WO2022095903A1 - 靶向pd-1h(vista)的抗肿瘤免疫治疗方法 - Google Patents

靶向pd-1h(vista)的抗肿瘤免疫治疗方法 Download PDF

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WO2022095903A1
WO2022095903A1 PCT/CN2021/128459 CN2021128459W WO2022095903A1 WO 2022095903 A1 WO2022095903 A1 WO 2022095903A1 CN 2021128459 W CN2021128459 W CN 2021128459W WO 2022095903 A1 WO2022095903 A1 WO 2022095903A1
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cells
tumor
immune cells
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黄纲雄
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福州拓新天成生物科技有限公司
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Definitions

  • the present invention relates to the field of biomedicine, in particular, to an anti-tumor immunotherapy method targeting PD-1H (VISTA).
  • VISTA anti-tumor immunotherapy method targeting PD-1H
  • PD-1H (also known as VISTA, DD1- ⁇ ) is a co-suppressor molecule of the CD28/B7 family with high homology to PD-1 and PD-L1.
  • PD-1H is mainly expressed on hematopoietic cells, and expressed on myeloid cells (including macrophages, dendritic cells, monocytes and neutrophils) and CD4+ T cells.
  • myeloid cells including macrophages, dendritic cells, monocytes and neutrophils
  • CD4+ T cells As a ligand on antigen-presenting cells and regulatory T cells, PD-1H can inhibit T cell proliferation and cytokine release, and has an important regulatory role in autoimmune diseases and tumor progression.
  • PD-1H can act as an inhibitory receptor on CD4+ T cells to regulate immune tolerance and tumor immunity involving T cells.
  • PD-1H-specific blocking antibodies can effectively inhibit tumor growth in multiple mouse tumor models (melanoma, bladder cancer, etc.).
  • the PD-1H-specific agonistic antibody can effectively alleviate the symptoms of autoimmune diseases (systemic lupus erythematosus, asthma, arthritis, etc.).
  • the purpose of the present invention is to provide a class of engineered immune cells that can significantly enhance the good killing effect of tumors.
  • a first aspect of the present invention provides an engineered immune cell in which the expression of PD-1H gene is silenced.
  • the "PD-1H gene expression is silenced" refers to no or low expression of PD-1H gene.
  • the "low expression” refers to the ratio of the expression level G1 of the PD-1H gene of the immune cells to the expression level G0 of the PD-1H gene of normal immune cells, that is, G1/G0 ⁇ 0.8, preferably G1/G0 ⁇ 0.5, more preferably ⁇ 0.2, more preferably ⁇ 0.1, and most preferably 0.
  • the silencing of PD-1H gene expression is achieved by a method selected from the group consisting of gene mutation, gene knockout, gene interruption, RNA interference technology, gene editing technology, and inhibitors of introduced genes or proteins , blocking of antibodies or proteins or polypeptides or compounds, cellular screening, or a combination thereof.
  • the inhibitor is selected from the group consisting of antisense nucleic acid, antibody, small molecule compound, Crispr reagent, small molecule ligand, or a combination thereof.
  • the gene editing technology is selected from the group consisting of CRISPR technology, TALEN technology, ZFN technology, or a combination thereof.
  • the engineered immune cells include CD8+T cells, CD3+T cells, CD4+T cells, B cells, NK cells, myeloid leukocytes or monocytes, antigen-presenting cells, or other Immune Cells.
  • the engineered immune cells have the following characteristics:
  • the engineered immune cells also have the following characteristics:
  • the immune cell expresses a chimeric antigen receptor CAR or an exogenous TCR, the CAR targets an antigen molecule or a tumor cell marker, and the exogenous TCR targets an antigen molecule or a tumor cell marker.
  • the engineered immune cells include:
  • an optional chimeric antigen receptor CAR comprising: an antigen binding domain, a hinge domain, a transmembrane domain and an intracellular domain, wherein the antigen binding domain is specific sexually binds to antigenic molecules or tumor cell surface antigens;
  • the inhibitory molecule is selected from the group consisting of inhibitory nucleic acid, small molecule compound, antibody (eg, single domain antibody), polypeptide, or a combination thereof.
  • the inhibitory nucleic acid includes RNA interference agent and Crispr agent.
  • the inhibitory nucleic acid is selected from the group consisting of siRNA, miRNA, shRNA, hairpin siRNA, tandemly expressed miRNA, microRNA-adapted shRNA, precursor microRNA, or a combination thereof.
  • sequence of the inhibitory nucleic acid is shown in any one or a combination of SEQ ID NO.:5-10.
  • the Crispr reagent includes a gene editing protein.
  • the gene editing protein is selected from the group consisting of CRISPR, TALEN, ZFN, or a combination thereof.
  • the CRISPR protein is selected from the group consisting of Cas9, nCas9, Cas10, Cas9a, Cas12, Cas12a, Cas12b, Cas13, Cas14, or a combination thereof.
  • the Crispr reagent further includes gRNA.
  • At least part of the sequence of the gRNA can be complementary to the target DNA, and the gRNA can form a functional complex with the CRISPR protein.
  • the gene editing enzyme is derived from Streptococcus pyogenes, Streptococcus thermophiles, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae Bacteria (Lachnospiraceae bacterium), or a combination thereof.
  • the gRNA includes sgRNA.
  • sequence of the gRNA is shown in any one or combination of SEQ ID NO.: 1-4, 12-18.
  • the inhibitory nucleic acid molecule comprises a sequence complementary to PD-1H mRNA (messaging nucleic acid) or a nucleic acid encoding PD-1H.
  • the inhibitory nucleic acid molecule comprises an antisense oligonucleotide complementary to PD-1H mRNA (information nucleic acid) or a nucleic acid encoding PD-1H.
  • the small molecule compound is selected from the group consisting of 1,2,4-oxadiazole compounds and derivatives thereof, oxadiazole compounds, thiadiazole compounds, sulfonamide compounds, Benzene compounds, or combinations thereof.
  • the tumor cell surface antigens include cell surface antigens of various solid tumors, solid tumors and hematological tumors.
  • the tumor cell surface antigen is selected from the group consisting of CD19, c-Met, PSMA, MUC-1, MUC16, CD7, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD38, PD-L1, CD276, B7-H4, mesothelin, EGFR, EGFRviii, GPC3, BCMA, ErbB2, ErbB3, ErbB4, NKG2D ligand, LMP1, EpCAM, VEGFR-1, Lewis-Y, ROR1, Claudin18 .2, CD7, CD5, CD2, CD3, CD19, CD20, CD22, CD24, CD25, CD28, CD123, CD47, CD52, CD56, CD80, CD86, CD81, CD138, CD33, CD38, CD30, CD133, CD97, CD99 , CD40, CD43, CD137, CD151, CD171, KIT(CD117), CD174, CD44V6, CD179a, B7-H3(CD
  • the tumor cell surface antigen includes CD19.
  • the tumor cell surface antigen includes B7-H3 (CD276).
  • the reducing or inhibiting the expression activity of PD-1H protein refers to reducing the expression activity of PD-1H protein by ⁇ 20%, preferably ⁇ 40%, more preferably ⁇ 60%, more preferably Ground, > 80%, more preferably > 90% or 100%.
  • the antigen-binding domain is an antibody or an antigen-binding fragment.
  • the antigen-binding fragment is a Fab or scFv or a single-domain antibody sdFv.
  • the engineered immune cells are selected from the following group:
  • CAR-T cells chimeric antigen receptor T cells
  • CAR-NK cells chimeric antigen receptor NK cells
  • chimeric antigen receptor phagocytes or monocytes (iii) chimeric antigen receptor phagocytes or monocytes (CAR-macrophage cells);
  • TCR Exogenous T cell receptor (TCR) T cells
  • the immune cells are autologous.
  • the immune cells are allogeneic.
  • the immune cells are derived from iPS.
  • the cells are mammalian cells, preferably human cells.
  • the immune cells also express inhibitory molecules that reduce or inhibit the expression activity of PD-1H protein.
  • the inhibitory molecule is expressed independently and/or co-expressed with a chimeric antigen receptor CAR targeting a tumor cell surface antigen.
  • the co-expression with a chimeric antigen receptor CAR targeting a tumor cell surface antigen includes the tandem expression of an inhibitory molecule and a chimeric antigen receptor CAR targeting a tumor cell surface antigen.
  • the engineered immune cells include T cells, NK cells or macrophages.
  • the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CTLA-4, PD-1, LAG-3, 2B4, BTLA, or a combination thereof.
  • the hinge domain is a hinge domain of a protein selected from the group consisting of CD8, CD28, CD137, CD80, CD86, or a combination thereof.
  • the intracellular domain includes a costimulatory signaling molecule and a cytoplasmic signaling sequence derived from CD3 ⁇ .
  • the costimulatory signal molecule is a costimulatory signal molecule of a protein selected from the group consisting of OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137) , PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.
  • the second aspect of the present invention provides a method for preparing the engineered immune cells described in the first aspect of the present invention, comprising the following steps:
  • step (B) includes introducing a second expression cassette for silencing the PD-1H gene into the immune cells.
  • step (B) it further comprises the steps of: (B1) introducing the first expression cassette expressing CAR into the immune cells; and (B2) expressing the first expression cassette for silencing the PD-1H gene Two expression cassettes are introduced into the immune cells,
  • step (A) when the immune cells to be engineered in step (A) already express a certain CAR, then in step (B), including (B2) expressing a second expression for silencing PD-1H
  • the cassettes are introduced into the immune cells.
  • the "order is not limited” means that any two steps can be performed sequentially, simultaneously, or in reverse order.
  • step (B1) can be performed before, after, at the same time, or alternately with the step (B2).
  • first expression cassette and the second expression cassette are located on the same or different vectors.
  • first expression cassette and the second expression cassette are located in the same vector.
  • the vector is a viral vector.
  • the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, other gene transfer systems, or a combination thereof.
  • the second expression cassette comprises CRISPR/Cas9 (gRNA and Cas9), antisense RNA, or a combination thereof.
  • the gRNA includes sgRNA.
  • the gRNA targets PD-1H, and the sequence of the gRNA is shown in any one or combination of SEQ ID NO.: 1-4, 12-18.
  • the antisense RNA includes miRNA, siRNA, shRNA, inhibitory mRNA, or dsRNA.
  • sequence of the antisense RNA is shown in any one or a combination of SEQ ID NO.:5-10.
  • the third aspect of the present invention provides a preparation, the preparation contains the engineered immune cells described in the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the preparation is a liquid preparation.
  • the preparation is an injection.
  • the concentration of the immune cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 10 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 8 cells/ml.
  • the fourth aspect of the present invention provides a use of the engineered immune cells described in the first aspect of the present invention for preparing a medicament or preparation for preventing and/or treating cancer or tumor.
  • the tumor is selected from the group consisting of hematological tumors, solid tumors, or a combination thereof.
  • the tumor includes a CD19-positive tumor.
  • the tumor includes a B7-H3 (CD276) positive tumor.
  • the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myelodysplastic/myeloproliferative disease (MDS/MPD), chronic myeloproliferative Sexual Disorders (SMPD); pre-B lymphoblastic leukemia/lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, B lymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cells Leukemia, plasma cell myeloma/plasmacytoma, MALT-type marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma/leukemia, T/NK Cell neoplasms, prelymphoblastic lymphoma/leukemia, T-lymph
  • AML
  • the solid tumor is selected from the group consisting of head and neck tumor, throat cancer, lung cancer, non-small cell lung cancer, bronchial cancer, gastric cancer, gastric cancer peritoneal metastases, esophageal cancer, bile duct cancer, pancreatic cancer, Colorectal cancer, colorectal cancer peritoneal metastases, small bowel cancer, kidney tumor, kidney cancer, bladder tumor, transitional epithelial malignant tumor, endocrine tumor, thyroid cancer, adrenal tumor, breast cancer, cervical cancer, ovarian cancer, ovarian cancer peritoneal Metastatic tumor, endometrial cancer, choriocarcinoma, prostate cancer, testicular tumor, germ cell tumor, seminoma, embryonal tumor, nervous system tumor, glioma, neuroblastoma, skin tumor, malignant Melanoma, lymphoma, thymic tumor, nasopharyngeal carcinoma, bone cancer, sarcoma, rhabdomyosarcom
  • a fifth aspect of the present invention provides a kit for preparing the engineered immune cells described in the first aspect of the present invention, the kit comprising a container, and in the container:
  • first and second nucleic acid sequences are independent or linked.
  • first and second nucleic acid sequences are located in the same or different containers.
  • first and second nucleic acid sequences are located in the same or different expression vectors.
  • the kit further contains: (4) a third nucleic acid sequence, the third nucleic acid sequence contains an expression cassette for expressing a gene editing protein; or a gene editing protein.
  • a sixth aspect of the present invention provides a method for regulating immune cell activity, comprising:
  • the activity of immune cells is regulated.
  • the activity of immune cells is enhanced by reducing or inhibiting the expression level of PD-1H in immune cells.
  • the activity of immune cells is reduced by enhancing the expression level of PD-1H in immune cells.
  • the "reducing or inhibiting the expression level of PD-1H in immune cells” refers to the ratio of the expression level G1 of the PD-1H gene in the immune cells to the expression level G0 of the PD-1H gene in normal immune cells , that is, G1/G0 ⁇ 0.8, preferably G1/G0 ⁇ 0.5, more preferably ⁇ 0.2, more preferably ⁇ 0.1, and optimally 0.
  • the "enhancing the expression level of PD-1H in immune cells” refers to the ratio of the expression level G1 of the PD-1H gene in the immune cells to the expression level G0 of the PD-1H gene in normal immune cells, namely G1/G0 ⁇ 2, preferably G1/G0 ⁇ 3, more preferably, G1/G0 ⁇ 4.
  • a seventh aspect of the present invention provides a method for judging or evaluating immune cell activity, comprising:
  • the activity of immune cells can be judged or evaluated.
  • the "increased expression level of PD-1H in immune cells” refers to the ratio of the expression level G1 of the PD-1H gene in the immune cells to the expression level G0 of the PD-1H gene in normal immune cells, That is, G1/G0 ⁇ 2, preferably G1/G0 ⁇ 3, more preferably, G1/G0 ⁇ 4.
  • the "decreased expression level of PD-1H in immune cells” refers to the ratio of the expression level G1 of the PD-1H gene in the immune cells to the expression level G0 of the PD-1H gene in normal immune cells, namely G1/G0 ⁇ 0.8, preferably G1/G0 ⁇ 0.5, more preferably ⁇ 0.2, more preferably ⁇ 0.1, and most preferably 0.
  • An eighth aspect of the present invention provides a nucleic acid molecule comprising a first nucleic acid and an optional second nucleic acid, wherein the first nucleic acid contains a first expression cassette encoding a PD-1H protein that reduces or inhibits An inhibitory molecule for expressing activity, the second nucleic acid contains a second expression cassette, the second expression cassette encodes a chimeric antigen receptor CAR, and the chimeric antigen receptor CAR includes: an antigen binding domain, a transmembrane domain and an intracellular domain, wherein the antigen binding domain specifically binds to a tumor cell surface antigen.
  • the first expression cassette and/or the second expression cassette further comprises a constitutive promoter or an inducible promoter.
  • the constitutive promoter is selected from the group consisting of CMV, EF1a, U6, SV40, PGK1, Ubc, CAG, H1, or a combination thereof.
  • the inducible promoter is selected from the group consisting of a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, a tetracycline promoter, or a combination thereof.
  • the ninth aspect of the present invention provides a vector containing the nucleic acid molecule of the eighth aspect of the present invention.
  • the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.
  • the vector is a lentiviral vector.
  • the tenth aspect of the present invention provides a host cell, which contains the vector of the ninth aspect of the present invention or the exogenous nucleic acid molecule of the eighth aspect of the present invention integrated into the chromosome.
  • the cells are isolated cells, and/or the cells are genetically engineered cells.
  • the cells are mammalian cells, preferably human cells.
  • the host cells include engineered immune cells.
  • the engineered immune cells are selected from the following group:
  • CAR-T cells chimeric antigen receptor T cells
  • CAR-NK cells chimeric antigen receptor NK cells
  • TCR Exogenous T cell receptor (TCR) T cells
  • the immune cells are autologous.
  • the immune cells are allogeneic.
  • the immune cells also express inhibitory molecules that reduce or inhibit the expression activity of PD-1H protein.
  • the inhibitory molecule is expressed independently and/or co-expressed with a chimeric antigen receptor CAR targeting a tumor cell surface antigen.
  • the co-expression with the chimeric antigen receptor CAR targeting the tumor cell surface antigen includes the tandem expression of the inhibitory molecule and the chimeric antigen receptor CAR targeting the tumor cell surface antigen.
  • the engineered immune cells include T cells, NK cells or macrophages.
  • the cells are T cells.
  • An eleventh aspect of the present invention provides a pharmaceutical composition, comprising:
  • the pharmaceutical composition is a liquid preparation.
  • the dosage form of the pharmaceutical composition is an injection.
  • the engineered immune cells are (i) chimeric antigen receptor T cells (CAR-T cells); or (ii) chimeric antigen receptor NK cells (CAR-NK cells).
  • the concentration of the cells is 1 ⁇ 10 3 -1 ⁇ 10 10 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 8 cells/ml .
  • the pharmaceutical composition further contains other drugs (such as antibody drugs, chemotherapeutic drugs or other CAR-T drugs) that selectively kill tumor cells.
  • drugs such as antibody drugs, chemotherapeutic drugs or other CAR-T drugs
  • a twelfth aspect of the present invention provides a method for preparing engineered immune cells, comprising:
  • the engineered immune cells are obtained by transfecting the nucleic acid molecule described in the eighth aspect of the present invention or the vector described in the ninth aspect of the present invention into immune cells.
  • the introduction includes simultaneous, sequential, or sequential introduction.
  • the immune cells are T cells or NK cells.
  • the method further includes the step of testing the function and effectiveness of the obtained engineered immune cells.
  • a thirteenth aspect of the present invention provides a reagent combination, the reagent combination comprising:
  • the second agent is an inhibitory molecule that reduces or inhibits the expression activity of PD-1H protein.
  • the immune cells include engineered immune cells.
  • the engineered immune cell contains a chimeric antigen receptor CAR, and the chimeric antigen receptor CAR includes: an antigen binding domain, a transmembrane domain and an intracellular domain, wherein the antigen The binding domain specifically binds to tumor cell surface antigens.
  • the inhibitory molecule is selected from the group consisting of inhibitory nucleic acid, small molecule compound, antibody (eg, single domain antibody), polypeptide, or a combination thereof.
  • the engineered immune cells also express inhibitory molecules that reduce or inhibit the expression or activity of PD-1H protein.
  • the engineered immune cells also express inhibitory nucleic acids for reducing or inhibiting the expression of PD-1H protein.
  • a fourteenth aspect of the present invention provides a method for enhancing the tumor killing efficiency of engineered immune cells, comprising:
  • the engineered immune cells are contacted with tumor cells, thereby enhancing the tumor killing efficiency of the engineered immune cells.
  • the method is in vitro.
  • the method is non-therapeutic and non-diagnostic.
  • the inhibitory molecule that reduces or inhibits the expression activity of PD-1H protein is endogenously produced or exogenously added.
  • the endogenous production refers to production by the engineered immune cells.
  • the exogenous addition refers to the exogenous addition of inhibitory molecules.
  • the engineered immune cells include CD8+T cells, CD3+T cells, CD4+T cells, B cells, NK cells, myeloid leukocytes or monocytes, antigen-presenting cells, or other Immune Cells.
  • the fifteenth aspect of the present invention provides a use of the host cell of the tenth aspect of the present invention or the pharmaceutical composition of the eleventh aspect of the present invention for preparing a drug or preparation for selectively killing tumor cells.
  • the tumor cells are derived from tumors selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myelodysplastic/myeloproliferative disease (MDS/MPD) , chronic myeloproliferative disease (SMPD); pre-B lymphoblastic leukemia/lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, B lymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma tumor, hairy cell leukemia, plasma cell myeloma/plasmacytoma, marginal zone B-cell lymphoma of MALT type, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma/leukemia , T/NK cell neoplasms, prolymphoblastic lymphoma/leukemia,
  • AML
  • a sixteenth aspect of the present invention provides a kit for selectively killing tumor cells, the kit comprising a container, and the host cell according to the tenth aspect of the present invention located in the container.
  • kit further contains labels or instructions for use.
  • a seventeenth aspect of the present invention provides a method for selectively killing tumor cells, comprising:
  • a safe and effective amount of the engineered immune cells described in the first aspect of the present invention, the host cells described in the tenth aspect of the present invention, or the pharmaceutical composition described in the eleventh aspect of the present invention is administered to a subject in need of treatment.
  • the subject includes a human or a non-human mammal.
  • non-human mammals include rodents (eg, mice, rats, rabbits), primates (eg, monkeys).
  • the method is non-therapeutic and non-diagnostic.
  • the eighteenth aspect of the present invention provides a method for treating cancer or tumor, characterized in that it includes:
  • a safe and effective amount of the engineered immune cells described in the first aspect of the present invention, the host cells described in the tenth aspect of the present invention, or the pharmaceutical composition described in the eleventh aspect of the present invention is administered to a subject in need of treatment.
  • the tumor cells are derived from tumors selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myelodysplastic/myeloproliferative disease (MDS/MPD) , chronic myeloproliferative disease (SMPD); pre-B lymphoblastic leukemia/lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, B lymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma tumor, hairy cell leukemia, plasma cell myeloma/plasmacytoma, marginal zone B-cell lymphoma of MALT type, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma/leukemia , T/NK cell neoplasms, prolymphoblastic lymphoma/leukemia,
  • AML
  • Figure 1 shows increased PD-1H expression on tumor-infiltrating CD8+ T cells.
  • B16-OVA mouse melanoma
  • EG7 lymphoma
  • Figure 2 shows that the addition of anti-CD3 and anti-CD28 stimulation to CD8+ T cells in vitro can up-regulate PD-1H expression on CD8+ T cells.
  • Figure 3 shows that PD-1H-depleted CD8+ T cells have enhanced antitumor effects and increased tumor infiltration in a mouse melanoma (B16-OVA) model.
  • Figure 4 shows enhanced antitumor effect of PD-1H-depleted CD8+ T cells and increased tumor infiltration in a mouse lymphoma (EG7) model.
  • Figure 5 shows that the expression of PD-1H on CD8+ T cells can be effectively knocked down using the CRISPR Cas9 system.
  • Figure 6 shows the enhanced antitumor effect of CD8+ T cells knocked out of PD-1H using the CRISPR Cas9 system in a mouse lymphoma (EG7) model.
  • Figure 7 shows that there is no PD-1H expression on the surface of T cells and CAR-T cells cultured in vitro, and the expression of PD-1H on the surface of T cells is increased in spleen and tumor tissue.
  • Figure 8 shows immunofluorescence co-localization showing PD-1H expression on tumor-infiltrating CD8+ T cells.
  • Figure 9 shows a schematic diagram of the vector construction of the chimeric antigen receptor co-expressing PD-1H shRNA.
  • Figure 10 shows the expression of different chimeric antigen receptors co-expressing PD-1H shRNA on T cells.
  • Figure 11 shows that candidate PD-1H shRNAs silence PD-1H expression on Jurkat-PD-1H cells.
  • Figure 12 shows that CD19 CAR-T cells co-expressing PD-1H shRNA can effectively kill CD19 positive tumor cells (CA-46).
  • Figure 13 shows that CD19 CAR-T cell therapy co-expressing PD-1H shRNA can effectively inhibit tumor growth in the NCG mouse lymphoma (CA-46) model.
  • Figure 14 shows that CD19 CAR-T cell therapy co-expressing PD-1H shRNA can effectively prolong the survival of mice.
  • Figure 15 shows that sgRNA1 and sgRNA2 can effectively knock down the expression of PD-1H on Jurkat-PD-1H cells, and the knockdown efficiency of sgRNA1 is higher.
  • Figure 16 shows the gene editing efficiency of sgRNA1 detected by T7E1 endonuclease on B7-H3 CAR-T cells.
  • Figure 17 shows TIDE analysis of the gene editing efficiency of sgRNA1 on B7-H3 CAR-T cells.
  • Figure 18 shows the editing of the PD-1H gene of B7-H3 CAR-T cells by sgRNA1.
  • CAR-T cells are taken as an example, and the engineered immune cells of the present invention are described in detail.
  • the engineered immune cells of the present invention are not limited to the CAR-T cells described above and below, and the engineered immune cells of the present invention have the same or similar technical features and beneficial effects as the CAR-T cells described above and below.
  • NK cells are equivalent to T cells (or T cells can replace NK cells);
  • TCR is equivalent to CAR (or CAR can be replaced by TCR) ).
  • antibody shall include, but is not limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigens thereof combined part.
  • Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region contains three constant domains, CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region contains one constant domain, CL.
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • a “chimeric antigen receptor (CAR)” is a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a different polypeptide from the extracellular domain, and at least one cellular internal domain.
  • CAR Chimeric Antigen Receptor
  • CIR Chomeric Immunoreceptor
  • the "extracellular domain capable of binding an antigen” refers to any oligopeptide or polypeptide capable of binding an antigen.
  • Extracellular domain refers to any oligopeptide or polypeptide known as a domain that transmits signals to activate or inhibit intracellular biological processes.
  • domain refers to a region of a polypeptide that folds into a specific structure independently of other regions.
  • tumor antigen refers to an antigenic biomolecule whose expression causes cancer.
  • administer refers to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid.
  • administering can refer to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contact of reagents with cells, as well as contact of reagents with fluids, and contact of fluids with cells.
  • administering and “treating” also mean in vitro and ex vivo treatment by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to humans, animals or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnosis.
  • treating refers to the administration of an internal or external therapeutic agent, comprising any one of the engineered immune cells and compositions thereof of the present invention, to a patient with one or more disease symptoms for which the Therapeutic agents have a therapeutic effect on these symptoms.
  • a patient is administered to a patient in an amount of the therapeutic agent effective to alleviate one or more symptoms of the disease (therapeutically effective amount).
  • the terms “optional” or “optionally” mean that the subsequently described event or circumstance can, but need not, occur.
  • “optionally comprising 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable region of a specific sequence may, but does not necessarily have, one, two or three.
  • Sequence identity refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions.
  • sequence identity between the sequences described in the present invention and the sequences with which they are identical may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ,100%.
  • PD-1H also known as VISTA, c10orf54, VSIR, SISP1, B7-H5, DD1 ⁇ , Gi24 and Dies1, is a 311 amino acid type 1 transmembrane protein.
  • PD-1H contains an N-terminal Ig V-type domain, a transmembrane domain, and an intracellular domain. It has homology with CD28 family member PD-1 and B7 family member PD-L1. Unlike other members of the family, the PD-1H intracellular segment does not contain the canonical ITIM/ITAM motif, but its ectodomain has a 21-residue C-C' loop and more histidines. Recent studies have found that the histidine residue on PD-1H is the primary binding site for its receptor PSGL-1, while the C-C' loop is the primary binding site for another receptor, VSIG3.
  • PD-1H is mainly expressed on hematopoietic cells, and expressed on myeloid cells (including macrophages, dendritic cells, monocytes and neutrophils) and CD4+ T cells.
  • PD-1H can act as both a ligand and a receptor to produce inhibitory effects.
  • PD-1H can inhibit T cell proliferation and cytokine release, and has an important regulatory role in autoimmune diseases and tumor progression.
  • PD-1H can act as an inhibitory receptor on CD4+ T cells to regulate immune tolerance and tumor immunity involving T cells.
  • PD-1H-specific blocking antibodies can effectively inhibit tumor growth in multiple mouse tumor models (melanoma, bladder cancer, etc.).
  • the PD-1H-specific agonistic antibody can effectively alleviate the symptoms of autoimmune diseases (systemic lupus erythematosus, asthma, arthritis, etc.).
  • the tumor antigens of the present invention include but are not limited to CD7, CD5, CD2, CD3, CD19, CD20, CD22, CD24, CD25, CD28, CD123, CD47, CD52, CD56, CD80, CD86, CD81, CD138, CD33, CD38, CD30, CD133, CD97, CD99, CD40, CD43, CD137, CD151, CD171, KIT(CD117), CD174, CD44V6, CD179a, B7-H3(CD276), B7-H4, HER2, HER3, HER4, c-Met, PSMA, PSCA, MUC16, MUC1, mesothelin, EGFR, VEGFR2, EGFR-VIII, VEGFR-1, GPC3, BCMA, ErbB2, ErbB3, ErbB4, NKG2D ligand, LMP1, EpCAM, Lewis-Y, ROR1, Claudin18 .2, LIGHT, NKG2C, CEA (carcinoembryonic antigen), FAP,
  • CD19 refers to the differentiation antigen cluster 19 protein, which is a detectable antigenic determinant on leukemia precursor cells.
  • CD19 described herein comprises proteins that are mutated (eg, point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type CD19).
  • CD19 is expressed on most B-lineage cancers, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. It is also an early marker of B cell progenitors.
  • the antigen binding domain of the chimeric antigen receptor CAR specifically binds to tumor cell surface antigens.
  • the antigen binding domain of the chimeric antigen receptor CAR of the present invention targets CD19, B7-H3 (CD276).
  • the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.
  • the transmembrane domain naturally associated with one of the domains in the CAR is used.
  • transmembrane domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with receptor complexes interactions with other members.
  • Transmembrane domains can be derived from natural or synthetic sources.
  • the domain can be derived from any membrane-bound or transmembrane protein.
  • the hinge region and the transmembrane region in the CAR of the present invention are the hinge region and the transmembrane region of CD8.
  • the intracellular domain or additional intracellular signaling domain of the CAR of the invention is responsible for the activation of at least one normal effector function of the immune cell in which the CAR has been placed.
  • effector function refers to the exclusive function of a cell.
  • the effector function of a T cell can be cytolytic activity or helper activity including cytokine secretion.
  • intracellular signaling domain thus refers to the portion of the protein that transduces effector function signals and directs the cell to perform a specific function. Although the entire intracellular signaling domain can generally be used, in many instances it is not necessary to use the entire chain.
  • intracellular signaling domain In the case of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used in place of the complete chain, so long as it transduces effector function signals.
  • the term intracellular signaling domain thus refers to any truncated portion that includes an intracellular signaling domain sufficient to signal effector function.
  • TCRs T cell receptors
  • co-receptors that act cooperatively to initiate signaling upon antigen receptor binding
  • the cytoplasmic domain of the CAR can be designed to include the CD3-zeta signaling domain itself, or can be combined with any other desired cytoplasmic domain (a or more) union.
  • the cytoplasmic domain of a CAR can include a CD3 ⁇ chain portion and a costimulatory signaling region.
  • the costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of the costimulatory molecule.
  • Costimulatory molecules are cell surface molecules, not antigen receptors or their ligands, that are required for effective lymphocyte responses to antigens.
  • 4-1BB CD137
  • 4-1BB CD137
  • cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention can be linked to each other randomly or in a prescribed order.
  • short oligopeptide or polypeptide linkers preferably between 2 and 10 amino acids in length, can form the link.
  • Glycine-serine doublets provide particularly suitable linkers.
  • the cytoplasmic domain in the CAR of the invention is designed to include the signaling domain of 4-1BB (costimulatory molecule) and the signaling domain of CD3 ⁇ .
  • Chimeric antigen receptors are composed of an extracellular antigen recognition region, usually a scFv (single-chain variable fragment), a transmembrane region and an intracellular costimulatory signal region.
  • the design of CARs has gone through the following process: the first-generation CAR has only one intracellular signal component, CD3 ⁇ or Fc ⁇ RI molecule. Since there is only one activation domain in the cell, it can only cause transient T cell proliferation and less cytokine secretion. , and can not provide long-term T cell proliferation signal and sustained anti-tumor effect in vivo, so it has not achieved good clinical efficacy.
  • the second-generation CARs introduce a costimulatory molecule on the basis of the original structure, such as CD28, 4-1BB, OX40, and ICOS. Compared with the first-generation CARs, the function is greatly improved, which further strengthens the persistence of CAR-T cells and promotes tumor cells. destructive ability. On the basis of second-generation CARs, some new immune costimulatory molecules such as CD27 and CD134 are connected in series to develop into third- and fourth-generation CARs.
  • the extracellular segment of CARs can recognize a specific antigen, and then transduce the signal through the intracellular domain, causing cell activation and proliferation, cytolytic toxicity, and secretion of cytokines, thereby eliminating target cells.
  • the patient's autologous cells or allogeneic donors
  • the patient's autologous cells are isolated, activated and genetically modified to produce CAR-producing immune cells, and then injected into the same patient. In this way, the probability of graft-versus-host disease is extremely low, and the antigen is recognized by immune cells in a non-MHC-restricted manner.
  • CAR-immune cell therapy has achieved a very high clinical response rate in the treatment of hematological malignancies. Such a high response rate is unattainable by any previous treatment method, and has triggered an upsurge in clinical research all over the world.
  • the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes target-specific binding elements (also referred to as antigen binding domains).
  • the intracellular domain includes a costimulatory signaling region and/or a zeta chain portion.
  • a costimulatory signaling region refers to a portion of an intracellular domain that includes a costimulatory molecule.
  • Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that are required for an efficient lymphocyte response to an antigen.
  • a linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.
  • the term "linker” generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain.
  • the linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.
  • the CAR of the present invention when expressed in T cells, is capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, it affects tumor cells, causing the tumor cells to not grow, being driven to die, or otherwise being affected, and resulting in a reduction or elimination of the patient's tumor burden.
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and/or the zeta chain.
  • the antigen binding domain is fused to the intracellular domain in combination with the 4-1BB signaling domain and/or the CD3 ⁇ signaling domain.
  • antigen-binding domain and “single-chain antibody fragment” each refer to a Fab fragment, Fab' fragment, F(ab')2 fragment, or a single Fv fragment having antigen-binding activity.
  • Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and are the smallest antibody fragment with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.
  • the antigen binding domain is usually a scFv (single-chain variable fragment). The size of scFv is generally 1/6 of that of a complete antibody.
  • Single chain antibodies are preferably one amino acid chain sequence encoded by one nucleotide chain.
  • the scFv comprises antibodies that specifically recognize the highly expressed tumor antigen CD47 and MSLN, preferably single-chain antibodies.
  • the scFv of the present invention also includes its conservative variants, which means that compared with the amino acid sequence of the scFv of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, most preferably Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide.
  • the number of added, deleted, modified and/or substituted amino acids is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33%, More preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • the number of amino acids added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, 1 is optimally.
  • the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.
  • the transmembrane domain naturally associated with one of the domains in the CAR is used.
  • transmembrane domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with receptor complexes interactions with other members.
  • the extracellular domain of the CAR of the present invention includes an antigen-binding domain that specifically binds to a tumor cell surface antigen, preferably an antigen-binding domain that specifically binds to CD19.
  • the intracellular domain in the CAR of the present invention includes an extracellular domain, a transmembrane region of CD8, a costimulator of 4-1BB, a signaling domain of CD3 ⁇ , wherein the extracellular domain includes a target- Specific binding elements (also known as antigen-binding domains)
  • CAR-T cells As used herein, the terms “CAR-T cells”, “CAR-T” and “CAR-T cells of the present invention” all refer to the CAR-T cells of the present invention, and the CAR-T cells of the present invention can target the surface of tumor cells Antigens (preferably CD19) are used to treat tumors with high expression or positivity of tumor cell surface antigens (such as CD19).
  • Antigens preferably CD19
  • tumor cell surface antigens such as CD19
  • CAR-T cells have the following advantages over other T-cell-based therapies: (1) the action of CAR-T cells is not restricted by MHC; (2) given that many tumor cells express the same tumor antigen, targeting a certain tumor Once the CAR gene construction of the antigen is completed, it can be widely used; (3) CAR can use both tumor protein antigens and glycolipid non-protein antigens, which expands the target range of tumor antigens; (4) uses patient autologous The cells reduce the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.
  • the CAR of the present invention comprises (i) an extracellular domain comprising an antigen-binding domain that specifically binds to a tumor cell surface antigen; (ii) a transmembrane domain; (iii) a costimulatory factor; and ( iv) The signaling domain of CD3 ⁇ .
  • CAR-NK cell As used herein, the terms “CAR-NK cell”, “CAR-NK”, “CAR-NK cell of the present invention” all refer to the CAR-NK cell of the present invention.
  • the CAR-NK cells of the present invention can target tumor cell surface antigens (preferably CD19) for the treatment of tumors with high expression or positive tumor cell surface antigens (such as CD19).
  • Natural killer (NK) cells are a major class of immune effector cells that protect the body from virus infection and tumor cell invasion through non-antigen-specific pathways.
  • Engineered (genetically modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and have enhanced anti-tumor cytotoxicity.
  • CAR-NK cells Compared with autologous CAR-T cells, CAR-NK cells also have the following advantages, such as: (1) they directly kill tumor cells by releasing perforin and granzyme, but have no killing effect on normal cells in the body; (2) they release A very small amount of cytokines reduces the risk of cytokine storm; (3) it is easy to expand in vitro and develop into "off-the-shelf" products. Other than that, it is similar to CAR-T cell therapy.
  • exogenous T cell receptor is the ⁇ chain and ⁇ chain of TCR cloned from tumor-reactive T cells by gene transfer technology, by means of genetic engineering, with lentivirus or Retroviruses are used as vectors to exogenously transfer TCRs into T cells.
  • T cells modified by exogenous TCR can specifically recognize and kill tumor cells, and by optimizing the affinity of TCR with tumor-specific antigens, the affinity of T cells with tumors can be improved, and the anti-tumor effect can be improved.
  • PD-1H gene expression is down-regulated or silenced
  • PD-1H gene expression is silenced refers to no or low expression of the PD-1H gene.
  • Low expression refers to the ratio of the expression level G1 of the PD-1H gene of the CAR-T cells to the expression level G0 of the PD-1H gene of normal immune cells, that is, G1/G0 ⁇ 0.8, preferably G1/G0 ⁇ 0.5, More preferably ⁇ 0.2, more preferably ⁇ 0.1, most preferably 0.
  • PD-1 gene expression down-regulation or silencing methods in the present invention include CRISPR/Cas9, RNA interference technology, transcription activator-like (TAL) effector nucleases TALENs (transcription activator-like (TAL) effector nucleases) and zinc finger nucleases Zinc finger nucleases ( ZFNs).
  • the present invention down-regulates or silences the PD-1H gene through CRISPR/Cas9 and RNA interference technology.
  • the PD-1H gene is down-regulated or silenced using CRISPR/Cas9 or shRNA.
  • CRISPR clustered regularly interspersed short palindromic repeats
  • Cas CRISPR-associated
  • Type II CRISPR/Cas systems have been successfully applied in many eukaryotes and prokaryotes as tools for direct RNA-mediated genome editing.
  • the development of the CRISPR/Cas9 system has revolutionized the ability to edit DNA sequences and regulate the expression levels of target genes, thereby providing a powerful tool for precise genome editing of organisms.
  • the simplified CRISPR/Cas9 system consists of two parts: Cas9 protein and sgRNA.
  • the principle of action is that the sgRNA forms a Cas9-sgRNA complex with the Cas9 protein through its own Cas9 handle, and the base complementary pairing region sequence of the sgRNA in the Cas9-sgRNA complex is paired with the target sequence of the target gene through the base complementary pairing principle.
  • Cas9 utilizes its own endonuclease activity to cleave target DNA sequences.
  • the CRISPR/Cas9 system has several distinct advantages: ease of use, simplicity, low cost, programmability, and the ability to edit multiple genes simultaneously.
  • an "expression cassette” or “expression cassette of the invention” includes an optional first expression cassette and a second expression cassette.
  • the first expression cassette comprises a nucleic acid sequence encoding a CAR.
  • the second expression cassette contains nucleic acid sequences for silencing PD-1H.
  • the present invention further includes a third expression cassette for expressing the gene editing protein.
  • the optional first expression cassette, second expression cassette and third expression cassette, respectively further comprise a promoter. In one embodiment, the optional first expression cassette, second expression cassette and third expression cassette, respectively, further comprise a terminator.
  • the optional first, second and third expression cassettes are located on the same or different vectors.
  • the optional first expression cassette, second expression cassette and third expression cassette are located in the same vector.
  • the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, other gene transfer systems, or a combination thereof.
  • the vector is a viral vector.
  • the third expression cassette comprises CRISPR/Cas9 (sgRNA and Cas9), antisense RNA, or a combination thereof.
  • the sgRNA targets PD-1H, and the sequence of the sgRNA is shown in any of SEQ ID NO.: 1-4, 12-18 or a combination thereof.
  • the antisense RNA includes miRNA, siRNA, shRNA, inhibitory mRNA, or dsRNA, and the sequence of the antisense RNA is shown in any one or combination of SEQ ID NO.:5-10.
  • Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by using standard technology to isolate directly from cells and tissues that contain the gene. Alternatively, the gene of interest can be produced synthetically.
  • the present invention also provides vectors into which the expression cassettes of the present invention are inserted.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer because they allow long-term, stable integration of the transgene and its proliferation in daughter cells.
  • Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus because they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.
  • an expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector.
  • the vector is suitable for replication and integration in eukaryotic cells.
  • Typical cloning vectors contain transcriptional and translational terminators, initial sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
  • the expression constructs of the present invention can also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, eg, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety.
  • the present invention provides gene therapy vectors.
  • the nucleic acid can be cloned into many types of vectors.
  • the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • expression vectors can be provided to cells in the form of viral vectors.
  • Viral vector techniques are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO01/96584; WO01/29058; and U.S. Patent No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo.
  • Many retroviral systems are known in the art.
  • adenoviral vectors are used.
  • Many adenoviral vectors are known in the art.
  • lentiviral vectors are used.
  • promoter elements can regulate the frequency of transcription initiation. Typically, these are located in a region of 30-110 bp upstream of the initiation site, although it has recently been shown that many promoters also contain functional elements downstream of the initiation site.
  • the spacing between promoter elements is often flexible so that promoter function is maintained when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline.
  • individual elements appear to act cooperatively or independently to initiate transcription.
  • a suitable promoter is the U6 promoter.
  • the promoter sequence is a constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is elongation growth factor-1 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences can also be used, including but not limited to the CMV promoter, the simian virus 40 (SV40) early promoter, the mouse breast cancer virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) ) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russell sarcoma virus promoter, and human gene promoters such as but not limited to muscle Actin promoter, myosin promoter, heme promoter and creatine kinase promoter.
  • the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention.
  • an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is not desired.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • the expression vector introduced into the cells may also contain either or both of a selectable marker gene or a reporter gene to facilitate the search for the transfected or infected cell population from the viral vector Identification and selection of expressing cells.
  • the selectable marker can be carried on a single piece of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.
  • Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is clearly indicated by some readily detectable property such as enzymatic activity. After the DNA has been introduced into the recipient cells, the expression of the reporter gene is measured at an appropriate time.
  • Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (eg, Ui-Tei et al., 2000 FEBS Letters 479:79). -82).
  • Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Typically, constructs with a minimum of 5 flanking regions showing the highest levels of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to assess the ability of an agent to modulate promoter-driven transcription.
  • an expression vector can be readily introduced into a host cell, eg, mammalian, bacterial, yeast or insect cells, by any method known in the art.
  • a host cell eg, mammalian, bacterial, yeast or insect cells
  • an expression vector can be transferred into a host cell by physical, chemical or biological means.
  • Physical methods of introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, eg, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). The preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.
  • Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors.
  • Viral vectors especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, eg, human cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, among others. See, eg, US Patent Nos. 5,350,674 and 5,585,362.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids plastid.
  • lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and lipids plastid.
  • Exemplary colloidal systems for use as in vitro and in vivo delivery vehicles are liposomes (eg, artificial membrane vesicles).
  • exemplary delivery vehicles are liposomes.
  • lipid formulations is contemplated to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo).
  • nucleic acid can be associated with a lipid.
  • Nucleic acids associated with lipids can be encapsulated into the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, attached via linker molecules associated with both liposomes and oligonucleotides to liposomes, entrapped in liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in micelles or Complex with micelles, or otherwise associated with lipids.
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may exist in bilayer structures, as micelles or have a "collapsed" structure.
  • Lipids are fatty substances, which can be naturally occurring or synthetic lipids.
  • lipids include lipid droplets, which occur naturally in the cytoplasm as well as in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes.
  • the vector is a lentiviral vector.
  • the pCDH-GFP-based CD19-CAR vector and B7-H3(CD276)-CAR vector designed in the present invention integrate the shRNA or sgRNA of PD-1H, but the content of the present invention is not limited to the CAR vector constructed with pCDH-GFP, It should be suitable for all other plasmid vectors suitable for the construction of CART; the target is not limited to CD19, B7-H3, and should be suitable for all CAR-T targets.
  • the present invention provides the engineered immune cell of the first aspect of the present invention, the host cell of the tenth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a liquid formulation.
  • the formulation is an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 9 cells/Kg body weight, more preferably 1 ⁇ 10 4 -1 ⁇ 10 8 cells/Kg body weight.
  • the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine ; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, etc.
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • preservatives e.g, aluminum hydroxide
  • the present invention includes therapeutic applications of cells (eg, T cells, more preferably PD-1H silenced T cells (eg, CD8+ T cells)) transduced with lentiviral vectors (LVs) encoding the expression cassettes of the present invention.
  • Transduced T cells can target tumor cell markers (such as CD19), synergistically activate T cells, and cause cellular immune responses, thereby significantly improving their killing efficiency against solid tumors and hematological tumors.
  • the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the steps of: administering to the mammal a CAR-T cell of the present invention.
  • the present invention includes a type of cell therapy wherein a patient's autologous T cells (or a heterologous donor) are isolated, activated and genetically engineered to produce CAR-T cells, and subsequently infused into the same patient.
  • a patient's autologous T cells or a heterologous donor
  • CAR-T can treat all cancers that express this antigen.
  • CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.
  • the CAR-T cells of the invention can undergo robust in vivo T cell expansion for extended amounts of time.
  • a CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR.
  • CD19 CAR-T cells elicit specific immune responses against CD19-expressing cells.
  • Cancers that can be treated include tumors that are not vascularized or substantially not vascularized, as well as tumors that are vascularized. Cancers may include non-solid tumors (such as hematological tumors, eg, leukemias and lymphomas) or may include solid tumors. Cancer types treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemic or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Also includes adult tumors/cancers and pediatric tumors/cancers.
  • Hematological cancers are cancers of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastoid, promyelocytic, myelomonocytic type) , monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (myeloid) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non- Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • acute leukemias such
  • Solid tumors are abnormal masses of tissue that typically do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell type that forms them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.
  • the CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals.
  • the mammal is a human.
  • CAR-modified cells are isolated from mammals (preferably human) and genetically modified (ie, transduced or transfected in vitro) with vectors expressing the CARs disclosed herein.
  • CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefit.
  • the mammalian recipient can be human, and the CAR-modified cells can be autologous to the recipient.
  • the cells may be allogeneic, syngeneic or xenogeneic with respect to the recipient.
  • the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.
  • the present invention provides methods of treating tumors comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the present invention.
  • the CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components or other cytokines or cell populations.
  • the pharmaceutical compositions of the present invention may include a target cell population as described herein in association with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelates Adjuvants such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • the compositions of the present invention are preferably formulated for intravenous administration.
  • compositions of the present invention can be administered in a manner appropriate to the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease - although appropriate doses may be determined by clinical trials.
  • the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the patient (subject ) individual differences in age, weight, tumor size, degree of infection or metastasis, and condition. It may generally be indicated that the pharmaceutical compositions comprising the T cells described herein may be administered at a dose of 104 to 109 cells/kg body weight, preferably 105 to 106 cells/kg body weight (including all integers within those ranges). value) application. The T cell composition can also be administered multiple times at these doses.
  • Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection.
  • the T cell composition of the present invention is preferably administered by i.v. injection.
  • the composition of T cells can be injected directly into tumors, lymph nodes or the site of infection.
  • cells activated and expanded using the methods described herein, or other methods known in the art to expand T cells to therapeutic levels are combined with any number of relevant therapeutic modalities (eg, previously , concurrently or subsequently) to a patient in a form of treatment including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or elfazizumab therapy for psoriasis patients or other treatments for PML patients.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or elfazizumab therapy for psoriasis patients or other treatments for PML patients.
  • the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutics.
  • the cellular composition of the invention is administered in combination with (eg, before, concurrently or after) bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an infusion of expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • the dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be carried out according to art-accepted practice. Typically, 1 x 106 to 1 x 1010 T cells of the invention (eg, CD19-CAR-T cells) can be administered to a patient per treatment or per course of treatment, eg, by intravenous infusion .
  • the present invention finds for the first time that PD-1H has an inhibitory immune regulation function in CD8+ T cells, and can negatively regulate the role of CD8+ T cells in tumor immunity.
  • the present invention finds for the first time that the expression of PD-1H on CD8+ T cells (including mouse-derived OT-1 cells and human-derived CAR-T cells) can enhance the anti-tumor effect of CD8+ T cells by RNA interference or gene knockout, promote its infiltration into the tumor microenvironment.
  • CD8+ T cells including mouse-derived OT-1 cells and human-derived CAR-T cells
  • PD-1H has a significant immunosuppressive function in CD8+ T cells.
  • PD-1H expression was significantly upregulated on tumor-infiltrating CD8+ T cells.
  • the anti-tumor effect of PD-1H-depleted CD8+ T cells was significantly enhanced, and the release of immune effector molecules (such as IFN- ⁇ , GranzmB, etc.) was increased, and PD-1H-depleted CD8+ T cells were better infiltrate the tumor microenvironment. Therefore, targeting PD-1H on T cells may be a potential tumor immunotherapy approach.
  • the present invention finds for the first time that targeting PD-1H (silencing/knockout PD-1H expression, specific antibody action or other methods) can be applied to CAR-T cells or other immune cells.
  • Example 1 PD-1H expression is increased in tumor-infiltrating CD8+ T cells and activated CD8+ T cells
  • mice Female, 8-week-old C57BL/6 mice were selected, and mouse lymphoma EG7 cells or mouse melanoma B16-ova cells were implanted subcutaneously on the ventral side. After 17 days of tumor seeding, the subcutaneous tumor and spleen of mice were taken to prepare a single cell suspension, anti-mFc receptor (clone: 2.4G2) antibody was added to incubate at 4°C for 10 min, and washed once with PBS containing 1% FBS.
  • anti-mFc receptor clone: 2.4G2
  • anti-mCD3, anti-mCD4, anti-mCD8, anti-mPD-1H antibodies were added and incubated at 4°C for 30 min, washed once with PBS containing 1% FBS, and the expression of PD-1H on T cells was detected by flow cytometry. express the situation.
  • PD-1H was expressed on both tumor-infiltrating T cells of mouse EG7 lymphoma and B16-OVA melanoma, and the expression of PD-1H on CD8+ T cells was higher than that on CD4+ T cells.
  • CD8+ T cells from PD-1H KO mice and their control wild-type mice were sorted by CD8+ T cell sorting kit, cultured in vitro, and added with anti-mCD3 (1 ⁇ g/ml) and anti-mCD28 (2 ⁇ g/ml) stimulation.
  • WT wild-type mice
  • anti-mCD3 1 ⁇ g/ml
  • anti-mCD28 2 ⁇ g/ml
  • T cells were collected and anti-mCD8 and anti-mPD-1H antibodies were added and incubated at 4°C for 30 min. After washing once with PBS containing 1% FBS, T cells were detected by flow cytometry. The expression of PD-1H.
  • Example 2 The anti-tumor effect of CD8+ T cells lacking PD-1H is enhanced
  • mice Female, 8-week-old C57BL/6 mice were selected and subcutaneously implanted with mouse melanoma B16-OVA cells on the ventral side. Six days after tumor seeding, PD-1H KO or WT OT-1 T cells were injected into the tail vein of tumor-bearing mice for treatment, respectively. Tumor size was measured every two days with an electronic vernier caliper. After 17 days, the subcutaneous tumor of the mouse was taken and prepared into a single cell suspension, anti-mFc receptor (clone: 2.4G2) antibody was added and incubated at 4°C for 10 min, and washed once with PBS containing 1% FBS. Anti-mCD3, anti-mCD45, and anti-mCD8 antibodies were added and incubated at 4°C for 30 min, washed once with PBS containing 1% FBS, and detected by flow cytometry.
  • anti-mFc receptor clone: 2.4G2
  • mice Female, 8-week-old C57BL/6 mice were selected and subcutaneously implanted with mouse lymphoma EG7 cells on the ventral side. Six days after tumor seeding, PD-1H KO or WT OT-1 T cells were transfused into the tail vein of tumor-bearing mice for treatment. Tumor size was measured every two days with an electronic vernier caliper. After 17 days, the subcutaneous tumor of the mouse was taken and prepared into a single cell suspension, anti-mFc receptor (clone: 2.4G2) antibody was added and incubated at 4°C for 10 min, and washed once with PBS containing 1% FBS. Anti-mCD3, anti-mCD45, and anti-mCD8 antibodies were added and incubated at 4°C for 30 min, washed once with PBS containing 1% FBS, and detected by flow cytometry.
  • anti-mFc receptor clone: 2.4G2
  • Anti-mCD3, anti-mCD45, and anti-mCD8 antibodies were added
  • Example 3 Using CRISPR Cas9 technology to knock down the PD-1H gene on CD8+ T cells can enhance the anti-tumor effect of CD8+ T cells.
  • the sgRNA sequence was designed.
  • the LentiCRISPR v2 plasmid replaces the original puromycin resistance sequence with a GFP protein sequence.
  • the synthetic mPD-1H sgRNA1 and mPD-1H sgRNA2 fragments were annealed into double-stranded DNA and ligated into the BsmBI site of the LentiCRISPR v2 vector (Addgene 52961). The clones were selected, sequenced and analyzed to confirm that the sgRNA fragment was successfully constructed into the LentiCRISPR v2 vector.
  • the lentiCRISPR mPD-1H sgRNA plasmids (sgRNA1 and sgRNA2 plasmids) were mixed with pRSV-Rev, pMDLg/pRRE and pCMV-VSVG helper plasmids in a certain proportion to co-transfect 293FT cells.
  • the cell culture supernatants containing mPD-1H sgRNA-H1 and mPD-1H sgRNA-H2 lentiviruses were collected and centrifuged at 4°C and 3000rpm for 5min. The supernatant was filtered through a 0.22uml filter and stored at -80°C for later use. 293T-mPD-1h cells were infected with lentivirus.
  • Lymphocytes from the spleen of 8-week-old female OT-1 mice were isolated by lymphocyte separation medium and density gradient centrifugation. The cells were labeled with magnetic beads using CD8+ T cell isolation Kit (Miltenyi), and CD8+ T lymphocytes were isolated and purified. The purified CD8+ T cells were then activated and proliferated by CD3/CD28 antibody. Activated T lymphocytes were harvested 24 hours after antibody stimulation and resuspended in RPMI 1640 medium. Activated CD8+ T lymphocytes were infected with mPD-1H sgRNA-H2 lentivirus, and the cell suspension was added to a 6-well plate and incubated overnight in a 37°C, 5% CO 2 incubator.
  • T cells were collected by centrifugation, resuspended in a suitable freezing medium, and frozen in liquid nitrogen for later use.
  • mice Female, 8-week-old C57BL/6 mice were selected and subcutaneously implanted with mouse lymphoma EG7 cells on the ventral side. Six days after tumor seeding, sgRNA OT-1, PD-1H KO or WT OT-1 T cells were transfused into the tail vein of tumor-bearing mice for treatment. Tumor size was measured every two days with an electronic vernier caliper. After 17 days, the subcutaneous tumor of the mouse was taken and prepared into a single cell suspension, anti-mFc receptor (clone: 2.4G2) antibody was added and incubated at 4°C for 10 min, and washed once with PBS containing 1% FBS. Anti-mCD3, anti-mCD45, and anti-mCD8 antibodies were added and incubated at 4°C for 30 min, washed once with PBS containing 1% FBS, and detected by flow cytometry.
  • anti-mFc receptor clone: 2.4G2
  • Example 4 Increased PD-1H expression in human T cells or human CAR-T cells in the tumor microenvironment of the mouse colon cancer PDX model
  • B7-H3-CAR-T cells obtained from Fuzhou Tuoxin Tiancheng Biotechnology Co., Ltd., the structure is: B7-H3scFv-CD8 hinge region-CD8 transmembrane region-41BB costimulatory molecule-CD3 ⁇ cytoplasmic signal transduction area), anti-hCD3, anti-hB7-H3-mFc and anti-hPD-1H antibodies were added and incubated at 4°C for 30 min, washed once with PBS containing 1% FBS, and detected by flow cytometry.
  • xenograft (PDX) colon cancer tumor model 8-10-week-old female NCG mice (NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju) were selected to construct a xenograft (PDX) colon cancer tumor model.
  • the patient's colon tumor tissue was cut into small pieces of 15 mm 3 and implanted subcutaneously in the bilateral axilla of NCG mice.
  • the tumor growth of the mice was observed daily, and the tumor size was measured weekly with a vernier caliper. When the tumor reached a size of about 500 mm 3 , the tumor tissue was taken for passage. Passaging Methods As described above, after five consecutive mouse-to-mouse passages, xenografted tumors were seeded subcutaneously in the bilateral axilla of NCG mice.
  • CAR-T cells were injected intratumorally for treatment.
  • the peripheral blood, spleen and tumor tissue of the mice were taken to prepare a single-cell suspension, labeled with fluorescent antibodies of specific molecules, and analyzed by flow cytometry. Part of the spleen and tumor tissue were made into paraffin sections for analysis.
  • the tumor tissue sections of the PDX mouse model were taken, dewaxed and rehydrated, and then kept in 1 mmol/L EDTA for 20 min in micro-boiling for antigen retrieval. Endogenous catalase was blocked by incubation for 20 min in 3% hydrogen peroxide in water. Immunofluorescence staining was performed with PD-1H antibody (clone D1L2G, CST) as the primary antibody, HRP-conjugated secondary antibody and tyramide amplification system (TSA Plus Fluorescence Kits). Cells were phenotyped with CD3 and CD8 antibodies and incubated with appropriate fluorescent secondary antibodies. Nuclei were labeled with DAPI. The stained sections were observed, scanned and photographed with EVOS FL Auto Cell Imaging System.
  • Example 5 Using short hairpin RNA (shRNA) to silence the expression of PD-1H on CAR-T cells can enhance the anti-tumor effect of CAR-T cells.
  • shRNA short hairpin RNA
  • the shRNA sequences designed according to the CDS sequence of PD-1H are shown in Table 1 as candidate PD-1H shRNA sequences.
  • the U6 promoter and PD-1H shRNA sequence were synthesized, and the U6-PD-1H shRNA sequence was constructed into the pCDH-EF1-CAR plasmid by restriction enzyme ligation technology, pCDH-U6-PD-1H shRNA-EF1 -CAR plasmid structure is shown in Figure 9.
  • the sequence of U6-PD-1H shRNA-EF1-CAR plasmid was confirmed to be correct by second-generation sequencing technology.
  • lentiviral vectors were packaged and prepared on 293T cells to express pCDH-U6-PD-1H shRNA-EF1-CAR (PD-1H shRNA1-shRNA6) and Control shRNA lentivirus.
  • PD-1H shRNA1-shRNA6 pCDH-U6-PD-1H shRNA-EF1-CAR
  • Control shRNA lentivirus pCDH-U6-PD-1H shRNA-EF1-CAR
  • Jurkat-PD-1H cells were infected with lentivirus. 72 hours after virus infection, the expression of PD-1H in Jurkat-PD-1H cells was detected by flow cytometry. The results are shown in Figure 10.
  • the pCDH-U6-PD-1H shRNA-EF1-CAR plasmid can express CAR molecules on the surface of T cells unaffected.
  • PD-1H shRNA1 can most effectively silence the expression of PD-1H, followed by PD-1H shRNA3, PD-1H shRNA2, PD-1H shRNA4, PD-1H shRNA5, PD-1H shRNA6 are more effective in silencing weak.
  • PBMC peripheral blood mononuclear cells
  • PD-1H shRNA-CD19-CAR an example of CD19-CAR structure is: CD19-targeted scFv-CD8 hinge region-CD8 transmembrane region-41BB costimulatory molecule-CD3 ⁇ cytoplasmic signaling region
  • Control shRNA-CD19-CAR lentivirus infects 1 ⁇ 10 6 activated T lymphocytes respectively, and the cell suspension is added to a G-Rex culture flask and incubated overnight in a 37°C, 5% CO 2 incubator, every 2 Add fresh medium every day and continue to expand the culture.
  • CAR-T cells (PD-1H shRNA-CD19-CAR T cells and Control shRNA-CD19-CAR T cells) were collected by centrifugation, resuspended in a suitable freezing medium, and frozen in liquid nitrogen for later use.
  • DAPI was applied and detected by flow cytometry. Killing effect of CAR T cells on target tumor cells.
  • NCG mice 8-10-week-old female NCG mice (NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju) were selected, and CD19-positive tumor cells (lymphoma cells CA-46) were injected into the tail vein to establish a tumor model. After 5 days of modeling, CAR-T cell therapy was transfused in the tail vein. Tumor growth was detected by a small animal in vivo imaging system.
  • PD-1H shRNA-CD19-CAR T cells can effectively inhibit the growth of lymphoma cells, and have a better therapeutic effect than control CAR-T cells. At the same time, it effectively prolongs the survival time of mice.
  • the sgRNA sequences were designed according to the CDS sequence of human PD-1H, and the candidate PD-1H sgRNA sequences are shown in Table 2.
  • the PD-1H sgRNA (single guide RNA) added with the RNA backbone was synthesized according to Table 2, and the sgRNA was formed by connecting the gRNA and the RNA backbone.
  • the above-synthesized PD-1H sgRNA1 and PD-1H sgRNA2 were mixed with cas9 protein in a certain proportion, and electroporated into Jurkat-PD-1H cells using a Lonza Amaxa TM 4D-Nucleofector TM electroporator. After 72 hours of electroporation, about 2 ⁇ 10 5 cells were added with anti-PD-1H antibody and incubated at 4°C for 30 min, washed once with PBS containing 1% FBS, and detected by flow cytometry.
  • PD-1H gRNA1 and gRNA2 can effectively knock down the expression of PD-1H on Jurkat-PD-1H cells, and the knockout effect of PD-1H gRNA1 is better.
  • PBMC peripheral blood mononuclear cells
  • the cells were labeled with magnetic beads using Pan T cell isolation Kit (Miltenyi), and T lymphocytes were isolated and purified. The purified T cells were then activated by anti-CD3/CD28 magnetic beads.
  • PD-1H gRNA1 or control sRNA was mixed with cas9 protein in a certain proportion, and electroporated into activated T lymphocytes using a Lonza Amaxa TM 4D-Nucleofector TM electroporator.
  • B7-H3-CAR 48h after electroporation, B7-H3-CAR was used (the structure of B7-H3-CAR is: scFv targeted by B7-H3-CD8 hinge region-CD8 transmembrane region-41BB costimulatory molecule-CD3 ⁇ cytoplasmic signal transduction region)
  • Infect 1 ⁇ 10 7 electroporated T lymphocytes with lentivirus add the cell suspension to a G-Rex culture flask, incubate overnight in a 37°C, 5% CO 2 incubator, and add fresh medium every 2 days , continue to expand the cultivation.
  • CAR-T cells were collected by centrifugation, resuspended in a suitable freezing medium, and frozen in liquid nitrogen for later use.
  • the genomic DNA of the prepared PD-1H knockout B7-H3 CAR-T cells was extracted, and the targeted gene fragments were amplified by PCR using the primers in Table 3.
  • the purified and recovered DNA fragments were mixed according to the annealing system in Table 4. After thorough mixing, they were heated to 95°C in a PCR machine, and taken out after 5 minutes and cooled to room temperature naturally. After the annealing was completed, 1 ⁇ L of T7EI endonuclease was added to the annealing system, and after reacting at 37°C for 15 min, the bands of T7EI endonuclease were detected by gel electrophoresis, and the editing efficiency was analyzed by ImageJ.
  • the genomic DNA of the prepared PD-1H knockout B7-H3 CAR-T cells was extracted, and the targeted gene fragments were amplified by PCR using the primers in Table 3, and the PCR products were purified and recovered. PCR products were sequenced by next-generation sequencing technology. The sequencing results were analyzed by the TIDE analysis system (shinyapps.datacurators.nl/tide/), and the editing efficiency was calculated. The results are shown in Figure 17. The TIDE analysis showed that the editing efficiency of PD-1H sgRNA1 to the target site was 81.9%, which was similar to the T7E1 restriction detection result.

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Abstract

提供了工程化免疫细胞及其制备方法、用途，所述免疫细胞或工程化免疫细胞中的PD-1H基因是不表达的或被敲除的，或基因的表达是被沉默的。还提供了调控免疫细胞活性的方法、判断或评估免疫细胞活性的方法、以及增强免疫细胞肿瘤杀伤效率的方法。还提供了含有该工程化免疫细胞的制剂、试剂和相关的核酸分子、载体、宿主细胞和药物组合物。

Description

靶向PD-1H(VISTA)的抗肿瘤免疫治疗方法 技术领域
本发明涉及生物医药领域，具体地，涉及靶向PD-1H(VISTA)的抗肿瘤免疫治疗方法。
背景技术
PD-1H(又名VISTA、DD1-α)是CD28/B7家族的一种共抑制分子，与PD-1和PD-L1高度同源。PD-1H主要表达再造血系细胞上，在髓系细胞(包括巨噬细胞、树突状细胞、单核细胞和中性粒细胞)和CD4+ T细胞上均有表达。在抗原提呈细胞和调节性T细胞上PD-1H作为配体能抑制T细胞增殖和细胞因子释放，对自身免疫性疾病和肿瘤进展中具有重要调控作用。PD-1H在CD4+ T细胞上能作为抑制性受体，调控T细胞参与的免疫耐受和肿瘤免疫。PD-1H特异的阻断抗体在多个小鼠肿瘤(黑色素瘤、膀胱癌等)模型中都能有效抑制肿瘤生长。而PD-1H特异的激动抗体能有效减轻自身免疫性疾病(系统性红斑狼疮、哮喘、关节炎等)的病症。
在正常状态下，T细胞上PD-1H低水平表达，并且小鼠T细胞在体外培养后PD-1H表达会迅速丢失。同样的，体外培养的人的T细胞通常也检测不到PD-1H的表达。之前关于PD-1H在T细胞中的功能的研究主要集中在CD4+T细胞上，而PD-1H在CD8+T细胞中的功能仍不明确，其在肿瘤免疫治疗中的作用尚未可知。
发明内容
本发明的目的在于提供一类能显著增强肿瘤良好杀伤效果的工程化的免疫细胞。
本发明第一方面提供了一种工程化免疫细胞，所述工程化免疫细胞中的PD-1H基因的表达是被沉默的。
在另一优选例中，所述“PD-1H基因表达是被沉默的”指PD-1H基因不表达或低表达。
在另一优选例中，所述“低表达”指所述免疫细胞PD-1H基因的表达量G1与正常免疫细胞PD-1H基因的表达量G0的比值，即G1/G0≤0.8，较佳地G1/G0≤0.5，更佳地≤0.2，更佳地≤0.1，最佳地为0。
在另一优选例中，所述沉默PD-1H基因的表达通过选自下组的方法实现：基因突变、基因敲除、基因中断、RNA干扰技术、基因编辑技术、导入基因或蛋白的抑制剂、抗体或蛋白或多肽或化合物的阻断、细胞筛选、或其组合。
在另一优选例中，所述抑制剂选自下组：反义核酸、抗体、小分子化合物、Crispr试剂、小分子配体、或其组合。
在另一优选例中，所述的基因编辑技术选自下组：CRISPR技术、TALEN技术、ZFN技术、或其组合。
在另一优选例中，所述工程化免疫细胞包括CD8+T细胞、CD3+T细胞、CD4+T细胞、B细胞、NK细胞、髓性白细胞或单核细胞、抗原提呈细胞、或其他免疫细胞。
在另一优选例中，所述工程化免疫细胞具有以下特征：
(a)所述免疫细胞中的PD-1H基因的表达是被沉默的。
在另一优选例中，所述工程化免疫细胞还具有如下特征：
(b)所述免疫细胞表达嵌合抗原受体CAR或外源TCR，所述CAR靶向抗原分子或肿瘤细胞的标志物，所述外源TCR靶向抗原分子或肿瘤细胞的标志物。
在另一优选例中，所述工程化免疫细胞包括：
(a)任选的嵌合抗原受体CAR，所述嵌合抗原受体CAR包括：抗原结合结构域、铰链结构域、跨膜结构域和胞内结构域，其中所述抗原结合结构域特异性结合于抗原分子或肿瘤细胞表面抗原；和
(b)降低或抑制PD-1H蛋白表达活性的抑制分子。
在另一优选例中，所述抑制分子选自下组：抑制性核酸、小分子化合物、抗体(如单域抗体)、多肽、或其组合。
在另一优选例中，所述抑制性核酸包括RNA干扰剂、Crispr试剂。
在另一优选例中，所述抑制性核酸选自下组：siRNA、miRNA、shRNA、发夹siRNA、串联表达的miRNA、微小RNA适应性shRNA、前体微小RNA、或其组合。
在另一优选例中，所述抑制性核酸的序列如SEQ ID NO.:5-10中任一或组合所示。
在另一优选例中，所述Crispr试剂包括基因编辑蛋白。
在另一优选例中，所述基因编辑蛋白选自下组：CRISPR、TALEN、ZFN、或其组合。
在另一优选例中，所述CRISPR蛋白选自下组：Cas9、nCas9、Cas10、Cas9a、Cas12、Cas12a、Cas12b、Cas13、Cas14、或其组合。
在另一优选例中，所述Crispr试剂还包括gRNA。
在另一优选例中，所述gRNA的至少部分序列能够与靶DNA互补，且gRNA能够与CRISPR蛋白形成功能性复合体。
在另一优选例中，所述基因编辑酶来源于酿脓链球菌(Streptococcus pyogenes)、嗜热链球菌(Streptococcus thermophiles)、葡萄球菌(Staphylococcus aureus)、氨基酸球菌属(Acidaminococcus sp)、毛螺科菌(Lachnospiraceae bacterium)、或其组合。
在另一优选例中，所述gRNA包括sgRNA。
在另一优选例中，所述gRNA的序列如SEQ ID NO.:1-4、12-18中任一或组合所示。
在另一优选例中，所述抑制性核酸分子包括与PD-1H mRNA(信息核酸)或编码PD-1H的核酸互补的序列。
在另一优选例中，所述抑制性核酸分子包括与PD-1H mRNA(信息核酸)或编码PD-1H的核酸互补的反义寡核苷酸。
在另一优选例中，所述小分子化合物选自下组：1，2，4-噁二唑化合物及其衍生物、噁二唑类化合物、噻二唑类化合物、磺酰胺类化合物、联苯类化合物、或其组合。
在另一优选例中，所述肿瘤细胞表面抗原包括各种实体瘤、固体肿瘤和血液肿瘤的细胞表面抗原。
在另一优选例中，所述肿瘤细胞表面抗原选自下组：CD19、c-Met、PSMA、MUC-1、MUC16、CD7、CD20、CD22、CD123、CD47、CD138、CD33、CD30、CD38、PD-L1、CD276、B7-H4、间皮素(mesothelin)、EGFR、EGFRviii、GPC3、BCMA、ErbB2、ErbB3、ErbB4、NKG2D配体、LMP1、EpCAM、VEGFR-1、Lewis-Y、ROR1、Claudin18.2、CD7、CD5、CD2、CD3、CD19、CD20、CD22、CD24、CD25、CD28、CD123、CD47、CD52、CD56、CD80、CD86、CD81、CD138、CD33、CD38、CD30、CD133、CD97、CD99、CD40、CD43、CD137、CD151、CD171、KIT(CD117)、CD174、CD44V6、CD179a、B7-H3(CD276)、B7-H4、HER2、HER3、HER4、c-Met、PSMA、PSCA，MUC16、MUC1、间皮素(mesothelin)、EGFR、VEGFR2、EGFR-VIII、VEGFR-1、GPC3、BCMA、ErbB2、ErbB3、ErbB4、NKG2D配体、LMP1、EpCAM、Lewis-Y、ROR1、Claudin18.2、LIGHT、NKG2C、CEA(癌胚抗原)，FAP，PSMA，CA125，EphA2，L1CAM，CS1，ROR1，EC，NY-ESO-1，GD2，EPG，DLL3，5T4，IL-13Ra2(或CD213A2)、IL-11Ra、PRSS21、PDGFR-β、SSEA-4、叶酸受体α(FRa或FR1)；叶酸受体β(FRb)、AFP/MHC复合物、NCAM、ELF2M、FAP、IGF-I受体、CAIX、sLe、神经节苷脂GM3(aNeu5Ac(2-3)bDClalp(1-4)bDGlcp(1-1)Cer)、TGS5、HMWMAA、OAcGD2、(TEM1/CD248)、TEM7R、CLDN6、TSHR、GPRC5D、ALK、HAVCR1、ADRB3、PANX3、GPR20、OR51E2、LAGE-1a、MAGE-A1、ETV6-AML、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、PCT A-1、MelanA或MARTI、hTERT、ML-IAP、CAIX、CEACAM6、IGF1R、O-acetyl GD2、O-acetyl GD3、GHRHR、GHR、Flt1、Flt3、KDR、Flt4、CTLA-4、GITR、BTLA、TGFBR2、TGFBR1、IL6R，gp130、Lewis，TNFR1、TNFR2、PD1、PD-L1、PD-L2，HVEM、MAGE-A、NY-ESO-1、NY-ESO-1/MHC I复合物、PSMA、RANK、TNFRSF4、TWEAK-R、LTPR、LIFRP、LRP5、TCRa、TCRp、TLR7、TLR9、PTCH1、WT-1、WT1/MHC I复合物、NA17、MYCN、RhoC、SART3、SSX2、RAGE-1、HPV E7、HPV E7/MHC I复合物；AFP/MHC I复合物、Ras/MHC I复合物、Robol、Frizzled、OX40、CD79a、CD79b、CD72、Notch-1-4、CLL-1(或CLECL1)、TAG72、LILRA2；CD300LF；CLEC12A；BST2；EMR2；LY75、FCRL5；IGLL1、MPL、生物素、c-MYC表位标签、CD34、LAMP1 TROP2、GFRα4、CDH17、CDH6、NYBR1、CDH19、CD200R、Slea；岩藻糖基GM1、PTK7、gpNMB、CDH1-CD324、DLL3、CD179b-IGLl1、TCRγ-δ、NKG2D、CD32(FCGR2A)、Tn ag、Tim1-/HVCR1、CSF2RA(GM-CSFR-α)、TGFβR2、Lews Ag、TCR-β1链、TCR-β2链、TCR-γ链、TCR-δ链、FITC、LHR、FSHR、CGHR或GR、CCR4、SLAMF6、SLAMF4、HIV1包膜糖蛋白、HTLV1-Tax、CMV pp65、EBV-EBNA3c、KSHV K8.1、KSHV-gH、甲型流感血凝素(HA)、GAD、PDL1、胍基环化酶C(GCC)、抗桥粒芯糖蛋白3(Dsg3)的自身抗体、Dsg1、HLA、HLA-A、HLA-A2、HLA-B、HLA-C、HLA-DP、HLA-DM、HLA-DOA、HLA-DOB、HLA-DQ、HLA-DR、HLA-G、IgE、CD99、RasG12V、组织因子1(TF1)、AFP、GPRC5D、P-糖蛋白、STEAP1、Liv1、粘连蛋白-4、Cripto、gpA33、BST1/CD157、低电导氯离子通道以及TNT抗体识别的抗原、或其组合。
在另一优选例中，所述肿瘤细胞表面抗原包括CD19。
在另一优选例中，所述肿瘤细胞表面抗原包括B7-H3(CD276)。
在另一优选例中，所述降低或抑制PD-1H蛋白表达活性指将PD-1H蛋白的表 达活性降低≥20％，较佳地，≥40％，更佳地，≥60％，更佳地，≥80％，更佳地，≥90％或100％。
在另一优选例中，所述抗原结合结构域为抗体或抗原结合片段。
在另一优选例中，所述抗原结合片段是Fab或scFv或单结构域抗体sdFv。
在另一优选例中，所述的工程化免疫细胞选自下组：
(i)嵌合抗原受体T细胞(CAR-T细胞)；
(ii)嵌合抗原受体NK细胞(CAR-NK细胞)；
(iii)嵌合抗原受体吞噬细胞或单核细胞(CAR-macrophage细胞)；
(iv)外源T细胞受体(TCR)T细胞(TCR-T细胞)。
在另一优选例中，所述免疫细胞为自体的。
在另一优选例中，所述免疫细胞为异体的。
在另一优选例中，所述免疫细胞为iPS来源的。
在另一优选例中，所述细胞为哺乳动物细胞，优选人细胞。
在另一优选例中，所述的免疫细胞还表达降低或抑制PD-1H蛋白表达活性的抑制分子。
在另一优选例中，所述的抑制分子是独立表达的和/或与靶向肿瘤细胞表面抗原的嵌合抗原受体CAR共表达的。
在另一优选例中，所述的与靶向肿瘤细胞表面抗原的嵌合抗原受体CAR共表达包括抑制分子与靶向肿瘤细胞表面抗原的嵌合抗原受体CAR的串联表达。
在另一优选例中，所述的工程化的免疫细胞包括T细胞、NK细胞或巨噬细胞。
在另一优选例中，所述跨膜结构域为选自下组的蛋白的跨膜结构域：CD28、CD3epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、CTLA-4、PD-1、LAG-3、2B4、BTLA、或其组合。
在另一优选例中，所述铰链结构域为选自下组的蛋白的铰链结构域：CD8、CD28、CD137、CD80、CD86、或其组合。
在另一优选例中，所述胞内结构域包括共刺激信号分子和源于CD3ζ的胞浆信号传导序列。
在另一优选例中，所述共刺激信号分子为选自下组的蛋白的共刺激信号分子：OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD1、Dap10、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、TLR2、或其组合。
本发明第二方面提供了一种制备本发明第一方面所述的工程化免疫细胞的方法，包括以下步骤：
(A)提供一经筛选或待改造的免疫细胞；和
(B)对所述的免疫细胞进行改造，从而使得所述的免疫细胞中PD-1H基因的表达沉默，从而获得权利要求1所述的免疫细胞。
在另一优选例中，步骤(B)中包括将表达用于沉默PD-1H基因的第二表达盒导入所述免疫细胞。
在另一优选例中，在步骤(B)中，还包括步骤：(B1)将表达CAR的第一表达盒导入所述免疫细胞；和(B2)将表达用于沉默PD-1H基因的第二表达盒导入所述免 疫细胞，
其中，所述的步骤(B1和(B2)的次序无任何限定。
在另一优选例中，当步骤(A)中的待改造的免疫细胞已经表达某一CAR时，则在步骤(B)中，包括(B2)将表达用于沉默PD-1H的第二表达盒导入所述免疫细胞。
在另一优选例中，所述的“次序无任何限定”指对于任何二个步骤而言，可以依次、同时、或以相反次序进行。
在另一优选例中，所述的步骤(B1)可在步骤(B2)之前、之后、同时、或交替进行。
在另一优选例中，所述的第一表达盒、第二表达盒位于相同或不同的载体上。
在另一优选例中，所述的第一表达盒、第二表达盒位于同一载体。
在另一优选例中，所述的载体为病毒载体。
在另一优选例中，所述的载体选自下组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子、其他基因转移系统、或其组合。
在另一优选例中，所述的第二表达盒包含CRISPR/Cas9(gRNA和Cas9)、反义RNA、或其组合。
在另一优选例中，所述gRNA包括sgRNA。
在另一优选例中，所述的gRNA靶向PD-1H，且gRNA的序列如SEQ ID NO.:1-4、12-18中任一或组合所示。
在另一优选例中，所述的反义RNA包括miRNA、siRNA、shRNA、抑制性mRNA、或dsRNA。
在另一优选例中，所述反义RNA的序列如SEQ ID NO.:5-10中任一或组合所示。
本发明第三方面提供了一种制剂，所述制剂含有本发明第一方面所述的工程化免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中，所述制剂为液态制剂。
在另一优选例中，所述制剂为注射剂。
在另一优选例中，所述制剂中所述免疫细胞的浓度为1×10 3-1×10 10个细胞/ml，较佳地1×10 4-1×10 8个细胞/ml。
本发明第四方面提供了一种本发明第一方面所述的工程化免疫细胞的用途，用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
在另一优选例中，所述肿瘤选自下组：血液肿瘤、实体瘤、或其组合。
在另一优选例中，所述肿瘤包括CD19阳性的肿瘤。
在另一优选例中，所述肿瘤包括B7-H3(CD276)阳性的肿瘤。
在另一优选例中，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、骨髓增生异常综合症(MDS)、骨髓增生异常/骨髓增殖性疾病(MDS/MPD)、慢性骨髓增殖性疾病(SMPD)；前B淋巴母细胞性白血病/淋巴瘤、慢性淋巴细胞白血病/小淋 巴细胞性淋巴瘤、B淋巴细胞白血病、淋巴浆细胞淋巴瘤、脾边缘区B细胞淋巴瘤、毛细胞白血病、浆细胞骨髓瘤/浆细胞瘤、MALT型边缘区B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、弥漫性大B细胞淋巴瘤、伯基特淋巴瘤/白血病、T/NK细胞肿瘤、前淋巴母细胞性淋巴瘤/白血病、T淋巴细胞白血病、T粒状淋巴细胞白血病、侵袭性NK细胞白血病、成熟T细胞淋巴瘤/白血病、结外鼻型NK/T细胞淋巴瘤、肠病型T细胞淋巴瘤、蕈样真菌病/sezary综合征(MF/SS)、周围T细胞淋巴瘤、间变性大细胞淋巴瘤、多发性骨髓瘤(MM)、急性淋巴白血病(ALL)；前T细胞淋巴瘤/白血病；霍奇金淋巴瘤；肥大细胞增多症、肥大细胞白血病(MCL)、肥大细胞肉瘤(MCS)；巨噬细胞/组织细胞性肿瘤、树突状细胞肿瘤、朗格汉斯细胞组织增生症(LCH)、朗格汉斯细胞肉瘤(LCS)、滤泡树突状细胞肉瘤/肿瘤、树突状细胞肉瘤、或其组合。
在另一优选例中，所述实体瘤选自下组：头颈部肿瘤、咽喉癌、肺癌、非小细胞肺癌、支气管癌、胃癌、胃癌腹膜转移肿瘤、食道癌、胆管癌、胰腺癌、结直肠癌、结直肠癌腹膜转移肿瘤、小肠癌、肾脏肿瘤、肾癌、膀胱肿瘤、移行性上皮恶性肿瘤、内分泌肿瘤、甲状腺癌、肾上腺肿瘤、乳腺癌、宫颈癌、卵巢癌、卵巢癌腹膜转移肿瘤、子宫内膜癌、绒毛膜癌、前列腺癌、睾丸肿瘤、生殖细胞肿瘤、精原细胞癌、胚胎原性肿瘤、神经系统肿瘤、脑胶质瘤、神经母细胞瘤、皮肤肿瘤、恶性黑色素瘤、淋巴癌、胸腺肿瘤、鼻咽癌、骨癌、肉瘤、横纹肌肉瘤、脂肪肉瘤、血管肉瘤、平滑肌肉瘤、纤维肉瘤、骨肉瘤、尤文氏肉瘤、实体瘤转移性肿瘤如腹腔、胸腔、盆腔、实质器官处等转移瘤、或其组合。
本发明第五方面提供了一种用于制备本发明第一方面所述的工程化免疫细胞的试剂盒，所述试剂盒含有容器，以及位于容器内的：
(1)任选的第一核酸序列，所述第一核酸序列含有用于表达CAR或外源TCR的第一表达盒；
(2)第二核酸序列，所述第二核酸序列含有用于沉默PD-1H的第二表达盒或靶向PD-1H的gRNA。
在另一优选例中，所述的第一、第二核酸序列为独立的或相连的。
在另一优选例中，所述的第一、第二核酸序列位于相同或不同的容器内。
在另一优选例中，所述的第一、第二核酸序列位于相同或不同的表达载体。
在另一优选例中，所述的试剂盒还含有：(4)第三核酸序列，所述第三核酸序列含有用于表达基因编辑蛋白的表达盒；或基因编辑蛋白。
本发明第六方面提供了一种调控免疫细胞活性的方法，包括：
通过调控免疫细胞中PD-1H的表达水平，从而调控免疫细胞的活性。
在另一优选例中，通过降低或抑制免疫细胞中PD-1H的表达水平，从而增强免疫细胞的活性。
在另一优选例中，通过增强免疫细胞中PD-1H的表达水平，从而降低免疫细胞的活性。
在另一优选例中，所述“降低或抑制免疫细胞中PD-1H的表达水平”指所述免疫细胞PD-1H基因的表达量G1与正常免疫细胞PD-1H基因的表达量G0的比值，即G1/G0≤0.8，较佳地G1/G0≤0.5，更佳地≤0.2，更佳地≤0.1，最佳地为0。
在另一优选例中，所述“增强免疫细胞中PD-1H的表达水平”指所述免疫细胞PD-1H基因的表达量G1与正常免疫细胞PD-1H基因的表达量G0的比值，即G1/G0≥2，较佳地G1/G0≥3，更佳地，G1/G0≥4。
本发明第七方面提供了一种判断或评估免疫细胞活性的方法，包括：
通过检测免疫细胞中PD-1H的表达水平，从而判断或评估免疫细胞的活性。
在另一优选例中，当免疫细胞中PD-1H的表达水平升高时，则免疫细胞的活性降低。
在另一优选例中，当免疫细胞中PD-1H的表达水平降低时，则免疫细胞的活性增强。
在另一优选例中，所述“免疫细胞中PD-1H的表达水平升高”指所述免疫细胞PD-1H基因的表达量G1与正常免疫细胞PD-1H基因的表达量G0的比值，即G1/G0≥2，较佳地G1/G0≥3，更佳地，G1/G0≥4。
在另一优选例中，所述“免疫细胞中PD-1H的表达水平降低”指所述免疫细胞PD-1H基因的表达量G1与正常免疫细胞PD-1H基因的表达量G0的比值，即G1/G0≤0.8，较佳地G1/G0≤0.5，更佳地≤0.2，更佳地≤0.1，最佳地为0。
本发明第八方面提供了一种核酸分子，包括第一核酸和任选的第二核酸，其中所述第一核酸含有第一表达盒，所述第一表达盒编码降低或抑制PD-1H蛋白表达活性的抑制分子，所述第二核酸含有第二表达盒，所述第二表达盒编码嵌合抗原受体CAR，所述嵌合抗原受体CAR包括：抗原结合结构域、跨膜结构域和胞内结构域，其中所述抗原结合结构域特异性结合于肿瘤细胞表面抗原。
在另一优选例中，所述第一表达盒和/或第二表达盒还包括组成型启动子或诱导型启动子。
在另一优选例中，所述组成型启动子选自下组：CMV、EF1a、U6、SV40、PGK1、Ubc、CAG、H1、或其组合。
在另一优选例中，所述诱导型启动子选自下组：金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子、四环素启动子、或其组合。
本发明第九方面提供了一种载体，所述载体含有本发明第八方面所述的核酸分子。
在另一优选例中，所述的载体选自下组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子、或其组合。
在另一优选例中，所述载体为慢病毒载体。
本发明第十方面提供了一种宿主细胞，所述的宿主细胞含有本发明第九方面所述的载体或染色体中整合有外源的本发明第八方面所述的核酸分子。
在另一优选例中，所述细胞为分离的细胞，和/或所述细胞为基因工程化的细胞。
在另一优选例中，所述细胞为哺乳动物细胞，优选人细胞。
在另一优选例中，所述的宿主细胞包括工程化的免疫细胞。
在另一优选例中，所述的工程化免疫细胞选自下组：
(i)嵌合抗原受体T细胞(CAR-T细胞)；
(ii)嵌合抗原受体NK细胞(CAR-NK细胞)；或
(iii)外源T细胞受体(TCR)T细胞(TCR-T细胞)。
在另一优选例中，所述免疫细胞为自体的。
在另一优选例中，所述免疫细胞为异体的。
在另一优选例中，所述的免疫细胞还表达降低或抑制PD-1H蛋白表达活性的抑制分子。
在另一优选例中，所述的抑制分子是独立表达的和/或与靶向肿瘤细胞表面抗原的嵌合抗原受体CAR共表达的。
在另一优选例中，所述的与靶向肿瘤细胞表面抗原的嵌合抗原受体CAR共表达包括抑制分子与靶向肿瘤细胞表面抗原的嵌合抗原受体CAR的串联表达。
在另一优选例中，所述的工程化的免疫细胞包括T细胞、NK细胞或巨噬细胞。
在另一优选例中，所述细胞为T细胞。
本发明第十一方面提供了一种药物组合物，包括：
(a)本发明第十方面所述的宿主细胞；和
(b)药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中，所述药物组合物为液态制剂。
在另一优选例中，所述药物组合物的剂型为注射剂。
在另一优选例中，所述的工程化免疫细胞是(i)嵌合抗原受体T细胞(CAR-T细胞)；或(ii)嵌合抗原受体NK细胞(CAR-NK细胞)。
在另一优选例中，所述药物组合物中，所述细胞的浓度为1×10 3-1×10 10个细胞/ml，较佳地1×10 4-1×10 8个细胞/ml。
在另一优选例中，所述药物组合物还含有选择性杀伤肿瘤细胞的其他药物(如抗体药物、化疗药物或其他CAR-T药物)。
本发明第十二方面提供了一种制备工程化免疫细胞的方法，包括：
将本发明第八方面所述的核酸分子或本发明第九方面所述的载体转导入免疫细胞内，从而获得所述工程化免疫细胞。
在另一优选例中，所述导入包括同时、先后、或依次导入。
在另一优选例中，所述免疫细胞为T细胞或NK细胞。
在另一优选例中，所述的方法还包括对获得的工程化免疫细胞进行功能和有效性检测的步骤。
本发明第十三方面提供了一种试剂组合，所述试剂组合包括：
(i)任选的第一试剂，所述第一试剂为免疫细胞；和
(ii)第二试剂，所述的第二试剂为降低或抑制PD-1H蛋白表达活性的抑制分子。
在另一优选例中，所述免疫细胞包括工程化免疫细胞。
在另一优选例中，所述工程化免疫细胞含有嵌合抗原受体CAR，所述嵌合抗原 受体CAR包括：抗原结合结构域、跨膜结构域和胞内结构域，其中所述抗原结合结构域特异性结合于肿瘤细胞表面抗原。
在另一优选例中，所述抑制分子选自下组：抑制性核酸、小分子化合物、抗体(如单域抗体)、多肽、或其组合。
在另一优选例中，所述的工程化免疫细胞还表达降低或抑制PD-1H蛋白表达量或活性的抑制分子。
在另一优选例中，所述的工程化免疫细胞还表达用于降低或抑制PD-1H蛋白表达的抑制性核酸。
本发明第十四方面提供了一种增强工程化免疫细胞的肿瘤杀伤效率的方法，包括：
在降低或抑制PD-1H蛋白表达活性的抑制分子存在下，将工程化免疫细胞与肿瘤细胞接触，从而增强工程化免疫细胞的肿瘤杀伤效率。
在另一优选例中，所述方法为体外的。
在另一优选例中，所述方法为非治疗和非诊断性的。
在另一优选例中，所述降低或抑制PD-1H蛋白表达活性的抑制分子为内源产生的或外源添加的。
在另一优选例中，所述内源产生指由所述工程化免疫细胞产生。
在另一优选例中，所述外源添加指外源添加抑制分子。
在另一优选例中，所述工程化免疫细胞包括CD8+T细胞、CD3+T细胞、CD4+T细胞、B细胞、NK细胞、髓性白细胞或单核细胞、抗原提呈细胞、或其他免疫细胞。
本发明第十五方面提供了一种本发明第十方面所述的宿主细胞、或本发明第十一方面所述的药物组合物的用途，用于制备选择性杀伤肿瘤细胞的药物或制剂。
在另一优选例中，所述肿瘤细胞来源于选自下组的肿瘤：急性髓细胞白血病(AML)、骨髓增生异常综合症(MDS)、骨髓增生异常/骨髓增殖性疾病(MDS/MPD)、慢性骨髓增殖性疾病(SMPD)；前B淋巴母细胞性白血病/淋巴瘤、慢性淋巴细胞白血病/小淋巴细胞性淋巴瘤、B淋巴细胞白血病、淋巴浆细胞淋巴瘤、脾边缘区B细胞淋巴瘤、毛细胞白血病、浆细胞骨髓瘤/浆细胞瘤、MALT型边缘区B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、弥漫性大B细胞淋巴瘤、伯基特淋巴瘤/白血病、T/NK细胞肿瘤、前淋巴母细胞性淋巴瘤/白血病、T淋巴细胞白血病、T粒状淋巴细胞白血病、侵袭性NK细胞白血病、成熟T细胞淋巴瘤/白血病、结外鼻型NK/T细胞淋巴瘤、肠病型T细胞淋巴瘤、蕈样真菌病/sezary综合征(MF/SS)、周围T细胞淋巴瘤、间变性大细胞淋巴瘤、多发性骨髓瘤(MM)、急性淋巴白血病(ALL)；前T细胞淋巴瘤/白血病；霍奇金淋巴瘤；肥大细胞增多症、肥大细胞白血病(MCL)、肥大细胞肉瘤(MCS)；巨噬细胞/组织细胞性肿瘤、树突状细胞肿瘤、朗格汉斯细胞组织增生症(LCH)、朗格汉斯细胞肉瘤(LCS)、滤泡树突状细胞肉瘤/肿瘤、树突状细胞肉瘤、头颈部肿瘤、咽喉癌、肺癌、非小细胞肺癌、支气管癌、胃癌、胃癌腹膜转移肿瘤、食道癌、胆管癌、胰腺癌、结直肠癌、结直肠癌腹膜转移肿瘤、小肠癌、肾脏肿瘤、肾癌、膀胱肿瘤、移行性上皮恶性肿瘤、内分泌肿瘤、甲状腺癌、肾上腺肿瘤、乳腺癌、宫颈癌、卵巢癌、卵巢癌腹膜转移肿瘤、子宫内膜癌、绒毛膜癌、前列腺癌、睾丸肿瘤、生殖细胞肿瘤、精原细胞癌、胚胎原性肿瘤、神经系统肿瘤、脑胶质瘤、神经母细胞瘤、皮肤肿瘤、恶性黑色素瘤、淋巴癌、胸腺肿瘤、鼻咽癌、骨癌、肉瘤、横纹肌肉瘤、脂肪肉瘤、血管肉瘤、平滑肌 肉瘤、纤维肉瘤、骨肉瘤、尤文氏肉瘤、实体瘤转移性肿瘤如腹腔、胸腔、盆腔、实质器官处等转移瘤、或其组合。
本发明第十六方面提供了一种用于选择性杀伤肿瘤细胞的试剂盒，所述试剂盒含有容器，以及位于容器内的本发明第十方面所述的宿主细胞。
在另一优选例中，所述试剂盒还含有标签或使用说明书。
本发明第十七方面提供了一种选择性杀伤肿瘤细胞的方法，包括：
给需要治疗的对象施用安全有效量的本发明第一方面所述的工程化免疫细胞、本发明第十方面所述的宿主细胞、或本发明第十一方面所述的药物组合物。
在另一优选例中，所述对象包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物(如小鼠、大鼠、兔)、灵长类动物(如猴)。
在另一优选例中，所述方法为非治疗性和非诊断性的。
本发明第十八方面提供了一种治疗癌症或肿瘤的方法，其特征在于，包括：
给需要治疗的对象施用安全有效量的本发明第一方面所述的工程化免疫细胞、本发明第十方面所述的宿主细胞、或本发明第十一方面所述的药物组合物。
在另一优选例中，所述肿瘤细胞来源于选自下组的肿瘤：急性髓细胞白血病(AML)、骨髓增生异常综合症(MDS)、骨髓增生异常/骨髓增殖性疾病(MDS/MPD)、慢性骨髓增殖性疾病(SMPD)；前B淋巴母细胞性白血病/淋巴瘤、慢性淋巴细胞白血病/小淋巴细胞性淋巴瘤、B淋巴细胞白血病、淋巴浆细胞淋巴瘤、脾边缘区B细胞淋巴瘤、毛细胞白血病、浆细胞骨髓瘤/浆细胞瘤、MALT型边缘区B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、弥漫性大B细胞淋巴瘤、伯基特淋巴瘤/白血病、T/NK细胞肿瘤、前淋巴母细胞性淋巴瘤/白血病、T淋巴细胞白血病、T粒状淋巴细胞白血病、侵袭性NK细胞白血病、成熟T细胞淋巴瘤/白血病、结外鼻型NK/T细胞淋巴瘤、肠病型T细胞淋巴瘤、蕈样真菌病/sezary综合征(MF/SS)、周围T细胞淋巴瘤、间变性大细胞淋巴瘤、多发性骨髓瘤(MM)、急性淋巴白血病(ALL)；前T细胞淋巴瘤/白血病；霍奇金淋巴瘤；肥大细胞增多症、肥大细胞白血病(MCL)、肥大细胞肉瘤(MCS)；巨噬细胞/组织细胞性肿瘤、树突状细胞肿瘤、朗格汉斯细胞组织增生症(LCH)、朗格汉斯细胞肉瘤(LCS)、滤泡树突状细胞肉瘤/肿瘤、树突状细胞肉瘤、头颈部肿瘤、咽喉癌、肺癌、非小细胞肺癌、支气管癌、胃癌、胃癌腹膜转移肿瘤、食道癌、胆管癌、胰腺癌、结直肠癌、结直肠癌腹膜转移肿瘤、小肠癌、肾脏肿瘤、肾癌、膀胱肿瘤、移行性上皮恶性肿瘤、内分泌肿瘤、甲状腺癌、肾上腺肿瘤、乳腺癌、宫颈癌、卵巢癌、卵巢癌腹膜转移肿瘤、子宫内膜癌、绒毛膜癌、前列腺癌、睾丸肿瘤、生殖细胞肿瘤、精原细胞癌、胚胎原性肿瘤、神经系统肿瘤、脑胶质瘤、神经母细胞瘤、皮肤肿瘤、恶性黑色素瘤、淋巴癌、胸腺肿瘤、鼻咽癌、骨癌、肉瘤、横纹肌肉瘤、脂肪肉瘤、血管肉瘤、平滑肌肉瘤、纤维肉瘤、骨肉瘤、尤文氏肉瘤、实体瘤转移性肿瘤如腹腔、胸腔、盆腔、实质器官处等转移瘤、或其组合。
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，在此不再一一赘述。
附图说明
图1显示了PD-1H在肿瘤浸润CD8+ T细胞上表达上升。在小鼠黑色素瘤(B16-OVA)模型和淋巴瘤(EG7)模型中，肿瘤浸润CD8+ T细胞上PD-1H的表达高于CD4+ T细胞。
图2显示了在体外培养的CD8+ T细胞中加入anti-CD3和anti-CD28刺激能使CD8+T细胞上PD-1H表达上调。
图3显示了在小鼠黑色素瘤(B16-OVA)模型中，PD-1H缺失的CD8+ T细胞抗肿瘤作用增强，肿瘤浸润增加。
图4显示了在小鼠淋巴瘤(EG7)模型中，PD-1H缺失的CD8+ T细胞抗肿瘤作用增强，肿瘤浸润增加。
图5显示了利用CRISPR Cas9系统能有效敲除CD8+ T细胞上PD-1H的表达。
图6显示了在小鼠淋巴瘤(EG7)模型中，利用CRISPR Cas9系统敲除PD-1H的CD8+ T细胞的抗肿瘤作用增强。
图7显示了体外培养的T细胞和CAR-T细胞表面无PD-1H表达，在脾脏和肿瘤组织中T细胞表面PD-1H表达上升。
图8显示了免疫荧光共定位显示肿瘤浸润CD8+ T细胞上有PD-1H的表达。
图9显示了共表达PD-1H shRNA的嵌合抗原受体的载体构造示意图。
图10显示了不同的共表达PD-1H shRNA的嵌合抗原受体在T细胞上的表达情况。
图11显示了候选的PD-1H shRNA沉默Jurkat-PD-1H细胞上PD-1H的表达。
图12显示了共表达PD-1H shRNA的CD19 CAR-T细胞能有效杀伤CD19阳性肿瘤细胞(CA-46)。
图13显示了共表达PD-1H shRNA的CD19 CAR-T细胞治疗能有效抑制NCG小鼠淋巴瘤(CA-46)模型中肿瘤的生长。
图14显示了共表达PD-1H shRNA的CD19 CAR-T细胞治疗能有效延长小鼠的生存期。
图15显示了sgRNA1和sgRNA2能有效敲除Jurkat-PD-1H细胞上PD-1H的表达，sgRNA1的敲除效率更高。
图16显示了T7E1核酸内切酶检测sgRNA1对B7-H3 CAR-T细胞的基因编辑效率。
图17显示了TIDE分析sgRNA1对B7-H3 CAR-T细胞的基因编辑效率。
图18显示了sgRNA1对B7-H3 CAR-T细胞的PD-1H基因的编辑情况。
具体实施方式
本发明人通过广泛而深入的研究，经过大量筛选，意外地发现在工程化的免疫细胞(如T细胞、NK细胞)中敲低PD-1H基因的表达，可显著提高免疫细胞的杀伤肿瘤作用。在此基础上，本发明人完成了本发明。
本发明以CAR-T细胞为例，代表性地对本发明的工程化的免疫细胞进行详细说明。本发明的工程化的免疫细胞不限于上下文所述的CAR-T细胞，本发明的工程化的免疫细胞具有与上下文所述的CAR-T细胞相同或类似的技术特征和有益效 果。具体地，当免疫细胞表达嵌合抗原受体CAR时，NK细胞等同于T细胞(或T细胞可替换NK细胞)；当免疫细胞为T细胞时，TCR等同于CAR(或CAR可替换为TCR)。
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。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
术语“抗体”(Ab)应包括但不限于免疫球蛋白，其特异性结合抗原并包含通过二硫键互连的至少两条重(H)链和两条轻(L)链，或其抗原结合部分。每条H链包含重链可变区(本文缩写为VH)和重链恒定区。重链恒定区包含三个恒定结构域CH1、CH2和CH3。每条轻链包含轻链可变区(本文缩写为VL)和轻链恒定区。轻链恒定区包含一个恒定结构域CL。VH和VL区可以进一步细分为称为互补决定区(CDR)的高变区，其散布有更保守的称为框架区(FR)的区域。每个VH和VL包含三个CDR和四个FR，从氨基末端到羧基末端按照以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。
如本文所用，“嵌合抗原受体(CAR)”是一种融合蛋白，其包含能够结合抗原的胞外结构域，与胞外结构域衍生自不同多肽的跨膜结构域，以及至少一个胞内结构域。“嵌合抗原受体(CAR)”也称为“嵌合受体”、“T-body”或“嵌合免疫受体(CIR)”。所述的“能够结合抗原的胞外结构域”是指能够结合某一抗原的任何寡肽或多肽。“胞内结构域”是指已知的作为传递信号以激活或抑制细胞内生物过程的结构域的任何寡肽或多肽。
如本文所用，“结构域”是指多肽中独立于其它区域且折叠成特异结构的区域。
如本文所用，“肿瘤抗原”是指具有抗原性的生物分子，其表达导致癌症。
如本文所用，术语“给予”和“处理”是指外源性药物、治疗剂、诊断剂或组合物应用于动物、人、受试者、细胞、组织、器官或生物流体。“给予”和“处理”可以指治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触、以及试剂与流体的接触、流体与细胞的接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理。“处理”当应用于人、动物或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断。
如本文所用，术语“治疗”指给予患者内用或外用治疗剂，包含本发明的任何一种工程化免疫细胞及其组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，以有效缓解一种或多种疾病症状的治疗剂的量(治疗有效量)给予患者。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。例如，“任选包含1-3个抗体重链可变区”是指特定序列的抗体重链可变区可以有但不是必须有，可以是1个、2个或3个。
本发明所述的“序列同一性”表示当具有适当的替换、插入或缺失等突变的情况下最佳比对和比较时，两个核酸或两个氨基酸序列之间的同一性程度。本发明中所述的序列和其具有同一性的序列之间的序列同一性可以至少为85％、90％或95％，优选至少为95％。非限制性实施例包括85％,86％,87％,88％,89％,90％,91％,92％,93％,94％,95％,96％,97％,98％,99％,100％。
PD-1H
PD-1H也称为VISTA、c10orf54、VSIR、SISP1、B7-H5、DD1α、Gi24和Dies1，含有311个氨基酸的1型跨膜蛋白。PD-1H包含一个N端Ig V型结构域、一个跨膜结构域和一个胞内结构域。与CD28家族成员PD-1和B7家族成员PD-L1具有同源性。不同于家族的其他成员，PD-1H胞内段不包含经典的ITIM/ITAM基序，但它的胞外域具有21个残基的C-C’环和更多的组氨酸。最近的研究发现，PD-1H上的组氨酸残基是与其受体PSGL-1结合主要位点，同时C-C’环是与另一受体VSIG3结合的主要位点。
PD-1H主要表达再造血系细胞上，在髓系细胞(包括巨噬细胞、树突状细胞、单核细胞和中性粒细胞)和CD4+ T细胞上均有表达。
本发明的研究表明，PD-1H即可作为配体，又可作为受体产生抑制性作用。在抗原提呈细胞和调节性T细胞上PD-1H作为配体能抑制T细胞增殖和细胞因子释放，对自身免疫性疾病和肿瘤进展中具有重要调控作用。PD-1H在CD4+ T细胞上能作为抑制性受体，调控T细胞参与的免疫耐受和肿瘤免疫。PD-1H特异的阻断抗体在多个小鼠肿瘤(黑色素瘤、膀胱癌等)模型中都能有效抑制肿瘤生长。而PD-1H特异的激动抗体能有效减轻自身免疫性疾病(系统性红斑狼疮、哮喘、关节炎等)的病症。
肿瘤抗原
本发明的肿瘤抗原包括不限于CD7、CD5、CD2、CD3、CD19、CD20、CD22、CD24、CD25、CD28、CD123、CD47、CD52、CD56、CD80、CD86、CD81、CD138、CD33、CD38、CD30、CD133、CD97、CD99、CD40、CD43、CD137、CD151、CD171、KIT(CD117)、CD174、CD44V6、CD179a、B7-H3(CD276)、B7-H4、HER2、HER3、HER4、c-Met、PSMA、PSCA，MUC16、MUC1、间皮素(mesothelin)、EGFR、VEGFR2、EGFR-VIII、VEGFR-1、GPC3、BCMA、ErbB2、ErbB3、ErbB4、NKG2D配体、LMP1、EpCAM、Lewis-Y、ROR1、Claudin18.2、LIGHT、NKG2C、CEA(癌胚抗原)，FAP，PSMA，CA125，EphA2，L1CAM，CS1，ROR1，EC，NY-ESO-1，GD2，EPG，DLL3，5T4，IL-13Ra2(或CD213A2)、IL-11Ra、PRSS21、PDGFR-β、SSEA-4、叶酸受体α(FRa或FR1)；叶酸受体β(FRb)、AFP/MHC复合物、NCAM、ELF2M、FAP、IGF-I受体、CAIX、sLe、神经节苷脂GM3(aNeu5Ac(2-3)bDClalp(1-4)bDGlcp(1-1)Cer)、TGS5、HMWMAA、 OAcGD2、(TEM1/CD248)、TEM7R、CLDN6、TSHR、GPRC5D、ALK、HAVCR1、ADRB3、PANX3、GPR20、OR51E2、LAGE-1a、MAGE-A1、ETV6-AML、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、PCT A-1、MelanA或MARTI、hTERT、ML-IAP、CAIX、CEACAM6、IGF1R、O-acetyl GD2、O-acetyl GD3、GHRHR、GHR、Flt1、Flt3、KDR、Flt4、CTLA-4、GITR、BTLA、TGFBR2、TGFBR1、IL6R，gp130、Lewis，TNFR1、TNFR2、PD1、PD-L1、PD-L2，HVEM、MAGE-A、NY-ESO-1、NY-ESO-1/MHC I复合物、PSMA、RANK、TNFRSF4、TWEAK-R、LTPR、LIFRP、LRP5、TCRa、TCRp、TLR7、TLR9、PTCH1、WT-1、WT1/MHC I复合物、NA17、MYCN、RhoC、SART3、SSX2、RAGE-1、HPV E7、HPV E7/MHC I复合物；AFP/MHC I复合物、Ras/MHC I复合物、Robol、Frizzled、OX40、CD79a、CD79b、CD72、Notch-1-4、CLL-1(或CLECL1)、TAG72、LILRA2；CD300LF；CLEC12A；BST2；EMR2；LY75、FCRL5；IGLL1、MPL、生物素、c-MYC表位标签、CD34、LAMP1 TROP2、GFRα4、CDH17、CDH6、NYBR1、CDH19、CD200R、Slea；岩藻糖基GM1、PTK7、gpNMB、CDH1-CD324、DLL3、CD179b-IGLl1、TCRγ-δ、NKG2D、CD32(FCGR2A)、Tn ag、Tim1-/HVCR1、CSF2RA(GM-CSFR-α)、TGFβR2、Lews Ag、TCR-β1链、TCR-β2链、TCR-γ链、TCR-δ链、FITC、LHR、FSHR、CGHR或GR、CCR4、SLAMF6、SLAMF4、HIV1包膜糖蛋白、HTLV1-Tax、CMV pp65、EBV-EBNA3c、KSHV K8.1、KSHV-gH、甲型流感血凝素(HA)、GAD、PDL1、胍基环化酶C(GCC)、抗桥粒芯糖蛋白3(Dsg3)的自身抗体、Dsg1、HLA、HLA-A、HLA-A2、HLA-B、HLA-C、HLA-DP、HLA-DM、HLA-DOA、HLA-DOB、HLA-DQ、HLA-DR、HLA-G、IgE、CD99、RasG12V、组织因子1(TF1)、AFP、GPRC5D、P-糖蛋白、STEAP1、Liv1、粘连蛋白-4、Cripto、gpA33、BST1/CD157、低电导氯离子通道以及TNT抗体识别的抗原。
以CD19为例。
CD19指分化抗原簇19蛋白，是白血病前体细胞上可检出的抗原决定簇。本文所述的CD19包含突变(例如，全长野生型CD19的点突变、片段、插入、缺失和剪接变体)的蛋白质。CD19在大部分B系癌上表达，包括急性淋巴母细胞白血病、慢性淋巴细胞白血病和非霍奇金淋巴瘤。它也是B细胞祖细胞的早期标志物。
抗原结合结构域
在本发明中，嵌合抗原受体CAR的抗原结合结构域特异性结合于肿瘤细胞表面抗原。
在一优选实施方式中，本发明的嵌合抗原受体CAR的抗原结合结构域靶向CD19、B7-H3(CD276)。
绞链区和跨膜区
对于绞链区和跨膜区(跨膜结构域)，CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中，使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中，可选择跨膜结构域，或通过氨基酸置换进行修饰，以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构域，从而最小化与受体复合物的其他成员的相互作用。
跨膜结构域可源于天然来源或合成来源。在天然来源中，该结构域可源于任何膜结合蛋白或跨膜蛋白。优选地，本发明的CAR中的绞链区和跨膜区为CD8的绞链区和跨膜区。
胞内结构域
本发明的CAR的胞内结构域或另外的细胞内信号传导结构域是造成其中已放置CAR的免疫细胞的至少一种正常效应子功能的活化的原因。术语“效应子功能”指的是细胞的专有功能。例如，T细胞的效应子功能可为包括细胞因子分泌的细胞溶解活性或辅助活性。因此术语“细胞内信号传导结构域”指的是转导效应子功能信号并指导细胞实施专有功能的蛋白部分。尽管通常可使用整个细胞内信号传导结构域，但在很多例子中，不必使用整个链。就使用细胞内信号传导结构域的截短部分而言，这种截短部分可用于代替完整的链，只要它转导效应子功能信号。术语细胞内信号传导结构域因此指包括足以转导效应子功能信号的细胞内信号传导结构域的任何截短部分。
用于本发明的CAR的细胞内信号传导结构域的优选例子包括T细胞受体(TCR)的胞浆序列和协同行动以在抗原受体结合后开始信号转导的共受体，以及这些序列的任何衍生物或变体和具有相同的功能能力的任何合成序列。
在优选的实施方式中，CAR的胞浆结构域可被设计以本身包括CD3-ζ信号传导结构域，或可与在本发明的CAR的内容中有用的任何其他期望的胞浆结构域(一个或多个)联合。例如，CAR的胞浆结构域可包括CD3ζ链部分和共刺激信号传导区。共刺激信号传导区指的是包括共刺激分子的细胞内结构域的一部分CAR。共刺激分子是淋巴细胞对抗原的有效应答所需的细胞表面分子，而不是抗原受体或它们的配体。优选地，包括4-1BB(CD137)等。
本发明的CAR的胞浆信号传导部分内的胞浆信号传导序列可以随机或以规定的顺序相互连接。任选地，短的寡肽或多肽连接体，优选长度在2和10个氨基酸，可形成该连接。甘氨酸-丝氨酸双联体提供了特别合适的连接体。
在一个实施方式中，本发明的CAR中的胞浆结构域被设计以包括4-1BB的信号传导结构域(共刺激分子)以及CD3ζ的信号传导结构域。
嵌合抗原受体(CAR)
嵌合免疫抗原受体(Chimeric antigen receptors，CARs)由胞外抗原识别区域，通常是scFv(single-chain variable fragment)，跨膜区以及胞内共刺激信号区域组成。CARs的设计经历了以下过程：第一代CAR只有一个胞内信号组份CD3ζ或者FcγRI分子，由于胞内只有一个活化结构域，因此它只能引起短暂的T细胞增殖和较少的细胞因子分泌，而并不能提供长时间的T细胞增殖信号和持续的体内抗肿瘤效应，所以并没有取得很好地临床疗效。第二代CARs在原有结构基础上引入一个共刺激分子，如CD28、4-1BB、OX40、ICOS，与一代CARs相比功能有很大提高，进一步加强CAR-T细胞的持续性和对肿瘤细胞的杀伤能力。在二代CARs基础上串联一些新的免疫共刺激分子如CD27、CD134，发展成为三代和四代CARs。
CARs的胞外段可识别一个特异的抗原，随后通过胞内结构域转导该信号，引起细胞的活化增殖、细胞溶解毒性和分泌细胞因子，进而清除靶细胞。首先分离病人自体细胞(或者异源供体)，激活并进行基因改造产生CAR的免疫细胞，随后注入同一病人体内。这种方式患移植物抗宿主病概率极低，抗原被免疫细胞以非MHC限制方式识别。
CAR-免疫细胞治疗在血液恶性肿瘤治疗中取得了非常高的临床反应率，这样的高反应率是以往任何一种治疗手段都无法达到的，在世界各引发了临床研究的 热潮。
具体地，本发明的嵌合抗原受体(CAR)包括细胞外结构域、跨膜结构域、和细胞内结构域。胞外结构域包括靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激信号传导区和/或ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子，而不是抗原受体或它们的配体。
在CAR的胞外结构域和跨膜结构域之间，或在CAR的胞浆结构域和跨膜结构域之间，可并入接头。如本文所用的，术语“接头”通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸，优选地2至100个氨基酸和最优选地3至50个氨基酸。
本发明的CAR当在T细胞中表达时，能够基于抗原结合特异性进行抗原识别。当其结合其关联抗原时，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和/或ζ链中的一个或多个的细胞内结构域融合。优选地，抗原结合结构域与4-1BB信号传导结构域和/或CD3ζ信号结构域组合的细胞内结构域融合。
如本文所用，“抗原结合结构域”“单链抗体片段”均指具有抗原结合活性的Fab片段，Fab'片段，F(ab')2片段，或单一Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。抗原结合结构域通常是scFv(single-chain variable fragment)。scFv的大小一般是一个完整抗体的1/6。单链抗体优选是由一条核苷酸链编码的一条氨基酸链序列。作为本发明的优选方式，所述scFv包含特异性识别肿瘤高表达抗原CD47和MSLN的抗体，较佳地为单链抗体。
在本发明中，本发明的scFv还包括其保守性变异体，指与本发明scFv的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。
在本发明中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
在本发明中，所述添加、缺失、修饰和/或取代的氨基酸数量通常是1、2、3、4或5个，较佳地为1-3个，更佳地为1-2个，最佳地为1个。
对于绞链区和跨膜区(跨膜结构域)，CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中，使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中，可选择跨膜结构域，或通过氨基酸置换进行修饰，以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构域，从而最小化与受体复合物的其他成员的相互作用。
本发明的CAR的胞外结构域包括特异性结合于肿瘤细胞表面抗原的抗原结合结构域，优选特异性结合于CD19的抗原结合结构域。
在本发明中，本发明的CAR中的胞内结构域包括细胞外结构域、CD8的跨膜区、4-1BB的共刺激因子、CD3ζ的信号传导结构域，其中细胞外结构域包括靶-特异性结合元件(也称为抗原结合结构域)
嵌合抗原受体T细胞(CAR-T细胞)
如本文所用，术语“CAR-T细胞”、“CAR-T”、“本发明CAR-T细胞”均指本发明所述的CAR-T细胞，本发明CAR-T细胞可靶向肿瘤细胞表面抗原(优选CD19)，用来治疗肿瘤细胞表面抗原(如CD19)高表达或阳性的肿瘤。
CAR-T细胞较其它基于T细胞的治疗方式存在以下优势：(1)CAR-T细胞的作用过程不受MHC的限制；(2)鉴于很多肿瘤细胞表达相同的肿瘤抗原，针对某一种肿瘤抗原的CAR基因构建一旦完成，便可以被广泛利用；(3)CAR既可以利用肿瘤蛋白质抗原，又可利用糖脂类非蛋白质抗原，扩大了肿瘤抗原的靶点范围；(4)使用患者自体细胞降低了排异反应的风险；(5)CAR-T细胞具有免疫记忆功能，可以长期在体内存活。
在本发明中，本发明的CAR包含(i)胞外结构域，其包含特异性结合于肿瘤细胞表面抗原的抗原结合结构域；(ii)跨膜域；(iii)共刺激因子；和(iv)CD3ζ的信号传导结构域。
嵌合抗原受体NK细胞(CAR-NK细胞)
如本文所用，术语“CAR-NK细胞”、“CAR-NK”、“本发明CAR-NK细胞”均指本发明所述的CAR-NK细胞。本发明CAR-NK细胞可靶向肿瘤细胞表面抗原(优选CD19)，用于治疗肿瘤细胞表面抗原(如CD19)高表达或阳性的肿瘤。
自然杀伤(NK)细胞是一类主要的免疫效应细胞，通过非抗原特异性途径去保护机体免受病毒感染和肿瘤细胞的侵袭。通过工程化(基因修饰)的NK细胞可能获得新的功能，包括特异性识别肿瘤抗原的能力及具有增强的抗肿瘤细胞毒作用。
与自体CAR-T细胞相比，CAR-NK细胞还具有一下优点，例如：(1)通过释放穿孔素和颗粒酶直接杀伤肿瘤细胞，而对机体正常的细胞没有杀伤作用；(2)它们释放很少量的细胞因子从而降低了细胞因子风暴的危险；(3)体外极易扩增及发展为“现成的”产品。除此之外，与CAR-T细胞治疗类似。
外源T细胞抗原受体
如本文所用，外源T细胞抗原受体(T cell receptor,TCR)为通过基因转移技术从肿瘤反应性T细胞中克隆出TCR的α链和β链，通过基因工程的手段，以慢病毒或逆转录病毒为载体，外源性转入到T细胞内的TCR。
外源TCR修饰的T细胞能够特异性识别和杀伤肿瘤细胞，并通过优化TCR与肿瘤性特异性抗原的亲和力，可以提高T细胞与肿瘤的亲和力，提高抗肿瘤效果。
PD-1H基因表达下调或沉默
如本文所用，“PD-1H基因表达是被沉默的”指PD-1H基因不表达或低表达。“低表达”指所述CAR-T细胞PD-1H基因的表达量G1与正常免疫细胞PD-1H基因的表达量G0的比值，即G1/G0≤0.8，较佳地G1/G0≤0.5，更佳地≤0.2，更佳地≤0.1，最佳地为0。
本发明中PD-1基因表达下调或沉默方法有CRISPR/Cas9、RNA干扰技术、转录激活因子样效应物核酸酶TALENs(transcription activator-like(TAL)effector  nucleases)和锌指核酸酶Zinc finger nucleases(ZFNs)。优选地，本发明通过CRISPR/Cas9、RNA干扰技术使PD-1H基因下调或沉默。在本发明的一个实施例中，采用CRISPR/Cas9或shRNA使PD-1H基因下调或沉默。
CRISPR/Cas系统
CRISPR(clustered regularly interspersed short palindromic repeats)/Cas(CRISPR-associated)系统是原核生物特有的一种天然免疫系统，用于抵抗病毒或外源性质粒的侵害。Ⅱ型CRISPR/Cas系统作为RNA直接介导的基因组编辑工具已经在许多真核生物和原核生物体内成功应用。CRISPR/Cas9系统的发展彻底改变了人们编辑DNA序列和调控目标基因表达水平的能力，从而为生物体的精确基因组编辑提供了有力的工具。简化后的CRISPR/Cas9系统由两部分组成：Cas9蛋白和sgRNA。其作用原理为sgRNA通过自身的Cas9把手与Cas9蛋白形成Cas9-sgRNA复合体，Cas9-sgRNA复合体中sgRNA的碱基互补配对区序列与目标基因的靶序列通过碱基互补配对原则进行配对结合，Cas9利用自身的核酸内切酶活性对目标DNA序列进行切割。与传统的基因组编辑技术相比，CRISPR/Cas9系统具有几大明显的优势：易用性、简便性、低成本、可编程性以及可同时编辑多个基因。
表达盒
如本文所用，“表达盒”或“本发明表达盒”包括任选的第一表达盒和第二表达盒。所述第一表达盒包含编码CAR的核酸序列。所述第二表达盒包含用于沉默PD-1H的核酸序列。在另一优选例中，本发明还包括用于表达基因编辑蛋白的第三表达盒。
在一个实施方式中，所述任选的第一表达盒、第二表达盒和第三表达盒分别还包括启动子。在一个实施方式中，所述任选的第一表达盒、第二表达盒和第三表达盒分别还包括终止子。
在一个实施方式中，所述的任选的第一表达盒、第二表达盒和第三表达盒位于相同或不同的载体上。优选地，所述的任选的第一表达盒、第二表达盒和第三表达盒位于同一载体。优选地，所述的载体选自下组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子、其他基因转移系统、或其组合。优选地，所述的载体为病毒载体。
在一个实施方式中，所述的第三表达盒包含CRISPR/Cas9(sgRNA和Cas9)、反义RNA、或其组合。优选地，所述的sgRNA靶向PD-1H，且sgRNA的序列如SEQ ID NO.:1-4、12-18中任一或其组合所示。优选地，所述的反义RNA包括miRNA、siRNA、shRNA、抑制性mRNA、或dsRNA，所述反义RNA的序列如SEQ ID NO.:5-10中任一或组合所示。
载体
编码期望分子的核酸序列可利用在本领域中已知的重组方法获得，诸如例如通过从表达基因的细胞中筛选文库，通过从已知包括该基因的载体中得到该基因，或通过利用标准的技术，从包含该基因的细胞和组织中直接分离。可选地，感兴趣的基因可被合成生产。
本发明也提供了其中插入本发明的表达盒的载体。源于逆转录病毒诸如慢病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因长期、稳定的整合并且其在子细胞中增殖。慢病毒载体具有超过源自致癌逆转录病毒诸如鼠科白血病病毒的载体的优点，因为它们可转导非增殖的细胞，诸如肝细胞。它们也具有低免疫原性的优点。
简单概括，通常可操作地连接本发明的表达盒或核酸序列至启动子，并将其并入表达载体。该载体适合于复制和整合真核细胞。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、初始序列和启动子。
本发明的表达构建体也可利用标准的基因传递方案，用于核酸免疫和基因疗法。基因传递的方法在本领域中是已知的。见例如美国专利号5,399,346、5,580,859、5,589,466，在此通过引用全文并入。在另一个实施方式中，本发明提供了基因疗法载体。
该核酸可被克隆入许多类型的载体。例如，该核酸可被克隆入如此载体，其包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。特定的感兴趣载体包括表达载体、复制载体、探针产生载体和测序载体。
进一步地，表达载体可以以病毒载体形式提供给细胞。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记(例如，WO01/96584；WO01/29058；和美国专利号6,326,193)。
已经开发许多基于病毒的系统，用于将基因转移入哺乳动物细胞。例如，逆转录病毒提供了用于基因传递系统的方便的平台。可利用在本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。许多逆转录病毒系统在本领域中是已知的。在一些实施方式中，使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一个实施方式中，使用慢病毒载体。
额外的启动子元件，例如增强子，可以调节转录开始的频率。通常地，这些位于起始位点上游的30-110bp区域中，尽管最近已经显示许多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔经常是柔性的，以便当元件相对于另一个被倒置或移动时，保持启动子功能。在胸苷激酶(tk)启动子中，启动子元件之间的间隔可被增加隔开50bp，活性才开始下降。取决于启动子，表现出单个元件可合作或独立地起作用，以启动转录。
合适的启动子的一个例子为U6启动子。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于CMV启动子、类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒 (MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关，其能够当这样的表达是期望的时，打开可操作地连接诱导型启动子的多核苷酸序列的表达，或当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
为了评估CAR多肽或其部分的表达，被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面，可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表达。有用的可选择标记包括例如抗生素抗性基因，诸如neo等等。
报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。通常地，报道基因为以下基因：其不存在于受体有机体或组织或由受体有机体或组织进行表达，并且其编码多肽，该多肽的表达由一些可容易检测的性质例如酶活性清楚表示。在DNA已经被引入受体细胞后，报道基因的表达在合适的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白的基因(例如，Ui-Tei等，2000FEBS Letters479:79-82)。合适的表达系统是公知的并可利用已知技术制备或从商业上获得。通常，显示最高水平的报道基因表达的具有最少5个侧翼区的构建体被鉴定为启动子。这样的启动子区可被连接至报道基因并用于评价试剂调节启动子-驱动转录的能力。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。在表达载体的内容中，载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。生产包括载体和/或外源核酸的细胞的方法在本领域中是公知的。见例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)。将多核苷酸引入宿主细胞的优选方法为磷酸钙转染。
将感兴趣的多核苷酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体，特别是逆转录病毒载体，已经成为最广泛使用的将基因插入哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。见例如美国专利号5,350,674和5,585,362。
将多核苷酸引入宿主细胞的化学手段包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体。用作体外和体内传递工具(delivery vehicle)的示例性胶体系统为脂质体(例如，人造膜囊)。
在使用非病毒传递系统的情况下，示例性传递工具为脂质体。考虑使用脂质制剂，以将核酸引入宿主细胞(体外、离体(ex vivo)或体内)。在另一方面，该核酸可与脂质相关联。与脂质相关联的核酸可被封装入脂质体的水性内部中，散布在脂质体的脂双层内，经与脂质体和寡核苷酸两者都相关联的连接分子附接至脂质体，陷入脂质体，与脂质体复合，分散在包含脂质的溶液中，与脂质混合，与脂质联合，作为悬浮液包含在脂质中，包含在胶束中或与胶束复合，或以其他方式与脂质相关联。与组合物相关联的脂质、脂质/DNA或脂质/表达载体不限于溶液中的任何具体结构。例如，它们可存在于双分子层结构中，作为胶束或具有“坍缩的(collapsed)”结构。它们也可简单地被散布在溶液中，可能形成大小或形状不均一的聚集体。脂质为脂肪物质，其可为天然发生或合成的脂质。例如，脂质包括脂肪小滴，其天然发生在细胞质以及包含长链脂肪族烃和它们的衍生物诸如脂肪酸、醇类、胺类、氨基醇类和醛类的该类化合物中。
在本发明的一个优选地实施方式中，所述载体为慢病毒载体。
本发明所设计的整合了PD-1H的shRNA或sgRNA的基于pCDH-GFP的CD19-CAR载体、B7-H3(CD276)-CAR载体，但本发明内容不限于用pCDH-GFP构建的CAR载体，应适合于所有适合于构建CART的其他质粒载体；靶点也不限于CD19、B7-H3，应适合于所有靶点的CAR-T。
制剂
本发明提供了一种本发明第一方面所述的工程化免疫细胞、本发明第十方面所述的宿主细胞、以及药学上可接受的载体、稀释剂或赋形剂。在一个实施方式中，所述制剂为液态制剂。优选地，所述制剂为注射剂。优选地，所述制剂中所述CAR-T细胞的浓度为1×10 3-1×10 9个细胞/Kg体重，更优地1×10 4-1×10 8个细胞/Kg体重。
在一个实施方式中，所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的制剂优选配制用于静脉内施用。
治疗性应用
本发明包括用编码本发明表达盒的慢病毒载体(LV)转导的细胞(例如，T细胞，更优选PD-1H沉默的T细胞(如CD8+T细胞))进行的治疗性应用。转导的T细胞可靶向肿瘤细胞的标志物(如CD19)，协同激活T细胞，引起细胞免疫应答，从而显著提高其对实体瘤和血液肿瘤的杀伤效率。
因此，本发明也提供了刺激对哺乳动物的靶细胞群或组织的T细胞-介导的免疫应答的方法，其包括以下步骤：给哺乳动物施用本发明的CAR-T细胞。
在一个实施方式中，本发明包括一类细胞疗法，分离病人自体T细胞(或者异源供体)，激活并进行基因改造产生CAR-T细胞，随后注入同一病人体内。这种方式患移植物抗宿主病概率极低，抗原被T细胞以无MHC限制方式识别。此外，一种CAR-T就可以治疗表达该抗原的所有癌症。不像抗体疗法，CAR-T细胞能够体内复制，产生可导致持续肿瘤控制的长期持久性。
在一个实施方式中，本发明的CAR-T细胞可经历稳固的体内T细胞扩展并可 持续延长的时间量。另外，CAR介导的免疫应答可为过继免疫疗法步骤的一部分，其中CAR-修饰T细胞诱导对CAR中的抗原结合结构域特异性的免疫应答。例如，CD19的CAR-T细胞引起抗表达CD19的细胞的特异性免疫应答。
尽管本文公开的数据具体公开了包括抗CD19的抗原结合结构域、铰链和跨膜区、和4-1BB和CD3ζ信号传导结构域的慢病毒载体，但本发明应被解释为包括对构建体组成部分中的每一个的任何数量的变化。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤，以及血管化的肿瘤。癌症可包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)或可包括实体瘤。用本发明的CAR治疗的癌症类型包括但不限于癌、胚细胞瘤和肉瘤，和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤，例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病，包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌卵巢癌。
本发明的CAR-修饰T细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地，哺乳动物为人。
对于离体免疫，以下中的至少一项在将细胞施用进入哺乳动物前在体外发生：i)扩增细胞，ii)将编码CAR的核酸引入细胞，和/或iii)冷冻保存细胞。
离体程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞从哺乳动物(优选人)中分离并用表达本文公开的CAR的载体进行基因修饰(即，体外转导或转染)。CAR-修饰的细胞可被施用给哺乳动物接受者，以提供治疗益处。哺乳动物接受者可为人，和CAR-修饰的细胞可相对于接受者为自体的。可选地，细胞可相对于接受者为同种异基因的、同基因的(syngeneic)或异种的。
除了就离体免疫而言使用基于细胞的疫苗之外，本发明也提供了体内免疫以引起针对患者中抗原的免疫应答的组合物和方法。
本发明提供了治疗肿瘤的方法，其包括施用给需要其的对象治疗有效量的本发明的CAR-修饰的T细胞。
本发明的CAR-修饰的T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞群，与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的组合物优选配制用于静脉内施用。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症、和患者疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的T细胞的药物组合物可以以10 4至10 9个细胞/kg体重的剂量，优选10 5至10 6个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，NewEng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
对象组合物的施用可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。在一个实施方式中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过i.v.注射施用。T细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将T细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄法珠单抗治疗或对PML患者的其他治疗。在进一步的实施方式中，本发明的T细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506，抗体或其他免疫治疗剂。在进一步的实施方式中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方式中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方式中，在移植后，对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方式中，扩展的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×10 6个至1×10 10个本发明的T细胞(如，CD19-CAR-T细胞)，通过例如静脉回输的方式，施用于患者。
本发明的主要优点包括：
(1)本发明首次发现，PD-1H在CD8+T细胞中有抑制性免疫调控功能，可负向调控CD8+ T细胞在肿瘤免疫中的作用。
(2)本发明首次发现，通过RNA干扰或基因敲除CD8+ T细胞(包括鼠源OT-1细胞和人源CAR-T细胞)上PD-1H的表达可以增强CD8+ T细胞的抗肿瘤作用，促进其浸润到肿瘤微环境中。
(3)本发明的研究发现，PD-1H在CD8+ T细胞中具有显著的免疫抑制功能。在肿瘤浸润的CD8+ T细胞上，PD-1H的表达显著上调。在小鼠肿瘤模型中，PD-1H缺失的CD8+ T细胞的抗肿瘤作用明显增强，免疫效应分子(如IFN-γ、GranzmB等)的释放增加，同时PD-1H缺失的CD8+ T细胞能更好地浸润到肿瘤微环境中。因此，靶向T细胞上PD-1H可能是潜在的肿瘤免疫治疗途径。
(4)本发明首次发现，靶向PD-1H(沉默/敲除PD-1H表达，特异性抗体作用或其他方法)可以应用在CAR-T细胞或其他免疫细胞上。
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。
除非特别说明，否则本发明实施例中所用材料和试剂均为市售产品。
实施例1 PD-1H在肿瘤浸润的CD8+ T细胞和激活的CD8+ T细胞中表达上升
(一)检测肿瘤浸润淋巴细胞上PD-1H的表达情况
选用雌性、8周龄的C57BL/6小鼠，在腹侧皮下种植小鼠淋巴瘤EG7细胞或小鼠黑色素瘤B16-ova细胞。种瘤17天后，取小鼠的皮下种瘤和脾脏，制备成单细胞悬液，加入anti-mFc receptor(clone:2.4G2)抗体4℃孵育10min，用含1％FBS的PBS洗1次。再加入anti-mCD3、anti-mCD4、anti-mCD8、anti-mPD-1H抗体4℃孵育30min，用含1％FBS的PBS洗1次后，利用流式细胞仪检测T细胞上PD-1H的表达情况。
结果如图1所示，在小鼠EG7淋巴瘤和B16-OVA黑色素瘤的种瘤浸润T细胞上均有PD-1H的表达，同时CD8+ T细胞上PD-1H的表达高于CD4+ T细胞。
(二)检测激活的CD8+ T细胞上PD-1H的表达情况
利用CD8+ T细胞分选试剂盒分选出PD-1H KO小鼠和其对照野生型小鼠(Wild-type，WT)的CD8+ T细胞，在体外培养，加入anti-mCD3(1μg/ml)和anti-mCD28(2μg/ml)刺激。分别于24、48、72小时时间点，收取T细胞加入anti-mCD8、anti-mPD-1H抗体4℃孵育30min，用含1％FBS的PBS洗1次后，利用流式细胞仪检测T细胞上PD-1H的表达情况。
结果如图2所示，加入anti-mCD3和anti-mCD28刺激的WT CD8+ T细胞上PD-1H表达增加。
实施例2 缺失PD-1H的CD8+ T细胞抗肿瘤作用增强
(一)PD-1H缺失的CD8+ T细胞在小鼠黑色素瘤模型中的作用
选用雌性、8周龄的C57BL/6小鼠，在腹侧皮下种植小鼠黑色素瘤B16-OVA细胞。种瘤6天后，分别给荷瘤小鼠尾静脉注射PD-1H KO或WT OT-1 T细胞进行治疗。用电子游标卡尺每两天测量一次肿瘤大小。17天后，取小鼠的皮下种瘤，制备成单细胞悬液，加入anti-mFc receptor(clone:2.4G2)抗体4℃孵育10min，用含1％FBS的PBS洗1次。再加入anti-mCD3、anti-mCD45、anti-mCD8抗体4℃ 孵育30min，用含1％FBS的PBS洗1次后，利用流式细胞仪检测。
结果如图3所示，转输PD-1H-KO OT-1细胞治疗的小鼠肿瘤生长明显受到抑制(左图)。转输PD-1H-KO OT-1细胞的小鼠的肿瘤浸润淋巴细胞中，CD8+ T细胞占比增加(右图)。
(二)PD-1H缺失的CD8+ T细胞在小鼠淋巴瘤模型中的作用
选用雌性、8周龄的C57BL/6小鼠，在腹侧皮下种植小鼠淋巴瘤EG7细胞。种瘤6天后，分别给荷瘤小鼠尾静脉转输PD-1H KO或WT OT-1 T细胞进行治疗。用电子游标卡尺每两天测量一次肿瘤大小。17天后，取小鼠的皮下种瘤，制备成单细胞悬液，加入anti-mFc receptor(clone:2.4G2)抗体4℃孵育10min，用含1％FBS的PBS洗1次。再加入anti-mCD3、anti-mCD45、anti-mCD8抗体4℃孵育30min，用含1％FBS的PBS洗1次后，利用流式细胞仪检测。
结果如图4所示，转输PD-1H-KO OT-1细胞治疗的小鼠肿瘤生长明显受到抑制(上图)。转输PD-1H-KO OT-1细胞的小鼠的肿瘤浸润淋巴细胞中，CD8+ T细胞占比明显增加(下图)。
实施例3 利用CRISPR Cas9技术敲除CD8+ T细胞上PD-1H基因能增强CD8+ T细胞的抗肿瘤作用。
(一)筛选能有效敲低mPD-1H的sgRNA序列
根据mPD-1H的CDS序列，设计出sgRNA序列。
Figure PCTCN2021128459-appb-000001
LentiCRISPR v2质粒用GFP蛋白序列替代了原有的嘌呤霉素抗性序列。将合成的mPD-1H sgRNA1和mPD-1H sgRNA2片段退火成双链DNA，连接到LentiCRISPR v2载体(Addgene 52961)的BsmBI位点上。挑选克隆，测序分析，确定sgRNA片段成功构建到LentiCRISPR v2载体上。分别将lentiCRISPR mPD-1H sgRNA质粒(sgRNA1、sgRNA2质粒)，与pRSV-Rev、pMDLg/pRRE及pCMV-VSVG辅助质粒按一定比例混合，共转染293FT细胞。转染48h后，分别收集含有mPD-1H sgRNA-H1和mPD-1H sgRNA-H2慢病毒的细胞培养上清，4℃、3000rpm离心5min。上清经0.22uml滤器过滤后，-80℃冻存备用。利用慢病毒侵染293T-mPD-1h细胞。
结果如图5所示mPD-1H sgRNA-H2序列能有效敲除PD-1H。
(二)利用CRISPR Cas9制备敲除PD-1H的CD8+ T细胞(sgRNA-OT-1)
通过淋巴细胞分离液、密度梯度离心方法分离8周龄的雌性OT-1小鼠脾脏的淋巴细胞。利用CD8+ T cell isolation Kit(美天旎)对细胞进行磁珠标记，并分离纯化出CD8+ T淋巴细胞。纯化后的CD8+ T细胞，再利用CD3/CD28抗体进行T淋巴细胞激活和增殖。抗体刺激24小时后收集激活的T淋巴细胞，重悬在RPMI 1640培养基中。用mPD-1H sgRNA-H2慢病毒感染活化的CD8+ T淋巴细胞，将细胞悬液加在6孔板中，置37℃、5％CO 2培养箱中孵育过夜。第二天，再次离心并更换新鲜培养基，每隔2天加入新鲜培养基，继续扩大培养。培养至第8天时，离心收集T细胞，并用适宜的冻存液重悬，液氮中冻存备用。
(三)敲除PD-1H的CD8+ T细胞在小鼠淋巴瘤(EG7)模型中的作用
选用雌性、8周龄的C57BL/6小鼠，在腹侧皮下种植小鼠淋巴瘤EG7细胞。种瘤6天后，分别给荷瘤小鼠尾静脉转输sgRNA OT-1、PD-1H KO或WT OT-1 T细胞进行治疗。用电子游标卡尺每两天测量一次肿瘤大小。17天后，取小鼠的皮下种瘤，制备成单细胞悬液，加入anti-mFc receptor(clone:2.4G2)抗体4℃孵育10min，用含1％FBS的PBS洗1次。再加入anti-mCD3、anti-mCD45、anti-mCD8抗体4℃孵育30min，用含1％FBS的PBS洗1次后，利用流式细胞仪检测。
结果如图6所示，转输sgRNA OT-1与PD-1H-KO OT-1细胞治疗的小鼠肿瘤生长明显受到抑制，通过CRISPR Cas9敲除CD8+ T细胞的PD-1H基因能提高CD8+ T细胞的抗肿瘤作用。
实施例4 在小鼠结肠癌PDX模型肿瘤微环境中人源T细胞或人源CAR-T细胞中PD-1H表达上升
(一)检测体外培养的T细胞和CAR-T细胞上PD-1H的表达
取PBMC和B7-H3-CAR-T细胞(获自福州拓新天成生物科技有限公司，结构为：B7-H3scFv-CD8铰链区-CD8跨膜区-41BB共刺激分子-CD3ζ胞浆信号转导区)，加入anti-hCD3、anti-hB7-H3-mFc和anti-hPD-1H抗体4℃孵育30min，用含1％FBS的PBS洗1次后，利用流式细胞仪检测。
结果如图7所示，在体外的T细胞和CAR-T细胞表面无PD-1H表达。
(二)NCG小鼠异种移植(PDX)结肠癌肿瘤模型的构建
选用8-10周龄、雌性NCG小鼠(NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju) 构建异种移植(PDX)结肠癌肿瘤模型。将患者的结肠肿瘤组织剪切成15mm 3的小碎块，种植到NCG小鼠的双侧腋窝皮下处。每日观察小鼠肿瘤生长情况，每周用游标卡尺测量肿瘤大小，当肿瘤达到约500mm 3大小时，即可取肿瘤组织进行传代。传代方式如上描述，经过五次连续的小鼠至小鼠传代后，将异种移植的肿瘤种植到NCG小鼠的双侧腋窝皮下处。当肿瘤平均半径达到约8mm时，瘤内注射CAR-T细胞进行治疗。治疗20天后，取小鼠外周血、脾脏和肿瘤组织，制备成单细胞悬液，标记特定分子的荧光抗体，进行流式细胞分析。部分脾脏和肿瘤组织则制作成石蜡切片进行分析。
(三)多重免疫荧光共定位
取PDX小鼠模型肿瘤组织切片，脱蜡复水，在1mmol/L EDTA中保持微沸20min进行抗原修复。在3％过氧化氢水溶液中，孵育20分钟阻断内源性过氧化氢酶。以PD-1H抗体(clone D1L2G,CST)为一抗，HRP偶联二抗和酪酰胺放大系统(TSA Plus Fluorescence Kits)进行免疫荧光染色。用CD3、CD8抗体标记细胞表型，再孵育适当的荧光二抗。用DAPI标记细胞核。染色完成的切片用EVOS FL Auto Cell Imaging System观察扫描拍照。
结果如图8所示，在PDX小鼠模型的肿瘤组织中，经过CAR-T细胞注射治疗后，肿瘤浸润的CD8+ CAR-T细胞上有PD-1H的表达。
实施例5 利用短发卡RNA(shRNA)沉默CAR-T细胞上PD-1H的表达能增强CAR-T细胞的抗肿瘤作用。
(一)构建共表达PD-1H shRNA的嵌合抗原受体表达载体
根据PD-1H的CDS序列设计的shRNA序列，如表1所示为候选PD-1H shRNA序列。
表1.PD-1H shRNA序列
Figure PCTCN2021128459-appb-000002
Figure PCTCN2021128459-appb-000003
根据表1序列，合成U6启动子和PD-1H shRNA序列，通过酶切酶连技术将U6-PD-1H shRNA序列构建到pCDH-EF1-CAR质粒上，pCDH-U6-PD-1H shRNA-EF1-CAR质粒结构如图9所示。通过第二代测序技术确定U6-PD-1H shRNA-EF1-CAR质粒序列正确。
(二)筛选有效沉默PD-1H表达的shRNA序列
通过PEI、磷酸钙沉淀或其他的转染方式，在293T细胞上进行慢病毒载体的包装制备，制备表达pCDH-U6-PD-1H shRNA-EF1-CAR(PD-1H shRNA1-shRNA6)和Control shRNA的慢病毒。利用慢病毒侵染Jurkat-PD-1H细胞。病毒侵染72小时后，利用流式细胞仪检测Jurkat-PD-1H细胞中PD-1H的表达。结果如图10所示，pCDH-U6-PD-1H shRNA-EF1-CAR质粒能在T细胞表面CAR分子表达不受影响。如图11所示，PD-1H shRNA1能最有效沉默PD-1H的表达，PD-1H shRNA3次之，PD-1H shRNA2、PD-1H shRNA4、PD-1H shRNA5、PD-1H shRNA6则沉默效果较弱。
(三)共表达PD-1H shRNA的CD19-CAR-T细胞的制备
取50ml志愿者新鲜外周血，通过淋巴细胞分离液、密度梯度离心方法分离外周血单核细胞(PBMC)。利用Pan T cell isolation Kit(美天旎)对细胞进行磁珠标记，并分离纯化出T淋巴细胞。纯化后的T细胞，再利用anti-CD3/CD28磁珠进行T淋巴细胞激活。用PD-1H shRNA-CD19-CAR(其中CD19-CAR结构的一个例子为：CD19靶向的scFv-CD8铰链区-CD8跨膜区-41BB共刺激分子-CD3ζ胞浆信号转导区)和Control shRNA-CD19-CAR慢病毒分别感染1×10 6个活化的T淋巴细胞，将细胞悬液加在G-Rex培养瓶中，置37℃、5％CO 2培养箱中孵育过夜，每隔2天加入新鲜培养基，继续扩大培养。培养至第8天时，离心收集CAR-T细胞(PD-1H shRNA-CD19-CAR T细胞和Control shRNA-CD19-CAR T细胞)，并用适宜的冻存液重悬，液氮中冻存备用。
(四)PD-1H shRNA-CD19-CAR T对肿瘤细胞的体外杀伤作用
将PD-1H shRNA-CD19-CAR T按不同的效靶比分别与标记的靶肿瘤细胞(淋巴瘤细胞CA-46)在37℃下共孵育18h后，应用DAPI染色，通过流式细胞仪检测CAR T细胞对靶肿瘤细胞的杀伤作用。
结果见图12，结果表明：PD-1H shRNA-CD19-CAR T和control shRNA-CD19-CAR T细胞均能有效杀伤表达CD19的肿瘤细胞，PD-1H shRNA-CD19-CAR T比control shRNA-CD19-CAR T细胞有更高的杀伤效率。
(五)PD-1H shRNA-CD19-CAR T细胞动物模型体内抗肿瘤的作用
选用8-10周龄、雌性NCG小鼠(NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju)，尾静脉注射CD19阳性的肿瘤细胞(淋巴瘤细胞CA-46)，建立肿瘤模型。在建模5天后，尾静脉转输CAR-T细胞治疗。肿瘤生长情况通过小动物活体成像系统检测。
结果如图13、14所示，PD-1H shRNA-CD19-CAR T细胞能有效抑制淋巴瘤细胞的生长，相对于对照CAR-T细胞有更好的治疗效果。同时有效延长小鼠的生存时间。
实施例6 利用CRISPR CAS9技术敲除T细胞上的PD-1H
(一)筛选能有效敲低hPD-1H的gRNA(guide RNA)序列
根据人源PD-1H的CDS序列设计sgRNA序列，如表2所示为候选PD-1H sgRNA序列。
表2.PD-1H sgRNA序列
Figure PCTCN2021128459-appb-000004
按照表2合成添加上RNA骨架的PD-1H sgRNA(single guide RNA)，sgRNA由gRNA和RNA骨架连接构成。
将上述合成好的PD-1H sgRNA1与PD-1H sgRNA2分别和cas9蛋白按一定比例混合，用Lonza Amaxa TM 4D-Nucleofector TM电转仪，电转至Jurkat-PD-1H细胞中。电转72小时后，取约2×10 5个细胞加入anti-PD-1H抗体抗体4℃孵育30min，用含1％FBS的PBS洗1次后，利用流式细胞仪检测。
结果如图15所示，PD-1H gRNA1和gRNA2能有效敲除Jurkat-PD-1H细胞上PD-1H的表达，PD-1H gRNA1的敲除效果更佳。
(二)敲除PD-1H的B7-H3 CAR-T细胞的制备
取50ml志愿者新鲜外周血，通过淋巴细胞分离液、密度梯度离心方法分离外周血单核细胞(PBMC)。利用Pan T cell isolation Kit(美天旎)对细胞进行磁珠标记，并分离纯化出T淋巴细胞。纯化后的T细胞，再利用anti-CD3/CD28磁珠进行T淋巴细胞激活。T淋巴细胞激活48h后，将PD-1H gRNA1或control sRNA与cas9蛋白按一定比例混合，用Lonza Amaxa TM 4D-Nucleofector TM电转仪，电转至激活的T淋巴细胞中。电转48h后，用B7-H3-CAR(其中B7-H3-CAR结构为：B7-H3靶向的scFv-CD8铰链区-CD8跨膜区-41BB共刺激分子-CD3ζ胞浆信号转导区)慢病毒感染1×10 7个电转后的T淋巴细胞，将细胞悬液加在G-Rex培养瓶中，置37℃、5％CO 2培养箱中孵育过夜，每隔2天加入新鲜培养基，继续扩大培养。培养至第8天时，离心收集CAR-T细胞，并用适宜的冻存液重悬，液氮中冻存备用。
(三)T7E1酶切检测靶位点编辑效率
提取制备好的敲除PD-1H的B7-H3 CAR-T细胞的基因组DNA，利用表3中的引物将靶向的目的基因片段进行PCR扩增。纯化回收后的DNA片段，按照表4退火体系进行混合，充分混匀后至于PCR仪中加热至95℃，5min后取出自然冷却至室温。退火完成后，向退火体系中加入1μL T7EI核酸内切酶，37℃反应15min后,使用凝胶电泳检测T7EI核酸内切酶酶切条带，并使用ImageJ分析编辑效率。
表3.PD-1H PCR引物序列
编号 序列(5’-3’)
Forward primer GCCGTATTCCCTGTATGTCTGT(SEQ ID NO.:21)
Reward primer CAGTAGAGGCCGCTATCCAG(SEQ ID NO.:22)
表4.DNA双链退火体系
10×T7E1 reaction buffer 2μL
DNA产物 200ng
DNase/RNase-free water Up to 19μL
结果如图16所示，靶向PD-1H的sgRNA1对CAR-T细胞的编辑效率达83.2％。
(四)TIDE分析靶位点编辑效率
提取制备好的敲除PD-1H的B7-H3 CAR-T细胞的基因组DNA，利用表3中的引物将靶向的目的基因片段进行PCR扩增，PCR产物进行纯化回收，然后通过通过第二代测序技术对PCR产物进行测序。测序结果通过TIDE分析系统(shinyapps.datacurators.nl/tide/)进行分析，计算出编辑效率。结果如图17所示，TIDE分析显示PD-1H sgRNA1对靶位点的编辑效率为81.9％，与T7E1酶切检测结果相近。
此外，我们将纯化后的PCR产物连接到PEASY-T1Simple Cloning vector上，然后转化至T1感受态细胞中，并将转化后菌液均匀涂布在LB固体选择培养基上，37℃细菌培养箱培养过夜。第二天挑取20个单克隆菌落进行测序。结果如图18所示，在挑选的20个克隆中成功测序出18个序列，其中11个克隆有1个碱基的缺失(11/18)，5个克隆有3个碱基的缺失(5/18)，2个克隆没被编辑(2/18)，结果与TIDE分析结果一致。
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 (60)

  1. 一种工程化免疫细胞，其特征在于，所述工程化免疫细胞中的PD-1H基因的表达是被沉默的。
  2. 如权利要求1所述的工程化免疫细胞，其特征在于，所述“PD-1H基因表达是被沉默的”指PD-1H基因不表达或低表达。
  3. 如权利要求2所述的工程化免疫细胞，其特征在于，所述“低表达”指所述免疫细胞PD-1H基因的表达量G1与正常免疫细胞PD-1H基因的表达量G0的比值，即G1/G0≤0.8，较佳地G1/G0≤0.5，更佳地≤0.2，更佳地≤0.1，最佳地为0。
  4. 如权利要求1所述的工程化免疫细胞，其特征在于，所述工程化免疫细胞包括CD8+T细胞、CD3+T细胞、CD4+T细胞、B细胞、NK细胞、髓性白细胞或单核细胞、抗原提呈细胞、或其他免疫细胞。
  5. 如权利要求1所述的工程化免疫细胞，其特征在于，所述工程化免疫细胞具有以下特征：
    (a)所述免疫细胞中的PD-1H基因的表达是被沉默的。
  6. 如权利要求5所述的工程化免疫细胞，其特征在于，所述工程化免疫细胞还具有如下特征：
    (b)所述免疫细胞表达嵌合抗原受体CAR或外源TCR，所述CAR靶向抗原分子或肿瘤细胞的标志物，所述外源TCR靶向抗原分子或肿瘤细胞的标志物。
  7. 如权利要求1所述的工程化免疫细胞，其特征在于，所述工程化免疫细胞包括：
    (a)任选的嵌合抗原受体CAR，所述嵌合抗原受体CAR包括：抗原结合结构域、铰链结构域、跨膜结构域和胞内结构域，其中所述抗原结合结构域特异性结合于抗原分子或肿瘤细胞表面抗原；和
    (b)降低或抑制PD-1H蛋白表达活性的抑制分子。
  8. 如权利要求7所述的工程化免疫细胞，其特征在于，所述抑制分子选自下组：抑制性核酸、小分子化合物、抗体(如单域抗体)、多肽、或其组合。
  9. 如权利要求8所述的工程化免疫细胞，其特征在于，所述抑制性核酸包括RNA干扰剂、Crispr试剂。
  10. 如权利要求9所述的工程化免疫细胞，其特征在于，所述抑制性核酸选自下组：siRNA、miRNA、shRNA、发夹siRNA、串联表达的miRNA、微小RNA适应性shRNA、前体微小RNA、或其组合。
  11. 如权利要求10所述的工程化免疫细胞，其特征在于，所述抑制性核酸的序列如SEQ ID NO.:5-10中任一或组合所示。
  12. 如权利要求9所述的工程化免疫细胞，其特征在于，所述Crispr试剂包括基因编辑蛋白。
  13. 如权利要求12所述的工程化免疫细胞，其特征在于，所述基因编辑蛋白选自下组：CRISPR、TALEN、ZFN、或其组合。
  14. 如权利要求13所述的工程化免疫细胞，其特征在于，所述CRISPR蛋白选自下组：Cas9、nCas9、Cas10、Cas9a、Cas12、Cas12a、Cas12b、Cas13、Cas14、或其组合。
  15. 如权利要求9所述的工程化免疫细胞，其特征在于，所述Crispr试剂还包括gRNA。
  16. 如权利要求15所述的工程化免疫细胞，其特征在于，所述gRNA的序列如SEQ ID NO.:1-4、12-18任一或组合所示。
  17. 如权利要求8所述的工程化免疫细胞，其特征在于，所述小分子化合物选自下组：1，2，4-噁二唑化合物及其衍生物、噁二唑类化合物、噻二唑类化合物、磺酰胺类化合物、联苯类化合物、或其组合。
  18. 如权利要求7所述的工程化免疫细胞，其特征在于，所述肿瘤细胞表面抗原包括各种实体瘤、固体肿瘤和血液肿瘤的细胞表面抗原。
  19. 如权利要求7所述的工程化免疫细胞，其特征在于，所述肿瘤细胞表面抗原选自下组：CD19、c-Met、PSMA、MUC-1、MUC16、CD7、CD20、CD22、CD123、CD47、CD138、CD33、CD30、CD38、PD-L1、CD276、B7-H4、间皮素(mesothelin)、EGFR、EGFRviii、GPC3、BCMA、ErbB2、ErbB3、ErbB4、NKG2D配体、LMP1、EpCAM、VEGFR-1、Lewis-Y、ROR1、Claudin18.2、CD7、CD5、CD2、CD3、CD19、CD20、CD22、CD24、CD25、CD28、CD123、CD47、CD52、CD56、CD80、CD86、CD81、CD138、CD33、CD38、CD30、CD133、CD97、CD99、CD40、CD43、CD137、CD151、CD171、KIT(CD117)、CD174、CD44V6、CD179a、B7-H3(CD276)、B7-H4、HER2、HER3、HER4、c-Met、PSMA、PSCA，MUC16、MUC1、间皮素(mesothelin)、EGFR、VEGFR2、EGFR-VIII、VEGFR-1、GPC3、BCMA、ErbB2、ErbB3、ErbB4、NKG2D配体、LMP1、EpCAM、Lewis-Y、ROR1、Claudin18.2、LIGHT、NKG2C、CEA(癌胚抗原)，FAP，PSMA，CA125，EphA2，L1CAM，CS1，ROR1，EC，NY-ESO-1，GD2，EPG，DLL3，5T4，IL-13Ra2(或CD213A2)、IL-11Ra、PRSS21、PDGFR-β、SSEA-4、叶酸受体α(FRa或FR1)；叶酸受体β(FRb)、AFP/MHC复合物、NCAM、ELF2M、FAP、IGF-I受体、CAIX、sLe、神经节苷脂GM3(aNeu5Ac(2-3)bDClalp(1-4)bDGlcp(1-1)Cer)、TGS5、HMWMAA、OAcGD2、(TEM1/CD248)、TEM7R、CLDN6、TSHR、GPRC5D、ALK、HAVCR1、ADRB3、PANX3、GPR20、OR51E2、LAGE-1a、MAGE-A1、ETV6-AML、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、PCT A-1、MelanA或MARTI、hTERT、ML-IAP、CAIX、CEACAM6、IGF1R、O-acetyl GD2、O-acetyl GD3、GHRHR、GHR、Flt1、Flt3、KDR、Flt4、CTLA-4、GITR、BTLA、TGFBR2、TGFBR1、IL6R，gp130、Lewis，TNFR1、TNFR2、PD1、PD-L1、PD-L2，HVEM、MAGE-A、NY-ESO-1、NY-ESO-1/MHC I复合物、PSMA、RANK、TNFRSF4、TWEAK-R、LTPR、LIFRP、LRP5、TCRa、TCRp、TLR7、TLR9、PTCH1、WT-1、WT1/MHC I复合物、NA17、MYCN、RhoC、SART3、SSX2、RAGE-1、HPV E7、HPV E7/MHC I复合物；AFP/MHC I复合物、Ras/MHC I复合物、Robol、Frizzled、OX40、CD79a、CD79b、CD72、Notch-1-4、CLL-1(或CLECL1)、TAG72、LILRA2；CD300LF；CLEC12A；BST2；EMR2；LY75、FCRL5；IGLL1、MPL、生物素、c-MYC表位标签、CD34、LAMP1 TROP2、GFRα4、CDH17、CDH6、NYBR1、CDH19、CD200R、Slea；岩藻糖基GM1、PTK7、gpNMB、CDH1-CD324、DLL3、CD179b-IGLl1、TCRγ-δ、NKG2D、CD32(FCGR2A)、Tn ag、Tim1-/HVCR1、CSF2RA(GM-CSFR-α)、TGFβR2、Lews Ag、TCR-β1链、TCR-β2链、TCR-γ链、TCR-δ链、FITC、LHR、FSHR、CGHR或GR、CCR4、SLAMF6、SLAMF4、HIV1包膜糖蛋白、HTLV1-Tax、CMV pp65、EBV-EBNA3c、 KSHV K8.1、KSHV-gH、甲型流感血凝素(HA)、GAD、PDL1、胍基环化酶C(GCC)、抗桥粒芯糖蛋白3(Dsg3)的自身抗体、Dsg1、HLA、HLA-A、HLA-A2、HLA-B、HLA-C、HLA-DP、HLA-DM、HLA-DOA、HLA-DOB、HLA-DQ、HLA-DR、HLA-G、IgE、CD99、RasG12V、组织因子1(TF1)、AFP、GPRC5D、P-糖蛋白、STEAP1、Liv1、粘连蛋白-4、Cripto、gpA33、BST1/CD157、低电导氯离子通道以及TNT抗体识别的抗原、或其组合。
  20. 如权利要求7所述的工程化免疫细胞，其特征在于，所述肿瘤细胞表面抗原包括CD19。
  21. 如权利要求7所述的工程化免疫细胞，其特征在于，所述肿瘤细胞表面抗原包括B7-H3(CD276)。
  22. 一种制备权利要求1所述的工程化免疫细胞的方法，其特征在于，包括以下步骤：
    (A)提供一经筛选或待改造的免疫细胞；和
    (B)对所述的免疫细胞进行改造，从而使得所述的免疫细胞中PD-1H基因的表达沉默，从而获得权利要求1所述的免疫细胞。
  23. 如权利要求22所述的方法，其特征在于，步骤(B)中包括将表达用于沉默PD-1H基因的第二表达盒导入所述免疫细胞。
  24. 如权利要求22所述的方法，其特征在于，在步骤(B)中，还包括步骤：(B1)将表达CAR的第一表达盒导入所述免疫细胞；和(B2)将表达用于沉默PD-1H基因的第二表达盒导入所述免疫细胞，
    其中，所述的步骤(B1和(B2)的次序无任何限定。
  25. 如权利要求23所述的方法，其特征在于，所述的第二表达盒包含CRISPR/Cas9(gRNA和Cas9)、反义RNA。
  26. 如权利要求25所述的方法，其特征在于，所述的gRNA靶向PD-1H，且gRNA的序列如SEQ ID NO.:1-4、12-18中任一或组合所示。
  27. 如权利要求25所述的方法，其特征在于，所述的反义RNA包括miRNA、siRNA、shRNA、抑制性mRNA、或dsRNA。
  28. 如权利要求27所述的方法，其特征在于，所述反义RNA的序列如SEQ ID NO.:5-10中任一或组合所示。
  29. 一种制剂，其特征在于，所述制剂含有权利要求1所述的工程化免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。
  30. 如权利要求29所述的制剂，其特征在于，所述制剂中所述免疫细胞的浓度为1×10 3-1×10 10个细胞/ml，较佳地1×10 4-1×10 8个细胞/ml。
  31. 一种权利要求1所述的工程化免疫细胞的用途，其特征在于，用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
  32. 如权利要求31所述的用途，其特征在于，所述癌症或肿瘤选自下组：血液肿瘤、实体瘤、或其组合。
  33. 如权利要求31所述的用途，其特征在于，所述癌症或肿瘤包括CD19阳性的肿瘤。
  34. 如权利要求31所述的用途，其特征在于，所述癌症或肿瘤包括B7-H3(CD276)阳性的肿瘤。
  35. 一种用于制备权利要求1所述的工程化免疫细胞的试剂盒，其特征在于，所述试剂盒含有容器，以及位于容器内的：
    (1)任选的第一核酸序列，所述第一核酸序列含有用于表达CAR或外源TCR的第一表达盒；
    (2)第二核酸序列，所述第二核酸序列含有用于沉默PD-1H的第二表达盒或靶向PD-1H的gRNA。
  36. 如权利要求35所述的试剂盒，其特征在于，所述的试剂盒还含有：(4)第三核酸序列，所述第三核酸序列含有用于表达基因编辑蛋白的表达盒；或基因编辑蛋白。
  37. 一种调控免疫细胞活性的方法，其特征在于，包括：
    通过调控免疫细胞中PD-1H的表达水平，从而调控免疫细胞的活性。
  38. 如权利要求37所述的方法，其特征在于，通过降低或抑制免疫细胞中PD-1H的表达水平，从而增强免疫细胞的活性。
  39. 如权利要求37所述的方法，其特征在于，通过增强免疫细胞中PD-1H的表达水平，从而降低免疫细胞的活性。
  40. 如权利要求38所述的方法，其特征在于，所述“降低或抑制免疫细胞中PD-1H的表达水平”指所述免疫细胞PD-1H基因的表达量G1与正常免疫细胞PD-1H基因的表达量G0的比值，即G1/G0≤0.8，较佳地G1/G0≤0.5，更佳地≤0.2，更佳地≤0.1，最佳地为0。
  41. 如权利要求39所述的方法，其特征在于，所述“增强免疫细胞中PD-1H的表达水平”指所述免疫细胞PD-1H基因的表达量G1与正常免疫细胞PD-1H基因的表达量G0的比值，即G1/G0≥2，较佳地G1/G0≥3，更佳地，G1/G0≥4。
  42. 一种判断或评估免疫细胞活性的方法，其特征在于，包括：
    通过检测免疫细胞中PD-1H的表达水平，从而判断或评估免疫细胞的活性。
  43. 如权利要求42所述的方法，其特征在于，当免疫细胞中PD-1H的表达水平升高时，则免疫细胞的活性降低。
  44. 如权利要求42所述的方法，其特征在于，当免疫细胞中PD-1H的表达水平降低时，则免疫细胞的活性增强。
  45. 如权利要求43所述的方法，其特征在于，所述“免疫细胞中PD-1H的表达水平升高”指所述免疫细胞PD-1H基因的表达量G1与正常免疫细胞PD-1H基因的表达量G0的比值，即G1/G0≥2，较佳地G1/G0≥3，更佳地，G1/G0≥4。
  46. 如权利要求44所述的方法，其特征在于，所述“免疫细胞中PD-1H的表达水平降低”指所述免疫细胞PD-1H基因的表达量G1与正常免疫细胞PD-1H基因的表 达量G0的比值，即G1/G0≤0.8，较佳地G1/G0≤0.5，更佳地≤0.2，更佳地≤0.1，最佳地为0。
  47. 一种核酸分子，其特征在于，包括第一核酸和任选的第二核酸，其中所述第一核酸含有第一表达盒，所述第一表达盒编码降低或抑制PD-1H蛋白表达活性的抑制分子，所述第二核酸含有第二表达盒，所述第二表达盒编码嵌合抗原受体CAR，所述嵌合抗原受体CAR包括：抗原结合结构域、跨膜结构域和胞内结构域，其中所述抗原结合结构域特异性结合于肿瘤细胞表面抗原。
  48. 如权利要求47所述的核酸分子，其特征在于，所述第一表达盒和/或第二表达盒还包括组成型启动子或诱导型启动子。
  49. 如权利要求48所述的核酸分子，其特征在于，所述组成型启动子选自下组：CMV、EF1a、U6、SV40、PGK1、Ubc、CAG、H1、或其组合。
  50. 如权利要求48所述的核酸分子，其特征在于，所述诱导型启动子选自下组：金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子、四环素启动子、或其组合。
  51. 一种载体，其特征在于，所述载体含有权利要求47所述的核酸分子。
  52. 一种宿主细胞，其特征在于，所述的宿主细胞含有权利要求51所述的载体或染色体中整合有外源的权利要求47所述的核酸分子。
  53. 一种药物组合物，其特征在于，包括：
    (a)权利要求52所述的宿主细胞；和
    (b)药学上可接受的载体、稀释剂或赋形剂。
  54. 如权利要求，所述药物组合物中，所述细胞的浓度为1×10 3-1×10 10个细胞/ml，较佳地1×10 4-1×10 8个细胞/ml。
  55. 一种制备工程化免疫细胞的方法，其特征在于，包括：
    将权利要求47所述的核酸分子或权利要求51所述的载体转导入免疫细胞内，从而获得所述工程化免疫细胞。
  56. 一种试剂组合，其特征在于，所述试剂组合包括：
    (i)任选的第一试剂，所述第一试剂为免疫细胞；和
    (ii)第二试剂，所述的第二试剂为降低或抑制PD-1H蛋白表达活性的抑制分子。
  57. 一种增强工程化免疫细胞的肿瘤杀伤效率的方法，其特征在于，包括：
    在降低或抑制PD-1H蛋白表达活性的抑制分子存在下，将工程化免疫细胞与肿瘤细胞接触，从而增强工程化免疫细胞的肿瘤杀伤效率。
  58. 如权利要求57所述的方法，其特征在于，所述方法为体外的。
  59. 一种权利要求52所述的宿主细胞、或权利要求53所述的药物组合物的用途，其特征在于，用于制备选择性杀伤肿瘤细胞的药物或制剂。
  60. 一种用于选择性杀伤肿瘤细胞的试剂盒，其特征在于，所述试剂盒含有容器，以及位于容器内的权利要求52所述的宿主细胞。
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