GPC3 targeting chimeric antigen receptor gamma delta T cell and application thereof in hepatocellular carcinoma treatment

Abstract

The invention discloses a GPC3 targeting chimeric antigen receptor gamma delta T cell and an application thereof in treatment of hepatocellular carcinoma. The CAR-gamma delta T cell has a third-generation CAR structure and has high targeting accuracy and killing efficiency, the in-vitro killing rate of GPC3 positive hepatoma carcinoma cells (HepG2 / Hep3B) is larger than 90% (the efficiency-target ratio is 10: 1) and is obviously improved compared with that of common gamma delta T cells, the tumor size is obviously reduced in a PDX mouse model, and the excellent treatment effect of the CAR-gamma delta T cell is proved.

Description

Technical Field

[0001] This invention relates to the fields of cell engineering and biomedicine, and in particular to a GPC3-targeting chimeric antigen receptor γδT cell and its application in the treatment of hepatocellular carcinoma. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide, and its incidence has been rising steadily in recent years. Although some progress has been made in treatment methods such as surgical resection and systemic chemotherapy, most patients are diagnosed at an advanced stage due to the insidious onset, rapid progression, and tendency to invade and metastasize early, making radical surgery impossible. This results in limited overall treatment outcomes, and clinical practice still faces serious challenges.

[0003] Chimeric antigen receptor T-cell (CAR-T) therapy is an immunotherapy method that uses genetically engineered T cells to express chimeric receptors that specifically recognize tumor antigens, thereby achieving targeted killing of tumors. Currently, CD19-targeted CAR-T therapy has shown significant efficacy in hematologic malignancies (such as leukemia and lymphoma), but its application in solid tumor treatment still faces many limitations. On the one hand, CAR-T cells may induce adverse reactions such as off-target toxicity, cytokine release syndrome, neurotoxicity, or graft-versus-host disease; on the other hand, the immunosuppressive microenvironment unique to solid tumors severely limits the infiltration and persistence of effector cells, resulting in CAR-T therapy being far less effective against solid tumors than against hematologic malignancies. Therefore, developing novel, highly efficient, and low-toxicity CAR-modified effector cells has significant clinical importance and application value.

[0004] Based on the type of T cell receptor, T cells can be divided into αβ-T cells and γδ-T cells. Traditional CAR-T therapy is mainly based on αβ-T cells, whose antigen recognition depends on the presentation of the major histocompatibility complex (MHC). Furthermore, αβ-T cells may induce graft-versus-host disease in allogeneic transplantation, limiting their widespread application. In contrast, γδ-T cells possess a unique anti-tumor mechanism: their antigen recognition is MHC-independent, allowing them to directly target various tumor-associated antigens. They are also less likely to induce graft-versus-host disease in allogeneic transplantation, exhibiting better safety and tolerability. These characteristics make γδ-T cells a highly promising vector for solid tumor immunotherapy and provide a theoretical basis for the development of CAR-modified γδ-T cells (CAR-γδT).

[0005] Glypican-3 (GPC3) is a highly specific tumor marker in hepatocellular carcinoma (HCC), highly expressed on the surface of 70-90% of HCC cells, but almost not expressed in normal adult tissues. GPC3 promotes tumor proliferation and invasion by activating signaling pathways such as Wnt / β-catenin, and its expression level is closely related to poor patient prognosis and increased risk of vascular invasion, thus it is considered an ideal target for HCC targeted therapy. However, the immunosuppressive properties of the HCC tumor microenvironment, the physical barrier of the tumor stroma, and the spatiotemporal heterogeneity of GPC3 expression severely limit the clinical translation efficacy and safety of GPC3-based targeted therapy strategies.

[0006] In summary, current CAR-T technology still faces the following key challenges in the treatment of solid tumors: 1. Weak targeting specificity and tumor heterogeneity: Traditional CAR-T relies on a single target, which makes it difficult to overcome the heterogeneity of antigen expression in solid tumors, easily leading to antigen escape and tumor recurrence.

[0007] 2. Insufficient cell activation and persistence: Hypoxia, immunosuppressive factors and other factors in the solid tumor microenvironment can easily lead to CAR-T cell depletion, and existing CAR structures (such as second-generation single co-stimulation designs) provide limited support for cell activation.

[0008] 3. Tumor microenvironment infiltration and immunosuppression: The dense matrix of solid tumors and the immunosuppressive microenvironment hinder the effective infiltration of CAR-T cells, and the synergistic mechanism between CAR-T cells and immune checkpoint inhibitors is still unclear.

[0009] 4. Safety and limitations of preparation process: Traditional CAR-T therapy has risks such as graft-versus-host disease and cytokine storm, and the preparation cycle of autologous cells is long and costly, making it difficult to achieve standardized "off-the-shelf" applications.

[0010] Therefore, there is an urgent need to develop a novel CAR-modified effector cell that combines high targeting, strong persistence, good safety, and adaptation to the tumor microenvironment, in order to break through the bottleneck of existing solid tumor immunotherapy and provide new treatment strategies for patients with advanced hepatocellular carcinoma. Summary of the Invention

[0011] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide a GPC3-targeting chimeric antigen receptor γδT cell and its application in the treatment of hepatocellular carcinoma. This invention overcomes the shortcomings of the prior art by developing a GPC3-targeting CAR-γδT cell and its preparation method, thereby enabling its use in the treatment of solid tumors.

[0012] In a first aspect, the present invention provides a chimeric antigen receptor (CAR) molecule, said CAR molecule being a CAR molecule targeting GPC3.

[0013] In some embodiments of the present invention, the CAR molecule comprises: (1) A CAR molecule containing, in tandem, a CD8 signal peptide, a single-chain antibody variable region of a GPC3 antibody, a CD8 hinge region, a CD28 transmembrane region and intracellular segment, a 4-1BB, a CD3ζ signal domain and a fluorescent protein sequence; or (2) Based on (1), a CAR molecular variant after amino acid substitution, addition or deletion, and the CAR molecular variant still has GPC3 targeting.

[0014] In some embodiments of the present invention, the CAR molecule comprises two different co-stimulatory molecules (CD28 and 4-1BB).

[0015] In some embodiments of the present invention, the CAR molecule is a third-generation CAR molecule.

[0016] In this field, third-generation CAR molecules refer to CAR molecules obtained by tandemly incorporating two different co-stimulatory molecules in the intracellular signaling region of the second-generation CAR structure (CD3ζ + a co-stimulatory domain (such as CD28 or 4-1BB)) and binding them to the CD3ζ signaling domain. The improvement of third-generation CAR molecules over second-generation CAR molecules lies in further enhancing the activation signal, survival ability and anti-tumor efficacy of T cells.

[0017] In some embodiments of the present invention, the amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO:1.

[0018] In some embodiments of the present invention, the amino acid sequence of the single-chain antibody variable region of the GPC3 antibody is shown in SEQ ID NO:3.

[0019] In some embodiments of the present invention, the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO:5.

[0020] In some embodiments of the present invention, the amino acid sequences of the transmembrane region and intracellular region of CD28 are shown in SEQ ID NO:7.

[0021] In some embodiments of the present invention, the amino acid sequence of 4-1BB is shown in SEQ ID NO:9.

[0022] In some embodiments of the present invention, the amino acid sequence of the CD3ζ signal domain is shown in SEQ ID NO:11.

[0023] In some embodiments of the present invention, the number of amino acid substitutions, additions, or deletions is 1-10.

[0024] In some embodiments of the present invention, the fluorescent protein includes enhanced green fluorescent protein. Of course, other types of fluorescent proteins or markers may also be used for ease of observation and detection.

[0025] In some embodiments of the present invention, the amino acid sequence of the fluorescent protein is shown in SEQ ID NO:15.

[0026] In some embodiments of the present invention, the CAR molecule further includes a linker.

[0027] In some embodiments of the present invention, the linker includes a self-cleaving peptide, such as 2A peptide.

[0028] In some embodiments of the present invention, the 2A peptide is as shown in SEQ ID NO:13.

[0029] In some embodiments of the present invention, the CAR molecule sequentially comprises the following portions: The GPC3 antibody contains a single-chain antibody variable region (scFv), a CD8 hinge region (CD8 Hinge), a CD28 transmembrane region (CD28 TM) and an intracellular region (CD28 IC), 4-1BB, a CD3ζ signal domain, a 2A peptide, and an enhanced green fluorescent protein (eGFP) for detection.

[0030] In some embodiments of the present invention, the CAR molecule comprises: a CAR molecule with an amino acid sequence as shown in SEQ ID NO:17.

[0031] In some embodiments of the present invention, the CAR molecule variant has GPC3 targeting that is substantially similar to or higher than that of the CAR molecule shown in SEQ ID NO:17.

[0032] In this invention, the term "substantially similar" means that the absolute value of the difference in targeting ability with the CAR molecule GPC3 shown in SEQ ID NO:17 is within 1%, 2%, 3%, 4%, or 5%. In this invention, targeting ability can be determined based on indicators such as binding specificity and efficiency.

[0033] In some embodiments of the present invention, the CAR molecule variant described in (2) has at least 90% sequence identity compared to the CAR molecule shown in SEQ ID NO:17.

[0034] In some embodiments of the present invention, the CAR molecule variant described in (2) has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with respect to the CAR molecule shown in SEQ ID NO:17.

[0035] In this invention, the term "sequence identity" or "homology" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, provided that the sequences are aligned and (if necessary) vacancies are introduced to achieve maximum sequence identity, and no conserved substitutions are considered part of the sequence identity. Sequence alignment used to determine the percentage of amino acid sequence identity can be performed by various methods known to those skilled in the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR). Those skilled in the art can determine appropriate parameters for determining the alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences.

[0036] In some embodiments of the present invention, the CAR molecular variant described in (2) is a CAR molecular variant obtained with the amino acid sequence shown in SEQ ID NO:17 as the core fragment.

[0037] In some embodiments of the present invention, the CAR molecule may also contain chemically modified or non-functional groups.

[0038] In some embodiments of the present invention, the nonfunctional groups include, but are not limited to, tag sequences, targeting peptides, dyes, biotin, and affinity ligands.

[0039] In some embodiments of the present invention, the tag sequence includes, but is not limited to: His-tag, GST-tag, maltose-binding protein tag, Strep-tag, c-Myc tag, HA tag, FLAG tag, V5 tag, AviTag, SNAP-tag, and SUMO tag.

[0040] In some embodiments of the present invention, the chemical modification includes: phosphorylation, acetylation, methylation, ubiquitination or ubiquitination-like processes, glycosylation, esterification, cyclization, enzymatic cleavage, formation of disulfide bonds, and hydroxylation.

[0041] In some embodiments of the present invention, the method of obtaining CAR molecules is not limited, and any conventional method in the art can be used, including but not limited to: solid-phase synthesis, biosynthesis, etc.

[0042] A second aspect of the present invention provides a nucleic acid molecule encoding the CAR molecule described above.

[0043] In some embodiments of the present invention, the nucleic acid molecule encoding the CAR molecule described above is shown in SEQ ID NO:18.

[0044] In some embodiments of the present invention, nucleic acid molecules encoding CD8 signal peptide (CD8SP), single-chain antibody variable region (scFv) of GPC3 antibody, CD8 hinge region (CD8 Hinge), CD28 transmembrane region (CD28 TM) and intracellular region (CD28 IC), 4-1BB, CD3ζ signal domain, 2A peptide, and enhanced green fluorescent protein (eGFP) for detection are shown in SEQ ID NO:2, 4, 6, 8, 10, 12, 14 and 16, respectively.

[0045] A third aspect of the present invention provides an expression vector containing the nucleic acid molecules described above.

[0046] In some embodiments of the present invention, the expression vector includes a plasmid.

[0047] In some embodiments of the present invention, the expression vector is the lentiviral expression plasmid pWPXLd.

[0048] In some embodiments of the present invention, a three-plasmid packaging system is used for lentivirus packaging, wherein the helper plasmid is a commercially available helper plasmid.

[0049] In some embodiments of the present invention, the auxiliary plasmids are pSPAX2 and pMD2.G.

[0050] Of course, the expression vector of the present invention can be any suitable expression vector capable of being used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably to express the genes or sequences in the host cell. Suitable vectors include those designed for expansion and amplification or for expression or both of the above, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0051] A third aspect of the present invention provides a transformant comprising the nucleic acid molecule and / or expression vector described in the above aspects.

[0052] In this invention, the term "transformant" refers to any type of host cell. In some embodiments of this invention, the host cell is derived from or obtained from a mammal, preferably a human. In this invention, the host cell may include cells in which exogenous nucleic acids have been introduced, as well as the progeny of these cells. The progeny may not be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. This invention also includes mutant progeny that have the same function or biological activity as cells screened or selected in the initially transformed cells.

[0053] In some embodiments of the present invention, the transformant comprises cytotoxic immune cells.

[0054] In some embodiments of the present invention, the cells include cytotoxic T cells.

[0055] In this invention, T cells can be any type of T cell, such as cultured T cells, like primary T cells, or T cells derived from cultured T cell lines, such as Jurkat, SupTl, etc., or T cells obtained from mammals. If obtained from mammals, the T cells can be derived from numerous sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or converted. T cells can be obtained by maturing hematopoietic stem cells into T cells in vitro or in vivo.

[0056] In some embodiments of the present invention, the T cells are human T cells.

[0057] In some embodiments of the present invention, the T cells are T cells isolated from humans.

[0058] In some embodiments of the present invention, the T cells are γδT cells.

[0059] A fourth aspect of the invention provides a pharmaceutical composition comprising the transformant described above and, optionally, a pharmaceutically acceptable excipient.

[0060] In some embodiments of the present invention, the pharmaceutically acceptable excipients are rationally selected based on factors such as the product form of the pharmaceutical composition, the intended use, and the route of administration, and include, but are not limited to: buffers, coenzymes, enzyme protectants, metal ions, catalysts, defoamers, diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, dicalcium phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), solvents, pH adjusters, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic adjusters (such as glucose, sodium chloride, etc.), chelating agents (such as disodium EDTA), etc.

[0061] In some embodiments of the present invention, the pharmaceutical composition further includes a second active substance.

[0062] In some embodiments of the present invention, the second active substance includes an immune checkpoint inhibitor.

[0063] In some embodiments of the present invention, the immune checkpoint inhibitor includes at least one of PD-1 and CTLA-4 antibodies.

[0064] In some embodiments of the present invention, the immune checkpoint inhibitors are pembrolizumab (PD-1 antibody) and texilimumab (CTLA-4 antibody).

[0065] A fifth aspect of the present invention provides the use of at least one of the CAR molecules, nucleic acid molecules, expression vectors, transformants, and pharmaceutical compositions described above in the preparation of an antitumor drug.

[0066] In some embodiments of the present invention, the tumor is a tumor expressing GPC3.

[0067] In some embodiments of the present invention, the tumor includes liver cancer expressing GPC3.

[0068] In some embodiments of the present invention, the liver cancer expressing GPC3 includes liver cancer based on HepG2 or Hep3B liver cancer cells.

[0069] In some embodiments of the present invention, the antitumor drug can be administered via intratumoral injection and tail vein injection.

[0070] In some embodiments of the present invention, the antitumor drug is administered via intratumoral injection.

[0071] The beneficial effects of this invention are: 1. This invention provides a GPC3-targeting CAR-γδT cell that integrates a third-generation CAR structure (CD28+4-1BB dual co-stimulatory domain) with γδT cells, achieving high targeting precision and killing efficiency. Its in vitro killing rate against GPC3-positive liver cancer cells (HepG2 / Hep3B) is >90% (effect-to-target ratio 10:1), significantly higher than that of ordinary γδT cells, and it significantly reduces tumor volume in a PDX mouse model, demonstrating its excellent therapeutic effect.

[0072] 2. This invention achieves efficient in vitro expansion of γδT cells based on ZOL, and further evaluates the effects of this method on the purity, expansion fold, and cytotoxicity of γδT cells, thus confirming the high efficiency and reliability of the method.

[0073] 3. In this invention, it is demonstrated that intratumoral injection significantly improves efficacy compared to tail vein injection, and this local administration avoids systemic CRS. Attached Figure Description

[0074] Figure 1 This is a schematic diagram of the third-generation CAR molecule targeting GPC3 in this invention.

[0075] Figure 2 The expression of GPC3 protein in HepG2, Hep3B and SK-HEP-1 cell lines was detected by Western blotting. In the figure, A represents the bands and B represents the corresponding statistical graph.

[0076] Figure 3 This study describes the changes in OD (dose distribution) of γδT cells after stimulation and expansion using ZOL in vitro.

[0077] Figure 4 Results of flow cytometry detection of the positivity rate of CAR-γδ T.

[0078] Figure 5 The in vitro killing effect of CAR-γδ T cells on liver cancer cell lines is shown in Figure A, where A represents the in vitro killing effect of the control group and experimental group at different effector-to-target ratios after 12 hours of co-culture, and B represents the secretion of cytokines after 12 hours of co-culture at an effector-to-target ratio of 10:1.

[0079] Figure 6 The graph shows the changes in PD-1 expression levels in CAR-γδ T cells after co-culturing with target cells. In the graph, A shows the changes in PD-1 expression levels after 3, 5, and 7 days of co-culturing, B shows the changes in PD-1 expression levels after co-culturing with a 5:1 effector-target ratio for different durations, and C and D are the corresponding line graphs of A and B.

[0080] Figure 7 The in vivo killing effect of CAR-γδ T cells on liver cancer cells is shown in Figure 1. A is the experimental flowchart, B is the growth curve of subcutaneous tumor volume in each group of mice over time, C is the physical image of subcutaneous tumor in each group of mice after the experiment, and D is the statistical results of subcutaneous tumor volume and weight in each group of mice after the experiment.

[0081] Figure 8 To illustrate the in vivo killing effect of CAR-γδ T cells targeting GPC3 on HepG2 subcutaneous tumors, the following diagrams are presented: A is the experimental flowchart; B is the growth curve of subcutaneous tumor volume in each group of mice over time; C is the physical image of subcutaneous tumors in each group of mice after the experiment; and D is the statistical results of the volume and weight of subcutaneous tumors in each group of mice after the experiment.

[0082] Figure 9The in vivo killing effect of CAR-γδ T cells combined with PD-1 antibody and CTLA-4 antibody on HepG2 subcutaneous tumors is shown in Figure A, which is the experimental flowchart; Figure B is the growth curve of subcutaneous tumor volume in each group of mice over time; and Figures C and D are the statistical results of subcutaneous tumor volume and weight in each group of mice after the experiment.

[0083] Figure 10 H&E staining images of organs (heart, lungs, liver, spleen, and kidney tissues) of mice in each group. Detailed Implementation

[0084] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0085] Example 1 In this embodiment, a GPC3-targeting CAR is provided, which is a third-generation CAR containing two different co-stimulatory molecules (CD28 and 4-1BB).

[0086] Specifically, such as Figure 1 As shown, the CAR gene sequence consists of the following parts in sequence: The GPC3 antibody contains a single-chain antibody variable region (scFv), a CD8 hinge region (CD8 Hinge), a CD28 transmembrane region (CD28 TM) and an intracellular region (CD28 IC), 4-1BB, a CD3ζ signal domain, a 2A peptide, and an enhanced green fluorescent protein (eGFP) for detection.

[0087] The sequence information for each part is shown in the table below.

[0088] Table 1. Sequence information of each component in the CAR molecule

[0089] The CAR molecule was constructed using conventional methods in the art. Specifically, the gene sequences of each domain were tandemly linked into a hybrid gene using molecular cloning technology to obtain a recombinant plasmid (using a commercially available pWPXLd plasmid containing GFP as a vector). This plasmid, along with helper plasmids (commercially available pSPAX2 and pMD2.G vectors), was then inserted into a lentiviral vector to obtain the CAR molecule. In this embodiment, the CAR molecule was directly synthesized by a biosynthetic company using the above method.

[0090] The full-length amino acid sequence and nucleotide sequence of the obtained CAR molecule are shown below.

[0091]

[0092] The titer of the obtained CAR molecule was determined to be 6.51 × 10⁻⁶. 8 TU / mL.

[0093] Example 2 In this embodiment, liver cancer cell lines are screened to obtain liver cancer cell lines expressing GPC3.

[0094] Specifically, in this embodiment, Western blotting and human recombinant GPC3 protein (purchased from Abcam) were used to detect the expression level of GPC3 protein in HCC cell lines HepG2, Hep3B and SK-HEP-1.

[0095] The results are as follows Figure 2 As shown.

[0096] It was observed that GPC3 protein was strongly expressed in the HepG2 and Hep3B cell lines, while no GPC3 protein expression was detected in the SK-HEP-1 cell line. Therefore, in subsequent experiments, the HepG2 and Hep3B cell lines expressing GPC3 protein were selected as the experimental groups, while the SK-HEP-1 cell line was used as the negative control group, to evaluate the targeting and cytotoxic functions of GPC3-targeting CAR-γδT cells.

[0097] Example 3 In this embodiment, a method for obtaining and expanding γδT cells is provided.

[0098] The specific steps include: (1) Extraction of PBMCs from healthy donors: Take an appropriate amount of peripheral blood sample from a healthy donor and dilute it with an equal volume of PBS to reduce blood viscosity and facilitate better separation with the lymphocyte separation medium. Add a certain amount of Ficoll lymphocyte separation medium (purchased from TBD) to a new centrifuge tube. The amount of Ficoll lymphocyte separation medium added should be approximately half the volume of the diluted blood, i.e., peripheral blood sample volume: PBS volume: Ficoll lymphocyte separation medium volume = 1:1:1. Slowly add the diluted peripheral blood sample along the wall of the centrifuge tube onto the surface of the Ficoll lymphocyte separation medium using a pipette. The addition should be slow to avoid disrupting the separation interface. Centrifuge. Centrifugation parameters can be adjusted appropriately based on centrifuge machine differences. In this example, the centrifugation force is 800 g, 4°C for the initial acceleration and 0°C for the final acceleration, and centrifugation for 30 minutes. After centrifugation, the centrifuge tube will roughly separate into a red blood cell layer, a separation medium layer, a PBMC layer, and a plasma layer from bottom to top. Carefully aspirate the PBMC layer using a pipette, avoiding the red blood cell layer as much as possible. Transfer the aspirated PBMC layer to a new centrifuge tube, add an appropriate amount of PBS, and centrifuge again (300 g, 10 min). Discard the supernatant. Repeat this step twice to remove residual lymphocyte separation fluid and other impurities. Add an appropriate amount of erythrocyte lysis buffer (purchased from Beyotime Biotechnology) according to the cell volume. Gently mix with a pipette and let stand at room temperature for 5-10 minutes. Add an appropriate amount of PBS to terminate the lysis reaction; the volume of PBS added should be approximately equal to the volume of erythrocyte lysis buffer added. Centrifuge again (300 g, 10 min). Discard the supernatant and resuspend the PBMC pellet in T cell expansion medium (purchased from Stemcell) + ZOL (zoledronic acid, final concentration 10 μM) + IL-2 (final concentration 500 IU / mL). ZOL (zoledronic acid) can effectively stimulate γδT cells in PBMCs, promoting their activation and expansion.

[0099] (2) Sorting of γδT cells: Using the TCR γδT cell isolation kit (purchased from Miltenyi Biotec), γδT cells were isolated from the PBMC cell suspension obtained in the above steps according to the instructions for use.

[0100] The specific steps are as follows: Resuspend the PBMC cell suspension in PBS buffer containing 2% FBS, adjusting the PBMC cell suspension volume appropriately based on the PBMC cell concentration. When the cell concentration is approximately 1×10⁻⁶ cells / mL... 8 When the cell concentration is <1×10⁶ cells / mL, the cell suspension volume is 100 µL - 2.5 mL; when the cell concentration is <1×10⁶ cells / mL, the cell suspension volume is 100 µL - 2.5 mL. 7 The cell suspension volume was 0.1 mL. If the PBMC cell content was less than 2%, the concentration was adjusted to 2 × 10⁻⁶ cells / mL. 8 cells / mL.

[0101] Add Anti-Human CD32 (Fc gamma RII) Blocker to the PBMC cell suspension at a ratio of 100 µL / mL (i.e., 100 µL of Anti-Human CD32 (Fc gamma RII) Blocker to 1 mL of cell suspension) and mix well. Then add PE anti-human TCRγ / δ (400 µg / mL) at a ratio of 0.3–3 µg / mL (relative to the cell suspension), mix well, and incubate at room temperature for 15 minutes. After incubation, add an excess of 10 volumes of buffer (PBS + 2% FBS + 1 mM EDTA), centrifuge at 300×g for 10 minutes at room temperature, centrifuge at 4°C and 0°C, carefully remove the supernatant, and resuspend to the initial volume. Add EasySep™ PE Selection Cocktail at a ratio of 100 µL / mL (i.e., 100 µL of Cocktail to 1 mL of cell suspension), mix well, and incubate at room temperature for 15 minutes. Vortex the magnetic beads (EasySep™ DextranRapidSpheres™) for 30 seconds to ensure uniform dispersion. Add the dispersed magnetic beads at a ratio of 50 µL / mL, mix thoroughly, and incubate at room temperature for 10 minutes. Adjust the volume to 2.5 mL using the above buffer solution and gently mix with a pipette. Place the flow cytometry tube (without the cap) into the magnet (inserting the tube to the bottom) and incubate at room temperature for 5 minutes. While maintaining the flow cytometry tube position, tilt the entire magnet and flow cytometry tube, holding this tilted position for 2-3 seconds to allow the liquid to flow naturally. Do not shake or aspirate with a pipette to preserve the target cells. Remove the flow cytometry tube from the magnet. Repeat twice to obtain the target cells. Resuspend the cells in the above T cell expansion medium to obtain purified TCR γδ+ cells. Continue culturing using T cell expansion medium.

[0102] The growth of TCR γδ+ cells was detected using the CCK-8 reagent. The specific steps were as follows: Prepare five 96-well plates, and precisely add 100 μL of purified γδT cell suspension (approximately 2000 cells / well) to each well. After seeding, place all five 96-well plates together in a cell culture incubator. On days 1, 3, 5, 7, and 9 of culture, remove one well from the incubator for CCK-8 assay. The CCK-8 assay must be performed in the dark. Specifically, slowly add 10 μL of CCK-8 solution to each well. Avoid introducing air bubbles into the wells during addition to prevent interference with absorbance (OD) measurement. Then return the plate to the incubator and continue incubation for 2.5 hours to ensure sufficient reaction between CCK-8 and the cells. After incubation, remove the plate from the incubator. Before reading the absorbance, gently place the plate on a shaker to mix and ensure uniform cell distribution. The absorbance of each well at a wavelength of 450 nm was measured using an ELISA reader.

[0103] (3) Detection of γδT cell purity based on flow cytometry: Based on the above steps, PBMCs were extracted from healthy donors, and γδT cells were sorted. 10 μM ZOL was added to stimulate γδT cell proliferation. Then, on days 3, 5, 7, 9, and 11 of culture, approximately 3 mL of cell suspension was aspirated from the culture medium. After centrifugation at 800 rpm for 3 minutes, the supernatant was discarded, and the cells were washed once with 2 mL PBS, followed by another centrifugation at 800 rpm for 3 minutes. The supernatant was discarded, and the cell pellet was resuspended in 200 μL PBS. The obtained cell suspension was divided into control tubes and experimental tubes. The control tubes were left untreated, while the experimental tubes contained 5 μL of PE-labeled anti-human TCR γδ flow cytometry antibody (purchased from Biolegend) and 5 μL of FITC-labeled anti-human CD3 flow cytometry antibody (purchased from Biolegend), and incubated at 4°C in the dark for 30 minutes. After incubation, the cells were centrifuged at 800 rpm for 3 minutes, washed once with PBS, and then resuspended in PBS for flow cytometry analysis. After the test was completed, the experimental data were recorded and exported. FlowJo software (version 10.8.1) and GraphPad Prism software (version 10.1.2) were used for data summarization, graphing, and statistical analysis.

[0104] (4) Construction of CAR-γδT cells targeting GPC3: Construction of GFP and Luciferase double-positive (GL) hepatocellular carcinoma cell lines: On day 1 of the experiment, SK-HEP-1, HepG2, and Hep3B cells were seeded into 6-well plates. The seeding density was adjusted according to the cell growth rate, with the standard being that the cells could grow to approximately 50% confluence by the next day. On the next day, the required viral load for each cell line was calculated based on the cell count and the multiplicity of infection (MOI) value. The corresponding amounts of luciferase-containing lentiviral plasmid (LV5-LUC-GFP-Puro) (purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.) and polybrene (final concentration 10 μg / mL) were added to the corresponding wells for cell transfection. Twelve hours after transfection, the cell status was observed under a microscope. The transfection time was extended appropriately according to the cell status, but generally, the total infection time was controlled within 24 hours. After transfection, the culture medium was replaced with fresh medium containing 2 μg / mL puromycin and cultured for 7 days to complete the selection. On day 7 of culture, cells were digested with trypsin, washed once with PBS, the supernatant was removed, and the cell pellet was resuspended in PBS to obtain GFP and Luciferase double-positive (GL) hepatocellular carcinoma cell lines. Flow cytometry was used to detect the GFP expression rate of each cell line. If the results showed a low expression rate, the above procedure could be repeated to obtain a cell population with a high positive rate.

[0105] Lentiviral infection of γδT cells: Following the GL cell line construction method, the lentiviral solution was replaced with the CAR molecules described in the above examples to obtain CAR-γδT cells targeting GPC3. On day 3 of the experiment, 10 μL of cell suspension and 10 μL of trypan blue solution were aspirated, mixed thoroughly, and then 10 μL of the mixture was added to a hemocytometer to confirm the number of viable cells. Based on the obtained number of viable cells and the multiplicity of infection (MOI) value, the total volume of lentiviral agent required for infection was calculated.

[0106] (5) In vitro killing effect of CAR-γδT cells on liver cancer cell lines: The in vitro killing effects of γδT cells (GL cells) and CAR-γδT cells were detected using a luciferase assay. Specifically, the corresponding target cells (the aforementioned liver cancer cells) were digested with trypsin and then counted, at a ratio of 1×10⁻⁶. 4Cells were seeded per well in 96-well plates, with experimental groups (CAR-γδT cells) and control groups (GL cells). Each group had three replicates to ensure accuracy and reproducibility. Based on the grouping, appropriate amounts of GL and CAR-γδT cell suspensions were centrifuged at 800 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in fresh culture medium and counted. The required cell number for each group was calculated based on different effector-to-target ratios (E:T) (1:1, 2.5:1, 5:1, and 10:1). The 96-well plates were gently rinsed once with PBS, and the prepared cell suspensions were added to the corresponding wells according to the different effector-to-target ratios. The 96-well plates were co-cultured in a cell culture incubator for 6 or 12 hours. The supernatant was discarded, and the 96-well plates were gently rinsed once with PBS. 100 μL of sodium fluorescein (to a final concentration of 150 μg / mL) was added to each well, and the reaction was carried out for 10 minutes in the dark. The fluorescence value was then detected using a microplate reader. The obtained data were organized and summarized, and the percentage of effector cell killing was calculated according to the formula.

[0107] Percentage of damage = ×100%.

[0108] The control wells are those containing only target cells and no effector cells (i.e., GL cells or CAR-γδT cells). GraphPad Prism software was used for data aggregation, graphing, and statistical analysis.

[0109] Meanwhile, the culture supernatant from the culture medium at different times was collected in centrifuge tubes for the detection of cytokines.

[0110] Specifically, cytokines were detected using enzyme-linked immunosorbent assay (ELISA): the supernatant (effect-to-target ratio of 10:1) was collected, centrifuged at 1000 g for 20 minutes at 4°C, and the supernatant was slowly aspirated for detection. The ELISA cytokine detection kit (purchased from Reddy's Biotechnology (Wuhan) Co., Ltd.) was used, and the detection was performed according to the instructions.

[0111] Simultaneously, flow cytometry was used to detect PD-1 expression levels after co-culturing γδT cells and hepatocellular carcinoma cells. The effector-to-target ratio was set at 5:1, and the number of γδT cells was 5 × 10⁶. 4 Cells / well were cultured for 0 hours, 6 hours, and 12 hours, respectively. The antibodies used for flow cytometry were PE-labeled anti-human TCR γδ flow cytometry antibody, FITC-labeled anti-human CD3 flow cytometry antibody, and APC-labeled anti-human PD-1 flow cytometry antibody, respectively.

[0112] The results are as follows Figure 3-6 As shown.

[0113] Figure 3This study shows the changes in OD (oxidative stress) of γδT cells after stimulation and expansion using ZOL (zolium hydroxide) in vitro. It was found that a ZOL concentration of 10 μM had the best stimulating effect on γδT cells.

[0114] Figure 4 The results of flow cytometry analysis of the CAR-γδ T positivity rate show that the method described in this embodiment can achieve a high CAR-γδ T positivity rate.

[0115] Figure 5 The study investigated the in vitro killing effect of CAR-γδ T cells on hepatocellular carcinoma cell lines. It was found that, except for the SK-HEP-1 cell line which does not express GPC3, the other two cell lines exhibited significant in vitro killing effects at four different effector-target ratios: 1:1, 2.5:1, 5:1, and 10:1. ELISA results also showed that, after co-culturing for 12 hours at an effector-target ratio of 10:1, there were significant differences in cytokine secretion between the two cell lines, except for the SK-HEP-1 cell line which does not express GPC3.

[0116] Figure 6 The study investigated the changes in PD-1 expression levels in cells after co-culturing with target cells. Significant differences in PD-1 expression levels were observed after co-culturing human PBMCs with ZOL for 3, 5, and 7 days. Furthermore, the upregulation of PD-1 expression levels was associated with prolonged culture time, showing a progressive increase with increasing culture time (n=3). Similarly, co-culturing CAR-γδ T cells with HepG2 cells at a 5:1 effector-target ratio for different durations also resulted in significant changes in PD-1 expression levels, which similarly increased with prolonged co-culture time.

[0117] Example 4 In this embodiment, the in vivo killing effect of CAR-γδT cells targeting GPC3 on hepatocellular carcinoma was further verified.

[0118] Experimental flowchart as follows Figure 7 , Figure 8 and Figure 9 As shown in A, the specific experimental steps include: (1) Construction of a Hep3B subcutaneous tumor model of liver cancer: Hep3B cells were cultured according to the requirements for animal experiments. When the tumor cells reached the logarithmic growth phase and approximately 80% confluence, the supernatant was discarded, and the cells were washed once with PBS. 1 mL of 0.25% trypsin was added to each 100 mm culture dish to digest the cells. The digested cells were then transferred to centrifuge tubes, centrifuged, and washed once with PBS. The supernatant was discarded, and the Hep3B cell pellet was resuspended in 1 mL of serum-free PBS. The cells were then counted, and the cells were prepared into 2 × 10⁶ cells / mL culture media. 6 One cell per 100 μL of cell suspension. Using a 1 mL syringe, 100 μL of cell suspension was drawn and injected subcutaneously approximately 1 cm below the right axilla of NSG mice. The injection site was visibly punctured, and a small wheal was observed. When the subcutaneous tumor in the mouse reached approximately 50 mm in size... 3 A successful model is considered to have been constructed. Typically, a subcutaneous tumor model can be constructed within 10 days.

[0119] (2) Treatment of hepatocellular carcinoma based on CAR-γδT cells targeting GPC3: The experimental mice were randomly divided into 3 groups of 5 mice each. The specific grouping details are as follows: Untreated group: Model mice that did not receive any injection treatment; The treatment groups included: a CAR-γδT cell tail vein injection group and an intratumoral injection group. Injections were administered every 5 days, with a CAR-γδT cell dosage of 1×10⁻⁶ cells. 7 One per animal per injection, for a total of 6 injections.

[0120] During the experiment, mice in each group were marked, and the longest and shortest diameters of the tumors were measured periodically (every 3 days) using calipers to calculate the volume of the subcutaneous tumors and record the experimental data.

[0121] After the entire experimental procedure was completed (day 40), mice in each group were euthanized, and subcutaneous tumors were dissected and isolated.

[0122] (3) Treatment of hepatocellular carcinoma with CAR-γδT cells combined with monoclonal antibodies (anti-PD-1 antibody) or bispecific antibodies (anti-PD-1 antibody and anti-CTLA-4 antibody): The Hep3B subcutaneous tumor model of liver cancer was constructed in the same manner as in (1). Experimental mice were randomly divided into 5 groups of 5 mice each. The specific groupings included: PBS group, γδT cell group, CAR-γδT cell (i.e., GL cell) group, CAR-γδT cell combined with anti-PD-1 antibody group, and CAR-γδT cell combined with anti-PD-1 antibody and CTLA-4 antibody group. The cell injection volume was 1×10⁻⁶ cells / mL. 750 μL / mouse / dose. The anti-PD-1 antibody was pembrolizumab (injection), administered at a dose of 130 μg / mouse. The CTLA-4 antibody was tesimumab (injection), administered at a dose of 200 μg / mouse. The CAR-γδT cell combination with anti-PD-1 antibody and the CAR-γδT cell combination with both anti-PD-1 and CTLA-4 antibody groups were administered with 1×10... 7 A mixture of CAR-γδT cells was prepared into a 50 μL solution for administration. Treatment was administered via injection every 5 days for a total of 3 injections.

[0123] Calculate the size of the subcutaneous tumor using the method shown in (2).

[0124] After the entire experimental procedure was completed (day 22), mice in each group were euthanized, and subcutaneous tumors, as well as important organs such as the heart, lungs, liver, spleen, and kidneys, were dissected, separated, and collected for subsequent analysis and research.

[0125] Mouse organs were stained with hematoxylin and eosin (HE).

[0126] The results are as follows Figure 7-9 As shown.

[0127] It can be observed that intratumoral injection of CAR-γδT cells in this embodiment of the invention has a better therapeutic effect on subcutaneous hepatocellular carcinoma than tail vein injection. CAR-γδT cells targeting GPC3 alone already showed a significant tumor-suppressing effect on HepG2 subcutaneous tumors, and this effect was further enhanced by combining them with anti-PD-1 and CTLA-4 antibodies.

[0128] To further evaluate the potential effects and safety of in vivo CAR-γδ T cell reinfusion on normal organs in mice, the inventors dissected and collected the hearts, lungs, livers, spleens, and kidneys of mice from each group and performed H&E staining analysis. Microscopic examination of organ sections was used to systematically assess the presence of tissue damage or pathological changes.

[0129] The results are as follows Figure 10 As shown.

[0130] The experimental results showed that no significant tissue damage or pathological changes were observed in the heart, lungs, liver, spleen, and kidneys of mice in any group, and no tumor formation was observed. This result also indicates that in vivo infusion of CAR-γδ T cells did not induce GvHD and has a high safety profile.

[0131] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A chimeric antigen receptor (CAR) molecule, characterized in that, The CAR molecule is a CAR molecule that targets GPC3; The CAR molecule includes: (1) A CAR molecule containing, in sequence, a CD8 signal peptide, a single-chain antibody variable region of a GPC3 antibody, a CD8 hinge region, a CD28 transmembrane region and intracellular segment, a 4-1BB, a CD3ζ signal domain and a fluorescent protein sequence; The amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO:1; the amino acid sequence of the variable region of the single-chain antibody of GPC3 is shown in SEQ ID NO:3; the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO:5; the amino acid sequences of the transmembrane region and intracellular region of CD28 are shown in SEQ ID NO:7; the amino acid sequence of 4-1BB is shown in SEQ ID NO:9; and the amino acid sequence of the CD3ζ signal domain is shown in SEQ ID NO:

11. (2) Based on (1), a CAR molecule variant after amino acid substitution, addition or deletion, and the CAR molecule variant still has GPC3 targeting; Preferably, the fluorescent protein includes enhanced green fluorescent protein, the amino acid sequence of which is shown in SEQ ID NO:

15.

2. The CAR molecule according to claim 1, characterized in that, The CAR molecule also includes: a linker; Preferably, the CAR molecule comprises a CAR molecule with an amino acid sequence as shown in SEQ ID NO:

17.

3. A nucleic acid molecule encoding the CAR molecule of any one of claims 1-2.

4. An expression vector containing the nucleic acid molecule of claim 3; Preferably, the expression vector includes a plasmid.

5. A transformant containing the nucleic acid molecule of claim 3 and / or the expression vector of claim 4; Preferably, the transformant comprises cytotoxic immune cells; Preferably, the cells comprise cytotoxic T cells; Preferably, the T cells are γδ-T cells.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes the transformant of claim 5, and optionally a pharmaceutically acceptable excipient.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition also includes a second active substance; Preferably, the second active substance includes an immune checkpoint inhibitor; Preferably, the immune checkpoint inhibitor includes at least one of PD-1 antibody and CTLA-4 antibody.

8. The use of at least one of the CAR molecule of any one of claims 1-2, the nucleic acid molecule of claim 3, the expression vector of claim 4, the transformant of claim 5, and the pharmaceutical composition of any one of claims 6-7 in the preparation of an antitumor drug; Preferably, the tumor is a solid tumor.

9. The application according to claim 8, characterized in that, The tumor is a tumor that expresses GPC3.

10. The application according to claim 9, characterized in that, The tumors include liver cancer expressing GPC3; Preferably, the liver cancer expressing GPC3 includes liver cancer based on HepG2 or Hep3B liver cancer cells.

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