CN114438033A

CN114438033A - Anti-tumor immunotherapy method targeting PD-1H (VISTA)

Info

Publication number: CN114438033A
Application number: CN202011212512.6A
Authority: CN
Inventors: 黄纲雄
Original assignee: Fuzhou Tuoxin Tiancheng Biotechnology Co ltd
Current assignee: Fuzhou Tuoxin Tiancheng Biotechnology Co ltd
Priority date: 2020-11-03
Filing date: 2020-11-03
Publication date: 2022-05-06
Also published as: WO2022095903A1

Abstract

The invention provides anti-tumor immunotherapy methods targeting PD-1h (vista). In particular, the invention provides immune cells or engineered immune cells in which the PD-1H gene is not expressed or knocked out, or in which the expression of the gene is silenced. The immune cell or the engineered immune cell has good tumor killing effect.

Description

Anti-tumor immunotherapy method targeting PD-1H (VISTA)

Technical Field

The invention relates to the field of biological medicines, in particular to an anti-tumor immunotherapy method targeting PD-1H (VISTA).

Background

PD-1H (also known as VISTA, DD 1-alpha) is a co-inhibitory molecule of the CD28/B7 family, with high homology to PD-1 and PD-L1. PD-1H is expressed predominantly on cells of the hematopoietic lineage, both myeloid (including macrophages, dendritic cells, monocytes and neutrophils) and CD4+ T cells. PD-1H on antigen presenting cells and regulatory T cells can inhibit T cell proliferation and cytokine release as ligand, and has important regulation and control effect on autoimmune diseases and tumor progression. PD-1H can be used as an inhibitory receptor on CD4+ T cells, and regulates the immune tolerance and tumor immunity involved by the T cells. The PD-1H specific blocking antibody can effectively inhibit tumor growth in a plurality of mouse tumor (melanoma, bladder cancer and the like) models. The specific PD-1H agonism antibody can effectively relieve the symptoms of autoimmune diseases (systemic lupus erythematosus, asthma, arthritis and the like).

Under normal conditions, PD-1H is expressed at low levels on T cells, and PD-1H expression is rapidly lost after mouse T cells are cultured in vitro. Similarly, expression of PD-1H is not usually detectable in human T cells cultured in vitro. Previous studies on the function of PD-1H in T cells have focused mainly on CD4+ T cells, while the function of PD-1H in CD8+ T cells has remained unclear and its role in tumor immunotherapy has not been known.

Disclosure of Invention

The invention aims to provide an engineered immune cell which can obviously enhance the good killing effect of tumors.

In a first aspect, the invention provides an engineered immune cell in which the expression of the PD-1H gene is silenced.

In another preferred embodiment, the phrase "PD-1H gene expression is silenced" means that the PD-1H gene is not expressed or is under-expressed.

In another preferred example, the "low expression" refers to the ratio of the expression amount G1 of the PD-1H gene of the immune cell to the expression amount G0 of the PD-1H gene of the normal immune cell, i.e., G1/G0 ≦ 0.8, preferably G1/G0 ≦ 0.5, more preferably ≦ 0.2, more preferably ≦ 0.1, and most preferably 0.

In another preferred embodiment, said silencing the expression of the PD-1H gene is effected by a method selected from the group consisting of: gene mutation, gene knockout, gene disruption, RNA interference techniques, gene editing techniques, inhibitors of introduced genes or proteins, blocking of antibodies or proteins or polypeptides or compounds, cell screening, or combinations thereof.

In another preferred embodiment, the inhibitor is selected from the group consisting of: an antisense nucleic acid, an antibody, a small molecule compound, a Crispr agent, a small molecule ligand, or a combination thereof.

In another preferred embodiment, the gene editing technique is selected from the group consisting of: CRISPR technology, TALEN technology, ZFN technology, or a combination thereof.

In another preferred embodiment, 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.

In another preferred embodiment, the engineered immune cell has the following characteristics:

(a) the expression of the PD-1H gene in the immune cell is silenced.

In another preferred embodiment, the engineered immune cell further has the following characteristics:

(b) the immune cell expresses a chimeric antigen receptor CAR that targets an antigenic molecule or a marker of a tumor cell or an exogenous TCR that targets an antigenic molecule or a marker of a tumor cell.

In another preferred embodiment, the engineered immune cell comprises:

(a) optionally, a chimeric antigen receptor CAR comprising: an antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds to an antigen molecule or a tumor cell surface antigen; and

(b) an inhibitory molecule that reduces or inhibits the activity of PD-1H protein expression.

In another preferred embodiment, the inhibitory molecule is selected from the group consisting of: inhibitory nucleic acids, small molecule compounds, antibodies (e.g., single domain antibodies), polypeptides, or combinations thereof.

In another preferred embodiment, the inhibitory nucleic acid comprises an RNA interfering agent, a criprpr agent.

In another preferred embodiment, the inhibitory nucleic acid is selected from the group consisting of: siRNA, miRNA, shRNA, hairpin siRNA, tandem expressed miRNA, microrna-adapted shRNA, precursor microrna, or a combination thereof.

In another preferred embodiment, the sequence of the inhibitory nucleic acid is as shown in any one or combination of SEQ ID No. 15-20.

In another preferred embodiment, the criprpr reagent comprises a gene editing protein.

In another preferred embodiment, the gene-editing protein is selected from the group consisting of: CRISPR, TALEN, ZFN, or a combination thereof.

In another preferred embodiment, the CRISPR protein is selected from the group consisting of: cas9, nCas9, Cas10, Cas9a, Cas12, Cas12a, Cas12b, Cas13, Cas14, or a combination thereof.

In another preferred embodiment, the criprpr reagent further comprises gRNA.

In another preferred embodiment, at least a portion of the sequence of the gRNA is capable of being complementary to a target DNA, and the gRNA is capable of forming a functional complex with a CRISPR protein.

In another preferred example, the gene-editing enzyme is derived from Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus thermophilus (Streptococcus thermophiles), Staphylococcus aureus (Staphylococcus aureus), Aminococcus sp, Mucor (Lachnospiricus sp), or a combination thereof.

In another preferred example, the gRNA includes a sgRNA.

In another preferred embodiment, the sequence of the gRNA is as shown in any one or combination of SEQ ID NO. 1-14, 22-23, 25-26.

In another preferred embodiment, the inhibitory nucleic acid molecule comprises a sequence complementary to PD-1H mRNA (information nucleic acid) or a nucleic acid encoding PD-1H.

In another preferred embodiment, the inhibitory nucleic acid molecule comprises an antisense oligonucleotide complementary to PD-1H mRNA (messenger nucleic acid) or a nucleic acid encoding PD-1H.

In another preferred embodiment, 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, biphenyl compounds, or combinations thereof.

In another preferred embodiment, the tumor cell surface antigens include cell surface antigens of various solid tumors, solid tumors and hematological tumors.

In another preferred embodiment, the tumor cell surface antigen is selected from the group consisting of: CD, c-Met, PSMA, MUC-1, MUC, CD123, CD138, CD, PD-L, CD276, B-H, mesothelin (mesothelin), EGFR, EGFRviii, GPC, BCMA, ErbB, NKG2 ligand, LMP, EpCAM, VEGFR-1, Lewis-, Claudin18.2, CD123, CD138, CD133, CD137, CD151, CD171, KIT (CD117), CD174, CD44V, CD179, B-H (CD276), B-H, HER, C-Met, PSMA, PSCA, PSMC, MUC, mesothelin (VEGFR), VEGFR, EGFR, CER, CEGE, CEGH-2, EGFR, CEGE, CEGR 2, CEGR, CEGE, CEGR 2, CEGE, VEGFR, CEGE, CER 2, CER, 2, CER, 2, CER, 2, CER, 2, and ERG, 2, ERG, 2, and ERG, 2, ERC, ERG, ERC, FAP, PSMA, CA125, EphA2, L1CAM, CS1, ROR1, EC, NY-ESO-1, GD2, EPG, DLL3, 5T4, IL-13Ra2 (or CD213A2), IL-11Ra, PRSS21, PDGFR-beta, SSEA-4, folate receptor alpha (FRa or FR 1); folate receptor beta (FRb), AFP/MHC complex, NCAM, ELF2M, FAP, IGF-I receptor, CAIX, sLe, ganglioside GM M (aNeu 5M (2-3) bDClalp (1-4) bDGlcp (1-1) Cer), TGS M, HMWMAA, OAcGD M, (TEM M/CD 248), TEM 7M, CLDN M, TSHR, GPRC 5M, ALK, HAZCR M, ADRB M, PANX M, GPR M, OR51E M, MARE-1 a, MAGE-A M, ETV M-AML, MADD-CT-1, MAD-CT-2, Fos-related antigen 1, p 4 mutant, MARA-1, MePCT A OR PD, hTERP, CTPI, CAIX-MHC M, ACAAC-M, ACAT M, FLT-M, FLT-D-M, FLT-D, FLT-M, FLT-D-M, FLT-D, FLS-M, FLT-M, FLG-M, FLT-D-M, FLT-D, FLT-D-M, FLT-D, FLT-M, FLT-M, FLT-D, FLT-M, FLG-D, FLG-M, FLT-M, FLG-M, FLT, FLG M, FLT-D-M, FLT-M, FLT-D, FLT-M, FLT-M, FLG-M, FLT-D, FLT-M, FLT-D, FLT-M, FLT-D, FLT-M, FLT-685, NY-ESO-1, NY-ESO-1/MHC I complex, PSMA, RANK, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, TCRa, TCRp, TLR7, TLR9, PTCH1, WT-1, WT1/MHC I complex, NA17, MYCN, RhoC, SART3, SSX2, RAGE-1, HPV E7, HPV E7/MHC I complex; AFP/MHC I complex, Ras/MHC I complex, Robol, Frizzled, OX40, CD79a, CD79b, CD72, Notch-1-4, CLL-1 (or CLECL1), TAG72, LILRA 2; CD300 LF; CLEC 12A; BST 2; EMR 2; LY75, FCRL 5; IGLL1, MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFR α 4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea; fucosyl GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD179b-IGLl1, TCR γ - δ, NKG2D, CD32(FCGR2A), Tn Ag, Tim1-/HVCR1, CSF2RA (GM-CSFR- α), TGF β R2, Lews Ag, TCR β 1 chain, TCR β 2 chain, TCR- γ chain, TCR- δ chain, FITC, LHR, FSHR, CGHR or GR, CCR4, SLAMF6, SLAMF 8, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3 65, HV K8.1, KSHV-gH, influenza A (HA), GAD, 65, guanidinium C (GCC) cyclase, anti-HLA core 65 (DsG) 3, HLA-DHDP 65, HLA-DHFR-DHHR-alpha, DHFR-DHHR-alpha, DHFR-alpha, DHFR-beta-alpha, DHF-beta-4, and their derivatives, IgE, CD99, RasG12V, tissue factor 1(TF1), AFP, GPRC5D, P-glycoprotein, STEAP1, Liv1, fibronectin-4, Cripto, gpA33, BST1/CD157, low conductance chloride channels, and antigens recognized by TNT antibodies, or combinations thereof.

In another preferred embodiment, the tumor cell surface antigen comprises CD 19.

In another preferred embodiment, the tumor cell surface antigen comprises B7-H3(CD 276).

In another preferred embodiment, the reduction or inhibition of the expression activity of the PD-1H protein means that the expression activity of the PD-1H protein is reduced by 20% or more, preferably 40% or more, more preferably 60% or more, still more preferably 80% or more, still more preferably 90% or 100% or more.

In another preferred embodiment, the antigen binding domain is an antibody or antigen binding fragment.

In another preferred embodiment, the antigen binding fragment is a Fab or scFv or a single domain antibody sdFv.

In another preferred embodiment, the engineered immune cell is selected from the group consisting of:

(i) a chimeric antigen receptor T cell (CAR-T cell);

(ii) chimeric antigen receptor NK cells (CAR-NK cells);

(iii) chimeric antigen receptor phagocytes or monocytes (CAR-macrophage cells);

(iv) exogenous T Cell Receptor (TCR) T cells (TCR-T cells).

In another preferred embodiment, the immune cells are autologous.

In another preferred embodiment, the immune cells are allogeneic.

In another preferred embodiment, the immune cell is iPS-derived.

In another preferred embodiment, the cell is a mammalian cell, preferably a human cell.

In another preferred embodiment, the immune cell further expresses an inhibitory molecule that reduces or inhibits the expression activity of the PD-1H protein.

In another preferred embodiment, the inhibitory molecule is independently expressed and/or co-expressed with a chimeric antigen receptor CAR that targets a tumor cell surface antigen.

In another preferred embodiment, said co-expression with a chimeric antigen receptor CAR targeting a tumor cell surface antigen comprises tandem expression of an inhibitory molecule and a chimeric antigen receptor CAR targeting a tumor cell surface antigen.

In another preferred embodiment, the engineered immune cells comprise T cells, NK cells or macrophages.

In another preferred embodiment, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of: CD28, CD3 epsilon, 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.

In another preferred embodiment, 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.

In another preferred embodiment, the intracellular domain comprises a costimulatory signaling molecule and a cytoplasmic signaling sequence derived from CD3 ζ.

In another preferred embodiment, the costimulatory signal molecule is a costimulatory signal molecule for 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.

In a second aspect, the present invention provides a method for preparing an engineered immune cell according to the first aspect of the invention, comprising the steps of:

(A) providing an immune cell that has been screened or is to be engineered; and

(B) engineering the immune cell such that expression of the PD-1H gene in the immune cell is silenced, thereby obtaining the immune cell of claim 1.

In another preferred embodiment, step (B) comprises introducing into said immune cell a second expression cassette expressing a gene for silencing PD-1H.

In another preferred example, in the step (B), the method further comprises the steps of: (B1) introducing a first expression cassette expressing a CAR into the immune cell; and (B2) introducing into the immune cell a second expression cassette expressing a gene for silencing PD-1H,

wherein the order of the steps (B1 and (B2) is not limited at all.

In another preferred embodiment, when the immune cell to be engineered in step (a) already expresses a certain CAR, then in step (B), it comprises (B2) introducing into said immune cell a second expression cassette expressing for silencing PD-1H.

In another preferred embodiment, the word "without any restriction on the order" means that any two steps can be performed sequentially, simultaneously, or in reverse order.

In another preferred embodiment, the step (B1) can be performed before, after, simultaneously with or alternatively to the step (B2).

In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same or different vectors.

In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same vector.

In another preferred embodiment, the vector is a viral vector.

In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, other gene transfer systems, or combinations thereof.

In another preferred example, the second expression cassette comprises CRISPR/Cas9(gRNA and Cas9), an antisense RNA, or a combination thereof.

In another preferred example, the gRNA includes a sgRNA.

In another preferred embodiment, the gRNA targets PD-1H, and the sequence of the gRNA is as shown in any one or combination of SEQ ID NO. 1-14, 22, 23, 25-26.

In another preferred embodiment, the antisense RNA comprises miRNA, siRNA, shRNA, inhibitory mRNA, or dsRNA.

In another preferred embodiment, the sequence of the antisense RNA is shown in any one or combination of SEQ ID NO. 15-20.

In a third aspect, the invention provides a formulation comprising an engineered immune cell according to the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the formulation is a liquid formulation.

In another preferred embodiment, the formulation is an injection.

In another preferred embodiment, the concentration of said immune cells in said formulation is 1X 10³-1×10¹⁰Individual cells/ml, preferably 1X 10⁴-1×10⁸Individual cells/ml.

In a fourth aspect, the invention provides a use of the engineered immune cell of the first aspect of the invention for preparing a medicament or a preparation for preventing and/or treating cancer or tumor.

In another preferred embodiment, the tumor is selected from the group consisting of: a hematologic tumor, a solid tumor, or a combination thereof.

In another preferred embodiment, the tumor comprises a tumor that is CD19 positive.

In another preferred embodiment, the tumor comprises a B7-H3(CD276) positive tumor.

In another preferred embodiment, the hematological tumor is selected from the group consisting of: acute Myeloid Leukemia (AML), myelodysplastic syndrome (MDS), myelodysplastic/myeloproliferative disorders (MDS/MPD), chronic myeloproliferative 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 cell leukemia, plasma cell myeloma/plasmacytoma, MALT type marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, Burkitt's lymphoma/leukemia, T/NK cell tumors, pre-lymphoblastic lymphoma/leukemia, T lymphocytic leukemia, T granular lymphocytic leukemia, aggressive NK cell leukemia, mature T cell lymphoma/leukemia, extranodal rhino type NK/T cell lymphoma, enteropathy type T cell lymphoma, mycosis fungoides/sezary syndrome (MF/SS), Peripheral T cell lymphoma, anaplastic large cell lymphoma, Multiple Myeloma (MM), Acute Lymphoblastic Leukemia (ALL); pre-T cell lymphoma/leukemia; hodgkin lymphoma; mastocytosis, Mast Cell Leukemia (MCL), Mast Cell Sarcoma (MCS); macrophage/histiocytic tumor, dendritic cell tumor, Langerhans Cell Histioproliferation (LCH), Langerhans Cell Sarcoma (LCS), follicular dendritic cell sarcoma/tumor, dendritic cell sarcoma, or a combination thereof.

In another preferred embodiment, the solid tumor is selected from the group consisting of: head and neck tumors, laryngeal carcinoma, lung cancer, non-small cell lung cancer, bronchial cancer, stomach cancer, gastric cancer peritoneal metastasis, esophageal cancer, bile duct cancer, pancreatic cancer, colorectal cancer peritoneal metastasis, small bowel cancer, kidney cancer, bladder tumor, transitional epithelial malignancy, endocrine tumor, thyroid cancer, adrenal tumor, breast cancer, cervical cancer, ovarian cancer peritoneal metastasis, endometrial cancer, choriocarcinoma, prostate cancer, testicular tumor, germ cell tumor, seminal cell carcinoma, embryonal tumor, nervous system tumor, brain glioma, neuroblastoma, skin tumor, malignant melanoma, lymphatic cancer, thymic tumor, nasopharyngeal carcinoma, bone cancer, sarcoma, rhabdomyosarcoma, liposarcoma, angiosarcoma, leiomyosarcoma, fibrosarcoma, osteosarcoma, ewing's sarcoma, neuroblastoma, skin tumor, malignant melanoma, lymph cancer, thymic tumor, thyroid tumor, nasopharyngeal carcinoma, bone cancer, sarcoma, rhabdomyosarcoma, liposarcoma, angiosarcoma, leiomyosarcoma, fibrosarcoma, osteosarcoma, ewing's sarcoma, and the like tumor of the head and neck, Solid tumor metastatic tumors such as metastases of abdominal cavity, thoracic cavity, pelvic cavity, solid organs, or combinations thereof.

In a fifth aspect, the present invention provides a kit for preparing the engineered immune cell of the first aspect of the invention, the kit comprising a container, and in the container:

(1) optionally a first nucleic acid sequence comprising a first expression cassette for expressing a CAR or exogenous TCR;

(2) a second nucleic acid sequence containing a second expression cassette for silencing PD-1H or a gRNA targeting PD-1H.

In another preferred embodiment, the first and second nucleic acid sequences are independent or linked.

In another preferred embodiment, the first and second nucleic acid sequences are located in the same or different containers.

In another preferred embodiment, the first and second nucleic acid sequences are located in the same or different expression vectors.

In another preferred embodiment, the kit further comprises: (4) a third nucleic acid sequence comprising an expression cassette for expression of a gene-editing protein; or a gene editing protein.

In a sixth aspect, the present invention provides a method for modulating immune cell activity, comprising:

the activity of the immune cells is regulated by regulating the expression level of PD-1H in the immune cells.

In another preferred embodiment, the activity of the immune cell is enhanced by reducing or inhibiting the expression level of PD-1H in the immune cell.

In another preferred embodiment, the activity of the immune cell is reduced by enhancing the expression level of PD-1H in the immune cell.

In another preferred example, the "decrease or inhibition of the expression level of PD-1H in an immune cell" refers to the ratio of the expression amount G1 of PD-1H gene of the immune cell to the expression amount G0 of PD-1H gene of a normal immune cell, i.e., G1/G0 ≦ 0.8, preferably G1/G0 ≦ 0.5, more preferably ≦ 0.2, more preferably ≦ 0.1, most preferably 0.

In another preferred embodiment, the expression level of PD-1H in the immune cells is enhanced by the ratio of the expression amount G1 of PD-1H gene of the immune cells to the expression amount G0 of PD-1H gene of normal immune cells, namely G1/G0 is more than or equal to 2, preferably G1/G0 is more than or equal to 3, and more preferably G1/G0 is more than or equal to 4.

In a seventh aspect, the present invention provides a method for determining or assessing the activity of an immune cell, comprising:

the activity of the immune cells is judged or evaluated by detecting the expression level of PD-1H in the immune cells.

In another preferred embodiment, the activity of the immune cell is decreased when the expression level of PD-1H in the immune cell is increased.

In another preferred embodiment, the activity of the immune cell is enhanced when the expression level of PD-1H in the immune cell is decreased.

In another preferred embodiment, the expression level of PD-1H in the immune cells is increased, which means the ratio of the expression amount G1 of PD-1H gene of the immune cells to the expression amount G0 of PD-1H gene of normal immune cells, namely G1/G0 is more than or equal to 2, preferably G1/G0 is more than or equal to 3, and more preferably G1/G0 is more than or equal to 4.

In another preferred example, the "expression level of PD-1H in the immune cell is decreased" means a ratio of the expression amount G1 of the PD-1H gene of the immune cell to the expression amount G0 of the PD-1H gene of the normal immune cell, that is, G1/G0. ltoreq.0.8, preferably G1/G0. ltoreq.0.5, more preferably. ltoreq.0.2, still more preferably. ltoreq.0.1, most preferably 0.

In an eighth aspect, the invention provides a nucleic acid molecule comprising a first nucleic acid and optionally a second nucleic acid, wherein the first nucleic acid comprises a first expression cassette encoding an inhibitory molecule that reduces or inhibits the expression activity of a PD-1H protein, and the second nucleic acid comprises a second expression cassette encoding a chimeric antigen receptor CAR comprising: an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds to a tumor cell surface antigen.

In another preferred embodiment, the first expression cassette and/or the second expression cassette further comprises a constitutive promoter or an inducible promoter.

In another preferred embodiment, the constitutive promoter is selected from the group consisting of: CMV, EF1a, U6, SV40, PGK1, Ubc, CAG, H1, or a combination thereof.

In another preferred embodiment, 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.

According to a ninth aspect of the invention there is provided a vector comprising a nucleic acid molecule according to the eighth aspect of the invention.

In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, or combinations thereof.

In another preferred embodiment, the vector is a lentiviral vector.

According to a tenth aspect of the present invention there is provided a host cell comprising a vector or chromosome according to the ninth aspect of the present invention integrated with an exogenous nucleic acid molecule according to the eighth aspect of the present invention.

In another preferred embodiment, the cell is an isolated cell, and/or the cell is a genetically engineered cell.

In another preferred embodiment, the host cell comprises an engineered immune cell.

(i) a chimeric antigen receptor T cell (CAR-T cell);

(ii) chimeric antigen receptor NK cells (CAR-NK cells); or

(iii) Exogenous T Cell Receptor (TCR) T cells (TCR-T cells).

In another preferred embodiment, the immune cells are autologous.

In another preferred embodiment, the immune cells are allogeneic.

In another preferred embodiment, the engineered immune cells include T cells, NK cells, or macrophages.

In another preferred embodiment, the cell is a T cell.

In an eleventh aspect, the present invention provides a pharmaceutical composition comprising:

(a) a host cell according to the tenth aspect of the present invention; and

(b) a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

In another preferred embodiment, the engineered immune cell is (i) a chimeric antigen receptor T cell (CAR-T cell); or (ii) a chimeric antigen receptor NK cell (CAR-NK cell).

In another preferred embodiment, the concentration of the cells in the pharmaceutical composition is 1 × 10³-1×10¹⁰Individual cells/ml, preferably 1X 10⁴-1×10⁸Individual cells/ml.

In another preferred embodiment, the pharmaceutical composition further comprises other agents that selectively kill tumor cells (e.g., antibody agents, chemotherapeutic agents, or other CAR-T agents).

In a twelfth aspect, the invention provides a method of preparing an engineered immune cell, comprising:

transducing the nucleic acid molecule of the eighth aspect of the invention or the vector of the ninth aspect of the invention into an immune cell, thereby obtaining the engineered immune cell.

In another preferred embodiment, the introducing includes introducing simultaneously, sequentially or sequentially.

In another preferred embodiment, the immune cell is a T cell or NK cell.

In another preferred embodiment, the method further comprises the step of performing functional and effective detection on the obtained engineered immune cells.

A thirteenth aspect of the invention provides a reagent combination comprising:

(i) optionally a first agent which is an immune cell; and

(ii) a second agent which is an inhibitory molecule that reduces or inhibits the activity of PD-1H protein expression.

In another preferred embodiment, the immune cell comprises an engineered immune cell.

In another preferred embodiment, the engineered immune cell contains a chimeric antigen receptor CAR comprising: an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds to a tumor cell surface antigen.

In another preferred embodiment, the engineered immune cell further expresses an inhibitory molecule that reduces or inhibits the expression level or activity of the PD-1H protein.

In another preferred embodiment, the engineered immune cell further expresses an inhibitory nucleic acid for reducing or inhibiting expression of the PD-1H protein.

In a fourteenth aspect, the invention provides a method of enhancing tumor killing efficiency of engineered immune cells, comprising:

contacting the engineered immune cell with a tumor cell in the presence of an inhibitory molecule that reduces or inhibits the activity of PD-1H protein expression, thereby enhancing the tumor killing efficiency of the engineered immune cell.

In another preferred embodiment, the method is in vitro.

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

In another preferred embodiment, the inhibitory molecule that reduces or inhibits the expression activity of a PD-1H protein is endogenously produced or exogenously added.

In another preferred embodiment, said endogenous production refers to production by said engineered immune cells.

In another preferred embodiment, the exogenous addition refers to the exogenous addition of an inhibitory molecule.

In another preferred example, 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.

In a fifteenth aspect, the present invention provides a use of the host cell of the tenth aspect, or the pharmaceutical composition of the eleventh aspect, for preparing a drug or a preparation for selectively killing tumor cells.

In another preferred embodiment, the tumor cell is derived from a tumor selected from the group consisting of: acute Myeloid Leukemia (AML), myelodysplastic syndrome (MDS), myelodysplastic/myeloproliferative disorders (MDS/MPD), chronic myeloproliferative 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 cell leukemia, plasma cell myeloma/plasmacytoma, MALT type marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, Burkitt's lymphoma/leukemia, T/NK cell tumors, pre-lymphoblastic lymphoma/leukemia, T lymphocytic leukemia, T granular lymphocytic leukemia, aggressive NK cell leukemia, mature T cell lymphoma/leukemia, extranodal rhino type NK/T cell lymphoma, enteropathy type T cell lymphoma, mycosis fungoides/sezary syndrome (MF/SS), Peripheral T cell lymphoma, anaplastic large cell lymphoma, Multiple Myeloma (MM), Acute Lymphoblastic Leukemia (ALL); pre-T cell lymphoma/leukemia; hodgkin lymphoma; mastocytosis, Mast Cell Leukemia (MCL), Mast Cell Sarcoma (MCS); macrophage/histiocytic tumor, dendritic cell tumor, Langerhans cell histoproliferative disorder (LCH), Langerhans Cell Sarcoma (LCS), follicular dendritic cell sarcoma/tumor, dendritic cell sarcoma, head and neck tumor, laryngeal cancer, lung cancer, non-small cell lung cancer, bronchial cancer, gastric cancer peritoneal metastatic tumor, esophageal cancer, bile duct cancer, pancreatic cancer, colorectal cancer peritoneal metastatic tumor, small intestine cancer, kidney tumor, kidney cancer, bladder tumor, transitional epithelial malignancy, endocrine tumor, thyroid cancer, adrenal tumor, breast cancer, cervical cancer, ovarian cancer peritoneal metastatic tumor, endometrial cancer, choriocarcinoma, prostate cancer, testicular tumor, germ cell tumor, seminal cell cancer, embryonal tumor, nervous system tumor, brain glioma, Neuroblastoma, skin tumor, malignant melanoma, lymphoma, thymic tumor, nasopharyngeal carcinoma, bone cancer, sarcoma, rhabdomyosarcoma, liposarcoma, angiosarcoma, leiomyosarcoma, fibrosarcoma, osteosarcoma, ewing's sarcoma, solid tumor metastatic tumor such as metastasis at abdominal cavity, thoracic cavity, pelvic cavity, parenchymal organ, etc., or a combination thereof.

In a sixteenth aspect, the present invention provides a kit for selectively killing tumor cells, the kit comprising a container, and a host cell according to the tenth aspect of the present invention in the container.

In another preferred embodiment, the kit further comprises a label or instructions for use.

In a seventeenth aspect, the present invention provides a method for selectively killing tumor cells, comprising:

administering to a subject in need of treatment a safe and effective amount of an engineered immune cell according to the first aspect of the invention, a host cell according to the tenth aspect of the invention, or a pharmaceutical composition according to the eleventh aspect of the invention.

In another preferred embodiment, the subject comprises a human or non-human mammal.

In another preferred embodiment, the non-human mammal includes a rodent (e.g., mouse, rat, rabbit), primate (e.g., monkey).

In an eighteenth aspect, the present invention provides a method for treating cancer or tumor, comprising:

administering to a subject in need thereof a safe and effective amount of an engineered immune cell according to the first aspect of the invention, a host cell according to the tenth aspect of the invention, or a pharmaceutical composition according to the eleventh aspect of the invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. For reasons of space, they will not be described in detail.

Drawings

FIG. 1 shows that PD-1H expression is elevated on tumor infiltrating CD8+ T cells. Expression of PD-1H was higher on tumor-infiltrating CD8+ T cells than on CD4+ T cells in both the mouse melanoma (B16-OVA) model and the lymphoma (EG7) model.

FIG. 2 shows that the addition of anti-CD3 and anti-CD28 to CD8+ T cells cultured in vitro results in up-regulation of PD-1H expression on CD8+ T cells.

FIG. 3 shows enhanced anti-tumor effect and increased tumor infiltration of PD-1H-depleted CD8+ T cells in a mouse melanoma (B16-OVA) model.

FIG. 4 shows enhanced anti-tumor effect and increased tumor infiltration of PD-1H-depleted CD8+ T cells in a mouse lymphoma (EG7) model.

Fig. 5 shows that the expression of PD-1H on CD8+ T cells can be effectively knocked out using the CRISPR Cas9 system.

Fig. 6 shows enhanced antitumor effect of PD-1H knockout CD8+ T cells using CRISPR Cas9 system in mouse lymphoma (EG7) model.

FIG. 7 shows that no PD-1H expression was observed on the surface of T cells and CAR-T cells cultured in vitro, and that PD-1H expression was elevated on the surface of T cells in spleen and tumor tissues.

FIG. 8 shows immunofluorescence co-localization showing PD-1H expression on tumor infiltrating CD8+ T cells.

FIG. 9 shows a schematic vector construct for a chimeric antigen receptor co-expressing PD-1H shRNA.

FIG. 10 shows expression of different chimeric antigen receptors co-expressing PD-1H shRNA on T cells.

FIG. 11 shows candidate PD-1H shRNA silences PD-1H expression on Jurkat-PD-1H cells.

FIG. 12 shows that CD19 CAR-T cell therapy co-expressing PD-1H shRNA is effective in inhibiting tumor growth in the NCG mouse lymphoma (CA-46) model.

FIG. 13 shows that CD19 CAR-T cell therapy co-expressing PD-1H shRNA is effective in extending survival in mice.

FIG. 14 shows that sgRNA3 and sgRNA4 were effective in knocking down the expression of PD-1H on Jurkat-PD-1H cells.

Detailed Description

The inventor unexpectedly discovers that the tumor killing effect of immune cells can be obviously improved by knocking down the expression of PD-1H gene in engineered immune cells (such as T cells and NK cells) through extensive and intensive research and a large amount of screening. On this basis, the present inventors have completed the present invention.

The present invention is representatively illustrated in detail for the engineered immune cells of the present invention, taking CAR-T cells as an example. The engineered immune cells of the invention are not limited to the CAR-T cells described above and below, and the engineered immune cells of the invention have the same or similar technical features and benefits as the CAR-T cells described above and below. Specifically, when the immune cell expresses the chimeric antigen receptor CAR, the NK cell is identical to a T cell (or a T cell can replace an NK cell); when the immune cell is a T cell, the TCR is identical to the CAR (or the CAR can be replaced with a TCR).

Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

The term "antibody" (Ab) 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, or antigen-binding portions thereof, interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a constant domain CL. The VH and VL regions may be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

As used herein, 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 than the extracellular domain, and at least one intracellular domain. "Chimeric Antigen Receptors (CARs)" are also referred to as "chimeric receptors", "T-bodies" or "Chimeric Immunoreceptors (CIRs)". The term "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding an antigen. "intracellular domain" refers to any oligopeptide or polypeptide known to be a domain that transmits signals to activate or inhibit biological processes in a cell.

As used herein, "domain" refers to a region of a polypeptide that is independent of other regions and folds into a specific structure.

As used herein, "tumor antigen" refers to an antigenic biomolecule, the expression of which results in cancer.

As used herein, the terms "administration" and "treatment" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells, and contacting the reagent with a fluid, and contacting the fluid with the cells. "administering" and "treating" also mean treating in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, animal or study subject refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnosis.

As used herein, the term "treatment" refers to the administration of a therapeutic agent, either internally or externally, comprising any of the engineered immune cells of the invention and compositions thereof to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount).

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but need not, be 1, 2 or 3.

"sequence identity" as referred to herein means 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. The sequence identity between a sequence described in the present invention and a sequence with which it is 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

PD-1H, also known as VISTA, c10orf54, VSIR, SISP1, B7-H5, DD1 α, Gi24, and Dies1, is a member of the B7 family, a type 1 transmembrane protein containing 311 amino acids. PD-1H contains an Ig-like V-type domain, a transmembrane domain and an intracellular domain, and through phylogenetic and proteomic analysis, the IgV region of PD-1H is highly homologous with PD-1 and PD-L1, so that the domain is called PD-1Homolog, called PD-1H for short. Unlike other members of the family, the PD-1H intracellular segment does not contain the classical ITIM/ITAM motif, but has a potential protein kinase C binding site and a proline-rich motif, which transduces downstream signals into the cell.

PD-1H is expressed predominantly on cells of the hematopoietic lineage, both myeloid (including macrophages, dendritic cells, monocytes and neutrophils) and CD4+ T cells.

The research of the invention shows that PD-1H can be used as a ligand and a receptor to generate inhibitory action. PD-1H on antigen presenting cells and regulatory T cells can inhibit T cell proliferation and cytokine release as ligand, and has important regulation and control effect on autoimmune diseases and tumor progression. PD-1H can be used as an inhibitory receptor on CD4+ T cells, and regulates the immune tolerance and tumor immunity involved by the T cells. The PD-1H specific blocking antibody can effectively inhibit tumor growth in a plurality of mouse tumor (melanoma, bladder cancer and the like) models. The specific PD-1H agonism antibody can effectively relieve the symptoms of autoimmune diseases (systemic lupus erythematosus, asthma, arthritis and the like).

Tumor antigens

Tumor antigens of the 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 (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, PSMA, CA125, EphA2, L1CAM, CS1, ROR1, EC, NY-ESO-1, GD2, EPG, DLL3, 5T4, IL-13Ra2 (or CD213A2), IL-11Ra, PRSS21, PDGFR-beta, SSEA-4, folate receptor alpha (FRa or FR 1); folate receptor beta (FRb), AFP/MHC complex, NCAM, ELF2M, FAP, IGF-I receptor, CAIX, sLe, ganglioside GM M (aNeu 5M (2-3) bDClalp (1-4) bDGlcp (1-1) Cer), TGS M, HMWMAA, OAcGD M, (TEM M/CD 248), TEM 7M, CLDN M, TSHR, GPRC 5M, ALK, HAZCR M, ADRB M, PANX M, GPR M, OR51E M, MARE-1 a, MAGE-A M, ETV M-AML, MADD-CT-1, MAD-CT-2, Fos-related antigen 1, p 4 mutant, MARA-1, MePCT A OR PD, hTERP, CTPI, CAIX-MHC M, ACAAC-M, ACAT M, FLT-M, FLT-D-M, FLT-D, FLT-M, FLT-D-M, FLT-D, FLS-M, FLT-M, FLG-M, FLT-D-M, FLT-D, FLT-D-M, FLT-D, FLT-M, FLT-M, FLT-D, FLT-M, FLG-D, FLG-M, FLT-M, FLG-M, FLT, FLG M, FLT-D-M, FLT-M, FLT-D, FLT-M, FLT-M, FLG-M, FLT-D, FLT-M, FLT-D, FLT-M, FLT-D, FLT-M, FLT-685, NY-ESO-1, NY-ESO-1/MHC I complex, PSMA, RANK, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, TCRa, TCRp, TLR7, TLR9, PTCH1, WT-1, WT1/MHC I complex, NA17, MYCN, RhoC, SART3, SSX2, RAGE-1, HPV E7, HPV E7/MHC I complex; AFP/MHC I complex, Ras/MHC I complex, Robol, Frizzled, OX40, CD79a, CD79b, CD72, Notch-1-4, CLL-1 (or CLECL1), TAG72, LILRA 2; CD300 LF; CLEC 12A; BST 2; EMR 2; LY75, FCRL 5; IGLL1, MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFR α 4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea; fucosyl GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD179b-IGLl1, TCR γ - δ, NKG2D, CD32(FCGR2A), Tn Ag, Tim1-/HVCR1, CSF2RA (GM-CSFR- α), TGF β R2, Lews Ag, TCR β 1 chain, TCR β 2 chain, TCR- γ chain, TCR- δ chain, FITC, LHR, FSHR, CGHR or GR, CCR4, SLAMF6, SLAMF 8, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3 65, HV K8.1, KSHV-gH, influenza A (HA), GAD, 65, guanidinium C (GCC) cyclase, anti-HLA core 65 (DsG) 3, HLA-DHDP 65, HLA-DHFR-DHHR-alpha, DHFR-DHHR-alpha, DHFR-alpha, DHFR-beta-alpha, DHF-beta-4, and their derivatives, IgE, CD99, RasG12V, tissue factor 1(TF1), AFP, GPRC5D, P-glycoprotein, STEAP1, Liv1, fibronectin-4, Cripto, gpA33, BST1/CD157, low-conductance chloride channels, and antigens recognized by TNT antibodies.

Take CD19 as an example.

CD19 refers to the differentiation epitope 19 protein, which is a detectable epitope on leukemia precursor cells. CD19 described herein comprises mutated (e.g., point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type CD19) proteins. 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.

Antigen binding domains

In the present invention, the antigen binding domain of the chimeric antigen receptor CAR specifically binds to a tumor cell surface antigen.

In a preferred embodiment, the antigen binding domain of the chimeric antigen receptor CAR of the invention targets CD19, B7-H3(CD 276).

Hinge region and transmembrane region

For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.

The transmembrane domain may be derived from natural sources or synthetic sources. In natural sources, the domain may be derived from any membrane bound or transmembrane protein. Preferably, the hinge and transmembrane regions of the CARs of the invention are those of CD 8.

Intracellular domains

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. The term "effector function" refers to a cell's exclusive function. For example, the effector function of a T cell may be cytolytic activity or helper activity involving secretion of cytokines. The term "intracellular signaling domain" thus refers to a portion of a protein that transduces effector function signals and directs a cell to perform a proprietary function. Although the entire intracellular signaling domain may generally be used, in many instances, the entire strand need not be used. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the entire chain, so long as it transduces effector function signals. The term intracellular signaling domain thus refers to any truncated portion of an intracellular signaling domain that includes sufficient signal transduction of effector function.

Preferred examples of intracellular signaling domains for the CARs of the invention include cytoplasmic sequences of the T Cell Receptor (TCR) and co-receptors that act synergistically to initiate signal transduction upon antigen receptor binding, as well as any derivative or variant of these sequences and any synthetic sequence with the same functional capacity.

In preferred embodiments, the cytoplasmic domain of the CAR can be designed to itself include the CD 3-zeta signaling domain, or can be associated with any other desired cytoplasmic domain(s) useful in the context of the CARs of the invention. For example, the cytoplasmic domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. A 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 required for effective response of lymphocytes to antigens, rather than antigen receptors or their ligands. Preferably, 4-1BB (CD137) and the like are included.

The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention can be linked to each other randomly or in a defined order. Optionally, short oligopeptide or polypeptide linkers, preferably between 2 and 10 amino acids in length, can form the linkage. Glycine-serine doublets provide particularly suitable linkers.

In one embodiment, the cytoplasmic domain in the CAR of the invention is designed to include the signaling domain of 4-1BB (co-stimulatory molecule) and the signaling domain of CD3 ζ.

Chimeric Antigen Receptor (CAR)

Chimeric immune antigen receptors (CARs) consist 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 goes through the following process: the first generation CARs had only one intracellular signaling component, CD3 ζ or Fc γ RI molecule, and due to the single intracellular activation domain, it caused only transient T cell proliferation and less cytokine secretion, and did not provide long-term T cell proliferation signaling and sustained in vivo anti-tumor effects, and thus did not achieve good clinical efficacy. The second generation CARs introduce a costimulatory molecule such as CD28, 4-1BB, OX40 and ICOS on the basis of the original structure, and compared with the first generation CARs, the function of the second generation CARs is greatly improved, and the persistence of CAR-T cells and the killing capability of the CAR-T cells on tumor cells are further enhanced. On the basis of the second generation CARs, a plurality of novel immune co-stimulatory molecules such as CD27 and CD134 are connected in series, and the third generation CARs and the fourth generation CARs are developed.

The extracellular domain of CARs recognizes a specific antigen and subsequently transduces this signal through the intracellular domain, causing activated proliferation, cytolytic toxicity and secretion of cytokines, which in turn clear the target cell. Autologous cells from the patient (or a heterologous donor) are first isolated, activated and genetically engineered to produce immune cells for CAR production, and then injected into the same patient. In this way, the probability of graft versus host disease is very low and antigens are recognized by immune cells in a non-MHC restricted manner.

CAR-immune cell therapy has achieved very high clinical response rates in the treatment of hematological malignancies, which rates were previously unattainable by any therapeutic approach, and have triggered a hot surge of clinical research in the world.

Specifically, the Chimeric Antigen Receptors (CARs) of the invention include an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding member (also referred to as an antigen-binding domain). The intracellular domain includes a costimulatory signaling region and/or a zeta chain moiety. The costimulatory signaling region refers to a portion of the intracellular domain that includes the costimulatory molecule. Costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.

A linker may 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. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to an ectodomain or a 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 CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds its associated antigen, it affects the tumor cells, causing the tumor cells to not grow, to be driven to death, or to otherwise be affected, and causing the patient's tumor burden to shrink or be eliminated. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecules and/or the zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the 4-1BB signaling domain and/or the CD3 zeta signaling domain combination.

As used herein, "antigen binding domain" and "single chain antibody fragment" each refer to an Fab fragment, Fab 'fragment, F (ab')2 fragment, or single Fv fragment having antigen binding activity. Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest antibody fragment of the entire antigen binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. The antigen binding domain is typically a scFv (single-chain variable fragment). The size of the scFv is typically 1/6 for a whole antibody. Single chain antibodies are preferably a sequence of amino acids encoded by a single nucleotide chain. In a preferred embodiment of the invention, the scFv comprises an antibody, preferably a single chain antibody, that specifically recognizes the tumor highly expressed antigens CD47 and MSLN.

In the present invention, the scFv of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced with amino acids having similar or similar properties as compared with the amino acid sequence of the scFv of the present invention to form a polypeptide.

In the present invention, the number of amino acids to be added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total number of amino acids in the original amino acid sequence.

In the present invention, the number of the amino acids to be added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or5, preferably 1 to 3, more preferably 1 to 2, and most preferably 1.

The extracellular domain of the CAR of the invention comprises an antigen binding domain that specifically binds to a tumor cell surface antigen, preferably an antigen binding domain that specifically binds to CD 19.

In the present invention, the intracellular domain in the CAR of the invention includes the extracellular domain comprising the target-specific binding element (also referred to as the antigen binding domain), the transmembrane region of CD8, the costimulatory factor for 4-1BB, the signaling domain of CD3 zeta

Chimeric antigen receptor T cells (CAR-T cells)

As used herein, the terms "CAR-T cell", "CAR-T cells of the invention" all refer to CAR-T cells of the invention, which can be targeted to a tumor cell surface antigen (preferably CD19) for the treatment of tumors that are highly expressed or positive for a tumor cell surface antigen (e.g., CD 19).

CAR-T cells have the following advantages over other T cell-based therapies: (1) the action process of the CAR-T cell is not limited by MHC; (2) given that many tumor cells express the same tumor antigen, CAR gene construction for a certain tumor antigen can be widely utilized once it is completed; (3) the CAR can utilize tumor protein antigens and glycolipid non-protein antigens, so that the target range of the tumor antigens is expanded; (4) the use of patient autologous cells reduces the risk of rejection; (5) the CAR-T cell has an immunological memory function and can survive in vivo for a long time.

In the present invention, the CAR of the 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 co-stimulatory factor; and (iv) the signaling domain of CD3 ζ.

Chimeric antigen receptor NK cells (CAR-NK cells)

As used herein, the terms "CAR-NK cell", "CAR-NK cell of the invention" all refer to a CAR-NK cell of the invention. The CAR-NK cells of the invention can target a tumor cell surface antigen (preferably CD19) for the treatment of tumors that are highly expressed or positive for a tumor cell surface antigen (e.g., CD 19).

Natural Killer (NK) cells are a major class of immune effector cells that protect the body from viral infection and tumor cell invasion through non-antigen specific pathways. By engineering (genetically modifying) NK cells it is possible to obtain new functions, including the ability to specifically recognize tumor antigens and having an enhanced anti-tumor cytotoxic effect.

CAR-NK cells also have the following advantages compared to autologous CAR-T cells, for example: (1) directly kills tumor cells by releasing perforin and granzyme, but has no killing effect on normal cells of an organism; (2) they release very small amounts of cytokines and thus reduce the risk of cytokine storm; (3) is easy to be amplified in vitro and can be developed into ready-made products. Otherwise, similar to CAR-T cell therapy.

Exogenous T cell antigen receptor

As used herein, a foreign T cell antigen receptor (TCR) is a TCR that is exogenously transferred into a T cell by means of genetic engineering, using lentivirus or retrovirus as a vector, by cloning the α chain and β chain of the TCR from a tumor-reactive T cell by gene transfer technique.

The exogenous TCR modified T cell can specifically recognize and kill tumor cells, and affinity of the T cell and tumor can be improved and anti-tumor effect can be improved by optimizing affinity of TCR and tumor specific antigen.

Down-regulation or silencing of PD-1H gene expression

As used herein, "PD-1H gene expression is silenced" means that the PD-1H gene is not expressed or is under-expressed. "Low expression" means the ratio of the expression level G1 of the PD-1H gene of the CAR-T cell to the expression level G0 of the PD-1H gene of a normal immune cell, i.e., G1/G0. ltoreq.0.8, preferably G1/G0. ltoreq.0.5, more preferably. ltoreq.0.2, still more preferably. ltoreq.0.1, most preferably 0.

The PD-1 gene expression down-regulation or silencing method of the invention comprises CRISPR/Cas9, RNA interference technology, transcription activator-like effector nucleases TALENs (transcription activator-like) (TAL) effectors) and Zinc finger nucleases Zinc Finger Nuclei (ZFNs). Preferably, the PD-1H gene is down-regulated or silenced by CRISPR/Cas9, RNA interference technology. In one embodiment of the invention, the PD-1H gene is down-regulated or silenced using CRISPR/Cas9 or shRNA.

CRISPR/Cas system

The CRISPR (clustered regularly interspersed short palindromic repeats)/Cas (CRISPR-associated) system is a natural immune system specific to prokaryotes and is used to protect against viruses or exogenous plasmids. The type ii CRISPR/Cas system has been successfully applied in many eukaryotes and prokaryotes as a tool for RNA-directly mediated genome editing. The development of the CRISPR/Cas9 system has drastically changed the ability of people to edit DNA sequences and regulate the expression level 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 action principle is that the sgRNA forms a Cas9-sgRNA complex with Cas9 protein through a Cas9 handle of the sgRNA, a base complementary pairing region sequence of the sgRNA in the Cas9-sgRNA complex is paired and combined with a target sequence of a target gene through a base complementary pairing principle, and the Cas9 cuts the target DNA sequence by utilizing the endonuclease activity of the self. Compared with traditional genome editing technology, the CRISPR/Cas9 system has several significant advantages: ease of use, simplicity, low cost, programmability, and the ability to edit multiple genes simultaneously.

Expression cassette

As used herein, "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 comprises a nucleic acid sequence for silencing PD-1H. In another preferred embodiment, the invention further comprises a third expression cassette for expression of a gene-editing protein.

In one embodiment, the optional first, second and third expression cassettes each further comprise a promoter. In one embodiment, the optional first, second and third expression cassettes each further comprise a terminator.

In one embodiment, the optional first, second and third expression cassettes are located on the same or different vectors. Preferably, the optional first, second and third expression cassettes are located on the same vector. Preferably, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, other gene transfer systems, or combinations thereof. Preferably, the vector is a viral vector.

In one embodiment, the third expression cassette comprises CRISPR/Cas9(sgRNA and Cas9), an antisense RNA, or a combination thereof. Preferably, the sgRNA targets PD-1H, and the sequence of the sgRNA is shown in any one or the combination of SEQ ID NO. 1-14, 22-23 and 25-26. Preferably, the antisense RNA comprises miRNA, siRNA, shRNA, inhibitory mRNA or dsRNA, and the sequence of the antisense RNA is shown in any one or the combination of SEQ ID NO. 15-20.

Carrier

Nucleic acid sequences encoding the desired molecule 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 vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

The present invention also provides a vector into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, since they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.

In brief summary, 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 into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence.

The expression constructs of the invention may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the invention provides a gene therapy vector.

The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and Molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp apart, and activity begins to decline. Depending on the promoter, it appears that the individual elements may function cooperatively or independently to initiate transcription.

An example of 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 α (EF-1 α). However, other constitutive promoter sequences may also be used, including, but not limited to, the CMV promoter, the simian virus 40(SV40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr (Epstein-Barr) virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the heme promoter, and the creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in a 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. Typically, the reporter gene is the following: which is not present in or expressed by the recipient organism or tissue and which 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 cell, the expression of the reporter gene is determined at an appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al, 2000FEBS Letters479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the minimum of 5 flanking regions that showed the highest level of reporter gene expression was identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of an agent to modulate promoter-driven transcription.

Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell by any method known in the art, e.g., mammalian, bacterial, yeast or insect cells. For example, the expression vector may be transferred into a host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing the polynucleotide into a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.

Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).

In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for use to introduce nucleic acids into host cells (ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, mixed with the lipid, associated with the lipid, contained as a suspension in the lipid, contained in or complexed with a micelle, or otherwise associated with the lipid. 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 be present in bilayer structures, either as micelles or with a "collapsed" structure. They may also simply be dispersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fatty droplets that occur naturally in the cytoplasm as well as such compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

In a preferred embodiment of the invention, the vector is a lentiviral vector.

The invention designs a pCDH-GFP based CD19-CAR vector and a B7-H3(CD276) -CAR vector which integrate shRNA or sgRNA of PD-1H, but the invention is not limited to the CAR vector constructed by pCDH-GFP and is suitable for all other plasmid vectors suitable for constructing CART; the targets are also not limited to CD19, B7-H3, and should be suitable for CAR-T of all targets.

Preparation

The invention provides an engineered immune cell according to the first aspect of the invention, a host cell according to the tenth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the CAR-T cells are present in the formulation at a concentration of 1X 10³-1×10⁹One cell/Kg body weight, more preferably 1X 10⁴-1×10⁸One cell/Kg body weight.

In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.

Therapeutic applications

The invention includes therapeutic applications of cells (e.g., T cells, more preferably PD-1H silenced T cells (e.g., CD8+ T cells)) transduced with a Lentiviral Vector (LV) encoding an expression cassette of the invention. The transduced T cells can target markers (such as CD19) of tumor cells, and the T cells are synergistically activated to cause cellular immune response, so that the killing efficiency of the T cells on solid tumors and hematologic tumors is remarkably improved.

Accordingly, the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal comprising the steps of: administering to the mammal the CAR-T cells of the invention.

In one embodiment, the invention includes a class of cell therapy in which autologous T cells (or allogeneic donors) from a patient are isolated, activated, genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. In this way, the probability of graft versus host disease is very low and antigens are recognized by T cells in an MHC-unrestricted manner. Furthermore, one CAR-T can treat all cancers expressing this antigen. Unlike antibody therapy, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can last for an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cell induces an immune response specific to the antigen binding domain in the CAR. For example, CAR-T cells of CD19 elicit a specific immune response against cells expressing CD 19.

Although the data disclosed herein specifically disclose lentiviral vectors comprising an antigen binding domain, hinge and transmembrane regions, and 4-1BB and CD3 zeta signaling domains of anti-CD 19, the invention should be construed to include any number of variations on each of the construct components.

Treatable cancers include tumors that are not vascularized or have not substantially vascularized, as well as vascularized tumors. The cancer may comprise a non-solid tumor (such as a hematological tumor, e.g., leukemia and lymphoma) or may comprise a solid tumor. The types of cancer treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematological) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythrocytic leukemias), chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma (indolent and higher forms), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

A solid tumor is an abnormal mass of tissue that generally does not contain cysts or fluid regions. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types that form 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, ovarian cancer.

The CAR-modified T cells of the invention may also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human.

For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells into a mammal: i) expanding the cell, ii) introducing a nucleic acid encoding the CAR into the cell, and/or iii) cryopreserving the cell.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous with respect to the recipient. Alternatively, the cells may be allogeneic, syngeneic (syngeneic), or xenogeneic with respect to the recipient.

In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

The invention provides a method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.

The CAR-modified T cells of the 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. Briefly, a pharmaceutical composition of the invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease-although the appropriate dosage may be determined by clinical trials.

When referring to an "immunologically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). It can be generally pointed out that: pharmaceutical compositions comprising T cells described herein can be in the range of 10⁴To 10⁹Dosage of individual cells/kg body weight, preferably 10⁵To 10⁶Doses of individual cells per kg body weight (including all integer values within those ranges) are administered. The T cell composition may also be administered multiple times at these doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., 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 the treatment accordingly.

Administration of the subject composition may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) any number of relevant treatment modalities, including but not limited to treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or efavirenz therapy for psoriasis patients or other therapy for PML patients. In further embodiments, 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 immunotherapeutic agents. In a further embodiment, the cell composition of the invention is administered to the patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation with a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an injection of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered pre-or post-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. The proportion of doses administered to a human can be effected in accordance with accepted practice in the art. Typically, 1X 10 may be administered per treatment or per course of treatment ⁶1 to 10¹⁰A T cell of the invention (e.g., a CD19-CAR-T cell) is administered to a patient, for example, by intravenous infusion.

The main advantages of the invention include:

(1) the invention discovers for the first time that PD-1H has an inhibitory immune regulation function in CD8+ T cells and can negatively regulate the function of CD8+ T cells in tumor immunity.

(2) The invention discovers for the first time that the anti-tumor effect of CD8+ T cells can be enhanced and the infiltration of the CD8+ T cells into a tumor microenvironment can be promoted through RNA interference or gene knockout of the expression of PD-1H on the CD8+ T cells (including murine OT-1 cells and human CAR-T cells).

(3) The research of the invention finds that PD-1H has a remarkable immunosuppressive function in CD8+ T cells. On tumor-infiltrating CD8+ T cells, PD-1H expression was significantly upregulated. In a mouse tumor model, the anti-tumor effect of the PD-1H-deleted CD8+ T cells is obviously enhanced, the release of immune effector molecules (such as IFN-gamma, granzmB and the like) is increased, and meanwhile, the PD-1H-deleted CD8+ T cells can better infiltrate into a tumor microenvironment. Thus, targeting PD-1H on T cells may be a potential tumor immunotherapy approach.

(4) The invention discovers for the first time that targeted PD-1H (silencing/knocking out PD-1H expression, specific antibody action or other methods) can be applied on CAR-T cells or other immune cells.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise specified, materials and reagents used in examples of the present invention are commercially available products.

Example 1 increased expression of PD-1H in tumor-infiltrating CD8+ T cells and activated CD8+ T cells

(I) detecting the expression of PD-1H on tumor infiltrating lymphocytes

Female, 8-week-old C57BL/6 mice were selected and mouse lymphoma EG7 cells or mouse melanoma B16-ova cells were implanted subcutaneously ventrally. After 17 days of tumor inoculation, subcutaneous tumors and spleens of mice were collected and prepared into single cell suspensions, and added with anti-mFc receptor (clone:2.4G2) antibody, incubated at 4 ℃ for 10min, and washed 1 time with PBS containing 1% FBS. And adding anti-mCD3, anti-mCD4, anti-mCD8 and anti-mPD-1H antibody, incubating for 30min at 4 ℃, washing for 1 time by PBS containing 1% FBS, and detecting the expression condition of PD-1H on T cells by using a flow cytometer.

The results are shown in FIG. 1, and both mouse EG7 lymphoma and B16-OVA melanoma have higher PD-1H expression on tumor-infiltrating T cells, while CD8+ T cells have higher PD-1H expression than CD4+ T cells.

(II) detecting the expression of PD-1H on activated CD8+ T cells

PD-1H KO mice and their control Wild type mice (Wild-type, WT) were sorted for CD8+ T cells using a CD8+ T cell sorting kit, cultured in vitro, and stimulated with the addition of anti-mCD3 (1. mu.g/ml) and anti-mCD28 (2. mu.g/ml). At the time points of 24 hours, 48 hours and 72 hours respectively, collecting T cells, adding anti-mCD8 and anti-mPD-1H antibody, incubating for 30min at 4 ℃, washing for 1 time by PBS containing 1% FBS, and detecting the expression condition of PD-1H on the T cells by using a flow cytometer.

The results are shown in FIG. 2, and the expression of PD-1H on WT CD8+ T cells stimulated with the addition of anti-mCD3 and anti-mCD28 was increased.

Example 2 depletion of PD-1H CD8+ T cells with enhanced antitumor Effect

(one) role of PD-1H deleted CD8+ T cells in mouse melanoma model

Female, 8-week-old C57BL/6 mice were selected and mouse melanoma B16-OVA cells were implanted subcutaneously ventrally. After 6 days of tumor implantation, the tumor-bearing mice were treated by tail vein injection of PD-1H KO or WT OT-1T cells, respectively. Tumor size was measured every two days with an electronic vernier caliper. After 17 days, subcutaneous hybridomas of mice were harvested and prepared as a single cell suspension, which was incubated with anti-mFc receptor (clone:2.4G2) antibody at 4 ℃ for 10min and washed 1 time with PBS containing 1% FBS. Then adding anti-mCD3, anti-mCD45 and anti-mCD8 antibodies, incubating for 30min at 4 ℃, washing for 1 time by PBS containing 1% FBS, and detecting by a flow cytometer.

The results are shown in FIG. 3, in which the tumor growth of the PD-1H-KO OT-1 cell-transfected mice was significantly inhibited (left panel). In tumor-infiltrating lymphocytes from mice transfused with PD-1H-KO OT-1 cells, the proportion of CD8+ T cells was increased (right panel).

(II) Effect of PD-1H deleted CD8+ T cells in mouse lymphoma model

Female, 8-week-old C57BL/6 mice were selected and murine lymphoma EG7 cells were implanted subcutaneously ventrally. After 6 days of tumor implantation, PD-1H KO or WT OT-1T 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, subcutaneous hybridomas of mice were harvested and prepared as a single cell suspension, which was incubated with anti-mFc receptor (clone:2.4G2) antibody at 4 ℃ for 10min and washed 1 time with PBS containing 1% FBS. Then adding anti-mCD3, anti-mCD45 and anti-mCD8 antibodies, incubating for 30min at 4 ℃, washing for 1 time by PBS containing 1% FBS, and detecting by a flow cytometer.

The results are shown in FIG. 4, in which the tumor growth of the mice treated with the transferred PD-1H-KO OT-1 cells was significantly inhibited (upper panel). The proportion of CD8+ T cells was significantly increased in tumor-infiltrating lymphocytes from mice transfused with PD-1H-KO OT-1 cells (lower panel).

Example 3 knocking out the PD-1H gene on CD8+ T cells by using CRISPR Cas9 technology can enhance the anti-tumor effect of CD8+ T cells.

Screening sgRNA sequence capable of effectively knocking down mPD-1H

Based on the CDS sequence of mPD-1H, a sgRNA sequence is designed.

The LentiCRISPR v2 plasmid replaces the original puromycin resistance sequence with a GFP protein sequence. The synthesized mPD-1H sgRNA1 and mPD-1H sgRNA2 fragments were annealed to double-stranded DNA and ligated to the BsmBI site of the LentiCRISPR v2 vector (Addgene 52961). And selecting clones, sequencing and analyzing to determine that the sgRNA fragment is successfully constructed on the LentiCRISPR v2 vector. The lentiCRISPR mPD-1H sgRNA plasmids (sgRNA1 and sgRNA2 plasmids) are mixed with pRSV-Rev, pMDLg/pRRE and pCMV-VSVG auxiliary plasmids according to a certain proportion respectively, and are cotransfected into 293FT cells. 48H after transfection, cell culture supernatants containing mPD-1H sgRNA-H1 and mPD-1H sgRNA-H2 lentivirus were collected and centrifuged at 3000rpm for 5min at 4 ℃. The supernatant was filtered through a 0.22uml filter and frozen at-80 ℃ for use. 293T-mPD-1h cells were infected with lentivirus.

The result is shown in FIG. 5 that the mPD-1H sgRNA-H2 sequence can effectively knock out PD-1H.

(II) preparing PD-1H knockout CD8+ T cell (sgRNA-OT-1) by using CRISPR Cas9

Lymphocytes of spleen of 8-week-old female OT-1 mice were isolated by lymphocyte separation medium and density gradient centrifugation method. Using CD8+ T cell isolation Kit (whirling in America)) The cells were labeled with magnetic beads and purified to isolate CD8+ T lymphocytes. And (3) carrying out T lymphocyte activation and proliferation on the purified CD8+ T cells by using a CD3/CD28 antibody. Activated T lymphocytes were collected 24 hours after antibody stimulation and resuspended in RPMI1640 medium. Infecting activated CD8+ T lymphocytes with mPD-1H sgRNA-H2 lentivirus, adding the cell suspension to a 6-well plate, standing at 37 deg.C and 5% CO₂Incubate overnight in the incubator. The next day, the medium was centrifuged again and replaced with fresh medium, and the culture was continued for another 2 days with addition of fresh medium. At day 8, the T cells were harvested by centrifugation and resuspended in a suitable cryopreservative solution and frozen in liquid nitrogen for use.

(III) Effect of PD-1H knockout CD8+ T cells in mouse lymphoma (EG7) model

Female, 8-week-old C57BL/6 mice were selected and murine lymphoma EG7 cells were implanted subcutaneously ventrally. After 6 days of tumor implantation, sgRNA OT-1, PD-1H KO or WT OT-1T cells are transfused to the tail vein of tumor-bearing mice for treatment. Tumor size was measured every two days with an electronic vernier caliper. After 17 days, subcutaneous hybridomas of mice were harvested and prepared as a single cell suspension, which was incubated with anti-mFc receptor (clone:2.4G2) antibody at 4 ℃ for 10min and washed 1 time with PBS containing 1% FBS. Then adding anti-mCD3, anti-mCD45 and anti-mCD8 antibodies, incubating for 30min at 4 ℃, washing for 1 time by PBS containing 1% FBS, and detecting by a flow cytometer.

The result is shown in fig. 6, the growth of the tumor of the mice treated by the sgRNA OT-1 and PD-1H-KO OT-1 cells is obviously inhibited, and the anti-tumor effect of the CD8+ T cells can be improved by knocking out the PD-1H gene of the CD8+ T cells through the CRISPR Cas 9.

Example 4 increased expression of PD-1H in human T cells or human CAR-T cells in the tumor microenvironment of the mouse colon cancer PDX model

(one) detecting the expression of PD-1H on T cells and CAR-T cells cultured in vitro

PBMC and B7-H3-CAR-T cells (obtained from Fuzhou Tuo Xin Tian Biotechnology Co., Ltd., structure: B7-H3scFv-CD8 hinge region-CD 8 transmembrane region-41 BB co-stimulatory molecule-CD 3 zeta cytoplasmic signaling region) were taken, and anti-hCD3, anti-hB7-H3-mFc and anti-hPD-1H antibody were added, incubated at 4 ℃ for 30min, washed 1 time with 1% FBS-containing PBS, and then detected by flow cytometry.

The results are shown in FIG. 7, no PD-1H expression was observed on the surface of T cells and CAR-T cells in vitro.

(II) construction of NCG mouse xenograft (PDX) colon cancer tumor model

A8-10 week old female NCG mouse (NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju) is selected to construct a xenograft (PDX) colon cancer tumor model. Shearing colon tumor tissue of patient into 15mm³The small pieces of (a) were implanted subcutaneously in the bilateral axilla of NCG mice. The growth of the tumor in the mice was observed daily, and the size of the tumor was measured with a vernier caliper every week when the tumor reached about 500mm³When the size is large, the tumor tissue can be taken for passage. The mode of passage was as described above, and after five consecutive mouse-to-mouse passages, xenografted tumors were implanted subcutaneously in the bilateral axilla of NCG mice. When the mean tumor radius reached about 8mm, CAR-T cells were injected intratumorally for treatment. After 20 days of treatment, peripheral blood, spleen and tumor tissues of the mice are taken to prepare single cell suspension, and fluorescent antibody of specific molecules is marked for flow cytometry analysis. A part of spleen and tumor tissue were prepared as paraffin sections for analysis.

(III) multiple immunofluorescence Co-localization

Taking a PDX mouse model tumor tissue section, dewaxing and rehydrating, and keeping slight boiling for 20min in 1mmol/L EDTA to carry out antigen retrieval. Endogenous catalase was blocked by incubation for 20min in 3% aqueous hydrogen peroxide. Immunofluorescent 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). The cell phenotype was labeled with CD3, CD8 antibodies, and appropriate fluorescent secondary antibodies were incubated. Nuclei were labeled with DAPI. The stained sections were photographed by scanning with an EVOS FL Auto Cel Imaging System.

Results as shown in figure 8, there was PD-1H expression on tumor-infiltrating CD8+ CAR-T cells in tumor tissues of the PDX mouse model after CAR-T cell injection treatment.

Example 5 silencing expression of PD-1H on CAR-T cells Using short hairpin RNA (shRNA) enhances the anti-tumor effect of CAR-T cells.

(I) construction of chimeric antigen receptor expression vector co-expressing PD-1H shRNA

shRNA sequences designed based on CDS sequences of PD-1H are candidate PD-1H shRNA sequences as shown in Table 1.

TABLE 1 PD-1H shRNA sequences

A U6 promoter and PD-1H shRNA sequence were synthesized according to the sequence in Table 1, and constructed on a pCDH-EF1-CAR plasmid through enzymatic ligation, and the structure of the pCDH-U6-PD-1H shRNA-EF1-CAR plasmid is shown in FIG. 9. Sequencing confirmed that the U6-PD-1H shRNA sequence was ligated to the plasmid.

(II) screening shRNA sequence for effectively silencing PD-1H expression

pCDH-U6-PD-1H shRNA-EF1-CAR plasmids (PD-1H shRNA1-shRNA6 and Control shRNA plasmids) are mixed with pRSV-Rev, pMDLg/pRRE and pCMV-VSVG auxiliary plasmids according to a certain proportion to transfect 293FT cells. 48H after transfection, cell culture supernatants containing mPD-1H sgRNA-H1 and mPD-1H sgRNA-H2 lentivirus were collected and centrifuged at 3000rpm for 5min at 4 ℃. The supernatant was filtered through a 0.22uml filter and frozen at-80 ℃ for use. Jurkat-mPD-1H cells were infected with lentivirus. After 72 hours of virus infection, about 2X 10 was taken⁵The cells were incubated with anti-PD-1H antibody at 4 ℃ for 30min, washed 1 time with PBS containing 1% FBS, and then detected by flow cytometry. The results are shown in FIG. 10, pCDH-U6-PD-1H shRNA-EF1-CAR plasmid is able to not affect the expression of CAR molecules on the surface of T cells. As shown in FIG. 11, PD-1H shRNA1 can silence PD-1H expression most effectively, PD-1H shRNA2, PD-1H shRNA4, PD-1H shRNA5 and PD-1H shRNA6 are less effective in 3 times.

(III) preparation of CD19-CAR-T cells Co-expressing PD-1H shRNA

50ml of fresh peripheral blood of the volunteers were taken and Peripheral Blood Mononuclear Cells (PBMC) were isolated by lymphocyte separation medium, density gradient centrifugation method. Cells were labeled with magnetic beads using the Pan T cell isolation Kit (whirlpool), and pure was isolatedT lymphocytes are differentiated. And (4) activating and proliferating the T lymphocytes by using the purified T cells and CD3/CD28 magnetic beads. Activated T lymphocytes were collected and resuspended in RPMI1640 medium. 1X 10 infection with PD-1H shRNA-CD19-CAR (where an example of CD19-CAR structure is scFv-CD8 hinge region targeted by CD 19-CD 8 transmembrane region-41 BB costimulatory molecule-CD 3 zeta cytoplasmic signaling region) and Control shRNA-CD19-CAR lentivirus⁶Activated T lymphocytes, the cell suspension was loaded into 6-well plates and placed at 37 ℃ in 5% CO₂Incubate overnight in the incubator. The next day, the medium was centrifuged again and replaced with fresh medium, and the culture was continued for another 2 days with addition of fresh medium. When the cells are cultured to the 8 th day, the CAR-T cells (PD-1H shRNA-CD19-CAR T cells and Control shRNA-CD19-CAR T cells) are collected by centrifugation and are resuspended in a suitable freezing medium and are frozen in liquid nitrogen for later use.

(tetra) anti-tumor effect of PD-1H shRNA-CD19-CAR T cell animal model in vivo

Female NCG mice (NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju) with the age of 8-10 weeks are selected, and tail vein injection is carried out on CD19 positive tumor cells (lymphoma cells CA-46) to establish a tumor model. After 5 days of modeling, tail vein transfused CAR-T cell therapy. Tumor growth was detected by a small animal in vivo imaging system.

The results are shown in fig. 12 and 13, and the PD-1H shRNA-CD19-CAR T cells can effectively inhibit the growth of lymphoma cells, and have better therapeutic effect compared with the control CAR-T cells. And the survival time of the mouse is effectively prolonged.

Example 6 knock-out of PD-1H on T cells using the CRISPR CAS9 technique

(I) construction of pSBSO sleeping beauty transposon (SP) plasmid containing PD-1H sgRNA sequence and B7-H3-CAR sequence

sgRNA sequences were designed based on the CDS sequence of PD-1H, and candidate PD-1H sgRNA sequences are shown in table 2.

TABLE 2 PD-1H sgRNA sequences

Synthesizing a U6 promoter and a PD-1H sgRNA sequence according to the table 2, amplifying an EF1 promoter and a B7-H3-CAR sequence by PCR, carrying out homologous recombination on the two fragments on a pSBSO vector, and carrying out sequencing and identification on the sequences accurately, wherein the pSBSO-PD-1H sgRNA-B7-H3-CAR plasmid is successfully constructed.

(II) screening sgRNA sequences capable of effectively knocking out PD-1H

Mixing the above-constructed pSBSO-PD-1H sgRNA-B7-H3-CAR plasmid, pSB11 plasmid and eSPCas9(1.1) plasmid at a certain ratio, and adding Lonza Amaxa^TM 4D-Nucleofector^TMAnd (4) electrotransfering to Jurkat-PD-1H cells by using an electrotransfer instrument. After 72 hours of electrotransfer, about 2X 10 is taken⁵The cells were incubated at 4 ℃ for 30min with anti-PD-1H antibody, washed 1 time with PBS containing 1% FBS, and then detected by flow cytometry.

The results are shown in fig. 14, and sgRNA3 and sgRNA4 were effective in knocking out PD-1H expression on Jurkat-PD-1H cells.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Claims

1. An engineered immune cell, wherein expression of the PD-1H gene in the engineered immune cell is silenced.

2. The engineered immune cell of claim 1, wherein "PD-1H gene expression is silenced" means that PD-1H gene is not expressed or is under-expressed.

3. The engineered immune cell of claim 2, wherein said "under-expression" is the ratio of the expression level of PD-1H gene G1 in said immune cell to the expression level of PD-1H gene G0 in a normal immune cell, i.e., G1/G0 ≦ 0.8, preferably G1/G0 ≦ 0.5, more preferably ≦ 0.2, more preferably ≦ 0.1, most preferably 0.

4. The engineered immune cell of claim 1, wherein the engineered immune cell comprises a CD8+ T cell, a CD3+ T cell, a CD4+ T cell, a B cell, an NK cell, a myeloid leukocyte or monocyte, an antigen-presenting cell, or other immune cell.

5. The engineered immune cell of claim 1, wherein said engineered immune cell has the following characteristics:

(a) the expression of the PD-1H gene in the immune cell is silenced.

6. The engineered immune cell of claim 5, further characterized by:

7. The engineered immune cell of claim 1, wherein the engineered immune cell comprises:

8. The engineered immune cell of claim 7, wherein the inhibitory molecule is selected from the group consisting of: inhibitory nucleic acids, small molecule compounds, antibodies (e.g., single domain antibodies), polypeptides, or combinations thereof.

9. The engineered immune cell of claim 8, wherein the inhibitory nucleic acid comprises an RNA interference agent, a Crispr agent.

10. The engineered immune cell of claim 9, wherein the inhibitory nucleic acid is selected from the group consisting of: siRNA, miRNA, shRNA, hairpin siRNA, tandem expressed miRNA, microrna-adapted shRNA, precursor microrna, or a combination thereof.

11. The engineered immune cell of claim 10, wherein the sequence of the inhibitory nucleic acid is set forth in any one or a combination of SEQ ID No. 15-20.

12. The engineered immune cell of claim 9, wherein the Crispr reagent comprises a gene-editing protein.

13. The engineered immune cell of claim 12, wherein the gene-editing protein is selected from the group consisting of: CRISPR, TALEN, ZFN, or a combination thereof.

14. The engineered immune cell of claim 13, wherein said CRISPR protein is selected from the group consisting of: cas9, nCas9, Cas10, Cas9a, Cas12, Cas12a, Cas12b, Cas13, Cas14, or a combination thereof.

15. The engineered immune cell of claim 9, wherein said Crispr reagent further comprises a gRNA.

16. The engineered immune cell of claim 15, wherein the gRNA has the sequence set forth in any one or combination of SEQ ID nos. 1-14, 22-23, 25-26.

17. The engineered immune cell of claim 8, wherein 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, biphenyl compounds, or combinations thereof.

18. The engineered immune cell of claim 7, wherein said tumor cell surface antigens comprise cell surface antigens of various solid tumors, and hematological tumors.

19. The engineered immune cell of claim 7, wherein said tumor cell surface antigen is selected from the group consisting of: CD, c-Met, PSMA, MUC-1, MUC, CD123, CD138, CD, PD-L, CD276, B-H, mesothelin (mesothelin), EGFR, EGFRviii, GPC, BCMA, ErbB, NKG2 ligand, LMP, EpCAM, VEGFR-1, Lewis-, Claudin18.2, CD123, CD138, CD133, CD137, CD151, CD171, KIT (CD117), CD174, CD44V, CD179, B-H (CD276), ClaB-H, HER, c-Met, PSMA, PSCA, MUC, mesothelin (EGFR), CEPV, EGFR, VEGF-2 ligands, EGFRcGMII, EGFRII, EGFRviii, FAP, PSMA, CA125, EphA2, L1CAM, CS1, ROR1, EC, NY-ESO-1, GD2, EPG, DLL3, 5T4, IL-13Ra2 (or CD213A2), IL-11Ra, PRSS21, PDGFR-beta, SSEA-4, folate receptor alpha (FRa or FR 1); folate receptor beta (FRb), AFP/MHC complex, NCAM, ELF2M, FAP, IGF-I receptor, CAIX, sLe, ganglioside GM M (aNeu 5M (2-3) bDClalp (1-4) bDGlcp (1-1) Cer), TGS M, HMWMAA, OAcGD M, (TEM M/CD 248), TEM 7M, CLDN M, TSHR, GPRC 5M, ALK, HAVC M, ADRB M, PANX M, GPR M, OR51E M, LAGE-1a, MAGE-A M, ETV M-AML, MAD-CT-1, MAD-CT-2, Fos-related antigen, p M mutant, PCT A-1, MePD OR LANTID, hTETA, TMALP-IAP, ACAIP, CAIX, CAFL 4, IGF-CT-M, FL4, FLT-M, FLR-M, FLT-M, TNFR-M, CTD-M, TGS-M, CTD-M, TGS-M, TGS-M-G-M-C, TGS-C-M, TGS-1, TGS-C-M-C, PLGA-C-M, TGS-M, PLGA-M, TGS-C, PLGA-C-1, TGS-C-D-C-M, PLGA-M, TGS-C-M, TGS-D-1, FL4, TGS-C-D-C-M, TGS-C-1, PLGA-C-D-C-D, FVB-C-M, TGS-C-M, p6854, pS-C, NY-ESO-1, NY-ESO-1/MHC I complex, PSMA, RANK, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, TCRa, TCRp, TLR7, TLR9, PTCH1, WT-1, WT1/MHC I complex, NA17, MYCN, RhoC, SART3, SSX2, RAGE-1, HPV E7, HPV E7/MHC I complex; AFP/MHC I complex, Ras/MHC I complex, Robol, Frizzled, OX40, CD79a, CD79b, CD72, Notch-1-4, CLL-1 (or CLECL1), TAG72, LILRA 2; CD300 LF; CLEC 12A; BST 2; EMR 2; LY75, FCRL 5; IGLL1, MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFR α 4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea; fucosyl GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD179b-IGLl1, TCR γ - δ, NKG2D, CD32(FCGR2A), Tn Ag, Tim1-/HVCR1, CSF2RA (GM-CSFR- α), TGF β R2, Lews Ag, TCR β 1 chain, TCR β 2 chain, TCR- γ chain, TCR- δ chain, FITC, LHR, FSHR, CGHR or GR, CCR4, SLAMF6, SLAMF 8, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3 65, HV K8.1, KSHV-gH, influenza A (HA), GAD, 65, guanidinium C (GCC) cyclase, anti-HLA core 65 (DsG) 3, HLA-DHDP 65, HLA-DHFR-DHHR-alpha, DHFR-DHHR-alpha, DHFR-alpha, DHFR-beta-alpha, DHF-beta-4, and their derivatives, IgE, CD99, RasG12V, tissue factor 1(TF1), AFP, GPRC5D, P-glycoprotein, STEAP1, Liv1, fibronectin-4, Cripto, gpA33, BST1/CD157, low conductance chloride channels, and antigens recognized by TNT antibodies, or combinations thereof.

20. The engineered immune cell of claim 7, wherein said tumor cell surface antigen comprises CD 19.

21. The engineered immune cell of claim 7, wherein said tumor cell surface antigen comprises B7-H3(CD 276).

22. A method of preparing the engineered immune cell of claim 1, comprising the steps of:

(A) providing an immune cell that has been screened or is to be engineered; and

23. The method of claim 22, wherein step (B) comprises introducing into the immune cell a second expression cassette that expresses a gene for silencing PD-1H.

24. The method of claim 22, wherein in step (B), further comprising the step of: (B1) introducing a first expression cassette expressing a CAR into the immune cell; and (B2) introducing into the immune cell a second expression cassette expressing a gene for silencing PD-1H,

wherein the order of the steps (B1 and (B2) is not limited at all.

25. The method of claim 23 wherein the second expression cassette comprises CRISPR/Cas9(gRNA and Cas9), antisense RNA.

26. The method of claim 25 wherein the gRNA targets PD-1H and the sequence of the gRNA is as set forth in any one or combination of SEQ ID nos. 1-14, 22, 23, 25-26.

27. The method of claim 25, wherein the antisense RNA comprises miRNA, siRNA, shRNA, inhibitory mRNA, or dsRNA.

28. The method of claim 27, wherein the sequence of the antisense RNA is set forth in any one or a combination of SEQ ID No. 15-20.

29. A formulation comprising the engineered immune cell of claim 1, and a pharmaceutically acceptable carrier, diluent, or excipient.

30. The formulation of claim 29, wherein said immune cells are present in said formulation at a concentration of 1 x 10³-1×10¹⁰Individual cells/ml, preferably 1X 10⁴-1×10⁸Individual cells/ml.

31. Use of an engineered immune cell according to claim 1 for the preparation of a medicament or formulation for the prevention and/or treatment of cancer or tumor.

32. The use of claim 31, wherein the cancer or tumor is selected from the group consisting of: a hematologic tumor, a solid tumor, or a combination thereof.

33. The use of claim 31, wherein the cancer or tumor comprises a CD19 positive tumor.

34. The use of claim 31, wherein said cancer or tumor comprises a B7-H3(CD276) positive tumor.

35. A kit for preparing the engineered immune cell of claim 1, comprising a container, and within the container:

36. The kit of claim 35, wherein the kit further comprises: (4) a third nucleic acid sequence comprising an expression cassette for expression of a gene-editing protein; or a gene editing protein.

37. A method of modulating immune cell activity, comprising:

38. The method of claim 37, wherein the activity of the immune cell is enhanced by reducing or inhibiting the expression level of PD-1H in the immune cell.

39. The method of claim 37, wherein the activity of the immune cell is decreased by increasing the expression level of PD-1H in the immune cell.

40. The method of claim 38, wherein the "reducing or inhibiting the expression level of PD-1H in an immune cell" is the ratio of the expression level of PD-1H gene in said immune cell G1 to the expression level of PD-1H gene in a normal immune cell G0, i.e. G1/G0 ≤ 0.8, preferably G1/G0 ≤ 0.5, more preferably ≤ 0.2, more preferably ≤ 0.1, and most preferably 0.

41. The method of claim 39, wherein the expression level of PD-1H in the immune cells is enhanced by the ratio of the expression level of PD-1H gene in the immune cells G1 to the expression level of PD-1H gene in normal immune cells G0, i.e., G1/G0 is 2 or more, preferably G1/G0 is 3 or more, and more preferably G1/G0 is 4 or more.

42. A method of determining or assessing immune cell activity, comprising:

43. The method of claim 42, wherein the activity of the immune cell is decreased when the expression level of PD-1H in the immune cell is increased.

44. The method of claim 42, wherein the activity of the immune cell is increased when the expression level of PD-1H in the immune cell is decreased.

45. The method of claim 43, wherein the expression level of PD-1H in the immune cells is increased as the ratio of the expression level of PD-1H gene in the immune cells G1 to the expression level of PD-1H gene in normal immune cells G0, that is, G1/G0 is 2 or more, preferably G1/G0 is 3 or more, and more preferably G1/G0 is 4 or more.

46. The method of claim 44, wherein the "reduced expression level of PD-1H in the immune cell" is the ratio of the expression level of PD-1H gene in the immune cell G1 to the expression level of PD-1H gene in a normal immune cell G0, i.e. G1/G0 ≦ 0.8, preferably G1/G0 ≦ 0.5, more preferably ≦ 0.2, more preferably ≦ 0.1, most preferably 0.

47. A nucleic acid molecule comprising a first nucleic acid and optionally a second nucleic acid, wherein the first nucleic acid comprises a first expression cassette encoding an inhibitory molecule that reduces or inhibits the expression activity of a PD-1H protein, and the second nucleic acid comprises a second expression cassette encoding a chimeric antigen receptor CAR comprising: an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds to a tumor cell surface antigen.

48. The nucleic acid molecule of claim 47, wherein said first expression cassette and/or said second expression cassette further comprises a constitutive promoter or an inducible promoter.

49. The nucleic acid molecule of claim 48, wherein said constitutive promoter is selected from the group consisting of: CMV, EF1a, U6, SV40, PGK1, Ubc, CAG, H1, or a combination thereof.

50. The nucleic acid molecule of claim 48, wherein said 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.

51. A vector comprising the nucleic acid molecule of claim 47.

52. A host cell comprising the vector of claim 51 or a nucleic acid molecule of claim 47 integrated into the chromosome.

53. A pharmaceutical composition, comprising:

(a) the host cell of claim 52; and

(b) a pharmaceutically acceptable carrier, diluent or excipient.

54. The pharmaceutical composition of claim, wherein the concentration of said cells is 1 x 10³-1×10¹⁰Individual cells/ml, preferably 1X 10⁴-1×10⁸Individual cells/ml.

55. A method of preparing an engineered immune cell, comprising:

transducing the nucleic acid molecule of claim 47 or the vector of claim 51 into an immune cell, thereby obtaining the engineered immune cell.

56. A reagent combination, characterized in that the reagent combination comprises:

(i) optionally a first agent which is an immune cell; and

57. A method of enhancing tumor killing efficiency of engineered immune cells, comprising:

58. The method of claim 57, wherein the method is in vitro.

59. Use of the host cell of claim 52 or the pharmaceutical composition of claim 53 for the preparation of a medicament or formulation for selective killing of tumor cells.

60. A kit for selectively killing tumor cells, comprising a container, and the host cell of claim 52 disposed within the container.