Target-switchable car-t cell linker system and use thereof

Abstract

The present invention relates to the field of biomedicine. Disclosed are a target-switchable CAR-T cell linker system and a use thereof, specifically a target-switchable linker for improving the activation and proliferation of a specific T cell. The linker comprises a domain capable of binding to an antigen-specific T cell and a targeting domain capable of recognizing a cell surface membrane protein. On the one hand, the linker can link the antigen-specific T cell to APC to mediate the activation, phenotypic change, and proliferation of the antigen-specific T cell, enhance killing capacity, and reduce or reverse exhaustion; on the other hand, the linker can switch the CAR-T cell to a FRβ-targeting functional state, so that the T cell can recognize and kill an FRβ-expressing myeloid cell or macrophage, thereby achieving the activation, proliferation, and functional enhancement of the antigen-specific T cell. The present invention can significantly improve the sustained activation capacity and anti-tumor effect of CAR-T cells in a tumor microenvironment, reduce exhaustion, and enhance therapeutic safety.

Description

A target-switched CAR-T cell linker system and its application Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a target-switched CAR-T cell linker system and its application. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) therapy, as a breakthrough cellular immunotherapy strategy, has achieved significant efficacy in various hematologic malignancies. However, CAR-T therapy for solid tumors still faces multiple challenges, including tumor antigen heterogeneity, relapse due to antigen loss, and the influence of immunosuppressive cell populations in the tumor microenvironment (TME). Traditional CAR-T cells typically target a single antigen (such as GPC3 or HER2), and their efficacy depends on the stable expression of this antigen on tumor cells. Once the antigen is downregulated or lost, CAR-T cells lose their recognition ability, leading to treatment failure. Furthermore, after long-term survival in vivo, CAR-T cells often lack continuous antigen stimulation, frequently resulting in functional exhaustion or inactivation, making it difficult to achieve a sustained anti-tumor immune response.

[0003] Recent studies have revealed that tumor-associated myeloid cells, especially macrophages expressing folate receptor β (FRβ), are widely present in the immune microenvironment of various solid tumors and play an important role in regulating T cell function and maintaining immune homeostasis. Based on this, utilizing FRβ-positive myeloid cells as an auxiliary activation platform may provide a new activation and expansion mechanism for CAR-T cells. While existing switchable or universal CAR systems can achieve target modulation through bridging molecules, they generally suffer from conformational limitations, high immunogenicity, or imprecise activation regulation. Therefore, a novel switchable target CAR-T linker system is urgently needed. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a target-switched CAR-T cell linker system and its application in the art.

[0005] The present invention achieves the above objectives using the following technical solution:

[0006] A first aspect of the present invention provides a target-switching linker, characterized in that the linker comprises a first component and a second component. The first component is an arbitrary amino acid sequence capable of binding to the TCR of T cells recognizing corresponding antigens or to cell surface membrane proteins expressed by gene-edited T cells. The second component is a domain having cell surface membrane protein targeting capability.

[0007] In an optional embodiment, the cell surface membrane proteins expressed by the gene-edited T cells include molecules of CAR, TruC, and STAR.

[0008] In this invention, CAR-T cells refer to T cells modified through genetic engineering that express chimeric antigen receptors (CARs). These receptors typically include: 1) an antigen recognition domain: such as a single-stranded variable fragment (scFv), used for specific recognition of target antigens; 2) a transmembrane region: connecting extracellular and intracellular structures; and 3) an intracellular signal transduction region: such as the CD3ζ chain, which may also contain co-stimulatory signaling domains (such as CD28 or 4-1BB) for activating T cell function.

[0009] In this invention, TRuC-T cells (TCR Fusion Construct T cells) refer to T cells expressing a T cell receptor fusion construct (TCR Fusion Construct, TRuC). The TRuC structure directly fuses an antigen recognition domain (e.g., scFv) to a subunit of the TCR complex (e.g., CD3ε, CD3γ, TCRα, etc.). This linker is embedded in the TCR complex and mediates T cell activation via the endogenous TCR-CD3 signaling pathway. TRuC does not depend on traditional co-stimulatory elements in the CAR domain; its activation relies on the TCR complex's own signal transduction mechanism.

[0010] In this invention, STAR-T cells (Synthetic TCR and Antigen Receptor T cells) refer to T cells that express a synthetic T cell receptor and antigen receptor fusion construct (Synthetic TCR and Antigen Receptor, STAR). The STAR structure consists of two polypeptide chains. The first chain is formed by the fusion of the antibody heavy chain variable region (VH) and the TCRα chain constant region (including the transmembrane region). The second chain is formed by the fusion of the antibody light chain variable region (VL) and the TCRβ chain constant region (including the transmembrane region). The two chains are linked by disulfide bonds to form a heterodimer, which can assemble with TCR complex subunits such as CD3ε, CD3γ, CD3δ, and CD3ζ to form a complete TCR-like structure, thereby initiating the TCR signaling activation process.

[0011] The aforementioned CAR-T, TruC-T, and STAR-T constructs also include all their structural equivalents, functional analogs, or derivatives, such as peptides, VHHs, ligand forms that recognize the domain, and various transmembrane and intracellular signaling modules (such as co-stimulatory structures, scaffold sequences, linker peptides, etc.), regardless of whether they adopt the specific forms expressed in this specification, and should be considered to be covered within the scope of protection of this invention.

[0012] In an optional embodiment, the first component is a specific antigen that specifically binds to T cells that target tumor antigens.

[0013] In this invention, the terms "specific," "binding," and "targeting" refer to binding interactions that are selective for antigens and can be distinguished from unintended or nonspecific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

[0014] In optional embodiments, the specific antigen is derived from single-chain antibodies, single-chain peptide-MHC, bacterial antigens, viral antigens, cancer-associated antigens, cancer-specific antigens, or fragments having antigenic determinants.

[0015] In some embodiments, the specific antigen can bind to CAR-T or TCR-T cells that specifically target GPC3, DLL3, HLA-PRAME, HLA-NY-ESO-1, CD19, or MSLN.

[0016] In some embodiments, the GPC3 is a protein fragment of GPC3 having an amino acid sequence as shown in SEQ ID NO:1, or having an amino acid sequence that is more than 80% identical to that in SEQ ID NO:1.

[0017] In some embodiments, the GPC3 is a protein fragment of GPC3 having an amino acid sequence as shown in SEQ ID NO:71-74, or having an amino acid sequence that is more than 80% identical to SEQ ID NO:71-74.

[0018] In some embodiments, the surface antigen comprises protein fragments from different regions of DLL3, including DLL3-EGF4 and DLL3(277).

[0019] In a specific embodiment of the present invention, the amino acid sequence of DLL3-EGF4 is as shown in SEQ ID NO:86, or has more than 80% identity with SEQ ID NO:86.

[0020] In a specific embodiment of the present invention, the amino acid sequence of DLL3 (277) is as shown in SEQ ID NO:87, or has more than 80% identity with SEQ ID NO:87.

[0021] In a specific embodiment of the present invention, the HLA-PRAME comprises an amino acid sequence as shown in SEQ ID NO:2, or having at least 80% identity with SEQ ID NO:2.

[0022] In a specific embodiment of the present invention, the HLA-NY-ESO-1 comprises an amino acid sequence as shown in SEQ ID NO:3, or having at least 80% identity with SEQ ID NO:3.

[0023] In a specific embodiment of the present invention, CD19 comprises an amino acid sequence as shown in SEQ ID NO:4, or having at least 80% identity with SEQ ID NO:4.

[0024] In a specific embodiment of the present invention, the MSLN comprises an amino acid sequence as shown in SEQ ID NO:5, or having at least 80% identity with SEQ ID NO:5.

[0025] In this invention, GPC3 is also known as SGB, DGSX, MXR7, SDYS, SGBS, OCI-5, SGBS1, and GTR2-2. This term encompasses full-length, unprocessed GPC3, as well as any form of GPC3 derived from cells or a portion of the GPC3 protein that can bind to GPC3 antibodies. This term encompasses naturally occurring variants of GPC3 (e.g., splice variants or allelic variants). This term encompasses, for example, the GPC3 gene, the GPC3 protein, human GPC3, and GPC3 from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats). As a preferred embodiment, in this invention, GPC3 is the human GPC3, with gene ID 2719.

[0026] In this invention, DLL3 is also known as SCDO1. The term encompasses full-length, unprocessed DLL3, as well as any form of DLL3 derived from cells or a portion of the DLL3 protein that can bind to DLL3 antibodies. The term encompasses naturally occurring variants of DLL3 (e.g., splice variants or allelic variants). The term encompasses, for example, the DLL3 gene, the DLL3 protein, human DLL3, and DLL3 from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats). As a preferred embodiment, in this invention, DLL3 is the human DLL3 with gene ID 10683.

[0027] In an optional embodiment, the domain having cell surface membrane protein targeting capability includes a domain targeting APC cells or macrophages, a domain targeting other cells, or a single-chain variable fragment of an antibody.

[0028] In this invention, APCs (Antigen-presenting cells), also known as antigen-presenting cells, are a type of immune cell capable of taking up, processing, and presenting antigens to T cells. They are widely distributed throughout the body. APCs are mainly divided into professional APCs and part-time APCs. Professional APCs include monocytes / macrophages, dendritic cells, and B lymphocytes, which take up and process immune antigens and present them to T cells by expressing MHC class II molecules. Part-time APCs include certain endothelial cells and epithelial-mesothelial cells, such as fibroblasts, glial cells, thymus, thyroid epithelial cells, vascular endothelial cells, and eosinophils.

[0029] In an optional embodiment, the domains targeting APC cells are selected from CD40, CLEC7A, LILRB4, and FAP.

[0030] In some embodiments, the domain having cell surface membrane protein targeting capability is a CD40 binding domain.

[0031] In some embodiments, the CD40 binding domain is derived from the extracellular domain of CD40L, other fragments of CD40L that can bind to CD40, or an antibody against CD40.

[0032] In some embodiments, the domain having cell surface membrane protein targeting capability is a domain that targets FRβ protein.

[0033] In this invention, FRβ (Folate Receptor Beta), also known as FR-β, FR4, FOLR2, and JTC-31, is a glycosylphosphatidylinositol (GPI)-anchored membrane protein, a member of the folate receptor family, and plays an important role in certain physiological and pathological processes. This term encompasses full-length, unprocessed FRβ, as well as any form of FRβ derived from cells or a portion of the FRβ protein that can bind to FRβ antibodies. This term encompasses naturally occurring variants of FRβ (e.g., splice variants or allelic variants). This term encompasses, for example, the FRβ gene, the FRβ protein, human FRβ, and FRβ from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats). As a preferred embodiment, in this invention, FRβ is the human FRβ, with gene ID 2356.

[0034] In some embodiments, the single-chain variable fragment of the antibody is selected from the CDR1, CDR2, and CDR3 regions of the light and heavy chains of any of the following antibodies: CD40L, anti-CD40 Selicrelumab, anti-CD40 (GILORALIMAB), anti-CLEC7A.2M24, anti-CLEC7A.15E2, anti-LILRB4.ATG034, anti-LILRB4.Hz5A7, or anti-FAP. Alternatively, the second component comprises a region having more than 80% amino acid identity with the CDR1, CDR2, and CDR3 regions defined by any of the aforementioned antibodies, wherein the CDR regions can be obtained according to any antibody numbering system such as Kabat, Chothia, IMGT, or AHo.

[0035] Furthermore, the linker also includes an exomembrane signal peptide.

[0036] In a specific embodiment of the present invention, the CD40L extracellular segment comprises an amino acid sequence as shown in SEQ ID NO:6, or having at least 80% identity with SEQ ID NO:6.

[0037] In a specific embodiment of the present invention, the structure of the antibody (GILORALIMAB) that recognizes CD40 includes an amino acid sequence such as SEQ ID NO:7 or having at least 80% identity with SEQ ID NO:7.

[0038] In a specific embodiment of the present invention, the structure of the antibody (Selicrelumab) that recognizes CD40 includes an amino acid sequence such as SEQ ID NO:8 or having at least 80% identity with SEQ ID NO:8.

[0039] .

[0040] In a specific embodiment of the present invention, the amino acid sequence of the anti-CLEC7A.2M24 is as shown in SEQ ID NO:59, or has more than 80% identity with SEQ ID NO:59.

[0041] In a specific embodiment of the present invention, the amino acid sequence of the anti-CLEC7A.15E2 is as shown in SEQ ID NO:60, or has more than 80% identity with SEQ ID NO:60.

[0042] In a specific embodiment of the present invention, the amino acid sequence of the anti-LILRB4.ATG034 is as shown in SEQ ID NO:60, or has more than 80% identity with SEQ ID NO:60.

[0043] In a specific embodiment of the present invention, the amino acid sequence of the anti-LILRB4.Hz5A7 is as shown in SEQ ID NO:61, or has more than 80% identity with SEQ ID NO:61.

[0044] In a specific embodiment of the present invention, the amino acid sequence of the anti-FAP is as shown in SEQ ID NO:77, or has more than 80% identity with SEQ ID NO:77.

[0045] In a specific embodiment of the present invention, the domain of the targeted FRβ protein is anti-FRβ, which has an amino acid sequence as shown in any of SEQ ID NO:94-95, or has an amino acid sequence that is more than 80% identical to any of SEQ ID NO:94-95.

[0046] In an optional embodiment, the exosome signal peptide is selected from any of the following molecules: CD8a, IL-2, CD33, CD5, HSA, TPA, IgKVIII, GM-CSF, GM-CSFRα, CD3ζ, CD28, HLA.

[0047] In some embodiments, the exophase signal peptide is the CD8a exophase signal peptide.

[0048] In a specific embodiment of the present invention, the amino acid sequence of the CD8a exosynthesis signal peptide is as shown in SEQ ID NO:11 or has at least 80% identity with SEQ ID NO:11.

[0049] In some embodiments, the exosome signal peptide is an HLA exosome signal peptide.

[0050] In a specific embodiment of the present invention, the amino acid sequence of the HLA exosynthesis signal peptide is such as SEQ ID NO:12 or an amino acid sequence having at least 80% identity with SEQ ID NO:12.

[0051] The term "identity" is used interchangeably with "sequence consistency" and "homology," referring to an amino acid or nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence provided in this invention. To determine sequence identity, sequence alignment can be performed using various methods known to those skilled in the art, such as BLAST, BLAST-2, ALIGN, NEEDLE, Megalign (DNASTAR), Snapgene, or DNAMAN software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0052] In this invention, sequences obtained through modification that have 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the linker described in the first aspect of this invention also fall within the scope of protection of this invention. The term "modification" refers to any form of modification to an amino acid sequence, such as substitution, deletion, insertion, and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids in the original amino acid sequence with a different amino acid. The term "deletion" refers to reducing one or more amino acids in the original amino acid sequence. The terms "insertion" or "addition" refer to changes in the amino acid sequence resulting in the addition of one or more amino acids compared to the original amino acid sequence.

[0053] Furthermore, the first component and the second component are connected via a linker or directly.

[0054] In some implementations, the linker, also referred to as a "connecting peptide," is a short peptide chain that links different functional domains or subunits of the TCR. Its core function is to maintain the spatial conformational stability of the complex and ensure the accuracy of signal transduction. Linkers are typically 10-30 amino acids long, rich in glycine (Gly) and / or proline (Pro), forming a random coil structure that imparts flexibility to the complex and avoids steric hindrance. Common linkers include flexible connecting peptides selected from (G4S). n Whitlow / 218 linker peptide (G3S) n Or (G2S) n Rigid linker peptides, selected from α-helical linkers such as EAAAK, C-helical linkers, and proline-containing rigid linkers (XP). n Cleavable linker peptides; mixed linker peptides.

[0055] In an optional embodiment, the linker is a flexible linker or a rigid linker.

[0056] In an optional embodiment, the linker is approximately 3 to 100 amino acid residues in length.

[0057] In an optional embodiment, the linker includes one or more functional tag sequences, the tags being selected from one or more of FLAG, Myc, and HA.

[0058] In an optional embodiment, the linker is a monomeric structure or a polymeric structure.

[0059] In an optional embodiment, the linker is selected from one or more of peptide linkers, polyethylene glycol linkers, polyamide linkers, carbohydrate derivative linkers, aliphatic linkers, aromatic linkers, and synthetic polymer linkers.

[0060] In some embodiments, the linker includes one or more Gly4Ser repeating units, denoted as (Gly-Gly-Gly-Gly-Ser). n , where n is from 1 to 10.

[0061] In a specific embodiment of the present invention, the linker is CD40L-flag-GPC3, which has an amino acid sequence as shown in SEQ ID NO:13, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:13.

[0062] In a specific embodiment of the present invention, the linker is CD40L-G4S-GPC3, which has an amino acid sequence as shown in SEQ ID NO:14, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:14.

[0063] In a specific embodiment of the present invention, the linker is CD40L-HLA-PRAME, which has an amino acid sequence as shown in SEQ ID NO:15, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:15.

[0064] In a specific embodiment of the present invention, the linker is CD40L-HLA-NY-ESO-1, which has an amino acid sequence as shown in SEQ ID NO:16, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:16.

[0065] In a specific embodiment of the present invention, the linker is CD40L-flag-CD19, which has an amino acid sequence as shown in SEQ ID NO:17, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:17.

[0066] In a specific embodiment of the present invention, the linker is CD40L-flag-MSLN, which has an amino acid sequence as shown in SEQ ID NO:18, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:18.

[0067] In a specific embodiment of the present invention, the linker is anti-CD40 scFv1-flag-GPC3, which has an amino acid sequence as shown in SEQ ID NO:19, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:19.

[0068] In a specific embodiment of the present invention, the linker is anti-CD40 scFv2-flag-GPC3, which has an amino acid sequence as shown in SEQ ID NO:20, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:20.

[0069] In a specific embodiment of the present invention, the linker is GPC3-anti-CLEC7A.2M24, and the amino acid sequence of GPC3-anti-CLEC7A.2M24 is as shown in SEQ ID NO:53, or has more than 80% identity with SEQ ID NO:53.

[0070] In a specific embodiment of the present invention, the linker is GPC3-anti-CLEC7A.15E2, and the amino acid sequence of GPC3-anti-CLEC7A.15E2 is as shown in SEQ ID NO:54, or has more than 80% identity with SEQ ID NO:54.

[0071] In a specific embodiment of the present invention, the linker is GPC3-anti-LILRB4.ATG034, and the amino acid sequence of GPC3-anti-LILRB4.ATG034 is as shown in SEQ ID NO:55, or has more than 80% identity with SEQ ID NO:55.

[0072] In a specific embodiment of the present invention, the linker is GPC3-anti-LILRB4.Hz5A7, and the amino acid sequence of GPC3-anti-LILRB4.Hz5A7 is as shown in SEQ ID NO:56, or has more than 80% identity with SEQ ID NO:56.

[0073] In a specific embodiment of the present invention, the linker is GPC3-anti-FAP, and the amino acid sequence of GPC3-anti-FAP is as shown in SEQ ID NO:67, or has more than 80% identity with SEQ ID NO:67.

[0074] In a specific embodiment of the present invention, the linker is DLL3-anti-FAP, and the amino acid sequence of DLL3-anti-FAP is as shown in SEQ ID NO:68, or has more than 80% identity with SEQ ID NO:68.

[0075] In a specific embodiment of the present invention, the linker is Linker C, and the amino acid sequence of Linker C is as shown in SEQ ID NO:80, or has more than 80% identity with SEQ ID NO:80.

[0076] In a specific embodiment of the present invention, the linker is Linker 277, and the amino acid sequence of Linker 277 is as shown in SEQ ID NO:81, or has more than 80% identity with SEQ ID NO:81.

[0077] In a specific embodiment of the present invention, the linker is Linker ATG034, and the amino acid sequence of Linker ATG034 is as shown in SEQ ID NO:82, or has more than 80% identity with SEQ ID NO:82.

[0078] In a specific embodiment of the present invention, the linker is Linker Hz5A7, and the amino acid sequence of Linker Hz5A7 is as shown in SEQ ID NO:83, or has more than 80% identity with SEQ ID NO:83.

[0079] In a specific embodiment of the present invention, the linker is Linker 2M24, and the amino acid sequence of Linker 2M24 is as shown in SEQ ID NO:84, or has more than 80% identity with SEQ ID NO:84.

[0080] In a specific embodiment of the present invention, the linker is Linker 15E2, and the amino acid sequence of Linker 15E2 is as shown in SEQ ID NO:85, or has more than 80% identity with SEQ ID NO:85.

[0081] In a specific embodiment of the present invention, the linker is GPC3-anti-FRβ(m909), which has an amino acid sequence as shown in SEQ ID NO:96, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:96.

[0082] In a specific embodiment of the present invention, the linker is GPC3-anti-FRβ(m923), which has an amino acid sequence as shown in SEQ ID NO:97, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:97.

[0083] A second aspect of the present invention provides an immunotherapeutic composition comprising: the linker described in the first aspect, and a combination of at least one of the following: (a) a domain for extending in vivo half-life, said domain being selected from one or more of Fc fragments, albumin-binding molecules, PEG-modified compounds, high molecular weight peptides, XTEN peptide chains, PAS sequences, or analogues thereof; and (b) at least one immunomodulatory component, said immunomodulatory component being selected from cytokines, chemokines, immune adjuvants, or functional analogues thereof.

[0084] The linker described in this invention can be a natural structure or a pharmacokinetic optimized form. For example, it can be fused with an Fc domain, an albumin-binding structure (such as an anti-albumin antibody, albumin-binding peptide), or other structures that prolong plasma half-life (such as PEG, PAS, XTEN peptides) to extend in vivo half-life or improve tissue distribution stability. The scope of this invention covers all such fused, modified, or derived forms.

[0085] In some embodiments, the cytokines are selected from one or more of interleukins, interferons, tumor necrosis factors, colony-stimulating factors, and transforming growth factors.

[0086] In some embodiments, the chemokine is selected from one or more of the CCL family, CXCL family, XCL family, CX3CL family and their functional analogs.

[0087] In some embodiments, the immune adjuvant or immune agonist is selected from one or more of the following: TLR agonists, STING agonists, RIG-I agonists, cGAMP, Poly(I:C), CpG oligonucleotides, QS-21, MF59, AS03, and aluminum adjuvants.

[0088] A third aspect of the present invention provides a biomaterial having any one of the following characteristics:

[0089] (1) A nucleic acid molecule comprising a nucleic acid molecule encoding the linker described in the first aspect, which can be used to express the linker described in the first aspect.

[0090] In some embodiments, the linker expressed in the first aspect is present in the form of a polypeptide, protein, fusion protein, or other gene expression product.

[0091] In some embodiments, the nucleic acid molecule is selected from DNA, RNA, or a modified form thereof.

[0092] In some embodiments, the RNA is selected from linear mRNA, circular RNA, self-amplifying RNA, or modified forms thereof.

[0093] In a further embodiment of the present invention, the mRNA used for expressing the linker is not limited to a linear structure, but also includes novel RNA forms such as circular RNA (circRNA) and self-amplifying RNA (saRNA). The circRNA can be formed through covalent circular closure, which has the advantages of enhanced stability and prolonged expression time; the saRNA includes replicase elements and can self-amplify in vivo to enhance expression efficiency. All of the above-mentioned RNA forms are within the scope of protection of the present invention.

[0094] In a further embodiment, the present invention also provides a nucleic acid delivery system for expressing the linker in vivo. The nucleic acid may be mRNA, DNA, or a modified form thereof, and the delivery system may include liposomes, LNPs (lipid nanoparticles), polymer nanoparticles, virus-like particles (VLPs), artificial exosomes, etc., for effectively delivering the nucleic acid to target tissue cells via intravenous, intramuscular, or local injection. Preferably, the LNP comprises ionized lipids, cholesterol, phospholipids, and PEGylated lipids, exhibiting high delivery efficiency and biocompatibility.

[0095] The nucleic acid molecules of this invention can be delivered to target tissues or cells in vivo via various delivery systems, including non-biological particle systems (such as LNPs, liposomes, and polymer nanoparticles) and biological carrier systems (such as engineered exosomes, virus-like particles, engineered bacteria, and their spore forms). The engineered bacteria can be selected from Escherichia coli, Salmonella, Clostridium, or Bacillus subtilis, etc. The spore form exhibits strong tolerance and tissue targeting ability, making it suitable for intratumoral injection, oral delivery, or intestinal targeted therapy.

[0096] (2) A recombinant vector containing the nucleic acid molecules described in (1).

[0097] In some embodiments, the recombinant vector is used to express the linker described in the first aspect, and is selected from one or more of DNA vectors, viral vectors, and mRNA vectors.

[0098] In this invention, the term "recombinant vector" or "vector" refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. The vectors of this invention can be expression vectors, viral vectors, etc. In some embodiments, the vector contains a target gene encoding the linker described in the first aspect of this invention, a promoter, a terminator, a signal peptide, or optionally, a marker gene. The vector can be a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, herpesvirus vectors, and other viral vectors. Other viral vectors may include bacteriophage vectors, baculovirus vectors, animal viral vectors, plant viral vectors, and may include lactoblastic viral vectors, herpesvirus vectors, poxvirus vectors, RNA virus vectors, bovine papillomavirus vectors, EB virus vectors, retroviral vectors, etc.

[0099] (3) A recombinant host cell comprising the nucleic acid molecule described in (1) and / or the recombinant vector described in (2).

[0100] In this invention, the term "recombinant host cell" refers to any cell type suitable for transformation, transfection, transduction, etc., using an expression vector containing the nucleic acid molecules provided by this invention. Recombinant host cells include any progeny of the parent cell that differs from the parent cell due to mutations occurring during replication. Preferably, the recombinant cells include prokaryotic cells and eukaryotic cells; more preferably, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsiae; even more preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells; most preferably, the recombinant host cell is an immune cell; most preferably, the immune cell includes T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, or any combination thereof.

[0101] A fourth aspect of the invention provides a delivery system for delivering the nucleic acid molecule described in the third aspect and / or the linker described in the first aspect, which is present in the form of a polypeptide, protein, fusion protein or other gene expression product, to a target tissue or cell in a mammal to express the linker described in the first aspect.

[0102] In some embodiments, the delivery carrier is selected from one or more of liposomes, lipid nanoparticles, polymer nanoparticles, exosomes, viruses, virus-like particles, engineered microorganisms or their spores, cells, inorganic nanoparticles, hydrogels, emulsions, polymer micelles, and biomimetic membrane-encapsulated nanoparticles.

[0103] In some embodiments, the lipid nanoparticles comprise one or more of ionized lipids, cofactor lipids, cholesterol, and PEG-modified lipids.

[0104] In some embodiments, the virus is an oncolytic virus, which is selected from one or more of adenovirus, adeno-associated virus, herpes simplex virus, varicella-zoster virus, vaccinia virus / vaccinia virus, measles virus, Newcastle disease virus, reovirus, poliovirus, and samba virus.

[0105] In some embodiments, the cells are selected from T cells, natural killer cells, dendritic cells, macrophages, monocytes, erythrocytes, mesenchymal stem cells, stromal cells, and tumor cells.

[0106] The fifth aspect of the present invention provides applications of the linker described in the first aspect, the immunotherapy composition described in the second aspect, the biomaterial described in the third aspect, and / or the delivery system described in the fourth aspect, said applications including any one of the following:

[0107] 1) Applications in the preparation of products that enhance the activation, expansion, and / or reduce and reverse the depletion of specific T cells; 2) Applications in the preparation of products that promote changes in T cell subtypes; 3) Applications in the preparation of drugs for treating tumors.

[0108] In some embodiments, the specific T cell is selected from T cells, CAR-T cells, STAR-T cells, TruC-T cells, and TCR-T cells.

[0109] In some embodiments, the specific T cells are CAR-T cells that target GPC3, DLL3, HLA-PRAME, HLA-NY-ESO-1, CD19, or MSLN.

[0110] In some embodiments, the GPC3-targeting CAR-T cells can be constructed from any of the following CARs: GPC3 CAR, 9F2GPC3 CAR, or GC33 GPC3 CAR (SEQ ID NO:75), or their corresponding scFv sequences. The scFv sequence includes CDR1, CDR2, and CDR3 of the light and heavy chains defined by any CAR or its corresponding scFv sequence, or regions having more than 80% amino acid sequence identity with said CDR1, CDR2, and CDR3. The CDR regions can be determined according to any of the antibody numbering systems Kabat, Chothia, IMGT, or Aho.

[0111] In some embodiments, the DLL3-targeting CAR-T cells can be constructed from any of the following CARs: DLL3.Juno CAR, DLL3.T3 CAR, or their corresponding scFv sequences. The scFv sequence includes CDR1, CDR2, and CDR3 of the light and heavy chains defined by any CAR or its corresponding scFv sequence, or regions having more than 80% amino acid sequence identity with said CDR1, CDR2, and CDR3. The CDR regions can be determined according to any of the antibody numbering systems Kabat, Chothia, IMGT, or Aho.

[0112] In some embodiments, promoting T cell subtype alteration refers to promoting CAR-T cell subtype alteration targeting GPC3, DLL3, HLA-PRAME, HLA-NY-ESO-1, CD19, or MSLN.

[0113] In some embodiments, the subtype change refers to a shift from a terminally differentiated cell subtype to an effector memory T cell subtype.

[0114] Furthermore, the tumor includes tumors expressing GPC3, DLL3, HLA-PRAME, HLA-NY-ESO-1, CD19, or MSLN.

[0115] In some embodiments, the tumors expressing GPC3 include liver cancer, hepatocellular carcinoma, clear cell ovarian carcinoma, yolk sac tumor, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma, and chronic myeloid leukemia.

[0116] In some embodiments, the tumor includes a tumor expressing DLL3.

[0117] In some embodiments, the tumors expressing DLL3 include small cell lung cancer, neuroendocrine tumors, and neuroendocrine carcinomas.

[0118] In some embodiments, the product includes a reagent kit, test strip, nucleic acid membrane strip, chip, system, or device.

[0119] In some embodiments, the drug may be used alone or in the form of a pharmaceutical composition.

[0120] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0121] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995), and these substances are used as needed to aid in the stability of the formulation or to help improve the activity or bioavailability of the active substance. In some embodiments, the pharmaceutical composition is used by administering a safe and effective amount of the pharmaceutical composition of the present invention to a human. There are no particular limitations on the dosage and route of administration of the pharmaceutical composition, and a skilled physician can usually readily determine the dosage and effectiveness of the prescription for the desired treatment and / or prevention, such as by injection or other treatment methods.

[0122] In some embodiments, the biological agent has a dosage form selected from: solution, suspension, emulsion, tablet, pill, powder, granule, capsule, syrup, sterile aqueous solution, non-aqueous solution, lyophilized preparation, suppository. Furthermore, it can be administered once or multiple times. In this case, the biological agent is administered in the form of a liquid preparation, powder, aerosol, capsule, or suppository. Routes of administration may include, but are not limited to: intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, local, intranasal, intrapulmonary, rectal, etc. When administered orally, it can be formulated with a coating to protect the active ingredient in the biological agent from degradation in the stomach. Furthermore, the active ingredient can be administered via any device capable of transfer to the target tissue. In specific embodiments, the biological agent provided by the present invention can be formulated into various dosage forms as needed, and the dosage beneficial to the patient can be determined by a clinician based on factors such as the subject's type, age, weight, general disease condition, and route of administration. The route of administration may include, for example, injection or any other suitable route of administration known to those skilled in the art.

[0123] In some embodiments, the biological agent further comprises buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates 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; and preservatives.

[0124] The compositions described in this invention can be administered to subjects in various ways, including intravenous injection, subcutaneous injection, intramuscular injection, or local injection. Preferably, local injection sites include, but are not limited to, intratumoral injection, peritumoral injection, and groin injection. Groin injection can utilize the local lymphatic drainage system to promote immune cell activation and chemotactic signal transduction, thereby enhancing antibody-mediated local or systemic immune responses. All of the above delivery methods and combinations thereof fall within the scope of protection of this invention.

[0125] A sixth aspect of the present invention provides a method comprising any one of the following:

[0126] 1) A method for promoting the binding of specific T cells and downstream cells, the method comprising the step of co-incubating the linker described in the first aspect with specific T cells and downstream cells.

[0127] 2) A method for enhancing the ability of T cells to specifically activate, expand, and / or reduce or reverse exhaustion, the method comprising the steps of co-incubating the linker described in the first aspect with specific T cells and downstream cells.

[0128] 3) A method for treating a tumor, the method comprising administering to a patient the linker described in the first aspect, the linker being present in the form of a polypeptide, protein, fusion protein or other gene expression product; or administering to a patient the linker described in the first aspect, the immunotherapy composition described in the second aspect, the biomaterial described in the third aspect and / or the delivery system described in the fourth aspect.

[0129] Furthermore, the tumor includes tumors expressing GPC3, DLL3, HLA-PRAME, HLA-NY-ESO-1, CD19, or MSLN.

[0130] In some embodiments, the tumors expressing GPC3 include liver cancer, hepatocellular carcinoma, clear cell ovarian carcinoma, yolk sac tumor, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma, and chronic myeloid leukemia.

[0131] In some embodiments, the tumor includes a tumor expressing DLL3.

[0132] In some embodiments, the tumors expressing DLL3 include small cell lung cancer, neuroendocrine tumors, and neuroendocrine carcinomas.

[0133] In some embodiments, the downstream cells are APC cells.

[0134] In some embodiments, the specific T cell is selected from T cells, CAR-T cells, STAR-T cells, TruC-T cells, and TCR-T cells.

[0135] In some implementations, the term "treatment" refers to any action aimed at improving a patient's health, such as treating, preventing, or delaying a disease. In some implementations, the term refers to improving or eradicating a disease or disease-related symptoms. In other implementations, the term refers to minimizing the spread or worsening of the disease as a result of administering one or more therapeutic agents to a subject suffering from such a disease.

[0136] Advantages and beneficial effects of the present invention: The present invention provides a novel switchable target CAR-T linker system. This linker can, on the one hand, link antigen-specific T cells to APCs, mediating antigen-specific T cell activation, subtyping alteration, expansion, and enhanced killing ability, while reducing or reversing exhaustion; on the other hand, it can switch CAR-T cells to a functional state targeting FRβ, enabling T cells to recognize and kill myeloid cells or macrophages expressing FRβ, thereby achieving activation, expansion, and functional enhancement of antigen-specific T cells. The present invention can significantly improve the sustained activation capacity and anti-tumor effect of CAR-T cells in the tumor microenvironment, reduce exhaustion, and enhance therapeutic safety. Attached Figure Description

[0137] Figure 1 is a schematic diagram of a linker design targeting CD40 and T cell recognition receptors.

[0138] Figure 2 shows the flow cytometry results of different types of linkers binding to CD40-mCherry indicator cells and CD40ΔICD-mCherry cells.

[0139] Figure 3 shows the flow cytometry results of the linker binding to different types of effector T cells.

[0140] Figure 4 shows the results of DC activation of effector cells corresponding to the target CAR / STAR / TRuC / TCR-T by the 24h linker.

[0141] Figure 5 shows the results of specific expansion and subtype alteration of CAR / STAR / TRuC / TCR-T effector cells achieved by the 72h linker.

[0142] Figure 6 shows the results of specific amplification and subtype alteration of CAR-T effector cells achieved by the antibody linker after 72 hours.

[0143] Figure 7 shows the flow cytometry results of M0, M1, and M2 cell identification and 24-hour CAR-T activation effect.

[0144] Figure 8 shows the difference between DC-activated CAR-T and CAR-T activated by other target cells achieved by the linker.

[0145] Figure 9 shows the results of DC autocrine linker activating CAR-T and increasing long-acting CAR-T killing effect.

[0146] Figure 10 is a schematic diagram of the linker structure.

[0147] Figure 11 is a statistical graph showing the results of the detection of CAR-T specific amplification and CD8+CAR+ cell memory cell typing changes after co-incubation of GPC3 linkers targeting different APC surface proteins with CAR-T cells and DC cells.

[0148] Figure 12 is a statistical chart showing the results of specific activation of CAR-T cells and changes in memory cell typing after co-incubation of CAR-T cells and DC cells with GPC3 linkers corresponding to other GPC3 antibodies.

[0149] Figure 13 is a statistical chart showing the CAR-T's ability to target new targets for killing by linking the GPC3 linker with the new target system.

[0150] Figure 14 is a statistical chart showing the results of the detection of the binding ability of different GPC3 fragment linkers with corresponding CAR-T cells.

[0151] Figure 15 is a schematic diagram of the linker structure.

[0152] Figure 16 shows the flow cytometry results of linkers expressing different DLL3 regions binding to DLL3 CAR-T cells.

[0153] Figure 17 is a statistical chart showing the results of CD69 and CD137 expression detection in DLL3 CAR-T cells.

[0154] Figure 18 is a statistical chart showing the expression results of CD45RA and CCR7 after CAR-T cells, DC cells and linkers were co-cultured for 72 hours.

[0155] Figure 19 is a statistical graph showing the expression of CD69 and CD137 after co-culturing linkers targeting different APC surface proteins with CAR-T cells and DC cells, and the results of detection of specific activation, amplification of CAR-T cells and changes in CD8+CAR+ cell memory cell typing.

[0156] Figure 20 is a statistical graph showing the expression of CD69 and CD137 in H82 tumor cell lines expressing DLL3 or the linker of the present invention after co-culturing with DLL3 CAR-T, and the results of detection of specific activation, amplification and CD8+CAR+ cell memory cell typing changes of CAR-T.

[0157] Figure 21 is a flow cytogram of the m909 and m923 linkers combined with GC33 CAR-T.

[0158] Figure 22 is a statistical chart showing the killing power of m909 and m923 on target cells.

[0159] Figure 23 shows the flow cytometry results of FRβ expression in PBMCs and differentiated macrophages. A represents the flow cytometry results of PBMCs and a negative control, and B represents the flow cytometry results of differentiated macrophages and a negative control.

[0160] Figure 24 shows the results of flow cytometry detection of CAR-T specific amplification using different linkers. Detailed Implementation

[0161] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0162] The reagents, raw materials, and experimental consumables used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are typically performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0163] Example 1: Linker targeting CD40 and T cell recognition receptors

[0164] 1. The supernatant of anti-CD40 / CD40L-antigen fusion protein (linker) produced by 293 cells can activate CD40 downstream signaling in reporter cells: construct CD40-mCherry expression lentiviral system plasmid, prepare lentivirus, and obtain CD40 / NF-κB reporter 293 cells. Plasmids expressing different proteins (protein linking sequence from N-terminus to C-terminus: Antigen-linker-CD40L / anti-CD40) were constructed as listed below (CD40L-flag-GPC3 SEQ ID NO:13, CD40L-(G4S)3-GPC3 SEQ ID NO:14, CD40L-flag-HLA-PRAME SEQ ID NO:15, CD40L-flag-HLA-NY-ESO-1 SEQ ID NO:16, anti-CD40 scFv1-flag-GPC3[GILORALIMAB ABBV-927] SEQ ID NO:19, anti-CD40 scFv2-flag-GPC3[Selicrelumab-Roche] SEQ ID NO:20, CD40L-flag-MSLN SEQ ID NO:18, CD40L-flag-CD19 SEQ ID NO:19). As shown in NO:17, the linker is flag(SEQ ID NO:9) / (G4S)n (n=3 as shown in SEQ ID NO:10). After transfection into 293 cells using the PEI system, the supernatant of 293 cells was collected after 24 hours to obtain cells containing the corresponding linker. After co-incubation with 293 reporter cells overnight, flow cytometry analysis showed strong red fluorescence expression in the 293 reporter cells. The positive control was CD40L protein (1 μg / ml, Recombinant Human CD40L, novoprotein, Cat.No.:CI56). This indicates that the linker containing both CD40L and anti-CD40 scFv can bind to CD40 on 293 reporter cells and activate downstream signaling. To rule out potential non-specific activation, negative control reporter cells related to CD40ΔICD-mCherry were constructed. The results are shown in Figure 2, indicating that when these negative reporter cells bind to the linker, mCherry expression cannot be initiated.

[0165] 2. The supernatant of the 293 production linker can bind to effector T cells corresponding to the target: the pCDH-EF1α lentiviral expression plasmid was constructed by synthesizing the sequences corresponding to GPC3 CAR-T (SEQ ID NO:41), CD19 CAR-T (SEQ ID NO:44), PR20 CAR-T (SEQ ID NO:45), and NY-ESO-1TCR-T (SEQ ID NO:46). The lentiviral system plasmid (pCDH-EF1α lentiviral expression plasmid, PsPAX2, and pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) was transfected into adherent 293T cells in logarithmic growth phase. The cell culture supernatant was harvested 48-72 hours after transfection, concentrated and filtered, and the lentivirus was stored at -80℃ for later use. Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood. T cells were isolated using human CD3 / 28 magnetic beads and transduced with the virus within 72 hours of activation. After 24 hours of transduction, the medium was changed and the cells were cultured until day 8. Cells were collected by centrifugation and resuspended in physiological saline. Flow cytometry was used to identify the G4S or EGFRt molecule fraction in T cell editing proteins to confirm the CAR-T / TCR-T positivity rate. The supernatant containing the linker was co-incubated with the corresponding edited T cells for 1 hour, followed by washing with PBS and flow cytometry staining. Specific flow cytometry parameters: 3 × 10⁻⁶ cells / mL. 5 Cell counts were determined by adding anti-flag (PE), anti-CD40L, and anti-G4S (CAR) to each well. After incubation for 30 minutes, the cells were washed with PBS and analyzed by flow cytometry. GPC3 CAR-T specifically recognized the GPC3 molecule linked to G4S / flag and CD40L / anti-CD40 scfv; CD19 CAR-T recognized the CD19 molecule; PR20 CAR-T recognized the molecular structure of PRAME (ALYVDSLFFL, amino acids 1-10 of SEQ ID NO:2) presented by HLA-A*0201; and NY-ESO-1 TCR-T recognized the molecular structure of NY-ESO-1 (SLLMWITQC, amino acids 1-9 of SEQ ID NO:3) presented by HLA-A*0201. The results are shown in Figure 3. The double-positive cell population was a population of editing T cells bound to the linker.

[0166] 3. The linker successfully activated target-specific CAR / STAR / TRUC / TCR-T cells in the DC-T system, promoting their expansion: The Attachment Solution Kit (Hycells, Shanghai; CAT:DCT01003-A) was used to promote the adhesion of monocytes from PBMCs to culture dishes. Then, the Dendritic Cells Differentiation Kit (Monocyte-Derived) (Hycells, Shanghai; CAT:DCT01003-kit) was used to induce the adherent monocytes into DCs. The DCs were used approximately 48 hours after being added to maturation medium. The corresponding GPC3CAR / STAR / TRUC-T cells were expanded to the plateau phase. After adjusting the positive rate to approximately 20%, they were co-incubated with DCs at a ratio of 10:1. Experimental groups were set up by adding supernatant from 293T cells of different sources. The culture media were all x-vivo medium containing 10 ng / ml IL-7 and 5 ng / ml IL-15, mixed 1:1 with linker 293 supernatant (or without linker 293 supernatant). After co-incubation for 24 hours, the activation status of CPC3 CAR-T (SEQ ID NO:41), STAR-T (SEQ ID NO:42), and TRuC-T (SEQ ID NO:43) cells was detected. Following the same protocol, the NY-ESO-1TCR-T (SEQ ID NO:46) group was set up to examine the effect of CD40L-flag-HLA-NY-ESO-1 on TCR-T. The results are shown in Figure 4, indicating that only the addition of the corresponding CAR / STAR / TRuC / TCR-T antigens can activate T cells by DCs, and that only the supernatant containing the linker cannot directly activate T cells.

[0167] After 72 hours of co-incubation, the positivity rate and cell population of CAR / STAR / TRuC / TCR-T cells were detected using CD45RA and CCR7 to obtain the fold change in the corresponding gene-edited T cell population. Subtype detection within the CD8+ cell population was performed using CCR7 and CD45RA, and the results are shown in Figure 5. This indicates that with the presence of dendritic cells (DCs) and corresponding linkers, the number of terminally differentiated T cell subtypes that specifically recognize antigens can be reduced, leading to the conversion to effector memory T cell subtypes. Furthermore, this protocol can also utilize a 40G3 (full-legth) (SEQ ID NO:76) linker constructed from the full-length GPC3 sequence to achieve corresponding CAR-T cell amplification and T cell subtyping changes.

[0168] When using the linker for anti-CD40 scfv1 / scfv2, as shown in Figure 6, after 72 hours, an increase in the total number of CAR-T cells and a decrease in the number of terminally differentiated cell subtypes can be observed.

[0169] 4. Function of the linker under co-incubation of different macrophages and GPC3 CAR-T (SEQ ID NO:41) cells: CD14-positive cells in PBMCs were sorted using a CD14 sorting kit and then cultured as follows: 1) M0 cells were cultured in 1640 medium containing 100 ng / mL M-CSF for 8 days; 2) M1 cells were cultured in 1640 medium containing 100 ng / mL M-CSF for 6 days, followed by stimulation with 100 ng / mL LPS and 50 ng / mL IFN-γ for 48 hours to induce differentiation; 3) M2 cells were cultured in 1640 medium containing 100 ng / mL M-CSF for 6 days, followed by stimulation with 50 ng / mL IL-4 and 50 ng / mL IL-13 for 48 hours to induce differentiation. The corresponding macrophage phenotypes were detected using CD80 and CD206. For different types of macrophages, CAR-T cells were added to CD40L-GPC3 (Figure 40G3), control CD40L-MSLN (Figure 40MN), blank control 293 supernatant (Figure 293T), and 1 μg / ml CD40L positive control (Figure 293T40L) and co-incubated for 24 hours. The activation of CD69 and CD137 in T cells was then measured. The results showed that only CAR-T cells were activated under the action of CD40L-GPC3 during co-incubation, and M0, M1, and M2 cells could all activate GPC3 CAR-T. As shown in Figure 7, after 72 hours of co-incubation, the number and type of CAR-T cells were detected. It was found that M0, M1, and M2 cells all increased CAR-T cell proliferation and could reduce the proportion of terminally differentiated cells in CAR-T cells.

[0170] 5. The linker preserves the ability of DCs to act on CAR-T cells, rather than simply providing target cells for killing: To verify that the linker can preserve the effect of DCs on GPC3 CAR-T (SEQ ID NO:41) cells, the project designed co-incubation of GPC3-expressing Huh7 tumor cells, 293CD40-mCherry reporter cells, and DCs with GPC CAR-T cells. The linker CD40L-GPC3 was added to the co-incubation system of 293CD40-mCherry reporter cells and DCs, while GPC3 CAR-T cells cultured alone with CD40L-GPC3 were used as a control group. The culture medium was x-vivo medium containing 10 ng / ml IL-7 and 5 ng / ml IL-15, mixed 1:1 with 293 linker supernatant (or without 293 linker supernatant). After 72 hours, CD8 CAR-T cells were collected for mRNA sequencing. The results are shown in Figure 8. Sequencing results indicated a significant difference in CAR-T activity between the DC linker-treated group and ordinary 293T cells containing only target cells or linker-connected cells (PCA grouping). Differentially expressed genes were also observed, primarily enriched in T cell pathways such as cytokines, chemokines, and JAK-STAT activation. This demonstrates that the linker can enhance CAR-T cell activation by linking with DC cells and CAR-T cells, rather than simply stimulating CAR-T cells through cellular protein expression.

[0171] 6. Linker and DC can reactivate CAR-T cells during the killing process: 1) GPC3 CAR-T cells prepared according to the above method were repeatedly co-incubated with GPC3-expressing target cells Huh-7 until the killing ratio of target cells remained unchanged after 24 hours of co-incubation. Depleted GPC3 CAR-T cells (SEQ ID NO:41) were then sorted to obtain depleted GPC3 CAR-T cells. These depleted CAR-T cells were co-incubated for 3 days with supernatant containing control supernatant, supernatant containing CD40L-GPC3 linker, linkers containing different target sites, and DC cells. GPC3 CAR-T cells were then co-incubated with Huh-7 target cells at a ratio of 1:10, and their long-term killing effect was detected in RTCA. The results are shown in Figure 9. In the group co-incubated with CD40L-GPC3 linker (Figure 40G3) and DC cells, the long-term killing effect of effector CAR-T cells was significantly increased in the later stage. 2) CHO cells overexpressing GPC3 were co-incubated with GPC3 CAR-T cells at a ratio of 30:1. Absolute CHO-GPC3 counts were performed daily, and the GPC3 positivity rate was detected by flow cytometry. The percentage of CD3+CAR+ positive cells was calculated by flow cytometry. DCs were infected with adenovirus overexpressing the CD40L-flag-GPC3 linker. On day 5 of co-incubation, the DCs were added to the co-incubation system (Figure 40G3). A no-treatment group (Figure T), a DC-infected group (Figure DCT), and a DC-infected group with adenovirus expressing MSLN (Figure 40MN) were set as negative controls. The results, shown in Figure 9, indicate that after adding DCs expressing the CD40L-flag-GPC3 linker (Figure 40G3), CHO-GPC3 levels decreased, and the proportion of CAR-T cells in this group increased after adding DCs, indicating that DCs expressing the corresponding linker can activate and expand GPC3 CAR-T cells during the killing process.

[0172] Example 2: GPC3 fragment fusion with APC surface protein to achieve T cell expansion and typing alteration

[0173] 1. Construction of plasmids expressing different linker proteins: GPC3-anti-CD40.Selicrelumab (Roche) (SEQ ID NO:20), GPC3-anti-CD40.ABBV-927 (GILORALIMAB) (SEQ ID NO:19), GPC3-anti-CLEC7A.2M24 (reference patent US20220127366A1) (SEQ ID NO:53), GPC3-anti-CLEC7A.15E2 (reference patent US20220127366A1) (SEQ ID NO:54), GPC3-anti-LILRB4.ATG034 (reference patent WO2023236891A1) (SEQ ID NO:55), GPC3-anti-LILRB4.Hz5A7 (reference patent US20240043533A1) (SEQ ID NO:20). NO:56) was transfected into 293 cells using the PEI system. The supernatant of 293 cells was collected 24 hours later to obtain the supernatant containing the corresponding linker. Using the above protocol, CAR-T cells and DC cells were co-incubated with and without the corresponding linker supernatant (the control was a non-GPC3 antigen linked to the corresponding APC antigen antibody). The results are shown in Figure 11. The detection revealed that CAR-T cells can interact with DC cells under the action of the corresponding linker, achieving specific amplification of CAR-T cells and alteration of CD8+CAR+ memory cell typing.

[0174] Example 3: GPC3 linker combined with other antibodies to achieve T cell expansion and typing alteration.

[0175] 1. As mentioned above, CAR-T cells corresponding to other GPC3 antibodies were constructed and cultured, namely GPC3.H93 CAR-T (refer to patent US20230088461A1, SEQ ID NO:57) and GPC3.9F2 CAR-T (refer to patent US20220056408A1, SEQ ID NO:58). Using the above method, CAR-T cells and DC cells were co-incubated with and without corresponding linker supernatant. The results are shown in Figure 12, indicating that CAR-T cells constructed with different antibody sequences can interact with DC cells under the action of corresponding linkers, achieving specific activation of CAR-T cells and alteration of memory cell typing.

[0176] 2. Linking GPC3 with other scfv antibody systems to achieve GPC3 CAR-T cell targeting for novel killing: GPC3 CAR-T cells were co-incubated with FAP-expressing 3T3 cells at a 1:1 ratio. Different groups were added with GPC3-anti-FAP (SEQ ID NO:67) and DLL3-anti-FAP (control group, SEQ ID NO:68). RTCA was used to detect cell death in 3T3 cells after 24 hours of co-culture. The results, shown in Figure 13, indicate that the GPC3-anti-FAP system successfully directed GPC3 CAR-T cells to kill FAP-expressing cells, demonstrating that linking the GPC3 linker with other antibody systems can achieve killing of novel targets.

[0177] Example 4: Optimization of GPC3 Link Fragment Truncation

[0178] 1. As mentioned above, CAR-T cells corresponding to different GPC3 fragments are constructed. By linking these cells with linkers containing different truncated GPC3 fragments, the selective binding of the linker and CAR-T cells can be further optimized. As shown in Figure 14, HYP7 CAR-T can bind GPC3 fragments 511-550 (SEQ ID NO:71), 521-540 (SEQ ID NO:72), and 521-550 (SEQ ID NO:73); H93 CAR-T can bind GPC3 fragments 511-550, 521-550, and 541-560 (SEQ ID NO:74); 9F2 CAR-T can bind GPC3 fragment 541-560; and GC33 CAR-T can bind GPC3 fragment 541-560.

[0179] Example 5 illustrates the linker construction and binding capability detection for different DLL3 regions.

[0180] 1. Cultivating DLL3-specific T cells: The pCDH-EF1α lentiviral expression plasmid was constructed by synthesizing the sequences of the corresponding DLL3.Juno CAR-T (refer to patent WO2024044779A2, SEQ ID NO:78) and DLL3.T3 CAR-T (refer to patent US20220249563A1, SEQ ID NO:79). The lentiviral system plasmid (pCDH-EF1α lentiviral expression plasmid, PsPAX2, and pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) was transfected into adherent 293T cells in logarithmic growth phase. The cell culture supernatant was harvested 48-72 hours after transfection, concentrated and filtered, and the lentivirus was stored at -80℃ for later use. Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood. T cells were isolated using human CD3 / 28 magnetic beads and transduced with the virus within 72 hours of activation. After 24 hours of transduction, the medium was changed and the cells were cultured until day 14. Cells were collected by centrifugation and resuspended in physiological saline. Flow cytometry was used to identify the G4S molecule in the T cell editing protein to confirm the positive rate of edited T cells. The supernatant containing the linker was co-incubated with the corresponding edited T cells for 0.5 hours, followed by washing with PBS and flow cytometry staining.

[0181] 2. Construct linker plasmids containing different DLL3 regions: Linker C (SEQ ID NO:80) and Linker 277 (SEQ ID NO:81).

[0182] Linker C is constructed by linking sCD40L (SEQ ID NO:6) to the C-end of DLL3-EGF4 (SEQ ID NO:86); Linker277 is constructed by linking sCD40L to the C-end of DLL3 (277) (SEQ ID NO:87).

[0183] After transfecting 293 cells with the above plasmid using the PEI system, the supernatant from the 293 cells was collected 24 hours later to obtain cells containing the corresponding linker. The supernatant containing the linker was co-incubated with the corresponding edited T cells for 0.5 hours, followed by washing the cells with PBS and performing flow cytometry staining. Specific parameters for flow cytometry detection: 3 × 10⁻⁶ cells / cells. 5 Cell counts were determined per well. Anti-CD40L (APC) and anti-EGFR (PE) (CAR-T marker) were added, and after incubation for 30 minutes, the cells were washed with PBS and analyzed by flow cytometry. The results are shown in Figure 16. Different DLL3 fragments showed inconsistent binding abilities to DLL3 CAR-T. Linker C and Linker 277 exhibited excellent binding abilities to DLL3.Juno CAR-T and DLL3.T3 CAR-T.

[0184] 3. Effects of the linker on T cell activation and subtype conversion: Attachment Solution Kit (Hycells, Shanghai; CAT:DCT01003-A) was used to promote the adhesion of monocytes from PBMCs to culture dishes. Then, Dendritic Cells Differentiation Kit (Monocyte-Derived) (Hycells, Shanghai; CAT:DCT01003-kit) was used to induce dendritic cells (DCs). DCs were used approximately 48 hours after being added to maturation medium. DLL3 CAR-T cells were expanded to the plateau phase, and after adjusting the positive rate to approximately 20%, they were co-incubated with DCs at a 10:1 ratio. Experimental groups were set up by adding 293T cell supernatant from different sources. The culture medium was x-vivo medium containing 10 ng / ml IL-7 and 5 ng / ml IL-15, mixed 1:1 with either 293T cell supernatant containing the linker or 293T cell supernatant without the linker (control, ctrl in the figure). After 24 hours of co-incubation, the expression of CD69 and CD137 in DLL3 CAR-T cells was detected to indicate their activation status. The results are shown in Figure 17. The results indicate that only with the addition of the corresponding DLL3 CAR-T binding linkers Linker C (Figure C) and Linker 277 (Figure 277) in the presence of DCs can DCs activate T cells.

[0185] After incubating the CAR-T cells and DCs in the above system with the linker for 72 hours, the proliferation of CAR-T cells and the differentiation of CD8+CAR+ cells were detected by CD45RA and CCR7. The results are shown in Figure 18. It shows that with DCs and corresponding linkers (293 supernatant without linker, ctrl in the figure; 293 supernatant with Linker C linker, C in the figure; 293 supernatant with Linker 277 linker, 277 in the figure), the number of T cells that specifically recognize antigens can be increased, and the number of terminally differentiated cell subtypes can be reduced, turning into effector memory T cell subtypes.

[0186] Example 6: Construction and Binding Ability Detection of Linkers Targeting Different APC Surface Proteins

[0187] 1. Constructing linker plasmids expressing different APC surface proteins: including two linkers each for the DLL3 fragment (as described in Example 7) and anti-LILRB4, and the DLL3 fragment and anti-CLEC7A: Linker ATG034 (SEQ ID NO:82), Linker Hz5A7 (SEQ ID NO:83), Linker 2M24 (SEQ ID NO:84), and Linker 15E2 (SEQ ID NO:85).

[0188] After transfection into 293 cells via the PEI system, the cell supernatant was collected 24 hours later to obtain supernatant containing the corresponding linker. Using the protocol described in Example 7, DLL3.T3 CAR-T cells were co-cultured with DC cells under conditions with and without the corresponding linker supernatant. The results showed that CAR-T cells could interact with DC cells under the action of the corresponding linker, achieving specific activation, amplification, and CD8+CAR+ cell memory cytotyping changes. The results are shown in Figure 19, with GPC3- used to identify control linkers not recognized by DLL3.

[0189] 2. H82 Tumor Cell Line vs. Linker: To further verify that the linker can enhance CAR-T function and alter cell typing by linking APCs to CAR-T, the experiment used the H82 tumor cell line expressing DLL3 to co-culture with DLL3 CAR-T cells. Compared with the DC system co-cultured with the linker, the results showed that H82 promoted DLL3 CAR-T activation but did not significantly affect CAR-T amplification and typing, indicating that the linker is the active agent in promoting CAR-T amplification and typing (Figure 20).

[0190] Example 7: Target-Switchable CAR-T Cell Linker System

[0191] 1. Integration of M909 and M923 Linkers with GC33 CAR-T

[0192] The sequence corresponding to GC33 GPC3 CAR-T (SEQ ID NO:100) was synthesized and constructed into the pCDH-EF1α lentiviral expression plasmid. The resulting plasmid was co-transfected into logarithmically growing adherent 293T cells using a four-plasmid lentiviral system (pCDH-EF1α expression vector, PsPAX2, pMD2.G, and helper plasmid pRev, mixed in a mass ratio of 4:3:2:1). Cell supernatant was collected after 48-72 hours, concentrated, filtered, and lentivirus was obtained and stored at -80℃ for later use. Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood, and T cells were isolated using the EasySep™ Human T Cell Isolation Kit (STEMCELL, #17951). X-vivo (lonza) medium containing 10 ng / mL IL-7 (nearshore protein, GMP-C086), 5 ng / mL IL-15 (nearshore protein, GMP-C016), and ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator (STEMCELL, #10970) antibody was used to activate T cells. After 2-3 days of normal activation, lentiviral transduction was performed. The medium was replaced with medium without the activation antibody, and the cells were amplified to day 14. CAR-T cell positivity was then measured. The supernatant containing the linker was co-incubated with the corresponding edited T cells for 0.5 hours, followed by washing with PBS and flow cytometry staining. Protein plasmids expressing different linkers with the Flag were constructed as follows:

[0193] m909: GPC3-anti-FRβ (SEQ ID NO:96); m923: GPC3-anti-FRβ (SEQ ID NO:97); control: GPC3-anti-FAP (SEQ ID NO:67).

[0194] After transfection into 293 cells using the PEI system, the cell supernatant was collected 24 hours later to obtain the supernatant containing the corresponding linker. The constructed CAR-T cells were co-incubated with the supernatant containing the linker protein particles for 1 hour. The linker was detected by Flag / His flow cytometry, and the CAR-T cells were detected by G4S flow cytometry. It was found that the linker protein in all groups could bind to CAR-T cells (as shown in Figure 22).

[0195] 2. Verification of the lethality of the M909 and M923 linkage

[0196] A K562 cell line overexpressing FRβ was constructed, which highly expressed FRβ but not GPC3. GC33GPC3 CAR-T or 9F2 GPC3 CAR-T (SEQ ID NO:58) prepared according to the above protocol were co-incubated with target cells (K562 cells overexpressing FRβ) under different linker supernatant conditions for 24 hours. The killing effect on target cells was then detected (effective cells:target cells = 1:1). It was found that the linker in the GPC3-anti-FRβ group could successfully convert GPC3-targeted CAR-T to FRβ-targeted CAR-T, thereby achieving killing, with m923 showing the best effect (Figure 23).

[0197] 3. Specific amplification of GPC3 CAR-T under linker-driven operation

[0198] Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors. CD14-positive cells were sorted from the PBMCs using a CD14 sorting kit. Macrophage differentiation medium (RPMI-1640 + 10% FBS) containing 100 ng / mL M-CSF was then prepared. The sorted monocytes were cultured at 1 × 10⁻⁶ cells / mL. 6 Cells / mL were seeded in differentiation medium and cultured at 37℃ and 5% CO2 for 5-7 days to induce differentiation into macrophages. PBMCs and differentiated macrophages were stained with PE anti-human Folate Receptorβ (FRβ) (Biolegend, 391704) antibody to confirm FRβ expression in the corresponding cells (Figure 24). The CD14 positivity rate of PBMCs was confirmed by FITC anti-human CD14 Antibody (Biolegend, 367116). The control group consisted of FITC Mouse IgG2b, κIsotype Ctrl Antibody (Biolegend, 400310) and PE Mouse IgG1, κIsotype Ctrl Antibody (Biolegend, 400112) with the same channel isotype antibodies.

[0199] The GPC3 CAR-T cells were expanded to the plateau phase (day 14 of culture) and cultured with macrophages at a 5:1 ratio. Different linkers (m923 or control GPC3-anti-FAP) were added to the supernatant secreted by 293T cell culture. After three days of co-culture, flow cytometry analysis revealed that CAR-T specific amplification was achieved only in groups where both macrophages and GPC3-anti-FRβ were present, indicating that this specific amplification was mediated by the linker.

[0200] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A target-switching linker, characterized in that, The linker includes a first component and a second component; The first component is any amino acid sequence, which can bind to the TCR of T cells that recognize the corresponding antigen or the cell surface membrane protein expressed by gene-edited T cells; The second component is a domain with targeting properties to cell surface membrane proteins; Preferably, the cell surface membrane proteins expressed by the gene-edited T cells include molecules of CAR, TruC, and STAR; Preferably, the first component is a specific antigen, which specifically binds to T cells that target tumor antigens; Preferably, the specific antigen is derived from a single-chain antibody, a single-chain peptide-MHC, a bacterial antigen, a viral antigen, a cancer-associated antigen, a cancer-specific antigen, or a fragment having an antigenic determinant.

2. The linker according to claim 1, characterized in that, The specific antigen can bind to CAR-T or TCR-T cells that specifically target GPC3, DLL3, HLA-PRAME, HLA-NY-ESO-1, CD19 or MSLN. Preferably, the GPC3 is a protein fragment of GPC3, the protein fragment of GPC3 having an amino acid sequence as shown in SEQ ID NO:1, or having an amino acid sequence that is more than 80% identical to SEQ ID NO:1; Preferably, the GPC3 is a protein fragment of GPC3, the protein fragment of GPC3 having an amino acid sequence as shown in SEQ ID NO:71-74, or having an amino acid sequence that has more than 80% sequence identity with SEQ ID NO:71-74; Preferably, the surface antigen comprises protein fragments from different regions of DLL3, wherein the different regions include DLL3-EGF4 and DLL3(277); Preferably, the amino acid sequence of DLL3-EGF4 is as shown in SEQ ID NO:86, or has more than 80% identity with SEQ ID NO:86; Preferably, the amino acid sequence of DLL3 (277) is as shown in SEQ ID NO:87, or has more than 80% identity with SEQ ID NO:87; Preferably, the HLA-PRAME comprises an amino acid sequence as shown in SEQ ID NO:2, or having at least 80% identity with SEQ ID NO:2; Preferably, the HLA-NY-ESO-1 comprises an amino acid sequence as shown in SEQ ID NO:3, or having at least 80% identity with SEQ ID NO:3; Preferably, CD19 comprises an amino acid sequence as shown in SEQ ID NO:4, or having at least 80% identity with SEQ ID NO:4; Preferably, the MSLN comprises an amino acid sequence as shown in SEQ ID NO:5, or having at least 80% identity with SEQ ID NO:

5.

3. The linker according to claim 1, characterized in that, The domains with cell surface membrane protein targeting capabilities include domains targeting APC cells or macrophages, domains targeting other cells, or single-chain variable fragments of antibodies. Preferably, the domains targeting APC cells are selected from CD40, CLEC7A, LILRB4, and FAP; Preferably, the domain with cell surface membrane protein targeting capability is a CD40 binding domain; Preferably, the CD40 binding domain is derived from the extracellular domain of CD40L, other fragments of CD40L that can bind to CD40, or an antibody against CD40; Preferably, the domain with cell surface membrane protein targeting capability is a domain that targets FRβ protein; Preferably, the single-chain variable fragment of the antibody is selected from the CDR1, CDR2, and CDR3 regions of the light and heavy chains of any of the following antibodies: CD40L, anti-CD40.Selicrelumab, anti-CD40.GILORALIMAB, anti-CLEC7A.2M24, anti-CLEC7A.15E2, anti-LILRB4.ATG034, anti-LILRB4.Hz5A7, or anti-FAP; or, the second component comprises a region having more than 80% amino acid identity with the CDR1, CDR2, and CDR3 regions defined by any of the above antibodies, wherein the CDR regions can be obtained according to any antibody numbering system such as Kabat, Chothia, IMGT, or AHo; Preferably, the CD40L extracellular segment comprises an amino acid sequence such as SEQ ID NO:6 or having at least 80% identity with SEQ ID NO:6; Preferably, the structure of the antibody (GILORALIMAB) that recognizes CD40 comprises an amino acid sequence such as SEQ ID NO:7 or having at least 80% identity with SEQ ID NO:7; Preferably, the structure of the antibody (Selicrelumab) that recognizes CD40 comprises an amino acid sequence such as SEQ ID NO:8 or having at least 80% identity with SEQ ID NO:8; Preferably, the amino acid sequence of anti-CLEC7A.2M24 is as shown in SEQ ID NO:59, or has more than 80% identity with SEQ ID NO:59; Preferably, the amino acid sequence of anti-CLEC7A.15E2 is as shown in SEQ ID NO:60, or has more than 80% identity with SEQ ID NO:60; Preferably, the amino acid sequence of anti-LILRB4.ATG034 is as shown in SEQ ID NO:61, or has more than 80% identity with SEQ ID NO:61; Preferably, the amino acid sequence of the anti-LILRB4.Hz5A7 is as shown in SEQ ID NO:62, or has more than 80% identity with SEQ ID NO:62; Preferably, the amino acid sequence of the anti-FAP is as shown in SEQ ID NO:77, or has more than 80% identity with SEQ ID NO:77; Preferably, the domain of the targeted FRβ protein is anti-FRβ, which has an amino acid sequence as shown in any of SEQ ID NO:94-95, or has an amino acid sequence that is more than 80% identical to any of SEQ ID NO:94-95.

4. The linker according to claim 1, characterized in that, The linker also includes an exomembrane signal peptide; Preferably, the exosome signal peptide is selected from any of the following molecules: CD8a, IL-2, CD33, CD5, HSA, TPA, IgKVIII, GM-CSF, GM-CSFRα, CD3ζ, CD28, HLA; Preferably, the exosome signal peptide is the CD8a exosome signal peptide; Preferably, the amino acid sequence of the exophase signal peptide of CD8a is as shown in SEQ ID NO:11 or has at least 80% identity with SEQ ID NO:

11. Preferably, the exosome signal peptide is an HLA exosome signal peptide; Preferably, the amino acid sequence of the HLA exosome signal peptide is as shown in SEQ ID NO:12 or an amino acid sequence having at least 80% identity with SEQ ID NO:

12.

5. The linker according to claim 1, characterized in that, The first component and the second component are connected via a linker or directly; Preferably, the linker is a flexible linker or a rigid linker; Preferably, the linker has a length of about 3 to 100 amino acid residues; Preferably, the linker includes one or more functional tag sequences, wherein the tags are selected from one or more of FLAG, Myc, and HA; Preferably, the linker has a monomeric structure or a polymeric structure; Preferably, the linker is selected from one or more of peptide linkers, polyethylene glycol linkers, polyamide linkers, carbohydrate derivative linkers, aliphatic linkers, aromatic linkers, and synthetic polymer linkers; More preferably, the linker includes one or more Gly4Ser repeating units, denoted as (Gly-Gly-Gly-Gly-Ser). n , where n is from 1 to 10.

6. The linker according to claim 1, characterized in that, The linker is CD40L-flag-GPC3, which has an amino acid sequence as shown in SEQ ID NO:13, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:

13. Preferably, the linker is CD40L-G4S-GPC3, which has an amino acid sequence as shown in SEQ ID NO:14, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:

14. Preferably, the linker is CD40L-HLA-PRAME, which has an amino acid sequence as shown in SEQ ID NO:15, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:15; Preferably, the linker is CD40L-HLA-NY-ESO-1, which has an amino acid sequence as shown in SEQ ID NO:16, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:

16. Preferably, the linker is CD40L-flag-CD19, which has an amino acid sequence as shown in SEQ ID NO:17, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:17; Preferably, the linker is CD40L-flag-MSLN, which has an amino acid sequence as shown in SEQ ID NO:18, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:18; Preferably, the linker is anti-CD40 scFv1-flag-GPC3, which has an amino acid sequence as shown in SEQ ID NO:19, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:19; Preferably, the linker is anti-CD40 scFv2-flag-GPC3, which has an amino acid sequence as shown in SEQ ID NO:20, or has an amino acid sequence that is more than 80% identical to that in SEQ ID NO:20; Preferably, the linker is GPC3-anti-CLEC7A.2M24, and the amino acid sequence of GPC3-anti-CLEC7A.2M24 is as shown in SEQ ID NO:53, or has more than 80% identity with SEQ ID NO:53; Preferably, the linker is GPC3-anti-CLEC7A.15E2, and the amino acid sequence of GPC3-anti-CLEC7A.15E2 is as shown in SEQ ID NO:54, or has more than 80% identity with SEQ ID NO:54; Preferably, the linker is GPC3-anti-LILRB4.ATG034, and the amino acid sequence of GPC3-anti-LILRB4.ATG034 is as shown in SEQ ID NO:55, or has more than 80% identity with SEQ ID NO:55; Preferably, the linker is GPC3-anti-LILRB4.Hz5A7, and the amino acid sequence of GPC3-anti-LILRB4.Hz5A7 is as shown in SEQ ID NO:56, or has more than 80% identity with SEQ ID NO:56; Preferably, the linker is GPC3-anti-FAP, and the amino acid sequence of GPC3-anti-FAP is as shown in SEQ ID NO:67, or has more than 80% identity with SEQ ID NO:67; Preferably, the linker is DLL3-anti-FAP, and the amino acid sequence of DLL3-anti-FAP is as shown in SEQ ID NO:68, or has more than 80% identity with SEQ ID NO:68; Preferably, the linker is Linker C, and the amino acid sequence of Linker C is as shown in SEQ ID NO:80, or has more than 80% identity with SEQ ID NO:80; Preferably, the linker is Linker 277, and the amino acid sequence of Linker 277 is as shown in SEQ ID NO:81, or has more than 80% identity with SEQ ID NO:81; Preferably, the linker is Linker ATG034, and the amino acid sequence of Linker ATG034 is as shown in SEQ ID NO:82, or has more than 80% identity with SEQ ID NO:82; Preferably, the linker is Linker Hz5A7, and the amino acid sequence of Linker Hz5A7 is as shown in SEQ ID NO:83, or has more than 80% identity with SEQ ID NO:83; Preferably, the linker is Linker 2M24, and the amino acid sequence of Linker 2M24 is as shown in SEQ ID NO:84, or has more than 80% identity with SEQ ID NO:84; Preferably, the linker is Linker 15E2, and the amino acid sequence of Linker 15E2 is as shown in SEQ ID NO:85, or has more than 80% identity with SEQ ID NO:

85. Preferably, the linker is GPC3-anti-FRβ, which has an amino acid sequence as shown in SEQ ID NO:96, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:96; Preferably, the linker is GPC3-anti-FRβ, which has an amino acid sequence as shown in SEQ ID NO:97, or has an amino acid sequence that is more than 80% identical to SEQ ID NO:

97.

7. An immunotherapy composition comprising: The linker according to any one of claims 1-6, and a combination of at least one of the following: (a) A domain that extends the in vivo half-life, wherein the domain that extends the in vivo half-life is selected from one or more of the following: Fc fragments, albumin-binding molecules, PEG-modified compounds, high molecular weight peptides, XTEN peptide chains, PAS sequences, or analogues thereof; (b) at least one immunomodulatory component, said immunomodulatory component being selected from cytokines, chemokines, immune adjuvants or their functional analogs; Preferably, the cytokines are selected from one or more of interleukins, interferons, tumor necrosis factors, colony-stimulating factors, and transforming growth factors; Preferably, the chemokine is selected from one or more of the CCL family, CXCL family, XCL family, CX3CL family and their functional analogs; Preferably, the immune adjuvant or immune agonist is selected from one or more of the following: TLR agonists, STING agonists, RIG-I agonists, cGAMP, Poly(I:C), CpG oligonucleotides, QS-21, MF59, ASO3, and aluminum adjuvants.

8. A biomaterial, characterized in that, The biomaterial has any one of the following characteristics: (1) A nucleic acid molecule comprising a nucleic acid molecule encoding the linker of any one of claims 1-6, which can be used to express the linker of any one of claims 1-6; Preferably, the linker expressing any one of claims 1-6 is present in the form of a polypeptide, protein, fusion protein, or other gene expression product; Preferably, the nucleic acid molecule is selected from DNA, RNA, or a modified form thereof; Preferably, the RNA is selected from linear mRNA, circular RNA, self-amplifying RNA, or modified forms thereof; (2) A recombinant vector comprising the nucleic acid molecules described in (1); Preferably, the recombinant vector is used to express the linker according to any one of claims 1-6, and is selected from one or more of DNA vectors, viral vectors, and mRNA vectors; (3) A recombinant host cell comprising the nucleic acid molecule described in (1) and / or the recombinant vector described in (2).

9. A delivery system, characterized in that, The delivery system is used to deliver the nucleic acid molecule of claim 8 and / or the linker of any one of claims 1-6 in the form of a polypeptide, protein, fusion protein or other gene expression product to a target tissue or cell in a mammal to express the linker of any one of claims 1-6. Preferably, the delivery carrier is selected from one or more of liposomes, lipid nanoparticles, polymer nanoparticles, exosomes, viruses, virus-like particles, engineered microorganisms or their spores, cells, inorganic nanoparticles, hydrogels, emulsions, polymer micelles, and biomimetic membrane-encapsulated nanoparticles. Preferably, the lipid nanoparticles comprise one or more of ionized lipids, cofactor lipids, cholesterol, and PEG-modified lipids; Preferably, the virus is an oncolytic virus, which is selected from one or more of adenovirus, adeno-associated virus, herpes simplex virus, varicella-zoster virus, vaccinia virus / vaccinia virus, measles virus, Newcastle disease virus, reovirus, poliovirus, and samba virus. Preferably, the cells are selected from T cells, natural killer cells, dendritic cells, macrophages, monocytes, erythrocytes, mesenchymal stem cells, stromal cells, and tumor cells.

10. The application of the linker according to any one of claims 1-6, the immunotherapy composition according to claim 7, the biomaterial according to claim 8, and / or the delivery system according to claim 9, characterized in that, The application includes any of the following: 1) Application in the preparation of products that enhance the activation, expansion and / or reduce and reverse the depletion ability of specific T cells; 2) Application in the preparation of products that promote changes in T cell subtypes; 3) Application in the preparation of drugs for treating tumors; Preferably, the specific T cells are selected from T cells, CAR-T cells, STAR-T cells, TruC-T cells, and TCR-T cells; Preferably, the specific T cells are CAR-T cells that target GPC3, DLL3, HLA-PRAME, HLA-NY-ESO-1, CD19, or MSLN; Preferably, the GPC3-targeting CAR-T cells can be constructed from any of the following CARs: GPC3 CAR, 9F2 GPC3 CAR, or GC33 GPC3 CAR, or their corresponding scFv sequences; the scFv sequence contains CDR1, CDR2, and CDR3 of the light and heavy chains defined by any CAR or its corresponding scFv sequence, or regions that have more than 80% amino acid identity with CDR1, CDR2, and CDR3, and the CDR regions can be determined according to any of the antibody numbering systems Kabat, Chothia, IMGT, or Aho; Preferably, the CAR-T cells targeting DLL3 can be constructed from any of the following CARs: DLL3.Juno CAR, DLL3.T3CAR, or their corresponding scFv sequences; the scFv sequence contains CDR1, CDR2, and CDR3 of the light and heavy chains defined by any CAR or its corresponding scFv sequence, or regions that have more than 80% amino acid identity with CDR1, CDR2, and CDR3, and the CDR regions can be determined according to any of the antibody numbering systems Kabat, Chothia, IMGT, or Aho; Preferably, the promotion of T cell subtype alteration refers to promoting the alteration of CAR-T cell subtypes targeting GPC3, DLL3, HLA-PRAME, HLA-NY-ESO-1, CD19, or MSLN; Preferably, the subtype change refers to the transformation from a terminally differentiated cell subtype to an effector memory T cell subtype; Preferably, the tumor includes tumors expressing GPC3, DLL3, HLA-PRAME, HLA-NY-ESO-1, CD19, or MSLN; Preferably, the tumors expressing GPC3 include liver cancer, hepatocellular carcinoma, ovarian clear cell carcinoma, yolk sac tumor, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma, and chronic myeloid leukemia. Preferably, the tumor includes a tumor expressing DLL3; Preferably, the tumors expressing DLL3 include small cell lung cancer, neuroendocrine tumors, and neuroendocrine carcinomas; Preferably, the product includes a reagent kit, test strip, nucleic acid membrane strip, chip, system, or device; Preferably, the drug can be used alone or in the form of a pharmaceutical composition; Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

11. A method, characterized in that, The method includes any one of the following: 1) A method for promoting the binding of specific T cells and downstream cells, the method comprising the step of co-incubating the linker according to any one of claims 1-6 with specific T cells and downstream cells; 2) A method for enhancing the specific activation, expansion, and / or reduction and reversal of exhaustion of T cells, the method comprising the steps of co-incubating the linker according to any one of claims 1-6 with specific T cells and downstream cells; 3) A method for treating a tumor, the method comprising administering to a patient the linker of any one of claims 1-6, the linker being present in the form of a polypeptide, protein, fusion protein or other gene expression product; or administering to a patient the linker of any one of claims 1-6, the immunotherapy composition of claim 7, the biomaterial of claim 8 and / or the drug prepared by the delivery system of claim 9; Preferably, the tumor includes tumors expressing GPC3, DLL3, HLA-PRAME, HLA-NY-ESO-1, CD19, or MSLN; Preferably, the tumors expressing GPC3 include liver cancer, hepatocellular carcinoma, ovarian clear cell carcinoma, yolk sac tumor, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma, and chronic myeloid leukemia. Preferably, the tumor includes a tumor expressing DLL3; Preferably, the tumors expressing DLL3 include small cell lung cancer, neuroendocrine tumors, and neuroendocrine carcinomas; preferably, the downstream cells are APC cells; Preferably, the specific T cells are selected from T cells, CAR-T cells, STAR-T cells, TruC-T cells, and TCR-T cells.

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