Chimeric antigen receptor targeting CD26 and application thereof

Abstract

The invention discloses a chimeric antigen receptor targeting CD26 and an application of the chimeric antigen receptor. According to the present invention, the binding recognition sequence of the natural ligand Caveolin-1 of CD26 is adopted as the antigen binding region to be constructed in the CAR structure, and the mRNA-LNP particle is adopted to instantaneously transfect the T cell so as to obtain the instantaneous CAR-T cell with the targeting CD26 killing function, such that the safety is further improved; the CAR-T cell provided by the invention has a good effect of removing CD26 positive senescence cells and hepatic fibrosis cells, and is a novel CAR-T cell which is expected to be applied to clinical anti-aging and hepatic fibrosis treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a chimeric antigen receptor targeting CD26 and its application. Background Technology

[0002] Aging is an emerging and important research field. With the accumulation of related knowledge and the advancement of technology, people have gradually realized that aging itself can be targeted for intervention, achieving the goal of extending lifespan and delaying the onset and progression of aging-related diseases, which has significant scientific and practical implications. Among the many factors causing individual aging, the accumulation of senescent cells is considered a major cause of organ degeneration and ultimately aging-related diseases. Multiple studies have shown that clearing senescent cells from the body can delay the onset of various aging-related diseases, directly proving that senescent cells are one of the important causes of aging-related diseases and providing new targets for the treatment of these diseases. Senescent cells undergo a series of changes in cell morphology, transcriptional profiles, protein homeostasis, epigenetics, and metabolism. Simultaneously, senescent cells resist apoptosis, leading to their accumulation in multiple organs and tissues throughout the body. Senescent cells activate inflammatory cytokine secretion pathways, resulting in a local non-infectious inflammatory microenvironment in tissues, which in turn leads to organ degeneration and the development of various aging-related diseases. Therefore, targeted killing of senescent cells has significant clinical application prospects for the treatment of various aging-related diseases and the improvement of healthy lifespan.

[0003] Cellular senescence signifies a stable arrest of the cell cycle. While senescent cells can continue to survive and retain metabolic capabilities, they lose their proliferative capacity and exhibit characteristic phenotypic changes. Epidemiological studies show that the progression of fibrosis is highly age-related; both its incidence and mortality rates increase with patient age. The accumulation of senescent cells due to aging is key to the development of fibrosis. In young bodies, damaged cells are detected and promptly cleared by the immune surveillance system by shifting towards senescence, thus maintaining normal tissue homeostasis. However, with aging, factors such as an increased number of damaged cells and a decline in immune surveillance capacity lead to a reduced clearance rate of senescent cells, resulting in their accumulation and the development of fibrosis. Targeting senescent cells as therapeutic targets for fibrosis has opened up new avenues for the development of fibrosis treatments. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides a chimeric antigen receptor targeting CD26 and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a chimeric antigen receptor targeting CD26, the chimeric antigen receptor comprising a binding recognition sequence of Caveolin-1, the natural ligand of CD26, as shown in SEQ ID NO:3.

[0006] Furthermore, the chimeric antigen receptor also includes a transmembrane domain.

[0007] Furthermore, the transmembrane domain comprises the transmembrane domains of the following molecules: IgG1, IgG4, CD8, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1, CD34, OX40, CD3ε, or variants thereof.

[0008] Furthermore, the transmembrane domain is a CD8 transmembrane domain.

[0009] Furthermore, the amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID NO:5.

[0010] Furthermore, the chimeric antigen receptor also includes a hinge region.

[0011] Furthermore, the hinge region comprises the hinge region of the following molecules: IgG1, IgG4, CD8, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1, CD34, OX40, CD3ε or variants thereof.

[0012] Furthermore, the hinge area is a CD8 hinge area.

[0013] Furthermore, the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO:4.

[0014] Furthermore, the chimeric antigen receptor also includes an intracellular signal transduction domain.

[0015] Furthermore, the intracellular signal transduction domain comprises intracellular signal transduction domains of the following molecules: FcγR, FcεR, FcαR, FcRn, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L, CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278, CD247ζ, CD247η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, Zap70, or variants thereof.

[0016] Furthermore, the intracellular signal transduction domain is the CD3ζ intracellular signal transduction domain.

[0017] Furthermore, the amino acid sequence of the CD3ζ intracellular signal transduction domain is shown in SEQ ID NO:7.

[0018] Furthermore, the chimeric antigen receptor also includes a co-stimulatory signaling domain.

[0019] Furthermore, the co-stimulatory signaling domain comprises the co-stimulatory signaling domains of the following molecules: CD19, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, ICAM, LFA-1, Lck, CD2, CD4, CD5, CD7, CD226, CD8α, CD8β, LIGHT, CD83, DAP10, DAP12, GITR, DR3, NKG2C, HVEM, B7-H3, or variants thereof.

[0020] Furthermore, the co-stimulation signal structure domain is a 4-1BB co-stimulation signal structure domain.

[0021] Furthermore, the amino acid sequence of the 4-1BB co-stimulatory signaling domain is shown in SEQ ID NO:6.

[0022] Furthermore, the chimeric antigen receptor also includes a signal peptide, a detection tag, and a linker peptide.

[0023] Furthermore, the signal peptide comprises signal peptides of the following molecules: α and β chains of T cell receptors, CD3ζ, CD3ε, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, IgG6 or variants thereof.

[0024] Furthermore, the signal peptide is the CD8α signal peptide.

[0025] Furthermore, the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO:1.

[0026] Furthermore, the detection tags include FLAG tags, c-Myc tags, HA tags, and His tags.

[0027] Furthermore, the detection tag is a FLAG tag.

[0028] Furthermore, the amino acid sequence of the FLAG tag is shown in SEQ ID NO:2.

[0029] Furthermore, the linker peptide includes P2A, T2A, E2A, or F2A.

[0030] Furthermore, the linker peptide is P2A.

[0031] Furthermore, the amino acid sequence of P2A is shown in SEQ ID NO:8.

[0032] Furthermore, the chimeric antigen receptor also includes RISR-RIAD.

[0033] Furthermore, the amino acid sequence of the RISR-RIAD is shown in SEQ ID NO:17.

[0034] Furthermore, the chimeric antigen receptor contains a binding recognition sequence, a hinge region, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signal transduction domain connected in sequence.

[0035] Furthermore, the chimeric antigen receptor contains a signal peptide, a FLAG tag, a binding recognition sequence, a hinge region, a transmembrane domain, a co-stimulatory signaling domain, an intracellular signal transduction domain, a linker peptide, and a RISR-RIAD, connected in sequence.

[0036] Furthermore, the chimeric antigen receptor contains a Caveolin-1 binding recognition sequence, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory signaling domain, and a CD3ζ intracellular signal transduction domain connected in sequence.

[0037] Furthermore, the chimeric antigen receptor contains, in sequence, a CD8 signal peptide, a FLAG tag, a Caveolin-1 binding recognition sequence, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory signaling domain, a CD3ζ intracellular signal transduction domain, P2A, and RISR-RIAD.

[0038] Furthermore, the amino acid sequence of the chimeric antigen receptor is an amino acid sequence formed by sequentially linking SEQ ID NO:3, 4, 5, 6, and 7.

[0039] Furthermore, the amino acid sequence of the chimeric antigen receptor is an amino acid sequence formed by sequentially linking SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, and 17.

[0040] A second aspect of the present invention provides an isolated nucleic acid molecule that encodes the chimeric antigen receptor described in the first aspect of the present invention.

[0041] Furthermore, the coding sequence of the chimeric antigen receptor includes a coding sequence sequentially connected to a binding recognition sequence, a coding sequence of a hinge region, a coding sequence of a transmembrane domain, a coding sequence of a co-stimulatory signaling domain, and a coding sequence of an intracellular signal transduction domain.

[0042] Furthermore, the coding sequence of the chimeric antigen receptor includes, in sequence, the coding sequences of a signal peptide, a FLAG tag, a binding recognition sequence, a hinge region, a transmembrane domain, a co-stimulatory signaling domain, an intracellular signal transduction domain, a linker peptide, and a RISR-RIAD.

[0043] Furthermore, the coding sequence of the chimeric antigen receptor includes a coding sequence sequentially linked to the Caveolin-1 binding recognition sequence, a coding sequence of the CD8 hinge region, a coding sequence of the CD8 transmembrane domain, a coding sequence of the 4-1BB co-stimulatory signaling domain, and a coding sequence of the CD3ζ intracellular signal transduction domain.

[0044] Furthermore, the coding sequence of the chimeric antigen receptor comprises, in sequence, the coding sequence of the CD8α signal peptide, the coding sequence of the FLAG tag, the coding sequence of the Caveolin-1 binding recognition sequence, the coding sequence of the CD8 hinge region, the coding sequence of the CD8 transmembrane domain, the coding sequence of the 4-1BB co-stimulatory signaling domain, the coding sequence of the CD3ζ intracellular signal transduction domain, the coding sequence of P2A, and the coding sequence of RISR-RIAD.

[0045] Furthermore, the coding sequence of the CD8α signal peptide is shown in SEQ ID NO:9.

[0046] Furthermore, the encoding sequence of the FLAG tag is shown in SEQ ID NO:10.

[0047] Furthermore, the encoded sequence of the Caveolin-1 binding recognition sequence is shown in SEQ ID NO:11.

[0048] Furthermore, the encoding sequence of the CD8 hinge region is shown in SEQ ID NO:12.

[0049] Furthermore, the coding sequence of the CD8 transmembrane domain is shown in SEQ ID NO:13.

[0050] Furthermore, the encoding sequence of the 4-1BB co-stimulation signal structure domain is shown in SEQ ID NO:14.

[0051] Furthermore, the coding sequence of the CD3ζ intracellular signal transduction domain is shown in SEQ ID NO:15.

[0052] Furthermore, the encoding sequence of P2A is shown in SEQ ID NO:16.

[0053] Furthermore, the encoding sequence of the RISR-RIAD is shown in SEQ ID NO:18.

[0054] A third aspect of the present invention provides a carrier comprising the nucleic acid molecule described in the second aspect of the present invention.

[0055] Furthermore, the vector comprises a DNA vector, an RNA vector, a plasmid, a lipid nanoparticle, a transposon vector, a CRISPR / Cas9 vector, or a viral vector.

[0056] Furthermore, the carrier is lipid nanoparticles.

[0057] A fourth aspect of the present invention provides an engineered host cell that contains or expresses the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, or the vector described in the third aspect of the present invention.

[0058] Furthermore, the host cells include immune cells.

[0059] Furthermore, the immune cells include T cells, B cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, myeloid cells, or any combination thereof.

[0060] Furthermore, the immune cells are T cells.

[0061] The fifth aspect of the present invention provides a method for preparing the host cell described in the fourth aspect of the present invention, the method comprising introducing the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention into the host cell.

[0062] A sixth aspect of the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor described in the first aspect of the present invention or the host cell described in the fourth aspect of the present invention.

[0063] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier, excipient, or diluent.

[0064] The seventh aspect of the present invention provides any of the following applications: 1) The use of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the carrier described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention in the preparation of anti-aging drugs.

[0065] 2) The use of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the carrier described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention in the preparation of a medicament for treating organ fibrosis.

[0066] 3) The use of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the carrier described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention in the preparation of a medicament for restoring muscle strength, improving endurance, and improving motor coordination.

[0067] Furthermore, the organ fibrosis includes liver fibrosis, lung fibrosis, kidney fibrosis, heart fibrosis, joint fibrosis, spleen fibrosis, and pancreatic fibrosis.

[0068] Furthermore, the organ fibrosis is liver fibrosis.

[0069] Advantages and beneficial effects of the present invention: This invention constructs a CAR structure by using the binding recognition sequence of Caveolin-1, the natural ligand of CD26, as the antigen-binding region. T cells are then transiently transfected with mRNA-LNP particles to obtain transient CAR-T cells with CD26-targeting killing function, further improving safety. The CAR-T cells provided by this invention have good effects on eliminating CD26-positive senescent cells and liver fibrosis cells, and are promising novel CAR-T cells for clinical anti-aging and liver fibrosis treatment. Attached Figure Description

[0070] Figure 1 This is a map of the chimeric antigen receptor (CAR) targeting CD26 in this invention.

[0071] Figure 2 The image shows the flow cytometry results of CAR-T cells in vivo after CAR treatment. In the image, A represents the proportion of CAR-T cells in vivo 8 hours after CAR treatment, and B represents the proportion of CAR-T cells in vivo 72 hours after CAR treatment.

[0072] Figure 3 The figure shows the results of an experiment on signs of aging in mice after CAR treatment. In the figure, A represents the gripping force of the mouse, B represents the running distance of the mouse, and C represents the maximum drop speed of the stick.

[0073] Figure 4 This is a graph showing the change in mouse body weight after CAR treatment.

[0074] Figure 5 This is a staining image of mouse adipose tissue after CAR treatment.

[0075] Figure 6The image shows the qPCR results of aging and inflammation markers in mice after CAR treatment.

[0076] Figure 7 HE staining of mouse muscle after CAR treatment.

[0077] Figure 8 This is a MASSON staining image of mouse liver after CAR treatment.

[0078] Figure 9 HE staining images of other major organs of mice after CAR treatment. Detailed Implementation

[0079] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation of the invention in any way.

[0080] In this invention, the term "targeting," when referring to protein expression, is intended to include, but is not limited to, directing a protein or peptide to a suitable destination inside or outside the cell. Targeting is typically achieved through a signal peptide or targeting peptide, which is a segment of amino acid residues in a polypeptide chain. These signal peptides can be located anywhere within the polypeptide sequence, but are typically located at the N-terminus. Polypeptides can also be engineered to have a signal peptide at the C-terminus. The signal peptide can direct the polypeptide to undergo extracellular cleavage and localize to the plasma membrane, Golgi apparatus, endosomes, endoplasmic reticulum, and other cellular compartments. As used herein, the term "targeting," when referring to tumor targeting, refers to the ability of a cell to recognize and kill tumor cells (i.e., target cells). In this context, the term "targeting" refers, for example, the ability of a cell-expressed CAR to recognize and bind to a cell surface antigen expressed by the tumor.

[0081] In this invention, the terms "chimeric antigen receptor" and "CAR" are used interchangeably, referring to a chimeric polypeptide comprising multiple functional domains, the sequence of which, from the amino terminus to the carboxyl terminus, is arranged as follows: (a) an extracellular domain comprising a target-binding domain and a hinge region; (b) a transmembrane domain; and (c) an intracellular region comprising a co-stimulatory signaling domain and one or more intracellular signal transduction domains, wherein the aforementioned domains may optionally be connected by one or more spacer subdomains. The CAR may further comprise a signal peptide sequence, which is typically removed during post-translational processing and presented on the surface of cells transformed with an expression vector containing a nucleic acid sequence encoding the CAR. The CAR can be prepared according to principles well known in the art.

[0082] In this invention, the term "transmembrane domain" is used interchangeably with "transmembrane region (TM)," referring to a portion of the CAR that fuses the extracellular and intracellular domains and anchors the CAR to the plasma membrane of immune effector cells. The transmembrane domain can be obtained from natural proteins or synthesized, semi-synthesized, or recombinantly. The chimeric antigen receptor of this invention also includes a hinge region, a sequence that facilitates the binding of the chimeric antigen receptor to CD26 via a ligand recognition sequence and enhances signal transduction to the cell. The hinge region can be positioned between the extracellular and transmembrane domains, or between the intracellular and transmembrane domains. The hinge region also refers to any oligopeptide or polypeptide that functions by connecting the transmembrane domain to the extracellular domain and / or the transmembrane domain to the intracellular domain. The hinge region contains up to 300 amino acids, for example, about 10-100 amino acids, or about 25-50 amino acids.

[0083] The transmembrane domain and hinge region include, but are not limited to, the transmembrane domains and hinge regions of the following molecules: T cell receptor α chain, β chain or ζ chain, CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD34, CD37, CD45, CD64, CD80, CD86, OX40, CD137, ICOS, CD152, CD154, IgG1, IgG4, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1, GITR or variants thereof. In a specific embodiment of the present invention, the transmembrane domain and hinge region is the CD8 transmembrane domain and hinge region.

[0084] In this invention, the term "intracellular signal transduction domain" is generally part of a CAR that participates in transducing information about the effective targeting of CD26 by the CAR to human CD26 into the interior of immune effector cells to induce effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cells or other cellular responses induced by antigens that bind to the extracellular domain of the CAR. In this invention, the intracellular signal transduction domain may include, but is not limited to, the intracellular signal transduction domains of the following molecules: FcγR, FcεR, FcαR, FcRn, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L, CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278, CD247ζ, CD247η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, Zap70, or variants thereof. In a specific embodiment of this invention, the intracellular signal transduction domain is the CD3ζ intracellular signal transduction domain.

[0085] In this invention, the term "co-stimulatory signaling domain" generally refers to the intracellular signal transduction domain of a co-stimulatory molecule. For example, a co-stimulatory molecule can be a cell surface molecule other than an antigen receptor or an Fc receptor, which can provide a second signal required for the efficient activation and function of T lymphocytes upon antigen binding. For example, the co-stimulatory signaling domain may be selected from the group consisting of: CARD11, CD2, CD4, CD5, CD7, CD8α, CD8β, CD19, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LFA-1, Lck, LAT, LIGHT, DAP10, DAP12, NKD2CSLP76, GITR, DR3, NKG2C, TRIM, B7-H3, ZAP70 or variants thereof, or co-stimulatory signaling domains derived from suicide immunoglobulin-like receptors (KIR). In a specific embodiment of the present invention, the co-stimulation signal structure domain is a 4-1BB co-stimulation signal structure domain.

[0086] In this invention, the term "RISR-RIAD" refers to the combined use of RISR (an immunomodulatory peptide) and RIAD (an anchoring interference peptide), or specifically refers to a module with similar function introduced into a CAR structure. Its core function is to significantly enhance the persistence, infiltration, and activity of T cells by blocking inhibitory signaling pathways.

[0087] In this invention, "CAR" includes, but is not limited to, first-generation CARs, second-generation CARs, third-generation CARs, or fourth-generation CARs. The term "first-generation CAR" refers to a CAR whose intracellular region contains only a single signal transduction domain, such as CD3ζ signaling. Second-generation CARs, in addition to a single signal transduction domain such as CD3ζ, also include a co-stimulatory signaling domain, including but not limited to CD28 and 4-1BB, which enhance CAR-T cell persistence, cytokine secretion, and antitumor activity. Third-generation CARs include two co-stimulatory signaling domains. Fourth-generation CARs, also known as "armored vehicles," further enhance CAR-T cell function by adding structures that can improve CAR-T cell function, such as receptor structures for cytokines or chemokines, building upon the third-generation CAR.

[0088] The term "isolated nucleic acid molecule" as used in this invention generally refers to an isolated form of nucleotide, deoxyribonucleotide or ribonucleotide of any length, or an analogue isolated from its natural environment or synthesized artificially.

[0089] As used in this invention, the term "encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes. These polymers and macromolecules have defined nucleotide sequences (e.g., rRNA, tRNA, and mRNA) or defined amino acid sequences and the biological properties derived therefrom. Therefore, if the transcription and translation of mRNA corresponding to the gene produces a protein in a cell or other biological system, then the gene, cDNA, or RNA encodes a protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically provided in the sequence listing, and the non-coding strand, which serves as a template for transcribing the gene or cDNA, can be referred to as the protein or other product encoding that gene or cDNA.

[0090] In this invention, the term "vector" generally refers to a nucleic acid delivery vehicle that can insert a polynucleotide encoding a protein into itself and enable the protein to be expressed. Vectors can be transformed, transduced, or transfected into host cells, allowing the genetic material elements they carry to be expressed within the host cells.

[0091] In this invention, the term "lipid nanoparticles," also known as LNPs, refers to nanoscale delivery carriers composed of lipid-like molecules. These carriers have an average diameter between tens and hundreds of nanometers, are spherical, and are formed by the self-assembly of components such as phospholipids, cholesterol, ionizable cationic lipids, and polyethylene glycol (PEG)-modified lipids. Compared to traditional viral vectors, LNPs, as non-viral vectors, exhibit excellent biocompatibility and controllability, making them an important representative of novel drug delivery systems. LNPs can encapsulate unstable nucleic acid molecules such as mRNA and siRNA in vitro, protecting them from enzymatic degradation after entering the body until they are delivered to the cytoplasm of target cells to exert their effects.

[0092] In this invention, the term "immune cell" refers to a cell capable of inducing an immune response, and "immune cell" and its other grammatical forms can refer to immune cells of any origin. The term "immune cell" can also be human or non-human. "Immune cells" include, but are not limited to, leukocytes, T cells, B cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, myeloid cells, or any combination thereof. Further, the immune cell is a T cell; even further, examples of the T cell include, but are not limited to, primary T cells ("lymphocyte progenitor cells"), central memory T cells, effector memory T cells, T memory stem cells (Tscm), iPSC-derived T cells, synthetic T cells, or combinations thereof.

[0093] In this invention, the terms "T cell" or "T lymphocyte" are terms understood by those skilled in the art and refer to a type of lymphocyte that develops in the thymus and plays a central role in the immune response. T cells can be distinguished from other lymphocytes by the presence of T cell receptors (TCRs) on their cell surface. The term "primary T cells" refers to T cells derived from an individual, distinct from T cells maintained in culture for an extended period. Therefore, primary T cells are particularly peripheral blood T cells derived from a subject. The population of primary T cells may consist primarily of a subset of T cells. Alternatively, the population of primary T cells may consist of different subsets of T cells.

[0094] As used herein, the term "introduction" refers to a method of delivering a vector containing a nucleic acid molecule encoding a CAR into a host cell. Such introduction can be performed by various methods known in the art, including but not limited to lipid transfection, DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, and protoplast fusion. Additionally, the vector can be introduced into the host cell via gene bombardment.

[0095] In this invention, a composition refers to any mixture of two or more products, substances, or compounds (including cells). It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof. As used herein, the term "pharmaceutical composition" refers to a formulation in which the biological activity of one or more active ingredients is effective and which does not contain other components that would have unacceptable toxicity to a subject to which the formulation is administered. Therefore, it is a composition suitable for pharmaceutical use in mammalian subjects (typically humans). Pharmaceutical compositions typically contain an effective amount of an active agent and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively. Such formulations may be sterile.

[0096] The term "pharmaceutically acceptable carrier, excipient, or diluent" as used herein includes any and all solvents, diluents or other liquid solvents, dispersants or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., provided they are suitable for the desired specific dosage form. The use of any conventional carrier medium is also considered within the scope of this invention unless it is incompatible with the compounds of this invention, for example, by producing any undesirable biological effects or otherwise interacting harmfully with one or more other components of the pharmaceutical composition.

[0097] Example 1: Construction of CAR-T cells targeting CD26 I. Experimental Methods 1. Constructing CD26-CAR-mRNA-LNP particles This invention constructs a chimeric antigen receptor (CAR) based on the binding recognition sequence of Caveolin-1, the natural ligand of CD26. The CAR sequence is formed by sequentially tandemly connecting the CD8 signal peptide, the FLAG tag, the Caveolin-1 binding recognition sequence, the CD8 hinge region (CD8Hinge), the CD8 transmembrane domain (CD8TM), the 4-1BB co-stimulatory signaling domain, the CD3ζ intracellular signal transduction domain, P2A, and RISR-RIAD, as shown in the diagram. Figure 1 As shown, the amino acid and nucleotide sequences corresponding to each part of the CAR are shown in Table 1 and Table 2, respectively.

[0098] CD26 CAR mRNA LNP particle preparation process: CD26 CAR mRNA expression includes a ligand-derived fragment with a flag tag, as well as mouse CD3ζ and CD28 cytoplasmic signaling domains. The complete sequence was cloned into an in vitro transcription template plasmid containing the T7 promoter, 5' and 3' untranslated regions (UTRs), and a poly(A) tail. First, a DNA template containing the HDV ribozyme sequence was transcribed in vitro using the HiScribe™ T7 ARCA mRNA Kit to synthesize a 5'Cap1-ORF main module. After self-cleavage and purification with HDV ribozyme (20 mM MgCl2, 37℃), a homogeneous 3' end main RNA was obtained. Finally, a poly(A) tail was precisely added using E. coli Poly(A) Polymerase, and the final product was obtained through preparative-grade HPLC purification.

[0099] LNP conjugation with CD5 antibody: Based on the reaction of DSPE-PEG-MAL lipids and SATA (-NH2 to -SH), the DSPE-PEG-MAL lipids contain maleimide groups at the end, which can specifically react with the thiol groups (-SH) exposed after antibody treatment with SATA (thioether bond formation), thereby conjugating the antibody to the LNP surface.

[0100] Purified mRNA was encapsulated into LNPs via a self-assembly process. CD5-coupled ionized lipids (SM102), structural lipids (cholesterol), accessory lipids (DSPC), and polyethylene glycol lipids (DMG-PEG-2k) were mixed with mRNA in acetate buffer at a 1:3 volume ratio using a microfluidic device. The mixture was neutralized with PBS pH 7.4 (containing sucrose cryoprotectant) and then aseptically filtered. The product was analyzed to determine particle size, polydispersity (PDI), zeta potential, pH, and endotoxin levels.

[0101] Table 1. Amino acid sequences corresponding to each part of the CAR

[0102] Table 2. Nucleotide sequences corresponding to each part of the CAR

[0103] 2. Construction of CAR-T cells Transfection procedure: LNP-encapsulated particles were directly transfected into mice via intraorbital vein injection.

[0104] Example 2: Study on the in vivo anti-aging effect of CD26-targeted CAR-T cells. I. Experimental Methods 1. Animals: Eight naturally aged C57 mice at 20 months of age were randomly divided into a control group (CTR group, C group) and a CAR group (CAR-T treatment group, D group).

[0105] 2. Flow cytometry of blood samples: Blood was collected from mice via orbital sampling. 500 mL of pre-chilled erythrocyte lysis buffer was added to a centrifuge tube containing a suspension of peripheral blood single cells. After gentle mixing, the tube was incubated on ice in the dark for 10 minutes (inverting gently every 3 minutes to ensure complete lysis of erythrocytes). After incubation, 500 μl of PBS was added to neutralize the lysate. The tube was centrifuged at 500×g for 5 minutes (4℃), and the supernatant was discarded, retaining the cell pellet at the bottom. The cells were washed twice, and an appropriate amount of diluted fluorescently labeled antibodies (APC-labeled CD3 antibody (Biolegend, 100236), PE-labeled CD4 antibody (Biolegend, 100421), and PE-labeled CD26 antibody (Biolegend, 137809)) were added to the cell suspension. After gentle mixing, the tube was incubated at 4℃ in the dark for 30 minutes to ensure sufficient binding of the antibodies to cell surface antigens.

[0106] 3. Detection of signs of aging: Grasping strength test: Using a grip strength tester, the mouse's forepaws grasp the test probe, and the probe is gently pulled backward until the mouse releases it. Each mouse is measured three times, and the average value is recorded (unit: N). Running distance test: The mouse is placed on a treadmill at a constant speed, and the distance the mouse runs continuously is recorded (unit: m), reflecting its endurance level. Maximum drop speed test: Using a rotundimeter, the rotation speed of the rotundi is gradually increased, and the maximum speed at which the mouse falls from the rotundi is recorded. Each mouse is tested three times, and the best value is recorded. The CAR-T treatment group (D1-D4) and the control group (C1-C4) mice are marked separately, placed in clean cages, and allowed to acclimatize to the experimental environment for 30 minutes. After pelting and weighing, the mice in groups C and D are weighed.

[0107] 4. qPCR: Total RNA extraction from animal liver tissue using the Trizol method: Mouse liver tissue was immersed in 1 ml of Trizol solution, then repeatedly pipetted using a Trizol-specific pipette before grinding in a tissue homogenizer until the tissue was completely dissolved by Trizol. 200 μl of chloroform was added to each centrifuge tube, the cap was tightened, and the tube was inverted for 15 seconds to mix. The tube was incubated at room temperature for 3 minutes. The tube was then centrifuged at 14,000 rpm for 15 minutes at 4°C. Upon removal, the liquid in the centrifuge tube was observed to separate into three layers: a lower red phenol-chloroform phase, an intermediate phase, and an upper aqueous phase. The total RNA was dissolved in the upper aqueous phase. The upper aqueous phase was carefully transferred to a new centrifuge tube, and 250 μl of isopropanol was added. The tube was gently inverted to mix, and incubated at room temperature for 10 minutes. The tube was then centrifuged at 14,000 rpm for 10 minutes at 4°C. A white gel-like precipitate, the RNA precipitate, was observed at the bottom of the centrifuge tube. Discard the supernatant, add 500 μl of 75% ethanol, mix manually by inverting, and centrifuge at 14000 rpm for 5 min in a 4°C centrifuge. Discard the ethanol in the centrifuge tube, invert the tube onto absorbent paper to blot and dry the remaining ethanol on the tube walls, add 20 μl of enzyme-free water, and gently pipette to dissolve the RNA. Incubate at 56°C in a metal bath for 10 min.

[0108] Total RNA was reverse transcribed into cDNA using the PrimerScript RT Master Mix kit (Takara), following the kit's instructions. Primer sequences are shown in Table 3. The reaction system was placed in a real-time quantitative PCR instrument, and the cDNA amplification process was initiated according to the pre-set system program.

[0109] Table 3 Primer Sequences

[0110] II. Experimental Results The positive rate of CAR-T expression in control (Ctr) and CAR-T treatment (CAR) mice at different time points (8 h and 72 h after treatment) was analyzed by flow cytometry. Figure 2As can be seen, in the early post-treatment period, the proportion of positive cells in the control group (Ctr) was extremely low (mean approximately 0.43% - 1.05%), while the proportion of positive cells in the CAR group increased significantly, with a mean of 31.65% - 54.38%, significantly higher than that in the control group. This demonstrates that the CAR-T delivery system can rapidly induce or enrich target positive cells in vivo, reflecting the high efficiency of CAR-T formulations in the early stages. After 72 hours, the proportion of positive cells in the control group remained at a low level (mean approximately 0.13% - 0.75%); although the proportion of positive cells in the CAR group decreased slightly compared to 8 hours (mean 10.21% - 15.2%), it was still significantly higher than that in the control group. CAR-T has a certain sustained effect in vivo, but the proportion of positive cells gradually decreases over time, which is consistent with the characteristic that "mRNA-mediated CAR-T expression is a short-term effect (mRNA is easily degraded, leading to the decay of CAR protein expression over time)," and also reflects the dynamic metabolic pattern of CAR-T in vivo. CAR-T expression in the treatment group was significantly higher than that in the control group; although it decreased slightly at 72 hours, it was still higher in the treatment group than in the control group. This result indicates that our designed CAR-T system can effectively induce a cell population with specific functions and maintain a high proportion for a certain period of time, thus preliminarily verifying the effectiveness of the system.

[0111] Results of signs of aging, such as Figure 3 As shown, the mean grip strength in the CTR group was approximately 1.55 N, and the mean grip strength in the CAR group was approximately 1.8 N. The difference between the two groups was significant (P<0.05), indicating that CAR-T intervention significantly enhanced the muscle strength of aging mice. The mean running distance in the CTR group was approximately 80 m, and the mean running distance in the CAR group was approximately 180 m. The difference between the two groups was extremely significant (P<0.01), indicating that CAR-T treatment significantly improved the endurance of aging mice. The mean distance in the CTR group was approximately 80 m, and the mean distance in the CAR group was approximately 180 m. The difference between the two groups was extremely significant (P<0.01), indicating that CAR-T intervention improved the motor coordination ability of aging mice and reduced the age-related decline in motor function. These results demonstrate that the CAR-T delivery system can effectively reverse the physical decline in aging mice, showing significant improvements in muscle strength, endurance, and motor coordination.

[0112] like Figure 4 As shown, during the 8-day observation period, the body weight of the control group showed a trend of "decreasing or remaining at a low level." Mice in groups C1 and C3 showed significant weight loss, which may reflect age-related metabolic decline or overall physiological deterioration. The body weight of the CAR-T treatment groups was more stable, and even showed an upward trend. The body weight of group D3 showed a continuous increase, and the body weight levels of groups D1, D2, and D4 were generally better than the control group.

[0113] Adipose tissue staining results as follows Figure 5 As shown, compared with the aging control group, the relative cell area of ​​adipocytes increased significantly after 1 month of CAR-T intervention (P<0.01), suggesting that CAR-T intervention can improve age-related adipocyte atrophy and make its morphology closer to that of young adipocytes.

[0114] qPCR was used to detect the expression levels of aging-related genes and inflammatory factors, including IL-1β, PI5, and TNF. Compared with the control group, the relative expression levels of these factors in mice treated with CAR-T were all reduced. Figure 6 This result suggests that CAR-T therapy may delay or reverse aging by downregulating the expression of aging-related genes and inhibiting the release of inflammatory factors.

[0115] Results of HE staining of muscle tissue as follows Figure 7 As shown, compared with the aging control group, the relative cell area of ​​muscle fibers in the CAR-T treatment group was significantly increased (P<0.0001), suggesting that CAR-T intervention can improve age-related muscle fiber atrophy and make its morphology closer to that of young muscle fibers.

[0116] Example 3: Study on the in vivo anti-fibrotic effect of CD26-targeted CAR-T cells I. Experimental Methods 1. Animals: Eight naturally aged C57 mice at 20 months of age were randomly divided into a control group (CTR group, C group) and a CAR group (CAR-T treatment group, D group).

[0117] 2. MASSON Staining: Dewaxing Paraffin Sections: Sections are sequentially immersed in environmentally friendly dewaxing solution I for 20 min, environmentally friendly dewaxing solution II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, then rinsed with tap water. Frozen Section Restoration and Fixation: Frozen sections are removed from the -20℃ freezer and brought to room temperature. They are fixed with tissue fixative for 15 min and then rinsed with running water. Sections are immersed in Masson A solution overnight and rinsed with running tap water. Sections are immersed in a mixture of equal parts Masson B and Masson C solutions for 1 min, rinsed with tap water, differentiated with differentiation solution for a few seconds, and rinsed with tap water. Sections are immersed in Masson D solution for 6 min and rinsed with tap water. Sections are immersed in Masson E solution for 1 min. Without rinsing, slightly drained, they are directly immersed in Masson F solution for 2-30 seconds. Sections are rinsed with 1% acetic acid for differentiation and dehydrated with two batches of anhydrous ethanol. Clearing and mounting: Sections were placed in anhydrous ethanol for 5 min, xylene for 5 min for clearing, and then mounted with neutral resin. Microscopic examination and image acquisition and analysis were performed. Collagen fibers appeared blue; muscle fibers, cellulose, and erythrocytes appeared red.

[0118] 3. HE staining: Dewaxing paraffin sections to water: Immerse sections sequentially in environmentally friendly dewaxing solution I for 20 min, environmentally friendly dewaxing solution II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, then rinse with tap water. Frozen sections are then thawed and fixed: Remove frozen sections from the -20℃ freezer and allow them to reach room temperature. Fix with tissue fixative for 15 min, then rinse with running water. Pretreatment: Immerse sections in high-resolution constant staining pretreatment solution for 1 min. Hematoxylin staining: Immerse sections in hematoxylin staining solution for 3-5 min, rinse with tap water, differentiate with differentiation solution, rinse with tap water, re-blue with blue solution, and rinse with running water. Eosin staining: Dehydrate sections in 95% ethanol for 1 min, then stain in eosin staining solution for 15 s. Dehydration and mounting: Sections were sequentially immersed in anhydrous ethanol I for 2 min, then anhydrous ethanol II for 2 min, then anhydrous ethanol III for 2 min, then n-butanol I for 2 min, then n-butanol II for 2 min, then xylene I for 2 min, and finally xylene II for 2 min. After clearing, the sections were mounted with neutral resin. Microscopic examination and image acquisition and analysis were performed. Cell nuclei appeared blue, and cytoplasm appeared red.

[0119] II. Experimental Results Masson staining is used to visualize liver collagen fibers (which appear blue). From the stained images ( Figure 8 In the control group (Group C), blue collagen fibers were widely distributed and covered a large area in the liver tissue, indicating significant liver fibrosis. In the CAR-T treatment group (Group D), blue collagen fibers were significantly reduced in the liver, and the degree of fibrosis was greatly alleviated. The quantitative bar chart on the right further confirmed that the proportion of collagen fibers in the liver of the control group was significantly higher than that in the CAR-T group, indicating that CAR-T intervention can effectively reduce collagen deposition in the liver and improve liver fibrosis.

[0120] HE staining results of major organs as follows Figure 9 As shown, there were no significant differences between the CAR-T treatment group and the control group (CTRL). No pathological changes were observed in the organs of the CAR-T treatment group, confirming that the CAR-T therapy in this study caused almost no damage to vital organs.

[0121] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A chimeric antigen receptor targeting CD26, characterized in that, The chimeric antigen receptor contains a binding recognition sequence for Caveolin-1, the natural ligand of CD26, as shown in SEQ ID NO:

3.

2. The chimeric antigen receptor according to claim 1, characterized in that, The chimeric antigen receptor also includes a transmembrane domain; Preferably, the transmembrane domain comprises the transmembrane domains of the following molecules: IgG1, IgG4, CD8, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1, CD34, OX40, CD3ε or variants thereof; Preferably, the transmembrane domain is a CD8 transmembrane domain; Preferably, the amino acid sequence of the CD8 transmembrane domain is as shown in SEQ ID NO:5; Preferably, the chimeric antigen receptor further includes a hinge region; Preferably, the hinge region comprises the hinge region of the following molecules: IgG1, IgG4, CD8, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1, CD34, OX40, CD3ε or variants thereof; Preferably, the hinge area is a CD8 hinge area; Preferably, the amino acid sequence of the CD8 hinge region is as shown in SEQ ID NO:4; Preferably, the chimeric antigen receptor further comprises an intracellular signal transduction domain; Preferably, the intracellular signal transduction domain comprises the intracellular signal transduction domains of the following molecules: FcγR, FcεR, FcαR, FcRn, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L, CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278, CD247ζ, CD247η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, Zap70, or variants thereof; Preferably, the intracellular signal transduction domain is the CD3ζ intracellular signal transduction domain; Preferably, the amino acid sequence of the CD3ζ intracellular signal transduction domain is shown in SEQ ID NO:7; Preferably, the chimeric antigen receptor further includes a co-stimulatory signaling domain; Preferably, the co-stimulatory signaling domain comprises the co-stimulatory signaling domains of the following molecules: CD19, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, ICAM, LFA-1, Lck, CD2, CD4, CD5, CD7, CD226, CD8α, CD8β, LIGHT, CD83, DAP10, DAP12, GITR, DR3, NKG2C, HVEM, B7-H3, or variants thereof; Preferably, the co-stimulation signal structure domain is a 4-1BB co-stimulation signal structure domain; Preferably, the amino acid sequence of the 4-1BB co-stimulatory signaling domain is shown in SEQ ID NO:6; Preferably, the chimeric antigen receptor further comprises a signal peptide, a detection tag, and a linker peptide; Preferably, the signal peptide comprises signal peptides of the following molecules: α and β chains of T cell receptors, CD3ζ, CD3ε, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, IgG6 or variants thereof; Preferably, the signal peptide is the CD8α signal peptide; Preferably, the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO:1; Preferably, the detection tags include FLAG tags, c-Myc tags, HA tags, and His tags; Preferably, the detection tag is a FLAG tag; Preferably, the amino acid sequence of the FLAG tag is as shown in SEQ ID NO:2; Preferably, the linker peptide includes P2A, T2A, E2A, or F2A; Preferably, the linker peptide is P2A; Preferably, the amino acid sequence of P2A is as shown in SEQ ID NO:8; Preferably, the chimeric antigen receptor further includes RISR-RIAD; Preferably, the amino acid sequence of the RISR-RIAD is shown in SEQ ID NO:17; Preferably, the chimeric antigen receptor comprises a binding recognition sequence, a hinge region, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signal transduction domain connected in sequence. Preferably, the chimeric antigen receptor comprises a signal peptide, a FLAG tag, a binding recognition sequence, a hinge region, a transmembrane domain, a co-stimulatory signaling domain, an intracellular signal transduction domain, a linker peptide, and a RISR-RIAD, connected in sequence. Preferably, the chimeric antigen receptor contains a Caveolin-1 binding recognition sequence, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory signaling domain, and a CD3ζ intracellular signal transduction domain connected in sequence. Preferably, the chimeric antigen receptor contains, in sequence, a CD8 signal peptide, a FLAG tag, a Caveolin-1 binding recognition sequence, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory signaling domain, a CD3ζ intracellular signal transduction domain, P2A, and RISR-RIAD. Preferably, the amino acid sequence of the chimeric antigen receptor is an amino acid sequence formed by sequentially linking SEQ ID NO: 3, 4, 5, 6, and 7; Preferably, the amino acid sequence of the chimeric antigen receptor is an amino acid sequence formed by sequentially linking SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, and 17.

3. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the chimeric antigen receptor as described in claim 1 or 2; Preferably, the coding sequence of the chimeric antigen receptor comprises, in sequence, a coding sequence of a binding recognition sequence, a coding sequence of a hinge region, a coding sequence of a transmembrane domain, a coding sequence of a co-stimulatory signaling domain, and a coding sequence of an intracellular signal transduction domain. Preferably, the coding sequence of the chimeric antigen receptor comprises, in sequence, the coding sequences of a signal peptide, a FLAG tag, a binding recognition sequence, a hinge region, a transmembrane domain, a co-stimulatory signaling domain, an intracellular signal transduction domain, a linker peptide, and a RISR-RIAD. Preferably, the coding sequence of the chimeric antigen receptor comprises, in sequence, the coding sequence of the Caveolin-1 binding recognition sequence, the coding sequence of the CD8 hinge region, the coding sequence of the CD8 transmembrane domain, the coding sequence of the 4-1BB co-stimulatory signaling domain, and the coding sequence of the CD3ζ intracellular signal transduction domain. Preferably, the coding sequence of the chimeric antigen receptor comprises, in sequence, the coding sequence of the CD8α signal peptide, the coding sequence of the FLAG tag, the coding sequence of the Caveolin-1 binding recognition sequence, the coding sequence of the CD8 hinge region, the coding sequence of the CD8 transmembrane domain, the coding sequence of the 4-1BB co-stimulatory signaling domain, the coding sequence of the CD3ζ intracellular signal transduction domain, the coding sequence of P2A, and the coding sequence of RISR-RIAD. Preferably, the coding sequence of the CD8α signal peptide is as shown in SEQ ID NO:9; Preferably, the encoding sequence of the FLAG tag is as shown in SEQ ID NO:10; Preferably, the coding sequence of the Caveolin-1 binding recognition sequence is as shown in SEQ ID NO:11; Preferably, the encoding sequence of the CD8 hinge region is as shown in SEQ ID NO:12; Preferably, the coding sequence of the CD8 transmembrane domain is shown in SEQ ID NO:13; Preferably, the coding sequence of the 4-1BB co-stimulation signal structure domain is shown in SEQ ID NO:14; Preferably, the coding sequence of the CD3ζ intracellular signal transduction domain is shown in SEQ ID NO:15; Preferably, the encoding sequence of P2A is as shown in SEQ ID NO:16; Preferably, the encoding sequence of the RISR-RIAD is as shown in SEQ ID NO:

18.

4. A carrier, characterized in that, The carrier comprises the nucleic acid molecule according to claim 3; Preferably, the vector comprises a DNA vector, an RNA vector, a plasmid, a lipid nanoparticle, a transposon vector, a CRISPR / Cas9 vector, or a viral vector. Preferably, the carrier is lipid nanoparticles.

5. An engineered host cell, characterized in that, The host cell contains or expresses the chimeric antigen receptor of claim 1 or 2, the nucleic acid molecule of claim 3, or the vector of claim 4; Preferably, the host cells include immune cells; Preferably, the immune cells include T cells, B cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, myeloid cells, or any combination thereof; Preferably, the immune cells are T cells.

6. A method for preparing the host cell according to claim 5, characterized in that, The method comprises introducing the nucleic acid molecule of claim 3 or the vector of claim 4 into a host cell.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the chimeric antigen receptor of claim 1 or 2 or the host cell of claim 5.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier, excipient, or diluent.

9. Any of the following applications: 1) The use of the chimeric antigen receptor of claim 1 or 2, the nucleic acid molecule of claim 3, the carrier of claim 4, the host cell of claim 5, or the pharmaceutical composition of claim 7 in the preparation of anti-aging drugs; 2) The use of the chimeric antigen receptor of claim 1 or 2, the nucleic acid molecule of claim 3, the carrier of claim 4, the host cell of claim 5, or the pharmaceutical composition of claim 7 in the preparation of a medicament for treating organ fibrosis; 3) The use of the chimeric antigen receptor of claim 1 or 2, the nucleic acid molecule of claim 3, the carrier of claim 4, the host cell of claim 5, or the pharmaceutical composition of claim 7 in the preparation of a medicament for restoring muscle strength, improving endurance, and enhancing motor coordination.

10. The application according to claim 9, characterized in that, The organ fibrosis includes liver fibrosis, pulmonary fibrosis, renal fibrosis, cardiac fibrosis, joint fibrosis, splenic fibrosis, and pancreatic fibrosis; Preferably, the organ fibrosis is liver fibrosis.

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