Chimeric antigen receptor and use thereof

Abstract

Provided are a chimeric antigen receptor and the use thereof. The chimeric antigen receptor specifically binds to an antigen and inhibits a pathogenic immune effector cell expressing the antigen. The chimeric antigen receptor of the present invention is expressed in a UCART cell, such that an ADR.UCAR-T cell has a better tumor killing effect and lower immunological rejection, thus preventing T and / or NK cell-mediated hosts in individuals from repelling allogeneic T cells, tissue or organs.

Description

Chimeric antigen receptors and their applications Technical Field

[0001] The present invention belongs to the field of cell therapy, and in particular relates to a chimeric antigen receptor and an application thereof. Background Art

[0002] Chimeric antigen receptor T (CAR-T) cell therapy is rapidly developing in the field of anti-tumor treatment, particularly for hematologic malignancies. Currently, both the US FDA and the China CDE have approved several CAR-T cell therapies for marketing. However, all approved CAR-Ts are autologous, and their clinical application still has certain limitations: long preparation time, high cost, and a narrow scope of application. To address these shortcomings of autologous CAR-T cells, scientists have proposed the concept of universal CAR-T (UCAR-T) cells.

[0003] UCAR-T cell therapy is a "one-to-many" relationship between healthy people and patients. To achieve this, it is necessary to solve the attack of allogeneic CAR-T on the receptor or host (GvHD) and the immune rejection of CAR-T cells by the receptor or host (HvGD). Researchers can now avoid GvHD by knocking out or destroying the expression of T cell receptors (TCR), but HvGD is still a difficult problem that needs to be broken through. In order to prevent the rejection of allogeneic T cells introduced into the host, the MHC molecules of UCAR-T are usually silenced, but the activation of NK cells caused by the lack of MHC and the subsequent rejection are still problems to be solved. Existing solution strategies can be divided into two types: passive defense and active attack.

[0004] Passive defense: Researchers loaded UCAR-T with CD47 molecules, which bind to signal regulatory protein α (SIPRα), sending a "don't eat me" signal to reduce macrophage phagocytosis and NK cell rejection. They also loaded non-classical MHC molecules such as HLA-E and HLA-G to bind to inhibitory receptors on NK cells. However, these inhibitory effects have limitations and uncertainties. For example, overexpression of HLA-G may enhance NK cell-mediated rejection.

[0005] Active attack: Before reinfusion of UCAR-T cells, lymphodepleting agents such as CD52 or CD38 monoclonal antibodies can be used to eliminate T and NK cells. However, this long-term pan-immunosuppression may increase the risk of infection. Therefore, specific approaches targeting alloimmune components may be better. For example, the ADR (auto / allo-immune defense receptor) strategy specifically targets activated T and NK cells in the host and has a short-term effect, avoiding permanent damage to important immune and non-immune tissues.

[0006] Existing ADR.UCAR-Ts use the 41BB molecule as their ADR target, but their ability to eliminate activated host NK cells is limited due to low 41BB expression on activated NK cells. Therefore, screening for more suitable ADR molecules and developing ADR.UCAR-Ts with enhanced targeted elimination of activated NK cells is expected to further broaden the applicability of UCAR-T and enhance its application value.

[0007] TIM3, short for T cell immunoglobulin and mucin domain-containing protein 3, was first discovered in 2002 and is a member of the TIM family of immunomodulatory proteins. In humans, TIM family members are encoded by three genes: TIM1 (encoded by HAVCR1), TIM3 (encoded by HAVCR2), and TIM4 (encoded by TIMD4). TIM3 has garnered the most attention due to its involvement in regulating immune responses in autoimmunity and cancer.

[0008] TIM3 was originally identified as a receptor for interferon-γ (FN-γ)-producing CD4 + T and CD8 + TIM3 is expressed by T cells. However, subsequent studies have revealed that other cell types, such as regulatory T cells (Treg cells), bone marrow cells, natural killer (NK) cells, and mast cells, also express TIM3. Therefore, in theory, therapeutic strategies targeting TIM3 may modulate immune responses by acting on multiple cell types. Currently, TIM3-blocking drugs are being investigated in clinical trials for their potential to treat cancer. Although the role of TIM3 in some of these cell types is still under investigation, accumulating data suggest that TIM3 also functions as an inhibitory receptor in some cells, including DCs, macrophages, bone marrow-derived suppressor cells, and NK cells. Studies have shown that TIM3 is highly expressed on NK cells and acts as an activating coreceptor in human NK cells, demonstrating that TIM3 can serve as a marker of NK cell activation or maturation, particularly on highly cytotoxic NK cells.

[0009] A large amount of evidence shows that TIM3 plays a role in transplant tolerance and autoimmunity, Mycobacterium tuberculosis infection, chronic viral infection and cancer. Taking cancer as an example, TIM3 also marks tumor-infiltrating CD8 + PD1 + The most dysfunctional subset of T cells. Antibodies that block TIM3 and PD-1 inhibit tumor growth and improve tumor antigen-specific CD8 + T cell responses have synergistic effects. In addition to TIM3 + CD8+ Outside T cells, TIM3 + Treg cells may also be targeted by TIM3 antibodies because TIM3 + Treg cells are the main Treg cell population in tumors (60% of Treg cells in lung cancer patients express TIM3; 70% of Treg cells in hepatocellular carcinoma patients express TIM3; in mouse colorectal cancer models, TIM3 + Treg cells account for more than 50% of total Treg cells), and their presence is associated with tumor severity and progression. TIM3 can also be used as a prognostic marker for solid tumors (such as colon cancer, gastric cancer, cervical cancer, non-small cell lung cancer, and clear cell renal carcinoma), and high levels of TIM3 expression are associated with lower survival.

[0010] TIM3 is also expressed in malignant hematopoietic cells. In AML cells, TIM3 is highly expressed in leukemia stem cells. Although the exact role of TIM3 in leukemia stem cells is still unclear, TIM3 antibodies can ameliorate the disease in AML xenograft models, indicating that TIM3 can serve as a biomarker and clinical target. In existing studies, researchers have prepared bispecific CAR-T cells targeting CD13 and TIM3 to treat AML. Early animal experiments have demonstrated its ability to effectively eliminate tumor cells, while also significantly reducing the toxicity of bispecific CAR-T cells to human bone marrow stem cells and peripheral myeloid cells.

[0011] TIM3 has become an attractive target for cancer immunotherapy, which is closely related to its co-expression with PD-1. + T cells and CD8 + IL-27, a key cytokine expressed by TIM3 in T cells, induces the activation of a transcriptional regulator of a co-inhibitory receptor module (including not only PD-1 and TIM3, but also TIGIT and LAG3). Therefore, there is a connection between TIM3 and other checkpoint receptors that regulate the function of effector T cells. As a single agent, TIM3 antibodies have not yet shown substantial clinical benefits. However, when combined with other checkpoint inhibitors, they can induce a stronger immune response, such as TIM3 antibodies + PD-1 antibodies.

[0012] Summary of the Invention

[0013] The technical problem to be solved by the present invention is to overcome the limited ability of ADR.UCAR-T to target and eliminate host activated NK cells in the prior art, and to provide a chimeric antigen receptor and its application. The chimeric antibody of the present invention not only improves the ability of ADR.UCAR-T to target and eliminate host activated NK cells and T cells, but also reduces the occurrence of immune rejection reactions, thus facilitating the widespread application of ADR.UCAR-T and having high clinical application value.

[0014] The present invention is committed to solving the rejection caused by NK in the host while solving GvHD, and adopts an autologous / allogeneic immune defense receptor (ADR) strategy that selectively targets pathogenic NK cells (NK cells activated in the host) to specifically kill alloimmune components. It was verified through preliminary experiments that universal CART can be rejected by NK, and a new potential target molecule TIM3 that can be used as ADR was subsequently obtained through experimental screening. TIM3 is highly expressed on activated NK cells, and its anti-rejection ability is better than the existing technology targeting 4-1BB molecules. At the same time, the TIM3 molecule is highly expressed on many tumor cells and has limited expression in normal tissues, which is a better choice for treatment. In the context of diseases with abnormal expression of TIM3, the ADR engineered cells of the present invention can be used alone as a means of treating the disease, or can be combined with other treatment methods, or can be coupled with other engineering designs to enhance its biological function. In the context of universal CAR cells, the ADR engineered cells of the present invention can prevent the NK cells in the patient's body from killing the introduced therapeutic CAR engineered immune cells. In this process, the therapeutic engineered immune cells can express only ADR or can express it in combination with other chimeric antigen receptors.

[0015] The present invention solves the above technical problems through the following technical solutions.

[0016] A first aspect of the present invention provides a chimeric antigen receptor that specifically binds to an antigen and inhibits pathogenic immune effector cells expressing the antigen;

[0017] The antigen is selected from NCR2, NTRK2, MAL, CD70, GPR15, CD40LG, CCR8, KCNA2, IL2RA, LAG3, TNFRSF9 (4-1BB), TNFRSF18 (GITR), HAVCR2 (TIM3), LAMP1, CTLA4, PDCD1, and TIGIT.

[0018] In some embodiments of the present invention, the antigen is selected from the group consisting of HAVCR2, GITR, PDCD1, LAG3 and CD70.

[0019] In some specific embodiments of the present invention, the antigen is HAVCR2 or GITR.

[0020] In some embodiments of the present invention, the pathogenic immune effector cells are selected from T cells and / or NK cells.

[0021] In some embodiments of the present invention, the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain connected in sequence;

[0022] The extracellular domain is an antigen-binding fragment that targets an antigen.

[0023] In some embodiments of the present invention, the intracellular signaling domain further comprises one or more costimulatory domains.

[0024] In some embodiments of the present invention, the transmembrane domain is selected from the transmembrane domains of CD4, CD3ζ, CD8α and CD28, for example, the transmembrane domain of CD8α.

[0025] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof is a scFv or a ligand-binding functional fragment of an antigen.

[0026] In the present invention, the ligand-binding functional fragment of the antigen preferably includes the extracellular segment of the antigen ligand.

[0027] In some preferred embodiments of the present invention, the antigen-binding fragment is a scFv targeting HAVCR2 or GITR.

[0028] In some preferred embodiments of the present invention, the antigen-binding fragment is the extracellular segment of CEACAM1 or GITRL.

[0029] In some embodiments of the present invention, the scFv comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region are preferably connected by a linker.

[0030] In some embodiments of the present invention, the costimulatory domain is selected from CD28, ICOS, 4-1BB, OX40, CD30, CD40, CD70, CD134, LIGHT, DAP10, CDS, ICAM-1 and CD27, or a combination thereof.

[0031] In some embodiments of the present invention, the extracellular domain and the transmembrane domain are connected by a hinge region, for example, a hinge region selected from CD8α, CD28 and IgG.

[0032] In some embodiments of the present invention, the N-terminus of the chimeric antigen receptor is further connected to a signal peptide, such as a CD8 signal peptide.

[0033] In some specific embodiments of the present invention, the signal peptide comprises the amino acid sequence shown in SEQ ID NO:19.

[0034] In some specific embodiments of the present invention, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 1.

[0035] In some specific embodiments of the present invention, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:2.

[0036] In some specific embodiments of the present invention, the hinge region comprises the amino acid sequence shown in SEQ ID NO:15.

[0037] In some specific embodiments of the present invention, when the antigen-binding fragment is a scFv targeting HAVCR2, the heavy chain variable region of the scFv comprises CDR1 as shown in SEQ ID NO:20, CDR2 as shown in SEQ ID NO:21, and CDR3 as shown in SEQ ID NO:22, and the light chain variable region comprises CDR1 as shown in SEQ ID NO:23, CDR2 as shown in SEQ ID NO:24, and CDR3 as shown in SEQ ID NO:25.

[0038] In some specific embodiments of the present invention, when the antigen-binding fragment is a ligand-binding functional fragment targeting HAVCR2, the antigen-binding fragment is the extracellular segment of CEACAM1.

[0039] In some specific embodiments of the present invention, the antigen-binding fragment is a protein comprising the amino acid sequence as shown in SEQ ID NO: 9, or a protein encoded by an amino acid sequence formed by substitution, deletion or addition of one or more (e.g., 1-10) amino acid residues in the amino acid sequence as shown in SEQ ID NO: 9, wherein the protein is a derivative protein having a ligand binding function targeting HAVCR2; or comprises an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence as shown in SEQ ID NO: 9 and the derivative protein encoded thereby has a ligand binding function targeting HAVCR2.

[0040] In some specific embodiments of the present invention, when the antigen-binding fragment is a scFv targeting GITR, the heavy chain variable region of the scFv comprises CDR1 as shown in SEQ ID NO:26, CDR2 as shown in SEQ ID NO:27, and CDR3 as shown in SEQ ID NO:28, and the light chain variable region comprises CDR1 as shown in SEQ ID NO:29, CDR2 as shown in SEQ ID NO:30, and CDR3 as shown in SEQ ID NO:31.

[0041] In some specific embodiments of the present invention, when the antigen-binding fragment is a ligand-binding functional fragment targeting GITR, the antigen-binding fragment is the extracellular segment of GITRL.

[0042] In some specific embodiments of the present invention, the antigen-binding fragment is a protein comprising the amino acid sequence as shown in SEQ ID NO: 12, or a protein encoded by an amino acid sequence formed by substitution, deletion or addition of one or more (e.g., 1-10) amino acid residues in the amino acid sequence as shown in SEQ ID NO: 12, wherein the protein is a derivative protein having a ligand binding function targeting GITR; or comprises an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence as shown in SEQ ID NO: 12 and the derivative protein encoded thereby has a ligand binding function targeting GITR.

[0043] In some specific embodiments of the present invention, the linker comprises the amino acid sequence shown in SEQ ID NO:3.

[0044] In some specific embodiments of the present invention, the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO:17.

[0045] In some embodiments of the present invention, the chimeric antigen receptor further comprises a transmembrane domain as shown in SEQ ID NO:16.

[0046] In some embodiments of the present invention, the chimeric antigen receptor further comprises an intracellular signaling domain as shown in SEQ ID NO:18.

[0047] The second aspect of the present invention provides a polypeptide comprising the chimeric antigen receptor according to the first aspect.

[0048] In some embodiments of the present invention, the polypeptide further comprises a protein functional molecule.

[0049] In some preferred embodiments of the present invention, the protein functional molecules are selected from protein tags and cytokines.

[0050] The third aspect of the present invention provides an isolated nucleic acid encoding the chimeric antigen receptor according to the first aspect or the polypeptide according to the second aspect.

[0051] The fourth aspect of the present invention provides an expression cassette, comprising a promoter and the nucleic acid according to the third aspect.

[0052] The fifth aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid as described in the third aspect or the expression cassette as described in the fourth aspect.

[0053] In some embodiments of the present invention, the starting vector of the recombinant expression vector is selected from plasmid, cosmid, phage, retroviral vector, lentiviral vector and adenoviral vector.

[0054] The sixth aspect of the present invention provides an engineered immune cell, wherein the engineered immune cell expresses the chimeric antigen receptor as described in the first aspect or the polypeptide as described in the second aspect.

[0055] In some embodiments of the present invention, the engineered immune cells are selected from one or more of T cells, NK cells, NKT cells, mast cells, macrophages, dendritic cells, CIK cells and stem cell-derived immune effector cells.

[0056] In some preferred embodiments of the present invention, the engineered immune cells are selected from T cells, NK cells and NKT cells.

[0057] In some embodiments of the present invention, the engineered immune cells are engineered to silence endogenous expression of a gene selected from the group consisting of PD1, LAG3, TIM3, CTLA4, GITR, and 4-1BB.

[0058] In some preferred embodiments of the present invention, the engineered immune cells are further engineered to inhibit or silence the expression of a gene selected from the group consisting of TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK and CIITA.

[0059] In some embodiments of the present invention, the engineered immune cell further comprises an antibody or antigen-binding fragment thereof targeting a second antigen.

[0060] In some preferred embodiments of the present invention, the second antigen is selected from the group consisting of TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, CS1, CD138, NCAM, and Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumin, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, CD155, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, or a combination thereof.

[0061] The seventh aspect of the present invention provides a method for preparing immune cells, comprising the steps of introducing the nucleic acid described in the third aspect, the expression cassette described in the fourth aspect, and the recombinant expression vector described in the fifth aspect into the immune cells.

[0062] In some embodiments of the present invention, the method further comprises the step of silencing the endogenous expression of PD1, LAG3, TIM3, CTLA4, GITR and / or 4-1BB in the immune cells.

[0063] In some embodiments of the present invention, the method further comprises the step of inhibiting or silencing the expression of TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK and / or CIITA in the immune cells.

[0064] In some embodiments of the present invention, the method further comprises the step of introducing into the immune cells or causing the immune cells to express an antibody or antigen-binding fragment thereof targeting a second antigen; the second antigen is preferably as described in the fifth aspect.

[0065] In some embodiments of the present invention, the introduction, inhibition, silencing or expression is achieved by gene editing.

[0066] In some preferred embodiments of the present invention, the gene editing is selected from CRISPR-based gene editing, zinc finger nuclease-based gene editing, TALE nuclease-based gene editing, base editors and shRNA.

[0067] The eighth aspect of the present invention provides a pharmaceutical composition comprising the engineered immune cells as described in the sixth aspect, and optionally a pharmaceutically acceptable carrier and / or adjuvant.

[0068] The ninth aspect of the present invention provides a pharmaceutical combination comprising the pharmaceutical composition as described in the eighth aspect and a second therapeutic agent.

[0069] The tenth aspect of the present invention provides a chimeric antigen receptor as described in the first aspect, the polypeptide as described in the second aspect, the nucleic acid as described in the third aspect, the expression cassette as described in the fourth aspect, the recombinant expression vector as described in the fifth aspect, the engineered immune cell as described in the sixth aspect, the pharmaceutical composition as described in the eighth aspect, or the drug combination as described in the ninth aspect for use in the preparation of a drug for treating a disease.

[0070] In some embodiments of the present invention, the disease is cancer or an autoimmune disease.

[0071] In some embodiments of the present invention, the cancer includes solid tumors and hematological tumors.

[0072] In the present invention, the blood tumor is preferably selected from leukemia, lymphoma and myeloma, such as multiple myeloma; the solid tumor is preferably selected from early, middle and late primary or metastatic lung cancer, breast cancer, ovarian cancer, colorectal cancer, cervical cancer, kidney cancer, liver cancer, bladder cancer, prostate cancer, pharyngeal cancer, central nervous system tumors, head and neck cancer, skin cancer, pancreatic cancer and esophageal cancer.

[0073] In some embodiments of the present invention, the lung cancer is non-small cell lung cancer.

[0074] In some embodiments of the present invention, the colorectal cancer is colon cancer, rectal cancer or small intestine cancer.

[0075] In some embodiments of the present invention, the renal cancer is renal cell carcinoma.

[0076] In the present invention, the autoimmune disease is selected from transplant rejection, graft-versus-host disease, type I diabetes, multiple sclerosis and autoimmune colitis.

[0077] The eleventh aspect of the present invention provides a method for treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the chimeric antigen receptor as described in the first aspect, the polypeptide as described in the second aspect, the nucleic acid as described in the third aspect, the expression cassette as described in the fourth aspect, the recombinant expression vector as described in the fifth aspect, the engineered immune cell as described in the sixth aspect, the pharmaceutical composition as described in the eighth aspect, or the drug combination as described in the ninth aspect.

[0078] In some embodiments of the present invention, the disease is cancer or an autoimmune disease.

[0079] In some embodiments of the present invention, the cancer includes solid tumors and hematological tumors.

[0080] In the present invention, the blood tumor is preferably selected from leukemia, lymphoma and myeloma, such as multiple myeloma; the solid tumor is preferably selected from early, middle and late primary or metastatic lung cancer, breast cancer, ovarian cancer, colorectal cancer, cervical cancer, kidney cancer, liver cancer, bladder cancer, prostate cancer, pharyngeal cancer, central nervous system tumors, head and neck cancer, skin cancer, pancreatic cancer and esophageal cancer.

[0081] In some embodiments of the present invention, the lung cancer is non-small cell lung cancer.

[0082] In some embodiments of the present invention, the colorectal cancer is colon cancer, rectal cancer or small intestine cancer.

[0083] In some embodiments of the present invention, the renal cancer is renal cell carcinoma.

[0084] In the present invention, the autoimmune disease is selected from transplant rejection, graft-versus-host disease, type I diabetes, multiple sclerosis and autoimmune colitis.

[0085] The reagents and raw materials used in the present invention are commercially available.

[0086] The positive progress effect of the present invention is:

[0087] The chimeric antigen receptor of the present invention is expressed in UCART cells, so that ADR.UCAR-T cells have stronger tumor killing effects and lower immune rejection reactions, avoiding T and / or NK cell-mediated host rejection of allogeneic T cells, tissues or organs in individuals, increasing the safety and effectiveness of CART cells, and having broad clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 shows the role of β2M in NK cell allogeneic killing;

[0089] A is the result of flow cytometry detection of NK cell CD107a release; B is the result of LDH detection of NK cell killing; C is the absolute count of NK cell killing T cells.

[0090] Figure 2 shows the sequencing and expression results of some membrane proteins and marker genes in NK cells;

[0091] A is the analysis result of RNA-seq sequencing data of activated NK cells; B is the qPCR experimental result of NK cells after 48 hours of stimulation.

[0092] Figure 3 shows the results of flow cytometry detection of TIM3 / GITR / 41BB protein expression in activated NK cells.

[0093] Figure 4 shows the results of single-cell analysis of the expression of each target gene in human bone marrow cells;

[0094] FIG4A shows the expression results of HAVCR2 and CD19 genes in human bone marrow cells; FIG4B shows the expression results of TNFRSF18 and TNFRSF9 genes in human bone marrow cells.

[0095] FIG5 is a plasmid schematic diagram of CD19 and exemplary ADRs of the present invention.

[0096] FIG6 shows the results of flow cytometry detection of CD19 and ADR virus titers.

[0097] FIG7 shows the knockout effect of each target detected by flow cytometry.

[0098] Figure 8 shows the CD19-CAR.ADR-T positive rate detection results.

[0099] Figure 9 shows the effect of Anti-TIM3-UCAR-T in clearing allogeneic activated NK cells; A is the NK ratio detected by flow cytometry on DAY2; B is the killing result of CD19-CAR.ADR-T cells on allogeneic NK cells; C is the flow cytometry results of co-incubation of TIM3-ADR-T, CD19-UCAR-T and allogeneic cells on DAY0-DAY2.

[0100] Figure 10 is the detection of tumor cell killing effect of Anti-TIM3-UCAR-T; among them, A is the killing result of 5 types of CD19-CAR.ADR-T co-incubated with Raji cells; B is the IVIS result of 5 types of CD19-CAR.ADR-T killing Raji cells.

[0101] FIG11 shows the proliferation detection results of CAR.ADR-T.

[0102] Figure 12 shows the NK cell property detection of NK92 cells; wherein A is the detection of the activation property of NK92 cells; B is the rejection of KO-β2MT cells by NK92 cells.

[0103] Figure 13 shows the results of Anti-TIM3-UCAR-T against NK cell rejection; A is the proportion of NK92 detected by flow cytometry on DAY0-DAY2 of co-incubation of CAR.ADR-T and NK92; B is the absolute count of NK92 after co-incubation of CAR.ADR-T and NK92 by flow cytometry.

[0104] Figure 14 shows the results of Anti-TIM3-UCAR-T cells resisting activated immune cell rejection; wherein, A is the result of flow cytometry detection of mixed lymphocyte reaction on DAY 0 and DAY 6; B is the number of NK cells counted on DAY 6 of the mixed lymphocyte reaction by flow cytometry.

[0105] Figure 15 shows the results of Anti-TIM3-UCAR-T targeting and killing tumor cells of CAR target; wherein, A is the flow cytometry detection killing result of CAR.ADR-T and Raji cells co-incubated; B is the flow cytometry absolute counting result of target cell Raji after CAR.ADR-T and Raji cells co-incubated. DETAILED DESCRIPTION

[0106] definition

[0107] Chimeric Antigen Receptor (CAR)

[0108] The term "chimeric antigen receptor (CAR)" as used herein is a fusion protein comprising an antigen binding domain (extracellular domain) capable of binding to an antigen, a transmembrane domain, and at least one intracellular signaling domain. "Chimeric antigen receptor (CAR)" is sometimes also referred to as a "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)." An "extracellular domain" capable of binding to an antigen refers to any oligopeptide or polypeptide capable of binding to a specific antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to act as a domain that transmits signals to activate or inhibit biological processes within a cell.

[0109] When a chimeric antigen receptor specifically binds an antigen and inhibits pathogenic immune effector cells expressing the antigen, the chimeric antigen receptor is also called an auto / allogeneic immune defense receptor (ADR).

[0110] Autologous / allogeneic immune defense receptor (ADR)

[0111] The term "autologous / allogeneic immune defense receptor (ADR)" as used herein is a synthetic, non-natural, artificial construct that can selectively target activated immune cells, such as NK cells or T cells. ADR can also be used as a chimeric antigen receptor (CAR), in which case it can selectively target pathogenic immune cells or tumor cells.

[0112] ADR comprises at least: (1) an extracellular domain: an antigen binding domain that selectively targets a compound present on activated immune cells (in a specific embodiment, the antigen binding domain is a protein or a functional fragment or derivative thereof that targets one or more compounds upregulated on activated NK cells or T cells); which is operatively linked to (2) a transmembrane domain, including a transmembrane domain selected from CD4, CD3ζ, CD8α and CD28; (3) an intracellular signaling domain: a signaling domain that promotes T cell activation, including, for example, a domain selected from or derived from CD28, ICOS, 4-1BB, OX40, CD30, CD40, CD70, CD134, LIGHT, DAP10, CDS, ICAM-1 and CD27 or a combination thereof, or a sequence comprising ITAM. The ADR molecule may comprise elements (1)-(3) or consist of elements (1)-(3) or consist essentially of elements (1)-(3). In at least some instances, the ADR comprises one or more components of a type I transmembrane protein and / or one or more components of a type II transmembrane protein.

[0113] In certain embodiments, the antigen binding domain of the ADR molecule comprises a protein that selectively binds to a related protein on activated NK cells or T cells. For example, the ADR antigen binding domain may comprise a specific antibody or ligand for a receptor on activated NK cells or T cells, or the ADR antigen binding domain may comprise a receptor for a ligand on activated NK cells or T cells. In some cases, the ADR molecule comprises two or more antigen binding domains to facilitate targeting of activated NK cells or T cells.

[0114] Antigen binding domains targeting activated NK cells or T cells and targeting pathogenic T cells or tumor cells comprise antibodies or their functional fragments or derivatives. The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. In the present invention, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only complete antibodies but also antigen-binding fragments of antibodies. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, or any immunogenic part of a complete immunoglobulin. Antibodies typically include two heavy chains and two light chains, with the light and heavy chains linked by disulfide bonds. The light and heavy chains are further divided into a variable region and a constant region. The antibodies of the present invention can exist in various forms, including, for example, full-length antibodies, Fab, Fab', F(ab')2, Fv, VHH, multispecific antibodies, single-chain antibodies, and humanized antibodies.

[0115] The antigen binding domain targeting activated immune cells and pathogenic immune cells or tumor cells comprises a ligand of the target or a functional fragment or derivative thereof. The term "ligand" as used herein refers to a substance that specifically binds to the target.

[0116] In the ADR molecule, the antigen binding domain is connected to one or more components. The component can be a protein that mediates downstream signal transduction during T cell activation. In a specific embodiment, the ADR comprises CD3ζ (also known as CD247, CD3ζ, CD3H, CD3Q, CD3Z, IMD25, T3Z or TCRZ) or a functional fragment or derivative thereof. CD3ζ mediates downstream signal transduction by ITAM during T cell activation. Other signal transduction domains containing ITAMs can include domains derived from DAP12, Fc receptors, other CD3 subunits, etc. The signal transduction domain can be non-covalently linked to the ADR through another domain.

[0117] In some cases, ADR can include one, two, three or more costimulatory domains that enhance the production of cytokines by cells expressing ADR. These costimulatory domains can be derived from intracellular signaling regions of costimulatory proteins including CD28, ICOS, 4-1BB, OX40, CD30, CD40, CD70, CD134, LIGHT, DAP10, CDS, ICAM-1 and CD27 or combinations thereof.

[0118] In some embodiments, ADR includes a transmembrane domain, and this transmembrane domain makes the CD3ζ component of ADR be located intracellularly, and the domain of the compound that targets one or more compounds that are upregulated in activated immune cells is located extracellularly. In some cases, ADR is a soluble protein that can bind to the corresponding ligand on activated T cells and promote cytotoxicity by cross-linking TCR (for example, ADR-CD3T cell binding protein). In some cases, the antigen binding domain of ADR may come from some surface proteins (such as CD40) with a transmembrane domain, so the transmembrane domain of ADR may include a transmembrane domain from a corresponding endogenous molecule. In the case where the ADR molecule includes one or more costimulatory domains, the transmembrane domain can be from an endogenous molecule with this costimulatory domain. The transmembrane domain includes a transmembrane domain from CD3, CD8α, CD27, CD28, 4-1BB, OX40, CD4, etc.

[0119] The ADR may also include one or more detectable labels, such as colorimetric labels, fluorescent labels, radioactive labels, and the like, for example, green fluorescent protein, blue fluorescent protein, and the like.

[0120] ADR-expressing cells

[0121] The cells expressing ADR may be one or more of T cells, NK cells, NKT cells, mast cells, macrophages, dendritic cells, CIK cells and stem cell-derived immune effector cells.

[0122] In some cases, the cells can be engineered to remove endogenous TCRs to suppress graft-versus-host disease (GvHD).

[0123] In some embodiments, the antigen binding domain of the ADR recognizes and binds to receptors expressed on activated immune cells to inhibit activated immune cells. The inhibition of activated immune cells refers to the antigen binding domain of the ADR specifically binding to the receptors of activated immune cells (such as NK cells and T cells), and under the action of the co-stimulatory domain and the signaling domain, promoting the activation of immune cells loaded with this ADR, targeting and killing activated host NK cells or T cells, thereby achieving inhibition of the activated immune cells.

[0124] In the present invention, cells expressing one or more CARs and / or one or more TCRs can be engineered to express one or more ADRs, or cells expressing one or more ADRs can be engineered to express one or more CARs and / or one or more TCRs. Therefore, in some cases, ADRs are expressed on different vectors, different from CARs and / or TCRs. In some cases, ADR molecules are expressed together with CARs and / or TCRs on the same vector. In the case where ADRs and CARs are expressed on the same vector, the expression of ADRs and CARs can be guided by the same or different regulatory elements. In any case, ADRs and CARs can be expressed as a single polypeptide with a cleavable element between them, such as 2A.

[0125] In the case where the ADR-expressing cell also expresses a CAR or TCR, the CAR or TCR can be directed against any specific antigen. In the case of using a CAR, the CAR can be in the form of a first-generation, second-generation, third-generation, etc. CAR. In some cases, the CAR may also be bispecific.

[0126] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0127] Example 1: Construction of CD19-CAR and ADR plasmid vectors

[0128] First, the ADR plasmid was constructed by referring to the 4-1BB-ADR plasmid in the article (DOI:10.1038 / s41587-020-0601-5). After obtaining the protein sequence from the article, it was delivered to General Bio for human optimization. After optimization, two restriction sites, Xhol and KpnI, were added to the front and back ends of TNFSF9 (41BBL). This sequence was then constructed into the existing pELPS plasmid vector. Its structure includes: single-chain antibodies or ligands for each target, CD8α signal peptide, CD3 intracellular domain, linker and GFP. Secondly, the sequences of other targets were obtained. The sequences of TIM3-scFv and GITR-scFv required for the experiment were on the plasmids already in the laboratory, so only the sequences of CEACAM1 and GITRL needed to be obtained. The inventors found the extracellular sequences of the CEACAM1 and GITRL genes on the Ensembl website, downloaded the sequences, and imported them directly into SnapGene. Amplification primers were designed for the aforementioned targets, incorporating the XhoI and KpnI restriction sites. These primers were synthesized by Shanghai Boshang Biotechnology Co., Ltd. Finally, through genetic recombination, the constructed TIM3-scFv / CEACAM1 / GITR-scFv / GITRL-ADR sequence was incorporated into the CD19-CAR expression vector backbone, completing the construction of the ADR plasmid. A reference plasmid backbone is shown in Figure 5.

[0129] Among them, the sequences of each element of the ADR plasmid are as follows:

[0130] The amino acid sequence of the spacer and transmembrane domain of the ADR plasmid is shown in SEQ ID NO: 1:

[0131] The amino acid sequence of the intracellular domain of CD3ζ of the ADR plasmid is shown in SEQ ID NO: 2:

[0132] The amino acid sequence of the linker between CD3ζ and GFP is shown in SEQ ID NO: 3:

[0133] The amino acid sequence of GFP is shown in SEQ ID NO: 4:

[0134] The base sequence of 4-1BBL is shown in SEQ ID NO: 5:

[0135] The amino acid sequence of 4-1BBL is shown in SEQ ID NO: 6:

[0136] The amino acid sequence of the TIM3 single-chain antibody heavy chain variable region (Anti-TIM3-scFv VH) is shown in SEQ ID NO: 7:

[0137] The amino acid sequence of the TIM3 single-chain antibody light chain variable region (Anti-TIM3-scFv VL) is shown in SEQ ID NO: 8:

[0138] The TIM3 single-chain antibody light chain variable region (Anti-TIM3-scFv VL) and the single-chain antibody heavy chain variable region (Anti-TIM3-scFv VH) are connected by a linker with an amino acid sequence as shown in SEQ ID NO: 3.

[0139] The amino acid sequence of the TIM3 ligand CEACAM1 is shown in SEQ ID NO: 9:

[0140] The amino acid sequence of the heavy chain variable region of the GITR single-chain antibody (Anti-GITR-scFv VH) is shown in SEQ ID NO: 10:

[0141] The amino acid sequence of the GITR single-chain antibody light chain variable region (Anti-GITR-scFv VL) is shown in SEQ ID NO: 11:

[0142] The amino acid sequence of the GITR ligand GITRL is shown in SEQ ID NO: 12:

[0143] The components of the CD19 CAR plasmid are as follows:

[0144] The amino acid sequence of the CD19 single-chain antibody heavy chain variable region (Anti-CD19-scFv VH) is shown in SEQ ID NO: 13:

[0145] The amino acid sequence of the CD19 single-chain antibody light chain variable region (Anti-CD19-scFv VL) is shown in SEQ ID NO: 14:

[0146] The amino acid sequence of the CD8 hinge region (Hinge) of the CD19 CAR plasmid is shown in SEQ ID NO: 15:

[0147] The amino acid sequence of the CD8 transmembrane region of the CD19 CAR plasmid is shown in SEQ ID NO: 16:

[0148] The amino acid sequence of the 4-1BB costimulatory signal is shown in SEQ ID NO: 17:

[0149] The amino acid sequence of the intracellular domain of CD3ζ of the CD19 CAR plasmid is shown in SEQ ID NO: 18:

[0150] The amino acid sequence of the CD8 signal peptide contained in the ADR and CD19 plasmids is shown in SEQ ID NO: 19:

[0151] The CDR amino acid sequences (Kabat nomenclature) of the TIM3 single-chain antibody heavy chain variable region (Anti-TIM3-scFv VH) are shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively:

[0152] SYNMH(CDR1),DIYPGQGDTSYNQKFKG(CDR2),VGGAFPMDY(CDR3)

[0153] The CDR amino acid sequences (Kabat nomenclature) of the TIM3 single-chain antibody light chain variable region (Anti-TIM3-scFv VL) are shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively:

[0154] RASESVEYYGTSLMQ(CDR1), AASNVES(CDR2), QQSRKDPST(CDR3)

[0155] The CDR amino acid sequences (Kabat nomenclature) of the GITR single-chain antibody heavy chain variable region (Anti-GITR-scFv VH) are shown in SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively:

[0156] SYGMH(CDR1)、VIWYEGSNKYYADSVKG(CDR2)、GGSMVRGDYYYGMDV(CDR3)

[0157] The CDR amino acid sequences (Kabat nomenclature) of the GITR single-chain antibody light chain variable region (Anti-GITR-scFv VL) are shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively:

[0158] RASQGISSALA(CDR1)、DASSLES(CDR2)、QQFNSYPYT(CDR3)

[0159] Example 2: Knockout of β2M in T cells causes rejection of allogeneic NK cells

[0160] To verify the rejection of allogeneic NK cells, this example selected non-knockout and knockout β2M (WT / KOβ2M) T cells and allogeneic NK cells for co-incubation. At the same time, a negative control group (Neg-Ctrl) was set up as NK cells, and a positive control group (Pos-Ctrl) was co-cultured with K562 cells and NK cells to detect the release of CD107a of NK cells. NK cells are mainly cytolytic immune cells, and their main mechanism of action depends on the process of degranulation (their cytoplasm contains pre-formed cytotoxic granules with perforin and granzymes). Lysosomal associated membrane protein-1 (LAMP-1 or CD107a) is a highly glycosylated protein that accounts for 50% of lysosomal membrane proteins. At the end of particle release, the outer membrane of the particles will fuse with the NK cell membrane, the CD107a molecule will be exposed to the surface, and its expression level will be upregulated in accordance with the secretion of perforin. Therefore, NK cells with high expression of CD107a positive can represent that they have killing activity. The cell-killing ability of WT and KOβ2M cells was assessed using a lactate dehydrogenase (LDH) assay. When cells are damaged or die, membrane damage releases a stable cytoplasmic enzyme, lactate dehydrogenase, which reacts with certain chemicals to form a red compound. The absorbance at 450 nm measured on a microplate reader indicates cell lysis and damage.

[0161] Afterwards, the killing of KOβ2M group cells by allogeneic NK cells was detected. During the detection, two groups of controls were added: a T cell group without β2M knockout and a T cell group with KOβ2M. The above two groups of cells were cultured separately under the same conditions without adding allogeneic NK cells. Magnetic beads were used to perform absolute cell counts on each group of T cells, and the two T cell groups cultured separately were compared to calculate the killing toxicity of allogeneic NK cells on T cells. The results showed that the T cells in the WT group proliferated normally, while the cells in the KOβ2M group decreased in number due to being killed by NK cells (AC in Figure 1), and the killing toxicity of NK cells was the strongest. It was proved that interfering with the expression of HLA-I molecules on T cells did indeed cause the activation of allogeneic NK cells.

[0162] Example 3: Screening found that TIM3 molecules are highly expressed on activated NK cells

[0163] An RNAseq sequencing dataset (GSE165498) for NK cell activation was downloaded from the GEO (GENE EXPRESSION OMNIBUS) database. Analysis revealed multiple membrane proteins and classic activation marker genes that are upregulated in NK cells after antigen stimulation (Figure 2A). NK cells were stimulated with cytokines and antigens, and key genes were detected by qPCR (Figure 2B). Finally, two targets were identified: TIM3 and GITR. After screening for three targets (TIM3, GITR, and 4-1BB), NK cells were stimulated with K562 target cells. The expression of each target was significantly elevated in activated NK cells, with TIM3 showing higher and longer-lasting expression (Figure 3). Bioinformatics analysis showed that each target had limited expression on human bone marrow cells (Figures 4A and 4B), making them suitable as targeting molecules.

[0164] Example 4: Construction and detection of CD19-CAR and ADR

[0165] (1) Construction of viruses expressing CD19-CAR and ADR

[0166] The method is as follows: The ADR and CD19-CAR plasmids, along with the lentiviral packaging helper plasmids pMD2.G and psPAX2, were amplified in E. coli. Plasmids were extracted and verified for plasmid accuracy by agarose gel electrophoresis and sequencing. 293T cells in good condition and at an early passage were selected as lentiviral packaging cells. The three plasmids were transfected into the 293T cells using the transfection reagent PEI. Transfections were performed in 10 mL of serum-free DMEM in a 10 cm dish. The transfection mixture was prepared in 1 mL of serum-free DMEM per dish, with a ratio of 5 μg psPAX2 plasmid: 3 μg pMD2.G plasmid: 5 μg PEI: 60 μL. The transfection mixture was mixed at room temperature, allowed to stand for 20 minutes, and then slowly added to 293T cells containing 9 mL of culture medium until the cells reached a cell density of 70-80%. After 6-8 hours, the culture medium was replaced with fresh DMEM (10% FBS + 1% P / S). The culture supernatant was harvested at 48 h and 72 h of culture, respectively, and viruses expressing ADR and CD19-CAR were obtained after ultrafiltration and ultracentrifugation concentration. The obtained viruses were named ADR and CD19-CAR viruses.

[0167] (2) Virus titer detection

[0168] Here’s how:

[0169] Select a 293T cell culture in good condition to test the viral titer. Inoculate 500 μl of cells at a density of 4 x 10^5 / mL in a 24-well plate. After the cells adhere, add different volumes of concentrated virus solution. After 48 hours of incubation, digest the cells. Incubate with biotinylated CAR protein, which can recognize and bind to CAR, at 4°C for 50 minutes, then wash. Then, stain with APC-streptavidin SA, which can bind to biotin, at 4°C for 30 minutes. After staining, wash the tubes and use flow cytometry to detect the CAR positivity rate. Select the virus volume with the appropriate positivity rate to calculate the viral titer. The titer calculation formula is: Titer (TU / mL) = (2 x 10^5 x CAR positivity rate) / virus volume. ADR virus titer detection does not require flow cytometry antibody staining because the plasmid itself has GFP fluorescence.

[0170] The titer test results are shown in Figure 6. The titer test was performed according to the above titer test method. After the cells were attached to the plate, two volume gradients of 1μl and 3μl were set for ADR and CD19-CAR viruses, respectively. To avoid false positives caused by nonspecific staining, CTRL was set for CAR positive gate. Cells falling within the APC positive gate were CAR positive cells, and the ratio value shown was the CAR positive rate. The titer of each virus was calculated using the 1μl volume gradient results. The results showed that the titers of various viruses were all between 9.1×10 7 Above, the titer is higher.

[0171] Example 5: Construction of ADR.UCAR-T and CD19-UCAR-T cells

[0172] Here’s how:

[0173] PBMCs were isolated from human blood using lymphocyte separation fluid, and then T cells were isolated using CD4 and CD8 magnetic bead sorting. After 48 hours of activation with CD3 / CD28 complexes, CRISPR Cas9 electroporation was used to knock out the β2M, TCR, and corresponding targets of the T cells. The knockout rates are shown in Figure 7. Subsequently, UCAR-T cells were infected with lentivirus, and 12-well plates were pre-coated with RetroNectin. Based on the viral titer, the packaged virus was centrifuged and infected at an MOI of 10. The medium was changed after 24 hours. The cells prepared above were named ADR.UCAR-T (dual virus infection, including ADR and CD19 CAR) and CD19-UCAR-T.

[0174] 48 hours after infection, the CAR and ADR expression levels of the ADR.UCAR-T cells were tested using a titer assay similar to that used in the previous studies. The results are shown in Figure 8. Each ADR.UCAR-T cell was successfully infected with both the CD19 CAR and ADR viruses, with the highest positive rate reaching 20% ​​and the lowest being approximately 6%. Meanwhile, the positive rate of CD19-UCAR-T cells infected with the CD19 CAR virus alone was approximately 30%. These results demonstrate the successful generation of triple-knockout and dual-virus-infected ADR.UCAR-T cells and CD19-UCAR-T cells, enabling subsequent functional evaluation experiments.

[0175] Example 6: Evaluation of the ability of five ADR.UCAR-Ts to target and eliminate allogeneic activated NK cells and kill tumor cells

[0176] On the basis of the previous work, the functional evaluation of the five ADR.UCAR-T prepared in Example 5 was carried out in order to screen out the ADR.UCAR-T that can most effectively prevent allogeneic NK cell rejection and accurately kill tumor cells targeting CD19. First, we co-incubated the above-mentioned ADR.UCAR-T and CD19-UCAR-T with allogeneic NK cells at an effector-target ratio of 1:1 for 48 hours, and took samples on day 0 and day 2, respectively, and used anti-human CD3 / CD56 (CD3 + CD56 + The cell population is NK cells) flow antibody staining and counting magnetic beads were added, and then the flow cytometry method was used for detection. The results are shown in AC of Figure 9. The results showed that CD19-UCAR-T, as a positive control group, was greatly killed by it during co-incubation with NK, so the killing of NK cells was shown as a negative value in the figure. Compared with CD19-UCAR-T, the five ADR.UCAR-Ts can effectively kill activated NK, among which the best effect is the Anti-TIM3-UCAR-T experimental group. Therefore, we selected this group and the CD19-UCAR-T group for further in-depth analysis. The results showed that Anti-TIM3-UCAR-T can kill NK cells, resulting in a significant decrease in the proportion of NK cells in the total system, while the proportion of NK cells in the control group CD19-UCAR-T increased, proving that NK cells began to proliferate in large numbers. At the same time, the experiment showed that β2M-ADR + The cell population is expanding, and β2M-ADR - The cell population is decreasing, which indirectly indicates that ADR is playing its defense and clearance function.

[0177] The ultimate goal of loading UCAR-T with ADRs is to enable them to more effectively kill tumor cells. Therefore, the next step in the experiment was to evaluate whether ADR loading affected UCAR-T cell killing and whether ADR.UCAR-T could target and kill CD19-targeted tumor cells. To verify these two points, the five ADR.UCAR-Ts and CD19-UCAR-Ts were co-incubated with Raji-luci cells in a 96-well plate at effector-to-target ratios of 8:1, 4:1, 2:1, and 1:2 in a volume of 100 μL. After 18 hours of co-incubation, the cells were transferred from the 96-well plate to a white plate and centrifuged at low speed to remove bubbles. Luci substrate was then added sequentially at 10 μL per well, ensuring that replicates were consistent. The cells were protected from light and read on a microplate reader. Images were then captured using an IVIS imager, protected from light. To ensure the validity of the experiment, controls included a CD19-UCAR-T group and an untreated T cell group (Ctrl-T). After 24 hours, samples were taken, substrate was added, and the samples were tested on an instrument. The experimental results are shown in Figure 10, A and B.

[0178] The results showed that all five ADR.UCAR-T groups could kill the target cells Raji, but compared with the CD19-UCAR-T group, only the Anti-TIM3-UCAR-T group and the 4-1BBL-UCAR-T group achieved more ideal killing effects, and their killing abilities were higher than those of the CD19-UCAR-T group.

[0179] Example 7: Evaluation of the proliferation capacity of CD19-UCAR-T, Anti-TIM3-UCAR-T, and 4-1BBL-UCAR-T

[0180] This example further evaluates the functions of CD19-UCAR-T, Anti-TIM3-UCAR-T, and 4-1BBL-UCAR-T. UCAR-T cells were prepared again according to Example 5, and their proliferation was detected: on the third day, a certain number of prepared CAR-T cells were taken out and cultured separately in a 48-well plate, and live cell counts were performed: the cells were blown evenly, 20 μL of cell suspension was taken with a pipette and added to a 1.5 mL EP tube, and 20 μL of 0.2% trypan blue was added after gentle pipetting to mix. 20 μL of the mixture was drawn into the cell counting plate, and the data was recorded in the cell counter. The final cell concentration was adjusted to 1×10 6 / mL, and the next step of culture was carried out. Cells were collected for proliferation counting on the 5th, 7th and 14th days, and the medium was replaced and replenished according to the cell growth.

[0181] As shown in Figure 11, the results showed that there was no significant difference in the proliferation degree among the above-mentioned UCAR-T cells, and all of them could be expanded well, which proved that loading ADR on CAR would not significantly affect the proliferation of UCAR-T cells.

[0182] Example 8: Evaluation of the ability of Anti-TIM3-UCAR-T, 4-1BBL-UCAR-T, and CD19-UCAR-T to target and eliminate NK92 cell lines

[0183] The properties of the NK92 cell line in activating NK cells were verified in the early stage, and the results are shown in the figure (A and B of Figure 12). Therefore, rejection experiments were further carried out. NK92 cells were incubated with Anti-TIM3-UCAR-T, 41BBL-UCAR-T, and CD19-UCAR-T at a 1:1 effector-target ratio for 48 hours. The same treatment mode as the previous co-incubation experiment with NK cells was used. Samples were taken on day 0 and day 2, stained, and counted with magnetic beads, and then detected by flow cytometry.

[0184] The results show that, as shown in Figure 13A and B, compared with the CD19-UCAR-T group, both Anti-TIM3-UCAR-T and 41BBL-UCAR-T effectively prevented the rejection of NK92 cells and killed NK92 cells. At the same time, from the proportion and counting results of NK92 cells in the total co-incubation system on the second day, it can be seen that Anti-TIM3-UCAR-T has a stronger ability to target and eliminate NK92 cells than 4-1BBL-UCAR-T, and can more effectively inhibit the killing of UCAR-T by NK92 cells.

[0185] Example 9: Evaluation of the ability of ADR.UCAR-T cells to eliminate alloreactive cytotoxic lymphocytes and resist immune rejection

[0186] This example uses a mixed lymphocyte reaction (MLR) model to evaluate the ability of ADR.UCAR-T cells to eliminate alloreactive cytotoxic lymphocytes and resist immune rejection. After staining and labeling, two types of ADR.UCAR-T and CD19-UCAR-T cells were co-incubated with allogeneic PBMC at a 2:1 ratio, and the cells were supplemented with 20U mL-1 of recombinant human IL-2 on day 0. On day 4, the culture medium was replaced with fresh X-VIVO + 10% FBS without cytokines. The culture medium (without cytokines) was then replaced every 2-3 days for a total of 7 days. At each time point, a certain volume of cells was collected and stained with flow cytometry antibodies CD3 / CD56, and absolute cell counts were performed after adding absolute counting magnetic beads. As expected, knocking out β2-microglobulin (β2M) on ADR.UCAR-T activated NK cells. The experiment showed that the β2M-edited control CD19-UCAR-T cells were eliminated by allogeneic PBMCs on day 6, leading to a significant expansion of recipient NK cells, as shown in Figure 14A and B. In contrast, the absolute count results on day 6 are shown in Figure 14A and B. These results indicate that ADR loading can resist NK cell rejection and inhibit NK cell expansion, and that Anti-TIM3-UCAR-T has a stronger ability to target and eliminate NK cells than 4-1BBL-UCAR-T.

[0187] Example 10: Evaluation of the killing ability of ADR.UCAR-T on tumor cells

[0188] Previous experiments have demonstrated that ADR.UCAR-T cells have superior tumor cell-killing ability to CD19-UCAR-T cells. Therefore, this example modified the detection method: after 24 hours of incubation of target and effector cells, tumor cells were labeled with flow cytometry antibodies and absolute tumor cell counts were performed using magnetic beads. The results, as shown in Figure 15A and B, show that both ADR.UCAR-T cells had a higher killing ability against Raji than CD19-UCAR-T, with no significant difference between the two.

[0189] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that, the chimeric antigen receptor specifically binds to an antigen and inhibits pathogenic immune effector cells expressing the antigen; the antigen is selected from NCR2, NTRK2, MAL, CD70, GPR15, CD40LG, CCR8, KCNA2, IL2RA, LAG3, TNFRSF9 (4-1BB), TNFRSF18 (GITR), HAVCR2 (TIM3), LAMP1, CTLA4, PDCD1, TIGIT.

2. The chimeric antigen receptor according to claim 1, characterized in that, the antigen is selected from HAVCR2, GITR, PDCD1, LAG3 and CD70; for example, HAVCR2 or GITR; and / or, the pathogenic immune effector cells are selected from T cells and / or NK cells.

3. The chimeric antigen receptor according to claim 1 or 2, characterized in that, the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain connected in sequence; the extracellular domain is an antigen-binding fragment targeting the antigen; preferably, the intracellular signaling domain further comprises one or more co-stimulatory domains; and / or, the transmembrane domain is selected from the transmembrane domains of CD4, CD3ζ, CD8α and CD28, for example, the transmembrane domain of CD8α; and / or, the antigen-binding fragment is an scFv or a ligand-binding functional fragment of the antigen; more preferably, the antigen-binding fragment is an scFv targeting HAVCR2 or GITR; and / or, the antigen-binding fragment is the extracellular segment of CEACAM1 or GITRL; and / or, the scFv comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region are preferably connected by a linker; and / or, the co-stimulatory domain is selected from CD28, ICOS, 4-1BB, OX40, CD30, CD40, CD70, CD134, LIGHT, DAP10, CDS, ICAM-1 and CD27 or a combination thereof; and / or, the extracellular domain and the transmembrane domain are connected by a hinge region, for example, selected from the hinge regions of CD8α, CD28 and IgG; and / or, the ligand-binding functional fragment of the antigen comprises the extracellular segment of the antigen ligand.

4. The chimeric antigen receptor according to any one of claims 1-3, characterized in that, a signal peptide is further connected to the N-terminus of the chimeric antigen receptor, for example, the signal peptide of CD8, and its amino acid sequence preferably comprises the amino acid sequence shown in SEQ ID NO:19; and / or, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:1; and / or, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:2; and / or, the hinge region comprises the amino acid sequence shown in SEQ ID NO:15; And / or, when the antigen-binding fragment is an scFv targeting HAVCR2, the heavy-chain variable region of the scFv comprises CDR1 as shown in SEQ ID NO:20, CDR2 as shown in SEQ ID NO:21, and CDR3 as shown in SEQ ID NO:22, and the light-chain variable region comprises CDR1 as shown in SEQ ID NO:23, CDR2 as shown in SEQ ID NO:24, and CDR3 as shown in SEQ ID NO:25; or, when the antigen-binding fragment is a ligand-binding functional fragment targeting HAVCR2, the antigen-binding fragment is the extracellular segment of CEACAM1, for example, a protein comprising the amino acid sequence as shown in SEQ ID NO:9, or a protein encoded by an amino acid sequence formed by substitution, deletion or addition of one or more (e.g., 1-10) amino acid residues on the amino acid sequence as shown in SEQ ID NO:9, and the protein is a derivative protein having the ligand-binding function targeting HAVCR2; or comprises an amino acid sequence having at least 90% or at least 95% identity with the amino acid sequence as shown in SEQ ID NO:9 and the encoded derivative protein has the ligand-binding function targeting HAVCR2; When the antibody or its antigen-binding fragment is an scFv targeting GITR, the heavy-chain variable region of the scFv comprises CDR1 as shown in SEQ ID NO:26, CDR2 as shown in SEQ ID NO:27, and CDR3 as shown in SEQ ID NO:28, and the light-chain variable region comprises CDR1 as shown in SEQ ID NO:29, CDR2 as shown in SEQ ID NO:30, and CDR3 as shown in SEQ ID NO:31; or, when the antigen-binding fragment is a ligand-binding functional fragment targeting GITR, the antigen-binding fragment is the extracellular segment of GITRL, for example, a protein comprising the amino acid sequence as shown in SEQ ID NO:12, or a protein encoded by an amino acid sequence formed by substitution, deletion or addition of one or more (e.g., 1-10) amino acid residues on the amino acid sequence as shown in SEQ ID NO:12, and the protein is a derivative protein having the ligand-binding function targeting GITR; or comprises an amino acid sequence having at least 90% (or at least 95%) identity with the amino acid sequence as shown in SEQ ID NO:12 and the encoded derivative protein has the ligand-binding function targeting GITR; And / or, the linker comprises the amino acid sequence as shown in SEQ ID NO:3; And / or, the co-stimulatory domain comprises the amino acid sequence as shown in SEQ ID NO:17; ​ Preferably, the chimeric antigen receptor further comprises a transmembrane domain as shown in SEQ ID NO: 16; and / or an intracellular signaling domain as shown in SEQ ID NO: 18, and the intracellular signaling domain preferably further comprises the amino acid sequence as shown in SEQ ID NO:

17.

5. A polypeptide, wherein, the polypeptide comprises the chimeric antigen receptor according to any one of claims 1-4; preferably, the polypeptide further comprises a protein functional molecule; more preferably, the protein functional molecule is selected from protein tags and cytokines.

6. An isolated nucleic acid, wherein, the nucleic acid encodes the chimeric antigen receptor according to any one of claims 1-4 or the polypeptide according to claim 5.

7. An expression cassette, wherein, the expression cassette comprises a promoter and the nucleic acid according to claim 6.

8. A recombinant expression vector, wherein, the recombinant expression vector comprises the nucleic acid according to claim 6 or the expression cassette according to claim 7; preferably, the starting vector of the recombinant expression vector is selected from plasmids, cosmids, phages, retroviral vectors, lentiviral vectors and adenoviral vectors.

9. An engineered immune cell, wherein, the engineered immune cell expresses the chimeric antigen receptor according to any one of claims 1-4 or the polypeptide according to claim 5.

10. The engineered immune cell according to claim 9, wherein, the engineered immune cell is selected from one or more of T cells, NK cells, NKT cells, mast cells, macrophages, dendritic cells, CIK cells and stem cell-derived immune effector cells; preferably, the engineered immune cell is selected from T cells, NK cells and NKT cells.

11. The engineered immune cell according to any one of claims 9-10, wherein, the engineered immune cell is engineered to silence the endogenous expression selected from PD1, LAG3, TIM3, CTLA4, GITR and 4-1BB; preferably, the engineered immune cell is further engineered to inhibit or silence the expression selected from TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK and CIITA.

12. The engineered immune cell according to any one of claims 9-11, wherein, the engineered immune cell further comprises an antibody or an antigen-binding fragment thereof targeting a second antigen; Preferably, the second antigen is selected from TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, podoplanin, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, CD155, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, or a combination thereof.

13. A method for preparing immune cells, characterized in that, the method comprises introducing into the immune cells the nucleic acid according to claim 6, the expression cassette according to claim 7, the recombinant expression vector according to claim 8; preferably, the method further comprises the step of silencing the endogenous expression of PD1, LAG3, TIM3, CTLA4, GITR, and / or 4-1BB in the immune cells; more preferably, the method further comprises the step of inhibiting or silencing the expression of TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, and / or CIITA in the immune cells; even more preferably, the method further comprises the step of introducing into the immune cells or causing the immune cells to express an antibody or an antigen-binding fragment thereof targeting a second antigen; the second antigen is preferably as described in claim 12.

14. The method according to claim 13, characterized in that, the introduction, inhibition, silencing, or expression is achieved by gene editing; preferably, the gene editing is selected from CRISPR-based gene editing, zinc finger nuclease-based gene editing, TALE nuclease-based gene editing, base editors, and shRNA.

15. A pharmaceutical composition, characterized in that, the pharmaceutical composition comprises the engineered immune cells according to any one of claims 9-12, and optionally a pharmaceutically acceptable carrier and / or adjuvant.

16. A drug combination, characterized in that, the drug combination comprises the pharmaceutical composition according to claim 15 and a second therapeutic agent.

17. Use of a chimeric antigen receptor according to any one of claims 1-4, a polypeptide according to claim 5, a nucleic acid according to claim 6, an expression cassette according to claim 7, a recombinant expression vector according to claim 8, an engineered immune cell according to any one of claims 9-12, a pharmaceutical composition according to claim 15, or a drug combination according to claim 16 in the preparation of a drug for treating a disease.

18. The use according to claim 17, characterized in that, the disease is cancer or an autoimmune disease; preferably, the cancer includes solid tumors and hematological tumors; the hematological tumors are preferably selected from leukemia, lymphoma, and myeloma, such as multiple myeloma; the solid tumors are preferably selected from primary or metastatic lung cancer, breast cancer, ovarian cancer, colorectal cancer, cervical cancer, kidney cancer, liver cancer, bladder cancer, prostate cancer, pharyngeal cancer, central nervous system tumors, head and neck cancer, skin cancer, pancreatic cancer, and esophageal cancer at an early, middle, or late stage; and / or, the autoimmune disease is selected from graft rejection, graft-versus-host disease, type I diabetes, multiple sclerosis, and autoimmune colitis. More preferably, the lung cancer is non-small cell lung cancer; and / or, the colorectal cancer is colon cancer, rectal cancer or small intestine cancer; and / or, the kidney cancer is renal cell carcinoma.

19. A method for treating a disease in a subject in need thereof, characterized in that the method comprises administering to the subject an effective amount of a chimeric antigen receptor as described in any one of claims 1-4, a polypeptide as described in claim 5, a nucleic acid as described in claim 6, an expression cassette as described in claim 7, a recombinant expression vector as described in claim 8, an engineered immune cell as described in any one of claims 9-12, a pharmaceutical composition as described in claim 15 or a pharmaceutical combination as described in claim 16; Preferably, the disease is cancer or an autoimmune disease; More preferably, the cancer includes solid tumors and hematological tumors; the hematological tumors are preferably selected from leukemia, lymphoma and myeloma, such as multiple myeloma; the solid tumors are preferably selected from primary or metastatic lung cancer, breast cancer, ovarian cancer, colorectal cancer, cervical cancer, kidney cancer, liver cancer, bladder cancer, prostate cancer, pharyngeal cancer, central nervous system tumors, head and neck cancer, skin cancer, pancreatic cancer and esophageal cancer at an early, middle or late stage; and / or, the autoimmune disease is selected from graft rejection, graft-versus-host disease, type I diabetes, multiple sclerosis and autoimmune colitis; Even more preferably, the lung cancer is non-small cell lung cancer; and / or, the colorectal cancer is colon cancer, rectal cancer or small intestine cancer; and / or, the kidney cancer is renal cell carcinoma.

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