Engineering type 2 cytokine signaling to enhance car-t cell effector function

Abstract

The present invention relates to chimeric antigen receptor constructs and their use for therapy and treatment. Further disclosed are methods of treating and / or preventing diseases, such as cancer, infectious diseases, inflammatory or inflammation-induced diseases, chronic diseases or autoimmune diseases.

Description

Technical Field

[0001] This invention relates to chimeric antigen receptor constructs and their use in treatment and therapy. Methods for treating and / or preventing diseases, such as cancer, infectious diseases, inflammatory or inflammation-induced diseases, chronic diseases, or autoimmune diseases, are also disclosed. Background Technology

[0002] Despite the success of CAR-T cell therapy in treating hematologic malignancies, several limitations exist in the application of CAR-T cells in the TME (tumor endothelial cell line). 1 Due to limitations in the persistence and activity of CAR-T cells, this therapy remains largely ineffective against solid tumors. These limitations include T cell dysfunction, heterogeneous expression of tumor antigens, and insufficient T cell infiltration into the tumor, leading to tumor progression beyond the reach of CAR-T cells. 2,3 Control.

[0003] To enhance the therapeutic efficacy against solid tumors, fourth-generation CAR-T cells have been engineered to express stimulating cytokines, known as redirected T cells for universal cytokine-mediated killing (TRUCK) or armored CARs. These cytokine-armored CAR-T cells are used to enhance the persistence of infused CAR-T cells or stimulate endogenous immune cells. For example, CAR-T cells are modified T cells to co-express CAR molecules and T cell-stimulating cytokines (such as IL-7 and IL-15), which improve the intrinsic activity of CAR-T cells without modulating adjacent endogenous immune populations. 4,5 In addition, CAR-T cells are engineered to express stimulating cytokines (such as IL-12 or IL-18) to activate CAR-T cells and local immune cells. 6-8 .

[0004] To date, the design of armored CAR-T cells has been primarily explored in the field of type 1 cytokines, but there has been little in-depth research in the field of type 2 cytokines, which can also assist in anti-tumor immunity.

[0005] Strategies that can overcome one or more of the above obstacles offer an opportunity to improve the response rate of CAR-T cells to cancer, especially solid tumors. Summary of the Invention

[0006] This invention provides a chimeric antigen receptor polypeptide comprising: -At least one antigen-binding domain, -Transmembrane domain, -At least one co-stimulatory domain, -At least one activation domain, and - At least one interleukin-4 (IL-4) molecule, its variant or a functional fragment thereof.

[0007] Further, isolated polynucleotides encoding the chimeric antigen receptor (CAR) of the present invention are provided.

[0008] A vector containing the isolated polynucleotides of the present invention is also provided.

[0009] It also provides host cells, preferably immune cells, which contain i) the chimeric antigen receptor polypeptide of the present invention, isolated polynucleotides and / or vectors, or ii) expressing the chimeric antigen receptor of the present invention.

[0010] A pharmaceutical composition is also provided comprising: i) the chimeric antigen receptor polypeptide of the present invention, ii) the isolated polynucleotide of the present invention, iii) the carrier of the present invention, or iv) the immune cell of the present invention, and pharmaceutically acceptable carriers, diluents and / or excipients.

[0011] Further, methods for treating cancer, infectious diseases, inflammatory or inflammation-induced diseases, chronic diseases, or autoimmune diseases are provided, comprising administering to a subject in need i) the chimeric antigen receptor polypeptide of the present invention, ii) the isolated polynucleotide of the present invention, iii) the carrier of the present invention, iv) the immune cells of the present invention, or v) the pharmaceutical composition of the present invention. Attached Figure Description

[0012] Figure 1. HER2-specific CAR-T cells co-expressing IL-4 (IL-4 HER2 CAR T) enhance the antitumor activity and pluripotency of CAR-T cells in LHDA-dependent glycolysis. a. Schematic diagram of a HER2-directed second-generation CAR (HER2CAR) and a HER2-directed second-generation CAR modified to express mouse IL-4 after a 2A element (HER2 CAR-IL-4). b. Transduction of HER2 CAR or HER2 CAR-IL-4 constructs using retroviral vectors. CAR expression levels were analyzed by flow cytometry. The numbers in the histogram represent the percentage of HER2 CAR-positive cells. Similar results were obtained in five independent experiments. c. IL-4 HER2 CAR-T or HER2 CAR-T cells were cultured for 2 days. The concentration of IL-4 in the culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). d. Cytotoxicity assay using MC38-HER2 (MC38 colon cancer cells expressing HER2) as target cells. Target cells were mixed with HER2 CAR T cells or IL-4 HER2 CAR T cells at a specified E / T ratio. For example, IL-4 HER2 CAR-T cells or HER2 CAR-T cells in the presence or absence of mouse IL-4 (20 ng / ml) were co-cultured with MC38-HER2 cells at an E / T ratio of 0.5:1 for 48 hours. The figure shows the percentage of lysed MC38-HER2 cells (e), the number of viable CAR-T cells (f), and granzyme B. + IFNγ + Frequency of multifunctional CAR-T cells (g). hk. Isolation of IL-4 HER2 CAR-T or HER2 CAR-T cells from culture for Seahorse assay. h, i. Mean basal OCR (h) and maximum OCR (i). j, k. Mean basal ECAR (j) and maximum ECAR (k). lo. Co-culture of IL-4 HER2 CAR-T or HER2 CAR-T cells with MC38-HER2 cells at an E / T ratio of 5:1 for 18 hours. Isolation of CAR-T cells for Seahorse assay. l, m. Mean basal OCR (l) and maximum OCR (m). n, o. Mean basal ECAR (n) and maximum ECAR (o). p. Co-culture of HER2 CAR-T or IL-4 HER2 CAR-T cells with MC38-HER2 cells in the presence of a specified inhibitor. Granzyme B is shown in the figure. + IFNγ + TNFα +Relative frequencies of pluripotent CAR-T cells (IL-4 HER2 CAR-T vs HER2 CAR-T). All data represent mean ± sem and were analyzed by unpaired Student's t-tests (c, d, ho) or ANOVA combined with Tukey's multiple comparison test (eg, p).

[0013] Figure 2. IL-4 HER2 CAR-T cells resist exhaustion and enrich type 2 T cell subsets in tumors. C57BL / 6 mice were inoculated with MC38-HER2 tumor cells (1 × 10⁻⁶). 6 Subcutaneous irradiation was performed on day 5 to induce sublethal lymphocyte clearance, followed by IL-4 HER2 CAR-T cell therapy (3 × 10⁻⁶ cells) on day 6. 6 HER2 CAR-T cells (3 × 10⁻⁶) were transferred intravenously via adoptive transfer or received with or without intravenous administration of IL-4 (1 µg). 6 Intravenous adoptive transfer of HER2 CAR-T cells. Mice were sacrificed on day 14 and the designated tissues were processed. ac. Counts of viable HER2 CAR-T cells in tumor (a), spleen (b), and tumor draining lymph nodes (TDLN) (c). d. Representative flow cytometry plots and mean composition showing the frequencies of four subsets of HER2 CAR-T cells gated based on PD-1 and TIM-3 expression. e. Representative flow cytometry plots and mean composition showing the frequencies of four subsets of IL-4 HER2 CAR-T cells gated based on PD-1 and TIM-3 expression. f. Frequency of progenitor-depleted CAR-T cells. g. MFI of PD-1 in CAR-T cells in tumors. h. Representative flow cytometry plots and bar charts showing granzyme B in tumors. + IFNγ + TNFα + Frequency of pluripotent CAR-T cells. i. Active IFNγ in tumors. + Granzyme B + j. Frequency of Tc2 in CAR-T cells in tumors. k. Frequency of Th2 in CAR-T cells in tumors. All data represent mean ± sem and were analyzed using unpaired Student's t-tests (ac, fh) and ANOVA combined with Tukey's multiple comparison test (ik).

[0014] Figure 3. IL-4 HER2 CAR-T therapy ablates established mouse MC38-HER2 colon adenocarcinoma, inducing durable anti-tumor immunity and broad immune activation. a, b. C57BL / 6 mice were subcutaneously inoculated with MC38-HER2 colon cancer cells (3 × 10⁻⁶).5 On day 5, sublethal lymphocytes were cleared via irradiation, and on day 6, IL-4 HER2 CAR-T cells (3 × 10⁻⁶) were administered. 6 ) or HER2 CAR-T cells (3 × 10 6 Intravenous adoptive transfer of MC38-HER2 (n=5 per group). a, b. The figure shows the mean tumor growth curve (a) and mouse survival curve (b) of the MC38-HER2 model. The number of long-term surviving mice in the total number of mice in the group is shown (b). c, d. On day 90 after primary tumor inoculation, MC38-HER2 (1 × 10⁻⁶) was used. 6 Mice that survived IL-4HER2 CAR-T cell therapy were subcutaneously challenged again. On day 130 post-inoculation of the primary tumor, MC38 (0.5 × 10⁻⁶) was used. 6 Mice that survived subcutaneous re-challenge to MC38-HER2 were subjected to further subcutaneous re-challenge. Untreated WT mice (n=5 per group) were inoculated with the same number of tumor cells as controls. c, d. The figures show the survival curves and number of long-term survivors who rejected the second tumor challenge in the MC38-HER2 (c) and MC38 (d) tumor models. The figures also show the survival curves and number of long-term survivors who resisted re-challenge (c, d). e, f. C57BL / 6 mice were inoculated with MC38-HER2 tumor cells (1 × 10⁻⁶). 6 Subcutaneous CAR-T cells were administered on day 5 via radiation for sublethal lymphocyte clearance, followed by HER2 CAR-T cell therapy (3 × 10⁻⁶ cells) on day 6. 6 ) or IL-4 HER2 CAR-T cells (3 × 10 6 Intravenous adoptive transfer was performed on mice. On day 14, mice were sacrificed, and phenotypic analysis of designated immune cells in the tumor was performed by flow cytometry. The figure shows CD45.2 in MC38-HER2 tumors from each treatment group. + Counts of cells (e) and mature dendritic cells (DCs) (f). All data represent mean ± sem and were analyzed by unpaired Student's t-test (e, f), ANOVA combined with Tukey's multiple comparison test (a) or log-rank test (bd).

[0015] Figure 4. Complete regression of a pre-established mouse B16F10 melanoma model by treatment with IL-4 TRP-1 CAR-T cells. a. Schematic diagram showing the constructs of a TRP-1-guided 4-1BB-based second-generation CAR (TRP-1 CAR) and a TRP-1 CAR secreting mouse IL-4 (IL-4 TRP-1 CAR). b. TRP-1 CAR expression level detected by c-Myc tag staining. Ctrl T: Untransduced T cells, as a control. c. IL-4 TRP-1 CAR-T or TRP-1 CAR-T cells were cultured for 2 days. The concentration of IL-4 in the culture supernatant was detected by ELISA. d, e. IL-4 TRP-1 CAR-T cells or TRP-1 CAR-T cells in the presence or absence of IL-4 (20 ng / ml) were co-cultured with B16F10 cells at an E / T ratio of 0.5:1 for 48 hours. The percentage of lysed tumor cells (d) and the viable cell count of CAR-T cells (e) were analyzed by flow cytometry. f, g. C57BL / 6 mice were subcutaneously inoculated with B16F10 melanoma cells (3 × 10⁻⁶). 5 Lymphocytes were cleared, and on day 6, IL-4 TRP-1 CAR-T cells (3 × 10⁻⁶) were administered. 6 Intravenous adoptive transfer of TRP-1 CAR-T cells (3 × 10⁻⁶) with or without intravenous administration of IL-4 (1 µg). 6 Intravenous adoptive transfer of the B16F10 model was performed (n=6 per group). The figure shows the mean tumor growth curve (f) and mouse survival curve (g) of the B16F10 model. The number of long-term surviving mice in the total number of mice in the group is shown (g). All data represent mean ± sem and were analyzed by unpaired Student's t-test (c), ANOVA combined with Tukey's multiple comparison test (d, e, f) or log-rank test (g).

[0016] Figure 5. IL-4 EGFRvIII CAR-T therapy prolongs survival in a pre-established mouse 4T1-Luc-EGFRvIII metastatic breast cancer model. a. Schematic diagram showing the constructs of the EGFRvIII-guided 4-1BB-based second-generation CAR (EGFRvIIICAR) and the EGFRvIII CAR secreting mouse IL-4 (IL-4 EGFRvIII CAR). b. EGFRvIII CAR expression level detected by c-Myc tag staining. Ctrl T: Untransduced T cells, as a control. c. IL-4 EGFRvIII CAR-T or EGFRvIII CAR-T cells were cultured for 2 days. The concentration of IL-4 in the culture supernatant was detected by ELISA. d, e. IL-4 EGFRvIII CAR-T cells or EGFRvIII CAR-T cells with or without IL-4 (20 ng / ml) were co-cultured with 4T1-EGFRvIII-Luc cells at an E / T ratio of 0.5:1 for 48 hours. The percentage of tumor cell lysis (d) and the viable cell count of CAR-T cells (e) were analyzed by flow cytometry. f, g. BALB / c mice were intravenously inoculated with 4T1-EGFRvIII-Luc breast cancer cells (5 × 10⁻⁶). 4 Lymphocytes were cleared, and on day 6, IL-4 EGFRvIII CAR-T cells (3 × 10⁻⁶) were administered. 6 Intravenous adoptive transfer, or receiving EGFRvIII CAR-T cells (3 × 10⁻⁶) with or without intravenous administration of IL-4 (1 µg). 6 f. Individual radiance (p / s / cm) of mice in different treatment groups. 2 / sr). g. Survival curves of the 4T1-EGFRvIII-Luc tumor model. The number of mice that survived long-term out of the total number of mice in the group is shown (g). All data represent mean ± sem and were analyzed by unpaired Student's t-test (c), ANOVA combined with Tukey's multiple comparison test (d, e) or log-rank test (g).

[0017] Figure 6. In vitro characterization of IL-4 CD19 human CAR-T cells. a. Schematic diagram of CD19-guided second-generation CAR (CD19 CAR) and CD19-guided second-generation CAR modified to express human IL-4. b. CAR expression level analysis by flow cytometry. d. NTD (non-transduced T cells), CD19 CAR-T cells, or CD19 IL-4 CAR-T cells were co-cultured with Nalm6-GFP cells at an E / T ratio of 1:5 for 48 hours, and the percentage of tumor cell killing was analyzed by flow cytometry. Data represent mean ± SEM. Detailed Implementation

[0018] Although similar or equivalent methods and materials may be used in the practice or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. The publications and applications mentioned herein are provided only because their publication date predates the filing date of this application. Nothing described herein should be construed as an admission that this invention is not entitled to precedence over such publications by virtue of a prior invention. Furthermore, the materials, methods, and examples described are for illustrative purposes only and are not intended to be limiting.

[0019] In case of any conflict, this specification (including definitions) shall prevail. Unless otherwise defined, all technical and scientific terms used in this specification shall have the meanings commonly understood by one of ordinary skill in the art to which this subject matter pertains. As used herein, the following definitions are provided to facilitate understanding of the invention.

[0020] The term "comprise / comprising" is generally used to mean "include / including," which implies that one or more features or components are permitted. The term also encompasses the narrower meaning of "composed of."

[0021] As used in the specification and claims, the singular indefinite / definite articles “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise.

[0022] The term "amino acid" includes all naturally occurring amino acids as well as modified amino acids.

[0023] As used in this article, "at least one / kind" refers to "one / kind or more / kinds", "two / kind or more / kinds", "three / kind or more / kinds", etc. For example, at least one costimulatory domain refers to one costimulatory domain, two costimulatory domains, three costimulatory domains, etc.

[0024] The term “about”, especially when referring to a given numerical value, means a deviation of ±10 percent.

[0025] "Homology" refers to the percentage of identity between two polynucleotides or two polypeptide motifs. Two nucleic acid sequences or two polypeptide sequences are "fundamentally homologous" to each other when they exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% or at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% to 98% sequence identity over a molecule of a defined length. As used herein, fundamental homology also refers to a sequence that is completely identical to a specified sequence. Alternatively, homology can be determined by readily available computer programs or by polynucleotide hybridization under conditions that form a stable double strand between homologous regions, followed by digestion with a single-strand-specific nuclease, and determination of the size of the digested fragment. Fundamentally homologous DNA sequences can be identified by Southern hybridization experiments under, for example, stringent conditions (conditions defined for the particular system). Determining suitable hybridization conditions is within the ordinary skill of those skilled in the art.

[0026] Generally, "identity" refers to the exact correspondence between nucleotides of two polynucleotide sequences or between amino acids of two polypeptide sequences. The percentage of identity can be determined by directly comparing the sequence information of two molecules: aligning the sequences, counting the exact matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. In some aspects, the nucleotide or amino acid sequence of the present invention, or a portion thereof, has at least 80% identity with the corresponding nucleotide or amino acid sequence (e.g., SEQ ID NO identifier), i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0027] As used herein, the terms “peptide,” “protein,” “polypeptide,” “polypeptide chain,” “polypeptide,” and “peptide” are used interchangeably to refer to a series of amino acid residues linked to another amino acid residue by a peptide bond between the α-amino and carboxyl groups of adjacent residues.

[0028] When referring to the polypeptides or nucleic acids of the present invention, the term "variant" refers to one or more bioactive derivatives of the polypeptide or nucleic acid. Generally, the term "variant" refers to a molecule having one or more additions, substitutions (usually of a conserved nature), and / or deletions of the native sequence compared to the native molecule, provided that these modifications do not impair its biological activity and that the variant is "fundamentally homologous" to the reference molecule (Gorby et al., Sci. Signal. 13, eabc0653, 2020; Saxton et al., Science 371, eabc8433, 2021). Generally, such variant sequences have the same biological activity. Generally, when the two sequences are aligned, such variants have a high degree of sequence homology or identity with the reference sequence, for example, greater than 25%, typically greater than 50% to 70%, and even more specifically 80% or 85% or higher, such as at least 90% or 95% or higher. Spencer, Juliet V et al. reported that although the splice variant of IL-10 has only 27% sequence identity with hIL-10, it still retains biological activity or properties (Spencer, Juliet V et al. “Stimulation of B lymphocytes by cmvIL-10 but not LAcmvIL-10.” Virology vol.374,1 (2008): 164-9. doi:10.1016 / j.virol.2007.11.031, the contents of which are incorporated herein by reference in their entirety).

[0029] As used herein, a “fragment” of the polypeptide or nucleic acid of the present invention refers to a sequence containing nucleotides shorter than the corresponding nucleic acid sequence, or containing amino acids shorter than the corresponding polypeptide sequence. Preferably, the nucleotide or amino acid length of the sequence or fragment is less than 90%, more preferably less than 60%, and particularly less than 30% of the corresponding nucleic acid sequence or corresponding polypeptide sequence. More preferably, a “fragment” of the polypeptide or nucleic acid of the present invention is a “functional fragment,” meaning that it shares the same biological function or property as the corresponding nucleic acid sequence or corresponding polypeptide sequence.

[0030] The inventors of this invention have demonstrated, surprisingly, that CAR-T cells engineered to express type 2 cytokines (e.g., IL-4) exhibit enhanced proliferative capacity, effector function, and protection against functional impairment in, for example, solid tumors, compared to CAR-T cells that do not express IL-4.

[0031] This invention provides a chimeric antigen receptor (CAR) polypeptide comprising at least one antigen-binding domain, a transmembrane domain, at least one co-stimulatory domain, at least one activation domain, and at least one type II cytokine, a variant thereof, or a functional fragment thereof. Preferably, the at least one type II cytokine is interleukin-4 (IL-4), a variant thereof, or a functional fragment thereof.

[0032] Both bispecific and multispecific CARs fall within the scope of this invention, as they are specific to more than one target.

[0033] Optionally, short linkers can form a link between any or some of the CAR's domains (i.e., antigen-binding domain, transmembrane domain, co-stimulatory domain, activation domain, intracellular domain) and the IL-4 molecule.

[0034] An antigen-binding domain typically consists of, or is contained within, an extracellular domain that binds to cell surface markers. Preferably, the antigen-binding domain is selected from the non-limiting group comprising: antigen-binding peptides, receptors, and natural ligands of target cell antigens or receptors.

[0035] Preferably, the antigen-binding polypeptide is an antibody or antibody fragment selected from the group consisting of: mouse antibodies, rabbit antibodies, human antibodies, humanized antibodies, single-chain variable region fragments (scFv), camel antibody variable region domains and humanized versions, shark antibody variable region domains and humanized versions, single-domain antibody variable region domains, nanobodies (VHH), and camel-derived antibody variable region domains.

[0036] In one respect, an antigen-binding domain is an extracellular antigen recognition or binding domain derived from a single-chain fragment variant (scFv) of an antibody that recognizes an antigen, ligand, or receptor.

[0037] In one respect, antigens recognized by antigen-binding domains are typically selected from the group consisting of cancer cell-associated antigens, infection-associated antigens, and autoantigens.

[0038] Preferably, the antigen recognized by the extracellular antigen-binding domain is selected from the group comprising: HER2, TRP-1, EGFRvIII, CD19, CD20, CD38, CD30, ERBB2, fibroblast activation protein (FAP), CA125, MUC-1, PSMA, PSA, CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), CEACAM5, CEACAM6, epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, carbonic anhydrase EX, alpha-fetoprotein, A3, A33 antibody-specific antigen, Ba 733, BrE3-antigen, CD1, CDIa, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138, colon-specific antigen-p (CSAp), CSAp, EGP-I, EGP-2, Ep-CAM, FIT-I, Flt-3, folate receptor, HLA-DR, Human chorionic gonadotropin (HCG) and its subunits, hypoxia-inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, insulin-like growth factor-1 (IGF-I), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, macrophage inhibitory factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, tyrosinase, PRAME, EBNA, KLK3, HPV E7, LMP2, NY-ESO-1, PAP, reverse transcriptase, nucleolar phosphatase, PRTN3 / ELANE, CT83 / KKLC1, MUC16, DNTT, PAM-4 antibody-specific antigen, placental growth factor, p53, prostatic acid phosphatase, RS5, S1OO, TAC, TAG-72, tendinin, TRAIL receptor, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, 17-1A antigen, NeuGcGM3, N-hydroxyacetyl GM3 ganglioside, Neu5Gc, GM3-ganglioside, GD3, GM2, carbohydrate antigen, ganglioside antigen, Lewis Y, Lewis B, MOG, MBP, aB-crystal protein, PLP, GlialCAM, β-synuclein, HLA-A2, TNP, CD123, immunoglobulin κ chain, or any combination thereof.

[0039] Non-limiting instances of ScFv are selected from the group consisting of HER2-scFv (SEQ ID NO: 3), TRP-1-scFv (SEQ ID NO: 4), and EGFRvIII-scFv (SEQ ID NO: 5), or fragments or variants of any of these sequences.

[0040] In some cases, the antigen-binding domain includes a hinge portion (also called a connector or interlimb). Various hinges or connectors can be used according to the invention described herein and inventions known in the art.

[0041] Those skilled in the art will understand that any suitable transmembrane domain may be used in this invention. In one aspect, the transmembrane domain may be selected from the group comprising: CD28, CD28T, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulatory factor (ICOS), CD1-1a / CD18, CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC. Class 1 molecules, TNF receptor proteins, immunoglobulins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAM F7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD (e.g., CD8α, CD8β), IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD1 The ligands selected include 1c, ITB1, CD29, ITB2, CD18, LFA-1, ITB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof. Preferably, the transmembrane domain is selected from those containing a CD8 transmembrane domain, its variants, or fragments.

[0042] If a linker region is inserted between the antigen-binding domain and the transmembrane domain, the linker region is selected from the group consisting of: i) an immunoglobulin hinge region or a linker region derived from CD8, CD8α or CD28, or any variant thereof; and ii) a (GnS)m linker, wherein G is glycine, S is serine, n is an integer from 2 to 12 (preferably 3 to 10, more preferably 3 to 5, even more preferably n=3), and m is an integer from 2 to 12 (preferably 3 to 10, more preferably 3 to 5, even more preferably m=3).

[0043] In one aspect, the CD8 transmembrane structural domain and hinge region are as shown in SEQ ID NO: 7, its variants or fragments.

[0044] The costimulatory domain is a signal transduction region (or other suitable portion) that contains or is composed of molecules selected from the following: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18)), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC. Class I molecules, TNF receptor protein, immunoglobulins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM protein), activated NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

[0045] Preferably, the co-stimulatory domain is selected from the group consisting of 4-1BB co-stimulatory domains, variants thereof, or fragments thereof. Most preferably, the 4-1BB co-stimulatory domain is as shown in SEQ ID NO: 9, variants thereof, or fragments thereof.

[0046] The appropriate source of the activation domain may be derived from (or correspond to) part or all of CD3.

[0047] In a preferred aspect, the activation domain is the CD3 domain, more preferably the CD3 zeta (CD3ζ) domain.

[0048] Most preferably, the CD3ζ domain is as shown in SEQ ID NO: 8, its variants or fragments.

[0049] The CAR of this invention also includes type 2 cytokines. Type 2 cytokines include interleukin-4 (IL-4), IL-5, IL-6, IL-9, IL-13, IL-25, and IL-33.

[0050] In one respect, type 2 cytokines are interleukin-4 (IL-4) molecules, their variants, or functional fragments thereof.

[0051] Preferably, IL-4 is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a variant or fragment of any of these sequences. More preferably, IL-4 is a human sequence as shown in SEQ ID NO: 2.

[0052] In one aspect, the connection between the activation domain and IL-4 is achieved, for example, by encoding a sequence of a self-cleaving peptide. Preferably, the self-cleaving domain or peptide contains the sequence DxExNPGP (where x is any amino acid).

[0053] Non-restricted examples of self-cleaving domains or peptides containing the sequence DxExNPGP are selected from the group consisting of:

[0054] Or a combination thereof.

[0055] For example, once the self-cleaving peptide is cleaved, IL-4 or fragments or variants thereof can be secreted by immune cells or bound to the membrane of immune cells, preferably in the tumor microenvironment.

[0056] It should be understood that adding an optional linker “GSG” (Gly-Ser-Gly) to the N-terminus of the 2A peptide of the present invention may be helpful for efficiency.

[0057] The present invention also relates to isolated polynucleotides or nucleic acids encoding the chimeric antigen receptor (CAR) of the present invention and fragments or variants thereof, including the domains or regions disclosed herein.

[0058] In one aspect, the isolated polynucleotide is selected from the group consisting of polynucleotides encoding the chimeric antigen receptor (CAR) of the present invention, said polynucleotide having at least 80%, at least 80%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 10, 11, or 12.

[0059] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably to refer to polymers of any type of deoxyribonucleotide (e.g., DNA, cDNA, etc.) or ribonucleotide (e.g., RNA, mRNA, etc.), or polymers of combinations of deoxyribonucleotides and ribonucleotides (e.g., DNA / RNA), which are linear or cyclic conformations and can be single-stranded or double-stranded. These terms should not be construed as limiting the length of the polymer and may cover known analogs of natural nucleotides, as well as nucleotides modified at the base, sugar, and / or phosphate moieties (e.g., phosphate thioester backbones). Typically, analogs of a particular nucleotide have the same base-pairing specificity; that is, an analog of A forms a base pair with T.

[0060] Further, this relates to vectors containing the polynucleotides isolated by the present invention. As used herein, the term "vector" refers to any vector known in the art suitable for the present invention. In some aspects, a vector refers to a viral vector or nucleic acid (DNA or RNA) molecule (such as a plasmid) or other vector containing one or more heterologous nucleic acid sequences of the present invention; and preferably, the vector is designed for transfer between different host cells and / or for amplification purposes. The terms "expression vector," "gene delivery vector," and "gene therapy vector" refer to any vector capable of efficiently integrating and expressing one or more nucleic acids of the present invention within cells, preferably under the regulation of a promoter. Cloning or expression vectors may contain additional elements, such as regulatory elements in addition to the promoter and / or post-transcriptional regulatory elements.

[0061] In one aspect, the vector is selected from the non-restrictive group comprising: retroviral vectors, DNA vectors, plasmids, RNA vectors, adenovirus vectors, adenovirus-associated vectors, lentiviral vectors, lipid nanoparticles (LNPs) targeting RNA (e.g., mRNA), liposomes, or any combination thereof.

[0062] Examples of viral vectors include: retroviruses (such as retroviridae viral vectors); adenoviruses (such as Ad5, Ad26, Ad34, Ad35, and Ad48); parvoviruses (such as adeno-associated virus); coronaviruses; negative-strand RNA viruses, such as orthomyxoviruses (such as influenza virus), rhabdoviruses (such as rabies virus and vesicular stomatitis virus), paramyxoviruses (such as measles virus and Sendai virus); positive-strand RNA viruses, such as picornaviruses and alphaviruses; and double-stranded DNA viruses, including adenoviruses, herpesviruses (such as herpes simplex virus type 1 and 2, Epstein-Barr virus, cytomegalovirus); and poxviruses (such as vaccinia virus, modified vaccinia virus Ankara strain (MVA), fowlpox virus, and canarypox virus). Other viruses include, for example, norovirus, cloacal virus, flavivirus, reovirus, papillomavirus, hepatotropic DNA virus, human papillomavirus, human foamy virus, and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma virus, avian type C virus, mammalian type C virus, type B virus, type D virus, oncoretrovirus, HTLV-BLV group viruses, lentivirus, alpha retrovirus, gamma retrovirus, and foam virus (Coffin, JM, Retroviridae: The viruses and their replication). , Virology, Third Edition (Lippincott-Raven, Philadelphia, 1996). Other examples include: murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus, Mason-Pfizer monkey virus, simmon immunodeficiency virus, simmon sarcoma virus, Rous sarcoma virus, and lentiviruses. Other examples of vectors are also described, such as U.S. Patent No. 5,801,030; the disclosure of that patent is incorporated herein by reference because it relates to viral vectors for gene therapy.

[0063] In some aspects, the polynucleotides described herein are integrated into rAAV vectors and / or viral particles to facilitate their introduction into cells. rAAV vectors that can be used in the compositions and methods described herein are recombinant polynucleotide constructs comprising: (1) a promoter, (2) a sequence to be expressed (e.g., a polynucleotide encoding a protein such as pendrin or Atoh1, or a polynucleotide that can be transcribed to produce an RNA molecule such as repressive RNA), and (3) a viral sequence that facilitates the integration and expression of the expressed sequence. The viral sequence may include the cis sequence (e.g., a functional ITR) required in the AAV for DNA replication and packaging into viral particles. In some embodiments, the rAAV vector also comprises at least one SLC26A4 enhancer. Such rAAV vectors may also contain a biomarker or reporter gene. Available rAAV vectors have one or more AAV WT genes that are partially or completely deleted but retain a functional flanking ITR sequence. The AAV ITR can be any serotype suitable for a particular application. When used in the methods and compositions described herein, the ITR may be an AAV2 ITR. Methods using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279 (2000) and Monahan and Samulski, Gene Delivery 7:24 (2000); the disclosures of each of these publications are incorporated herein by reference because they relate to AVV vectors used for gene delivery.

[0064] The polynucleotides and vectors described herein can be integrated into rAAV viral particles to facilitate the delivery of polynucleotides or vectors into cells. The capsid proteins of AAV constitute the outer non-nucleic acid portion of the viral particle and are encoded by the AAV cap gene. The cap gene encodes three viral capsid proteins required for viral particle assembly: VP1, VP2, and VP3. Methods for constructing rAAV viral particles are contained, for example, in US 5,173,414, US 5,139,941, US 5,863,541, US 5,869,305, US 6,057,152, and US 6,376,237, as well as Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003); the disclosures of each of these publications are incorporated herein by reference because they relate to AVV vectors used for gene delivery.

[0065] The rAAV viral particles that can be used in conjunction with the compositions and methods described herein include viral particles derived from a variety of AAV serotypes, including: AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, and PHP.S. For targeting SLC26A4-expressing cells, AAV1, AAV2, AAV2quad(YF), AAV6, AAV8, AAV9, Anc80, Anc80L65, AAV-DJ, AAV-DJ / 9, 7m8, and PHP.B may be particularly useful. Serotypes evolved for retinal transduction can also be used in the methods and compositions described herein. Construction and application of AAV vectors and AAV proteins of different serotypes are described in, for example: Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al., J. Virol. 72:2224 (1998); Halbert et al., J. Virol. 74:1524 (2000); Halbert et al., J. Virol. 75:6615 (2001) and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001). Because these publications relate to AVV vectors used for gene delivery, the contents of each of these publications are incorporated herein by reference.

[0066] Pseudotyped rAAV vectors can also be used in conjunction with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) that are pseudotyped using capsid genes derived from serotypes other than that given serotype (e.g., AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). Techniques relating to the construction and use of pseudotyped rAAV viral particles are known in the art and are documented in, for example: Duan et al., J. Virol. 75:7662 (2001); Halbert et al., J. Virol. 74:1524 (2000); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001).

[0067] Compared to non-mutated capsid viral particles, AAV viral particles with mutations within their capsids can be used to infect specific cell types more effectively. For example, suitable AAV mutants can carry ligand insertion mutations to facilitate AAV targeting to specific cell types. Wu et al., J. Virol. 74:8635 (2000) describes the construction and characterization of AAV capsid mutants, including insertion mutants, alanine scan mutants, and epitope tag mutants. Other rAAV viral particles that can be used in the methods described in this invention include capsid hybrids generated through viral molecular breeding and exon shuffling. See, for example, Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

[0068] In one aspect, the cell is a mammalian cell, whether autologous or allogeneic. Preferably, the cell is selected from the group consisting of cytotoxic cells, immune cells, stem cells, progenitor cells, cell lines, or cells derived from stem cells or progenitor cells.

[0069] When the cell is an immune cell, the immune cell is selected from the non-restricted group comprising: T cells, tumor-infiltrating lymphocytes (TILs), NK cells, regulatory T cells (Treg cells), macrophages, TCR-expressing cells, eosinophils, basophils, neutrophils, myeloid cells, B cells, plasma cells, regulatory B cells (Breg cells), type 1 innate lymphocytes (ICL1), ILC2, ICL3, dendritic cells, or NK-T cells.

[0070] Various techniques are known in the prior art for the isolation and enrichment of T cells (e.g., PBMCs obtained from peripheral blood samples). Preferably, the immune cells are selected from the group consisting of: T cells, tumor-infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, or NK-T cells.

[0071] When the immune cells are T cells or NK cells, they are autologous or allogeneic T cells or NK cells. In some aspects, the immune cells are T cell lines or NK cell lines. Any cell line derived from any primary cell can be used in this invention. NK cells can be engineered, for example, from a variety of NK sources, such as NK cells derived from iPSCs, NK cells isolated from blood (PB-NK), NK cells isolated from umbilical cord blood, or NK cell lines including NK92, YTS, or KHYG1 cell lines.

[0072] When the cells are derived from stem cells, they are either natural or induced stem cells (e.g., cells derived from iPSCs).

[0073] According to one aspect of the invention, the immune cells express interleukin-4, fragments thereof, or variants thereof. Preferably, the immune cells comprise one or more recombinant constructs, wherein at least one recombinant construct encodes interleukin-4 (IL-4), fragments thereof, or variants thereof.

[0074] The at least one recombinant construct encodes the chimeric antigen receptor (CAR), T-cell receptor (TCR), or any other synthetic tumor-targeting motif described herein.

[0075] According to one aspect, the construct encoding IL-4, its fragments or variants encodes: i) a sequence comprising or consisting of SEQ ID NO:1, its variants or functional fragments thereof; or ii) a sequence comprising or consisting of SEQ ID NO:2, its variants or functional fragments thereof.

[0076] Preferably, a recombinant construct encoding IL-4, its variants, or functional fragments is linked to a second recombinant construct encoding a CAR, TCR, or any other synthetic tumor-targeting motif. In one aspect, the linking is achieved via a sequence encoding a self-cleaving peptide (e.g., peptide 2A selected from P2A, T2A, E2A, and F2A). In another aspect, the sequence encoding the self-cleaving peptide is selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0077] Alternatively, the host cell (e.g., an immune cell) contains and / or expresses at least one expression vector of the present invention and at least one CAR, TCR, or any other synthetic tumor-targeting motif of the present invention. In this case, the expression vector expresses a first recombinant construct encoding IL-4, a variant thereof, or a functional fragment thereof, while a second recombinant construct encodes a CAR, TCR, or any other synthetic tumor-targeting motif, but does not encode IL-4, a variant thereof, or a functional fragment thereof.

[0078] The present invention provides a composition comprising: i) the chimeric antigen receptor polypeptide of the present invention, ii) the isolated polynucleotide or nucleic acid of the present invention, iii) the vector of the present invention, or iv) a host cell, such as an immune cell expressing the chimeric antigen receptor of the present invention.

[0079] The present invention also provides pharmaceutical compositions comprising therapeutically effective amounts of: i) the chimeric antigen receptor polypeptide of the present invention, ii) the isolated polynucleotide or nucleic acid of the present invention, iii) the carrier of the present invention, or iv) host cells, such as immune cells expressing the chimeric antigen receptor of the present invention, and pharmaceutically acceptable carriers, diluents and / or excipients.

[0080] The present invention also provides the use of pharmaceutical compositions in the preparation of medicaments for treating cancer, infectious diseases, inflammatory or inflammation-induced diseases, chronic diseases or autoimmune diseases, said pharmaceutical compositions comprising therapeutically effective amounts of: i) the chimeric antigen receptor polypeptide of the present invention, ii) the isolated polynucleotide or nucleic acid of the present invention, iii) the carrier of the present invention, or iv) host cells, such as immune cells expressing the chimeric antigen receptor of the present invention, and pharmaceutically acceptable carriers, diluents and / or excipients.

[0081] As used herein, the term “therapeutic effective dose” refers to an amount of immune cells, CAR peptides, nucleic acids, plasmids, or vectors that is high enough to significantly and positively alter the symptoms and / or condition to be treated, but low enough to avoid serious side effects (within a reasonable risk / benefit ratio) and within reasonable medical judgment.

[0082] The therapeutically effective amount of host cells (e.g., immune cells), CAR peptides, nucleic acids, plasmids, or vectors described herein is determined based on a variety of factors, including: the type, species, age, weight, sex, and medical condition of the patient or subject; the severity of the condition or disease to be treated (e.g., cancer, infection, or autoimmune disease); the route of administration; and the patient's or subject's renal and hepatic function. Those skilled in the art can readily determine and prescribe the effective amount of immune cells, nucleic acids, plasmids, or vectors required to prevent, combat, or halt the progression of diseases such as cancer, infectious diseases, inflammatory or inflammation-induced diseases, chronic diseases, or autoimmune diseases.

[0083] "Pharmaceutically acceptable carriers or diluents" refers to carriers or diluents that are generally safe, non-toxic, and desirable for use in the preparation of pharmaceutical compositions, including carriers or diluents acceptable for human pharmaceutical use.

[0084] Such pharmaceutical compositions may contain: buffer solutions (such as neutral buffered saline, phosphate buffered saline, etc.), sugars such as glucose, mannose, sucrose or dextran, mannitol, proteins, peptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives.

[0085] Any composition provided herein may be provided as any suitable pharmaceutical composition and may be administered via any suitable route of administration. Suitable routes of administration include, but are not limited to, inhalation, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes. The pharmaceutical compositions of the present invention are preferably formulated for intravenous administration.

[0086] Pharmaceutical compositions (solutions, suspensions, etc.) may contain one or more of the following: sterile diluents, such as water for injection, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride, fixed oils (e.g., synthetic monoglycerides or diglycerides) that can serve as solvents or suspension media, polyethylene glycol, glycerol, propylene glycol, or other solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and tonic modifiers, such as sodium chloride or dextran. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0087] The pharmaceutical compositions of the present invention may further comprise at least one additional therapeutic agent or therapy. A variety of other additional therapeutic agents may be used in combination with the compositions described herein.

[0088] In one aspect, the at least one additional therapeutic agent or therapy is an anticancer agent or anticancer therapy that can be used to treat cancer, preferably solid cancer. Preferably, the one or more anticancer therapies are selected from the group consisting of: radiotherapy, chemotherapy, immune checkpoint inhibitors, immunotherapy, and hormone therapy, or a combination of one or more thereof.

[0089] Additional therapeutic agents suitable for use in combination with this invention include, but are not limited to: ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), and trastuzumab emtansine (trastuzumab).emtansine (KADCYLA®), imatinib (Gleevec®), cetuximab (Erbitux®), panitumumab (Vectibix®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib (gefitinib), vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, ninteda nib), pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib Bosutinib, lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adiponectin peptide, and denileukin interleukin-diphtheria toxin conjugate.diftitox, mTOR inhibitors such as everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, and CDK inhibitors such as palbociclib.

[0090] In other respects, additional treatment agents may be immune checkpoint blockade therapies selected from the group comprising: PD-1 inhibitors, PD-L1 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, BTLA inhibitors and CTLA-4 inhibitors, or one or more combinations thereof (e.g., CTLA-4 inhibitors and PD-1 inhibitors, or LAG-3 inhibitors and PD-1 inhibitors).

[0091] In a preferred aspect, ICBT-based treatment includes treatment with a monoclonal antibody (mAb) specifically targeting or designed to bind to PD-1, PD-L1, LAG-3, TIM-3, TIGIT, BTLA, or CTLA-4, or a combination thereof.

[0092] Non-limiting examples of mAbs specifically targeting PD-1 include nivolumab, pembrolizumab, dostarlimab, retifanlimab, and cemiplimab.

[0093] Non-limiting examples of mAbs specifically targeting PDL-1 include atezolizumab, avelumab, and durvalumab.

[0094] Non-limiting examples of mAbs specifically targeting LAG-3 include favezelimab and relatlimab.

[0095] Non-limiting examples of mAbs specifically targeting TIM-3 include cobolimab, LY3321367, or sabatolimab.

[0096] Non-limiting examples of mAbs specifically targeting TIGIT include tiragolumab, EOS-448 (developed by GSK and iTeos Therapeutics), and Domvanalimab (Gilead / Arcus).

[0097] Non-restrictive examples of mAbs specifically targeting BTLA include the IND (junishi biosciences) and talquetamab-tgvs.

[0098] Non-limiting examples of mAbs specifically targeting CTLA-4 include those consisting of ipilimumab and tremelimumab.

[0099] In other respects, additional treatment agents may be anti-inflammatory agents. Anti-inflammatory agents or drugs include, but are not limited to: steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone); nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate mofetil. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol, or dextropropoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include: molecules that target cell surface markers (e.g., CD4, CD5, etc.); cytokine inhibitors, such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®)); chemokine inhibitors; and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and molecules in recombinant forms. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (orally (auronofen) and intramuscularly), and minocycline.

[0100] The present invention also covers methods for treating and / or preventing cancer, infectious diseases, inflammatory or inflammation-induced diseases, chronic diseases or autoimmune diseases, said methods comprising administering to a subject in need: i) the isolated polynucleotide of the present invention, ii) the carrier of the present invention, iii) the host cell of the present invention, such as an immune cell, or iv) the pharmaceutical composition of the present invention.

[0101] The term "treatment" or "to perform treatment" refers to any administration of the compositions, pharmaceutical compositions, therapeutic agents, compounds, etc. of this disclosure to a subject for the following purposes: (i) Suppress the disease, i.e., prevent the development of clinical symptoms; and / or (ii) Relieve the disease, even if clinical symptoms subside.

[0102] As used herein, the terms “prevention” or “prevention” mean any use of any composition, pharmaceutical composition, therapeutic agent, compound, etc. of this disclosure to a subject for the purpose of preventing disease, i.e., preventing the development of clinical symptoms of the disease.

[0103] In the context of this invention, a disease is cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease, or an autoimmune disease.

[0104] As used herein, the terms “subject” / “subject in need” or “patient” / “patient in need” are well known in the art; these terms are used interchangeably herein to refer to mammals, including dogs, cats, rats, mice, monkeys, cattle, horses, goats, sheep, pigs, camels, and most preferably, humans. In some cases, a subject is a subject in need of treatment or a subject suffering from a disease or condition. However, in other respects, a subject may be a healthy subject. The term does not indicate a specific age or sex. Therefore, it is intended to cover adult and neonatal subjects, whether male or female. Preferably, a subject is a person; most preferably, a person who has or is at risk of having cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease, or an autoimmune disease.

[0105] In one aspect, a method for treating and / or preventing cancer, infection, or autoimmune disease in a patient or subject includes: (i) removing and isolating immune cells, preferably immune cells, more preferably native T cells, from the patient or subject; (ii) genetically engineering the T cells using a recombinant construct (e.g., a vector, plasmid, or polynucleotide) encoding the chimeric antigen receptor (CAR) of the present invention; (iii) expanding the engineered immune cells, such as T cells, in vitro into a larger population; and (iv) reintroducing the engineered immune cells, such as T cells, into the patient or subject. After reintroduction of the immune cells, such as T cells, into the patient or subject, they perform their functions, which vary depending on the type of immune cells used. Examples of functions include triggering cell death of target cells (T cells and NK cells), phagocytosis of target cells (macrophages), or inducing an immunosuppressive environment (Treg) around the target cells.

[0106] Alternatively, methods for treating and / or preventing cancer, infectious diseases, inflammatory or inflammation-induced diseases, chronic diseases, or autoimmune diseases in patients or subjects may include: (i) providing genetically engineered immune cells, such as T cells, having a recombinant construct (e.g., a vector, plasmid, or polynucleotide of the present invention) encoding the chimeric antigen receptor (CAR) of the present invention; (ii) expanding the engineered immune cells, such as T cells, into a larger population in vitro; and (iii) reintroducing the engineered immune cells, such as T cells, into the patient or subject.

[0107] When the disease to be treated is inflammatory or inflammation-induced, it is selected from the non-restricted group including the following: fibrosis (pulmonary or cardiac fibrosis), chronic obstructive pulmonary disease, cardiovascular disease, diabetes, asthma, fatty liver disease, gout, and scleroderma.

[0108] When the disease to be treated is a chronic disease, it is selected from the non-restricted group that includes the following: fibrosis, Alzheimer's disease, lupus, and chronic kidney disease.

[0109] When the disease to be treated is an infectious disease, it is selected from the non-restricted group including the following: HIV, hepatitis C, and human cytomegalovirus.

[0110] When the disease to be treated is cancer, it is selected from solid cancer or liquid cancer.

[0111] When the cancer to be treated is a solid cancer, it is selected from the non-restricted group including: lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, and skin cancer (especially melanoma), or one or more combinations thereof.

[0112] When the cancer to be treated is a liquid cancer, it refers to cancer cells present in bodily fluids such as blood, lymph, and bone marrow. Liquid cancers are selected from the non-restricted group that includes the following: leukemia, myeloma, myelodysplastic syndromes (MDS), and liquid lymphoma. For example, a liquid cancer can be acute myeloid leukemia (AML). Liquid lymphomas include lymphomas containing cysts or fluid-filled areas.

[0113] When the disease to be treated is an autoimmune disease, it is selected from the non-restricted group including: rheumatoid arthritis (RA), multiple sclerosis (MS), endometriosis, inflammatory bowel disease (IBD), psoriasis and psoriatic arthritis, or one or more combinations thereof.

[0114] The polypeptides disclosed herein, or nucleic acids encoding such polypeptides, can be introduced into host cells using transfection and / or transduction techniques known in the art. The nucleic acids may integrate into the host cell DNA or be maintained in an extrachromosomal form. The nucleic acids may be transiently maintained or stably introduced. Transfection can be performed using a variety of methods known in the art, including, but not limited to: calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and gene gun methods. Transduction refers to the delivery of genes using viral or retroviral vectors via viral infection rather than transfection. In some embodiments, retroviral vectors are transduced by packaging the vector into viral particles prior to contact with cells. For example, nucleic acids encoding transmembrane polypeptides carried by a retroviral vector can be introduced into cells via infection and proviral integration.

[0115] In some respects, nucleic acids or vectors such as viral vectors are transferred via in vitro transformation. Methods for transfecting cells and tissues taken from an organism in an in vitro setting are known to those skilled in the art. Therefore, it is conceivable that cells (or tissues) are taken and transfected in vitro using the polynucleotides described herein. In certain respects, transplanted cells or tissues can be placed into an organism. Thus, isolating antigen-presenting cells (such as T cells or NK cells) from an animal (e.g., a human), transfecting these cells with expression vectors, and then administering the transfected or transformed cells back to the animal (e.g., a human) is entirely within the knowledge of those skilled in the art.

[0116] In some cases, nucleic acids or vectors are transferred via injection. In others, polynucleotides are introduced into organelles, cells, tissues, or organisms via electroporation. In still others, polynucleotides are delivered into cells using DEAE-glucan followed by polyethylene glycol.

[0117] In one aspect, methods of treating and / or preventing cancer, infectious diseases, inflammatory or inflammation-induced diseases, chronic diseases, or autoimmune diseases in a subject include administering the pharmaceutical composition of the present invention to the subject in need.

[0118] In one aspect, the aforementioned methods of treatment and / or prevention may further include the application of at least one additional therapeutic agent or therapy.

[0119] In one aspect, at least one additional therapeutic agent or therapy is an anticancer agent or anticancer therapy, more preferably a therapeutically effective amount or dose of an anticancer agent or anticancer therapy. The one or more anticancer agents or therapies are selected from the non-limiting group comprising: radiotherapy, chemotherapy, immune checkpoint inhibitors, immunotherapy, and hormone therapy, or combinations thereof, as described herein.

[0120] In another respect, additional treatments can be anti-inflammatory agents as described in this article.

[0121] This invention also relates to kits for treating and / or preventing diseases of this invention. In one aspect of the invention, the kit comprises: i) the CAR polypeptide of the invention; ii) the isolated polynucleotide of the invention; iii) the vector of the invention; iv) the host cell of the invention, preferably an immune cell; or v) the pharmaceutical composition of the invention.

[0122] The kit of the present invention may also include a container and a label or packaging insert affixed to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be made of a variety of materials, such as glass or plastic. The container contains a composition effective for treating the disease or condition of the present invention and may be equipped with a sterile access port (e.g., the container may be an intravenous infusion bag or vial with a stopper that can be punctured by a hypodermic needle). Alternatively, or in addition, the kit may also include a second (or third) container containing a pharmaceutically acceptable buffer solution (e.g., antibacterial water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution) and / or at least one additional therapeutic agent (e.g., the ligand of the present invention). It may also contain other materials desired from a commercial and user perspective, such as other buffers, diluents, filters, needles, and syringes.

[0123] Labels or packaging inserts may contain instructions for use. These instructions may be affixed to the packaging material or incorporated as a packaging insert. While these instructions are typically written or printed materials, they are not limited to such forms. This disclosure covers any medium capable of storing and communicating such instructions for use to the end user.

[0124] Unless otherwise stated, the implementation of this disclosure will employ conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology within the scope of the art. These techniques have been described in detail in the literature. See, for example: TE Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991); Carey and Sundberg Advanced Organic Chemistry 3rd Edition (Plenum Press), Volumes A and B (1992).

[0125] Therefore, this disclosure should be regarded as illustrative rather than restrictive in all respects, the scope of protection of the invention is defined by the appended claims, and all modifications in the equivalent sense and scope are intended to be included in the invention.

[0126] sequence SEQ ID NO: 1--Mouse IL-4 amino acid sequence

[0127] SEQ ID NO: 2 -- Human IL-4 amino acid sequence

[0128] SEQ ID NO: 3--HER2 scFv amino acid sequence

[0129] SEQ ID NO: 4--TRP-1 scFv amino acid sequence

[0130] SEQ ID NO: 5--EGFRvIII scFv amino acid sequence

[0131] SEQ ID NO: 6--CD19 scFv amino acid sequence

[0132] SEQ ID NO: 7--CD8 transmembrane structural domain and hinge region amino acid sequence

[0133] SEQ ID NO: 8--CD3ζ amino acid sequence

[0134] SEQ ID NO: 9--4-1BB amino acid sequence

[0135] SEQ ID NO: 10--HER2 CAR-P2A-IL4 Polynucleotide sequence

[0136] SEQ ID NO: 11--TRP1 CAR-P2A-IL4 Polynucleotide sequence

[0137] SEQ ID NO: 12--EGFR CAR-P2A-IL4 Polynucleotide sequence

[0138] SEQ ID NO: 13--CD19 CAR-T2A-IL4 Polynucleotide sequence

[0139] Example 1. Materials and Methods Mice.Female C57BL / 6 (C57BL / 6J) and BALB / c (BALB / cByJ) mice, aged six to eight weeks, were purchased from Charles River Laboratories (Lyon, France) and housed at the Animal Research Center (CPG) of the Swiss Federal Institute of Technology in Lausanne (EPFL). The experimental procedures for the mouse studies were approved by the Swiss authorities (Canton of Vaud, Laboratory Animal Research Program ID: 3533) and conducted in accordance with the guidelines of the University of Lausanne Animal Research Center (EPFL CPG).

[0140] Cell and tumor models. Based on the methods of previous literature 18,19 HER2-transduced MC38 mouse colon cancer cells (MC38-HER2) were generated. B16F10 melanoma cells and Phoenix-Eco cells were originally purchased from the American Type Culture Collection (ATCC). 4T1-EGFRvIII-Luc mouse breast cancer cells were provided by Professor Darrell J. Irvine (MIT). All mouse tumor cells were cultured in complete Duchenne Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) (10% v / v, Gibco / Thermo Fisher Scientific) and penicillin / streptomycin (1% v / v, Gibco / Thermo Fisher Scientific). MC38-HER2 cells (3 × 10⁻⁶) were cultured in DMEM supplemented with fetal bovine serum (FBS) (10% v / v, Gibco / Thermo Fisher Scientific). 5 ) and B16F10 tumor cells (3×10 5 Subcutaneous (sc) implantation was performed into the right flank of C57BL / 6 wild-type (WT) mice to establish a syngeneic tumor model. 4T-EGFRvIII-Luc tumor cells (5 × 10⁻⁶) were implanted. 4 ) Intravenous injection into BALB / c WT mice.

[0141] Building CAR As previously stated 20A HER2-targeting CAR construct was generated. In short, the complete CAR sequence consists of a mouse CD8 signal peptide, an antigen-specific scFv (TA99 scFv for the monospecific TRP-1 CAR and 139 scFv for the EGFRvIII CAR), a mouse CD8a hinge region and transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular domain. To facilitate CAR detection by flow cytometry, a c-Myc tag was inserted between the scFv and the CD8a hinge region of the EGFRvIII CAR and TRP-1 CAR. The full-length mouse IL-4 gene (accession number: NP_034678.1) was obtained by PCR amplification from cDNA clones custom-purchased from Twist Bioscience. The IL-4 CAR construct was prepared by fusing the 2A self-cleaving peptide with the IL-4 gene fragment into a CAR-containing viral vector (pMSGV for HER2 CAR, and pMSCV for TRP-1 CAR and EGFRvIII CAR).

[0142] Preparation of retroviruses. Retroviruses were produced using the calcium phosphate method according to the manufacturer's (Clonetech) protocol. In short, Phoenix cells were seeded in 10 cm culture dishes and cultured for 18 hours prior to transfection, with 10 ml of pre-warmed medium added undisturbed before transduction. For each transfection, 14 µg of plasmid (8.5 µg CAR plasmid and 5.5 µg pCL-Eco packaging plasmid) was added to 628 µl of ultrapure water, followed by 72 µl of CaCl2 solution (2 M, Sigma-Aldrich). Hepes buffered saline (700 µl × 2, Sigma-Aldrich) was then added dropwise under gentle vortexing. After incubation at 25°C for 20 minutes, the transfection mixture was gently added to the Phoenix cells, and 10 ml of pre-warmed medium was added after 16 hours. After incubation for 36 to 72 hours, the virus-containing supernatant was collected and passed through a 0.45 µm filter (Merck Millipore) to remove cell debris. The virus-containing supernatant was then aliquoted and stored at -80°C.

[0143] Lentiviral preparation. Lentiviral viruses are produced using the polyethyleneimine (PEI) process, according to the manufacturer's (Polysciences, Inc.) protocol. In short, 1×10 7Human embryonic kidney (HEK) cells were seeded in T150 cell culture flasks and cultured for 24 hours prior to transfection. For each transfection, 15 µg of CAR plasmid, 18 µg of Δ8.9 plasmid, and 7 µg of VSV-G plasmid were added to 2 mL of serum-free medium, followed by the addition of 100 µg of PEI. The transfection mixture was incubated at 25 °C for 20 min, during which time the medium for HEK cells was aspirated. The mixture was then slowly added to the HEK cells and vortexed to mix. 14 mL of serum-containing medium was added to the HEK cells, and the culture flasks were incubated at 37 °C for 2 days. After 48 hours of incubation, the virus-containing supernatant was collected and passed through a 0.45 µm filter (Merck Millipore) to remove cell debris. The virus-containing supernatant was then aliquoted and stored at -80 °C.

[0144] Preparation of mouse CAR-T cells. The spleens of WT mice were mechanically lysed and filtered through a 70 μm sieve (Fisher Scientific). Red blood cells (RBCs) were lysed for 5 min at 25°C with ACK lysis buffer (2 ml / spleen, Gibco / Thermo Fisher Scientific). Spleen cells were washed once with cold, complete RPMI medium containing RPMI-1640 (Gibco), FBS (10% v / v, Gibco / Thermo Fisher Scientific), HEPES (1% v / v, Gibco / Thermo Fisher Scientific), penicillin / streptomycin (1% v / v, Gibco / Thermo Fisher Scientific), sodium pyruvate (1% v / v, Gibco / Thermo Fisher Scientific), and 2-mercaptoethanol (0.1% v / v, Gibco / Thermo Fisher Scientific), followed by 2 × 10⁻⁶ ppm. 6 Cells were resuspended at a density of 10 cells / ml in complete RPMI medium supplemented with IL-2 (10 ng / ml, PeproTech) and IL-7 (10 ng / ml, PeproTech). For WT T cells, 6-well plates were pre-coated with 1 ml anti-CD3 antibody (1 µg / ml, 17A2, BioXCell) and anti-CD28 antibody (5 µg / ml, 37.51, BioXCell) / well for 18 hours at 4°C. T cells were enriched using Ficoll-Paque PLUS (GE Healthcare) and subsequently cultured at 3 × 10⁻⁶ cells / well. 6Cells were seeded at a density of 3 ml / well into pre-coated 6-well plates in complete RPMI medium. Cells were cultured at 37°C for 48 hours under undisturbed conditions. Before transduction, the 6-well plates were coated with protamine (10 µg / ml, Sigma-Aldrich) for 24 hours. On day 2, the protamine-coated plates were blocked with PBS containing (0.5% v / v) FBS for 30 minutes before use. Viral supernatant was first added to each well of the blocked plates (1 ml / well), followed by centrifugation at 2000g for 2 hours at 32°C. Activated T cells were collected, enriched again using Ficoll-Paque PLUS, and cultured at 2 × 10⁻⁶ cells / well. 6 Cells / ml were resuspended in complete RPMI medium supplemented with IL-2 (10 ng / ml). The cell suspension was added to virus-enriched culture plates (1 ml per well) and thoroughly mixed by gentle shaking. Transfection was performed at 32°C with 500g rotation for 30 minutes. The culture plates were then transferred to an incubator and incubated overnight. On day 3, a second transduction was performed, and T cells were collected and added to a virus-containing plate after centrifugation. After overnight incubation, T cells were collected and expanded in fresh complete RPMI medium supplemented with IL-2 (10 ng / ml). On day 5, transduction efficiency was determined by flow cytometry. For HER2 CAR-T, CAR expression was detected using biotinylated human Her2 / ErbB2 protein (Acro Biosystems) and the secondary staining reagent streptavidin-PE / Cy7 (BioLegend). The expression of TRP-1 CAR or EGFRvIII CAR was assessed by surface staining of the c-Myc tag using anti-Myc antibody (9B11, Cellsignaling). Untransduced T cells, simultaneously activated and cultured in virus-free medium, were used as controls. CAR-T cells were used for in vitro and in vivo experiments on day 5. After co-culturing for 2 days following CAR-T cell generation, the IL-4 concentration in the culture supernatant was determined using ELISA MAX™ Standard Set Mouse IL-4 (BioLegend).

[0145] Preparation of human CAR-T cells. Human peripheral blood mononuclear cells were obtained from anonymous healthy donors (prepared as erythrocyte sedimentation rate brown layer). CD3+ T cells were isolated using Miltenyi Biotec's Human Pan T Cell Isolation Kit (catalog number: 130-096-535). In the presence of human IL-2 (50 IU / mL), cells were expanded and activated in vitro using Dynabead for T cell proliferation and activation. TMHuman T-Activator CD3 / CD28 (Catalog No.: 11161D) activates CD3+ T cells for 5 days. On day 1, T cells are injected at a dose of 1×10⁻⁶. 6 Dynabeads were seeded at a concentration of 1 cell / mL in 24-well plates, 2 mL per well. 1.4 mL of culture medium was removed, and lentivirus was added to the plate at an MOI of 2–3 in the presence of 20 μg / mL protamine. Human IL-2 was added to bring the total concentration to 30 IU / mL. Cells were centrifuged at 800 × g for 30 min at 32°C. Cells were incubated at 37°C for 4 h, followed by incubation at 37°C for 48 h with 1 mL of pre-warmed R10 medium containing human IL-2 (30 IU / mL) in each well. On day 5, Dynabeads were removed using magnetic cell separation. TM On day 10, transduction efficiency was determined by flow cytometry. For CD19 CAR-T, CAR expression was detected using Acro Biosystems' monoclonal anti-FMC63 antibody mouse IgG1 (Y45) (catalog number: FM3-Y45). Non-transduced T cells (NTDs) that were simultaneously activated and cultured in virus-free medium served as controls. On day 10, CAR-T cells were used in vitro. After co-culturing for 2 days following CAR-T cell generation, the IL-4 concentration in the culture supernatant was determined using ELISA MAX™ Standard Set MouseIL-4 (BioLegend).

[0146] Flow cytometry analysis.For surface marker staining, cells were collected in U-bottom 96-well plates (ThermalFischer Scientific), blocked with anti-mouse CD16 / 32 antibody (BioLegend), and incubated with the specified antibody at 4°C for 20 min. Live / dead cell staining was then performed using 4',6-diamidinyl-2-phenylindole (DAPI, Sigma-Aldrich). Cells were then washed and resuspended in PBS containing bovine serum albumin (BSA, 0.2% wt / v, Sigma-Aldrich) for flow cytometry analysis. Further surface marker and DAPI staining was then performed for flow cytometry analysis. For intracellular cytokine staining, cells were first stimulated at 37°C for 5 h with a cell stimulation mixture (Invitrogen / ThermoFisher Scientific). Following stimulation, cells were first stained with surface markers and Zombie AquaFixable Dye (BioLegend), followed by fixation and permeabilization using the Cytofix / Cytoperm™ Fixation / Permeabilization Kit (BDBiosciences). Intracellular staining was performed using the specified antibodies according to the manufacturer's protocol. Cells were analyzed using an Attune NxT flow cytometer and Attune NxT software v.3 (Invitrogen / Thermal FischerScientific). Analyses were performed using FlowJo 10.6.1 (Tree Star). Gate margins were determined using isotype and fluorescence minus one control.

[0147] Antibodies and reagents used in flow cytometry. Purchase the following antibodies or staining reagents from BioLegend: CD16 / 32 (93, 101302), CD45.2 (104, 109814), CD8β (YTS256.7.7, 126606), CD4 (RM4-5, 100526), ​​CD3ε (17A2, 100306), CD11c (N418, 117348), IA / IE (MHC-II, M5 / 114.15.2, 107643), granzyme B (GB11, 515403), IFNγ (XMG1.2, 505826), TNFα (MP6-XT22, 506308), IL-4 (11B11, 504133), and streptavidin-PE / Cy7 (405206). Biotinylated human Her2 / ErbB2 protein (HE2-H822R) was purchased from ACROBiosystems. Myc-tag antibody (9B11, 3739) was purchased from Cell Signaling Technology.

[0148] Antitumor therapy and re-attack experiments. On day 5, C57BL / 6 or BALB / c WT mice with established tumors underwent sublethal lymphocyte depletion via whole-body irradiation (5 Gy). On day 6, mice received intravenous adoptive transfer of CAR-T cells (HER2 CAR-T, TRP-1 CAR-T, or EGFRvIII CAR-T, 3 × 10⁻⁶ cells). 6 CAR + T cells), with or without intravenous administration of IL-4 (1 µg); IL-4 CAR-T cells (IL-4 HER2CAR-T, IL-4 TRP-1 CAR-T, or IL-4 EGFRvIII CAR-T, 3 × 10⁻⁶ cells). 6 CAR + T cells); or PBS. For the MC38-HER2 or B16F10 subcutaneous model in C57BL / 6 mice, tumor area and body weight were measured every other day. Tumor area was calculated using the formula “Area = Length x Width”, where length and width were two orthogonal diameters measured with calipers. For the 4T1-EGFRvIII-Luc metastatic tumor model, BALB / c mice were anesthetized and intraperitoneally injected with 100 µl of D-fluorescein potassium salt (pre-diluted to 30 mg / ml with 1×PBS). Ten minutes after injection, mice were imaged using the Xenogen IVIS fluorescence / bioluminescence imaging system to monitor tumor growth. In the tumor cell re-challenge experiment, MC38-HER2 (1 × 10⁻⁶ T cells) were used 3 months after adoptive CAR-T cell transfer. 6 B16F10 (1 × 10) 5 Tumor cells were re-inoculated subcutaneously into the left flank of surviving mice in each treatment group. Untreated WT mice were subcutaneously inoculated with the same number of tumor cells as a control. Survival of re-challenged mice was monitored for at least another 60 days. Mice were considered viable after re-challenge when body weight loss exceeded 15% of baseline body weight and tumor area reached 150 mm². 2 Mice were euthanized when researchers detected any signs of discomfort or when a veterinarian monitoring the mice every other day made a recommendation.

[0149] Analysis of tumor-infiltrating immune cells. C57BL / 6 mice were subcutaneously inoculated with MC38-HER2 tumor cells (1 × 10⁻⁶). 6 On day 5, sublethal lymphocytes were eliminated by irradiation, and on day 6 post-tumor inoculation, the following material was received via intravenous adoptive transfer: HER2 CAR-T cells (3 × 10⁻⁶). 6 CAR+ T cells), in the presence or absence of intravenous administration of IL-4 (1 µg); IL-4 HER2 CAR-T (3 × 10⁻⁶ cells). 6 CAR + T cells); or PBS control. On day 14, tumors were collected, weighed, mechanically disrupted, and digested at 37°C for 60 min in RPMI-1640 medium supplemented with type IV collagenase (1 mg / ml, Gibco / Thermo Fisher Scientific), dispersin II (100 μg / ml, Sigma-Aldrich), hyaluronidase (100 μg / ml, Sigma-Aldrich), and DNase I (100 μg / ml, Sigma-Aldrich). RBCs in the digested tumor samples were lysed with ACK lysis buffer. Tumor-infiltrating leukocytes were then enriched by Percoll (GEHEALTHCARE) density gradient centrifugation, resuspended in PBS containing BSA (0.2%, wt / v), stained with designated antibodies, and analyzed by flow cytometry.

[0150] Analysis of spleen-derived CAR-T cells. On day 5, C57BL / 6 or BALB / c WT mice with established tumors underwent sublethal lymphocyte depletion via whole-body irradiation (5 Gy). On day 6, mice received intravenous adoptive transfer of CAR-T cells (HER2 CAR-T or EGFRvIII CAR-T, 3 × 10⁻⁶ cells). 6 CAR + T cells); IL-4 CAR-T cells (IL-4 HER2 CAR-T or IL-4 EGFRvIII CAR-T, 3 × 10⁻⁶) 6 CAR + T cells); or PBS. Spleens were collected on days 14 and 18, respectively, for analysis of HER2 CAR-T and EGFRvIII CAR-T cells. The spleen was dissected from the surrounding tissue, milled, and filtered through a 70 μm sieve (Fisher Scientific). RBCs in spleen samples were lysed with ACK lysis buffer (2 ml per spleen, Gibco / Thermo Fisher Scientific) and then resuspended in PBS containing BSA (0.2%, wt / v). RBCs were lysed with ACK lysis buffer, and RBC fragments were removed with Percoll (GE Healthcare) and then resuspended in PBS containing BSA (0.2%, wt / v). Cells collected from the above samples were stained with designated antibodies and analyzed by flow cytometry.

[0151] Co-culture of CAR-T cells and tumor cells in vitro. MC38-HER2, B16F10, 4T1-EGFRvIII-Luc, and NALM6-GFP tumor cells (1 × 10⁻⁶) were used. 5 Cells were seeded in complete DMEM medium in 12-well plates (Thermal Fischer Scientific) and incubated overnight. After aspirating the tumor culture medium, HER2 CAR-T, TRP-1 CAR-T, EGFRvIII CAR-T, or human CD19 CAR-T cells were suspended in complete RPMI medium supplemented with IL-2 (10 ng / ml) (mice) or human IL-2 (30 IU / mL) (humans) on day 5 (mice) or day 10 (humans), and then added to the tumor cell culture. For mice, the E:T ratio was 0.5:1 in the presence or absence of IL-4 (145 ng / ml), and the E:T ratio was 1:5. After co-culturing for 1 day, cells were collected from the culture plates and analyzed by flow cytometry to determine the cytotoxic phenotype of CAR-T cells. After co-culturing for 2 days, cells were collected from the culture plates and analyzed by flow cytometry to determine the viability of tumor cells, the proliferation and exhaustion phenotype of CAR-T cells.

[0152] Seahorse measurement. MC38-HER2 tumor cells (1 × 10⁻⁶) 6 HER2 CAR-T cells were seeded in complete DMEM medium in T25 culture flasks (Thermal Fischer Scientific) and incubated overnight. After aspirating the tumor cell culture medium, HER2 CAR-T or IL-4 HER2 CAR-T cells in complete RPMI medium supplemented with IL-2 (10 ng / ml) were added to the tumor cell culture at an E:T ratio of 5:1. After co-culturing for 16 hours, the CAR-T cells in the culture flasks were collected and isolated using Ficoll-PaquePLUS for seahorse assays. Seahorse assays were performed to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of CAR-T cells. CAR-T cells (3 × 10⁶ cells / vial) under normal culture conditions or after co-culture were seeded in complete DMEM medium in T25 culture flasks (Thermal Fischer Scientific) and incubated overnight. 5Cells were seeded in / well / wells in Seahorse culture plates (Seahorse Bioscience) and incubated at 37°C for 40 minutes in a CO2-free incubator. OCR and ECAR were measured using a Seahorse XFe96 analyzer (Seahorse Bioscience) according to the manufacturer's instructions. In the Seahorse assay, cells were treated with oligomycin (1 μM, Sigma-Aldrich), 4-(trifluoromethoxy)phenylhydrazone (FCCP, 2 μM, Sigma-Aldrich), rotenone (0.5 μM, Sigma-Aldrich), antimycin A (0.5 μM, Sigma-Aldrich), glucose (10 mM, Sigma-Aldrich), and 2-deoxy-D-glucose (2-DG, 50 mM, Sigma-Aldrich). Each condition was set up in 3 to 6 replicates per experiment. Treatment was based on previous literature reports. 21 Calculate the basics or maximum OCR and ECAR.

[0153] Treatment with metabolic inhibitors. MC38-HER2 cells (1 × 10⁻⁶) 5 CAR-T cells were seeded in 12-well plates (Thermal Fischer Scientific) and incubated overnight. As described above, on day 6, HER2 CAR-T or IL-4 HER2 CAR-T cells were co-cultured with pre-seeded MC38-HER2 cells at an E:T ratio of 0.5:1 in complete RPMI medium containing IL-2 (10 ng / ml) and a specified inhibitor for 2 days. The phenotype of CAR-T cells was determined by flow cytometry.

[0154] Statistical analysis. Statistical analysis was performed using GraphPad Prism 9 (GraphPad software). Unless otherwise stated, data are expressed as mean ± sem. Comparisons between two groups were performed using a two-tailed unpaired Student's t-test. Comparisons among multiple groups were performed using one-way or two-way ANOVA combined with Tukey's multiple comparison test. The Log-rank test was used to analyze survival data. A p-value greater than 0.05 was considered statistically significant (NS) for all groups.

[0155] 2. Results 2.1 We hypothesize that CAR-T cells armored with type 2 cytokines can enhance the cytotoxicity of CAR-T cells and stimulate immune activation that counteracts immunosuppression in the tumor microenvironment (TME) of solid tumors. To test this hypothesis, we first constructed mouse IL-4-expressing CAR-T cells based on a conventional second-generation CAR (HER2 scFv-CD8 transmembrane (TM)-4-1BB-CD3ζ) (Fig. 1a). The cell surface expression of HER2 CAR in IL-4 HER2 CAR-T cells was almost equivalent to or slightly lower than that in conventional HER2 CAR-T cells (Fig. 1b). During in vitro culture, HER2 CAR-T cells engineered to express IL-4 produced IL-4 (Fig. 1c). Compared with HER2 CAR-T cells, IL-4-expressing HER2 CAR-T cells showed a higher tumor lysis potential against MC38-HER2 cells (Fig. 1d).

[0156] To predict in vivo activity in the TME, we co-cultured MC38-HER2 cells with HER2CAR-T cells expressing or not expressing IL-4 at a low E:T ratio for 2 days. In co-culture with MC38-HER2 cells, IL-4 HER2 CAR-T cells exhibited significantly enhanced proliferative capacity, pluripotency, and killing efficiency against target cells (Figure 1e-g), results consistent with our previously described tumor lysis assay.

[0157] 2.2 Next, we evaluated whether IL-4 expression could reprogram CAR-T cell metabolism. Compared to HER2 CAR-T cells, IL-4-expressing HER2 CAR-T cells exhibited significantly elevated basal and maximum ECAR values, while the OCR value remained essentially unchanged (Fig. 1h-k). This finding was consistently observed when persistent tumor antigen stimulation was present (Fig. 1l-o). These results indicate that IL-4 expression reprograms CAR-T cell metabolism toward glycolysis in a process independent of antigen.

[0158] 2.3 Next, we used multiple pathway-specific inhibitors to explore the molecular basis of secreted IL-4's metabolic regulation of CAR-T cells. By applying specific inhibitors targeting glycolysis-related metabolic pathways, we found that inhibiting glucose-to-pyruvate conversion using 2-deoxy-D-glucose (2-DG), 3-bromopyruvate (3BP), 6-aminonicotinamide (6-AN), and sodium fluoride (NaF) did not impair IL-4-induced cytotoxic CAR-T cell proliferation (Fig. 1p). Notably, inhibition of lactate dehydrogenase A (LDHA) by FX11 significantly eliminated IL-4-mediated cytotoxic CAR-T cell enrichment (Fig. 1p). Given the central role of LDHA in the conversion of pyruvate to lactate, our results suggest that IL-4 expression enhances the effector function of CAR-T cells by enhancing glycolysis in an LDHA-dependent manner.

[0159] 2.4 Next, we investigated whether IL-4-armored HER2 CAR-T cells could combat functional impairment and maintain effector function in tumors. We intravenously transferred IL-4 HER2 CAR-T cells (3 × 10⁻⁶ cells). 6 ), or intravenously transferred HER2 CAR-T cells (3 × 10⁻⁶) under conditions with or without intravenously administered free mouse IL-4. 6 IL-4 HER2 CAR-T cells were used to treat subcutaneous MC38-HER2 murine colonic adenocarcinoma tumors established in a mouse model. Compared with conventional CAR-T cells, IL-4 HER2 CAR-T cells showed an approximately 4.1-fold increase in cell count in the tumor, indicating that IL-4 expression significantly promoted CAR-T cell proliferation in the tumor mesenchymal stem cell (TME) (Fig. 2a). Furthermore, compared with conventional HER2 CAR-T cells, IL-4 HER2 CAR-T cells also showed a significantly increased cell count in secondary lymphoid organs (including the spleen and tumor-draining lymph nodes) (Fig. 2b, c). IL-4 expression promotes TIM-3... 高 PD-1 高 CAR-T cells convert to TIM-3 高 PD-1 低Phenotypic subset differentiation was observed, but progenitor cell exhaustion subsets were not altered (Fig. 2d-f). Importantly, IL-4 HER2 CAR-T cells also exhibited enhanced cytotoxicity and pluripotency, but decreased expression of the inhibitory marker PD-1 (Fig. 2g-i). Since IL-4 is a type 2 cytokine that can repolarize T cells to promote potent type 2 immunity, we then investigated whether IL-4-expressing CAR-T cells could alter the composition of T cell subset distributions. We observed that IL-4-expressing HER2 CAR-T cells were enriched in Th2 cells and type 2 cytotoxic (Tc2) cells in tumor CAR-T cells (Fig. 2j, k). These results suggest that IL-4-expressing CAR-T cells possess unique antitumor characteristics compared to conventional CAR-T cells, including excessive proliferative capacity, enhanced cytotoxicity, and an activated type 2 T cell population.

[0160] The discovery that IL-4 HER2 CAR-T cells enhance expansion and effector function prompted us to evaluate their efficacy against solid tumors. To treat pre-established MC38-HER2 subcutaneous solid tumors in mice, we administered IL-4 HER2 CAR-T cells (3 × 10⁻⁵ cells) intravenously. 6 This resulted in a 40% tumor clearance rate and a radical response (Fig. 3a, b). To investigate whether IL-4-expressing HER2 CAR-T cells form anti-tumor immune memory, we re-challenged surviving mice 3 months after adoptive CAR-T cell transfer. Notably, long-term survivors treated with IL-4 HER2 CAR-T cells rejected the second challenge with the original tumor cells in 100% of cases (Fig. 3c). One of the main reasons for failure of CAR-T cell therapy in solid tumors is antigen downregulation or loss. Therefore, we next evaluated whether IL-4-expressing CAR-T cells could resist the challenge of antigen-negative MC38 tumors in long-term surviving mice after re-challenging with MC38-HER2 cells (Fig. 3d). Interestingly, we found that surviving mice with IL-4-expressing CAR-T cells not only rejected the growth of HER2-positive tumors but also completely resisted the challenge of HER2-negative tumors (Fig. 3d). This finding prompted us to determine whether endogenous cell types are involved in the IL-4-CAR-T cell-induced anti-tumor response during the rejection of antigen-negative tumors. To identify changes in immune cells within tumors, we performed flow cytometry analysis on tumor-infiltrating cells purified from MC38-HER2 tumor-bearing mice treated with HER2 CAR-T cells. Compared to HER2 CAR-T cells, IL-4 HER2 CAR-T cell therapy significantly increased CD45.2 levels. +The number of cells was increased, leading to the enrichment of mature dendritic cells (DCs) in the tumor (Fig. 3e, f). In summary, these changes appear to promote the control of antigen-negative tumors by IL-4-expressing CAR-T cells, suggesting that IL-4 may have pleiotropic effects, shifting the balance of the immune population towards durable anti-tumor immune metastasis against both antigen-positive and pre-existing antigen-negative tumor cells.

[0161] 2.6 We then extended this strategy to CARs targeting the melanoma-associated antigen TRP-1 and prepared IL-4 TRP-1 CAR-T cells (Figs. 4a-c). Compared to conventional TRP-1 CAR-T cells, IL-4 TRP-1 CAR-T cells exhibited enhanced expansion and killing efficiency against TRP-1-expressing B16F10 melanoma cells in vitro (Figs. 4d, e). In a therapeutic setting of low-immunogenicity and highly invasive mouse B16F10 melanoma in pretreated mice with lymphocyte depletion, adoptive transfer of IL-4 TRP-1 CAR-T cells (3 × 10⁻⁶ cells) was performed. 6 In 50% of treated mice, IL-4 induced complete tumor remission and durable cure (Fig. 4f, g). In contrast, adoptive transfer of TRP-1 CAR-T cells alone or in combination with intravenous IL-4 failed to induce any durable tumor regression (Fig. 4f, g). We then evaluated whether IL-4-expressing CAR-T cells could control metastatic tumors. We prepared IL-4-expressing CAR-T cells targeting EGFRvIII (Fig. 5a-c). Compared with EGFRvIII CAR-T cells, IL-4 EGFRvIII CAR-T cells exhibited enhanced proliferation and in vitro killing efficiency against 4T1-EGFRvIII-Luc target cells (Fig. 5d, e). To establish a metastatic tumor model, BALB / c mice received intravenous injection of 4T1-EGFRvIII-Luc cells. Mice with metastatic lung tumors underwent sublethal lymphocyte depletion and were then adopted into EGFRvIII CAR-T cells, either alone or in combination with free mouse IL-4 infusion, or into IL-4-expressing EGFRvIII CAR-T cells. Tumor growth was monitored using bioluminescence imaging. Notably, IL-4-expressing EGFRvIII CAR-T cells exhibited superior anti-metastatic activity and led to durable cure in 20% of treated mice, while EGFRvIII CAR-T cells showed only limited tumor burden control regardless of whether exogenous IL-4 infusion was combined (Fig. 5f, g). Overall, these results suggest that IL-4-expressing CAR-T cells represent an effective immunotherapy for solid tumors.

[0162] In summary, we have demonstrated that CAR-T cells engineered to express IL-4 exhibit enhanced proliferative capacity, effector function, and protection against functional impairment in solid tumors. Metabolic analysis showed that IL-4 expression promotes glycolysis in CAR-T cells in an LDHA-dependent manner. Adoptive transfer of IL-4-expressing CAR-T cells cleared established solid tumors in three syngeneic mouse models, including a metastatic tumor model. Furthermore, IL-4 secretion from CAR-T cells induces the maturation of dendritic cells (DCs) in tumors, which may contribute to broad-spectrum immune protection against re-attacks on both antigen-positive and antigen-negative tumors in treated mice. IL-4-armored CAR-T cells represent a promising therapeutic strategy that preserves T cell effector function and induces effective, broad-spectrum anti-tumor immunity.

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Claims

1. An immune cell that expresses interleukin-4, a fragment thereof, or a variant thereof, said immune cell comprising one or more recombinant constructs, wherein at least one recombinant construct encodes interleukin-4 (IL-4), a fragment thereof, or a variant thereof.

2. The immune cells of claim 1, wherein at least one recombinant construct encodes a chimeric antigen receptor (CAR), a T-cell receptor (TCR), or any other synthetic tumor-targeting motif.

3. The immune cell according to claim 1 or 2, wherein the immune cell is a T cell, a chimeric antigen receptor (CAR)-T cell, a T cell receptor (TCR)-transgenic T cell, a tumor-infiltrating lymphocyte (TIL), an NK cell, an NK-T cell, a CAR-NK cell, a CAR-NKT cell, a TCR-transgenic NK cell, a TCR-transgenic NK-T cell, a dendritic cell, a macrophage, a CAR-macrophage, or any synthetic tumor-specific immune cell.

4. The immune cell according to any one of the preceding claims, wherein the construct encoding IL-4, a fragment thereof, or a variant thereof encodes: i) a sequence comprising or consisting of SEQ ID NO:1, a variant thereof having at least 80% sequence identity with it, or a functional fragment thereof; or ii) a sequence comprising or consisting of SEQ ID NO:2, a variant thereof having at least 80% sequence identity with it, or a functional fragment thereof.

5. The immune cell according to any one of the preceding claims, wherein the recombinant construct encoding IL-4, a variant thereof, or a functional fragment thereof is linked to a second recombinant construct encoding CAR, TCR, or any other synthetic tumor-targeting motif.

6. The immune cell of claim 5, wherein the recombinant construct encoding IL-4, a variant thereof, or a functional fragment thereof is linked by a sequence encoding a self-cleaving peptide (e.g., peptide 2A) to at least one recombinant construct encoding a CAR, TCR, or any other synthetic tumor-targeting motif.

7. The immune cell according to claim 5 or 6, wherein the at least one recombinant construct encoding the CAR comprises -At least one antigen-binding domain, -Transmembrane domain, - At least one co-stimulatory domain, and - At least one active structural domain.

8. The immune cell of claim 7, wherein the at least one antigen-binding domain is an extracellular antigen recognition domain derived from a single-chain fragment variant (scFv) of an antibody that recognizes an antigen selected from the group consisting of: HER2, TRP-1, EGFRvIII, CD19, CD20, CD38, CD30, ERBB2, fibroblast activation protein (FAP), CA125, MUC-1, PSMA, PSA, CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), CEACAM5, CEACAM6, epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, carbonic anhydrase EX, alpha-fetoprotein, A3, A33 antibody-specific antigen, Ba 733, BrE3-antigen, CD1, CDIa, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138, colon-specific antigen-p (CSAp), CSAp, EGP-I, EGP-2, Ep-CAM, FIT-I, Flt-3, folate receptor, HLA-DR, Human chorionic gonadotropin (HCG) and its subunits, hypoxia-inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, insulin-like growth factor-1 (IGF-I), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, macrophage inhibitory factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, tyrosinase, PRAME, EBNA, KLK3, HPV E7, LMP2, NY-ESO-1, PAP, reverse transcriptase, nucleolar phosphatase, PRTN3 / ELANE, CT83 / KKLC1, MUC16, DNTT, PAM-4 antibody-specific antigen, placental growth factor, p53, prostatic acid phosphatase, RS5, S1OO, TAC, TAG-72, tendinin, TRAIL receptor, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, 17-1A antigen, NeuGcGM3, N-hydroxyacetylGM3 ganglioside, Neu5Gc, GM3-ganglioside, GD3, GM2, carbohydrate antigen, ganglioside antigen, Lewis Y, Lewis B, MOG, MBP, aB-crystal protein, PLP, GlialCAM, β-synuclein, HLA-A2, TNP, CD123, immunoglobulin κ chain, or any combination thereof.

9. The immune cell according to claim 7 or 8, wherein the transmembrane domain is selected from the group comprising: CD28, CD28T, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulatory factor (ICOS), CD1-1a / CD18, CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC. Class 1 molecules, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

10. The immune cell according to any one of claims 7 to 9, wherein the co-stimulatory domain is a signal transduction region (or other suitable portion) comprising: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulatory factor (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class I molecules, TNF receptor protein, immunoglobulins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM protein), activated NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

11. The immune cell according to any one of claims 7 to 10, wherein the at least one activation domain is a T cell activation domain, preferably a CD3 domain, more preferably a CD3 zeta (CD3ζ) domain.

12. A CAR polypeptide comprising -At least one antigen-binding domain, -Transmembrane domain, -At least one co-stimulatory domain, -At least one activation domain, and - At least one interleukin-4 (IL-4) molecule, its variant or a functional fragment thereof.

13. The CAR polypeptide of claim 12, wherein the at least one IL-4 molecule comprises a sequence selected from the group consisting of: i) SEQ ID NO:1, a variant thereof having at least 80% sequence identity with it, or a functional fragment thereof; or ii) SEQ ID NO:2, a variant thereof having at least 80% sequence identity with it, or a functional fragment thereof. 14.According to claim 12 or 13, the at least one antigen-binding domain is an extracellular antigen recognition domain derived from a single-chain fragment variant (scFv) of an antibody that recognizes an antigen selected from the group consisting of: HER2, TRP-1, EGFRvIII, CD19, CD20, CD38, CD30, ERBB2, fibroblast activation protein (FAP), CA125, MUC-1, PSMA, PSA, CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), CEACAM5, CEACAM6, epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, carbonic anhydrase EX, alpha-fetoprotein, A3, A33 antibody-specific antigen, Ba 733, BrE3-antigen, CD1, CDIa, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138, colon-specific antigen-p (CSAp), CSAp, EGP-I, EGP-2, Ep-CAM, FIT-I, Flt-3, folate receptor, HLA-DR, Human chorionic gonadotropin (HCG) and its subunits, hypoxia-inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, insulin-like growth factor-1 (IGF-I), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, macrophage inhibitory factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, tyrosinase, PRAME, EBNA, KLK3, HPV E7, LMP2, NY-ESO-1, PAP, reverse transcriptase, nucleolar phosphatase, PRTN3 / ELANE, CT83 / KKLC1, MUC16, DNTT, PAM-4 antibody-specific antigen, placental growth factor, p53, prostatic acid phosphatase, RS5, S1OO, TAC, TAG-72, tendinin, TRAIL receptor, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, 17-1A antigen, NeuGcGM3, N-hydroxyacetylGM3 ganglioside, Neu5Gc, GM3-ganglioside, GD3, GM2, carbohydrate antigen, ganglioside antigen, Lewis Y, Lewis B, MOG, MBP, aB-crystallin, PLP, GlialCAM, β-synuclein, HLA-A2, TNP, CD123, immunoglobulin κ chain, or any combination thereof. .

15. The CAR polypeptide according to any one of claims 12 to 14, wherein the transmembrane domain is selected from the group comprising: CD28, CD28T, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulatory factor (ICOS), CD1-1a / CD18, CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class 1 molecules, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

16. The CAR peptide according to any one of claims 12 to 15, wherein the co-stimulatory domain is a signal transduction region (or other suitable portion) comprising: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulatory factor (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class I molecules, TNF receptor protein, immunoglobulins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM protein), activated NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

17. The CAR peptide according to any one of claims 12 to 16, wherein the at least one activation domain is a T cell activation domain, preferably a CD3 domain, more preferably a CD3 zeta (CD3ζ) domain.

18. The CAR polypeptide according to any one of claims 12 to 17, further comprising a self-cleaving domain, said self-cleaving domain being contained between said at least one activation domain and said at least one interleukin-4 (IL-4) molecule, wherein said self-cleaving domain comprises the sequence DxExNPGP, where x is any amino acid.

19. An isolated polynucleotide encoding a chimeric antigen receptor (CAR) according to any one of claims 12 to 18.

20. The isolated polynucleotide according to claim 19, having at least 80% sequence identity with SEQ ID NO: 10, 11, 12 or 13.

21. A vector comprising the isolated polynucleotide according to claim 19 or 20.

22. The vector of claim 21, wherein the vector is selected from the group consisting of: viral vectors, DNA vectors, plasmids, RNA vectors, adenovirus vectors, adenovirus-associated vectors, lentiviral vectors, lipid nanoparticles (LNPs) targeting RNA (e.g., mRNA), liposomes, or any combination thereof.

23. The vector of claim 22, wherein the viral vector is selected from the group comprising: retroviruses (e.g., retroviral vectors); adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48); parvoviruses (e.g., adeno-associated virus); coronaviruses; negative-strand RNA viruses, such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus); positive-strand RNA viruses, such as picornaviruses and alphaviruses; and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia virus, modified vaccinia virus Ankara strain (MVA), fowlpox virus, and canarypox virus).

24. The vector of claim 23, wherein the vector is selected from the group consisting of: norovirus, cloacal virus, flavivirus, reovirus, papillomavirus, hepatotropic DNA virus, human papillomavirus, human foamy virus, and hepatitis virus.

25. The vector according to claim 22, wherein the retrovirus is selected from the group comprising: avian leukosis-sarcoma virus, avian type C virus, mammalian type C virus, type B virus, type D virus, carcinogenic retrovirus, HTLV-BLV group virus, lentivirus, alpha retrovirus, gamma retrovirus and foam virus.

26. The vector of claim 22, wherein the vector is selected from the group consisting of: murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus, Mason-Pfizer monkey virus, simmon immunodeficiency virus, simmon sarcoma virus, Lauer's sarcoma virus, and lentivirus.

27. A pharmaceutical composition comprising a therapeutically effective amount of: i) a CAR polypeptide according to any one of claims 12 to 18, ii) a separated polynucleotide according to any one of claims 19 to 20, iii) a carrier according to any one of claims 21 to 26, or iv) immune cells according to any one of claims 1 to 11, and optionally a pharmaceutically acceptable carrier, diluent, and / or excipient.

28. The pharmaceutical composition according to claim 27, for the treatment and / or prevention of cancer, infectious diseases, inflammatory or inflammation-induced diseases, chronic diseases or autoimmune diseases.

29. A method of treating and / or preventing cancer, infectious diseases, inflammatory or inflammation-induced diseases, chronic diseases, or autoimmune diseases in a subject in need, the method comprising administering the pharmaceutical composition of claim 27 to the subject in need.

30. The treatment and / or prevention method according to claim 29, wherein the inflammatory or inflammation-induced disease is selected from the group comprising: fibrosis (pulmonary or cardiac fibrosis), chronic obstructive pulmonary disease, cardiovascular disease, diabetes, asthma, fatty liver disease, gout, and scleroderma.

31. The treatment and / or prevention method according to claim 29, wherein the chronic disease is selected from the group comprising: fibrosis, Alzheimer's disease, lupus erythematosus, and chronic kidney disease.

32. The treatment and / or prevention method according to claim 29, wherein the infectious disease is selected from the non-limiting group comprising: HIV, hepatitis C, and human cytomegalovirus.

33. The treatment and / or prevention method according to claim 29, wherein the cancer is selected from solid cancer or liquid cancer.

34. The treatment and / or prevention method according to claim 33, wherein the solid cancer is selected from the group comprising: lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, and skin cancer (e.g., melanoma), or one or more combinations thereof.

35. The treatment and / or prevention method according to claim 33, wherein the liquid cancer is selected from the group comprising: leukemia, myeloma, myelodysplastic syndrome (MDS) and liquid lymphoma.

36. The treatment and / or prevention method according to claim 29, wherein the autoimmune disease is selected from the non-limiting group comprising: rheumatoid arthritis (RA), multiple sclerosis (MS), endometriosis, inflammatory bowel disease (IBD), psoriasis and psoriatic arthritis, or one or more combinations thereof.

37. A kit comprising: i) a CAR polypeptide according to any one of claims 12 to 18, ii) an isolated polynucleotide according to any one of claims 19 to 20, iii) a vector according to any one of claims 21 to 26, iv) immune cells according to any one of claims 1 to 11, or v) a pharmaceutical composition according to any one of claims 27 to 28.

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