Modified cells expressing a trimer complex

Abstract

This application generally relates to modified cells, such as CAR-T cells, that express trimer complexes. Furthermore, it provides fusion polypeptides involved in the formation of trimer complexes.

Description

[Technical Field]

[0001] This application is based on the international patent application PCT / CN2023 / 112027, which claims priority. The entire contents of the said international patent application are incorporated herein by reference.

[0002] This disclosure generally relates to modified cells expressing a trimer complex capable of binding to FasL. Furthermore, this disclosure provides fusion polypeptides involved in the formation of the trimer complex. [Background technology]

[0003] Fas (also known as CD95) is a cell surface receptor belonging to the tumor necrosis factor (TNF) receptor family. When activated by binding to FasL, which uses Fas as a ligand, it activates caspases and induces a signaling cascade that ultimately leads to cell death (10.1038 / sj.cdd.4401305). In the context of cancer, tumor cells may utilize the Fas / FasL pathway to evade immune surveillance. Tumor cells expressing FasL can induce activation-induced cell death (AICD) in invasive T cells, thus limiting the immune response to the tumor (10.1007 / s002620050505, 10.1016 / s0167-5699(98)01382-6).

[0004] In addition to FasL expression on tumors, when CAR T cells encounter cancer cells, FasL is upregulated on the CAR T cells, which can result in early apoptosis and limit their persistence in the patient's body (10.1016 / s1074-7613(00)80566-x). Therefore, developing strategies to promote the persistence of CAR T cells while mitigating activation-induced cell death is a crucial research challenge in the field of CAR T cell therapy. [Overview of the project]

[0005] This disclosure provides novel modified cells expressing a trimer complex. The trimer complex enhances the persistence and proliferation of the modified cells, particularly in the tumor microenvironment, without the need for exogenous administration of antibodies or cytokines. Furthermore, the expression of the trimer complex mitigates toxicity issues associated with systemic administration by limiting cytokine signaling to within the modified cells and minimizing its impact on the surrounding environment.

[0006] This disclosure provides a modified cell expressing a trimer complex comprising at least one fusion polypeptide, the fusion polypeptide comprising, in order from its N-terminus to its C-terminus, a) an extracellular component comprising a Fas ectodomain; b) a transmembrane component; and c) an intracellular component comprising an IL-7R endodomain, a CD2 endodomain, or a combination thereof, but not comprising a Fas endodomain. In one embodiment, the trimer complex is capable of binding to a Fas ligand. In one embodiment, the trimer complex is capable of inducing signal transduction of the intracellular component independently of binding to a Fas ligand.

[0007] The Disclosure further provides a fusion polypeptide comprising: a) an extracellular component comprising a Fas ectodomain; b) a transmembrane component; and c) an intracellular component comprising an IL-7R endodomain, a CD2 endodomain, or a combination thereof, but not comprising a Fas endodomain. The Disclosure also provides a trimer complex comprising at least one fusion polypeptide comprising: a) an extracellular component comprising a Fas ectodomain; b) a transmembrane component; and c) an intracellular component comprising an IL-7R endodomain, a CD2 endodomain, or a combination thereof, but not comprising a Fas endodomain. In one embodiment, each component in the fusion polypeptide is arranged sequentially from the N-terminus to the C-terminus.

[0008] In one embodiment, the extracellular component includes or consists of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1. In one embodiment, the extracellular component includes cysteine-rich domain 1, cysteine-rich domain 2, and cysteine-rich domain 3 of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the extracellular component includes or consists of the amino acid sequence of SEQ ID NO: 1.

[0009] In one embodiment, the transmembrane component is self-oligomerizable. In one embodiment, the transmembrane component includes or comprises a transmembrane domain that includes a gain-of-function insertion compared to SEQ ID NO: 10. In one embodiment, the transmembrane component includes or comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 33. In one embodiment, the transmembrane component includes or comprises the amino acid sequence of SEQ ID NO: 11 (PILLTCPTIX1IX2SX3X4X5X6X7X8X9X10X11LX12X13X14LW). Here, X1 is S or L; X2 is L or S; X3 is F or L; X4 is F or A; X5 is S or I; X6 is V or L; X7 is A or L; X8 is L or M; X9 is L or V; X10 is V or S; X11 is I or L; X12 is A or L; X13 is C or L; X14 is V or S. In one embodiment, the transmembrane component includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 to 39.

[0010] In one embodiment, the intracellular component comprises or consists of the IL-7Rα endodomain. In one embodiment, the intracellular component comprises or consists of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4. In one embodiment, the intracellular component comprises or consists of the amino acid sequence of SEQ ID NO: 4.

[0011] In one embodiment, the intracellular component includes or consists of a CD2 end domain. In one embodiment, the intracellular component includes or consists of a full-length CD2 end domain or a functional fragment thereof. In one embodiment, the functional fragment has its C-terminus cleaved up to 65 amino acids compared to the full-length CD2 end domain. In one embodiment, the intracellular component includes or consists of an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 5 to 8. In one embodiment, the intracellular component includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs. 5 to 8.

[0012] In one embodiment, the transmembrane component is self-oligomerizable, and the intracellular component includes an IL-7Rα endodomain fused to the N-terminus of the transmembrane component, or consists of such a sequence. In one embodiment, the intracellular component includes an IL-7Rα endodomain, or consists of such a sequence, and the transmembrane component includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 33, or consists of such a sequence. In one embodiment, the intracellular component includes the amino acid sequence of SEQ ID NO: 4, or consists of such a sequence, and the transmembrane component includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 to 39, or consists of such a sequence.

[0013] In one embodiment, the intracellular component comprises or consists of an IL-7Rα end-domain and a CD2 end-domain, the transmembrane component is self-oligomerizable, the IL-7R end-domain has the C-terminus of the transmembrane component fused to its N-terminus, and the IL-7R end-domain has the N-terminus of the CD2 end-domain fused to its C-terminus. In one embodiment, the intracellular component comprises or consists of a full-length CD2 end-domain or a functional fragment thereof, wherein the functional fragment has its C-terminus cleaved to a maximum of 65 amino acids compared to the full-length CD2 end-domain; and the transmembrane component comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs. 11 to 33. In one embodiment, the intracellular component comprises or consists of the amino acid sequence of SEQ ID NOs. 4 and an amino acid sequence selected from the group consisting of SEQ ID NOs. 5 to 8, and the transmembrane component comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs. 34 to 39.

[0014] In one aspect, the transmembrane component comprises, or consists of, a transmembrane domain selected from the group consisting of Fas, CD2, CD3s, CD35, CD3C, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, OX40, 4-IBB, SLAMF1, CTLA4, CD200R, LAG3, HVEM, BTLA, PD-L2, PD-L1, ICOS, PD-1, CD300, GITR, A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, KG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPa, pTa, TCRa, TCRβ, TIM3, TRIM, LPA5, and Zap70, and the intracellular component comprises, or consists of, a CD2 endodomain in which the C-terminus of the transmembrane component is fused to its N-terminus. In one aspect, the CD2 endodomain is a full-length CD2 endodomain or a functional fragment thereof, where the functional fragment has its C-terminus truncated by up to 65 amino acids compared to the full-length CD2 endodomain, and the transmembrane component comprises, or consists of, a Fas transmembrane domain. In one aspect, the intracellular component comprises, or consists of, an amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 8, and the transmembrane component comprises, or consists of, the amino acid sequence of SEQ ID NO: 9.

[0015] In one aspect, the trimeric complex is expressed on the surface of the modified cell. In one aspect, the trimeric complex is a homotrimer or a heterotrimer, where the heterotrimer comprises endogenous Fas. In one aspect, when the intracellular component comprises an IL-7R endodomain, the trimeric complex comprises two or three fusion polypeptides.

[0016] In one aspect, the modified cell is an immune cell. In one aspect, the modified cell is a lymphocyte. In one aspect, the modified cell is a NK cell, a T cell, or a combination thereof.

[0017] In one embodiment, the modified cells include an antigen receptor. In one embodiment, the antigen receptor is a chimeric antigen receptor or an antigen-specific TCR. In one embodiment, the chimeric antigen receptor is CD19, CD20, CD22, CD30, BCMA, AFP, ALK-, GPC3, HER2, EGFRα folate receptor, 5T4, avβ6 integrin, B7-H3, B7-H6, CAIX, CD16, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, claudin 18.2, CSPG4, DLL3, EGFR, HER 2. Targets EGFRvlll, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, Fetal AchR, FRa, GD2, GD3, MAGE-1, NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesoserine, Muc1, Muc16, NCAM, NKG2D ligand, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TAG72, TEMs, VEGFR2, or WT-1.

[0018] In one embodiment, the modified cells express exogenous FasL. In one embodiment, the exogenous FasL is secreted or membrane-bound. In one embodiment, the exogenous FasL contains or consists of an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 40 to 47, or contains or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs. 40 to 47.

[0019] This disclosure further provides the use of the fusion polypeptide described herein to enhance the persistence and proliferation of the modified cells. In one embodiment, the fusion polypeptide can be involved in the formation of the trimer complex. In one embodiment, the trimer complex can induce signaling of the intracellular components independently of the binding of the Fas ligand.

[0020] This disclosure further provides isolated nucleic acids encoding the fusion polypeptide described herein. In one embodiment, the isolated nucleic acid encodes the fusion polypeptide and the antigen receptor. In one embodiment, the fusion polypeptide and the antigen receptor are linked via a viral autocleavage polypeptide.

[0021] This disclosure further provides vectors comprising nucleic acids as described herein. In one embodiment, the vector is a viral vector. In one embodiment, the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated viral vector.

[0022] This disclosure further provides pharmaceutical formulations comprising modified cells, fusion polypeptides, isolated nucleic acids or vectors as described herein, and pharmaceutically acceptable carriers.

[0023] This disclosure further provides a method for treating a disease of interest, comprising administering a modified cell, fusion polypeptide, isolated nucleic acid, vector, or pharmaceutical formulation described herein to a subject. Furthermore, this disclosure provides the use of a modified cell, fusion polypeptide, isolated nucleic acid, vector, or pharmaceutical formulation described herein in the manufacture of a pharmaceutical for the treatment of a disease or disorder. Furthermore, this disclosure provides a modified cell, fusion polypeptide, isolated nucleic acid, vector, or pharmaceutical formulation described herein for use as a pharmaceutical. In one embodiment, the disease or disorder is cancer or an autoimmune disease. In one embodiment, the disease is selected from the group consisting of leukemia, lymphoma, lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, and rhabdomyosarcoma. In one embodiment, the disease is leukemia or lymphoma. [Brief explanation of the drawing]

[0024] [Figure 1] Figure 1 shows T cell proliferation without additional stimulation.

[0025] [Figure 2] Figure 2A shows the proliferation, survival rate, and enrichment of EGFR+ T cells under 100 IU / mL IL2 supplementation. Figure 2B shows the proliferation, survival rate, and enrichment of EGFR+ T cells under conditions lacking exogenous stimulation or cytokines. Figure 2C shows the proliferation, survival rate, and enrichment of EGFR+ T cells when stimulated with 50 ng / mL trimer FasL in the presence of 100 IU / mL IL2 supplementation. [Figure 3] Figure 3 shows that T cells into which various fusion proteins (sharing the same extracellular domain of human Fas) were introduced exhibited enhanced persistence against FasL-induced apoptosis, and that cells expressing F1 showed slightly higher proliferation levels than cells with C1 and F2. [Figure 4] Figure 4 shows that T cells introduced with C-3 were unable to proliferate for 7 days in the absence of cytokines (C-3 vs. F-1, p=0.0277 (day 3), p=0.0060 (day 7)). [Modes for carrying out the invention]

[0026] I. Definition Unless otherwise defined herein, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which the present invention pertains.

[0027] The terms "a" and "an" refer to one or more (i.e., at least one) grammatical objects (such as verbs) to which the articles are attached. For example, "a module" refers to one module or two or more modules.

[0028] As used herein, “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” or “containing,” and “contains,” and are open-ended, not excluding any additional components, elements, or processes not described herein. “Comprising” also encompasses “consisting of.”

[0029] Percent (%) amino acid sequence identity and homology for polypeptides are defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a specific polypeptide sequence, after aligning the candidate sequence with the specific polypeptide sequence and introducing gaps as necessary to achieve the maximum percentage sequence identity. However, conservative substitutions are not considered as part of sequence identity. Alignment for determining the amino acid sequence identity percentage can be achieved by various means within the scope of the art, such as BLAST, BLAST-2, ALIGN, or MEGALIGN. 登録商標 Known computer software such as DNASTAR can be used. Those skilled in the art can determine appropriate parameters for measuring alignment, including the algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0030] The terms "trimeric complex" and "trimer" are interchangeable terms used herein and refer to a complex formed by the non-covalent or covalent bonding of three monomer polypeptides. Each monomer polypeptide may be identical or different. The term "homotrimer" refers to a complex containing three monomers having the same amino acid sequence. The term "heterotrimer" refers to a complex containing three monomers in which at least one monomer has a sequence difference from the others.

[0031] The term "fusion polypeptide" refers to a hybrid (e.g., chimeric, recombinant) polypeptide that contains protein domains derived from at least two different naturally occurring proteins.

[0032] The terms "extracellular domain" and "ectodomain" are interchangeable terms used herein and refer to the region located outside the vesicle membrane in membrane proteins such as transmembrane proteins. Ectodomains often include a binding domain that specifically binds to a ligand or cell surface receptor (for example, a binding domain that specifically binds to the ligand or cell surface receptor). "Fas ectodomain" refers to a binding domain that may be involved in the formation of a trimer that binds to the Fas ligand (FasL).

[0033] The terms "endodomain," "intracellular domain," or "cytoplasmic domain" are interchangeable terms used herein and refer to the region of certain membrane proteins, such as transmembrane proteins, that extends into the internal space defined by the cell surface membrane. In some cells, the endodomain can interact with intracellular components and participate in signal transduction, and therefore may in some cases become an intracellular signal transduction domain.

[0034] As used herein, the term "transmembrane domain" refers to a domain in a membrane protein that substantially or completely penetrates a lipid bilayer found in biological membranes such as mammalian cells, or a lipid bilayer found in artificial structures such as liposomes. A transmembrane protein may pass through both layers of the lipid bilayer once or multiple times.

[0035] The term "functional fragment" refers to a truncated polypeptide that retains the function of the full-length polypeptide.

[0036] As used herein, the terms “specifically binds” or “bind” are interchangeable and refer to the ability of a protein to bind to a target protein under specific binding conditions. The affinity or avidity is at least five times the average affinity or avidity that the same protein exhibits to a statistically sufficient-sized random peptide or polypeptide assembly, and may optionally be at least 10, 20, 30, 40, 50, 100, 250, or 500 times, or even at least 1000 times. A specifically binding protein does not need to bind exclusively to a single target molecule; it may also specifically bind to a non-target molecule due to structural conformational similarity between the target and non-target molecules (e.g., paralogs or orthologues).

[0037] The term "endogenous" refers to proteins or nucleic acids that are naturally present within cells or naturally produced by cells. The term "exogenous" refers to proteins or nucleic acids that are not derived from cells but are introduced into cells by DNA transduction.

[0038] The term "autologous" refers to any material originating from the same individual that is later reintroduced into the individual in question. The term "allogeneic" refers to any material originating from an animal of the same species as the individual into which the material is introduced, but from a different individual. Two or more individuals are said to be of the same species if they do not have identical genes at one or more gene loci. In some embodiments, allogeneic material from individuals of the same species may be genetically sufficiently different to be antigenically interactable.

[0039] The term "gain-of-function" refers to any mutation, such as substitution, deletion, or insertion, that causes the mutant protein to acquire a function not typically associated with the non-mutated protein.

[0040] The term "expression" refers to the process by which polypeptides are produced based on the coding sequence of nucleic acid molecules such as genes. This process may include transcription, post-transcriptional regulation, post-transcriptional modification, translation, post-translational regulation, post-translational modification, or any combination thereof.

[0041] The term "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to function as a template for the biosynthesis of defined nucleotide sequences (e.g., rRNA, tRNA, and mRNA) or defined amino acid sequences, as well as other polymers and macromolecules possessing the resulting biological properties. Therefore, a gene, cDNA, or RNA encodes a protein when the transcription and translation of the mRNA corresponding to that gene produces that protein in a cell or other biological system.

[0042] The terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a peptide sequence. The term "polypeptide" is also used to include post-expression modified products, including modifications by glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-natural amino acids. Polypeptides in this disclosure may have sizes of approximately 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, or 2000 or more amino acids. Polypeptides may have a distinct three-dimensional structure, but are not required to have such a structure.

[0043] The term "polynucleotide" refers to an isolated nucleic acid molecule or structure, such as messenger RNA (mRNA), viral RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or atypical bonds (e.g., amide bonds found in peptide nucleic acids (PNA)). A "nucleic acid molecule" refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA or RNA fragments.

[0044] An "isolated" nucleic acid molecule is a nucleic acid molecule that has been separated from its natural environment. An isolated nucleic acid molecule includes, even if it is normally contained within a cell, cases where the nucleic acid molecule exists outside of a chromosome or at a chromosomal location different from its natural chromosomal location. An "isolated polynucleotide (or nucleic acid) encoding a fusion polypeptide" means one or more polynucleotide molecules encoding a fusion polypeptide, including cases where the polynucleotide molecules exist in a single vector or in separate vectors, and cases where the polynucleotide molecules exist at one or more locations within a host cell.

[0045] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid it has bound to. This term includes not only vectors as self-replicating nucleic acid structures, but also vectors integrated into the genome of a host cell. Certain vectors can induce the expression of a functionally linked nucleic acid.

[0046] The terms "subject" or "individual" are intended to include organisms capable of eliciting an immune response (e.g., mammals, humans).

[0047] As used herein, “treatment” (and grammatical forms such as “treat” and “treating”) refers to a clinical intervention aimed at altering the natural course of a disease in an individual being treated. This may be performed for preventive purposes or during the course of a clinical condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, improvement or mitigation (variation) of the disease state, and remission or improved prognosis.

[0048] The term "pharmaceutical composition" refers to a formulation in which the biological activity of the active ingredient contained in the composition is effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. A pharmaceutical composition usually contains one or more pharmaceutically acceptable carriers. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than the active ingredient that is non-toxic to the subject. pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0049] An "effective amount" refers to the quantity of a drug, such as a pharmaceutical composition, that is effective when administered in the dose and duration necessary to achieve the desired therapeutic or preventive effect.

[0050] II. Trimeric Complexes and Fusion Polypeptides This disclosure provides novel modified cells expressing a trimer complex. The trimer complex comprises at least one fusion polypeptide, the fusion polypeptide comprising a) an extracellular component containing a Fas ectodomain, b) a transmembrane component, and c) an intracellular component. Compared to cells that do not express the trimer complex or the fusion polypeptide, the modified cells exhibit improved persistence and / or proliferation, as well as reduced toxicity.

[0051] 2.1 Extracellular components of fusion polypeptides

[0052] In one embodiment, a fusion polypeptide as defined above is provided herein, wherein the extracellular component comprises a Fas ectodomain. In one embodiment, the Fas ectodomain is a full-length Fas ectodomain or a functional fragment thereof. In one embodiment, the functional fragment of the full-length Fas ectodomain can be involved in the formation of a trimer that binds to a Fas ligand (FasL). In one embodiment, the Fas ectodomain does not contain a signal peptide (typically located at amino acids 1-25 of wild-type Fas having a signal peptide). In one embodiment, the Fas ectodomain is a human Fas ectodomain.

[0053] In one embodiment, the Fas ectodomain is cleaved by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids at its N-terminus, corresponding to the wild-type Fas ectodomain (e.g., SEQ ID NO: 1). In one embodiment, the Fas ectodomain includes substitutions or deletions at positions 6, 7, 8, and / or 9. In one embodiment, the Fas ectodomain includes a deletion at position 6 or 7. In one embodiment, the Fas ectodomain includes a point substitution at position 7. In one embodiment, the Fas ectodomain includes a point substitution S7A. In one embodiment, the Fas ectodomain is cleaved by only 1, 2, 3, 4, 5, 6, or 7 amino acids at its C-terminus. In one embodiment, the Fas ectodomain includes a human Fas ectodomain. In one embodiment, the Fas ectodomain comprises cysteine-rich domain 1, cysteine-rich domain 2, and cysteine-rich domain 3 of a human Fas ectodomain. In one embodiment, the Fas ectodomain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 1. In one embodiment, the Fas ectodomain comprises or consists of the amino acid sequence of SEQ ID NO: 1.

[0054] 2.2 Transmembrane components of fusion polypeptides

[0055] In one embodiment, the fusion polypeptide comprises a transmembrane component operably linked to the extracellular component and the intracellular component. The transmembrane component comprises a native or unnative transmembrane domain. In a fusion polypeptide comprising an IL-7α end domain, the transmembrane domain comprises one or more mutations that enable the transmembrane protein to self-activate compared to its corresponding wild-type peptide. In a fusion polypeptide comprising only a CD2 end domain, the transmembrane domain comprises a naturally occurring transmembrane domain.

[0056] In one embodiment, the transmembrane component is self-oligomerizable. Such a transmembrane component may fuse its C-terminus to the N-terminus of an IL-7R endodomain. The transmembrane domain of the transmembrane component enables homodimerization of two separate fusion polypeptides, each containing the transmembrane domain. In one embodiment, the transmembrane domain induces structural twisting of the transmembrane region and endodomain of the fusion polypeptide to form a self-activating helix structure. In one embodiment, the transmembrane component contains one or more gain-of-function mutations corresponding to naturally occurring proteins in healthy subjects. The mutations may be substitutions, insertions, deletions, or combinations thereof. In one embodiment, the one or more mutations include the introduction of at least one cysteine ​​and / or at least one proline. In one embodiment, the mutant polypeptide containing the cysteine ​​insertion induces disulfide bond formation in the transmembrane domain. In one embodiment, the mutation is a gain-of-function insertion. In one embodiment, the gain-of-function insertion enables homodimerization of the polypeptide. In one embodiment, the gain-of-function insertion is a CPT or PPCL. In one embodiment, the insertion is located after the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, or 24th amino acid in SEQ ID NO: 10. In one embodiment, the transmembrane domain of the transmembrane component consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 33.

[0057] In one embodiment, if the intracellular component includes an IL-7R endodomain, the transmembrane component consists of the amino acid sequence of SEQ ID NO: 11 (PILLTCPTIX1IX2SX3X4X5X6X7X8X9X10X11LX12X13X14LW), where X1 is S or L; X2 is L or S; X3 is F or L; X4 is F or A; X5 is S or I; X6 is V or L; X7 is A or L; X8 is L or M; X9 is L or V; X10 is V or S; X11 is I or L; X12 is A or L; X13 is C or L; and X14 is V or S. In one embodiment, X1 is S; X2 is L; X3 is F or L; X4 is F or A; X5 is S or I; X6 is V or L; X7 is A or L; X8 is L or M; X9 is L or V; X10 is V or S; X11 is I or L; X12 is A or L; X13 is C or L; X14 is V or S. In one embodiment, X1 is S; X2 is L; X3 is L; X4 is A; X5 is I; X6 is L; X7 is A or L; X8 is L or M; X9 is L or V; X10 is V or S; X11 is I or L; X12 is A or L; X13 is C or L; X14 is V or S. In one embodiment, X1 is S; X2 is L; X3 is F; X4 is F; X5 is S; X6 is V; X7 is A or L; X8 is L or M; X9 is L or V; X10 is V or S; X11 is I or L; X12 is A or L; X13 is C or L; X14 is V or S. In one embodiment, X1 is S; X2 is L; X3 is L; X4 is A; X5 is I; X6 is L; X7 is L; X8 is M; X9 is V; X10 is S; X11 is I or L; X12 is A or L; X13 is C or L; X14 is V or S.In one embodiment, X1 is S; X2 is L; X3 is F; X4 is F; X5 is S; X6 is V; X7 is A; X8 is L; X9 is L; X10 is V; X11 is I or L; X12 is A or L; X13 is C or L; X14 is V or S. In one embodiment, X1 is S; X2 is L; X3 is F; X4 is F; X5 is S; X6 is V; X7 is A; X8 is L; X9 is L; X10 is V; X11 is I; X12 is A; X13 is C; X14 is V or S. In one embodiment, the transmembrane component consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 to 39.

[0058] In one embodiment, the transmembrane component is Fas, CD2, CD3s, CD35, CD3C, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, OX40, 4-IBB, SLAMF1, CTLA4, CD200R, LAG3, HVEM, BTLA, PD-L2, PD-L1, ICOS, PD-1, CD300, GITR, A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, KG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPa, pTa, TCRa, TCRβ, TIM3, TRIM, LPA5 The transmembrane component consists of a transmembrane domain selected from the group consisting of and Zap70. The C-terminus of such a transmembrane component may be fused to the N-terminus of a CD2 end domain. In one embodiment, the transmembrane domain may be modified compared to naturally occurring transmembrane domains, as long as the original function is maintained. In one embodiment, the transmembrane component consists of a Fas transmembrane domain. In one embodiment, the Fas transmembrane domain is a human Fas transmembrane domain. In one embodiment, the transmembrane component consists of an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 9. In one embodiment, the transmembrane component consists of the amino acid sequence of SEQ ID NO: 9.

[0059] 2.3 Intracellular components of fusion polypeptides

[0060] In one embodiment, the fusion polypeptide comprises an intracellular component including an IL-7R (IL7 receptor) endodomain, a CD2 endodomain, or a combination thereof, wherein the intracellular component does not include a Fas endodomain. In one embodiment, the intracellular component includes an IL-7R endodomain or consists of such a sequence. In one embodiment, the intracellular component includes a CD2 endodomain or consists of such a sequence. In one embodiment, the intracellular component includes an IL-7R endodomain and a CD2 endodomain or consists of such a sequence. In one embodiment, the Fas endodomain not included in the intracellular component is a full-length Fas endodomain or a functional fragment thereof.

[0061] In one embodiment, the IL-7R end domain is an IL-7Rα end domain. In one embodiment, the IL-7Rα end domain is a human IL-7Rα end domain. In one embodiment, the IL-7R end domain may be modified compared to a naturally occurring transmembrane domain, provided that signal transduction activity is maintained. In one embodiment, the IL-7R end domain is a full-length IL-7R end domain or a functional fragment thereof. In one embodiment, the IL-7R end domain consists of 70-195 amino acids, 70-175 amino acids, 70-150 amino acids, 70-125 amino acids, 70-100 amino acids, 80-195 amino acids, 80-175 amino acids, 80-150 amino acids, 80-100 amino acids, 90-195 amino acids, 90-175 amino acids, 90-150 amino acids, 90-125 amino acids, 90-100 amino acids, 100-195 amino acids, 100-175 amino acids, 100-150 amino acids, 100-125 amino acids, 125-195 amino acids, 125-175 amino acids, 125-150 amino acids, 150-195 amino acids, 150-175 amino acids, or 175-195 amino acids. In one embodiment, the intracellular component includes or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 4. In one embodiment, the intracellular component includes or consists of the amino acid sequence of SEQ ID NO: 4.

[0062] In one embodiment, the CD2 end domain is a human CD2 end domain. In one embodiment, the CD2 end domain may be modified compared to a naturally occurring CD2 end domain, provided that signal transduction activity is maintained. In one embodiment, the CD2 end domain is a full-length CD2 end domain or a functional fragment thereof. In one embodiment, the functional fragment has its C-terminal end cleaved by up to 65 amino acids compared to a full-length CD2 end domain. In one embodiment, the functional fragment is cleaved by 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid at its C-terminal end compared to the full-length CD2 end domain. In one embodiment, each intracellular component contains or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NOs. 5 to 8. In one embodiment, the intracellular component contains or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs. 5 to 8.

[0063] 2.4 Structure of fusion polypeptide

[0064] In one embodiment, the fusion polypeptide comprises, or consists of, an extracellular component containing a Fas ectodomain, a transmembrane component, and an IL-7R endodomain, in that order from its N-terminus to its C-terminus. In one embodiment, the fusion polypeptide comprises, or consists of, an extracellular component containing a Fas ectodomain, a transmembrane component, and a CD2 endodomain, in that order from its N-terminus to its C-terminus. In one embodiment, the fusion polypeptide comprises, or consists of, an extracellular component containing a Fas ectodomain, a transmembrane component, and an IL-7R endodomain and a CD2 endodomain, in that order from its N-terminus to its C-terminus. In one embodiment, each of the components is fused to one another directly or via a flexible peptide. In one embodiment, each of the components is fused to one another directly. Exemplary fusion polypeptides are shown in Table 4.

[0065] In one embodiment, the extracellular component may further contain ectodomains of proteins other than Fas, insofar as it maintains the function of a fusion polypeptide having only Fas ectodomains. In one embodiment, the function is to be involved in the formation of a trimer complex. In one embodiment, the proteins other than Fas are selected from the group consisting of PD-1, CD30, HER2, EGFR, CD19, CD34, TGFβR, IL-4R, IL-13R, IL-8R, IL-10R, LAG3, TIGIT, CTLA4, CD19, CD27, CD28, CD52, CD134, CD137, HER2, EGFR, and NGFR.

[0066] In one embodiment, the intracellular component may further contain the end domains of proteins other than IL-7R or CD2, insofar as the intracellular component maintains the signal transduction activity of a fusion polypeptide having only the IL-7R and / or CD2 end domains. In one embodiment, the proteins other than IL-7R or CD2 are selected from the group consisting of IL-2R, IL-6R, IL-12R, IL-21R, IL-23R, and CD122.

[0067] 2.5 Trimeric complex containing fusion polypeptide

[0068] This disclosure provides a trimer complex comprising the fusion polypeptide described herein. In one embodiment, the trimer complex is capable of binding to a Fas ligand. In one embodiment, the trimer complex is capable of inducing signal transduction of the intracellular component independently of binding to the Fas ligand.

[0069] In one embodiment, the trimer complex is formed on the surface of a modified cell expressing the fusion polypeptide. In one embodiment, the trimer complex is formed automatically on the surface of a modified cell expressing the fusion polypeptide. In one embodiment, the extracellular component of the fusion polypeptide includes cysteine-rich domain 1, cysteine-rich domain 2, and cysteine-rich domain 3. Cysteine ​​residues in these cysteine-rich domains contribute to the formation of the trimer complex. In one embodiment, the trimer complex is formed via disulfide bridges between the cysteine-rich domains. In one embodiment, the trimer complex is formed via disulfide bridges between cysteine-rich domains 1.

[0070] In one embodiment, the trimer complex is a homotrimer comprising three identical fusion polypeptides. In one embodiment, the trimer complex is a heterotrimer comprising at least one fusion polypeptide. In one embodiment, the one fusion polypeptide comprises a CD2 endodomain. In one embodiment, the trimer complex is a heterotrimer comprising two or three identical fusion polypeptides. In one embodiment, the two or three identical fusion polypeptides each comprise an IL-7R endodomain and / or a CD2 endodomain. In one embodiment, the two or three identical fusion polypeptides each comprise an IL-7R endodomain. In one embodiment, the two or three identical fusion polypeptides each comprise an IL-7R endodomain and a CD2 endodomain. In one embodiment, the two or three identical fusion polypeptides each comprise a CD2 endodomain. In one embodiment, the two or three identical fusion polypeptides each comprise a self-oligomerizable transmembrane component. Surprisingly, self-oligomerization of the transmembrane component does not affect the formation and function of the trimer complex. In one embodiment, the heterotrimer comprises one or two endogenous Fas monomers.

[0071] III. Modified cells and their preparation 3.1 Cells

[0072] In one embodiment, the present disclosure provides modified cells, such as modified immune cells, comprising the fusion polypeptide or trimer complex described herein. The origin of the modified immune cells of the present disclosure may be the patient being treated (i.e., autologous cells) or a donor other than the patient being treated (e.g., allogeneic cells).

[0073] In one aspect, the cell is an immunoresponsive cell. In one aspect, the cell is a cell of the lymphoid lineage. In one aspect, the modified immune cell is a modified T cell. In one aspect, the T cell is derived from a mammalian individual. In one aspect, the T cell is derived from a primate individual such as a human individual. Examples of T cells and / or subtypes and subpopulations of CD4+ and / or CD8+ T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes (stem cell-like memory T (TSCM), central memory T (TCM), effector memory T (TEM), terminally differentiated effector memory T cells, etc.), tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal associated invariant T (MAIT) cells, natural and adaptive regulatory T (Treg) cells, helper T cells (TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, etc.), α / β T cells, and δ / γ T cells, etc. Non-limiting examples of commercially available T cell lines include BCL2(AAA) Jurkat (ATCC 登録商標 CRL-2902 商標 ), BCL2(S70A) Jurkat (ATCC 登録商標 CRL-2900 商標 ), BCL2(S87A) Jurkat (ATCC 登録商標 CRL-2901 商標 ), BCL2 Jurkat (ATCC 登録商標 CRL-2899 商標 ), Neo Jurkat (ATCC 登録商標 CRL-2898 商標Examples include the TALL-104 cytotoxic human T cell line (ATCC # CRL-11386), etc. Furthermore, there are mature T cell lines (e.g., Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki 225, My-La, Se-Ax, SKW-3, SMZ-1, T34, etc.) and immature T cell lines (e.g., ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37, KOPT-K1, K-T1, L-KAW, Luucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13, MOLT-16, MT-1, MT-ALL, P12 / Ichikawa, Peer, PER0117, PER-255, PF-382, PFI-285, RPMI-8402, ST-4, SUP-T1~T14, TALL-1, TALL-101, T ALL-103 / 2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, TK-6, TLBR-1, -2, -3, and -4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCC TIB-153), J45.01 (ATCC CRL-1990), J.CaM1.6 (ATCC CRL-2063), RS4;11 (ATCC Examples of commercially available cell lines include, but are not limited to, CRL-1873, CCRF-CEM (ATCC CRM-CCL-119), and cutaneous T-cell lymphoma lines (e.g., HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), HuT102 (ATCC TIB-162), etc.). Non-limited sources of such commercially available cell lines include the American Type Culture Collection (ATCC) (Manassas, Virginia, USA) and the German Collection of Microorganisms and Cell Cultures.

[0074] In one embodiment, the cells are cytotoxic T cells (TC, also known as cytotoxic T lymphocytes (CTLs), T killer cells, lytic T cells, CD8+ T cells, or killer T cells). In one embodiment, the T cells are CD4+ T cells. In one embodiment, the T cells may be CD4+ T cells or CD8+ T cells. In one embodiment, the cells are tumor-specific T cells.

[0075] In one embodiment, the cells are natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer (LAK) cells, etc. NK cells may be isolated or obtained from a commercially available source. A non-limiting example of a commercially available NK cell line is NK-92 (ATCC). 登録商標 CRL-2407 商標 ), NK-92MI (ATCC 登録商標 CRL-2408 商標 Examples include ) and, but are not limited to, NK cell lines such as HANK1, KHYG-1, NKL, NK-YS, NOI-90, and YT. Non-limited sources of such commercially available cell lines include ATCC (Manassas, Virginia, USA) and the German Collection of Microorganisms and Cell Cultures.

[0076] In one embodiment, the cells are B cells, monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and / or basophils.

[0077] In one embodiment, the cells are myeloid lineage cells. Non-limiting examples of myeloid lineage cells include monocytes, macrophages, basophils, neutrophils, eosinophils, mast cells, erythrocytes, megakaryocytes, platelets, and stem cells that can differentiate into myeloid cells. In one embodiment, the stem cells are pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells).

[0078] 3.2 Antigen receptors contained in modified cells

[0079] In one embodiment, the disclosure provides modified cells comprising a fusion polypeptide or trimer complex described herein and an antigen receptor (e.g., a chimeric antigen receptor (CAR) or antigen-specific TCR) that binds to an antigen. The antigen-recognizing receptor may bind to a tumor antigen or a pathogen antigen. In one embodiment, the antigen-recognizing receptor binds to a tumor antigen. In one embodiment, the tumor antigen is a tumor-specific antigen or a tumor-associated antigen. In one embodiment, the fusion polypeptide results in an enhancement of the function of the antigen receptor.

[0080] CARs can be chimeric type I transmembrane proteins that link an extracellular antigen-recognition domain (binder) to an intracellular signaling domain (endodomain). A spacer domain may be necessary to isolate the binder from the membrane and enable proper orientation. Depending on the antigen, a more compact spacer may suffice, such as the stalk of CD8α or even just the hinge of IgG1. The transmembrane domain fixes the protein to the cell membrane and links the spacer to the endodomain.

[0081] 3.2.1 Antigen-binding domain of CAR

[0082] In one embodiment, the antigen-binding domain may include a single-chain variable fragment (scFv) derived from a monoclonal antibody, a natural ligand of the target antigen, a peptide having sufficient affinity for the target, a single-domain conjugate such as a single-domain conjugate derived from a camelid, an artificial conjugate such as Darpin, or a single chain derived from a T cell receptor. In one embodiment, the antigen-binding domain may include a monospecific, bispecific, or multispecific antibody molecule.

[0083] In one embodiment, the chimeric antigen receptor is CD19, CD20, CD22, CD30, BCMA, AFP, ALK, GPC3, HER2, EGFR, 5T4, avβ6 integrin, B7-H3, B7-H6, CA-125, CAIX, CD5, CD13, CD16, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD160, CD171, CEA, claudin 18.2, CSPG4, DLL3, EGFR, HER2, EGFRvlll, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, folate binding protein, fetal carbonic anhydrase IX The target is IX), AchR, FRa, G250, GD2, GD3, MAGE-1, NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesoserine, Muc1, Muc16, NCAM, NKG2D ligand, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TAG72, TEMs, VEGFR2, Vimentin, or WT-1. Several tumor-associated antigens are known in Table 1 below. Furthermore, the antigen-binding domain used in the present invention may be a domain capable of binding to the tumor-associated antigens (TAAs) shown in the table. [Table 1]

[0084] 3.2.2 CAR Spacer Domain

[0085] The CAR may include a spacer sequence for linking the antigen-binding domain to the transmembrane domain. The flexible spacer allows the antigen-binding domain to be oriented in different directions, thereby facilitating binding. In one embodiment, the spacer sequence includes the Fc region of IgG1, the IgG1 hinge, the IgG4 hinge, a human CD8 stalk, or a mouse CD8 stalk. In one embodiment, the spacer includes a linker sequence having a length and / or domain spacing characteristics similar to that of the Fc region of IgG1, the IgG1 hinge, or the CD8 stalk.

[0086] 3.2.3 Transmembrane domain of CAR

[0087] The transmembrane domain is a transmembrane sequence in classical CARs. In one embodiment, the transmembrane domain may be obtained from either natural or recombinant sources. In one embodiment, the transmembrane domain is a natural peptide derived from any membrane-binding protein or transmembrane protein. In one embodiment, the transmembrane domain can transmit a signal to an intracellular domain(s) when the CAR binds to a target. Particularly useful transmembrane domains in the present invention include, for example, those containing at least the transmembrane regions of the α, β, or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8 (e.g., CD8α, CD8β), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, etc.

[0088] 3.2.4 Intracellular signaling domain of CAR

[0089] Intracellular signaling domains are generally involved in the activation of at least one normal effector function. In one embodiment, the intracellular signaling domain comprises two distinct classes of cytoplasmic signaling sequences, namely, a sequence that initiates antigen-dependent primary activation via the TCR (primary intracellular signaling domain) and a sequence that acts antigen-independently to confer secondary or co-stimulatory signals (secondary cytoplasmic domain, e.g., co-stimulatory domain).

[0090] The primary signaling domain modulates the primary activation of the TCR complex either stimulatively or repressively. Stimulative primary intracellular signaling domains may include signaling motifs known as immune receptor tyrosine-dependent activation motifs (ITAMs). Examples of primary intracellular signaling domains containing ITAMs that are particularly useful in the present invention include those of TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, DAP10, DAP12, and CD66d. In one embodiment, the CAR includes an intracellular signaling domain such as the CD3-ζ primary signaling domain. In one embodiment, the primary signaling domain includes a modified ITAM domain, such as a mutant ITAM domain whose activity is altered (e.g., enhanced or reduced) compared to a native ITAM domain. In one embodiment, the primary signaling domain includes a modified ITAM-containing primary intracellular signaling domain, such as an optimized and / or cleaved ITAM-containing primary intracellular signaling domain. In one embodiment, the signaling domain of CD3-ζ is mutant CD3ζ or wild-type human CD3ζ. In one embodiment, the primary signaling domain contains one, two, three, four or more ITAM motifs.

[0091] In one embodiment, the intracellular signaling domain of CAR may include a primary signaling domain and a co-stimulatory signaling domain. In one embodiment, the co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand, which is necessary for the efficient response of lymphocytes to an antigen. Examples of such molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SEAM proteins), activated NK cell receptors, BTFA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, FFA-1 (CD11a / CD18), 4-1BB (CD1 37), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, FIGHT, HVEM, KIRDS2, SFAMF7, NKp80 (KFRF1), NKp44, NKp30, N Kp46, CD19, CD4, CD8α, CD8β, IF2Rβ, IF2Rγ, IF7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VFA-6, CD49f, ITG AD, CD11d, ITGAE, CD103, ITGAF, FFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, FFA-1, ITGB7, NKG2 D, NKG2C, TNFR2, TRANCE / RANKF, DNAM1(CD226), SFAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Fy9(C Examples of ligands that specifically bind to CD83 include, but are not limited to, D229, CD160(BY55), PSGF1, CD100(SEMA4D), CD69, SFAMF6(NTB-A, Fy108), SEAM(SFAMF1, CD150, IPO-3), BFAME(SFAMF8), SEPFPG(CD162), FTBR, FAT, GADS, SFP-76, PAG / Cbp, CD19a, and CD83. The intracellular signal sequences within the cytoplasmic portion of the CAR of the present invention can be linked to each other in a random or specific order.In one embodiment, the intracellular signaling domain is designed to include two or more, for example, two, three, four, five, or more co-stimulatory signaling domains.

[0092] In one embodiment, the modified cells include recombinant T cell receptors (TCRs) and / or TCRs cloned from naturally occurring T cells. The TCRs or their antigen-binding moieties include those that recognize peptide epitopes or T cell epitopes of target polypeptides such as tumor antigens, virus-derived proteins, or autoimmune-related proteins. In one embodiment, the TCRs have binding specificity to tumor-related antigens, such as carcinoembryonic antigen (CEA), GP100, T cell-recognized melanoma antigen 1 (MART1), melanoma antigen A3 (MAGEA3), melanoma antigen A4 (MAGEA4), or p53.

[0093] 3.3 Exogenous FasL contained in modified cells

[0094] In one embodiment, the disclosure provides modified cells comprising the fusion polypeptide or trimer complex described herein and exogenous FasL. In one embodiment, the disclosure provides modified cells comprising the fusion polypeptide or trimer complex, antigen receptor, and exogenous FasL described herein. FasL binds to Fas and induces apoptosis in both target cells and immune-responsive cells. Surprisingly, the fusion polypeptide or trimer complex of the disclosure can protect modified cells from fratricide or suicide-type killing caused by exogenous Fas. Therefore, co-expression of the fusion polypeptide or trimer complex and FasL can improve at least the cytotoxic activity, persistence, and / or proliferation of modified cells.

[0095] In one embodiment, the modified cells overexpress FasL. In one embodiment, FasL is constitutively expressed on the surface of modified cells (e.g., T cells or NK cells). In one embodiment, the exogenous FasL is human FasL. In one embodiment, the exogenous FasL is mouse FasL. In one embodiment, the exogenous FasL is secreted. In one embodiment, the exogenous FasL is membrane-bound. In one embodiment, FasL may be modified compared to naturally occurring FasL (e.g., a polypeptide containing the amino acid sequence of SEQ ID NO: 40) insofar as apoptotic activity is maintained. In one embodiment, FasL is full-length FasL or a functional fragment thereof. In one embodiment, FasL includes or consists of an amino acid sequence comprising amino acids 1-281, 1-80, 81-102, 1-102, 1-110, 1-127, 81-102, 103-281, 132-281, 134-281, or 135-281 of the amino acid sequence of SEQ ID NO: 40. In one embodiment, FasL includes or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 40 to SEQ ID NOs: 47. In one embodiment, (as in the original text) the intracellular component includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 40 to SEQ ID NOs: 47.

[0096] 3.4 Methods of genetic engineering modification

[0097] In one embodiment, the modified cells are prepared by various methods of introducing a fusion polypeptide, an antigen receptor (e.g., CAR), and / or a polynucleotide encoding FasL. Physical methods include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. Biological methods include the use of DNA and RNA vectors, such as viral vectors and lentiviral vectors. Chemical methods include colloidal dispersions such as polymer complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. Exemplary methods are shown in Table 2. [Table 2]

[0098] In one embodiment, recombinant polynucleotides are introduced into cells using recombinant infectious viral particles (e.g., adenovirus vectors, AAV vectors, lentiviral vectors, retroviral vectors (gamma-retroviral vectors, etc.)). In one embodiment, the retroviral vector or lentiviral vector has a long-end repeat sequence (LTR). In one embodiment, the vector is self-inactivating (SIN). In one embodiment, the vector is a conditionally replicating (mobilizable) vector. In one embodiment, the lentiviral vector is derived from a human, feline, or monkey lentivirus. In one embodiment, the retroviral vector is derived from a mouse retrovirus. In one embodiment, the lentivirus or retrovirus includes those derived from any avian or mammalian cell. In one embodiment, the lentivirus or retrovirus is amphotropic, meaning it can infect multiple host cell species, including humans. In one embodiment, the gene to be expressed substitutes the gag, pol, and / or env sequences of the retrovirus.

[0099] In one embodiment, a vector comprising a polynucleotide encoding a fusion polypeptide may include a promoter and / or enhancer or regulatory element for regulating the expression of the encoded fusion polypeptide. In one embodiment, the promoter and / or enhancer or regulatory element may be a condition-dependent promoter, enhancer, and / or regulatory element. In one embodiment, the polynucleotide encoding the fusion polypeptide may be operably linked to a constitutive promoter. In one embodiment, the promoter is selected from the group consisting of the cytomegalovirus (CMV) promoter, the elongation factor 1α (EF1α) promoter, the ubiquitin C promoter (UbiC), the phosphoglycerokinase promoter (PGK), the monkey virus 40 initial promoter (SV40), and the chicken β-actin promoter (CAGG) linked to the CMV initial enhancer.

[0100] In one embodiment, the polynucleotide is operably linked to an inducible promoter. The inducible promoter can be induced by one or more conditions, such as physical conditions, the microenvironment of modified immunoeffector cells, the physiological state of modified immunoeffector cells, an inducer (i.e., an inducer), or a combination thereof. In one embodiment, the inducible conditions do not induce the expression of endogenous genes in modified mammalian cells and / or in subjects receiving the pharmaceutical composition. In one embodiment, the inducible conditions are selected from the group consisting of an inducer, irradiation (ionizing radiation, light, etc.), temperature (heat, etc.), redox state, tumor environment, and the activation state of modified mammalian cells.

[0101] In one embodiment, the polynucleotide is operably linked to a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) located downstream of the polynucleotide.

[0102] In one embodiment, the vector may comprise a single promoter that drives the expression of one or more nucleic acid molecules. In one embodiment, such nucleic acid molecules may be multi-cistronic. For example, in one embodiment, the transcription unit may be designed as a dicistronic unit including an internal ribosome entry site (IRES), thereby allowing a single message from a single promoter to co-express a gene product (e.g., encoding a fusion polypeptide, optionally encoding CAR and FasL). In one embodiment, the single promoter may comprise two or three genes within a single open reading frame (ORF), which may direct the expression of RNAs separated from each other by sequences encoding self-cleaving peptides or sequences encoding protease recognition sites. In one embodiment, the self-cleaving peptides are selected from the group consisting of foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), and porcine tesiovirus-1 (P2A).

[0103] In one embodiment, the polynucleotide is introduced into T cells by electroporation. In one embodiment, the polynucleotide is introduced into T cells by transposition. In one embodiment, the polynucleotide is delivered by transposons including the Sleeping Beauty transposon system (SB) and / or the piggyBac (PB) transposon system.

[0104] The polynucleotide encoding the fusion polypeptide may be associated with an additional coding region encoding a secretory or signal peptide that directs the secretion of the fusion polypeptide. For example, if secretion of the fusion polypeptide is desired, DNA encoding a signal sequence may be placed upstream of the fusion polypeptide. Those skilled in the art understand that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, and that the secretory or “mature” polypeptide is produced by cleaving the signal peptide from the translated polypeptide. In one embodiment, the signal peptide includes the sequence of signal peptides for human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, CD37, CD45, 4-1BB, GM-CSFR, IL-2, CD33, human IgKVIII, human IgG2 H, chymotrypsinogen, trypsinogen-2, HSA, insulin, or tPA.

[0105] In one embodiment, the polynucleotide encoding the fusion polypeptide comprises nucleic acid sequences encoding one or more markers. In one embodiment, the one or more markers are a transduction marker, a surrogate marker, and / or a selection marker. In one embodiment, the polynucleotide encoding the fusion polypeptide comprises nucleic acid sequences encoding one or more additional modules that enhance and / or suppress the cellular response during adoptive transfer and ligand encounter.

[0106] In one embodiment, the polynucleotide may encode one or more surrogate markers. In one embodiment, the surrogate markers may include truncated forms of cell surface polypeptides, for example, truncated forms that are non-functional and do not transmit or are unable to transmit signals or signals normally transmitted by the full-length cell surface polypeptide, and / or truncated forms that do not internalize or are unable to internalize. In one embodiment, the truncated cell surface polypeptides include truncated forms of growth factors or other receptors (e.g., truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (tEGFR), truncated prostate-specific membrane antigen (PSMA), or variants thereof). tEGFR can be used to identify or select cells modified with tEGFR and the encoded exogenous protein, and / or to remove or isolate cells expressing the encoded exogenous protein.

[0107] In one embodiment, the marker is a fluorescent protein such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP) (e.g., superfold GFP (sfGFP)), red fluorescent protein (RFP) (e.g., tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2), cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), and their variants (including species-differential variants, monomeric variants, and codon-optimized and / or improved variants). In one embodiment, the marker is or includes an enzyme such as luciferase, the E. coli-derived lacZ gene, alkaline phosphatase, secretory embryonic alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT). Examples of luminescence reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or their variants.

[0108] In one embodiment, the marker is a selection marker. In one embodiment, the selection marker is a polypeptide that confers resistance to an exogenous agent or drug. In one embodiment, the selection marker is an antibiotic resistance gene. In one embodiment, the selection marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In one embodiment, the selection marker is selected from the group consisting of a pueomycin resistance gene, a hygromycin resistance gene, a blastosidine resistance gene, a neomycin resistance gene, a genetisin resistance gene, a zeosin resistance gene, or a variant thereof.

[0109] 3.5 Preparation of Modified Cells

[0110] In one embodiment, the present disclosure provides a process for producing modified cells. In one embodiment, any known method for preparation can be used. In a particular embodiment, the method comprises transducing an isolated cell population with a polynucleotide encoding a fusion polypeptide, and selecting a subpopulation of the isolated cells that have been successfully transduced with the fusion polynucleotide, thereby producing genetically modified cells as described above.

[0111] In one embodiment, the method includes the steps of acquisition, isolation, transduction, and propagation. In a particular embodiment, the method includes the following steps: (i) Acquisition of an immune cell population (e.g., blood cells); (ii) Isolation of a specific cell population (e.g., T cells and / or NK cells); (iii) Transduction of an isolated cell population by a polynucleotide encoding a fusion polypeptide; and (iv)(iii) The subpopulation of the isolated cells that have been successfully transduced by the nucleic acid sequence of (iv)(iii) is propagated to produce genetically modified cells. Each of these steps will be explained in more detail below.

[0112] 3.5.1 Cell acquisition

[0113] In one embodiment, the subjects from whom cells are obtained for the introduction of a fusion polypeptide are subjects having the disease or condition, or subjects requiring cell therapy, or subjects receiving cell therapy. In one embodiment, the cells may be derived from a healthy donor.

[0114] In one embodiment, the cells may be obtained from a sample such as a biological sample. In one embodiment, the sample is selected from whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, intestinal lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organs and / or cells derived therefrom. In one embodiment, the cells are primary cells. In one embodiment, cells are obtained from the circulating blood of the subject, for example, by apheresis or leukapheresis. The obtained sample includes lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and / or platelets, and in some embodiments includes cells other than erythrocytes and platelets.

[0115] 3.5.2 Cell Isolation

[0116] Various methods for isolating immune cells from a sample are readily available, for example, Life Technologies Dynabeads 登録商標 System; EasySep by STEMcell Technologies 商標 RoboSep 商標 RosetteSep 商標 SepMate 商標 MACS by Miltenyi Biotec 商標Cell isolation kits; these include those based on cell surface marker expression and other commercially available cell isolation kits (e.g., ISOCELL from Pierce, Inc., Rockford, Illinois, USA). Specific subpopulations of immune cells can be isolated using beads or other binders included in the kit, specifically targeting unique cell surface markers. For example, MACS. 商標 CD4+ and CD8+ T cells can be isolated using CD4+ and CD8+ MicroBeads.

[0117] In one embodiment, the isolation of the cells comprises one or more non-affinity-based cell separation steps. In one embodiment, the cells are washed, centrifuged and / or incubated in the presence of one or more reagents for purposes such as removing unwanted components, concentrating desired components, or lysing or removing cells sensitive to specific reagents. In one embodiment, the cells are separated based on one or more properties such as density, adhesion, size, sensitivity and / or resistance to specific components.

[0118] In one embodiment, blood cells collected from a subject are washed to remove, for example, the plasma fraction and replace the cells with a buffer or medium suitable for the subsequent step. In one embodiment, the cells are washed with phosphate-buffered saline (PBS). In one embodiment, the washing solution is calcium-free, magnesium-free, or lacks many, or possibly all, divalent cations. The initial activation step in the absence of calcium may result in enhanced activation. In one embodiment, the washing step is carried out using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In one embodiment, the washing step is carried out by tangential flow filtration (TFF) according to the manufacturer's instructions. In one embodiment, after washing, the cells are treated with, for example, Ca 2+ / Mg 2+The cells are resuspended in various biocompatible buffers, such as free PBS. In one embodiment, components of the blood cell sample are removed, and the cells are directly resuspended in culture medium. In one embodiment, isolation includes density-based cell separation methods, such as erythrocyte lysis via a density gradient (Percoll or Ficoll gradient) and leukocyte preparation from peripheral blood by centrifugation.

[0119] In one embodiment, the isolation method includes separating different cell types based on the expression or presence of one or more specific molecules in cells, such as surface markers (surface proteins, etc.), intracellular markers, or nucleic acids. In one embodiment, the separation is based on affinity or immunoaffinity. Separation may be based on positive selection, which retains cells bound to a reagent for further use, and / or negative selection, which retains cells that did not bind to the antibody or binding partner. Separation does not necessarily result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker.

[0120] In one embodiment, a single separation step may simultaneously deplete cells expressing multiple markers by incubating cells with multiple antibodies or binding partners specific to each marker targeted for negative selection, for example. Similarly, multiple cell types may be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed on various cell types. In one embodiment, multiple separation steps are performed, and fractions positively or negatively selected in one step are subjected to the next separation step (e.g., subsequent positive or negative selection).

[0121] In one embodiment, a specific subpopulation, such as T cells that are positive for or highly express one or more surface markers, such as CD3+, CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, is isolated by positive or negative selection techniques. In one embodiment, the T cells are treated with anti-CD3 / anti-CD28 binding particles or beads (e.g., DYNABEADS). 登録商標 M-450 CD3 / CD28 T Cell Expander, MACSiBeads商標 They are isolated by incubation with (etc.). In one embodiment, the positive selection period is about 30 minutes. In further embodiments, the period is at least 1, 2, 3, 4, 5, or 6 hours. In one embodiment, the period is 10 to 24 hours. In one embodiment, the incubation time is 24 hours. The concentrations of cells and particles may vary to isolate the desired cell population by positive or negative selection. In one embodiment, it may be desirable to significantly reduce the volume of bead-cell mixing (i.e., increase the cell concentration) to ensure maximum contact between beads and cells. In one embodiment, concentrations greater than 100 million cells / mL are used.

[0122] In one embodiment, T cells are isolated from the sample by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes such as CD14. In another embodiment, a CD4 or CD8 selection step is used to separate CD4+ helper T cells from CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations may be further sorted into subpopulations by positive or negative selection for markers expressed, or expressed at relatively high levels, in one or more naive, memory, and / or effector T cell subpopulations.

[0123] In one embodiment, CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, etc., by positive or negative selection based on surface antigens associated with each subpopulation. In one embodiment, central memory T (TCM) cells are enriched to improve efficacy, such as improving long-term survival, proliferation, and / or engraftment after administration. In one embodiment, efficacy is further enhanced by combining TCM-enriched CD8+ T cells with CD4+ T cells.

[0124] In one embodiment, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted in terms of the CD62L-CD8+ and / or CD62L+CD8+ fractions using anti-CD8 antibodies and anti-CD62L antibodies, etc.

[0125] In one embodiment, enrichment of TCM cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127. In another embodiment, this is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In another embodiment, isolation of a CD8+ population rich in TCM cells is carried out by depleting cells expressing CD4, CD14, and CD45RA, and positively selecting or enriching cells expressing CD62L. In one embodiment, enrichment of TCM cells is initiated with a negative fraction of cells selected based on CD4 expression, and then negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L are sequentially performed on this fraction.

[0126] In one embodiment, NK cell enrichment is based on positive or high surface expression of CD56 and CD16, negative expression of CD3, and / or optionally the presence of NKp46 or NKp30 receptors.

[0127] In one embodiment, the sample or cell composition to be separated is a magnetizable or magnetic field-responsive small material, such as magnetic field-responsive particles or microparticles, such as paramagnetic beads (e.g., Dynabeads). 登録商標 or MACS 登録商標The material (e.g., beads) is incubated with the material. The magnetic field-responsive material (e.g., particles) is typically directly or indirectly bound to a binding partner (e.g., antibody) that specifically binds to molecules (e.g., surface markers) present on cells, cell groups, or cell populations that are to be separated (negative or positive selection). In one embodiment, the sample is placed in a magnetic field, and cells to which the magnetic field-responsive or magnetizable particles are attached are attracted to a magnet and separated from unlabeled cells. In positive selection, cells attracted to the magnet are retained; in negative selection, cells not attracted (unlabeled cells) are retained. In one embodiment, a combination of positive and negative selection is performed during the same selection step, and the positive and negative fractions are retained and further processed or subjected to further separation steps.

[0128] In one embodiment, the magnetic field-responsive particles remain attached to cells to be subsequently incubated, cultured, and / or modified; in another embodiment, the particles remain attached to cells for administration to a patient. In one embodiment, the magnetic field-responsive particles are removed from the cells. Methods for removing magnetic field-responsive particles from cells are known and include, for example, the use of competing unlabeled antibodies, or antibodies conjugated to magnetic field-responsive particles or cleavable linkers. In one embodiment, the magnetic field-responsive particles are biodegradable.

[0129] In one embodiment, the affinity-based selection is performed using magnetically activated cell sorting (MACS). 登録商標 (Miltenyi Biotec, Auburn, California, USA) MACS 登録商標 The system can select cells to which magnetized particles are attached with high purity. In one embodiment, MACS 登録商標 The system operates in a mode in which non-target and target species are sequentially eluted after the application of an external magnetic field. That is, cells to which magnetized particles are attached are retained in place, while species without attached particles are eluted. Subsequently, after the completion of the first elution step, species that were trapped in the magnetic field and whose elution was prevented are released by some means, eluted, and recovered. In one embodiment, non-target cells are labeled and depleted from a heterogeneous cell population.

[0130] In one embodiment, the cell population described herein is recovered and concentrated (or depleted) by flow cytometry, in which cells stained with multiple cell surface markers are transported in a fluid stream. In another embodiment, the cell population described herein is recovered and concentrated (or depleted) by preparation-scale accelerometry (FACS) sorting. In one embodiment, the cell population described herein is recovered and concentrated (or depleted) by the use of a microelectromechanical system (MEMS) chip combined with a FACS-based detection system. In any of these cases, since the cells can be labeled with multiple markers, isolation of a highly pure and well-defined T cell subset is possible.

[0131] In one embodiment, the preparation method includes a step of freezing (e.g., cryopreserving) the cells before or after isolation, incubation, and / or modification. In one embodiment, the freezing and subsequent thawing steps remove granulocytes and, to some extent, monocytes from the cell population. In one embodiment, the cells are suspended in a cryopreservation solution after a washing step to remove, for example, plasma and platelets. Various known cryopreservation solutions and parameters may be used. As an example, PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium may be used. This is diluted 1:1 with the medium to a final concentration of 10% DMSO and 4% HSA. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. In one embodiment, the cryopreserved cells are thawed and washed as described herein and allowed to rest at room temperature for 1 hour before activation.

[0132] 3.5.3 Cell proliferation

[0133] In one embodiment, the method provided herein comprises the steps of cultivation, incubation, culture, and / or genetic modification. In one embodiment, cells are incubated and / or cultured before or in connection with the genetic modification. The incubation step may include culture, cultivation, stimulation, activation, and / or propagation. In one embodiment, cells are incubated under stimulating conditions or in the presence of stimulants. The conditions may include a specific medium, temperature, oxygen content, carbon dioxide content, time, and one or more of nutrients, amino acids, antibiotics, ions, and / or stimulants (e.g., cytokines, chemokines, antigens, binding partners, fusion polypeptides, recombinant soluble receptors, and any other agents designed to activate cells).

[0134] In one embodiment, the stimulating condition or stimulant comprises one or more agents (e.g., ligands) capable of stimulating or activating the intracellular signaling domain of the TCR complex. In one embodiment, the agent turns on or initiates the TCR / CD3 intracellular signaling cascade in T cells. Examples of such agents include antibodies specific to TCR components and / or costimulatory receptors (e.g., anti-CD3, anti-CD28, etc.), and beads (e.g., Dynabeads). 登録商標 ) may be bound to a solid support such as; and / or may contain one or more cytokines. In one embodiment, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 80, 85, 90, 95, 100, 125, or 150 million cells / mL are used.

[0135] In one embodiment, the mixture may be cultured for a period of several hours (about 3 hours) to about 14 days, or any integer period in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment, the beads and the T cells are co-cultured for about 8 days. In another embodiment, the beads and the cells are co-cultured for 2-3 days. Multiple stimulation cycles may be desired, in which case the T cell culture time may be 60 days or more. Suitable conditions for T cell culture include suitable media that may contain factors necessary for proliferation and survival (e.g., interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives). Other additives for cell proliferation include, but are not limited to, surfactants, plasmanates, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Examples of culture media include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, X-Vivo 20, and optimizers, which may be supplemented with amino acids, sodium pyruvate, and vitamins, may be serum-free, or supplemented with an appropriate amount of serum (or plasma) or a defined hormone set, and / or may contain sufficient amounts of cytokines (or more) for T cell proliferation and expansion. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures and not in the culture of cells to be injected into the target. Target cells are maintained under conditions necessary to support proliferation, such as appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0136] In one embodiment, the NK cell population can be proliferated in vitro using interleukin-2 (IL-2), IL-15, IL-15 / IL-15RA complex, IL-18, and IL-12. In one embodiment, the NK cells are proliferated ex vivo for at least about 5 days, for example, about 10 days or more, about 15 days or more, or about 20 days or more, before administration to the patient.

[0137] IV. Pharmaceutical Compositions The pharmaceutical compositions of this disclosure may include modified cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline or phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In one embodiment, the compositions of this disclosure are formulated for intravenous administration.

[0138] In one embodiment, the pharmaceutical composition is substantially free of contaminants. That is, contaminants selected from the group consisting of, for example, endotoxins, mycoplasmas, reproducible lentiviruses (RCLs), p24, VSV-G nucleic acids, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture medium components, vector packaging cells or plasmid components, bacteria, and fungi. In one embodiment, the bacterium is at least one species selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenzae[e], Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia[e], and Streptococcus pyogenes group A.

[0139] Where “immunologically effective dose,” “antitumor effective dose,” “tumor inhibitory effective dose,” or “therapeutic dose” is indicated, the exact dose of the composition of this disclosure can be determined by a physician taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and the patient's (subject's) condition. Generally, the pharmaceutical composition containing T cells described herein is 10 per kg of body weight. 4 ~10 9 It may be administered in cellular doses, and in some cases up to 10 per kg of body weight. 5 ~10 6 These can be cells and include any integer values ​​within these ranges. The T cell composition may be administered once or multiple times in these doses. The cells can be administered using infusion techniques, which are generally known in immunotherapy.

[0140] V. Treatment method The modified cells or pharmaceutical compositions described herein may be administered in a manner suitable for the disease to be treated (or prevented). The aforementioned diseases include solid tumors such as sarcomas and carcinomas, for example fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and central nervous system (CNS) tumors (for example gliomas (brainstem gliomas and mixed gliomas, etc.), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, embryonic tumors, etc.). Examples of non-solid tumors include germinoma, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases; and non-solid tumors such as leukemia, for example, acute leukemia (acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia, etc.), chronic leukemia (chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia, etc.), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (low-grade and high-grade types), multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0141] The aforementioned diseases also include inflammatory reactions such as inflammatory skin diseases including psoriasis and dermatitis (e.g., atopic dermatitis), systemic sclerosis and sclerosis, reactions associated with inflammatory bowel disease (Crohn's disease and ulcerative colitis, etc.), respiratory distress syndrome (including adult respiratory distress syndrome (ARDS)), dermatitis, meningitis, encephalitis, uveitis, colitis, glomerulonephritis, eczema and asthma, and other conditions involving T-cell infiltration and chronic inflammatory reactions, arteriosclerosis, leukocyte adhesion disorders, rheumatoid arthritis, systemic lupus erythematosus (SLE), diabetes (e.g., type 1 diabetes or insulin-dependent diabetes), multiple sclerosis, Raynaud's syndrome, and autoimmune diseases. Immune responses associated with cytokine and T lymphocyte-mediated acute and delayed-type hypersensitivity, typically seen in thyroiditis, allergic encephalomyelitis, Sjögren's syndrome, juvenile-onset diabetes, and tuberculosis, sarcoidosis, polymyositis, granulomatous diseases, and vasculitis, including pernicious anemia (Addison's disease), diseases including leukocyte extravasation (diapedesis), central nervous system (CNS) inflammatory disorders, multiple organ dysfunction syndromes, hemolytic anemia, myasthenia gravis, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Graves' disease, Lambert-Eaton myasthenic syndrome, and bullous pemphigoid. The pharmacopoeia may include autoimmune diseases selected from the group consisting of bullous, pemphigus, autoimmune polyendocrine disorders, Reiter's disease, Stiffman syndrome, Behçet's disease, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immune thrombocytopenic purpura (ITP), or autoimmune thrombocytopenia. In one embodiment, the pharmacopoeia is for the treatment of multiple sclerosis. In one preferred embodiment, the pharmacopoeia is for the treatment of secondary progressive multiple sclerosis.

[0142] Dosage and frequency of administration may be determined by clinical trials, but the appropriate dose will depend on the patient's condition and the type and severity of the patient's disease. The compositions described herein may be administered by any convenient method, such as aerosol injection, oral ingestion, blood transfusion, implantation, or transplantation. The compositions described herein may be administered to the patient by intra-arterial, subcutaneous, intradermal, intratumoral, intralymph node, intramedullary, intramuscular, intravenous (iv) injection, or intraperitoneal injection. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous administration. In one embodiment, the composition is directly injected into the organ of interest (e.g., an organ affected by a neoplasm). Alternatively, the composition is delivered indirectly to the organ of interest, for example, by administration to the circulatory system (e.g., the tumor vascular system). Growth and differentiation factors may be provided before, during, or after administration of cells or compositions to increase T cell or NK cell production in vitro or in vivo.

[0143] In one embodiment, lymphodepletion is performed on the subject before administering, for example, one or more cells described herein. In one embodiment, the lymphodepletion includes administering one or more selected from the group consisting of melphalan, cytoxan, cyclophosphamide, and fludarabine.

[0144] In one embodiment, the modified cells are administered as part of a combination therapy carried out concurrently or sequentially, in any order, with other therapeutic interventions (e.g., antibodies, modified cells, receptors, or agents, e.g., cytotoxic agents or therapeutic agents). In one embodiment, the cells or antibodies are administered concurrently or sequentially, in any order, with one or more additional therapeutic agents or in connection with other therapeutic interventions. In one embodiment, the cells are administered in combination with other therapies at a time sufficiently close to the other therapy so that the cell population enhances (or vice versa) the effect of one or more additional therapeutic agents. In one embodiment, the cells or antibodies are administered prior to the one or more additional therapeutic agents. In one embodiment, the cells or antibodies are administered after the one or more additional therapeutic agents, such as anticancer agents. In the context of this disclosure, it is assumed that cell therapy may be used in combination with chemotherapy, radiotherapy, or immunotherapeutic interventions, as well as pre-apoptotic agents or cell cycle regulators such as immune checkpoint inhibitors.

[0145] Alternatively, this treatment may be administered prior to or following treatment with other agents, at intervals ranging from minutes to weeks. In embodiments where other agents and this disclosure are applied separately to an individual, it is generally ensured that no significant time elapses between each administration so that the agents and treatments can still exert a favorable synergistic effect on the cells. In such cases, it is envisioned that both modalities can treat the cells within approximately 12 to 24 hours, more preferably within approximately 6 to 12 hours. In some situations, it may be desirable to significantly extend the treatment period, in which case the interval between each administration may range from several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks). Treatment cycles are expected to be repeated as needed. It is also envisioned that various standard treatments and surgical interventions may be applied in combination with cell therapy. VI. Exemplary Sequence [Table 3-1] [Table 3-2] [Table 3-3] [Examples]

[0146] VIII. Examples The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0147] Example 1: Preparation of fused polypeptide

[0148] The plasmid construct was designed to encode a transduction marker (cleaved EGFR: tEGFR) at the 3' end of the ORF, followed by a sequence encoding a self-cleaved 2A peptide derived from equine rhinitis A virus. This was followed by sequences encoding the signal peptide (SP-1) and the extracellular domain of human Fas, as well as sequences encoding transmembrane sequences (TM1-1 to TM1-6, Fas TM), and sequences encoding the end-domains of human IL7Rα and / or human CD2 (F-1, F-3 to F-7; F-2). In some cases, sequences encoding the cleaved portion of the CD2 end-domain were placed after the IL7Rα end-domain sequence (F-8 to F-11). As a functional negative control, a plasmid encoding only the tEGFR transduction marker (Ctrl) was constructed. As a positive control for protection against FasL-mediated apoptosis, a plasmid (construct containing C-1) was designed, containing tEGFR followed by a 2A sequence derived from equine rhinitis A virus, and further including a signal peptide (SP-1), the extracellular domain, transmembrane domain, and cleaved endodomain of human Fas. As a positive control for cytokine receptor signaling, a plasmid (construct containing C-2) was designed, containing tEGFR followed by a 2A sequence derived from equine rhinitis A virus, and further including a signal peptide (SP-2), a sequence encoding the extracellular domain of human CD34, the transmembrane sequence TM1-1, and a sequence encoding the endodomain of human IL7Rα. The construct containing C-3 was designed to contain tEGFR followed by a 2A sequence derived from equine rhinitis A virus, and further including a signal peptide (SP-2), a sequence encoding the extracellular domain of human TGFβR, the transmembrane sequence TM1-1, and a sequence encoding the endodomain of human IL7Rα. [Table 4]

[0149] Example 2: Apoptosis resistance of modified T cells

[0150] Excessive activation / stimulation of T cells by surrounding tumors can induce Fas / FasL-mediated activation-induced cell death (AICD), resulting in a decrease in the number of viable T cells. T cells were transduced using a lentiviral vector expressing the fusion polypeptide described in Example 1. Transduced T cells (5 × 10⁻⁶) 5 T cells were added to the wells and cultured in an incubator for 7 days without any further stimulation.

[0151] At the time of seeding in 24-well plates (day 0), the cells were spun by centrifugation and stained with EGFR (Biolegend, #352908). A High Speed, High Throughput Cell Counter (Nexcelom) was used to measure the number of viable T cells.

[0152] On days 1, 3, 7, 8, 10, and 15 of incubation, T cells were gently mixed, and a 60 μL cell suspension was collected. The number of viable T cells was measured using a High Speed, High Throughput Cell Counter (Nexcelom). On days 3, 7, and 10, after measuring the number of viable T cells, the T cell density was 1 × 10⁶. 6 If the cell / mL exceeds 5 × 10 5 The cells were adjusted to the specified cell / mL. The number of surviving T cells during incubation was calculated as a relative value to the analysis results on day 0 in order to measure the difference in proliferation rate. Figure 1 shows the initial seeding (5 × 10⁶). 5 This shows the proliferation of transduced T cells (from 7 healthy donors) when maintained without additional stimulation after initial T cell transfection. Here, compared to the control constructs C-1 (p=0.0135, F-1 vs. C-1) and Ctrl (p=0.0063, F-1 vs. Ctrl), the proliferation of T cells transduced with F-1 was significantly improved. These data indicate that F-1 has high proliferative capacity under unstimulated conditions.

[0153] Example 3: Persistence of modified T cells

[0154] Improved persistence of CAR T cells correlates with clinical efficacy in many situations (10.1186 / s40364-022-00434-9). To proliferate and maintain remission for extended periods, T cells need to persist even in environments with low levels of stimulation and cytokines. To evaluate the ability of cells to survive under such low-stimulus / cytokine (quiet) conditions, transduced T cells were subjected to a starvation assay. In summary, primary T cells from four healthy donors were transduced using either Ctrl, C-2, or F-1. All transduced T cells were spun by centrifugation and stained with EGFR to evaluate transduction efficiency. Subsequently, non-transduced T cells were added to normalize the transduction rate of transduced T cells to 50% EGFR-positive, and then seeded at the same density (1 × 10^6 T cells) and cultured in an incubator for 7 days. On days 3 and 7 of incubation, the cell suspension was collected, and the number of viable T cells and the enrichment of EGFR-positive T cells were evaluated.

[0155] Figure 2B shows that, under conditions without cytokines or stimuli, transduced T cells with Ctrl could not proliferate, and their cell viability dramatically decreased to approximately 40% over 7 days (p=0.0002, C-2 vs. Ctrl; p=0.0003, F-1 vs. Ctrl). Both C-2 and F-1 cells showed a significant viability advantage compared to Ctrl (p<0.0001, C-2 vs. Ctrl; p=0.0002, F-1 vs. Ctrl), exhibiting linear proliferation over 7 days (p=0.0008, C-2 vs. Ctrl; p=0.0014, F-1 vs. Ctrl), and demonstrating sustained viability comparable to the same conditions with exogenous IL2 (100 IU / mL, Cytiva, #29062790) supplementation (Figures 2A and 2B). Furthermore, C-2 and F-1 cells were enriched in the transduced T cell population during this period compared to Ctrl (Figure 2B) or compared to IL2 (100 IU / mL) supplementation conditions (Figure 2A). This indicates that T cells transduced with F-1 can deliver cytokine survival signals and promote proliferation and viability in the absence of exogenous stimuli or cytokines. To further evaluate the construct's ability to withstand Fas / FasL-mediated apoptosis, transduced T cells were exposed to trimer FasL stimulation (50 ng / mL, Adipogene, AG-40B-0130-3010) in the presence of IL2 supplementation (100 IU / mL), and cell proliferation, viability, and enrichment were evaluated over 7 days. Here, T cells transduced with Ctrl or C-2 were unable to proliferate, showed decreased viability, and exhibited reduced enrichment of the transduced population. In contrast, T cells transduced at F-1 continued to proliferate, reaching levels comparable to those under FasL-free conditions (p=0.0035, F-1 vs. Ctrl; p=0.0037, F-1 vs. C-2). Significant improvements in survival rate (p=0.0007, F-1 vs. Ctrl; p=0.0146, F-1 vs. C-2) and enrichment (p=0.0003, F-1 vs. Ctrl; p=0.0003, F-1 vs. C-2) were also observed over a 7-day period (Figure 2C).

[0156] Example 4: Module persistence after FasL stimulation

[0157] To further investigate the resistance of fusion proteins (sharing the same extracellular domain of human Fas and possessing either a different endodomain of human IL7Rα or CD2) to Fas / FasL-mediated apoptosis, transduced T cells (from 7 healthy donors) were exposed to 50 ng / mL of trimer FasL stimulation in the presence of IL-2 supplementation (100 IU / mL) and cultured in an incubator for 7 days. On days 1, 3, and 7 of incubation, the T cells were gently mixed and collected to measure the number of viable T cells using a High Speed, High Throughput Cell Counter (Nexcelom). To measure the difference in proliferation ratios, the viable T cell density during incubation was measured from the initial seeding on day 0 (5 × 10⁶). 5 The values ​​were calculated as relative to the number of T cells / mL. As shown in Figure 3, T cells transduced with various fusion proteins (sharing the same extracellular domain of human Fas) showed enhanced persistence against FasL-induced apoptosis, with cells expressing F1 showing slightly higher proliferation levels than cells with C1 and F2.

[0158] Example 5: Module proliferation under IL-2 non-supplement (starvation) conditions

[0159] To further investigate the proliferation ability of fusion proteins (sharing different extracellular domains derived from either human Fas or TGFβR, and possessing identical transmembrane and endodomains) under cytokine starvation conditions, transduced T cells were subjected to IL-2-free conditions. In summary, primary T cells from four healthy donors were transduced using either Ctrl, C-3, or F-1. All transduced T cells were spun by centrifugation and stained with EGFR to evaluate transduction efficiency. Subsequently, non-transduced T cells were added to normalize the proportion of EGFR-positive T cells to 50%, and then seeded at the same density (1 × 10^6 T cells) and cultured in an incubator for 7 days. On days 3 and 7 of incubation, the cell suspension was collected, and the number of viable T cells was measured using a Cell Counter, and EGFR-positive T cells were detected by flow cytometry. The number of EGFR-positive T cells during incubation was calculated as a relative value to the analysis result on day 0 to measure the difference in proliferation ratio.

[0160] Figure 4 shows that T cells transduced with C-3 were unable to proliferate for 7 days in the absence of cytokines (C-3 vs. F-1: day 3, p=0.0277; day 7, p=0.0060). These results suggest that the function of the transmembrane-endodomain (TM-endo) of IL-7Rα can be inhibited by specific extracellular domains.

Claims

1. A modified cell expressing a trimer complex comprising at least one fusion polypeptide, wherein the fusion polypeptide is arranged sequentially from its N-terminus to its C-terminus. a) Extracellular components containing Fas ectodomain; b) Transmembrane components; and c) Intracellular components comprising an IL-7R endodomain, a CD2 endodomain, or a combination thereof, but not comprising a Fas endodomain; Including, Modified cells in which the aforementioned trimer complex is capable of binding to Fas ligand.

2. The extracellular component contains, or consists of, an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; Preferably, the extracellular component comprises cysteine-rich domain 1, cysteine-rich domain 2, and cysteine-rich domain 3 of the amino acid sequence of SEQ ID NO: 1; More preferably, the extracellular component contains or consists of the amino acid sequence of SEQ ID NO: 1; Modified cells according to claim 1.

3. The aforementioned transmembrane component is self-oligomerizable; Preferably, the transmembrane component consists of a transmembrane domain that includes a gain-of-function insertion compared to SEQ ID NO: 10; More preferably, the transmembrane component consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 33. Modified cells according to claim 1 or 2.

4. The transmembrane component consists of the amino acid sequence of SEQ ID NO: 11 (PILLTCPTIX1IX2SX3X4X5X6X7X8X9X10X11LX12X13X14LW), where X1 is S or L; X2 is L or S; X3 is F or L; X4 is F or A; X5 is S or I; X6 is V or L; X7 is A or L; X8 is L or M; X9 is L or V; X10 is V or S; X11 is I or L; X12 is A or L; X13 is C or L; X14 is V or S; Preferably, the transmembrane component consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 to 39. Modified cells according to claim 3.

5. The intracellular component comprises an IL-7Rα endodomain or consists of a sequence thereof; Preferably, the intracellular component contains or consists of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4; More preferably, the intracellular component contains the amino acid sequence of SEQ ID NO: 4, or consists of the sequence. A modified cell according to any one of claims 1 to 4.

6. The intracellular component comprises a CD2 endodomain or consists of a sequence thereof; Preferably, the intracellular component comprises a full-length CD2 end domain or a functional fragment thereof, or consists of such sequence, wherein the functional fragment has its C-terminus cleaved by up to 65 amino acids compared to the full-length CD2 end domain; More preferably, the intracellular component contains or consists of an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 5 to 8, or contains or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs. 5 to 8. A modified cell according to any one of claims 1 to 5.

7. The transmembrane component is self-oligomerizable, and the intracellular component contains an IL-7Rα endodomain with the C-terminus of the transmembrane component fused to its N-terminus, or consists of such a sequence; Preferably, the transmembrane component consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 33; More preferably, the intracellular component comprises or consists of the amino acid sequence of SEQ ID NO: 4, and the transmembrane component consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 to 39. A modified cell according to any one of claims 1 to 6.

8. The intracellular component comprises an IL-7Rα end-domain and a CD2 end-domain, or consists of such a sequence, the transmembrane component is self-oligomerizable, the IL-7R end-domain has the C-terminus of the transmembrane component fused to its N-terminus, and the IL-7R end-domain has the N-terminus of the CD2 end-domain fused to its C-terminus; Preferably, the intracellular component consists of a full-length CD2 end domain or a functional fragment thereof, wherein the functional fragment has its C-terminus cleaved to a maximum of 65 amino acids compared to the full-length CD2 end domain; and the transmembrane component consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 33; More preferably, the intracellular component comprises or consists of the amino acid sequence of SEQ ID NO: 4 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 8; the transmembrane component consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 to 39. A modified cell according to any one of claims 1 to 7.

9. The transmembrane component is Fas, CD2, CD3s, CD35, CD3C, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, OX40, 4-IBB, SLAMF1, CTLA. 4, CD200R, LAG3, HVEM, BTLA, PD-L2, PD-L1, ICOS, PD-1, CD300, GITR, A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, The intracellular component consists of a transmembrane domain selected from the group consisting of Lck, LAT, LRP, KG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPa, pTa, TCRa, TCRβ, TIM3, TRIM, LPA5, and Zap70, wherein the intracellular component includes a CD2 end domain in which the C-terminus of the transmembrane component is fused to its N-terminus, or consists of such a sequence; Preferably, the CD2 end domain is a full-length CD2 end domain or a functional fragment thereof, wherein the functional fragment has its C-terminus cleaved to a maximum of 65 amino acids compared to the full-length CD2 end domain, and the transmembrane component consists of a Fas transmembrane domain; More preferably, the intracellular component consists of an amino acid sequence selected from the group consisting of SEQ ID NOs. 5 to 8, and the transmembrane component consists of the amino acid sequence of SEQ ID NO.

9. A modified cell according to any one of claims 1, 2, and 6.

10. The trimer complex is expressed on the surface of the modified cells; Preferably, the trimer complex is a homotrimer or a heterotrimer, where the heterotrimer contains endogenous Fas; More preferably, if the intracellular component includes an IL-7R endodomain, the trimer complex includes two or three fusion polypeptides. A modified cell according to any one of claims 1 to 9.

11. The modified cell according to any one of claims 1 to 10, wherein the modified cell is an immune cell, preferably a lymphocyte; more preferably, the modified cell is an NK cell, a T cell, or a combination thereof.

12. The modified cells express an antigen receptor; preferably, the antigen receptor is a chimeric antigen receptor or an antigen-specific TCR; more preferably, the chimeric antigen receptor is CD19, CD20, CD22, CD30, BCMA, AFP, ALK, GPC3, HER2, EGFRα folate receptor, 5T4, avβ6 integrin, B7-H3, B7-H6, CAIX, CD16, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, chloro Targeting -din 18.2, CSPG4, DLL3, EGFR, HER2, EGFRvlll, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, Fetal AchR, FRa, GD2, GD3, MAGE-1, NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesoserine, Muc1, Muc16, NCAM, NKG2D ligand, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TAG72, TEMs, VEGFR2, or WT-1, A modified cell according to any one of claims 1 to 11.

13. The modified cells express exogenous FasL; preferably, the exogenous FasL is secreted or membrane-bound; more preferably, the exogenous FasL contains or consists of an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 40 to 47, or contains or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 40 to 47. A modified cell according to any one of claims 1 to 12.

14. A fusion polypeptide contained in a modified cell according to any one of claims 1 to 13.

15. A use of the fusion polypeptide of claim 14 for enhancing the persistence and proliferation of the modified cells; preferably, the fusion polypeptide is capable of participating in the formation of the trimer complex; more preferably, the trimer complex is capable of inducing the signaling of the intracellular components independently of the binding of the Fas ligand.

16. An isolated nucleic acid encoding the fusion polypeptide of claim 14, preferably the isolated nucleic acid encoding the fusion polypeptide and the antigen receptor, and more preferably the fusion polypeptide and the antigen receptor linked via a viral self-cleaving polypeptide.

17. A vector comprising the nucleic acid of claim 16, wherein the vector is preferably a viral vector, and more preferably the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated viral vector.

18. A pharmaceutical formulation comprising a modified cell according to any one of claims 1 to 13, a fusion polypeptide according to claim 14, an isolated nucleic acid according to claim 16, or a vector according to claim 17, and a pharmaceutically acceptable carrier.

19. A method for treating a target disease, comprising administering to a target a modified cell according to any one of claims 1 to 13, a fusion polypeptide according to claim 14, an isolated nucleic acid according to claim 16, a vector according to claim 17, or a pharmaceutical formulation according to claim 18; preferably, the disease is selected from the group consisting of leukemia, lymphoma, lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, and rhabdomyosarcoma; more preferably, the disease is leukemia or lymphoma.

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