WO2023179789A1 - 干扰趋化素样因子超家族成员6(cmtm6)表达的基因治疗载体的制备和抗肿瘤应用 - Google Patents

干扰趋化素样因子超家族成员6(cmtm6)表达的基因治疗载体的制备和抗肿瘤应用 Download PDF

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WO2023179789A1
WO2023179789A1 PCT/CN2023/083888 CN2023083888W WO2023179789A1 WO 2023179789 A1 WO2023179789 A1 WO 2023179789A1 CN 2023083888 W CN2023083888 W CN 2023083888W WO 2023179789 A1 WO2023179789 A1 WO 2023179789A1
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tumor
expression
cmtm6
vector
cells
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French (fr)
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宫丽崑
龙益如
孙建华
陈润秋
尉骁璐
童永亮
秦秋平
刘婷婷
徐俊玖
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中国科学院上海药物研究所
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Definitions

  • the present invention relates to the field of biomedicine, specifically to the preparation and application of gene therapy vectors that interfere with the expression of chemokine-like factor superfamily member 6 (CMTM6), especially as adeno-associated virus and lentiviral gene drugs targeting CMTM6. Use in the treatment of tumors.
  • CMTM6 chemokine-like factor superfamily member 6
  • immune checkpoint blockade therapy shows two obvious trends: the discovery of new immune checkpoint molecules and combination therapy.
  • new immune checkpoint molecules such as LAG3, TIM3, TIGIT and Siglec-15 have been gradually discovered and applied, expanding our understanding and regulation of T cell activity.
  • Target selection range for tumor immunotherapy is still low. Exploring new treatment targets and combination drug regimens is still one of the effective ways to improve treatment effects.
  • CMTM6 Chemokine-like factor superfamily member 6 (CKLF-like MARVEL transmembrane domain-containing protein 6, CMTM6) is a member of the human chemokine-like factor superfamily (CMTM family). CMTM6 contains a four-times-spanning MARVEL domain. The biological functions of CMTM6 and this family of proteins are currently poorly studied. On the surface of tumor cells, CMTM6 can serve as a post-translational modification regulatory molecule for PD-L1, interacting with PD-L1 and inhibiting its ubiquitin-proteasome degradation pathway and endosome-lysosomal degradation pathway to achieve PD-L1 Maintenance of cell membrane expression. CMTM6 is a potential tumor immunotherapy target, as well as a tumor diagnostic and prognostic biomarker.
  • Gene therapy is a method of introducing target genes into target cells to achieve therapeutic purposes.
  • Existing gene therapy vectors include liposomes, nanocarriers, naked DNA, adenovirus vectors, retroviral vectors, lentiviral vectors and adeno-associated virus vectors.
  • tumor immunotherapy based on gene therapy is still in its infancy: on the one hand, the effect of existing tumor immune gene therapy is still unsatisfactory, and the efficacy of preclinical and clinical trials needs to be improved; on the other hand, the existing tumor immune gene therapy options
  • Most of the targets are commonly used immune checkpoint molecules or cytokines. These targets have often been developed with antibody drugs or recombinant protein drugs. Gene therapy for these targets has not shown obvious advantages. Therefore, tumor immune gene therapy not only needs to improve the therapeutic effect and response rate, but also needs to explore targets that are difficult to develop with traditional antibodies and recombinant proteins. Both of these points require the application of new tumor immunotherapy targets in gene therapy, and attempts to use combination drugs , to improve the therapeutic effect.
  • CMTM6 serves as a potential tumor immunotherapy target. Its special membrane protein structural characteristics determine other types of The difficulty of drug development can be effectively utilized through gene therapy vectors. Therefore, developing a new gene therapy vector that interferes with CMTM6 expression and achieving good tumor treatment effects, as well as being widely used in the anti-tumor field, can improve the means and prospects of gene therapy in the field of tumor immunotherapy.
  • the purpose of the present invention is to provide a new cancer treatment target and corresponding treatment methods for the new target.
  • Another object of the present invention is to provide a gene therapy vector with anti-tumor effect that interferes with CMTM6 expression and its derivatives and compositions.
  • a gene therapy vector for targeted down-regulation of CMTM6, for preparing a composition or preparation said composition or preparation being used for: (a) prevention and/or or treat tumors; and/or (b) inhibit tumor cells.
  • the prevention and/or treatment of the gene therapy vector includes inhibiting tumor growth and/or metastasis.
  • the inhibition of the gene therapy vector includes inhibition of tumor cell growth and/or metastasis.
  • the gene therapy vector is combined with a drug selected from the following group: immune checkpoint antibodies, immune agonists, chemotherapy drugs, lipid metabolism regulating drugs, glucose metabolism regulating drugs, additional gene therapy carrier, or a combination thereof.
  • the combined medication regimen is to combine the gene therapy vector with chemotherapy drugs and/or lipid metabolism regulating drugs.
  • the tumor is a tumor of mammals (including human and non-human mammals).
  • the non-human mammal is a mouse.
  • the tumor is a human tumor.
  • the tumor is a tumor for which immune checkpoint antibody or immune checkpoint inhibitor treatment has failed or failed, or a tumor that is not suitable for treatment with immune checkpoint antibody or immune checkpoint inhibitor.
  • the tumor is a tumor for which PD-L1 antibody or PD-L1 inhibitor treatment fails or fails, or a tumor that is not suitable for treatment with PD-L1 antibody or PD-L1 inhibitor.
  • the tumor or tumor cell is a tumor or tumor cell with high expression of CMTM6.
  • the immune checkpoint is selected from the following group: PD-1, PD-L1, CTLA-4, B7-H3, LAG-3, VISTA, CD47, TIM-3, TIGIT, BTLA, Siglec -15 etc.
  • the immune checkpoint antibodies are PD-L1 antibodies and CTLA-4 antibodies.
  • the additional gene therapy vector refers to a gene therapy vector targeting immune checkpoints.
  • the tumors are tumors that express PD-L1 and tumors that do not express PD-L1.
  • the tumor is selected from the following group: tumors with high expression of PD-L1, tumors with medium expression of PD-L1, and tumors with low expression of PD-L1.
  • the tumor is a tumor with medium expression of PD-L1 or a tumor with low expression of PD-L1.
  • the tumor is a tumor with low expression of PD-L1.
  • high expression of PD-L1 means that the ratio of the amount of PD-L1 expressed by the tumor E1 to the amount of PD-L1 expressed by the normal tumor E0 (E1/E0)>1, more preferably ⁇ 1.5, preferably ⁇ 2.0.
  • “moderate expression of PD-L1” means that the ratio of the amount E1 of PD-L1 expressed by the tumor to the amount E0 of PD-L1 expressed by the normal tumor (E1/E0) is 0.5-1.1, more Optimally, it is 0.7-1.0, and more preferably, it is 0.8-0.9.
  • low expression of PD-L1 means that the ratio of the amount of PD-L1 expressed by the tumor E1 to the amount of PD-L1 expressed by the normal tumor E0 (E1/E0) ⁇ 1/2, more Best land ⁇ 1/3, better land ⁇ 1/4.
  • the gene therapy vector has a very significant inhibitory effect on tumors that low express PD-L1 or do not express PD-L1.
  • CMTM6 in the tumor or tumor cells is down-regulated or its activity is significantly reduced after administration of the gene therapy vector.
  • the expression of PD-L1 in the tumor or tumor cells is down-regulated or its activity is significantly reduced after administration of the gene therapy vector.
  • the tumors include but are not limited to: breast cancer, liver cancer, gastric cancer, colorectal cancer, melanoma, leukemia, lung cancer, kidney tumor, small intestine cancer, prostate cancer, colorectal cancer, prostate cancer, cervical cancer cancer, lymphoma, bone cancer, adrenal gland tumor, or bladder tumor.
  • the tumor is an in situ tumor or metastasis of the above-mentioned tumor.
  • the tumor cells are located in vitro or in vivo.
  • the tumor cells include but are not limited to: breast cancer cells, liver cancer cells, colorectal cancer cells, melanoma cells, non-small cell lung cancer cells, etc.
  • the gene therapy vector includes viral vectors and non-viral vectors.
  • the viral vector includes but is not limited to: lentivirus, adenovirus, retrovirus, adeno-associated virus, etc.
  • non-viral vectors include but are not limited to: naked DNA, liposomes, nanocarriers, etc.
  • the viral vector is a lentivirus.
  • the viral vector is an adeno-associated virus.
  • the gene therapy vector is selected from the following group:
  • the gene therapy vector carries or contains a coding sequence targeting DNA or RNA of CMTM6 and/or PD-L1.
  • the gene therapy vector selected from (Z1) and (Z2) carries or contains a coding sequence targeting DNA or RNA of CMTM6.
  • the gene therapy vector selected from (Z3) and (Z4) carries or contains a coding sequence that includes (a) DNA or RNA targeting CMTM6; and (b) DNA or RNA targeting PD-L1 or RNA.
  • the coding sequence carried or contained by the gene therapy vector is an oligonucleotide sequence that can target the expression of the CMTM6 gene in cells for degradation.
  • the oligonucleotide sequence is shRNA, siRNA, miRNA, sgRNA or lncRNA, most preferably shRNA or sgRNA.
  • the oligonucleotide sequence is shRNA or sgRNA.
  • the length of the shRNA is 17-62nt, preferably 18-23nt, most preferably 19-21nt.
  • the shRNA contains a hairpin structure.
  • the length of the sgRNA is 18-23nt, preferably 19-21nt, and most preferably 20nt.
  • the shRNA is selected from one or more of SEQ ID NO: 1-12, preferably 1-3.
  • the shRNA includes a derivative sequence that substitutes, adds or deletes 1-3 nucleotides to any one of SEQ ID NO: 1-12.
  • the sgRNA is selected from one or more of SEQ ID NO: 13-15, preferably 1-3.
  • the sgRNA includes a derivative sequence that substitutes, adds or deletes 1-3 nucleotides to any one of SEQ ID NO: 13-15.
  • the coding sequence carried or contained by the gene therapy vector is an oligonucleotide sequence that can target the degradation of PD-L1 gene expression in cells.
  • the oligonucleotide sequence is shRNA, siRNA, miRNA, sgRNA or lncRNA, most preferably shRNA or sgRNA.
  • the oligonucleotide sequence is shRNA.
  • the length of the shRNA is 17-62nt, preferably 18-23nt, most preferably 19-21nt.
  • the shRNA contains a hairpin structure.
  • the shRNA is selected from one or more of SEQ ID NO: 16-20, preferably There are 1-3 lands.
  • the shRNA includes a derivative sequence that substitutes, adds or deletes 1-3 nucleotides to any one of SEQ ID NO: 16-20.
  • the coding sequence carried or contained by the gene therapy vector includes a dual-targeting nucleotide sequence that can simultaneously target CMTM6 and PD-L1, and the dual-targeting nucleotide sequence includes:
  • the derivative sequence described in (a) is a derivative sequence in which 1-3 nucleotides are substituted, added or deleted to any one of SEQ ID NO: 1-15.
  • the derivative sequence described in (b) is a derivative sequence in which 1-3 nucleotides are substituted, added or deleted in any one of SEQ ID NO: 16-20.
  • sequences in group (a) and the sequences in group (b) have a synergistic inhibitory effect.
  • the dual-targeting nucleotide sequence has a very significant inhibitory effect on tumors that low express PD-L1 or do not express PD-L1.
  • a viral vector or a non-viral vector that can target the expression of CMTM6 in tumors and/or cells is provided, and the vector carries or contains a coding sequence that inhibits the expression of CMTM6.
  • the viral vector or non-viral vector has a very significant inhibitory effect on tumors that low express PD-L1 or do not express PD-L1.
  • low expression of PD-L1 means that the ratio of the amount of PD-L1 expressed by the tumor E1 to the amount of PD-L1 expressed by the normal tumor E0 (E1/E0) ⁇ 1/2, more Best land ⁇ 1/3, better land ⁇ 1/4.
  • the viral vector includes lentivirus and adeno-associated virus.
  • the non-viral vector is selected from the following group: naked DNA, liposomes, nanocarriers, etc.
  • the coding sequence that inhibits the expression of CMTM6 targets the DNA or RNA of CMTM6.
  • the coding sequence that inhibits CMTM6 expression is an oligonucleotide sequence that can target the degradation of CMTM6 gene expression in tumors and/or cells.
  • the oligonucleotide sequence is shRNA, siRNA, miRNA, sgRNA or lncRNA, most preferably shRNA or sgRNA.
  • the oligonucleotide sequence is shRNA or sgRNA.
  • the coding sequence that inhibits CMTM6 expression is an sgRNA or shRNA that targets CMTM6 inhibition, including:
  • the derived sequence described in (i) is obtained by subjecting any one of SEQ ID NOs: 1-12 to 1-3 nuclei. Derived sequences with substitutions, additions or deletions of nucleotides.
  • the derivative sequence described in (ii) is a derivative sequence in which 1-3 nucleotides are substituted, added or deleted to any one of SEQ ID NO: 13-15.
  • the length of the shRNA is 17-62nt, preferably 18-23nt, most preferably 19-21nt.
  • the length of the sgRNA is 18-23nt, preferably 19-21nt, and most preferably 20nt.
  • the shRNA is selected from one or more of SEQ ID NO: 1-12, preferably 1-3.
  • the sgRNA is selected from one or more of SEQ ID NO: 13-15, preferably 1-3.
  • the viral vector or non-viral vector can also inhibit the expression of PD-L1.
  • the viral vector or non-viral vector can also be used for combined medication.
  • the combined drug regimen is to combine the lentivirus with drugs including but not limited to selected from the following group: immune checkpoint antibodies, immune agonists, chemotherapy drugs, and lipid metabolism regulating drugs. , glucose metabolism regulating drugs, or combinations thereof.
  • the species of the cells is human or mouse.
  • the cells are located in vitro or in vivo.
  • the cells are tumor cells or non-tumor cells.
  • the lentivirus has an inhibitory effect on tumors and/or tumor cells.
  • the inhibitory effect can occur both in vivo and in vitro.
  • the tumor cells include but are not limited to: breast cancer cells, liver cancer cells, colorectal cancer cells, melanoma cells, non-small cell lung cancer cells, etc.
  • CMTM6 is down-regulated, or its activity is significantly reduced.
  • the expression of PD-L1 is down-regulated, or its activity is significantly reduced.
  • a dual-targeting viral vector that can simultaneously inhibit the expression of CMTM6 and PD-L1 in tumors and/or cells.
  • the dual-targeting viral vector carries or contains a selected coding sequence. Group from below:
  • the dual-targeting viral vector includes lentivirus and adeno-associated virus.
  • the derived sequence described in (i) is obtained by subjecting any one of SEQ ID NO: 1-15 to 1-3 nuclei. Derived sequences with substitutions, additions or deletions of nucleotides.
  • the derivative sequence described in (ii) is a derivative sequence in which 1-3 nucleotides are substituted, added or deleted to any one of SEQ ID NO:16-20.
  • sequences of group (i) and the sequence of group (ii) have a synergistic inhibitory effect.
  • the dual-targeting viral vector has a very significant inhibitory effect on tumors that express little or no PD-L1.
  • the dual-targeting viral vector can also be used for combined medication.
  • the combined drug regimen is to combine the dual-targeted viral vector with drugs including but not limited to selected from the following group: immune checkpoint antibodies, immune agonists, chemotherapy drugs, lipids Metabolism-modulating drugs, glucose metabolism-modulating drugs, or combinations thereof.
  • drugs including but not limited to selected from the following group: immune checkpoint antibodies, immune agonists, chemotherapy drugs, lipids Metabolism-modulating drugs, glucose metabolism-modulating drugs, or combinations thereof.
  • the coding sequence carried or contained by the dual-targeting viral vector includes (a) DNA or RNA targeting CMTM6; and (b) DNA or RNA targeting PD-L1.
  • the dual-targeting viral vector has an inhibitory effect on tumors and/or tumor cells.
  • the inhibitory effect can occur both in vivo and in vitro.
  • a polynucleotide encoding the genome of a vector selected from the group consisting of a vector as described in the second aspect of the present invention, or a vector as described in the third aspect of the present invention.
  • the polynucleotide includes DNA, RNA or cDNA.
  • an expression vector is provided, the expression vector containing the polynucleotide according to the fourth aspect of the present invention.
  • the expression vector includes a plasmid vector, a viral vector, a liposome, a nanovector or a combination thereof.
  • the viral vector includes a baculovirus vector, a lentivirus expression vector, an adenovirus expression vector, a transposon expression vector, or a combination thereof.
  • the lentiviral expression vector is a lentiCRISPR lentiviral expression vector.
  • the lentiviral expression vector is pLKO.1 lentiviral expression vector.
  • the adeno-associated virus vector is a pscAAV-EGFP-shRNA vector.
  • the adeno-associated virus vector is a pscAAV-EGFP-shRNA2 vector.
  • the expression vector further includes a modified expression vector.
  • the modification includes but is not limited to modification of the viral shell.
  • the modification is an RGD polypeptide modification.
  • amino acid sequence of the RGD polypeptide is CDCRGDCFC.
  • the expression vector can also be used for combined medicine.
  • the combined drug regimen is to combine the expression vector with drugs including, but not limited to, selected from the following group: immune checkpoint antibodies, immune agonists, chemotherapy drugs, and lipid metabolism regulating drugs. , glucose metabolism regulating drugs, or combinations thereof.
  • drugs including, but not limited to, selected from the following group: immune checkpoint antibodies, immune agonists, chemotherapy drugs, and lipid metabolism regulating drugs. , glucose metabolism regulating drugs, or combinations thereof.
  • the expression vector has a 5'-3' structure represented by Formula I: Z0-Z1-Z2-Z3(I)
  • each “-” is independently a bond or nucleotide connecting sequence
  • Z0 is none or enhancer
  • Z1 is the promoter element
  • Z2 is the first nucleotide molecule that reduces the expression of the first target gene
  • Z3 is an optional second nucleotide molecule that reduces expression of a second target gene.
  • the expression vector contains a promoter, a replication origin and a marker gene.
  • the promoter element includes a constitutive promoter, an inducible promoter, and a specific promoter.
  • the promoter element is selected from the following group: U6, CMV, EFl or a combination thereof.
  • the first target gene and the second target gene are different.
  • the first target gene is CMTM6.
  • the second target gene is PD-L1.
  • sequence of the first nucleotide molecule is selected from one or two of SEQ ID NO: 1-15 or its derivative sequences.
  • the derivative sequence is a derivative sequence in which 1-3 nucleotides are substituted, added or deleted to any one of SEQ ID NO: 1-15.
  • sequence of the second nucleotide molecule is selected from one or both of SEQ ID NO: 16-20 or its derivative sequences.
  • the derivative sequence is a derivative sequence in which 1-3 nucleotides are substituted, added or deleted in any one of SEQ ID NO: 16-20.
  • the expression vector includes a plasmid vector, a viral vector, a liposome, a nanovector or a combination thereof.
  • the viral vector includes a baculovirus vector, a lentivirus expression vector, an adenovirus expression vector, a transposon expression vector, or a combination thereof.
  • the expression vector is a lentiviral vector.
  • the expression vector is an adeno-associated virus vector.
  • the adeno-associated virus is a self-complementary adeno-associated virus.
  • a host cell in the sixth aspect of the present invention, contains the expression vector as described in the fifth aspect of the present invention, or the polynucleotide as described in the fourth aspect of the present invention is integrated into its genome.
  • the host cells include prokaryotic cells or eukaryotic cells.
  • the host cell is selected from the following group: Escherichia coli, yeast cells, HEK293T, HEK293F cells, CHO cells, etc.
  • the seventh aspect of the present invention there is provided a method for producing the vector as described in the second aspect of the present invention, or the dual-targeting viral vector as described in the third aspect of the present invention, comprising the steps:
  • step (c) Optionally, purify and/or modify the vector or dual-targeting viral vector obtained in step (b).
  • nucleic acid conjugate in an eighth aspect of the present invention, includes:
  • the other coupling moieties are selected from the following group: small molecule compounds, PEG, fluorescein, radioactive isotopes, fatty acid chains, protein fragments, polypeptides, or combinations thereof.
  • the components (a) and (b) are operably connected.
  • the coupling moiety includes chemical markers and biological markers.
  • the chemical label is selected from isotopes, immunotoxins and/or chemical drugs.
  • the biomarker is selected from biotin, avidin or enzyme markers.
  • the small molecule compound is selected from drugs or toxins for treating tumors or autoimmune diseases.
  • the radioactive isotopes include:
  • Diagnostic isotope the said diagnostic isotope is selected from the following group: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and/or
  • Therapeutic isotope the therapeutic isotope is selected from the following group: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb- 177, or a combination thereof.
  • the radioactive isotopes include but are not limited to iodine-131, indium-111 and lutetium-177.
  • the protein fragments include but are not limited to antibody Fc, biotin, avidin, HRP, antibodies, enzymes, cytokines and other biologically active proteins or polypeptides.
  • the coupling moiety is a detectable label.
  • the coupling moiety is selected from the group consisting of fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or is capable of producing Enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, and nanomagnetic particles that can detect products , prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or any form of nanoparticle.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the polypeptide molecules or fragments include but are not limited to: targeting PD-1, IL-4R, IL-4R ⁇ , TNF- ⁇ , VEGF, 4-1BB, CD47, TIM3, CTLA4, IL -17A, CD19, CD22, CD28, CD38, CD40, CD47, B7-H3, TSLP, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, HER2 polypeptide molecules or fragments.
  • polypeptide molecule is an RGD polypeptide or a derivative thereof.
  • the polypeptide molecule or fragment with therapeutic function includes a single-chain antibody (scFv), a diabody, a monoclonal antibody, or a chimeric antibody.
  • scFv single-chain antibody
  • diabody diabody
  • monoclonal antibody monoclonal antibody
  • chimeric antibody a single-chain antibody
  • the fusion protein also contains a tag sequence to assist expression and/or purification.
  • the tag sequence is selected from the following group: 6His tag, GGGS sequence, and FLAG tag.
  • the fusion protein includes bispecific antibodies and chimeric antibodies.
  • a pharmaceutical preparation which pharmaceutical preparation contains:
  • the preparation is a liquid dosage form.
  • the preparation is an injection.
  • the expression vector includes a lentiviral vector, an adeno-associated virus vector, or a combination thereof.
  • a pharmaceutical composition which pharmaceutical composition includes:
  • the pharmaceutical composition includes single drugs, compound drugs, or synergistic drugs.
  • the other biologically active drugs include immune checkpoint antibodies or gene therapy vectors, immune agonistic drugs, chemotherapy drugs, lipid metabolism regulating drugs, and glucose metabolism regulating drugs.
  • the gene therapy vector refers to a gene therapy vector targeting immune checkpoints.
  • the immune checkpoint is selected from the following group: PD-1, PD-L1, CTLA-4, B7-H3, LAG-3, VISTA, CD47, TIM-3, TIGIT, BTLA, Siglec -15 etc.
  • the targets of the immune checkpoint antibody include but are not limited to: PD-1, PD-L1, CTLA-4, B7-H3, LAG-3, VISTA, CD47, TIM-3, TIGIT, BTLA, Siglec-15, etc.
  • the immune agonist drugs are TLR receptor agonists, CD40 agonistic antibodies, STING agonists, CD3 antibodies, CD28 antibodies, etc.
  • the TLR receptor agonist is a TLR7 agonist.
  • the chemotherapy drug is selected from the group consisting of doxorubicin, paclitaxel, cisplatin, carboplatin, gemcitabine, pemetrexed, methotrexate, oxaliplatin, fluorouracil, etc.
  • the lipid metabolism regulating drug is selected from statins, mAb, etc.
  • statin is fluvastatin.
  • the glucose metabolism regulating drug is metformin.
  • the pharmaceutical composition is used for anti-tumor treatment.
  • the tumor is selected from, but is not limited to: breast cancer, liver cancer, gastric cancer, colorectal cancer, leukemia, lung cancer, kidney tumor, small intestine cancer, prostate cancer, colorectal cancer, prostate cancer, cervical cancer, Lymphoma, bone cancer, adrenal gland tumors, or bladder tumors.
  • the tumor is an in situ tumor or metastasis of the above-mentioned tumor.
  • the tumor is a tumor resistant to immune checkpoint antibody therapy.
  • the targets of the immune checkpoint antibody include but are not limited to: PD-1, PD-L1, CTLA-4, B7-H3, LAG-3, VISTA, CD47, TIM-3, TIGIT, BTLA, Siglec-15, etc.
  • the tumors are tumors that express PD-L1 and tumors that do not express PD-L1.
  • the tumor is selected from the following group: tumors with high expression of PD-L1, tumors with medium expression of PD-L1, and tumors with low expression of PD-L1.
  • the tumor is a tumor with medium expression of PD-L1 or a tumor with low expression of PD-L1.
  • the tumor is a tumor with low expression of PD-L1.
  • high expression of PD-L1 means that the ratio of the amount of PD-L1 expressed by the tumor E1 to the amount of PD-L1 expressed by the normal tumor E0 (E1/E0)>1, more preferably ⁇ 1.5, preferably ⁇ 2.0.
  • “moderate expression of PD-L1” means that the ratio of the amount E1 of PD-L1 expressed by the tumor to the amount E0 of PD-L1 expressed by the normal tumor (E1/E0) is 0.5-1.1, more Optimally, it is 0.7-1.0, and more preferably, it is 0.8-0.9.
  • low expression of PD-L1 means that the ratio of the amount of PD-L1 expressed by the tumor E1 to the amount of PD-L1 expressed by the normal tumor E0 (E1/E0) ⁇ 1/2, more Best land ⁇ 1/3, better land ⁇ 1/4.
  • the pharmaceutical preparation and pharmaceutical composition are administered in a manner selected from the following group: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal cavity. Spray and aerosol inhalation.
  • the pharmaceutical preparation and pharmaceutical composition are administered in a dosage form selected from the following group: liquid, solid, or gel.
  • a method for preventing and/or treating tumors comprising the steps of: administering to a subject in need a carrier as described in the second aspect of the present invention, a method as described in the third aspect of the present invention Dual-targeting viral vector, the expression vector as described in the fifth aspect of the present invention, the nucleic acid conjugate as described in the eighth aspect of the present invention, the pharmaceutical preparation as described in the ninth aspect of the present invention, or the tenth aspect of the present invention The pharmaceutical composition described in the aspect.
  • the subject is a human or non-human mammal.
  • the non-human mammal is a rodent, such as a mouse.
  • the expression vector is a viral vector or a non-viral vector.
  • the viral vector includes but is not limited to: lentivirus, adenovirus, retrovirus, adeno-associated virus, etc.
  • non-viral vectors include but are not limited to: naked DNA, liposomes, nanocarriers, etc.
  • the administration includes injection of a lentiviral vector, an adeno-associated viral vector, or a combination thereof.
  • the tumor is a tumor resistant to immune checkpoint antibody therapy.
  • the targets of the immune checkpoint antibody include but are not limited to: PD-1, PD-L1, CTLA-4, B7-H3, LAG-3, VISTA, CD47, TIM-3, TIGIT, BTLA, Siglec-15, etc.
  • the immune checkpoint antibodies are PD-L1 antibodies and CTLA-4 antibodies.
  • the tumors are tumors that express PD-L1 and tumors that do not express PD-L1.
  • the tumor is selected from the following group: tumors with high expression of PD-L1, tumors with medium expression of PD-L1, and tumors with low expression of PD-L1.
  • the tumor is a tumor with medium expression of PD-L1 or a tumor with low expression of PD-L1.
  • the tumor is a tumor with low expression of PD-L1.
  • high expression of PD-L1 means that the ratio of the amount of PD-L1 expressed by the tumor E1 to the amount of PD-L1 expressed by the normal tumor E0 (E1/E0)>1, more preferably ⁇ 1.5, preferably ⁇ 2.0.
  • “moderate expression of PD-L1” means that the ratio of the amount E1 of PD-L1 expressed by the tumor to the amount E0 of PD-L1 expressed by the normal tumor (E1/E0) is 0.5-1.1, more Optimally, it is 0.7-1.0, and more preferably, it is 0.8-0.9.
  • low expression of PD-L1 means that the ratio of the amount of PD-L1 expressed by the tumor E1 to the amount of PD-L1 expressed by the normal tumor E0 (E1/E0) ⁇ 1/2, more Best place ⁇ 1/3, better place ⁇ 1/4.
  • the tumors include but are not limited to: breast cancer, liver cancer, gastric cancer, colorectal cancer, melanoma, leukemia, lung cancer, kidney tumor, small intestine cancer, prostate cancer, colorectal cancer, prostate cancer, cervical cancer cancer, lymphoma, bone cancer, adrenal gland tumor, or bladder tumor.
  • the tumor is an in situ tumor or metastasis of the above-mentioned tumor.
  • the method of administration is selected from the group consisting of: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal spray and aerosol inhalation.
  • the dosage form for administration is selected from the group consisting of liquid, solid, or gel.
  • Figure 1 shows the coding pattern of the lentivirus knocking out CMTM6 expression of the present invention.
  • Figure 2 is a Western blot exposure picture showing that the sgRNA for knocking out CMTM6 expression of the present invention can delete CMTM6 expression in tumor cells in vitro.
  • Figure 3 shows the mouse tumor growth curve and end-point tumor weight.
  • the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the growth of CT26 colorectal cancer tumors in vivo.
  • Figure 4 shows the mouse tumor growth curve and end-point tumor weight.
  • the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the growth of MC38 colorectal cancer tumors in vivo.
  • Figure 5 shows the mouse tumor growth curve and end-point tumor weight.
  • the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the growth of B16F10 melanoma in vivo.
  • Figure 6 shows the mouse tumor growth curve and end-point tumor weight.
  • the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the growth of Hepa1-6 liver cancer tumors in vivo.
  • Figure 7 shows the mouse tumor growth curve and end-point tumor weight.
  • the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the growth of LLC non-small cell lung cancer tumors in vivo.
  • Figure 8 shows the coding pattern diagram of the lentivirus knocking out CMTM6 and PD-L1 expression of the present invention.
  • Figure 9 shows the mouse tumor growth curve.
  • the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the growth of PD-L1-deficient CT26 colorectal cancer tumors in vivo.
  • Figure 10 shows the mouse tumor growth curve and end-point tumor weight.
  • the lentivirus knocking out CMTM6 expression of the present invention can induce anti-tumor immune memory in vivo.
  • Figure 11 shows the mouse tumor growth curve and end-point tumor weight.
  • the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the growth of PD-L1-deficient B16F10 melanoma in vivo.
  • Figure 12 shows the mouse tumor growth curve and end-point tumor weight.
  • the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the growth of PD-1 and PD-L1 deficient non-responsive MC38 colorectal cancer in vivo.
  • Figure 13 shows the mouse tumor growth curve and end-point tumor weight.
  • the lentivirus knocking out CMTM6 expression of the present invention can significantly increase the growth of 4T1 breast cancer that is resistant to immune checkpoint therapy with PD-L1 antibodies in vivo.
  • Figure 14 shows the mouse tumor growth curve and end-point tumor weight.
  • the lentivirus knocking out CMTM6 expression of the present invention can significantly improve LLC non-small cells resistant to immune checkpoint therapy by PD-L1 antibodies and CTLA-4 antibodies in vivo. Growth of lung cancer.
  • Figure 15 shows photos of mouse lungs and lung coefficients.
  • the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the lung metastasis of B16F10 melanoma in vivo.
  • Figure 16 shows photos of mouse lungs and lung coefficients.
  • the lentivirus knocking out CMTM6 expression of the present invention in vivo It can significantly inhibit the lung metastasis of 4T1 breast cancer.
  • Figure 17 shows the coding pattern of the lentivirus interfering with CMTM6 expression of the present invention.
  • Figure 18 shows the mouse tumor growth curve and end-point tumor weight.
  • the lentivirus that interferes with CMTM6 expression and the lentivirus that interferes with PD-L1 expression of the present invention can significantly inhibit the in vivo growth of CT26 colorectal cancer tumors.
  • Figure 19 shows the coding pattern of the adeno-associated virus that interferes with CMTM6 expression of the present invention.
  • Figure 20 shows the coding pattern of the adeno-associated virus that knocks out CMTM6 expression of the present invention.
  • Figure 21 is a flow chart showing that the adeno-associated virus of the present invention can infect a variety of tumor cells in vitro.
  • Figure 22 fluorescence imaging photos show that the adeno-associated virus of the present invention can infect a variety of tumor cells in vitro.
  • Figure 23 The relative quantitative results of western blotting show that the adeno-associated virus of the present invention can reduce the expression of CMTM6 in tumor cells in vitro.
  • Figure 24 shows the flow cytometry diagram showing that the adeno-associated virus of the present invention can infect tumor cells in vivo.
  • Figure 25 shows the mouse tumor growth curve and end-point tumor weight.
  • the adeno-associated virus that interferes with CMTM6 expression of the present invention can inhibit the growth of CT26 colorectal cancer tumors in vivo.
  • Figure 26 shows the mouse tumor growth curve and end-point tumor weight.
  • the adeno-associated virus that interferes with CMTM6 expression of the present invention can inhibit the growth of B16 melanoma tumors in vivo.
  • Figure 27 shows the mouse tumor growth curve and end-point tumor weight.
  • the adeno-associated virus that interferes with CMTM6 expression of the present invention can inhibit the growth of Hepa1-6 liver cancer tumors in vivo.
  • Figure 28 shows the coding pattern of the adeno-associated virus that simultaneously interferes with CMTM6 and PD-L1 expression of the present invention.
  • Figure 29 shows that shRNA that interferes with PD-L1 expression reduces PD-L1 levels in CT26 tumor cells analyzed by flow cytometry.
  • Figure 30 shows the mouse tumor growth curve and end-point tumor weight.
  • the adeno-associated virus that interferes with the expression of CMTM6, the adeno-associated virus that interferes with the expression of PD-L1, and the adeno-associated virus that interferes with the expression of CMTM6 and PD-L1 simultaneously can be used in vivo. Inhibits CT26 colorectal cancer tumor growth.
  • Figure 31 shows tumor anatomy photos.
  • the adeno-associated virus that interferes with CMTM6 expression, the adeno-associated virus that interferes with PD-L1 expression, and the adeno-associated virus that interferes with the expression of CMTM6 and PD-L1 simultaneously can inhibit CT26 colorectal cancer tumors in vivo. grow.
  • Figure 32 shows the mouse tumor growth curve.
  • the adeno-associated virus that interferes with the expression of CMTM6 combined with the PD-L1 antibody drug of the present invention can inhibit the growth of CT26 colorectal cancer tumors in vivo.
  • Figure 33 shows the mouse tumor growth curve.
  • the adeno-associated virus that interferes with CMTM6 expression of the present invention combined with chemotherapy drugs can inhibit the growth of CT26 colorectal cancer tumors in vivo.
  • Figure 34 shows the mouse tumor growth curve.
  • the adeno-associated virus that interferes with CMTM6 expression of the present invention combined with immune agonist drugs can inhibit the growth of CT26 colorectal cancer tumors in vivo.
  • Figure 35 shows the mouse tumor growth curve.
  • the adeno-associated virus that interferes with CMTM6 expression of the present invention combined with sugar metabolism regulating drugs can inhibit the growth of CT26 colorectal cancer tumors in vivo.
  • Figure 36 shows the mouse tumor growth curve.
  • the adeno-associated virus that interferes with CMTM6 expression of the present invention is combined with lipolysis. Metabolism-modulating drugs can inhibit CT26 colorectal cancer tumor growth in vivo.
  • Figure 37 shows that the adeno-associated virus that interferes with CMTM6 expression of the present invention can improve the tumor immune microenvironment.
  • Figure 38 shows the coding pattern of the RGD polypeptide-modified adeno-associated virus of the present invention that interferes with CMTM6 expression.
  • Figure 39 shows the tumor anatomical endpoint tumor weight and tumor growth curve in mice.
  • the RGD polypeptide-modified adeno-associated virus of the present invention that interferes with CMTM6 expression can inhibit the growth of CT26 colorectal cancer tumors when administered systemically in vivo.
  • Figure 40 shows the growth curve of human RKO tumors.
  • the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the growth of RKO colon cancer tumors in vivo.
  • a gene therapy vector including lentivirus and adeno-associated virus
  • the lentivirus and adeno-associated virus constructed in the present invention can be used as gene vectors for sgRNA or shRNA that can degrade CMTM6 gene expression and can reduce the expression of CMTM6 in vitro and in vivo.
  • the inventors also selected colorectal cancer, melanoma, liver cancer, breast cancer, non-small cell lung cancer and other tumor models and metastasis models (including mouse tumors and human tumors) to evaluate the developed lentivirus that interferes with CMTM6 expression.
  • lentivirus and adeno-associated virus were modified and combined with drug regimens to test, and the developed lentivirus and adeno-associated virus that interfered with or down-regulated CMTM6 expression were evaluated for low PD-L1 expression.
  • the gene therapy vector of the present invention has a significant inhibitory effect on the growth and metastasis of various tumors, and the combination of drugs also shows excellent anti-tumor effects, showing broad clinical application prospects.
  • the gene therapy vector of the present invention has excellent therapeutic effects (including inhibiting tumor growth and/or metastasis) for tumors that are not suitable for treatment with conventional immune checkpoint drugs such as PD-L1 antibodies, and can be applied to certain tumors with Drug-resistant tumors, especially difficult-to-treat tumors that fail to respond to PD-L1 antibody therapy.
  • the term “contains” or “includes” can be open, semi-closed and closed. In other words, the term also includes “consisting essentially of,” or “consisting of.”
  • Sequence identity is determined by comparing two aligned sequence and determine the number of positions where identical residues occur. Typically, this is expressed as a percentage.
  • the measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.
  • the terms “subject” and “subject in need” refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cattle, horses, dogs, cats, pigs, sheep, goats.
  • gene therapy vector As used herein, the terms "gene therapy vector”, “gene therapy vector of the present invention”, “gene therapy vector that interferes with CMTM6 expression”, “gene vector”, etc. are used interchangeably, and all refer to the construct in this application that can be used in vitro and gene therapy vectors that reduce CMTM6 expression in vivo.
  • nucleic acid conjugates and fusion expression products include: drugs, toxins, cytokines (Cytokine), radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the nucleic acid molecules of the present invention or fragments thereof. the conjugate formed.
  • the present invention also includes cell surface markers or antigens that bind to the nucleic acid molecules or fragments thereof.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of the nucleotide molecules of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having substituent groups in one or more amino acid residues, or (iii) a mature polypeptide combined with another compound (such as a compound that extends the half-life of the polypeptide, e.g.
  • polyethylene glycol or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a protein sequence, or with Fusion protein formed by 6His tag).
  • additional amino acid sequence such as a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a protein sequence, or with Fusion protein formed by 6His tag.
  • Nucleotide molecules of the present invention may be in the form of DNA or RNA.
  • Forms of DNA include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • Biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.
  • sequence of the nucleotide molecule (or fragment thereof, or derivative thereof) of the present invention can be obtained entirely through chemical synthesis.
  • the sequence can then be introduced into a variety of existing nucleic acid molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the nucleotide sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate sequences as described above and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant nucleic acids can be performed using conventional techniques well known to those skilled in the art.
  • competent cells capable of taking up nucleic acid molecules can be harvested after the exponential growth phase and treated with the CaCl2 method, using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in culture can be selected from various conventional media. Cultivate under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced using an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed within the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • nucleic acids or vectors of the invention may be used alone, or may be combined or coupled with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties, or any combination thereof.
  • detectable markers for diagnostic purposes
  • therapeutic agents for therapeutic purposes
  • PK protein kinase
  • Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or those capable of producing a detectable product Enzymes.
  • Therapeutic agents that can be combined or coupled with the nucleic acid or carrier of the present invention include but are not limited to: 1. Radionuclides; 2. Biotoxicants; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5 Virus particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • Gene therapy is a method of introducing target genes into target cells to achieve therapeutic purposes.
  • Existing gene therapy vectors include adenovirus vectors, retroviral vectors, lentiviral vectors and adeno-associated virus vectors.
  • Adeno-associated virus AAV is a type of virus with simple structure, non-envelope and DNA defect. The life cycle of AAV relies on replicating viruses, such as adenovirus and herpes simplex virus. AAV has good tissue specificity, effective diffusion, low immunogenicity, high safety and stability. Therefore, recombinant adeno-associated virus is one of the promising viral vectors.
  • Lentiviral vectors are modified from type I human acquired immunodeficiency virus HIV-I. They can infect dividing cells and non-dividing cells. The encoded genes can be integrated into the genome of target cells. They have large coding capacity and broad application potential. One of the promising viral vectors.
  • CMTM6 protein As used herein, the terms "CMTM6 protein”, “polypeptide”, “protein of the invention” and “human CMTM6 protein” have the same meaning and are used interchangeably herein.
  • CMTM6 is a member of the human chemokine-like factor superfamily (CMTM family). CMTM6 contains a four-transmembrane MARVEL domain. On the surface of tumor cells, CMTM6 can serve as a post-translational modification regulatory molecule for PD-L1, interacting with PD-L1 and inhibiting its ubiquitin-proteasome degradation pathway and endosome-lysosomal degradation pathway to achieve PD-L1 Maintenance of cell membrane expression.
  • the inventor unexpectedly discovered that by down-regulating the expression of the protein of the present invention, the growth of tumor cells can be effectively inhibited. Especially for tumor cells with low expression or no expression of PD-L1, the present invention can more effectively inhibit tumors by inhibiting the expression of CMTM6.
  • the first nucleotide molecule refers to a nucleotide molecule capable of reducing the expression of the first target gene (ie, CMTM6).
  • sequence of the first nucleotide molecule is as shown in SEQ ID NO: 1-15.
  • the second nucleotide molecule refers to a nucleotide molecule capable of reducing the expression of the second target gene (ie, PD-L1).
  • sequence of the second nucleotide molecule is as shown in SEQ ID NO: 16-20.
  • the present invention also provides an expression vector, which contains the polynucleotide of the present invention.
  • the expression vector usually also contains a promoter, replication origin and/or marker gene, etc. Methods well known to those skilled in the art can be used to construct the expression vector required by the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as carbimycin, gentamicin, hygromycin, and ampicillin resistance.
  • representative promoters include (but are not limited to): U6, CMV, EFl promoter or combinations thereof.
  • the present invention also provides a method for treating tumors, that is, using a safe and effective amount of the expression vector according to the seventh aspect of the present invention, the nucleic acid conjugate according to the tenth aspect of the present invention, and the tenth aspect of the present invention.
  • the pharmaceutical preparation described in one aspect, or the pharmaceutical composition described in the twelfth aspect of the present invention is administered to a desired subject to treat tumors.
  • the present invention provides for the first time a gene therapy vector that can interfere with CMTM6 expression and its anti-tumor application.
  • the gene therapy vectors that interfere with CMTM6 expression such as lentivirus and adeno-associated virus of the present invention are effective in resisting the growth and development of tumors such as colorectal cancer, melanoma, liver cancer, breast cancer, and non-small cell lung cancer (including mouse tumors and human tumors). It has significant curative effect in metastasis.
  • the gene therapy vectors that interfere with CMTM6 expression such as lentiviruses and adeno-associated viruses provided by the present invention, have significant efficacy on tumors that are resistant to immune checkpoint antibody treatment such as PD-1/PD-L1/CTLA-4, and are effective for Tumors with low or no expression of PD-L1 also have significant drug effects, showing clinical application prospects.
  • the present invention provides for the first time the anti-tumor application of a combined drug regimen of a gene therapy vector that can interfere with CMTM6 expression.
  • the test results show that the gene therapy vector that can interfere with CMTM6 expression and immune checkpoint antibodies/immune agonists/chemotherapy drugs/lipids
  • the combination of metabolism-regulating drugs/glucose metabolism-regulating drugs has excellent anti-tumor effects.
  • the present invention provides for the first time a gene therapy vector that can simultaneously interfere with the expression of CMTM6 and PD-L1, showing an excellent compound anti-tumor effect that can reduce the in vivo growth of tumors on the one hand and stimulate anti-tumor immune memory on the other. , showing broad clinical application prospects.
  • the present invention provides for the first time a modified derivative of a gene therapy vector that interferes with CMTM6 expression, successfully achieving more convenient systemic administration.
  • the gene therapy vector that interferes with CMTM6 expression provided by the present invention for the first time can reshape the tumor immune microenvironment and stimulate anti-tumor immune responses of CD8 + T and NK cells, etc., revealing the value of CMTM6 as a tumor immune regulatory target.
  • the gene therapy vector of the present invention has a greatly improved therapeutic effect and an improved response rate, providing new methods for existing tumor treatment methods. technical means.
  • Example 1 Construction of lentiviral vector to knock out CMTM6 expression and evaluation of in vitro activity
  • the inventor first used software to design sgRNA oligonucleotides with a length of 18-24 nucleotides targeting the CMTM6 coding sequence in the human or mouse genome. The actual matching results were checked by NCBI database sequence comparison, and the preferred SEQ ID NO: 13-15.
  • the sgRNA short gene sequence used to construct a lentiviral vector for knocking out CMTM6 expression was obtained through in vitro DNA chemical synthesis; the synthesized sgRNA short gene sequence was cloned into lentiCRISPR lentiviral vector using enzyme digestion and enzyme ligation methods. Viral expression vector plasmid, gene sequencing to verify the accuracy of the insertion sequence.
  • the lentiviral vector is transformed into E. coli Stbl3 competent cells, and the amplification and extraction of the vector plasmid are completed; after the lentiviral vector plasmid is purified and extracted, the lentiviral vector plasmid and virus packaging plasmid system (pSPAX2 and pMD2.G) were co-transfected into HEK293T cells.
  • pSPAX2 and pMD2.G virus packaging plasmid system
  • CMTM6 Three days after the virus is packaged in HEK293T, use PEG concentration reagent to shake and concentrate overnight. After high-speed centrifugation (4000 ⁇ g, 30 minutes), the concentrated and purified lentivirus is obtained, which is the successfully constructed lentiviral vector that can be used to knock out CMTM6.
  • the lentiviral solution obtained above was infected into the following tumor cells adherently cultured in vitro: Hepa1-6 liver cancer cells, CT26 colorectal cancer cells, and B16F10 melanoma cells; after virus infection and antibiotic screening, 1 million cells were counted and passed through RIPA lysis and sonication were performed, and cell lysates were prepared for Western blotting to verify that the constructed lentivirus could be used to knock down CMTM6 expression in vitro.
  • the classic immunoblotting method was used to sequentially perform polyacrylamide gel electrophoresis, wet transfer, blocking, primary antibody incubation, secondary antibody incubation, and exposure to obtain the final immunoblot exposure picture, which is shown in Figure 2.
  • Example 2 Evaluation of in vivo anti-tumor activity of lentiviral vectors knocking out CMTM6 expression
  • the inventors evaluated its anti-tumor activity in various transplanted tumor models.
  • CT26 colorectal cancer tumor cells Resuscitate CT26 colorectal cancer tumor cells and passage them to ensure that the tumor cells have been passaged for at least 3 generations at the time of tumor bearing; inoculate the CT26 tumor cells subcutaneously into female BALB/c mice at an inoculation volume of 5 ⁇ 10 5 cells/mouse; and The mice were randomly divided into 2 groups (10 mice in each group), namely: Cas9 control lentivirus treatment group (Cas9 control) and lentivirus treatment group knocking out CMTM6 expression (CMTM6 KO).
  • Cas9 control Cas9 control
  • CMTM6 KO lentivirus treatment group knocking out CMTM6 expression
  • the animal is euthanized and the animal is dissected, and the subcutaneous tumor is removed; the mouse tumor growth curve and endpoint tumor weight are recorded and statistically recorded.
  • the results show that in vivo, the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the growth of CT26 colorectal cancer tumors, with a tumor inhibition rate as high as 73%.
  • MC38 colorectal cancer tumor cells Resuscitate and passage MC38 colorectal cancer tumor cells to ensure that the tumor cells have been passaged for at least 3 generations when bearing tumors; subcutaneously inoculate MC38 tumor cells into female C57BL/6 mice at an inoculation volume of 5 ⁇ 10 5 cells/mouse; and Will Mice were randomly divided into 2 groups (10 mice in each group), namely: Cas9 control lentivirus treatment group (Cas9 control) and lentivirus treatment group knocking out CMTM6 expression (CMTM6 KO).
  • Cas9 control Cas9 control
  • CMTM6 KO lentivirus treatment group knocking out CMTM6 expression
  • the animal is euthanized and the animal is dissected, and the subcutaneous tumor is removed; the mouse tumor growth curve and endpoint tumor weight are recorded and statistically recorded.
  • the results show that in vivo, the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the growth of MC38 colorectal cancer tumors, with a tumor inhibition rate of 33.4%.
  • B16F10 melanoma tumor cells Resuscitate B16F10 melanoma tumor cells and passage them to ensure that the tumor cells have been passaged for at least 3 generations when bearing tumors; subcutaneously inoculate B16F10 tumor cells into female C57BL/6 mice at an inoculation volume of 5 ⁇ 10 5 cells/mouse; and Mice were randomly divided into 2 groups (10 mice in each group), namely: Cas9 control lentivirus treatment group (Cas9 control) and lentivirus treatment group knocking out CMTM6 expression (CMTM6 KO).
  • Cas9 control Cas9 control
  • CMTM6 KO lentivirus treatment group knocking out CMTM6 expression
  • the animal is euthanized and the animal is dissected, and the subcutaneous tumor is removed; the mouse tumor growth curve and endpoint tumor weight are recorded and statistically recorded.
  • the results show that in vivo, the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the growth of B16F10 melanoma tumors, with a tumor inhibition rate of 57.8%.
  • Hepa1-6 liver cancer tumor cells Resuscitate Hepa1-6 liver cancer tumor cells and passage them to ensure that the tumor cells have been passed down for at least 3 generations when bearing tumors; Hepa1-6 tumor cells are subcutaneously inoculated into female C57BL/6 mice, and the inoculation volume is 5 ⁇ 10 5 cells/mouse. ; And the mice were randomly divided into 2 groups (10 mice in each group), namely: Cas9 control lentivirus treatment group (Cas9 control) and lentivirus treatment group knocking out CMTM6 expression (CMTM6 KO).
  • Cas9 control Cas9 control
  • CMTM6 KO lentivirus treatment group knocking out CMTM6 expression
  • the animal is euthanized and the animal is dissected, and the subcutaneous tumor is removed; the mouse tumor growth curve and endpoint tumor weight are recorded and statistically recorded.
  • the results show that in vivo, the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the growth of Hepa1-6 liver cancer tumors, with a tumor inhibition rate as high as 64.4%.
  • Resuscitate LLC non-small cell lung cancer tumor cells and passage them to ensure that the tumor cells have been passaged for at least 3 generations when bearing tumors; inoculate LLC tumor cells subcutaneously into female C57BL/6 mice at an inoculation volume of 5 ⁇ 10 5 cells/mouse; The mice were randomly divided into 2 groups (10 mice in each group), namely: Cas9 control lentivirus treatment group (Cas9 control) and lentivirus treatment group knocking out CMTM6 expression (CMTM6 KO).
  • Cas9 control Cas9 control
  • CMTM6 KO lentivirus treatment group knocking out CMTM6 expression
  • the animal is euthanized and the animal is dissected, and the subcutaneous tumor is removed; the mouse tumor growth curve and endpoint tumor weight are recorded and statistically recorded.
  • the results show that in vivo, the lentivirus knocking out CMTM6 expression of the present invention can significantly inhibit the growth of LLC non-small cell lung cancer tumors, with a tumor inhibition rate of 52%.
  • the lentiviral vector for knocking out CMTM6 expression of the present invention has excellent anti-tumor effects in vivo, and has been fully verified in colorectal cancer, melanoma, liver cancer, and non-small cell lung cancer, and its tumor growth inhibition effect is extremely high. It is good and has good application prospects.
  • Example 3 Evaluation of the activity of lentiviral vectors knocking out CMTM6 expression against PD-L1-deficient tumors
  • CMTM6 knocking out CMTM6 will reduce PD-L1 expression in tumors.
  • many patients have low or no expression of PD-L1, so it is very important to test whether the lentiviral vector of the present invention that knocks out CMTM6 expression can mediate in vivo growth inhibition of tumors with low or no expression of PD-L1.
  • Example 2 of the present invention the various transplanted tumor models used by the inventor include two tumors with low expression of PD-L1, CT26 and B16F10, and the lentiviral vector of the present invention that knocks out CMTM6 expression can The extremely significant inhibitory effects of 73% and 57.8% limited tumor growth in vivo, indicating that the lentiviral vector of the present invention is not affected by the low expression of PD-L1 in tumors.
  • the lentiviral vector of the present invention that knocks out CMTM6 expression shows the same tumor growth inhibition effect on ordinary CT26 cells as in Example 2;
  • the newly constructed lentiviral vector of the present invention that knocks out PD-L1 expression also has a good tumor inhibitory effect on ordinary CT26 cells;
  • the lentiviral vector of the present invention to knock out CMTM6 expression showed better anti-tumor effect. Specifically, the tumor inhibition rate was 100%, and the tumor inhibition rate of all tumor-bearing mice was 100%. Tumor regression, that is, an unexpected anti-tumor effect with a tumor regression rate of 100%.
  • the present invention constructed a lentiviral vector for simultaneous knockout of PD-L1 and CMTM6, and its coding pattern is shown in Figure 8.
  • the newly constructed lentiviral vector of the present invention that simultaneously knocks out CMTM6 and PD-L1 was used to treat CT26 colorectal cancer tumors, and results consistent with the above description were found, and the tumor regression rate reached 100%.
  • the inventors once again loaded the surviving mice with tumor regression on the opposite side of the limb with CT26 colorectal cancer cells, and observed and recorded the tumor growth.
  • the results show that the tumor regression of surviving mice treated with lentiviral vectors that simultaneously knock out CMTM6 and PD-L1 has unexpected anti-tumor immune memory for CT26 colorectal cancer tumors, and the tumors that bear tumors again The regression rate was still as high as 70%, and the volume and size of the unresolved tumors were also significantly lower than those in the control mice.
  • the lentiviral vector of the present invention can also stimulate anti-tumor immune memory in the body.
  • the inventors used the B16F10 melanoma xenograft tumor model to test the therapeutic effect of lentiviral vectors that simultaneously knock out CMTM6 and PD-L1. During the experimental period, the tumor growth curve and end-point anatomical tumor weight statistics were observed.
  • the anti-tumor effect of this lentiviral vector is significant, and is significantly better than the lentiviral vector constructed by the present invention to knock out PD-L1.
  • MC38 colorectal cancer xenograft tumor model In order to further test the therapeutic effect of the CMTM6 knockout lentiviral vector on PD-1/PD-L1 axis-deficient tumors, the inventors used the MC38 colorectal cancer xenograft tumor model.
  • MC38 colorectal cancer is a tumor insensitive to PD-L1 deficiency, that is, knocking out PD-L1 of MC38 has no significant effect on its growth in vivo.
  • the CMTM6 knockout lentiviral vector of the present invention had a significant effect, indicating that the CMTM6 knockout lentiviral vector of the present invention can effectively inhibit PD- L1-deficient non-responsive tumors.
  • the lentiviral vector of the present invention still has a good anti-tumor effect on PD-1/PD-L1-deficient tumors, and still has an inhibitory effect on tumors that are unresponsive to PD-L1 deficiency.
  • Example 4 Evaluation of the activity of lentiviral vectors knocking out CMTM6 expression in anti-immune checkpoint antibody-resistant tumors
  • Immune checkpoint antibody therapy represented by PD-1/PD-L1/CTLA-4 antibodies, has good results in the treatment of various tumors, but there are also many tumors that do not respond to immune checkpoint antibody therapy. For these unresponsive tumors, it is necessary to use combined drugs to improve the response rate and achieve therapeutic effects. Therefore, the inventors used two tumor models that are resistant to immune checkpoint antibody treatment: the 4T1 breast cancer xenograft tumor model and the LLC non-small cell lung cancer xenograft tumor model to test the lentiviral vector of the present invention for knocking out CMTM6 expression. Whether tumors can break immune checkpoint antibody resistance and non-response.
  • 4T1 tumor cells were subcutaneously inoculated into female BALB/c mice at an inoculation volume of 5 ⁇ 10 5 cells/mouse, and the mice were randomly divided into groups, namely: Cas9 control lentivirus + IgG administration group (Cas9 control + IgG), Cas9 control lentivirus + PD-L1 antibody administration group (Cas9 control + ⁇ PD-L1), CMTM6 knockout lentivirus + IgG administration group (CMTM6KO+IgG), CMTM6 knockout lentivirus + PD-L1 antibody administration group Drug group (CMTM6 KO+ ⁇ PD-L1).
  • Cas9 control lentivirus + IgG administration group Cas9 control + IgG
  • Cas9 control lentivirus + PD-L1 antibody administration group Cas9 control + ⁇ PD-L1
  • CMTM6 knockout lentivirus + IgG administration group CMTM6KO+IgG
  • the animal is euthanized and the animal is dissected, and the subcutaneous tumor is removed; the mouse tumor growth curve and endpoint tumor weight are recorded and statistically recorded.
  • the results show that the PD-L1 antibody does not have a significant inhibitory effect on 4T1 tumors, and the lentiviral vector of the present invention that knocks out CMTM6 expression can significantly inhibit the growth of 4T1 tumors.
  • the PD-L1 antibody combined with this The lentiviral vector of the invention can better and significantly antagonize the in vivo growth of 4T1, that is, the lentiviral vector of the invention that knocks out CMTM6 expression can respond to immune checkpoint-resistant tumors and improve the response of immune checkpoint-resistant/unresponsive tumors to Checkpoint antibody response.
  • LLC non-small cell lung cancer tumor cells were resuscitated and passaged to ensure that the tumor cells had been passaged for at least 3 generations at the time of tumor bearing; LLC tumor cells were subcutaneously inoculated into female C57BL/6 mice at an inoculation volume of 5 ⁇ 10 5 cells /only and randomly divided into groups, namely: Cas9 control lentivirus + IgG administration group (Cas9 control + IgG), Cas9 control lentivirus + PD-L1 antibody administration group (Cas9 control + ⁇ PD-L1), Cas9 control lentivirus + CTLA-4 antibody administration group (Cas9 control + ⁇ CTLA-4), CMTM6 knockout lentivirus + IgG administration group (CMTM6 KO+IgG), CMTM6 knockout lentivirus + PD-L1 antibody administration group (CMTM6 KO+ ⁇ PD-L1), CMTM6 knockout lentivirus+CTLA-4 administration group (CMTM6 KO+
  • the animal is euthanized and the animal is dissected, and the subcutaneous tumor is removed; the mouse tumor growth curve and endpoint tumor weight are recorded and statistically recorded.
  • the results show that PD-L1 antibodies and CTLA-4 antibodies indeed have no obvious inhibitory effect on LLC tumors, while the lentiviral vector of the present invention that knocks out CMTM6 expression can significantly inhibit LLC tumor growth.
  • PD -L1 antibody or CTLA-4 antibody combined with the lentiviral vector of the present invention can better and significantly antagonize the in vivo growth of LLC, that is, the lentiviral vector of the present invention that knocks out CMTM6 expression can respond to immune checkpoint-resistant tumors and improve Immune checkpoint-resistant/non-responsive tumors respond to checkpoint antibodies.
  • Example 5 Evaluation of anti-tumor metastasis activity of lentiviral vectors that knock out CMTM6 expression
  • the inventors used the lung metastasis model of B16F10 melanoma and the lung metastasis model of 4T1 breast cancer. To evaluate the effect of the lentiviral vector knocking out CMTM6 expression of the present invention in limiting tumor metastasis.
  • B16F10 melanoma tumor cells are resuscitated and passaged to ensure that the tumor cells have been passed down for at least 3 generations when bearing tumors; B16F10 tumor cells are injected into female C57BL/6 mice through the tail vein, and the cells are treated with Cas9 control lentiviral vector or the present invention's
  • the cell volume is 1 ⁇ 10 6 and the cell suspension volume is 200 ⁇ L.
  • the tumors were dissected, and the complete lung tissue was taken, weighed and photographed.
  • the lungs of the control group mice are already covered with melanoma metastases, while the lungs of the mice of the present invention are After treatment with lentiviral vectors that knock out CMTM6 expression, only a few melanoma metastases were seen in the lungs of mice, and their lung coefficients were also significantly lower than those of the control group, indicating that the pathological damage to their lungs was significantly lower than that of the control group.
  • CMTM6 expression of the present invention can limit the lung metastasis of melanoma.
  • 4T1 breast cancer tumor cells Resuscitate 4T1 breast cancer tumor cells and pass them down to ensure that the tumor cells have been passed down for at least 3 generations when bearing tumors; 4T1 tumor cells are injected into female BALB/c mice through the tail vein, and the cells are treated with Cas9 control lentiviral vector or the present invention's
  • the cell volume is 1 ⁇ 10 6 and the cell suspension volume is 200 ⁇ L.
  • the tumors were dissected, and the complete lung tissue was taken, weighed and photographed.
  • CMTM6 expression of the present invention can limit the lung metastasis of breast cancer tumors.
  • the lentiviral vector knocking out CMTM6 expression of the present invention showed an excellent inhibitory effect on tumor metastasis, indicating its application in tumor immunotherapy to prevent or limit tumor metastasis.
  • Example 6 Construction of lentiviral vectors that interfere with CMTM6 expression and evaluation of anti-tumor activity in vivo
  • Examples 1-5 have shown the in vivo and in vitro anti-tumor activities of the lentiviral vector for knocking out CMTM6 expression of the present invention.
  • the inventors also constructed a short hairpin RNA (shRNA) oligonucleotide-based vector for interfering with CMTM6 expression.
  • shRNA short hairpin RNA
  • Lentiviral vector is used to degrade CMTM6 transcribed RNA to interfere with the expression of CMTM6.
  • the inventor first used software to design shRNA oligonucleotides of 18-24 nucleotides in length targeting the CMTM6 RNA sequence in the human or mouse transcriptome. The actual matching results were checked by NCBI database sequence comparison, and the preferred are SEQ ID NO: 1-12.
  • shRNA short gene sequence used to construct a lentiviral vector that interferes with CMTM6 expression was obtained through in vitro DNA chemical synthesis; the synthesized shRNA short gene sequence was cloned into the pLKO.1 lentiviral expression vector plasmid through enzyme digestion and enzyme ligation, and gene sequencing verified the insertion. Sequence accuracy.
  • the lentiviral vector is transformed into E. coli Stbl3 competent cells, and the amplification and extraction of the vector plasmid are completed; after the lentiviral vector plasmid is purified and extracted, the lentiviral vector plasmid and virus packaging plasmid system (pSPAX2 and pMD2.G) were co-transfected into HEK293T cells.
  • pSPAX2 and pMD2.G virus packaging plasmid system
  • HEK293T Three days after the virus is packaged in HEK293T, use PEG concentration reagent to shake and concentrate overnight. After high-speed centrifugation (4000 ⁇ g, 30 minutes), the concentrated and purified lentivirus is obtained, which is the successfully constructed lentiviral vector that can be used to interfere with CMTM6.
  • the present invention also constructed a lentiviral vector for interfering with PD-L1 expression, and the construction process was the same as above.
  • the inventors evaluated its anti-tumor activity in the CT26 colorectal cancer xenograft tumor model.
  • CT26 colorectal cancer tumor cells Resuscitate CT26 colorectal cancer tumor cells and passage them to ensure that the tumor cells have been passaged for at least 3 generations when bearing tumors; inoculate CT26 tumor cells subcutaneously into female BALB/c mice at an inoculation volume of 5 ⁇ 10 5 cells/mouse; The mice were randomly divided into 3 groups (12 mice in each group), namely: unrelated shRNA lentivirus treatment group (shNT), lentivirus treatment group that interferes with CMTM6 expression (shCMTM6), and lentivirus treatment group that interferes with PD-L1 expression. (shPD-L1).
  • shNT unrelated shRNA lentivirus treatment group
  • shCMTM6 lentivirus treatment group that interferes with CMTM6 expression
  • lentivirus treatment group that interferes with PD-L1 expression lentivirus treatment group that interferes with PD-L1 expression.
  • the animal is euthanized and the animal is dissected, and the subcutaneous tumor is removed; the mouse tumor growth curve and endpoint tumor weight are recorded and statistically recorded.
  • the results show that in vivo, the lentivirus interfering with CMTM6 expression of the present invention can significantly inhibit the growth of CT26 colorectal cancer tumors, with a tumor inhibition rate as high as 74%, and the effect of the lentiviral vector interfering with PD-L1 expression. quite.
  • CMTM6 lentiviral vector constructed in the present invention that interferes with CMTM6 expression also has excellent anti-tumor activity in vivo.
  • Example 7 Construction of adeno-associated viral vector for interfering and knocking out CMTM6 expression
  • the inventors In addition to using lentivirus-based gene therapy methods to knock out or interfere with CMTM6 expression to inhibit tumor growth, the inventors also constructed a gene therapy method using adeno-associated virus (AAV) as a vector to knock out or knock out CMTM6 expression. .
  • AAV adeno-associated virus
  • the inventor first uses software to design shRNA oligonucleotides of 18-24 nucleotides in length targeting the CMTM6 RNA sequence in the human or mouse transcriptome.
  • the actual matching results are checked by NCBI database sequence comparison, preferably SEQ ID NO. :1-12;
  • the actual matching results are checked by NCBI database sequence comparison, preferably SEQ ID NO: 13-15.
  • the short gene sequence used to construct an adeno-associated virus vector that interferes with or knocks out CMTM6 expression is obtained through in vitro DNA chemical synthesis; the synthesized shRNA/sgRNA short gene sequence is cloned into the pscAAV-EGFP-shRNA vector plasmid through enzyme digestion and enzyme ligation. Gene sequencing verified insert sequence accuracy.
  • the pscAAV-EGFP-shRNA vector is transformed into E. coli DH5 ⁇ competent cells, and the amplification and extraction of the vector plasmid are completed; after the vector plasmid is purified and extracted, the pscAAV-EGFP-shRNA vector plasmid and virus packaging plasmid are The system (pAAV-RC9 and pHelper) were co-transfected into HEK293T-AAV cells.
  • a concentration reagent to obtain the concentrated and purified adeno-associated virus, which is a successfully constructed adeno-associated virus vector that can be used to interfere with or knock out CMTM6. Its genome encoding pattern is shown in Figure 19 and Figure 19. 20 shown.
  • Example 8 In vitro evaluation of adeno-associated virus vectors that interfere with CMTM6 expression
  • the inventors evaluated the ability of the constructed adeno-associated virus vector to interfere with CMTM6 expression in vitro to invade tumor cells and reduce the expression of CMTM6 in tumor cells.
  • the infection MOI of the adeno-associated virus is 1:10000; after 48 hours of virus infection, cells were taken to evaluate the invention's interference with CMTM6 expression at the flow cytometry, immunofluorescence and western blot levels.
  • the FITC fluorescence channel was used to detect the EGFP protein expressed in the cells.
  • the adeno-associated virus of the present invention can effectively enter tumor cells, with an infection efficiency of 46.85% in CT26 and as high as 84.49% in B16F10 cells. %.
  • the adeno-associated virus of the present invention effectively entered CT26 and B16F10 tumor cells, and efficiently expressed EGFP green fluorescent protein in the tumor cells.
  • the inventors In order to evaluate the ability of the adeno-associated virus to reduce the expression of CMTM6 in tumor cells in vitro, the inventors extracted the total protein and prepared immunoblotting samples from infected CT26 and B16F10 cells. After polyacrylamide gel electrophoresis, electroporation membrane, primary antibody and For secondary antibody incubation and exposure, Na+K ATPase was selected as the membrane protein internal reference. As shown in Figure 23, it was found that the adeno-associated virus of the present invention can significantly interfere with CMTM6 expression.
  • adeno-associated virus vector constructed by the present invention to interfere with CMTM6 expression has good entry Invasive tumor cell activity, and can effectively reduce the expression of CMTM6 in tumor cells in vitro.
  • Example 9 In vitro distribution evaluation of adeno-associated virus vectors that interfere with CMTM6 expression
  • the inventors confirmed that the constructed adeno-associated virus that interferes with CMTM6 expression can infect tumor cells in vitro and verified the infection effect in vivo.
  • CT26 colorectal cancer tumor cells Resuscitate CT26 colorectal cancer tumor cells and passage them to ensure that the tumor cells have been passaged for at least 3 generations when bearing tumors; inoculate CT26 tumor cells subcutaneously into female BALB/c mice at an inoculation volume of 5 ⁇ 10 5 cells/mouse; wait for After the tumor volume grows to 200 mm 3 , 1 ⁇ 10 10 vg of adeno-associated virus of the present invention is injected around the tumor.
  • mice Four days later, the mice were dissected, and the tumor tissues were taken and digested with type IV collagenase and hyaluronidase.
  • the cell digestion solution was filtered through a 200 ⁇ m cell mesh and washed twice with PBS buffer.
  • the tissue cell suspension was removed with red blood cell lysis solution. red blood cells in; mouse Fc blocker was used to block the Fc receptors of single cells.
  • the obtained single cell suspension was stained and labeled with fluorescent antibodies, and the EGFP fluorescence signal level of the CD45-negative tumor cells was detected by flow cytometry.
  • Example 10 Evaluation of anti-tumor activity of adeno-associated virus vectors that interfere with CMTM6 expression
  • the inventors evaluated its anti-tumor activity in various transplanted tumor models.
  • CT26 colorectal cancer tumor cells Resuscitate CT26 colorectal cancer tumor cells and passage them to ensure that the tumor cells have been passaged for at least 3 generations at the time of tumor bearing; inoculate the CT26 tumor cells subcutaneously into female BALB/c mice at an inoculation volume of 5 ⁇ 10 5 cells/mouse; and The mice were randomly divided into 2 groups, namely: control shRNA adeno-associated virus treatment group (shNC scAAV9) and adeno-associated virus vector treatment group that interferes with CMTM6 expression (shCMTM6 scAAV9).
  • shNC scAAV9 shRNA adeno-associated virus treatment group
  • shCMTM6 scAAV9 adeno-associated virus vector treatment group that interferes with CMTM6 expression
  • the animal is euthanized and the animal is dissected, and the subcutaneous tumor is removed; the mouse tumor growth curve and endpoint tumor weight are recorded and statistically recorded.
  • the results show that in vivo, the adeno-associated virus vector that interferes with CMTM6 expression of the present invention can significantly inhibit the growth of CT26 colorectal cancer tumors, with a tumor inhibition rate of 48.9%.
  • B16F10 melanoma tumor cells Resuscitate B16F10 melanoma tumor cells and passage them to ensure that the tumor cells have been passaged for at least 3 generations when bearing tumors; subcutaneously inoculate B16F10 tumor cells into female C57BL/6 mice at an inoculation volume of 5 ⁇ 10 5 cells/mouse; and Mice were randomly divided into 2 groups, namely: control shRNA adeno-associated virus treatment group (shNC scAAV9) and adeno-associated virus vector treatment group that interferes with CMTM6 expression (shCMTM6 scAAV9).
  • shNC scAAV9 shRNA adeno-associated virus treatment group
  • shCMTM6 scAAV9 adeno-associated virus vector treatment group that interferes with CMTM6 expression
  • the animal is euthanized and the animal is dissected, and the subcutaneous tumor is removed; the mouse tumor growth curve and endpoint tumor weight are recorded and statistically recorded.
  • the results show that in vivo, the adeno-associated virus vector that interferes with CMTM6 expression of the present invention can significantly inhibit the growth of B16F10 melanoma tumors, with a tumor inhibition rate of 58.8%.
  • Hepa1-6 liver cancer tumor cells Resuscitate Hepa1-6 liver cancer tumor cells and passage them to ensure that the tumor cells have been passed down for at least 3 generations when bearing tumors; Hepa1-6 tumor cells are subcutaneously inoculated into female C57BL/6 mice, and the inoculation volume is 5 ⁇ 10 5 cells/mouse. ; And the mice were randomly divided into 2 groups, namely: control shRNA adeno-associated virus treatment group (shNC scAAV9) and adeno-associated virus vector treatment group that interferes with CMTM6 expression (shCMTM6 scAAV9).
  • shNC scAAV9 shRNA adeno-associated virus treatment group
  • shCMTM6 scAAV9 adeno-associated virus vector treatment group that interferes with CMTM6 expression
  • the animal is euthanized and the animal is dissected, and the subcutaneous tumor is removed; the mouse tumor growth curve and endpoint tumor weight are recorded and statistically recorded.
  • the results show that in vivo, the adeno-associated virus vector that interferes with CMTM6 expression of the present invention can significantly inhibit the growth of Hepa1-6 liver cancer tumors, with a tumor inhibition rate as high as 78.8%.
  • adeno-associated virus vector that interferes with CMTM6 expression of the present invention has excellent in vivo anti-tumor effects and has been fully verified in colorectal cancer, melanoma, and liver cancer. It has excellent tumor growth inhibition effects and has application prospects.
  • Example 11 Construction of an adeno-associated virus vector that simultaneously interferes with the expression of CMTM6 and PD-L1 and evaluation of anti-tumor activity price
  • the inventors also constructed a gene therapy method using adeno-associated virus as a vector that can simultaneously interfere with the expression of CMTM6 and PD-L1.
  • the inventor first used software to design shRNA oligonucleotides of 18-24 nucleotides in length targeting the RNA sequences of CMTM6 and PD-L1 in human or mouse transcriptomes. The actual matching results were verified by NCBI database sequence comparison. , wherein targeting CMTM6 is preferably SEQ ID NO:1-15, and targeting PD-L1 is preferably SEQ ID NO:16-20.
  • the short gene sequence used to construct the adeno-associated virus vector was obtained through in vitro DNA chemical synthesis; the synthesized shRNA short gene sequence was cloned into the pscAAV-EGFP-shRNA2 vector plasmid simultaneously through enzyme digestion and enzyme ligation in pairs, and the insertion was verified by gene sequencing. Sequence accuracy.
  • the pscAAV-EGFP-shRNA2 vector is transformed into E. coli DH5 ⁇ competent cells, and the amplification and extraction of the vector plasmid are completed; after the vector plasmid is purified and extracted, the pscAAV-EGFP-shRNA2 vector plasmid and virus packaging plasmid are system (pAAV-RC9 and pHelper) together transfected into HEK293T-AAV cells.
  • a concentrated reagent is used to obtain the concentrated and purified adeno-associated virus, which is a successfully constructed adeno-associated virus vector that can be used to interfere with the expression of CMTM6 and PD-L1 at the same time. Its genome coding pattern is as shown in the figure. 28 shown.
  • shRNA interfering with CMTM6 expression has been evaluated in the previous embodiments of the present invention.
  • an adeno-associated virus was expressed to interfere with PD-L1 expression, and the PD-L1 level on the surface of colorectal cancer cell CT26 was analyzed by flow cytometry.
  • the target of the present invention was found.
  • shRNA or adeno-associated virus targeting PD-L1 can effectively reduce PD-L1 expression, and can still function effectively even in the presence of interferon to stimulate PD-L1 expression.
  • the inventors evaluated its anti-tumor activity in the CT26 colorectal cancer xenograft tumor model.
  • CT26 colorectal cancer tumor cells Resuscitate CT26 colorectal cancer tumor cells and passage them to ensure that the tumor cells have been passaged for at least 3 generations at the time of tumor bearing; inoculate the CT26 tumor cells subcutaneously into female BALB/c mice at an inoculation volume of 5 ⁇ 10 5 cells/mouse; and Mice were randomly divided into 4 groups, namely: control shRNA adeno-associated virus treatment group (shNC scAAV) and adeno-associated virus vector treatment group that interferes with CMTM6 expression (shCMTM6 scAAV); adeno-associated virus vector treatment group that interferes with PD-L1 expression (shPD-L1 scAAV); adeno-associated virus vector treatment group that simultaneously interferes with the expression of CMTM6 and PD-L1 (shCMTM6&PD-L1 scAAV); the dosage is 1 ⁇ 10 10 vg.
  • shNC scAAV control shRNA
  • mice After the subcutaneous tumors of the mice grow to a certain length, the animals are euthanized, the animals are dissected, and the subcutaneous tumors are removed; the mouse tumor growth curve and end-point tumor weight are recorded and statistically recorded.
  • the results show that in vivo, the adeno-associated virus vector that interferes with CMTM6 expression of the present invention can still significantly inhibit the growth of CT26 colorectal cancer tumors, and its anti-tumor effect is relatively better than interfering with PD-L1 expression.
  • the effect of adeno-associated virus vector; in addition, the adeno-associated virus vector constructed by the present invention that simultaneously interferes with the expression of CMTM6 and PD-L1 shows significantly better effects than the adeno-associated virus that interferes with CMTM6 expression and PD-L1 expression alone.
  • Example 12 Evaluation of anti-tumor activity of various combination regimens of adeno-associated virus vectors that interfere with CMTM6 expression price
  • the inventors also evaluated the combined medication regimen and effect of using the adeno-associated virus vector that interferes with CMTM6 expression of the present invention in combination with immune checkpoint antibodies, immune agonists, chemotherapy drugs, lipid metabolism regulating drugs, and glucose metabolism regulating drugs.
  • CT26 tumor cells were subcutaneously inoculated into female BALB/c mice, with an inoculation volume of 5 ⁇ 10 5 cells/mouse; mice were randomly divided into 4 groups in five combination drug regimens, namely: saline control, Adeno-associated virus vector treatment group (AAV) that interferes with CMTM6 expression; candidate combination drug treatment group (PD-L1 antibody or imiquimod or doxorubicin or fluvastatin or metformin); adeno-associated virus vector that interferes with CMTM6 expression Combination drug treatment (AAV+combination drug).
  • AAV Adeno-associated virus vector treatment group
  • the dosage of AAV is 1 ⁇ 10 10 vg;
  • PD-L1 antibody is 100 ⁇ g/animal, administered three times, once every three days; imiquimod is 50 ⁇ g/animal, administered five times, every three days.
  • Administer once; metformin is 200 ⁇ g/animal, administered five times, once every two days;
  • adriamycin is 100 ⁇ g/animal, administered three times, and administered once every three days;
  • fluvastatin is 200 ⁇ g/animal, administered once every three days; The medicine was administered five times, once every two days.
  • the animal is euthanized and the animal is dissected, and the subcutaneous tumor is removed; the mouse tumor growth curve and endpoint tumor weight are recorded and statistically recorded.
  • the results show that in the CT26 colorectal cancer xenograft tumor model, the adeno-associated virus that interferes with CMTM6 expression of the present invention can be widely used in combination with other drugs to achieve better results.
  • Excellent anti-tumor effect; the adeno-associated virus that interferes with CMTM6 expression of the present invention combined with immune checkpoint antibodies, immune agonists, chemotherapy drugs, lipid metabolism regulating drugs, and glucose metabolism regulating drugs can effectively improve the anti-tumor effect, and the anti-tumor effect of the combined drug
  • the highest tumor inhibition rate can reach 95.02%; among them, the tumor inhibition effect is especially good when combined with chemotherapy drugs and lipid metabolism regulating drugs.
  • Example 13 Evaluation of the effect of adeno-associated viral vectors that interfere with CMTM6 expression on the tumor immune microenvironment
  • Example 10 At the end of mouse anatomy of the CT26 transplanted tumor model in Example 10, subcutaneous tumors were removed, and 150 mg of tumor tissue was chopped into a pulp, and digested with hyaluronidase and collagenase IV at 37°C and 180 rpm for 1.5 hours. , processed into single cell suspension.
  • the pretreated cell suspension was divided into two parts, one was used for immunophenotyping analysis of myeloid cells and tumor cells, and the other was used for immunophenotyping analysis of lymphocytes.
  • cells are treated with 2-4 mL of sterile red blood cell lysis solution for 8 minutes and terminated with PBS buffer to remove red blood cells and debris in the sample suspension; cell samples are washed and set aside.
  • Lymphocyte sample suspension processing The cells are centrifuged with lymphocyte separation solution to obtain the lymphocyte separation layer, which is the tumor lymphocyte suspension. Wash and set aside.
  • the flow immunophenotyping results are shown in Figure 37.
  • the CMTM6 expression-interfering adeno-associated virus of the present invention targets the tumor immune microenvironment, can reshape the tumor immune microenvironment, and coordinate innate immunity and adaptive immunity to exert anti-tumor activity.
  • the activity of cytotoxic cells such as CD8+ T cells and NK cells, is mobilized, which can promote them to secrete powerful anti-tumor factors TNF- ⁇ , granzymes, etc.
  • the AAV of the present invention can also reduce CD4+ T cells Immunosuppressive analysis of cell expression, such as PD-1, CTLA-4, etc.; at the same time, the AAV of the present invention also reduces PD-L1 expressed by tumor cells.
  • the adeno-associated virus that interferes with CMTM6 expression of the present invention can achieve anti-tumor growth effects by targeting tumor tissue, regulating the tumor immune environment, mobilizing anti-tumor immune responses, and down-regulating inhibitory tumor immunity.
  • Example 14 Construction of RGD polypeptide-modified adeno-associated virus that interferes with CMTM6 expression
  • the optimal administration method for the adeno-associated virus constructed and used in the above embodiments of the present invention is peritumoral administration, which helps to improve its anti-tumor effect.
  • peritumoral administration helps to improve its anti-tumor effect.
  • the inventor constructed an RGD polypeptide-modified adeno-associated virus that interferes with CMTM6 expression.
  • the construction method is generally consistent with the construction method of the AAV genome vector in Example 7. The difference is that its viral shell needs to be modified, and the viral packaging plasmid pAAV -Insert an amino acid sequence at position 589 of RC9, the representative one is CDCRGDCFC, as shown in Figure 38.
  • the AAV packaged using pAAV-RC9-RGD has an RGD polypeptide sequence on its outer shell, which can specifically target tumor cells that highly express integrins and can be used for systemic administration.
  • Example 15 Evaluation of in vivo anti-tumor activity of RGD polypeptide-modified adeno-associated virus that interferes with CMTM6 expression
  • Example 14 of the present invention In order to evaluate the anti-tumor activity of systemic administration of the RGD polypeptide-modified adeno-associated virus that interferes with CMTM6 expression constructed in Example 14 of the present invention, the inventors used the CT26 transplanted tumor model for evaluation.
  • CT26 colorectal cancer tumor cells Resuscitate CT26 colorectal cancer tumor cells and passage them to ensure that the tumor cells have been passaged for at least 3 generations at the time of tumor bearing; inoculate the CT26 tumor cells subcutaneously into female BALB/c mice at an inoculation volume of 5 ⁇ 10 5 cells/mouse; and The mice were randomly divided into 2 groups, namely: control shRNA adeno-associated virus treatment group (shNC AAV-RGD) and RGD polypeptide-modified adeno-associated virus vector treatment group that interferes with CMTM6 expression (shCMTM6 AAV-RGD).
  • shNC AAV-RGD control shRNA adeno-associated virus treatment group
  • RGD polypeptide-modified adeno-associated virus vector treatment group that interferes with CMTM6 expression shCMTM6 AAV-RGD
  • the dosage was 1 ⁇ 10 10 vg, intraperitoneally. Record and count the endpoint tumor volume and endpoint tumor weight of mouse tumors.
  • the results show that in vivo, the RGD polypeptide-modified adeno-associated virus vector that interferes with CMTM6 expression of the present invention can still significantly inhibit the growth of CT26 colorectal cancer tumors through systemic administration, which expands the scope of the present invention.
  • Example 16 Evaluation of anti-tumor activity of lentiviral vectors that knock out CMTM6 expression in immune humanized systems
  • the inventor conducted an experiment to evaluate the anti-tumor activity of human RKO colon cancer tumors in an immune humanized system.
  • mice Female NSG mice were randomly divided into 4 groups, namely 1Cas9 control group, 2CMTM6 KO group, 3Cas9 Control+PBMCs group and 4CMTM6 KO+PBMCs group. Three days before tumor bearing, pet electric clippers were used to remove the hair in and around the armpit of the right forelimb of mice in the four groups.
  • human colorectal cancer tumor RKO cells are cultured for at least three generations after recovery. When the cells are in good condition and in the logarithmic growth phase, the tumor cells are digested with trypsin solution containing 0.05% EDTA and centrifuged, and then treated with cold PBS. Wash twice, resuspend in cold PBS and count;
  • PBMCs cells isolated from the same person were recovered and used immediately. PBMCs and RKO cells were mixed so that the RKO density was 5 ⁇ 10 7 cells/mL and the PBMCs density was 1 ⁇ 10 7 cells/mL.
  • the inoculation amount/tumor-bearing amount of group 12 is: 5 ⁇ 10 6 cells; the inoculation amount/tumor-bearing amount of group 34 is: 5 ⁇ 10 6 RKO cells and 1 ⁇ 10 6 PBMCs cells.
  • mice After tumor-bearing, the mice were monitored for tumor volume every 5 days for a total of 5 time points.
  • the lentiviral vector for knocking out CMTM6 expression of the present invention also has a significant inhibitory effect on human tumors.
  • Tumor immunotherapy has achieved breakthrough results in the field of cancer treatment, but there are still problems such as poor efficacy and low response rate that need to be solved.
  • PD-1/PD-L1 immune checkpoint antibody therapy as an example, there are many non-responsive tumor types in clinical practice, and its application in some tumor indications may even lead to tumor hyperprogression, which greatly limits this type of antibody drugs. Applications.
  • biomarkers for their efficacy is also a major research direction.
  • Currently, such as PD-L1 expression level and tumor mutation load, etc. have been used for screening and administration. patients, however the issue of medication for non-compliant patients remains to be resolved.
  • Trying to apply new types of targets may be one of the effective ways to solve the above problems.
  • the application of new targets should consider directly targeting the pain points of PD-1/PD-L1 antibodies, and try to reduce PD-L1 low expression, cold tumors, and PD-1/PD-L1 antibody treatment resistance when used alone or in combination. Patients can benefit from the drug.
  • the present invention provides for the first time a gene therapy vector that interferes with the expression of CMTM6, and also provides modifications and multi-type combination drugs for the gene therapy vector.
  • the gene therapy vector of the present invention targets the new anti-tumor target CMTM6, regulates the in vivo growth of tumor cells by interfering with CMTM6 gene expression, and can inhibit the in vivo growth of colorectal cancer, melanoma, breast cancer, non-small cell lung cancer, liver cancer, etc. , and achieved significant therapeutic effects on tumors with low or no expression of PD-L1, as well as tumors that are resistant to PD-1/PD-L1/CTLA-4 antibodies.
  • the gene therapy vector that interferes with the expression of CMTM6 provided by the present invention can fully stimulate the intratumoral anti-tumor immune response, especially the activation of CD8 + T cells and NK cell activity, showing that this target is a new target for tumor immunotherapy. point potential.

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Abstract

本发明公开了干扰趋化素样因子超家族成员6(CMTM6)表达的基因治疗载体的制备和抗肿瘤应用。具体地，本发明公开了编码靶向CMTM6的基因序列的基因治疗载体及其衍生物、载体编码的基因序列、制备方法、载体单独和联合其他药物***的作用。这些基因治疗载体包括腺相关病毒和慢病毒等，可抑制肿瘤组织内CMTM6的表达，通过改善肿瘤免疫抑制微环境，有效抑制小鼠及人源结直肠癌、黑色素瘤、肝癌、乳腺癌、非小细胞肺癌等的体内生长，展现出和免疫检查点抗体、化疗药物、免疫激动药物及代谢调节药物的联合作用来发挥强效抗肿瘤作用，并对PD-L1缺陷的肿瘤和免疫检查点抗体耐药的肿瘤具有显著药效。

Description

干扰趋化素样因子超家族成员6(CMTM6)表达的基因治疗载体的制备和抗肿瘤应用 技术领域
本发明涉及生物医药领域，具体地，涉及干扰趋化素样因子超家族成员6(CMTM6)表达的基因治疗载体的制备和应用，特别是作为靶向CMTM6的腺相关病毒和慢病毒基因药物在***中的用途。
背景技术
免疫检查点阻断疗法的发展表现出新型免疫检查点分子的发现和联合治疗两个明显趋势。除经典的CTLA-4和PD-1/PD-L1外，LAG3、TIM3、TIGIT和Siglec-15等新型免疫检查点分子被陆续发现并走向应用，扩展了我们对T细胞活性调节的认知和肿瘤免疫疗法的靶点选择范围。然而目前以免疫检查点阻断疗法为代表的肿瘤免疫治疗的疗效和响应率仍较低，探索新的治疗靶点和联合用药方案仍是提升治疗效果的有效途径之一。
趋化素样因子超家族成员6(CKLF-like MARVEL transmembrane domain-containing protein 6，CMTM6)为人类趋化素样因子超家族(CMTM家族)成员之一。CMTM6含有一个四次跨膜的MARVEL结构域，CMTM6及该家族蛋白的生物功能目前均研究较少。在肿瘤细胞表面，CMTM6可作为PD-L1的翻译后修饰调控分子，与PD-L1相互作用并通过阻遏其泛素-蛋白酶体降解途径和内体-溶酶体降解途径，实现对PD-L1细胞膜表达量的维持。CMTM6是潜在的肿瘤免疫治疗靶点，以及肿瘤诊断及预后生物标志物。
基因治疗是将目标基因导入靶细胞中以达到治疗目的的手段。现有的基因治疗载体包括脂质体、纳米载体、裸DNA、腺病毒载体、逆转录病毒载体、慢病毒载体和腺相关病毒载体等。
目前，基于基因治疗的肿瘤免疫治疗发展尚属起步阶段：一方面现有肿瘤免疫基因治疗的效果仍难以令人满意，临床前和临床试验疗效有待提高；另一方面现有肿瘤免疫基因治疗选用的靶点多为常用的免疫检查点分子或细胞因子等，这些靶点往往已有抗体药物或重组蛋白药物开发，针对这些靶点的基因治疗并未展现明显优势。因此，肿瘤免疫基因治疗既需要提高治疗效果和响应率也需要开拓传统抗体和重组蛋白难以开发的靶点，这两点都需要将新型肿瘤免疫治疗靶点应用于基因治疗，并可尝试联合用药，以提高治疗效果。
CMTM6作为潜在的肿瘤免疫治疗靶点其特殊的膜蛋白结构特征决定了其他类型 药物开发的难度，而通过基因治疗载体即可实现有效利用。因此，开发一种干扰CMTM6表达的新型基因治疗载体并达到良好的肿瘤治疗效果，且实现在抗肿瘤领域的广泛应用，可提高基因治疗在肿瘤免疫治疗领域的手段和前景。
发明内容
本发明目的就是提供一种新的癌症治疗靶点，以及相应的针对新靶点的治疗方法。
本发明的又一目的在于提供一种具有抗肿瘤作用的干扰CMTM6表达的基因治疗载体及其衍生物和组合物。
在本发明的第一方面，提供了一种用于靶向下调CMTM6的基因治疗载体的用途，用于制备一组合物或制剂，所述的组合物或制剂用于：(a)预防和/或***；和/或(b)抑制肿瘤细胞。
在另一优选例中，所述基因治疗载体的预防和/或治疗包括抑制肿瘤的生长和/或转移。
在另一优选例中，所述基因治疗载体的抑制包括对肿瘤细胞的生长和/或转移的抑制。
在另一优选例中，所述的基因治疗载体与选自下组的药物进行联用：免疫检查点抗体、免疫激动剂、化疗药物、脂代谢调节药物、糖代谢调节药物、额外的基因治疗载体，或其组合。
在另一优选例中，所述联合用药方案为将所述基因治疗载体与化疗药物和/或脂代谢调节药物联用。
在另一优选例中，所述肿瘤为哺乳动物(包括人和非人哺乳动物)的肿瘤。
在另一优选例中，所述非人哺乳动物为小鼠。
在另一优选例中，所述肿瘤为人源肿瘤。
在另一优选例中，所述的肿瘤是免疫检查点抗体或免疫检查点抑制剂治疗失效或失败的肿瘤，或不适合用免疫检查点抗体或免疫检查点抑制剂治疗的肿瘤。
在另一优选例中，所述的肿瘤是PD-L1抗体或PD-L1抑制剂治疗失效或失败的肿瘤，或不适合用PD-L1抗体或PD-L1抑制剂治疗的肿瘤。
在另一优选例中，所述的肿瘤或肿瘤细胞是CMTM6高表达的肿瘤或肿瘤细胞。
在另一优选例中，所述免疫检查点选自下组：PD-1、PD-L1、CTLA-4、B7-H3、LAG-3、VISTA、CD47、TIM-3、TIGIT、BTLA、Siglec-15等。
在另一优选例中，所述的免疫检查点抗体为PD-L1抗体和CTLA-4抗体。
在另一优选例中，所述额外的基因治疗载体指靶向针对免疫检查点的基因治疗载体。
在另一优选例中，所述的肿瘤为表达PD-L1的肿瘤和不表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤选自下组：高表达PD-L1的肿瘤、中表达PD-L1的肿瘤、低表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤为中表达PD-L1的肿瘤或低表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤为低表达PD-L1的肿瘤。
在另一优选例中，“高表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)＞1，更佳地≥1.5，更佳地≥2.0。
在另一优选例中，“中表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)为0.5-1.1，更佳地为0.7-1.0，更佳地为0.8-0.9。
在另一优选例中，“低表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)≤1/2，更佳地≤1/3，更佳地≤1/4。
在另一优选例中，所述基因治疗载体对低表达PD-L1或不表达PD-L1的肿瘤具有极显著的抑制效果。
在另一优选例中，所述的肿瘤或肿瘤细胞在施用所述基因治疗载体后，其CMTM6的表达是下调的，或其活性显著降低。
在另一优选例中，所述的肿瘤或肿瘤细胞在施用所述基因治疗载体后，其PD-L1的表达是下调的，或其活性显著降低。
在另一优选例中，所述的肿瘤包括但不限于：乳腺癌、肝癌、胃癌、大肠癌、黑色素瘤、白血病、肺癌、肾脏肿瘤、小肠癌、***癌、结直肠癌、***癌、***、淋巴癌、骨癌、肾上腺肿瘤、或***。
在另一优选例中，所述的肿瘤为上述肿瘤的原位瘤或转移瘤。
在另一优选例中，所述肿瘤细胞位于体外或体内。
在另一优选例中，所述肿瘤细胞包括但不限于：乳腺癌细胞、肝癌细胞、结直肠癌细胞、黑色素瘤细胞、非小细胞肺癌细胞等。
在另一优选例中，所述基因治疗载体包括病毒载体和非病毒载体。
在另一优选例中，所述的病毒载体包括但不限于：慢病毒、腺病毒、逆转录病毒、腺相关病毒等。
在另一优选例中，所述的非病毒载体包括但不限于：裸DNA、脂质体、纳米载体等。
在另一优选例中，所述的病毒载体为慢病毒。
在另一优选例中，所述的病毒载体为腺相关病毒。
在另一优选例中，所述的基因治疗载体选自下组：
(Z1)靶向抑制CMTM6表达的慢病毒；
(Z2)靶向抑制CMTM6表达的腺相关病毒；
(Z3)同时靶向抑制CMTM6表达和PD-L1表达的慢病毒；
(Z4)同时靶向抑制CMTM6表达和PD-L1表达的腺相关病毒；
(Z5)上述Z1～Z4的任意组合。
在另一优选例中，所述基因治疗载体携带或含有的编码序列靶向CMTM6和/或PD-L1的DNA或RNA。
在另一优选例中，选自(Z1)、(Z2)的基因治疗载体携带或含有的编码序列靶向CMTM6的DNA或RNA。
在另一优选例中，选自(Z3)、(Z4)的基因治疗载体携带或含有的编码序列包含(a)靶向CMTM6的DNA或RNA；和(b)靶向PD-L1的DNA或RNA。
在另一优选例中，所述基因治疗载体携带或含有的编码序列为可靶向降解细胞CMTM6基因表达的寡核苷酸序列。
在另一优选例中，所述的寡核苷酸序列为shRNA、siRNA、miRNA、sgRNA或lncRNA，最佳地为shRNA或sgRNA。
在另一优选例中，所述的寡核苷酸序列为shRNA或sgRNA。
在另一优选例中，所述的shRNA长度为17-62nt，较佳地为18-23nt，最佳地为19-21nt。
在另一优选例中，所述的shRNA包含发夹结构。
在另一优选例中，所述的sgRNA长度为18-23nt，较佳地为19-21nt，最佳地为20nt。
在另一优选例中，所述的shRNA选自SEQ ID NO:1-12中的一个或多个，最佳地为1-3个。
在另一优选例中，所述的shRNA包括对SEQ ID NO:1-12中任一进行1-3个核苷酸的替换、增添或删除的衍生序列。
在另一优选例中，所述的sgRNA选自SEQ ID NO:13-15中的一个或多个，最佳地为1-3个。
在另一优选例中，所述的sgRNA包括对SEQ ID NO:13-15中任一进行1-3个核苷酸的替换、增添或删除的衍生序列。
在另一优选例中，所述基因治疗载体携带或含有的编码序列为可靶向降解细胞PD-L1基因表达的寡核苷酸序列。
在另一优选例中，所述的寡核苷酸序列为shRNA、siRNA、miRNA、sgRNA或lncRNA，最佳地为shRNA或sgRNA。
在另一优选例中，所述的寡核苷酸序列为shRNA。
在另一优选例中，所述的shRNA长度为17-62nt，较佳地为18-23nt，最佳地为19-21nt。
在另一优选例中，所述的shRNA包含发夹结构。
在另一优选例中，所述的shRNA选自SEQ ID NO:16-20中的一个或多个，最佳 地为1-3个。
在另一优选例中，所述的shRNA包括对SEQ ID NO:16-20中任一进行1-3个核苷酸的替换、增添或删除的衍生序列。
在另一优选例中，所述基因治疗载体携带或含有的编码序列包括可同时靶向CMTM6和PD-L1的双靶向核苷酸序列，所述双靶向核苷酸序列包含：
(a)选自SEQ ID NO:1-15或其衍生序列中的一个或多个；和
(b)选自SEQ ID NO:16-20或其衍生序列中的一个或多个。
在另一优选例中，(a)中所述衍生序列为对SEQ ID NO:1-15中任一进行1-3个核苷酸的替换、增添或删除的衍生序列。
在另一优选例中，(b)中所述衍生序列为对SEQ ID NO:16-20中任一进行1-3个核苷酸的替换、增添或删除的衍生序列。
在另一优选例中，(a)组序列与(b)组序列具有协同抑制的效果。
在另一优选例中，所述双靶向核苷酸序列对低表达PD-L1或不表达PD-L1的肿瘤具有极显著的抑制效果。
在本发明的第二方面，提供了一种可靶向抑制肿瘤和/或细胞中CMTM6表达的病毒载体或非病毒载体，所述载体携带或含有抑制CMTM6表达的编码序列。
在另一优选例中，所述病毒载体或非病毒载体对低表达PD-L1或不表达PD-L1的肿瘤具有极显著的抑制效果。
在另一优选例中，“低表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)≤1/2，更佳地≤1/3，更佳地≤1/4。
在另一优选例中，所述病毒载体包括慢病毒和腺相关病毒。
在另一优选例中，所述的非病毒载体选自下组：裸DNA、脂质体、纳米载体等。
在另一优选例中，所述抑制CMTM6表达的编码序列靶向CMTM6的DNA或RNA。
在另一优选例中，所述抑制CMTM6表达的编码序列为可靶向降解肿瘤和/或细胞中CMTM6基因表达的寡核苷酸序列。
在另一优选例中，所述的寡核苷酸序列为shRNA、siRNA、miRNA、sgRNA或lncRNA，最佳地为shRNA或sgRNA。
在另一优选例中，所述的寡核苷酸序列为shRNA或sgRNA。
在另一优选例中，所述抑制CMTM6表达的编码序列为靶向抑制CMTM6的sgRNA或shRNA，包括：
(i)选自SEQ ID NO:1-12或其衍生序列中的一个或多个；或
(ii)选自SEQ ID NO:13-15或其衍生序列中的一个或多个。
在另一优选例中，(i)中所述衍生序列为对SEQ ID NO:1-12中任一进行1-3个核 苷酸的替换、增添或删除的衍生序列。
在另一优选例中，(ii)中所述衍生序列为对SEQ ID NO:13-15中任一进行1-3个核苷酸的替换、增添或删除的衍生序列。
在另一优选例中，所述的shRNA长度为17-62nt，较佳地为18-23nt，最佳地为19-21nt。
在另一优选例中，所述的sgRNA长度为18-23nt，较佳地为19-21nt，最佳地为20nt。
在另一优选例中，所述的shRNA选自SEQ ID NO:1-12中的一个或多个，最佳地为1-3个。
在另一优选例中，所述的sgRNA选自SEQ ID NO:13-15中的一个或多个，最佳地为1-3个。
在另一优选例中，所述的病毒载体或非病毒载体还可抑制PD-L1的表达。
在另一优选例中，所述的病毒载体或非病毒载体还可用于联合用药。
在另一优选例中，所述联合用药的方案为将所述慢病毒与包括但不限于选自下组的药物进行联用：免疫检查点抗体、免疫激动剂、化疗药物、脂代谢调节药物、糖代谢调节药物，或其组合。
在另一优选例中，所述细胞的种属为人或小鼠。
在另一优选例中，所述细胞位于体外或体内。
在另一优选例中，所述细胞为肿瘤细胞或非肿瘤细胞。
在另一优选例中，所述的慢病毒对肿瘤和/或肿瘤细胞具有抑制作用。
在另一优选例中，所述抑制作用在体内和体外均可发生。
在另一优选例中，所述肿瘤细胞包括但不限于：乳腺癌细胞、肝癌细胞、结直肠癌细胞、黑色素瘤细胞、非小细胞肺癌细胞等。
在另一优选例中，所述的肿瘤或细胞在施用所述慢病毒后，其CMTM6的表达是下调的，或其活性显著降低。
在另一优选例中，所述的肿瘤或细胞在施用所述慢病毒后，其PD-L1的表达是下调的，或其活性显著降低。
在本发明的第三方面，提供了一种可同时靶向抑制肿瘤和/或细胞中CMTM6和PD-L1表达的双靶向病毒载体，所述双靶向病毒载体携带或含有的编码序列选自下组：
(i)选自SEQ ID NO:1-15或其衍生序列中的一个或两个；和
(ii)选自SEQ ID NO:16-20或其衍生序列中的一个或两个。
在另一优选例中，所述双靶向病毒载体包括慢病毒和腺相关病毒。
在另一优选例中，(i)中所述衍生序列为对SEQ ID NO:1-15中任一进行1-3个核 苷酸的替换、增添或删除的衍生序列。
在另一优选例中，(ii)中所述衍生序列为对SEQ ID NO:16-20中任一进行1-3个核苷酸的替换、增添或删除的衍生序列。
在另一优选例中，(i)组序列与(ii)组序列具有协同抑制的效果。
在另一优选例中，所述双靶向病毒载体对低表达或不表达PD-L1的肿瘤具有极显著的抑制效果。
在另一优选例中，所述的双靶向病毒载体还可用于联合用药。
在另一优选例中，所述联合用药的方案为将所述双靶向病毒载体与包括但不限于选自下组的药物进行联用：免疫检查点抗体、免疫激动剂、化疗药物、脂代谢调节药物、糖代谢调节药物，或其组合。
在另一优选例中，所述的双靶向病毒载体携带或含有的编码序列包含(a)靶向CMTM6的DNA或RNA；和(b)靶向PD-L1的DNA或RNA。
在另一优选例中，所述的双靶向病毒载体对肿瘤和/或肿瘤细胞具有抑制作用。
在另一优选例中，所述抑制作用在体内和体外均可发生。
在本发明的第四方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的载体的基因组：如本发明第二方面所述的载体、或如本发明第三方面所述的双靶向病毒载体。
在另一优选例中，所述的多核苷酸包括DNA或RNA或cDNA。
在本发明的第五方面，提供了一种表达载体，所述表达载体含有如本发明第四方面所述的多核苷酸。
在另一优选例中，所述的表达载体包括质粒载体、病毒载体、脂质体、纳米载体或其组合。
在另一优选例中，所述的病毒载体包括杆状病毒载体、慢病毒表达载体、腺病毒表达载体、转座子表达载体、或其组合。
在另一优选例中，所述慢病毒表达载体为lentiCRISPR慢病毒表达载体。
在另一优选例中，所述慢病毒表达载体为pLKO.1慢病毒表达载体。
在另一优选例中，所述腺相关病毒载体为pscAAV-EGFP-shRNA载体。
在另一优选例中，所述腺相关病毒载体为pscAAV-EGFP-shRNA2载体。
在另一优选例中，所述表达载体还包括进行修饰的表达载体。
在另一优选例中，所述修饰包括但不限于对病毒外壳的改造。
在另一优选例中，所述修饰为RGD多肽修饰。
在另一优选例中，所述RGD多肽的氨基酸序列为CDCRGDCFC。
在另一优选例中，所述的表达载体还可用于联合用药。
在另一优选例中，所述联合用药的方案为将所述表达载体与包括但不限于选自下组的药物进行联用：免疫检查点抗体、免疫激动剂、化疗药物、脂代谢调节药物、糖代谢调节药物，或其组合。
在另一优选例中，所述表达载体具有5’-3’的式I所示的结构：
Z0-Z1-Z2-Z3(I)
式中，各“-”独立地为键或核苷酸连接序列；
其中，Z0为无或增强子；
Z1为启动子元件；
Z2为降低第一靶基因的表达的第一核苷酸分子；
Z3为任选的降低第二靶基因的表达的第二核苷酸分子。
在另一优选例中，所述的表达载体含有启动子、复制起点和标记基因。
在另一优选例中，所述启动子元件包括组成型启动子、诱导型启动子、特异型启动子。
在另一优选例中，所述启动子元件选自下组：U6、CMV、EF1或其组合。
在另一优选例中，所述第一靶基因和第二靶基因不同。
在另一优选例中，所述第一靶基因为CMTM6。
在另一优选例中，所述第二靶基因为PD-L1。
在另一优选例中，所述第一核苷酸分子的序列选自SEQ ID NO:1-15或其衍生序列中的一个或两个。
在另一优选例中，所述衍生序列为对SEQ ID NO:1-15中任一进行1-3个核苷酸的替换、增添或删除的衍生序列。
在另一优选例中，所述第二核苷酸分子的序列选自SEQ ID NO:16-20或其衍生序列中的一个或两个。
在另一优选例中，所述衍生序列为对SEQ ID NO:16-20中任一进行1-3个核苷酸的替换、增添或删除的衍生序列。
在另一优选例中，所述的表达载体包括质粒载体、病毒载体、脂质体、纳米载体或其组合。
在另一优选例中，所述的病毒载体包括杆状病毒载体、慢病毒表达载体、腺病毒表达载体、转座子表达载体、或其组合。
在另一优选例中，所述的表达载体为慢病毒载体。
在另一优选例中，所述的表达载体为腺相关病毒载体。
在另一优选例中，所述腺相关病毒为自身互补腺相关病毒。
在本发明的第六方面，提供了一种宿主细胞，所述宿主细胞含有如本发明第五方面所述的表达载体，或其基因组中整合有如本发明第四方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞、HEK293T、HEK293F细胞、CHO细胞等。
在本发明的第七方面，提供了一种生产如本发明第二方面所述的载体、或如本发明第三方面所述的双靶向病毒载体的方法，包括步骤：
(a)在适合的条件下，将如本发明的第四方面所述的多核苷酸导入一宿主细胞或培养如本发明第六方面所述的宿主细胞，从而获得所述载体或双靶向病毒载体的培养物；
(b)从所述培养物中分离和/或回收所述的载体或双靶向病毒载体；
(c)任选地，对步骤(b)获得的所述载体或双靶向病毒载体进行纯化和/或修饰。
在本发明的第八方面，提供了一种核酸偶联物，所述核酸偶联物包括：
(a)如本发明第四方面所述的多核苷酸；和
(b)其他偶联部分。
在另一优选例中，所述的其他偶联部分选自下组：小分子化合物、PEG、荧光素、放射性同位素、脂肪酸链、蛋白片段、多肽、或其组合。
在另一优选例中，所述的组分(a)和(b)可操作性连接。
在另一优选例中，所述的偶联部分包括化学标记和生物标记。
在另一优选例中，所述化学标记选自同位素、免疫毒素和/或化学药物。
在另一优选例中，所述生物标记选自生物素、亲和素或酶标记。
在另一优选例中，所述小分子化合物选自***或自身免疫性疾病药物或毒素。
在另一优选例中，所述的放射性同位素包括：
(i)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188，或其组合；和/或
(ii)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、Xe-133、Yb-169、Yb-177，或其组合。
在另一优选例中，所述的放射性同位素包括但不限于碘131、铟111和镥177。
在另一优选例中，所述的蛋白片段包括但不限于抗体Fc、生物素、亲和素、HRP、抗体、酶、细胞因子及其他生物活性蛋白或多肽。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联部分选自下组：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂，或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))或任何形式的纳米颗粒。
在另一优选例中，所述的多肽分子或片段包括但不限于：靶向PD-1、IL-4R、IL-4Rα、TNF-α、VEGF、4-1BB、CD47、TIM3、CTLA4、IL-17A、CD19、CD22、CD28、CD38、CD40、CD47、B7-H3、TSLP、BCMA、GLP-1、Trop2、TIGIT、LAG-3、FGL1、HER2的多肽分子或片段。
在另一优选例中，所述的多肽分子为RGD多肽或其衍生物。
在另一优选例中，所述具有治疗功能的多肽分子或片段包括单链抗体(scFv)、双链抗体、单克隆抗体、或嵌合抗体。
在另一优选例中，所述融合蛋白还包含协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列选自下组：6His标签、GGGS序列、FLAG标签。
在另一优选例中，所述的融合蛋白包括双特异性抗体、嵌合抗体。
在本发明的第九方面，提供了一种药物制剂，所述药物制剂含有：
(a)如本发明第五方面所述的表达载体或如本发明第八方面所述的核酸偶联物；和
(b)药学上可接受的载体。
在另一优选例中，所述的制剂为液体剂型。
在另一优选例中，所述的制剂为注射剂。
在另一优选例中，所述的表达载体包括慢病毒载体、腺相关病毒载体，或其组合。
在本发明的第十方面，提供了一种药物组合物，所述的药物组合物包括：
(a)如本发明第九方面所述的药物制剂；和
(b)其他生物活性的药物，如***的药物。
在另一优选例中，所述的药物组合物包括单方药物、复方药物、或协同药物。
在另一优选例中，所述的其他生物活性的药物包括免疫检查点抗体或基因治疗载体、免疫激动药物、化疗药物、脂质代谢调节药物、糖代谢调节药物。
在另一优选例中，所述的基因治疗载体指靶向针对免疫检查点的基因治疗载体。
在另一优选例中，所述免疫检查点选自下组：PD-1、PD-L1、CTLA-4、B7-H3、LAG-3、VISTA、CD47、TIM-3、TIGIT、BTLA、Siglec-15等。
在另一优选例中，所述的免疫检查点抗体的靶点包括但不限于：PD-1、PD-L1、CTLA-4、B7-H3、LAG-3、VISTA、CD47、TIM-3、TIGIT、BTLA、Siglec-15等。
在另一优选例中，所述的免疫激动药物为TLR受体激动剂、CD40激动型抗体、STING激动剂、CD3抗体、CD28抗体等。
在另一优选例中，所述的TLR受体激动剂为TLR7激动剂。
在另一优选例中，所述的化疗药物选自阿霉素、紫杉醇、顺铂、卡铂、吉西他滨、培美曲塞、甲氨蝶玲、奥沙利铂、氟尿嘧啶等。
在另一优选例中，所述的脂代谢调节药物选自他汀类药物、麦布类药物等。
在另一优选例中，所述的他汀类药物为氟伐他汀。
在另一优选例中，所述的糖代谢调节药物为二甲双胍。
在另一优选例中，所述的药物组合物用于抗肿瘤治疗。
在另一优选例中，所述的肿瘤选自但不限于：乳腺癌、肝癌、胃癌、大肠癌、白血病、肺癌、肾脏肿瘤、小肠癌、***癌、结直肠癌、***癌、***、淋巴癌、骨癌、肾上腺肿瘤、或***。
在另一优选例中，所述的肿瘤为上述肿瘤的原位瘤或转移瘤。
在另一优选例中，所述的肿瘤为免疫检查点抗体治疗耐药的肿瘤。
在另一优选例中，所述的免疫检查点抗体的靶点包括但不限于：PD-1、PD-L1、CTLA-4、B7-H3、LAG-3、VISTA、CD47、TIM-3、TIGIT、BTLA、Siglec-15等。
在另一优选例中，所述的肿瘤为表达PD-L1的肿瘤和不表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤选自下组：高表达PD-L1的肿瘤、中表达PD-L1的肿瘤、低表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤为中表达PD-L1的肿瘤或低表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤为低表达PD-L1的肿瘤。
在另一优选例中，“高表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)＞1，更佳地≥1.5，更佳地≥2.0。
在另一优选例中，“中表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)为0.5-1.1，更佳地为0.7-1.0，更佳地为0.8-0.9。
在另一优选例中，“低表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)≤1/2，更佳地≤1/3，更佳地≤1/4。
在另一优选例中，所述的药物制剂与药物组合物以选自下组的方式施用：皮下注射、皮内注射、肌肉注射、静脉注射、腹腔注射、微针注射、口服、或口鼻腔喷入和雾化吸入。
在另一优选例中，所述的药物制剂与药物组合物以选自下组的剂型施用：液态、固体、或凝胶态。
在本发明的第十一方面，提供了一种预防和/或***的方法，包括步骤：向需要的对象施用如本发明第二方面所述的载体、如本发明第三方面所述的双靶向病毒载体、如本发明第五方面所述的表达载体、如本发明第八方面所述的核酸偶联物、如本发明第九方面所述的药物制剂、或如本发明第十方面所述的药物组合物。
在另一优选例中，所述的对象为人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物为啮齿类动物，如小鼠。
在另一优选例中，所述的表达载体为病毒载体和非病毒载体。
在另一优选例中，所述的病毒载体包括但不限于：慢病毒、腺病毒、逆转录病毒、腺相关病毒等。
在另一优选例中，所述的非病毒载体包括但不限于：裸DNA、脂质体、纳米载体等。
在另一优选例中，所述施用包括注射慢病毒载体、腺相关病毒载体，或其组合。
在另一优选例中，所述的肿瘤为免疫检查点抗体治疗耐药的肿瘤。
在另一优选例中，所述的免疫检查点抗体的靶点包括但不限于：PD-1、PD-L1、CTLA-4、B7-H3、LAG-3、VISTA、CD47、TIM-3、TIGIT、BTLA、Siglec-15等。
在另一优选例中，所述的免疫检查点抗体为PD-L1抗体和CTLA-4抗体。
在另一优选例中，所述的肿瘤为表达PD-L1的肿瘤和不表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤选自下组：高表达PD-L1的肿瘤、中表达PD-L1的肿瘤、低表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤为中表达PD-L1的肿瘤或低表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤为低表达PD-L1的肿瘤。
在另一优选例中，“高表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)＞1，更佳地≥1.5，更佳地≥2.0。
在另一优选例中，“中表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)为0.5-1.1，更佳地为0.7-1.0，更佳地为0.8-0.9。
在另一优选例中，“低表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)≤1/2，更佳地≤1/3，更佳地≤1/4。
在另一优选例中，所述的肿瘤包括但不限于：乳腺癌、肝癌、胃癌、大肠癌、黑色素瘤、白血病、肺癌、肾脏肿瘤、小肠癌、***癌、结直肠癌、***癌、***、淋巴癌、骨癌、肾上腺肿瘤、或***。
在另一优选例中，所述的肿瘤为上述肿瘤的原位瘤或转移瘤。
在另一优选例中，所述施用的方式选自下组：皮下注射、皮内注射、肌肉注射、静脉注射、腹腔注射、微针注射、口服、或口鼻腔喷入和雾化吸入。
在另一优选例中，所述施用的剂型选自下组：液态、固体、或凝胶态。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了本发明的敲除CMTM6表达的慢病毒的编码模式图。
图2免疫印迹曝光图片显示了本发明的敲除CMTM6表达的sgRNA在体外可以删除肿瘤细胞的CMTM6表达。
图3显示了小鼠肿瘤生长曲线和终点瘤重，本发明的敲除CMTM6表达的慢病毒在体内可以显著抑制CT26结直肠癌肿瘤生长。
图4显示了小鼠肿瘤生长曲线和终点瘤重，本发明的敲除CMTM6表达的慢病毒在体内可以显著抑制MC38结直肠癌肿瘤的生长。
图5显示了小鼠肿瘤生长曲线和终点瘤重，本发明的敲除CMTM6表达的慢病毒在体内可以显著抑制B16F10黑色素瘤的生长。
图6显示了小鼠肿瘤生长曲线和终点瘤重，本发明的敲除CMTM6表达的慢病毒在体内可以显著抑制Hepa1-6肝癌肿瘤的生长。
图7显示了小鼠肿瘤生长曲线和终点瘤重，本发明的敲除CMTM6表达的慢病毒在体内可以显著抑制LLC非小细胞肺癌肿瘤的生长。
图8显示了本发明的敲除CMTM6和PD-L1表达的慢病毒的编码模式图。
图9显示了小鼠肿瘤生长曲线，本发明的敲除CMTM6表达的慢病毒在体内可以显著抑制PD-L1缺陷的CT26结直肠癌肿瘤的生长。
图10显示了小鼠肿瘤生长曲线和终点瘤重，本发明的敲除CMTM6表达的慢病毒在体内可以诱发抗肿瘤免疫记忆。
图11显示了小鼠肿瘤生长曲线和终点瘤重，本发明的敲除CMTM6表达的慢病毒在体内可以显著抑制PD-L1缺陷的B16F10黑色素瘤的生长。
图12显示了小鼠肿瘤生长曲线和终点瘤重，本发明的敲除CMTM6表达的慢病毒在体内可以显著抑制PD-1和PD-L1缺陷非响应性的MC38结直肠癌的生长。
图13显示了小鼠肿瘤生长曲线和终点瘤重，本发明的敲除CMTM6表达的慢病毒在体内可以显著提高PD-L1抗体对免疫检查点疗法耐药的4T1乳腺癌的生长。
图14显示了小鼠肿瘤生长曲线和终点瘤重，本发明的敲除CMTM6表达的慢病毒在体内可以显著提高PD-L1抗体和CTLA-4抗体对免疫检查点疗法耐药的LLC非小细胞肺癌的生长。
图15显示了小鼠肺部照片和肺系数情况，本发明的敲除CMTM6表达的慢病毒在体内可以显著抑制B16F10黑色素瘤的肺部转移。
图16显示了小鼠肺部照片和肺系数情况，本发明的敲除CMTM6表达的慢病毒在体 内可以显著抑制4T1乳腺癌的肺部转移。
图17显示了本发明的干扰CMTM6表达的慢病毒的编码模式图。
图18显示了小鼠肿瘤生长曲线和终点瘤重，本发明的干扰CMTM6表达的慢病毒和干扰PD-L1表达的慢病毒在体内可以显著抑制CT26结直肠癌肿瘤的体内生长。
图19显示了本发明的干扰CMTM6表达的腺相关病毒的编码模式图。
图20显示了本发明的敲除CMTM6表达的腺相关病毒的编码模式图。
图21流式图显示了本发明的腺相关病毒在体外可以感染多种肿瘤细胞。
图22荧光成像照片显示了本发明的腺相关病毒在体外可以感染多种肿瘤细胞。
图23免疫印迹相对定量结果显示了本发明的腺相关病毒在体外可以降低肿瘤细胞CMTM6表达。
图24流式细胞图显示了本发明的腺相关病毒在体内可以感染肿瘤细胞。
图25显示了小鼠肿瘤生长曲线和终点瘤重，本发明的干扰CMTM6表达的腺相关病毒在体内可以抑制CT26结直肠癌肿瘤生长。
图26显示了小鼠肿瘤生长曲线和终点瘤重，本发明的干扰CMTM6表达的腺相关病毒在体内可以抑制B16黑色素瘤肿瘤生长。
图27显示了小鼠肿瘤生长曲线和终点瘤重，本发明的干扰CMTM6表达的腺相关病毒在体内可以抑制Hepa1-6肝癌肿瘤生长。
图28显示了本发明的同时干扰CMTM6和PD-L1表达的腺相关病毒的编码模式图。
图29显示了流式细胞术分析的干扰PD-L1表达的shRNA可降低CT26肿瘤细胞的PD-L1水平。
图30显示了小鼠肿瘤生长曲线和终点瘤重，本发明的干扰CMTM6表达的腺相关病毒、干扰PD-L1表达的腺相关病毒、同时干扰CMTM6和PD-L1表达的腺相关病毒在体内可以抑制CT26结直肠癌肿瘤生长。
图31显示了肿瘤解剖照片，本发明的干扰CMTM6表达的腺相关病毒、干扰PD-L1表达的腺相关病毒、同时干扰CMTM6和PD-L1表达的腺相关病毒在体内可以抑制CT26结直肠癌肿瘤生长。
图32显示了小鼠肿瘤生长曲线，本发明的干扰CMTM6表达的腺相关病毒联合PD-L1抗体药物在体内可以抑制CT26结直肠癌肿瘤生长。
图33显示了小鼠肿瘤生长曲线，本发明的干扰CMTM6表达的腺相关病毒联合化疗药物在体内可以抑制CT26结直肠癌肿瘤生长。
图34显示了小鼠肿瘤生长曲线，本发明的干扰CMTM6表达的腺相关病毒联合免疫激动药物在体内可以抑制CT26结直肠癌肿瘤生长。
图35显示了小鼠肿瘤生长曲线，本发明的干扰CMTM6表达的腺相关病毒联合糖代谢调节药物在体内可以抑制CT26结直肠癌肿瘤生长。
图36显示了小鼠肿瘤生长曲线，本发明的干扰CMTM6表达的腺相关病毒联合脂代 谢调节药物在体内可以抑制CT26结直肠癌肿瘤生长。
图37显示了本发明的干扰CMTM6表达的腺相关病毒可以改善肿瘤免疫微环境。
图38显示了本发明的RGD多肽修饰的干扰CMTM6表达的腺相关病毒的编码模式图。
图39显示了小鼠肿瘤解剖终点瘤重和肿瘤生长曲线，本发明的RGD多肽修饰的干扰CMTM6表达的腺相关病毒在体内***给药，可以抑制CT26结直肠癌肿瘤生长。
图40显示了人源RKO肿瘤的生长曲线，本发明的敲除CMTM6表达的慢病毒在体内可以显著抑制RKO结肠癌肿瘤的生长。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次开发了一种干扰CMTM6表达的基因治疗载体(例如包括慢病毒和腺相关病毒)。本发明构建的慢病毒和腺相关病毒作为可降解CMTM6基因表达的sgRNA或shRNA的基因载体可在体外和体内降低CMTM6的表达。此外，本发明人还选用结直肠癌、黑色素瘤、肝癌、乳腺癌、非小细胞肺癌等肿瘤模型及转移模型(包括小鼠肿瘤和人源肿瘤)，评价了开发的干扰CMTM6表达的慢病毒和腺相关病毒的抗肿瘤效果，同时还对慢病毒和腺相关病毒进行修饰改造和联合用药方案测试，并评价了开发的干扰或下调CMTM6表达的慢病毒和腺相关病毒对于PD-L1低表达或不表达的肿瘤和免疫检查点治疗耐药的肿瘤的治疗效果和免疫微环境调节作用。在此基础上完成了本发明。
实验表明，本发明的基因治疗载体对多种肿瘤的生长和转移均具有显著的抑制作用，联合用药也表现出极佳的抗肿瘤效果，显示了广泛的临床应用前景。
此外，本发明的基因治疗载体对于不适合采用PD-L1抗体等常规免疫检查点药物进行治疗的肿瘤有优良的治疗效果(包括抑制肿瘤的生长和/或转移)，并且可适用于某些具有耐药性的肿瘤，尤其是经PD-L1抗体治疗无效的难治疗性肿瘤。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
序列同一性通过沿着预定的比较窗(其可以是参考核苷酸序列或蛋白的长度的50％、60％、70％、80％、90％、95％或100％)比较两个对齐的序列，并且确定出现相同的残基的位置的数目来确定。通常地，这表示为百分比。核苷酸序列的序列同一性的测量是本领域技术人员熟知的方法。
如本文使用的，术语“受试者”、“需要的对象”指任何哺乳动物或非哺乳动物。哺乳动物包括但不限于人类、脊椎动物诸如啮齿类、非人类灵长类、牛、马、狗、猫、猪、绵羊、山羊。
如本文所用，术语“基因治疗载体”、“本发明的基因治疗载体”、“干扰CMTM6表达的基因治疗载体”、“基因载体”等可互换使用，均指本申请中构建的可在体外和体内降低CMTM6表达的基因治疗载体。
如本领域技术人员所知，核酸偶联物及融合表达产物包括：药物、毒素、细胞因子(Cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的核酸分子或其片段结合而形成的偶联物。本发明还包括与所述的核酸分子或其片段结合的细胞表面标记物或抗原。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明核苷酸分子相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明的核苷酸分子可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到本发明核苷酸分子(或其片段，或其衍生物)的序列。然后可将该序列引入本领域中已知的各种现有的核酸分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明核苷酸序列中。
本发明还涉及包含上述的适当序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组核酸转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原 核生物如大肠杆菌时，能吸收核酸分子的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的核酸或载体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明核酸或载体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))等。
基因治疗及其载体
基因治疗是将目标基因导入靶细胞中以达到治疗目的的手段。现有的基因治疗载体包括腺病毒载体、逆转录病毒载体、慢病毒载体和腺相关病毒载体等。腺相关病毒(Adeno-associated virus，AAV)是一类结构简单、无包膜、DNA缺陷型病毒。AAV的生命周期依赖复制病毒，如腺病毒和单纯疱疹病毒。AAV具有良好的组织特异性、有效的扩散性、低免疫原性、高安全性和稳定性。因此，重组腺相关病毒是具有前景的病毒载体之一。慢病毒载体是以Ⅰ型人类获得性免疫缺陷病毒HIV-Ⅰ改造而来的，可感染***期细胞和非***期细胞，编码的基因可整合至靶细胞基因组，且编码容量大，也是具有广阔前景的病毒载体之一。
CMTM6
如本文所用，术语“CMTM6蛋白”、“多肽”、“本发明蛋白”、“人CMTM6蛋白”具有相同的意义，在本文中可互换使用。
CMTM6为人类趋化素样因子超家族(CMTM家族)成员之一。CMTM6含有一个四次跨膜的MARVEL结构域。在肿瘤细胞表面，CMTM6可作为PD-L1的翻译后修饰调控分子，与PD-L1相互作用并通过阻遏其泛素-蛋白酶体降解途径和内体-溶酶体降解途径，实现对PD-L1细胞膜表达量的维持。
本发明人意外地发现，通过下调本发明蛋白的表达，可以有效抑制肿瘤细胞的生长。尤其是对于低表达或者不表达PD-L1的肿瘤细胞，本发明通过抑制CMTM6的表达，可以更有效地抑制肿瘤。
第一核苷酸分子
在本发明中，第一核苷酸分子指能够降低第一靶基因(即CMTM6)的表达的核苷酸分子。
在一优选实施方式中，第一核苷酸分子的序列如SEQ ID NO:1-15所示。
第二核苷酸分子
在本发明中，第二核苷酸分子指能够降低第二靶基因(即PD-L1)的表达的核苷酸分子。
在一优选实施方式中，第二核苷酸分子的序列如SEQ ID NO:16-20所示。
表达载体
本发明还提供一种表达载体，它含有本发明所述的多核苷酸。所述的表达载体通常还含有启动子、复制起点和/或标记基因等。本领域的技术人员熟知的方法能用于构建本发明所需的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如卡拉霉素、庆大霉素、潮霉素、氨苄青霉素抗性。
在本发明中，代表性的启动子包括(但并不限于)：U6、CMV、EF1启动子或其组合。
治疗方法
本发明还提供了一种***的方法，即，将安全有效量的如本发明第七方面所述的表达载体、如本发明第十方面所述的核酸偶联物、如本发明第十一方面所述的药物制剂、或如本发明第十二方面所述的药物组合物施用于所需对象，从而***。
本发明的主要优点包括：
(a)本发明首次提供了可干扰CMTM6表达的基因治疗载体及其抗肿瘤应用。本发明的慢病毒和腺相关病毒等干扰CMTM6表达的基因治疗载体在抗结直肠癌、黑色素瘤、肝癌、乳腺癌、非小细胞肺癌(包括小鼠肿瘤和人源肿瘤)等肿瘤的生长和转移中具有显著疗效。
(b)本发明提供的慢病毒和腺相关病毒等干扰CMTM6表达的基因治疗载体对于PD-1/PD-L1/CTLA-4等免疫检查点抗体治疗耐药的肿瘤具有显著药效，并对PD-L1低表达或不表达的肿瘤同样具有显著药效，显示了临床应用前景。
(c)本发明首次提供了可干扰CMTM6表达的基因治疗载体的联合用药方案的抗肿瘤应用，测试结果显示可干扰CMTM6表达的基因治疗载体与免疫检查点抗体/免疫激动剂/化疗药物/脂代谢调节药物/糖代谢调控药物的联合用药抗肿瘤效果极佳。
(d)本发明首次提供了可同时干扰CMTM6和PD-L1表达的基因治疗载体，显示出了一方面可消退肿瘤的体内生长和另一方面还激发抗肿瘤免疫记忆的极佳复合抗肿瘤作用，显示了广泛的临床应用前景。
(e)本发明首次提供了干扰CMTM6表达的基因治疗载体的修饰改造衍生物，成功实现更为便利的***给药。
(f)本发明首次提供的干扰CMTM6表达的基因治疗载体可重塑肿瘤免疫微环境，激发CD8+T和NK细胞等的抗肿瘤免疫反应，揭示了CMTM6作为肿瘤免疫调节靶点的价值。
(g)本发明的基因治疗载体相比目前以免疫检查点阻断疗法为代表的肿瘤免疫治疗，疗效得到了较大的提高，还提高了响应率，为现有的肿瘤治疗方法提供了新的技术手段。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
核苷酸序列
>SEQ ID NO:1 mCMTM6_shRNA_1
>SEQ ID NO:2 mCMTM6_shRNA_2
>SEQ ID NO:3 mCMTM6_shRNA_3
>SEQ ID NO:4 hCMTM6_shRNA_1
>SEQ ID NO:5 hCMTM6_shRNA_2
>SEQ ID NO:6 hCMTM6_shRNA_3
>SEQ ID NO:7 mCMTM6_shRNA_4
>SEQ ID NO:8 mCMTM6_shRNA_5
>SEQ ID NO:9 mCMTM6_shRNA_6
>SEQ ID NO:10 hCMTM6_shRNA_4
>SEQ ID NO:11 hCMTM6_shRNA_5
>SEQ ID NO:12 hCMTM6_shRNA_6
>SEQ ID NO:13 mCMTM6_sgRNA
>SEQ ID NO:14 hCMTM6_sgRNA_1
>SEQ ID NO:15 hCMTM6_sgRNA_2
>SEQ ID NO:16 mPD-L1_shRNA
>SEQ ID NO:17 hPD-L1_shRNA_1
>SEQ ID NO:18 hPD-L1_shRNA_2
>SEQ ID NO:19 hPD-L1_shRNA_1
>SEQ ID NO:20 hPD-L1_shRNA_2
实施例1.敲除CMTM6表达的慢病毒载体的构建和体外活性评价
发明人首先通过软件设计靶向人或小鼠基因组中CMTM6编码序列的18-24个核苷酸长度的sgRNA寡核苷酸，实际匹配结果由NCBI数据库序列比对检验，优选为SEQ ID NO:13-15。
经由体外DNA化学合成获得用于构建敲除CMTM6表达的慢病毒载体的sgRNA短基因序列；通过酶切酶连方法将合成的sgRNA短基因序列克隆入lentiCRISPR慢 病毒表达载体质粒，基因测序验证***序列准确性。
验证完毕后，通过将慢病毒载体转化入大肠杆菌Stbl3感受态细胞中，并完成载体质粒的扩增和提取；纯化提取慢病毒载体质粒后，将慢病毒载体质粒和病毒包装质粒***(pSPAX2和pMD2.G)一同转染入HEK293T细胞。
在HEK293T中病毒包装三天后，使用PEG浓缩试剂摇晃浓缩过夜，高速离心(4000×g，30分钟)后获得浓缩提纯后的慢病毒，即为构建成功的可用于敲除CMTM6的慢病毒载体。
敲除CMTM6表达的慢病毒的编码模式如图1所示。
将以上获得的慢病毒液浸染体外贴壁培养的以下肿瘤细胞：Hepa1-6肝癌细胞、CT26结直肠癌细胞、B16F10黑色素瘤细胞；病毒浸染并经过抗生素筛选后，计数取100万个细胞，经由RIPA裂解和超声破碎，制备细胞裂解液用于免疫印迹验证构建的慢病毒可以用于体外敲除CMTM6表达。
采用经典的免疫印迹方法，依次进行聚丙烯酰胺凝胶电泳、湿转转膜、封闭、一抗孵育、二抗孵育、曝光，获取最终的免疫印迹曝光图片即图2。
如图2所示的结果显示，本发明构建的慢病毒在体外可以基本完全敲除Hepa1-6肝癌细胞、CT26结直肠癌细胞和B16F10黑色素瘤细胞表达的CMTM6，提示了其高水平的生物活性。
实施例2.敲除CMTM6表达的慢病毒载体的体内抗肿瘤活性评价
为了评价本发明构建的敲除CMTM6表达的慢病毒载体的体内抗肿瘤活性，发明人在多种移植瘤模型中评价了其抗肿瘤活性。
(1)对CT26结直肠癌肿瘤的抗肿瘤活性评价：
复苏CT26结直肠癌肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将CT26肿瘤细胞皮下接种至雌性BALB/c小鼠中，接种量为5×105细胞/只；并将小鼠随机分组为2组(每组10只)，即：Cas9对照慢病毒处理组(Cas9对照)和敲除CMTM6表达的慢病毒处理组(CMTM6 KO)。
观察并记录小鼠体重和肿瘤长径(L)与短径(W)，直至解剖终点，计算瘤体积V＝(L×W×W)/2，绘制肿瘤生长曲线，计算肿瘤抑制率。
小鼠皮下肿瘤生长到一定长度后，执行安乐死并解剖动物，取出皮下肿瘤；记录统计小鼠肿瘤生长曲线和终点瘤重。
如图3所示，结果表明，在体内，本发明的敲除CMTM6表达的慢病毒可以显著抑制CT26结直肠癌肿瘤的生长，抑瘤率高达73％。
(2)对MC38结直肠癌肿瘤的抗肿瘤活性评价：
复苏MC38结直肠癌肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将MC38肿瘤细胞皮下接种至雌性C57BL/6小鼠中，接种量为5×105细胞/只；并将 小鼠随机分组为2组(每组10只)，即：Cas9对照慢病毒处理组(Cas9对照)和敲除CMTM6表达的慢病毒处理组(CMTM6 KO)。
观察并记录小鼠体重和肿瘤长径(L)与短径(W)，直至解剖终点，计算瘤体积V＝(L×W×W)/2，绘制肿瘤生长曲线，计算肿瘤抑制率。
小鼠皮下肿瘤生长到一定长度后，执行安乐死并解剖动物，取出皮下肿瘤；记录统计小鼠肿瘤生长曲线和终点瘤重。
如图4所示，结果表明，在体内，本发明的敲除CMTM6表达的慢病毒可以显著抑制MC38结直肠癌肿瘤的生长，抑瘤率达到了33.4％。
(3)对B16F10黑色素瘤肿瘤的抗肿瘤活性评价：
复苏B16F10黑色素瘤肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将B16F10肿瘤细胞皮下接种至雌性C57BL/6小鼠中，接种量为5×105细胞/只；并将小鼠随机分组为2组(每组10只)，即：Cas9对照慢病毒处理组(Cas9对照)和敲除CMTM6表达的慢病毒处理组(CMTM6 KO)。
观察并记录小鼠体重和肿瘤长径(L)与短径(W)，直至解剖终点，计算瘤体积V＝(L×W×W)/2，绘制肿瘤生长曲线，计算肿瘤抑制率。
小鼠皮下肿瘤生长到一定长度后，执行安乐死并解剖动物，取出皮下肿瘤；记录统计小鼠肿瘤生长曲线和终点瘤重。
如图5所示，结果表明，在体内，本发明的敲除CMTM6表达的慢病毒可以显著抑制B16F10黑色素瘤肿瘤的生长，抑瘤率达到了57.8％。
(4)对Hepa1-6肝癌肿瘤的抗肿瘤活性评价：
复苏Hepa1-6肝癌肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将Hepa1-6肿瘤细胞皮下接种至雌性C57BL/6小鼠中，接种量为5×105细胞/只；并将小鼠随机分组为2组(每组10只)，即：Cas9对照慢病毒处理组(Cas9对照)和敲除CMTM6表达的慢病毒处理组(CMTM6 KO)。
观察并记录小鼠体重和肿瘤长径(L)与短径(W)，直至解剖终点，计算瘤体积V＝(L×W×W)/2，绘制肿瘤生长曲线，计算肿瘤抑制率。
小鼠皮下肿瘤生长到一定长度后，执行安乐死并解剖动物，取出皮下肿瘤；记录统计小鼠肿瘤生长曲线和终点瘤重。
如图6所示，结果表明，在体内，本发明的敲除CMTM6表达的慢病毒可以显著抑制Hepa1-6肝癌肿瘤的生长，抑瘤率高达64.4％。
(5)对LLC非小细胞肺癌肿瘤的抗肿瘤活性评价：
复苏LLC非小细胞肺癌肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将LLC肿瘤细胞皮下接种至雌性C57BL/6小鼠中，接种量为5×105细胞/只；并将小鼠随机分组为2组(每组10只)，即：Cas9对照慢病毒处理组(Cas9对照)和敲除CMTM6表达的慢病毒处理组(CMTM6 KO)。
观察并记录小鼠体重和肿瘤长径(L)与短径(W)，直至解剖终点，计算瘤体积V＝(L×W×W)/2，绘制肿瘤生长曲线，计算肿瘤抑制率。
小鼠皮下肿瘤生长到一定长度后，执行安乐死并解剖动物，取出皮下肿瘤；记录统计小鼠肿瘤生长曲线和终点瘤重。
如图7所示，结果表明，在体内，本发明的敲除CMTM6表达的慢病毒可以显著抑制LLC非小细胞肺癌肿瘤的生长，抑瘤率达到了52％。
以上结果显示，本发明的敲除CMTM6表达的慢病毒载体具有极佳的体内抗肿瘤作用，在结直肠癌、黑色素瘤、肝癌、非小细胞肺癌中均得到充分验证，其抑制肿瘤生长作用极佳，具有良好的应用前景。
实施例3.敲除CMTM6表达的慢病毒载体的抗PD-L1缺陷型肿瘤的活性评价
现有研究已知，敲除CMTM6会降低肿瘤的PD-L1表达。但是，在临床中许多患者低表达或者不表达PD-L1，因此检验本发明的敲除CMTM6表达的慢病毒载体能否介导对低表达和不表达PD-L1肿瘤的体内生长抑制十分重要。
一方面，在本发明的实施例2中，发明人采用的多种移植瘤模型中包含了CT26和B16F10两种低表达PD-L1的肿瘤，而本发明的敲除CMTM6表达的慢病毒载体可以以73％和57.8％的极其显著的抑制作用限制肿瘤体内生长，说明了本发明的慢病毒载体并不受肿瘤低表达PD-L1的影响。
进一步地，发明人采用PD-L1缺陷的CT26结直肠癌肿瘤细胞皮下荷瘤。结果如图9所示，发现：
1)作为对照的本发明的敲除CMTM6表达的慢病毒载体对于普通CT26细胞表现出和实施例2中一致的抑制肿瘤生长作用；
2)作为对照的，本发明新构建的敲除PD-L1表达的慢病毒载体对于普通的CT26细胞也有良好的抑制肿瘤作用；
3)在CT26细胞PD-L1缺陷的基础上，本发明的敲除CMTM6表达的慢病毒载体表现出了更佳的抗肿瘤作用，具体地，抑瘤率为100％，全部荷瘤小鼠的肿瘤消退，即肿瘤消退率为100％的令人意外的抗肿瘤效果。
以上结果表明，本发明的敲除CMTM6表达的慢病毒载体对于不表达PD-L1的肿瘤仍具有极佳的抗肿瘤效果。
基于此本发明又构建了PD-L1和CMTM6同时敲除的慢病毒载体，其编码模式图如图8所示。
使用本发明新构建的同时敲除CMTM6和PD-L1的慢病毒载体治疗CT26结直肠癌肿瘤，发现了和以上描述一致的结果，肿瘤消退率达100％。
为了进一步验证这样的抗肿瘤作用是否具有免疫记忆，本发明人将肿瘤消退的存活小鼠在肢体对侧再一次荷瘤CT26结直肠癌细胞，观察并记录肿瘤生长。
如图10所示，结果表明原同时敲除CMTM6和PD-L1的慢病毒载体治疗后的肿瘤消退存活小鼠对于CT26结直肠癌肿瘤具有令人意外的抗肿瘤免疫记忆，再次荷瘤的肿瘤消退率仍高达70％，且未消退的肿瘤体积和大小也大幅低于对照组小鼠的肿瘤。
以上表明，本发明的慢病毒载体除具有极佳的抗肿瘤药效以外，还可以激发体内抗肿瘤免疫记忆。
类似地，发明人使用B16F10黑色素瘤的移植瘤模型测试了同时敲除CMTM6和PD-L1的慢病毒载体的治疗效果，实验周期内观测肿瘤生长曲线和终点解剖肿瘤重量统计。
如图11所示，该慢病毒载体抗肿瘤效果显著，并且明显优于本发明另构建的敲除PD-L1的慢病毒载体效果。
为了进一步测试敲除CMTM6的慢病毒载体对于PD-1/PD-L1轴缺陷的肿瘤的治疗效果，发明人使用了MC38结直肠癌的移植瘤模型。MC38结直肠癌是一种对于PD-L1缺陷不敏感的肿瘤，即敲除MC38的PD-L1对于其体内生长无明显影响。
从本发明的实验结果来看，如图12所示，在野生型小鼠中，对比Cas9对照组和PD-L1KO组，两组小鼠肿瘤生长无差异，这和已有研究基础一致，说明了我们实验体系的可靠性。
接着，在野生型小鼠中，对比Cas9对照组和CMTM6 KO组，意外地，本发明的敲除CMTM6的慢病毒载体作用显著，说明本发明的敲除CMTM6的慢病毒载体可以有效抑制PD-L1缺陷不响应的肿瘤。
另一方面，在PD-1缺陷的小鼠中，PD-1的缺陷同样也对肿瘤生长无明显影响，但此时对比Cas9对照组和CMTM6 KO组，再次发现本发明的敲除CMTM6表达的慢病毒载体作用显著。
以上表明，本发明的慢病毒载体对于PD-1/PD-L1缺陷的肿瘤仍具有良好的抗肿瘤效果，对于PD-L1缺陷不响应的肿瘤依旧具备抑制作用。
实施例4.敲除CMTM6表达的慢病毒载体的抗免疫检查点抗体耐药肿瘤的活性评
以PD-1/PD-L1/CTLA-4抗体为代表的免疫检查点抗体疗法在多种肿瘤治疗中效果良好，但是也存在很多肿瘤对于免疫检查点抗体治疗不响应。对于这部分不响应的肿瘤需要通过联合用药的方式，提高响应率，达到治疗效果。因此，本发明人采用了两种对于免疫检查点抗体治疗耐药的肿瘤模型：4T1乳腺癌移植瘤模型和LLC非小细胞肺癌移植瘤模型，来测试本发明的敲除CMTM6表达的慢病毒载体是否可以打破肿瘤的免疫检查点抗体耐药和不响应。
(1)4T1乳腺癌移植瘤模型：
复苏4T1乳腺癌肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将 4T1肿瘤细胞皮下接种至雌性BALB/c小鼠中，接种量为5×105细胞/只，并将小鼠随机分组，即：Cas9对照慢病毒+IgG给药组(Cas9对照+IgG)、Cas9对照慢病毒+PD-L1抗体给药组(Cas9对照+αPD-L1)、敲除CMTM6的慢病毒+IgG给药组(CMTM6KO+IgG)、敲除CMTM6的慢病毒+PD-L1抗体给药组(CMTM6 KO+αPD-L1)。
在给药程序中，观察并记录小鼠体重和肿瘤长径(L)与短径(W)，直至解剖终点，计算瘤体积V＝(L×W×W)/2，绘制肿瘤生长曲线，计算肿瘤抑制率。
小鼠皮下肿瘤生长到一定长度后，执行安乐死并解剖动物，取出皮下肿瘤；记录统计小鼠肿瘤生长曲线和终点瘤重。
如图13所示，结果表明，PD-L1抗体确实对于4T1肿瘤无明显抑制作用，而本发明的敲除CMTM6表达的慢病毒载体可以显著抑制4T1肿瘤生长，意外地，PD-L1抗体联合本发明的慢病毒载体可以更佳显著地拮抗4T1的体内生长，即本发明的敲除CMTM6表达的慢病毒载体可响应免疫检查点耐药的肿瘤，并提高免疫检查点耐药/不响应肿瘤对于检查点抗体的响应。
(2)LLC非小细胞肺癌移植瘤模型：
类似地，复苏LLC非小细胞肺癌肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将LLC肿瘤细胞皮下接种至雌性C57BL/6小鼠中，接种量为5×105细胞/只并随机分组，即：Cas9对照慢病毒+IgG给药组(Cas9对照+IgG)、Cas9对照慢病毒+PD-L1抗体给药组(Cas9对照+αPD-L1)、Cas9对照慢病毒+CTLA-4抗体给药组(Cas9对照+αCTLA-4)、敲除CMTM6的慢病毒+IgG给药组(CMTM6 KO+IgG)、敲除CMTM6的慢病毒+PD-L1抗体给药组(CMTM6 KO+αPD-L1)、敲除CMTM6的慢病毒+CTLA-4给药组(CMTM6 KO+αCTLA-4)。
在给药程序中，观察并记录小鼠体重和肿瘤长径(L)与短径(W)，直至解剖终点，计算瘤体积V＝(L×W×W)/2，绘制肿瘤生长曲线，计算肿瘤抑制率。
小鼠皮下肿瘤生长到一定长度后，执行安乐死并解剖动物，取出皮下肿瘤；记录统计小鼠肿瘤生长曲线和终点瘤重。
如图14所示，结果表明，PD-L1抗体和CTLA-4抗体确实对于LLC肿瘤无明显抑制作用，而本发明的敲除CMTM6表达的慢病毒载体可以显著抑制LLC肿瘤生长，意外地，PD-L1抗体或CTLA-4抗体联合本发明的慢病毒载体可以更佳显著地拮抗LLC的体内生长，即本发明的敲除CMTM6表达的慢病毒载体可响应免疫检查点耐药的肿瘤，并提高免疫检查点耐药/不响应肿瘤对于检查点抗体的响应。
实施例5.敲除CMTM6表达的慢病毒载体的抗肿瘤转移的活性评价
肿瘤细胞从原发部位经***、血管或临近组织转移到继发部位，这往往是恶性肿瘤治疗失败的原因。因此，在肿瘤免疫治疗中预防或限制肿瘤细胞的转移是亟需关注的内容。本发明人采用了B16F10黑色素瘤的肺转移模型和4T1乳腺癌的肺转移模 型来评价本发明的敲除CMTM6表达的慢病毒载体限制肿瘤转移的作用。
(1)B16F10黑色素瘤的肺转移模型：
复苏B16F10黑色素瘤肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将B16F10肿瘤细胞尾静脉注射至雌性C57BL/6小鼠中，细胞经由Cas9对照慢病毒载体处理或本发明的敲除CMTM6表达的慢病毒载体处理，细胞量为1×106个，细胞悬液体积为200μL。
肿瘤生长25天后，解剖，取完整肺组织，称重拍照。
从解剖终点的肺部照片和肺系数(小鼠肺重与小鼠体重的比值)统计结果来看，如图15所示，对照组小鼠肺部已经遍布黑色素瘤转移灶，而本发明的敲除CMTM6表达的慢病毒载体处理后，小鼠肺部仅见少数黑色素瘤转移灶，同时其肺系数也显著低于对照组，说明其肺部病理损伤明显低于对照组。
以上表明，本发明的敲除CMTM6表达的慢病毒载体可以限制黑色素瘤的肺部转移。
(2)4T1乳腺癌的肺转移模型：
复苏4T1乳腺癌肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将4T1肿瘤细胞尾静脉注射至雌性BALB/c小鼠中，细胞经由Cas9对照慢病毒载体处理或本发明的敲除CMTM6表达的慢病毒载体处理，细胞量为1×106个，细胞悬液体积为200μL。
肿瘤生长25天后，解剖，取完整肺组织，称重拍照。
从解剖终点的肺部照片和肺系数(小鼠肺重与小鼠体重的比值)统计结果来看，如图16所示，对照组小鼠肺部已经遍布乳腺癌肿瘤转移灶，而本发明的敲除CMTM6表达的慢病毒载体处理后，小鼠肺部仅见少数乳腺癌肿瘤转移灶，同时其肺系数也显著低于对照组，说明其肺部病理损伤明显低于对照组。
以上表明，本发明的敲除CMTM6表达的慢病毒载体可以限制乳腺癌肿瘤的肺部转移。
综上，在两种肿瘤转移模型中，本发明的敲除CMTM6表达的慢病毒载体都表现出极佳地抑制肿瘤转移作用，表明了其用于预防或限制肿瘤转移的肿瘤免疫治疗的应用。
实施例6.干扰CMTM6表达的慢病毒载体的构建和体内抗肿瘤活性评价
实施例1-5已显示了本发明的敲除CMTM6表达的慢病毒载体的体内外抗肿瘤活性，发明人又构建了基于短发夹RNA(shRNA)寡核苷酸的用于干扰CMTM6表达的慢病毒载体，用于实现对CMTM6转录RNA的降解以干扰CMTM6的表达。
发明人首先通过软件设计靶向人或小鼠转录组中CMTM6 RNA序列的18-24个核苷酸长度的shRNA寡核苷酸，实际匹配结果由NCBI数据库序列比对检验，优选 为SEQ ID NO:1-12。
经由体外DNA化学合成获得用于构建干扰CMTM6表达的慢病毒载体的shRNA短基因序列；通过酶切酶连方法将合成的shRNA短基因序列克隆入pLKO.1慢病毒表达载体质粒，基因测序验证***序列准确性。
验证完毕后，通过将慢病毒载体转化入大肠杆菌Stbl3感受态细胞中，并完成载体质粒的扩增和提取；纯化提取慢病毒载体质粒后，将慢病毒载体质粒和病毒包装质粒***(pSPAX2和pMD2.G)一同转染入HEK293T细胞。
在HEK293T中病毒包装三天后，使用PEG浓缩试剂摇晃浓缩过夜，高速离心(4000×g，30分钟)后获得浓缩提纯后的慢病毒，即为构建成功的可用于干扰CMTM6的慢病毒载体。
干扰CMTM6表达的慢病毒的编码模式如图17所示。
同时，本发明也构建了用于干扰PD-L1表达的慢病毒载体，构建过程同上。
为了评价本发明构建的干扰CMTM6表达的慢病毒载体的体内抗肿瘤活性，发明人在CT26结直肠癌移植瘤模型中评价了其抗肿瘤活性。
复苏CT26结直肠癌肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代，；将CT26肿瘤细胞皮下接种至雌性BALB/c小鼠中，接种量为5×105细胞/只；并将小鼠随机分组为3组(每组12只)，即：无关shRNA慢病毒处理组(shNT)、干扰CMTM6表达的慢病毒处理组(shCMTM6)和干扰PD-L1表达的慢病毒处理组(shPD-L1)。
观察并记录小鼠体重和肿瘤长径(L)与短径(W)，直至解剖终点，计算瘤体积V＝(L×W×W)/2，绘制肿瘤生长曲线，计算肿瘤抑制率。
小鼠皮下肿瘤生长到一定长度后，执行安乐死并解剖动物，取出皮下肿瘤；记录统计小鼠肿瘤生长曲线和终点瘤重。
如图18所示，结果表明，在体内，本发明的干扰CMTM6表达的慢病毒可以显著抑制CT26结直肠癌肿瘤的生长，抑瘤率高达74％，和干扰PD-L1表达的慢病毒载体效果相当。
以上表明，本发明构建的干扰CMTM6表达的慢病毒载体同样具备极佳的体内抗肿瘤活性。
实施例7.干扰和敲除CMTM6表达的腺相关病毒载体的构建
发明人除采用慢病毒为载体的基因治疗方法来敲除或干扰CMTM6表达以达到抑制肿瘤生长作用外，还构建了以腺相关病毒(AAV)为载体的干扰和敲除CMTM6表达的基因治疗方法。
发明人首先通过软件设计靶向人或小鼠转录组中CMTM6 RNA序列的18-24个核苷酸长度的shRNA寡核苷酸，实际匹配结果由NCBI数据库序列比对检验，优选为SEQ ID NO:1-12；或者，本发明人设计靶向人或鼠基因组CMTM6基因的18-24 个核苷酸长度的sgRNA寡核苷酸，实际匹配结果由NCBI数据库序列比对检验，优选为SEQ ID NO:13-15。
经由体外DNA化学合成获得用于构建干扰或敲除CMTM6表达的腺相关病毒载体的短基因序列；通过酶切酶连方法将合成的shRNA/sgRNA短基因序列克隆入pscAAV-EGFP-shRNA载体质粒，基因测序验证***序列准确性。
验证完毕后，通过将pscAAV-EGFP-shRNA载体转化入大肠杆菌DH5α感受态细胞中，并完成载体质粒的扩增和提取；纯化提取载体质粒后，将pscAAV-EGFP-shRNA载体质粒和病毒包装质粒***(pAAV-RC9和pHelper)一同转染入HEK293T-AAV细胞。
在HEK293T-AAV中病毒包装三天后，使用浓缩试剂获得浓缩提纯后的腺相关病毒，即为构建成功的可用于干扰或敲除CMTM6的腺相关病毒载体，其基因组编码模式分别如图19和图20所示。
实施例8.干扰CMTM6表达的腺相关病毒载体的体外评价
发明人在体外评价了已构建的干扰CMTM6表达的腺相关病毒载体的侵入肿瘤细胞活性和降低肿瘤细胞CMTM6表达的能力。
发明人在24孔板中铺CT26和B16F10细胞板，细胞密度为3×105个/mL，每孔400μL；待细胞过夜贴壁后，加入生理盐水对照、对照shRNA腺相关病毒(shNC AAV)和干扰CMTM6表达的腺相关病毒(shCMTM6 AAV)，腺相关病毒的浸染MOI为1:10000；病毒浸染48小时后，取细胞在流式、免疫荧光和免疫印迹水平评价本发明的干扰CMTM6表达的腺相关病毒载体的体外活性。
通过胰酶消化贴壁细胞、PBS缓冲液清洗两遍后，重悬为单细胞悬液，上机由流式细胞仪检测；选用FITC荧光通道，检测细胞中表达地EGFP蛋白。
从流式细胞结果来看，如图21所示，在体外，本发明的腺相关病毒可以有效地进入肿瘤细胞中，在CT26中的感染效率为46.85％，在B16F10细胞中的感染效率高达84.49％。
将贴壁细胞用PBS缓冲液清洗两遍后，去除细胞培养板上盖，置于倒置荧光显微镜下，在明场显微镜中确定视野后，选用FITC荧光镜头，拍摄若干照片。
从免疫荧光的结果来看，如图22所示，类似地，本发明的腺相关病毒有效地进入了CT26和B16F10肿瘤细胞，并在肿瘤细胞中高效表达EGFP绿色荧光蛋白。
为了评价该腺相关病毒在体外降低肿瘤细胞表达CMTM6的能力，发明人对感染后的CT26和B16F10细胞进行总蛋白提取和免疫印迹样品制备，经过聚丙烯酰胺凝胶电泳、电转膜、一抗和二抗孵育及曝光，选用Na+K ATPase作为膜蛋白内参，如图23所示，发现本发明的腺相关病毒可以明显地干扰CMTM6表达。
以上结果表明，本发明构建地干扰CMTM6表达地腺相关病毒载体具有良好地入 侵肿瘤细胞活性，且在体外可有效降低肿瘤细胞表达CMTM6。
实施例9.干扰CMTM6表达的腺相关病毒载体的体外分布评价
发明人在确认了构建的干扰CMTM6表达的腺相关病毒在体外可以感染肿瘤细胞，又在体内验证了感染效果。
复苏CT26结直肠癌肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将CT26肿瘤细胞皮下接种至雌性BALB/c小鼠中，接种量为5×105细胞/只；待肿瘤体积生长至200mm3后，瘤周注射1×1010vg的本发明腺相关病毒。
4天后，解剖小鼠，取肿瘤组织用Ⅳ型胶原酶和透明质酸酶消化组织，经过200μm细胞筛网过滤细胞消化液并用PBS缓冲液清洗两遍后，用红细胞裂解液去除组织细胞悬液中的红细胞；用小鼠Fc blocker封闭单细胞的Fc受体。
最终，获得的单细胞悬液经过荧光抗体的染色标记，通过流式细胞术检测其中CD45阴性肿瘤细胞的EGFP荧光信号水平。
结果如图24，瘤周注射了本发明腺相关病毒的肿瘤内，肿瘤细胞高表达EGFP，说明了本发明的腺相关病毒具备良好的体内感染活性。
实施例10.干扰CMTM6表达的腺相关病毒载体的抗肿瘤活性评价
为了评价本发明构建的干扰CMTM6表达的腺相关病毒载体的体内抗肿瘤活性，发明人在多种移植瘤模型中评价了其抗肿瘤活性。
(1)对CT26结直肠癌肿瘤的抗肿瘤活性评价：
复苏CT26结直肠癌肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将CT26肿瘤细胞皮下接种至雌性BALB/c小鼠中，接种量为5×105细胞/只；并将小鼠随机分组为2组，即：对照shRNA腺相关病毒处理组(shNC scAAV9)和干扰CMTM6表达的腺相关病毒载体处理组(shCMTM6 scAAV9)。
观察并记录小鼠体重和肿瘤长径(L)与短径(W)，直至解剖终点，计算瘤体积V＝(L×W×W)/2，绘制肿瘤生长曲线，计算肿瘤抑制率。
小鼠皮下肿瘤生长到一定长度后，执行安乐死并解剖动物，取出皮下肿瘤；记录统计小鼠肿瘤生长曲线和终点瘤重。
如图25所示，结果表明，在体内，本发明的干扰CMTM6表达的腺相关病毒载体可以显著抑制CT26结直肠癌肿瘤的生长，抑瘤率为48.9％。
(2)对B16F10黑色素瘤肿瘤的抗肿瘤活性评价：
复苏B16F10黑色素瘤肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将B16F10肿瘤细胞皮下接种至雌性C57BL/6小鼠中，接种量为5×105细胞/只；并将小鼠随机分组为2组，即：对照shRNA腺相关病毒处理组(shNC scAAV9)和干扰CMTM6表达的腺相关病毒载体处理组(shCMTM6 scAAV9)。
观察并记录小鼠体重和肿瘤长径(L)与短径(W)，直至解剖终点，计算瘤体积V＝(L×W×W)/2，绘制肿瘤生长曲线，计算肿瘤抑制率。
小鼠皮下肿瘤生长到一定长度后，执行安乐死并解剖动物，取出皮下肿瘤；记录统计小鼠肿瘤生长曲线和终点瘤重。
如图26所示，结果表明，在体内，本发明的干扰CMTM6表达的腺相关病毒载体可以显著抑制B16F10黑色素瘤肿瘤的生长，抑瘤率为58.8％。
(3)对Hepa1-6肝癌肿瘤的抗肿瘤活性评价：
复苏Hepa1-6肝癌肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将Hepa1-6肿瘤细胞皮下接种至雌性C57BL/6小鼠中，接种量为5×105细胞/只；并将小鼠随机分组为2组，即：对照shRNA腺相关病毒处理组(shNC scAAV9)和干扰CMTM6表达的腺相关病毒载体处理组(shCMTM6 scAAV9)。
观察并记录小鼠体重和肿瘤长径(L)与短径(W)，直至解剖终点，计算瘤体积V＝(L×W×W)/2，绘制肿瘤生长曲线，计算肿瘤抑制率。
小鼠皮下肿瘤生长到一定长度后，执行安乐死并解剖动物，取出皮下肿瘤；记录统计小鼠肿瘤生长曲线和终点瘤重。
如图27所示，结果表明，在体内，本发明的干扰CMTM6表达的腺相关病毒载体可以显著抑制Hepa1-6肝癌肿瘤的生长，抑瘤率高达78.8％。
以上结果显示，本发明的干扰CMTM6表达的腺相关病毒载体具有极佳的体内抗肿瘤作用，在结直肠癌、黑色素瘤、肝癌中得到充分验证，其抑制肿瘤生长作用极佳，具有应用前景。
实施例11.同时干扰CMTM6和PD-L1表达的腺相关病毒载体的构建和抗肿瘤活性评 价
发明人还构建了以腺相关病毒为载体的可同时干扰CMTM6和PD-L1表达的基因治疗方法。
发明人首先通过软件分别设计靶向人或小鼠转录组中CMTM6和PD-L1的RNA序列的18-24个核苷酸长度的shRNA寡核苷酸，实际匹配结果由NCBI数据库序列比对检验，其中靶向CMTM6优选为SEQ ID NO:1-15，靶向PD-L1优选为SEQ ID NO:16-20。
经由体外DNA化学合成获得用于构建腺相关病毒载体的短基因序列；通过酶切酶连方法将合成的shRNA短基因序列两条一组同时克隆入pscAAV-EGFP-shRNA2载体质粒，基因测序验证***序列准确性。
验证完毕后，通过将pscAAV-EGFP-shRNA2载体转化入大肠杆菌DH5α感受态细胞中，并完成载体质粒的扩增和提取；纯化提取载体质粒后，将pscAAV-EGFP-shRNA2载体质粒和病毒包装质粒***(pAAV-RC9和pHelper)一同 转染入HEK293T-AAV细胞。
在HEK293T-AAV中病毒包装三天后，使用浓缩试剂获得浓缩提纯后的腺相关病毒，即为构建成功的可用于同时干扰CMTM6和PD-L1表达的腺相关病毒载体，其基因组编码模式分别如图28所示。
本发明前述实施例已经评价了干扰CMTM6表达的shRNA的效果。这里，在体外肿瘤细胞水平，通过浸染表达干扰PD-L1表达的腺相关病毒，通过流式细胞术分析其结直肠癌细胞CT26表面PD-L1水平，如图29所示，发现本发明的靶向PD-L1的shRNA或腺相关病毒能够有效降低PD-L1表达，即使存在干扰素刺激PD-L1表达仍能够有效发挥作用。
为了评价本发明构建的同时干扰CMTM6和PD-L1表达的腺相关病毒载体的体内抗肿瘤活性，发明人在CT26结直肠癌移植瘤模型中评价了其抗肿瘤活性。
复苏CT26结直肠癌肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将CT26肿瘤细胞皮下接种至雌性BALB/c小鼠中，接种量为5×105细胞/只；并将小鼠随机分组为4组，即：对照shRNA腺相关病毒处理组(shNC scAAV)和干扰CMTM6表达的腺相关病毒载体处理组(shCMTM6 scAAV)；干扰PD-L1表达的腺相关病毒载体处理组(shPD-L1 scAAV)；同时干扰CMTM6和PD-L1表达的腺相关病毒载体处理组(shCMTM6&PD-L1 scAAV)；给药剂量均为1×1010vg。
观察并记录小鼠体重和肿瘤长径(L)与短径(W)，直至解剖终点，计算瘤体积V＝(L×W×W)/2，绘制肿瘤生长曲线，计算肿瘤抑制率。
小小鼠皮下肿瘤生长到一定长度后，执行安乐死并解剖动物，取出皮下肿瘤；记录统计小鼠肿瘤生长曲线和终点瘤重。
如图30和图31所示，结果表明，在体内，本发明的干扰CMTM6表达的腺相关病毒载体仍旧可以显著抑制CT26结直肠癌肿瘤的生长，其抗肿瘤作用相对优于干扰PD-L1表达的腺相关病毒载体作用；另外，本发明构建的同时干扰CMTM6和PD-L1表达的腺相关病毒载体表现出显著优于单独干扰CMTM6表达和干扰PD-L1表达的腺相关病毒作用。
以上说明，本发明同时干扰CMTM6和PD-L1表达的腺相关病毒具有极佳的抗肿瘤作用，其应用价值甚至高于干扰CMTM6表达的腺相关病毒。
实施例12.干扰CMTM6表达的腺相关病毒载体的多种联合用药方案的抗肿瘤活性评 价
发明人还评价了以本发明的干扰CMTM6表达的腺相关病毒载体联合免疫检查点抗体、免疫激动剂、化疗药物、脂代谢调节药物、糖代谢调节药物的联合用药方案和效果。
复苏CT26结直肠癌肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代； 将CT26肿瘤细胞皮下接种至雌性BALB/c小鼠中，接种量为5×105细胞/只；在五种联合用药方案中，分别将小鼠随机分组为4组，即：生理盐水对照、干扰CMTM6表达的腺相关病毒载体处理组(AAV)；候选联用药物处理组(PD-L1抗体或咪喹莫特或阿霉素或氟伐他汀或二甲双胍)；干扰CMTM6表达的腺相关病毒载体联用药物处理(AAV+联用药物)。
AAV的给药剂量均为1×1010vg；PD-L1抗体为100μg/只，给药三次，每三天给药一次；咪喹莫特为50μg/只，给药五次，每三天给药一次；二甲双胍为200μg/只，给药五次，每两天给药一次；阿霉素为100μg/只，给药三次，每三天给药一次；氟伐他汀为200μg/只，给药五次，每两天给药一次。
观察并记录小鼠体重和肿瘤长径(L)与短径(W)，直至解剖终点，计算瘤体积V＝(L×W×W)/2，绘制肿瘤生长曲线，计算肿瘤抑制率。
小鼠皮下肿瘤生长到一定长度后，执行安乐死并解剖动物，取出皮下肿瘤；记录统计小鼠肿瘤生长曲线和终点瘤重。
如图32、33、34、35和36所示，结果表明，在CT26结直肠癌移植瘤模型中，本发明的干扰CMTM6表达的腺相关病毒可广泛用于和其他药物的联合用药以达到更佳的抗肿瘤效果；本发明的干扰CMTM6表达的腺相关病毒联合免疫检查点抗体、免疫激动剂、化疗药物、脂代谢调节药物、糖代谢调节药物均能有效提高抗肿瘤效果，联合用药的抗肿瘤效果最高抑瘤率可达95.02％；其中，尤以联合化疗药物和脂代谢调节药物的抑制肿瘤效果为佳。
实施例13.干扰CMTM6表达的腺相关病毒载体的对肿瘤免疫微环境影响的作用评价
在实施例10中的CT26移植瘤模型的小鼠解剖终点时，取皮下肿瘤，取150mg肿瘤组织剪碎成肉泥状，于透明质酸酶和胶原酶Ⅳ中37℃下180rpm震荡消化1.5h，处理成单细胞悬液。预处理后的细胞悬液分为两份，一份用于髓系细胞和肿瘤细胞的免疫分型分析，一份用于淋巴细胞的免疫分型分析。
髓系细胞和肿瘤细胞样品悬液处理：细胞用2-4mL无菌红细胞裂解液处理8min并用PBS缓冲液终止，去除样品悬液中的红细胞及碎片；细胞样品清洗待用。
淋巴细胞样品悬液处理：细胞用淋巴细胞分离液离心处理，获取淋巴细胞分离层，即为肿瘤淋巴细胞悬液，清洗待用。
将髓系细胞和肿瘤细胞样品和淋巴细胞样品用Fc受体封闭液4℃封闭1小时，去除Fc受体带来的非特异性染色；进行细胞表面蛋白染色，随后于冰上染色20min；接着进行胞内蛋白染色，在细胞经受固定破膜处理后，对细胞进行破膜染色，随后冰上染色20min；清洗后重悬样品细胞液，流式细胞仪上机检测。
流式免疫分型结果如图37所示。本发明的干扰CMTM6表达腺相关病毒靶向肿瘤免疫微环境，可重塑肿瘤免疫微环境，协调先天免疫和适应性免疫来发挥抗肿瘤活 性；其中尤以细胞毒细胞，如CD8+T细胞和NK细胞的活性被调动起来，可促进他们分泌强力的抗肿瘤因子TNF-α、颗粒酶等；本发明的AAV还可以降低CD4+T细胞表达的免疫抑制性分析，如PD-1、CTLA-4等；同时，本发明的AAV还降低了肿瘤细胞表达的PD-L1。
总结来说，本发明的干扰CMTM6表达的腺相关病毒可以通过靶向肿瘤组织，调节肿瘤免疫环境，调动抗肿瘤免疫应答，下调抑制性肿瘤免疫，达到对抗肿瘤生长作用。
实施例14.RGD多肽修饰的干扰CMTM6表达的腺相关病毒的构建
本发明上述实施例中构建并应用的腺相关病毒最优施用方式为瘤周给药，这有助于提高其抗肿瘤效果。虽然目前肿瘤原位给药技术成熟，但是相对于静脉注射等***给药方式较为不便，且患者依从性不高。
因此，发明人构建了一种RGD多肽修饰的干扰CMTM6表达的腺相关病毒，其构建方法总体和实施例7中AAV基因组载体的构建方式一致，区别在于需要改造其病毒外壳，在病毒包装质粒pAAV-RC9的589位点***一段氨基酸序列，代表性的为CDCRGDCFC，如图38所示。
这样，使用pAAV-RC9-RGD包装出来的AAV外壳上具有RGD多肽序列，可以特异性的靶向高表达整合素的肿瘤细胞，可用于体统给药。
实施例15.RGD多肽修饰的干扰CMTM6表达的腺相关病毒的体内抗肿瘤活性评价
为了评价本发明实施例14构建的RGD多肽修饰的干扰CMTM6表达的腺相关病毒的***给药抗肿瘤活性，发明人采用了CT26移植瘤模型来评价。
复苏CT26结直肠癌肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代；将CT26肿瘤细胞皮下接种至雌性BALB/c小鼠中，接种量为5×105细胞/只；并将小鼠随机分组为2组，即：对照shRNA腺相关病毒处理组(shNC AAV-RGD)和RGD多肽修饰的干扰CMTM6表达的腺相关病毒载体处理组(shCMTM6 AAV-RGD)。
给药剂量均为1×1010vg，腹腔注射。记录统计小鼠肿瘤终点瘤体积和终点瘤重。
如图39所示，结果表明，在体内，本发明的RGD多肽修饰的干扰CMTM6表达的腺相关病毒载体通过***给药的方式仍旧可以显著抑制CT26结直肠癌肿瘤的生长，扩展了本发明的用到的基因治疗方法的施用手段的便利性。
实施例16.敲除CMTM6表达的慢病毒载体在免疫人源化体系中的抗肿瘤活性评价
在本实施例中，发明人在免疫人源化体系中，进行了对人源RKO结肠癌肿瘤的抗肿瘤活性评价实验。
将雌性NSG小鼠随机分为4组，即①Cas9对照组、②CMTM6 KO组、③Cas9 对照+PBMCs组以及④CMTM6 KO+PBMCs组。在荷瘤3天前利用宠物电推剪剔除4组小鼠右前肢腋下及周围的毛发。
对于组①②：人结直肠癌肿瘤RKO细胞复苏后至少培养三代以上，在细胞状态良好且处于对数生长期时，将肿瘤细胞用含0.05％EDTA的胰酶溶液消化并离心后，用冷PBS清洗两遍后用冷PBS重悬后计数；
对于组③④：分离自同一人的PBMCs细胞被复苏并立即使用，将PBMCs与RKO细胞混合，使得RKO密度为5×107个/mL，PBMCs密度为1×107个/mL。
利用胰岛素针在4组NSG小鼠右侧前肢腋下分别皮下接种以上细胞100μL，细胞注意保存于冰上；
其中组①②的接种量/荷瘤量为：5×106个细胞；组③④的接种量/荷瘤量为：5×106个RKO细胞和1×106个PBMCs细胞。
荷瘤后，小鼠被每5天监测肿瘤体积，共监测5个时间点。
实验结果如图40所示，表明：在RKO/人PBMCs混合荷瘤的人源化肿瘤模型中，CMTM6的敲除显著抑制了人RKO肿瘤的体内生长。
因而，本发明的敲除CMTM6表达的慢病毒载体对人源肿瘤也有显著的抑制作用。
讨论
肿瘤免疫疗法已在癌症治疗领域取得突破性成果，然而其依旧存在疗效欠佳和响应率低等问题有待解决。以PD-1/PD-L1免疫检查点抗体治疗为例，其在临床中存在诸多不响应的瘤种，应用于部分肿瘤适应症中甚至可能导致肿瘤超进展，极大限制了这类抗体药物的应用。另一方面，为了提高PD-1/PD-L1抗体的响应率，发现和应用其疗效生物标志物也是一大研究方向，目前诸如PD-L1表达水平和肿瘤突变负荷等已被用于筛选施用患者，然而对于不符合的患者的用药问题仍待解决。
尝试应用新型靶点可能是解决上述问题的有效途径之一。同时，新型靶点的应用应考虑直击PD-1/PD-L1抗体的痛点，尝试在单独或联合用药的情况下使得PD-L1低表达、冷肿瘤、PD-1/PD-L1抗体治疗耐药的患者可以获益。
本发明首次提供了一种干扰CMTM6表达的基因治疗载体，同时提供了针对所述基因治疗载体的修饰改造和多类型联合用药。本发明的基因治疗载体靶向新型抗肿瘤靶点CMTM6，以干扰CMTM6基因表达的方式调控肿瘤细胞体内生长，可抑制结直肠癌、黑色素瘤、乳腺癌、非小细胞肺癌、肝癌等的体内生长，并实现了对PD-L1低表达或不表达肿瘤的显著疗效，以及对PD-1/PD-L1/CTLA-4抗体耐药的肿瘤的显著疗效。另外，本发明提供的干扰CMTM6表达的基因治疗载体，可以充分激发瘤内抗肿瘤免疫反应，尤以CD8+T细胞和NK细胞活性的激活为显著，显示了该靶点作为肿瘤免疫治疗新靶点的潜力。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种用于靶向下调CMTM6的基因治疗载体的用途，用于制备一组合物或制剂，其特征在于，所述的组合物或制剂用于：(a)预防和/或***；和/或(b)抑制肿瘤细胞。
  2. 如权利要求1所述的用途，其特征在于，所述基因治疗载体选自下组：
    (Z1)靶向抑制CMTM6表达的慢病毒；
    (Z2)靶向抑制CMTM6表达的腺相关病毒；
    (Z3)同时靶向抑制CMTM6表达和PD-L1表达的慢病毒；
    (Z4)同时靶向抑制CMTM6表达和PD-L1表达的腺相关病毒；
    (Z5)上述Z1～Z4的任意组合。
  3. 如权利要求1所述的用途，其特征在于，所述的基因治疗载体与选自下组的药物进行联用：免疫检查点抗体、免疫激动剂、化疗药物、脂代谢调节药物、糖代谢调节药物、额外的基因治疗载体，或其组合。
  4. 如权利要求1所述的用途，其特征在于，所述的肿瘤为免疫检查点抗体或免疫检查点抑制剂治疗失效或失败的肿瘤，或不适合用免疫检查点抗体或免疫检查点抑制剂治疗的肿瘤。
  5. 一种可靶向抑制肿瘤和/或细胞中CMTM6表达的病毒载体或非病毒载体，其特征在于，所述载体携带或含有抑制CMTM6表达的编码序列。
  6. 如权利要求5所述的载体，其特征在于，所述抑制CMTM6表达的编码序列为靶向抑制CMTM6的sgRNA或shRNA，包括：
    (i)选自SEQ ID NO:1-12或其衍生序列中的一个或多个；或
    (ii)选自SEQ ID NO:13-15或其衍生序列中的一个或多个。
  7. 一种可同时靶向抑制肿瘤和/或细胞中CMTM6和PD-L1表达的双靶向病毒载体，其特征在于，所述双靶向病毒载体携带或含有的编码序列选自下组：
    (i)选自SEQ ID NO:1-15或其衍生序列中的一个或两个；和
    (ii)选自SEQ ID NO:16-20或其衍生序列中的一个或两个。
  8. 一种多核苷酸，其特征在于，所述多核苷酸编码选自下组的载体的基因组：如权利要求5所述的载体、或如权利要求7所述的双靶向病毒载体。
  9. 一种表达载体，其特征在于，所述表达载体含有如权利要求8所述的多核苷酸。
  10. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求9所述的表达载体，或其基因组中整合有如权利要求8所述的多核苷酸。
PCT/CN2023/083888 2022-03-25 2023-03-24 干扰趋化素样因子超家族成员6(cmtm6)表达的基因治疗载体的制备和抗肿瘤应用 WO2023179789A1 (zh)

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