Engineered immune cell expressing chemokine 10 and interleukin 15

Abstract

Provided are an engineered immune cell expressing CXCL10 and IL15 and the use thereof. The immune cell can further express a chimeric antigen receptor or a T cell receptor. The engineered immune cell can enhance the recruitment and accumulation of immune cells, enhance the killing efficiency of immune cells on target cells, and enhance the treatment effect of immune cells on tumors, such as solid tumors.

Description

Engineered immune cells expressing chemokine 10 and interleukin 15

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application No. 202311869165.8 filed with the China Patent Office on December 29, 2023, the entire text of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of immunotherapy, in particular to engineered immune cells, and more specifically to engineered immune cells expressing chemokine 10 and interleukin 15. Background Art

[0004] CAR T therapy is one of the most important areas of tumor immune cell therapy. It edits T lymphocytes to express specific antigen receptors, enhancing their ability to recognize tumor cells and generate specific immune responses, thereby achieving efficient and precise anti-tumor effects. CAR T has achieved remarkable success in preclinical and clinical trials for hematological B-cell malignancies. However, compared with hematological malignancies, CAR T has limited efficacy in solid tumors. The collagen matrix and immunosuppressive microenvironment of solid tumors hinder T cell infiltration, and the effective accumulation of T cells in solid tumors is a key determinant of the safety and efficacy of adoptive T cell therapy.

[0005] The CXC subgroup of chemokines and their related receptors are important members of the chemokine family. Among them, the chemokine receptor CXCR3 is mainly expressed in activated T cells, B cells and natural killer cells. CXCR3 regulates the body's immune response by binding to its ligands, inducing the directional movement and accumulation of specific immune cells. The CXCL9 / 10 / CXCL11-CXCR3 axis is associated with the progression of various solid tumors. Studies have shown that increasing the secretion of CXCL10 in tumor tissues, combined with CXCR3, can regulate the immune response of the body. + However, the secretion of chemokines in tumor tissues varies, and regulating the secretion of chemokines in tumor cells is difficult.

[0006] IL15 is a member of the γ-chain co-receptor family of cytokines. It regulates the survival, proliferation, and function of T cells and NK cells and has a wide range of immunomodulatory activities. Furthermore, IL15 can prevent the activation of regulatory T cells (Tregs). Studies have shown that the combination of chemokines and interleukins to modify CAR T cells facilitates immune cell infiltration into solid tumors. However, the combination of CAR T cells with CXCL10 and IL15 has not been reported. Summary of the Invention

[0007] In one aspect, the present invention provides engineered immune cells that express CXCL10 and IL15.

[0008] In some embodiments, the immune cell is a T cell, a B cell, a macrophage, a dendritic cell, a monocyte, a granulocyte, a NK cell, or a NKT cell.

[0009] In some embodiments, the immune cells express a fusion protein of CXCL10 and IL15. In some embodiments, in the fusion protein, CXCL10 and IL15 are connected by a linker, preferably a self-cleavable linker, more preferably T2A, F2A, or P2A. In some embodiments, CXCL10 comprises the amino acid sequence set forth in SEQ ID NO:9, and IL15 comprises the amino acid sequence set forth in SEQ ID NO:10.

[0010] In some embodiments, the immune cells further express a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the immune cells express a fusion protein of a chimeric antigen receptor, CXCL10, and IL15. In some embodiments, in the fusion protein, the chimeric antigen receptor, CXCL10, and IL15 are connected by a linker, which is preferably a self-cleaving linker, more preferably selected from T2A, F2A, or P2A.

[0011] In some embodiments, the chimeric antigen receptor targets an antigen expressed by a pathogen, a tumor-associated antigen, or an autoimmune disease-associated antigen. In some embodiments, the tumor-associated antigen is selected from CD123, CD7, BCMA, CD19, CEA, CD17L, CD20, IL13RA2, G02, PSMA, ERBB2, IgKappa, CD174, VEGFR2, CD30, mesothelin, CD138, CD33, c-Met, EGFR, CD22, FAP, CD133, EPCAM, GPC3, IL1RAP, MUC1, NKG2D-L, ROR1, CD70, LMP1, MG7, NY-ESO-1, CD276, GD2, B7H3, KRAS mutation, AFP, GPC3, and DLL3.

[0012] In some embodiments, the chimeric antigen receptor comprises a binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In some embodiments, the binding domain is a single-chain antibody (scFv). In some embodiments, the binding domain is an anti-Claudin18.2scFv or an anti-EGFRVIIIscFv. In some embodiments, the binding domain comprises an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 6.

[0013] In some embodiments, the hinge region is a CD8α hinge region. In some embodiments, the transmembrane domain is a transmembrane domain of CD28. In some embodiments, the costimulatory domain is a CD28 costimulatory domain or a 41BB costimulatory domain. In some embodiments, the intracellular signaling domain is a CD3ζ intracellular signaling domain. In some embodiments, the chimeric antigen receptor further comprises a signal peptide, preferably a CD8 signal peptide.

[0014] Another aspect of the present invention provides isolated polynucleotides encoding CXCL10 and IL15. In some embodiments, the isolated polynucleotides encode a fusion protein comprising CXCL10 and IL15. In some embodiments, in the fusion protein, CXCL10 and IL15 are connected by a linker, preferably a self-cleavable linker, more preferably a T2A, F2A, or P2A linker. In some embodiments, CXCL10 comprises the amino acid sequence set forth in SEQ ID NO:9, and IL15 comprises the amino acid sequence set forth in SEQ ID NO:10.

[0015] In some embodiments, the isolated polynucleotide further encodes a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the isolated polynucleotide encodes a fusion protein comprising the chimeric antigen receptor, CXCL10 and IL15. In some embodiments, in the fusion protein, the chimeric antigen receptor, CXCL10 and IL15 are connected by a linker, and the linker is preferably a self-cleaving linker, more preferably selected from T2A, F2A or P2A. In some embodiments, the chimeric antigen receptor or T cell receptor targets an antigen expressed by a pathogen, a tumor-associated antigen or an autoimmune disease-associated antigen. In some embodiments, the tumor-associated antigen is selected from CD123, CD7, BCMA, CD19, CEA, CD17L, CD20, IL13RA2, G02, PSMA, ERBB2, IgKappa, CD174, VEGFR2, CD30, mesothelin, CD138, CD33, c-Met, EGFR, CD22, FAP, CD133, EPCAM, GPC3, IL1RAP, MUC1, NKG2D-L, ROR1, CD70, LMP1, MG7, NY-ESO-1, CD276, GD2, B7H3, KRAS mutation, AFP, GPC3 and DLL3 and any combination thereof. In some embodiments, the chimeric antigen receptor comprises a binding domain, a hinge region, a transmembrane domain, a costimulatory domain and an intracellular signaling domain. In some embodiments, the binding domain is a single-chain antibody (scFv). In some embodiments, the binding domain is an anti-Claudin18.2scFv or an anti-EGFRVIIIscFv. In some embodiments, the binding domain comprises an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 6. In some embodiments, the hinge region is a CD8α hinge region. In some embodiments, the transmembrane domain is the transmembrane domain of CD28. In some embodiments, the costimulatory domain is a CD28 costimulatory domain or a 41BB costimulatory domain. In some embodiments, the intracellular signaling domain is a CD3ζ intracellular signaling domain. In some embodiments, the chimeric antigen receptor further comprises a signal peptide, preferably a CD8 signal peptide.

[0016] Another aspect of the present invention provides an expression vector comprising any one of the above-mentioned polynucleotides.

[0017] Another aspect of the present invention provides a host cell comprising any one of the above polynucleotides or the above expression vectors. In some embodiments, the host cell is an immune cell. In some embodiments, the immune cell is a T cell, a B cell, a macrophage, a dendritic cell, a monocyte, a granulocyte, a NK cell, or a NKT cell.

[0018] Another aspect of the present invention provides a pharmaceutical composition comprising any one of the above-mentioned engineered immune cells or any one of the above-mentioned host cells.

[0019] Another aspect of the present invention provides a method for treating a disease in a subject, comprising administering any one of the above-mentioned engineered immune cells, any one of the above-mentioned host cells, or the above-mentioned pharmaceutical composition to a subject in need thereof.

[0020] In some embodiments, the disease is characterized by the expression of a specific antigen, and the chimeric antigen receptor or T cell receptor contained in the immune cell is capable of specifically binding to the specific antigen. In some embodiments, the disease is an infectious disease, a tumor, or an autoimmune disease. In some embodiments, the infectious disease is a viral, bacterial, fungal, or parasitic infection. In some embodiments, the tumor is a blood tumor or a solid tumor. In some embodiments, the tumor expresses Claudin18.2 or EGFRVIII. In some embodiments, the blood tumor is acute myeloid leukemia AML, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, lymphoma, multiple myeloma, or myeloproliferation. In some embodiments, the solid tumor is a gastrointestinal tumor, gastric cancer, esophageal cancer, bile duct cancer, neuroblastoma, glioma, glioblastoma, mesothelioma, lung cancer, ovarian cancer, breast cancer, liver cancer, sarcoma, pancreatic cancer, non-small cell lung cancer, or prostate cancer. In some embodiments, the autoimmune disease is inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Sjögren's syndrome, and systemic lupus erythematosus.

[0021] Another aspect of the present invention provides a kit comprising: (1) an immune cell; and (2) any one of the aforementioned polynucleotides or the aforementioned expression vectors. In some embodiments, the immune cell is a T cell, a B cell, a macrophage, a dendritic cell, a monocyte, a granulocyte, a NK cell, or a NKT cell.

[0022] Another aspect of the present invention provides the use of any of the aforementioned immune cells, any of the aforementioned polynucleotides, any of the aforementioned expression vectors, any of the aforementioned host cells, any of the aforementioned pharmaceutical compositions, or any of the aforementioned kits in the preparation of a medicament for treating a disease in a subject. In some embodiments, the disease is characterized by the expression of a specific antigen, and the chimeric antigen receptor or T cell receptor contained in the immune cell is capable of specifically binding to the specific antigen. In some embodiments, the disease is an infectious disease, a tumor, or an autoimmune disease. In some embodiments, the infectious disease is a viral, bacterial, fungal, or parasitic infection. In some embodiments, the tumor is a hematological tumor or a solid tumor. In some embodiments, the tumor expresses Claudin18.2 or EGFRVIII. In some embodiments, the hematological tumor is acute myeloid leukemia AML, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, lymphoma, multiple myeloma, or myeloproliferation. In some embodiments, the solid tumor is a gastrointestinal tumor, gastric cancer, esophageal cancer, bile duct cancer, neuroblastoma, glioma, glioblastoma, mesothelioma, lung cancer, ovarian cancer, breast cancer, liver cancer, sarcoma, pancreatic cancer, non-small cell lung cancer or prostate cancer. In some embodiments, the autoimmune disease is inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Sjögren's syndrome and systemic lupus erythematosus.

[0023] Another aspect of the present invention provides any of the aforementioned immune cells, any of the aforementioned polynucleotides, any of the aforementioned expression vectors, any of the aforementioned host cells, any of the aforementioned pharmaceutical compositions, or any of the aforementioned kits, for treating a disease in a subject. In some embodiments, the disease is characterized by the expression of a specific antigen, and the chimeric antigen receptor or T cell receptor contained in the immune cell is capable of specifically binding to the specific antigen. In some embodiments, the disease is an infectious disease, a tumor, or an autoimmune disease. In some embodiments, the infectious disease is a viral, bacterial, fungal, or parasitic infection. In some embodiments, the tumor is a hematological tumor or a solid tumor. In some embodiments, the tumor expresses Claudin18.2 or EGFRVIII. In some embodiments, the hematological tumor is acute myeloid leukemia AML, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, lymphoma, multiple myeloma, or myeloproliferation. In some embodiments, the solid tumor is a gastrointestinal tumor, gastric cancer, esophageal cancer, bile duct cancer, neuroblastoma, glioma, glioblastoma, mesothelioma, lung cancer, ovarian cancer, breast cancer, liver cancer, sarcoma, pancreatic cancer, non-small cell lung cancer or prostate cancer. In some embodiments, the autoimmune disease is inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Sjögren's syndrome and systemic lupus erythematosus.

[0024] Another aspect of the present invention provides the use of polynucleotides encoding CXCL10 and IL15 in improving the therapeutic effect of immune cells in treating diseases in subjects. In some embodiments, the polynucleotide encodes a fusion protein comprising CXCL10 and IL15. In some embodiments, in the fusion protein, CXCL10 and IL15 are connected by a linker, and the linker is preferably a self-cleaving linker, more preferably T2A, F2A or P2A. In some embodiments, CXCL10 comprises the amino acid sequence shown in SEQ ID NO: 9, and IL15 comprises the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the polynucleotide encoding CXCL10 and the polynucleotide encoding IL15 are contained in an expression vector. In some embodiments, the immune cells are T cells, B cells, macrophages, dendritic cells, monocytes, granulocytes, NK cells or NKT cells. In some embodiments, the immune cells express chimeric antigen receptors or T cell receptors. In some embodiments, the immune cells are CAR T cells or TCR T cells. In some embodiments, the chimeric antigen receptor or T cell receptor targets an antigen expressed by a pathogen, a tumor-associated antigen, or an autoimmune disease-associated antigen. In some embodiments, the tumor-associated antigen is selected from CD123, CD7, BCMA, CD19, CEA, CD17L, CD20, IL13RA2, G02, PSMA, ERBB2, IgKappa, CD174, VEGFR2, CD30, mesothelin, CD138, CD33, c-Met, EGFR, CD22, FAP, CD133, EPCAM, GPC3, IL1RAP, MUC1, NKG2D-L, ROR1, CD70, LMP1, MG7, NY-ESO-1, CD276, GD2, B7H3, KRAS mutation, AFP, GPC3, and DLL3, and any combination thereof. In some embodiments, the chimeric antigen receptor comprises a binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In some embodiments, the binding domain is a single-chain antibody (scFv). In some embodiments, the binding domain is an anti-Claudin18.2scFv or an anti-EGFRVIIIscFv. In some embodiments, the binding domain comprises an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 6. In some embodiments, the hinge region is a CD8α hinge region. In some embodiments, the transmembrane domain is a transmembrane domain of CD28. In some embodiments, the costimulatory domain is a CD28 costimulatory domain or a 41BB costimulatory domain. In some embodiments, the intracellular signaling domain is a CD3ζ intracellular signaling domain.In some embodiments, the chimeric antigen receptor further comprises a signal peptide, preferably a CD8 signal peptide. In some embodiments, the disease is characterized by the expression of a specific antigen, and the chimeric antigen receptor or T cell receptor contained in the immune cell is capable of specifically binding to the specific antigen. In some embodiments, the disease is an infectious disease, a tumor, or an autoimmune disease. In some embodiments, the infectious disease is a viral, bacterial, fungal, or parasitic infection. In some embodiments, the tumor is a blood tumor or a solid tumor. In some embodiments, the tumor expresses Claudin18.2 or EGFRVIII. In some embodiments, the blood tumor is acute myeloid leukemia AML, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, lymphoma, multiple myeloma, or myeloproliferation. In some embodiments, the solid tumor is a gastrointestinal tumor, gastric cancer, esophageal cancer, bile duct cancer, neuroblastoma, glioma, glioblastoma, mesothelioma, lung cancer, ovarian cancer, breast cancer, liver cancer, sarcoma, pancreatic cancer, non-small cell lung cancer, or prostate cancer. In some embodiments, the autoimmune disease is inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Sjögren's syndrome, and systemic lupus erythematosus.

[0025] In some embodiments, the enhancing the therapeutic effect of immune cells in treating a disease in a subject comprises enhancing the infiltration of immune cells into a tumor, increasing immune cell recruitment, reducing tumor volume, and / or increasing the proliferation of immune cells.

[0026] Another aspect of the present invention provides a method for preparing engineered immune cells, comprising: (i) expressing CXCL10 and IL15 in the immune cells; and (ii) expressing a chimeric antigen receptor and / or a T cell receptor in the immune cells. In some embodiments, step (i) comprises introducing polynucleotides encoding CXCL10 and IL15 into the immune cells to express CXCL10 and IL15. In some embodiments, the polynucleotide encodes a fusion protein comprising CXCL10 and IL15. In some embodiments, in the fusion protein, CXCL10 and IL15 are connected by a linker, which is preferably a self-cleavable linker, more preferably T2A, F2A or P2A. In some embodiments, CXCL10 comprises the amino acid sequence shown in SEQ ID NO: 9, and IL15 comprises the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the polynucleotide encoding CXCL10 and the polynucleotide encoding IL15 are contained in an expression vector. In some embodiments, step (ii) includes introducing a polynucleotide encoding the chimeric antigen receptor and / or T cell receptor into the immune cell to express the chimeric antigen receptor and / or T cell receptor. In some embodiments, step (i) and step (ii) can be performed separately, successively or simultaneously. In some embodiments, the order of step (i) and step (ii) is not limited, and step (i) can be performed first and then step (ii), or step (ii) can be performed first and then step (i). In some embodiments, the method includes introducing a polynucleotide encoding a fusion protein comprising the chimeric antigen receptor, CXCL10 and IL15 into the immune cell to express the chimeric antigen receptor, CXCL10 and IL15. In some embodiments, the polynucleotide encoding the fusion protein comprising the chimeric antigen receptor, CXCL10 and IL15 is contained in an expression vector. In some embodiments, in the fusion protein, the chimeric antigen receptor, CXCL10 and IL15 are connected by a linker, and the linker is preferably a self-cleaving linker, more preferably selected from T2A, F2A or P2A. In some embodiments, the immune cell is a T cell, a B cell, a macrophage, a dendritic cell, a monocyte, a granulocyte, a NK cell, or a NKT cell. In some embodiments, the immune cell expresses a chimeric antigen receptor or a T cell receptor. In some embodiments, the immune cell is a CAR T cell or a TCR T cell. In some embodiments, the chimeric antigen receptor or T cell receptor targets an antigen expressed by a pathogen, a tumor-associated antigen, or an autoimmune disease-associated antigen.In some embodiments, the tumor-associated antigen is selected from CD123, CD7, BCMA, CD19, CEA, CD17L, CD20, IL13RA2, G02, PSMA, ERBB2, IgKappa, CD174, VEGFR2, CD30, mesothelin, CD138, CD33, c-Met, EGFR, CD22, FAP, CD133, EPCAM, GPC3, IL1RAP, MUC1, NKG2D-L, ROR1, CD70, LMP1, MG7, NY-ESO-1, CD276, GD2, B7H3, KRAS mutation, AFP, GPC3 and DLL3 and any combination thereof. In some embodiments, the chimeric antigen receptor comprises a binding domain, a hinge region, a transmembrane domain, a costimulatory domain and an intracellular signaling domain. In some embodiments, the binding domain is a single-chain antibody (scFv). In some embodiments, the binding domain is an anti-Claudin18.2scFv or an anti-EGFRVIIIscFv. In some embodiments, the binding domain comprises an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 6. In some embodiments, the hinge region is a CD8α hinge region. In some embodiments, the transmembrane domain is the transmembrane domain of CD28. In some embodiments, the costimulatory domain is a CD28 costimulatory domain or a 41BB costimulatory domain. In some embodiments, the intracellular signaling domain is a CD3ζ intracellular signaling domain. In some embodiments, the chimeric antigen receptor further comprises a signal peptide, preferably a CD8 signal peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1: Schematic diagram of the structure of CAR-CXCL10-IL15.

[0028] Figure 2: CAR expression of CAR T-CXCL10-IL15. CONTROL T refers to activated T cells without lentivirus transduction, CLDN18.2CAR and EGFRVIIICAR refer to T cells transduced with control lentivirus, i.e., control CAR T cells, and CLDN18.2CAR-CXCL10-IL-15 and EGFRVIIICAR-CXCL10-IL-15 refer to T cells transduced with experimental lentivirus, i.e., experimental CAR T cells.

[0029] Figure 3: CXCL10 expression by CAR T-CXCL10-IL15.

[0030] Figure 4: IL15 expression by CAR T-CXCL10-IL15.

[0031] Figure 5: Killing effect of CAR T-CXCL10-IL15, where the empty group is activated T cells without lentivirus transduction, and the experimental group and control group are experimental group CAR T cells and control group CAR T cells, respectively.

[0032] Figure 6: Proliferative effect of CAR T-CXCL10-IL15.

[0033] Figure 7: Migration effect of CAR T-CXCL10-IL15.

[0034] Figure 8: Tumor volume curve of mice.

[0035] Figure 9: CD3 infiltration of anti-CLDN18.2 CAR T-CXCL10-IL15. DETAILED DESCRIPTION

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0037] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0038] The terms "about" and "approximately" when used in connection with a numerical variable generally mean that the value of the variable and all values ​​of the variable are within measurement or experimental error (e.g., 95% confidence interval for the mean) or within the wider range of the specified value (e.g., ±5%).

[0039] The term "comprise" or its variations such as "contains," "has," and "includes" means including the stated steps or elements but not excluding any other steps or elements. "Consisting of" means excluding unrecited steps or elements. "Consisting essentially of" means not excluding steps or elements that do not materially affect the basic and novel characteristics of the claimed invention. The term "comprising" specific steps or elements and its variations also include "consisting of" and "consisting essentially of" specific steps or elements.

[0040] When a numerical range is mentioned, it should be considered that the specific values ​​of its upper and lower limits are specifically disclosed, as well as all intermediate ranges included therein, such as intermediate ranges between its upper or lower limit and any intermediate value, or intermediate ranges between any two intermediate values. In addition, any intermediate ranges, subranges and all individual values ​​described in the numerical range can be excluded from the numerical range.

[0041] The term "and / or" should be understood to refer to any one element or any combination of several elements connected by this term.

[0042] The term "immune cell" refers to cells that participate in the immune response and produce immune effects. Immune cells can be activated to affect the viability of target cells. They have hematopoietic origin and play a direct role in the immune response against targets (such as pathogens, cancer cells or foreign substances). Immune cells include T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, granulocytes (including neutrophils, eosinophils, basophils, mast cells), dendritic cells, etc.

[0043] The term "CXCL10" refers to CXC motif chemokine ligand 10, also known as interferon gamma-induced protein 10 (IP-10) or small inducible cytokine B10. CXCL10 is a small cytokine belonging to the CXC chemokine family. Native CXCL10 is an 8.7 kDa protein that is encoded by the CXCL10 gene in humans. The term "CXCL10" includes CXCL10 from different species, such as CXCL10 from mammals (such as humans), and includes native forms of CXCL10 as well as functional variants or isoforms thereof, such as splice variants thereof.

[0044] The term "IL15" refers to interleukin-15 protein, also known as MGC9721. Native IL15 is a proinflammatory cytokine of 14 to 15 kDa that is encoded by the IL-15 gene in humans. The term "IL15" includes IL15 from different species, such as IL15 from mammals (such as humans), and includes native forms of IL15 as well as functional variants or isoforms thereof, such as splice variants thereof.

[0045] The term "native form" refers to a form that occurs naturally in nature, such as a protein (such as CXCL10 or IL15) that is naturally expressed by a cell.

[0046] The term "functional variant" refers to a variant that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the parent amino acid sequence and retains the biological activity of the parent amino acid sequence.

[0047] The term "engineered" refers to one or more artificially designed changes to a nucleic acid (e.g., a nucleic acid within the genome of an organism). The term can refer to the alteration, addition, and / or deletion of a gene. An engineered cell can refer to a cell with an added, deleted, and / or altered gene. In the present invention, the engineered cell can be genetically modified to express one or more proteins described herein, such as a chimeric antigen receptor, CXCL10, and / or IL15.

[0048] The term "exogenous" refers to a polynucleotide that does not naturally occur in a cell, or a protein that is not expressed by a polynucleotide that naturally occurs in a cell. Artificially introducing a polynucleotide that does not naturally occur in a cell, or introducing one or more copies of a polynucleotide that naturally occurs in a cell to increase the number of copies of the polynucleotide in the cell, is considered to be introducing an exogenous polynucleotide into the cell. Proteins encoded by exogenous polynucleotides are referred to as exogenous proteins.

[0049] The term "autologous" refers to any material derived from an individual (eg, a subject) that is subsequently reintroduced into that same individual.

[0050] The term "allogeneic" means that the material introduced into an individual (eg, a subject, also referred to as a recipient in this definition) is derived from another individual (also referred to as a donor) of the same species as the individual.

[0051] The term "chimeric antigen receptor" or "CAR" refers to a fusion protein comprising an extracellular binding domain capable of binding to an antigen, a transmembrane domain, and an intracellular domain comprising one or more intracellular signaling domains derived from signal transduction proteins. These intracellular signaling domains are generally different from the polypeptides from which the extracellular domains come. The extracellular binding domain can be any protein molecule or a portion thereof that can specifically bind to a predetermined ligand (such as an antigen). In some embodiments, the intracellular signaling domain can be any known oligopeptide or polypeptide domain, the function of which is to transmit signals, causing activation or inhibition of intracellular biological processes, for example, activating immune cells such as T cells or NK cells. The intracellular signaling domain generally includes an immunoreceptor tyrosine activation motif (ITAM), such as a signaling domain from a CD3ζ molecule, which is responsible for activating immune cells and producing a killing effect. In addition, the chimeric antigen receptor may also include a signal peptide at the amino terminus, which is responsible for the intracellular localization of the fusion protein, and a hinge region between the extracellular domain and the transmembrane domain. The intracellular domain may also include a costimulatory domain from, for example, the 41BB or CD28 molecule.

[0052] The term "antibody" is used in its broadest sense and includes immunoglobulins or other types of molecules that contain one or more antigen-binding domains that specifically bind to an antigen, and are proteins or polypeptides that exhibit binding specificity to a specific antigen. Specific examples of antibodies may include complete antibodies (e.g., classic four-chain antibody molecules), single-chain antibodies, single-domain antibodies, multispecific antibodies, and the like. Classic four-chain antibody molecules include two identical heavy chains (H) and two identical light chains (L), which are interconnected by disulfide bonds to form a tetramer. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH, including domains CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). VH and VL form an antigen-binding site, and each VH and VL consists of three CDRs and four FRs.

[0053] The term "antigen-binding fragment" refers to one or more portions of an intact antibody that retain the ability to specifically bind to the antigenic epitope to which the antibody is directed, for example, see Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd Edition, Raven Press, NY (1989). Examples of antigen-binding fragments include: (1) Fab fragment: an antigen-binding fragment produced by papain digestion of an intact antibody, consisting of the complete light chain (including the VL domain and the CL domain) of a full-length antibody and the heavy chain variable region VH and heavy chain constant region domain CH1 of a full-length antibody, without a hinge region; (2) F(ab')2 fragment: an antigen-binding fragment produced by pepsin digestion of an intact antibody, comprising the complete light chains (including the VL domain and the CL domain) of two full-length antibodies and the heavy chain variable region VH and heavy chain constant region domain CH1 of two full-length antibodies and a hinge region, which can be regarded as having two (1) a fragment formed by pairing two Fab' fragments with disulfide bonds; (2) a Fab' fragment formed by treating the hinge region of F(ab')2 with a reducing agent to break the disulfide bonds; (3) a Fab' fragment formed by treating the hinge region of F(ab')2 with a reducing agent to break the disulfide bonds; (4) an Fd fragment consisting of a VH domain and a CH1 domain; and (5) an Fv fragment consisting of a VL domain and a VH domain of a single antibody arm. The term "antigen-binding fragment" generally comprises at least one or both of the heavy chain variable region and the light chain variable region of an intact antibody, and may also comprise a portion or all of the light chain constant region and / or heavy chain constant region of an intact antibody, for example, one or two or more of the CL domain, CH1 domain, CH2 domain, and CH3 domain.

[0054] The term "single domain antibody (sdAb)" refers to an antigen-binding polypeptide with a single variable domain that includes three complementary determining regions (CDRs). Single domain antibodies are able to bind to antigens alone without pairing with another polypeptide containing CDRs. Common single domain antibodies include heavy chains but lack light chains that are typically found in antibodies, such as camelid sdAbs (see eg, Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)). Single domain antibodies can be artificially engineered from camel heavy chain antibodies, called V H H antibody, V H The H antibody has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single-domain antibodies can also be considered a special "antigen-binding fragment" of a full-length antibody.

[0055] The term "single-chain antibody (scFv)" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, 113, Roseburg & Moore, Springer-Verlag, New York, 269-315 (1994)). Such scFv molecules typically have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0056] The term "T cell receptor" or "TCR" typically refers to a heterodimeric receptor composed of paired αβ or γδ chains on the surface of T cells. Each α, β, γ and δ chain is composed of two Ig-like domains, including a variable domain (V) and a constant domain (C) containing a complementary determining region (CDR), which is anchored to the cell membrane by a connecting peptide and a transmembrane (TM) region. The term "T cell receptor" herein includes natural TCRs as well as TCR variants or fragments.

[0057] The term "CAR T cell" or "CAR T" refers to a T cell that can express or produce a CAR. CART cells are typically obtained by modifying T cells to contain a nucleic acid molecule encoding a CAR. For a subject, CART cells can be obtained by modifying autologous T cells or allogeneic T cells.

[0058] The term "TCR T cell" or "TCR T" refers to a T cell that expresses an exogenous T cell receptor.

[0059] The term "tumor-associated antigen," which may also be referred to herein as a "tumor antigen," refers to an antigen expressed by tumor cells, including antigens expressed only by tumor cells and not by other cells, as well as antigens highly expressed in tumor cells. Tumor-associated antigens may be antigens expressed within or on the surface of tumor cells. In some embodiments, tumor-associated antigens refer to antigens expressed within or on the surface of tumor cells that can be recognized by the immune system and / or immunotherapeutic agents.

[0060] The term "high expression" refers to the expression level of a specific antigen in or on the surface of tumor cells being higher than that in other cells (eg, non-tumor cells or other types of tumor cells).

[0061] The term "polynucleotide encoding CXCL10 and IL15" can also be understood as a polynucleotide encoding CXCL10 and a polynucleotide encoding IL15. The term encompasses the following situations: a single polynucleotide sequence encoding both CXCL10 and IL15; and two independent polynucleotide sequences (i.e., not covalently linked, such as by a phosphate bond), encoding CXCL10 and IL15, respectively.

[0062] The term "polynucleotide encoding a chimeric antigen receptor, CXCL10, and IL-15" can also be understood as a polynucleotide encoding a chimeric antigen receptor, a polynucleotide encoding CXCL10, and a polynucleotide encoding IL15. The term covers the following situations: one polynucleotide sequence encoding a chimeric antigen receptor, CXCL10, and IL15; two independent polynucleotide sequences (i.e., they are not covalently linked, such as not covalently linked by a phosphate bond) encoding any two of the chimeric antigen receptor, CXCL10, and IL-15 and the remaining one; and three independent polynucleotide sequences (i.e., they are not covalently linked, such as not covalently linked by a phosphate bond) encoding a chimeric antigen receptor, CXCL10, and IL15.

[0063] The terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a sequence composed of bases, sugars, and a phosphate backbone. A polynucleotide can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and can include natural or non-natural bases, can be single-stranded or double-stranded, and can include coding and / or non-coding sequences.

[0064] The term "isolated" refers to a nucleic acid molecule, peptide, polypeptide, or protein that is separated from its natural environment or from other components with which it is originally present. The term "substantially isolated" means that the nucleic acid molecule, peptide, polypeptide, or protein comprises greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95% of the total nucleic acid molecule, peptide, polypeptide, or protein in the isolated material, for example, by weight.

[0065] The term "vector" refers to any molecule capable of transporting a heterologous polynucleotide contained therein into a host cell (either in a free form or integrated into the host cell genome). Vectors used to express a gene of interest in a host cell are called "expression vectors" or "recombinant expression vectors." Vectors can be linear or circular molecules, and can be RNA or DNA molecules.

[0066] The term "expression" refers to the production of a target protein (in the present invention, chimeric antigen receptor, CXCL10 and / or IL-15) in a host cell, which includes transcription and / or translation of the target protein.

[0067] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence, such as a promoter, a leader sequence, a selection marker, an origin of replication and / or polyadenylation, operably linked to a nucleotide sequence to be expressed.

[0068] The term "host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. In the present invention, a host cell can be an immune cell, such as a T cell. Host cells include the progeny of a single host cell, and the progeny may not be completely identical to the original parent cell, for example, in morphology or genomic DNA complement, due to natural, accidental, or intentional mutations. Host cells can be isolated cells or cell lines, and also include cells transfected in vivo with a polynucleotide or expression vector provided herein.

[0069] The term "targeting" or "specific binding" refers to the formation of a relatively stable complex between the binding domain of a chimeric antigen receptor and its target (e.g., a tumor antigen or a cell expressing a tumor antigen) under physiological conditions, and preferably does not exhibit significant binding to other undesired targets. Specific binding can be detected, for example, by flow cytometry.

[0070] The term "tumor" refers to the growth and proliferation of neoplastic cells, which can be benign or malignant (i.e., cancer), or a precancerous lesion. The term "cancer" refers to a tumor that exhibits uncontrolled growth and division, can invade adjacent tissues, and can metastasize to other locations in the body.

[0071] Unless otherwise indicated, nucleic acids are written herein in 5' to 3' orientation left to right and amino acid sequences are written left to right in amino to carboxyl orientation.

[0072] The inventors have discovered that expressing CXCL10 and IL15 in immune cells can increase the accumulation of CXCL10 in tumor tissues, enhance the recruitment and accumulation of immune cells (such as T cells), increase the infiltration of immune cells (such as T cells) into solid tumors, promote the proliferation of immune cells (such as CAR T cells), ensure the survival rate of immune cells (such as T cells) in tumor tissues, increase the killing efficiency of immune cells (such as CAR T cells) on target cells, improve the anti-tumor effect of immune cells (such as CAR T cells), and / or enhance the therapeutic effect of immune cells (such as CAR T cells) on solid tumors.

[0073] The present invention relates to engineered immune cells that express CXCL10 and IL-15.

[0074] In some embodiments, the immune cell can be a T cell, a B cell, a macrophage, a dendritic cell, a monocyte, a granulocyte (including a neutrophil, an eosinophil, a basophil, a mast cell), a NK cell or a NKT cell. The immune cell can be an autologous or allogeneic cell. The T cell can be, can be a helper T cell (Th) or a cytotoxic T cell (CTL) or a mixture thereof, can be a CD4+ T cell, a CD8+ T cell or a mixture thereof.

[0075] Engineered immune cells refer to cells obtained by genetically manipulating the source cells of immune cells and engineering them. The source cells of immune cells can be cells of natural origin, such as immune cells isolated from mammals (including non-human mammals and humans), or can be cells obtained by differentiation, passage or genetic engineering in vitro from cells of natural origin. Source cells of immune cells (such as T cells) can be obtained from a variety of sources, including but not limited to peripheral blood mononuclear cells (PBMC), bone marrow, lymph, umbilical cord blood, thymus tissue, tumor, infected tissue, stem cells (such as pluripotent stem cells), etc. In some embodiments, the immune cells of the present invention are derived from cells separated from peripheral blood, such as PBMC.

[0076] In some embodiments, the CXCL10 and IL15 expressed in the engineered immune cells are exogenous to the immune cells. In some embodiments, the immune cells are engineered to express exogenous CXCL10 and exogenous IL15. In some embodiments, the immune cells are engineered to introduce polynucleotides encoding CXCL10 and IL15 to express CXCL10 and IL15.

[0077] In some embodiments, CXCL10 and IL-15 are expressed in the engineered immune cells as a fusion protein. The relative positions of CXCL10 and IL-15 are not particularly limited and can be such that the C-terminus of CXCL10 is linked to the N-terminus of IL-15, or vice versa. In some embodiments, the fusion protein comprises CXCL10 and IL-15 from the N-terminus to the C-terminus. In some embodiments, CXCL10 and IL-15 in the fusion protein are connected by a linker. The linker can be a self-cleavable linker, such as a 2A linker, e.g., T2A, P2A, or F2A. In some embodiments, the fusion protein of CXCL10 and IL-15 has a structure of "CXCL10-linker-IL15" from the N-terminus to the C-terminus. In some embodiments, CXCL10 comprises the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, IL-15 comprises the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the linker is a T2A linker comprising the amino acid sequence set forth in SEQ ID NO:8.

[0078] In some embodiments, the engineered immune cell further expresses a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the engineered immune cell is a CAR T cell or a TCR T cell.

[0079] In some embodiments, the engineered immune cells express a fusion protein comprising any two or three of a chimeric antigen receptor, CXCL10, and IL-15. In some embodiments, the engineered immune cells express a fusion protein comprising a chimeric antigen receptor, CXCL10, and IL-15. The relative positions of the chimeric antigen receptor, CXCL10, and IL-15 are not particularly limited, and any one can be placed at the N-terminus or C-terminus. In some embodiments, the fusion protein comprises a chimeric antigen receptor and a fusion protein comprising CXCL10 and IL-15 from the N-terminus to the C-terminus. In some embodiments, the fusion protein comprises a chimeric antigen receptor, CXCL10, and IL-15 from the N-terminus to the C-terminus. In some embodiments, any two or three of the chimeric antigen receptor, CXCL10, and IL-15 in the fusion protein are connected to each other by a linker. The linker can be a self-cleaving linker, such as a 2A linker, for example, T2A, P2A, or F2A. In some embodiments, the fusion protein of CXCL10 and IL15 comprises a structure of "chimeric antigen receptor-linker-CXCL10-linker-IL15" from the N-terminus to the C-terminus. In some embodiments, the linker is a T2A linker comprising the amino acid sequence shown in SEQ ID NO:8.

[0080] In the present invention, the chimeric antigen receptor (CAR) or T cell receptor (TCR) can target different antigens, such as antigens expressed by pathogens (such as viruses, bacteria, fungi or parasites), tumor-associated antigens (including cancer-associated antigens), autoimmune disease-associated antigens, etc. Engineered immune cells expressing the chimeric antigen receptor (CAR) and / or T cell receptor (TCR), CXCL10 and IL15 can target the pathogens, tumor cells (including cancer cells) and / or autoimmune disease cells and are used to treat infection by the pathogen, tumors (including cancer) and / or autoimmune diseases.

[0081] In some embodiments, the chimeric antigen receptor (CAR) may include a binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain may further include a costimulatory domain.

[0082] In some embodiments, the extracellular binding domain comprises an antibody or an antigen-binding fragment thereof that targets a specific antigen, such as an antigen expressed by a pathogen (such as a virus, bacteria, fungus, or parasite), a tumor-associated antigen (including a cancer-associated antigen), or an autoimmune disease-associated antigen. In some embodiments, the antibody is a single-chain antibody (scFv). In some embodiments, the binding domain targets a tumor-associated antigen. In some embodiments, the tumor-associated antigens include but are not limited to CD123, CD7, BCMA, CD19, CEA, CD17L, CD20, IL13RA2, G02, PSMA, ERBB2, IgKappa, CD174, VEGFR2, CD30, mesothelin, CD138, CD33, c-Met, EGFR, CD22, FAP, CD133, EPCAM, GPC3, IL1RAP, MUC1, NKG2D-L, ROR1, CD70, LMP1, MG7, NY-ESO-1, CD276, GD2, B7H3, KRAS mutation, AFP, GPC3, DLL3, or any combination thereof.

[0083] In some embodiments, the chimeric antigen receptor comprises a binding domain that specifically binds to a tumor antigen. In some embodiments, the chimeric antigen receptor targets Claudin18.2 or EGFRVⅢ. In some embodiments, the chimeric antigen receptor comprises a binding domain that specifically binds to Claudin18.2 or EGFRVⅢ. In some embodiments, the binding domain is an antibody. In some embodiments, the antibody is a single-chain antibody (scFv). In some embodiments, the binding domain is an anti-Claudin18.2scFv or an anti-EGFRVⅢscFv. In some embodiments, the binding domain comprises an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 6.

[0084] In some embodiments, the hinge region of CAR may include the hinge region of CD8, CD28, IgG1, 41BB, CD4, CD27, ICOS, OX40 or NKG2D, preferably a CD8 hinge region, such as a CD8α hinge region. In some embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO: 3.

[0085] In some embodiments, the transmembrane domain of CAR may include CD28, CD8, CD3ε (CD3e), 41BB, CD4, CD27, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4 or SLAM transmembrane domain, preferably CD28 transmembrane (TM) domain. In some embodiments, the intracellular signaling domain of CAR may include CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ or DAP-12 intracellular signaling domain, preferably CD3ζ intracellular signaling domain. In some embodiments, CAR further includes a costimulatory domain, such as a costimulatory domain of CD28, 41BB (CD137), CD27, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3 or MyD88, preferably a costimulatory domain of CD28 or 41BB. In some embodiments, the transmembrane domain and the costimulatory domain in the CAR are the transmembrane domain and the costimulatory domain of CD28. In some embodiments, the transmembrane domain and the costimulatory domain in the CAR are the transmembrane domain and the costimulatory domain of CD28 and the costimulatory domain of 41BB. In some embodiments, the transmembrane domain and the costimulatory domain in the CAR comprise the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 7. In some embodiments, the intracellular signaling domain in the CAR comprises the amino acid sequence shown in SEQ ID NO: 5

[0086] In some embodiments, the chimeric antigen receptor (CAR) further comprises a signal peptide at its N-terminus. In some embodiments, the signal peptide may include a CD8 signal peptide. In some embodiments, the CD8 signal peptide comprises an amino acid sequence as shown in SEQ ID NO: 1.

[0087] The present invention also provides polynucleotides encoding CXCL10 and IL15. In some embodiments, the polynucleotides encode a fusion protein of CXCL10 and IL15. The present invention also provides polynucleotides encoding the chimeric antigen receptor, CXCL10 and IL-15 or polynucleotides encoding T cell receptor, CXCL10 and IL-15. In some embodiments, the polynucleotides encode a fusion protein of the chimeric antigen receptor, CXCL10 and IL15. The polynucleotides can be DNA or RNA, can be double-stranded or single-stranded, can be a coding strand (sense strand) or a non-coding strand (antisense strand). The polynucleotides can be isolated or substantially isolated. In some embodiments, the polynucleotides encoding CXCL10 and IL15, the polynucleotides encoding the chimeric antigen receptor, CXCL10 and IL-15 or the polynucleotides encoding the T cell receptor, CXCL10 and IL-15 are contained in a vector.

[0088] Therefore, the present invention further relates to a carrier, wherein the carrier includes polynucleotides encoding CXCL10 and IL15, or includes polynucleotides encoding chimeric antigen receptors, CXCL10 and IL-15, or includes polynucleotides encoding T cell receptors, CXCL10 and IL-15. The carrier can express CXCL10 and IL15 in the host cell when introduced into a host cell (e.g., an immune cell, such as a T cell), or expresses chimeric antigen receptors, CXCL10 and IL15, or expresses T cell receptors, CXCL10 and IL15. In some embodiments, the carrier includes polynucleotides encoding the fusion protein of CXCL10 and IL15, or includes polynucleotides encoding the fusion protein of chimeric antigen receptors, CXCL10 and IL15. In some embodiments, the carrier comprising polynucleotides encoding CXCL10 and IL15 can be introduced into immune cells (e.g., T cells) expressing CAR and / or TCR, so that the immune cells expressing CAR and / or TCR express CXCL10 and IL15. In some embodiments, a vector comprising a polynucleotide encoding a chimeric antigen receptor, CXCL10, and IL-15 can be introduced into an immune cell (e.g., a T cell) to cause the immune cell to express the chimeric antigen receptor, CXCL10, and IL-15. In some embodiments, a vector comprising a polynucleotide encoding a T cell receptor, CXCL10, and IL-15 can be introduced into an immune cell (e.g., a T cell) to cause the immune cell to express the T cell receptor, CXCL10, and IL-15.

[0089] The vector may be one or more vectors, each comprising a polynucleotide encoding a chimeric antigen receptor, a polynucleotide encoding a T cell receptor, a polynucleotide encoding CXCL10, and / or a polynucleotide encoding IL15. In some embodiments, one or more vectors may be used to introduce polynucleotides encoding a chimeric antigen receptor, CXCL10, and IL15, or polynucleotides encoding a T cell receptor, CXCL10, and IL-15 into immune cells (e.g., T cells) so that the immune cells (e.g., T cells) express chimeric antigen receptors, CXCL10, and IL-15, or express T cell receptors, CXCL10, and IL-15.

[0090] In the present invention, the vector used can be DNA, RNA (such as mRNA), circular RNA, etc. In some embodiments, the vector can be an episomal vector or an integrative vector. In some embodiments, the vector can be a plasmid, a viral vector, etc. In some embodiments, the viral vector can be a lentiviral vector, an adenoviral vector, a retroviral vector, an adeno-associated viral vector, etc. The vector can be an expression vector. When two or more vectors are included in the composition of the present invention, or two or more vectors are used in the method of the present invention, the two or more vectors can be vectors of the same type or different types, and can be independently selected from the above-mentioned types of vectors.

[0091] The above-mentioned engineered immune cells (such as T cells) can be constructed in any appropriate manner.For example, polynucleotides encoding chimeric antigen receptors and / or T cell receptors, polynucleotides encoding CXCL10, and polynucleotides encoding IL-15 can be introduced into the immune cells separately or together.For example, in some embodiments, polynucleotides encoding CXCL10 and IL15 can be introduced into immune cells (such as T cells) expressing CAR or TCR, so that the cells express CXCL10 and IL15.In some embodiments, polynucleotides encoding CXCL10 and IL15 can be introduced into immune cells (such as T cells) so that the immune cells express CXCL10 and IL15, and then polynucleotides encoding chimeric antigen receptors or T cell receptors are introduced into the immune cells so that they express chimeric antigen receptors or T cell receptors.In some embodiments, polynucleotides encoding fusion proteins comprising chimeric antigen receptors, CXCL10, and IL-15 can be introduced into T cells so that they express chimeric antigen receptors, CXCL10, and IL-15.

[0092] In some embodiments, the engineered immune cells of the present invention comprise any of the polynucleotides described above or any of the vectors described above to express chimeric antigen receptors and / or T cell receptors, CXCL10, and / or IL-15 in the immune cells. In some embodiments, the polynucleotides encoding CXCL10 and / or IL-15 are integrated into the genome of the immune cells or are contained in a free vector.

[0093] In some embodiments, the present invention also relates to immune cells (such as T cells) expressing CXCL10 and IL-15, which can be used as chassis cells to prepare immune cells expressing chimeric antigen receptors or T cell receptors. Expression of CXCL10 and IL-15 in immune cells (such as T cells) can be achieved by a variety of appropriate means, for example, the aforementioned polynucleotides encoding CXCL10 and IL-15 or vectors comprising the polynucleotides can be introduced into immune cells (such as T cells). The immune cells (such as T cells) can then be engineered to express chimeric antigen receptors or T cell receptors.

[0094] The present invention also relates to a composition or a kit comprising: (1) an immune cell (such as a T cell); (2) a polynucleotide encoding a chimeric antigen receptor and / or a T cell receptor; and (3) a polynucleotide encoding CXCL10 and IL15. In some embodiments, the polynucleotide encoding CXCL10 and IL15 is a polynucleotide encoding a fusion protein of CXCL10 and IL15. In some embodiments, the composition or kit comprises a polynucleotide encoding a chimeric antigen receptor, a fusion protein of CXCL10 and IL15. In some embodiments, the polynucleotide encoding a chimeric antigen receptor and / or a T cell receptor, the polynucleotide encoding CXCL10 and IL15 are contained in a vector (e.g., one, two, or three vectors). When they are respectively contained in two or three vectors, the vectors used can be vectors of the same type or different types. In some embodiments, the composition or kit comprises an immune cell (such as a T cell), and a vector comprising a polynucleotide encoding a fusion protein of a chimeric antigen receptor, CXCL10, and IL15.

[0095] The present invention also relates to a composition or a kit comprising: (1) an immune cell (such as a T cell) expressing a chimeric antigen receptor or a T cell receptor; (2) a polynucleotide encoding CXCL10 and IL15. In some embodiments, the composition or kit comprises a CAR T cell or a TCR T cell. In some embodiments, the composition or kit comprises a polynucleotide encoding a fusion protein of CXCL10 and IL15. In some embodiments, the composition or kit comprises a polynucleotide encoding a fusion protein of a chimeric antigen receptor, CXCL10, and IL15. In some embodiments, the polynucleotide encoding CXCL10 and IL15 is contained in a vector (e.g., one or two vectors). When they are respectively contained in two vectors, the vectors used can be vectors of the same type or different types. In some embodiments, the composition or kit comprises an immune cell (such as a T cell) expressing a chimeric antigen receptor or a T cell receptor, and a vector comprising a polynucleotide encoding a fusion protein of CXCL10 and IL15.

[0096] In some embodiments, the kit further comprises instructions for introducing polynucleotides encoding CXCL10 and IL15 into the immune cells to cause them to express CXCL10 and IL15.

[0097] Methods for introducing the above-mentioned polynucleotides or vectors into host cells (e.g., immune cells, such as T cells, such as CAR T cells or TCR T cells) are well known in the art, for example, by electroporation, calcium phosphate precipitation, DEAE-dextran transfection, virus-like particle infection, and the like.

[0098] The present invention also relates to a method for preparing an engineered immune cell (e.g., a T cell), comprising: (i) expressing CXCL10 and IL15 in the immune cell (e.g., a T cell); and (ii) expressing a chimeric antigen receptor in the immune cell (e.g., a T cell). It should be understood that in the method, the order of steps (i) and (ii) is not limited, and step (i) can be performed first, followed by step (ii), or step (ii) can be performed first, followed by step (i), or steps (i) and (ii) can be performed simultaneously.

[0099] The expression of CXCL10 and IL-15 in immune cells can be achieved in various appropriate ways, for example, the aforementioned polynucleotides encoding CXCL10 and IL-15 or vectors containing the polynucleotides can be introduced into the immune cells.

[0100] The present invention also relates to a pharmaceutical composition comprising the engineered immune cells described above. The pharmaceutical composition may optionally comprise a pharmaceutically acceptable carrier.

[0101] The term "pharmaceutically acceptable carrier" refers to any carrier included in a pharmaceutical composition as an inactive ingredient that gives the composition a form and properties suitable for administration. Pharmaceutically acceptable carriers, such as stabilizers, diluents, additives, adjuvants, and excipients, have substantially no long-term or permanent adverse effects when administered to a subject. A "pharmaceutically acceptable carrier" should be a pharmaceutically inert material that is substantially biologically inactive and constitutes the majority of the formulation.

[0102] The engineered immune cells (such as CAR-T cells or TCR T cells) or pharmaceutical compositions of the present invention can be formulated into various modes of administration according to known techniques. For example, see Remington, The Science and Practice of Pharmacy (9th Ed. 1995). When manufacturing a pharmaceutical composition, the active agent is usually mixed with a pharmaceutically acceptable carrier, etc. Of course, a pharmaceutically acceptable carrier must be acceptable, i.e., compatible with any other ingredients in the formula, and must be harmless to the subject. Pharmaceutically acceptable carriers may include but are not limited to buffers, excipients, stabilizers, preservatives, wetting agents, surfactants, emulsifiers, or combinations thereof. Examples of buffers include but are not limited to acetic acid, citric acid, histidine, boric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, Tris buffer, HEPPSO, HEPES, neutral buffered saline, phosphate buffered saline, etc.

[0103] The engineered immune cells (e.g., CAR-T cells or TCR T cells) or pharmaceutical compositions of the present invention can be administered in any manner suitable for the disease to be treated (or prevented) and the subject. In certain embodiments, the mode of administration may include, but is not limited to, parenteral routes, injections, which may be administered to the subject by intravenous (iv), intraperitoneal, intracranial, intrathecal, intratumoral injection, etc. The pharmaceutical composition can be injected directly into a tumor, lymph node, tissue, organ, or site of infection.

[0104] The present invention also relates to a method for treating a disease in a subject, comprising administering the engineered immune cells or pharmaceutical compositions described above to a subject in need thereof. The subject may benefit or be expected to benefit from the engineered immune cells. The disease may, for example, be characterized by expression of a specific antigen by the patient's cells, to which the chimeric antigen receptor or T cell receptor contained in the engineered immune cells is capable of specifically binding. The specific antigen is a disease-associated antigen that is expressed only in the diseased cells or tissues and not in other cells or tissues, or is highly expressed in the diseased cells or tissues.

[0105] The disease can be a pathogen (e.g., virus or bacteria) infection, a tumor (e.g., cancer) or an autoimmune disease. The tumor can be, for example, a hematologic tumor or a solid tumor. In some embodiments, the specific antigen is a tumor-associated antigen, including but not limited to CD123, CD7, BCMA, CD19, CEA, CD17L, CD20, IL13RA2, G02, PSMA, ERBB2, IgKappa, CD174, VEGFR2, CD30, mesothelin, CD138, CD33, c-Met, EGFR, CD22, FAP, CD133, EPCAM, GPC3, IL1RAP, MUC1, NKG2D-L, ROR1, CD70, LMP1, MG7, NY-ESO-1, CD276, GD2, B7H3, KRAS mutation, AFP, GPC3, DLL3 and any combination thereof. In some embodiments, the pathogen includes but is not limited to viruses, bacteria, fungi and parasites. In some embodiments, the blood tumor includes but is not limited to acute myeloid leukemia AML, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, lymphoma (including follicular lymphoma, Hodgkin's lymphoma, diffuse large B-cell lymphoma), multiple myeloma and myeloproliferation. In some embodiments, the solid tumor includes but is not limited to: gastrointestinal tumors, gastric cancer, esophageal cancer, bile duct cancer, neuroblastoma, glioma (such as glioblastoma), mesothelioma, lung cancer, ovarian cancer, breast cancer, liver cancer, sarcoma, pancreatic cancer, non-small cell lung cancer and prostate cancer. In some embodiments, the tumor-associated antigen can be Claudin18.2 and EGFRVⅢ. Diseases expressing Claudin18.2 can include gastrointestinal tumors, gastric cancer, pancreatic cancer, esophageal cancer, bile duct cancer, ovarian cancer, lung cancer, etc. Diseases expressing EGFRVⅢ can include non-small cell lung cancer, glioma (such as glioblastoma). In some embodiments, the autoimmune disease includes but is not limited to inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Sjögren's syndrome, and systemic lupus erythematosus.

[0106] The present invention also relates to the use of the engineered immune cells in the preparation of a medicament for treating the above-mentioned disease in a subject. The present invention also relates to the use of the engineered immune cells in the treatment of the above-mentioned disease in a subject.

[0107] The terms "subject" and "patient" are used interchangeably herein. The term "subject" as used herein refers to any organism to which the engineered immune cells of the present invention can be administered, for example, for experimental, diagnostic, preventive and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, such as chimpanzees and other apes, and humans). The subject can be a mammal, particularly a human, including females (women) or males (men), and includes newborns, infants, teenagers, youth, adults or the elderly, and further includes various races and ethnicities. In some instances, a subject refers to an individual in need of diagnosis, treatment or prevention of a disease or condition, and the subject may suffer from the disease or condition, or have the risk of suffering from the disease or condition.

[0108] The term "treatment" refers to providing a beneficial or desired clinical outcome to a disease, such as eliminating the disease, alleviating symptoms, reducing the extent of the disease, stabilizing, improving or alleviating the state of the disease, or slowing down the progression of the disease. Measurement of treatment outcome can be based on, for example, the results of a physical examination, pathological test, and / or diagnostic test known in the art. Treatment can also refer to extending survival compared to the expected survival of the subject if he or she does not receive treatment. Treatment can also refer to reducing the incidence or morbidity of the disease, or its recurrence, compared to the disease that would occur if the measure were not taken. Clinically, this treatment can also be referred to as prevention.

[0109] The engineered immune cells (e.g., CAR T cells or TCR T cells) of the present invention can be administered to a subject in a therapeutically effective amount. The terms "therapeutically effective amount" and "effective amount" as used herein are used interchangeably and refer to an amount that is effective within the necessary dosage and time period to achieve the amount of the active ingredient (e.g., engineered immune cells of the present invention) for the desired therapeutic effect. The therapeutically effective amount can vary according to different factors, such as disease state, age, sex, and personal weight, as well as the ability of a combination of treatment methods or treatment methods to cause a desired response in an individual. An effective amount can refer to an amount that causes a detectable change in biological or chemical activity. Detectable changes can be detected and / or further quantified by personnel in related fields. In addition, an "effective amount" can specify an amount that maintains a desired physiological state, i.e., reduces or prevents a significant decline and / or promotes improvement in the condition of a disease.

[0110] The amount and frequency of administration will be determined by factors such as the subject's condition (such as age, weight, sex, and response to the drug) and the type and severity of the subject's disease, although appropriate dosages may be determined by clinical trials.

[0111] The engineered immune corresponding cells (e.g., CAR T cells or TCR T cells) or pharmaceutical compositions of the present invention can be administered to a subject in a therapeutically effective amount of about 0.5 to about 250 mg / kg, for example, about 1 to about 250 mg / kg, about 2 to about 200 mg / kg, about 3 to about 120 mg / kg, about 5 to about 250 mg / kg, about 10 to about 200 mg / kg, or about 20 to about 120 mg / kg.

[0112] An effective amount of the engineered immune cells (e.g., CAR T cells or TCR T cells) or pharmaceutical compositions of the present invention can be, for example, 1×10 3 cells to 1×10 9 cells, for example 1×10 4 cells to 1×10 8 cells, 1×10 5 cells to 1×10 7 cells.

[0113] The engineered immune cells (eg, CAR T cells or TCR T cells) or pharmaceutical compositions of the present invention can be administered once or twice a day; or once every 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, once every 1, 2, 3, 4, 5 or 6 weeks, or once every 1, 2, 3, 4, 5 or 6 months or longer. The pharmaceutical composition can also be administered several times a week (eg, 1, 2, 3, 4 or 5 times) or several times a month (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times).

[0114] The engineered immune cells (e.g., CAR T cells or TCR T cells) or pharmaceutical compositions of the present invention can be used in combination with other drugs and treatment methods, such as other anti-tumor drugs, chemotherapy, or radiotherapy. "Combined use" as referred to herein refers to the administration of two (or more) different drugs and / or therapies to a subject during treatment. The two or more drugs and / or therapies in the combination can be administered by different routes and regimens. Two or more drugs and / or treatment methods can be administered to the subject simultaneously or sequentially. In some embodiments, when the administration of the second drug or therapy begins, the administration of one drug or therapy is still ongoing, so there is overlap in administration. Such a regimen may be referred to as "simultaneously" herein. When administered simultaneously, two or more drugs and / or therapies can be formulated together into a single dosage form, or formulated into two or more independent dosage forms.

[0115] The present invention also relates to the use of polynucleotides encoding CXCL10 and polynucleotides encoding IL-15 in improving the therapeutic effects of immune cells expressing CAR or TCR (e.g., CAR T or TCR T cells) on the above-mentioned diseases. The "improvement" can be relative to the therapeutic effects of immune cells (which may be referred to as control cells) that express the same CAR and / or TCR but do not express CXCL10 and IL-15. Improving the therapeutic effect can, for example, refer to eliminating the disease, alleviating symptoms, reducing the extent of the disease, stabilizing, improving or alleviating the state of the disease, or slowing down the progression of the disease to achieve better results than control cells, such as obtaining better measurements or evaluation results than control cells on one or more measurements or evaluation indicators of the disease. Improving the therapeutic effect of immune cells can, for example, include increasing the infiltration of immune cells (e.g., T cells) into tumors, increasing the recruitment of immune cells (e.g., T cells), reducing tumor volume, and / or increasing the proliferation of immune cells (e.g., CAR T or TCR T cells) expressing CAR or TCR. The polynucleotides encoding CXCL10 and the polynucleotides encoding IL-15 are introduced into the immune cells expressing CAR or TCR (e.g., CAR T or TCR T cells) to express CXCL10 and IL-15 in the immune cell cells. In some embodiments, the polynucleotides encoding the fusion protein of CXCL10 and IL-15 are introduced into the immune cells expressing CAR or TCR (e.g., CAR T or TCR T cells). In some embodiments, the polynucleotides encoding the fusion protein of CXCL10 and IL-15 are contained in a vector and the vector is introduced into the immune cells expressing CAR or TCR (e.g., CAR T or TCR T cells). The present invention also relates to the use of polynucleotides encoding CXCL10 and polynucleotides encoding IL-15 in the preparation of reagents for improving the therapeutic effects of immune cells expressing CAR or TCR (e.g., CAR T or TCR T cells) on the above-mentioned diseases.

[0116] The present invention also relates to the use of the aforementioned immune cells, polynucleotides, expression vectors, host cells, pharmaceutical compositions, kits, or compositions in the preparation of medicaments for treating the aforementioned diseases in a subject. The present invention also relates to the use of the aforementioned immune cells, polynucleotides, expression vectors, host cells, pharmaceutical compositions, kits, or compositions in the preparation of medicaments for treating the aforementioned diseases in a subject.

[0117] The present invention is further described by the following examples, which should not be construed as limiting the present invention.

[0118] Unless otherwise specified, all reagents used in the following examples are commercially available. Molecular biology experimental methods not specifically described in the examples were performed according to the specific methods listed in J. Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition, or according to the kits and product instructions.

[0119] Example 1

[0120] Targeting Claudin18.2 (also referred to as CLDN 18.2 in the present invention) and EGFRVⅢ, CAR T cells expressing CXCL10 and IL15 were constructed, which express CD8αsignal peptide-anti-Claudin 18.2scFv-CD8hinge-CD28transmembrane-CD28costimulatory-CD3ζ-T2A-CXCL10-T2A-IL15 (hereinafter referred to as anti-CLDN18.2CAR-CXCL10-IL15) and CD8αsignal peptide-anti-EGFRVⅢscFv-CD8hinge-CD8transmembrane-41BB costimulatory-CD3ζ-T2A-CXCL10-T2A-IL15 (hereinafter referred to as anti-EGFRVⅢCAR-CXCL10-IL15), respectively.

[0121] The amino acid sequences of the individual elements are as follows:

[0122] 1. Claudin18.2CAR structure:

[0123] (1) Human CD8a signal peptide: MALPVTALLLPLALLLHAARP (SEQ ID NO: 1)

[0124] (2) Anti-human Claudin 18.2 single-chain variable region (anti-Claudin 18.2 scFv):

[0125] (3)CD8 hinge region (CD8hinge):

[0126] (4)CD28 transmembrane-CD28 costimulatory domain:

[0127] (5)CD3ζ:

[0128] 2.EGFRVⅢCAR structure:

[0129] (1) Human CD8a signal peptide: MALPVTALLLPLALLLHAARP (SEQ ID NO: 1) (same as Claudin18.2CAR)

[0130] (2) Anti-human EGFRVIII single-chain variable region (anti-EGFRVIII scFv):

[0131] (3)CD8 hinge region (CD8hinge):

[0132] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:3)(Same as Claudin18.2CAR)

[0133] (4)CD8 transmembrane-41BB costimulatory domain:

[0134] (5)CD3ζ:

[0135] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:5)(Same as Claudin18.2CAR)

[0136] 3.2A peptide (T2A):

[0137] 4.CXCL10:

[0138] 5.IL15:

[0139] The coding polynucleotide sequences of each element are as follows:

[0140] 1. Claudin18.2CAR structure:

[0141] (1) Human CD8a signal peptide:

[0142] (2) Anti-human Claudin 18.2 single-chain variable region (anti-Claudin 18.2 scFv):

[0143] (3) CD8 hinge region:

[0144] (4)CD28 transmembrane and co-stimulatory domain:

[0145] (5)CD3ζ:

[0146] 2.EGFRVⅢCAR structure:

[0147] (1) Human CD8a signal peptide:

[0148] (2) Anti-human EGFRVIII single-chain variable region (anti-EGFRVIII scFv):

[0149] (3) CD8 hinge region:

[0150] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT(SEQ ID NO:13)(Same as Claudin18.2CAR)

[0151] (4) CD8 transmembrane and 41BB co-stimulatory domain:

[0152] (5)CD3ζ:

[0153] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:15)(Same as Claudin18.2CAR)

[0154] 3.2A peptide:

[0155] 4.CXCL10:

[0156] 5.IL15:

[0157] Therefore, the coding polynucleotide sequence of anti-CLDN18.2CAR-CXCL10-IL15 (2379 bp) is:

[0158] The coding polynucleotide sequence of anti-EGFRVIIICAR-CXCL10-IL15 (2367 bp):

[0159] Methods and Results

[0160] 1.CAR structure design

[0161] T2A-CXCL10 and T2A-IL15 were synthesized by GENEWIZ using pZX-K-CD8αsignal peptide-anti-Claudin 18.2scFv-CD8hinge-CD28transmembrane-CD28TM-CD3ζ and pZX-K-CD8αsignal peptide-anti-EGFRVⅢscFv-CD8hinge-CD8transmembrane-41BB-CD3ζ vectors. The target fragments were inserted into the vectors by homologous recombination to form pZX-K-CD8αsignal peptide-anti-Claudin 18.2scFv-CD8hinge-CD28transmembrane-CD28TM-CD3ζ-T2A-CXCL10-T2A-IL15 and pZX-K-CD8αsignal peptide-anti-EGFRVⅢscFv-CD8hinge-CD8transmembrane-41BB-CD3ζ-T2A-CXCL10-T2A-IL15.

[0162] The coding polynucleotide sequence of T2A-CXCL10:

[0163] The coding polynucleotide sequence of T2A-IL15:

[0164] Vector pZX-K-CD8αsignal peptide-anti-Claudin 18.2scFv-CD8hinge-CD28transmembrane-CD28TM-CD3ζ sequence (including the stop codon):

[0165] pZX-KCD8αsignal peptide-anti-Claudin 18.2scFv-CD8hinge-CD28transmembrane-CD28TM-CD3ζ-T2A-CXCL10-T2A-IL15 (contains a stop codon at the end):

[0166] Vector pZX-K-CD8αsignal peptide-anti-EGFRVⅢscFv-CD8hinge-CD8transmembrane-41BB-CD3ζ sequence (including stop codon):

[0167] pZX-K-CD8αsignal peptide-anti-EGFRVⅢscFv-CD8hinge-CD8transmembrane-41BB-CD3ζ-T2A-CXCL10-T2A-IL15 (contains the stop codon at the end):

[0168] A schematic diagram of the structure of the plasmids constructed for expressing anti-CLDN18.2CAR-CXCL10-IL15 and anti-EGFRVIIICAR-CXCL10-IL15 (also referred to as CAR-CXCL10-IL15 in the text) is shown in Figure 1.

[0169] 2. Lentivirus Production

[0170] 2.1 Materials

[0171] 293T cells

[0172] Main plasmid: experimental group CAR plasmid and control group CAR plasmid, including:

[0173] CAR plasmids in the experimental group: The plasmids constructed above for expressing CAR-CXCL10-IL15 are shown in the two right panels of Figure 1 ;

[0174] Control group CAR plasmid: a plasmid containing the polynucleotide encoding the above-mentioned anti-CLDN18.2CAR or anti-EGFRVⅢCAR, but excluding the plasmid encoding the polynucleotides of CXCL10 and IL15.

[0175] Helper plasmid: Four-plasmid system (pLenti-P3A / B / C)

[0176] 2.2 Methods

[0177] (1) Using the four-plasmid system method, the auxiliary plasmid and the main plasmid were co-infected into 293T cells to obtain the experimental group lentivirus and the control group lentivirus;

[0178] (2) Two days after transfection, the supernatant was collected and centrifuged, treated with virus concentration reagent, and stored at -80°C for later use.

[0179] 3. CAR T cell construction method

[0180] 3.1 Materials

[0181] Ficoll centrifuge tube: Ficoll-PaqueTW DREMIUM, Cytiva

[0182] Human CD4 magnetic beads: CD4MicroBeads human, Miltenyi Biotec, Germany

[0183] Human CD8 magnetic beads: CD8MicroBeads human, Miltenyi Biotec, Germany

[0184] LS Columns: LS Columns, Miltenyi Biotec, Germany

[0185] CD3 / CD28 activating magnetic beads: Dynabeads TM CD3 / CD28, Thermo Fisher Scientific

[0186] X VIVO15 medium: TheraPEAK™ X-Vivo™ 15, LONZA

[0187] 3.2 Methods

[0188] 3.2.1 Peripheral PBMC Isolation

[0189] (1) Slowly add peripheral blood into a Ficoll centrifuge tube and centrifuge at 400g for 30 minutes.

[0190] (2) Collect the middle layer PBMC suspension in a centrifuge tube and wash twice with PBS.

[0191] 3.2.2 T cell sorting

[0192] (1) Human CD4 and CD8 magnetic beads were incubated with PBMC cells for 15 minutes.

[0193] (2) Separation of CD4+ and CD8+ cells using LS columns;

[0194] 3.2.3 T cell activation

[0195] (1) Cultivate the isolated CD4+ and CD8+ cells using X VIVO15 medium;

[0196] (2) Add CD3 / CD28 activated magnetic beads for culture.

[0197] 3.2.4 T cell transduction

[0198] (1) Adjust the T cell density to 1×10 6 cells / ml;

[0199] (2) Add the lentivirus obtained in 2.2 to the infected cells overnight to obtain experimental group CAR T cells and control group CAR T cells, where the experimental group CAR T cells may also be referred to as CAR T-CXCL10-IL 15 in this article.

[0200] 4. Element Expression Detection

[0201] 4.1 CAR Expression Detection

[0202] Methods: iF647 protein L (GenScript) was used to detect the expression rate of CAR elements in CAR T cells.

[0203] Results: As shown in Figure 2, there was no significant difference in CAR expression between the experimental group and the control group.

[0204] 4.2 CXCL10 expression detection

[0205] Methods: The Human CXCL10 ELISA kit (Solarbio) was used to detect the concentration of CXCL10 in the cell culture supernatant of the experimental group and the control group.

[0206] Results: As shown in Figure 3, the concentration of CXCL10 in the supernatant of CAR T cells in the experimental group was significantly higher than that in the control group, indicating that CAR T-CXCL10-IL15 in the experimental group could secrete CXCL10.

[0207] 4.3 IL15 expression detection

[0208] Methods: The Human IL15 ELISA kit (Ecosai) was used to detect the concentration of IL15 in the cell culture supernatant of the experimental group and the control group.

[0209] Results: As shown in Figure 4, the concentration of IL15 in the supernatant of CAR T cells in the experimental group was significantly higher than that in the control group, indicating that CAR T-CXCL10-IL15 in the experimental group could secrete IL15.

[0210] 5. CAR T function testing

[0211] 5.1 Killing

[0212] 5.1.1 Materials:

[0213] Target cells:

[0214] NUGC4-18.2, NUGC4 cells overexpressing claudin 18.2, were purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.

[0215] U87-EGFRVⅢ is U87 cells that overexpress EGFRVⅢ. U87 cells were purchased from ATCC and genetically modified to overexpress EGFRVⅢ.

[0216] Effector cells: CAR T cells in the experimental group and CAR T cells in the control group

[0217] Microplate reader: SpectraMax i3x

[0218] Luciferase substrate: Vazyme

[0219] 5.1.2 Methods:

[0220] (1) Two groups of CAR T cells were added to target cells in 96-well plates at the corresponding effector-target ratios (5:1, 2.5:1, and 1.25:1) and incubated for 7 hours;

[0221] (2) Remove the plate, centrifuge, and wash once with PBS;

[0222] (3) Discard PBS and add substrate to react for 5 minutes;

[0223] (4) Read the plate using an enzyme-linked microplate reader.

[0224] 5.1.3 Results (as shown in Figure 5):

[0225] (1) Under different effector-target ratios, both the experimental and control groups had a killing effect on target cells;

[0226] (2) The CAR T cells in the experimental and control groups had the same killing effects under the same CAR expression conditions, which indicated that the addition of cytokines did not affect the killing effect of CAR.

[0227] 5.2 Proliferation

[0228] 5.2.1 Materials:

[0229] (1) Effector cells: CAR T cells in experimental group and control group

[0230] (2) Culture medium: X VIVO15 (LONZA) culture medium + 10% fetal bovine serum

[0231] (3) Cellometer K2 counter

[0232] (4) ViaStain AOPI stain solution CS2-0106

[0233] 5.2.2 Methods:

[0234] (1) Two groups of CAR T cells were injected at a rate of 1.5x10 6The number of cells is the starting amount and should be cultured in the culture medium;

[0235] (2) After 4 days, the cell suspension was mixed and the cell numbers of the two groups were detected;

[0236] 5.2.3 Results:

[0237] As shown in Figure 6, after 4 days of culture, the number of cells in the experimental group was significantly higher than that in the control group, and CART cells that secreted IL15 and CXCL10 were beneficial to the proliferation of CART cells.

[0238] 5.3 Migration

[0239] 5.3.1 Materials:

[0240] Transwell plate (corning, 3421)

[0241] Transwell upper chamber cells: activated T cells that were not transduced with lentivirus after steps 3.2.1-3.2.3

[0242] Transwell lower chamber cells: CAR T culture supernatant of experimental group and control group

[0243] Culture medium: X VIVO15 (LONZA) medium

[0244] Cellometer K2 counter

[0245] viastain AOPI stain solution cell staining solution CS2-0106

[0246] 5.3.2 Methods:

[0247] (1) A transwell chamber model was used to detect the recruitment of T cells by CAR T cell supernatant. T cells were added to the upper chamber of the transwell, and the experimental or control CAR T cell supernatant was added to the lower chamber and incubated for 5 hours.

[0248] (2) Detect the total number of cells in the lower chamber;

[0249] 5.3.3 Results: As shown in Figure 7, the number of T cells in the lower chamber of the experimental group was significantly higher than that of the control group, and CAR T cells secreting IL15 and CXCL10 enhanced the recruitment of T cells.

[0250] 6. Pharmacodynamics

[0251] 6.1 Materials:

[0252] NOG mice (Viton River);

[0253] NUGC4-18.2, U87-EGFRVⅢ;

[0254] Effector cells: CAR T cells

[0255] CD3+ epsilon antibody (ab52959, abcam)

[0256] 6.2 Methods:

[0257] (1) NOG mice were subcutaneously loaded with NUGC4-18.2 and U87-EGFRVⅢ tumors;

[0258] (2) When the subcutaneous tumor reaches 80-110 mm 3 When CAR T cell therapy was enrolled, the groups were as follows;

[0259] (3) Observe the eating status of mice, measure the subcutaneous tumor volume of mice and record it;

[0260] (4) 29 days after CAR T treatment, the tumor was removed and IHC was used to detect CD3+ T cell infiltration;

[0261] 6.3 Results:

[0262] 6.3.1 Tumor formation curve (as shown in Figure 8):

[0263] (1) The tumor volumes of the two groups of CAR T mice were significantly smaller than those of the blank group;

[0264] (2) The tumor volume of the experimental group CAR T was significantly smaller than that of the control group CAR T;

[0265] This indicates that the pharmacodynamics of CAR T in the experimental group is better than that in the control group.

[0266] 6.3.2 Cell Infiltration: As shown in Figure 9, the proportion of CD3+ cells in the tumor tissue of the CLDN18.2 experimental group was significantly higher than that of the control group. This indicates that the proportion of T cells in the tumor tissue of the experimental group was higher than that of the control group. In other words, after CAR T-CXCL10-IL15 treatment in the experimental group, the accumulation of T cells in the tumor tissue increased.

[0267] in conclusion

[0268] CAR T-CXCL10-IL15 was constructed, which can enhance the pharmacodynamics of CAR T cells, improve the recruitment of T cells in solid tumors, and improve the anti-tumor effect of CAR T.

[0269] The embodiments of the present invention are not limited to the above embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and details, and these are all considered to fall within the scope of protection of the present invention.

Claims

1. Engineered immune cells that express CXCL10 and IL15.

2. The engineered immune cells according to claim 1, wherein the immune cells are T cells, B cells, macrophages, dendritic cells, monocytes, granulocytes, NK cells or NKT cells.

3. The engineered immune cells according to claim 1 or 2, which express a fusion protein of CXCL10 and IL15.

4. The engineered immune cells according to claim 3, wherein in the fusion protein, CXCL10 and IL15 are linked by a linker, and the linker is preferably a self-cleaving linker, more preferably T2A, F2A or P2A.

5. The engineered immune cells according to any one of claims 1-4, wherein CXCL10 comprises the amino acid sequence shown in SEQ ID NO:9, and IL15 comprises the amino acid sequence shown in SEQ ID NO:

10.

6. The engineered immune cells according to any one of claims 1-5, wherein the cells further express a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

7. The engineered immune cells according to claim 6, which express a fusion protein of a chimeric antigen receptor, CXCL10 and IL15.

8. The engineered immune cells according to claim 7, wherein in the fusion protein, the chimeric antigen receptor (CAR), CXCL10 and IL15 are linked by a linker, and the linker is preferably a self-cleaving linker, more preferably selected from T2A, F2A or P2A.

9. The engineered immune cells according to claim 7 or 8, wherein the chimeric antigen receptor targets an antigen expressed by a pathogen, a tumor-associated antigen or an antigen associated with an autoimmune disease.

10. The engineered immune cells according to claim 9, wherein the tumor-associated antigen is selected from CD123, CD7, BCMA, CD19, CEA, CD17L, CD20, IL13RA2, G02, PSMA, ERBB2, IgKappa, CD174, VEGFR2, CD30, mesothelin, CD138, CD33, c-Met, EGFR, CD22, FAP, CD133, EPCAM, GPC3, IL1RAP, MUC1, NKG2D-L, ROR1, CD70, LMP1, MG7, NY-ESO-1, CD276, GD2, B7H3, KRAS mutation, AFP, GPC3 and DLL3 and any combination thereof.

11. The engineered immune cells according to any one of claims 6-10, wherein the chimeric antigen receptor comprises a binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain.

12. The engineered immune cells according to claim 11, wherein the binding domain is a single-chain antibody (scFv).

13. The engineered immune cells according to claim 12, wherein the binding domain is an anti-Claudin18.2 scFv or an anti-EGFRVⅢ scFv.

14. The engineered immune cell according to claim 13, wherein the binding domain comprises the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:

6.

15. The engineered immune cell according to any one of claims 11-14, wherein the hinge region is the CD8α hinge region.

16. The engineered immune cell according to any one of claims 11-15, wherein the transmembrane domain is the transmembrane domain of CD28.

17. The engineered immune cell according to any one of claims 11-16, wherein the co-stimulatory domain is the CD28 co-stimulatory domain or the 4-1BB co-stimulatory domain.

18. The engineered immune cell according to any one of claims 11-17, wherein the intracellular signaling domain is the CD3ζ intracellular signaling domain.

19. The engineered immune cell according to any one of claims 11-18, wherein the chimeric antigen receptor further comprises a signal peptide, preferably the CD8 signal peptide.

20. An isolated polynucleotide encoding CXCL10 and IL15.

21. The isolated polynucleotide according to claim 20, encoding a fusion protein comprising CXCL10 and IL15.

22. The isolated polynucleotide according to claim 21, wherein in the fusion protein, CXCL10 and IL15 are linked by a linker, preferably a self-cleaving linker, more preferably T2A, F2A or P2A.

23. The isolated polynucleotide according to any one of claims 20-22, wherein CXCL10 comprises the amino acid sequence shown in SEQ ID NO:9, and IL15 comprises the amino acid sequence shown in SEQ ID NO:

10.

24. The isolated polynucleotide according to any one of claims 20-23, further encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

25. The isolated polynucleotide according to claim 24, encoding a fusion protein comprising the chimeric antigen receptor, CXCL10 and IL15.

26. The isolated polynucleotide according to claim 25, wherein in the fusion protein, the chimeric antigen receptor (CAR), CXCL10 and IL15 are linked by a linker, preferably a self-cleaving linker, more preferably selected from T2A, F2A or P2A.

27. The isolated polynucleotide according to claim 25 or 26, wherein the chimeric antigen receptor targets an antigen expressed by a pathogen, a tumor-associated antigen or an antigen associated with an autoimmune disease.

28. The isolated polynucleotide according to claim 27, wherein the tumor-associated antigen is selected from CD123, CD7, BCMA, CD19, CEA, CD17L, CD20, IL13RA2, G02, PSMA, ERBB2, IgKappa, CD174, VEGFR2, CD30, mesothelin, CD138, CD33, c-Met, EGFR, CD22, FAP, CD133, EPCAM, GPC3, IL1RAP, MUC1, NKG2D-L, ROR1, CD70, LMP1, MG7, NY-ESO-1, CD276, GD2, B7H3, KRAS mutation, AFP, GPC3, and DLL3, and any combination thereof.

29. The isolated polynucleotide according to any one of claims 24-28, wherein the chimeric antigen receptor comprises a binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

30. The isolated polynucleotide according to claim 29, wherein the binding domain is a single-chain antibody (scFv).

31. The isolated polynucleotide according to claim 30, wherein the binding domain is an anti-Claudin18.2 scFv or an anti-EGFRVⅢ scFv.

32. The isolated polynucleotide according to claim 31, wherein the binding domain comprises the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:

6.

33. The isolated polynucleotide according to any one of claims 29-32, wherein the hinge region is a CD8α hinge region.

34. The isolated polynucleotide according to any one of claims 29-33, wherein the transmembrane domain is the transmembrane domain of CD28.

35. The isolated polynucleotide according to any one of claims 29-34, wherein the co-stimulatory domain is a CD28 co-stimulatory domain or a 41BB co-stimulatory domain.

36. The isolated polynucleotide according to any one of claims 29-35, wherein the intracellular signaling domain is a CD3ζ intracellular signaling domain.

37. The isolated polynucleotide according to any one of claims 29-36, wherein the chimeric antigen receptor further comprises a signal peptide, preferably a CD8 signal peptide.

38. An expression vector comprising the polynucleotide according to any one of claims 20-37.

39. A host cell comprising the polynucleotide according to any one of claims 20-37 or the expression vector according to claim 38.

40. The host cell according to claim 39, which is an immune cell.

41. The host cell according to claim 40, wherein the immune cell is a T cell, B cell, macrophage, dendritic cell, monocyte, granulocyte, NK cell, or NKT cell.

42. A pharmaceutical composition comprising the engineered immune cell according to any one of claims 1-19 or the host cell according to any one of claims 39-41.

43. A method for treating a disease in a subject, comprising administering to a subject in need thereof an engineered immune cell as described in any one of claims 1-19, a host cell as described in any one of claims 39-41, or a pharmaceutical composition as described in claim 42.

44. The method according to claim 43, wherein the disease is characterized by the expression of a specific antigen, and the chimeric antigen receptor or T cell receptor contained in the immune cell is capable of specifically binding to the specific antigen.

45. The method according to claim 43 or 44, wherein the disease is an infectious disease, a tumor, or an autoimmune disease.

46. The method according to claim 45, wherein the infectious disease is a viral, bacterial, fungal, or parasitic infection.

47. The method according to claim 45, wherein the tumor is a hematological tumor or a solid tumor.

48. The method according to claim 45, wherein the tumor expresses Claudin18.2 or EGFRVⅢ.

49. The method according to claim 47, wherein the hematological tumor is acute myeloid leukemia AML, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, multiple myeloma, or myeloproliferation.

50. The method according to claim 47, wherein the solid tumor is a gastrointestinal tumor, gastric cancer, esophageal cancer, cholangiocarcinoma, neuroblastoma, glioma, glioblastoma, mesothelioma, lung cancer, ovarian cancer, breast cancer, liver cancer, sarcoma, pancreatic cancer, non-small cell lung cancer, or prostate cancer.

51. The method according to claim 45, wherein the autoimmune disease is inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Sjogren's syndrome, and systemic lupus erythematosus.

52. Kit, comprising: (1) An immune cell; and (2) a polynucleotide as described in any one of claims 20-37 or an expression vector as described in claim 38.

53. The kit according to claim 52, wherein the immune cell is a T cell, B cell, macrophage, dendritic cell, monocyte, granulocyte, NK cell, or NKT cell.

54. Use of an immune cell as described in any one of claims 1-19, a polynucleotide as described in any one of claims 20-37, an expression vector as described in claim 38, a host cell as described in any one of claims 39-41, a pharmaceutical composition as described in claim 42, or a kit as described in claim 52 or 53 in the preparation of a drug for treating a disease in a subject.

55. The use according to claim 54, wherein the disease is characterized by the expression of a specific antigen, and the chimeric antigen receptor or T cell receptor contained in the immune cell is capable of specifically binding to the specific antigen.

56. The use according to claim 54 or 55, wherein the disease is an infectious disease, a tumor, or an autoimmune disease.

57. The use according to claim 56, wherein the infectious disease is a viral, bacterial, fungal, or parasitic infection.

58. The use according to claim 56, wherein the tumor is a hematological tumor or a solid tumor.

59. The use according to claim 56, wherein the tumor expresses Claudin18.2 or EGFRVⅢ.

60. The use according to claim 58, wherein the hematological tumor is acute myeloid leukemia AML, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, multiple myeloma or myeloproliferation.

61. The use according to claim 58, wherein the solid tumor is a gastrointestinal tumor, gastric cancer, esophageal cancer, cholangiocarcinoma, neuroblastoma, glioma, glioblastoma, mesothelioma, lung cancer, ovarian cancer, breast cancer, liver cancer, sarcoma, pancreatic cancer, non-small cell lung cancer or prostate cancer.

62. The use according to claim 56, wherein the autoimmune disease is inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Sjogren's syndrome and systemic lupus erythematosus.

63. Use of a vector comprising a polynucleotide encoding CXCL10 and a polynucleotide encoding IL15 in enhancing the therapeutic effect of immune cells in treating a disease in a subject.

64. The use according to claim 63, wherein the immune cells are T cells, B cells, macrophages, dendritic cells, monocytes, granulocytes, NK cells or NKT cells.

65. The use according to claim 64, wherein the disease is characterized by the expression of a specific antigen, and the chimeric antigen receptor or T cell receptor comprised in the immune cells is capable of specifically binding to the specific antigen.

66. The use according to claim 64 or 65, wherein the disease is an infectious disease, a tumor or an autoimmune disease.

67. The use according to claim 66, wherein the infectious disease is a viral, bacterial, fungal or parasitic infection.

68. The use according to claim 66, wherein the tumor is a hematological tumor or a solid tumor.

69. The use according to claim 66, wherein the tumor expresses Claudin18.2 or EGFRVⅢ.

70. The use according to claim 68, wherein the hematological tumor is acute myeloid leukemia AML, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, multiple myeloma or myeloproliferation.

71. The use according to claim 68, wherein the solid tumor is a gastrointestinal tumor, gastric cancer, esophageal cancer, cholangiocarcinoma, neuroblastoma, glioma, glioblastoma, mesothelioma, lung cancer, ovarian cancer, breast cancer, liver cancer, sarcoma, pancreatic cancer, non-small cell lung cancer or prostate cancer.

72. The use according to claim 66, wherein the autoimmune disease is inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Sjogren's syndrome and systemic lupus erythematosus.

73. The use according to any one of claims 63 - 72, wherein enhancing the therapeutic effect of immune cells in treating a disease in a subject comprises enhancing the infiltration of immune cells into the tumor, increasing immune cell recruitment, reducing tumor volume and / or increasing the proliferation of immune cells.

74. A method for preparing engineered immune cells, comprising: (i) Express CXCL10 and IL15 in the immune cells; and (ii) expressing a chimeric antigen receptor and / or a T cell receptor in the immune cells.

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