Composition and method for cellular immunotherapy

Abstract

The present application relates to a fusion protein having a function of resisting transplantation rejection, and an engineering cell for expressing the fusion protein, further relates to a method for resisting transplant immune rejection, and in particular to a method for resisting NK cell immune rejection. The present application relates to a cellular therapy.

Description

Compositions and methods of cellular immunotherapy TECHNICAL FIELD

[0001] The present application relates to a fusion protein, an engineered cell expressing the fusion protein, and a method of applying the fusion protein and the engineered cell to immune cell therapy.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of Chinese Patent Application No. CN2024104474157, filed on April 12, 2024, the contents of which are incorporated herein in their entirety.

[0004] SEQUENCE LISTING FILE SUBMITTED CONCURRENTLY

[0005] The contents of the following XML file are incorporated herein by reference in their entirety: Computer readable format (CRF) sequence listing (Name: FH01054PCT-SequenceListing.xml, Date: 2024614, Size: 68.3 KB). BACKGROUND

[0006] Due to the immunogenetic differences between the donor and the recipient, when performing exogenous donor transplantation, the donor can also be recognized and attacked by the immune cells in the body of the recipient as an exogenous graft, thereby inhibiting or eliminating the exogenous graft, resulting in host versus graft reaction (HVGR). By knocking out the MHC molecules in the graft cells, the rejection reaction of the host T cells to the graft can be effectively resisted, but it may cause rejection reactions of other immune cells in the host. For example, in allogeneic cell transplantation, the deletion of MHC class I molecules of allogeneic cells will lead to rejection reactions of NK cells in the host, enhancing the elimination of allogeneic cells. Therefore, how to effectively prevent the immune rejection reaction of host NK cells is crucial for the development of allogeneic cell transplantation therapy.

[0007] SUMMARY

[0008] It should be understood that, within the scope of the present application, each of the above technical features of the present application and the technical features specifically described below (such as examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here.

[0009] In a first aspect, a fusion protein, wherein, from N-terminus to C-terminus, it comprises an extracellular domain, a transmembrane domain, and the extracellular domain comprises a domain binding CD300A.

[0010] Further, the fusion protein further comprises an intracellular signal transduction domain.

[0011] Further, the intracellular signaling domain comprises a costimulatory signal, or a primary signaling domain, or a costimulatory signal and a primary signaling domain.

[0012] Further, the domain that binds CD300A comprises a CD300A ligand or an extracellular segment thereof, an antigen binding unit that binds CD300A.

[0013] Further, the domain that binds CD300A comprises an anti-CD300A antibody or fragment thereof.

[0014] Further, the anti-CD300A antibody or fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0015] Further, the VH and VL have, respectively, (1) the amino acid sequences set forth in SEQ ID NOs: 2 and 1.

[0016] Further, the anti-CD300A antibody is selected from the group consisting of: a whole antibody, an scFv, a single domain antibody, a Fab fragment, a Fab’ fragment, a Fv fragment, a F(ab’)2 fragment, a Fd fragment, a sdAb, a multi-functional antibody, a DDPP antibody, an scFv-Fc antibody, or an IgG4 antibody.

[0017] Further, the anti-CD300A antibody is an scFv.

[0018] Further, the anti-CD300A antibody is a single domain antibody.

[0019] Further, the anti-CD300A antibody is a DDPP antibody.

[0020] Further, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif or signaling motif of an ITAM.

[0021] Further, the intracellular signaling domain comprises an intracellular signaling domain selected from the group consisting of: TCRa, TCRp, TCRy, TCRd, CD3y, CD3d, CD3s, CD3z, CD5, CD22, CD79a, CD79b, CD278, or CD66d, or a combination thereof.

[0022] Further, the intracellular signaling domain comprises a CD3z intracellular signaling domain (SEQ ID NO: 10).

[0023] Further, the intracellular signaling domain comprises only a costimulatory domain.

[0024] Further, wherein the co-stimulatory domain is selected from the intracellular signaling domain of CD137 (4-1BB), CD28, CD27, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, or CD83, or a combination thereof.

[0025] Further, wherein the co-stimulatory domain is selected from the intracellular signaling domain of CD137 (4-1BB) (SEQ ID NO: 8) or CD28 (SEQ ID NO: 7).

[0026] Further, wherein the transmembrane domain of the fusion protein is selected from the transmembrane domain of CD2, CD3 epsilon, CD3 delta, CD3 zeta, CD8, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcR alpha, FcR beta, FcR gamma, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRP alpha, pT alpha, TCR alpha, TCR beta, TIM3, TRIM, LPA5, PDGFR, or Zap70.

[0027] Further, wherein the transmembrane domain is the transmembrane domain of CD28 (SEQ ID NO: 6).

[0028] Further, wherein the transmembrane domain is the transmembrane domain of CD8 (SEQ ID NO: 4).

[0029] Further, wherein the extracellular domain and the transmembrane domain are connected by a connecting polypeptide.

[0030] Further, wherein the connecting polypeptide is selected from the hinge region of CD28, the hinge region of CD8, an IgG spacer or fragment thereof, or a combination thereof.

[0031] Further, the connecting polypeptide is a CD8 hinge region (SEQ ID NO: 9).

[0032] Further, the transmembrane domain is a PDGFR transmembrane domain (SEQ ID NO: 3).

[0033] Further, the connecting polypeptide is an IgG4 spacer region (SEQ ID NO: 14).

[0034] Further, the fusion protein further comprises a domain that binds to a target antigen on a pathological cell, or further comprises a domain that binds to another NK cell marker.

[0035] Further, the pathological cell is selected from a malignant cell or an infected cell.

[0036] Further, the pathological cell is selected from a solid tumor cell, a hematological tumor cell, a pathological cell of an autoimmune disease.

[0037] Further, the solid tumor is selected from: esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, sarcoma; the hematological tumor is selected from: leukemia, lymphoma, myeloma; the autoimmune disease is selected from: multiple sclerosis, autoimmune liver disease, type I diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), polymyositis / dermatomyositis.

[0038] Further, the target antigen is selected from: CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin 18.2, FAP, Mesothelin, NKG2D ligand, NKG2A, CD94, FCRH5, EGFR and its mutants, or a combination thereof.

[0039] In a second aspect, an engineered cell is provided, wherein the engineered cell comprises the fusion protein of the first aspect.

[0040] Further, the engineered cell further comprises a chimeric receptor 1 that binds to a target antigen on a pathological cell; or the engineered cell further comprises a chimeric receptor 2 that binds to another NK cell marker.

[0041] Further, wherein the pathological cells are selected from malignant cells or infected cells.

[0042] Further, wherein the pathological cells are selected from solid tumor cells, blood tumor cells, pathological cells of autoimmune diseases.

[0043] Further, wherein the solid tumor is selected from: esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, sarcoma; the blood tumor is selected from: leukemia, lymphoma, myeloma; the autoimmune disease is selected from: multiple sclerosis, autoimmune liver disease, type I diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), polymyositis / dermatomyositis.

[0044] Further, wherein the target antigen is selected from: CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin 18.2, FAP, Mesothelin, NKG2D ligand, NKG2A, CD94, FCRH5, EGFR and its mutants, or a combination thereof.

[0045] Further, wherein the engineered cell further comprises: endogenous TCR, B2M low expression or no expression.

[0046] Further, wherein the engineered cell further comprises: endogenous TCR and no expression of B2M.

[0047] Further, wherein the engineered cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, a stem cell, or a combination thereof.

[0048] Further, wherein the immune cell is an autologous or allogeneic cell selected from: a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a T cell, a NKT cell, a stem cell-derived immune effector cell, or a combination thereof.

[0049] Further, wherein the engineered cell is an allogeneic T cell.

[0050] Further, wherein the engineered cell has an extended survival time or an enhanced expansion capacity in the presence of host immune cells.

[0051] Further, the engineered cell has inhibitory or killing function on immune cells of the host.

[0052] Further, the engineered cell has inhibitory or killing function on T cells, NK cells of the host.

[0053] Further, the engineered cell can enhance survival, proliferation of another engineered cell introduced into the subject before, simultaneously, or after, and killing of pathological cells by the another engineered cell.

[0054] In a third aspect, a polynucleotide is provided, which is a nucleic acid molecule encoding the fusion protein constructed according to the first aspect.

[0055] In a fourth aspect, a vector is provided, which comprises the polynucleotide according to the third aspect.

[0056] In a fifth aspect, a virus is provided, which comprises the vector according to the fourth aspect.

[0057] In a sixth aspect, a pharmaceutical composition is provided, which comprises an effective amount of the fusion protein according to the first aspect, the engineered cell according to the second aspect, the polynucleotide according to the third aspect, the vector according to the fourth aspect, or the virus according to the fifth aspect, and a pharmaceutically acceptable carrier.

[0058] In a seventh aspect, the fusion protein according to the first aspect, or the engineered cell according to the second aspect, is used for preparing a medicament for preventing or resisting immune rejection of transplantation.

[0059] In an eighth aspect, the fusion protein according to the first aspect, or the engineered cell according to the second aspect, is used for preventing or resisting immune rejection of transplantation.

[0060] In a ninth aspect, the fusion protein according to the first aspect, or the engineered cell according to the second aspect, is used for preparing a medicament for treating autoimmune diseases or inflammatory diseases.

[0061] In a tenth aspect, the fusion protein according to the first aspect, or the engineered cell according to the second aspect, is used for treating, preventing, or ameliorating autoimmune diseases or inflammatory diseases in a subject in need thereof.

[0062] Further, the autoimmune disease is selected from the group consisting of multiple sclerosis, autoimmune liver disease, type I diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), and polymyositis / dermatomyositis.

[0063] In an eleventh aspect, the fusion protein of the first aspect or the engineered cell of the second aspect is used for preparing a medicament for treating a tumor.

[0064] In a twelfth aspect, the fusion protein of the first aspect or the engineered cell of the second aspect is used for treating, preventing or ameliorating a tumor in a subject in need thereof.

[0065] Further, the tumor is a solid tumor or a hematological tumor.

[0066] Further, the solid tumor is selected from the group consisting of esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, renal cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, sarcoma; and the hematological tumor is selected from the group consisting of leukemia, lymphoma, myeloma.

[0067] The present application also provides:

[0068] In a first aspect, the present application provides a fusion protein, wherein from N-terminus to C-terminus, it comprises an extracellular domain, a transmembrane domain, and the extracellular domain comprises a CD300A binding domain.

[0069] Preferably, the fusion protein further comprises an intracellular signal transduction domain; preferably, the intracellular signal transduction domain comprises a costimulatory signal, or a primary signal domain, or a costimulatory signal and a primary signal domain.

[0070] Preferably, the CD300A binding domain comprises a CD300A ligand or an extracellular segment thereof, an antigen binding unit binding CD300A; preferably, the CD300A binding domain comprises an anti-CD300A antibody or a fragment thereof; preferably, the anti-CD300A antibody is selected from the group consisting of a whole antibody, an scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, an sdAb, a multifunctional antibody, a DDPP antibody, an scFv-Fc antibody, an IgG4 antibody, a hybridoma antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a monoclonal antibody; preferably, the anti-CD300A antibody or the fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL); more preferably, wherein the VH and VL have the amino acid sequences shown in SEQ ID NO: 2 and 1, respectively.

[0071] Preferably, the intracellular signaling domain comprises an intracellular signaling domain selected from the group consisting of TCRa, TCRp, TCRy, TCR5, FcRy, FcRp, CD3y, CD35, CD3e, CD3z, CD5, CD22, CD79a, CD79b, CD278, or CD66d, or a combination thereof; preferably, the intracellular signaling domain comprises a CD3z intracellular signaling domain (SEQ ID NO: 10, 36); preferably, the intracellular signaling domain comprises only a costimulatory domain; preferably, the costimulatory domain is selected from the group consisting of an intracellular signaling domain of CD137 (4-1BB), CD28, CD27, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, CARD11, CD30, CD54, OX40, CD150, CD152, CD223, CD270, PD-L2, PD-L1, DAP10, LAT, NKD2C, SLP76, TRIM, FceRIy, MyD88, ICAM-1, LFA-1 (CD11a / CD18), 41BBL, or CD83, or a combination thereof; preferably, the costimulatory domain is selected from the group consisting of an intracellular signaling domain of CD137 (4-1BB) (SEQ ID NO: 8) or CD28 (SEQ ID NO: 7).

[0072] Preferably, the transmembrane domain of the fusion protein is selected from the group consisting of: CD2, CD3 epsilon, CD3 delta, CD3 zeta, CD8, CD9, CD16, CD22, CD25, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD154, CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcR alpha, FcR beta, FcR gamma, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRP alpha, pT alpha, TCR alpha, TCR beta, TIM3, TRIM, LPA5, PDGFR, or Zap70.

[0073] Preferably, the transmembrane domain is a transmembrane domain of CD28 (SEQ ID NO: 6 or 29), a CD8 transmembrane domain (SEQ ID NO: 4), or a PDGFR transmembrane domain (SEQ ID NO: 3, 41, 42, 43, 46, 47, or 48).

[0074] Preferably, the extracellular domain and the transmembrane domain are connected by a connecting polypeptide; preferably, the connecting polypeptide is selected from the group consisting of: a CD28 hinge region, a CD8 hinge region, an IgG spacer or a fragment thereof, or a combination thereof; preferably, the connecting polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 9, 14, 37, or 44.

[0075] Preferably, the fusion protein comprises an amino acid sequence as set forth in SEQ ID NO: 1, 2 and 3, or comprises an amino acid sequence as set forth in SEQ ID NO: 1, 2 and 47, or comprises an amino acid sequence as set forth in SEQ ID NO: 1, 2 and 48, or comprises an amino acid sequence as set forth in SEQ ID NO: 1, 2 and 41, or comprises an amino acid sequence as set forth in SEQ ID NO: 1, 2, 44 and 43, or comprises an amino acid sequence as set forth in SEQ ID NO: 1, 2, 44 and 46, or comprises an amino acid sequence as set forth in SEQ ID NO: 1, 2, 44 and 42, or comprises an amino acid sequence as set forth in SEQ ID NO: 1, 2, 9 and 43, or comprises an amino acid sequence as set forth in SEQ ID NO: 1, 2, 9 and 46, or comprises an amino acid sequence as set forth in SEQ ID NO: 1, 2, 9 and 42.

[0076] Preferably, the fusion protein comprises an amino acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence as set forth in any one of SEQ ID NO: 16, 18 or 21; or the fusion protein comprises an amino acid sequence encoded by a nucleic acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence as set forth in any one of SEQ ID NO: 15, 17, 19, 20, 24, 25, 26 or 38; or the fusion protein is an amino acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence as set forth in any one of SEQ ID NO: 16, 18 or 21; or the fusion protein is encoded by a nucleic acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence as set forth in any one of SEQ ID NO: 15, 17, 19, 20, 24, 25, 26 or 38.

[0077] Preferably, the fusion protein further comprises a domain that binds to a target antigen on a pathological cell, or further comprises a domain that binds to another NK cell marker.

[0078] The second aspect of the present application provides an engineered cell, wherein the engineered cell comprises the fusion protein of the first aspect.

[0079] Preferably, the engineered cell further comprises a chimeric receptor 2 that binds to a target antigen on a pathological cell; or the engineered cell further comprises a chimeric receptor 1 that binds to another NK cell marker.

[0080] Preferably, said pathological cells are selected from malignant cells or infected cells; preferably, said pathological cells are selected from solid tumor cells, blood tumor cells, pathological cells of autoimmune diseases; preferably, said solid tumor is selected from: esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, sarcoma; blood tumor is selected from: leukemia, lymphoma, myeloma; autoimmune diseases are selected from: multiple sclerosis, autoimmune liver disease, type I diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), polymyositis / dermatomyositis.

[0081] Preferably, said target antigen is selected from: CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin 18.2, FAP, Mesothelin, NKG2D ligand, NKG2A, CD94, FCRH5, EGFR and its mutants, ASGPR1, IL13RA2, WT1, CLL1 or a combination thereof.

[0082] Preferably, the other NK cell marker is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD138, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A), CD159c (NKG2C), FasL, NKG2E, CD279, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRa, TCRb, TIGIT, TRAIL, SLAMF7, NKG2F, NKG2H, NKp30, NKp44, NKp46, NKp80, SLAM family members, L-selectin, natural cytotoxicity receptors NCR1, NCR2, NCR3, or a combination thereof; preferably, the chimeric receptor 1 comprises an amino acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in any one of SEQ ID NOs: 27, 28, 39, or 45; or the chimeric receptor 1 is an amino acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in any one of SEQ ID NOs: 27, 28, 39, or 45.

[0083] Preferably, the chimeric receptor 2 comprises an amino acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in any one of SEQ ID NOs: 30, 31, 32, 33, 39, 40, or 45; or the chimeric receptor 2 is an amino acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in any one of SEQ ID NOs: 30, 31, 32, 33, 39, 40, or 45.

[0084] Preferably, the engineered cell comprises an amino acid sequence encoded by a nucleic acid sequence having 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 15, 17, 19, 20, 24, 25, 26, or 38; or the fusion protein is an amino acid sequence having 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 16, 18, or 21; or the fusion protein is encoded by a nucleic acid sequence having 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 15, 17, 19, 20, 24, 25, 26, or 38; or the engineered cell comprises: an amino acid sequence of SEQ ID NOs: 21 and 27; an amino acid sequence of SEQ ID NOs: 18 and 28; an amino acid sequence of SEQ ID NOs: 18, 28, and 39; an amino acid sequence of SEQ ID NOs: 18 and 40; an amino acid sequence of SEQ ID NOs: 18 and 30; an amino acid sequence of SEQ ID NOs: 18 and 31; or an amino acid sequence of SEQ ID NOs: 18, 32, and 33.

[0085] Preferably, the engineered cell further comprises: low or no expression of endogenous TCR / B2M, TCR / B2M / FAS, TCR / B2M / NKG2A, or TCR / B2M / FAS / NKG2A; or the engineered cell is low or no expression of endogenous TCR / B2M, TCR / B2M / FAS, TCR / B2M / NKG2A, or TCR / B2M / FAS / NKG2A.

[0086] Preferably, the engineered cell is an autologous or allogeneic cell selected from the group consisting of: an immune cell, a neuron, an epithelial cell, an endothelial cell, a stem cell, or a combination thereof; preferably, the engineered cell is selected from the group consisting of: a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a T cell, a NKT cell, a stem cell-derived immune effector cell, or a combination thereof; preferably, the engineered cell is an allogeneic T cell.

[0087] Preferably, the engineered cell has an extended survival time or an enhanced expansion capacity in the presence of a host immune cell; preferably, the host immune cell is an NK cell.

[0088] Preferably, the engineered cell is capable of enhancing the survival, proliferation, and killing of pathological cells of another engineered cell introduced prior to, simultaneously with, or after the engineered cell into a subject.

[0089] The third aspect of the present application provides a polynucleotide, which is a nucleic acid molecule encoding the fusion protein of the first aspect, or a nucleic acid molecule encoding the fusion protein, the chimeric receptor 1 and / or the chimeric receptor 2 comprised by the engineered cell of the second aspect.

[0090] The fourth aspect of the present application provides a vector comprising the polynucleotide of the third aspect.

[0091] The fifth aspect of the present application provides a virus comprising the vector of the fourth aspect.

[0092] The sixth aspect of the present application provides a pharmaceutical composition comprising an effective amount of the fusion protein of the first aspect, the engineered cell of the second aspect, the polynucleotide of the third aspect, the vector of the fourth aspect, or the virus of the fifth aspect, and a pharmaceutically acceptable carrier.

[0093] The fusion protein of the first aspect, the engineered cell of the second aspect, is used for preparing a drug for preventing or resisting immune rejection of transplantation, or for preventing or resisting immune rejection of transplantation, or for preparing a drug for treating autoimmune diseases or inflammatory diseases, or for treating, preventing or ameliorating autoimmune diseases or inflammatory diseases in a subject in need, or for preparing a drug for treating tumors, or for treating, preventing or ameliorating tumors in a subject in need.

[0094] Preferably, the autoimmune disease is selected from the group consisting of multiple sclerosis, autoimmune liver disease, type I diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), polymyositis / dermatomyositis.

[0095] Preferably, the tumor is a solid tumor or a hematological tumor; preferably, the solid tumor is selected from the group consisting of esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, sarcoma; the hematological tumor is selected from the group consisting of leukemia, lymphoma, myeloma. BRIEF DESCRIPTION OF DRAWINGS

[0096] FIGS. 1A-1B show the expression of CD300A in primary NK (FIG. 1A), activated NK (aNK, FIG. 1B).

[0097] FIG. 2 shows a schematic diagram of the structure of the CD300A fusion protein.

[0098] FIG. 3A-3B show the CAR positive rate of CD300A-28z (FIG. 3A), and the percentage of CD300A-28z-dko cells after magnetic bead sorting (FIG. 3B).

[0099] FIG. 4A-4B show that CD300A-28z-dko cells can kill aNK (FIG. 4A) and resist aNK killing and improve T cell survival (FIG. 4B) at an effector to target ratio of 1:1.5 and 1:5.

[0100] FIG. 5A-5B show that CD300A-28z-dko cells can kill aNK (FIG. 5A) and resist aNK killing and improve T cell survival (FIG. 5B) at an effector to target ratio of 1:1.5 and 1:5.

[0101] FIG. 6A-6B show the CD300A-PDGFR positive rate in the prepared T cells (FIG. 6A), and the percentage of CD300A-PDGFR-dko cells after magnetic bead sorting (FIG. 6B).

[0102] FIG. 7A-7B-7C show that CD300A-PDGFR-dko cells significantly resist killing by human primary NK cells and improve T cell survival (FIG. 7A) at an effector to target ratio of 1:2, CD300A-PDGFR-dko cells do not affect the survival of human primary NK cells (FIG. 7B), and CD300A-PDGFR-dko cells can significantly resist killing by human primary NK cells and improve T cell survival (FIG. 7C) compared to TIM3-PDGFR-dko.

[0103] FIG. 8A-8B-8C show that CD300A-PDGFR-dko cells can significantly resist killing by aNK cells and improve T cell survival (FIG. 8A) at an effector to target ratio of 1:1.5, CD300A-PDGFR-dko cells do not affect the survival of aNK cells (FIG. 8B), and CD300A-PDGFR-dko does not affect the survival of human aNK cells but can significantly resist killing by aNK cells and improve T cell survival (FIG. 8C) compared to TIM3-PDGFR-dko at an effector to target ratio of 1:2.

[0104] FIG. 9A-9B show that CD300A / FasL-CAR-T-dko cells do not affect human primary NK cell survival, but can significantly resist primary NK cell killing, increase T cell survival (FIG. 9A) at effector to target ratio 1:2 co-incubation; CD300A / NKG2A-CAR-T-tko1 cells significantly kill aNK cells, and can significantly resist aNK cell killing, increase T cell survival (FIG. 9B top) at effector to target ratio 1:2 co-incubation; CD300A / CD38-NKG2A-CAR-T-tko1 cells can significantly resist aNK cell killing, increase T cell survival (FIG. 9B bottom) at effector to target ratio 1:1 or 1:2.

[0105] FIG. 10A-10B show that CD300A / BCMA-NKG2A-CAR-T-tko1 cells can significantly resist aNK killing, increase T cell survival (FIG. 10A) at effector to target ratio 1:1 or 1:2 co-incubation; CD300A / BCMA-NKG2A-CAR-T-tko1 cells can significantly resist primary NK killing, increase T cell survival (FIG. 10B) at effector to target ratio 1:2 co-incubation.

[0106] FIG. 11 shows that CD300A / CD19-CD20-CAR-T-dko cells can significantly resist aNK killing, increase T survival (FIG. 11) at effector to target ratio 1:1 or 1:2 co-incubation, compared to CD19-CD20-BBZ-dko. DETAILED DESCRIPTION

[0107] The present application relates to a cell binding CD300A with anti-transplant rejection function, and to a method for resisting anti-transplant immune rejection.

[0108] Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of gene therapy, biochemistry, genetics, and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, with the methods and materials being described herein as suitable for that purpose. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations and modifications will become apparent to those skilled in the art once the application is better understood in connection with the following detailed description. The present application is intended to cover any and all variations of the specific embodiments described herein which become apparent to those skilled in the art and fall within the scope of the application. The application is not limited in scope by the exemplified embodiments which are intended as illustrations only. Equivalent methods and components can be substituted for those illustrated and this application is intended to embrace modifications and variations of these specific embodiments along with all of their equivalents. The application is intended to cover all alternatives, modifications and equivalents.

[0109] The practice of the present application will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature.

[0110] In this application, descriptions in terms of a range format are used as shorthand for describing endpoints that are included in the range. Unless otherwise stated, the description in terms of a range format is also intended to include ranges using fractions, decimals, and endpoints. For example, when describing a range that includes 3-5, it is intended to disclose also 3-4, 3.1-4.9, 3.15, 4, and 5, individually, and any other subsets of these ranges, as well as the individual endpoints 3 and 5.

[0111] Definitions

[0112] The term "about" refers to the usual error range of values well known to those skilled in the art. Reference to "about" a value or parameter herein includes embodiments that are directed to that value or parameter per se. For example, description of "about X" includes description of "X." In this context, "about" can indicate an acceptable range of error for the described technology. For example, "about" a value or parameter can mean a range of values plus or minus 10% of that value, e.g., about 5 uM can include any number between 4.5 uM and 5.5 uM.

[0113] The term "CD300A" (Gene ID: 11314) is a CD300 family protein, a class of transmembrane glycoproteins. Consists of an IgV-like extracellular segment, a transmembrane region, and an intracellular segment. The natural receptor of CD300A is phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell membrane. CD300A is mainly expressed on cells such as NK cells, DC cells, monocytes, etc.

[0114] The term "PDGFR" refers to platelet-derived growth factor receptor. PDGFR receptor has two components, PDGFR-A and PDGFR-B. PDGFRA (Gene ID: 5156) is the alpha subunit of PDGFR, and PDGFRB (Gene ID: 5159) is the beta subunit of PDGFR. The receptor is a type I transmembrane protein, consisting of an extracellular domain, a transmembrane region, and an intracellular segment. In one example, PDGFR refers to PDGFR-A. In one example, PDGFR refers to PDGFR-B. For example, PDGFR transmembrane includes the amino acid sequence set forth in SEQ ID NO: 3, 41, 42, 43, 46, 47, or 48.

[0115] The term "host versus graft reaction (HVGR)" generally refers to: due to the immunogenetic differences between the donor and the recipient (or called host), when performing exogenous donor transplantation, the donor as the exogenous graft is recognized and attacked by the immune cells (such as NK cells) in the host, and then inhibited or cleared by the host immune cells.

[0116] The term "graft versus host disease (GVHD)" generally refers to: due to the diversity of TCR of the exogenous transplantation donor T lymphocytes, and the incompatibility with the host HLA molecules, the donor T lymphocytes recognize the antigens on the normal tissues of the host, amplify and release a series of cytokines, and attack the host cells.

[0117] The term "antibody" is used herein in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), domain antibodies, and antibody fragments thereof that can specifically bind to an antigen or antigenic determinant, so long as they exhibit the desired antigen-binding activity. The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to (i) a Fab fragment consisting of VL, VH, CL, and CH1 domains, including Fab' and Fab'-SH, (ii) a Fd fragment consisting of VH and CH1 domains, (iii) a Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a single variable region; (v) a F(ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vi) a single chain Fv molecule, a monospecific antibody fragment; (vii) a bispecific single chain Fv dimer; (viii) a "diabody" or "triabody", a multivalent or multispecific fragment constructed by gene fusion; and (ix) an scFv genetically fused to the same or different antibody. For example, the antibody is selected from the group consisting of: whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, sdAb, multifunctional antibody, scFv-Fc antibody, or IgG4 antibody. In an example, the antibody of the present application comprises a typical antibody, scFv, and combinations thereof, wherein, for example, a DDpp is covalently linked (e.g., by a peptide bond or by a chemical linker) to the N-terminus of the heavy chain and / or light chain of a typical intact (full-length) antibody, or inserted into the H chain and / or L chain of the full-length antibody. For example, the DDpp antibody recognizing CD300A is prepared according to the preparation method of CN111727250A.

[0118] The terms "recognize", "bind", "target" are used interchangeably and refer to selective binding to a target antigen. For example, binding to a target cell refers to binding to a target antigen (e.g., a molecule of interest) on a target cell.

[0119] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are contiguous (e.g., joined by a synthetic linker, such as a short, flexible polypeptide linker), and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody comprising the scFv. Unless specified, as used herein, an scFv can have the VL and VH variable regions in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), an scFv can comprise VL-linker-VH or can comprise VH-linker-VL. The antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody. These fragments are collectively referred to as "antigen binding fragments." The term "antigen binding fragment" also includes any molecular structure with polypeptide chains that has a specific shape that is suitable and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope.

[0120] The term "DDpp" refers to a target-binding D-domain (DD) polypeptide based on a non-traditional antibody structural scaffold. D-domain polypeptides (DDpp) are characterized by high target-binding affinity and a non-antibody structural scaffold. DDpp can be monovalent or multivalent. In some embodiments, DDpp is monospecific or multispecific. In further embodiments, DDpp is monospecific and multivalent. In other embodiments, DDpp is multispecific and multivalent. See CN111727250A for more information.

[0121] The term "variable region or variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of an antibody to an antigen. The variable domains of the heavy chain (VH) and light chain (VL) of a native antibody generally have similar structures, with each domain comprising four conserved FRs and three CDRs. An individual VH or VL domain can confer antigen binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated using a library of complementary VL or VH domains from antibodies that bind the antigen to screen for a library of complementary VL or VH domains.

[0122] The term "hypervariable region" or "complementarity determining region" or "CDR" refers to each of the regions of an antibody variable domain that exhibit significant sequence variability and / or form structurally defined loops ("hypervariable loops") and / or contain residues involved in antigen contact ("antigen contacts"). Generally, antibodies comprise six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3).

[0123] The term "Fc region" or "Fc" is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0124] "Framework" (FR) refers to the variable domain residues other than the hypervariable sequence (CDR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. CDR and FR sequences in a VH (or VL) generally appear in the following order: FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

[0125] Unless otherwise indicated, in this document, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered according to Kabat et al.

[0126] The term "native antibody" refers to naturally occurring immunoglobulin molecules with various structures. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), which is also referred to as a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), which is also referred to as a variable light domain or a light chain variable domain, followed by a light chain constant (CL) domain. The light chains of antibodies can be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequence of their constant domains.

[0127] The terms "whole antibody," "full-length antibody," "intact antibody" are used interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that contains an Fc region as defined herein or includes a complete full-length antibody having an antigen binding region.

[0128] The term "single domain antibody" (sdAb) can also be referred to as "VHH," or "VHH polypeptide," comprises one variable VHH domain responsible for antigen recognition. Antigen binding by a VHH domain is mediated by three CDRs flanked by four relatively constant framework regions (FRs). A class of antibodies that lack the light chain and have only the heavy chain variable region are referred to as Nanobodies due to their small molecular weight. In certain embodiments, a VHH can be truncated at the N- or C-terminus such that it contains only part of FR1 and / or FR4, or lacks one or both of those framework regions, as long as the VHH substantially maintains antigen binding and specificity.

[0129] The term "single domain antibody" refers to a heavy chain variable domain, or all or a portion of a light chain variable domain, comprising all or a portion of an antibody. A single domain antibody can be a human single domain antibody.

[0130] The terms "monoclonal antibody," "monoclonal" refer to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical and / or bind the same epitope, except for possible variants that can arise during production of the monoclonal antibody preparation, such variants are typically minor in nature. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant of the antigen. Thus, the designation "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and transgenic animals containing all or a portion of immunoglobulin loci that in response to an antigen receptor generate human antibodies.

[0131] The term "fully human antibody" or "fully human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or from an antibody derived from a non-human source that utilizes a human antibody repertoires or other human antibody encoding sequences. The definition of fully human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Fully human antibodies can be generated by phage display technology. Fully human antibodies can be produced by engineered strains and / or engineered cells.

[0132] The term “fusion polypeptide” or “fusion protein” refers to a fusion molecule that links DNA fragments, or the corresponding cDNAs or peptide segments of proteins, of different origins. For example, the fusion protein includes any protein or protein domain that binds to CD300A, including a natural protein or domain, or an artificially synthesized protein or domain. For example, the fusion protein includes a secreted protein that binds to CD300A. For example, the fusion protein includes a bispecific molecule or a multispecific molecule that binds to CD300A. For example, the fusion protein includes a membrane protein that binds to CD300A. For example, the fusion protein includes a synthetic binding domain that binds to CD300A. For example, the fusion protein includes a D-domain polypeptide (DDpp, CN111727250A). For example, the fusion protein includes an antibody or a fragment thereof that binds to CD300A. For example, the fusion protein includes an extracellular domain, a transmembrane domain, and an intracellular domain. For example, the fusion protein includes, but is not limited to, a chimeric antigen receptor (CAR), a recombinant TCR receptor. For example, the extracellular domain of the fusion protein includes a binding domain that binds to CD300A. For example, the binding domain includes a CD300A antibody. For example, the binding domain includes a synthetic binding domain that binds to CD300A. For example, the binding domain includes an artificially synthesized D-domain polypeptide that binds to CD300A. For example, the extracellular domain further includes a linker polypeptide. For example, the linker polypeptide includes a multimerization domain. For example, the linker polypeptide includes a hinge region / spacing region. For example, the linker polypeptide includes a CD8 hinge region, a CD28 hinge region, or an IgG spacing region. For example, the linker polypeptide includes an IgG1, 2, 3, or 4 spacing region or a fragment thereof. For example, the transmembrane domain is selected from the group consisting of a transmembrane domain of CD2, CD3 epsilon, CD3 delta, CD3 zeta, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcR alpha, FcR beta, FcR gamma, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRP alpha, pT alpha, TCR alpha, TCR beta, TIM3, TRIM, LPA5, PDGFR, or Zap70.For example, the endodomain comprises an intracellular signaling domain of CD3 epsilon, CD3 delta, CD3 zeta, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD278 (ICOS), CD357 (GITR), CARD11, DAP10, DAP12, FcR alpha, FcR beta, FcR gamma, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pT alpha, TCR alpha, TCR beta, TRIM, Zap70, PTCH2, or a combination thereof.

[0133] The term "variant" with respect to a protein or polypeptide having a specified sequence characteristic ("control protein" or "reference polypeptide") denotes a different protein or polypeptide having one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions as compared to the control protein or reference polypeptide. The change in the amino acid sequence can be an amino acid substitution. The change in the amino acid sequence can be a conservative amino acid substitution. A functional fragment or functional variant of a protein or polypeptide maintains the essential structural and functional properties of the control protein or polypeptide.

[0134] The term "cell marker", also known as cell surface molecule, cell surface protein, is a molecule that is preferably present on the membrane surface of an immune cell. For example, "marker of T cells and / or NK cells" refers to a marker that is present on T cells or NK cells, respectively, or on both T cells and NK cells, including but not limited to: CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD160, CD161 CD178, CD218, CD226, CD244, CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, SLAMF7, TIGIT, FasL. For example, an NK cell marker is selected from the group consisting of: the NKG2 receptor family, the killer immunoglobulin-like receptor (KIR) family, the natural cytotoxicity receptors (NCR), and / or other antigens specifically expressed by NK cells. The NKG2 receptor family includes NKG2A, NKG2D, NKG2C. The KIR family includes KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1. The NCR includes NKP30, NKP44, NKP46, NKp80. Other antigens specifically expressed by NK cells include CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161, TIGIT, CS1, IL-15R, FasL.

[0135] The term “NK inhibitory receptor (NKIR),” refers to a class of receptors on NK cells that can transduce a killing inhibitory signal, inhibiting the killing function of the NK cell. Includes HLA-specific and non-HLA-specific inhibitory receptors. NKIRs include, but are not limited to, having an immunoreceptor tyrosine-based inhibitory motif (ITIM). For example, NKIRs include: an NKG2 / CD94 component, a KIR family member, a LIR family member, an NKR-P1 family member, an immune checkpoint receptor, an immune checkpoint inhibitor, a SIGLEC family member, a Ly49 family member, or a combination thereof. For example, the NKG2 / CD94 component is selected from the group consisting of NKG2A, NKG2C, and CD94. For example, the KIR family member is selected from the group consisting of KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3. For example, the LIR family member is selected from the group consisting of LIR1, LIR2, LIR3, LIR5, and LIR8. For example, the NKR-P1 family member is selected from the group consisting of NKR-P1B and NKR-P1D. For example, the immune checkpoint inhibitor includes: (a) one or more antagonists of checkpoint molecules comprising PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR; (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lifirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof; or (c) at least one of atezolizumab, nivolumab, and pembrolizumab, or comprising one or more of venetoclax, azacitidine, and pomalidomide. For example, the immune checkpoint receptor is selected from the group consisting of PD-1, TIGIT, CD96, TIM3, and LAG3. For example, the SIGLEC family member is selected from the group consisting of SIGLEC7 and SIGLEC9. For example, the Ly49 family member is selected from the group consisting of Ly49A, Ly49C, Ly49F, Ly49G1, and Ly49G4.

[0136] The term "pathological cell" generally refers to any type of cell that is present in a patient that is believed to cause a worsening of the health condition, or to a malignant or infected cell that needs to be reduced or eliminated for remission of the patient. The present application relates to methods of new adoptive immunotherapy strategies for treating diseases associated with the development of pathological cells, such as cancer (tumors), infections and autoimmune diseases.

[0137] The term "tumor antigen" refers to an antigen that is newly appeared or overexpressed during the development and progression of a hyperproliferative disease. In certain aspects, the hyperproliferative disorder of the present application refers to cancer. The hyperproliferative disorder is referred to as cancer or tumor. Tumor antigens include solid tumor antigens, hematological tumor antigens. The tumor described in the present application is a solid tumor, and can also be a hematological tumor.

[0138] The term "chimeric receptor" refers to a fusion molecule that is formed by linking DNA fragments or corresponding cDNAs of proteins of different origins using genetic recombination technology, including an extracellular domain, a transmembrane domain and an intracellular domain. The chimeric receptor includes but is not limited to: a chimeric antigen receptor (CAR), a recombinant TCR receptor.

[0139] The term "chimeric antigen receptor" (CAR) includes at least one extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain. The intracellular signaling domain includes a functional signaling domain of a stimulatory molecule and / or a costimulatory molecule. For example, the stimulatory molecule is a zeta chain associated with the T cell receptor complex (e.g., CD3Z). For example, the intracellular signaling domain further includes a functional signaling domain of one or more costimulatory molecules, such as 4-1BB (i.e., CD137), CD27 and / or CD28. For example, the polypeptides are linked to each other. For example, the intracellular signaling domain (or referred to as the structural region) can be selected from the intracellular costimulatory domain of any one or more of the following polypeptides: CD27, CD28, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, CD83.

[0140] The term "recombinant T cell receptor (recombinant TCR)" includes one or more chimeric receptors derived from TCR subunits. For example, a recombinant TCR includes at least a portion of an extracellular domain, a transmembrane domain, and an intracellular domain of a TCR subunit operably linked to an antigen binding domain. For example, a TCR subunit in a recombinant TCR includes a CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, TCR alpha, TCR beta, TCR gamma, and / or TCR delta subunit. For example, a recombinant TCR can integrate into a TCR / CD3 complex expressed on a T cell. For example, a recombinant TCR includes a constant region and an intracellular domain of a TCR alpha and TCR beta subunit, and the constant region of the subunits is operably linked to an antigen binding domain. For example, a recombinant TCR includes a constant region and an intracellular domain of a TCR gamma and TCR delta subunit, and the constant region of the subunits is operably linked to an antigen binding domain. For example, a recombinant TCR includes a CD3 zeta, CD3 epsilon, CD3 gamma, or CD3 delta subunit, and the extracellular domain of the subunit is operably linked to an antigen binding domain.

[0141] The term "primary signaling domain" or "primary signaling domain" regulates the initial activation of a TCR complex in a stimulatory manner. In one aspect, a primary signaling domain is triggered by, for example, the binding of a TCR / CD3 complex to a peptide-loaded MHC molecule, thereby mediating a T cell response (including but not limited to, proliferation, activation, differentiation, etc.). A primary signaling domain that acts in a stimulatory manner can include an immunoreceptor tyrosine-based activation motif or an ITAM signaling motif. For example, fragments of a primary signaling domain that include an ITAM include, but are not limited to, an intracellular signaling domain derived from CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD5, CD79a, CD79b, CD278, and CD66d.

[0142] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting a signal within a cell to regulate the activity of the cell through a defined signaling pathway by generating a second messenger or by acting as an effector in response to such a messenger. An intracellular signaling domain can include all of the intracellular portion of a molecule, or all of a native intracellular signaling domain, or a functional fragment or derivative thereof.

[0143] The term“co-stimulatory signaling domain” or“co-stimulatory molecule”: generally refers to the intracellular domain of a co-stimulatory molecule capable of combining with a cell stimulatory signaling molecule, such as TCR / CD3, to result in a signal that leads to T cell proliferation and / or up- or down-regulation of key molecules. Co-stimulatory molecules are often cognate binding partners on T cells that specifically bind to co-stimulatory ligands, mediating a co-stimulatory response by the T cell, including but not limited to proliferation. Co-stimulatory molecules are cell surface molecules or their ligands other than antigen receptors that are required for efficient immune responses. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137).

[0144] The term“immune inhibitory signaling domain” or“immune inhibitory receptor molecule”: generally refers to a molecule capable of negatively regulating inhibitory immune cell activation signals, such as classical ITIM domain-containing receptors. Also includes non-classical ITIM domains, such as CD300A receptor. Also includes other non-ITIM dependent inhibitory receptors or enzymatic molecules, such as CTLA-4, IDO1 and IDO2 molecules.

[0145] The term“CD3 zeta” (also known as“CD3 Zeta”) includes the protein provided in GenBank Accession No. BAG36664.1, or equivalent residues in non-human species such as mouse, rodent, monkey, ape, etc. In this application,“CD3 zeta” is used interchangeably with“CD3z” and“CD3Z”.

[0146] The term“T cell receptor” (TCR) mediates the recognition of specific major histocompatibility complex (MHC)-restricted peptide antigens by T cells, including classical TCR receptors and optimized TCR receptors. A classical TCR receptor is composed of two peptide chains, alpha and beta, each of which can be further divided into variable (V), constant (C), transmembrane, and cytoplasmic regions, with antigen specificity residing in the V region (Vα, Vβ), which in turn has three hypervariable regions, CDR1, CDR2, and CDR3. In one aspect, a T cell expressing a classical TCR can be induced to respond specifically to a target antigen by the TCR of the T cell through, for example, antigen stimulation.

[0147] In one example, the fusion protein is a chimeric receptor. For example, the fusion protein is a CAR or a recombinant TCR. In one example, the fusion protein is used in combination with a chimeric receptor that binds to a pathological cell. In one example, the fusion protein is used in combination with a chimeric receptor that binds to an NK cell marker. In one example, the fusion protein is used in combination with a chimeric receptor that binds to a pathological cell and a chimeric receptor that binds to an NK cell marker. In one example, a chimeric receptor that binds to a pathological cell and an NK cell marker is used in combination with a fusion protein.

[0148] The term "cell" refers to a cell from a human or non-human, or animal.

[0149] The term "host" or "subject" refers to a recipient who receives a transplant, for example, an individual, such as a human, who receives an implant of foreign cells. For example, a "subject" can be a clinical patient, a clinical trial volunteer, an experimental animal, and the like. A subject can be suspected of having a disease characterized by cell proliferation or diagnosed as having a disease characterized by cell proliferation. For example, a subject is suffering from or can be suffering from an immune disease, such as an autoimmune disease, or suffering from a tumor, or suffering from an inflammatory disease.

[0150] The term "engineered" or "engineering" refers to the application of principles and methods of cell biology and molecular biology to alter genetic material within a cell or to obtain a cell product at the whole cell level or organelle level by some engineering means according to people's will. For example, "engineered" refers to one or more changes in a nucleic acid (e.g., a nucleic acid within a genome of an organism). "Engineered" can refer to changes, additions, and / or deletions of genes. An "engineered cell" can also refer to a cell with added, deleted, and / or altered genes.

[0151] In one example, the engineered cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell. Stem cells include human pluripotent stem cells (including human induced pluripotent stem cells (iPSCs) and human embryonic stem cells). For example, the engineered cell is an immune cell. For example, the engineered cell is a primary cell. For example, the engineered cell is a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a T cell, an NKT cell, or a combination thereof. The engineered cell can be an autologous cell or an allogeneic cell. For example, the engineered cell is a T cell that is genetically engineered from a human PBMC cell.

[0152] The term "immune cell" refers to a cell involved in immune response, producing immune effect, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, macrophages, mast cells, and the like. For example, the immune cell is a T cell, a NK cell, a NKT cell. For example, the T cell can be an autologous T cell, an allogeneic T cell, a syngeneic T cell. For example, the NK cell can be an autologous NK cell or a syngeneic NK cell. For example, the immune cell is obtained by sorting donor peripheral blood mononuclear cells (PBMCs).

[0153] The term "T cell" can be a PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and natural T cells obtained from an infection site, ascites, pleural effusion, spleen tissue, tumor tissue, and can also be a cell population with specific phenotypic characteristics obtained by sorting, etc., or a mixed cell population with different phenotypic characteristics, such as "T cell" can be a cell comprising at least one T cell subpopulation: stem cell-like memory T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (Tregs), and / or effector memory T cells (Tem). In some cases, "T cell" can be a certain specific subtype of T cell, such as alpha beta T cell, gamma delta T cell. In some cases, T cells can be obtained from blood collected from an individual using any number of techniques known to those skilled in the art, such as Ficoll™ separation and / or apheresis. For example, T cells are obtained by inducing differentiation of pluripotent stem cells. For example, cells of the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. For example, the cells collected by apheresis can be washed to remove plasma molecules and place the cells in a suitable buffer or medium for subsequent processing steps. The T cells can be derived from a healthy donor, or a subject diagnosed with cancer. The T cells can be autologous T cells or allogeneic T cells. The T cells can be primary T cells. In one example, "T cell" can also be a T cell carrying a fusion protein binding CD300A. For example, CAR T cell, recombinant TCR-T cell.

[0154] The term "cell composition" generally refers to a combination of at least two types of cells, wherein the first type of cells is capable of binding at least CD300A; the second type of cells binds to a target antigen on an NK cell marker and / or a pathologic cell (e.g., a tumor cell, a pathologic cell of an autoimmune disease). For example, each type of cells can be present in a different container, and can be formulated into a desired formulation with appropriate adjuvants at the same time or separately when needed; each type of cells can be of different origin (e.g., prepared, produced or sold by different manufacturers; e.g., a naturally occurring T cell isolated from a donor and a T cell derived from a stem cell, respectively); each type of cells can be prepared into an independent formulation (solid, liquid, gel, etc.) respectively; each type of cells can be present in a mixed form. The cell composition can further comprise an effective amount of an antibody, an immunological conjugate, a chimeric receptor, a nucleic acid or a host cell, and can further comprise a pharmaceutically acceptable carrier.

[0155] The term "MHC" is a major histocompatibility complex. In human cells, MHC is known as HLA antigen, which plays an important role in transplantation reactions, and is mediated by T cells that react to histocompatibility antigens on the surface of the transplanted tissue.

[0156] The term "human leukocyte antigen" (HLA) is a gene encoding the major histocompatibility complex of humans, which is closely related to the function of the immune system of humans. HLA includes I, II and III gene parts. HLA class I is a heterodimer composed of a heavy chain (alpha chain) and a light chain beta2 microglobulin (B2M). The term "B2M" is beta-2 microglobulin, also known as B2M, which is the light chain of MHC class I molecules. HLA class II genes include HLA-D family, mainly HLA-DP, HLA-DQ and HLA-DR, etc., which are mainly distributed on the surface of professional antigen presenting cells such as B lymphocytes, macrophages and dendritic cells.

[0157] The term "exogenous" refers to a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not expressed endogenously in the organism itself, or is expressed at a level insufficient to achieve overexpression.

[0158] The term "endogenous" refers to a nucleic acid molecule or polypeptide of the organism itself.

[0159] The terms "activate" and "activation" are used interchangeably to refer to the process by which a cell transitions from a quiescent state to an active state. The process can include a response to an antigen, a migratory, and / or a phenotypic or genetic change to a state of functional activity. For example, the term "activation" can refer to the process by which a T cell is progressively activated. The activation process is co-regulated by a primary stimulus signal and a costimulatory signal. Activation of a T cell is a dynamic process, the duration and strength of which is influenced by external conditions. "T cell activation" or "T cell activation" refers to the state of a T cell that is stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector function. The degree and duration of T cell activation can be influenced by the use of CD3 / CD28 magnetic beads, in vitro antigen stimulation, or in vivo antigen stimulation. For example, the engineered T cells are activated upon co-incubation with tumor cells containing a specific target antigen or viral infection.

[0160] The term "genome editing" or "gene editing" refers to a genetic engineering technique that uses site-specific nucleases to make DNA insertions, knockouts, modifications, or replacements at specific locations in the genome of an organism, altering the DNA sequence. Gene editing can be used to achieve precise, efficient gene knockouts or knockins. Gene knockout techniques using nucleases include CRISPR / Cas technology, ZFN technology, TALEN technology, and TALEN-CRISPR / Cas technology, single-base editor technology, Prime Editor technology, Meganuclease technology. A guide sequence (gRNA) is a polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence, the gRNA being capable of directing specific binding of a CRISPR complex to the target sequence. Whenever the present application refers to a sequence of a gRNA, it can be a DNA sequence targeted, or the complete Cas9 guide sequence formed by the DNA corresponding ribonucleotide and crRNA, TracrRNA. The gRNA is used to guide, bind, or recognize the Cas enzyme. For example, the degree of complementarity between a guide sequence and its corresponding target sequence is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. For example, the endogenous TCR / B2M knockout, or endogenous TCR / B2M / FAS knockout, or endogenous TCR / B2M / NKG2A knockout, or endogenous TCR / B2M / FAS / NKG2A knockout engineered cells are constructed using CRISPR technology. The gRNA sequences targeting TCR, B2M, NKG2A, FAS are shown in SEQ ID NO: 13, 12, 22, 23, respectively.

[0161] “Low expression” as described herein refers to the level of protein and / or RNA expression of a gene of interest in an engineered cell that is lower than the level of expression prior to the engineering of the cell. For example, low expression of B2M, TCR, FAS, NKG2A refers to at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% reduction in the expression of B2M, TCR, FAS, NKG2A in the cell. The expression or amount of a particular protein in a cell can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, immunoblotting, or flow cytometry using specific antibodies. CRISPR library screening technology has been widely used for screening of various biological problems, including screening of tumor drug resistance regulatory genes, screening of cell therapy product functional enhancement genes, etc.

[0162] The term “transfection” refers to the introduction of foreign nucleic acid into a eukaryotic cell. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0163] The terms “nucleic acid molecule encodes,” “coding DNA sequence,” and “coding DNA” refer to the sequence or order of deoxyribonucleotides along a deoxyribonucleic acid chain. For example, a nucleic acid sequence encodes an amino acid sequence. When referring to a nucleotide sequence, “sequence” can include DNA or RNA, and can be single-stranded or double-stranded.

[0164] The term “individual” refers to any animal, such as a mammal or a marsupial. Individuals of the present application include, but are not limited to, humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any species of poultry.

[0165] The term “peripheral blood mononuclear cell” (PBMC) refers to cells with a single nucleus in the peripheral blood, including lymphocytes, monocytes, etc. PBMCs can be prepared by density-based cell separation methods, for example, by lysing red blood cells or not lysing red blood cells and centrifuging peripheral blood or apheresis samples or leukapheresis samples through a Percoll Ficoll gradient.

[0166] The term "effective amount" or "therapeutically effective amount" refers to the amount of the agent that is sufficient to prevent or treat a disease (cancer) in an individual. The effective amount for therapeutic or prophylactic use will depend on the stage and severity of the disease being treated, the age, body weight, and general health of the subject, and the judgment of the prescribing physician. The size of the dose will also depend on the active agent chosen, the method of administration, the time and frequency of administration, the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular active agent, and the desired physiological effect. One or more rounds, or multiple administrations, can be required according to the judgment of the prescribing physician or person of skill in the art.

[0167] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors comprise sufficient cis-acting elements for expression; additional elements for expression can be supplied by the host cell or an in vitro expression system. Expression vectors include plasmids, viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0168] The term "vector" is a composition comprising an isolated nucleic acid and is useful for delivering the isolated nucleic acid into the interior of a cell. Included, but not limited to, linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. For example, self-replicating plasmids or viruses are included. Also included are non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like.

[0169] The term "modulate" refers to either an increase or a decrease. Examples of modulation include a 1%, 2%, 10%, 25%, 50%, 75%, or 100% change. In a specific embodiment, it refers to a decrease.

[0170] The terms "homology" or "identity" refer to the sequence identity of subunits between two polymeric molecules, for example, between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. The terms "substantial identity" or "substantial homology" refer to a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity with a reference amino acid sequence or nucleic acid sequence. In an example, such a sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homologous or identical to the amino acid or nucleic acid sequence used for comparison. Polypeptides that are modified from the amino acid sequences provided herein, and / or have one or more amino acid substitutions, and / or deletions and / or additions of one or more amino acids, and have 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more identity to the amino acid sequences provided herein, and have the same function, are within the scope of the present application.

[0171] The term "treatment" refers to an intervention performed with the intention of altering the natural course of a disease, whether or not it is successful. Treatment effects include, but are not limited to, preventing or delaying the onset of a disease or recurrence, alleviating symptoms, lessening any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the progression of a disease, ameliorating or palliating a disease state, improving or slowing the progression of a disease, and / or otherwise improving the prognosis of a disease.

[0172] The term "prevention" refers to an intervention performed with the intention to prevent, or slow the onset of a disease (such as rejection of a cell transplant).

[0173] The term "transplant immune rejection" refers to the immunological reaction in which a host recognizes a transplanted tissue, organ, or cell, etc., as a "non-self component" and mounts an attack, destruction, and elimination of the transplanted tissue, organ, or cell, etc. The present application provides cells and methods for combating transplant immune rejection.

[0174] The term "transplant" refers to a biological material or preparation that is isolated from an individual other than the host and is implanted into the host. A transplant can be isolated from any animal, such as a mammalian source, preferably from a human. The transplant can be isolated from the host, such as a cell isolated from the host that is cultured in vitro and / or modified for reimplantation into the host. The transplant can be isolated from another individual of the same species, such as a cell isolated from another human that is cultured in vitro and / or modified for implantation into the host. The transplant can be isolated from an individual of a different species, such as an organ from another species (e.g., mouse, pig, monkey) implanted into a human. Xenotransplantation includes, but is not limited to, vascularized xenotransplantation, partially vascularized xenotransplantation, non-vascularized xenotransplantation, xenodressing, xenobandage, and xenosturcture.

[0175] The term "autologous" refers to originating from the same organism. For example, a sample (e.g., a cell) can be removed from a subject, treated, and returned to the subject (e.g., a patient) at a later time. Autologous procedures are distinguished from allogeneic procedures, in which the donor and recipient are different subjects.

[0176] The term "autologous transplant" includes any procedure involving the transplantation, implantation, or infusion of a cell, tissue, or organ into a recipient, wherein the subject and the donor are the same individual. The transplantation of the cells, organs, and / or tissues described herein can be used in autologous transplantation into a human. Autologous transplantation includes, but is not limited to, vascularized autotransplantation, partially vascularized autotransplantation, non-vascularized autotransplantation, autodressing, autobandage, and autosturcture.

[0177] The term "allogeneic transplantation" includes any procedure involving the transplantation, implantation, or infusion of cells, tissues, or organs into a subject, wherein the subject and the donor are different individuals of the same species. Transplantation of cells, organs, and / or tissues described herein can be used in allogeneic transplantation into a human. Allogeneic transplantation includes, but is not limited to, vascularized allogeneic transplantation, partially vascularized allogeneic transplantation, non-vascularized allogeneic transplantation, allogeneic dressing, allogeneic bandage, and allogeneic structure.

[0178] Statistical analysis used in this application: t-test (also known as Student's t-test) is a tool that uses hypothesis testing to assess the mean of one or two populations. The P value refers to the probability of a result more extreme than the sample observation obtained when the null hypothesis is true, and a small p-value indicates that the probability of observing the result under the null hypothesis is small. In this application, *: P-value < 0.05; **: P-value < 0.01; ***: P-value < 0.001.

[0179] Fusion proteins binding CD300A

[0180] The present application provides a fusion protein targeting CD300A, which has an anti-transplant rejection function. The engineered cells (such as T, NKT cells) expressing the fusion protein can resist transplant rejection, such as NK cell-mediated immune rejection, especially allogeneic host NK cell-mediated transplant rejection. For example, the engineered cells (such as T, NKT cells) expressing the fusion protein have high survival rate in the presence of NK cells. For example, the engineered cells (such as T, NKT cells) expressing the fusion protein can significantly resist the killing of NK cells, but do not affect the survival of NK cells. For example, the engineered cells (such as T, NKT cells) expressing the fusion protein can significantly resist the killing of NK cells and can kill NK cells. For example, the CAR T cells expressing the fusion protein can effectively kill allogeneic host NK cells. For example, the CAR T cells expressing the fusion protein do not affect the survival of NK cells. For example, the CAR T cells expressing the fusion protein do not affect the survival of NK cells, but can significantly resist the killing of NK cells, and the CAR T cells have high survival rate. For example, the CAR T cells expressing the fusion protein can kill NK cells, can significantly resist the killing of NK cells, and the CAR T cells have high survival rate.

[0181] In one example, the fusion protein is a membrane protein that targets CD300A. In one example, the fusion protein comprises, from N-terminus to C-terminus, an extracellular domain and a transmembrane domain, and does not comprise an intracellular signaling domain. In one example, the fusion protein comprises, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, and a costimulatory signaling domain. In one example, the fusion protein comprises, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, a costimulatory signaling domain, and a primary signaling domain. In one example, the fusion protein comprises, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, and a primary signaling domain. In one example, the extracellular domain and the transmembrane domain are directly connected. In one example, the extracellular domain and the transmembrane domain are connected by a connecting polypeptide. In one example, the connecting polypeptide is selected from the group consisting of a CD28 hinge region, a CD8 hinge region, an IgG spacer or a fragment thereof, or a combination thereof. The domain that binds CD300A comprises a native CD300A ligand extracellular segment or a fragment or variant thereof that can bind CD300A, an antibody or a fragment thereof against CD300A, or a synthetic binding domain. In one example, the domain that targets CD300A comprises an antigen binding unit that binds CD300A.

[0182] In one example, the fusion protein comprises, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, and an intracellular signaling domain, the extracellular domain comprising a domain that binds CD300A. In one example, the extracellular domain and the transmembrane domain are directly connected. In one example, the extracellular domain and the transmembrane domain are connected by a connecting polypeptide. In one example, the connecting polypeptide is selected from the group consisting of a CD28 hinge region, a CD8 hinge region, an IgG spacer or a fragment thereof, or a combination thereof. The domain that binds CD300A comprises a native CD300A ligand extracellular segment or a fragment or variant thereof that can bind CD300A, an antibody or a fragment thereof against CD300A, or a synthetic binding domain. In one example, the domain that targets CD300A comprises an antigen binding unit that binds CD300A.

[0183] In one example, the fusion protein comprises, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, the extracellular domain comprising a domain that binds CD300A. In one example, the fusion protein does not comprise an intracellular signaling domain. In one example, the extracellular domain and the transmembrane domain are directly connected. In one example, the extracellular domain and the transmembrane domain are connected by a connecting polypeptide. In one example, the connecting polypeptide is selected from the group consisting of a CD28 hinge region, a CD8 hinge region, an IgG spacer or a fragment thereof, or a combination thereof. The domain that binds CD300A comprises a native CD300A ligand extracellular segment or a fragment or variant thereof that can bind CD300A, an antibody or a fragment thereof against CD300A, or a synthetic binding domain. In one example, the domain that targets CD300A comprises an antigen binding unit that binds CD300A.

[0184] Any antibody described in this application or known in the art that has high affinity to CD300A can be used as the domain that binds to CD300A in the fusion protein in this application. In one example, the anti-CD300A antibody is selected from the group consisting of a whole antibody, a scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, a sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody, or an IgG4 antibody. In one example, the anti-CD300A antibody is a scFv. In one example, the anti-CD300A antibody is a single domain antibody. In one example, the anti-CD300A antibody is a DDPP antibody.

[0185] In one example, the antibody or fragment thereof against CD300A comprises a heavy chain variable region (VH1) and a light chain variable region (VL1). In one embodiment, the VH1 comprises a VH CDR1, a VH CDR2, and a VH CDR3, and the VL1 comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 are from a VH having the amino acid sequence of SEQ ID NO: 2, and the VL CDR1, the VL CDR2, and the VL CDR3 are from a VL having the amino acid sequence of SEQ ID NO: 1. In one embodiment, the VH1 and the VL1 have the amino acid sequences of SEQ ID NOs: 2 and 1, respectively, or are each at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical thereto.

[0186] It is well known in the art that the sequences of VH and VL regions can have some substitutions of amino acids while the CDR sequences remain the same and retain their affinity to the target; the sequences of CDRs can also have some substitutions of amino acids while the amino acids that maintain the contact with the target remain the same and retain their affinity to the target.

[0187] In one example, the antibody or fragment thereof against CD300A is a scFv (see FIG. 2). The VH (heavy chain variable region) and the VL (light chain variable region) in the scFv can be connected by a linker peptide, and the positions can be interchanged. In one example, the antibody against CD300A comprises, from N-terminus to C-terminus, a VH, a linker peptide, and a VL. In one example, the antibody against CD300A comprises, from N-terminus to C-terminus, a VL, a linker peptide, and a VH. Any linker peptide that connects the VH and the VL together so that they become a functionally complete single chain antibody can be used. In one example, the linker peptide is a GS linker peptide, for example, GGGGS, (GGGGS)3, or (GGGGS)4.

[0188] In one example, the extracellular domain of the fusion protein that binds a domain of CD300A comprises an extracellular segment of a CD300A ligand or a variant thereof that can bind CD300A.

[0189] The fusion proteins provided herein include an extracellular domain, a transmembrane domain, and an intracellular signaling domain, from N-terminus to C-terminus. For example, the intracellular signaling domain comprises one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM), a kinase domain, a costimulatory domain, and the like, which can directly promote a cellular response. In other embodiments, the intracellular signaling domain will indirectly promote a cellular response by associating with one or more other proteins that in turn directly promote a cellular response. For example, the intracellular signaling domain or functional fragment thereof comprises an intracellular signaling domain of CD3 epsilon, CD3 delta, CD3 zeta, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD278 (ICOS), CD357 (GITR), CARD11, DAP10, DAP12, FcR alpha, FcR beta, FcR gamma, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pT alpha, TCR alpha, TCR beta, TRIM, Zap70, PTCH2, or any combination thereof. For example, the fusion proteins provided herein include an intracellular signaling domain. For example, the fusion proteins provided herein do not include an intracellular signaling domain. In one example, the intracellular signaling domain comprises a signaling motif of an immunoreceptor tyrosine-based activation motif or ITAM.

[0190] In one example, the intracellular signaling domain comprises an intracellular signaling domain of TCR alpha, TCR beta, TCR gamma, TCR delta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD5, CD22, CD79a, CD79b, CD278, or CD66d, or a combination thereof. In one example, the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain (SEQ ID NO: 10, 36).

[0191] In one example, the intracellular signaling domain included in the fusion protein provided herein further comprises a costimulatory domain. In one example, the costimulatory domain is selected from the group consisting of intracellular signaling domain of CD137 (4-1BB), CD28, CD27, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, CARD11, CD30, CD54, OX40, CD150, CD152, CD223, CD270, PD-L2, PD-L1, DAP10, LAT, NKD2C, SLP76, TRIM, Fc epsilon R1 gamma, MyD88, ICAM-1, LFA-1 (CD11a / CD18), 41BBL, or CD83, or a combination thereof. In one example, the costimulatory domain is the intracellular signaling domain of CD137 (4-1BB) (SEQ ID NO: 8). In one example, the costimulatory domain is the intracellular signaling domain of CD28 (SEQ ID NO: 7).

[0192] The fusion proteins provided herein include a transmembrane domain. In one example, the transmembrane domain is selected from the group consisting of a transmembrane domain of CD2, CD3 epsilon, CD3 delta, CD3 zeta, CD8, CD9, CD16, CD22, CD25, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD154, CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcR alpha, FcR beta, FcR gamma, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRP alpha, pT alpha, TCR alpha, TCR beta, TIM3, TRIM, LPA5, PDGFR, or Zap70. In one example, the transmembrane domain is a transmembrane domain of CD28 (SEQ ID NO: 6, 29). In one example, the transmembrane domain is a transmembrane domain of PDGFR (SEQ ID NO: 3, 41, 42, 43, 46, 47, or 48). In one example, the transmembrane domain is a transmembrane domain of PDGFR (SEQ ID NO: 3, 41, 42, 43, 46, 47, or 48) that is a variant of no more than three amino acid mutations. In one example, the transmembrane domain is a transmembrane domain of CD28 (SEQ ID NO: 6, 29) that is a variant of no more than three amino acid mutations. In one example, the transmembrane domain is a CD8 transmembrane domain (SEQ ID NO: 4). In one example, the transmembrane domain is a CD8 transmembrane domain (SEQ ID NO: 4) that is a variant of no more than three amino acid mutations. In one example, the transmembrane domain of PDGFR includes a hinge region or a spacer region.

[0193] In one example, the extracellular domain and the transmembrane domain in the fusion protein provided herein are connected by a connecting polypeptide. In one example, the connecting polypeptide is selected from the group consisting of a CD28 hinge region, a CD8 hinge region, an IgG spacer region or a fragment thereof, or a combination thereof. In one example, the connecting polypeptide can be an IgG1 spacer region or a fragment thereof, an IgG2 spacer region or a fragment thereof, an IgG3 spacer region or a fragment thereof, or an IgG4 spacer region or a fragment thereof. In one example, the connecting polypeptide includes an amino acid sequence as set forth in SEQ ID NO: 9, 14, 37, or 44.

[0194] In one example, the fusion protein includes a signal peptide. The signal peptide is a peptide sequence that is often found at the N-terminus of newly synthesized proteins, directing them into the secretory pathway. The signal peptide is covalently attached to the N-terminus of the extracellular antigen binding domain of the CAR as a fusion protein. As is well known in the art, any suitable signal peptide can be applied to the CAR to provide cell surface expression in immune cells (see Gierasch Biochem. 28:923-930 (1989); von Heijne, J. Mol. Biol. 184(l):99-105 (1985)). For example, the signal peptide includes an amino acid sequence as set forth in SEQ ID NO: 5 or 34

[0195] In one example, the fusion protein provided herein comprises, from N-terminus to C-terminus, (1) a domain that binds CD300A (e.g., an extracellular segment of CD300A or an antibody that binds CD300A), (2) a CD8 hinge region (SEQ ID NO: 9, 44) or an IgG4 spacer fragment (SEQ ID NO: 14) or a new linker (SEQ ID NO: 37), (3) a CD8 transmembrane domain (SEQ ID NO: 4) or a CD28 transmembrane domain (SEQ ID NO: 6, 29), and (4) a CD28 intracellular signaling domain (SEQ ID NO: 7) and / or a CD137 intracellular signaling domain (SEQ ID NO: 8); and (5) a CD3 zeta intracellular signaling domain (SEQ ID NO: 10, 36).For example, the fusion protein includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 9, 6, and 10, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 9, 4, and 10, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 9, 4, and 36, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 9, 6, and 7, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 9, 4, and 8, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 9, 6, 7, and 10, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 9, 6, 7, and 36, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 9, 6, 8, and 10, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 9, 4, 8, and 36, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 14, 6, and 7, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 14, 6, and 8, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 14, 6, 7, and 10, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 14, 6, 8, and 10, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 14, 6, 7, and 36, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 14, 6, 8, and 36, or as SEQ ID NO: 1, 2, 14, 29, and 7, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 14, 29, and 8, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 14, 29, 7, and 10, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 14, 29, 8, and 10, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 14, 29, 7, and 36, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 14, 29, 8, and 36, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 9, 29, and 36, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 14, 6, and 10, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 44, 6, and 10, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 44, 4, and 10, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 44, 4, and 36, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 44, 6, and 7, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 44, 4, and 8, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 44, 6, 7, and 10, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 44, 6, 7, and 36, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 44, 6, 8, and 10, or includes the amino acid sequence set forth as SEQ ID NO: 1, 2, 44, 29, and 36.

[0196] In one example, the fusion protein provided herein comprises, in N-terminal to C-terminal order, (1) a domain comprising an anti-CD300A antibody (VH / VL selected from SEQ ID NOs: 2, 1), (2) a CD8 hinge region (SEQ ID NO: 9, 44), an IgG4 spacer segment (SEQ ID NO: 14), or a newlinker (SEQ ID NO: 37), and (3) a PDGFR transmembrane domain (SEQ ID NO: 3, 41, 42, 43, 46, 47, or 48). The extracellular region thereof can comprise any of the domains that bind CD300A mentioned above, such as an extracellular segment of a CD300A ligand or an antibody that binds CD300A. For example, the fusion protein further comprises an intracellular signaling domain. For example, the fusion protein does not comprise an intracellular signaling domain. For example, the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, 44, and 43, or comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, 44, and 46, or comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, 44, and 42, or comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, 9, and 43, or comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, 9, and 46, or comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, 9, and 42.

[0197] In one example, the fusion protein provided herein comprises, in N-terminal to C-terminal order, (1) a domain comprising an anti-CD300A antibody (VH / VL selected from SEQ ID NOs: 2, 1), and (2) a PDGFR transmembrane domain (SEQ ID NO: 3, 41, 42, 43, 46, 47, or 48). The extracellular region thereof can comprise any of the domains that bind CD300A mentioned above, such as an extracellular segment of a CD300A ligand or an antibody that binds CD300A. For example, the fusion protein further comprises an intracellular signaling domain. For example, the fusion protein does not comprise an intracellular signaling domain. For example, the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, and 3, or comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, and 42, or comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, and 43, or comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, and 46, or comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, and 47, or comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, and 48, or comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, and 41.

[0198] The extracellular domain of the fusion protein that binds CD300A can have different conformations, such as some of those illustrated in FIG. 2 or Table 1. For example, the fusion protein includes an amino acid sequence that is about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 16, 18, or 21. For example, the fusion protein includes an amino acid sequence encoded by a nucleotide sequence that is about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 15, 17, 19, 20, 24, 25, 26, or 38.

[0199] The fusion protein variants in these examples include, but are not limited to, for example, a fusion protein in which the VH and VL positions are interchanged in the scFv structure of the anti-CD300A antibody moiety; a fusion protein in which the linker peptide is replaced by another linker peptide; a fusion protein in which the anti-CD300A antibody moiety is replaced by another antibody structure (such as Fab, single domain antibody, etc.); and a fusion protein in which the sequences of the VH and VL of the anti-CD300A antibody moiety can be changed but do not affect the binding of the anti-CD300A antibody to CD300A; and the like. Those skilled in the art can obtain the above-mentioned possible variants by common general knowledge and routine experiments.

[0200] In an example, the fusion protein includes a domain that binds an immune cell marker other than CD300A, which can be together with the domain that binds CD300A to form the extracellular region of the fusion protein. In an example, the extracellular domain of the fusion protein further includes a domain that binds an immune cell marker other than CD300A.

[0201] In an example, the domain that binds the immune cell marker includes a ligand that binds the immune cell marker, a synthetic binding domain, an antibody, or a fragment thereof. In an example, the extracellular domain includes an antibody or a fragment thereof that binds the immune cell marker. In an example, the antibody or fragment that binds the immune cell marker is selected from the group consisting of a whole antibody, an scFv, a single domain antibody, a Fab fragment, a Fab’ fragment, a Fv fragment, a F(ab’)2 fragment, a Fd fragment, an sdAb, a multifunctional antibody, a DDPP antibody, an scFv-Fc antibody, or an IgG4 antibody. In an example, the antibody or fragment that binds the immune cell marker is an scFv. In an example, the antibody or fragment that binds the immune cell marker is a single domain antibody. In an example, the antibody or fragment that binds the immune cell marker is a Fab fragment.

[0202] In one example, the antibody or fragment thereof that binds to the immune cell marker comprises a heavy chain variable region (VH2) and a light chain variable region (VL2). In one example, the extracellular domain of the fusion protein comprises an antibody or fragment thereof that binds to CD300A, and an antibody or fragment thereof that binds to the immune cell marker; wherein the antibody or fragment thereof that binds to CD300A comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), and the antibody or fragment thereof that binds to the immune cell marker comprises a heavy chain variable region (VH2) and a light chain variable region (VL2). The different variable regions are linked by a linker peptide chain. In one example, the linker peptide chain is a GS linker peptide chain, for example, GGGGS, (GGGGS)3, or (GGGGS)4.

[0203] In one example, the fusion protein comprises a domain that binds to a target antigen on a pathological cell. The domain that binds to a target antigen on a pathological cell can together with the domain that binds to CD300A comprise the extracellular region of the fusion protein.

[0204] In one example, the extracellular domain of the fusion protein further comprises a domain that binds to a target antigen on a pathological cell. In one example, the domain that binds to a target antigen comprises a ligand that binds to a target antigen, a synthetic binding domain, an antibody or fragment thereof. In one example, the extracellular domain comprises an antibody or fragment thereof that binds to the target antigen. In one example, the antibody or fragment is selected from the group consisting of a whole antibody, a scFv, a single domain antibody, a Fab fragment, a Fab’ fragment, a Fv fragment, a F(ab’)2 fragment, a Fd fragment, a sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody, or an IgG4 antibody.

[0205] In one example, the antibody or fragment thereof that binds to a target antigen on a pathological cell comprises a heavy chain variable region (VH3) and a light chain variable region (VL3). In one example, the extracellular domain of the fusion protein comprises an antibody or fragment thereof that binds to CD300A, and an antibody or fragment thereof that binds to a target antigen on a pathological cell; wherein the antibody or fragment thereof that binds to CD300A comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), and the antibody or fragment thereof that binds to a target antigen on a pathological cell comprises a heavy chain variable region (VH3) and a light chain variable region (VL3). The different variable regions are linked by a linker peptide chain. In one example, the linker peptide chain is a GS linker peptide chain, for example, GGGGS, (GGGGS)3, or (GGGGS)4.

[0206] In one example, the pathological cells are selected from malignant cells or infected cells. In one example, the pathological cells are selected from solid tumor cells, blood tumor cells, pathological cells of autoimmune diseases. In one example, the solid tumor is selected from: esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, sarcoma; the blood tumor is selected from: leukemia, lymphoma, myeloma; the autoimmune disease is selected from: multiple sclerosis, autoimmune liver disease, type I diabetes, systemic lupus erythematosus (LE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren's syndrome (SS), polymyositis / dermatomyositis.

[0207] In one example, the target antigen is selected from the group consisting of: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); Interleukin-13 receptor subunit alpha (IL-13Ra); Interleukin-11 receptor alpha (IL-11Ra); Prostate stem cell antigen (PSCA); Prostate specific membrane antigen (PSMA); Carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; Tyrosinase; Mesothelin; EpCAM; Protease serine 21 (PRSS21); Vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); Mucin 1 (MUC1), MUC6; Epidermal growth factor receptor family and mutants thereof (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); Neural cell adhesion molecule (NCAM); Carbonic anhydrase IX (CAIX); LMP2; Ephrin type-A receptor 2 (EphA2); Fucosyl GM1; Sialyl Lewis adhesion molecule (sLe); Ganglioside GM3; TGS5; High molecular weight-melanoma-associated antigen (HMWMAA); O-Acetyl GD2 ganglioside (OAcGD2); Folate receptor; Tumor endothelial marker 1 (TEM1 / CD248); Tumor endothelial marker 7 related (TEM7R); Claudin 6, Claudin 18.2, Claudin 18.1; ASGPR1; CDH16; 5T4; 8H9; ανβ6 integrin; B-cell maturation antigen (BCMA); CA9; Kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; Embryonic AchR; HLA-A1, HLA-A2; MAGE A1, MAGE3; KDR; MCSP; NKG2D ligands; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; Fibronectin; Tenascin; Carcinoembryonic variant of the tumor necrosis zone; G protein-coupled receptor class C group 5-member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1);hexose portion of GloboH glycoceramide (GloboH); breast cancer differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternate reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchoring protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5);Immunoglobulin lambda-like polypeptide 1 (IGLL1). Preferably, the tumor antigen is BCMA or CD19. For example, the tumor antigen is selected from the group consisting of: CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin 18.2, FAP, Mesothelin, NKG2D ligand, CD94, FCRH5, EGFR and mutants thereof, or a combination thereof.

[0208] In one example, the target antigen is a pathogenic cell. In one example, the target antigen is selected from the group consisting of: a viral, bacterial, fungal, protozoal, or parasitic antigen. In one example, the target antigen is a viral antigen. The viral antigen is selected from the group consisting of: a cytomegalovirus antigen, an Epstein-Barr virus antigen, a human immunodeficiency virus antigen, or an influenza virus antigen.

[0209] In one example, the fusion protein also binds to a target antigen on a pathological cell. For example, the pathological cell comprises a malignant cell or an infected cell. For example, the pathological cell is a solid tumor cell, a hematological tumor cell, a pathological cell of an autoimmune disease.

[0210] In one example, the target antigen is selected from the group consisting of: CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin 18.2, FAP, Mesothelin, NKG2D ligand, CD94, FCRH5, EGFR and mutants thereof, ASGPR1, IL13RA2, WT1, CLL1, or a combination thereof.

[0211] The present disclosure provides fusion proteins that bind CD300A as shown in Table 1. Chimeric receptors that bind immune cell markers as shown in Table 2. Chimeric receptors that bind target antigens on pathological cells as shown in Table 3.

[0212] Table 1. Examples of fusion proteins that bind CD300A

[0213] Table 2. Chimeric receptors that target immune cell markers

[0214] Table 3. Chimeric receptors that target target antigens on pathological cells

[0215] In Tables 1, 2, and 3 above: CD300A_scFv: VH of anti-CD300A antibody (SEQ ID NO: 2), VL of anti-CD300A antibody (SEQ ID NO: 1); PDGFR(TM): PDGFR transmembrane region (SEQ ID NO: 3, 41, 42, 43, 46, 47, or 48); CD8(TM): CD8 transmembrane region (SEQ ID NO: 4); CD8 signal peptide (SEQ ID NO: 5); CD28(TM): CD28 transmembrane region (SEQ ID NO: 6, 29); CD28(C): CD28 intracellular domain (SEQ ID NO: 7); CD137(C): CD137 intracellular domain (SEQ ID NO: 8); CD8(H): CD8 hinge (SEQ ID NO: 9, 44); IgG4(H): IgG4 hinge (SEQ ID NO: 14); CD3 zeta(C): CD3 zeta intracellular domain (SEQ ID NO: 10, 36).

[0216] The present application also provides a polynucleotide encoding the fusion protein and / or chimeric receptor disclosed herein. The present application also provides a vector comprising the polynucleotide disclosed herein. The present application also provides a virus comprising the vector disclosed herein.

[0217] Engineered cell

[0218] The present application provides an engineered cell, wherein the engineered cell comprises the fusion protein and / or chimeric receptor disclosed herein.

[0219] In one example, the engineered cell further comprises a chimeric receptor 1 that binds at least one or more immune cell markers that are different from CD300A. In one example, the immune cell marker is selected from the group consisting of: a T cell marker and / or an NK cell marker. In one example, the immune cell marker is an NK inhibitory receptor (NKIR). In one example, the immune cell marker is selected from the group consisting of: an NKG2 / CD94, a KIR family member, an LIR family member, an NKR-P1 family member, an immune checkpoint receptor, a SIGLEC family member, a Ly49 family member, or a combination thereof. In one example, the NKG2 / CD94 component is selected from the group consisting of NKG2A, NKG2C, and CD94; the KIR family member is selected from the group consisting of KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3; the LIR family member is selected from the group consisting of LIR1, LIR2, LIR3, LIR5, and LIR8; the NKR-P1 family member is selected from the group consisting of NKR-P1B and NKR-P1D; the immune checkpoint receptor is selected from the group consisting of PD-1, TIGIT, CD96, TIM3, and LAG3; the SIGLEC family member is selected from the group consisting of SIGLEC7 and SIGLEC9; and the Ly49 family member is selected from the group consisting of Ly49A, Ly49C, Ly49F, Ly49G1, and Ly49G4. In one example, the immune cell marker is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD138, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD279, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRa, TCRp, TIGIT, TRAIL, SLAMF7, NKG2F, NKG2H, NKp30, NKp44, NKp46, NKp80, a SLAM family member, L-selectin, a natural cytotoxicity receptor NCR1, NCR2, NCR3, or a combination thereof.

[0220] In one example, the engineered cell comprises a fusion protein binding CD300A and a chimeric receptor 1. The chimeric receptor 1 binds at least one or more immune cell markers different from CD300A. In one example, the fusion protein binding CD300A has a structure as shown in FIG. 2 or Table 1. For example, the chimeric receptor 1 has a structure as shown in Table 2. For example, the fusion protein binding CD300A is CD300A-PDGFR, CD300A-28Z, CD300A-BB. In one example, the engineered cell comprises a fusion protein binding CD300A and a chimeric receptor 1, wherein the fusion protein binding CD300A comprises an extracellular domain of a domain binding CD300A, a transmembrane domain, and an intracellular signaling domain; the chimeric receptor 1 comprises an extracellular domain of a domain binding an immune cell marker, a transmembrane domain, and an intracellular signaling domain. In one example, the fusion protein binding CD300A and the chimeric receptor 1 are constructed in one vector. In one example, the fusion protein binding CD300A and the chimeric receptor 1 are connected by a hydrolysable linker. In one example, the hydrolysable linker is P2A. In one example, the hydrolysable linker is T2A. In one example, the hydrolysable linker is E2A. In one example, the hydrolysable linker is F2A. In one example, the fusion protein binding CD300A and the chimeric receptor 1 are constructed in different vectors, respectively. In one example, the chimeric receptor 1 comprises a ligand, a synthetic binding domain, an antibody or a fragment thereof binding the immune cell marker. In one example, the chimeric receptor 1 comprises an antibody or a fragment thereof binding the immune cell marker. In one example, the antibody or fragment binding the immune cell marker is selected from the group consisting of a whole antibody, a scFv, a single domain antibody, a Fab fragment, a Fab’ fragment, a Fv fragment, a F(ab’)2 fragment, a Fd fragment, a sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody, or an IgG4 antibody. In one example, the antibody or fragment binding the immune cell marker is a scFv. In one example, the antibody or fragment binding the immune cell marker is a single domain antibody. In one example, the antibody or fragment binding the immune cell marker is a Fab fragment. In one example, the chimeric receptor 1 further binds a target antigen on a pathological cell.

[0221] In one example, the engineered cell further expresses a chimeric receptor 2 that binds a target antigen on a pathological cell. In one example, the engineered cell comprises a fusion protein that binds CD300A and a chimeric receptor 2 that comprises a domain that binds a target antigen on a pathological cell. In one example, the fusion protein that binds CD300A has a structure as shown in FIG. 2 or Table 1. For example, the fusion protein that binds CD300A is CD300A-PDGFR, CD300A-28Z, CD300A-BB. For example, the chimeric receptor 2 has a structure as shown in Table 3. In one example, the fusion protein that binds CD300A and the chimeric receptor 2 are constructed in one vector. In one example, the fusion protein that binds CD300A and the chimeric receptor 2 are connected by a cleavable linker. In one example, the cleavable linker is P2A, T2A, E2A, or F2A. In one example, the fusion protein that binds CD300A and the chimeric receptor 2 are constructed in different vectors.

[0222] In one example, the engineered cell comprises a fusion protein that binds CD300A, a chimeric receptor 1 that comprises a domain that binds an immune cell marker, and a chimeric receptor 2 that comprises a domain that binds a target antigen on a pathological cell. In one example, the fusion protein that binds CD300A, the chimeric receptor 1, and the chimeric receptor 2 are constructed in one vector. In one example, the fusion protein that binds CD300A and the chimeric receptor 1 are constructed in one vector, and the chimeric receptor 2 is constructed in a different vector. In one example, the chimeric receptor 1 and the chimeric receptor 2 are constructed in one vector, and the fusion protein that binds CD300A is constructed in a different vector. In one example, the cleavable linker is P2A, T2A, E2A, or F2A. In one example, the fusion protein that binds CD300A, the chimeric receptor 1, and the chimeric receptor 2 are constructed in different vectors.

[0223] The CD300A / FasL-CAR-T cells provided herein include a FasL-CAR and a fusion protein that binds CD300A. For example, the FasL-CAR includes FasL-28Z, FasL-s28Z, or FasL-BBZ; and the fusion protein that binds CD300A includes a fusion protein shown in Table 1. For example, the CD300A / FasL-CAR-T cell includes FasL-28Z and CD300A-PDGFR; FasL-28Z and CD300A-BB; or FasL-28Z and CD300A-28Z. For example, the CD300A / FasL-CAR-T cell includes an amino acid sequence as set forth in SEQ ID NO: 16 and 27, or an amino acid sequence as set forth in SEQ ID NO: 18 and 27, or an amino acid sequence as set forth in SEQ ID NO: 21 and 27. For example, the CD300A / FasL-CAR includes a nucleic acid sequence as set forth in SEQ ID NO: 15, 17, 19, or 20.

[0224] The CD300A / CD38-CAR-T cells provided herein include a CD38-CAR and a fusion protein that binds CD300A. For example, the CD38-CAR includes CD38-28Z, CD38-s28Z, CD38-BBZ, or CD38-sBBZ; and the fusion protein that binds CD300A includes a fusion protein shown in Table 1. For example, the CD300A / CD38-CAR-T cell includes CD38-28Z and CD300A-PDGFR; CD38-s28Z and CD300A-PDGFR; CD38-BBZ and CD300A-PDGFR; or CD38-sBBZ and CD300A-PDGFR. For example, the CD300A / CD38-CAR-T cell includes an amino acid sequence as set forth in SEQ ID NO: 18 and 38, or an amino acid sequence as set forth in SEQ ID NO: 18 and 45, or an amino acid sequence as set forth in SEQ ID NO: 16 and 38, or an amino acid sequence as set forth in SEQ ID NO: 16 and 45, or an amino acid sequence as set forth in SEQ ID NO: 21 and 38, or an amino acid sequence as set forth in SEQ ID NO: 21 and 45.

[0225] The CD300A / TIGIT-CAR-T cells provided in the present application include TIGIT-CAR and fusion proteins binding CD300A. For example, the TIGIT-CAR includes TIGIT-28Z, TIGIT-s28Z, TIGIT-BBZ or TIGIT-sBBZ; the fusion proteins binding CD300A include the fusion proteins shown in Table 1. For example, the CD300A / TIGIT-CAR-T cells include TIGIT-28Z and CD300A-PDGFR; TIGIT-s28Z and CD300A-PDGFR; TIGIT-BBZ and CD300A-PDGFR; or TIGIT-sBBZ and CD300A-PDGFR. The CD300A / NKG2A-CAR-T cells provided in the present application include NKG2A-CAR and fusion proteins binding CD300A. For example, the NKG2A-CAR includes NKG2A-28Z, NKG2A-s28Z or NKG2A-BBZ; the fusion proteins binding CD300A include the fusion proteins shown in Table 1. For example, the CD300A / NKG2A-CAR-T cells include NKG2A-28Z and CD300A-PDGFR; NKG2A-28Z and CD300A-BB; or NKG2A-28Z and CD300A-28Z. For example, the CD300A / NKG2A-CAR-T cells include the amino acid sequences shown as SEQ ID NO: 16 and 28, or the amino acid sequences shown as SEQ ID NO: 18 and 28, or the amino acid sequences shown as SEQ ID NO: 21 and 28. For example, the CD300A / NKG2A-CAR includes the amino acid sequence encoded by the nucleic acid sequence shown as SEQ ID NO: 15, 17, 20, 24 or 25.

[0226] The CD300A / CD38-NKG2A-CAR-T cells provided in the application include CD38-CAR, NKG2A-CAR and fusion proteins binding CD300A. For example, the CD38-CAR includes CD38-28Z, CD38-s28Z, CD38-BBZ or CD38-sBBZ; the NKG2A-CAR includes NKG2A-28Z, NKG2A-s28Z or NKG2A-BBZ; and the fusion proteins binding CD300A include the fusion proteins shown in Table 1. For example, the CD300A / CD38-NKG2A-CAR-T cells include CD38-s28Z, NKG2A-28Z and CD300A-PDGFR; CD38-sBBZ, NKG2A-28Z and CD300A-PDGFR; CD38-s28Z, NKG2A-28Z and CD300A-BB; CD38-sBBZ, NKG2A-28Z and CD300A-BB; CD38-s28Z, NKG2A-28Z and CD300A-28Z; or CD38-sBBZ, NKG2A-28Z and CD300A-28Z. For example, the CD300A / CD38-NKG2A-CAR-T cells include the amino acid sequences as shown in SEQ ID NO: 16, 28 and 39; or the amino acid sequences as shown in SEQ ID NO: 16, 28 and 45; or the amino acid sequences as shown in SEQ ID NO: 18, 28 and 39; or the amino acid sequences as shown in SEQ ID NO: 18, 28 and 45; or the amino acid sequences as shown in SEQ ID NO: 21, 28 and 39; or the amino acid sequences as shown in SEQ ID NO: 21, 28 and 45. For example, the CD300A / CD38-NKG2A-CAR includes the nucleic acid sequences as shown in SEQ ID NO: 15, 17, 20, 24 or 38.

[0227] The present application provides engineered cell cells binding to target cells including: (1) CD300A / CD19-CAR-T cells, including CD19-sBBZ shown in Table 3, and CD300A-PDGFR. (2) CD300A / CD19-NKG2A-CAR-T cells, including CD19-sBBZ shown in Table 3, NKG2A-28Z or NKG2A-s28Z shown in Table 2, and CD300A-PDGFR. (3) CD300A / CD19-CD38-CAR-T cells, including CD19-sBBZ shown in Table 3, CD38-28Z or CD38-s28Z shown in Table 2, and CD300A-PDGFR. (4) CD300A / CD19-CD20-CAR-T cells, including CD19 / CD20-sBBZ shown in Table 3, and CD300A-PDGFR. (5) CD300A / CD19-CD20-NKG2A-CAR-T cells, including CD19 / CD20-sBBZ shown in Table 3, NKG2A-28Z or NKG2A-s28Z shown in Table 2, CD300A-PDGFR. (6) CD300A / CD19-CD20-CD38-CAR-T cells, including CD19 / CD20-sBBZ shown in Table 3, CD38-28Z or CD38-s28Z shown in Table 2, CD300A-PDGFR. (7) CD300A / BCMA-CAR-T cells, including BCMA-BBZ shown in Table 3, and CD300A-PDGFR. (8) CD300A / GPRC5D-CAR-T cells, including GPRC5D-BBZ shown in Table 3, and CD300A-PDGFR. (9) CD300A / BCMA-NKG2A-CAR-T cells, including BCMA-NKG2A-BBZ shown in Table 3, and CD300A-PDGFR. (10) CD300A / BCMA-GPRC5D-CAR-T1 cells, including BCMA-GPRC5D-BBZ shown in Table 3, and CD300A-PDGFR. (11) CD300A / BCMA-GPRC5D-CAR-T2 cells, including BCMA-BBZ, GPRC5D-BBZ shown in Table 3, and CD300A-PDGFR. (12) CD300A / BCMA-GPRC5D-NKG2A-CAR-T1 cells, including BCMA-GPRC5D-BBZ shown in Table 3, NKG2A-28Z or NKG2A-s28Z shown in Table 2, and CD300A-PDGFR.(13) CD300A / BCMA-GPRC5D-CD38-CAR-T1 cells comprising BCMA-GPRC5D-BBZ as shown in Table 3, CD38-28Z or CD38-s28Z as shown in Table 2, and CD300A-PDGFR. (14) CD300A / BCMA-GPRC5D-NKG2A-CAR-T2 cells comprising BCMA-BBZ and GPRC5D-BBZ as shown in Table 3, NKG2A-28Z or NKG2A-s28Z as shown in Table 2, and CD300A-PDGFR. (15) CD300A / BCMA-GPRC5D-CD38-CAR-T2 cells comprising BCMA-BBZ and GPRC5D-BBZ as shown in Table 3, CD38-28Z or CD38-s28Z as shown in Table 2, and CD300A-PDGFR. (16) CD300A / GPC3-CAR-T cells comprising GPC3-28Z or GPC3-BBZ as shown in Table 3, and CD300A-PDGFR. (17) CD300A / GPC3-NKG2A-CAR-T cells comprising GPC3-2 or GPC3-BBZ as shown in Table 3, NKG2A-28Z or NKG2A-s28Z as shown in Table 2, and CD300A-PDGFR. (18) CD300A / GPC3-CD38-CAR-T cells comprising GPC3-2 or GPC3-BBZ as shown in Table 3, CD38-28Z or CD38-s28Z as shown in Table 2, and CD300A-PDGFR. (19) CD300A / Claudin18.2-CAR-T cells comprising Claudin18.2-28Z or Claudin18.2-BBZ as shown in Table 3, and CD300A-PDGFR. (20) CD300A / Claudin18.2-NKG2A-CAR-T cells comprising Claudin18.2-28Z or Claudin18.2-BBZ as shown in Table 3, NKG2A-28Z or NKG2A-s28Z as shown in Table 2, and CD300A-PDGFR. (21) CD300A / Claudin18.2-CD38-CAR-T cells comprising Claudin18.2-28Z or Claudin18.2-BBZ as shown in Table 3, CD38-28Z or CD38-s28Z as shown in Table 2, and CD300A-PDGFR.

[0228] The embodiments of the present application provide: the CD300A / CD19-CAR-T cell comprises the amino acid sequence shown in SEQ ID NO: 18 and 49. The CD300A / CD19-NKG2A-CAR-T cell comprises the amino acid sequences shown in SEQ ID NO: 18, 28 and 49. The CD300A / CD19-CD38-CAR-T cell comprises the amino acid sequences shown in SEQ ID NO: 18, 45 and 49. The CD300A / BCMA-GPRC5D-CAR-T1 cell comprises the amino acid sequence shown in SEQ ID NO: 18 and 31. The CD300A / BCMA-GPRC5D-NKG2A-CAR-T1 cell comprises the amino acid sequences shown in SEQ ID NO: 18, 28 and 31. The CD300A / BCMA-GPRC5D-CAR-T2 cell comprises the amino acid sequences shown in SEQ ID NO: 18, 32 and 33. The CD300A / BCMA-GPRC5D-NKG2A-CAR-T2 cell comprises the amino acid sequences shown in SEQ ID NO: 18, 28, 32 and 33. The CD300A / BCMA-NKG2A-CAR-T cell comprises the amino acid sequence shown in SEQ ID NO: 18 and 40. The CD300A / BCMA-NKG2A-CAR-T2 cell comprises the amino acid sequences shown in SEQ ID NO: 18, 28, 32. The CD300A / BCMA-CD38-CAR-T cell comprises the amino acid sequences shown in SEQ ID NO: 18, 32 and 45. The CD300A / GPRC5D-NKG2A-CAR-T cell comprises the amino acid sequences shown in SEQ ID NO: 18, 28 and 33. The CD300A / GPRC5D-CD38-CAR-T cell comprises the amino acid sequences shown in SEQ ID NO: 18, 45 and 33. The CD300A / CD19-CD20-CAR-T cell comprises the amino acid sequence shown in SEQ ID NO: 18 and 30. The CD300A / BCMA-CAR-T cell comprises the amino acid sequence shown in SEQ ID NO: 18 and 32. The CD300A / GPRC5D-CAR-T cell comprises the amino acid sequence shown in SEQ ID NO: 18 and 33.

[0229] In one example, the pathological cells are selected from malignant cells or infected cells. In one example, the pathological cells are selected from solid tumor cells, blood tumor cells, pathological cells of autoimmune diseases. In one example, the solid tumor is selected from: esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, sarcoma; the blood tumor is selected from: leukemia, lymphoma, myeloma; the autoimmune disease is selected from: multiple sclerosis, autoimmune liver disease, type I diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), polymyositis / dermatomyositis.

[0230] In one example, the present application provides an engineered cell, wherein the engineered cell comprises a polynucleotide disclosed herein. In one example, the present application provides an engineered cell, wherein the engineered cell comprises a vector disclosed herein. In one example, the present application provides an engineered cell, wherein the engineered cell comprises a virus disclosed herein.

[0231] In one example, the engineered cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, a stem cell, or a combination thereof. In one example, the engineered cell is an immune cell. In one example, the immune cell is an autologous cell. In one example, the immune cell is an allogeneic cell. In one example, the immune cell is selected from the group consisting of a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a T cell, a NKT cell, a stem cell-derived immune effector cell, or a combination thereof. In one example, the engineered cell is a T cell. In one example, the engineered cell is an allogeneic T cell. In one example, the engineered cell is a stem cell-derived T cell. The T cell can be a cytotoxic T cell, a helper T cell, or an alpha beta T, gamma delta T, CD4+ / CD8+double positive T cell, CD4+T cell, CD8+T cell, CD4 / CD8 double negative T cell, CD3+T cell, naive T cell, effector T cell, cytotoxic T cell, helper T cell, memory T cell, regulatory T cell, ThO cell, Thl cell, Th2 cell, Th3 (Treg) cell, Th9 cell, Thl 7 cell, Th alpha beta helper cell, Tfh cell, stem cell-like central memory T (TSCM) cell, central memory T (TCM) cell, effector memory T (TEM) cell, effector memory T (TEMRA) cell, or gamma delta T cell. In one example, the T cell is a cytotoxic T cell. In some embodiments, the genetically engineered T cell provided herein is isolated. In one example, the genetically engineered T cell provided herein is substantially purified. In one example, the engineered cell provided herein is obtained by genetically engineering a cell isolated from a subject. As used herein, the engineered cell of the application is obtained by genetically manipulating a cell isolated from a subject. Alternatively, the engineered cell of the application is obtained by performing the genetic manipulation associated with the application on a cell isolated from a subject, which has been passaged in vitro or otherwise manipulated genetically.

[0232] In one example, the genetically engineered cell provided herein is obtained by genetically engineering a cell isolated from a human. The immune effector cell (e.g., T cell) can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In one example, a T cell line available in the art can be used. In one example, the genetically engineered cell provided herein is obtained by genetically engineering a cell isolated from peripheral blood. In one example, the genetically engineered cell provided herein is obtained by genetically engineering a cell isolated from bone marrow. In one example, the genetically engineered cell provided herein is obtained by genetically engineering a cell isolated from peripheral blood mononuclear cells (PBMCs).

[0233] In one example, the engineered cell provided herein is obtained by genetically engineering a stem cell or progenitor cell in vitro. In one example, the stem cell or progenitor cell is selected from the group consisting of a T cell progenitor cell, a hematopoietic stem / progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell. In one example, the engineered cell provided herein is obtained by genetically engineering a T cell progenitor cell in vitro. In one example, the engineered cell provided herein is obtained by genetically engineering a hematopoietic stem / progenitor cell in vitro. In one example, the engineered cell provided herein is obtained by genetically engineering a hematopoietic multipotent progenitor cell in vitro. In one example, the engineered cell provided herein is obtained by genetically engineering an embryonic stem cell in vitro. In one example, the engineered cell provided herein is obtained by genetically engineering an induced pluripotent cell in vitro.

[0234] Due to the immunogenetic differences between the donor and the recipient (or host), the donor as an exogenous graft is recognized and attacked by the immune cells (e.g., NK cells) in the host when the exogenous donor is transplanted, which in turn inhibits or eliminates the donor, resulting in host versus graft reaction (HVGR). As in allogeneic cell transplantation, the absence of HLA class I molecules in allogeneic cells can reduce host CD8+ mediated cellular immune rejection; in one example, the absence of HLA class I molecules in allogeneic cells enhances host NK cell mediated cellular immune rejection. In one example, the engineered cell provided herein has low expression or no expression of endogenous TCR and / or B2M.

[0235] Graft versus host disease (GVHD) is due to the diversity of TCR of the exogenous transplant donor T lymphocytes, and the incompatibility with host HLA molecules, the donor T lymphocytes can recognize the antigen on the host normal tissue, expand and release a series of cytokines, greatly enhance the immune response of the graft to the host antigen, attack the host cells, knock out the TCR of the exogenous transplant donor (such as T cells) to avoid graft versus host disease. In an example, the present application provides an engineered cell with low or no expression of endogenous HLA-I / TCR. In an example, the present application uses a CRISPR system to knock out the gene TRAC of the alpha chain of the endogenous TCR to prepare a cell with low or no expression of endogenous TCR. In an example, the present application uses a CRISPR system to knock out endogenous B2M to prepare a cell with deletion of endogenous HLA-I. In an example, the present application provides a cell with low or no expression of HLA-A and HLA-B and / or TCR. In an example, the present application provides an engineered cell with low or no expression of endogenous HLA-I / TCR and / or HLA-II. In an example, the present application provides an engineered cell with low or no expression of endogenous HLA-I / TCR. For example, the engineered cell has low or no expression of endogenous TCR / B2M, TCR / B2M / FAS, TCR / B2M / NKG2A, or TCR / B2M / FAS / NKG2A.

[0236] In an example, the present application provides a method of genetically engineering a cell by using gene editing to transfer a polynucleotide provided by the present application into the cell. If necessary, techniques such as nuclease, transcription activator-like effector nuclease (TALEN), zinc finger nuclease (ZFN), clustered regularly interspaced short palindromic repeats (CRISPR), homologous recombination, non-homologous end joining, microhomology-mediated end joining, homology-mediated end joining, etc. can be used to achieve site-specific integration (Gersbach et al., Nucl. Acids Res. 39:7868-7878 (2011); Vasileva, et al. Cell Death Dis. 6:el831. (Jul 23 2015); Sontheimer, Hum. Gene Ther. 26(7):413-424 (2015); Yao et al. Cell Research volume 27, 801-814 (2017)).

[0237] In an example, the endogenous TCR / B2M is knocked out by CRISPR / Cas9 technology. For example, the sgRNA sequences targeting TRAC, B2M, NKG2A, FAS are respectively as shown in SEQ ID NO: 13, 12, 22, 23.

[0238] For example, in some embodiments, the present application provides an engineered T cell expressing a fusion protein, the engineered T cell comprising no expression of endogenous TCR / B2M; the fusion protein comprising, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, optionally further comprising an intracellular signaling domain; wherein the extracellular domain comprises an antibody that binds to CD300A as disclosed herein, the transmembrane domain comprises a transmembrane domain of, e.g., CD28, wherein the intracellular signaling domain comprises, e.g., a CD3 zeta intracellular signaling domain and a CD28 intracellular signaling domain. The extracellular domain and the transmembrane domain thereof can be connected by, e.g., a CD8 hinge region, a CD28 hinge region, or an IgG spacer or fragment thereof. The fusion protein can further comprise a domain that binds to an immune cell marker other than CD300A and / or a domain that binds to a target antigen on a pathological cell.

[0239] In an example, the engineered cell expressing a fusion protein of the present application can bind to a target cell expressing CD300A. For example, the engineered cell of the present application can bind to an NK cell. In an example, the engineered cell of the present application can bind to a target cell expressing CD300A and an immune cell marker. For example, the engineered cell of the present application can bind to a target cell expressing CD300A and an NK cell marker. For example, the engineered cell of the present application can bind to a target cell expressing CD300A and an NK inhibitory receptor.

[0240] In an example, the engineered cell expressing a fusion protein of the present application does not elicit a host rejection graft response. In an example, the engineered cell expressing a fusion protein of the present application has a longer survival time and / or an enhanced expansion capacity in the presence of a host immune cell, e.g., an NK cell. For example, the engineered cell can kill a host immune cell. For example, the engineered cell can kill a host NK cell. For example, the engineered cell can kill an allogeneic NK cell. For example, the engineered cell can resist killing by a host NK cell. For example, the engineered cell can resist killing by an allogeneic NK cell. For example, the engineered cell expressing a fusion protein of the present application has a significantly enhanced ability to kill a pathological cell relative to a cell not expressing a fusion protein of the present application.

[0241] In an example, the engineered cell has an extended survival time or an enhanced expansion capacity in the presence of a host immune cell. In an example, the engineered cell has an inhibitory or killing function against a host immune cell. In an example, the engineered cell has an inhibitory or killing function against a host T cell, an NK cell. In an example, the engineered cell has an inhibitory or killing function against a host NK cell. In an example, the engineered cell can enhance the survival, proliferation, and killing of a pathological cell of another engineered cell previously, simultaneously, or subsequently introduced into a subject.

[0242] In one example, the engineered cells expressing a binding to CD300A of the present application have a longer survival time and / or expansion capacity in the presence of host immune cells (e.g., NK cells). For example, the engineered cells are more potent at killing host NK cells. For example, the engineered cells are more potent at killing allogeneic NK cells. For example, the engineered cells are more resistant to killing by host NK cells. For example, the engineered cells are more resistant to killing by allogeneic NK cells.

[0243] In one example, the dual-target engineered cells expressing a binding to CD300A and a binding to a target antigen on a pathological cell of the present application have a longer survival time and / or expansion capacity in the presence of host immune cells (e.g., NK cells). For example, the engineered cells exhibit a stronger cell killing effect on pathological cells carrying the target antigen in vivo and in vitro.

[0244] In one example, the engineered cell expressing the fusion protein also expresses an immune checkpoint inhibitor. Immune checkpoint inhibitors include any agent that blocks, inhibits, or reduces the activity or function of an inhibitory pathway of the immune system. Such inhibitors can include small molecule inhibitors or can include antibodies or antigen-binding fragments thereof that bind to and block or inhibit an immune checkpoint receptor, ligand, and / or receptor-ligand interaction. In some embodiments, modulation, enhancement, and / or stimulation of a particular receptor can prevail over immune checkpoint pathway components. Exemplary immune checkpoint molecules that can be targeted for blocking, inhibiting, modulating, enhancing, and / or stimulating include, but are not limited to: PD-1 (CD279), PD-L1 (CD274, B7-H1), PDL2 (CD273, B7-DC), CTLA-4, LAG-3 (CD223), TIM-3, 4-1BB (CD137), 4-1BBL (CD137L), GITR (TNFRSF18, AITR), CD40, OX40 (CD134, TNFRSF4), CXCR2, tumor associated antigen (TAA), B7-H3, B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, gd and memory CD8+(aP) T cells), CD160 (also known as BY55), CGEN-15049, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and transforming growth factor receptors (TGFR; e.g., TGFR beta). Immune checkpoint inhibitors include antibodies or antigen-binding fragments thereof or other binding proteins that bind to and block or inhibit and / or enhance or stimulate the activity of one or more of any of the described molecules.

[0245] Compositions

[0246] The present application also provides pharmaceutical compositions comprising the fusion proteins described above. The present application also provides cell compositions comprising the engineered cells described above. The present application also provides pharmaceutical compositions comprising the engineered cells described above. In one example, a pharmaceutical composition comprises an effective amount of a fusion protein disclosed herein and a pharmaceutically acceptable carrier. In one example, a pharmaceutical composition comprises an effective amount of an engineered cell disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is useful in inhibiting host rejection of a graft reaction. In some embodiments, the pharmaceutical composition is useful in immunotherapy. In some embodiments, the pharmaceutical composition is useful in immuno-oncology. In one example, the pharmaceutical composition is useful in inhibiting tumor growth in a subject (e.g., a human patient). In one example, the pharmaceutical composition is useful in treating cancer in a subject (e.g., a human patient). In one example, the pharmaceutical composition is useful in treating viral infection. In one example, the pharmaceutical composition is useful in treating autoimmune disease.

[0247] In one example, the present application provides a cell composition comprising a first engineered cell and a second engineered cell. In one example, the first engineered cell in the cell composition provided by the present application comprises a fusion protein disclosed herein that binds to a domain of CD300A, and the second engineered cell binds to a target antigen (e.g., CD19, BCMA) on a pathological cell (e.g., a tumor cell, a pathological cell of an autoimmune disease). The second engineered cell can be an engineered immune cell for tumor treatment in the prior art. In one example, the second engineered cell can be a CAR-T cell in the prior art.

[0248] In one example, the present application provides that the species of cells in the cell composition can be the same or different. For example, the first engineered cell or the second engineered cell can be any suitable immune cell known, and the species of the first engineered cell does not affect the species of the second engineered cell. For example, the first engineered cell can be a T, NK, NKT cell, and the second engineered cell can be a T, NK, NKT cell or any other suitable immune cell.

[0249] In one example, the present application provides a pharmaceutical composition comprising a fusion protein or a cell provided by the present application. In one example, the composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or topical administration (e.g., by injection or infusion). In one example, the composition is suitable for local administration. In some aspects, local administration includes intratumoral injection, peritumoral injection, juxtatumoral injection, intralesional injection, and / or injection into tumor-draining lymph nodes, or essentially any tumor-targeted injection in which the anti-tumor agent is expected to leak into the primary lymph nodes adjacent to the targeted solid tumor.

[0250] Depending on the route of administration, the active ingredient (i.e., the fusion protein or engineered cell) can be coated in a material to protect the active ingredient from the action of acids and other natural conditions that can inactivate the active ingredient. Pharmaceutically acceptable carriers that can be used in the compositions or formulations provided herein include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with physiological conditions. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or topical administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (i.e., the genetically engineered fusion protein or cell) can be coated in a material to protect the active ingredient from the action of acids and other natural conditions that can inactivate the active ingredient.

[0251] The present application also provides kits for preparing a pharmaceutical composition containing a fusion protein disclosed herein. In some embodiments, the kit includes a fusion protein disclosed herein and a pharmaceutically acceptable excipient in one or more containers. In another embodiment, the kit can include a fusion protein disclosed herein for administration to a subject. In particular embodiments, the kit includes instructions for the preparation and / or administration of the fusion protein.

[0252] The present application also provides kits for preparing a cell disclosed herein. In one embodiment, the kit includes one or more vectors for producing an engineered cell (e.g., a T cell) expressing a fusion protein disclosed herein. The kit can be used to produce a genetically engineered cell from an autologous or non-autologous cell for administration to a compatible subject. In another embodiment, the kit can include a cell disclosed herein for administration to a subject. In particular embodiments, the kit includes a cell disclosed herein in one or more containers. In particular embodiments, the kit includes instructions for the preparation and / or administration of the genetically engineered cell.

[0253] The present application also provides kits for inducing and / or enhancing an immune response and / or treating and / or preventing a tumor or pathogenic infection, an autoimmune disease, an inflammatory disease in a subject. For example, the kit includes an effective amount of a fusion protein and / or a composition of a chimeric receptor that binds to a pathologic cell of the present application and a pharmaceutical composition. For example, the kit includes a sterile container; such container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such container can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments. For example, the kit includes a molecule encoding a fusion protein, a chimeric receptor, a recombinant TCR, an exogenous cytokine, and / or a therapeutic mAb of the present application, which can optionally be included in one or more vectors.

[0254] Methods and uses

[0255] The present application provides uses of the fusion proteins or engineered cells disclosed herein. In one example, the fusion proteins or engineered cells disclosed herein are used in the manufacture of a medicament for preventing or combating transplant immune rejection. In one example, the fusion proteins or engineered cells disclosed herein are used for preventing or combating transplant immune rejection.

[0256] In one example, the present application provides a method for preventing, alleviating and / or treating a tumor, an autoimmune disease, an inflammatory disease, comprising administering to a subject in need thereof the fusion protein, the engineered cell, or the pharmaceutical composition described herein. The pharmaceutical composition provided herein has been described above, and the method for preventing, alleviating and / or treating a tumor, an autoimmune disease, an inflammatory disease provided herein includes all the technical solutions thereof.

[0257] In one example, the fusion proteins or engineered cells disclosed herein can induce and / or increase an immune response in a subject. The present application provides a method for inhibiting the activity of a pathological cell in a subject, the method comprising: administering to the subject a therapeutically effective amount of an engineered cell expressing a fusion protein of the present application, the engineered cell inhibiting the activity of and / or killing the pathological cell in the subject. For example, the pathological cell is a tumor cell. For example, the pathological cell includes, but is not limited to, an acute myeloma leukemia cell, an anaplastic lymphoma cell, an astrocytoma cell, a B-cell cancer cell, a breast cancer cell, a colon cancer cell, an ependymoma cell, an esophageal cancer cell, a glioblastoma cell, a glioma cell, a leiomyosarcoma cell, a liposarcoma cell, a liver cancer cell, a lung cancer cell, a mantle cell lymphoma cell, a melanoma cell, a neuroblastoma cell, a non-small cell lung cancer cell, an oligodendroglioma cell, an ovarian cancer cell, a pancreatic cancer cell, a peripheral T-cell lymphoma cell, a renal cancer cell, a sarcoma cell, a stomach cancer cell, a liver cancer cell, a mesothelioma cell, or a sarcoma cell. For example, the target cell is an immune cell.

[0258] In one example, the fusion proteins or engineered cells disclosed herein can be used to treat and / or prevent a tumor in a subject. The disclosed fusion proteins or engineered cells can be used to prolong the survival of a subject having a tumor. The disclosed fusion proteins or engineered cells can also be used to treat and / or prevent a pathogen infection or other infectious disease in a human subject, such as an immunocompromised subject. Such methods include administering an effective amount of a composition of the present application to achieve the desired effect, whether alleviating an existing condition or preventing recurrence. For treatment, the amount administered is an amount effective to produce the desired effect. An effective amount can be provided in one or more administrations. An effective amount can be provided in a bolus or by continuous infusion.

[0259] In one example, the tumor is a solid tumor or a hematological tumor. In one example, the solid tumor is selected from the group consisting of: esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, sarcoma; the hematological tumor is selected from the group consisting of: leukemia, lymphoma, myeloma.

[0260] In one example, the fusion protein or the engineered cell disclosed herein is used for preparing a medicament for treating or preventing an autoimmune disease or an inflammatory disease. In one example, the fusion protein or the engineered cell disclosed herein is used for treating, preventing or ameliorating an autoimmune disease or an inflammatory disease in a subject in need thereof.

[0261] In one example, the autoimmune disease is selected from the group consisting of: multiple sclerosis, autoimmune liver disease, type I diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), polymyositis / dermatomyositis.

[0262] In one example, the autoimmune disease-related inflammatory disease is selected from, such as arthritis (e.g., rheumatoid arthritis, arthritis chronica progrediente, and arthritis deformans) and rheumatic diseases, including inflammatory conditions and rheumatic diseases involving bone loss, inflammatory pain, spondyloarthropathies (including ankylosing spondylitis), Reiter's syndrome, reactive arthritis, psoriatic arthritis, juvenile idiopathic arthritis, and enteropathic arthritis, enthesitis, hypersensitivity reactions (including airway hypersensitivity and skin hypersensitivity), and allergy. The engineered T cells provided herein are used to treat and prevent autoimmune hematological disorders (including, e.g., hemolytic anemia, aplastic anemia, pure red cell anemia, and idiopathic thrombocytopenia), systemic lupus erythematosus (SLE), lupus nephritis, inflammatory muscle disease (dermatomyositis), periodontitis, polychrondritis, scleroderma, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel disease (including, e.g., ulcerative colitis, Crohn's disease, and irritable bowel syndrome), endocrine ophthalmopathy, Graves' disease, sarcoidosis, multiple sclerosis, systemic sclerosis, fibrotic disorders, primary biliary cirrhosis, juvenile onset diabetes (Type I diabetes), uveitis, keratoconjunctivitis sicca, and vernal keratoconjunctivitis, interstitial lung fibrosis, periprosthetic osteolysis, glomerulonephritis (with and without nephrotic syndrome, including idiopathic nephrotic syndrome or minimal change nephropathy), multiple myeloma, other types of tumors, inflammatory diseases of the skin and cornea, myositis, loosening of bone implants, metabolic disorders (such as obesity, atherosclerosis and other cardiovascular diseases, including dilated cardiomyopathy, myocarditis, Type II diabetes, and dyslipidemia), and autoimmune thyroid disease (including Hashimoto's thyroiditis), small and medium vessel primary vasculitis, large vessel vasculitis including giant cell arteritis, hidradenitis suppurativa, neuromyelitis optica, Sjogren's syndrome, Behcet's disease, atopic and contact dermatitis, bronchiolitis, inflammatory muscle disease, autoimmune peripheral neuropathy, immune renal, liver, and thyroid disease, inflammation and atherosclerosis, autoinflammatory fever syndromes, immune hematological disorders, and bullous diseases of the skin and mucosa. For example, AID refers to a group of diseases that affect multiple tissues organs or systems, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), polymyositis / dermatomyositis, etc.

[0263] In one example, the fusion protein or the engineered cell disclosed herein can be used to treat, prevent or ameliorate asthma, bronchitis, bronchiolitis, idiopathic interstitial pneumonia, pneumoconiosis, emphysema, and other obstructive or inflammatory diseases of the airways.

[0264] In one example, the present application provides a method of treating a disease in a subject having a tumor, an immune disease (e.g., an autoimmune disease), or an inflammatory disease, the method comprising: i) genetically modifying T cells, NK cells and / or NKT cells obtained from autologous or allogeneic with a vector comprising a nucleic acid molecule encoding a fusion protein of the present application, wherein the fusion protein is capable of specifically binding to an immune cell (e.g., a NK cell) and a pathological cell (e.g., a tumor cell, a pathological cell of an autoimmune disease, a pathological cell of an inflammatory disease) in the subject; ii) introducing the genetically modified T cells, NK cells and / or NKT cells into the subject, wherein the genetically modified T cells, NK cells and / or NKT cells recognize and kill the tumor cells, and the genetically modified cells can resist attack by immune cells of the subject.

[0265] In the present application, the methods described in the present application can also be interpreted as therapeutic uses, i.e., the methods described in the present application can all be considered as therapeutic uses of the compositions, nucleic acid molecules or engineered cells of the present application or pharmaceutical uses for preparing the corresponding therapeutic uses. For example, the present application relates to the use of the above-mentioned engineered cells or nucleic acid molecules for modulating the activity of the engineered cells or the use of the above-mentioned engineered cells for the preparation of a medicament for modulating the activity of the engineered cells; the present application relates to the use of the above-mentioned engineered cells or nucleic acid molecules for activating the engineered cells or the use of the above-mentioned engineered cells for the preparation of a medicament for activating the engineered cells; the present application relates to the use of the above-mentioned engineered cells or nucleic acid molecules for inhibiting the activity of target cells in a subject or the use for the preparation of a medicament for inhibiting the activity of target cells in a subject; the present application relates to the use of the above-mentioned engineered cells or nucleic acid molecules for improving or treating the health condition of a subject in need thereof or the use for the preparation of a medicament for improving or treating the health condition of a subject in need thereof. All the above-mentioned contents described in the present application can also be equally applicable to the therapeutic uses or pharmaceutical uses.

[0266] The engineered cells of the application can be administered as the sole active ingredient or in combination with other drugs, e.g., immunosuppressive or immunomodulatory agents or other anti-inflammatory agents or other cytotoxic or anticancer agents, e.g., as an adjuvant or in combination with them, e.g., to treat or prevent an immune disorder-related disease. For example, the antibodies of the application can be used in combination with DMARDs, e.g., gold salts, sulfasalazine, antimalarials, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, tacrolimus, sirolimus, minocycline, leflunomide, glucocorticoids; calcineurin inhibitors, e.g., cyclosporin A or FK 506; modulators of lymphocyte recirculation, e.g., FTY720 and FTY720 analogs; mTOR inhibitors, e.g., rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573 or TAFA-93; ascomycins with immunosuppressive properties, e.g., ABT-281, ASM981, etc.; corticosteroids; cyclophosphamide; azathioprine; leflunomide; mizoribine; mycophenolate mofetil; 15-deoxyspergualine or immunosuppressive homologs, analogs or derivatives thereof; immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies against leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86 or their ligands; other immunomodulatory compounds.

[0267] The application will be further illustrated by 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 application. The experimental methods in the following examples, where no specific conditions are indicated, were generally performed according to routine conditions, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory, 2002, or as suggested by the manufacturer. All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

[0268] Examples

[0269] Example 1. Flow cytometry detection of CD300A expression in human primary NK, aNK

[0270] The peripheral blood PBMC cells isolated from donors were collected, and human primary NK cells were screened out using CD56 magnetic beads (Miltenyi, 130-050-401) according to the manufacturer's instructions. 1 × 10 5NK cells, resuspend sample cells with 100 μL MACS solution (Miltenyi, 130-091-221), each flow sample add 1 μL CD300A antibody (Thermo Fisher, Cat# MA5-44131) and 1 μL CD56 antibody (eBioscience, Cat# 25-0567-42) at 1:100, while the sample with CD56 antibody alone is negative control, incubate at room temperature for 30 min, and detect the expression of CD300A by flow cytometry. Figure 1A shows that about 98% of NK cells isolated from different donors express CD300A.

[0271] PBMC cells isolated from donors are collected, and NK cells are screened according to the manufacturer's instructions using CD56 magnetic beads (Miltenyi, 130-050-401). After stimulation and expansion by cytokines IL2 and IL15, activated NK (aNK) cells are obtained. Add 1 μL CD300A antibody (Thermo Fisher, Cat# MA5-44131) and 1 μL CD56 antibody (eBioscience, Cat# 25-0567-42) to 1 x 10 5 PBMC cells isolated from donors are collected, and NK cells are screened according to the manufacturer's instructions using CD56 magnetic beads (Miltenyi, 130-050-401). After stimulation and expansion by cytokines IL2 and IL15, activated NK (aNK) cells are obtained. Add 1 μL CD300A antibody (Thermo Fisher, Cat# MA5-44131) and 1 μL CD56 antibody (eBioscience, Cat# 25-0567-42) to 1 x 10

[0272] Example 2. Vector construction

[0273] The vector expressing the fusion protein disclosed in the present application is constructed using conventional molecular biology techniques (Figure 2). The sequence comprising the extracellular domain (antibody binding CD300A or extracellular segment of CD300A ligand), hinge region, transmembrane region, and optionally, intracellular domain fragment (Suzhou Anshengda Biotechnology Co., Ltd.) is integrated into the pRRLSIN vector (Addgene) to construct. The CD300A binding domain can be an anti-CD300A antibody (such as scFv, single domain antibody (sdAb), or DDpp antibody), or an extracellular segment of CD300A ligand. The corresponding lentivirus of the above-mentioned expression vector is prepared using conventional molecular biology techniques. CD300A-28z, CD300A-PDGFR, CD300A-BB, comprising the sequences shown in SEQ ID NO: 15, 17, 20, respectively.

[0274] Example 3. Preparation of cells

[0275] The CAR-T cells were prepared by the method of the art: collecting donor PBMC cells, activating with magnetic beads of anti-CD3 and CD28 antibodies (Life Technologies, Cat#40203D), and then culturing to obtain T cells; infecting the T cells with the lentivirus containing the above-mentioned vector to obtain T cells containing the CD300A fusion protein. 1 x 10 5 The cells were collected, and the sample of the UTD group was used as a negative control. The cells were incubated with an anti-mouse Fab antibody (Jackson ImmunoResearch, Cat#115-545-006) at room temperature for 30 min, and the positive rate of CD300A-28z was about 98% (Fig. 3A) as detected by flow cytometry.

[0276] Example 4. Preparation of endogenous TCR / B2M knockout cells

[0277] The sgRNA sequences (as shown in SEQ ID NO: 13 and 12, respectively) targeting TRAC and B2M, respectively, were synthesized in vitro (Kai Xing Diagnostics), and dko cells with TRAC and B2M knocked out were prepared by CRISPR / Cas9 technology (Cas9 protein, Kai Xing Biotechnology (Shanghai), Cat#CAS-EE109). The endogenous TCR / B2M of the CD300A-28z cells constructed in Example 3 was knocked out by CRISPR / Cas9 technology, and CD300A-28z-dko cells were obtained by magnetic bead sorting (Miltenyi, Cat#130-048-801). UTD-dko cells with TCR / B2M knocked out but not transduced were used as a control. The same method was used to prepare tko1 cells with endogenous TRAC, B2M and NKG2A knocked out, and tko2 cells with endogenous TRAC, B2M and Fas knocked out. The sgRNA sequences targeting TRAC, B2M, NKG2A and Fas are shown in SEQ ID NO: 13, 12, 22 and 23, respectively.

[0278] Specifically, the cells prepared in Example 3 were collected, and TCR / B2M was knocked out by CRISPR / Cas9 technology on day 0. The knockout efficiency and CAR positive rate were determined on day 3, and CD300A-28z-dko cells were obtained by magnetic bead sorting on day 4. Fig. 3B shows that the proportion of CD300A-28z-dko cells was 99%.

[0279] Example 5. In vitro killing of human aNK cells by T cells containing CD300A fusion protein

[0280] On day 0, 5 x 10 4CD300A-28z-dko cells and aNK cells (effector to target ratio 1:1.5, 1:5) were co-incubated at 37°C in incubator, B2M knockout cells (B2M KO-T cells) were labeled with HLA-ABC (Thermo Fisher, Cat#17-9983-42) and NK cells were labeled with CD56 (Cat#BD 555516) at D0-D3 days respectively; 7-AAD fluorescent dye (BD, Cat#559925) was used to distinguish dead cells and live cells, and the number of aNK cells was detected by flow cytometry.

[0281] Fig. 4A, B shows that CD300A-28z-dko cells significantly kill aNK at effector to target ratio 1:1.5 and 1:5, and can resist aNK killing and improve T cell survival; CD300A-28z-dko cells were killed by aNK cells to the least number at D1, and then rapidly recovered growth at D2, and exceeded the number of starting CAR-T cells at D3. Similarly, when aNK cells isolated from different donors were used to replace the aNK cells in the experiment, similar results were obtained (Fig. 5A, B).

[0282] Example 6. Preparation of cells

[0283] CD300A-PDGFR-dko cells were prepared according to the preparation method of CD300A-PDGFR cells in Example 3 and Example 4, and the positive rate was about 84.1% (Fig. 6A), and the proportion of CD300A-PDGFR-dko cells after magnetic bead sorting was 99% (Fig. 6B). CD300A / FasL-CAR-T, CD300A / NKG2A-CAR-T, CD300A / BCMA-NKG2A-CAR-T, CD300A / CD19-CD20-CAR-T, CD300A / CD38-NKG2A-CAR-T cells were prepared, and the sequences contained in the cells were shown in SEQ ID NO: 19, 24, 25, 26, 38, respectively. CD300A / FasL-CAR-T-dko, CD300A / FasL-CAR-T-tko2, CD300A / NKG2A-CAR-T-dko, CDA300A / NKG2A-CAR-T-tko1, CD300A / BCMA-NKG2A-CAR-T-dko, CD300A / BCMA-NKG2A-CAR-T-tko1, CD300A / CD19-CD20-CAR-T-dko, CD300A / CD38-NKG2A-CAR-T-tko1 cells were prepared.

[0284] Briefly, donor PBMCs were collected on day 0 and T cells were activated for 24 hours (CD3 / 28 Dynabeads). T cells were infected with lentivirus on day 1. On day 4, demagnetization was performed and B2M / TRAC, B2M / TRAC / NKG2A, or B2M / TRAC / FAS were knocked down using RNP. On day 7, knockout efficiency and CAR positivity were detected using HLA-ABC (APC) and CD3 (BV421). B2M and CD3 double-negative cells were sorted using B2M (PE) and CD3 (PE). After sorting, sorting efficiency was further detected using HLA-ABC (APC) and CD3 (BV421). FasL-CAR positivity was detected using FasL-His antigen, NKG2A-CAR positivity was detected using NKG2A-biotin antigen, and CD300A-CAR positivity was detected using anti-mFab antibody (Jackson Immuno Research, 115-605-072). The CAR positivity rate of FasL-28z-dko was 98.8%, and the CD300A / FasL-CAR double-positive rate was 96%. The CAR positivity rate of NKG2A-28z-tko1 was 92.5%, and the CD300A / NKG2A-CAR-tko1 double-positive rate was 90.6%.

[0285] Example 7. T cells containing CD300A fusion protein have the ability to resist killing by human primary NK cells

[0286] On D0, 5×10 4 CD300A-PDGFR-dko cells and primary human NK cells from different donors (effector-target ratio 1:2) were co-incubated in an incubator at 37°C. On days D0-D3, B2M knockout cells (B2M KO-T cells) were labeled with HLA-ABC (Thermo Fisher, Cat#17-9983-42), and NK cells were labeled with CD56 (BD, Cat#555516). 7-AAD fluorescent dye (BD, Cat#559925) was used to distinguish dead cells from live cells, and the number of primary NK cells was detected by flow cytometry.

[0287] Figure 7A shows that compared with the UTD group, CD300A-PDGFR-dKO cells can significantly resist killing by primary human NK cells from different donors and have a high T cell survival rate. Figure 7B shows that compared with the NK group, the CD300A-PDGFR-dKO group has no effect on NK cell survival. Figure 7C shows that in the presence of primary human NK cells, the CD300A-PDGFR-dKO group can significantly improve T cell survival compared with TIM3-PDGFR-dKO.

[0288] Example 8. T cells comprising CD300A fusion proteins have the ability to resist aNK killing

[0289] On day 0, 5 x 105CD300A-PDGFR-dko cells and different donor human aNK cells (effector to target ratio 1 : 1.5, 1 :2) were co-incubated at 37 °C in incubator, B2M-KO T cells were labeled with HLA-ABC (Thermo Fisher, Cat# 17-9983-42) and NK cells were labeled with CD56 (BD, Cat# 555516) on day 0, 1, 2 and 3, respectively; 7-AAD fluorescent dye (BD, Cat# 559925) was used to distinguish dead cells from live cells, and the number of NK cells was detected by flow cytometry. 4

[0290] Figure 8A shows that CD300A-PDGFR-dko cells can significantly resist aNK cell killing and T cell survival is high compared to the UTD group. Figure 8B shows that CD300A-PDGFR-dko group does not affect NK cell survival compared to the NK group. Figure 8C shows that CD300A-PDGFR-dko group can significantly improve B2M-KO-T cell survival in the presence of aNK cells compared to TIM3-PDGFR-dko.

[0291] Example 9. T cells comprising CD300A / FasL-CAR, CD300A / NKG2A-CAR, CD300A / CD38-NKG2A-CAR have the ability to resist NK cell killing

[0292] Referring to Examples 7, 8. UCAR-T and NK cells were co-incubated at an effector to target ratio of 1 :2 for 24 or 72 hours. Figure 9A shows that CD300A / FasL-CAR-T-dko does not affect primary NK cell survival, but can significantly resist primary NK cell killing and improve T cell survival. Figure 9B shows that CD300A / NKG2A-CAR-T-tko1 cells significantly kill aNK cells and can significantly resist aNK cell killing and improve T cell survival. Figure 9B shows that CD300A / CD38-NKG2A-CAR-T-tko1 cells can significantly resist aNK cell killing and improve T cell survival.

[0293] Example 10. T cells comprising chimeric receptors targeting target antigens on pathological cells, and fusion proteins binding CD300A have the ability to resist NK cell killing

[0294] ​Referring to Example 7, 8. UCAR-T cells were co-incubated with NK cells at an effector to target ratio of 1:1 or 1:2 for 24 or 72 hours. FIG. 10A shows that CD300A / BCMA-NKG2A-CAR-T-tko1 cells can significantly resist the killing of aNK and improve T cell survival. FIG. 10B shows that CD300A / BCMA-NKG2A-CAR-T-tko1 cells can significantly resist the killing of primary NK and improve T cell survival. FIG. 11 shows that CD300A / CD19-CD20-CAR-T-dko can significantly resist the killing of aNK cells and has high T cell survival rate.

[0295] Example 11. Engineered cells containing CD300A-PDGFR and binding FasL, CD38 and / or TIGIT chimeric receptors have the ability to resist NK cell killing

[0296] Referring to Example 3, 4, 6, CD300A-PDGFR / FasL-CAR-T, CD300A-PDGFR / CD38-CAR-T, CD300A-PDGFR / TIGIT-CAR-T cells were constructed.

[0297] UCAR-T cells were co-incubated with NK cells at an effector to target ratio of 1:1 or 1:2 for 24 or 72 hours, and CD300A-PDGFR / FasL-CAR-T-dko, CD300A-PDGFR / FasL-CAR-T-tko2, CD300A-PDGFR / CD38-CAR-T-dko, CD300A-PDGFR / TIGIT-CAR-T-dko cells can significantly resist the killing of primary NK cells or aNK cells and have high T cell survival rate.

[0298] Example 12. CD300A / CD19-CAR-T, CD300A / CD19-NKG2A-CAR-T, CD300A / CD19-CD38-CAR-T, CD300A / BCMA-CAR-T, CD300A / BCMA-CD38-CAR-T, CD300A / GPRC5D-CAR-T, CD300A / GPRC5D-CD38-CAR-T, CD300A / GPRC5D-NKG2A-CAR-T, CD300A / GPC3-CAR-T, CD300A / GPC3-NKG2A-CAR-T, CD300A / GPC3-CD38-CAR-T, CD300A / Claudin18.2-CAR-T, CD300A / Claudin18.2-NKG2A-CAR-T, CD300A / Claudin18.2-CD38-CAR-T cells have the ability to resist NK cell killing

[0299] CD300A / CD19-NKG2A-CAR-T-tko1, CD300A / CD19-CD38-CAR-T-dko, CD300A / BCMA-CAR-T-dko, CD300A / BCMA-CD38-CAR-T-dko, CD300A / GPRC5D-CAR-T-dko, CD300A / GPRC5D-CD38-CAR-T-dko, CD300A / GPRC5D-NKG2A-CAR-T-tko1, CD300A / GPC3-CAR-T-dko, CD300A / GPC3-NKG2A-CAR-T-tko1, CD300A / GPC3-CD38-CAR-T-dko, CD300A / Claudin18.2-CAR-T-dko, CD300A / Claudin18.2-NKG2A-CAR-T-tko1, CD300A / Claudin18.2-CD38-CAR-T-dko cells were constructed according to the methods of Example 3, 4, 6.

[0300] UCAR-T cells and NK cells were co-incubated at an effector-target ratio of 1:1 or 1:2 for 24 or 72 hours, and CD300A / CD19-NKG2A-CAR-T-tko1, CD300A / CD19-CD38-CAR-T-dko, CD300A / BCMA-CAR-T-dko, CD300A / BCMA-CD38-CAR-T-dko, CD300A / GPRC5D-CAR-T-dko, CD300A / GPRC5D-CD38-CAR-T-dko, CD300A / GPRC5D-NKG2A-CAR-T-tko1, CD300A / GPC3-CAR-T-dko, CD300A / GPC3-NKG2A-CAR-T-tko1, CD300A / GPC3-CD38-CAR-T-dko, CD300A / Claudin18.2-CAR-T-dko, CD300A / Claudin18.2-NKG2A-CAR-T-tko1, CD300A / Claudin18.2-CD38-CAR-T-dko cells can significantly resist killing by primary NK cells or aNK cells, and T cells have high survival rate.

[0301] Example 13. T cells comprising a chimeric receptor targeting a target antigen on a pathological cell, and a fusion protein binding CD300A have anti-tumor effect in vivo and in vitro

[0302] CD300A / BCMA-NKG2A-CAR-T-tko1, CD300A / CD19-CD20-CAR-T-dko, CD300A / CD19-CAR-T-dko, CD300A / BCMA-GPRC5D-CAR-T1-dko, CD300A / BCMA-GPRC5D-CAR-T2-dko, CD300A / CD19-NKG2A-CAR-T-tko1, CD300A / CD19-CD38-CAR-T-dko, CD300A / BCMA-CAR-T-dko, CD300A / BCMA-CD38-CAR-T-dko, CD300A / GPRC5D-CAR-T-dko, CD300A / GPRC5D-CD38-CAR-T-dko, CD300A / GPRC5D-NKG2A-CAR-T-tko1, CD300A / GPC3-CAR-T-dko, CD300A / GPC3-NKG2A-CAR-T-tko1, CD300A / GPC3-CD38-CAR-T-dko, CD300A / Claudin18.2-CAR-T-dko, CD300A / Claudin18.2-NKG2A-CAR-T-tko1, CD300A / Claudin18.2-CD38-CAR-T-dko cells, all can significantly kill tumor cells expressing the corresponding target antigen.

[0303] The embodiments described herein include the embodiments as any single embodiment or in combination with any other embodiment or portions thereof. Moreover, it is to be understood that various modifications or changes can be made to the embodiments described herein by those skilled in the art, and such equivalents are intended to be within the scope of the claims.

[0304] Sequence information

Claims

1. A fusion protein, wherein From N-terminus to C-terminus, it includes an extracellular domain and a transmembrane domain, wherein the extracellular domain includes a domain that binds to CD300A.

2. The fusion protein according to claim 1, wherein The fusion protein further comprises an intracellular signal transduction domain; preferably, the intracellular signal transduction domain comprises a co-stimulatory signal, or a primary signaling domain, or a co-stimulatory signal and a primary signaling domain.

3. The fusion protein according to claim 1 or 2, wherein The CD300A-binding domain comprises a CD300A ligand or an extracellular segment thereof, and an antigen-binding unit that binds to CD300A; preferably, the CD300A-binding domain comprises an anti-CD300A antibody or a fragment thereof; preferably, the anti-CD300A antibody is selected from the group consisting of: a whole antibody, scFv, a single-domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, an sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody, an IgG4 antibody, a hybridoma antibody, a chimeric antibody, a humanized antibody, a fully human antibody or a monoclonal antibody; preferably, the anti-CD300A antibody or a fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL); more preferably, the VH and VL have the amino acid sequences shown in SEQ ID NOs: 2 and 1, respectively.

4. The fusion protein according to any one of claims 1 to 3, wherein The intracellular signal transduction domain comprises an intracellular signal transduction domain selected from: TCRα, TCRβ, TCRγ, TCRδ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD278 or CD66d, or a combination thereof; preferably, the intracellular signal transduction domain comprises a CD3ζ intracellular signal transduction domain (SEQ ID NO: 10, 36); preferably, the intracellular signal transduction domain comprises only a costimulatory domain; preferably, the costimulatory domain is selected from CD137 (4-1BB), CD28, CD27, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, CARD11, CD30, C D54, OX40, CD150, CD152, CD223, CD270, PD-L2, PD-L1, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, ICAM-1, LFA-1 (CD11a / CD18), 41BBL or CD83 intracellular signaling domain or a combination thereof; preferably, the co-stimulatory structure is selected from the intracellular signaling domain of CD137 (4-1BB) (SEQ ID NO: 8) or the intracellular signaling domain of CD28 (SEQ ID NO: 7).

5. The fusion protein according to any one of claims 1 to 4, wherein The transmembrane domain of the fusion protein is selected from the group consisting of: CD2, CD3ε, CD3δ, CD3ζ, CD8, CD9, CD16, CD22, CD25, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD154, CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), C D273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, transmembrane domain of PDGFR or Zap70.

6. The fusion protein according to any one of claims 1 to 5, wherein The transmembrane domain is the transmembrane domain of CD28 (SEQ ID NO: 6 or 29), the transmembrane domain of CD8 (SEQ ID NO: 4), or the transmembrane domain of PDGFR (SEQ ID NO: 3, 41, 42, 43, 46, 47 or 48).

7. The fusion protein according to any one of claims 1 to 6, wherein The extracellular domain and the transmembrane domain are connected by a connecting polypeptide; preferably, the connecting polypeptide is selected from: CD28 hinge region, CD8 hinge region, IgG spacer region or fragments thereof, or a combination thereof; preferably, the connecting polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 9, 14, 37 or 44.

8. The fusion protein according to any one of claims 1 to 7, wherein The fusion protein includes the amino acid sequence shown in SEQ ID NOs: 1, 2 and 3, or includes the amino acid sequence shown in SEQ ID NOs: 1, 2 and 47, or includes the amino acid sequence shown in SEQ ID NOs: 1, 2 and 48, or includes the amino acid sequence shown in SEQ ID NOs: 1, 2 and 41, or includes the amino acid sequence shown in SEQ ID NOs: 1, 2, 44 and 43, or includes the amino acid sequence shown in SEQ ID NOs: 1, 2, 44 and 46, or includes the amino acid sequence shown in SEQ ID NOs: 1, 2, 44 and 42, or includes the amino acid sequence shown in SEQ ID NOs: 1, 2, 9 and 43, or includes the amino acid sequence shown in SEQ ID NOs: 1, 2, 9 and 46, or includes the amino acid sequence shown in SEQ ID NOs: 1, 2, 9 and 42.

9. The fusion protein according to any one of claims 1 to 8, wherein The fusion protein comprises an amino acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 16, 18 or 21; or the fusion protein comprises an amino acid sequence encoded by a nucleic acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence shown in any one of SEQ ID NOs: 15, 17, 19, 20, 24, 25, 26 or 38; or the fusion protein is an amino acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 16, 18 or 21; or the fusion protein is a nucleic acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: The nucleic acid sequence shown in any one of NO: 15, 17, 19, 20, 24, 25, 26 or 38 is encoded by a nucleic acid sequence having 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity.

10. The fusion protein according to any one of claims 1 to 9, wherein The fusion protein further comprises a domain that binds to a target antigen on a pathological cell, or further comprises a domain that binds to another NK cell marker.

11. An engineered cell, wherein: The invention comprises the fusion protein according to any one of claims 1 to 10.

12. The engineered cell according to claim 11, wherein The engineered cell further comprises a chimeric receptor 2, which binds to a target antigen on a pathological cell; or the engineered cell further comprises a chimeric receptor 1 that binds to another NK cell marker.

13. The engineered cell according to claim 12, wherein The pathological cells are selected from malignant cells or infected cells; preferably, the pathological cells are selected from solid tumor cells, blood tumor cells, and pathological cells of autoimmune diseases; preferably, the solid tumors are selected from: esophageal cancer, gastric cancer, liver cancer, biliary tract tumors, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, and sarcoma; blood tumors are selected from: leukemia, lymphoma, and myeloma; and autoimmune diseases are selected from: multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren syndrome (SS), and polymyositis / dermatomyositis.

14. The engineered cell according to claim 12 or 13, wherein The target antigen is selected from: CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin18.2, FAP, Mesothelin, NKG2D ligand, NKG2A, CD94, FCRH5, EGFR and its mutants, ASGPR1, IL13RA2, WT1, CLL1 or a combination thereof.

15. The engineered cell according to any one of claims 12 to 14, wherein The other NK cell marker is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD138, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG 2A), CD159c (NKG2C), FasL, NKG2E, CD279, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRα, TCRβ, TIGIT, TRAIL, SLAMF7, NKG2F, NKG2H, NKp30, NKp44, NKp46, NKp80, SLAM family members, L-selectin, natural cytotoxicity receptor NCR1, NCR2, NCR3, or a combination thereof; preferably, the chimeric receptor 1 comprises a chimeric receptor 1 having a chimeric structure similar to SEQ An amino acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 27, 28, 39 or 45; or chimeric receptor 1 is an amino acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 27, 28, 39 or 45.

16. The engineered cell according to any one of claims 12 to 15, wherein The chimeric receptor 2 comprises an amino acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 30, 31, 32, 33, 39, 40 or 45; or the chimeric receptor 2 is an amino acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 30, 31, 32, 33, 39, 40 or 45.

17. The engineered cell according to any one of claims 12 to 16, wherein: The engineered cell comprises an amino acid sequence encoded by a nucleic acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence shown in any one of SEQ ID NOs: 15, 17, 19, 20, 24, 25, 26 or 38; or the fusion protein is an amino acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 16, 18 or 21; or the fusion protein is encoded by a nucleic acid sequence that is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence shown in any one of SEQ ID NOs: 15, 17, 19, 20, 24, 25, 26 or 38; or the engineered cell comprises the amino acid sequences shown in SEQ ID NOs: 21 and 27; SEQ ID NOs: the amino acid sequence shown in SEQ ID NOs: 18 and 28; the amino acid sequence shown in SEQ ID NOs: 18, 28 and 39; the amino acid sequence shown in SEQ ID NOs: 18 and 40; the amino acid sequence shown in SEQ ID NOs: 18 and 30; the amino acid sequence shown in SEQ ID NOs: 18 and 31; or the amino acid sequence shown in SEQ ID NOs: 18, 32 and 33.

18. The engineered cell according to any one of claims 11 to 17, wherein The engineered cells further include: low expression or no expression of endogenous TCR / B2M, TCR / B2M / FAS, TCR / B2M / NKG2A, or TCR / B2M / FAS / NKG2A; or, the engineered cells have low expression or no expression of endogenous TCR / B2M, TCR / B2M / FAS, TCR / B2M / NKG2A, or TCR / B2M / FAS / NKG2A.

19. The engineered cell according to any one of claims 11 to 18, wherein The engineered cells are autologous or allogeneic cells selected from: immune cells, neurons, epithelial cells, endothelial cells, stem cells or a combination thereof; preferably, the engineered cells are selected from: B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, T cells, NKT cells, stem cell-derived immune effector cells or a combination thereof; preferably, the engineered cells are allogeneic T cells.

20. The engineered cell according to any one of claims 11 to 19, wherein The engineered cells have a prolonged survival time or enhanced proliferation capacity in the presence of host immune cells; preferably, the host immune cells are NK cells.

21. The engineered cell according to any one of claims 11 to 20, wherein The engineered cells can enhance the survival, proliferation, and killing of pathological cells by another engineered cell that is previously, simultaneously, or subsequently introduced into a subject.

22. A polynucleotide, which is a nucleic acid molecule encoding the fusion protein constructed as described in any one of claims 1 to 10, or a nucleic acid molecule encoding the fusion protein, chimeric receptor 1 and / or chimeric receptor 2 contained in the engineered cell constructed as described in any one of claims 11 to 21. A vector comprising the polynucleotide according to claim 22 .

24. A virus comprising the vector of claim 23.

25. A pharmaceutical composition comprising an effective amount of the fusion protein according to any one of claims 1-10, the engineered cell according to any one of claims 11-21, the polynucleotide according to claim 22, the vector according to claim 23, or the virus according to claim 24, and a pharmaceutically acceptable carrier.

26. The fusion protein according to any one of claims 1-10 or the engineered cell according to any one of claims 11-21, which is used to prepare a drug for preventing or combating transplant rejection, or for preventing or combating transplant rejection, or for preparing a drug for treating an autoimmune disease or an inflammatory disease, or for treating, preventing or ameliorating an autoimmune disease or an inflammatory disease in a subject in need thereof, or for preparing a drug for treating tumors, or for treating, preventing or ameliorating tumors in a subject in need thereof.

27. The use according to claim 26, wherein The autoimmune disease is selected from the group consisting of: multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren's syndrome (SS), and polymyositis / dermatomyositis.

28. The use according to claim 26, wherein The tumor is a solid tumor or a blood tumor; preferably, the solid tumor is selected from: esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, sarcoma; the blood tumor is selected from: leukemia, lymphoma, myeloma.

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