：The nucleotide sequence encoding the No. 4 heavy chain of the recombinant antibody:
Figure PCTCN2021132785-appb-000015
Figure PCTCN2021132785-appb-000015
编码重组抗体6号重链的核苷酸序列：The nucleotide sequence encoding the No. 6 heavy chain of the recombinant antibody:
Figure PCTCN2021132785-appb-000016
Figure PCTCN2021132785-appb-000016
Figure PCTCN2021132785-appb-000017
Figure PCTCN2021132785-appb-000017
编码重组抗体3号重链的核苷酸序列：The nucleotide sequence encoding the No. 3 heavy chain of the recombinant antibody:
Figure PCTCN2021132785-appb-000018
Figure PCTCN2021132785-appb-000018
Figure PCTCN2021132785-appb-000019
Figure PCTCN2021132785-appb-000019
编码重组抗体5号重链的核苷酸序列：The nucleotide sequence encoding the No. 5 heavy chain of the recombinant antibody:
Figure PCTCN2021132785-appb-000020
Figure PCTCN2021132785-appb-000020
在本发明的第四方面，本发明提出了一种表达载体。根据本发明的实施例，所述表达载体包括：第一核酸分子，所述第一核酸分子编码抗PD-1的抗体轻链；第二核酸分子，所述第二核酸分子编码抗PD-1的抗体重链；第三核酸分子，所述第三核酸分子编码人抗VEGF的抗体、人抗VEGFR的抗体或人VEGF诱捕蛋白。根据本发明实施例的表达载体所表达获得的重组抗体可以有效与PD-1、VEGF或VEGFR进行结合，克服了PD-1单抗耐药的问题以及VEGF单抗导致的机体出现免疫应答抑制的问题，显著提高了各肿瘤适应症的响应率，抗肿瘤效果较单抗得到显著提升。In the fourth aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector includes: a first nucleic acid molecule encoding an anti-PD-1 antibody light chain; a second nucleic acid molecule encoding an anti-PD-1 the heavy chain of the antibody; a third nucleic acid molecule encoding a human anti-VEGF antibody, a human anti-VEGFR antibody, or a human VEGF trap. The recombinant antibody expressed by the expression vector according to the embodiment of the present invention can effectively bind to PD-1, VEGF or VEGFR, and overcome the problem of drug resistance of PD-1 monoclonal antibody and the suppression of the immune response of the body caused by VEGF monoclonal antibody The response rate of various tumor indications has been significantly improved, and the anti-tumor effect has been significantly improved compared with monoclonal antibodies.
根据本发明的实施例，上述表达载体还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned expression vector may further include at least one of the following additional technical features:
根据本发明的实施例，进一步包括：第四核酸分子，所述第四核酸分子设置在所述第二核酸分子与所述第三核酸分子之间，并且所述第四核酸分子编码连接肽。在将上述核酸分子连接到载体上时，可以将核酸分子与载体上的控制元件直接或者间接相连，只要这些控制元件能够控制核酸分子的翻译和表达等即可。当然这些控制元件可以直接来自于载体本身，也可以是外源性的，即并非来自于载体本身。当然，核酸分子与控制元件进行可操作地连接即可。According to an embodiment of the present invention, it further includes: a fourth nucleic acid molecule, the fourth nucleic acid molecule is arranged between the second nucleic acid molecule and the third nucleic acid molecule, and the fourth nucleic acid molecule encodes a connecting peptide. When the above-mentioned nucleic acid molecule is linked to the carrier, the nucleic acid molecule can be directly or indirectly linked to the control elements on the carrier, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, it is sufficient that the nucleic acid molecule is operably linked to the control element.
根据本发明的实施例，所述第一核酸分子具有如下所示的核苷酸序列：According to an embodiment of the present invention, the first nucleic acid molecule has a nucleotide sequence as shown below:
Figure PCTCN2021132785-appb-000021
Figure PCTCN2021132785-appb-000021
根据本发明的实施例，所述第二核酸分子具有SEQ ID NO：13所示的核苷酸序列。According to an embodiment of the present invention, the second nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 13.
Figure PCTCN2021132785-appb-000022
Figure PCTCN2021132785-appb-000022
Figure PCTCN2021132785-appb-000023
Figure PCTCN2021132785-appb-000023
根据本发明的实施例，所述第三核酸分子具有SEQ ID NO：14、15、16或29所示的核苷酸序列。According to an embodiment of the present invention, the third nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 14, 15, 16 or 29.
Figure PCTCN2021132785-appb-000024
Figure PCTCN2021132785-appb-000024
根据本发明的实施例，所述第四核酸分子具有SEQ ID NO：17所示的核苷酸序列。According to an embodiment of the present invention, the fourth nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 17.
Figure PCTCN2021132785-appb-000025
Figure PCTCN2021132785-appb-000025
根据本发明的实施例，所述表达载体包括但不限于重组慢病毒、腺病毒、逆转录病毒，电刺激或者脂质体转染表达前面所述蛋白的DNA载体进入到细胞中形成具有治疗价值的细胞治疗产品，所述细胞包括但不限于CAR-T、TCR-T、TIL、CAR-NK、CAR-M或CAR-DC等。According to an embodiment of the present invention, the expression vectors include but are not limited to recombinant lentiviruses, adenoviruses, and retroviruses. Electrical stimulation or liposome transfection of DNA vectors expressing the above-mentioned proteins into cells has therapeutic value Cell therapy products, including but not limited to CAR-T, TCR-T, TIL, CAR-NK, CAR-M or CAR-DC, etc.
在本发明的第五方面，本发明提出了一种制备第一方面所述的重组抗体的方法。根据本发明的实施例，包括：将第四方面所述的表达载体引入到真核细胞中；将所述真核细胞在适于蛋白表达和分泌的条件下进行培养，以便获得所述重组抗体。根据本发明实施例的方法所获得的重组抗体可以有效与PD-1、VEGF或VEGFR进行结合，克服了PD-1单抗耐药的问题以及VEGF单抗导致的机体出现免疫应答抑制的问题，显著提高了各肿瘤适应症的响应率，抗肿瘤效果较单抗得到显著提升。In the fifth aspect of the present invention, the present invention proposes a method for preparing the recombinant antibody described in the first aspect. According to an embodiment of the present invention, it includes: introducing the expression vector described in the fourth aspect into eukaryotic cells; culturing the eukaryotic cells under conditions suitable for protein expression and secretion, so as to obtain the recombinant antibody . The recombinant antibody obtained according to the method of the embodiment of the present invention can effectively bind to PD-1, VEGF or VEGFR, overcome the problem of PD-1 monoclonal antibody drug resistance and the problem of immune response suppression caused by VEGF monoclonal antibody, The response rate of various tumor indications has been significantly improved, and the anti-tumor effect has been significantly improved compared with monoclonal antibodies.
根据本发明的实施例，上述方法还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:
根据本发明的实施例，所述真核细胞为哺乳动物细胞。According to an embodiment of the present invention, the eukaryotic cells are mammalian cells.
根据本发明的实施例，所述哺乳动物细胞包括选自CHO-K1、CHOS、293F、293T、CAR-T、TCR-T、TIL、CAR-NK、CAR-M和CAR-DC中的至少之一。According to an embodiment of the present invention, the mammalian cells include at least one selected from CHO-K1, CHOS, 293F, 293T, CAR-T, TCR-T, TIL, CAR-NK, CAR-M and CAR-DC. one.
根据本发明的实施例，所述哺乳动物细胞不包括动物生殖细胞、受精卵或胚胎干细胞。According to an embodiment of the present invention, the mammalian cells do not include animal germ cells, fertilized eggs or embryonic stem cells.
在本发明的第六方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞携带第三方面所述的核酸，或第四方面所述的表达载体。根据本发明实施例的重组细胞在一定条件下分泌所获得的重组抗体可以有效与PD-1、VEGF或VEGFR进行结合，克服了PD-1单抗耐药的问题以及VEGF单抗导致的机体出现免疫应答抑制的问题，显著提高了各肿瘤适应症的响应率，抗肿瘤效果较单抗得到显著提升。In the sixth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the nucleic acid described in the third aspect, or the expression vector described in the fourth aspect. According to the embodiment of the present invention, the recombinant antibody secreted by recombinant cells under certain conditions can effectively bind to PD-1, VEGF or VEGFR, and overcome the problem of PD-1 monoclonal antibody resistance and the emergence of the body caused by VEGF monoclonal antibody The problem of immune response suppression has significantly improved the response rate of various tumor indications, and the anti-tumor effect has been significantly improved compared with monoclonal antibodies.
在本发明的第七方面，本发明提出了一种用于治疗癌症的组合物。根据本发明的实施例，包括：第一或第二方面所述的重组抗体、第三方面所述的核酸、第四方面所述的表达载体或第六方面所述的重组细胞。根据本发明实施例的组合物可以有效与PD-1、VEGF或VEGFR进行结合，克服了PD-1单抗耐药的问题以及VEGF单抗导致的机体出现免疫应答抑制的问题，显著提高了各肿瘤适应症的响应率，抗肿瘤效果较单抗得到显著提升。In the seventh aspect of the present invention, the present invention proposes a composition for treating cancer. According to the embodiments of the present invention, it includes: the recombinant antibody described in the first or second aspect, the nucleic acid described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cell described in the sixth aspect. The composition according to the embodiment of the present invention can effectively bind to PD-1, VEGF or VEGFR, overcome the problem of PD-1 monoclonal antibody drug resistance and the problem of immune response suppression caused by VEGF monoclonal antibody, and significantly improve each The response rate and anti-tumor effect of tumor indications have been significantly improved compared with monoclonal antibodies.
根据本发明的实施例，上述组合物还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above composition may further include at least one of the following additional technical features:
根据本发明的实施例，所述癌症包括选自肺癌，胃癌，肝癌，肠癌，食管癌，乳腺癌，宫颈癌，恶性淋巴瘤，鼻咽癌，白血病,皮肤癌和肾癌中的至少之一。According to an embodiment of the present invention, the cancer includes at least one selected from lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, leukemia, skin cancer and kidney cancer. one.
根据本发明的实施例，所述组合物进一步包括：药学上可接受的辅剂。According to an embodiment of the present invention, the composition further includes: a pharmaceutically acceptable adjuvant.
根据本发明的实施例，所述药学上可接受的辅剂包括稳定剂、湿润剂、乳化剂、粘合剂、等渗剂的至少之一。According to an embodiment of the present invention, the pharmaceutically acceptable adjuvant includes at least one of a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent.
根据本发明的实施例，所述药物呈片剂、颗粒剂、散剂、胶囊剂、溶液剂、悬浮剂、冻干制剂的至少一种。According to an embodiment of the present invention, the medicine is in the form of at least one of tablet, granule, powder, capsule, solution, suspension, and freeze-dried preparation.
在本发明的第八方面，本发明提出了第一或第二方面所述的重组抗体、第三方面所述的核酸、第四方面所述的表达载体、第六方面所述的重组细胞或第七方面所述的组合物在制备药物中的用途。根据本发明的实施例，所述药物用于治疗或者预防癌症。根据本发明实施例的的重组抗体、核酸、表达载体、重组细胞或组合物在一定条件下可以有效与PD-1、VEGF或VEGFR进行结合，可以有效治疗或预防癌症。In the eighth aspect of the present invention, the present invention provides the recombinant antibody described in the first or second aspect, the nucleic acid described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the sixth aspect or Use of the composition described in the seventh aspect in the preparation of medicaments. According to an embodiment of the present invention, the medicament is used for treating or preventing cancer. The recombinant antibody, nucleic acid, expression vector, recombinant cell or composition according to the embodiments of the present invention can effectively bind to PD-1, VEGF or VEGFR under certain conditions, and can effectively treat or prevent cancer.
根据本发明的实施例，上述用途还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above use may further include at least one of the following additional technical features:
根据本发明的实施例，所述癌症包括选自肺癌，胃癌，肝癌，肠癌，食管癌，乳腺癌，宫颈癌，恶性淋巴瘤，鼻咽癌，白血病,皮肤癌，肾癌的至少之一。According to an embodiment of the present invention, the cancer includes at least one selected from lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, leukemia, skin cancer, and kidney cancer .
在本发明的第九方面，本发明提出了一种预防或治疗癌症的方法。根据本发明的实施例，包括向受试者施用有效剂量的以下中的至少之一：前面所述的重组抗体、核酸、表达载体、重组细胞以及组合物。在一定条件下所述重组抗体、核酸、表达载体、重组细胞以及组合物可以有效与PD-1、VEGF或VEGFR进行结合，一方面阻断肿瘤细胞利用PD-1/PD-L1信号通路的免疫抑制作用，另一方面，阻断VEGF与VEGFR的结合，抑制肿瘤血管的生成，从而有效治疗或预防癌症。因此，根据本发明实施例的方法可以有效预防或治疗癌症。In the ninth aspect of the present invention, the present invention provides a method for preventing or treating cancer. According to the embodiments of the present invention, it includes administering to the subject an effective dose of at least one of the following: the aforementioned recombinant antibody, nucleic acid, expression vector, recombinant cell and composition. Under certain conditions, the recombinant antibodies, nucleic acids, expression vectors, recombinant cells, and compositions can effectively bind to PD-1, VEGF, or VEGFR. Inhibition, on the other hand, blocks the binding of VEGF to VEGFR and inhibits tumor angiogenesis, thereby effectively treating or preventing cancer. Therefore, the method according to the embodiment of the present invention can effectively prevent or treat cancer.
根据本发明的实施例，上述方法还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:
根据本发明的实施例，所述癌症包括选自肺癌，胃癌，肝癌，肠癌，食管癌，乳腺癌，宫颈癌，恶性淋巴瘤，鼻咽癌，白血病，皮肤癌，肾癌的至少之一。According to an embodiment of the present invention, the cancer includes at least one selected from lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, leukemia, skin cancer, and kidney cancer .
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
附图说明Description of drawings
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：The above and/or additional aspects and advantages of the present invention will become apparent and understandable from the description of the embodiments in conjunction with the following drawings, wherein:
图1是根据本发明实施例的重组抗体与靶点分子人PD-1的ELISA结合活性结果图，其中，HX008为抗PD-1的抗体，HX016-1为PD-1/VEGF重组抗体的第1种结构，HX016-2为PD-1/VEGF重组抗体的第2种结构，HX016-4为PD-1/VEGF重组抗体的第4种结构，HX016-6为PD-1/VEGF重组抗体的第6种结构，293F-HX016-4-0919AC表示用293F细胞瞬转表达出来的批号为0919AC的样品，HX016-6-0929UF为批号为0929UF的HX016-6样品，R2是对数据进行四参数拟合之后的总体拟合优度，Abs为450nm处的吸光度，Antibody Conc.为待测抗体的浓度；Figure 1 is a graph showing the ELISA binding activity results of the recombinant antibody and the target molecule human PD-1 according to an embodiment of the present invention, wherein HX008 is an anti-PD-1 antibody, and HX016-1 is the first PD-1/VEGF recombinant antibody. 1 structure, HX016-2 is the second structure of PD-1/VEGF recombinant antibody, HX016-4 is the fourth structure of PD-1/VEGF recombinant antibody, HX016-6 is the structure of PD-1/VEGF recombinant antibody The sixth structure, 293F-HX016-4-0919AC represents the sample with batch number 0919AC expressed transiently by 293F cells, HX016-6-0929UF is the sample of HX016-6 with batch number 0929UF, R2 is the four-parameter simulation of the data The overall goodness of fit after integration, Abs is the absorbance at 450nm, Antibody Conc. is the concentration of the antibody to be tested;
图2是根据本发明实施例的重组抗体与靶点分子人VEGF165的ELISA结合活性结果图，其中，HX006为VEGF抗体，Avastin为已上市VEGF单克隆抗体产品，HX016-2为PD-1/VEGF重组抗体的第2种结构，HX016-4为PD-1/VEGF重组抗体的第4种结构，HX016-6为PD-1/VEGF重组抗体的第6种结构，Abs为450nm处的吸光度，Antibody Conc.为待测抗体的浓度；Figure 2 is a graph showing the ELISA binding activity results of the recombinant antibody and the target molecule human VEGF165 according to an embodiment of the present invention, wherein HX006 is a VEGF antibody, Avastin is a marketed VEGF monoclonal antibody product, and HX016-2 is PD-1/VEGF The second structure of the recombinant antibody, HX016-4 is the fourth structure of the PD-1/VEGF recombinant antibody, HX016-6 is the sixth structure of the PD-1/VEGF recombinant antibody, Abs is the absorbance at 450nm, Antibody Conc. is the concentration of the antibody to be tested;
图3是根据本发明实施例的不同批次的HX016-1与靶点分子人VEGFR2的ELISA结合活性结果图，其中，HX016-1为PD-1/VEGF重组抗体的第1种结构，括号中代表HX016-1样品的不同批次(Lot)，如HX016-1-5K-2-E2(Lot20190419)表示第20190419批次生产的HX016-1，Abs为450nm处的吸光度，Antibody Conc.为待测抗体的浓度；Figure 3 is a graph showing the results of ELISA binding activity between different batches of HX016-1 and the target molecule human VEGFR2 according to an embodiment of the present invention, wherein, HX016-1 is the first structure of the PD-1/VEGF recombinant antibody, in brackets Represents different batches (Lot) of HX016-1 samples, such as HX016-1-5K-2-E2 (Lot20190419) represents the HX016-1 produced in the 20190419 batch, Abs is the absorbance at 450nm, Antibody Conc. is to be tested Antibody concentration;
图4是根据本发明实施例的重组抗体竞争性阻断PD-1/PD-L1结合的ELISA实验结果图，其中，HX008为PD-1抗体，HX016-1为PD-1/VEGF重组抗体的第1种结构，HX016-2为PD-1/VEGF重组抗体的第2种结构，HX016-4为PD-1/VEGF重组抗体的第4种结构，HX016-6为PD-1/VEGF重组抗体的第6种结构，括号中代表HX016各结构的样品的批次(Lot)，如HX016-1(Lot20190103)表示第20190103批次生产的HX016-1，Abs为450nm处的吸光度，Antibody Conc.为待测抗体的浓度；Figure 4 is a graph showing the results of an ELISA experiment in which recombinant antibodies competitively block PD-1/PD-L1 binding according to an embodiment of the present invention, wherein HX008 is a PD-1 antibody, and HX016-1 is a PD-1/VEGF recombinant antibody The first structure, HX016-2 is the second structure of PD-1/VEGF recombinant antibody, HX016-4 is the fourth structure of PD-1/VEGF recombinant antibody, HX016-6 is PD-1/VEGF recombinant antibody The sixth structure of HX016 in parentheses represents the batch (Lot) of each structure of HX016, such as HX016-1 (Lot20190103) represents the HX016-1 produced in the 20190103 batch, Abs is the absorbance at 450nm, Antibody Conc. is The concentration of the antibody to be tested;
图5是根据本发明实施例的重组抗体竞争性阻断VEGF/VEGFR2结合的ELISA实验结果图，其中，HX006为VEGF抗体，Avastin为已上市VEGF单克隆抗体产品，HX016-2为PD-1/VEGF重组抗体的第2种结构，HX016-4为PD-1/VEGF重组抗体的第4种结构，HX016-6为PD-1/VEGF重组抗体的第6种结构，括号中代表HX016各结构的样品的批次(Lot)，如HX016-1(Lot20190103)表示第20190103批次生产的HX016-1，Abs为450nm处的吸光度，Antibody Conc.为待测抗体的浓度；Figure 5 is a diagram showing the results of an ELISA experiment in which recombinant antibodies competitively block the binding of VEGF/VEGFR2 according to an embodiment of the present invention, wherein HX006 is a VEGF antibody, Avastin is a marketed VEGF monoclonal antibody product, and HX016-2 is a PD-1/VEGFR2 antibody product. The second structure of VEGF recombinant antibody, HX016-4 is the fourth structure of PD-1/VEGF recombinant antibody, HX016-6 is the sixth structure of PD-1/VEGF recombinant antibody, and the brackets represent the structures of HX016 The lot (Lot) of the sample, such as HX016-1 (Lot20190103) means the HX016-1 produced in the 20190103 batch, Abs is the absorbance at 450nm, and Antibody Conc. is the concentration of the antibody to be tested;
图6是根据本发明实施例的重组抗体HX016-2结合表面表达人PD-1的293T细胞实验结果图，其中，HX008为PD-1抗体，HX016-2为PD-1/VEGF重组抗体的第2种结构，横坐标Conc.为待测物的浓度，纵坐标MFI表示平均荧光强度；Figure 6 is a graph showing the experimental results of recombinant antibody HX016-2 binding to 293T cells expressing human PD-1 on the surface according to an embodiment of the present invention, wherein HX008 is the PD-1 antibody, and HX016-2 is the first PD-1/VEGF recombinant antibody Two structures, the abscissa Conc. is the concentration of the analyte, and the ordinate MFI is the average fluorescence intensity;
图7是根据本发明实施例的重组抗体HX016-2阻断PD-L1和细胞膜表面表达PD-1的293T细胞结合的实验结果图，其中，HX008为PD-1抗体，HX016-2为PD-1/VEGF重组抗体的第2种结构，横坐标Conc.为待测物的浓度，纵坐标MFI表示平均荧光强度；Figure 7 is a graph showing the experimental results of the recombinant antibody HX016-2 blocking the binding of PD-L1 to 293T cells expressing PD-1 on the cell membrane surface according to an embodiment of the present invention, wherein HX008 is a PD-1 antibody, and HX016-2 is a PD- 1/The second structure of VEGF recombinant antibody, the abscissa Conc. is the concentration of the analyte, and the ordinate MFI indicates the average fluorescence intensity;
图8是根据本发明实施例的重组抗体阻断CHO-scOKT3-PD-L1细胞对Jurkat-NFAT-luc细胞表达荧光素酶的抑制作用，恢复荧光素酶的表达的实验结果图，其中，PD-1 mAb为PD-1抗体，PD-1/VEGFR2bsAb-1为PD-1/VEGF重组抗体的第1种结构(HX016-1)，PD-1/VEGFbsAb-2为PD-1/VEGF重组抗体的第2种结构(HX016-2)，PD-1/VEGFbsAb-4为PD-1/VEGF重组抗体的第4种结构(HX016-4)，Fluorescent Intensity为荧光强度，Antibody Conc.为待测抗体的浓度；Figure 8 is a graph showing the experimental results of blocking the expression of luciferase by CHO-scOKT3-PD-L1 cells on Jurkat-NFAT-luc cells according to an embodiment of the present invention, and restoring the expression of luciferase, wherein, PD -1 mAb is PD-1 antibody, PD-1/VEGFR2bsAb-1 is the first structure of PD-1/VEGF recombinant antibody (HX016-1), PD-1/VEGFbsAb-2 is PD-1/VEGF recombinant antibody PD-1/VEGFbsAb-4 is the fourth structure of PD-1/VEGF recombinant antibody (HX016-4), Fluorescent Intensity is the fluorescence intensity, Antibody Conc. is the antibody to be tested concentration;
图9是根据本发明实施例的重组抗体阻断人VEGF165对293T-VEGFR2-NFAT-luc细胞表达荧光素酶 的激活作用，抑制荧光素酶的表达的实验结果图，其中，VEGF mAb为VEGF抗体，PD-1/VEGFR2bsAb-1为PD-1/VEGF重组抗体的第1种结构(HX016-1)，PD-1/VEGFbsAb-2为PD-1/VEGF重组抗体的第2种结构(HX016-2)，PD-1/VEGFbsAb-4为PD-1/VEGF重组抗体的第4种结构(HX016-4)，Fluorescent Intensity为荧光强度，Antibody Conc.为待测抗体的浓度；Figure 9 is a graph showing the experimental results of the recombinant antibody blocking the activation of luciferase expressed by human VEGF165 on 293T-VEGFR2-NFAT-luc cells and inhibiting the expression of luciferase according to an embodiment of the present invention, wherein VEGF mAb is a VEGF antibody , PD-1/VEGFR2bsAb-1 is the first structure of PD-1/VEGF recombinant antibody (HX016-1), PD-1/VEGFbsAb-2 is the second structure of PD-1/VEGF recombinant antibody (HX016- 2), PD-1/VEGFbsAb-4 is the fourth structure of PD-1/VEGF recombinant antibody (HX016-4), Fluorescent Intensity is the fluorescence intensity, Antibody Conc. is the concentration of the antibody to be tested;
图10是根据本发明实施例的重组抗体HX016-2在混合淋巴反应中刺激T细胞分泌IFN-γ的结果图，其中，HX008表示PD-1抗体，PD-1/VEGFR2bsAb-1为PD-1/VEGF重组抗体的第1种结构(HX016-1)，HX016-2为PD-1/VEGF重组抗体的第2种结构(HX016-2)，PD-1/VEGFbsAb-4为PD-1/VEGF重组抗体的第4种结构(HX016-4)，Fluorescent Intensity为荧光强度，Conc.为待测物浓度；Figure 10 is a graph showing the results of recombinant antibody HX016-2 stimulating T cells to secrete IFN-γ in a mixed lymphatic reaction according to an embodiment of the present invention, wherein HX008 represents the PD-1 antibody, and PD-1/VEGFR2bsAb-1 is PD-1 The first structure of /VEGF recombinant antibody (HX016-1), HX016-2 is the second structure of PD-1/VEGF recombinant antibody (HX016-2), PD-1/VEGFbsAb-4 is PD-1/VEGF The fourth structure of the recombinant antibody (HX016-4), Fluorescent Intensity is the fluorescence intensity, Conc. is the concentration of the analyte;
图11是根据本发明实施例的重组抗体HX016-2在SEB刺激试验中重新激活T细胞分泌IL-2的结果图，其中，横坐标Conc.为待测物浓度，纵坐标表示IL-2的含量；Figure 11 is a graph showing the results of recombinant antibody HX016-2 reactivating T cells to secrete IL-2 in the SEB stimulation test according to an embodiment of the present invention, wherein the abscissa Conc. is the concentration of the analyte, and the ordinate indicates the concentration of IL-2 content;
图12是根据本发明实施例的重组抗体HX016-2和HX016-6抑制人VEGF165介导的HUVEC细胞增殖的结果图，其中，横坐标Conc.为待测物浓度，纵坐标FLU表示荧光强度；以及Figure 12 is a graph showing the results of recombinant antibodies HX016-2 and HX016-6 inhibiting human VEGF165-mediated HUVEC cell proliferation according to an embodiment of the present invention, wherein the abscissa Conc. is the concentration of the analyte, and the ordinate FLU indicates the fluorescence intensity; as well as
图13-A是根据本发明实施例的不同组别BALB/c-hPD1小鼠皮下CT26.WT结肠癌模型的肿瘤体积变化图，其中，横坐标Days Post Treatment表示药物或抗体处理时间，纵坐标Tumor Volume表示肿瘤体积(mm2)，G1为IgG4同型阴性对照5mg/kg组，G2为抗PD-1单抗Keytruda 10mg/kg组，G3为抗PD-1单抗10mg/kg组，G4为抗PD-1单抗5mg/kg组，G5为抗VEGF单抗5mg/kg组，G6为VEGFR1D2结合域5mg/kg组，G7为HX016-2即PD-1/VEGF重组抗体的第2种结构5mg/kg组，G8为HX016-4即PD-1/VEGF重组抗体的第4种结构5mg/kg组，G9为HX016-6即PD-1/VEGF重组抗体的第6种结构5mg/kg组；Figure 13-A is a diagram of the tumor volume change of the subcutaneous CT26.WT colon cancer model of different groups of BALB/c-hPD1 mice according to an embodiment of the present invention, wherein the abscissa Days Post Treatment represents the time of drug or antibody treatment, and the ordinate Tumor Volume indicates the tumor volume (mm2), G1 is the IgG4 isotype negative control 5mg/kg group, G2 is the anti-PD-1 monoclonal antibody Keytruda 10mg/kg group, G3 is the anti-PD-1 monoclonal antibody 10mg/kg group, G4 is the anti-PD-1 monoclonal antibody 10mg/kg group, and G4 is the anti-PD-1 monoclonal antibody 10mg/kg group. PD-1 monoclonal antibody 5mg/kg group, G5 is anti-VEGF monoclonal antibody 5mg/kg group, G6 is VEGFR1D2 binding domain 5mg/kg group, G7 is HX016-2, the second structure of PD-1/VEGF recombinant antibody 5mg /kg group, G8 is HX016-4, the fourth structure 5mg/kg group of PD-1/VEGF recombinant antibody, G9 is HX016-6, the sixth structure 5mg/kg group of PD-1/VEGF recombinant antibody;
图13-B是根据本发明实施例的G1-G9不同组别中每只小鼠肿瘤体积变化图，其中，横坐标Days Post Treatment表示药物或抗体处理时间，纵坐标Tumor Volume表示肿瘤体积(mm2)；Figure 13-B is a graph showing the change in tumor volume of each mouse in different groups of G1-G9 according to an embodiment of the present invention, wherein the abscissa Days Post Treatment represents the time of drug or antibody treatment, and the ordinate Tumor Volume represents the tumor volume (mm2 );
图13-C是根据本发明实施例的G1-G9不同组别BALB/c-hPD1小鼠皮下CT26.WT结肠癌模型的肿瘤重量变化图，其中，横坐标表示不同组别，纵坐标Tumor Weight表示肿瘤重量(g)；以及Figure 13-C is a graph showing the change in tumor weight of the BALB/c-hPD1 mouse subcutaneous CT26.WT colon cancer model in different groups of G1-G9 according to an embodiment of the present invention, wherein the abscissa represents different groups, and the ordinate represents Tumor Weight Indicates tumor weight (g); and
图13-D是根据本发明实施例的不同组别BALB/c-hPD1小鼠皮下CT26.WT结肠癌模型小鼠的体重变化图，其中，横坐标Days Post Treatment表示药物或抗体处理时间，纵坐标Body Weight表示小鼠体重(g)。Fig. 13-D is a diagram of body weight changes of subcutaneous CT26.WT colon cancer model mice of different groups of BALB/c-hPD1 mice according to an embodiment of the present invention, wherein the abscissa Days Post Treatment represents the time of drug or antibody treatment, and the ordinate Coordinate Body Weight represents the mouse body weight (g).
发明详细描述Detailed description of the invention
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产 品。Embodiments of the present invention are described in detail below. The embodiments described below are exemplary only for explaining the present invention and should not be construed as limiting the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。In addition, the terms "first" and "second" are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.
术语“任选地”仅用于描述目的，而不能理解为指示或暗示相对重要性。由此，限定有“任选地”的特征可以明示或者隐含地包括或不包括该特征。The term "optionally" is used for descriptive purposes only and is not to be understood as indicating or implying relative importance. Thus, a feature defined as "optionally" may be expressly or implicitly included or excluded.
本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。"Operably linked" herein refers to linking the exogenous gene to the vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended role in regulating the transcription and translation of the exogenous gene function.
本文中“VEGF诱捕蛋白”是指能够与VEGF蛋白进行结合的蛋白，该蛋白不受特别限制，任何可与VEGF结合的蛋白均在该范围内，如VEGF受体的结构域，本发明中所述的VEGFR1D2即为VEGF受体1的结构域2。Herein, "VEGF trapping protein" refers to a protein capable of binding to VEGF protein, which is not particularly limited, and any protein that can bind to VEGF is within this scope, such as the structural domain of VEGF receptor, as described in the present invention VEGFR1D2 mentioned above is domain 2 of VEGF receptor 1.
目前，抗肿瘤药物以单克隆抗体为主，尤其以抗人PD-1/PD-L1单抗和抗人CTLA-4单抗为代表的第一代免疫检查点阻断性抗体疗法，在抗肿瘤治疗中取得了重大成功。PD-1/PD-L1单抗的免疫调节作用机理赋予了其具有广谱抗肿瘤活性。PD-1/PD-L1单抗已经被大量的临床研究结果证明在治疗各类实体瘤和血液肿瘤时都显示了优于现有标准治疗方案的疗效和安全性。At present, anti-tumor drugs are mainly monoclonal antibodies, especially the first-generation immune checkpoint blocking antibody therapy represented by anti-human PD-1/PD-L1 monoclonal antibody and anti-human CTLA-4 monoclonal antibody. Significant success has been achieved in cancer therapy. The immunomodulatory mechanism of PD-1/PD-L1 monoclonal antibody endows it with broad-spectrum anti-tumor activity. PD-1/PD-L1 monoclonal antibody has been proved by a large number of clinical research results to show efficacy and safety superior to existing standard treatment regimens in the treatment of various solid tumors and blood tumors.
但是，单一的PD-1/PD-L1单抗仍然具有以下缺点：针对有效人群的响应率仍然较低，针对不同肿瘤适应症总体响应率不同，但是平均不到30％，另有70％的人无法获益；为了提高人群响应率，PD-1/PD-L1单抗需要借助诊断试剂(Biomarker)帮助鉴别，单一Biomarker人群筛选存在弊端，多Biomarker则测量成本较高；容易产生耐药：PD-1治疗有效的病人，截至目前，15％-20％已经出现了耐药，癌症有了复发，其耐药的机制可能有多种，例如肿瘤又产生了新的免疫逃逸通路，或者抗体药物因免疫原性产生了抗药抗体；接受PD-1/PD-L1单抗治疗的肿瘤病人当中，会有一部分病人出现超进展的情况，即用药后肿瘤生长超过50％。而且在多种肿瘤中均报道出此类情况，其发生率、结局和预测因素仍尚不清楚。且为增大各肿瘤适应症的响应率，克服PD-1单抗耐药的问题以及更好的调节免疫抑制性肿瘤微环境，更大发挥PD-1靶点的免疫调节作用，发明人通过不同的组合方式构建了多种重组双特异性抗体分子，并对其进行体内、外的活性检测。However, a single PD-1/PD-L1 monoclonal antibody still has the following disadvantages: the response rate for the effective population is still low, and the overall response rate for different tumor indications is different, but the average is less than 30%, and another 70% People cannot benefit; in order to improve the response rate of the population, PD-1/PD-L1 monoclonal antibodies need to be identified with the help of diagnostic reagents (Biomarker). There are disadvantages in population screening with a single Biomarker, and the measurement cost of multiple Biomarkers is high; it is easy to produce drug resistance: Up to now, 15%-20% of patients with effective PD-1 therapy have developed drug resistance, and the cancer has relapsed. There may be multiple mechanisms of drug resistance, such as the tumor has produced a new immune escape pathway, or antibody Drugs produce anti-drug antibodies due to immunogenicity; among tumor patients receiving PD-1/PD-L1 monoclonal antibody therapy, some patients will experience hyperprogression, that is, tumor growth exceeds 50% after treatment. Moreover, this condition has been reported in a variety of tumors, and its incidence, outcome, and predictors remain unclear. In order to increase the response rate of various tumor indications, overcome the problem of PD-1 monoclonal antibody resistance, better regulate the immunosuppressive tumor microenvironment, and give greater play to the immune regulation effect of PD-1 targets, the inventors adopted A variety of recombinant bispecific antibody molecules were constructed in different combinations, and their activities were tested in vivo and in vitro.
下面将对实施例作具体介绍。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The embodiments will be described in detail below. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.
实施例1 双特异性抗体分子的构建Example 1 Construction of bispecific antibody molecules
本实施例中构建6种双特异性抗体分子(重组抗体)，6种双特异性抗体分子为PD-1 mAb分子通过连接肽与选自下列之一的蛋白分子进行连接获得：Ramucirumab单链抗体、VEGF单链抗体、 VEGFR1D2，连接的方式为PD-1 mAb分子的重链通过连接肽与上述蛋白分子进行连接。其中，所述连接肽(linker)具有如SEQ ID NO：01所示的氨基酸序列，所述PD-1 mAb的轻链即为所述6种重组蛋白分子的轻链，具有如SEQ ID NO：02所示的氨基酸序列，所述PD-1 mAb的重链具有如SEQ ID NO：21所示的氨基酸序列：In this example, 6 bispecific antibody molecules (recombinant antibodies) were constructed, and the 6 bispecific antibody molecules were PD-1 mAb molecules obtained by linking the peptide with a protein molecule selected from one of the following: Ramucirumab single-chain antibody , VEGF single-chain antibody, VEGFR1D2, the connection method is that the heavy chain of the PD-1 mAb molecule is connected to the above-mentioned protein molecule through a connecting peptide. Wherein, the connecting peptide (linker) has the amino acid sequence shown in SEQ ID NO: 01, and the light chain of the PD-1 mAb is the light chain of the 6 recombinant protein molecules, having the sequence as SEQ ID NO: The amino acid sequence shown in 02, the heavy chain of the PD-1 mAb has the amino acid sequence shown in SEQ ID NO: 21:
连接肽(Linker)的氨基酸序列如下：The amino acid sequence of the connecting peptide (Linker) is as follows:
Figure PCTCN2021132785-appb-000026
Figure PCTCN2021132785-appb-000026
PD-1 mAb轻链(6种双特异性重组抗体轻链)的氨基酸序列如下：The amino acid sequences of PD-1 mAb light chains (six types of bispecific recombinant antibody light chains) are as follows:
Figure PCTCN2021132785-appb-000027
Figure PCTCN2021132785-appb-000027
PD-1 mAb重链的氨基酸序列如下：The amino acid sequence of the PD-1 mAb heavy chain is as follows:
Figure PCTCN2021132785-appb-000028
Figure PCTCN2021132785-appb-000028
编码所述连接肽的核苷酸序列如下：The nucleotide sequence encoding the connecting peptide is as follows:
Figure PCTCN2021132785-appb-000029
Figure PCTCN2021132785-appb-000029
编码所述PD-1 mAb轻链(6种双特异性重组抗体轻链)的核苷酸序列如下：The nucleotide sequences encoding the PD-1 mAb light chains (6 kinds of bispecific recombinant antibody light chains) are as follows:
Figure PCTCN2021132785-appb-000030
Figure PCTCN2021132785-appb-000030
编码所述PD-1 mAb重链的核苷酸序列如下：The nucleotide sequence encoding the heavy chain of the PD-1 mAb is as follows:
Figure PCTCN2021132785-appb-000031
Figure PCTCN2021132785-appb-000031
所述双特异性抗体分子制备的具体实验操作如下所示：The specific experimental operation for the preparation of the bispecific antibody molecule is as follows:
1、PD-1/VEGFR2bsAb-1(HX016-1)1. PD-1/VEGFR2bsAb-1 (HX016-1)
HX016-1重链的氨基酸序列如SEQ ID NO：3所示，为人源化抗PD-1抗体(PD-1 mAb)的重链C端用linker连接Ramucirumab单链抗体(SEQ ID NO：22)N端。The amino acid sequence of the heavy chain of HX016-1 is shown in SEQ ID NO: 3, which is the C-terminal of the heavy chain of the humanized anti-PD-1 antibody (PD-1 mAb) and the linker is used to connect the Ramucirumab single-chain antibody (SEQ ID NO: 22) N-terminal.
全基因合成编码PD-1 mAb轻链、重链、linker-Ramucirumab单链抗体的核酸序列，以及构建表达载体1、2、3的具体实验操作参考CN 105330739，将编码PD-1 mAb的轻链的核酸序列先连接到表达载体中获得表达载体1；直接把编码PD-1 mAb的重链的核酸序列连接到表达载体1中获得表达抗PD1的单克隆抗体HX008的表达载体2；通过Over-lap PCR将PD-1 mAb重链C端与linker(所述linker的C端连接有Ramucirumab单链抗体)的N末端的核苷酸序列融合后获得编码所述HX016-1的重链的核苷酸序列(SEQ ID NO：8)，将编码所述HX016-1的重链的核苷酸序列连接到表达载体1中获得表达双特异性抗体HX016-1的表达载体3。提取表达载体1和3的DNA，分别转染至哺乳动物细胞293细胞，质粒DNA终浓度4μg/mL，转染试剂PEI终浓度8μg/mL转染，室温静置5分钟后缓慢滴加至293细胞中，边滴加边混匀。细胞转染后，抗体在哺乳动物细胞内表达，并分泌到细胞外。然后，通过蛋白A亲和层析柱，纯化表达的抗体，即获得HX016-1蛋白。The whole gene synthesis codes the nucleic acid sequence of PD-1 mAb light chain, heavy chain, linker-Ramucirumab single-chain antibody, and the specific experimental operation of constructing expression vectors 1, 2, and 3. Please refer to CN 105330739 to encode the light chain of PD-1 mAb The nucleic acid sequence of the expression vector was first connected to the expression vector to obtain the expression vector 1; the nucleic acid sequence encoding the heavy chain of PD-1 mAb was directly connected to the expression vector 1 to obtain the expression vector 2 expressing the anti-PD1 monoclonal antibody HX008; by Over- Lap PCR fused the C-terminal of the heavy chain of PD-1 mAb to the nucleotide sequence of the N-terminal of the linker (the C-terminal of the linker is connected with Ramucirumab single-chain antibody) to obtain the nucleotide sequence encoding the heavy chain of HX016-1 acid sequence (SEQ ID NO: 8), the nucleotide sequence encoding the heavy chain of HX016-1 was connected to the expression vector 1 to obtain the expression vector 3 expressing the bispecific antibody HX016-1. Extract the DNA of expression vectors 1 and 3, and transfect them into mammalian 293 cells respectively. The final concentration of plasmid DNA is 4 μg/mL, and the final concentration of transfection reagent PEI is 8 μg/mL. In the cells, mix well while adding dropwise. After cell transfection, antibodies are expressed in mammalian cells and secreted extracellularly. Then, the expressed antibody was purified through a protein A affinity chromatography column to obtain the HX016-1 protein.
重组抗体1号(HX016-1)重链的氨基酸序列如下：The amino acid sequence of the heavy chain of recombinant antibody No. 1 (HX016-1) is as follows:
Figure PCTCN2021132785-appb-000032
Figure PCTCN2021132785-appb-000032
Figure PCTCN2021132785-appb-000033
Figure PCTCN2021132785-appb-000033
Ramucirumab单链抗体的氨基酸序列如下：The amino acid sequence of Ramucirumab single-chain antibody is as follows:
Figure PCTCN2021132785-appb-000034
Figure PCTCN2021132785-appb-000034
编码重组抗体1号(HX016-1)重链核苷酸序列：Nucleotide sequence encoding recombinant antibody No. 1 (HX016-1) heavy chain:
Figure PCTCN2021132785-appb-000035
Figure PCTCN2021132785-appb-000035
Figure PCTCN2021132785-appb-000036
Figure PCTCN2021132785-appb-000036
2、PD-1/VEGFbsAb-2(HX016-2)2. PD-1/VEGFbsAb-2 (HX016-2)
HX016-2重链的氨基酸序列如SEQ ID NO：4所示，为VEGFR1D2氨基酸序列(SEQ ID NO：20)的C端用linker连接PD-1 mAb的重链N端；The amino acid sequence of the heavy chain of HX016-2 is shown in SEQ ID NO: 4, which is the C-terminal of the amino acid sequence of VEGFR1D2 (SEQ ID NO: 20), which is connected to the N-terminal of the heavy chain of PD-1 mAb with a linker;
实际操作中，分别全基因合成编码人源化抗PD-1抗体(PD-1 mAb)的轻链、重链、VEGFR1D2-linker的核苷酸序列，以及构建表达载体1、2、4的具体实验操作参考CN 105330739，将PD-1 mAb的轻链的核酸序列先连接到表达载体中获得表达载体1；把PD-1 mAb的重链序列连接到表达载体1中获得表达抗PD1的单克隆抗体HX008的表达载体2；通过Over-lap PCR将PD-1 mAb重链N端与linker(所述linker的N端连接有VEGFR1D2)的C端核苷酸序列融合后获得编码所述HX016-2的重链的核苷酸序列(SEQ ID NO：9)，连接到表达载体1中获得表达双特异性抗体HX016-2的表达载体4。提取表达载体1和4的DNA，分别转染至哺乳动物细胞293细胞，质粒DNA终浓度4μg/mL，转染试剂PEI终浓度8μg/mL转染，室温静置5分钟后缓慢滴加至293细胞中，边滴加边混匀。细胞转染后，抗体在哺乳动物细胞内表达，并分泌到细胞外。然后，通过蛋白A亲和层析柱，纯化表达的抗体，即获得HX016-2蛋白。合成表达载体1、2、4以及蛋白纯化的具体实验操作参考合成HHX016-1的实验操作。In actual operation, the nucleotide sequences encoding the light chain, heavy chain, and VEGFR1D2-linker of the humanized anti-PD-1 antibody (PD-1 mAb) were synthesized in the whole gene, and the specific procedures for constructing expression vectors 1, 2, and 4 were carried out. The experimental operation refers to CN 105330739, the nucleic acid sequence of the light chain of PD-1 mAb is first connected to the expression vector to obtain expression vector 1; the heavy chain sequence of PD-1 mAb is connected to expression vector 1 to obtain a monoclonal expression of anti-PD1 Expression vector 2 of antibody HX008; the N-terminal of the heavy chain of PD-1 mAb was fused to the C-terminal nucleotide sequence of the linker (the N-terminal of the linker is connected with VEGFR1D2) by Over-lap PCR to obtain the HX016-2 encoding The nucleotide sequence of the heavy chain (SEQ ID NO: 9) was connected to the expression vector 1 to obtain the expression vector 4 expressing the bispecific antibody HX016-2. Extract the DNA of expression vectors 1 and 4, and transfect them into mammalian 293 cells respectively. The final concentration of plasmid DNA is 4 μg/mL, and the final concentration of transfection reagent PEI is 8 μg/mL. In the cells, mix well while adding dropwise. After cell transfection, antibodies are expressed in mammalian cells and secreted extracellularly. Then, the expressed antibody is purified through a protein A affinity chromatography column to obtain the HX016-2 protein. For the specific experimental operation of synthesizing expression vectors 1, 2, 4 and protein purification, refer to the experimental operation of synthesizing HHX016-1.
重组抗体2号(HX016-2)的重链的氨基酸序列如下：The amino acid sequence of the heavy chain of recombinant antibody No. 2 (HX016-2) is as follows:
Figure PCTCN2021132785-appb-000037
Figure PCTCN2021132785-appb-000037
Figure PCTCN2021132785-appb-000038
Figure PCTCN2021132785-appb-000038
VEGFR1D2具有如下所示的氨基酸序列：VEGFR1D2 has the amino acid sequence shown below:
Figure PCTCN2021132785-appb-000039
Figure PCTCN2021132785-appb-000039
编码重组抗体2号(HX016-2)重链的核苷酸序列如下：The nucleotide sequence encoding the heavy chain of recombinant antibody No. 2 (HX016-2) is as follows:
Figure PCTCN2021132785-appb-000040
Figure PCTCN2021132785-appb-000040
3、PD-1/VEGFbsAb-3(HX016-3)3. PD-1/VEGFbsAb-3 (HX016-3)
HX016-3重链的氨基酸序列如SEQ ID NO：23所示，为VEGFR1D2的C端连接VEGFR2D3(SEQ ID NO：24)的N端，然后VEGFR2D3的C端用linker连接PD-1 mAb的重链N端。The amino acid sequence of the HX016-3 heavy chain is shown in SEQ ID NO: 23. The C-terminal of VEGFR1D2 is connected to the N-terminal of VEGFR2D3 (SEQ ID NO: 24), and then the C-terminal of VEGFR2D3 is connected to the heavy chain of PD-1 mAb with a linker N-terminal.
实际操作中，全基因合成编码人源化抗PD-1抗体的轻链、重链、VEGFR1D2-VEGFR2D3-linker的核苷酸序列，以及构建表达载体1、2、5的具体实验操作参考CN 105330739，将编码所述人源化抗PD-1抗体的轻链先连接到表达载体中获得表达载体1；把PD-1 mAb的重链序列连接到表达载体1 中获得表达抗PD1的单克隆抗体HX008的表达载体2；通过Over-lap PCR将PD-1 mAb重链N端与linker(所述linker的N端连接有VEGFR1D2-VEGFR2D3)的C端核苷酸序列融合后获得编码所述HX016-3的中重链的核苷酸序列，将编码所述HX016-3的重链的核苷酸序列连接到表达载体1中获得表达双特异性抗体HX016-3的表达载体5。提取表达载体1和5的DNA，分别转染至哺乳动物细胞293细胞，质粒DNA终浓度4μg/mL，转染试剂PEI终浓度8μg/mL转染，室温静置5分钟后缓慢滴加至293细胞中，边滴加边混匀。细胞转染后，抗体在哺乳动物细胞内表达，并分泌到细胞外。然后，通过蛋白A亲和层析柱，纯化表达的抗体，即获得HX016-3蛋白。In actual operation, the nucleotide sequences encoding the light chain, heavy chain, and VEGFR1D2-VEGFR2D3-linker of the humanized anti-PD-1 antibody by whole gene synthesis, and the specific experimental operation of constructing expression vectors 1, 2, and 5 refer to CN 105330739 , the light chain encoding the humanized anti-PD-1 antibody was first connected to the expression vector to obtain expression vector 1; the heavy chain sequence of PD-1 mAb was connected to expression vector 1 to obtain the monoclonal antibody expressing anti-PD1 Expression vector 2 of HX008; the HX016- 3, the nucleotide sequence encoding the heavy chain of HX016-3 was connected to the expression vector 1 to obtain the expression vector 5 expressing the bispecific antibody HX016-3. Extract the DNA of expression vectors 1 and 5, and transfect them into mammalian 293 cells respectively. The final concentration of plasmid DNA is 4 μg/mL, and the final concentration of transfection reagent PEI is 8 μg/mL. In the cells, mix well while adding dropwise. After cell transfection, antibodies are expressed in mammalian cells and secreted extracellularly. Then, the expressed antibody is purified through a protein A affinity chromatography column to obtain the HX016-3 protein.
重组抗体3号(HX016-3)的重链的氨基酸序列如下：The amino acid sequence of the heavy chain of recombinant antibody No. 3 (HX016-3) is as follows:
Figure PCTCN2021132785-appb-000041
Figure PCTCN2021132785-appb-000041
VEGFR2D3的氨基酸序列如下所示：The amino acid sequence of VEGFR2D3 is shown below:
Figure PCTCN2021132785-appb-000042
Figure PCTCN2021132785-appb-000042
编码VEGFR2D3的核苷酸序列如下：The nucleotide sequence encoding VEGFR2D3 is as follows:
Figure PCTCN2021132785-appb-000043
Figure PCTCN2021132785-appb-000043
编码重组抗体3号(HX016-3)重链的核苷酸序列如下：The nucleotide sequence encoding the heavy chain of recombinant antibody No. 3 (HX016-3) is as follows:
Figure PCTCN2021132785-appb-000044
Figure PCTCN2021132785-appb-000044
Figure PCTCN2021132785-appb-000045
Figure PCTCN2021132785-appb-000045
4、PD-1/VEGFbsAb-4(HX016-4)4. PD-1/VEGFbsAb-4 (HX016-4)
HX016-4的重链(SEQ ID NO：5)为PD-1 mAb的重链C端用linker连接VEGFR1D2的N端；The heavy chain of HX016-4 (SEQ ID NO: 5) is the C-terminal of the heavy chain of PD-1 mAb connected to the N-terminal of VEGFR1D2 with a linker;
实际操作中，全基因合成编码PD-1 mAb的轻链、重链、linker-VEGFR1D2的核苷酸序列，以及构建表达载体1、2、6的具体实验操作同前所述。先将PD-1 mAb的轻链的核酸序列先连接到表达载体中获得表达载体1；把PD-1 mAb的重链序列连接到表达载体1中获得表达抗PD1的单克隆抗体HX008的表达载体2；通过Over-lap PCR将PD-1 mAb重链C端与linker(所述linker的C端连接有VEGFR1D2)的N端核苷酸序列融合后获得核苷酸序列(SEQ ID NO：10)，连接到表达载体1中获得表达双特异性抗体HX016-4的表达载体6。提取表达载体1和6的DNA，分别转染至哺乳动物细胞293细胞，质粒DNA终浓度4μg/mL，转染试剂PEI终浓度8μg/mL转染，室温静置5分钟后缓慢滴加至293细胞中，边滴加边混匀。细胞转染后，抗体在哺乳动物细胞内表达，并分泌到细胞外。然后，通过蛋白A亲和层析柱，纯化表达的抗体，即获得HX016-4蛋白。In practice, the whole gene synthesis codes the light chain, heavy chain, and linker-VEGFR1D2 nucleotide sequences of PD-1 mAb, and the specific experimental operations for constructing expression vectors 1, 2, and 6 are the same as described above. First connect the nucleic acid sequence of the light chain of PD-1 mAb to the expression vector to obtain expression vector 1; connect the heavy chain sequence of PD-1 mAb to expression vector 1 to obtain the expression vector expressing anti-PD1 monoclonal antibody HX008 2. The C-terminal of the PD-1 mAb heavy chain was fused to the N-terminal nucleotide sequence of the linker (the C-terminal of the linker is connected with VEGFR1D2) by Over-lap PCR to obtain the nucleotide sequence (SEQ ID NO: 10) , connected to the expression vector 1 to obtain the expression vector 6 expressing the bispecific antibody HX016-4. Extract the DNA of expression vectors 1 and 6, and transfect them into mammalian 293 cells respectively. The final concentration of plasmid DNA is 4 μg/mL, and the final concentration of transfection reagent PEI is 8 μg/mL. In the cells, mix well while adding dropwise. After cell transfection, antibodies are expressed in mammalian cells and secreted extracellularly. Then, the expressed antibody was purified through a protein A affinity chromatography column to obtain the HX016-4 protein.
重组抗体4号(HX016-4)的重链的氨基酸序列如下：The amino acid sequence of the heavy chain of recombinant antibody No. 4 (HX016-4) is as follows:
Figure PCTCN2021132785-appb-000046
Figure PCTCN2021132785-appb-000046
Figure PCTCN2021132785-appb-000047
Figure PCTCN2021132785-appb-000047
编码重组抗体4号(HX016-4)重链的核苷酸序列如下：The nucleotide sequence encoding the heavy chain of recombinant antibody No. 4 (HX016-4) is as follows:
Figure PCTCN2021132785-appb-000048
Figure PCTCN2021132785-appb-000048
5、PD-1/VEGFbsAb-5(HX016-5)5. PD-1/VEGFbsAb-5 (HX016-5)
HX016-5重链的氨基酸序列如SEQ ID NO：25所示，为PD-1 mAb的重链C端用linker连接VEGFR1D2的N端，然后VEGFR1D2的C端连接VEGFR2D3的N端；The amino acid sequence of the heavy chain of HX016-5 is shown in SEQ ID NO: 25. The C-terminal of the heavy chain of PD-1 mAb is connected to the N-terminal of VEGFR1D2 with a linker, and then the C-terminal of VEGFR1D2 is connected to the N-terminal of VEGFR2D3;
实际操作中，全基因合成编码PD-1 mAb的轻链、重链、linker-VEGFR1D2-VEGFR2D3的核酸序列，以及构建表达载体1、2、7的具体实验操作同上，先将PD-1 mAb的轻链的核酸序列先连接到表达载体中获得表达载体1；把PD-1 mAb的重链序列连接到表达载体1中获得表达抗PD1的单克隆抗体HX008的表达载体2；通过Over-lap PCR将PD-1 mAb重链C端与linker(所述linker的C端来连接有VEGFR1D2-VEGFR2D3)的N端核苷酸序列融合后，获得编码所述HX016-5重链的核苷酸序列(SEQ ID NO：28)，将编码所述HX016-5重链的核苷酸序列连接到表达载体1中获得表达双特异性抗体HX016-5的表达载体7。提取表达载体1和7的DNA，分别转染至哺乳动物细胞293细胞，质粒DNA终浓度4μg/mL，转染试剂PEI终浓度8μg/mL转染，室温静置5分钟后缓慢滴加至293细胞中，边滴加边混匀。细胞转染后，抗体在哺乳动物细胞内表达，并分泌到细胞外。然后，通过蛋白A亲和层析柱，纯化表达的抗体，即获得HX016-5蛋白。In actual operation, the whole gene synthesis encodes the light chain, heavy chain, linker-VEGFR1D2-VEGFR2D3 nucleic acid sequence of PD-1 mAb, and the specific experimental operation of constructing expression vectors 1, 2, and 7 is the same as above. First, the PD-1 mAb The nucleic acid sequence of the light chain was first connected to the expression vector to obtain the expression vector 1; the heavy chain sequence of PD-1 mAb was connected to the expression vector 1 to obtain the expression vector 2 expressing the anti-PD1 monoclonal antibody HX008; by Over-lap PCR After fusing the C-terminal of the PD-1 mAb heavy chain with the N-terminal nucleotide sequence of the linker (the C-terminal of the linker is connected with VEGFR1D2-VEGFR2D3), the nucleotide sequence encoding the HX016-5 heavy chain was obtained ( SEQ ID NO: 28), the nucleotide sequence encoding the heavy chain of HX016-5 was connected to the expression vector 1 to obtain the expression vector 7 expressing the bispecific antibody HX016-5. Extract the DNA of expression vectors 1 and 7, and transfect them into mammalian 293 cells respectively. The final concentration of plasmid DNA is 4 μg/mL, and the final concentration of transfection reagent PEI is 8 μg/mL. In the cells, mix well while adding dropwise. After cell transfection, antibodies are expressed in mammalian cells and secreted extracellularly. Then, the expressed antibody was purified through a protein A affinity chromatography column to obtain the HX016-5 protein.
重组抗体5号(HX016-5)的重链的氨基酸序列如下：The amino acid sequence of the heavy chain of recombinant antibody No. 5 (HX016-5) is as follows:
Figure PCTCN2021132785-appb-000049
Figure PCTCN2021132785-appb-000049
编码重组抗体5号(HX016-5)重链的核苷酸序列：Nucleotide sequence encoding the heavy chain of recombinant antibody No. 5 (HX016-5):
Figure PCTCN2021132785-appb-000050
Figure PCTCN2021132785-appb-000050
Figure PCTCN2021132785-appb-000051
Figure PCTCN2021132785-appb-000051
6、PD-1/VEGFbsAb-6(HX016-6)6. PD-1/VEGFbsAb-6 (HX016-6)
HX016-6的重链(SEQ ID NO：6)为PD-1 mAb的重链C端用linker连接能结合VEGF的单链抗体(SEQ ID NO：26)的N端。The heavy chain of HX016-6 (SEQ ID NO: 6) is the C-terminal of the heavy chain of PD-1 mAb, and the N-terminal of the single-chain antibody (SEQ ID NO: 26) that can bind to VEGF is connected with a linker.
实际操作中，全基因合成编码PD-1 mAb轻链、重链、linker+VEGF单链抗体的核酸序列，以及构建载体1、2、8的具体实验操作同上。先将编码PD-1 mAb轻链的核酸序列连接到表达载体中获得表达载体1；把PD-1 mAb的重链序列连接到表达载体1中获得表达抗PD1的单克隆抗体HX008的表达载体2。通过Over-lap PCR将PD-1 mAb重链C端与linker(所述linker的C端连接有VEGF单链抗体)的N端核苷酸序列融合后获得编码所述HX016-6重链的核苷酸序列(SEQ ID NO：11)，连接到表达载体1中获得表达双特异性抗体HX016-6的表达载体8。提取表达载体1和8的DNA，分别转染至哺乳动物细胞293细胞，质粒DNA终浓度4μg/mL，转染试剂PEI终浓度8μg/mL转染，室温静置5分钟后缓慢滴加至293细胞中，边滴加边混匀。细胞转染后，抗体在哺乳动物细胞内表达，并分泌到细胞外。然后，通过蛋白A亲和层析柱，纯化表达的抗体，即获得HX016-6蛋白。In actual operation, the nucleic acid sequences encoding PD-1 mAb light chain, heavy chain, linker+VEGF single-chain antibody, and the specific experimental operations for constructing vectors 1, 2, and 8 are the same as above. First connect the nucleic acid sequence encoding the light chain of PD-1 mAb to the expression vector to obtain expression vector 1; connect the heavy chain sequence of PD-1 mAb to expression vector 1 to obtain the expression vector 2 expressing the anti-PD1 monoclonal antibody HX008 . The nucleus encoding the HX016-6 heavy chain was obtained by fusing the C-terminal of the heavy chain of PD-1 mAb with the N-terminal nucleotide sequence of the linker (the C-terminal of the linker is linked with VEGF single-chain antibody) by Over-lap PCR The nucleotide sequence (SEQ ID NO: 11) was connected to the expression vector 1 to obtain the expression vector 8 expressing the bispecific antibody HX016-6. Extract the DNA of expression vectors 1 and 8, and transfect them into 293 mammalian cells respectively, with a final concentration of plasmid DNA of 4 μg/mL and a final concentration of transfection reagent PEI of 8 μg/mL for transfection. In the cells, mix well while adding dropwise. After cell transfection, antibodies are expressed in mammalian cells and secreted extracellularly. Then, the expressed antibody is purified through a protein A affinity chromatography column to obtain the HX016-6 protein.
重组抗体6号(HX016-6)的重链的氨基酸序列如下：The amino acid sequence of the heavy chain of recombinant antibody No. 6 (HX016-6) is as follows:
Figure PCTCN2021132785-appb-000052
Figure PCTCN2021132785-appb-000052
Figure PCTCN2021132785-appb-000053
Figure PCTCN2021132785-appb-000053
VEGF单链抗体的氨基酸序列如下：The amino acid sequence of the VEGF single-chain antibody is as follows:
Figure PCTCN2021132785-appb-000054
Figure PCTCN2021132785-appb-000054
编码重组抗体6号(HX016-6)重链的核苷酸序列：Nucleotide sequence encoding the heavy chain of recombinant antibody No. 6 (HX016-6):
Figure PCTCN2021132785-appb-000055
Figure PCTCN2021132785-appb-000055
Figure PCTCN2021132785-appb-000056
Figure PCTCN2021132785-appb-000056
实施例2重组抗体分子的结合活性测定Example 2 Determination of Binding Activity of Recombinant Antibody Molecules
1、重组抗体分子与PD-1结合的结合活性检测1. Detection of binding activity of recombinant antibody molecule binding to PD-1
本实验共设5组，分别为HX008组(anti-PD-1 mAb)、HX016-1组、HX016-2组、HX016-4组、HX016-6组，通过ELISA法，以HX008组为对照组，检测HX016-1组、HX016-2组、HX016-4组、HX016-6组与PD-1的结合活性，There are 5 groups in this experiment, which are HX008 group (anti-PD-1 mAb), HX016-1 group, HX016-2 group, HX016-4 group, HX016-6 group. By ELISA method, HX008 group was used as the control group , to detect the binding activity of HX016-1 group, HX016-2 group, HX016-4 group, HX016-6 group and PD-1,
HX008(PD-1 mAb)轻链的氨基酸序列如下：The amino acid sequence of the light chain of HX008 (PD-1 mAb) is as follows:
Figure PCTCN2021132785-appb-000057
Figure PCTCN2021132785-appb-000057
HX008(PD-1 mAb)重链的氨基酸序列如下：The amino acid sequence of the heavy chain of HX008 (PD-1 mAb) is as follows:
Figure PCTCN2021132785-appb-000058
Figure PCTCN2021132785-appb-000058
具体实验操作如下：The specific experimental operation is as follows:
取0.25μg/mL的PD-1-his加入酶标板孔内，4℃包被过夜包被至，次日加入200μL PBST震荡洗涤10min，洗涤3次，再加入200μL PBS震荡洗涤10min，洗涤3次，以洗去多余抗原后加入1％BSA封闭液，于37℃封闭1h，洗涤3次，去除封闭液，将待测样品用浓度为1‰的BSA稀释至10000ng/mL，再5倍梯度稀释6个稀释度上样，使上样浓度分别为10000、2000、400、80、16、3.2、0.64ng/mL，设复孔空白，孵育1h后加入15000X稀释的Goat anti-Human二抗孵育1h，洗去 多余二抗后加入新配显色液，根据显色情况调整显色时间不超过15min，加入终止液于酶标仪中读取OD值。Add 0.25 μg/mL PD-1-his into the wells of the microtiter plate, coat at 4°C overnight, and add 200 μL PBST the next day to wash for 10 minutes by shaking, wash 3 times, then add 200 μL PBS to wash by shaking for 10 minutes, wash 3 times After washing away excess antigen, add 1% BSA blocking solution, block at 37°C for 1 hour, wash 3 times, remove the blocking solution, dilute the sample to be tested with BSA with a concentration of 1‰ to 10,000 ng/mL, and then use a 5-fold gradient Dilute the sample at 6 dilutions, so that the sample concentration is 10000, 2000, 400, 80, 16, 3.2, 0.64ng/mL respectively, set the duplicate hole blank, and add 15000X diluted Goat anti-Human secondary antibody after incubation for 1h 1h, after washing off the excess secondary antibody, add a new color developing solution, adjust the color developing time according to the color developing condition to no more than 15min, add stop solution and read the OD value in a microplate reader.
根据ELISA结果中的OD值计算EC50，具体结果如图1所示，其中HX016-1、HX016-4、HX016-6与PD-1的结合活性较接近均在25.0ng/mL左右，活性稳定，结果较好，HX016-2与PD-1结合活性为36.6ng/mL。The EC50 was calculated according to the OD value in the ELISA results. The specific results are shown in Figure 1. Among them, the binding activities of HX016-1, HX016-4, HX016-6 and PD-1 are relatively close to 25.0ng/mL, and the activities are stable. The results were good, the binding activity of HX016-2 to PD-1 was 36.6ng/mL.
2、重组抗体分子与VEGF结合的结合活性检测2. Detection of binding activity of recombinant antibody molecule binding to VEGF
本实施例共设4组，分别为HX006组(anti-VEGF mAb)、HX016-2组、HX016-4组、HX016-6组，通过ELISA法，以HX006组为对照组，检测HX016-2组、HX016-4组、HX016-6组与VEGF165的结合活性，In this example, there are 4 groups in total, which are HX006 group (anti-VEGF mAb), HX016-2 group, HX016-4 group, and HX016-6 group. By ELISA method, the HX006 group is used as the control group to detect the HX016-2 group. , HX016-4 group, HX016-6 group and VEGF165 binding activity,
HX006重链的氨基酸序列如下：The amino acid sequence of the HX006 heavy chain is as follows:
Figure PCTCN2021132785-appb-000059
Figure PCTCN2021132785-appb-000059
HX006轻链的氨基酸序列如下：The amino acid sequence of the HX006 light chain is as follows:
Figure PCTCN2021132785-appb-000060
Figure PCTCN2021132785-appb-000060
具体实验操作如下：The specific experimental operation is as follows:
取0.25μg/mL的VEGF165加入酶标板孔内，4℃包被过夜包被至，次日加入200μL PBST震荡洗涤10min，洗涤3次，再加入200μL PBS震荡洗涤10min，洗涤3次，以洗去多余抗原后加入1％BSA封闭液，于37℃封闭1h，洗涤3次，去除封闭液，将待测样品用浓度为1‰的BSA稀释至2μg/mL，再5倍梯度稀释6个稀释度上样，使上样浓度分别为2000、400、80、16、3.2、0.64、0.128ng/mL。设复孔空白，孵育1h后加入15000X稀释的Goat anti-Human二抗孵育1h，洗去多余二抗后加入新配显色液，根据显色情况调整显色时间不超过15min，加入终止液于酶标仪中读取OD值。Add 0.25 μg/mL VEGF165 into the wells of the microplate, coat at 4°C overnight, and add 200 μL PBST the next day to wash with shaking for 10 minutes, wash 3 times, then add 200 μL PBS to wash with shaking for 10 minutes, wash 3 times, to wash After removing excess antigen, add 1% BSA blocking solution, block at 37°C for 1 hour, wash 3 times, remove the blocking solution, dilute the sample to be tested with BSA with a concentration of 1‰ to 2 μg/mL, and then dilute 6 times in a 5-fold gradient Load the samples at different degrees, so that the loading concentrations are 2000, 400, 80, 16, 3.2, 0.64, and 0.128 ng/mL. Set the duplicate well as blank, incubate for 1 hour, add 15000X diluted Goat anti-Human secondary antibody and incubate for 1 hour, wash off excess secondary antibody, add new color development solution, adjust the color development time according to the color development situation to no more than 15 minutes, add stop solution in Read the OD value in a microplate reader.
根据ELISA结果中的OD值计算EC50，结果如图2所示，其中HX016-2和HX016-4与VEGF165的结合活性较接近，均在25.0ng/mL左右，活性较好，HX016-6与VEGF165结合活性为93.5ng/mL。The EC50 was calculated according to the OD value in the ELISA results. The results are shown in Figure 2. Among them, the binding activities of HX016-2 and HX016-4 to VEGF165 are relatively close, both at about 25.0ng/mL, and the activity is better. HX016-6 and VEGF165 The binding activity was 93.5 ng/mL.
3、不同批次HX016-1与分子靶点VEGFR2结合的结合活性检测3. Detection of the binding activity of different batches of HX016-1 binding to the molecular target VEGFR2
本实施例共设5组，分别为HX016-1(Lot.20190103)组、HX016-1-5K-2-E2组、HX016-1-7.5K-3-F4组、HX016-1-7.5K-4-F6组、HX016-1-7.5K-5-F10组，通过ELISA法，以HX016-1(Lot.20190103)组为对照组，检测HX016-1各批次所述样品与VEGFR2-his的结合活性，具体实验操作如下：In this embodiment, there are 5 groups in total, which are HX016-1 (Lot.20190103) group, HX016-1-5K-2-E2 group, HX016-1-7.5K-3-F4 group, HX016-1-7.5K- 4-F6 group, HX016-1-7.5K-5-F10 group, by ELISA method, with HX016-1 (Lot.20190103) group as the control group, detect the difference between the samples of each batch of HX016-1 and VEGFR2-his Combined activity, the specific experimental operation is as follows:
取0.5μg/mL的VEGFR2-his加入酶标板孔内，4℃包被过夜包被至，次日加入200μL PBST震荡洗涤10min，洗涤3次，再加入200μLPBS震荡洗涤10min，洗涤3次，以洗去多余抗原后加入1％BSA封闭液，于37℃封闭1h，洗涤3次，去除封闭液，将待测样品用浓度为1‰的BSA稀释至10μg/mL，再5倍梯度稀释6个稀释度共7个梯度上样，设复孔空白，孵育1h后加入15000X稀释的Goat anti-Human二抗孵育1h，洗去多余二抗后加入新配显色液，根据显色情况调整显色时间不超过15min，加入终止液于酶标仪中读取OD值。Add 0.5 μg/mL VEGFR2-his into the wells of the microtiter plate, coat at 4°C overnight, and add 200 μL PBST to shake and wash for 10 minutes the next day, wash 3 times, then add 200 μL PBS to shake and wash for 10 minutes, wash 3 times. After washing away excess antigen, add 1% BSA blocking solution, block at 37°C for 1 hour, wash 3 times, remove the blocking solution, dilute the sample to be tested with BSA at a concentration of 1‰ to 10 μg/mL, and then dilute 6 samples in a 5-fold gradient A total of 7 gradients of dilution were loaded, and the duplicate wells were set as blank. After incubation for 1 hour, 15000X diluted Goat anti-Human secondary antibody was added to incubate for 1 hour. After the excess secondary antibody was washed away, a new color development solution was added, and the color development was adjusted according to the color development situation. After no more than 15 minutes, add stop solution and read the OD value in a microplate reader.
根据ELISA结果中的OD值计算EC50，具体结果如图3所示，其中HX016-1(Lot.20190103)与VEGFR2的结合活性为83.6ng/mL，HX016-1-5K-2-E2与VEGFR2结合活性为59.7ng/mL，HX016-1-7.5K-3-F4与VEGFR2结合活性为78.7ng/mL，HX016-1-7.5K-4-F6与VEGFR2结合活性为84.3ng/mL，HX016-1-7.5K-5-F10与VEGFR2结合活性为130.4ng/mL。HX016-1各批次与VEGFR2的结合活性较接近，范围为59.7～130.4ng/mL。The EC50 was calculated according to the OD value in the ELISA results, and the specific results are shown in Figure 3, where the binding activity of HX016-1 (Lot.20190103) to VEGFR2 was 83.6 ng/mL, and the binding activity of HX016-1-5K-2-E2 to VEGFR2 The activity is 59.7ng/mL, the binding activity of HX016-1-7.5K-3-F4 and VEGFR2 is 78.7ng/mL, the binding activity of HX016-1-7.5K-4-F6 and VEGFR2 is 84.3ng/mL, HX016-1 The binding activity of -7.5K-5-F10 to VEGFR2 was 130.4ng/mL. The binding activity of each batch of HX016-1 to VEGFR2 was relatively close, ranging from 59.7 to 130.4 ng/mL.
实施例3 重组抗体分子的相对竞争活性检测Example 3 Detection of Relative Competitive Activity of Recombinant Antibody Molecules
1、重组抗体分子与靶点PD-1/PD-L1结合的相对竞争活性检测1. Detection of relative competitive activity of recombinant antibody molecules binding to target PD-1/PD-L1
本实施例共设5组，分别为HX008组(anti-PD-1 mAb)、HX016-1组、HX016-2组、HX016-4组、HX016-6组，通过ELISA法，以HX008组为对照组，检测HX016-1组、HX016-2组、HX016-4组、HX016-6组与PD-1/PD-L1的结合的相对竞争活性，具体实验操作如下：In this example, there are 5 groups in total, namely HX008 group (anti-PD-1 mAb), HX016-1 group, HX016-2 group, HX016-4 group, HX016-6 group, by ELISA method, with HX008 group as the control group, to detect the relative competitive activity of HX016-1 group, HX016-2 group, HX016-4 group, HX016-6 group in combination with PD-1/PD-L1, the specific experimental operation is as follows:
取0.5μg/mL的PD-1-his加入酶标板孔内，4℃包被过夜包被至，次日加入200μL PBST震荡洗涤10min，洗涤3次，再加入200μL PBS震荡洗涤10min，洗涤3次，以洗去多余抗原后加入1％BSA封闭液，于37℃封闭1h，洗涤3次，去除封闭液，将待测样品用浓度为1‰的BSA稀释至6μg/mL，3倍梯度稀释8个稀释度，第2与第8个梯度舍弃，共7个梯度上样，使上样浓度分别为6000，666.6，222.2，74.07，24.69，8.230，2.743ng/mL，将稀释后的待测样品与0.6μg/mL PD-L1-mFc以1:1的体积比进行混匀，设复孔空白，孵育1h后加入5000X稀释的Goat anti-Mouse二抗孵育1h，洗去多余二抗后加入新配显色液，根据显色情况调整显色时间不超过20min，加入终止液于酶标仪中读取 OD值。Add 0.5 μg/mL PD-1-his into the wells of the microplate, coat at 4°C overnight, and add 200 μL PBST to wash for 10 minutes the next day, wash 3 times, then add 200 μL PBS to wash for 10 minutes, wash 3 times After washing away excess antigen, add 1% BSA blocking solution, block at 37°C for 1 hour, wash 3 times, remove the blocking solution, dilute the sample to be tested with BSA with a concentration of 1‰ to 6 μg/mL, and make a 3-fold serial dilution For 8 dilutions, the second and eighth gradients were discarded, and a total of 7 gradients were loaded, so that the loading concentrations were 6000, 666.6, 222.2, 74.07, 24.69, 8.230, 2.743ng/mL, and the diluted samples to be tested were Mix the sample with 0.6 μg/mL PD-L1-mFc at a volume ratio of 1:1, set a duplicate hole blank, incubate for 1 hour, add 5000X diluted Goat anti-Mouse secondary antibody and incubate for 1 hour, wash off the excess secondary antibody and add Newly prepare the color developing solution, adjust the color developing time to no more than 20 minutes according to the color developing condition, add the stop solution and read the OD value in the microplate reader.
根据ELISA结果中的OD值计算IC50，具体结果如如图4所示，其中HX016-1(Lot.20190103)的IC50为97.5ng/mL，HX016-2(Lot.20190121)的IC50为121ng/mL，HX016-4(Lot.20190311)的IC50为64.9ng/mL，HX016-4(Lot.20190726)的IC50为63.7ng/mL，HX016-6(Lot.20190726)的IC50为83.5ng/mL，HX008(Lot.A103C20171201)的IC50为53.1ng/mL，因此，系列重组抗体能够阻断PD-1/PD-L1结合的活性与PD-1单抗的活性较一致。The IC50 is calculated according to the OD value in the ELISA result, as shown in Figure 4, where the IC50 of HX016-1 (Lot.20190103) is 97.5ng/mL, and the IC50 of HX016-2 (Lot.20190121) is 121ng/mL , the IC50 of HX016-4 (Lot.20190311) is 64.9ng/mL, the IC50 of HX016-4 (Lot.20190726) is 63.7ng/mL, the IC50 of HX016-6 (Lot.20190726) is 83.5ng/mL, HX008 (Lot.A103C20171201) has an IC50 of 53.1ng/mL. Therefore, the activity of a series of recombinant antibodies that can block the binding of PD-1/PD-L1 is more consistent with the activity of PD-1 monoclonal antibody.
2、重组抗体分子与靶点VEGF/VEGFR2结合的相对竞争活性检测2. Detection of the relative competitive activity of the recombinant antibody molecule binding to the target VEGF/VEGFR2
本实施例共设4组，分别为HX006组(anti-VEGF mAb)、HX016-1组、HX016-2组、HX016-4组、HX016-6组，通过ELISA法，以HX006组为对照组，检测HX016-1组、HX016-2组、HX016-4组、HX016-6组与VEGF165/VEGFR2结合的相对竞争活性，具体实验操作如下：In this embodiment, there are 4 groups, which are HX006 group (anti-VEGF mAb), HX016-1 group, HX016-2 group, HX016-4 group, HX016-6 group. By ELISA method, HX006 group is used as the control group. Detect the relative competitive activity of HX016-1 group, HX016-2 group, HX016-4 group, HX016-6 group combined with VEGF165/VEGFR2, the specific experimental operation is as follows:
取0.5μg/mL的VEGF165加入酶标板孔内，4℃包被过夜包被至次日加入200μL PBST震荡洗涤10min，洗涤3次，再加入200μL PBS震荡洗涤10min，洗涤3次，以洗去多余抗原后加入1％BSA封闭液，于37℃封闭1h，洗涤3次，去除封闭液，将HX016-1用1‰BSA稀释至6μg/mL，HX016-2用1‰BSA稀释至20μg/mL，HX006、HX016-4和HX016-6用1‰BSA稀释至60μg/mL后，3倍梯度稀释6个稀释度上样，使上样浓度分别为2000、400、80、16、3.2、0.64、0.128ng/mL，各稀释浓度的待测样品均与0.6μg/mL VEGFR2-mFc以1:1体积比混匀，设复孔空白，孵育1h后加入5000X稀释的Goat anti-Mouse二抗孵育1h，洗去多余二抗后加入新配显色液，根据显色情况调整显色时间不超过20min，加入终止液于酶标仪中读取OD值。Add 0.5 μg/mL VEGF165 into the wells of the microplate, coat at 4°C overnight, add 200 μL PBST to shake and wash for 10 minutes the next day, wash 3 times, then add 200 μL PBS to shake and wash for 10 minutes, wash 3 times to wash away Add 1% BSA blocking solution after excess antigen, block at 37°C for 1 hour, wash 3 times, remove the blocking solution, dilute HX016-1 with 1‰BSA to 6μg/mL, and dilute HX016-2 with 1‰BSA to 20μg/mL , HX006, HX016-4 and HX016-6 were diluted with 1‰BSA to 60μg/mL, and then 3-fold serially diluted 6 dilutions to load the samples, so that the loading concentrations were 2000, 400, 80, 16, 3.2, 0.64, 0.128ng/mL, the samples to be tested at each dilution concentration were mixed with 0.6μg/mL VEGFR2-mFc at a volume ratio of 1:1, set a duplicate hole blank, incubated for 1h, added 5000X diluted Goat anti-Mouse secondary antibody and incubated for 1h , after washing off the excess secondary antibody, add a new color development solution, adjust the color development time according to the color development situation to no more than 20 minutes, add the stop solution and read the OD value in a microplate reader.
根据ELISA结果中的OD值计算IC50，具体结果如图5所示。其中，HX016-1(Lot.20190103)的IC50为136.7ng/mL，HX016-2(Lot.20190121)的IC50为944.1ng/mL，HX016-4(Lot.20190311)的IC50为1345.4ng/mL，HX016-4(Lot.20190726)的IC50为6258.4ng/mL，HX006(Lot.20170614)的IC50为1097.3ng/mL，因此，HX016和HX006都具有阻断VEGF/VEGFR的结合的活性，其中HX016-1的活性最佳。The IC50 was calculated according to the OD value in the ELISA results, and the specific results are shown in Figure 5. Among them, the IC50 of HX016-1 (Lot.20190103) is 136.7ng/mL, the IC50 of HX016-2 (Lot.20190121) is 944.1ng/mL, the IC50 of HX016-4 (Lot.20190311) is 1345.4ng/mL, The IC50 of HX016-4 (Lot.20190726) is 6258.4ng/mL, and the IC50 of HX006 (Lot.20170614) is 1097.3ng/mL. Therefore, both HX016 and HX006 have the activity of blocking the binding of VEGF/VEGFR, among which HX016- 1 has the best activity.
实施例4 HX016-2结合表面表达PD-1的293T细胞Example 4 HX016-2 binds to 293T cells expressing PD-1 on the surface
HX016-2是一种抗PD-1/VEGF双特异性的免疫抗体，本试验采用293T-PD-1稳定转染细胞株细胞作为实验细胞，FACS方法检测HX016-2与细胞膜表面表达PD-1蛋白的细胞结合情况，具体操作如下：HX016-2 is an anti-PD-1/VEGF bispecific immune antibody. In this experiment, 293T-PD-1 stably transfected cell line cells were used as experimental cells. FACS method was used to detect HX016-2 and the expression of PD-1 on the cell membrane surface. Protein cell binding, the specific operation is as follows:
将293T-PD-1-5E11细胞调整密度至2x10 6个/mL，100μL/孔加入V底96孔板中，4℃保藏备用。用FACS buffer稀释样品HX016-2(20201225，8.11mg/mL)，10个稀释浓度(20μg/mL、10μg/mL、5μg/mL、1μg/mL、0.5μg/mL、0.2μg/mL、0.1μg/mL、20ng/mL、2ng/mL、0.2ng/mL)。细胞450xg条件下离心处理 5min，弃上清，依次加入10个稀释样品，100μL/孔。随行FACS buffer空白，4℃条件下孵育1h，450xg条件下离心处理5min，弃上清；250μL/孔FACS buffer洗2遍，100μL/孔加入200X稀释Alexa
Figure PCTCN2021132785-appb-000061
647荧光标记的抗人二抗，4℃条件下孵育30min后，于450x g条件下离心处理5min，弃上清；将沉淀物用250μL/孔FACS buffer洗2遍，用200μL/孔FACS buffer重悬，转入流式管中。用CytoFLEX流式细胞仪检测荧光强度，Alexa
Figure PCTCN2021132785-appb-000062
647采用APC通道。 Adjust the density of 293T-PD-1-5E11 cells to 2x10 6 cells/mL, add 100 μL/well into a V-bottom 96-well plate, and store at 4°C for future use. Dilute sample HX016-2 (20201225, 8.11mg/mL) with FACS buffer, 10 dilution concentrations (20μg/mL, 10μg/mL, 5μg/mL, 1μg/mL, 0.5μg/mL, 0.2μg/mL, 0.1μg /mL, 20ng/mL, 2ng/mL, 0.2ng/mL). The cells were centrifuged at 450×g for 5 min, the supernatant was discarded, and 10 diluted samples were added in turn, 100 μL/well. Accompanying FACS buffer blank, incubate at 4°C for 1 hour, centrifuge at 450xg for 5 minutes, discard the supernatant; wash 2 times with 250 μL/well FACS buffer, add 100 μL/well to 200X diluted Alexa
Figure PCTCN2021132785-appb-000061
647 fluorescently-labeled anti-human secondary antibody, incubated at 4°C for 30 minutes, centrifuged at 450x g for 5 minutes, discarded the supernatant; washed the precipitate twice with 250 μL/well FACS buffer, and reconstituted with 200 μL/well FACS buffer. Hang and transfer to flow tube. Detection of fluorescence intensity with CytoFLEX flow cytometer, Alexa
Figure PCTCN2021132785-appb-000062
647 uses APC channel.
FACS方法检测HX016-2与细胞膜表面表达PD-1蛋白的细胞结合情况，其中，HX008为anti-PD-1mAb，具体结果如图6所示，HX016-2与PD-1的结合的EC50值为0.2111ug/mL。与HX008相比，结合活性稍弱，但两者差异并不显著。The FACS method was used to detect the binding of HX016-2 to cells expressing PD-1 protein on the cell membrane surface. Among them, HX008 is an anti-PD-1 mAb. The specific results are shown in Figure 6. The EC50 value of the binding of HX016-2 to PD-1 is 0.2111ug/mL. Compared with HX008, the binding activity is slightly weaker, but the difference between the two is not significant.
实施例5 HX016-2阻断PD-L1和细胞膜表面表达PD-1的293T细胞结合实验Example 5 Binding experiment of HX016-2 blocking PD-L1 and expressing PD-1 on the cell membrane surface in 293T cells
HX016-2是一种抗PD-1/VEGF双特异性的免疫抗体，本试验采用293T-PD-1稳定转染细胞株细胞作为实验细胞，FACS方法检测HX016-2能否阻断PD-L1与细胞膜表面表达PD-1的细胞的结合情况，具体操作如下：HX016-2 is an anti-PD-1/VEGF bispecific immune antibody. In this experiment, 293T-PD-1 stably transfected cell line cells were used as experimental cells. FACS method was used to detect whether HX016-2 could block PD-L1 The specific operation of binding to cells expressing PD-1 on the cell membrane surface is as follows:
将293T-PD1-5E11细胞调整密度至2x10 6个/mL，100μL/孔加入V底96孔板中，4℃保藏备用。用FACS buffer稀释样品HX016-2(20201225，8.11mg/mL)，8个稀释浓度(20μg/mL，10μg/mL，2μg/mL，2μg/mL，0.2μg/mL，40ng/mL，4ng/mL，0.4ng/mL)。细胞450g条件下离心处理5min，弃去上清液后，依次加入8个稀释样品，50μL/孔。2个随行FACS buffer空白，4℃条件下孵育1h。用FACS buffer稀释样品PD-L1-mFC(Akesobio，20μg/mL)到10μg/mL，50μL/孔加到8个稀释样本孔和一个空白孔，4℃条件下孵育1h。450g条件下离心处理5min，弃去上清液；所得沉淀物用250μL/孔FACS buffer洗2遍，100μL/孔加入200X稀释Alexa
Figure PCTCN2021132785-appb-000063
647荧光标记的抗鼠二抗，4℃条件下孵育30min。450xg条件下离心处理5min，弃去上清；将所得沉淀物用250μL/孔FACS buffer洗涤2遍，用200μL/孔FACS buffer重悬，转入流式管中，用CytoFLEX流式细胞仪检测荧光强度，Alexa
Figure PCTCN2021132785-appb-000064
647采用APC通道。 Adjust the density of 293T-PD1-5E11 cells to 2x10 6 cells/mL, add 100 μL/well into a V-bottom 96-well plate, and store at 4°C for future use. Dilute sample HX016-2 (20201225, 8.11mg/mL) with FACS buffer, 8 dilution concentrations (20μg/mL, 10μg/mL, 2μg/mL, 2μg/mL, 0.2μg/mL, 40ng/mL, 4ng/mL , 0.4ng/mL). The cells were centrifuged at 450 g for 5 min, and after the supernatant was discarded, 8 diluted samples were sequentially added, 50 μL/well. Two accompanying FACS buffer blanks were incubated at 4°C for 1 hour. Dilute the sample PD-L1-mFC (Akesobio, 20 μg/mL) to 10 μg/mL with FACS buffer, add 50 μL/well to 8 diluted sample wells and one blank well, and incubate at 4 °C for 1 h. Centrifuge at 450g for 5min, discard the supernatant; wash the resulting precipitate twice with 250μL/well FACS buffer, add 100μL/well of 200X diluted Alexa
Figure PCTCN2021132785-appb-000063
647 fluorescently labeled anti-mouse secondary antibody, incubated at 4°C for 30min. Centrifuge at 450xg for 5 min, discard the supernatant; wash the obtained precipitate twice with 250 μL/well FACS buffer, resuspend with 200 μL/well FACS buffer, transfer to a flow tube, and detect fluorescence with a CytoFLEX flow cytometer Strength, Alexa
Figure PCTCN2021132785-appb-000064
647 uses APC channel.
FACS方法检测HX016-2与竞争PD-L1-mFc与细胞膜表面PD-1蛋白的细胞结合情况，其中，HX008为anti-PD-1 mAb，具体结果如图7所示，HX016-2与PD-1的结合EC50值为0.2735ug/mL，HX008(anti-PD-1 mAb)与PD-1的结合EC5值为0.0301ug/mL因此，HX016和HX008都有阻断PD-1/PD-L1结合的活性，其阻断活性有差异。The FACS method was used to detect the cell binding of HX016-2 and competing PD-L1-mFc to the PD-1 protein on the cell membrane surface. Among them, HX008 is anti-PD-1 mAb. The specific results are shown in Figure 7. HX016-2 and PD-1 The binding EC50 value of 1 is 0.2735ug/mL, and the binding EC5 value of HX008 (anti-PD-1 mAb) and PD-1 is 0.0301ug/mL. Therefore, both HX016 and HX008 can block the binding of PD-1/PD-L1 activity, and its blocking activity varies.
实施例6 目标蛋白阻断CHO-scOKT3-PD-L1细胞对Jurkat-NFAT-luc细胞表达荧光素酶的抑制实验Example 6 Inhibition of luciferase expression in Jurkat-NFAT-luc cells by target protein blocking CHO-scOKT3-PD-L1 cells
HX016是一种抗PD-1/VEGF双特异性的免疫抗体，本试验采用CHO-scOKT3-PD-L1和Jurkat-NFAT-luc稳定转染细胞株细胞作为实验细胞，荧光素酶报告基因检测HX016-2阻断CHO-scOKT3-PD-L1细胞对Jurkat-NFAT-luc细胞表达荧光素酶的抑制作用，恢复荧光素酶的表达，以检测PD-1端的生物学活性，具体操作如下：HX016 is an anti-PD-1/VEGF bispecific immune antibody. In this experiment, CHO-scOKT3-PD-L1 and Jurkat-NFAT-luc stably transfected cell lines were used as experimental cells. The luciferase reporter gene was used to detect HX016 -2 Block the inhibitory effect of CHO-scOKT3-PD-L1 cells on the expression of luciferase in Jurkat-NFAT-luc cells, restore the expression of luciferase, and detect the biological activity of PD-1, the specific operation is as follows:
将CHO-PDL1-CD3L细胞密度调整到5x10 5cells/mL，第一天，将100μL密度为5x10 5cells/mL的CHO-PDL1-CD3L细胞加入到白底96孔板中。于37℃、5％CO 2条件下置于培养箱中孵育16±2h。 Adjust the density of CHO-PDL1-CD3L cells to 5x10 5 cells/mL. On the first day, add 100 μL of CHO-PDL1-CD3L cells with a density of 5x10 5 cells/mL into a white-bottomed 96-well plate. Incubate in an incubator at 37°C, 5% CO 2 for 16±2h.
培养结束后，用分析培养基稀释样品HX016-1、HX016-2、HX016-4至2倍浓度，共设置10个稀释梯度(60μg/mL，20μg/mL，6.666μg/mL，2.222μg/mL，0.741μg/mL，0.247μg/mL，82.305ng/mL，27.435ng/mL，9.145ng/mL，3.048ng/mL)。After culturing, dilute samples HX016-1, HX016-2, and HX016-4 to 2-fold concentration with analytical medium, and set up 10 dilution gradients (60 μg/mL, 20 μg/mL, 6.666 μg/mL, 2.222 μg/mL , 0.741 μg/mL, 0.247 μg/mL, 82.305 ng/mL, 27.435 ng/mL, 9.145 ng/mL, 3.048 ng/mL).
Jurkat-PD1-NFAT细胞用分析培养基将其密度调整至2x10 6cells/mL，取出培养有CHO-PDL1-CD3L细胞的96孔白板，吸出细胞上清培养基，每孔加入50μL不同浓度的HX016溶液和50μL的Jurkat-PD1-NFAT细胞悬液，将细胞混合液置于37℃、5％CO 2的培养箱中孵育6h。荧光素酶检测液提前拿出，并避光预平衡至室温。细胞混合液6h孵育完毕后，每孔加入100μL的荧光素酶检测液，避光常温孵育1min后用Perkin Elmer荧光酶标仪读取自发光，Protocol中检测波长为560nm，检测高度为6.5cm，检测时间为5s，选择合适的检测孔后读取数值。 Use assay medium for Jurkat-PD1-NFAT cells to adjust the density to 2x10 6 cells/mL, take out the 96-well white plate cultured with CHO-PDL1-CD3L cells, suck out the cell supernatant medium, and add 50 μL of different concentrations of HX016 to each well solution and 50 μL of Jurkat-PD1-NFAT cell suspension, and the cell mixture was incubated in an incubator at 37°C and 5% CO 2 for 6h. The luciferase assay solution was taken out in advance and pre-equilibrated to room temperature in the dark. After the cell mixture was incubated for 6 hours, add 100 μL of luciferase detection solution to each well, incubate at room temperature in the dark for 1 min, and then read the autoluminescence with a Perkin Elmer fluorescence microplate reader. The detection wavelength in the Protocol is 560 nm, and the detection height is 6.5 cm. The detection time is 5s, and the value is read after selecting a suitable detection hole.
具体结果如图8所示，对荧光信号值和待测抗体浓度的对数值作出曲线，然后按照四参数方程进行拟合，得到半效应浓度值(EC50)。PD-1单抗HX008的EC50为51.7ng/mL，HX016-1的EC50为100.9ng/mL，HX016-2的EC50为208.8ng/mL，HX016-4的EC50为46.7ng/mL，因此，荧光素酶报告基因检测HX016各抗体均可阻断CHO-scOKT3-PD-L1细胞对Jurkat-NFAT-luc细胞表达荧光素酶的抑制作用。The specific results are shown in Figure 8. A curve was drawn between the fluorescence signal value and the logarithmic value of the antibody concentration to be tested, and then fitted according to the four-parameter equation to obtain the half-effect concentration value (EC50). The EC50 of PD-1 monoclonal antibody HX008 is 51.7ng/mL, the EC50 of HX016-1 is 100.9ng/mL, the EC50 of HX016-2 is 208.8ng/mL, and the EC50 of HX016-4 is 46.7ng/mL. Therefore, the fluorescence The luciferase reporter gene detection HX016 antibodies can block the inhibitory effect of CHO-scOKT3-PD-L1 cells on the expression of luciferase in Jurkat-NFAT-luc cells.
实施例7 目标蛋白阻断VEGF对293T-VEGFR2-NFAT-luc细胞表达荧光素酶的激活实验Example 7 The target protein blocks the activation experiment of VEGF on the expression of luciferase in 293T-VEGFR2-NFAT-luc cells
HX01为抗PD-1/VEGF双特异性的免疫抗体，本试验采用293T-VEGFR2-CNFAT-5F8稳定转染细胞株细胞作为实验细胞，荧光素酶报告基因检测HX016-2阻断VEGF对293T-VEGFR2-CNFAT-5F8细胞表达荧光素酶的激活，抑制荧光素酶的表达，以检测VEGF端的生物学活性，具体操作如下：HX01 is an anti-PD-1/VEGF bispecific immune antibody. In this experiment, 293T-VEGFR2-CNFAT-5F8 stably transfected cell line cells were used as experimental cells. The luciferase reporter gene detected HX016-2 to block the effect of VEGF on 293T- VEGFR2-CNFAT-5F8 cells express the activation of luciferase, and inhibit the expression of luciferase to detect the biological activity of VEGF terminal. The specific operation is as follows:
将293T-VEGFR2-CNFAT-5F8细胞密度调整到2x10 6cells/mL，将VEGF蛋白用分析培养基稀释至400ng/mL。 The 293T-VEGFR2-CNFAT-5F8 cell density was adjusted to 2x10 6 cells/mL, and the VEGF protein was diluted to 400ng/mL with assay medium.
用分析培养基稀释样品HX016-1、HX016-2、HX016-4至2倍浓度，共设9个稀释浓度(10μg/mL，5μg/mL，2.5μg/mL，1.25μg/mL，0.625μg/mL，0.313μg/mL，0.156μg/mL，78.125ng/mL，39.063ng/mL)。将细胞密度调整至2x10 6cells/mL的293T-VEGFR2-CNFAT-5F8细胞与400ng/mL的rhVEGF165单抗按1：1体积比进行混合，白底96孔培养板中每孔加入50uL上述混合液，每孔加入50μL不 同浓度的HX016溶液，随行分析培养基空白，置于37℃、5％CO 2培养箱中孵育18h。荧光素酶检测液避提前拿出避光预平衡至室温。混合液18h孵育完毕后，每孔加入100μL的荧光素酶检测液；避光常温孵育1min后用Perkin Elmer荧光酶标仪读取自发光，Protocol中检测波长为560nm，检测高度为6.5cm，检测时间为4s，选择合适的检测孔后读取数值。 Dilute samples HX016-1, HX016-2, HX016-4 to 2 times concentration with analysis medium, and set up 9 dilution concentrations (10μg/mL, 5μg/mL, 2.5μg/mL, 1.25μg/mL, 0.625μg/mL mL, 0.313 μg/mL, 0.156 μg/mL, 78.125 ng/mL, 39.063 ng/mL). Adjust the cell density to 2x10 6 cells/mL 293T-VEGFR2-CNFAT-5F8 cells and 400ng/mL rhVEGF165 monoclonal antibody in a 1:1 volume ratio, add 50uL of the above mixture to each well of a 96-well culture plate with a white bottom , add 50 μL of HX016 solution of different concentrations to each well, analyze the medium blank along with it, and incubate in a 37°C, 5% CO 2 incubator for 18h. The luciferase detection solution should be taken out in advance and pre-equilibrated to room temperature in the dark. After the mixture was incubated for 18 hours, 100 μL of luciferase detection solution was added to each well; after incubation at room temperature in the dark for 1 min, the autoluminescence was read with a Perkin Elmer fluorescence microplate reader. The detection wavelength in the Protocol was 560 nm, and the detection height was 6.5 cm. The time is 4s, select the appropriate detection hole and read the value.
具体结果如图9所示，对荧光信号值和待测抗体浓度的对数值作出曲线，然后按照四参数方程进行拟合，得到半效应浓度值(EC50)。VEGF单抗HX006的IC50为51.7ng/mL，HX016-1的IC50为1163.1ng/mL，HX016-2的IC50为39.0ng/mL，HX016-4的IC50为63.2ng/mL。因此，荧光素酶报告基因检测HX016各抗体均可以阻断VEGF对293T-VEGFR2-CNFAT-5F8细胞表达荧光素酶的激活，抑制荧光素酶的表达。The specific results are shown in Figure 9. A curve was drawn between the fluorescent signal value and the logarithmic value of the antibody concentration to be tested, and then fitted according to the four-parameter equation to obtain the half-effect concentration value (EC50). The IC50 of VEGF monoclonal antibody HX006 is 51.7ng/mL, the IC50 of HX016-1 is 1163.1ng/mL, the IC50 of HX016-2 is 39.0ng/mL, and the IC50 of HX016-4 is 63.2ng/mL. Therefore, each antibody of luciferase reporter gene detection HX016 can block the activation of VEGF on the expression of luciferase in 293T-VEGFR2-CNFAT-5F8 cells, and inhibit the expression of luciferase.
实施例8 HX016-2混合淋巴反应中刺激T细胞分泌IFN-γ实验验证Example 8 Experimental verification of stimulating T cells to secrete IFN-γ in HX016-2 mixed lymphatic reaction
HX016-2是一种抗PD-1/VEGF双特异性的免疫抗体，混合淋巴细胞反应(MLR)采用一个人的DC细胞刺激另一个人CD4+T细胞，激活CD4+T细胞使其表面PD-1高表达，而DC细胞表面表达的PD-L1与CD4+T细胞表面的PD-1结合抑制其下游的PI3k信号通路，从而抑制了T细胞功能(细胞因子分泌)。本实验主要验证HX016-2能否阻断DC对T细胞的抑制来增强免疫功能。利用CBA检测IFN-γ的分泌水平以确定HX016-2的药效，具体操作如下：HX016-2 is an anti-PD-1/VEGF bispecific immune antibody. The mixed lymphocyte reaction (MLR) uses one person's DC cells to stimulate another person's CD4+T cells, and activates CD4+T cells to make their surface PD -1 is highly expressed, while PD-L1 expressed on the surface of DC cells binds to PD-1 on the surface of CD4+ T cells to inhibit its downstream PI3k signaling pathway, thereby inhibiting T cell function (cytokine secretion). This experiment mainly verifies whether HX016-2 can block the inhibition of DC on T cells to enhance immune function. Use CBA to detect the secretion level of IFN-γ to determine the efficacy of HX016-2, the specific operation is as follows:
第1天，分离第一个人的PBMC细胞后用磁珠分选分选出monocyte(CD14+)，用DC诱导培养基+1000IU/mL GM-CSF+1000IU/mL IL-4调整细胞密度到1x10 6cell/mL，按2mL/孔的量接种于6孔板中，将其置于37℃、5％CO 2条件进行下培养，培养至第3天、第5天以半换液方式加入新鲜的DC诱导培养基+1000IU/mL GM-CSF+1000IU/mL IL-4；继续放置于37℃、5％CO 2条件下进行培养；培养至第7天，从各孔分别吸取上清液1mL弃去，向各孔分别补加诱导培养液A 1mL，并加入100IU/mL TNF-ɑ，轻柔晃匀；继续放置于37℃、5％CO 2条件下进行培养；培养至第8天、第9天：向各孔分别补加100IU/mL的TNF-ɑ，轻柔晃匀；继续放置于放37℃、5％CO 2条件下进行培养；培养至第10天，分离另一个人的PBMC细胞后用磁珠分选分选出CD4+T cell，用MLR培养基调整密度到4x10 6cell/mL。用MLR共培养培养基将HX016-2稀释到40μg/mL，然后依次进行10倍梯度稀释，共设置5个浓度梯度(具体浓度为40,4,0.4,0.04,0.004μg/mL)。DC吹下来，用MLR共培养培养基调整细胞密度为2x10 5/mL，100μL的DC和50μL的T细胞铺到平底的96孔板中，每孔加入然后50μL不同浓度的HX016-2稀释样本，置于37℃、5％CO 2条件下培养5天，5天后收集上清用CBA试剂盒检测IFN-γ。 On the 1st day, after the first human PBMC cells were isolated, monocytes (CD14+) were sorted out by magnetic bead sorting, and the cell density was adjusted to 1x10 with DC induction medium + 1000IU/mL GM-CSF + 1000IU/mL IL-4 6 cells/mL, inoculated in a 6-well plate at 2 mL/well, cultured at 37°C and 5% CO 2 , and added fresh DC induction medium + 1000IU/mL GM-CSF + 1000IU/mL IL-4; continue to culture at 37°C and 5% CO 2 ; culture until the 7th day, draw 1mL of supernatant from each well Discard, add 1mL of induction medium A to each well, and add 100IU/mL TNF-ɑ, shake gently; continue to culture at 37°C, 5% CO2 ; culture until the 8th day, Day 9: Add 100IU/mL TNF-ɑ to each well, shake gently; continue to culture at 37°C and 5% CO 2 ; culture until the 10th day, separate PBMC cells from another person Afterwards, CD4+T cells were sorted out by magnetic bead sorting, and the density was adjusted to 4x10 6 cell/mL with MLR medium. Dilute HX016-2 to 40 μg/mL with MLR co-cultivation medium, and then carry out 10-fold serial dilution, and set 5 concentration gradients in total (the specific concentrations are 40, 4, 0.4, 0.04, 0.004 μg/mL). Blow down the DC, adjust the cell density to 2x10 5 /mL with MLR co-cultivation medium, spread 100 μL of DC and 50 μL of T cells into a flat-bottomed 96-well plate, add 50 μL of different concentrations of HX016-2 diluted samples to each well, Cultured at 37°C and 5% CO 2 for 5 days, collected the supernatant after 5 days and detected IFN-γ with a CBA kit.
CBA检测细胞因子，V底96孔板中每孔加入50μL原每孔中的细胞上清液(各含有待测物10、1、0.1、0.01、0.001μg/mL)，每孔再加入50μL混合捕获微球，置于室温孵育1小时后，每孔中再加入50μL 用PE标记的检测抗体置于室温孵育2h后，用200μL洗液清洗样本1次后重悬，仪器调试校验完毕后上机检测。样本收集完毕(FCS 2.0格式)后，用CBA专业分析软件FCAP Array v1.0进行标准曲线绘制及数据分析。To detect cytokines by CBA, add 50 μL of the original cell supernatant in each well of the V-bottom 96-well plate (each containing 10, 1, 0.1, 0.01, 0.001 μg/mL of the analyte), and add 50 μL to each well to mix Capture the microspheres, incubate at room temperature for 1 hour, add 50 μL of PE-labeled detection antibody to each well and incubate at room temperature for 2 hours, wash the sample with 200 μL of washing solution once, resuspend, and install the instrument after debugging and calibration. machine detection. After the samples are collected (FCS 2.0 format), use the CBA professional analysis software FCAP Array v1.0 to draw the standard curve and analyze the data.
通过CBA方法检测MLR体系中IFN-γ的分泌水平来判断HX016-2能否阻断DC对T细胞的抑制，具体结果如图10所示，随着HX016-2剂量的增加，IFN-γ的浓度升高，存在剂量依赖效应；在HX016-2待测浓度为1ug/mL的浓度时，HX016-2和HX008的IFN-γ分泌水平相近，且在10ug/mL的浓度时，HX016-2的IFN-γ分泌水平比HX008的IFN-γ分泌水平高。The secretion level of IFN-γ in the MLR system was detected by the CBA method to determine whether HX016-2 could block the inhibition of DCs on T cells. The concentration increases, and there is a dose-dependent effect; when the tested concentration of HX016-2 is 1ug/mL, the IFN-γ secretion levels of HX016-2 and HX008 are similar, and at the concentration of 10ug/mL, the IFN-γ secretion level of HX016-2 The secretion level of IFN-γ was higher than that of HX008.
实施例9 应用细胞模型评价重组抗体分子的体外结合活性Example 9 Application of Cell Model to Evaluate the In Vitro Binding Activity of Recombinant Antibody Molecules
1、应用SEB细胞模型评价重组抗体分子的体外结合活性1. Using the SEB cell model to evaluate the in vitro binding activity of recombinant antibody molecules
HX016-2是一种抗PD-1/VEGF双特异性的免疫抗体。SEB(Staphylococcal enterotoxin B，葡萄球菌肠毒素B)是一种超抗原，极低的浓度就可以激活T细胞，能够刺激PBMC(Peripheral BloodMononuclear Cell，外周血单核细胞)中的T细胞持续性激活，使T细胞处于一种耗竭的状态，免疫系统功能下降。本实施例主要验证HX016-2在SEB的刺激体系下能否通过再次激活T细胞来增强免疫系统的能力。利用CBA检测IL-2的分泌水平以确定HX016-2的PD-1端的生物学活性，具体操作如下：HX016-2 is an anti-PD-1/VEGF bispecific immune antibody. SEB (Staphylococcal enterotoxin B, staphylococcal enterotoxin B) is a superantigen that can activate T cells at a very low concentration, and can stimulate the continuous activation of T cells in PBMC (Peripheral Blood Mononuclear Cell, peripheral blood mononuclear cells), The T cells are in a state of exhaustion, and the function of the immune system is reduced. This example mainly verifies whether HX016-2 can enhance the ability of the immune system by reactivating T cells under the stimulation system of SEB. Use CBA to detect the secretion level of IL-2 to determine the biological activity of the PD-1 end of HX016-2, the specific operation is as follows:
分离一个人的PBMC细胞后用PBMC完全培养基重悬，SEB浓度稀释至100ng/mL，PBMC细胞悬液于450xg条件下离心处理10min，用100ng/mL SEB重悬细胞至密度为2x10 6cell/mL，平底的96孔板中每孔加入100μL。 After isolating a human PBMC cell, resuspend it with PBMC complete medium, dilute the SEB concentration to 100ng/mL, centrifuge the PBMC cell suspension at 450xg for 10min, and resuspend the cell with 100ng/mL SEB to a density of 2x10 6 cell/ mL, add 100 μL to each well of a flat-bottomed 96-well plate.
用PBMC完全培养基将HX016-2浓度稀释到20μg/mL，然后依次进行10倍梯度稀释，共设6个浓度梯度(浓度为20000，2000，200，20，2，0.2ng/mL)。每孔中加入不同浓度HX016-2稀释样本100μL，于37℃、5％CO 2条件下培养3天，3天后收集上清用CBA试剂盒检测IFN-r。 The concentration of HX016-2 was diluted to 20 μg/mL with complete PBMC medium, and then 10-fold serial dilution was performed successively, and a total of 6 concentration gradients (20000, 2000, 200, 20, 2, 0.2 ng/mL) were established. Add different concentrations of HX016-2 to each well to dilute 100 μL of samples, and culture them at 37°C and 5% CO 2 for 3 days. After 3 days, collect the supernatant and use the CBA kit to detect IFN-r.
CBA检测细胞因子：样本稀释好之后，V底96孔板中每孔加入50μL和加入50μL混合捕获微球，室温孵育1小时。加入50μL/孔PE标记的检测抗体，室温孵育2小时。用200μL洗液清洗样本一次后重悬，仪器调试校验完毕后上机检测；样本收集完毕(FCS 2.0格式)后，用CBA专业分析软件FCAP Array v1.0进行标准曲线绘制及数据分析。CBA detection of cytokines: After the sample is diluted, add 50 μL and 50 μL of mixed capture microspheres to each well of the V-bottom 96-well plate, and incubate at room temperature for 1 hour. Add 50 μL/well PE-labeled detection antibody and incubate at room temperature for 2 hours. Wash the sample once with 200 μL of washing solution and resuspend it. After the instrument is debugged and verified, it is tested on the machine; after the sample is collected (FCS 2.0 format), the standard curve is drawn and data analysis is performed with the CBA professional analysis software FCAP Array v1.0.
通过CBA方法检测HX016-2对SEB刺激的PBMC中IL-2的分泌水平，具体结果如图11所示，随着HX016-2剂量的增加，IL-2的浓度升高，存在剂量依赖效应，且在10ug/mL的浓度时，HX016-2与HX008的IL-2分泌水平基本一致。The secretion level of IL-2 in PBMC stimulated by HX016-2 was detected by CBA method. The specific results are shown in Figure 11. With the increase of the dose of HX016-2, the concentration of IL-2 increased, and there was a dose-dependent effect. And at the concentration of 10ug/mL, the IL-2 secretion levels of HX016-2 and HX008 were basically the same.
2、应用HUVEC细胞模型评价重组抗体分子的体外结合活性2. Using HUVEC cell model to evaluate the in vitro binding activity of recombinant antibody molecules
HX016是一种抗PD-1/VEGF双特异性的免疫抗体，人脐静脉内皮细胞(Human Umbilical Vein  Endothelial Cells，HUVEC)在进行血管内皮细胞实验时，通常选用其为细胞模型，VEGF(血管血管内皮生长因子)可以介导的HUVEC细胞增殖。本试验采用HUVEC细胞作为实验细胞，检测HX016-2和HX016-6是否能够能够抑制VEGF介导的HUVEC细胞增殖，具体操作如下：HX016 is an anti-PD-1/VEGF bispecific immune antibody. Human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cells, HUVEC) are usually used as cell models when performing vascular endothelial cell experiments. VEGF (vascular Endothelial growth factor) can mediate the proliferation of HUVEC cells. In this experiment, HUVEC cells are used as experimental cells to detect whether HX016-2 and HX016-6 can inhibit VEGF-mediated HUVEC cell proliferation. The specific operations are as follows:
HUVEC细胞用接种培养基将细胞浓度调整为5×10 4细胞/mL，混合均匀。按照每孔100μL的体积用排枪将细胞悬液均匀加入96孔板中，置于37℃、5％CO 2条件下培养18～24h。 Adjust the cell concentration to 5×10 4 cells/mL with inoculation medium for HUVEC cells, and mix well. According to the volume of 100 μL per well, the cell suspension was evenly added to the 96-well plate with a discharge gun, and cultured at 37° C. and 5% CO 2 for 18-24 hours.
用稀释培养基将HX016-2和HX016-6分别先稀释到20ug/mL，在此基础上进行5倍稀释、在前述基础上继续进行5倍稀释，以此类推，继续进行5倍、5倍、2倍、2倍、4倍稀释，共得到8个梯度。用稀释培养基将rhVEGF165稀释到100ng/mL。Dilute HX016-2 and HX016-6 to 20ug/mL with dilution medium first, then perform 5-fold dilution on this basis, continue to perform 5-fold dilution on the previous basis, and so on, continue to 5-fold, 5-fold , 2 times, 2 times, 4 times dilution, a total of 8 gradients were obtained. Dilute rhVEGF165 to 100 ng/mL with dilution medium.
将HX016-2和HX016-6分别和100ng/mL的rhVEGF165单抗按1：1的体积比进行混合，随行rhVEGF165单独孔，置于37℃培养箱中孵育2h。将细胞培养板取出，按100μL/孔的量小心吸出细胞上清，再将孵育过的样品溶液按每孔100μL加入到细胞板中，随行空白孔，于37℃培养箱中孵育70h～72h；孵育结束后，从培养箱中取出细胞板，每孔加入10μL荧光试剂，于37℃孵育4h后用多功能酶标仪检测，激发波长570nm，发射波长585nm。HX016-2 and HX016-6 were mixed with 100ng/mL rhVEGF165 monoclonal antibody at a volume ratio of 1:1, followed by individual rhVEGF165 wells, and incubated in a 37°C incubator for 2h. Take out the cell culture plate, carefully suck out the cell supernatant according to the amount of 100 μL/well, then add the incubated sample solution into the cell plate at 100 μL per well, follow the blank well, and incubate in a 37°C incubator for 70h to 72h; After the incubation, the cell plate was taken out from the incubator, 10 μL of fluorescent reagent was added to each well, incubated at 37°C for 4 hours, and then detected with a multi-functional microplate reader, the excitation wavelength was 570 nm, and the emission wavelength was 585 nm.
检测HX016-2和HX016-6是否能够能够抑制VEGF介导的HUVEC细胞增殖，具体结果如图12所示，HX016-2抑制HUVEC细胞增殖实验的EC50为2940ng/mL，HX016-6抑制HUVEC细胞增殖实验的EC50为4620ng/mL。与HX006相比，活性较弱，HX016-2比HX016-6活性强。Detect whether HX016-2 and HX016-6 can inhibit VEGF-mediated HUVEC cell proliferation, the specific results are shown in Figure 12, the EC50 of HX016-2 inhibiting HUVEC cell proliferation experiment is 2940ng/mL, HX016-6 inhibits HUVEC cell proliferation The EC50 of the experiment was 4620ng/mL. Compared with HX006, the activity is weaker, and HX016-2 is more active than HX016-6.
实施例10 重组抗体的体内验证实验Example 10 In vivo verification experiment of recombinant antibody
本实验研究不同测试药物在免疫检查点人源化小鼠BALB/c-hPD1皮下接种小鼠结肠癌细胞CT26.WT肿瘤模型中的抗肿瘤作用，具体操作如下：This experiment studies the anti-tumor effect of different test drugs in the immune checkpoint humanized mouse BALB/c-hPD1 subcutaneously inoculated mouse colon cancer cell CT26.WT tumor model, the specific operation is as follows:
将小鼠结肠癌CT26.WT细胞复苏，并收集对数生长期的CT26.WT细胞，去除培养液并用PBS洗两次后接种至54只小鼠体内，接种量为：5×10 5cells/100μL/只。接种后第11天，平均肿瘤体积达到79.53mm 3时，将54只小鼠根据肿瘤体积随机分成9组，每组6只。分组当天定义为D0天，并于D0天开始给药，给药按照图中所述剂量一周两次，共给药7次。开始给药后，于D0、D3、D6、D9、D13、D15、D17、D20、D22、D24、D26天观测肿瘤大小并称量小鼠体重。具体结果如表1所示，瘤体积计算方式为：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)，表1中展示的为瘤体积平均值+标准差(每组n＝6)。 Mouse colon cancer CT26.WT cells were revived, and the CT26.WT cells in the logarithmic growth phase were collected, the culture medium was removed, washed twice with PBS, and then inoculated into 54 mice at an inoculation volume of 5×10 5 cells/ 100μL/piece. On the 11th day after inoculation, when the average tumor volume reached 79.53 mm 3 , 54 mice were randomly divided into 9 groups according to the tumor volume, 6 mice in each group. The day of grouping was defined as D0 day, and the administration started on D0 day, and the administration was given twice a week according to the dose mentioned in the figure, and a total of 7 administrations were administered. After the start of administration, the tumor size was observed and the body weight of the mice was weighed on days D0, D3, D6, D9, D13, D15, D17, D20, D22, D24 and D26. The specific results are shown in Table 1. The calculation method of tumor volume is: tumor volume (mm 3 )=0.5×(tumor long diameter×tumor short diameter 2 ), and the tumor volume average value + standard deviation (in each group) is shown in Table 1. n=6).
由图13和表1可以看出，在当前的测试系统下，测试药HX006(5mpk)对小鼠肿瘤体积及终点小鼠瘤重均未表现出明显的肿瘤抑制效果；R1D2(5mpk)、HX016-2(5mpk)对终点小鼠瘤重有抑制作用；Keytruda(10mpk)、HX008(5mpk)、HX016-6(5mpk)对小鼠肿瘤体积和终点小鼠瘤重均有的抑制作用， 但观测时间不同，显著性存在差异；G3组HX008(10mpk)、G7组HX016-2(5mpk)对小鼠肿瘤体积和终点小鼠瘤重均具有显著抑制作用。It can be seen from Figure 13 and Table 1 that under the current test system, the test drug HX006 (5mpk) did not show significant tumor inhibitory effect on the tumor volume and endpoint mouse tumor weight; R1D2 (5mpk), HX016 -2 (5mpk) has inhibitory effect on end-point mouse tumor weight; Keytruda (10mpk), HX008 (5mpk), HX016-6 (5mpk) have inhibitory effect on mouse tumor volume and end-point mouse tumor weight, but observed There are differences in significance at different times; HX008 (10mpk) in G3 group and HX016-2 (5mpk) in G7 group have significant inhibitory effects on the tumor volume of mice and the tumor weight of end mice.
表1:Table 1:
Figure PCTCN2021132785-appb-000065
Figure PCTCN2021132785-appb-000065
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the described specific features, structures, materials or characteristics may be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples and features of different embodiments or examples described in this specification without conflicting with each other.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and those skilled in the art can make the above-mentioned The embodiments are subject to changes, modifications, substitutions and variations.

Claims (17)

  1. 一种重组抗体，其特征在于，包括：A recombinant antibody, characterized in that it comprises:
    抗PD-1的抗体，所述抗PD-1的抗体包含重链和轻链；以及an anti-PD-1 antibody comprising a heavy chain and a light chain; and
    人抗VEGF的抗体、人抗VEGFR的抗体或人VEGF诱捕蛋白，Human anti-VEGF antibody, human anti-VEGFR antibody or human VEGF Trap,
    所述人抗VEGF的抗体的N端与所述抗PD-1的抗体的重链的C端相连；或The N-terminal of the human anti-VEGF antibody is connected to the C-terminal of the heavy chain of the anti-PD-1 antibody; or
    所述人VEGF诱捕蛋白的N端与所述抗PD-1的抗体的重链的C端相连或所述人VEGF诱捕蛋白的C端与所述抗PD-1的抗体的重链的N端相连；或The N-terminal of the human VEGF-trap protein is connected to the C-terminal of the heavy chain of the anti-PD-1 antibody or the C-terminal of the human VEGF-trap protein is connected to the N-terminal of the heavy chain of the anti-PD-1 antibody connected; or
    所述人抗VEGFR的抗体的N端与所述抗PD-1的抗体的重链的C端相连。The N-terminal of the human anti-VEGFR antibody is connected to the C-terminal of the heavy chain of the anti-PD-1 antibody.
  2. 根据权利要求1所述的重组抗体，其特征在于，所述人抗VEGFR的抗体包含Ramucirumab单链抗体；The recombinant antibody according to claim 1, wherein the human anti-VEGFR antibody comprises a Ramucirumab single-chain antibody;
    任选地，所述人VEGF诱捕蛋白包含VEGFR1D2或VEGFR2D3。Optionally, the human VEGF Trap comprises VEGFR1D2 or VEGFR2D3.
  3. 根据权利要求1所述的重组抗体，其特征在于，所述抗PD-1的抗体为抗PD-1的IgG类抗体，The recombinant antibody according to claim 1, wherein the anti-PD-1 antibody is an anti-PD-1 IgG antibody,
    任选地，进一步包括连接肽，所述连接肽的N端与所述抗PD-1的抗体的重链的C端相连，所述连接肽的C端与所述人抗VEGF的抗体的N端相连；Optionally, a connecting peptide is further included, the N-terminal of the connecting peptide is connected to the C-terminal of the heavy chain of the anti-PD-1 antibody, and the C-terminal of the connecting peptide is connected to the N-terminal of the human anti-VEGF antibody. end connected;
    任选地，所述连接肽的N端与所述抗PD-1的抗体的重链的C端相连，所述连接肽的C端与所述Ramucirumab单链抗体或VEGFR1D2的N端相连；Optionally, the N-terminus of the connecting peptide is connected to the C-terminus of the heavy chain of the anti-PD-1 antibody, and the C-terminus of the connecting peptide is connected to the N-terminus of the Ramucirumab single-chain antibody or VEGFR1D2;
    任选地，所述连接肽的N端与所述VEGFR1D2的C端相连，所述连接肽的C端与所述抗PD-1的抗体的重链的N端相连；Optionally, the N-terminus of the connecting peptide is connected to the C-terminus of the VEGFR1D2, and the C-terminus of the connecting peptide is connected to the N-terminus of the heavy chain of the anti-PD-1 antibody;
    任选地，所述连接肽的C端与所述抗PD-1的抗体的重链的N端相连，所述连接肽的N端与所述VEGFR2D3的C端相连，所述VEGFR2D3的N端与所述VEGFR1D2的C端相连；Optionally, the C-terminal of the connecting peptide is connected to the N-terminal of the heavy chain of the anti-PD-1 antibody, the N-terminal of the connecting peptide is connected to the C-terminal of the VEGFR2D3, and the N-terminal of the VEGFR2D3 connected to the C-terminus of the VEGFR1D2;
    任选地，所述连接肽的N端与所述抗PD-1的抗体的重链的C端相连，所述连接肽的C端与所述VEGFR1D2的N端相连，所述VEGFR1D2的C端与所述VEGFR2D3的N端相连；Optionally, the N-terminus of the connecting peptide is connected to the C-terminus of the heavy chain of the anti-PD-1 antibody, the C-terminus of the connecting peptide is connected to the N-terminus of the VEGFR1D2, and the C-terminus of the VEGFR1D2 connected to the N-terminus of the VEGFR2D3;
    任选地，所述连接肽具有SEQ ID NO：1所示的氨基酸序列。Optionally, the connecting peptide has the amino acid sequence shown in SEQ ID NO: 1.
  4. 一种重组抗体，其特征在于，所述重组抗体的轻链具有SEQ ID NO：2所示的氨基酸序列，所述重组抗体的重链选自SEQ ID NO：3-6、SEQ ID NO：23和SEQ ID NO：25所示的氨基酸序列的至少之一。A recombinant antibody, characterized in that the light chain of the recombinant antibody has the amino acid sequence shown in SEQ ID NO: 2, and the heavy chain of the recombinant antibody is selected from SEQ ID NO: 3-6, SEQ ID NO: 23 and at least one of the amino acid sequences shown in SEQ ID NO:25.
  5. 一种核酸，其特征在于，所述核酸编码权利要求1～4任一项所述的重组抗体。A nucleic acid, characterized in that the nucleic acid encodes the recombinant antibody according to any one of claims 1-4.
  6. 根据权利要求5所述的核酸，其特征在于，所述核酸具有SEQ ID NO：7所示的核苷酸序列，所述核酸进一步包括SEQ ID NO：8-11、SEQ ID NO：27和SEQ ID NO：28所示的核苷酸序列的至少之一。The nucleic acid according to claim 5, wherein the nucleic acid has a nucleotide sequence shown in SEQ ID NO: 7, and the nucleic acid further comprises SEQ ID NO: 8-11, SEQ ID NO: 27 and SEQ ID NO: 27 and SEQ ID NO: ID NO: at least one of the nucleotide sequences shown in 28.
  7. 一种表达载体，其特征在于，所述表达载体包括：An expression vector, characterized in that, the expression vector comprises:
    第一核酸分子，所述第一核酸分子编码抗PD-1的抗体轻链；A first nucleic acid molecule encoding an anti-PD-1 antibody light chain;
    第二核酸分子，所述第二核酸分子编码抗PD-1的抗体重链；a second nucleic acid molecule encoding an anti-PD-1 antibody heavy chain;
    第三核酸分子，所述第三核酸分子编码人抗VEGF的抗体、抗VEGFR的抗体或VEGF诱捕蛋白。A third nucleic acid molecule, the third nucleic acid molecule encodes a human anti-VEGF antibody, an anti-VEGFR antibody or a VEGF trap protein.
  8. 根据权利要求7所述的表达载体，其特征在于，进一步包括：The expression vector according to claim 7, further comprising:
    第四核酸分子，所述第四核酸分子设置在所述第二核酸分子与所述第三核酸分子之间，并且所述第四核酸分子编码连接肽，a fourth nucleic acid molecule, said fourth nucleic acid molecule being disposed between said second nucleic acid molecule and said third nucleic acid molecule, and said fourth nucleic acid molecule encoding a connecting peptide,
    任选地，所述第一核酸分子具有SEQ ID NO：7所示的核苷酸序列，Optionally, the first nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 7,
    任选地，所述第二核酸分子具有SEQ ID NO：13所示的核苷酸序列，Optionally, the second nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 13,
    任选地，所述第三核酸分子具有SEQ ID NO：14、15、16或29所示的核苷酸序列，Optionally, the third nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 14, 15, 16 or 29,
    任选地，所述第四核酸分子具有SEQ ID NO：17所示的核苷酸序列；Optionally, the fourth nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 17;
    任选地，所述表达载体包括慢病毒、腺病毒、逆转录病毒。Optionally, the expression vector includes lentivirus, adenovirus, retrovirus.
  9. 一种制备权利要求1～4任一项所述的重组抗体的方法，其特征在于，包括：A method for preparing the recombinant antibody according to any one of claims 1 to 4, characterized in that it comprises:
    将权利要求7或8所述的表达载体引入到真核细胞中；Introducing the expression vector according to claim 7 or 8 into eukaryotic cells;
    将所述真核细胞在适于蛋白表达和分泌的条件下进行培养，以便获得所述重组抗体。The eukaryotic cells are cultured under conditions suitable for protein expression and secretion, so as to obtain the recombinant antibody.
  10. 根据权利要求9所述的方法，其特征在于，所述真核细胞为哺乳动物细胞，The method according to claim 9, wherein the eukaryotic cells are mammalian cells,
    任选地，所述哺乳动物细胞包括选自CHO-K1、CHOS、293F、293T、CAR-T、TCR-T、TIL、CAR-NK、CAR-M和CAR-DC中的至少之一。Optionally, the mammalian cells include at least one selected from CHO-K1, CHOS, 293F, 293T, CAR-T, TCR-T, TIL, CAR-NK, CAR-M and CAR-DC.
  11. 一种重组细胞，其特征在于，所述重组细胞携带权利要求5或6所述的核酸，或权利要求7或8所述的表达载体。A recombinant cell, characterized in that the recombinant cell carries the nucleic acid according to claim 5 or 6, or the expression vector according to claim 7 or 8.
  12. 一种用于治疗癌症的组合物，其特征在于，包括：A composition for treating cancer, characterized in that it comprises:
    权利要求1～4任一项所述的重组抗体、权利要求5或6所述的核酸、权利要求7或8所述的表达载体或权利要求11所述的重组细胞。The recombinant antibody of any one of claims 1-4, the nucleic acid of claim 5 or 6, the expression vector of claim 7 or 8, or the recombinant cell of claim 11.
  13. 根据权利要求12所述的组合物，其特征在于，所述癌症包括选自肺癌，胃癌，肝癌，肠癌，食管癌，乳腺癌，宫颈癌，恶性淋巴瘤，鼻咽癌，白血病，皮肤癌和肾癌中的至少之一。The composition according to claim 12, wherein the cancer comprises cancers selected from lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, leukemia, skin cancer and at least one of kidney cancer.
  14. 权利要求1～4任一项所述的重组抗体、权利要求5或6所述的核酸、权利要求7或8所述的表达载体、权利要求11所述的重组细胞或权利要求12或13所述的组合物在制备药物中的用途，所述药物用于治疗或者预防癌症。The recombinant antibody of any one of claims 1 to 4, the nucleic acid of claim 5 or 6, the expression vector of claim 7 or 8, the recombinant cell of claim 11 or the nucleic acid of claim 12 or 13 Use of the composition in the preparation of medicaments for treating or preventing cancer.
  15. 根据权利要求14所述的用途，其特征在于，所述癌症包括选自肺癌，胃癌，肝癌，肠癌，食管癌，乳腺癌，宫颈癌，恶性淋巴瘤，鼻咽癌，白血病，皮肤癌和肾癌的中至少之一。The use according to claim 14, wherein the cancer comprises cancers selected from lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, leukemia, skin cancer and At least one of kidney cancer.
  16. 一种预防或治疗癌症的方法，其特征在于，包括向受试者施用有效剂量的以下中的至少之一：A method for preventing or treating cancer, comprising administering to a subject an effective dose of at least one of the following:
    权利要求1～4任一项所述的重组抗体；The recombinant antibody according to any one of claims 1 to 4;
    权利要求5或6所述的核酸；the nucleic acid of claim 5 or 6;
    权利要求7或8所述的表达载体；The expression vector according to claim 7 or 8;
    权利要求11所述的重组细胞；以及The recombinant cell of claim 11; and
    权利要求12或13所述的组合物。The composition of claim 12 or 13.
  17. 根据权利要求16所述的方法，其特征在于，所述癌症包括选自肺癌，胃癌，肝癌，肠癌，食管癌，乳腺癌，宫颈癌，恶性淋巴瘤，鼻咽癌，白血病，皮肤癌和肾癌中的至少之一。The method according to claim 16, wherein the cancer comprises cancers selected from lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, leukemia, skin cancer and At least one of the kidney cancers.
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