Genetically modified immune cell, and construction and use thereof

Abstract

The present application relates to the technical field of biomedicine, and in particular to a genetically modified immune cell, and construction and use thereof. The genetically modified immune cell expresses a target protein, the target protein comprises a chimeric antigen receptor and a membrane-bound interleukin, the chimeric antigen receptor comprises an antigen-binding domain that specifically binds to GPC3, and the membrane-bound interleukin comprises a membrane-bound IL-7.

Description

Genetically Modified Immune Cells, Their Construction and Application Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a genetically modified immune cell and its construction and application. Background Technology

[0002] Tumor-infiltrating lymphocytes (TILs) are composed of CD3+ cells. + A heterogeneous cell population composed of αβT cells, B cells, NK cells, γδT cells, and innate lymphocytes (ILCs). After in vitro expansion and culture, CD3... + T cells became the dominant cell population (accounting for more than 90%). CD3 + T cells express TCR receptors on their surface, playing a crucial role in mediating the immune killing of cancerous cells. Different TCR receptors recognize different antigenic peptides presented by the Major Histocompatibility Complex (MHC), thus conferring antigen specificity to T cells. Therefore, once T cells specific to a particular antigen are stimulated by that antigen, these cells can promote programmed cell death of the target cells through mechanisms such as the release of cytotoxins and receptor-mediated mechanisms.

[0003] Unlike CAR-T (Chimeric Antigen Receptor T-Cell) and TCR-T cell therapy products, TILs typically exert their anti-tumor effects by recognizing tumor-associated antigens, such as neoantigens, presented by tumor cells. Therefore, TIL therapy can achieve its anti-tumor therapeutic goals by recognizing and targeting multiple tumor-associated antigens.

[0004] However, due to the regulatory effects of various immunosuppressive factors in the tumor microenvironment, tumor-associated lymphoid tissue (TILs) highly express immune checkpoint-related molecules such as PD-1, CTLA4, TIM3, LAG3, and TIGIT. Simultaneously, due to continuous stimulation by tumor-associated antigens, TILs exhibit a highly depleted state, as evidenced by high expression of depletion-related transcription factors such as Tox and Id3. Even after TIL isolation and in vitro reactivation, their cytotoxic function remains limited. Therefore, enhancing the specificity and sustained anti-tumor function of TILs is a pressing technical challenge. Summary of the Invention

[0005] Based on this, one or more embodiments of this application provide a genetically modified immune cell and its construction and application, including the following technical solutions:

[0006] One or more embodiments of this application provide a genetically modified immune cell that expresses a target protein, the target protein including a chimeric antigen receptor and a membrane-bound interleukin.

[0007] The chimeric antigen receptor includes an antigen-binding domain that specifically binds to GPC3.

[0008] The membrane-bound interleukins include membrane-bound IL-7.

[0009] One or more embodiments of this application also provide a nucleic acid molecule that encodes the target protein defined above.

[0010] One or more embodiments of this application provide a carrier comprising the nucleic acid molecule described above.

[0011] One or more embodiments of this application further provide a method for constructing the genetically modified immune cells, the method comprising introducing the nucleic acid molecule or the vector into the immune cells to be modified to achieve the expression of the target protein.

[0012] One or more embodiments of this application further provide the use of the genetically modified immune cells, the nucleic acid molecules, or the vectors in the preparation of antitumor drugs.

[0013] One or more embodiments of this application further provide an anti-tumor drug, said drug comprising the genetically modified immune cells and a pharmaceutically acceptable carrier.

[0014] One or more embodiments of this application further provide a method for treating tumors, the method comprising administering an effective dose of the antitumor drug to a subject.

[0015] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0016] Figure 1 shows the molecular structures of GPC3-specific CAR and membrane-bound human IL-7 expressed in tandem (structural code: BN108-6P4);

[0017] Figure 2 shows the kill specificity verification of GPC3 CAR-TIL;

[0018] Figure 3 shows the validation of co-expression factors that enhance GPC3 CAR TIL killing;

[0019] Figure 4 shows the optimized molecular structure of GPC3 CAR;

[0020] Figure 5 shows the structural optimization of membrane-bound IL-7 for enhancing the killing function of TIL;

[0021] Figure 6 shows the structural optimization of GPC3 CAR tandem membrane-bonded IL-7;

[0022] Figure 7 shows the in vitro efficacy data of BN108-6P4;

[0023] Figure 8 shows the efficacy data of BN108-6P4 in Hep3B tumor-bearing mice. Detailed Implementation

[0024] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0025] A first aspect of this application provides a genetically modified immune cell that expresses a target protein, the target protein including a chimeric antigen receptor and a membrane-bound interleukin.

[0026] The chimeric antigen receptor includes an antigen-binding domain that specifically binds to GPC3.

[0027] The membrane-bound interleukin includes membrane-bound IL-7. This application enhances the tumor-killing ability of TILs by simultaneously overexpressing GPC3 CAR and membrane-bound IL-7, while also improving the proliferation capacity and sustained tumor-killing ability of immune cells in vivo and in vitro, which is more conducive to improving the therapeutic effect of immune response on GPC3-positive tumors such as hepatocellular carcinoma.

[0028] This application does not specifically limit IL-7, which can be as shown in SEQ ID NO.6 568aa-719aa, or a conserved mutant of that fragment.

[0029] A "conservative mutant" refers to a mutant modified based on IL-7 as shown in SEQ ID NO. 6, 568aa-719aa, such that the activity or function (including affinity for the receptor) of the resulting mutant is consistent with that of IL-7 as shown in SEQ ID NO. 6, 568aa-719aa. Engineered immune cells possessing a "conservative mutant" are biologically consistent with engineered immune cells possessing IL-7 as shown in SEQ ID NO. 6, 568aa-719aa.

[0030] Compared to IL-7 as shown in SEQ ID NO. 6 (568aa-719aa), the IL-7 mutant has up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids replaced by amino acids of similar or analogous properties. These mutant peptides are preferably generated by amino acid substitutions according to the table below.

[0031] Table A

[0032] For example, mutants of IL-7 are prepared by substituting, deleting, or adding at least one amino acid in IL-7 as shown in SEQ ID NO. 6 568aa-719aa without altering the function of IL-7, or conserved mutants of IL-7 are prepared by substituting, deleting, or adding at least one amino acid in IL-7 without altering the function of IL-7. These conserved mutants have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with their corresponding wild-type IL-7.

[0033] Traditionally, conserved mutants, relative to wild-type subunits, may contain some conserved amino acid substitutions. These substitutions have little or no effect on the overall protein charge, i.e., polarity or hydrophobicity. The following table lists conserved amino acid substitutions.

[0034] Table B

[0035] For each amino acid, additional conserved substitutions include the amino acid's "homolog." Specifically, a "homolog" is an amino acid obtained by inserting a methylene (CH2) group into the β-side chain of the amino acid's side chain. Examples of "homologs" may include, but are not limited to, homophenylalanine, homoarginine, homoserine, etc.

[0036] This application does not specifically limit the species source of IL-7, which can be mammals (humans, mice, rats, guinea pigs, rabbits, macaques, goats, sheep, horses, cattle, pigs, dogs, etc.) or poultry such as chickens, geese, and ducks. In some embodiments, IL-7 is derived from mammals. Preferably, IL-7 is derived from humans.

[0037] This application does not impose particular limitations on the expression methods of chimeric antigen receptors and membrane-bound interleukins in genetically modified immune cells, including but not limited to: CAR and membrane-bound IL-7 expressed using independent vectors, through one or more combinations of viral vectors, gene knock-in, transposon insertion, plasmid transfection, or mRNA transfection; CAR and membrane-bound IL-7 expressed using independent vectors via dual promoters or IRES; or CAR and membrane-bound IL-7 expressed using the same vector. In some embodiments of this application, the genetically modified immune cells include a fusion gene expression cassette containing the coding gene for the chimeric antigen receptor and the coding gene for the membrane-bound IL-7.

[0038] In some embodiments of this application, the genetically modified immune cells include tandemly expressed genes encoding the chimeric antigen receptor and the membrane-bound IL-7. Optionally, the genes encoding the chimeric antigen receptor and the membrane-bound IL-7 are expressed by a polycistronic expression; alternatively, the genes encoding the chimeric antigen receptor and the membrane-bound IL-7 are each expressed by two monocistronic expressions.

[0039] In some embodiments of this application, the gene encoding the chimeric antigen receptor and the gene encoding the membrane-bound IL-7 are linked together by a linker fragment to form a polycistronic peptide. Optionally, the linker peptide encoded by the linker fragment includes, but is not limited to, a self-cleaving peptide. In this application, the self-cleaving peptide includes, but is not limited to, P2A, T2A, E2A, and F2A, and may be one of them or a combination of several. In some embodiments of this application, P2A and T2A are selected, and further, P2A and T2A are selected to be sequentially linked; it is understood that this application is not limited to this. In some embodiments of this application, the linker fragment includes an internal ribosome entry site. The sequence of the internal ribosome entry site includes, but is not limited to, SEQ ID NO. 17 as shown below:

[0040] This application does not impose any particular limitation on the relative positions of the coding gene for the chimeric antigen receptor and the coding gene for the membrane-bound IL-7. For example, the coding gene for the chimeric antigen receptor and the coding gene for the membrane-bound IL-7 may be located at the 5' end and the 3' end, respectively; or the coding gene for the chimeric antigen receptor and the coding gene for the membrane-bound IL-7 may be located at the 3' end and the 5' end, respectively. In some embodiments of this application, the coding gene for the chimeric antigen receptor is located at the 5' end of the coding gene for the membrane-bound IL-7.

[0041] This application does not specifically limit the structure of the chimeric antigen receptor, including but not limited to the chimeric antigen receptor having the structure shown in formula (Ⅰ):

[0042] SP1-antigen-binding domain-hinge region-transmembrane region-intracellular signal transduction region (Ⅰ),

[0043] In the formula, - represents a peptide bond or a linking peptide, and SP1 represents a peptide without a signal or a signal peptide.

[0044] This application does not specifically limit the signal peptide in the chimeric antigen receptor. Optionally, the signal peptide includes the signal peptide of at least one of the following proteins: human CD8, human IgG, human GM-CSF, human IgE, human CD4, human CD28, and human CD137;

[0045] This application does not specifically limit the hinge region in the chimeric antigen receptor. Optionally, the hinge region includes all or part of the extracellular region of at least one of the following proteins: CD8, IgG1, IgG4, IgG4m, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154.

[0046] This application does not specifically limit the transmembrane region in the chimeric antigen receptor. Optionally, the transmembrane region includes all or part of the transmembrane region of at least one of the following proteins: CD3ζ, α chain of T cell receptor, β chain of T cell receptor, ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, C D49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, TN FR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and NKG2C.

[0047] This application does not specifically limit the intracellular signal transduction region in the chimeric antigen receptor. Optionally, the intracellular signal transduction region includes the intracellular signal transduction regions of one or more of the following proteins: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, CD66d, 4-1BB, CD28, CD3ζ, CD134, ICOS, 2B4, DAP10, and DAP12; optionally, the intracellular signal transduction region includes a combination of the intracellular signal transduction regions of the following proteins: CD3ζ and 4-1BB.

[0048] This application does not specifically limit the structure of the membrane-bound IL-7. In some embodiments of this application, the membrane-bound IL-7 has the structure shown in formula (II):

[0049] SP2-IL-7-hinge region-cell membrane anchoring protein domain (II),

[0050] In the formula, - represents a peptide bond or a linking peptide, and SP2 represents a peptide without a signal or a signal peptide.

[0051] This application does not specifically limit the hinge region of membrane-bound IL-7. Optionally, the hinge region includes all or part of the extracellular region of at least one of the following proteins: CD8, IgG1, IgG4, IgG4m, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154;

[0052] This application does not specifically limit the cell membrane anchoring protein domain of membrane-bound IL-7. Optionally, the cell membrane anchoring protein domain is the transmembrane domain of a transmembrane protein or the anchoring signal peptide of a glycosylphosphatidylinositol anchoring protein; more preferably, the transmembrane domain of the transmembrane protein includes all or part of the transmembrane region of at least one of the following proteins: CD3ζ, α chain of T cell receptor, β chain of T cell receptor, ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL 2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and NKG2C. Further optionally, the anchoring signal peptide of the glycosylphosphatidylinositol anchoring protein includes all or part of the anchoring signal peptide of at least one of the following proteins: CD48, CD55, and CD90.

[0053] This application does not specifically limit the signal peptide in the membrane-bound IL-7. Optionally, the signal peptide includes the signal peptide of at least one of the following proteins: human IL-7, human CD8, human IgG, human GM-CSF, human IgE, human CD4, human CD28, and human CD137.

[0054] This application does not specifically limit the membrane-bound IL-7. In conjunction with the foregoing, the membrane-bound IL-7 includes all or part of human IL-7 and its mutants.

[0055] The antigen-binding domain described herein, relative to the aforementioned CDR sequence, may contain one or more substituted, deleted, or inserted amino acids, for example, the number of amino acid insertions, substitutions, or deletions not exceeding three, preferably one. Substitutions, deletions, or insertions can be introduced into the nucleic acid molecule encoding the binding protein of the present invention using conventional techniques such as site-directed mutagenesis or PCR-mediated mutagenesis. In some embodiments, conserved amino acid substitutions are performed at one or more sites. "Conserved amino acid substitution" is the case where one amino acid residue is replaced by an amino acid residue having a similar side chain. The families of amino acids with similar side chains are defined in the prior art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, aspartamine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0056] The antigen-binding domain is a component of immunoglobulins (antibodies) or immune cell surface receptors.

[0057] This application does not specifically limit the CDRs of the antigen-binding domain in chimeric antigen receptors, including but not limited to: HCDR1 shown in SEQ ID NO.7, HCDR2 shown in SEQ ID NO.8, and HCDR3 shown in SEQ ID NO.9, as well as LCDR1 shown in SEQ ID NO.10, LCDR2 shown in EQ ID NO.11, and LCDR3 shown in EQ ID NO.12. In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.14. It is understood that this application is not limited to these.

[0058] This application does not impose any particular limitation on the connection method between the heavy chain variable region and the light chain variable region, including but not limited to indirect connection, such as indirect connection through a flexible linker peptide. In some embodiments of this application, the flexible linker peptide is a G4S linker peptide; however, it should be understood that this application is not limited to this.

[0059] This application does not impose any particular limitation on the relative positions of the heavy chain variable region and the light chain variable region in the chimeric antigen receptor; they can be located at the C-terminus and N-terminus, or at the N-terminus and C-terminus, respectively.

[0060] Based on the degeneracy of codons, this application does not impose any particular limitation on the nucleic acid molecule expressing the target protein. In some embodiments of this application, the genetically modified immune cells have nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 15 (or with at least 90% identity to SEQ ID NO. 15, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity).

[0061] In some embodiments of this application, the genetically modified immune cells include one or more of TIL cells, T cells, and NK cells.

[0062] A second aspect of this application provides a nucleic acid molecule that encodes the target protein defined above.

[0063] Optionally, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.15 (or has at least 90% identity with SEQ ID NO.15, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity).

[0064] A third aspect of this application provides a carrier comprising the aforementioned nucleic acid molecule.

[0065] A fourth aspect of this application provides a method for constructing genetically modified immune cells, the method comprising introducing the nucleic acid molecule or the vector into immune cells to be modified to achieve expression of the target protein.

[0066] Import methods can include, but are not limited to, transfection.

[0067] A fifth aspect of this application provides the use of the genetically modified immune cells, the nucleic acid molecules, or the carriers in the preparation of antitumor drugs.

[0068] The types of tumors for which this anti-tumor drug is applicable are not specifically limited, including but not limited to the treatment of GPC3-positive tumors. Tumors can be solid tumors, such as hepatocellular carcinoma, lung cancer, ovarian cancer, and melanoma. (Shimizu Y, Suzuki T, Yoshikawa T, Endo I, Nakatsura T. Next-Generation Cancer Immunotherapy Targeting Glypican-3. Front Oncol. 2019 Apr 10; 9:248. doi:10.3389 / fonc.2019.00248.PMID:31024850; PMCID:PMC6469401; Rodakowska E, Walczak-Drzewiecka A, Boroyec M, Gorzkiewicz M, Grzesik J, Ratajewski M, Rozanski M, Dastych J, Ginalski K, Rychlewski L. Recombinant immunotoxin targeting) GPC3 is cytotoxic to H446 small cell lung cancer cells.Oncol Lett.2021 Mar;21(3):222.doi:10.3892 / ol.2021.12483.Epub 2021Jan 21.PMID:33613711;PMCID:PMC7859473.).

[0069] A sixth aspect of this application provides an antitumor drug, the drug comprising the genetically modified immune cells and a pharmaceutically acceptable carrier.

[0070] A seventh aspect of this application provides a method for treating tumors, the method comprising administering an effective dose of the antitumor drug to a subject.

[0071] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0072] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0073] This application overexpresses GPC3CAR and membrane-bound IL-7 in TILs, and enhances their antigen recognition spectrum and sustained antitumor killing function (independent of IL-2 concomitant injection).

[0074] (1) Enhance the antigen recognition spectrum of TILs: In addition to some TILs being tumor antigen-specific T cells, by expressing CAR molecules that recognize GPC3, the proportion of T cells in TILs that can recognize / kill tumor cells is increased, that is, the targeting of TILs to T cells is enhanced.

[0075] (2) The molecular structure of membrane-anchored IL-7 was optimized by using the human IgG4 hinge region + CH3 domain to connect IL-7 and CD8 transmembrane domain, so that IL-7 is anchored on the cell membrane surface and cannot exist in a free form, thereby overcoming the cytotoxic effect of high concentration of free IL-7 in T cell anti-tumor immune response.

[0076] (3) The molecular structure of GPC3CAR and membrane-bound IL-7 was designed and optimized. GPC3CAR and mIL-7 were expressed simultaneously through the P2A-T2A linker peptide, which enhanced the killing ability of TIL against tumor cells. At the same time, the overexpression of mIL-7 also enhanced the sustained anti-tumor ability of TIL.

[0077] Therefore, this application enhances the tumor-killing ability of TIL by simultaneously overexpressing GPC3CAR and membrane-bound IL-7, while also improving the in vivo and in vitro expansion capacity and sustained tumor-killing ability of TIL, which is more conducive to improving the therapeutic effect of TIL on GPC3-positive tumors such as hepatocellular carcinoma.

[0078] The design of each domain is shown in Figure 1. The tandem expression of the GPC3-specific CAR and the membrane-bound human IL-7 molecule is named BN108-6P4. BN108-6P4 is composed of the following structures linked together in sequence: CD8 signal peptide (CD8 SP), light chain variable region (VL), G4S linker peptide, heavy chain variable region (VH), CD8 hinge region, CD3ζ transmembrane region, CD3ζ intracellular signal transduction region, 4-1BB intracellular domain (ICD), P2A-T2A linker peptide, human IL-7 signal peptide (hIL-7SP), human IL-7, IgG4 hinge region and CH3 domain, and CD8 transmembrane region.

[0079] The amino acid sequence of BN108-6P4 (SEQ ID NO.6) is as follows: MALPVTALLLPLALLLHAARPSDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSP QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFARQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRAKRGSGATNFSLLKQAGDVEENPGPGSGEGRGSLLTCGDVEENPGPMGMFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHFVESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCDIYIWAPLAGTCGVLLLSLVITLYCNHRNR。Among them: human CD8 signal peptide: 1aa-22aa; light chain variable region: 23aa-135aa; G4S linker peptide: 136aa-150aa; heavy chain variable region: 151aa-265aa; CD8 hinge region: 266aa-308aa; CD3ζ transmembrane region: 309aa-339aa; CD3ζ intracellular signal transduction region: 340aa-451aa; 4-1BB intracellular domain: 452aa-493aa; P2A-T2A linker peptide: 494aa-540aa; Human IL-7 signal peptide: 541aa-567aa; Human IL-7: 568aa-719aa; Human IgG4 hinge region +CH3 domain IgG4: 720aa-840aa; Human CD8 transmembrane domain: 841aa-871aa.

[0080] The sequence of the nucleic acid molecule encoding BN108-6P4 is shown in SEQ ID NO.15:

[0081] 1. Experimental Materials

[0082] Table 1

[0083] 2 Experimental Methods

[0084] 2.1 Lentiviral Preparation

[0085] (1) 293T cells were transfected with lentiviral vector plasmid.

[0086] Each lentiviral expression vector was constructed separately. The plasmids of each vector were mixed with the lentiviral packaging plasmids pMDLg-pRRE, pRSV-Rev, and pMD2.G respectively using polyethyleneimine transfection reagent and co-transfected into 293T cells. The medium was replaced with complete culture medium 6 hours after transfection. After 72 hours of culture, the viral supernatant was collected, centrifuged at 3000 rpm for 10–15 min at 4°C, filtered through a 0.45 μm pore size filter, and concentrated. The collected viral concentrate was stored at -80°C.

[0087] (2) Lentiviral titer determination

[0088] Add 300 μL of complete culture medium (RPMI 1640 medium + 10% fetal bovine serum + 100 U / mL penicillin + 100 μg / mL streptomycin) and Jurkat cells to each well of a 24-well plate to achieve a cell density of 1 × 10⁻⁶ cells / well. 5Cells / well. Lentiviral concentrate was serially diluted 5-fold with Opti-MEM medium. Each dilution of lentivirus was added at a rate of 200 μL / well to each well of the 24-well plate to infect Jurkat cells (human PBMCs in the negative control group were only in Opti-MEM medium), and the plates were incubated in a cell culture incubator (37°C, 5% CO2). After 3 days of incubation, the cells in each well were gently mixed and transferred to 1.5 mL centrifuge tubes for viral titer determination using qPCR.

[0089] 2.2 Lentiviral transfection and detection of TILs, and preparation of TILs overexpressing GPC3 CAR / mIL-7 related structures.

[0090] (1) T cell preparation

[0091] CD3 isolated / amplified from surgically removed / biopsy samples of liver / colorectal cancer tumor tissue + TIL, or cryopreserved TIL, adjusted to 2×10 after thawing. 6 Cells were cultured at a density of 1 / mL in TIL amplification medium and incubated in a cell culture incubator for 24 hours (culture temperature: 37℃, carbon dioxide concentration: 5%).

[0092] (2) Lentiviral transduction of TIL

[0093] The obtained TILs were washed and the cell density was adjusted to 3 × 10⁻⁶. 6 -4×10 6 / mL. Lentiviral virus was added at an MOI of 1-10TU / mL for transduction, and cultured in TIL amplification medium in a cell culture incubator (culture temperature 37℃, carbon dioxide concentration 5%).

[0094] After 4–8 hours, the transfected TILs were co-cultured with gamma-ray irradiated feeder cells (PBMCs) at a ratio of 1:100, with a TIL cell density of 1 × 10⁻⁶ cells / year during co-culture. 4 The sample was incubated in X-vivo15 + 5% SR + 600 IU / mL IL-2 + 5 ng / mL IL-7 + 5 ng / mL IL-15, with 30 ng / mL OKT3 and 3 ng / mL TGFβ1 added.

[0095] Seven days later, the cells were rehydrated with an equal volume of fluid, and OKT3 was not added again.

[0096] Ten days later, the cells were centrifuged and the medium was changed; TGFβ1 was no longer added to the culture system. Cells were counted, and based on the cell count, a suitable container was selected for cell expansion. Subsequent cell replenishment or expansion was performed every 3 days. During rapid expansion (days 21-24), cells were harvested and cryopreserved in liquid nitrogen using cryopreservation medium (containing 5% human serum albumin: physiological saline = 1:1) for later use. The obtained TILs were named according to their corresponding molecular structures; TIL cells not transduced with lentivirus were named Mock TILs. After thawing, the cryopreserved cells were used for subsequent in vitro and in vivo experiments.

[0097] (3) Expression detection of GPC3 CAR

[0098] The mock TILs and TILs transfected with GPC3 CAR were washed twice with PBS and resuspended in FACS buffer. Following the antibody manufacturer's instructions, PE-labeled anti-G4S Linker antibody and BV421-labeled anti-human CD3 antibody were added to the cell suspension and incubated at 4°C for 45 min. Mock TILs not transfected with lentivirus were used as a negative control. The expression rate of GPC3 CAR was detected by flow cytometry. Analysis was performed using FlowJo software.

[0099] (4) Detection of membrane-bound IL-7 expression

[0100] The mock TILs and transfected TILs were washed twice with PBS and resuspended in FACS buffer. Anti-Human IgG AF647 (mouse) and BV421-labeled anti-human CD3 antibodies were added to the cell suspension according to the antibody manufacturer's instructions and incubated at 4°C for 45 min. Mock TILs not transfected with lentivirus were used as a negative control. The expression rate of IgG on the cell membrane surface of the transfected cells was detected by flow cytometry, and the expression rate of membrane-anchored IL-7 was also measured. FlowJo software was used for analysis.

[0101] 2.3 Effects of GPC3 CAR overexpression on membrane-bound IL-7 structure: Experiment 1 (In vitro functional experiment)

[0102] (1) Overexpression of GPC3 in SK-Hep-1 (SK-Hep-1-GPC3) OE Cell line construction

[0103] Based on the wild-type SK-Hep-1 cell line, GPC3 was overexpressed by infecting the SK-Hep-1 cell line with a lentiviral vector (viral vector preparation method is described in 2.1). Forty-eight hours after transfection, SK-Hep-1-GPC3 expressing GPC3 was purified by flow cytometry. OEThe cells (Figure B in Figure 2) were cultured on a larger scale and used in subsequent in vitro experiments.

[0104] (2) Damage effect detection (Incucyte)

[0105] Three GPC3-positive hepatocellular carcinoma cell lines (Hep3B, Huh7, and PLC-PRF / 5) were selected to assess the cytotoxic effects of GPC3-overexpressing CARs and membrane-bound IL-7 TILs. Hep3B and Huh7 were GPC3-high expression cell lines, with positive rates of 97.9% and 60.4%, respectively; PLC-PRF / 5 was a GPC3-weak expression cell line, with a positive rate of 38.8%. Target cells were digested and counted at a concentration of 1×10⁻⁶ cells / cells. 4 The target cells were seeded into 96-well plates at a density of 1×10⁶ cells / well, and 1×10⁶ cells / well were added according to experimental requirements. 4 / hole or 2×10 4 T cells were co-cultured in / wells. The long-term killing effect was then assessed using an Incucyte instrument (3-7 days).

[0106] (3) ELISA detection of IFNγ release level in TIL cells

[0107] Mock TILs and transfected TILs were co-cultured with Hep3B, Huh7, and PLC-PRF / 5 target cells (Figure 2, B) in IL-2-free X-vivo medium (effect-to-target ratio 1:1). After 24 h of co-culture, the supernatant was collected by centrifugation at 500 g for 5 min for IFN-γ detection. Following the LEGEND MAX... TM The Human IFN-γ ELISA Kit is used to detect the release of IFN-γ in the co-culture supernatant. The absorbance at 450 nm and 570 nm is read using a microplate reader, and the IFN-γ value is calculated by fitting a 5-parameter model using the difference (OD450nm-OD570nm).

[0108] 2.4 Effects of GPC3 CAR overexpression on membrane-bound IL-7 (In vivo tumor suppression experiment)

[0109] Target cells in the logarithmic growth phase and in good growth condition were collected using trypsin digestion. After washing once with physiological saline, the cell density was adjusted to 2.0 × 10⁻⁶ cells / year. 7 / mL. 100 μL of cell suspension was subcutaneously injected into the right axillary region of NOG mice, i.e., each mouse was inoculated with 2.0 × 10⁹ cells / mL. 6 The target cells were inoculated, and the inoculation diary was dated day 0.

[0110] Day 14 after inoculation of target cells (or when the average tumor volume is approximately 50 mm) 3At that time, TIL (1×10⁻⁶) of the transfection group was injected via the tail vein. 7 / each), Mock TIL (1×10) 7 The NOG mice were injected with a solvent (400 μL / mouse, physiological saline), and the day of injection was recorded as day 0 of treatment. Tumor size and mouse weight were measured 2-3 times per week. Peripheral blood was collected from mice on days 7, 14, 21, 28, and 35 after treatment for flow cytometry analysis to detect the number of TIL-derived T cells in the blood.

[0111] 3. Results Data

[0112] 3.1 Validation of specific killing effect targeting GPC3 CAR TIL

[0113] In the scFv used to construct the CAR molecule in this application, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.13, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.14, the amino acid sequences of LCDR1 to LCDR3 are shown in SEQ ID NO.7 to SEQ ID NO.9, and the amino acid sequences of HCDR1 to HCDR3 are shown in SEQ ID NO.10 to SEQ ID NO.12.

[0114] LCDR1 shown in SEQ ID NO.7: RSSQSLVHSNANTYLH;

[0115] LCDR2 shown in SEQ ID NO.8: KVSNRFS;

[0116] LCDR3 shown in SEQ ID NO.9: SQNTHVPPT;

[0117] HCDR1 shown in SEQ ID NO.10: DYEMH;

[0118] HCDR2 shown in SEQ ID NO.11: ALDPKTGDTAYSQKFKG;

[0119] HCDR3:FYSYTY, as shown in SEQ ID NO.12.

[0120] The heavy chain variable region shown in SEQ ID NO.13:

[0121] The light chain variable region shown in SEQ ID NO.14:

[0122] First, this application constructed the structure of the GPC3 scFv-based CAR molecule BN108-1P4 (Figure 2A), and overexpressed it in liver cancer-derived TILs via lentiviral transfection to verify the killing specificity of the GPC3 CAR TIL. In Figure 2, the CAR molecule structure is CD8SP-VL-G4S linker-VH-CD8 hinge-CD8TM-41BB ICD-CD3ζICD, and the amino acid sequence is SEQ ID. NO.1: MALPVTALLLPLALLLHAARPSDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNT HVPPTFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVY YCTRFYSYTYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. Human CD8 signal peptide: 1aa-22aa; light chain variable region: 23aa-135aa; G4S linker peptide: 136aa-150aa; heavy chain variable region: 151aa-265aa; CD8 hinge region: 266aa-308aa; CD8 transmembrane region: 309aa-339aa; 4-1BB intracellular domain: 340aa-381aa; CD3ζ intracellular signal transduction region: 382aa-493aa.

[0123] On the other hand, this application used flow cytometry to detect the expression level of GPC3 in various human hepatocellular carcinoma cell lines (Figure 2B); among them, SK-Hep-1, SNU449, and HuCC-T1 are GPC3-negative hepatocellular carcinoma cell lines; and an overexpressing GPC3 cell line SK-Hep-1-GPC3 was constructed based on SK-Hep-1. OEThe positive rate was 96.9%; PLC / PRF / 5, a cell line with weak GPC3 expression, had a positive rate of 38.8%; Hep3B and Huh7, cell lines with high GPC3 expression, had positive rates of 97.9% and 80.4%, respectively. The killing results (Figure 2, C) showed that BN108-1P4 had no killing effect on GPC3-negative cell lines, such as SK-Hep-1, SNU449, and HuCC-T1; while it had no killing effect on GPC3-positive cell lines, such as Hep3B, Huh7, PLC-PRF / 5, and SK-Hep-1-GPC3. OE It exhibits significant killing effect. These results demonstrate that this GPC3 scFv can specifically target human GPC3 protein to initiate BN108-1P4 CAR-mediated T-cell killing.

[0124] Figure 2. Validation of the killing specificity of GPC3 CAR-TIL. A. Schematic diagram of the molecular structure of GPC3 CAR used for specificity validation; B. Detection of GPC3 expression level in liver cancer cell lines using flow cytometry; C. Killing effect of BN108-1P4 CAR TIL on GPC3+ or GPC3- liver cancer cell lines, with the killing of target cells monitored using a real-time fluorescence imaging system; Target cell only, target cell only group; Mock, control TIL transfection group; BN108-1P4, BN108-1P4 CAR TIL transfection group.

[0125] 3.2 Validation of co-expression factors enhancing GPC3 CAR TIL killing

[0126] This application enhances the sustained cytotoxic effect of GPC3CAR TIL by co-transfecting it with factors that enhance T-cell immune responses. Based on previous screening data, this experiment co-transfected GPC3CAR with LTBR or membrane-bound IL-7 (Figure 3A) to compare the sustained cytotoxic effect of GPC3CAR TIL in vitro and in vivo.

[0127] The amino acid sequence of LTBR overexpression is shown in SEQ ID NO.16: MLLPWATSAPGLAWGPLVLGLFGLLAASQPQAVPPYASENQTCRDQEKEYYEPQHRICCSRCPPGTYVSA KCSRIRDTVCATCAENSYNEHWNYLTICQLCRPCDPVMGLEEIAPCTSKRKTQCRCQPGMFCAAWALECTHCELLSDCPPGTEAELKDEVGKGNNHCVPCKAGHFQNTSSPSARCQPHTRCENQGLVEAAPGTAQSDTTCKNPLEPLPPEMSGTMLMLAVLLPLAFFLLLATVFSCIWKSHPS LCRKLGSLLKRRPQGEGPNPVAGSWEPPKAHPYFPDLVQPLLPISGDVSPVSTGLPAAPVLEAGVPQQQSPLDLTREPQLEPGEQSQVAHGTNGIHVTGGSMTITGNIYIYNGPVLGGPPGPGDLPATPEPPYPIPEEGDPGPPGLSTPHQEDGKAWHLAETEHCGATPSNRGPRNQFITHD. Human LTBR signal peptide: 1aa-31aa; human LTBR: 32aa-435aa.

[0128] Previous studies have indicated that LTBR, ​​a membrane protein receptor, can enhance the sustained killing effect of TILs in vitro and in vivo. On the other hand, IL-7 is believed to enhance the killing function of T cells by increasing the formation of memory T cells. First, co-expression of LTBR (BN138P4) or mIL-7 (BWm7P4, membrane-bound IL-7) on the basis of BN108-1P4 did not significantly affect the expression rate of BN108-1P4 in TILs (Figure 3, upper B), and the expression rates of LTBR and mIL-7 in BN108-1P4 CAR TILs could be detected normally (Figure 3, lower B). In vitro killing assays showed that, compared to the TIL group expressing only GPC3 CAR (BN108-1P4), co-expression of LTBR (BN138P4) tended to inhibit the sustained killing effect of CAR TILs (Figure 3, C, left panel); while co-expression of mIL-7 (BWm7P4) significantly enhanced the sustained killing effect of BN108-1P4 CAR TILs (Figure 3, C, right panel). Correspondingly, in the hepatocellular carcinoma cell line SK-Hep-1-GPC3... OEIn a tumor-bearing, severely immunodeficient mouse model, expression of LTBR (BN138P4) significantly attenuated the antitumor effect of BN108-1P4 CAR TIL; while co-expression of mIL-7 (BWm7P4) significantly enhanced the antitumor activity of BN108-1P4 TIL (Figure 3, D). This result was unexpected. Although both LTBR and IL7 can enhance the killing function of TILs or T cells in existing technologies, only overexpression of membrane-bound IL-7 significantly enhanced the sustained killing effect of TILs in vitro and in vivo when co-expressed with BN108-1P4 CAR. Current theories have not yet adequately explained this difference.

[0129] Figure 3. Validation of co-expression factors enhancing GPC3 CAR-TIL killing. A. Schematic diagram of the molecular structure used to validate the enhancement of GPC3 CAR killing by co-expression factors; B. Flow cytometry was used to detect the CAR positivity rate, LTBR (BN138P4) positivity rate, and membrane-bound IL-7 (BWm7P4) positivity rate after TIL transfection of GPC3 CAR molecules and co-transfection with different factors; C. The killing effects of BN108-1P4, BN108-1P4+BN138P4, and BN108-1P4+BWm7P4 on GPC3-positive hepatocellular carcinoma cell line (Huh7) were monitored using a real-time fluorescence imaging system; (D) The killing effects of BN108-1P4, BN108-1P4+BN138P4, and BN108-1P4+BWm7P4 on SK-Hep-1-GPC3 cells. OE Tumor suppression in tumor-bearing, severely immunodeficient mice. BN108-1P4 transfected with BN108-1P4 CAR TIL; BN108-1P4+BN138P4 transfected with BN108-1P4 and BN138P4 CAR TIL; BN108-1P4+BWm7P4 transfected with BN108-1P4 and BWm7P4 CAR TIL.

[0130] 3.3 Optimization of GPC3 CAR structure overexpression

[0131] This application screens for molecular structures with stronger killing effects on target cells by adjusting the structural order of different intra-sib signaling domains in CAR molecules (Figure 4A). Specifically, BN108-1P4 is described under section 3.1, and the molecular structure of BN108-2P4 is CD8SP-VL-G4S linker-VH-CD8hinge-CD3ζTM-CD3ζICD-41BB ICD, with the amino acid sequence SEQ ID NO.2: MALPVTALLLPLALLLHAARPSDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSP QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGGGSGGGGSGGGGS QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFA QSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL. Human CD8 signal peptide: 1aa-22aa; light chain variable region: 23aa-135aa; G4S linker peptide: 136aa-150aa; heavy chain variable region: 151aa-265aa; CD8 hinge region: 266aa-308aa; CD3ζ transmembrane region: 309aa-338aa; CD3ζ intracellular signal transduction region: 339aa-450aa; 4-1BB intracellular domain: 451aa-492aa.

[0132] The CAR expression rate results (Figure 4, B) showed that the expression rate of BN108-1P4 (44.2%) was significantly higher than that of BN108-2P4 (28.4%). Simultaneously, the killing effects of the two CAR TILs on GPC3-positive hepatocellular carcinoma cell lines were compared. CAR TILs were co-cultured with Huh7 and Hep3B tumor cells at an effector-to-target ratio of 1:1 or 1:2, and the killing effect of CAR TILs on tumor cells was recorded in real time using the IncuCyte live-cell imaging analysis system. Since all tumor cells overexpressed mCherry fluorescent protein, the fluorescence changes of tumor cells could be analyzed using IncuCyte software to characterize the degree of CAR TIL killing on tumor cells. Lower fluorescence signal values ​​indicated lower tumor cell activity and quantity in that group, and a more significant TIL killing effect. Figure 4, C, showed that the killing effect of BN108-2P4 on Hep3B and Huh7 was significantly stronger than that on BN108-1P4. Although the expression rate of BN108-2P4 was lower than that of BN108-1P4, its tumor-killing effect was stronger, suggesting that the BN108-2P4 CAR molecule had a more significant activating effect on TIL cells. Therefore, BN108-2P4 was finally selected for further research.

[0133] Figure 4. Optimization of GPC3 CAR molecular structure. A. Schematic diagram of GPC3 CAR molecular structure used for structure screening; B. CAR positivity rate after TIL transfection with different GPC3 CAR molecules, detected by flow cytometry; C. Killing effect of BN108-1P4 and BN108-2P4 on GPC3 positive liver cancer cell lines, monitored by real-time fluorescence imaging system; Target cell only, target cell only group; Mock, control TIL transfection group; BN108-1P4, BN108-1P4 CAR TIL transfection group; BN108-2P4, BN108-2P4 CAR TIL transfection group.

[0134] 3.4 Optimization of membrane-bound IL-7 related structures

[0135] To further enhance the sustained killing effect of CAR TILs, this application proposes to co-express membrane-bound IL-7 in addition to BN108-2P4 expression. Since the difference in sequence length used to anchor IL-7 may affect the effect of IL-7 on TILs, this application designs a molecular structure for anchoring IL-7 to the cell membrane surface (Figure 5A), using a shorter hinge region (BWm7P4, SEQ ID NO.3) and a longer hinge region (BWm7-GP4, SEQ ID NO.4) to anchor IL-7 to the cell membrane surface.

[0136] SEQ ID NO.3: MGMFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKR HICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNR. Human IL-7 signal peptide: 1aa-27aa; Human IL-7: 28aa-179aa; Human CD8 hinge region: 180aa-232aa; Human CD8 transmembrane domain: 233aa-263aa.

[0137] SEQ ID NO.4: MGMFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKR HICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHFVESKYGPPCPPCPGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCDIYIWAPLAGTCGVLLLSLVITLYCNHRNR. Human IL-7 signal peptide: 1aa-27aa; Human IL-7: 28aa-179aa; Human IgG4 hinge region +CH3 domain IgG4: 180aa-300aa; Human CD8 transmembrane domain: 301aa-331aa.

[0138] The expression rate detection results showed that the positive rate of BWm7P4 overexpression was significantly higher than that of BWm7-GP4 in the two different batches of TIL (Figure 5, B). However, the killing data showed that under different target cell conditions and different effector-to-target ratios, the killing function of BWm7-GP4 was stronger than that of BWm7P4 (Figure 5, C).

[0139] The above results demonstrate that although membrane-bound IL-7 can activate TIL cells, different molecular structures have drastically different effects. The molecular structure of BWm7-GP4, with its longer hinge region, exhibits a stronger activating effect on TIL cells. In this application, BWm7-GP4 and BN108-2P4 will be co-expressed in TILs for subsequent research.

[0140] Figure 5. Structural optimization of membrane-bound IL-7 used to enhance the killing function of TIL. A. Schematic diagram of the molecular structure of membrane-bound IL-7 used for structural screening; B. Expression rate of different membrane-bound IL-7 molecules after TIL transfection, detected by flow cytometry; C. Killing effect of BWm7P4 and BWm7-GP4TIL on SK-BN105 hepatocellular carcinoma cell line, respectively, monitored by real-time fluorescence imaging system; Target cell only, target cell only group; Mock, control TIL transfection group; BWm7P4, BWm7P4 TIL transfection group; BWm7-GP4, BWm7-GP4 TIL transfection group.

[0141] 3.5 Structural optimization for co-expression of GPC3 CAR and mIL-7

[0142] Based on the above results, this application uses a P2A-T2A structure to tandemly express BN108-2P4 and BWm7-GP4 in TIL. Since the cleavage structure of P2A-T2A may affect the expression intensity of the molecules before and after tandem expression, this application designed two molecular structures: BN108-5P4 and BN108-6P4, with the BN108-2P4 sequence located at the 5' end and the BWm7-GP4 sequence at the 3' end, and the BWm7-GP4 sequence located at the 5' end and the BN108-2P4 sequence at the 3' end (Figure 6A). The amino acid sequences of BN108-5P4 and BN108-6P4 are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0143] The amino acid sequence of the BN108-5P4 molecule (SEQ ID NO.5) is as follows: MGMFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHFVESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRAKRGSGATNFSLLKQAGDVEENPGPGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPSDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL。Human IL-7 signal peptide: 1aa-27aa; Human IL-7: 28aa-179aa; Human IgG4 hinge region + CH3 domain IgG4: 180aa-300aa; Human CD8 transmembrane domain: 301aa-331aa; P2A-T2A linker peptide: 332aa-378aa; Human CD8 signal peptide: 379aa-400aa; Light chain Variable regions: 401aa-513aa; G4S linker peptide: 514aa-528aa; Heavy chain variable region: 528aa-643aa; CD8 hinge region: 644aa-686aa; CD3ζ transmembrane region: 687aa-716aa; CD3ζ intracellular signal transduction region: 717aa-828aa; 4-1BB intracellular domain: 829aa-870aa. The expression rate results show that the CAR molecule expression positivity rate of BN108-6P4 is closer to that of BN108-2P4 alone, while the CAR molecule expression rate of BN108-5P4 is the lowest. On the other hand, the mIL-7 expression rate of BN108-5P4 is higher than that of BN108-6P4 (Figure 6, B). Furthermore, this application compared the in vitro proliferation of TILs in the transfection control group, BN108-2P4 group, BWm7-GP4 group, BN108-5P4 group, and BN108-6P4 group (Figure 6, C). The results showed that, compared to the transfection control group, the cell number in the BWm7-GP4 group was increased by an average of 5.43-fold (P<0.05), the BN108-2P4 group by an average of 2.11-fold, the BN108-5P4 group by an average of 3.94-fold, and the BN108-6P4 group by an average of 13.44-fold (P<0.0001). The average fold increase in cell number in the BN108-6P4 group was greater than the product of the fold increases in the BN108-2P4 and BWm7-GP4 groups, which was 11.46-fold (P<0.05). This indicates that BN108-6P4 has a synergistic effect compared to the BN108-2P4 and BWm7-GP4 groups; on the other hand, this application unexpectedly found that BN108-5P4 did not show a synergistic effect, suggesting that different structural sequences have a key impact on the synergistic effect of GPC3 CAR and membrane-bound IL-7.

[0144] Meanwhile, this application compared the IFNγ release levels when TILs in the transfected control group, BN108-2P4 group, BWm7-GP4 group, BN108-5P4 group, and BN108-6P4 group killed the Hep3B liver cancer cell line (Figure 6, D). Compared with the transfected control group, the IFNγ release level in the BWm7-GP4 group was increased by an average of 2.88 times, the IFNγ release level in the BN108-2P4 group was increased by an average of 15515.43 times (P<0.05), the IFNγ release level in the BN108-5P4 group was increased by an average of 27686.48 times, and the IFNγ release level in the BN108-6P4 group was increased by an average of 77356.88 times (P<0.0001). Among them, the average upregulation of IFNγ release level in the BN108-6P4 group was 44684.43 times greater than the product of the upregulation folds of the BN108-2P4 group and the BWm7-GP4 group (P<0.05). This indicates that BN108-6P4 has a synergistic effect compared to the BN108-2P4 and BWm7-GP4 groups. On the other hand, the average upregulation of IFNγ release level in BN108-6P4 was significantly higher than that in the BN108-5P4 group (P<0.05), suggesting that the structure of BN108-6P4 is superior to that of BN108-5P4, and also indicating that different structural sequences have a key influence on the synergistic effect of GPC3 CAR and membrane-bound IL-7.

[0145] Furthermore, comparing the in vitro killing effects of TILs on Hep3B and Huh7 liver cancer cell lines in the transfected control group, BN108-2P4 group, BN108-5P4 group, and BN108-6P4 group, it was found that: compared with BN108-2P4 overexpression alone, TILs overexpressing BN108-6P4 significantly enhanced the killing effect on target cells; while the killing effect of BN108-5P4 overexpression was only enhanced in some batches of TILs, and was not as good as BN108-6P4 (Figure 6, E). Based on the above data, this application will select the molecular structure of BN108-6P4 for gene modification of TILs and conduct in vivo anti-tumor efficacy experiments in mouse models to verify the effect.

[0146] Figure 6. Structural optimization of GPC3 CAR tandem membrane-bound IL-7. A. Schematic diagram of the molecular structure of the CAR tandem membrane-bound IL-7 structure used for structure screening; B. Expression rates of CAR and membrane-bound IL-7 after TIL transfection with different molecular structures, detected by flow cytometry; C. Proliferation of TIL after transfection with different molecular structures in vitro; D. IFNγ release level after TIL transfection with different molecular structures and co-cultured with Hep3B liver cancer cell lines for 16 hours, detected by ELISA; E. Killing effect of CAR expressing different structures combined with membrane-bound IL-7 structure TIL on positive liver cancer cell lines Hep3B and Huh7, monitored by real-time fluorescence imaging system; Target cell only, target cell only group; Mock, control TIL transfection group; BN108-2P4, BN108-2P4 CAR TIL transfection group; BN108-5P4, BN108-5P4 CAR transfection group. TIL group; BN108-6P4, transfected BN108-6P4 CAR TIL group; Statistical analysis: C. Two-way ANOVA, corrected by Dunnet algorithm; D. One-way ANOVA, corrected by Dunnet algorithm; *, P<0.05, ***, P<0.0001.

[0147] 3.6BN108-6P4 CAR-TIL Antitumor Function Validation (In Vitro Experiment)

[0148] This application validates the in vitro killing function of BN108-6P4 CAR TIL using different hepatocellular carcinoma cell lines. BN108-6P4 TIL was co-cultured with three GPC3-positive hepatocellular carcinoma cell lines for 24 hours at an effector-to-target ratio of 1:1. After culture, the culture supernatant was collected, and the level of IFNγ secreted by TIL was detected by ELISA. Figure 7A shows that after co-culturing with target cells, the level of IFNγ secreted by the Mock group was close to 0 pg / mL; the level of IFNγ secreted by BN108-2P4 TIL was significantly increased (Hep3B, 8.6–12.7 ng / mL; Huh7, 2.8–3.6 ng / mL; PLC / PRF / 5, 1.6–2.4 ng / mL); while the level of IFNγ secreted by BN108-6P4 TIL was significantly increased (Hep3B, 14.1–16.5 ng / mL; Huh7, 4.1–4.8 ng / mL; PLC / PRF / 5, 3.3–4.1 ng / mL). Furthermore, the data above show that in the PLC / PRF / 5 cell line model, compared with the transfection control group, the IFNγ release level in the BWm7-GP4 group was increased by an average of 1.56-fold, the IFNγ release level in the BN108-2P4 group was increased by an average of 90.21-fold (P<0.0001), and the IFNγ release level in the BN108-6P4 group was increased by an average of 163.45-fold (P<0.0001). The average upregulation of IFNγ release level in the BN108-6P4 group was greater than the product of the upregulation folds of the BN108-2P4 and BWm7-GP4 groups, which was 140.71-fold (P<0.05). This indicates that BN108-6P4 has a synergistic effect compared with the BN108-2P4 and BWm7-GP4 groups.

[0149] Meanwhile, monitoring of the killing results using the IncuCyte live-cell imaging analysis system showed that BN108-6P4 TIL had significant killing effects on Hep3B, Huh 7, and PLC-PRF / 5 cells (Figure 7, B). Furthermore, in the PLC / PRF / 5 cell line model, compared to the target cell-only group (mean fluorescence area: 2.36±0.12), the fluorescence area at the killing endpoint was 2.16±0.08 in the transfected control group (P<0.05), 1.95±0.12 in the BWm7-GP4 group (P<0.05), 0.26±0.03 in the BN108-2P4 group (P<0.001), and 0.11±0.03 in the BN108-6P4 group (P<0.001). Among them, the fluorescence area of ​​the BN108-6P4 group decreased by 21.45 times at the killing endpoint, which was greater than the product of the decrease folds of the BN108-2P4 and BWm7-GP4 groups (9.13 times) (P<0.05). This indicates that BN108-6P4 has a synergistic effect compared to the BN108-2P4 and BWm7-GP4 groups. In conclusion, BN108-6P4 TIL has significant specific killing ability against GPC3 positive hepatocellular carcinoma tumor cells.

[0150] Figure 7. In vitro efficacy data of BN108-6P4. A. IFNγ release level during BN108-6P4 killing various hepatocellular carcinoma target cells, detected by ELISA; B. Killing effect of BN108-6P4 on various hepatocellular carcinoma target cells, monitored by real-time fluorescence imaging system; Target cell only, target cell only group; Mock, transfected control TIL group; BN108-6P4, transfected BN108-6P4 TIL group. Statistical analysis: A. One-way ANOVA, Dunnet algorithm correction, **, P<0.01; ***, P<0.001.

[0151] 3.7BN108-6P4 CAR-TIL Antitumor Function Validation (In vivo Experiment)

[0152] Furthermore, this application investigated the in vivo antitumor efficacy of GPC3 CAR and mIL-7 expression. This application used a NOG mouse model with Hep3B subcutaneous xenograft liver cancer cells to observe the antitumor effect of GPC3 CAR TIL (BN108-6P4). First, this application compared the in vivo efficacy results of the BWm7-GP4 group, BN108-2P4 group, and BN108-6P4 group. As shown in Figure 8A, the body weight of mice in the vehicle control group and the BWm7-GP4 group gradually decreased, while the body weight of mice given BN108-2P4 and BN108-6P4 CAR-TIL remained relatively stable, suggesting that the decrease in body weight in the vehicle control group and the BWm7-GP4 group was related to the gradual increase in tumor burden. On the other hand, compared with the solvent group (tumor volume change fold: 82.66±8.05), the tumor volume change fold in the BWm7-GP4 group (77.52±10.28) did not decrease significantly; in contrast, the tumor volume change fold in the BN108-2P4 group was significantly downregulated (34.15±12.72, P<0.001); while the tumor in the BN108-6P4 group was completely cleared (Figure 8, Figure B), indicating the specific synergistic effect of BWm7-GP4 and BN108-2P4.

[0153] Furthermore, this application compared the antitumor efficacy of different doses of BN108-6P4. After TIL reinfusion, observation of mouse body weight revealed that the body weight of mice in the vehicular (Veh) control group and the mock group gradually decreased, while the body weight of mice given different doses of GPC3 CAR TIL (BN108-6P4) remained relatively stable, suggesting that the decrease in body weight in the vehicular (Veh) control group and the mock group was related to the gradual increase in tumor burden (Figure 8, C). After treatment with GPC3 CAR TIL (BN108-6P4), tumor volume continued to decrease from day 3 to day 20, and remained at a low level after day 20 (Figure 8, D); correspondingly, from day 3 to day 20, tumor volume continued to increase in the vehicular (Veh) control group and the mock group. These results demonstrate that GPC3 CAR TIL (BN108-6P4) has a significant antitumor effect (Figure 8, E). Meanwhile, this experiment compared the antitumor effects of different doses of BN108-6P4 TIL. As shown in Figure 8, D, compared with other dose groups, a single dose of 1×10 7 BN108-6P4 TIL exhibits the strongest antitumor effect; it is noteworthy that at a total dose of 1×10⁻⁶, the antitumor effect is relatively strong. 7 Under the premise of BN108-6P4 TIL, a single injection of 1×10 7 Cells were injected twice at a ratio of 5×10 6The cells showed a stronger anti-tumor effect, indicating that for BN108-6P4 TILs, a single dose of 1×10⁻⁶ was more effective. 7 Cell reinfusion yielded the best therapeutic effect. Furthermore, as shown in Figure 8E, on days 7 and 14 after reinfusion, the number of TILs in the peripheral blood of the GPC3 CAR TIL group showed an increasing trend, suggesting that GPC3 CAR TIL (BN108-6P4) was amplified in vivo under anti-tumor effects. Correspondingly, the number of TILs in the peripheral blood of the Mock group mice showed a decreasing trend and was significantly lower than that of the GPC3 CAR TIL (BN108-6P4) group, suggesting a correlation with the anti-tumor effect of TILs.

[0154] Figure 8 shows the efficacy data of BN108-6P4 in Hep3B tumor-bearing mice. A. Comparison of in vivo efficacy results among BWm7-GP4, BN108-2P4, and BN108-6P4 groups. Figures A and B show the statistical results of the relative fold changes in body weight and tumor volume of mice in each group, respectively. C. Body weight monitoring data of tumor-bearing mice in each group after infusion of different doses of TIL. D. Monitoring data of the relative fold changes in tumor volume of tumor-bearing mice in each group. E. Statistical analysis of the relative number of TILs in the peripheral blood of mice in the Mock and BN108-1P4 groups on days 7 and 14 after TIL infusion, using flow cytometry. Veh, solvent (physiological saline) control group; Mock, transfection control group; BWm7-GP4, transfection BWm7-GP4 TIL group; BN108-2P4, transfection BN108-2P4 TIL group; BN108-6P4, transfection BN108-6P4 TIL group.

[0155] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0156] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Genetically modified immune cells, characterized in that, The genetically modified immune cells express target proteins, including chimeric antigen receptors and membrane-bound interleukins. The chimeric antigen receptor includes an antigen-binding domain that specifically binds to GPC3. The membrane-bound interleukins include membrane-bound IL-7.

2. The genetically modified immune cells according to claim 1, characterized in that, The genetically modified immune cells include the gene encoding the chimeric antigen receptor and the gene encoding the membrane-bound IL-7 expressed in tandem. Optionally, the gene encoding the chimeric antigen receptor and the gene encoding the membrane-bound IL-7 are expressed by a polycistronic expression. Optionally, the gene encoding the chimeric antigen receptor and the gene encoding the membrane-bound IL-7 are each expressed by two monocistronic molecules.

3. The genetically modified immune cells according to claim 2, characterized in that, The gene encoding the chimeric antigen receptor and the gene encoding the membrane-bound IL-7 are linked together by a linker fragment to form a polycistronic group; Optionally, the linker fragment encodes a linker peptide that includes a self-cleaving peptide; Optionally, the connecting segment includes an internal ribosome entry site.

4. The genetically modified immune cells according to claim 3, characterized in that, The gene encoding the chimeric antigen receptor is located at the 5' end of the gene encoding the membrane-bound IL-7.

5. The genetically modified immune cells according to claim 1, characterized in that, The chimeric antigen receptor has the structure shown in formula (Ⅰ): SP1-antigen binding domain-hinge region-transmembrane region-intracellular signal transduction region (Ⅰ), In the formula, - represents a peptide bond or a linking peptide, and SP1 represents a peptide with no signal or a signal peptide; Optionally, the signal peptide includes the signal peptide of at least one of the following proteins: human CD8, human IgG, human GM-CSF, human IgE, human CD4, human CD28, and human CD137; Optionally, the hinge region includes all or part of the extracellular region of at least one of the following proteins: CD8, IgG1, IgG4, IgG4m, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; Optionally, the transmembrane region includes all or part of the transmembrane region of at least one of the following proteins: CD3ζ, α chain of T cell receptor, β chain of T cell receptor, ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, C D49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, TN FR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and NKG2C; Optionally, the intracellular signal transduction region includes the intracellular signal transduction regions of one or more of the following proteins: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, CD66d, 4-1BB, CD28, CD3ζ, CD134, ICOS, 2B4, DAP10, and DAP12; or, optionally, the intracellular signal transduction region includes a combination of the intracellular signal transduction regions of the following proteins: CD3ζ and 4-1BB.

6. The genetically modified immune cells according to claim 1, characterized in that, The membrane-bound IL-7 has the structure shown in formula (II): SP2-IL-7-hinge region-cell membrane anchoring protein domain (II), In the formula, - represents a peptide bond or a linking peptide, and SP2 represents a peptide with no signal or a signal peptide; Optionally, the hinge region includes all or part of the extracellular region of at least one of the following proteins: CD8, IgG1, IgG4, IgG4m, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; Optionally, the cell membrane anchoring protein domain is a transmembrane domain of a transmembrane protein or an anchoring signal peptide of a glycosylphosphatidylinositol anchoring protein; Optionally, the transmembrane domain of the transmembrane protein includes all or part of the transmembrane region of at least one of the following proteins: CD3ζ, α chain of T cell receptor, β chain of T cell receptor, ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA 1. CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, T NFR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and NKG2C; Optionally, the anchoring signal peptide of the glycosylphosphatidylinositol anchoring protein includes all or part of the anchoring signal peptide of at least one of the following proteins: CD48, CD55, and CD90; Optionally, the signal peptide includes the signal peptide of at least one of the following proteins: human IL-7, human CD8, human IgG, human GM-CSF, human IgE, human CD4, human CD28, and human CD137; Optionally, the membrane-bound IL-7 includes all or part of human IL-7 and its mutants.

7. The genetically modified immune cells according to any one of claims 1 to 6, characterized in that, The antigen-binding domain includes HCDR1 shown in SEQ ID NO.7, HCDR2 shown in SEQ ID NO.8, and HCDR3 shown in SEQ ID NO.9, as well as LCDR1 shown in SEQ ID NO.10, LCDR2 shown in EQ ID NO.11, and LCDR3 shown in EQ ID NO.12; Optionally, the antigen-binding domain has a heavy chain variable region as shown in SEQ ID NO.13 and a light chain variable region as shown in SEQ ID NO.

14.

8. The genetically modified immune cells according to any one of claims 1 to 6, characterized in that, The genetically modified immune cells include one or more types of TIL cells, T cells, and NK cells.

9. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the target protein as defined in any one of claims 1 to 8.

10. A carrier, characterized in that, The carrier comprises the nucleic acid molecule according to claim 9; Optionally, the vector includes one or more of lentiviruses, gamma retrovirus vectors, and lipid nanoparticles.

11. The method for constructing genetically modified immune cells according to any one of claims 1 to 8, characterized in that, The construction method includes introducing the nucleic acid molecule of claim 9 or the vector of claim 10 into the immune cells to be modified to achieve the expression of the target protein.

12. The use of the genetically modified immune cells of any one of claims 1 to 8, the nucleic acid molecule of claim 9, or the vector of claim 10 in the preparation of antitumor drugs; Optionally, the antitumor drug is used to treat GPC3-positive tumors; Optionally, the GPC3-positive tumors include solid tumors; Optionally, the GPC3-positive tumors include one or more of liver cancer, melanoma, ovarian cancer, and lung cancer.

13. An antitumor drug, characterized in that, The drug comprises the genetically modified immune cells according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier.

14. A treatment method for a tumor, the treatment method comprising administering to a subject an effective dose of the antitumor drug of claim 13.

Images