WO2017177907A1 - Vaccins contre les tumeurs pd-l1 et pd-l2 de point de contrôle immunitaire - Google Patents

Vaccins contre les tumeurs pd-l1 et pd-l2 de point de contrôle immunitaire Download PDF

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WO2017177907A1
WO2017177907A1 PCT/CN2017/080143 CN2017080143W WO2017177907A1 WO 2017177907 A1 WO2017177907 A1 WO 2017177907A1 CN 2017080143 W CN2017080143 W CN 2017080143W WO 2017177907 A1 WO2017177907 A1 WO 2017177907A1
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cell
recombinant protein
patient
cells
construct
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陈思毅
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北京普纳生物科技有限公司
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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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
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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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • 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
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of bioengineering, and in particular, to recombinant proteins and uses thereof.
  • Targeted therapies mainly include monoclonal antibodies (sometimes classified as passive cell transfusion and tumor vaccines, immunotherapy through the immune system of the motivational body, enhanced tumor microenvironment anti-tumor immunotherapy) and small molecule targeted drugs, while immunotherapy It mainly includes cytokine therapy, immunoassay monoclonal antibody, and adoptive immunotherapy to control and kill tumor cells. Therefore, it has the advantages of high efficiency, high specificity and good tolerance, and has broad prospects in cancer therapy.
  • Tumor immunotherapy vaccines mainly include tumor cell vaccines, dendritic cell (DC cell) vaccines, protein & peptide vaccines, nucleic acid vaccines, genetically engineered vaccines and anti-idiotype tumor vaccines.
  • the main mechanism by which these vaccines can kill tumors is through The patient is directed against a tumor-specific antigen immune response, including antibody response and cytotoxic T lymphocyte (CTL) specific killing.
  • CTL cytotoxic T lymphocyte
  • tumor vaccines target tumor-associated antigens, and their clinical efficacy is weak, and further research and development are needed to enhance clinical efficacy.
  • Tumor cells highly express immune checkpoint molecules PD-L1 or PD-L2, and activated cytotoxic T lymphocytes on the surface
  • PD-1 which inhibits the T lymphocyte response of the tumor, escapes the immune killing of cytotoxic T lymphocytes.
  • CTL immune cells-cytotoxic T lymphocytes
  • an object of the present invention is to provide a recombinant PD-L1 protein having an immune response which causes a tumor-specific antigen, which is actively stimulated to produce an anti-PD-L1 antibody in a patient, and the patient has been mobilized.
  • the presence of spontaneously induced immune cell CTLs is induced and stimulates the production of new anti-PD-L1 CTLs, which in turn specifically kill tumor cells.
  • the active immune killing effect on tumor cells caused by the recombinant protein proposed by the invention is remarkable.
  • the invention proposes a recombinant protein.
  • the recombinant protein comprises: an immunological checkpoint molecule fragment; a helper T cell epitope fragment; and an immunostimulatory molecule fragment.
  • the recombinant protein proposed in the embodiments of the present invention can continuously stimulate the production of anti-immunization checkpoint antibodies in vivo, mobilize the spontaneously induced immune cell CTLs existing in the body, and stimulate the production of CTLs against the immune checkpoints, thereby specifically killing the tumor cells. .
  • the active immune killing effect on tumor cells caused by the recombinant protein proposed by the embodiments of the present invention is remarkable.
  • the recombinant protein may further comprise at least one of the following additional technical features:
  • the immune checkpoint molecule is PD-L1 or PD-L2.
  • PD-L1 or PD-L2 is specifically expressed in tumor cells, and the specificity of the tumor antigen immune response caused by the recombinant protein proposed in the examples of the present invention is stronger.
  • the immunological checkpoint molecule fragment is an extracellular molecular fragment of the PD-L1 or PD-L2 removal transmembrane region.
  • the extracellular molecular fragment of PD-L1 or PD-L2 in the transmembrane region has only tumor antigenicity and does not have tumor immunosuppressive function, and thus the extracellular molecular fragment of PD-L1 or PD-L2 in the transmembrane region can be further removed.
  • the specificity is further improved.
  • the helper T cell epitope is a broad spectrum PADRE helper T cell epitope.
  • Broad-spectrum PADRE-assisted T cell epitopes can effectively activate helper T cells, thereby further enhancing the specific killing of cytotoxic T lymphocytes (CTLs) caused by recombinant proteins.
  • CTLs cytotoxic T lymphocytes
  • the immunostimulatory molecule is a flagellin.
  • the immunostimulatory molecule has biological activity, can significantly enhance the antigen presenting function of dendritic cells (DC cells) and enhance the activity of cytotoxic T lymphocytes (CTLs) and B lymphocytes, and further the recombinant protein of the embodiments of the present invention can be further To effectively cause tumor antigen immune response.
  • DC cells dendritic cells
  • CTLs cytotoxic T lymphocytes
  • B lymphocytes cytotoxic T lymphocytes
  • the N-terminus of the immunological checkpoint molecule fragment is linked to the C-terminus of the immunostimulatory molecule fragment, and the C-terminus of the immunological checkpoint molecular fragment and the N-terminus of the helper T cell epitope fragment Connected.
  • the related molecular fragment in the recombinant protein of the embodiment of the present invention is beneficial to the tumor antigen PD-L1 or PD-L2 in the above-mentioned linked state.
  • the presentation of the fragment in the DC cell facilitates the function of the helper T cell epitope and the immunostimulatory molecule to activate the immune cell, and the recombinant protein of the embodiment of the present invention can more effectively induce the tumor antigen immune response.
  • the invention proposes a recombinant protein.
  • the recombinant protein has the amino acid sequence of SEQ ID NOS: 1-3.
  • the recombinant protein proposed in the embodiment of the present invention can cause a tumor-specific antigen immune response, and the protein stimulates the production of anti-PD-L1 or PD-L2 antibody in the patient by active immunization, and mobilizes the spontaneous induction of the existing in the patient.
  • the cells are immunized with CTL and stimulated to produce anti-PD-L1 or PD-L2 CTL, thereby specifically killing tumor cells.
  • the active immune killing effect on tumor cells caused by the recombinant protein proposed by the invention is remarkable.
  • the invention proposes a nucleic acid.
  • the nucleic acid encodes the recombinant protein described above, and the nucleic acid has the nucleotide sequence shown in SEQ ID NOS: 4-6.
  • the recombinant protein encoded by the nucleic acid according to the embodiment of the present invention stimulates the production of anti-PD-L1 or PD-L2 antibody in the patient by active immunization, mobilizes the spontaneously induced immune cell CTL which has already existed in the patient, and stimulates the production of anti-drug.
  • PD-L1 or PD-L2CTL which specifically kills tumor cells.
  • the active immune killing effect on tumor cells caused by the recombinant protein proposed by the invention is remarkable.
  • the invention proposes a construct.
  • the construct carries a nucleic acid as described above.
  • the construct introduced into the recipient cell of the present invention can achieve high-efficiency expression of the nucleic acid described above, and thereby efficiently express the recombinant protein described above in the recipient cell.
  • the construct may further comprise at least one of the following additional technical features:
  • the vector of the construct is a pET series vector, a pPIC series vector, a BacPAK, a pSV series vector or a pCMV series vector.
  • the above vector of the embodiment of the present invention can achieve further efficient expression of the above recombinant protein in prokaryotic cells or eukaryotic cells.
  • the invention proposes a construct.
  • the construct carries the following nucleic acid molecule: (1) a nucleic acid molecule encoding a fragment of an immunological checkpoint molecule having the amino acid sequence set forth in SEQ ID NOS: 7-9, The nucleic acid molecule encoding the immunological checkpoint molecule fragment has the nucleotide sequence shown in SEQ ID NOS: 10 to 12; (2) the nucleic acid molecule encoding the helper T cell epitope fragment, the helper T cell epitope The fragment has the amino acid sequence set forth in SEQ ID NO: 13, the nucleic acid molecule encoding the helper T cell epitope fragment having the nucleotide sequence set forth in SEQ ID NO: 14; and (3) encoding the immunostimulatory A nucleic acid molecule of a molecular fragment having the amino acid sequence of SEQ ID NO: 15, the nucleic acid molecule encoding the immunostimulatory molecule fragment having the
  • the construct according to the embodiment of the present invention efficiently expresses a recombinant protein containing an immunological checkpoint molecular fragment, a helper T cell epitope fragment and an immunostimulatory molecule fragment in a recipient cell, and the recombinant protein is actively immunized in a patient. Stimulate the production of anti-PD-L1 antibody, mobilize the spontaneously induced immune cell CTL already existing in the patient, and stimulate the production of anti-PD-L1CTL, thereby specifically killing the tumor cells.
  • the invention provides a transgenic cell.
  • the transgenic cell carries a construct as described above.
  • the transgenic cells proposed in the embodiments of the present invention can express the recombinant protein as described above, and the obtained recombinant protein stimulates an anti-immunological checkpoint, such as PD-L1 or PD-L2 antibody, by active immunization, and mobilizes.
  • an anti-immunological checkpoint such as PD-L1 or PD-L2 antibody
  • Spontaneously induced immune cell CTLs already present in the patient and stimulate the production of anti-immunological checkpoints, such as PD-L1 or PD-L2 CTL, which specifically kill tumor cells.
  • the transgenic cell may further comprise at least one of the following additional technical features:
  • the transgenic cells are BL21, BL21 (DE3), BL21 (DE3) pLysS, DH10B, XL1-Blue, Pichia pastors, Kluyveromyces lactis, Sf9, Sf21, High-Five T, CHO cell line, HEK cell line, Hela cell line or COS cell line.
  • the transgenic cell can efficiently express the recombinant protein described above, and then the recombinant protein obtained by protein purification can be administered to a patient, and the patient can be further effectively stimulated to produce anti-PD in the patient by active immunization.
  • the -L1 or PD-L2 antibody mobilizes spontaneously induced immune cell CTLs already present in the patient and stimulates the production of anti-PD-L1 or PD-L2CTL, thereby specifically killing the tumor cells.
  • the transgenic cell is an antigen presenting cell.
  • the antigen presenting cell is derived from a patient, and the antigen presenting cell carrying the aforementioned construct can be further input into the patient, thereby realizing the continuous expression of the recombinant protein described above in the patient, and further Actively immunizing in the body to produce anti-PD-L1 or PD-L2 antibodies in vivo, mobilizing the spontaneously induced immune cell CTLs already present in the patient, and stimulating the production of anti-PD-L1 or PD-L2CTL, Specific killing of tumor cells.
  • the transgenic cell is a DC cell.
  • the DC cells have an antigen-presenting function, and the DC cells derived from the patient themselves carry the aforementioned construct and are returned to the patient, thereby realizing the high-efficiency expression of the recombinant protein described above in the patient and the tumor antigen PD-L1.
  • PD-L2 is efficiently presented on the surface of DC cells, thereby further effectively stimulating the production of anti-PD-L1 or PD-L2 antibodies, mobilizing the spontaneously induced immune cell CTLs already present in the patient, and stimulating the production of anti-PD-L1 or PD -L2CTL, which further effectively kills tumor cells specifically.
  • the invention provides the use of the aforementioned recombinant protein for the preparation of a medicament,
  • the drug is used to prevent or treat a tumor.
  • the recombinant protein proposed by the embodiments of the present invention can cause a significant tumor-specific antigen immune response in a tumor patient, and effectively stimulates the production of an anti-immunological checkpoint, such as PD-L1 or PD-L2 antibody, to mobilize the spontaneous induction already existing in the patient.
  • the resulting immune cell CTL is stimulated to produce an anti-immunological checkpoint, such as PD-L1 or PD-L2CTL, which effectively kills the tumor cells specifically.
  • the recombinant protein proposed in the examples of the present invention has utility in the preparation of a medicament effective for preventing or treating a tumor.
  • the invention provides the use of a recombinant protein as described above for the preparation of a vaccine for the prevention or treatment of a tumor.
  • the recombinant protein proposed by the embodiments of the present invention can cause a significant tumor-specific antigen immune response in a tumor patient, and effectively stimulates the production of an anti-immunological checkpoint, such as PD-L1 or PD-L2 antibody, to mobilize the spontaneous induction already existing in the patient.
  • the resulting immune cell CTL is stimulated to produce an anti-immunological checkpoint, such as PD-L1 or PD-L2CTL, which effectively kills the tumor cells specifically.
  • the recombinant protein proposed in the examples of the present invention has a use in preparing a vaccine effective for preventing or treating a tumor.
  • the invention provides the use of a recombinant protein as described above for the preparation of a vaccine for the treatment of a viral infection.
  • the inventors have found that HBV, HCV, HIV, EBV virus-infected cells express PD-L1, and the vaccine prepared by the recombinant protein of the present invention can stimulate anti-PD-L1 in a patient.
  • the antibody mobilizes spontaneously induced immune cell CTLs already present in the patient and stimulates the production of anti-immunological checkpoints, such as PD-L1 CTL, to effectively kill cells infected with the above virus.
  • the invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises: the recombinant protein described above; and a pharmaceutically acceptable adjuvant.
  • the recombinant protein in the pharmaceutical composition proposed in the examples of the present invention can cause a significant specific antigen immune response, and the function of the adjuvant to enhance the immune response.
  • the pharmaceutical composition provided by the embodiment of the present invention effectively stimulates an anti-immunization checkpoint, such as a PD-L1 or PD-L2 antibody, to mobilize an already existing spontaneously induced immune cell in a patient. CTL, and stimulate the production of anti-immunological checkpoints, such as PD-L1 or PD-L2CTL, to effectively kill tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
  • viruses HBV, HCV, HIV, EBV
  • the invention proposes a DC cell.
  • the DC cells are loaded with the recombinant protein described above.
  • the DC cells proposed in the embodiments of the present invention can present antigens in the recombinant protein (such as the immunological checkpoint molecular fragment described above), helper T cell epitope fragments, and immunostimulatory molecule fragments, respectively.
  • anti-immunological checkpoints such as PD-L1 or PD-L2 antibodies
  • mobilizes spontaneously induced immune cell CTLs already present in patients and stimulates the production of anti-immune checkpoints such as PD-L1 or PD-L2CTL, in turn, effectively kills tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
  • viruses HBV, HCV, HIV, EBV
  • the invention provides a targeted immune cell population.
  • the targeted immune cell population is obtained by co-culture of DC cells with lymphocytes as described above. According to this issue
  • the targeted immune cell population proposed in the embodiments of the present invention can specifically kill tumor cells, secrete antibodies that specifically bind tumor antigens, and achieve specific removal of tumor cells.
  • the invention proposes a vaccine.
  • the vaccine comprises a recombinant protein as described above, a DC cell as described above or a targeted immune cell population as described above.
  • the recombinant protein, DC cell, and targeted immune cell population proposed in the examples of the present invention can cause a significant specific antigen immune response in a patient.
  • the vaccine according to the embodiment of the present invention can effectively stimulate the production of an anti-immunization checkpoint, such as a PD-L1 or PD-L2 antibody, mobilize the spontaneously induced immune cell CTL already existing in the patient, and stimulate An anti-immunization checkpoint, such as PD-L1 or PD-L2CTL, is generated to effectively kill tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
  • an anti-immunization checkpoint such as a PD-L1 or PD-L2 antibody
  • the invention provides an antibody.
  • the antibody specifically recognizes the recombinant protein described above, and the antibody proposed in the embodiment of the present invention specifically recognizes a tumor antigen.
  • the present invention finds that the antibody specifically recognizes an antigen, specifically binds to a tumor cell or a cell infected by a virus (HBV, HCV, HIV, EBV), thereby causing a tumor cell or a virus (HBV) Cells infected with HCV, HIV, EBV) are engulfed by phagocytic cells to achieve specific clearance of tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
  • a virus HCV, HCV, HIV, EBV
  • the invention provides a method of preparing an antibody.
  • the method comprises: immunizing an animal with the recombinant protein described above; collecting serum of the immunized animal; and purifying the antibody of interest from the serum.
  • the method for preparing an antibody proposed in the embodiments of the present invention is simple and convenient, and the antibody can specifically recognize the recombinant protein.
  • the invention provides a therapeutic composition.
  • the therapeutic composition comprises: a recombinant protein as described above, a nucleic acid as described above, a construct as described above, a transgenic cell as described above, a pharmaceutical composition as described above, a front The DC cells, the aforementioned targeted immune cell population, the vaccine described above or the antibodies described above.
  • the therapeutic composition proposed by the embodiments of the present invention can directly or indirectly cause a specific antigen immune response, and achieve specificity to tumor cells or cells infected by viruses (HBV, HCV, HIV, EBV). Kill and clear.
  • the invention provides a method of stimulating anti-PD-L1 antibody production or cytotoxic T lymphocyte response in a patient.
  • the method is achieved by at least one of the following: 1) the recombinant protein described above is co-cultured with DC cells taken from a patient, and the DC of the recombinant protein described above is loaded The cells are returned to the patient; 2) the patient is administered the pharmaceutical composition described above; 3) the previously described construct is introduced into the DC cells taken from the patient, and the DC cells introduced into the construct are returned to the patient. In vivo; and 4) administering to the patient a construct as described above.
  • the manner proposed in the examples of the present invention can significantly stimulate anti-PD-L1 antibody production or cytotoxic T lymphocyte reaction in a patient.
  • the invention provides a method of treating cancer. According to an embodiment of the present invention, it is achieved by at least one of the following methods: 1) the recombinant protein described above is co-cultured with DC cells taken from a cancer patient, and the DC cells loaded with the recombinant protein described above are returned. Loss to a cancer patient; 2) administration of the aforementioned pharmaceutical composition to a cancer patient; 3) introduction of the aforementioned construct into DC cells taken from a cancer patient, and returning DC cells introduced into the construct to In vivo in a cancer patient; and 4) administration of the aforementioned construct to a cancer patient.
  • the method proposed by the embodiments of the present invention can directly or indirectly cause a specific antigen immune response, and achieve specific killing and elimination of tumor cells.
  • the invention provides a method of treating a patient infected with a virus.
  • it is achieved by at least one of the following methods: 1) the recombinant protein described above is co-cultured with DC cells taken from a patient, and the DC cells loaded with the recombinant protein described above are returned.
  • the patient is administered the construct described above.
  • the method proposed by the embodiments of the present invention can directly or indirectly cause a specific antigen immune reaction, and achieve specific killing and elimination of cells infected by viruses (HBV, HCV, HIV, EBV), thereby effectively treating patients infected with the virus.
  • the above method may further include at least one of the following additional technical features:
  • the virus comprises at least one selected from the group consisting of HBV, HCV, HIV and EBV. Further effective treatment of patients infected with HBV, HCV, HIV or EBV can be achieved using the above method according to an embodiment of the invention.
  • FIG. 1 is a schematic view showing the structure of a fusion protein (Flagellin-human PD-L1/L2 ⁇ -PADRE Th) according to an embodiment of the present invention
  • fusion protein Flagellin-human PD-L1 ⁇ -PADRE Th
  • a DC vaccine loaded with a fusion protein Flagellin-human PD-L1 ⁇ -PADRE Th capable of significantly inducing an anti-PD-L1 CTL reaction according to an embodiment of the present invention
  • PD-L1 + lung cancer is a DC vaccine loaded with a fusion protein (Flagellin-Human PD-L1 ⁇ -PADRE Th) capable of significantly controlling the growth of PD-L1 + lung cancer according to an embodiment of the present invention.
  • a fusion protein Flagellin-Human PD-L1 ⁇ -PADRE Th
  • first and second are used for descriptive purposes only, and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, features defining “first” and “second” may include one or more of the features either explicitly or implicitly. Further, in the description of the present invention, the meaning of "a plurality" is two or more unless otherwise specified.
  • the invention proposes a recombinant protein.
  • the recombinant protein comprises: an immunological checkpoint molecule fragment; a helper T cell epitope fragment; and an immunostimulatory molecule fragment.
  • the recombinant protein proposed in the embodiments of the present invention stimulates the production of anti-immunological checkpoint antibodies in the patient, mobilizes the spontaneously induced immune cell CTLs already existing in the body, and stimulates the production of CTLs against the immune checkpoints, thereby specifically killing the tumor cells.
  • the active immune killing effect on tumor cells caused by the recombinant protein proposed by the embodiments of the present invention is remarkable.
  • the immune checkpoint molecule may be selected from, but not limited to, at least one of PD-L1 and PD-L2.
  • PD-L1 or PD-L2 is specifically expressed in tumor cells, and the specificity of the tumor antigen immune response caused by the recombinant protein proposed in the examples of the present invention is stronger.
  • the immunological checkpoint molecule fragment is an extracellular molecular fragment (PD-L1 ⁇ /PD-L2 ⁇ ) of the transmembrane region of the PD-L1 or PD-L2.
  • the extracellular molecular fragment of the PD-L1 or PD-L2 removal transmembrane region has only tumor antigenicity and does not have tumor immunosuppressive function, and then the extracellular molecular fragment of the transmembrane region of PD-L1 or PD-L2 is antigen-removed.
  • Presenting cells such as DC cells are presented on the surface of the cells, which can effectively elicit a tumor antigen immune response, and the specificity is further improved.
  • the helper T cell epitope can be a broad spectrum PADRE helper T cell epitope (PADRE Th).
  • PADRE Th PADRE helper T cell epitope
  • the broad-spectrum PADRE helper T cell epitope is an epitope peptide that binds to a broad spectrum of human leukocyte antigen DR (HLA-DR) with high affinity or intermediate affinity and 16 of the most prevalent HLA- 15 of the DR types are combined. Because of its universal binding, PADRE needs to overcome the problems caused by the diversity of HLA-DR molecules in the population. PADRE acts as a helper T cell epitope and binds to the antigen. After binding, it activates the Ag-specific antigen reaction efficiently and for a long time.
  • HLA-DR human leukocyte antigen DR
  • the PADRE peptide is specifically processed to immunologically activate helper T lymphocyte 1 (Th1) in humans to assist in the activation of killer immune T cells and to activate helper T lymphocyte 2 (Th2) to assist B lymphocytes to secrete antibodies, thereby Further enhancing the antigenic immune response caused by the recombinant protein.
  • Th1 helper T lymphocyte 1
  • Th2 helper T lymphocyte 2
  • the immunostimulatory molecule may be selected from Flagellin.
  • the immunostimulatory molecule is biologically active.
  • Flagellin (toll-like receptor 5 ligand, TLR5L) is the major protein of bacterial flagella and is a highly conserved bacterial protein. Flagellin regulates TLR-5-dependent signaling in mammalian cells and induces immune responses, including the production of pro-immune cytokines and co-stimulatory molecules that upregulate antigen presenting cells (APS).
  • Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) expressed in various cells, especially in immune cells such as monocytes and macrophages. In cells, DC cells and lymphocytes. TLRs detect pathogen-associated molecules (PAMPs) and bind to innate immune cells to cause maturation and activation of these immune cells, activating autoimmunity.
  • PAMPs pathogen-associated molecules
  • Flagellin can be mixed or genetically fused with a recombinant protein or peptide vaccine. Flagellin also causes an effective immune response at low concentrations. In addition, early exposure of Flagellin does not impair its function as an adjuvant, however, the antigen sequence can be inserted into the amino acid sequence and inserted into the C-terminus, or within its hypervariable region without affecting the activity of Flagellin. Flagellin can also be prepared in large quantities in a simple manner in a GMP environment. For these reasons, flagellin has a significant role as a vaccine adjuvant and immunotherapy.
  • the ligation sequence of the helper T cell epitope fragment and the immunostimulatory molecule fragment is as follows: the N-terminus of the immunological checkpoint molecule fragment is linked to the C-terminus of the immunostimulatory molecule fragment, and the immunological checkpoint molecular fragment C The terminus is ligated to the N-terminus of the helper T cell epitope fragment.
  • the corresponding molecular fragment in the recombinant protein of the present invention can be presented on the surface of the DC cell, respectively.
  • APCs main antigen-presenting cells
  • the recombinant protein proposed by the embodiments of the present invention allows DC cells to present intracellular antigens to the cell surface, which is presented to the MHC class II by endocytic pathway, and is presented to the MHC class I by a cross-priming pathway, thereby leading to the production of antigen-specific Th cells. Reacts with CTL cells.
  • the recombinant protein presented by the embodiments of the present invention causes a strong antibody response on the surface of DC cells. It has long been believed that the activation of humoral responses by DC cells is achieved by the interaction of T lymphocytes and B lymphocytes by CD4+ Th cells. However, existing in vitro and in vivo experiments have demonstrated that DC cells activate a humoral response as a direct mode of action.
  • DC cells have been shown to strongly promote cell differentiation and production of antibodies to CD40-activated B lymphocytes. Inoculation of antigen-loaded DC cells is capable of inducing a protective humoral immune response.
  • the recombinant protein of the embodiments of the present invention can more effectively cause a tumor antigen immune response.
  • the above recombinant protein has the amino acid sequence of SEQ ID NOS: 1-3.
  • SEQ ID NO: 1 is the amino acid sequence of Flagellin-human PD-L1 ⁇ -PADRE Th recombinant protein
  • SEQ ID NO: 2 is the amino acid sequence of Flagellin-human PD-L2 ⁇ -PADRE Th recombinant protein
  • SEQ ID NO: 3 is Flagellin - Amino acid sequence of human PD-L1-PD-L2 ⁇ -PADRE Th recombinant protein.
  • the recombinant protein proposed in the embodiment of the invention can cause tumor-specific antigen immune response, can cause specific killing of cytotoxic T lymphocytes (CTL) and secrete specific antibodies of B cells, and achieve specific killing of tumor cells.
  • CTL cytotoxic T lymphocytes
  • the present invention proposes a nucleic acid encoding the recombinant protein described above, and according to an embodiment of the present invention, the nucleic acid encoding the recombinant protein described above has the nucleotide sequences shown in SEQ ID NOS: 4 to 6.
  • SEQ ID NO: 4 is the nucleotide sequence of the nucleic acid encoding the Flagellin-human PD-L1 ⁇ -PADRE Th recombinant protein
  • SEQ ID NO: 5 Is the nucleotide sequence of the nucleic acid encoding the Flagellin-human PD-L2 ⁇ -PADRE Th recombinant protein
  • SEQ ID NO: 6 is the nucleotide sequence of the nucleic acid encoding the Flagellin-human PD-L1-PD-L2 ⁇ -PADRE Th recombinant protein.
  • the recombinant protein encoded by the nucleic acid proposed in the embodiments of the present invention can cause tumor-specific antigen immune response, cause specific killing of cytotoxic T lymphocytes (CTL) and secrete specific antibodies of B cells, and achieve specificity to tumor cells. Killing.
  • CTL cytotoxic T lymphocytes
  • the present invention proposes a construct which carries the nucleic acid described above.
  • the construct introduced into the recipient cell of the embodiment of the present invention can achieve high-efficiency expression of the nucleic acid described above, thereby efficiently expressing the recombinant protein described above in the recipient cell.
  • the vector of the construct is a pET series vector, a pGEX series vector, a pPIC series vector, a BacPAK, a pSV series vector or a pCMV series vector, wherein the pET series vector is in the T7 promoter in E.
  • coli Down-regulated expression of recombinant protein pGEX series vectors were used to regulate the expression of recombinant proteins in E. coli under the tac promoter, pPIC series vectors were used to regulate the expression of recombinant proteins in yeast under the AOX1 promoter, and BacPAK vectors were used in Recombinant proteins are expressed in baculovirus under the control of the pPolh promoter, and the pSV series vector or pCMV series vectors are used to express recombinant proteins in mammalian cells under the control of the CMV, SV40 and (EF)-1 promoters.
  • the above vector of the embodiment of the present invention can achieve further efficient expression of the above recombinant protein in prokaryotic cells or eukaryotic cells.
  • the above construct carries the following nucleic acid molecule: (1) a nucleic acid molecule encoding an immunological checkpoint molecule fragment having the amino acid sequence of SEQ ID NOS: 7-9
  • SEQ ID NO: 7 is the amino acid sequence of human PD-L1 ⁇
  • SEQ ID NO: 8 is the amino acid sequence of human PD-L2 ⁇
  • SEQ ID NO: 9 is the amino acid sequence of human PD-L1-PD-L2 ⁇
  • the nucleic acid molecule encoding the immunological checkpoint molecule fragment has the nucleotide sequence shown in SEQ ID NOs: 10 to 12, wherein SEQ ID NO: 10 is the nucleotide sequence of the nucleic acid molecule encoding human PD-L1 ⁇ , SEQ ID NO: 11 is a nucleotide sequence of a nucleic acid molecule encoding human PD-L2 ⁇ , SEQ ID NO: 12 is a nucleotide sequence of a nucleic acid molecule
  • the vector of the construct is a prokaryotic or eukaryotic protein expression vector.
  • the construct according to the embodiment of the present invention efficiently expresses a recombinant protein containing an immunological checkpoint molecular fragment, a helper T cell epitope fragment and an immunostimulatory molecule fragment in a recipient cell, and the recombinant protein can be significantly caused in a tumor patient.
  • the tumor-specific antigen immunoreaction which in turn causes specific killing of cytotoxic T lymphocytes (CTL) and specific antibodies against B lymphocyte secretion, achieves specific killing of tumor cells.
  • CTL cytotoxic T lymphocytes
  • the present invention also proposes a transgenic cell.
  • the transgenic cell carries the construct described above, and the transgenic cell proposed in the embodiment of the present invention can express the recombinant protein as described above to actively stimulate the anti-immunoassay in the patient body by active immunization.
  • Point such as antibodies to PD-L1 or PD-L2, mobilize spontaneously induced immune cell CTLs already present in the patient, and stimulate the production of anti-immunological checkpoints, such as PD-L1 or PD-L2 CTL, thereby specifically killing Tumor cells.
  • the transgenic cells are BL21, BL21 (DE3), BL21 (DE3) pLysS, DH10B, XL1-Blue, Pichia pastors, Kluyveromyces lactis, Sf9, Sf21, High-Five T, CHO cell line , HEK cell line, Hela cell line or COS cell line.
  • BL21, BL21 (DE3), BL21 (DE3) pLysS, DH10B and XL1-Blue are E. coli cells
  • Pichia pastors and Kluyveromyces lactis are yeast cells
  • Sf9, Sf21, and High-Five T are used for baculovirus.
  • CHO cell line, HEK cell line, Hela cell line or COS cell line is a mammalian cell line.
  • the transgenic cell can efficiently express the recombinant protein described above, and then the recombinant protein obtained by protein purification can be administered to a patient, and the patient can be further effectively stimulated to produce anti-PD in the patient by active immunization.
  • the -L1 or PD-L2 antibody mobilizes spontaneously induced immune cell CTLs already present in the patient and stimulates the production of anti-PD-L1 or PD-L2CTL, thereby specifically killing the tumor cells.
  • the transgenic cell can be an antigen presenting cell and the transgenic cell is a DC cell.
  • the antigen presenting cell is derived from a patient, and the antigen presenting cell carrying the aforementioned construct can be further input into the patient, thereby realizing the continuous expression of the recombinant protein described above in the patient, and further Actively immunizing in the body to produce anti-PD-L1 or PD-L2 antibodies in vivo, mobilizing the spontaneously induced immune cell CTLs already present in the patient, and stimulating the production of anti-PD-L1 or PD-L2CTL, Specific killing of tumor cells.
  • the inventors have proposed the use of the aforementioned recombinant protein for the preparation of a medicament or a vaccine for preventing or treating a tumor.
  • the recombinant protein proposed by the embodiments of the present invention can cause a significant tumor-specific antigen immune response in a tumor patient, and effectively stimulates the production of an anti-immunological checkpoint, such as PD-L1 or PD-L2 antibody, to mobilize the spontaneous induction already existing in the patient.
  • the resulting immune cell CTL is stimulated to produce an anti-immunological checkpoint, such as PD-L1 or PD-L2CTL, which effectively kills the tumor cells specifically.
  • the recombinant protein proposed in the examples of the present invention has a use in the preparation of a medicament or vaccine effective for preventing or treating a tumor.
  • the present invention also proposes the use of the aforementioned recombinant protein for the preparation of a vaccine for the treatment of a viral infection.
  • the inventors have found that HBV, HCV, HIV, EBV virus-infected cells express PD-L1, and the vaccine prepared by the recombinant protein of the present invention can stimulate anti-PD-L1 in a patient.
  • the invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises: the recombinant protein described above; and a pharmaceutically acceptable adjuvant.
  • the recombinant protein in the pharmaceutical composition proposed in the examples of the present invention can cause a significant specific antigen immune response, and the function of the adjuvant to enhance the immune response.
  • the pharmaceutical composition provided by the embodiment of the present invention effectively stimulates an anti-immunological checkpoint, such as a PD-L1 or PD-L2 antibody, in a tumor patient, and mobilizes the patient already.
  • viruses HBV, HCV, HIV, EBV
  • the invention provides a DC cell.
  • the DC cells are loaded with the recombinant protein described above.
  • the DC cells proposed in the embodiments of the present invention can respectively distinguish a tumor antigen (such as the immunological checkpoint molecular fragment described above), a helper T cell epitope group and an immunostimulatory molecule fragment in the recombinant protein.
  • anti-immunological checkpoints such as PD-L1 or PD-L2 antibodies
  • mobilizes spontaneously induced immune cell CTLs already present in patients and stimulates the production of anti-immune checkpoints, such as PD-L1 Or PD-L2CTL, which in turn effectively kills tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
  • viruses HBV, HCV, HIV, EBV
  • the invention provides a targeted immune cell population.
  • the targeted immune cell population is obtained by co-culture of DC cells with lymphocytes as described above.
  • the targeted immune cell population proposed in the embodiments of the present invention can specifically kill tumor cells, secrete antibodies that specifically bind tumor antigens, and achieve specific removal of tumor cells.
  • the invention provides a vaccine.
  • the vaccine comprises a recombinant protein as described above, a DC cell as described above or a targeted immune cell population as described above.
  • the recombinant protein, DC cell, and targeted immune cell population proposed in the examples of the present invention can cause a significant specific antigen immune response in a patient.
  • the vaccine according to the embodiment of the present invention can effectively stimulate the production of an anti-immunization checkpoint, such as a PD-L1 or PD-L2 antibody, mobilize the spontaneously induced immune cell CTL already existing in the patient, and stimulate An anti-immunization checkpoint, such as PD-L1 or PD-L2CTL, is generated to effectively kill tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
  • an anti-immunization checkpoint such as a PD-L1 or PD-L2 antibody
  • the invention provides an antibody.
  • the antibody specifically recognizes the recombinant protein described above, and the antibody proposed in the embodiment of the present invention specifically recognizes a tumor antigen.
  • the present invention finds that the antibody specifically recognizes an antigen, specifically binds to a tumor cell or a cell infected by a virus (HBV, HCV, HIV, EBV), thereby causing a tumor cell or a virus (HBV) , HCV, HIV, EBV) infected cells are engulfed by phagocytic cells to achieve tumor cells or by viruses (HBV, HCV, HIV, EBV) Specific clearance of infected cells.
  • a virus HBV, HCV, HIV, EBV
  • the present invention proposes a method of preparing an antibody.
  • the method comprises: immunizing an animal with the recombinant protein described above; collecting serum of the immunized animal; and purifying the antibody of interest from the serum.
  • the method for preparing an antibody proposed in the embodiments of the present invention is simple and convenient, and the antibody can specifically recognize the recombinant protein.
  • the therapeutic composition proposed by the present invention may comprise the recombinant protein described above, the nucleic acid described above, the aforementioned construct, the above-described transgenic cell, the aforementioned pharmaceutical composition
  • the therapeutic composition proposed by the embodiments of the present invention can directly or indirectly cause a specific antigen immune response, and achieve specificity to tumor cells or cells infected by viruses (HBV, HCV, HIV, EBV). Kill and clear.
  • the patient is administered a therapeutically effective amount of the recombinant protein described above, a pharmaceutical composition as described above, a DC cell as described above, a previously described targeted immune cell population, a vaccine as described above, or a front
  • the antibodies are effective for treating or preventing tumors expressing PD-L1 or PD-L2.
  • administering refers to introducing a predetermined amount of a substance into a patient in some suitable manner.
  • the recombinant protein, pharmaceutical composition, DC cell, targeted immune cell population, vaccine or antibody in the embodiments of the present invention can be administered by any common route as long as it can reach the intended tissue.
  • Various modes of administration are contemplated, including peritoneal, venous, muscular, subcutaneous, cortical, oral, topical, nasal, pulmonary, and rectal, but the invention is not limited to these exemplary modes of administration.
  • the active ingredient of the orally administered composition should be coated or formulated to prevent its degradation in the stomach.
  • the compositions of the invention may be administered as an injectable preparation.
  • the pharmaceutical compositions of the invention may be administered using a particular device that delivers the active ingredient to the target cells.
  • the frequency and dose of administration of the recombinant protein, pharmaceutical composition, DC cell, targeted immune cell population, vaccine or antibody in the examples of the present invention can be determined by a plurality of related factors including the type of disease to be treated. , route of administration, patient age, sex, weight and severity of the disease, and the type of drug as the active ingredient.
  • the daily dose may be divided into 1 dose, 2 doses or multiple doses in a suitable form for administration once, twice or more times throughout the time period, as long as a therapeutically effective amount is achieved. .
  • terapéuticaally effective amount refers to an amount sufficient to significantly ameliorate certain symptoms associated with a disease or condition, that is, an amount that provides a therapeutic effect for a given condition and dosage regimen.
  • treatment is used to mean obtaining the desired pharmacological and/or physiological effect.
  • treatment encompasses administration of a recombinant protein, pharmaceutical composition, DC cell, targeted immune cell population, vaccine or antibody in an embodiment of the invention to a subject, including, but not limited to, administration comprising the agents described herein. The individual in need.
  • the recombinant protein and the use thereof, the pharmaceutical composition, the DC cell, the targeted immune cell population, the vaccine, the antibody, the method and system for treating and diagnosing cancer according to the embodiment of the present invention are the inventors of the present application. tough Creative labor and optimization work are discovered and completed.
  • the method of isolating DC cells from mouse bone marrow is as follows: bone marrow is punched out from the limbs of the mouse, and the bone marrow is passed through a nylon mesh, and red blood cells are removed with ammonium chloride. The cells were then thoroughly washed with RPMI-1640 medium, and then cultured in 2.5 ml of RPMI-1640 medium containing 10% FBS, 20 ng/ml recombinant mouse GM-CSF (rmGM-CSF) and 20 ng/ml. Recombinant mouse IL-4 (rmIL-4) (available from PeproTech, Inc., Rocky Hill, NJ).
  • the cell supernatant was removed and fresh medium was replaced with 20 ng/ml of rmGM-CSF and 20 ng/ml of rmIL-4.
  • the cells were cultured in an incubator at 37 ° C in a 5% CO 2 atmosphere. At 48 hours of the culture process, non-adherent granulocytes were removed and fresh medium was replaced. After 7 days of cell culture, about 80% or more of the cells expressed DC cell-specific markers by FACS analysis.
  • Recombinant protein was added to bone marrow-derived DC cells (obtained after 5-7 days of bone marrow cell culture) to activate DC cells, and after 8 hours, the cells were washed with PBS three times, and after further culture for 136 hours, DC cells were used as Immunization model.
  • antigen-activated DC cells were stimulated with 100 ng/ml LPS (Sigma, St. Louis, MO) for 24 hours, and the cells were washed with PBS and injected into mice via the soles of mice (C57BL/6, Jackson). Laboratory) In vivo.
  • rodent lung cancer cells CMT167 (C57BL) (purchased from the European Collection of Authenticated Cell Cultures (ECAC)) were introduced into the right abdominal cavity of syngeneic C57BL/6 mice by subcutaneous injection. After tumor inoculation, mice were randomized and injected into antigen-activated DC cells or PBS in different groups on different days. Tumor volume was measured 2 or 3 times a week using a caliper.
  • the CD8+ CTL response was assessed by standard chromium release assays.
  • Standard chromium release assessment experiments were performed by measuring the ability of spleen cells to lyse target cells in vitro. Splenocytes from immunized mice were restimulated in vitro with RPMI containing the polypeptide for 4 to 6 days. Target cells and control cells were labeled with 51Cr sodium chromate solution for 90 min. Different numbers of effector cells were co-cultured with a certain number (1 ⁇ 104/well) of target cells for 3 hours at 37° C. in a 96-well plate v-bottom plate (200 microliters of medium per well). 100 microliters of supernatant was collected from every 3 wells.
  • the dissolution rate is calculated by the following formula: (Experimental chrome release Flux - spontaneous chromium release) / (maximum chromium release - spontaneous chromium release) X 100.
  • the amount of chromium released was achieved by co-cultivation, placing the wells on a centrifuge and centrifuging, and calculating the radiation activity in the supernatant by a gamma counter (purchased from Beckman Coulter, Inc., Fullerton, CA) ( Chromium release).
  • Synthetic fusion gene one contains the complete Flagellin sequence, partial human PD-L1 sequence or PD-L2 sequence (Accession number GenBank: AF177937.1), and the complete helper T cell PADRE epitope sequence and flanking cloning site sequence ( The fusion gene-structure is shown in Figure 1 (synthesized by GENEWIZ, South Plainfield, NJ, USA).
  • Synthetic Fusion Gene 2 Contains a partial human PD-L1 sequence or a PD-L2 sequence (GenBank: AF177937.1) and an intact helper T cell PADRE epitope sequence.
  • the target recombinant plasmid was electroporated into Escherichia coli BL21(DE3) (Novagen) competent cells, and then Escherichia coli BL21 (DE3) was seeded on LB agar plates (containing 50 ⁇ g/ml of ampicillin). Amplification culture is carried out.
  • the method of recombinant protein expression described below is one of a series of experiments under different experimental conditions.
  • the frozen cell pellet was resuspended in lysate (50 mM Tris, pH 8.0, 1 mM EDTA and 1 mM PMSF) with a mass to volume ratio of cell pellet to lysate of 1:10.
  • the inclusion bodies containing the recombinant protein were restored to activity under the conditions of French Pressure (Constant Systems LTD) of 137.9 MPa.
  • French Pressure Constant Systems LTD
  • an equal volume of lysate is added to dilute to reduce viscosity, which is more advantageous for obtaining inclusion bodies.
  • the lysed solution was centrifuged at 48,000 x g for 30 min to cause inclusion bodies to precipitate.
  • the supernatant was discarded and the pellet was washed three times to remove endotoxin, protein and DNA from the host cells.
  • the solution used for the first pass of cleaning contained 50 mM Tris, pH 8.0, 5 mM EDTA and 2% Triton x-100.
  • the solution used for the second pass of cleaning contained 50 mM Tris, pH 8.0, 5 mM EDTA, 1% sodium deoxycholate.
  • the solution used for the third cleaning consisted of 50 mM Tris, pH 8.0, 5 mM EDTA, and 1 M NaCl. After washing, the pellet was resuspended at room temperature with a special lysate (mass to volume ratio of 1:40), stirred for 30 min and centrifuged.
  • the solubilized protein was further purified by a Ni-NTA Fast Start Kit (Qiagen).
  • the eluted proteins were analyzed by 12% SDS-PAGE gel electrophoresis, and the protein concentration was determined by Bradford et al. (Bio-Rad Laboratories). Recombinant proteins with a purity greater than 90% were stored at -20 °C for subsequent studies.
  • Example 4 DC cells loaded with a fusion protein containing PD-L1 were effective in inducing anti-PD-L1 antibody production and CTL responses in mice.
  • the levels of PD-L1-specific IgG in the serum of each group of rats were determined by ELISA, and the recombinant PD-L1 protein (Abeam, Cambridge, MA, USA) was plated every 3 wells of the ELISA plate, and the ELISA value was passed through serum (1). : 100 times dilution) The mean value of OD450nm values ⁇ SD was obtained.
  • Figure 2 shows that PD-L1 ⁇ -FljB-loaded DC cells can induce significant anti-PD-L1 antibody responses, whereas PD-L1 ⁇ protein-loaded DC cells can only induce weak anti-PD- L1 antibody reaction.
  • spleen cells were isolated from tumor cell suspensions vaccinated with immunized mice.
  • the isolated T cells were re-stimulated by PD-L1 recombinant protein-activated DC cells (10 ⁇ g/ml), and then the in vitro 51Cr release test was performed, and the 51Cr release test was performed according to the specified T/E (target cell: effector cell) ratio.
  • the target cell was the PD-L1+ murine lung cancer cell line CMT167 (C57BL) (available from the European Collection of Authenticated Cell Cultures (ECACC)).
  • Figure 3 shows that PD-L1 ⁇ -FljB-loaded DC cells can induce significant anti-PD-L1CTL responses, whereas DC cells loaded with PD-L1 ⁇ protein induce only weak anti-PD-L1CTL reaction.
  • Fig. 4 shows that the immunization of mice with PD-L1 ⁇ -FljB DC was able to significantly inhibit the growth of PD-L1 + lung cancer, but was immunized with DC loaded with protein PD-L1 ⁇ or PBS. Vaccination of mice did not inhibit the growth of PD-L1 + lung cancer.
  • the inventors examined DC cells loaded with recombinant protein (Flagellin-human PD-L2 ⁇ -PADRE Th or Flagellin-human PD-L1/L2 ⁇ -PADRE Th) to induce anti-PD-L1 antibodies in mice.
  • recombinant protein Flagellin-human PD-L2 ⁇ -PADRE Th or Flagellin-human PD-L1/L2 ⁇ -PADRE Th
  • the load Recombinant proteins (Flagellin-human PD-L2 ⁇ -PADRE Th or Flagellin-human PD-L1/L2 ⁇ -PADRE Th) DC cells are capable of inducing significant anti-PD-L1 antibody or anti-PD-L2 antibody responses and inducing significant anti-PD -L1 or anti-PD-L2CTL reaction, after immunization of mice with DC cells loaded with recombinant protein (Flagellin-human PD-L2 ⁇ -PADRE Th or Flagellin-human PD-L1/L2 ⁇ -PADRE Th), can significantly inhibit PD- Growth of L1 + or PD-L2 + lung cancer.
  • the load Recombinant proteins (Flagellin-human PD-L2 ⁇ -PADRE Th or Flagellin-human PD-L1/L2 ⁇ -PADRE Th) DC cells are capable of inducing significant anti-PD-L1 antibody or anti-PD-L2 antibody responses and inducing significant anti-PD -L1

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Abstract

L'invention concerne une protéine recombinée et ses utilisations. La protéine recombinée comprend un segment de molécule de point de contrôle immunitaire, un segment d'épitope de lymphocyte T auxiliaire et un segment de molécule immunostimulante.
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