Target-switchable CAR-T cell linker system and application thereof

Abstract

The invention discloses a target-switchable CAR-T cell linker system and application thereof, belongs to the field of biological medicine, and particularly relates to a target-switchable linker system for improving activation and amplification of specific T cells. The linker comprises a domain capable of binding to an antigen-specific T cell (such as CAR-T), and a targeting domain capable of recognizing a cell surface membrane protein (such as FRbeta). By means of the linker, CAR-T cells can be switched to be in a targeted FRbeta functional state, so that the T cells can recognize and kill myeloid cells or macrophages expressing FRbeta, and activation, amplification and function enhancement of the antigen-specific T cells are achieved. According to the invention, the continuous activation capability and the anti-tumor effect of the CAR-T cells in a tumor microenvironment can be obviously improved, the exhaustion is reduced, and the treatment safety is enhanced.

Description

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 could potentially provide a novel 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, the linker comprising a first component and a second component: the first component is an arbitrary amino acid sequence capable of binding to a TCR of a T cell that recognizes a corresponding antigen or a cell surface membrane protein expressed by a gene-edited T cell; the second component is a domain having cell surface membrane protein targeting capability.

[0007] In some embodiments, the cell surface membrane proteins expressed by the gene-edited T cells include molecules such as 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), for specifically recognizing 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 fusion protein 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 specified in this specification, and should be considered to be covered within the scope of protection of this invention.

[0012] In some embodiments, 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 some embodiments, the specific antigen binds to CAR-T cells that specifically target GPC3.

[0015] 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.

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

[0017] The term "sequence identity" is used interchangeably with "identity" 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 algorithm required to achieve optimal alignment across the compared full-length sequences. In this invention, sequences modified to have 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence 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 a given amino acid sequence with a different amino acid. The term "deletion" refers to reducing one or more amino acids in a given amino acid sequence. The terms "insertion" or "addition" refer to changes in the amino acid sequence that result in the addition of one or more amino acids compared to the original amino acid sequence.

[0018] In some embodiments, the domains with cell surface membrane protein targeting capabilities include domains targeting APC cells or macrophages, and domains targeting other cells.

[0019] In this invention, APCs (Antigen-presenting cells), also known as antigen-presenting cells, refer to a type of immune cell capable of taking up, processing, and presenting the processed 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. Non-professional APCs include certain endothelial cells and epithelial-mesothelial cells, such as fibroblasts, glial cells, thymus, thyroid epithelial cells, vascular endothelial cells, and eosinophils.

[0020] Furthermore, the domain with cell surface membrane protein targeting capability is a domain that targets FRβ protein.

[0021] 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.

[0022] Furthermore, the domain of the target FRβ protein is anti-FRβ, which has an amino acid sequence as shown in any of SEQ ID NO:2-3, or has an amino acid sequence that is more than 80% identical to any of SEQ ID NO:2-3.

[0023] Furthermore, the linker also includes an exomembrane signal peptide.

[0024] In some embodiments, the exophase signal peptide is selected from any of the following molecules: CD8a, IL-2, CD33, CD5, HSA, TPA, IgKVIII, GM-CSF, GM-CSFRα, CD3ζ, CD28.

[0025] In some embodiments, the exophase signal peptide is the CD8a exophase signal peptide.

[0026] In some embodiments, the amino acid sequence of the exophase signal peptide of CD8a is shown in SEQ ID NO:4.

[0027] In some embodiments, the first component and the second component are connected via a linker or directly.

[0028] In some implementations, the linker, also known 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). nWhitlow / 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.

[0029] Furthermore, the linker can be a flexible linker or a rigid linker.

[0030] Furthermore, the linker is approximately 3 to 100 amino acid residues in length.

[0031] In some embodiments, the linker includes one or more functional tag sequences, the tags being selected from one or more of FLAG, Myc, and HA.

[0032] In some embodiments, the linker is a monomeric structure or a polymeric structure.

[0033] Furthermore, the linker is selected from one or more of the following: peptide linker, polyethylene glycol linker, polyamide linker, carbohydrate derivative linker, aliphatic linker, aromatic linker, and synthetic polymer linker.

[0034] Furthermore, the linker contains one or more Gly4Ser repeating units, denoted as (Gly-Gly-Gly-Gly-Ser). n , where n is from 1 to 10.

[0035] Furthermore, the linker is (Gly-Gly-Gly-Gly-Ser)2.

[0036] Furthermore, the linker is GPC3-anti-FRβ, which is composed of the exosome signal peptide as described above, the protein fragment of GPC3 as described above, and the anti-FRβ as described above.

[0037] Furthermore, the GPC3-anti-FRβ is composed of the exosome signal peptide as described above, the protein fragment of GPC3 as described above, the linker as described above, the anti-FRβ as described above, and the flag tag sequentially linked together.

[0038] In some embodiments, the GPC3-anti-FRβ has an amino acid sequence as shown in SEQ ID NO:5, or has an amino acid sequence that is more than 80% identical to that in SEQ ID NO:5.

[0039] In some embodiments, the GPC3-anti-FRβ has an amino acid sequence as shown in SEQ ID NO:6, or has an amino acid sequence that is more than 80% identical to that of SEQ ID NO:6.

[0040] 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.

[0041] 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.

[0042] In some embodiments, the cytokines are selected from one or more of interleukins, interferons, tumor necrosis factors, colony-stimulating factors, and transforming growth factors.

[0043] 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.

[0044] 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.

[0045] A third aspect of the present invention provides a biomaterial having any one of the following characteristics:

[0046] (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.

[0047] 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.

[0048] In some embodiments, the nucleic acid molecule is selected from DNA, RNA, or a modified form thereof.

[0049] In some embodiments, the RNA is selected from linear mRNA, circular RNA, self-amplifying RNA, or modified forms thereof.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] In some embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding GPC3-anti-FRβ, the nucleic acid sequence encoding GPC3-anti-FRβ having a nucleotide sequence as shown in SEQ ID NO:7, or having a nucleotide sequence having more than 80% sequence identity with SEQ ID NO:7.

[0054] In some embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding GPC3-anti-FRβ, the nucleic acid sequence encoding GPC3-anti-FRβ having a nucleotide sequence as shown in SEQ ID NO:8, or having a nucleotide sequence having more than 80% sequence identity with SEQ ID NO:8.

[0055] (2) A recombinant vector containing the nucleic acid molecules described in (1).

[0056] 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.

[0057] 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.

[0058] (3) A recombinant host cell comprising the nucleic acid molecule described in (1) and / or the recombinant vector described in (2).

[0059] 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.

[0060] 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.

[0061] 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.

[0062] In some embodiments, the lipid nanoparticles comprise one or more of ionized lipids, cofactor lipids, cholesterol, and PEG-modified lipids.

[0063] 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.

[0064] 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.

[0065] 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:

[0066] 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.

[0067] 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.

[0068] In some embodiments, the specific T cells are CAR-T cells that target GPC3.

[0069] In some embodiments, the GPC3-targeting CAR-T cells can be constructed from any of the following CARs: GPC3CAR, 9F2 GPC3 CAR, or GC33 GPC3 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.

[0070] In this invention, GPC3 CAR refers to patent CN2025108024271 (SEQ ID NO:12), 9F2 GPC3 CAR refers to patent US20220056408A1 (SEQ ID NO:11), and GC33 GPC3 CAR refers to patent US7919086B2 (SEQ ID NO:10).

[0071] In some embodiments, promoting T cell subtype alteration refers to promoting GPC3-targeted CAR-T cell subtype alteration.

[0072] In some embodiments, the subtype change refers to a shift from a terminally differentiated cell subtype to an effector memory T cell subtype.

[0073] Furthermore, the tumor includes tumors expressing GPC3.

[0074] 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.

[0075] In some embodiments, the product includes a reagent kit, test strip, nucleic acid membrane strip, chip, system, or device.

[0076] In some embodiments, the drug may be used alone or in the form of a pharmaceutical composition.

[0077] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] A sixth aspect of the present invention provides a method comprising any one of the following:

[0083] 1) A method for promoting the binding of specific T cells and downstream cells in vitro, the method comprising the steps of co-incubating the linker described in the first aspect with specific T cells and downstream cells.

[0084] 2) A method for enhancing, expanding and / or reducing, reversing the depletion capacity of T cells in vitro, the method comprising the steps of co-incubating the linker described in the first aspect with specific T cells and downstream cells.

[0085] 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.

[0086] Furthermore, the tumor includes tumors expressing GPC3.

[0087] 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.

[0088] In some embodiments, the downstream cells are APC cells.

[0089] 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.

[0090] 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.

[0091] Advantages and benefits of the present invention: The present invention provides a novel switchable target CAR-T linker system that can convert CAR-T cells originally targeting GPC3 into a functional state that recognizes FRβ-positive myeloid cells through a specific linker, thereby achieving secondary activation and expansion of T cells in the tumor microenvironment, enhancing the anti-tumor effect and improving the durability and safety of treatment. Attached Figure Description

[0092] Figure 1 Flowcharts of m909 and m923 linkers combined with GC33 CAR-T.

[0093] Figure 2 Statistical chart showing the killing power of m909 and m923 on target cells.

[0094] Figure 3 Figure 1 shows the flow cytometry results of FRβ expression in PBMCs and differentiated macrophages. Figure A shows the flow cytometry results of PBMCs and a negative control, and Figure B shows the flow cytometry results of differentiated macrophages and a negative control.

[0095] Figure 4 Figure showing the results of flow cytometry detection of CAR-T specific amplification using different linkers. Detailed Implementation

[0096] 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.

[0097] 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.

[0098] Example

[0099] 1. Integration of M909 and M923 Linkers with GC33 CAR-T

[0100] The sequence corresponding to GC33 GPC3 CAR-T (SEQ ID NO:10) 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 lacking 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:

[0101] m909: GPC3-anti-FRβ (SEQ ID NO:5).

[0102] m923: GPC3-anti-FRβ (SEQ ID NO:6).

[0103] Control: GPC3-anti-FAP (SEQ ID NO:9).

[0104] 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 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 (e.g., ...). Figure 1 (As shown).

[0105] 2. Verification of the lethality of the M909 and M923 linkage

[0106] A K562 cell line overexpressing FRβ was constructed, which highly expressed FRβ but not GPC3. GC33 GPC3 CAR-T or 9F2 GPC3 CAR-T (SEQ ID NO:11) 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 assessed (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. Among these, m923 showed the best effect. Figure 2 ).

[0107] 3. Specific amplification of GPC3 CAR-T under linker-driven operation

[0108] 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 PBMCs were seeded at a density of [number] cells / mL in differentiation medium and cultured at 37°C and 5% CO2 for 5-7 days to induce differentiation into macrophages. PBMCs and differentiated macrophages were then stained with PE anti-human Folate Receptor β (FRβ) antibody (Biolegend, 391704) to confirm FRβ expression in the corresponding cells. Figure 3The CD14 positivity rate of PBMCs was confirmed by FITC anti-human CD14 Antibody (Biolegend, 367116), and the control group consisted of FITC Mouse IgG2b,κ Isotype Ctrl Antibody (Biolegend, 400310) and PE Mouse IgG1,κ Isotype Ctrl Antibody (Biolegend, 400112) with the same channel isotype antibody group.

[0109] The GPC3 CAR-T cells were expanded to the plateau phase (day 14 of culture) and cultured with macrophages at a ratio of 5:1. 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.

[0110] Table 1. Sequence List

[0111]

[0112]

[0113]

[0114]

[0115] 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 binds to CAR-T cells that specifically target GPC3; Preferably, the GPC3 is a protein fragment of GPC3, and the protein fragment of GPC3 has an amino acid sequence as shown in SEQ ID NO:1, or has an amino acid sequence that is more than 80% identical to that of SEQ ID NO:

1. Preferably, the domains with cell surface membrane protein targeting capabilities include domains targeting APC cells or macrophages, and domains targeting other cells; Preferably, the domain with cell surface membrane protein targeting capability is a domain that targets FRβ protein; 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:2-3, or has an amino acid sequence that is more than 80% identical to any of SEQ ID NO:2-3.

2. 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; Preferably, the exosome signal peptide is the CD8a exosome signal peptide; Preferably, the amino acid sequence of the exophase signal peptide of CD8a is shown in SEQ ID NO:

4.

3. 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 the following: peptide linker, polyethylene glycol linker, polyamide linker, carbohydrate derivative linker, aliphatic linker, aromatic linker, and synthetic polymer linker; Preferably, the linker contains one or more Gly4Ser repeating units, denoted as (Gly-Gly-Gly-Gly-Ser). n , where n is from 1 to 10; Preferably, the linker is (Gly-Gly-Gly-Gly-Ser)2.

4. The linker according to claim 1, characterized in that, The linker is GPC3-anti-FRβ, which is composed of the exosome signal peptide of claim 2, the protein fragment of GPC3 of claim 1, and the anti-FRβ of claim 1. Preferably, the GPC3-anti-FRβ is the membrane exit signal peptide of claim 2, the protein fragment of GPC3 of claim 1, the linker of claim 3, the anti-FRβ of claim 1, and the flag tag sequentially linked together; Preferably, the GPC3-anti-FRβ has an amino acid sequence as shown in SEQ ID NO:5, or has an amino acid sequence that is more than 80% identical to that in SEQ ID NO:5; Preferably, the GPC3-anti-FRβ has an amino acid sequence as shown in SEQ ID NO:6, or has an amino acid sequence that is more than 80% identical to that in SEQ ID NO:

6.

5. An immunotherapy composition comprising: The linker according to any one of claims 1-4, 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.

6. 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 1-4, which can be used to express the linker of any one of claims 1-4; Preferably, the linker expressing any one of claims 1-4 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; Preferably, the nucleic acid molecule includes a nucleic acid sequence encoding GPC3-anti-FRβ, the nucleic acid sequence encoding GPC3-anti-FRβ having a nucleotide sequence as shown in SEQ ID NO:7, or having a nucleotide sequence having more than 80% sequence identity with SEQ ID NO:7; Preferably, the nucleic acid molecule includes a nucleic acid sequence encoding GPC3-anti-FRβ, the nucleic acid sequence encoding GPC3-anti-FRβ having a nucleotide sequence as shown in SEQ ID NO:8, or having a nucleotide sequence having more than 80% sequence identity with SEQ ID NO:8; (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 1-4, 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).

7. A delivery system, characterized in that, The delivery system is used to deliver the nucleic acid molecule of claim 6 and / or the linker of any one of claims 1-4, which is 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 1-4. 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.

8. The application of the linker according to any one of claims 1-4, the immunotherapy composition according to claim 5, the biomaterial according to claim 6, and / or the delivery system according to claim 7, 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 targeting GPC3; Preferably, the GPC3-targeting CAR-T cells can be constructed from any of the following CARs: GPC3 CAR, 9F2 GPC3CAR, 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 sequence 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; Preferably, the subtype change refers to the transformation from a terminally differentiated cell subtype to an effector memory T cell subtype.

9. The application according to claim 8, characterized in that, The tumors include those expressing GPC3; 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 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.

10. 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 in vitro, the method comprising the steps of co-incubating the linker according to any one of claims 1-4 with specific T cells and downstream cells; 2) A method for enhancing, expanding and / or reducing and reversing the depletion ability of T cells in vitro, the method comprising the steps of co-incubating the linker according to any one of claims 1-4 with specific T cells and downstream cells; 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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