A cell expressing a fusion protein and use thereof

Abstract

A cell expressing a fusion protein and its applications. This cell possesses enhanced capabilities, such as cell proliferation, cytokine secretion, and tumor cell killing.

Description

A cell expressing fusion protein and its application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a cell expressing a fusion protein and an application thereof. Background Art

[0002] Currently, immunotherapy is an effective treatment for patients with poor prognosis. However, the immune cells used in immunotherapy suffer from poor function, proliferation, and survival after infusion. Therefore, developing modified immune cells and robust and reliable immune cell culture methods are urgent challenges.

[0003] Therefore, there is an urgent need in the art for a cell expressing a fusion protein, which can be modified to improve the function of the cell.

[0004] Summary of the Invention

[0005] The present invention provides an isolated cell comprising a member of the peptidase C14B protein family or a functional fragment thereof, or a member of the basic helix-loop-helix (bHLH) protein family or a functional fragment thereof. The immune cell of the present invention has one or more of the following characteristics: (1) improved cell proliferation ability; (2) enhanced cytokine secretion ability; (3) improved tumor cell killing ability; (4) improved or stabilized exogenous gene transduction efficiency; (5) improved downstream signaling pathway strength; and / or (6) improved cell subpopulation ratios, such as an increased proportion of activated cells, a decreased proportion of regulatory cells, a decreased proportion of exhausted cells, an increased proportion of central memory cells and / or naive cells, a decreased proportion of apoptotic cells, and an increased proportion of stem-like cells.

[0006] On the other hand, the present invention provides a method for producing the cell of the present invention, which comprises causing the cell to artificially express the peptidase C14B protein family member or a functional fragment thereof, or the basic helix-loop-helix (bHLH) protein family member or a functional fragment thereof.

[0007] In another aspect, the present invention provides a composition comprising the cells of the present invention, and optionally a pharmaceutically acceptable carrier.

[0008] In another aspect, the present invention provides a kit comprising the cells of the present invention and / or the composition of the present invention.

[0009] In another aspect, the present invention provides a use of the cell of the present invention, the composition of the present invention and / or the kit of the present invention in preparing a medicament for preventing, alleviating and / or treating a disease, preferably a tumor.

[0010] In another aspect, the present invention provides a medicament for preventing and / or treating a disease and / or symptom, preferably a tumor, comprising the cell of the present invention, the composition of the present invention and / or the kit of the present invention.

[0011] In another aspect, the present invention provides a method for preventing and / or treating a disease and / or condition, preferably a tumor, comprising administering the cell of the present invention, the composition of the present invention and / or the kit of the present invention to a subject in need thereof.

[0012] In another aspect, the present invention provides the cell of the present invention, the composition of the present invention and / or the kit of the present invention for use in preventing and / or treating a disease and / or symptom, preferably a tumor.

[0013] Those skilled in the art will readily appreciate other aspects and advantages of the present invention from the detailed description below. The detailed description below only shows and describes exemplary embodiments of the present invention. As will be appreciated by those skilled in the art, the disclosure of the present invention enables those skilled in the art to modify the disclosed embodiments without departing from the spirit and scope of the invention to which the present invention relates. Accordingly, the descriptions in the drawings and specification of the present invention are intended to be exemplary only and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The specific features of the present invention are shown in the appended claims. The features and advantages of the present invention can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. The drawings are briefly described as follows:

[0015] Figures 1A-1B show the transduction results of TIL cells from different donors.

[0016] 2A-2B show the results of the killing ability test of TIL cells transduced with the target polypeptide of the present invention.

[0017] FIG3 shows the in vivo tumor control effect of TIL cells transduced with the polypeptide of interest of the present invention.

[0018] Figures 4A-4C show the transduction results of TIL cells from different donors.

[0019] 5A-5D show the results of the killing ability test of TIL cells transduced with the target polypeptide of the present invention.

[0020] FIG6 shows the in vivo tumor control effect of TIL cells transduced with the polypeptide of interest of the present invention. DETAILED DESCRIPTION

[0021] The following describes the embodiments of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0022] Definition of terms

[0023] In the present invention, the term "peptidase C14B protein family member" generally includes peptidase C14B protein family members or functionally active fragments thereof. For example, the peptidase C14B protein family member may include MALT1. For example, the UniProt number of the peptidase C14B protein family member may be Q9UDY8. The peptidase C14B protein family members of the present invention may also include functionally active fragments thereof, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants thereof, active fragments thereof, or variants thereof comprising the functionally active fragments produced after processing and / or modification thereof in cells.

[0024] In the present invention, the term "basic helix-loop-helix (bHLH) protein family member" generally includes basic helix-loop-helix (bHLH) protein family members or functionally active fragments thereof. For example, a basic helix-loop-helix (bHLH) protein family member may include TFAP4. For example, the UniProt number of a basic helix-loop-helix (bHLH) protein family member may be Q01664. The basic helix-loop-helix (bHLH) protein family member of the present invention may also encompass functionally active fragments thereof, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or variants comprising the functionally active fragments thereof produced after processing and / or modification in cells.

[0025] In the present invention, the term "peptide linker" is generally used interchangeably with the term "linker." For example, a peptide linker is a peptide that covalently or non-covalently links two or more molecules or peptides to form a larger complex consisting of all molecules or peptides including the peptide linker.

[0026] In the present invention, the term "fusion protein" generally refers to a protein composed of two or more molecules or peptides that are not normally bound in their native state, but whose respective head and terminal ends can be directly or indirectly bound together to form a continuous polypeptide. For example, one portion of a fusion protein can be referred to as a "first polypeptide." For example, another portion of the fusion protein can be referred to as a "second polypeptide." For example, a fusion protein can contain two or more parts, and the "first polypeptide" or "second polypeptide" does not limit the number of components of the fusion protein.

[0027] In the present invention, the amino acid residues may be naturally occurring amino acids and / or artificially modified amino acids. For example, naturally occurring amino acids may be selected from the group consisting of alanine (three-letter code: ala, single-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0028] In the present invention, the term "immune cell" generally refers to cells that participate in an immune response, such as cells that promote immune effector responses. Examples of immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, NKT cells, mast cells, granulocytes, monocytes, lymphocytes, and macrophages. For example, the immune cells of the present invention may include cells derived from artificial pluripotent stem (iPS) cells, PBMC cells, and / or tumor infiltrating lymphocytes. For example, the immune cells of the present invention may be obtained by differentiation of iPS cells. The term also includes engineered immune cells, such as immune cells that are genetically modified by adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.

[0029] In the present invention, the term "killing ability" refers to the killing of cells by contacting the cells with an effective amount of antibodies, T cells, immunoconjugates, bispecific / multispecific molecules or compositions. For example, the fusion polypeptide of the present invention can enhance the killing ability of cells. For example, cells expressing the fusion polypeptide of the present invention can exhibit enhanced killing ability. For example, a combination of cells expressing the fusion polypeptide of the present invention and other immune cells, antibodies, immunoconjugates (e.g., conjugates of binding molecules targeting immune checkpoints with other active molecules), and / or bispecific / multispecific molecules can exhibit enhanced killing ability. The killing ability after the above-mentioned combination of substances can be manifested as cells expressing positive killing antigens, optionally in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis or by a combination of two or more of these mechanisms.

[0030] In the present invention, the term "directly or indirectly linked" refers to direct linkage via a peptide bond, or indirect linkage via a linker or a non-peptide linkage.

[0031] In the present invention, the term "PBMC" or "human peripheral blood mononuclear cell" generally refers to cells in peripheral blood that have a single nucleus. For example, any blood cell with a round nucleus (i.e., lymphocyte, monocyte, or macrophage). These blood cells are key components of the immune system to fight infection and adapt to invaders. The lymphocyte population is composed of CD4 + and CD8 + T cells, B cells and natural killer cells, CD14 + Monocytes and basophils / neutrophils / eosinophils / dendritic cells. Usually, FICOLL TM (a hydrophilic polysaccharide that separates blood), these cells are separated from whole blood, where monocytes and lymphocytes form the buffy coat below the plasma layer. For example, "PBMC" refers to a cell population that contains at least T cells, and optionally NK cells, NKT cells, and antigen-presenting cells.

[0032] In the present invention, the term "proliferation" refers to an increase in cell division (symmetrical or asymmetrical division of cells). "Proliferation" can refer to symmetrical or asymmetrical division of T cells. "Increased proliferation" occurs when there is an increase in the number of cells in a treated sample compared to cells in an untreated sample.

[0033] In the present invention, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, and can be mammals, such as non-human primates, sheep, dogs, cats, cows and horses.

[0034] In the present invention, the term "therapeutically effective amount" refers to an amount of a fusion protein of the present invention or an amount of cells expressing a fusion protein of the present invention sufficient to prevent or alleviate symptoms associated with a disease or condition (e.g., cancer). The therapeutically effective amount depends on the disease being treated, and those skilled in the art can readily determine the actual effective amount.

[0035] In the present invention, the term "drug" generally refers to a chemical compound or composition that is capable of inducing a desired therapeutic effect when properly administered to a patient.

[0036] As used herein, the term "composition" refers to a mixture containing one or more compounds of the present invention, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity. Therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage.

[0037] In the present invention, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid connected to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which other DNA segments can be connected. Another type of vector is a viral vector, in which other DNA segments can be connected to a viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (e.g., non-additive mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, thereby replicating together with the host genome, such as naked RNA polynucleotides that cannot autonomously replicate, naked DNA polynucleotides, polynucleotides composed of DNA and RNA in the same chain, poly-lysine-coupled DNA or RNA, peptide-coupled DNA or RNA, liposome-coupled DNA, etc. In addition, certain vectors can direct the expression of genes effectively connected to them. This type of vector is referred to as a "recombinant expression vector" (or simply "expression vector") in the present invention. Generally speaking, the expression vector used in recombinant DNA technology is typically in the form of a plasmid. In this specification, "plasmid" and "vector" are used interchangeably, as plasmid is the most commonly used form of vector.

[0038] In the present invention, the term "adjuvant" generally refers to any substance that assists or regulates the effects of drugs, including but not limited to immunological adjuvants, which enhance or diversify the immune response to antigens.

[0039] As used herein, the term "tumor" or "tumor cell" generally refers to or describes a physiological condition in mammals that is generally characterized by unregulated cell growth. Examples of tumors include, but are not limited to, carcinomas, lymphomas, blastomas (including medulloblastomas and retinoblastomas), sarcomas (including liposarcoma and synovial cell sarcomas), neuroendocrine tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas), mesotheliomas, schwannomas (including acoustic neuromas), meningiomas, adenocarcinomas, and melanomas. "Tumor" may further include "solid tumors," which refer to tumors selected from the group consisting of gastrointestinal cancer, pancreatic cancer, glioblastomas, cervical cancer, ovarian cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumors, and head and neck cancer.

[0040] In the present invention, the terms "about" and "approximately" generally refer to a statistically significant numerical range. Such a range can be within an order of magnitude of a given value or range, can be included within 50%, preferably included within 20%, more preferably included within 10%, and most preferably included within 5%. The permissible variations encompassed by the terms "about" or "approximately" may depend on the specific system under study and can be readily understood by those of ordinary skill in the art.

[0041] In the present invention, the terms "above", "below", "at most" and "at least" include the number.

[0042] In the present invention, the terms "include", "comprise" or "contain" are open expressions, while "consisting of" is a closed expression. The former covers the latter, and the latter is a special form of the former.

[0043] Detailed Description of the Invention

[0044] In one aspect, the present invention provides an isolated cell comprising a peptidase C14B protein family member or a functional fragment thereof.

[0045] For example, wherein the cell comprises an immune cell. For example, wherein the immune cell comprises a phagocyte, a lymphocyte, a neutrophil, an eosinophil and / or a basophil. For example, wherein the immune cell comprises a monocyte, a macrophage and / or a dendritic cell.

[0046] For example, wherein the immune cell is derived from an immune cell differentiated from stem cells.For example, wherein the stem cell comprises induced pluripotent stem cells (iPSCs), embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells and / or peripheral blood stem cells.

[0047] For example, wherein the immune cells comprise B cells, T cells, natural killer cells and / or natural killer-like T cells (NKT). For example, wherein the immune cells comprise αβT cells and / or γδT cells. For example, wherein the immune cells comprise tumor infiltrating lymphocytes (TIL). For example, wherein the TIL is derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of paracancerous tissue, pleural effusion and / or ascites and / or TIL recovered after cryopreservation. For example, the volume of the fragments is about 1 cubic millimeter to about 27 cubic millimeters, such as about 1 cubic millimeter, about 2 cubic millimeters, about 3 cubic millimeters, about 4 cubic millimeters, about 5 cubic millimeters, about 6 cubic millimeters, about 7 cubic millimeters, about 8 cubic millimeters, about 9 cubic millimeters, about 10 cubic millimeters, about 11 cubic millimeters, about 12 cubic millimeters, about 13 cubic millimeters, about 14 cubic millimeters, about 15 cubic millimeters, about 16 cubic millimeters, about 17 cubic millimeters, about 18 cubic millimeters, about 19 cubic millimeters, about 20 cubic millimeters, about 21 cubic millimeters, about 22 cubic millimeters, about 23 cubic millimeters, about 24 cubic millimeters, about 25 cubic millimeters, about 26 cubic millimeters, or about 27 cubic millimeters.

[0048] For example, wherein the immune cell comprises an engineered immune receptor displayed on the cell surface. For example, wherein the engineered immune receptor specifically binds to an antigen expressed on a target cell. For example, wherein the immune cell comprises a chimeric antigen receptor and / or a T cell receptor.

[0049] For example, the peptidase C14B protein family member comprises a caspase-like domain. For example, the peptidase C14B protein family member comprises MALT1. For example, the amino acid sequence of the peptidase C14B protein family member is shown in SEQ ID NO: 1. For example, the MALT1 of the present invention may comprise a truncation of MALT1. For example, the truncation of MALT1 may comprise the immunoglobulin-like 1 domain, immunoglobulin-like 2 domain, caspase-like domain, and / or immunoglobulin-like 3 domain of MALT1. For example, the truncation of MALT1 may comprise the sequence of amino acids 1 to 809 of wild-type MALT1.

[0050] For example, the sequences mentioned herein include sequences that are at least about 80% homologous to the sequence. For example, the sequences mentioned herein include sequences that are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% homologous to the sequence.

[0051] For example, the cell expresses a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 1.

[0052] For example, the fusion protein further comprises a marker peptide. For example, the fusion protein comprises EGFR or a functional fragment thereof. For example, the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 3.

[0053] For example, the marker peptide is linked to the peptidase C14B protein family member via a cleavable peptide.

[0054] For example, the cleavable peptide can be used to connect two or more components, such as molecules or peptides. For example, the cleavable peptide can be used to connect two or more non-peptide molecules. For example, the cleavable peptide can be used to connect two or more peptide molecules. For example, the cleavable peptide can be used to connect different types of components, such as small molecules in which one or some components are non-peptide substances and another or some components are peptides. For example, the substance connected by the cleavable peptide of the present invention can be called a fusion protein or a fusion polypeptide (the two terms are used interchangeably in the present invention).

[0055] For example, the cleavable peptide comprises T2A and / or P2A, and optionally further comprises a furin peptide. For example, the furin peptide is extended based on T2A and / or P2A. When T2A and / or P2A are cleaved, the furin peptide can act as a buffer to prevent cleavage of the active ingredient peptide to which T2A and / or P2A are attached. For example, the amino acid sequence of the cleavable peptide is set forth in SEQ ID NO: 2. For example, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0056] In one aspect, the present invention provides a method for producing a cell of the present invention, comprising causing the cell to artificially express a peptidase C14B protein family member or a functional fragment thereof. For example, the method of the present invention can improve the function and / or enhance the activity of the peptidase C14B protein family member or its functional fragment in the cell. For example, the cell of the present invention can reduce the activity of TRAF6, inhibit the interaction between MALT1 and TRAF6, and / or reduce the activity of the TRAF6 binding domain of MALT1 (e.g., T6BM1, T6BM2).

[0057] In one aspect, the present invention provides a composition comprising the cells of the present invention, and optionally a pharmaceutically acceptable carrier.

[0058] In one aspect, the invention provides a kit comprising a cell of the invention and / or a composition of the invention.

[0059] In one aspect, the present invention provides a use of a cell, a composition, and / or a kit of the present invention in the preparation of a medicament for preventing, alleviating, and / or treating a disease, for example, a tumor.

[0060] In one aspect, the present invention provides a medicament for preventing and / or treating a disease and / or symptom, comprising the cell of the present invention, the composition of the present invention, and / or the kit of the present invention. For example, the disease comprises a tumor.

[0061] In one aspect, the present invention provides a method for preventing and / or treating a disease and / or symptom, comprising administering a cell of the present invention, a composition of the present invention, and / or a kit of the present invention to a subject in need thereof. For example, the disease comprises a tumor.

[0062] In one aspect, the present invention provides a cell of the present invention, a composition of the present invention, and / or a kit of the present invention for use in preventing and / or treating a disease and / or symptom. For example, the disease comprises a tumor.

[0063] In another aspect, the present invention provides an isolated cell comprising a basic helix-loop-helix (bHLH) protein family member or a functional fragment thereof.

[0064] For example, wherein the cell comprises an immune cell. For example, wherein the immune cell comprises a phagocyte, a lymphocyte, a neutrophil, an eosinophil and / or a basophil. For example, wherein the immune cell comprises a monocyte, a macrophage and / or a dendritic cell.

[0065] For example, wherein the immune cell is derived from an immune cell differentiated from stem cells.For example, wherein the stem cell comprises induced pluripotent stem cells (iPSCs), embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells and / or peripheral blood stem cells.

[0066] For example, wherein the immune cells comprise B cells, T cells, natural killer cells and / or natural killer-like T cells (NKT). For example, wherein the immune cells comprise αβT cells and / or γδT cells. For example, wherein the immune cells comprise tumor infiltrating lymphocytes (TIL). For example, wherein the TIL is derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of paracancerous tissue, pleural effusion and / or ascites and / or TIL recovered after cryopreservation. For example, the volume of the fragments is about 1 cubic millimeter to about 27 cubic millimeters, such as about 1 cubic millimeter, about 2 cubic millimeters, about 3 cubic millimeters, about 4 cubic millimeters, about 5 cubic millimeters, about 6 cubic millimeters, about 7 cubic millimeters, about 8 cubic millimeters, about 9 cubic millimeters, about 10 cubic millimeters, about 11 cubic millimeters, about 12 cubic millimeters, about 13 cubic millimeters, about 14 cubic millimeters, about 15 cubic millimeters, about 16 cubic millimeters, about 17 cubic millimeters, about 18 cubic millimeters, about 19 cubic millimeters, about 20 cubic millimeters, about 21 cubic millimeters, about 22 cubic millimeters, about 23 cubic millimeters, about 24 cubic millimeters, about 25 cubic millimeters, about 26 cubic millimeters, or about 27 cubic millimeters.

[0067] For example, wherein the immune cell comprises an engineered immune receptor displayed on the cell surface. For example, wherein the engineered immune receptor specifically binds to an antigen expressed on a target cell. For example, wherein the immune cell comprises a chimeric antigen receptor and / or a T cell receptor.

[0068] For example, the basic helix-loop-helix (bHLH) protein family member comprises a basic helix-loop-helix domain. For example, the basic helix-loop-helix (bHLH) protein family member comprises TFAP4. For example, the amino acid sequence of the basic helix-loop-helix (bHLH) protein family member is set forth in SEQ ID NO: 7. For example, the TFAP4 of the present invention may comprise a truncation of TFAP4. For example, the truncation of TFAP4 may comprise the bHLH domain, the leucine zipper 1 domain, and / or the leucine zipper 2 domain of TFAP4. For example, the truncation of TFAP4 may comprise the sequence of amino acids 48 to 179 of wild-type TFAP4. For example, the sequence is set forth in SEQ ID NO: 11. For example, the truncation of TFAP4 may comprise the sequence of amino acids 48 to 338 of wild-type TFAP4. For example, the sequence is set forth in SEQ ID NO: 12.

[0069] For example, the sequences mentioned herein include sequences that are at least about 80% homologous to the sequence. For example, the sequences mentioned herein include sequences that are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% homologous to the sequence.

[0070] For example, the cell expresses a fusion protein comprising the amino acid sequence shown in SEQ ID NO:7.

[0071] For example, the fusion protein further comprises a marker peptide. For example, the fusion protein comprises EGFR or a functional fragment thereof. For example, the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 3.

[0072] For example, the marker peptide is linked to the basic helix-loop-helix (bHLH) protein family member via a cleavable peptide.

[0073] For example, the cleavable peptide can be used to connect two or more components, such as molecules or peptides. For example, the cleavable peptide can be used to connect two or more non-peptide molecules. For example, the cleavable peptide can be used to connect two or more peptide molecules. For example, the cleavable peptide can be used to connect different types of components, such as small molecules in which one or some components are non-peptide substances and another or some components are peptides. For example, the substance connected by the cleavable peptide of the present invention can be called a fusion protein or a fusion polypeptide (the two terms are used interchangeably in the present invention).

[0074] For example, the cleavable peptide comprises T2A and / or P2A, and optionally further comprises a furin peptide. For example, the furin peptide is extended based on T2A and / or P2A. When T2A and / or P2A are cleaved, the furin peptide can act as a buffer to prevent cleavage of the active ingredient peptide to which T2A and / or P2A are attached. For example, the amino acid sequence of the cleavable peptide is set forth in SEQ ID NO: 8 and / or 2. For example, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 9 and / or 10.

[0075] In one aspect, the present invention provides a method for producing a cell of the present invention, comprising artificially expressing a basic helix-loop-helix (bHLH) protein family member or a functional fragment thereof in the cell. For example, the method of the present invention can improve the function and / or enhance the activity of a basic helix-loop-helix (bHLH) protein family member or a functional fragment thereof in the cell.

[0076] In one aspect, the present invention provides a composition comprising the cells of the present invention, and optionally a pharmaceutically acceptable carrier.

[0077] In one aspect, the invention provides a kit comprising a cell of the invention and / or a composition of the invention.

[0078] In one aspect, the present invention provides a use of a cell, a composition, and / or a kit of the present invention in the preparation of a medicament for preventing, alleviating, and / or treating a disease, for example, a tumor.

[0079] In one aspect, the present invention provides a medicament for preventing and / or treating a disease and / or symptom, comprising the cell of the present invention, the composition of the present invention, and / or the kit of the present invention. For example, the disease comprises a tumor.

[0080] In one aspect, the present invention provides a method for preventing and / or treating a disease and / or symptom, comprising administering a cell of the present invention, a composition of the present invention, and / or a kit of the present invention to a subject in need thereof. For example, the disease comprises a tumor.

[0081] In one aspect, the present invention provides a cell of the present invention, a composition of the present invention, and / or a kit of the present invention for use in preventing and / or treating a disease and / or symptom. For example, the disease comprises a tumor.

[0082] Technical Solution

[0083] An isolated cell comprising a member of the basic helix-loop-helix (bHLH) protein family or a functional fragment thereof.

[0084] The cell according to technical solution 1, wherein the cell comprises an immune cell.

[0085] The cell according to technical solution 2, wherein the immune cells comprise phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils.

[0086] The cell according to any one of technical solutions 2-3, wherein the immune cells comprise monocytes, macrophages and / or dendritic cells.

[0087] The cell according to any one of technical solutions 2-4, wherein the immune cell is derived from an immune cell differentiated from a stem cell.

[0088] The cell according to technical solution 5, wherein the stem cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells and / or peripheral blood stem cells.

[0089] The cell according to any one of technical solutions 2-6, wherein the immune cell comprises a B cell, a T cell, a natural killer cell and / or a natural killer-like T cell (NKT).

[0090] The cell according to any one of technical solutions 2 to 7, wherein the immune cells comprise αβT cells and / or γδT cells.

[0091] A cell according to any one of technical solutions 2-8, wherein the immune cells comprise tumor infiltrating lymphocytes (TIL).

[0092] The cells according to technical solution 9, wherein the TILs are TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and / or ascites and / or TILs revived after cryopreservation.

[0093] The cell according to technical solution 10, wherein the volume of the fragments is about 1 cubic millimeter to about 27 cubic millimeters.

[0094] A cell according to any one of technical solutions 2-11, wherein the immune cell comprises an engineered immune receptor displayed on the cell surface.

[0095] A cell according to technical solution 12, wherein the engineered immune receptor specifically binds to an antigen expressed on a target cell.

[0096] The cell according to any one of technical solutions 2-13, wherein the immune cell comprises a chimeric antigen receptor and / or a T cell receptor.

[0097] A cell as described in any of technical solutions 1-14, wherein the basic helix-loop-helix (bHLH) protein family member comprises a basic helix-loop-helix domain.

[0098] A cell as described in any of technical solutions 1-15, wherein the basic helix-loop-helix (bHLH) protein family member comprises TFAP4.

[0099] The cell as described in any one of technical solutions 1-16, wherein the amino acid sequence of the basic helix-loop-helix (bHLH) protein family member is shown in SEQ ID NO: 7.

[0100] A cell as described in any one of technical solutions 1-17, wherein the cell expresses a fusion protein, and the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 7.

[0101] A cell as described in technical solution 18, wherein the fusion protein further comprises a marker peptide.

[0102] A cell as described in any of technical solutions 18-19, wherein the fusion protein comprises EGFR or a functional fragment thereof.

[0103] A cell as described in any one of technical solutions 18 to 20, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 3.

[0104] A cell as described in any of technical solutions 19-21, wherein the marker peptide is linked to the basic helix-loop-helix (bHLH) protein family member via a cleavable peptide.

[0105] A cell as described in technical solution 22, wherein the cleavable peptide comprises T2A and / or P2A, and the cleavable peptide optionally further comprises a Furin peptide.

[0106] A cell as described in any one of technical solutions 22-23, wherein the amino acid sequence of the cleavable peptide is shown as SEQ ID NO: 8 and / or 2.

[0107] A cell as described in any one of technical solutions 18-24, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 9 and / or 10.

[0108] A method for producing the cell of any one of technical solutions 1-25, comprising causing the cell to artificially express the basic helix-loop-helix (bHLH) protein family member or a functional fragment thereof.

[0109] A composition comprising the cell according to any one of technical solutions 1 to 25, and optionally a pharmaceutically acceptable carrier.

[0110] A kit comprising the cells according to any one of technical solutions 1 to 25 and / or the composition according to technical solution 27.

[0111] A use of the cell according to any one of technical solutions 1 to 25, the composition according to technical solution 27, and / or the kit according to technical solution 28 in the preparation of a medicament for preventing, alleviating, and / or treating a disease (preferably a tumor).

[0112] A drug for preventing and / or treating a disease and / or symptom (preferably a tumor), comprising the cell according to any one of technical solutions 1 to 25, the composition according to technical solution 27, and / or the kit according to technical solution 28.

[0113] A method for preventing and / or treating a disease and / or symptom (preferably a tumor), comprising administering the cell of any one of Technical Solutions 1-25, the composition of Technical Solution 27, and / or the kit of Technical Solution 28 to a subject in need thereof.

[0114] The cell according to any one of technical solutions 1 to 25, the composition according to technical solution 27, and / or the kit according to technical solution 28 are used for preventing and / or treating diseases and / or symptoms (preferably tumors).

[0115] Without intending to be bound by any theory, the following examples are merely intended to illustrate the products, preparation methods and uses of the present invention, and are not intended to limit the scope of the present invention.

[0116] Example

[0117] Example 1 Tumor Infiltrating Lymphocyte (TIL) Cell Culture Method

[0118] 1.1 Tumor tissue receipt and processing

[0119] 1.1.1 Organization reception

[0120] Receive tumor tissue and blood samples from donors, verify and record sample information, and print corresponding sample labels.

[0121] 1.1.2 Tissue processing and culture

[0122] Use 75% alcohol to disinfect the sample tube and blood collection tube and transfer them to a biosafety cabinet. Isolate PBMC cells from the blood sample and freeze them according to the above-mentioned PBMC manual isolation and freezing procedures. Take a culture flask or culture bag with a breathable surface, such as a culture bag (Origen), and add 300 mL of thawed complete culture medium. The complete culture medium can be arbitrarily selected from X-vivo15 culture medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and can be supplemented with essential amino acids and antibiotics, and IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, such as 6000 IU / mL). Take several 10 cm culture dishes, add an appropriate amount of culture medium, use sterile ophthalmic forceps to remove the tumor tissue from the sample tube into a 10 cm culture dish, wash the tissue and change the culture dish. Use ophthalmic scissors and forceps to perform initial shearing, removing adipose and necrotic tissue. Each tissue block is then minced to approximately 27 cubic millimeters. A non-suspended tumor tissue block is obtained. A 20 mL syringe is used to remove the internal stopcock and connect to a culture bag. Using a pipette, approximately 1 g of tissue is transferred from the syringe into the culture bag. The culture bag is placed in a CO2 incubator for incubation. The scissors and forceps are cleaned and initially disinfected with 75% alcohol. After ultrasonic cleaning, they are sterilized to obtain the first TIL population.

[0123] 1.2 Step (A) In vitro expansion and harvesting

[0124] 1.2.1 Step (A) In vitro amplification

[0125] Depending on the cell growth status, the medium should be replenished or half-replaced every 3-7 days to ensure cell nutrition. Complete culture medium can be arbitrarily selected from X-vivo 15 culture medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and essential amino acids and antibiotics can be added, and IL-2 (double heron and / or tetracycline) at a concentration of 300-9000 IU / mL (e.g. 1000-9000 IU / mL, such as 6000 IU / mL) can be added. 3-14 days in step (A), for example, samples can be taken and counted on the 13th or 14th day. If the cell number is between 5×10 5 to 5×10 8 During this time, the harvesting step of step (A) is entered.

[0126] 1.2.2 Results of Step (A)

[0127] Collect the cells after in vitro expansion in step (A), centrifuge, discard the culture medium, wash the cells once with PBS or normal saline, obtain the TILs (second TIL population) expanded in vitro in step (A), and take samples for counting and retain about 5×10 5 to 2×10 8 cells into the subsequent in vitro expansion step; about 5×10 5 The remaining cells can be added to the cryopreservation medium and cryopreserved as cryopreserved preREP TIL in vitro cells.

[0128] 1.3 Step (B) TIL activation

[0129] Continue to culture the TILs (second TIL population) expanded in vitro in step (A), or recover the frozen preREP TIL cells in vitro and perform TIL activation in step (B).

[0130] Complete culture medium can be selected from X-vivo 15 medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi Biotech, etc. Essential amino acids and antibiotics can be added to adjust the cell density to 5×10 5 to 2×10 6Cells are suspended in a 24-well culture plate at a concentration of 1 mL / well and IL-2 is added at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, for example, 6000 IU / mL). T cell activators, such as CD3 agonists and / or CD28 agonists, can be added to the culture medium of each TIL population, for example, approximately 30 ng / mL of CD3 antibody (Miltenyi Biotech, OKT3), approximately 30 ng / mL of CD28 antibody (Merck, 15E8), magnetic beads (Dynabeads, approximately 1 to 10 μm diameter, Thermo Fisher) at a ratio of approximately 1:2-2:1 beads to TILs, and / or transACT (approximately 100 to 500 nm diameter, Miltenyi) at a ratio of approximately 1:100-1:2000 transACT to TILs. Culture the cells for approximately 0-4 days to obtain a third TIL population.

[0131] 1.4(I) TIL cells transduced with MALT1

[0132] Transduce the TIL activated in step (B) above. One day before transduction, coat a 24-well suspension culture plate with recombinant human fibrin fragment (Retronectin, Takara) at a final concentration of 15 μg / mL. Protect from light and store at 4°C overnight. Remove the coated 24-well plate, discard the coating solution, add a blocking solution containing 2% BSA and block at room temperature for 30 minutes. Discard the blocking solution, wash the plate twice with a plate washing solution containing 2.5% HEPES, and discard the plate washing solution. Each experimental group is transduced with a retrovirus carrying a nucleic acid encoding the target polypeptide of the present invention (comprising the MALT1 peptide segment shown in SEQ ID NO: 1; for expression testing only, a fusion protein comprising the MALT1 peptide segment and a marker peptide as shown in SEQ ID NO: 4 can optionally be expressed).

[0133] The retroviral plasmid construction method is as follows: synthesize a nucleic acid fragment encoding the target polypeptide of the present invention, digest it with EcoRI and NotI, and recover the exogenous fragment. Digest plasmid MP71 with EcoRI and NotI to recover the vector fragment. Ligate the exogenous fragment and the vector fragment using T4 Ligase to obtain a retroviral plasmid capable of expressing the target polypeptide of the present invention.

[0134] Add 0.25-2 mL of retroviral solution to each well and centrifuge at 32°C, 2000 g for 2 hours. The blank control group is not transduced, or the GS linker control group is transduced with the control virus. Discard the supernatant of the 24-well plate and add the TIL group activated in step 1.3 above to each well of the 24-well plate in a volume of 300-500 μL, with a cell concentration of approximately 1×106 1000 g for 10 minutes at 30-32°C. After centrifugation, the culture plate was placed in a 37°C, 5% CO2 incubator for approximately 0-4 days to obtain the transduced TIL population and the fourth TIL population.

[0135] Alternatively, the above step 1.4(I) can be optionally replaced by the following step 1.4(II):

[0136] 1.4(II) TIL cell transduction with TFAP4

[0137] Transduce the activated TILs from step (B) above. One day prior to transduction, coat a 24-well suspension culture plate with recombinant human fibrin fragment (Retronectin, Takara) at a final concentration of 15 μg / mL. Protect from light and incubate at 4°C overnight. Remove the coated 24-well plate, discard the coating solution, and add a blocking solution containing 2% BSA to block the plate at room temperature for 30 minutes. Discard the blocking solution, wash the plate twice with a plate washer containing 2.5% HEPES, and discard the plate washer solution. Each experimental group was transduced with a retrovirus carrying a nucleic acid encoding the target polypeptide of the present invention (comprising the TFAP4 peptide segment shown in SEQ ID NO: 7; for expression testing only, a fusion protein comprising the TFAP4 peptide segment and a marker protein as shown in SEQ ID NO: 9 or 10 can optionally be expressed).

[0138] The retroviral plasmid construction method is as follows: synthesize a nucleic acid fragment encoding the target polypeptide of the present invention, digest it with EcoRI and NotI, and recover the exogenous fragment. Digest plasmid MP71 with EcoRI and NotI to recover the vector fragment. Ligate the exogenous fragment and the vector fragment using T4 Ligase to obtain a retroviral plasmid capable of expressing the target polypeptide of the present invention.

[0139] Add 0.25-2 mL of retroviral solution to each well and centrifuge at 32°C, 2000 g for 2 hours. The blank control group is not transduced, or the GS linker control group is transduced with the control virus. Discard the supernatant of the 24-well plate and add the TIL group activated in step 1.3 above to each well of the 24-well plate in a volume of 300-500 μL, with a cell concentration of approximately 1×10 6 1000 g for 10 minutes at 30-32°C. After centrifugation, the culture plate was placed in a 37°C, 5% CO2 incubator for approximately 0-4 days to obtain the transduced TIL population and the fourth TIL population.

[0140] 1.5 Step (D) TIL cell culture after transduction

[0141] Feeder cells (irradiated healthy donor PBMC T cells) are added to the fourth TIL cell population for culture. The time for contacting TIL with feeder cells needs to be several times T after the TIL is contacted with IL-2 and T cell activator (such as CD3 antibody or a nanomatrix containing CD3 antibody and CD28 antibody, such as transACT) in step (B). n Afterwards (T n The time period can be from 0 hours to 12 days, for example, 24 hours or 48 hours. First, resuscitate the mixed feeder cells from 1-5 donors; mix the activated TIL cells and feeder cells at a ratio of approximately 1:200, transfer them to a G-Rex100 culture flask or breathable bag, and supplement with complete culture medium. Samples are taken and counted every 1-3 days, and the medium is replenished or half-filled depending on the cell status until the total number of cells is greater than 1×10 9 Alternatively, the in vitro expansion culture of step (D) is carried out for about 5 days to about 14 days, and the in vitro expansion culture of step (D) is terminated.

[0142] 1.6 Harvesting of Tumor-Infiltrating Lymphocytes

[0143] Take the cells amplified in step (D), centrifuge and discard the culture supernatant, and wash three times with PBS or saline or compound electrolyte solution to obtain TILs amplified in step (D) (fifth TIL population). Samples are counted during the third wash. According to the counting results, after the final centrifugation, discard the supernatant and take 3×10 6 The cells were sent for quality control testing; all the remaining cells were used as the final cell product. Optional freezing medium can be added to adjust the cell density to 1-3×10 8 cells / mL for cryopreservation.

[0144] Example 2 TCR T cell preparation

[0145] T cells from various sources such as peripheral blood and stem cell differentiation, preferably PBMC cells, are cultured. A nucleic acid encoding a TCR (e.g., a TCR targeting NY-ESO-1) is transferred into the above-mentioned T cells according to methods known in the art, such as viral transduction, LNP transduction, electroporation, etc., thereby obtaining TCR-T cells. Before or after the TCR is transduced into the above-mentioned T cells, each test group is transduced with a retrovirus carrying a nucleic acid encoding the target polypeptide of the present invention to obtain TCR T cells transduced with the target polypeptide of the present invention. T cells that are not transduced with the target polypeptide are used as a blank control group (denoted as: Control).

[0146] Functional assay of cells transduced with MALT1

[0147] Example 3 Cell transduction efficiency detection

[0148] Referring to Example 1 of the present application, the MALT1-transduced TIL population obtained on the 7th or 8th day after gene editing was used to detect cytokine expression by flow cytometry.

[0149] Testing steps:

[0150] TILs from each experimental group were centrifuged in a 96-well V-bottom plate. Cell surface staining was performed using a cocktail of antibodies (BV421 anti-CD3, Biolegend 317344; PE anti-CD4, Biolegend 300508; FITC anti-CD8, Biolegend 344704; APC anti-EGFR, Biolegend 352906) at a 1:100 dilution. Cells were resuspended in 100 μL / well of the antibody cocktail and incubated at 2-8°C in the dark for 30 minutes. Following staining, cells were washed with an appropriate amount of PBS and centrifuged at 600 g for 3 minutes. The supernatant was discarded. Cells were resuspended in 100 μL / well of PBS and analyzed by flow cytometry.

[0151] Figures 1A-1B show the transduction results of TIL cells derived from different donors. The gray unfilled dashed line, gray unfilled solid line, and black filled solid line in Figures 1A and 1B represent the fluorescence subtraction control (FMO), the blank control (untransduced with the target peptide), and TIL expressing MALT1 (SEQ ID NO: 4), respectively. Figure 1A shows TIL derived from a cervical tumor, and Figure 1B shows TIL derived from a melanoma tumor.

[0152] By detecting the staining results of the EGFR tag, the results showed that the EGFR tag of the TIL cells from the two donors without gene editing was negative, while the EGFR tag of the TIL cells from the two donors after gene editing was partially positive. Because the target polypeptide of the present invention and EGFR are encoded by the same plasmid and are located upstream of EGFR, the present invention successfully expressed the target polypeptide of the present invention in TIL cells.

[0153] Example 4 Cell Killing Ability Detection

[0154] The MALT1-transduced TIL population obtained on the 7th or 8th day after gene editing was tested for its ability to kill tumor cells.

[0155] Exemplary tumor organoid culture method: Tumor tissue is obtained from surgery, transported and stored at 2-8°C after ex vivo, mechanically dissociated within 24 hours, and minced into approximately 0.5 mm 3The organoids were then digested at 37°C using a dedicated tissue digestion solution (bioGenous) and terminated with 10% FBS. The tissue suspension was filtered through a 100μm filter, washed, and thoroughly mixed with Martrigel on ice. The mixture was then applied to the bottom of a cell culture plate. The plate was placed in a 37°C incubator. After the Martrigel fully solidified, complete culture medium was carefully added for incubation. Once the organoids reached sufficient size and density, they were passaged or frozen to obtain a PDO (patient-derived organoid) model.

[0156] Use the tip of a pipette to scrape the Matrigel and organoid mixture and transfer it to a centrifuge tube containing basal medium. Pipet to separate the organoids and Matrigel. After centrifugation, add tissue digestion buffer for passage, mix well, and place in a 37°C incubator for digestion. Add basal medium to dilute the digestion buffer and wash by centrifugation. Take a small amount of the cell suspension and add 8 times the volume of digestion buffer to digest to single cells. Stop the reaction with FBS and count. Based on the count results, aspirate the required volume of organoids and centrifuge and resuspend them in T cell culture medium. Count the cells to be tested (e.g., TIL cells or TCR-T cells) and resuspend the cells to be tested to the desired density at different effector-target ratios. Add 100 μl of PDO target cells and test cells to a flat-bottom 96-well plate, and set up three replicates for each group for PDO co-culture testing. Set up several different effector-target ratios. At the same time, set up a group containing only PDO target cells and a control group (NT) for co-culturing unedited test cells with PDO target cells.

[0157] Testing steps:

[0158] Tumor target cells were plated in a 96-well flat-bottom plate one day in advance. The next day, each group of TIL cells were co-cultured with target cells at an effector-target ratio of 1:1, with 100 μL of target cells and TIL cells respectively. Three replicate wells were set up for each group, and a control group containing only target cells was also set up.

[0159] According to the instructions of the apoptosis detection reagent (SuperViewTM 488 Caspase-3 Assay Kit, UEL S6007L), the apoptosis detection reagent was added at 1.5 μL / well and diluted with culture medium at 18.5 μL / well. The activity of Caspase 3 was recorded using an Incucyte recorder (Sartorius) to analyze the killing ability of TIL cells on target cells. The activity was recorded every 3 hours for a total of approximately 5 days.

[0160] Figures 2A-2B show the results of the cytotoxicity test of TIL cells transduced with the target polypeptide of the present invention. The significance tests in the figures are compared with untransduced TIL cells. Figures 2A-2B show the cytotoxicity curves of TIL cells against A375 cells based on Caspase 3 activity.

[0161] The results showed that compared with the non-transduced control group, the TIL cells transduced with the target polypeptide of the present invention had more significant target cell killing ability.

[0162] Example 5: Detection of the Proliferation Ability of TIL

[0163] The MALT1-transduced TIL population obtained in step 1.5 of Example 1 above was plated onto 96-well plates after transduction and expansion. Untransduced T cells served as a blank control group. For the stimulated groups, stimulation was performed using TransACT (diameter approximately 100 to 500 nm, Miltenyi). The unstimulated group was labeled "medium," and the stimulated group was labeled "transact." After 2-3 days of stimulation, cell proliferation in each group was analyzed using a CTG kit, using the same assay as in Example 4.

[0164] The results showed that cells expressing the target polypeptide of the present invention can have better proliferation ability.

[0165] Example 6 Detection of cytokine secretion ability of TIL

[0166] The MALT1-transduced TIL population obtained in step 1.5 of Example 1 was plated in a 96-well plate after transduction and amplification. The supernatant was collected 24-48 hours later and cytokine secretion was analyzed using a CBA kit (BD) according to the instructions.

[0167] The results showed that cells expressing the target polypeptide of the present invention can have better cytokine secretion ability.

[0168] Example 7 Detection of the proliferation ability of TCRT cells

[0169] Anti-CD3 antibodies (e.g., OKT3) were plated in 96-well plates (those plated with antibodies were marked as CD3 coating, and those not plated with antibodies were marked as medium), incubated at 37 degrees Celsius for 2 hours, and then the MALT1-transduced PBMC-TCR T cells obtained in the above example were plated in 96-well plates after transduction and amplification. After 3 days, each group was analyzed for cell proliferation using a CTG kit. According to the instructions of the CTG kit (CellTiter-Glo Luminescent Cell Viability Assay, Promega), CTG substrate (CellTiter-Glo Substrate) and CTG buffer (CellTiter-Glo Buffer) were mixed to prepare a CTG reaction solution. The test cell suspension was added to a 96-well microplate at 50 μL / well, and a well containing only culture medium was set as the background value of fluorescence. An equal volume of CTG reaction solution was added to each well, shaken on a horizontal shaker for 2 minutes, and allowed to stand at room temperature for 10 minutes to stabilize the fluorescence signal before reading the fluorescence value.

[0170] The results showed that cells expressing the target polypeptide of the present invention can have better proliferation ability.

[0171] Example 8 Detection of cytokine secretion ability of TCRT cells

[0172] Anti-CD3 was plated in a 96-well plate and incubated at 37°C for 2 hours. Then, the MALT1-transduced PBMC-TCR T cells obtained in the above example were plated in a 96-well plate after transduction and expansion. The supernatant was collected after 24-48 hours and the secretion of cytokines IL-2, TNF, and IFN-γ was analyzed using a Cytometric Bead Array (CBA) kit (BD).

[0173] Refer to the cytokine detection kit instructions for specific methods. Specifically, reconstitute the lyophilized powder of the human Th1 / Th2 / Th17 cytokine standards with 2 mL of Assay Diluent (the standard stock solution contains 5000 pg / mL of each cytokine) and serially dilute in the following order: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, and 1:1024. Label this tube as the "Standard Tube." Remove one tube containing only Assay Diluent as a reference. Add 2 μL / Beads / well of each Capture Beads (BD), then add 10 μL / well of PE Detection Reagent and mix to prepare a mixture. Add 22 μL / well to a V-bottom 96-well plate, then add 10 μL / well of each standard and experimental group supernatant, mix, and incubate at room temperature in the dark for 3 hours. After incubation, add 200 μL Wash Buffer (BD) to each well and centrifuge at 500 g for 3 minutes. After centrifugation, add 100 μL Wash Buffer (BD) to each well and resuspend the cells for flow cytometry analysis.

[0174] The results showed that cells expressing the target polypeptide of the present invention can have enhanced cytokine secretion capacity.

[0175] Example 9 Detection of Tumor Cell Killing Ability of TCRT Cells

[0176] GFP-labeled A375 tumor cells (A375-GFP) were plated in 96-well plates. After the cells adhered, PBMC-TCR T cells transduced with MALT1 obtained in the above example were added to each well for co-culture. According to the instructions of the apoptosis detection reagent (Incucyte Caspase-3 / 7 Green Dye for Apoptosis, Sartorius), 0.2 μL / well was added. Caspase-3 / 7 Green Dye for Apoptosis was added to the wells at a rate of 25 μL / well. Caspase 3 / 7 Green Dye was diluted with culture medium. Caspase 3 / 7 activity was recorded using an Incucyte recorder (Sartorius) every 3 hours for approximately 3 days to analyze tumor cell cytotoxicity.

[0177] The results showed that cells expressing the target polypeptide of the present invention can have enhanced tumor cell killing ability.

[0178] Example 10 In vivo drug efficacy detection

[0179] The non-small cell lung cancer cell line HCC827 was revived under sterile conditions and stably expanded in vitro for 3-5 passages until the cells reached the logarithmic growth phase. The cells were harvested and counted to adjust the cell density. HCC827 cells were subcutaneously inoculated into NOG mice (Vitamin B, strain code 408) in an SPF animal room at a rate of 2.3E6 tumor cells per mouse. Tumor size was measured regularly until the tumor grew to approximately 50 mm. 3 Afterwards, the mice were randomly divided into groups.

[0180] The NT group served as a negative control, and mice were intravenously injected with non-gene-edited TIL cells; mice in each experimental group were intravenously injected with the gene-edited TIL cells of the present invention, and received adjuvant therapy with intraperitoneal injection of IL-2.

[0181] Figure 3 shows that the experimental group of mice supported by a low dose of IL-2 achieved superior tumor control compared to the NT group, which received a 3-fold higher dose of IL-2. Specifically, TILs expressing MALT1 (SEQ ID NO: 4) in the MALT1 group significantly inhibited tumor growth compared to the NT group (P = 0.037 (2-way ANOVA). This demonstrates that the MALT1-overexpressing cells of the present invention exhibit significantly greater tumor suppression than the NT group.

[0182] Functional assay of cells transduced with TFAP4

[0183] Example 11 Cell transduction efficiency detection

[0184] Referring to Example 1 of the present application, the TFAP4-transduced TIL population obtained on the 7th or 8th day after gene editing was used to detect cytokine expression by flow cytometry.

[0185] Testing steps:

[0186] TILs from each experimental group were centrifuged in a 96-well V-bottom plate. Cell surface staining was performed using a cocktail of antibodies (BV421 anti-CD3, Biolegend 317344; PE anti-CD4, Biolegend 300508; FITC anti-CD8, Biolegend 344704; APC anti-EGFR, Biolegend 352906) at a 1:100 dilution. Cells were resuspended in 100 μL / well of the antibody cocktail and incubated at 2-8°C in the dark for 30 minutes. Following staining, cells were washed with an appropriate amount of PBS and centrifuged at 600 g for 3 minutes. The supernatant was discarded. Cells were resuspended in 100 μL / well of PBS and analyzed by flow cytometry.

[0187] Figures 4A-1C show the transduction results of TIL cells derived from different donors. In Figure 4A, the gray unfilled dashed line, gray unfilled solid line, black filled dashed line, and black filled solid line represent the fluorescence subtraction control FMO, a control blank not transduced with the target polypeptide, TIL expressing TFAP4-1 (SEQ ID NO: 9), and TIL expressing TFAP4-2 (SEQ ID NO: 10), respectively. Figure 4A shows TIL derived from cervical tumors, Figure 4B shows TIL derived from lung tumors, and Figure 4C shows TIL derived from lung tumors. The gray unfilled solid line and black unfilled solid line in Figures 4B and 4C represent the control blank not transduced with the target polypeptide and TIL expressing TFAP4-2 (SEQ ID NO: 10), respectively.

[0188] By detecting the staining results of the EGFR tag, the results showed that the EGFR tag of the TIL cells from the two donors without gene editing was negative, while the EGFR tag of the TIL cells from the two donors after gene editing was partially positive. Because the target polypeptide of the present invention and EGFR are encoded by the same plasmid and are located upstream of EGFR, the present invention successfully expressed the target polypeptide of the present invention in TIL cells.

[0189] Example 12 Cell Killing Ability Detection

[0190] The TFAP4-transduced TIL population obtained on the 7th or 8th day after gene editing was tested for its ability to kill tumor cells.

[0191] Exemplary tumor organoid culture method: Tumor tissue is obtained from surgery, transported and stored at 2-8°C after ex vivo, mechanically dissociated within 24 hours, and minced into approximately 0.5 mm 3 The organoids were then digested at 37°C using a dedicated tissue digestion solution (bioGenous) and terminated with 10% FBS. The tissue suspension was filtered through a 100μm filter, washed, and thoroughly mixed with Martrigel on ice. The mixture was then applied to the bottom of a cell culture plate. The plate was placed in a 37°C incubator. After the Martrigel fully solidified, complete culture medium was carefully added for incubation. Once the organoids reached sufficient size and density, they were passaged or frozen to obtain a PDO (patient-derived organoid) model.

[0192] Use the tip of a pipette to scrape the Matrigel and organoid mixture and transfer it to a centrifuge tube containing basal medium. Pipet to separate the organoids and Matrigel. After centrifugation, add tissue digestion buffer for passage, mix well, and place in a 37°C incubator for digestion. Add basal medium to dilute the digestion buffer and wash by centrifugation. Take a small amount of the cell suspension and add 8 times the volume of digestion buffer to digest to single cells. Stop the reaction with FBS and count. Based on the count results, aspirate the required volume of organoids and centrifuge and resuspend them in T cell culture medium. Count the cells to be tested (e.g., TIL cells or TCR-T cells) and resuspend the cells to be tested to the desired density at different effector-target ratios. Add 100 μl of PDO target cells and test cells to a flat-bottom 96-well plate, and set up three replicates for each group for PDO co-culture testing. Set up several different effector-target ratios. At the same time, set up a group containing only PDO target cells and a control group (NT) for co-culturing unedited test cells with PDO target cells.

[0193] Testing steps:

[0194] Tumor target cells were plated in a 96-well flat-bottom plate one day in advance. The next day, each group of TIL cells were co-cultured with target cells at an effector-target ratio of 1:1, with 100 μL of target cells and TIL cells respectively. Three replicate wells were set up for each group, and a control group containing only target cells was also set up.

[0195] According to the instructions of the apoptosis detection reagent (SuperViewTM 488 Caspase-3 Assay Kit, UEL S6007L), the apoptosis detection reagent was added at 1.5 μL / well and diluted with culture medium at 18.5 μL / well. The activity of Caspase 3 was recorded using an Incucyte recorder (Sartorius) to analyze the killing ability of TIL cells on target cells. The activity was recorded every 3 hours for a total of approximately 5 days.

[0196] Figures 5A-2D show the results of the killing ability test of TIL cells transduced with the target polypeptide of the present invention. The significance tests in the figure are all compared with untransduced TIL. Figures 5A-2B are killing curves made based on the activity of Caspase 3 in the killing test of TIL cells against A375 (Figure 5A) or Caski (Figure 5B). Figures 5C-2D are killing curves made based on the activity of Caspase 3 in the killing test of TIL cells derived from tumor tissue in situ (Figure 5C) or lymph node (Figure 5D) against patient-derived tumor organoids.

[0197] The results showed that compared with the non-transduced control group, TIL cells transduced with the target polypeptide of the present invention had more significant target cell killing ability against A375 and Caski cells, as well as Caski cells and patient-derived tumor organoids.

[0198] Example 13: Detection of the Proliferation Ability of TIL

[0199] The TFAP4-transduced TIL population obtained in step 1.5 of Example 1 above was plated onto 96-well plates after transduction and expansion. Untransduced T cells served as a blank control group. For the stimulated groups, stimulation was performed with the addition of TransACT (diameter approximately 100 to 500 nm, Miltenyi). The unstimulated group was labeled "medium," and the stimulated group was labeled "transact." After 2-3 days of stimulation, cell proliferation in each group was analyzed using a CTG kit, using the same assay method as in Example 4.

[0200] The results showed that cells expressing the target polypeptide of the present invention can have better proliferation ability.

[0201] Example 14 Detection of cytokine secretion ability of TIL

[0202] The TFAP4-transduced TIL population obtained in step 1.5 of Example 1 was plated in a 96-well plate after transduction and amplification. The supernatant was collected 24-48 hours later and cytokine secretion was analyzed using a CBA kit (BD) according to the instructions.

[0203] The results showed that cells expressing the target polypeptide of the present invention can have better cytokine secretion ability.

[0204] Example 15 Detection of the proliferation ability of TCRT cells

[0205] Anti-CD3 antibodies (e.g., OKT3) were plated in 96-well plates (those plated with antibodies were marked as CD3 coating, and those not plated with antibodies were marked as medium), incubated at 37 degrees Celsius for 2 hours, and then the PBMC-TCR T cells transduced with TFAP4 obtained in the above example were plated in 96-well plates after transduction and amplification. After 3 days, each group was analyzed for cell proliferation using a CTG kit. According to the instructions of the CTG kit (CellTiter-Glo Luminescent Cell Viability Assay, Promega), CTG substrate (CellTiter-Glo Substrate) and CTG buffer (CellTiter-Glo Buffer) were mixed to prepare a CTG reaction solution. The test cell suspension was added to a 96-well microplate at 50 μL / well, and a well containing only culture medium was set as the background value of fluorescence. An equal volume of CTG reaction solution was added to each well, shaken on a horizontal shaker for 2 minutes, and allowed to stand at room temperature for 10 minutes to stabilize the fluorescence signal before reading the fluorescence value.

[0206] The results showed that cells expressing the target polypeptide of the present invention can have better proliferation ability.

[0207] Example 16 Detection of cytokine secretion ability of TCRT cells

[0208] Anti-CD3 was plated in a 96-well plate and incubated at 37°C for 2 hours. Then, the PBMC-TCR T cells transduced with TFAP4 obtained in the above example were plated in a 96-well plate after transduction and expansion. After 24-48 hours, the supernatant was collected and the secretion of cytokines IL-2, TNF, and IFN-γ was analyzed using a Cytometric Bead Array (CBA) kit (BD).

[0209] Refer to the cytokine detection kit instructions for specific methods. Specifically, reconstitute the lyophilized powder of the human Th1 / Th2 / Th17 cytokine standards with 2 mL of Assay Diluent (the standard stock solution contains 5000 pg / mL of each cytokine) and serially dilute in the following order: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, and 1:1024. Label this tube as the "Standard Tube." Remove one tube containing only Assay Diluent as a reference. Add 2 μL / Beads / well of each Capture Beads (BD), then add 10 μL / well of PE Detection Reagent and mix to prepare a mixture. Add 22 μL / well to a V-bottom 96-well plate, then add 10 μL / well of each standard and experimental group supernatant, mix, and incubate at room temperature in the dark for 3 hours. After incubation, add 200 μL Wash Buffer (BD) to each well and centrifuge at 500 g for 3 minutes. After centrifugation, add 100 μL Wash Buffer (BD) to each well and resuspend the cells for flow cytometry analysis.

[0210] The results showed that cells expressing the target polypeptide of the present invention can have enhanced cytokine secretion capacity.

[0211] Example 17 Detection of Tumor Cell Killing Ability of TCRT Cells

[0212] GFP-labeled A375 tumor cells (A375-GFP) were plated in 96-well plates. After the cells adhered, PBMC-TCR T cells transduced with TFAP4 obtained in the above example were added to each well for co-culture. According to the instructions of the apoptosis detection reagent (Incucyte Caspase-3 / 7 Green Dye for Apoptosis, Sartorius), 0.2 μL / well was added. Caspase-3 / 7 Green Dye for Apoptosis was added to the wells at a rate of 25 μL / well. Caspase 3 / 7 Green Dye was diluted with culture medium. Caspase 3 / 7 activity was recorded using an Incucyte recorder (Sartorius) every 3 hours for approximately 3 days to analyze tumor cell cytotoxicity.

[0213] The results showed that cells expressing the target polypeptide of the present invention can have enhanced tumor cell killing ability.

[0214] Example 18 In vivo drug efficacy detection

[0215] The non-small cell lung cancer cell line HCC827 was revived under sterile conditions and stably expanded in vitro for 3-5 passages until the cells reached the logarithmic growth phase. The cells were harvested and counted to adjust the cell density. HCC827 cells were subcutaneously inoculated into NOG mice (Vitamin B, strain code 408) in an SPF animal room at a rate of 2.3E6 tumor cells per mouse. Tumor size was measured regularly until the tumor grew to approximately 50 mm. 3 Afterwards, the mice were randomly divided into groups.

[0216] The NT group served as a negative control, and mice were intravenously injected with non-gene-edited TIL cells; mice in each experimental group were intravenously injected with the gene-edited TIL cells of the present invention, and received adjuvant therapy with intraperitoneal injection of IL-2.

[0217] Figure 6 shows that the experimental group of mice supported by a low dose of IL-2 achieved superior tumor control compared to the NT group supported by a 3-fold higher dose of IL-2. Specifically, the TFAP4 group, which contained TIL expressing TFAP4-2 (SEQ ID NO: 10), significantly inhibited tumor growth on day 13 compared to the NT group (P = 0.032 (t tests). This demonstrates that the TFAP4-overexpressing cells of the present invention exhibit significantly greater tumor suppression than the NT group.

[0218] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments of the present invention will be apparent to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

[0219] Sequence information of the present invention

[0220] The underlined amino acids represent the linker portion.

Claims

1. An isolated cell comprising a member of the peptidase C14B protein family or a functional fragment thereof.

2. The cell of claim 1, wherein the cell comprises an immune cell.

3. The cell according to claim 2, wherein the immune cell comprises a phagocyte, a lymphocyte, a neutrophil, an eosinophil and / or a basophil.

4. The cell according to any one of claims 2-3, wherein the immune cell comprises a monocyte, a macrophage and / or a dendritic cell.

5. The cell according to any one of claims 2 to 4, wherein the immune cell is derived from an immune cell differentiated from a stem cell.

6. The cell according to claim 5, wherein the stem cell comprises an induced pluripotent stem cell (iPSC), an embryonic stem cell, a bone marrow stem cell, an umbilical cord blood stem cell and / or a peripheral blood stem cell.

7. The cell according to any one of claims 2-6, wherein the immune cell comprises a B cell, a T cell, a natural killer cell and / or a natural killer-like T cell (NKT).

8. The cell according to any one of claims 2-7, wherein the immune cell comprises an αβ T cell and / or a γδ T cell.

9. The cell of any one of claims 2-8, wherein the immune cell comprises a tumor infiltrating lymphocyte (TIL).

10. The cell according to claim 9, wherein the TIL is TIL derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and / or peritoneal effusion and / or TIL derived from cryopreservation and resuscitation.

11. The cells of claim 10, wherein the volume of the fragments is about 1 cubic millimeter to about 27 cubic millimeters.

12. The cell of any one of claims 2-11, wherein the immune cell comprises an engineered immune receptor displayed on the cell surface.

13. The cell of claim 12, wherein the engineered immune receptor specifically binds to an antigen expressed on a target cell.

14. The cell according to any one of claims 2-13, wherein the immune cell comprises a chimeric antigen receptor and / or a T cell receptor.

15. The cell of any one of claims 1-14, wherein the peptidase C14B family member comprises a caspase-like domain.

16. The cell of any one of claims 1-15, wherein the peptidase C14B family member comprises MALT1.

17. The cell of any one of claims 1-16, wherein the amino acid sequence of the peptidase C14B family member is shown in SEQ ID NO:

1.

18. The cell according to any one of claims 1 to 17, wherein the cell expresses a fusion protein, and the fusion protein comprises the amino acid sequence shown in SEQ ID NO:

1.

19. The cell of claim 18, wherein the fusion protein further comprises a marker peptide.

20. The cell of any one of claims 18-19, wherein the fusion protein comprises EGFR or a functional fragment thereof.

21. The cell of any one of claims 18-20, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:

3.

22. The cell of any one of claims 19-21, wherein the marker peptide is linked to the peptidase C14B family member via a cleavable peptide.

23. The cell of claim 22, wherein the cleavable peptide comprises T2A and / or P2A, and the cleavable peptide optionally further comprises a Furin peptide.

24. The cell of any one of claims 22-23, wherein the amino acid sequence of the cleavable peptide is as shown in SEQ ID NO:

2.

25. The cell of any one of claims 18-24, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:

4.

26. A method for producing the cell of any one of claims 1 to 25, comprising causing the cell to artificially express the peptidase C14B family member or a functional fragment thereof.

27. A composition comprising the cell of any one of claims 1-25, and optionally a pharmaceutically acceptable carrier.

28. A kit comprising the cell of any one of claims 1-25 and / or the composition of claim 27.

29. Use of the cell according to any one of claims 1 to 25, the composition according to claim 27 and / or the kit according to claim 28 in the preparation of a medicament for preventing, alleviating and / or treating a disease (preferably a tumor).

30. A medicament for preventing and / or treating a disease and / or symptom (preferably a tumor), comprising the cell according to any one of claims 1 to 25, the composition according to claim 27 and / or the kit according to claim 28.

31. A method for preventing and / or treating a disease and / or symptom (preferably a tumor), comprising administering the cell of any one of claims 1 to 25, the composition of claim 27, and / or the kit of claim 28 to a subject in need thereof.

32. The cell according to any one of claims 1 to 25, the composition according to claim 27 and / or the kit according to claim 28, for use in preventing and / or treating a disease and / or symptom (preferably a tumor).

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