Preparation method for and use of immune cells

Abstract

The present invention relates to the field of biomedicine, and specifically provides a preparation method for and a use of immune cells. Immune cells obtained using the preparation method of the present invention can increase or promote the proliferative capability, cytotoxic ability, and / or exogenous nucleic acid expression level of immune cells.

Description

A method for preparing immune cells and its use Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for preparing immune cells and uses thereof. Background Art

[0002] Natural killer cells (NKT) are a special type of T lymphocyte subset that has the dual properties of T cells and NK cells. NKT cells can express two receptors, TCR of T cells and NKR-P1 of NK cells. Under the mediation of TCR and NKR, NKT cells can produce a large number of cytokines. Among them, the T cell receptor (TCR) mediates the recognition of peptide epitopes bound to major histocompatibility complex (MHC) molecules to exert adaptive cellular immunity. Normally, the half-life of infused NKT cells in the patient's body is about 2 weeks, the effective period is short, and repeated infusions are required. In addition, NKT cells themselves lack specific antibodies and are not enough to be enriched around tumors or in tumor nests, which restricts the targeted effect of NKT cell treatment. On the other hand, NKTs derived from peripheral blood are relatively rare, accounting for only 0.1-0.5%, and are not easy to use for cell therapy.

[0003] Therefore, there is an urgent need in the art for a method for preparing NKT cells to obtain a large number of NKT cells and to engineer the NKT cells to obtain stably engineered NKT cells that can be used for cell therapy. Summary of the Invention

[0004] The present invention provides a method for preparing immune cells and uses thereof, which may have one or more of the following advantages: improving or promoting the proliferation ability, killing ability, and / or the expression level of exogenous nucleic acids in the immune cells.

[0005] In one aspect, the present invention provides a method for preparing immune cells, comprising causing natural killer T cells derived from stem cells or progenitor cells to express chimeric antigen receptors and / or cytokines.

[0006] In another aspect, the present invention provides an immune cell obtained by the preparation method of the present invention.

[0007] In another aspect, the present invention provides a method for influencing tumor cell growth, comprising administering the immune cell of the present invention.

[0008] In another aspect, the present invention provides use of the immune cells of the present invention in the preparation of a medicament for preventing and / or treating a disease and / or symptom.

[0009] In another aspect, the present invention provides the immune cell of the present invention for use in preventing and / or treating a disease and / or symptom.

[0010] In another aspect, the present invention provides a method for preventing and / or treating a disease and / or symptom, comprising administering the immune cell of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] 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 accompanying drawings are briefly described as follows:

[0012] FIG1 shows the killing results of effector cells in each group.

[0013] Figures 2A-2B show the flow cytometry expression results of iTCR, CD3, IL-15Ra and CAR in each group of cells before and after cell killing assay. DETAILED DESCRIPTION

[0014] The following describes the embodiments of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0015] Definition of terms

[0016] In the present invention, the term "stem cell" generally refers to a cell that is capable of self-replication and pluripotency or multipotency. The stem cell of the present invention can be, but is not limited to, an embryonic stem (ES) cell, an induced pluripotent stem cell, or a tissue stem cell (also referred to as a tissue-specific stem cell or somatic stem cell). "Induced pluripotent stem cells," usually abbreviated as iPS cells or iPSCs, refer to a class of pluripotent stem cells artificially prepared from non-pluripotent cells by introducing certain factors (called reprogramming factors), and non-pluripotent cells are generally adult cells or terminally differentiated cells, such as fibroblasts, hematopoietic cells, muscle cells, neurons, epidermal cells, etc.

[0017] In the present invention, the term "hematopoietic stem and progenitor cells" or "hematopoietic precursor cells" generally refers to cells that are characterized by the hematopoietic lineage but are capable of further hematopoietic differentiation. Hematopoietic stem cells, multipotent hematopoietic stem cells (hematopoietic cells), bone marrow progenitor cells, megakaryocyte progenitor cells, erythrocyte progenitor cells and lymphocyte progenitor cells can be included. "Hematopoietic stem cells (HSC)" are generally multipotent stem cells that produce all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells). In the present invention, HSC can include "hematopoietic stem and progenitor cells" or "hematopoietic precursor cells" or a combination of the above.

[0018] Hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) are a type of stem cell and / or progenitor cell, typically isolated from umbilical cord blood or G-CSF-mobilized peripheral blood, or derived from embryonic stem cells or induced pluripotent stem cells (ES-HSCs or iPS-HSCs). Hematopoietic stem cells and progenitor cells can express CD34.

[0019] In the present invention, the term "natural killer T cell" generally refers to a T cell or T cell population that exhibits both conventional T cell characteristics and natural killer (NK) cell characteristics. For example, in some embodiments, NKT cells are mature lymphocytes that have both T cell receptors and NK cell receptors. The term "invariant NKT cell" or "iNKT cell" refers to an NKT cell or NKT cell population that expresses an invariant or semi-invariant TCR repertoire and binds to the glycosphingolipid α-galactosylceramide (α-GalCer) associated with class I MHC-like CD1d molecules.

[0020] In the present invention, the term "cytokine" generally refers to a substance secreted by a cell that has a variety of effects on other cells. Cytokines act by binding to their cell-specific receptors located on the cell membrane, allowing a unique signal transduction cascade to be initiated within the cell, ultimately leading to biochemical and phenotypic changes in the target cell. Cytokines can act locally and away from the release site. They include type I cytokines, which cover many interleukins and several hematopoietic growth factors; type II cytokines, including interferons and interleukin-10; tumor necrosis factor ("TNF") related molecules, including TNFα and lymphotoxin; members of the immunoglobulin superfamily, including interleukin 1 ("IL-1"); and chemokines, a family of molecules that play a key role in a variety of immune and inflammatory functions. Depending on the state of the cell, the same cytokine can have different effects on the cell. Cytokines often regulate the expression of other cytokines and trigger cascade reactions of other cytokines. Non-limiting examples of cytokines include, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 / IL-23P4 0. IL13, IL-15, IL-15 / IL15-RA, IL-17, IL-18, IL-21, IL-23, TGF-β, IFNγ, GM-CSF, Groα, MCP-1 and TNF-α.

[0021] In the present invention, the terms "IL-15" and "interleukin-15" generally refer to wild-type IL-15 or IL-15 derivatives. As used herein, the terms "wild-type IL-15" and "wild-type interleukin-15" in the context of a protein or polypeptide refer to any mammalian interleukin-15 amino acid sequence, including immature or precursor forms and mature forms. Non-limiting examples of GeneBank accession numbers for amino acid sequences of wild-type mammalian interleukin-15 from various species include NP_000576 (human). In some embodiments, IL-15 is an immature or precursor form of mammalian IL-15. In other embodiments, IL-15 is a mature form of mammalian IL-15. In a specific embodiment, IL-15 is a precursor form of human IL-15. In another embodiment, IL-15 is a mature form of human IL-15. In one embodiment, the IL-15 protein / polypeptide is isolated or purified. For example, the target cell is caused to express membrane-bound IL-15. For example, the target cells are caused to express a recombinant sequence comprising a fusion of an IL-15 sequence and a transmembrane sequence. In another embodiment, the target cells are caused to express a recombinant sequence comprising a fusion of an IL-15 sequence and a sequence of the transmembrane domain of the IL-15 receptor. In another embodiment, the target cells are caused to express a recombinant sequence comprising a fusion of an IL-15 sequence and a sequence of the IL-15 receptor.

[0022] In the present invention, the term "chimeric antigen receptor" or "CAR" generally refers to an engineered receptor. For example, the chimeric antigen receptor of the present invention can be expressed on immune cells. These receptors are called chimeric because they are composed of parts from different sources. The most common form of these molecules is a single-chain variable fragment (scFv) fusion derived from a monoclonal antibody, fused to the CD3-ζ transmembrane and intracellular domain, CD28 or 41BB intracellular domain or a combination thereof. Such molecules result in the transmission of signals in response to the recognition of their targets by scFv.

[0023] As used herein, the term "T cell receptor" or "TCR" generally refers to an endogenous or engineered T cell receptor. For example, a TCR may include an extracellular antigen-binding domain that binds to a specific antigen epitope within an MHC molecule. A TCR may include a TCRα polypeptide chain and a TCRβ polypeptide chain. A "TCR cell" refers to a cell expressing a recombinant TCR. The "antigen-binding domain" of a TCR refers to a polypeptide that binds to an antigen with a high degree of specificity due to its primary, secondary, or tertiary sequence and / or post-translational modifications and / or charge. The antigen-binding domain can be derived from any portion or fragment of a TCR that, as part of a TCR, retains the antigen-binding activity of the TCR. The antigen-binding portion encompasses a portion capable of detecting, treating, or preventing cancer. The antigen-binding domain of a TCR may comprise approximately 10-95% or more, for example, approximately 10%, approximately 25%, approximately 30%, approximately 50%, approximately 68%, approximately 80%, approximately 90%, approximately 95%, or more, of the full-length TCR. The antigen binding domain may comprise the antigen recognition portion of either or both of the α and β chains of the TCR, for example, it may comprise one or more portions of the complementary determining regions CDR1, CDR2, and CDR3 of the variable regions of the α and / or β chains of the TCR. The antigen binding domain may comprise the following amino acid sequence: CDR1 of the α chain, CDR2 of the α chain, CDR3 of the α chain, CDR1 of the β chain, CDR2 of the β chain, CDR3 of the β chain, or any combination thereof. Preferably, the antigen binding domain comprises the amino acid sequence of CDR1, CDR2, and CDR3 of the α chain of the TCR or the amino acid sequence of CDR1, CDR2, and CDR3 of the β chain; or the amino acid sequence of CDR1, CDR2, and CDR3 of all α and β chains. In one embodiment of the present invention, the antigen binding portion may include, for example, a variable region of a TCR comprising a combination of the above-mentioned CDR regions. In this regard, the antigen binding portion may comprise the amino acid sequence of the variable region (Vα) of the α chain of TCR, the amino acid sequence of the variable region (Vβ) of the β chain, or the amino acid sequence of both Vα and Vβ. In one embodiment of the invention, the antigen binding portion may comprise a combination of a variable region and a constant region. In this regard, the antigen binding portion may comprise the full length α or β chain of TCR, or both α and β chains.

[0024] For example, the α and β chains of a TCR are generally considered to each have two "domains" or "regions" referred to as variable and constant domains / regions. The terms "domain" and "region" are used interchangeably herein. The variable domain consists of a concatenation of a variable region and a connecting region. In the present invention, the term "TCRα variable domain" therefore refers to the concatenation of the TRAV and TRAJ regions, and the term TCRα constant domain refers to the extracellular TRAC region or to the TRAC sequence with a C-terminal truncation. Similarly, the term "TCRβ variable domain" refers to the concatenation of the TRBV and TRBD / TRBJ regions, and the term TCRβ constant domain refers to the extracellular TRBC region or to the TRBC sequence with a C-terminal truncation. Similarly, the connecting region of a TCR is defined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant region is defined by the IMGT TRAC and TRBC nomenclature. The β chain variable region is referred to by the abbreviation TRBD in the IMGT nomenclature, and the concatenated TRBD / TRBJ regions are generally considered together to be the connecting region.

[0025] The example of TCR includes but is not limited to full-length TCR, TCR antigen binding fragment, lack of transmembrane region and cytoplasmic region soluble TCR, containing TCR by the variable region connected by flexible joint single chain TCR, by the TCR chain connected by the disulfide bond through transformation, monospecific TCR, multispecific TCR (including bispecific TCR), TCR fusion, human TCR, humanized TCR, chimeric TCR, recombinant TCR and synthetic TCR.The term covers wild-type TCR and genetically modified TCR (for example, comprising chimeric TCR chain, the chimeric TCR chain comprising the first part of the TCR from the first species and the second part of the TCR from the second species).In certain embodiments, TCR includes transmembrane region.In certain embodiments, TCR includes costimulatory signaling region.

[0026] In the present invention, the term "vector" generally refers to a vector by which a polynucleotide sequence (such as a foreign gene) can be introduced into a host cell to obtain the desired gene expression of the introduced nucleotide sequence. Cloning vectors can include, for example, plasmids, phages, viruses, etc. Another type of vector is a viral vector that is connected to the nucleic acid construct to be transported in the viral genome. Viral vectors can replicate autonomously in the host cell into which they are introduced, or can integrate themselves into the genome of the host cell, thereby replicating together with the host genome. In addition, some vectors can instruct the expression of genes operably connected to them. Such vectors are referred to as "recombinant expression vectors" or simply "expression vectors" in this article. In some embodiments, the vector is a viral vector (such as a replication-defective retrovirus, adenovirus, and adeno-associated virus).

[0027] In the present invention, the term "cancer" or "cancer cell" generally refers to a cell that divides in an uncontrolled manner. Examples of such cells include cells with an abnormal state or condition characterized by rapidly proliferating cell growth. The term is intended to include cancerous growths (e.g., tumors), oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of the histopathological type or infiltration stage. Cancer cells may include tumor cells in the blood (e.g., hematological cancers), or may include tumor cells in solid tumors. Alternatively or additionally, it may include all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or infiltration stage.

[0028] In the present invention, the term "pharmaceutically acceptable" or "pharmacologically compatible" generally refers to a material that does not have biological or other undesirable effects. For example, the material can be incorporated into a pharmaceutical composition administered to a subject without causing any significant undesirable biological effects or interacting in a deleterious manner with any other component of the composition in which it is contained. Pharmaceutically acceptable adjuvants or excipients preferably have met the required toxicological and manufacturing testing standards and / or are included in the inactive ingredient guidelines established by the Food and Drug Administration.

[0029] 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.

[0030] 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, NKT 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.

[0031] In the present invention, the term "killing ability" refers to the killing of cells by contacting the cells with an effective amount of active substances.

[0032] In the present invention, the terms "about" and "approximately" generally refer to a statistically significant numerical range. Such a range may be within an order of magnitude of a given value or range, may be included within 50%, may be included within 20%, may be included within 10%, may be 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.

[0033] In the present invention, the terms "above", "below", "at most" and "at least" may include the number.

[0034] Detailed Description of the Invention

[0035] In one aspect, the present invention provides a method for preparing immune cells, comprising causing natural killer T cells derived from stem cells or progenitor cells to express chimeric antigen receptors and / or cytokines.

[0036] For example, the stem cells or progenitor cells include hematopoietic stem cells. For example, the natural killer T cells derived from stem cells or progenitor cells include natural killer T cells obtained by causing the stem cells or progenitor cells to express a T cell receptor. For example, the T cell receptor includes an invariant T cell receptor (iTCR) that specifically binds to α-galactosylceramide (α-GC).

[0037] For example, the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. For example, the antigen binding domain and the transmembrane domain further comprise a hinge domain. For example, the intracellular domain further comprises a costimulatory domain and / or a signal transduction domain.

[0038] For example, the cytokine comprises an interleukin family member protein or a functionally active fragment thereof. For example, the interleukin family member is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, and IL-21. For example, the cytokine comprises non-membrane-bound IL-15 and / or membrane-bound IL-15, wherein the non-membrane-bound IL-15 does not have a transmembrane domain, and the membrane-bound IL-15 has a transmembrane domain. For example, the cytokine comprises IL-15 or a functionally active fragment thereof, and IL-15 receptor alpha (IL-15Ra) or a functional fragment thereof, wherein the IL-15 or its functionally active fragment is directly or indirectly linked to the IL-15 receptor alpha or its functionally active fragment (e.g., linked via a connecting peptide).

[0039] For example, the method comprises introducing nucleic acids encoding the chimeric antigen receptor and the non-membrane-bound IL-15 and / or the membrane-bound IL-15 into natural killer T cells derived from stem cells or progenitor cells.

[0040] In one aspect, the present invention provides a method for preparing immune cells, comprising the following steps: (1) differentiating stem cells or progenitor cells into natural killer T cells, and (2) causing the natural killer T cells to express chimeric antigen receptors and / or cytokines.

[0041] For example, the stem cell or progenitor cell comprises a hematopoietic stem cell. For example, the stem cell or progenitor cell is expressed in a T cell receptor to obtain the natural killer T cell. For example, the T cell receptor comprises an invariant T cell receptor (iTCR), and the iTCR specifically binds to α-galactosylceramide (α-GC). For example, the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. For example, a hinge domain is further included between the antigen binding domain and the transmembrane domain. For example, the intracellular domain further comprises a costimulatory domain and / or a signal transduction domain.

[0042] For example, the cytokine comprises an interleukin family member protein or a functionally active fragment thereof. For example, the interleukin family member is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, and IL-21. For example, the cytokine comprises non-membrane-bound IL-15 and / or membrane-bound IL-15, wherein the non-membrane-bound IL-15 does not have a transmembrane domain, and the membrane-bound IL-15 has a transmembrane domain. For example, the cytokine comprises IL-15 or a functionally active fragment thereof, and IL-15 receptor alpha (IL-15Ra) or a functional fragment thereof, wherein the IL-15 or its functionally active fragment is directly or indirectly linked to the IL-15 receptor alpha or its functionally active fragment (e.g., linked via a connecting peptide).

[0043] For example, the method comprises further introducing nucleic acids encoding the chimeric antigen receptor and the non-membrane-bound IL-15 and / or the membrane-bound IL-15 into the natural killer T cells differentiated and obtained in step (1).

[0044] In one aspect, the present invention provides an immune cell obtained by the preparation method of the present invention.

[0045] In one aspect, the present invention provides use of the immune cells obtained by the preparation method of the present invention in the preparation of a drug for preventing and / or treating a disease and / or symptom (eg, tumor).

[0046] In one aspect, the present invention provides a pharmaceutical composition comprising the immune cells obtained by the preparation method of the present invention as an active ingredient.

[0047] In one aspect, the present invention provides a method for preventing and / or treating a disease and / or symptom (eg, tumor), comprising administering the immune cells obtained by the preparation method of the present invention to a subject in need thereof.

[0048] In one aspect, the present invention provides immune cells obtained by the preparation method of the present invention or the pharmaceutical composition of the present invention, which are used to prevent and / or treat diseases and / or symptoms (such as tumors).

[0049] Cell culture

[0050] (1) The first step is iNKT cell differentiation: human CD34-positive HSCs are collected from healthy donors, and the nucleic acid encoding the iTCR is transduced with a vector. The iNKT-transduced cells are cultured for 40 days and efficiently differentiated into iNKT cells.

[0051] (1) The second step is iNKT cell transduction: after the differentiation stage, the differentiated iNKT cells are activated, and the nucleic acid encoding CAR and the nucleic acid encoding the IL-15 sequence (such as the full length of IL-15 or its functionally active fragment, or the fusion polypeptide of IL-15 and IL-15Ra) are transduced with a vector to introduce the antigen-targeting CAR structure and IL-15 into the differentiated iNKT cells. After transduction, PBMC / α-GalCer is used as a nutrient cell and further expanded for about 2 weeks to obtain the immune cells of the present invention.

[0052] For example, iNKT cells are generated from less differentiated cells (e.g., embryonic stem cells, umbilical cord blood-derived stem cells, peripheral blood-derived stem cells, embryonic stem cells, pluripotent stem cells, hematopoietic stem cells or progenitor cells, induced pluripotent stem cells (iPS) cells, or stem or progenitor cells). For example, the donor hematopoietic stem cells can be obtained from the donor's bone marrow, peripheral blood, amniotic fluid, or umbilical cord blood. The donor can be an autologous donor, i.e., a subject to be treated with hematopoietic stem cell-iNKT cells; or an allogeneic donor, i.e., a human donor different from the subject to be treated with hematopoietic stem cell-iNKT cells.

[0053] For example, HSCs are transduced with one or more exogenous iNKT TCR nucleic acid molecules. "iNKT TCR nucleic acid molecules" include nucleic acid molecules encoding the α chain (TCR-α) of the iNKT T cell receptor, the β chain (TCR-β) of the iNKT T cell receptor, or both. For example, iNKT cells are generated from HSC cells.

[0054] For example, the vector can be a non-viral vector (e.g., a plasmid) or a viral vector (e.g., a lentivirus, a retrovirus, adeno-associated virus (AAV), herpes virus, or adenovirus). For example, after transduction, the cells can be activated and expanded by any suitable means, such as activation with α-galactosylceramide (α-GC). For example, the transduced cells can be co-cultured with irradiated feeder cells.

[0055] For example, the sequence of an exemplary TCR-α of the present invention may be shown as SEQ ID NO: 1. For example, the sequence of an exemplary TCR-β of the present invention may be shown as SEQ ID NO: 2.

[0056] Chimeric Antigen Receptor (CAR)

[0057] For example, the chimeric antigen receptor (CAR) may comprise an antigen binding domain, an optional hinge domain, a transmembrane domain, an intracellular domain (e.g., a costimulatory signaling domain and / or a signal transduction domain) sequentially from the N-terminus to the C-terminus. The chimeric antigen receptor comprises an antigen binding domain. The antigen binding domain may be a single chain variable fragment (scFv) or other antigen binding fragment derived from an antigen-specific antibody. In some embodiments, the antigen binding region is a BCMA binding domain. In some embodiments, the antigen binding region is a CD19 binding domain. In some embodiments, the antigen binding region is a NY-ESO-1 binding domain. A "single chain Fv" or "scFv" antibody fragment comprises the V domain of an antibody. H and V L domains, wherein these domains are present in a single polypeptide chain. In some embodiments, the antigen binding domain further comprises a V H With V LThe peptide linker between the domains can promote the formation of the desired structure of scFv for antigen binding. In the present invention, antigen binding domain is synonymous with antigen binding domain or antigen binding region and is used interchangeably.

[0058] For example, examples of antigens that the antigen binding domain of CAR can target include at least 5T4, 8H9, α v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, folate receptor-a, FAP, FBP, fetal AchR, FRα, G D2, G250 / CAIX, GD3, Glypican-3 (GPC3), Her2, IL-13Rα2, Lambda, Lewis-Y, Kappa, KDR, MAGE, MCSP, Mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TEM, Carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, Tyrosinase, MAGE, Laminin receptor, HPV E6, E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, EphA3, telomerase, SAP-1, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PAME, SSX-2, Melan-A / MART-1, GP100 / pmel17, TRP-1 / -2, P. polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CML66, fibronectin, MART-2, TGF-βRII or VEGF receptor (e.g., VEGFR2). CAR can be a first generation, second generation, third generation or more generation CAR. CAR can have bispecificity for any two different antigens, or it can have specificity for more than two different antigens.

[0059] For example, the chimeric antigen receptor comprises one or more of the intracellular domains. For example, the chimeric antigen receptor may comprise 1 to 5, such as 1, 2, 3, 4 or 5, intracellular domains. For example, the intracellular domain may comprise a costimulatory signaling domain and / or a signaling domain. For example, the intracellular domain may be located at the C-terminus of the transmembrane domain. For example, the intracellular domain may comprise a signaling domain of a protein or a functional fragment thereof selected from the group consisting of CD28, 2B4 and DAP10. For example, the intracellular domain of the present invention may comprise an intracellular domain of a protein or a functional fragment thereof selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1, CD2, CD7, LIGHT, NKG2C and B7-H3. For example, the intracellular domain of the present invention may comprise an intracellular domain of a protein or a functional fragment thereof selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.

[0060] For example, the chimeric antigen receptor further comprises a transmembrane domain, which is directly or indirectly connected to the costimulatory signaling domain. For example, the transmembrane domain is located at the N-terminus of the costimulatory signaling domain. For example, the transmembrane domain of the present invention comprises a transmembrane domain structure for a chimeric antigen receptor known in the art. For example, the transmembrane domain of the present invention comprises a transmembrane domain of a protein or a functional fragment thereof selected from the group consisting of CD4, CD8, CD28, and IL receptor family proteins. For example, the transmembrane domain of the present invention comprises a transmembrane domain of a protein or a functional fragment thereof selected from the group consisting of α, β or ζ chain of a cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C. For example, the transmembrane domain can recruit a protein with a costimulatory effect; the costimulatory protein comprises DAP10 protein. For example, the transmembrane domain comprises a transmembrane domain of a protein or functional fragment thereof selected from the group consisting of NKG2D and CD8a.

[0061] For example, the chimeric antigen receptor further comprises a hinge domain, which is directly or indirectly connected to the costimulatory signaling domain. For example, the hinge domain is located between the transmembrane domain and the antigen binding domain. For example, the hinge domain of the present invention comprises a hinge domain structure for a chimeric antigen receptor known in the art. For example, the hinge domain of the present invention comprises a hinge domain of a protein or a functional fragment thereof selected from the group consisting of: CD8, CD28, and CD4. For example, the hinge domain of the present invention comprises a hinge domain of a protein or a functional fragment thereof selected from the group consisting of: CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154. For example, the hinge domain comprises a hinge domain of CD8a or a functional fragment thereof.

[0062] For example, the sequence of an exemplary chimeric antigen receptor of the present invention can be shown as SEQ ID NO:3.

[0063] cytokines

[0064] For example, the cytokines of the present invention include interleukin family members. For example, the interleukin family members are selected from the group consisting of IL-2, IL-7, IL-12, IL-15, and IL-21.

[0065] For example, the cytokines of the present invention include non-membrane-bound IL-15. For example, the IL-15 of the present invention includes IL-15 without a transmembrane domain sequence. For example, the IL-15 of the present invention includes full-length IL-15 or a functionally active fragment thereof (e.g., as non-membrane-bound IL-15). For example, the cytokines of the present invention may further include a signal peptide (e.g., a TPA signal peptide or a CD58 signal peptide). For example, the sequence of an exemplary cytokine of the present invention may be as shown in SEQ ID NO: 4.

[0066] For example, the cytokine of the present invention comprises membrane-bound IL-15 (mbIL-15). For example, the IL-15 of the present invention comprises an IL-15 domain (having the full length of IL-15 or a functionally active fragment thereof), and a transmembrane domain. For example, the IL-15 domain in the membrane-bound IL-15 of the present invention comprises the full length of IL-15 or a functionally active fragment thereof. For example, the IL-15 of the present invention further comprises any cytokine receptor domain. For example, the IL-15 of the present invention further comprises any interleukin family member receptor as a cytokine receptor domain. For example, the interleukin family member receptor is selected from: IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, and IL-21 receptor. For example, the cytokine of the present invention can be a fusion protein (e.g., as membrane-bound IL-15) consisting of any interleukin family member and any interleukin family member receptor.

[0067] For example, the cytokine fusion protein of the present invention comprises interleukin-15 receptor alpha (IL-15Ra) or a functional fragment thereof. For example, the cytokine of the present invention may comprise an IL-15 domain and an IL-15 receptor or a functionally active fragment thereof. For example, the cytokine may further comprise an IL-15 receptor or a functionally active fragment thereof. For example, the IL-15 receptor may comprise IL-15Rα. For example, the IL-15 receptor of the present invention may comprise the full length of the IL-15 receptor or a functionally active fragment thereof. For example, the IL-15 receptor of the present invention may comprise the sushi domain (also known as the sushi domain), hinge domain, transmembrane domain and / or intracellular domain of the IL-15 receptor. For example, the IL-15 domain and the IL-15 receptor or a functionally active fragment thereof may be directly or indirectly linked. For example, the indirect link may comprise a link via a connecting peptide. For example, the connecting peptide may comprise a polypeptide known in the art for linking two moieties to be linked. For example, the cytokine fusion protein of the present invention may further comprise a signal peptide (e.g., a TPA signal peptide or a CD58 signal peptide). For example, the transmembrane domain of the IL-15 receptor α subunit contained in the fusion polypeptide of the present invention has amino acid mutations, which can increase the membrane anchoring ratio of the fusion protein of the present invention while maintaining or improving the activity of the fusion protein of the present invention. For example, the sequence of an exemplary cytokine of the present invention can be shown as SEQ ID NO: 5.

[0068] The cells obtained by the preparation method of the present invention can be further engineered using gene editing tools (CRISPR, TALEN, zinc fingers, etc.) to destroy genes that deplete the function of immune cells. For example, the destroyed genes encode cellular immune checkpoint inhibitors.

[0069] In some embodiments, the cell may also optionally include a genomic mutation. In some embodiments, the genomic mutation includes a mutation in one or more endogenous genes in the cell genome, wherein the one or more endogenous genes include B2M, CIITA, TRAC, TRBC1, or TRBC2 genes. In some embodiments, the mutation includes a loss-of-function mutation. In some embodiments, the inhibitor is an expression inhibitor. In some embodiments, the inhibitor includes an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid includes one or more of siRNA, shRNA, miRNA, or antisense molecules. In some embodiments, the cell includes an activity inhibitor.

[0070] The cells obtained by the preparation method of the present invention can also be further engineered using gene editing tools to knock out or overexpress HLA-E genes, MHC molecules, B2M, CIITA, etc. For example, to make the cells resistant to host cell-mediated depletion.

[0071] On the other hand, the present invention provides a kind of cell, and the cell may comprise the cell obtained by the preparation method of the present invention. For example, the cell may comprise an immune cell. For example, the cell may comprise NK cells, NKT cells and / or T cells. For example, the cell may comprise cells derived from artificial pluripotent stem (iPS) cells, totipotent stem cells, pluripotent stem cells, PBMC cells and / or tumor infiltrating lymphocytes. For example, the cell of the present invention may be obtained by differentiation of iPS cells. For example, the cell of the present invention may be HSC (hematopoietic stem cell). For example, the cell of the present invention may be obtained by differentiation of HSC cells. In some embodiments, the cell may also comprise T cells, such as type 1 T helper cells and type 2 T helper cells. In some embodiments, the cell may also comprise T cells, such as αβ-T cells. In alternative embodiments, the cell may also comprise T cells, such as γδ-T cells.

[0072] For example, compared to cells transduced with iTCR and CAR and IL-15 at the same time, when HSC cells are first transduced with iTCR to become iNKT and then transduced with CAR and / or IL-15, the cells have improved CAR and / or IL-15 expression levels, exhibit higher functional activity (e.g., cell killing activity), and / or exhibit higher anti-tumor activity.

[0073] For example, compared to cells transduced with iTCR, CAR, and IL-15 simultaneously, HSC cells are first transduced with iTCR to become iNKT and then transduced with CAR and / or IL-15, which can increase the expression level of CAR and / or IL-15 by at least about 80% to 120%, such as at least about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120%. The expression level is measured by detecting the proportion of CAR and / or IL-15 expressing cells using flow cytometry.

[0074] In some embodiments, HSC cells are first transduced with iTCR to become iNKT and then transduced with CAR and / or IL-15, compared to cells transduced with iTCR and CAR and IL-15 at the same time, which can increase the immune response, activity or number by at least 10% to 20 times, such as at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times or 20 times.

[0075] In some embodiments, compared to cells that are transduced with iTCR and CAR and IL-15 at the same time, HSC cells are first transduced with iTCR to become iNKT and then transduced with CAR and / or IL-15, which can induce specific killing of target cells by at least 10% to 5 times, such as at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 times, 2 times, 3 times, 4 times or 5 times. In some embodiments, the specific killing of target cells is determined by the killing efficacy of target cells using the methods described herein.

[0076] In some embodiments, compared to iNKT transduction CAR and / or IL-15 isolated from peripheral blood, HSC cells are first transduced with iTCR to become iNKT and then transduced with CAR and / or IL-15, which can have a higher iNKT cell ratio, for example, the iNKT cell ratio is increased by at least about 80% to 120%, such as at least about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120%. The expression level is determined by detecting the proportion of CAR and / or IL-15 expressing cells by flow cytometry.

[0077] In one aspect, the present invention provides the use of cells obtained by the preparation method of the present invention in the preparation of a medicament for preventing and / or treating a disease and / or symptom, for example, a tumor or an autoimmune disease.

[0078] In one aspect, the present invention provides cells obtained by the preparation method of the present invention, which can be used to prevent, alleviate and / or treat diseases. For example, the disease comprises a tumor.

[0079] In one aspect, the present invention provides a method for preventing, alleviating and / or treating a disease, the method comprising administering cells obtained by the preparation method of the present invention to a subject in need thereof. For example, the disease comprises a tumor or an autoimmune disease.

[0080] According to the present invention, the tumor is selected from the group consisting of lymphoma, melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer and kidney cancer.

[0081] Without intending to be bound by any theory, the following examples are merely intended to illustrate the proteins, methods and uses of the present invention and are not intended to limit the scope of the present invention.

[0082] Sequence information

[0083] Example

[0084] Example 1 Preparation of iNKT cells

[0085] Human CD34-positive HSCs are collected from healthy donors or umbilical cord blood of newborns, transduced with the lentiviral vector Lenti / iTCR, and then cultured in vitro. The iTCR-transduced human hematopoietic stem cells are induced to differentiate in vitro for 40 days, efficiently differentiating into human hematopoietic stem cell-derived iNKT cells. After the differentiation stage, CB-iNKT cells are activated and transduced with the Retro / CAR-mbIL-15 viral vector to introduce the antigen-targeting CAR structure and mbIL-15 into the CB-iNKT cells. After transduction, the cells are further expanded for about 2 weeks using PBMC / α-GalCer as feeder cells to obtain the CAR-mbIL-15-iNKT cells of the present invention.

[0086] In the pre-differentiation transduction control group, hematopoietic stem cells were transduced with the Lenti / iTCR-CAR-IL-15 lentiviral vector and cultured to obtain control group CAR-IL-15-iNKT (pre-differentiation control) cells.

[0087] In the PBMC-derived control group, human PBMCs were collected from healthy donors and the resulting iNKT cells (PB-iNKT) were isolated and expanded. The PB-iNKT cells were transduced with the Retro / CAR-mbIL-15 viral vector to introduce the antigen-targeting CAR structure and mbIL-15 into the PB-iNKT cells, and the control group CAR-mbIL-15-iNKT (PBMC-derived control) cells were cultured.

[0088] Example 2 Functional Experiment of iNKT Cells

[0089] Cell expansion

[0090] The CAR-mbIL-15-iNKT cells of the present invention and the CAR-IL-15-iNKT cells transduced before differentiation of the control group were harvested, and PBMC feeder cells with a cell number twice that of the control group were added (the feeder cells were incubated with α-GalCer for four hours and irradiated before addition). After one week of culture, the cell number in each group was detected and the expansion multiple was calculated.

[0091] The results showed that the CAR-mbIL-15-iNKT cells transduced after differentiation of the present invention were comparable to the cells transduced before differentiation of the control group in terms of cell expansion.

[0092] Cell killing

[0093] The CAR-mbIL-15-iNKT cells of the present invention and the CAR-IL-15-iNKT cells transduced before differentiation of the control group were harvested and co-cultured with the target cell NALM-6-eGFP cell line at an iNKT effector cell to tumor target cell ratio (effective target ratio) of E: T = 1: 4. The number of target cells remaining was detected 48 hours after co-culture, and the killing efficiency (number of target cells killed / total number of target cells added per round) was calculated. On the same day, the same number of tumor target cells at the start was added, and samples were taken for detection 48 hours later, and multiple rounds of killing were repeated.

[0094] Figure 1 shows the killing results of effector cells in each group. The results show that the CAR-mbIL-15-iNKT cells transduced after differentiation of the present invention have greater advantages in cell killing than the cells transduced before differentiation of the control group.

[0095] Cell flow cytometry characteristics

[0096] The CAR-mbIL-15-iNKT cells of the present invention, pre-differentiation control cells, and PBMC-derived control cells were harvested and flow cytometry was performed for the expression of iNKT TCR, CD3, IL15Ra, and CAR before the cell killing assay. After six rounds of cell killing assays in co-culture with target cells, the expression of iNKT TCR, CD3, IL15Ra, and CAR was again detected by flow cytometry.

[0097] Figures 2A-2B show the flow cytometry expression of iTCR, CD3, IL-15Ra, and CAR in each group of cells before and after cell killing assays. The results show that the differentiated and transduced CAR-mbIL-15-iNKT cells of the present invention maintain a high proportion of iNKT TCR-positive cells, as well as mbIL-15 and CAR expression levels after multiple rounds of killing.

[0098] In the control cells before differentiation, the expression levels of IL-15 and CAR were low, and after multiple killing of target cells, the expression level of CAR was basically undetectable.

[0099] In the PBMC-derived control group cells, after multiple killing of target cells, the proportion of NKT TCR-positive cells decreased significantly, so the final major cell population in the PBMC-derived control group cells was ordinary T cells.

[0100] Therefore, the CAR-mbIL-15-iNKT cells of the present invention have greater advantages than the pre-differentiation control group cells and the PBMC-derived control group cells in increasing the expression level of IL-15 and the expression level of CAR, and maintaining the proportion of iNKT TCR-positive cells.

[0101] 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.

Claims

1. A method for preparing immune cells, comprising the step of causing natural killer T cells derived from stem cells or progenitor cells to express chimeric antigen receptors and / or cytokines.

2. The method of claim 1, wherein the stem or progenitor cells comprise hematopoietic stem cells.

3. The method of any one of claims 1-2, wherein the natural killer T cells comprise natural killer T cells obtained after the stem cells or progenitor cells express a T cell receptor.

4. The method of claim 3, wherein the T cell receptor comprises an invariant T cell receptor (iTCR) that specifically binds α-galactosylceramide.

5. The method of any one of claims 1-4, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. The method of claim 5 , wherein the antigen binding domain and the transmembrane domain further comprise a hinge domain.

7. The method of any one of claims 5-6, wherein the intracellular domain further comprises a co-stimulatory domain and / or a signal transduction domain.

8. The method of any one of claims 1-7, wherein the cytokine comprises an interleukin family member protein or a functionally active fragment thereof.

9. The method of claim 8, wherein the interleukin family member is selected from the group consisting of IL-2, IL-7, IL-12, IL-15 and IL-21.

10. The method of any one of claims 1-9, wherein the cytokine comprises non-membrane-bound IL-15 and / or membrane-bound IL-15, wherein the non-membrane-bound IL-15 does not have a transmembrane domain and the membrane-bound IL-15 has a transmembrane domain.

11. The method of any one of claims 1 to 10, wherein the cytokine comprises IL-15 or a functionally active fragment thereof, and IL-15 receptor alpha (IL-15Ra) or a functional fragment thereof, and wherein the IL-15 or a functionally active fragment thereof is directly or indirectly linked to the IL-15 receptor alpha or a functionally active fragment thereof (e.g., linked via a connecting peptide).

12. The method of any one of claims 10-11, comprising introducing nucleic acids encoding the chimeric antigen receptor and the non-membrane-bound IL-15, and / or introducing nucleic acids encoding the chimeric antigen receptor and the membrane-bound IL-15 into natural killer T cells derived from stem cells or progenitor cells.

13. A method for preparing immune cells, comprising the following steps: (1) differentiating stem cells or progenitor cells into natural killer T cells, and (2) allowing the natural killer T cells to express chimeric antigen receptors and / or cytokines.

14. The method of claim 13, wherein the stem or progenitor cells comprise hematopoietic stem cells.

15. The method according to any one of claims 13 to 14, wherein the step (1) comprises causing the stem cells or progenitor cells to express a T cell receptor, thereby obtaining the natural killer T cells.

16. The method of claim 15, wherein the T cell receptor comprises an invariant T cell receptor (iTCR) that specifically binds α-galactosylceramide.

17. The method of any one of claims 13-16, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.

18. The method of claim 17, wherein the antigen binding domain and the transmembrane domain further comprise a hinge domain.

19. The method of any one of claims 17-18, wherein the intracellular domain further comprises a co-stimulatory domain and / or a signal transduction domain.

20. The method of any one of claims 13-19, wherein the cytokine comprises an interleukin family member protein or a functionally active fragment thereof.

21. The method of claim 20, wherein the interleukin family member is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, and IL-21.

22. The method of any one of claims 13-21, wherein the cytokine comprises non-membrane-bound IL-15 and / or membrane-bound IL-15, wherein the non-membrane-bound IL-15 does not have a transmembrane domain and the membrane-bound IL-15 has a transmembrane domain.

23. The method of any one of claims 13 to 22, wherein the cytokine comprises IL-15 or a functionally active fragment thereof, and IL-15 receptor alpha (IL-15Ra) or a functional fragment thereof, and wherein the IL-15 or a functionally active fragment thereof is directly or indirectly linked to the IL-15 receptor alpha or a functionally active fragment thereof (e.g., linked via a linker peptide).

24. The method according to any one of claims 22-23, comprising further introducing nucleic acids encoding the chimeric antigen receptor and the non-membrane-bound IL-15, and / or introducing nucleic acids encoding the chimeric antigen receptor and the membrane-bound IL-15 into the natural killer T cells differentiated in step (1).

25. An immune cell obtained by the preparation method according to any one of claims 1 to 24.

26. Use of the immune cell according to claim 25 in the preparation of a medicament, wherein the medicament is used to prevent and / or treat a disease and / or symptom (such as a tumor).

27. A pharmaceutical composition comprising the immune cell according to claim 25 as an active ingredient.

28. A method for preventing and / or treating a disease and / or symptom (eg, tumor), comprising administering the immune cell of claim 25 or the pharmaceutical composition of claim 27 to a subject in need thereof.

29. The immune cell according to claim 25 or the pharmaceutical composition according to claim 27, for use in preventing and / or treating a disease and / or symptom (such as a tumor).

30. A method for influencing tumor cell growth, comprising administering the immune cell of claim 25 or the pharmaceutical composition of claim 27.

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