WO2023124973A1 - 一种利用外源性抗原和治疗剂联合治疗肿瘤的方法 - Google Patents

一种利用外源性抗原和治疗剂联合治疗肿瘤的方法 Download PDF

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WO2023124973A1
WO2023124973A1 PCT/CN2022/138691 CN2022138691W WO2023124973A1 WO 2023124973 A1 WO2023124973 A1 WO 2023124973A1 CN 2022138691 W CN2022138691 W CN 2022138691W WO 2023124973 A1 WO2023124973 A1 WO 2023124973A1
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cells
virus
cancer
vector
protein
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周超
安鸿
周玲
杜永彪
廖鹏云
王桃希
尹海滨
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广州安捷生物医学技术有限公司
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Definitions

  • the invention belongs to the technical field of gene therapy, and in particular relates to a method for treating tumors in combination with exogenous antigens and therapeutic agents.
  • Cancer immunotherapy is a treatment method to control and eliminate tumors by restarting and maintaining the recognition and killing of tumor cells by the immune system and restoring the body's normal anti-tumor immune response. Cancer treatment research still faces many problems.
  • Tumor heterogeneity is the primary challenge to achieve precise diagnosis and treatment and overcome tumors.
  • Tumor heterogeneity refers to the huge difference from genotype to phenotype of the same malignant tumor among different patients and among tumor cells in different parts of the same patient.
  • Tumor heterogeneity is a pervasive and crucial performance feature that emerges during tumor evolution and plays an important role in tumor formation, progression, and drug resistance.
  • Antigen heterogeneity is an important factor causing tumor recurrence.
  • CAR-T cell therapy has significantly improved the treatment of B-cell leukemia and lymphoma, but post-treatment relapse remains a barrier, and up to 50% of patients treated with CAR19 T-cell therapy relapse within the first year of treatment , a significant proportion of these relapsed patients exhibited CD19 antigen loss.
  • researchers have previously developed a tandem bispecific CAR20-19 construct targeting CD19 and CD20 antigens, and showed efficacy in phase I clinical trials. Prospect for efficacy and tolerability.
  • CART-BiTE BiTE against wild-type EGFR
  • multi-targeted drugs such as double CARs, multi-specific antibodies, etc.
  • Tumors that express drug-targeted tumor antigens can be eliminated, but other tumor cells that do not express the target antigen are insensitive to the drug and continue to grow. Targeting tumor cells with complex and diverse antigens with limited drugs seems to be stretched.
  • neoantigens Another challenge in cancer therapy is the lack of specific targets.
  • the targets of current tumor therapeutic drugs are mainly tumor-associated antigens, which have potential off-target toxicity. Although some neoantigens caused by mutations are found, they are only expressed in a small number of tumor cells and are ineffective for most tumor cells that do not express neoantigens.
  • the discovery of neoantigens requires high-throughput sequencing, which is highly individualized and expensive, so it is limited Development of drugs targeting neoantigens.
  • the purpose of the first aspect of the present invention is to provide a method for expressing exogenous antigens in tumor cells.
  • the object of the second aspect of the present invention is to provide a composition.
  • the purpose of the third aspect of the present invention is to provide the application of the composition of the second aspect in the preparation of antitumor drugs.
  • the purpose of the fourth aspect of the present invention is to provide a method for treating tumors.
  • the first aspect of the present invention provides a method for expressing exogenous antigens in tumor cells, introducing exogenous antigens into tumor cells, and the exogenous antigens are any of (1) to (2) kind:
  • Non-human protein or polypeptide that is, a protein or polypeptide not expressed in the human body
  • the nucleic acid molecule comprises DNA and RNA; further comprising DNA and mRNA.
  • the non-human protein or polypeptide includes but not limited to: bacteria, yeast, protozoa, virus and artificially synthesized proteins or polypeptides.
  • the non-human protein or polypeptide is the F protein of respiratory syncytial virus, and its amino acid sequence is shown in SEQ ID NO: 2.
  • nucleotide sequence of the nucleic acid molecule encoding the non-human protein or polypeptide is shown in SEQ ID NO:1.
  • the exogenous antigen is transfected into tumor cells by delivery vector or electroporation.
  • the delivery vehicle is a tumor-selective delivery vehicle.
  • the tumor-selective delivery vehicle is a natural polymer, a synthetic polymer, a cationic peptide, a cell penetrating peptide, a biodegradable nanoparticle, a liposome, a lipoplex, a polyplex, a micelle, Dendrimers, gels, mucoadhesives, silicon nanoneedles, gold nanoparticles, exosomes, viruses or pseudoviruses.
  • the viruses include lentiviruses, adenoviruses and adeno-associated viruses.
  • the virus is an oncolytic virus.
  • the oncolytic virus is adenovirus, vaccinia virus, Sindbis virus, Senega Valley virus, Coxsackie virus, measles virus, reovirus, vaccinia virus, Newcastle disease virus, vesicular stomatitis virus , at least one of herpes simplex virus, poliovirus, influenza virus, mumps virus and parvovirus, further adenovirus.
  • said tumors include lung cancer, liver cancer, breast cancer, gastric cancer, esophageal cancer, melanoma, head and neck cancer, prostate cancer and pancreatic cancer.
  • a method for expressing exogenous antigens in tumor cells is to infect tumor cells with an oncolytic adenovirus expressing exogenous antigen RSV F protein.
  • the preparation method of the oncolytic adenovirus comprises the following steps:
  • S1 inserting the target gene containing the F protein of respiratory syncytial virus into the vector to obtain the vector containing the target gene, and single-digesting the vector containing the target gene to obtain a linearized vector containing the target gene;
  • S2 Transform competent cells with the linearized vector containing the target gene and the pAdEasy-1 plasmid containing the backbone of adenovirus type 5 to obtain a recombinant adenovirus vector, and single-digest the recombinant adenovirus vector to obtain a linearized recombinant adenovirus viral vector;
  • S3 Transfect the linearized recombinant adenovirus vector into cells to obtain oncolytic adenovirus.
  • the target gene in S1 further comprises a human telomerase reverse transcriptase promoter (hTERTp), adenovirus E1A, and an internal ribosome entry site sequence (Internal ribosome entry site, IRES).
  • hTERTp human telomerase reverse transcriptase promoter
  • IRES internal ribosome entry site sequence
  • nucleotide sequence of the target gene described in S1 is shown in SEQ ID NO:5.
  • the vector described in S1 is a pShuttle vector.
  • the target gene in S1 is inserted between the NotI and SalI restriction sites of the vector.
  • the single enzyme cut in S1 and S2 is cut by Pme I.
  • the competent cell in S2 is the competent cell BJ5183.
  • the cells in S3 are trex293 cells.
  • the tumor is prostate cancer or lung cancer.
  • the method is a non-disease treatment method.
  • the second aspect of the present invention provides a composition comprising: exogenous antigen and therapeutic agent;
  • the exogenous antigen is any one of (1) to (2):
  • Non-human protein or polypeptide that is, a protein or polypeptide not expressed in the human body
  • the therapeutic agent takes the exogenous antigen as a target, kills tissues or cells containing exogenous antigens, and does not act on tissues or cells not containing exogenous antigens, thereby specifically killing exogenous antigen-containing tissues or cells. Tissues or cells from which antigens are derived.
  • the exogenous antigen and therapeutic agent are each independently present in the composition without mixing with each other.
  • the tissue or cells are tumor tissues or cells.
  • the nucleic acid molecule comprises DNA and RNA; further comprising DNA and mRNA.
  • the non-human protein or polypeptide includes but not limited to: bacteria, yeast, protozoa, virus and artificially synthesized proteins or polypeptides.
  • the non-human protein or polypeptide is the F protein of respiratory syncytial virus, and its amino acid sequence is shown in SEQ ID NO: 2.
  • the exogenous antigen is transfected into the tissue or cell by a delivery vehicle or electroporation.
  • said exogenous antigen is encapsulated within said delivery vehicle.
  • the delivery vehicle is a tumor-selective delivery vehicle.
  • the tumor-selective delivery vehicle is a natural polymer, a synthetic polymer, a cationic peptide, a cell penetrating peptide, a biodegradable nanoparticle, a liposome, a lipoplex, a polyplex, a micelle, Dendrimers, gels, mucoadhesives, silicon nanoneedles, gold nanoparticles, exosomes, viruses or pseudoviruses.
  • the viruses include lentiviruses, adenoviruses and adeno-associated viruses.
  • the virus is an oncolytic virus.
  • the oncolytic virus is adenovirus, vaccinia virus, Sindbis virus, Senega Valley virus, Coxsackie virus, measles virus, reovirus, vaccinia virus, Newcastle disease virus, vesicular stomatitis virus , at least one of herpes simplex virus, poliovirus, influenza virus, mumps virus and parvovirus, further adenovirus.
  • the tumor selective delivery vehicle comprises a tumor targeting agent.
  • the therapeutic agent includes CAR-T cells (chimeric antigen receptor T cells), TCR-T cells (T cell receptor modified T cells), CAR-NK cells, antigen-specific T cells, antigen-specific DC cells, small molecule targeted drugs and monoclonal antibodies; further, the therapeutic agent is CAR-T cells (chimeric antigen receptor T cells) and/or monoclonal antibodies; further, the therapeutic agent is CAR-T cells (chimeric antigen receptor T cells).
  • the CAR-T cells are obtained by introducing a chimeric antigen receptor (CAR) into T lymphocytes.
  • CAR chimeric antigen receptor
  • the method for introducing chimeric antigen receptor (chimeric antigen receptor, CAR) into T lymphocytes includes lentivirus or retrovirus infection.
  • the CAR-T cells are obtained by infecting T lymphocytes with a lentivirus loaded with a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • said chimeric antigen receptor comprises an antigen binding domain targeted to an exogenous antigen.
  • the chimeric antigen receptor further comprises a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain.
  • the exogenous antigen is the F protein of respiratory syncytial virus, and its amino acid sequence is shown in SEQ ID NO: 2.
  • amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:4.
  • nucleotide sequence of the chimeric antigen receptor is shown in SEQ ID NO: 3.
  • the exogenous antigen is an oncolytic adenovirus expressing RSV F protein.
  • the preparation method of the oncolytic adenovirus comprises the following steps:
  • S1 inserting the target gene containing the F protein of respiratory syncytial virus into the vector to obtain the vector containing the target gene, and single-digesting the vector containing the target gene to obtain a linearized vector containing the target gene;
  • S2 Transform competent cells with the linearized vector containing the target gene and the pAdEasy-1 plasmid containing the backbone of adenovirus type 5 to obtain a recombinant adenovirus vector, and single-digest the recombinant adenovirus vector to obtain a linearized recombinant adenovirus viral vector;
  • S3 Transfect the linearized recombinant adenovirus vector into cells to obtain oncolytic adenovirus.
  • the target gene in S1 further comprises a human telomerase reverse transcriptase promoter (hTERTp), adenovirus E1A, and an internal ribosome entry site sequence (Internal ribosome entry site, IRES).
  • hTERTp human telomerase reverse transcriptase promoter
  • IRES internal ribosome entry site sequence
  • nucleotide sequence of the target gene described in S1 is shown in SEQ ID NO:5.
  • the vector described in S1 is a pShuttle vector.
  • the target gene in S1 is inserted between the NotI and SalI restriction sites of the vector.
  • the single enzyme cut in S1 and S2 is cut by Pme I.
  • the competent cell in S2 is the competent cell BJ5183.
  • the cells in S3 are trex293 cells.
  • the therapeutic agent is a CAR-T cell targeting RSV F protein.
  • the preparation method of the CAR-T cell targeting RSV F protein comprises the steps of:
  • a chimeric antigen receptor (CAR) containing an antigen-binding domain targeting RSV F protein was inserted into a lentiviral vector to obtain a chimeric antigen receptor containing an antigen-binding domain targeting RSV F protein Body lentiviral vector;
  • S2 Mix the lentiviral vector obtained in S1 with the packaging plasmid to obtain the packaging system; transfect the packaging system into HEK 293T cells for culture to obtain the lentivirus;
  • S3 Infect T lymphocytes with lentivirus to obtain CAR-T cells targeting RSV F protein.
  • nucleotide sequence of the chimeric antigen receptor containing the antigen-binding domain targeting RSV F protein described in step S1 is shown in SEQ ID NO:3.
  • the lentiviral vector in step S1 is pRRLSIN.
  • the preparation method of the pRRLSIN is as follows: the ampicillin resistance gene (Amp R) of pRRLSIN.cPPT.PGK-GFP.WPRE is replaced by the Kanna resistance gene (KanR), and at the same time, the XhoI and SalI are inserted between Multiple cloning sites, obtained.
  • Am R ampicillin resistance gene
  • KanR Kanna resistance gene
  • a chimeric antigen receptor (chimeric antigen receptor, CAR) containing an antigen-binding domain targeting RSV F protein is inserted between BamHI and MluI of the lentiviral vector pRRLSIN.
  • step S1 also includes the following steps: inserting the EF1 ⁇ promoter and c-Myc tag into the lentiviral vector containing the chimeric antigen receptor targeting the antigen-binding domain of the RSV F protein, including the following steps:
  • the packaging system in step S2 includes a second-generation three-plasmid packaging system and a third-generation four-plasmid packaging system.
  • the packaging system described in step S2 is a third-generation four-plasmid packaging system, comprising: the lentiviral vector obtained in S1, pMDLg/pRRE, pRSV-REV and pMD2.G.
  • the transfection method described in step S2 includes but not limited to electroporation transfection, liposome transfection, PEI transfection and the like.
  • the titer of the lentivirus in step S2 is (3-5) ⁇ 10 8 IU/mL.
  • the T lymphocytes described in step S3 are obtained by activating peripheral blood mononuclear cells (PBMCs), and the specific steps are as follows: culturing peripheral Blood mononuclear cells (PBMC), obtained.
  • PBMCs peripheral blood mononuclear cells
  • the lymphocyte culture medium is KBM 581 Serum-free Cell Medium.
  • the peripheral blood mononuclear cells are separated by a blood cell separator or Ficoll.
  • the step of infecting T lymphocytes with lentivirus in step S3 is as follows: mixing lentivirus, T lymphocytes and polybrene, centrifuging infection, and culturing to obtain.
  • the third aspect of the present invention provides the application of the composition of the second aspect in the preparation of antitumor drugs.
  • the tumors include lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, lymphatic cancer, gastric cancer, esophageal cancer, kidney cancer cancer, prostate cancer, pancreatic cancer and leukemia; further prostate cancer and/or lung cancer.
  • the fourth aspect of the present invention provides a method for treating tumors.
  • the exogenous antigen in the composition of the second aspect of the present invention is administered to the subject, so that the tumor tissue or cells of the subject contain the exogenous antigen ; administering to a subject a therapeutic agent in the composition of the second aspect of the invention.
  • the invention provides a method for expressing exogenous antigens in tumor cells, introducing exogenous antigens into tumor cells, so that tumor cells with heterogeneous antigens in tumor tissues can express the same specific antigen, and solve the problem of tumor treatment.
  • the problem of antigen heterogeneity, the exogenous antigen provided by the target has high specificity and strong immunogenicity, which solves the problem of lack of specific targets in tumor treatment, which is equivalent to turning the treatment of tumors (endogenous diseases) into An "infectious" disease (presence of xenoantigens), which facilitates the development of safe and effective antineoplastic drugs.
  • the present invention provides a composition comprising an exogenous antigen and a therapeutic agent, the therapeutic agent takes the exogenous antigen as a target, kills tissues or cells containing the exogenous antigen, and does not act on Tissues or cells with exogenous antigens, so as to specifically kill tissues or cells, can effectively kill tumor cells, and provide new ideas for tumor treatment.
  • exogenous antigens can be expressed in different types of tumors in different individuals, Therefore, it is a "broad-spectrum" anti-tumor method with huge economic value and social significance.
  • Fig. 1 is the plasmid map of shuttle vector pShuttle-hTERTp-E1A-IRES-RSV F in embodiment 1.
  • Fig. 2 is the electrophoresis result figure of Pac I digestion identification recombinant adenovirus vector pAd-hTERTp-E1A-IRES-RSV F in embodiment 1: Wherein, (A) is after the plasmid of clone 1 ⁇ 10 is digested by Pac I Electrophoresis result graph; (B) is the electrophoresis result graph after Pac I digestion of the plasmid of clone 8.
  • Figure 3 is a diagram of the electrophoresis results of PCR identification of P2 recombinant oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F in Example 1.
  • Fig. 4 is the result graph of the expression of RSV F protein after the P2 generation recombinant oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F infecting trex293 cells in Example 1 by flow cytometry.
  • FIG. 5 is a diagram showing the results of flow cytometry detection of exogenous antigen RSV F protein expression after oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F infection of prostate cancer cell line LnCap-FGC in Example 1.
  • FIG. 6 is a diagram showing the results of flow cytometry detection of exogenous antigen RSV F protein expression after oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F infection of non-small cell lung cancer cell line A549 in Example 1.
  • Example 7 is a graph showing the results of exogenous antigen RSV F protein expression in Example 1 after the oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F was infected with the non-small cell lung cancer cell line A549 by western blotting.
  • Fig. 8 is a graph showing the change of virus titer over time after the oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F infects the non-small cell lung cancer cell line A549 in Example 1.
  • Figure 9 is a graph showing the results of the oncolytic effect of the oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F on the non-small cell lung cancer cell line A549 in Example 1.
  • Fig. 10 is a diagram of electrophoresis results of PCR amplified NheI-EF1 ⁇ -Myc fragment and Myc-Palivizumab-BB-MIuI fragment in Example 2.
  • FIG. 11 is a diagram of the electrophoresis results of the PCR amplified EF1 ⁇ -Myc-Palivizumab-BB fragment in Example 2.
  • Fig. 12 is an electrophoresis result diagram of colony PCR identification lentiviral expression vector pRRLSIN-EF1 ⁇ -myc-Palivizumab-BB in Example 2.
  • Figure 13 is the plasmid map of the lentiviral expression vector pRRLSIN-EF1 ⁇ -myc-Palivizumab-BB in Example 2.
  • Example 14 is a graph showing the results of the positive rate of myc-Palivizumab-BB CAR-T cells detected by flow cytometry in Example 2.
  • Figure 15 is the results of the killing effect of Palivizumab-BB CAR-T cells targeting RSV F combined with oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F on lung cancer in Example 3: where (A) is the target The result graph of the killing effect of Palivizumab-BB CAR-T cells targeting RSV F combined with oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F (5 TCID50) on lung cancer; (B) is Palivizumab-BB targeting RSV F The results of the killing effect of BB CAR-T cells combined with oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F(10TCID50) on lung cancer.
  • Figure 16 is a graph showing the specificity of tumor killing of Palivizumab-BB CAR-T cells targeting RSV F combined with oncolytic adenovirus OAd-hTERTp-E1A-I RES-RSV F in Example 4.
  • the materials, reagents, etc. used in this example are reagents and materials obtained from commercial sources unless otherwise specified.
  • the shuttle vector was constructed with the pShuttle vector (purchased from Shanghai Jiying Biotechnology Co., Ltd.) as the backbone, and the inserted target fragments included human telomerase reverse transcriptase promoter (hTERTp), adenovirus E1A, internal ribosome entry site sequence ( Internal ribosome entry site, IRES), respiratory syncytial virus F protein (RSV-F, nucleotide shown in SEQ ID NO: 1; its amino acid shown in SEQ ID NO: 2) and bGH poly(A).
  • the nucleotide sequence of the target fragment in this example is shown in SEQ ID NO: 5, which was synthesized by Universal Biotechnology and cloned between the NotI and SalI restriction sites of the pShuttle vector.
  • the constructed shuttle vector was named pShuttle-hTERTp -E1A-IRES-RSV F, the plasmid map is shown in Figure 1.
  • the plasmid was extracted, linearized by Pme I digestion, and stored in a -20°C refrigerator.
  • the linearized shuttle plasmid obtained in step (1) and the pAdEasy-1 plasmid containing the backbone of adenovirus type 5 (purchased from Shanghai Jiying Biotechnology Co., Ltd.) were transformed into competent cells BJ5183, and the recombinant adenovirus vector was constructed by homologous recombination pAd-hTERTp-E1A-IRES-RSV F. Pick 10 single colonies and inoculate them into 3mL medium for culture, extract the plasmid, and identify it by Pac I digestion. If the recombination is successful, it should be cut into 2 fragments, one of which is a larger fragment of ⁇ 30kb, and the other is a smaller fragment.
  • the fragment should be 4.5kb or 3kb, as shown in (A) in Figure 2, the plasmids of clones No. 3, 4, 6, 7, 8, and 9 appear 2 bands after Pac I digestion and the band size is correct. It shows that all may be positive clones. Randomly select No.
  • Trex293 cells were digested and prepared into a single cell suspension, counted, seeded into a 6-well plate at 1 ⁇ 10 6 cells/well, and cultured overnight in a 37°C, 5% CO 2 incubator. The next day, take a 1.5mL EP tube, add 100 ⁇ L of serum-free DMEM culture medium, add 20 ⁇ L of the plasmid solution obtained in (2) (digested with PacI, the amount of the digested plasmid is about 1 ⁇ g) and 8 ⁇ L of PEI 40K solution, and use a pipette tip Gently blow and mix, and let stand at room temperature for 20 min.
  • the transfection reagent and the plasmid take out the 6-well plate that has been laid one day in advance from the CO2 incubator, remove the culture medium in the well, add 1 mL/well serum-free culture medium, let it stand for 1 min, and discard it. Then add 0.9 mL of serum-free culture medium/well, and put the culture plate into a CO2 incubator to continue culturing. After incubating for 20 minutes, take out the culture plate from the incubator, add the plasmid-transfection reagent PEI mixture, and gently shake the culture plate in the shape of " ⁇ " to mix the liquid in the well plate, and put the culture plate into the CO2 incubator Incubate overnight.
  • the trex293 cells were seeded in 10 cm culture dishes, and when the cells grew to a confluence of 70-80%, an appropriate amount of P1 generation virus solution was added to each culture dish. Observe the cells. After 2 to 3 days of infection, when most of the cells are diseased, centrifuge to collect the cell pellet, add serum-free DMEM culture medium to resuspend the cells, freeze and thaw repeatedly 3 times, and centrifuge at 3000g for 10 minutes to collect the supernatant to obtain the P2 generation virus liquid.
  • Lane1 uses the genomic DNA extracted from the P2 generation virus liquid as a template for PCR
  • lane2 uses the genomic DNA extracted from the freeze-thawed supernatant of trex293 cells as a template for PCR
  • the exogenous gene RSV F band was amplified from the template and the size was correct ( ⁇ 1700bp), indicating that the oncolytic adenovirus was successfully packaged in trex293 cells.
  • Take the P2 generation virus to infect trex293 cells collect the cells after 48 hours and detect the expression of RSV F by flow cytometry, and use trex293 cells without virus infection as a control, as shown in Figure 4, RSV F can be detected on the surface of OAd-infected trex293 cells.
  • the protein expression further indicated that the oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F could be successfully packaged in trex293 cells and RSV F could be expressed normally in the cells.
  • the P1-P4 generation viruses are all used as virus seeds for subsequent virus production.
  • the trex293 cells were expanded and inoculated into a 10-layer cell factory, and the P4 virus was taken for infection. After 2 to 3 days, most of the cells showed lesions, the culture was harvested and centrifuged, and the cell culture supernatant was temporarily placed in a refrigerator at 4 to 8°C. The precipitate was repeatedly frozen and thawed three times, the supernatant was collected by centrifugation, and the supernatant was mixed with the cell culture supernatant to obtain the oncolytic virus stock solution.
  • the virus stock solution was purified through clarification, nuclease treatment, hollow fiber column concentration and buffer replacement, chromatography, hollow fiber column concentration, sterile filtration, etc., and the purified oncolytic adenovirus OAd-hTERTp-E1A-IRE S- RSV F was subpackaged and stored in a -80°C refrigerator. Take a tube of virus purification solution, and detect the virus titer by Reed-Muench method, and the virus titer is 5.13 ⁇ 10 9 TCID50/mL.
  • the prostate cancer cell line LnCap-FGC was inoculated into a 6-well cell culture plate, infected with an oncolytic virus with an MOI of 10 TCID50, and the expression of RSV F protein on the cell surface was detected by flow cytometry.
  • FGC cells served as controls. 48 hours after infection, a large number of cells floated, and the cells were collected for detection. The results are shown in Figure 5. After infection with oncolytic adenovirus, there was RSV F protein on the surface of prostate cancer cells, and the positive rate reached ⁇ 35%.
  • Non-small cell lung cancer cell line A549 was inoculated into 24-well cell culture plates, and infected with oncolytic virus with MOI of 1 TCID50, 5 TCID50, and 10 TCID50 respectively, and RSV F on the surface of A549 cells was detected by flow cytometry at different time points.
  • A549 cells not infected with the virus were used as a control. The results are shown in Figure 6. After OAd infected tumor cells A549, the expression of exogenous antigen RSV F protein on the surface of tumor cells increased with the increase of virus dose and infection time.
  • the positive rate reached ⁇ 20%, and on the 5th day after oncolytic virus infection, the positive rate increased significantly at 5 TCID50 and 10 TCID50, indicating that the oncolytic virus can transfer foreign antigens to lung cancer cells Successful expression, especially on the 5th day ⁇ 90% of A549 cells express RSV F protein on the surface, so that tumor cells with original antigen heterogeneity express the same antigen.
  • A549 cells and trex293 cells were inoculated into 6-well plates and infected with oncolytic virus with an MOI of 5 TCID50. After 48 hours, the cells were collected to extract total protein and detect the expression of RSV F protein by western blotting.
  • the results are shown in Figure 7, lane1 is A549-OAd, lane2 is A549-NC, lane3 is trex293-OAd, lane4 is trex293-NC, and lane5 is PageRuler prestained protein Marker (Thermo).
  • RSV F protein After OAd infection, A549 and The expression of RSV F protein can be detected in trex293 cells, but the target band cannot be detected in uninfected cells, which further proves that cells infected with oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F can express exogenous antigens.
  • A549 cells were inoculated into 6-well cell culture plates, and A549 cells were infected with OAd-hTERTp-E1A-IRES-RSV F with MOI of 1 TCID50 and 5 TCID50 respectively, and the virus liquid was collected at different time points, passed through Adeno-X TM Rapid Titer Kit (Takara) was used to detect virus titers.
  • the results are shown in Figure 8. As time increases, the virus titer increases significantly, and at 5 TCID50, the virus titer at 96h (1.93 ⁇ 10 7 IFU/mL) is 6h (7.11 ⁇ 10 5 IFU/mL) 27 times that of OAd, indicating that OAd can replicate in A549 cells.
  • A549 cells were inoculated into 6-well cell culture plates, and A549 cells were infected with OAd-hTERTp-E1A-IRES-RSV F at MOIs of 10 TCID50, 20 TCID50 and 30 TCID50, respectively. 48h and 72h after infection, the growth of the cells was observed under a microscope, and then stained with crystal violet. The results are shown in Figure 9.
  • the CAR structure is composed of an antigen-binding domain, a transmembrane domain, a co-stimulatory signal transduction region, and an intracellular signal transduction domain.
  • Biologically synthesized and cloned into the lentiviral vector pRRLSIN (pRRLSIN is obtained by transforming pRRLSIN.cPPT.PGK-GFP.WPRE, as follows: first replace the ampicillin resistance gene (AmpR) in pRRLSIN.cPPT.PGK-GFP.WPRE For the Kanna resistance gene (KanR), obtain pRRLSIN.cPPT.PGK-GFP.WPRE (KanR); then insert a multiple cloning site between XhoI and SalI of pRRLSIN.cPPT.PGK-GFP.WPRE (KanR), Between the BamHI and MluI restriction sites of pRRLSIN), the constructed recombinant lentiviral expression vector was named pRRLS
  • NheI-EF1 ⁇ -F/SP-Myc-R was used as primers (the sequence of NheI-EF1a-F is: CTAGCTAGCGCTCCGGTGCCCGTCAGT, SEQ ID NO: 6; the sequence of SP-Myc-R is: GAGGTCCTTTCAGAGATAAGTTTTTGCTCCGGCCTGGCGGCGTGGA, SEQ ID NO: 7) to amplify NheI -EF1 ⁇ -Myc fragment; with pRRLSIN-Palivizumab-BB as template, Myc-RSV CAR-F/CD3-MIuI-R as primer (the sequence of Myc-RSV CAR-F is: GAGCAAAAACTTATCTCTGAAGAGGACCTCCAAGTGACC
  • the target fragment EF1 ⁇ -Myc-Palivizumab-BB and the carrier pRRLSIN-Palivizumab-BB were digested with NheI and MIuI, and the digested product was reintroduced for ligation, and the ligated product was transformed into trans5 ⁇ chemically competent cells (Beijing Quanshijin Biotechnology Co., Ltd.) . Eight single colonies were picked and identified by colony PCR. The results are shown in Figure 12. Clones No. 3 and No. 8 amplified the target bands and the bands were obvious, indicating that they may be positive clones. After shaking the bacteria, the plasmid was extracted, and the plasmid was sequenced, and the sequence was found to be correct.
  • Plasmids were extracted with an endotoxin-free plasmid maxi kit (Endo-free Plasmid Maxi Kit, Omega) and stored in a -20°C refrigerator.
  • HEK293T cells were recovered and subcultured with DMEM complete medium (DMEM medium + 10% FBS).
  • DMEM complete medium DMEM medium + 10% FBS.
  • HEK293T cells were inoculated into a 10-layer cell factory at a density of 3 ⁇ 10 6 /mL, and the volume of DMEM complete medium was added to 1 L. After overnight culture, the cells could reach 80-90% confluence, and plasmid transfection was performed.
  • PBMCs peripheral blood from healthy volunteers, anticoagulate with heparin, and the serum obtained after centrifugation is inactivated at 56°C for later use.
  • Precipitated cells were diluted with normal saline and added to a centrifuge tube filled with Ficoll solution, separated by density gradient centrifugation to obtain PBMCs, washed twice with normal saline, and counted for later use.
  • PBMC lymphocyte medium KBM 581 Serum-free Cell Medium (Corning)
  • KBM 581 Serum-free Cell Medium Adjust the cell density to 1-2 ⁇ 10 6 /mL
  • inoculate into T75 cell culture flask add anti-human CD3 monoclonal antibody (OKT-3) to activate PBMC were supplemented with 500 IU/mL recombinant human interleukin-2 (rhIL-2), 5-10% plasma at the same time, and cultured in a 37° C., 5% CO 2 incubator.
  • rhIL-2 human interleukin-2
  • PBMCs peripheral blood mononuclear cells
  • KBM 581 Serum-free Cell Medium recombinant human interleukin-2 (rhIL-2)
  • rhIL-2 human interleukin-2
  • Example 3 The killing effect of Palivizumab-BB CAR-T cells targeting RSV F combined with oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F on lung cancer
  • the non-small cell lung cancer cell line A549 was inoculated into a 6-well cell culture plate and cultured in a 37°C, 5% CO2 incubator .
  • replace the fresh medium RPMI-1640+10% FBS
  • add the lentivirus plv-LUC-IRES-neo (carrying firefly luciferase Luciferase and neomycin resistance gene neo, preserved by our company, specific preparation method
  • first insert firefly luciferase Luciferase between the XhoI and XbaI restriction sites of the pLVX-IRES-Neo vector to obtain the lentiviral expression vector pLVX-LUC-IRES-neo
  • equimolar concentration of pLVX-LUC-IRES -neo, lentiviral packaging plasmid pSPAX2 and lentiviral envelope plasmid pMD2.G were co-transfected into HEK
  • A549-LUC cells were digested to prepare a single cell suspension as target cells. The cells were washed twice with RPMI1640 culture medium, and the cells were counted. Divide the A549-LUC cells into 2 parts, add the oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F produced according to Example 1, the MOIs are 10 TCID50 and 5 TCID50 respectively, and divide the cells at 1 ⁇ 10 5 cells/ mL for resuspension (first mix the virus and culture medium that need to be added, and then resuspend the cells).
  • the Palivizumab-BB CAR-T cells and T cells prepared according to Example 2 were taken out from the incubator as effector cells.
  • the cell pellet was collected by centrifugation at 500 g for 3 min, the cells were washed twice with RPMI1640 culture medium, and the cell density was counted and adjusted.
  • the effector cells were added to the above-mentioned 96-well plate as the experimental group according to the effect-to-target ratio of 5, 10, and 20, that is, 0.5 ⁇ 10 5 cells/50 ⁇ L/well, 1 ⁇ 10 5 cells/50 ⁇ L/well, 2 ⁇ 10 5 pieces/50 ⁇ L/well.
  • MinCPS group minimum lumen value group
  • MaxCPS group maximum lumen value group
  • specific killing efficiency (%) (MaxCPS-sample CPS)/(MaxCPS-MinCPS) ⁇ 100%.
  • the results are shown in Figure 15, the killing effect of immune cells combined with 5 TCID50 or 10 TCID50 oncolytic adenovirus OAd-hTERTp-E1A-IRES-RS V F on A549-LUC was enhanced with the increase of effector-target ratio, and CAR -T combined with oncolytic virus was significantly stronger than T cells combined with oncolytic virus.
  • Palivizumab-BB CAR-T In order to prove the specificity of Palivizumab-BB CAR-T cells combined with oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F, Palivizumab-BB CAR-T (targeting RSV F protein, CART RSV), CART 5E5 ( Targeting the MUC1 Tn antigen, A549 cells do not express MUC1 Tn.
  • RSV F protein CART RSV
  • CART 5E5 Targeting the MUC1 Tn antigen
  • A549 cells do not express MUC1 Tn.
  • T cells perform cell killing experiments on A549-LUC and A549-LUC infected by oncolytic adenovirus.
  • A549-LUC cells were digested according to Example 3 to prepare a single cell suspension as target cells. The cells were washed twice with RPMI1640 culture medium, and the cells were counted. The A549-LUC cells were divided into 2 parts, one of which was added with the oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F produced according to Example 1, the MOI was 10 TCID50, and the other part was not added with the oncolytic virus. All cells were resuspended at 1 ⁇ 10 5 cells/mL. Two copies of cells were inoculated in a 96-well plate at 100 ⁇ L/well (cell density: 1 ⁇ 104 cells/well), and the culture plate was cultured in a 37°C, 5% CO 2 incubator.
  • the CART (RSV), CART (5E5) and T cells prepared according to Example 2 were taken out from the incubator as effector cells.
  • the cell pellet was collected by centrifugation at 500 g for 3 min, the cells were washed twice with RPMI1640 culture medium, and the cell density was counted and adjusted.
  • the effector cells were added to the above-mentioned 96-well plate according to 1.5 ⁇ 10 5 cells/50 ⁇ L/well as the experimental group.
  • MinCPS group and MaxCPS group at the same time, in which MinCPS wells remove the original culture medium, add 150 ⁇ L/well of 10% FBS RPMI1640 culture medium containing 1% Tween20, and add 50 ⁇ L/well of 10% FBS RPMI1640 culture medium in MaxCPS wells. All groups were set up with 3 replicate holes. After the culture plate was centrifuged at 300 g for 3 min, it was placed in an incubator to continue culturing.
  • CAR-T (RSV) combined with 10 TCID50 oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F has a significantly stronger killing effect on A549-LUC than CAR-T (5E5) and 10 TCID50 oncolytic Adenovirus combination group, CAR-T (RSV) group, oncolytic adenovirus group, while CAR-T (RSV) group, CAR-T (5E5) group and T cell group had no difference in killing effect on A549-LUC, indicating that CAR -T(RSV) has no specific cytotoxicity to A549-LUC cells that do not express exogenous antigen RSV F, while oncolytic adenovirus OAd-hTERTp-E1A-IRES-RSV F can make

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Abstract

本发明属于基因治疗技术领域，公开了一种利用外源性抗原和治疗剂联合治疗肿瘤的方法。本发明公开了一种组合物，该组合物包含外源性抗原和治疗剂，所述治疗剂以所述外源性抗原为靶点，对含外源性抗原的组织或细胞进行杀伤，并且不作用于不含外源性抗原的组织或细胞，从而特异性杀死组织或细胞，例如肿瘤细胞。基于所述外源性抗原可在不同个体不同类型的肿瘤中表达，因此本发明的方法是一种广谱的抗肿瘤方法。

Description

一种利用外源性抗原和治疗剂联合治疗肿瘤的方法 技术领域
本发明属于基因治疗技术领域，具体涉及一种利用外源性抗原和治疗剂联合治疗肿瘤的方法。
背景技术
癌症治疗的常规治疗方法包括手术、放疗、化疗和单克隆抗体等。近年来，肿瘤免疫治疗(Cancer Immunotherapy)因其对于常规疗法无效的晚期肿瘤具有显著疗效而受到极大关注，并于2013年被《Science》评为年度十大科技突破之首，为晚期肿瘤患者带来希望。肿瘤免疫治疗是通过重新启动并维持免疫系统对肿瘤细胞的识别和杀伤，恢复机体正常的抗肿瘤免疫反应，从而控制与清除肿瘤的一种治疗方法。肿瘤治疗研究仍面临诸多问题。
肿瘤异质性是实现精准诊治及攻克肿瘤的首要挑战。肿瘤异质性是指同一种恶性肿瘤在不同患者个体间以及同一患者个体内不同部位肿瘤细胞间从基因型到表型上的巨大差异。肿瘤异质性是肿瘤演化过程中出现的一个普遍而又至关重要的表现特征，在肿瘤形成、发展和耐药方面具有重要作用。抗原异质性是引起肿瘤复发重要因素。CAR-T细胞治疗对B细胞白血病和淋巴瘤的治疗有了明显的改善，但是治疗后复发仍是一个障碍，而且有多达50％的接受CAR19 T细胞治疗的患者在治疗后第一年内复发，这些复发患者的相当一部分人表现出CD19抗原丢失。为了解决肿瘤抗原异质性所引起的抗原逃逸的问题，研究人员之前已经开发了一种靶向CD19和CD20抗原的串联双特异性CAR20-19构建体，并在I期临床试验中显示出有前景的疗效和耐受性。Marcela为解决肿瘤抗原异质性问题，提高机体对GBM的抗肿瘤效应，在CART-EGFRvIII中引入抗野生型EGFR的BiTE(CART-BiTE)。尽管通过开发多靶点的药物(如双CAR、多特异性抗体等)在一定程度能提高对抗原异质性肿瘤的治疗效果，但是不能从根本上解决问题，因为在药物选择压力之下，能够清除表达药物靶向肿瘤抗原的肿瘤，但是其他不表达靶向抗原的肿瘤细胞对药物不敏感，能够继续生长。以有限的药物针对抗原复杂多样的肿瘤细胞未免显得捉襟见肘。
肿瘤治疗面临的另一挑战是缺乏特异性靶点。当前肿瘤治疗药物的针对的靶点主要是肿瘤相关抗原，存在潜在的脱靶毒性。尽管发现一些突变引起的新抗原，但是仅在少量肿瘤细胞表达，对大多数不表达新抗原的肿瘤细胞无效，此外新抗原的发现需要高通量测序，高度个体化、成本较高，因此限制了以新抗原作为靶点的药物的开发。
因此，解决肿瘤抗原异质性和缺乏特异性靶点的问题是开发安全有效的抗肿瘤药物的关键。
发明内容
本发明的第一方面的目的，在于提供一种在肿瘤细胞中表达外源性抗原的方法。
本发明的第二方面的目的，在于提供一种组合物。
本发明的第三方面的目的，在于提供第二方面的组合物在制备抗肿瘤药物中的应用。
本发明的第四方面的目的，在于提供一种治疗肿瘤的方法。
为了实现上述目的，本发明所采取的技术方案是：
本发明的第一个方面，提供一种在肿瘤细胞中表达外源性抗原的方法，向肿瘤细胞中导入外源性抗原，所述外源性抗原为(1)～(2)中任一种：
(1)非人蛋白或多肽，即人体内不表达的蛋白或多肽；
(2)编码(1)中所述非人蛋白或多肽的核酸分子。
优选地，所述核酸分子包含DNA和RNA；进一步包含DNA和mRNA。
优选地，所述非人蛋白或多肽包括但不限于：细菌、酵母、原生动物、病毒以及人工合成的蛋白或多肽。
进一步优选地，所述非人蛋白或多肽为呼吸道合胞病毒的F蛋白，其氨基酸序列为SEQ ID NO：2所示。
进一步优选地，编码所述非人蛋白或多肽的核酸分子的核苷酸序列如SEQ ID NO：1所示。
优选地，所述外源性抗原通过递送载体或电穿孔转染到肿瘤细胞。
优选地，所述递送载体为肿瘤选择性递送载体。
优选地，所述肿瘤选择性递送载体是天然聚合物、合成聚合物、阳离子肽、细胞穿透肽、生物可降解的纳米颗粒、脂质体、脂质复合物、聚合复合物、胶束、树枝状聚合物、凝胶、粘膜粘着剂、硅纳米针、纳米金颗粒、外泌体、病毒或假病毒。
优选地，所述病毒包括慢病毒、腺病毒和腺相关病毒。
优选地，所述病毒为溶瘤病毒。
优选地，所述溶瘤病毒为腺病毒、痘苗病毒、辛德毕斯病毒、塞内加谷病毒、柯萨奇病毒、麻疹病毒、呼肠孤病毒、牛痘病毒、新城疫病毒、水疱性口炎病毒、单纯疱疹病毒、脊髓灰质炎病毒、流感病毒、腮腺炎病毒和细小病毒中的至少一种，进一步为腺病毒。
优选地，所述肿瘤包括肺癌、肝癌、乳腺癌、胃癌、食管癌、黑色素瘤、头颈部癌、前列腺癌和胰腺癌。
优选地，一种在肿瘤细胞中表达外源性抗原的方法，用表达外源性抗原RSV F蛋白的溶瘤腺病毒感染肿瘤细胞。
优选地，所述溶瘤腺病毒的制备方法包括如下步骤：
S1：将包含呼吸道合胞病毒F蛋白的目的基因插入载体中，得到包含目的基因的载体，单酶切所述包含目的基因的载体，得到线性化的包含目的基因的载体；
S2：将线性化的包含目的基因的载体和含5型腺病毒骨架的pAdEasy-1质粒转化感受态细胞，得到重组腺病毒载体，单酶切所述重组腺病毒载体，得到线性化的重组腺病毒载体；
S3：将线性化的重组腺病毒载体转染至细胞，得到溶瘤腺病毒。
优选地，S1中所述目的基因还包含人端粒酶逆转录酶启动子(hTERTp)、腺病毒E1A、内部核糖体进入位点序列(Internal ribosome entry site，IRES)。
优选地，S1中所述目的基因的核苷酸序列如SEQ ID NO：5所示。
优选地，S1中所述载体为pShuttle载体。
优选地，S1中所述目的基因插入所述载体的NotI和SalI酶切位点之间。
优选地，S1、S2中所述单酶切通过Pme I酶切。
优选地，S2中所述感受态细胞为感受态细胞BJ5183。
优选地，S3中所述细胞为trex293细胞。
优选地，所述肿瘤为前列腺癌或肺癌。
优选地，所述方法为非疾病治疗方法。
本发明的第二个方面，提供一种组合物，包含：外源性抗原和治疗剂；
所述外源性抗原为(1)～(2)中任一种：
(1)非人蛋白或多肽，即人体内不表达的蛋白或多肽；
(2)编码(1)中所述非人蛋白或多肽的核酸分子；
所述治疗剂以所述外源性抗原为靶点，对含外源性抗原的组织或细胞进行杀伤，并且不作用于不含外源性抗原的组织或细胞，从而特异性杀死含外源性抗原的组织或细胞。
优选地，所述外源性抗原和治疗剂各自独立地存在于所述组合物中而互不混合。
优选地，所述组织或细胞为肿瘤组织或细胞。
优选地，所述核酸分子包含DNA和RNA；进一步包含DNA和mRNA。
优选地，所述非人蛋白或多肽包括但不限于：细菌、酵母、原生动物、病毒以及人工合成的蛋白或多肽。
进一步优选地，所述非人蛋白或多肽为呼吸道合胞病毒的F蛋白，其氨基酸序列为SEQ ID NO：2所示。
优选地，所述外源性抗原通过递送载体或电穿孔转染到所述组织或细胞。
优选地，所述外源性抗原包封在所述递送载体内。
优选地，所述递送载体为肿瘤选择性递送载体。
优选地，所述肿瘤选择性递送载体是天然聚合物、合成聚合物、阳离子肽、细胞穿透肽、生物可降解的纳米颗粒、脂质体、脂质复合物、聚合复合物、胶束、树枝状聚合物、凝胶、粘膜粘着剂、硅纳米针、纳米金颗粒、外泌体、病毒或假病毒。
优选地，所述病毒包括慢病毒、腺病毒和腺相关病毒。
优选地，所述病毒为溶瘤病毒。
优选地，所述溶瘤病毒为腺病毒、痘苗病毒、辛德毕斯病毒、塞内加谷病毒、柯萨奇病毒、麻疹病毒、呼肠孤病毒、牛痘病毒、新城疫病毒、水疱性口炎病毒、单纯疱疹病毒、脊髓灰质炎病毒、流感病毒、腮腺炎病毒和细小病毒中的至少一种，进一步为腺病毒。
优选地，所述肿瘤选择性递送载体包含肿瘤靶向剂。
优选地，所述治疗剂包括CAR-T细胞(嵌合抗原受体T细胞)、TCR-T细胞(T细胞受体修饰T细胞)、CAR-NK细胞、抗原特异性T细胞、抗原特异性DC细胞、小分子靶向药和单克隆抗体；进一步地，所述治疗剂为CAR-T细胞(嵌合抗原受体T细胞)和/或单克隆抗体；更进一步地，所述治疗剂为CAR-T细胞(嵌合抗原受体T细胞)。
优选地，所述CAR-T细胞通过将嵌合抗原受体(chimeric antigen receptor，CAR)导入T淋巴细胞得到。
优选地，所述嵌合抗原受体(chimeric antigen receptor，CAR)导入T淋巴细胞的方法包括慢病毒或逆转录病毒感染。
进一步优选地，所述CAR-T细胞通过负载嵌合抗原受体(chimeric antigen receptor，CAR)的慢病毒感染T淋巴细胞获得。
优选地，所述嵌合抗原受体包含靶向外源性抗原的抗原结合结构域。
优选地，所述嵌合抗原受体还包含跨膜结构域，共刺激结构域和细胞内信号传导结构域。
优选地，所述外源性抗原为呼吸道合胞病毒的F蛋白，其氨基酸序列为SEQ ID NO：2所示。
优选地，所述嵌合抗原受体的氨基酸序列如SEQ ID NO：4所示。
进一步优选地，所述嵌合抗原受体的核苷酸序列如SEQ ID NO：3所示。
优选地，所述外源性抗原为表达RSV F蛋白的溶瘤腺病毒。
优选地，所述溶瘤腺病毒的制备方法，包括如下步骤：
S1：将包含呼吸道合胞病毒F蛋白的目的基因插入载体中，得到包含目的基因的载体，单酶切所述包含目的基因的载体，得到线性化的包含目的基因的载体；
S2：将线性化的包含目的基因的载体和含5型腺病毒骨架的pAdEasy-1质粒转化感受态细胞，得到重 组腺病毒载体，单酶切所述重组腺病毒载体，得到线性化的重组腺病毒载体；
S3：将线性化的重组腺病毒载体转染至细胞，得到溶瘤腺病毒。
优选地，S1中所述目的基因还包含人端粒酶逆转录酶启动子(hTERTp)、腺病毒E1A、内部核糖体进入位点序列(Internal ribosome entry site，IRES)。
优选地，S1中所述目的基因的核苷酸序列如SEQ ID NO：5所示。
优选地，S1中所述载体为pShuttle载体。
优选地，S1中所述目的基因插入所述载体的NotI和SalI酶切位点之间。
优选地，S1、S2中所述单酶切通过Pme I酶切。
优选地，S2中所述感受态细胞为感受态细胞BJ5183。
优选地，S3中所述细胞为trex293细胞。
优选地，所述治疗剂为靶向RSV F蛋白的CAR-T细胞。
优选地，所述靶向RSV F蛋白的CAR-T细胞的制备方法，包括如下步骤：
S1：将含有靶向RSV F蛋白的抗原结合结构域的嵌合抗原受体(chimeric antigen receptor，CAR)插入慢病毒载体，得到插入含有靶向RSV F蛋白的抗原结合结构域的嵌合抗原受体的慢病毒载体；
S2：将S1得到的慢病毒载体与包装质粒混合，得到包装系统；将包装系统转染入HEK 293T细胞培养，得到慢病毒；
S3：将慢病毒感染T淋巴细胞，得到靶向RSV F蛋白的CAR-T细胞。
优选地，步骤S1中所述含有靶向RSV F蛋白的抗原结合结构域的嵌合抗原受体的核苷酸序列如SEQ ID NO：3所示。
优选地，步骤S1中所述慢病毒载体为pRRLSIN。
优选地，所述pRRLSIN的制备方法如下：将pRRLSIN.cPPT.PGK-GFP.WPRE的氨苄抗性基因(Amp R)替换为卡那抗性基因(KanR)，同时，在XhoI和SalI之间插入多克隆位点，得到。
优选地，步骤S1中将含有靶向RSV F蛋白的抗原结合结构域的嵌合抗原受体(chimeric antigen rec eptor，CAR)插入慢病毒载体pRRLSIN的BamHI和MluI之间。
优选地，步骤S1还包括如下步骤：在插入含有靶向RSV F蛋白的抗原结合结构域的嵌合抗原受体的慢病毒载体中插入EF1α启动子及c-Myc标签，包括如下步骤：
S11：以pLVX-EF1α-CAR 5E5为模板，NheI-EF1α-F/SP-Myc-R为引物，扩增NheI-EF1α-Myc片段；以pRRLSIN-Palivizumab-BB为模板，Myc-RSV CAR-F/CD3-MIuI-R为引物扩增Myc-Palivizumab-BB-MIuI片段；
S12：以NheI-EF1α-Myc片段、Myc-Palivizumab-BB-MIuI片段为模板，以NheI-EF1α-F/CD3-MIuI-R为引物，扩增得到EF1α-Myc-Palivizumab-BB；
S13：以NheI和MIuI酶切EF1α-Myc-Palivizumab-BB及插入含有靶向RSV F蛋白的抗原结合结构域的嵌合抗原受体的慢病毒载体，连接，得到慢病毒载体。
优选地，步骤S2中所述包装系统包括第二代三质粒包装系统和第三代四质粒包装系统。
进一步优选地，步骤S2中所述包装系统为第三代四质粒包装系统，包含：S1得到的慢病毒载体、p MDLg/pRRE、pRSV-REV和pMD2.G。
优选地，步骤S2中所述的转染的方法包括但不限于电击转染法、脂质体转染法、PEI转染法等。
优选地，步骤S2中所述慢病毒的滴度为(3～5)×10 8IU/mL。
优选地，步骤S3中所述T淋巴细胞通过激活外周血单核细胞(PBMC)获得，具体步骤如下：用含 有抗人CD3单抗、重组人白细胞介素2、血浆的淋巴细胞培养基培养外周血单核细胞(PBMC)，得到。
优选地，所述淋巴细胞培养基为KBM 581 Serum-free Cell Medium。
优选地，所述外周血单核细胞(PBMC)通过血细胞分离机或Ficoll分离得到。
优选地，步骤S3中慢病毒感染T淋巴细胞的步骤如下：将慢病毒、T淋巴细胞与聚凝胺混合，离心感染，培养，得到。
本发明的第三方面，提供第二方面的组合物在制备抗肿瘤药物中的应用。
优选地，所述肿瘤包括肺癌、黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、宫颈癌、淋巴癌、胃癌、食道癌、肾癌、前列腺癌、胰腺癌和白血病；进一步为前列腺癌和/或肺癌。
本发明的第四方面，提供一种治疗肿瘤的方法，对受试者施用本发明第二方面的组合物中的外源性抗原，使得受试者的肿瘤组织或细胞中含有外源性抗原；向受试者施用本发明第二方面的组合物中的治疗剂。
本发明的有益效果是：
本发明提供了一种在肿瘤细胞中表达外源性抗原的方法，向肿瘤细胞中导入外源性抗原，使肿瘤组织中抗原异质性的肿瘤细胞表达同一特异性抗原，解决了肿瘤治疗面临的抗原异质性难题，所提供的外源性抗原靶点特异性高，免疫原性强，解决了肿瘤治疗缺乏特异性靶点的难题，相当于把肿瘤(内生性疾病)的治疗变成一种“感染性”疾病(存在异种抗原)，有利于开发安全有效的抗肿瘤药物。
本发明提供了一种组合物，包含外源性抗原和治疗剂，治疗剂以所述外源性抗原为靶点，对含外源性抗原的组织或细胞进行杀伤，并且不作用于不含外源性抗原的组织或细胞，从而特异性杀死组织或细胞，可以有效的杀死肿瘤细胞，为肿瘤治疗提供了新思路，另外外源性抗原可在不同个体不同类型的肿瘤中表达，因此是一种“广谱”的抗肿瘤方法，具有巨大的经济价值和社会意义。
附图说明
图1是实施例1中穿梭载体pShuttle-hTERTp-E1A-IRES-RSV F的质粒图谱。
图2是实施例1中Pac I酶切鉴定重组腺病毒载体pAd-hTERTp-E1A-IRES-RSV F的电泳结果图：其中，(A)是克隆1～10的质粒经Pac I酶切后的电泳结果图；(B)是克隆8的质粒经Pac I酶切后的电泳结果图。
图3是实施例1中PCR鉴定P2重组溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F的电泳结果图。
图4是实施例1中流式细胞术检测P2代重组溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F感染trex293细胞后RSV F蛋白表达的结果图。
图5是实施例1中流式细胞术检测溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F感染前列腺癌细胞系LnCap-FGC后外源抗原RSV F蛋白表达的结果图。
图6是实施例1中流式细胞术检测溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F感染非小细胞肺癌细胞系A549后外源抗原RSV F蛋白表达的结果图。
图7是实施例1中western blotting检测溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F感染非小细胞肺癌细胞系A549后外源抗原RSV F蛋白表达的结果图。
图8是实施例1中溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F感染非小细胞肺癌细胞系A549后的病毒滴度随时间变化的结果图。
图9是实施例1中溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F对非小细胞肺癌细胞系A549的溶瘤作用的结果图。
图10是实施例2中PCR扩增NheI-EF1α-Myc片段和Myc-Palivizumab-BB-MIuI片段的电泳结果图。
图11是实施例2中PCR扩增EF1α-Myc-Palivizumab-BB片段的电泳结果图。
图12是实施例2中菌落PCR鉴定慢病毒表达载体pRRLSIN-EF1α-myc-Palivizumab-BB的电泳结果图。
图13是实施例2中慢病毒表达载体pRRLSIN-EF1α-myc-Palivizumab-BB质粒图谱。
图14是实施例2中流式细胞术检测myc-Palivizumab-BB CAR-T细胞的阳性率的结果图。
图15是实施例 3中靶向RSV F的Palivizumab-BB CAR-T细胞联合溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F对肺癌的杀伤作用的结果图：其中，(A)是靶向RSV F的Palivizumab-BB CAR-T细胞联合溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F(5 TCID50)对肺癌的杀伤作用的结果图；(B)是靶向RSV F的Palivizumab-BB CAR-T细胞联合溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F(10TCID50)对肺癌的杀伤作用的结果图。
图16是实施例4中靶向RSV F的Palivizumab-BB CAR-T细胞联合溶瘤腺病毒OAd-hTERTp-E1A-I RES-RSV F对肿瘤杀伤的特异性的结果图。
具体实施方式
以下通过具体的实施例对本发明的内容作进一步详细的说明。
本实施例中所使用的材料、试剂等，如无特别说明，为从商业途径得到的试剂和材料。
实施例1外源抗原呼吸道合胞病毒F蛋白(RSV F)在肿瘤细胞的表达
1.溶瘤病毒OAd-hTERTp-E1A-IRES-RSV F的构建
(1)构建穿梭载体pShuttle-hTERTp-E1A-IRES-RSV F
以pShuttle载体(购于上海吉荧生物技术有限公司)为骨架构建穿梭载体，插入的目的片段包括人端粒酶逆转录酶启动子(hTERTp)、腺病毒E1A、内部核糖体进入位点序列(Internal ribosome entry site，IRES)、呼吸道合胞病毒F蛋白(RSV-F，核苷酸如SEQ ID NO：1所示；其氨基酸如SEQ ID NO：2所示)和bGH poly(A)。本实施例中目的片段的核苷酸序列如SEQ ID NO：5所示，由通用生物合成，并克隆到pShuttle载体的NotI和SalI酶切位点之间，构建的穿梭载体命名为pShuttle-hTERTp-E1A-IRES-RSV F，质粒图谱如图1所示。提取质粒，通过Pme I酶切进行线性化，-20℃冰箱保存。
(2)构建重组腺病毒载体pAd-hTERTp-E1A-IRES-RSV F
将步骤(1)得到的线性化的穿梭质粒和含5型腺病毒骨架的pAdEasy-1质粒(购于上海吉荧生物技术有限公司)转化感受态细胞BJ5183，通过同源重组构建重组腺病毒载体pAd-hTERTp-E1A-IRES-RSV F。挑取10个单菌落接种到3mL培养基进行培养，提取质粒，通过Pac I酶切进行鉴定，如若重组成功应被切成2个片段，其中一个较大的片段为～30kb，另一较小的片段应为4.5kb或3kb，如图2中(A)所示，克隆3、4、6、7、8、9号的质粒在Pac I酶切以后出现2条带且条带大小正确，说明可能均为阳性克隆。任意选择其中的8号克隆，将其质粒转化trans5α化学感受态细胞(北京全式金生物技术有限公司)，挑取单菌落摇菌培养，提取质粒通过Pac I酶切进一步鉴定，如图2中(B)所示出现2条带且条带大小正确，说明成功构建重组腺病毒载体pAd-hTERTp-E1A-IRES-RSV F，将菌液扩大培养并冻存。
(3)包装溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F
将trex293细胞消化制备成单细胞悬液、计数，以1×10 6个/孔接种到6孔板，37℃、5％CO 2培养箱中过夜培养。次日，取一支1.5mL EP管，加入100μL无血清DMEM培养液，加入20μL(2)得到的质粒溶液(PacI酶切，酶切质粒量为1μg左右)和8μL PEI 40K溶液，用枪头轻轻吹打混匀，室温静置20m in。在转染试剂和质粒共孵育过程中，从CO 2培养箱中取出提前一天铺好板的6孔板，去掉孔中培养液，加入1mL/孔无血清培养液，静置1min后弃掉，再加入0.9mL无血清培养液/孔，将培养板放入CO 2培养箱中继续培养。孵育20min后，从培养箱中取出培养板，加入质粒-转染试剂PEI混合液，并呈“∞”字形 轻轻晃动培养板使孔板中液体混匀，将培养板放入CO 2培养箱中过夜培养。第二天更换5％FBS的DMEM培养液。持续观察细胞，并根据细胞状态和培养液颜色进行板换液，直至细胞病变效应(CPE)出现(此过程一般需要10d左右)。以细胞培养液直接收集细胞并轻轻吹打混匀，-80℃/室温反复冻融3次，3000g离心10min收集上清得到P1代病毒液。
(4)病毒的扩增与纯化
将trex293细胞接种于10cm培养皿，待细胞生长至融合度70～80％时，向每个培养皿中加入合适量的P1代病毒液。观察细胞，感染2～3天后待细胞大部分部病变时，离心收集细胞沉淀，加入无血清DMEM培养液重悬细胞并反复冻融3次，3000g离心10min收集上清得到P2代病毒液。取100μL P2代病毒液提取基因组DNA，通过PCR扩增外源基因RSV F从而对重组溶瘤腺病毒进行鉴定，同时设置对照组，取等比例的trex293细胞进行反复冻融，并取上清液提取基因组DNA。结果如图3所示，lane1是以P2代病毒液提取基因组DNA为模板进行PCR，lane2是对照trex293细胞冻融上清液提取的基因组DNA为模板进行PCR，以P2代病毒液提取基因组DNA为模板扩增出外源基因RSV F条带且大小正确(～1700bp)，说明在trex293细胞中成功包装出溶瘤腺病毒。取P2代病毒感染trex293细胞，48h后收集细胞通过流式检测RSV F的表达，以未进行病毒感染的trex293细胞作为对照，如图4所示，在OAd感染的trex293细胞表面能检测到RSV F蛋白的表达，进一步说明在trex293细胞中能成功包装出溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F且RSV F能在细胞中正常表达。将P2代病毒在trex293细胞中继续扩增出P3代病毒并保存，再用P3代病毒扩增出P4代病毒，P1～P4代病毒均作为病毒种子，用于后续病毒的生产。
将trex293细胞扩大培养并接种到10层细胞工厂，取P4代病毒进行感染，2～3天大多数细胞出现病变，收获培养物离心，将细胞培养上清暂置于4～8℃冰箱，细胞沉淀反复冻融3次，离心收集上清液，将上清液和细胞培养上清混合，得到溶瘤病毒原液。将病毒原液经过澄清、核酸酶处理、中空纤维柱浓缩和缓冲液置换、层析、中空纤维柱浓缩、除菌过滤等纯化过程，将纯化的溶瘤腺病毒OAd-hTERTp-E1A-IRE S-RSV F进行分装，置于-80℃冰箱保存。取一管病毒纯化液，通过Reed-Muench法检测病毒滴度，病毒滴度为5.13×10 9 TCID50/mL。
2.溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F感染肿瘤细胞
(1)检测溶瘤腺病毒感染前列腺癌细胞系LnCap-FGC后外源抗原的表达
将前列腺癌细胞系LnCap-FGC接种到6孔细胞培养板，以MOI为10 TCID50的溶瘤病毒进行感染，通过流式细胞术检测细胞表面RSV F蛋白的表达，以未进行病毒感染的LnCap-FGC细胞作为对照。感染后48h细胞大量漂浮，收集细胞进行检测，结果如图5所示，溶瘤腺病毒感染后前列腺癌细胞表面有RSV F蛋白，阳性率达到～35％。
(2)溶瘤腺病毒感染非小细胞肺癌细胞系A549后外源抗原的表达
将非小细胞肺癌细胞系A549接种到24孔细胞培养板，分别以MOI为1 TCID50、5 TCID50、10 TCID50的溶瘤病毒进行感染，在不同时间点通过流式细胞术检测A549细胞表面RSV F蛋白的表达，以未进行病毒感染的A549细胞作为对照。结果如图6所示，OAd感染肿瘤细胞A549以后，肿瘤细胞表面外源抗原RSV F蛋白的表达与随着病毒剂量和感染时间的增加而增加，溶瘤病毒感染后第3天，在5 TCID50和10 TCID50时阳性率达到～20％，而在溶瘤病毒感染后第5天，在5 TCID50和10 TCID50时阳性率达明显增加，说明能通过溶瘤病毒能将外源抗原在肺癌细胞中成功表达，特别是在第5天～90％的A549细胞表面表达RSV F蛋白，使本来抗原异质性的肿瘤细胞表达同一抗原。
将A549细胞和trex293细胞分别接种到6孔板，以MOI为5 TCID50的溶瘤病毒进行感染，48h后收 集细胞提取总蛋白通过western blotting检测RSV F蛋白的表达。结果如图7所示，lane1为A549-OAd、l ane2为A549-NC、lane3为trex293-OAd、lane4为trex293-NC、lane5为PageRuler预染蛋白Marker(The rmo)，OAd感染后在A549和trex293细胞都能检测到RSV F蛋白的表达，而在未感染细胞不能检测到目的条带，进一步证明了溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F感染细胞后能表达外源抗原。
(3)溶瘤腺病毒在肺癌细胞系A549中的复制
将A549细胞接种到6孔细胞培养板，分别以MOI为1 TCID50和5 TCID50的OAd-hTERTp-E1A-IRES-RSV F感染A549细胞，在不同时间点收集病毒液，通过Adeno-X TM Rapid Titer Kit(Takara)检测病毒滴度。结果如图8所示，随着时间增加，病毒滴度明显升高，其中在5 TCID50下，96h的病毒滴度(1.93×10 7IFU/mL)是6h(7.11×10 5IFU/mL)的27倍，说明OAd能在A549细胞中复制。
(4)溶瘤腺病毒对肺癌细胞系A549的溶瘤作用
将A549细胞接种到6孔细胞培养板，分别以MOI为10 TCID50、20 TCID50和30 TCID50的OAd-hTERTp-E1A-IRES-RSV F感染A549细胞。感染后48h和72h在显微镜下观察细胞的生长情况，然后进行结晶紫染色。结果如图9所示，图9中“48h”为病毒感染后48h细胞的显微镜下图片，“72h”为病毒感染后72h细胞的显微镜下图片，“72h”为病毒感染后72h结晶紫染色后细胞的显微镜下图片，对照组细胞生长良好，细胞铺满视野，细胞呈梭型、膜结构完整、折光性好，而溶瘤病毒感染组细胞密度下降，细胞皱缩、细胞膜破碎；在同一时间点，高剂量组(20 TCID50和30 TCID50)比低剂量组(10 TCID50)细胞密度更低、细胞状态更差，而在相同剂量下，72h的细胞比48h细胞密度更低、细胞状态更差，说明溶瘤腺病毒对肺癌细胞系A549的溶瘤作用与剂量和时间呈正相关；此外通过结晶紫染色发现，溶瘤病毒感染组镜下观察到的活细胞明显减少，高剂量组几乎无活细胞存在。以上结果说明溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F对肺癌细胞系A549的具有显著的杀伤作用。
实施例2靶向RSV F的抗肿瘤药物组合Palivizumab-BB CAR-T细胞的制备
1.慢病毒pRRLSIN-EF1α-myc-Palivizumab-BB生产
(1)构建慢病毒表达载体pRRLSIN-EF1α-myc-Palivizumab-BB
CAR结构由抗原结合结构域、跨膜结构域、共刺激信号传导区和细胞内信号传导结构域串联而成，本实施例中CAR的核苷酸序列如SEQ ID NO：3所示，由通用生物人工合成，并克隆到慢病毒载体pRRLSIN(pRRLSIN通过pRRLSIN.cPPT.PGK-GFP.WPRE改造获得，具体如下：先将pRRLSIN.cPPT.PGK-GFP.WPRE中的氨苄抗性基因(AmpR)替换为卡那抗性基因(KanR)，获得pRRLSIN.cPPT.PGK-GFP.WPRE(KanR)；然后在pRRLSIN.cPPT.PGK-GFP.WPRE(KanR)的XhoI和SalI之间插入多克隆位点，得到pRRLSIN)的BamHI和MluI酶切位点之间，构建的重组慢病毒表达载体命名为pRRLSIN-Palivizumab-BB。在此基础上我们在Palivizumab-BB前面插入EF1α启动子，为了在后续方便检测CAR-T细胞的阳性率，我们在慢病毒表达质粒Palivizumab-BB的信号肽序列之后插入c-Myc标签，从而构建pRRLSIN-EF1α-Myc-Palivizumab-BB。
首先以pLVX-EF1α-CAR 5E5(具体构建方法已在专利文献CN112940137A：一种PD-1基因敲除的靶向MUC1的CAR-T细胞及其制备方法和应用，中公开)为模板，NheI-EF1α-F/SP-Myc-R为引物(NheI-EF1a-F的序列为：CTAGCTAGCGCTCCGGTGCCCGTCAGT，SEQ ID NO：6；SP-Myc-R的序列为：GAGGTCCTCTTCAGAGATAAGTTTTTGCTCCGGCCTGGCGGCGTGGA，SEQ ID NO：7)扩增得到NheI-EF1α-Myc片段；以pRRLSIN-Palivizumab-BB为模板，Myc-RSV CAR-F/CD3-MIuI-R为引物(Myc-RSV CAR-F的序列为：GAGCAAAAACTTATCTCTGAAGAGGACCTCCAAGTGACCCTGAGAGAGTCT，SEQ ID NO：8；CD3-MIuI-R的序列为：CGACGCGTTTAGCGAGGGGGCAGGGCCT，SEQ ID NO：9)扩增 得到Myc-Palivizumab-BB-MIuI片段，结果如图10所示，其中lane1为NheI-EF1α-Myc片段，lane2为Myc-Palivizumab-BB-MIuI片段。
再以图10中的lane1和lane2割胶回收产物模板(按照相同摩尔浓度配成)，以NheI-EF1α-F/CD3-MIuI-R为引物，通过融合PCR扩增得到的EF1α-Myc-Palivizumab-BB，结果如图11所示。
目的片段EF1α-Myc-Palivizumab-BB和载体pRRLSIN-Palivizumab-BB经NheI和MIuI酶切后，回输酶切产物进行连接，连接产物转化trans5α化学感受态细胞(北京全式金生物技术有限公司)。挑取8个单菌落通过菌落PCR进行鉴定，结果如图12所示，3号和8号克隆扩增出目的条带且条带明显，说明可能是阳性克隆。摇菌后提取质粒，将质粒测序，发现序列均正确。以上结果说明成功构建慢病毒表达载体pRRLSIN-EF1α-myc-Palivizumab-BB，质粒图谱如图13所示。用无内毒素质粒大提试剂盒(Endo-free Plasmid Maxi Kit，Omega)提取质粒，置于-20℃冰箱保存。
(2)慢病毒pRRLSIN-EF1α-myc-Palivizumab-BB包装与纯化
取冻存的HEK293T细胞复苏，用DMEM完全培养基(DMEM培养基+10％FBS)传代培养。将HEK293T细胞以3×10 6/mL密度接种至10层细胞工厂，加入DMEM完全培养基体积为1L，过夜培养后细胞能达到80～90％的融合度，进行质粒转染。准备1个T75培养瓶(A瓶)，按照终浓度为等摩尔浓度加入慢病毒表达质粒pRRLSIN-EF1α-myc-Palivizumab-BB、慢病毒包装质粒pMDLg/pRRE(Kan +)和pRSV-REV(Kan+)、慢病毒包膜质粒pMD2.G(Kan +)，加入无血清DMEM补至60mL。再准备1个T75培养瓶(B瓶)，加入1mg/mL的PEI(polysciences)5.25mL，加入无血清DMEM补至60mL。分别将A、B瓶液体混匀，静置5min。将B液加入A液中，充分混匀，静置20min，形成DNA-PEI复合物。将DNA-PEI复合物加入1L含5％FBS的DMEM培养基中，充分混匀，替换掉10层细胞工厂中培养液。在转染后的48h收集培养上清液约1L，放入2～8℃冰箱中保存。同时向10层细胞工厂中加入1L新鲜的含5％FBS的DMEM培养基，24h后收集培养上清液约1L，放入2～8℃冰箱中保存，重复该过程一次。将3次收集的约3L培养上清液混合，使用囊式滤器(Sartorius)去掉细胞和细胞碎片。将澄清过滤的慢病毒上清通过仕必纯切向流过滤系统
Figure PCTCN2022138691-appb-000001
浓缩到200～300mL。经过0.45μm滤膜过滤后，进行层析纯化。将纯化的慢病毒经过0.22μm滤器(Sartorius)除菌过滤，分装，-80℃冰箱保存。经过检测纯化后慢病毒的滴度为3.41×10 8IU/mL。
2.慢病毒pRRLSIN-EF1α-myc-Palivizumab-BB感染T细胞制备CAR-T细胞
取健康志愿者外周血20mL，肝素抗凝，离心分离后获得的血清，经56℃灭活备用。沉淀细胞用生理盐水稀释后加至装有Ficoll溶液的离心管中，以密度梯度离心法分离得PBMC，生理盐水洗涤2次，计数备用。用淋巴细胞培养基KBM 581Serum-free Cell Medium(Corning)重悬PBMC，调整细胞密度为1～2×10 6/mL，接种至T75细胞培养瓶，加入抗人CD3单抗(OKT-3)激活PBMC，同时补加500IU/mL的重组人白细胞介素2(rhIL-2)，5～10％血浆，在37℃、5％CO 2培养箱培养。PBMC经刺激培养过夜后，计数、离心、淋巴细胞培养基KBM 581Serum-free Cell Medium(重组人白细胞介素2(rhIL-2))重悬，使T淋巴细胞密度达到2～5×10 6/mL，将T淋巴细胞等分至6孔板中，加入纯化的慢病毒液(MOI＝5)和聚凝胺(polybrene，终浓度6μg/mL)。离心感染，700g，1.5h，置于37℃、5％CO 2培养箱培养。慢病毒感染24h后离心换液，重悬于KBM581培养基中，加入5～10％血浆和500IU/mL rhIL-2，于37℃、5％CO 2培养箱继续扩增培养。慢病毒感染后72h通过流式细胞术检测CAR-T细胞的阳性率，结果如图14，myc-Palivizumab-BB CAR-T细胞的阳性率达到47.0％。
实施例3靶向RSV F的Palivizumab-BB CAR-T细胞联合溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F对肺癌的杀伤作用
1.靶细胞A549-LUC的构建
将非小细胞肺癌细胞系A549接种到6孔细胞培养板，37℃、5％CO 2培养箱培养。第2天更换新鲜培养基(RPMI-1640+10％FBS)并加入慢病毒plv-LUC-IRES-neo(携带萤火虫荧光素酶Luciferase和新霉素抗性基因neo，本公司保存，具体制备方法如下：首先将萤火虫荧光素酶Luciferase插入pLVX-IRES-Neo载体的XhoI和XbaI酶切位点之间得到慢病毒表达载体pLVX-LUC-IRES-neo，然后将等摩尔浓度的pLVX-LUC-IRES-neo、慢病毒包装质粒pSPAX2和慢病毒包膜质粒pMD2.G共转染HEK293T细胞，转染后48h和72h收集细胞培养上清，10000g离心5min，所得上清即为慢病毒pLVX-LUC-IRES-neo，分装，-80℃冰箱保存)，4～6h后更换培养基，继续传代培养。感染后72h，加入G418进行筛选。5～7天后通过有限稀释法筛选单克隆细胞A549-LUC。
2.基于荧光素酶的细胞毒性实验
(1)消化A549-LUC细胞制备单细胞悬液作为靶细胞。用RPMI1640培养液清洗细胞2次，对细胞进行计数。将A549-LUC细胞分成2份，加入按照实施例1生产的溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F，MOI分别为10 TCID50和5 TCID50，将细胞按照1×10 5个细胞/mL进行重悬(先分别将需要加入的病毒和培养液混合，再重悬细胞)。
(2)取出2块96孔细胞培养板，将2份细胞分别按照100μL/孔接种于96孔板中(细胞密度为1×10 4个细胞/孔)，将培养板放入37℃、5％CO 2培养箱培养。
(3)溶瘤病毒与靶细胞孵育48h后，从培养箱中取出按照实施例2制备的Palivizumab-BB CAR-T细胞和T细胞作为效应细胞。500g离心3min收集细胞沉淀，用RPMI1640培养液清洗细胞2次，计数并调整细胞密度。将效应细胞按照效靶比分别为5、10、20加入上述96孔板中作为实验组，即分别加入0.5×10 5个/50μL/孔、1×10 5个/50μL/孔、2×10 5个/50μL/孔。同时设置靶细胞最小流明值组(MinCPS组)和最大流明值组(MaxCPS组)，其中MinCPS孔去掉原有培养液，加入150μL/孔含1％Tween20的10％FBS RPMI1640培养液，MaxCPS孔中则加入50μL/孔含10％FBS RPMI1640培养液。所有分组均设置3个复孔。将培养板300g离心3min后，放入培养箱中继续培养。
(4)次日，取出培养板300g离心4min后，按照75μL/孔去掉培养液。将解冻的SteadyGlo Reagent混匀，按照75μl/孔加入培养板中。避光孵育5min，按照100μl/孔将细胞裂解上清转移至检测白板中，在多功能酶标仪上检测各组的荧光强度。
根据下列公式计算杀伤效率，特异性杀伤效率(％)＝(MaxCPS-样品CPS)/(MaxCPS-MinCPS)×100％。结果如图15所示，免疫细胞联合5 TCID50或10 TCID50溶瘤腺病毒OAd-hTERTp-E1A-IRES-RS V F对A549-LUC的杀伤作用随着效靶比的升高而增强，并且CAR-T联合溶瘤病毒明显强于T细胞联合溶瘤病毒。
实施例4靶向RSV F的Palivizumab-BB CAR-T细胞联合溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F对肿瘤杀伤的特异性
为了证明Palivizumab-BB CAR-T细胞联合溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F的特异性，分别用Palivizumab-BB CAR-T(靶向RSV F蛋白，CART RSV)、CART 5E5(靶向MUC1 Tn抗原，A549细胞不表达MUC1 Tn，制备方法参见专利文献CN112940137A中的实施例2)、T细胞对A549-LUC和溶瘤腺病毒感染的A549-LUC进行细胞杀伤实验。
(1)按照实施例3消化A549-LUC细胞制备单细胞悬液作为靶细胞。用RPMI1640培养液清洗细胞2次，对细胞进行计数。将A549-LUC细胞分成2份，其中一份加入按照实施例1生产的溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F，MOI为10 TCID50，另一份不加溶瘤病毒，将两份细胞均按照1×10 5个细胞/ mL进行重悬。将2份细胞分别按照100μL/孔接种于96孔板中(细胞密度为1×10 4个细胞/孔)，将培养板放入37℃、5％CO 2培养箱培养。
(2)24h后，从培养箱中取出按照实施例2制备的CART(RSV)、CART(5E5)和T细胞作为效应细胞。500g离心3min收集细胞沉淀，用RPMI1640培养液清洗细胞2次，计数并调整细胞密度。将效应细胞分别按照1.5×10 5个/50μL/孔加入上述96孔板中作为实验组。同时设置MinCPS组和MaxCPS组，其中MinCPS孔去掉原有培养液，加入150μL/孔含1％Tween20的10％FBS RPMI1640培养液，MaxCPS孔中则加入50μL/孔含10％FBS RPMI1640培养液。所有分组均设置3个复孔。将培养板300g离心3min后，放入培养箱中继续培养。
(3)次日，取出培养板300g离心4min后，按照75μl/孔去掉培养液。将解冻的SteadyGlo Reagent混匀，按照75μL/孔加入培养板中。避光孵育5min，按照100μL/孔将细胞裂解上清转移至检测白板中，在多功能酶标仪上检测各组的荧光强度。
根据下列公式计算杀伤效率，特异性杀伤效率(％)＝(MaxCPS-样品CPS)/(MaxCPS-MinCPS)×100％。结果如图16所示，CAR-T(RSV)联合10 TCID50溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F对A549-LUC杀伤作用明显强于CAR-T(5E5)和10 TCID50溶瘤腺病毒联合组、CAR-T(RSV)组、溶瘤腺病毒组，而CAR-T(RSV)组、CAR-T(5E5)组和T细胞组对A549-LUC杀伤作用没有差异，说明CAR-T(RSV)对不表达外源抗原RSV F的A549-LUC细胞无特异性细胞毒性，而溶瘤腺病毒OAd-hTERTp-E1A-IRES-RSV F可以使A549-LUC细胞表达外源抗原RSV F，并且可以被靶向该抗原的CAR-T(RSV)特异性杀伤。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种组合物，包含：外源性抗原和治疗剂；
    所述外源性抗原为(1)～(2)中任一种：
    (1)非人蛋白或多肽；
    (2)编码(1)中所述非人蛋白或多肽的核酸分子；
    所述治疗剂以所述外源性抗原为靶点，作用于含所述外源性抗原的组织或细胞。
  2. 根据权利要求1所述的组合物，其特征在于：
    所述非人蛋白或多肽包括但不限于：细菌、酵母、原生动物、病毒以及人工合成的蛋白或多肽；
    优选地，所述非人蛋白或多肽为呼吸道合胞病毒的F蛋白。
  3. 根据权利要求2所述的组合物，其特征在于：
    所述外源性抗原通过递送载体或电穿孔转染到所述组织或细胞；
    优选地，所述外源性抗原包封在递送载体内；
    优选地，所述递送载体为肿瘤选择性递送载体；
    优选地，所述肿瘤选择性递送载体为天然聚合物、合成聚合物、阳离子肽、细胞穿透肽、生物可降解的纳米颗粒、脂质体、脂质复合物、聚合复合物、胶束、树枝状聚合物、凝胶、粘膜粘着剂、硅纳米针、纳米金颗粒、外泌体、病毒或假病毒。
  4. 根据权利要求3所述的组合物，其特征在于：
    所述病毒为溶瘤病毒；
    优选地，所述溶瘤病毒为腺病毒、痘苗病毒、辛德毕斯病毒、塞内加谷病毒、柯萨奇病毒、麻疹病毒、呼肠孤病毒、牛痘病毒、新城疫病毒、水疱性口炎病毒、单纯疱疹病毒、脊髓灰质炎病毒、流感病毒、腮腺炎病毒和细小病毒中的至少一种，进一步为腺病毒。
  5. 根据权利要求1所述的组合物，其特征在于：
    所述治疗剂包括CAR-T细胞、TCR-T细胞、CAR-NK细胞、抗原特异性T细胞、抗原特异性DC细胞、小分子靶向药和单克隆抗体；
    优选地，所述治疗剂为CAR-T细胞和/或单克隆抗体；
    优选地，所述治疗剂为CAR-T细胞。
  6. 根据权利要求5所述的组合物，其特征在于：
    所述CAR-T细胞的嵌合抗原受体包含靶向外源性抗原的抗原结合结构域；
    优选地，所述嵌合抗原受体还包含跨膜结构域，共刺激结构域和细胞内信号传导结构域；
    优选地，所述嵌合抗原受体的氨基酸序列如SEQ ID NO：4所示。
  7. 根据权利要求1～6任一项所述的组合物，其特征在于：
    所述组合物包含表达呼吸道合胞病毒的F蛋白的溶瘤腺病毒和靶向呼吸道合胞病毒的F蛋白的CAR-T细胞。
  8. 根据权利要求7所述的组合物，其特征在于：
    所述溶瘤腺病毒的制备方法，包括如下步骤：
    S1：将包含呼吸道合胞病毒F蛋白的目的基因插入载体中，得到包含目的基因的载体，单酶切所述包含目的基因的载体，得到线性化的包含目的基因的载体；
    S2：将线性化的包含目的基因的载体和含5型腺病毒骨架的pAdEasy-1质粒转化感受态细胞，得到重组腺病毒载体，单酶切所述重组腺病毒载体，得到线性化的重组腺病毒载体；
    S3：将线性化的重组腺病毒载体转染至细胞，得到溶瘤腺病毒；
    优选地，所述靶向呼吸道合胞病毒F蛋白的CAR-T细胞的制备方法，包括如下步骤：
    S1：将含有靶向呼吸道合胞病毒F蛋白的抗原结合结构域的嵌合抗原受体插入慢病毒载体，得到插入含有靶向呼吸道合胞病毒F蛋白的抗原结合结构域的嵌合抗原受体的慢病毒载体；
    S2：将S1得到的慢病毒载体与包装质粒混合，得到包装系统；将包装系统转染入HEK 293T细胞培养，得到慢病毒；
    S3：将慢病毒感染T淋巴细胞，得到靶向呼吸道合胞病毒F蛋白的CAR-T细胞。
  9. 权利要求1～8中任一项所述的组合物在制备抗肿瘤药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于：
    所述肿瘤包括肺癌、黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、宫颈癌、淋巴癌、胃癌、食道癌、肾癌、前列腺癌、胰腺癌和白血病。
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