Targeted GPC3 chimeric antigen receptor macrophage and pharmaceutical composition thereof

Abstract

The invention belongs to the technical field of cellular immunotherapy, and particularly relates to a targeted GPC3 chimeric antigen receptor macrophage and a pharmaceutical composition thereof. According to the present invention, the chimeric antigen receptor modified engineering immune cell specifically targeting GPC3 is prepared by using the chimeric antigen receptor modified macrophage technology, the preparation method has simple steps, and the obtained novel engineering immune cell can specifically recognize tumor cells, can more effectively target and attack the tumor cells, has a high tumor killing rate, and can be used for preparing the GPC3-targeted engineering immune cell. And the compound can be used for preparing anti-tumor products, especially for preparing medicines for treating GPC3 positive tumors, so that a novel tumor treatment means with an application prospect is provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell immunotherapy, and particularly relates to a chimeric antigen receptor macrophage targeting GPC3 and a pharmaceutical composition thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) accounts for approximately 90% of all liver cancers. According to statistics, the number of HCC patients worldwide reached 776,000 in 2019. This number is expected to increase to 876,600 in 2024 and 1 million in 2030, with a compound annual growth rate of 2.3% from 2024 to 2030. my country has a high incidence of liver cancer, accounting for over 45% of HCC cases.

[0003] Existing treatments for liver cancer generally include surgery, drug therapy, or radiotherapy. Surgery is currently the most mainstream treatment method, which involves physically removing the tumor from the human body. However, surgery carries certain risks, and if the surgical resection is not complete, there is a high possibility of recurrence, making it not the best option.

[0004] With the rapid development of biotechnology, in addition to surgical removal of tumors, naturally occurring immune cells in the human body can also be used to eliminate tumor cells, especially macrophages. Macrophages can not only phagocytize and digest cell debris or pathogens, but also activate lymphocytes or other immune cells to kill pathogens. How to overcome the low specificity and safety of effector cells in the tumor microenvironment facing existing tumor clinical technologies, and provide a method for targeted phagocytosis of liver cancer cells to achieve the purpose of treating liver cancer, has become an urgent problem to be solved. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for targeted phagocytosis of liver cancer cells to achieve the treatment of liver cancer. The present application discloses a macrophage modified with a targeted GPC3 chimeric antigen receptor and a preparation method thereof. One advantage of the macrophage modified with a targeted GPC3 chimeric antigen receptor is that it can phagocytose and digest tumor cells expressing GPC3 in hepatocellular carcinoma, and further activate other immune cells to kill the GPC3-expressing tumor cells, thereby achieving targeted phagocytosis and digestion of tumor cells in hepatocellular carcinoma by macrophages.

[0006] More specifically, the GPC3-targeted chimeric antigen receptor modified macrophages disclosed in the present application express the GPC3-targeted chimeric antigen receptor, wherein the chimeric antigen receptor comprises an antigen binding domain, a hinge region, a transmembrane domain, and a signaling domain, and the antigen binding domain is an anti-GPC3 single-chain antibody.

[0007] Preferably, the hinge region comprises CD8.

[0008] Preferably, the transmembrane domain comprises the CD8 transmembrane region.

[0009] Preferably, the signaling domain comprises CD3ζ.

[0010] Preferably, the signaling domain further comprises any one or a combination of at least two of 4-1BB, CD28 intracellular region, DAP10 or OX40.

[0011] Preferably, the chimeric antigen receptor comprises an anti-GPC3 single-chain antibody, a CD8 hinge region, a CD8 transmembrane region, and CD3ζ.

[0012] Preferably, the antigen-binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 2.

[0013] Preferably, the hinge region of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 1.

[0014] Preferably, the transmembrane domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 3.

[0015] Preferably, the signaling domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:4.

[0016] Furthermore, the present application also discloses a method for preparing macrophages modified with a GPC3-targeting chimeric antigen receptor, wherein the method for preparing macrophages modified with a GPC3-targeting chimeric antigen receptor specifically comprises: Synthesizing the nucleic acid sequences of each structural domain of the transmembrane expression protein or polypeptide macromolecule targeting the GPC3 chimeric antigen receptor to obtain the nucleic acid molecule of the target fragment targeting the GPC3 chimeric antigen receptor; Connecting the target fragment to the overexpression vector to obtain a recombinant plasmid vector; amplifying the recombinant plasmid vector and the helper plasmid vector; Obtaining a packaged virus containing the target fragment based on the recombinant plasmid vector and the helper plasmid vector; Macrophages are infected with the virus to obtain modified macrophages expressing a chimeric antigen receptor targeting GPC3.

[0017] Furthermore, the overexpression vector is an adenovirus or lentivirus overexpression vector.

[0018] Furthermore, the present application also discloses a pharmaceutical composition, one advantage of which is that the pharmaceutical composition can achieve phagocytosis and digestion of tumor cells in liver cancer patients.

[0019] Furthermore, the pharmaceutical composition includes macrophages modified with the GPC3-targeting chimeric antigen receptor disclosed in the present application.

[0020] More specifically, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention utilizes chimeric antigen receptor-modified macrophage technology to prepare engineered immune cells modified with chimeric antigen receptors that specifically target GPC3. This preparation method is simple and highly targeted. The resulting novel engineered immune cells can specifically recognize tumor cells, effectively overcoming the problem faced by existing clinical tumor treatment techniques of the lack of specificity of effector cells in the tumor microenvironment in binding to and killing tumor cells. This provides a method for targeted phagocytosis of liver cancer cells, thereby achieving the goal of treating liver cancer. The GPC3-targeted chimeric antigen receptor-modified macrophages disclosed in this application have a high killing rate and can be used to prepare anti-tumor products, especially drugs for treating GPC3-positive tumors. The invention is expected to be used to prepare anti-tumor products, especially for treating liver cancer. In addition to liver cancer, it is also expected to be applied to prepare drugs for treating GPC3-positive lung adenocarcinoma, gastric cancer, pancreatic cancer, brain cancer, prostate cancer, lymphoma, leukemia, intestinal cancer, lung cancer, ovarian cancer, or breast cancer, showing good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic structural diagram of the anti-GPC3 CAR vector of a preferred embodiment of the present application.

[0023] Figure 2 This is a plasmid map of the CAR-GPC3 adenovirus vector (pAd5F35-CAR-GPC3-mCherry) in a preferred embodiment of the present application.

[0024] Figure 3 This is a map of the GPC3 overexpression vector (pCDH-CMV-MCS-GPC3-V5-HA-puro) of a preferred embodiment of the present application.

[0025] Figure 4This is the experimental data that Huh7 / HepG2 / C3A was screened as GPC3-positive target cells through WB verification.

[0026] Figure 5 The data are from the experiment after RAW264.7 cells transduced with ad5f35 were co-cultured with C3A / Huh7 / HepG2 / 293FT for 6 h.

[0027] Figure 6 These are the results of the Fluc killing assay and LDH killing experiment of the engineered immune cells' ability to kill GPC3-positive cells disclosed in this application. Implementation Method

[0028] The present invention is further described below by way of examples, but the present invention is not limited to these specific embodiments. Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] The term "scFv" as used herein refers to an antibody fragment that is a recombinant protein comprising a heavy chain variable region (VH) and a light chain variable region (VL) connected by a linker. The linker allows these two domains to associate to ultimately form an antigen-binding site. The size of an scFv is generally one-sixth that of an intact antibody. An scFv is preferably an amino acid sequence encoded by a single nucleotide chain. The scFv used in the present invention can be further modified using conventional techniques known in the art, such as amino acid deletion, insertion, substitution, addition, and / or recombination and / or other modification methods, alone or in combination. Methods for introducing such modifications into the DNA sequence of an antibody based on its amino acid sequence are well known to those skilled in the art (see, for example, Sambrook Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.). The modifications are preferably performed at the nucleic acid level. The above-mentioned scFv may also include derivatives thereof. scFv can be expressed as a single-chain polypeptide. scFv retains the specificity of the intact antibody from which it is derived. The light and heavy chains can be in any order, for example, VH-linker-VL or VL-linker-VH, as long as the specificity of the scFv for the target antigen is preserved. In the present invention, the linker can be a flexible linker peptide chain rich in glycine and serine (with the amino acid sequence GGGGSGGGGSGGGGS).

[0030] As is well known to those skilled in the art, "complementarity determining regions" refer to amino acid sequences within the variable regions of antibodies that confer specificity and binding affinity. Typically, there are three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3), and three CDRs in each heavy chain variable region (HCDR1, HCR2, and HCDR3). While the amino acid sequences of the light chain variable region and the heavy chain variable region are defined in the present invention, the amino acid sequences of the six CDR regions are also fixed, and the specificity and binding affinity of the antibody can be predicted accordingly.

[0031] The term "specific recognition" as used herein means that the antigen recognition region of the present invention does not or substantially does not cross-react with any polypeptide other than the target antigen. The degree of specificity can be determined by immunological techniques, including but not limited to immunoblotting, immunoaffinity chromatography, flow cytometry, etc.

[0032] The term "antigen recognition domain" used in the present invention refers to the portion of the CAR that specifically binds to an antigen on a target cell. It will be understood by those skilled in the art that in some embodiments, almost any molecule that binds to a given antigen with high affinity can be used as an antigen recognition domain. In some embodiments, the antigen recognition domain comprises a T cell receptor (TCR) or a portion thereof.

[0033] The term "costimulatory signal domain" used in the present invention refers to the part of CAR that enhances the proliferation, survival and / or development of memory cells. The CAR of the present invention may include one or more costimulatory domains. Each costimulatory domain includes any one or more of the following costimulatory domains: for example, members of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, or a combination thereof.

[0034] The term "signal domain" used in the present invention refers to CAR transduction effector function signals and instructs cells to perform the part of their specialized functions. The example of the domain of transduction effector function signals includes but is not limited to the z chain of cell receptor complex or any homologue thereof (for example, h chain, FceR1g and b chain, MB1 (Iga) chain, B29 (Igb) chain etc.), human CD3 ζ chain, CD3 polypeptide (D, d and e), syk family tyrosine kinase (Syk, ZAP 70 etc.), src family tyrosine kinase (Lck, Fyn, Lyn etc.) and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In the present invention, CD3 ζ signal domains are preferably used.

[0035] The term "hinge region" as used in the present invention refers to the hydrophilic region between the antigen recognition domain and the transmembrane domain. The hinge region can be the hinge region of various antibodies or antigen receptors, in particular the hinge region of CD molecules. In a specific embodiment, the hinge region can be selected from the hinge region of proteins such as CD8 or CD28. CD8 or CD28 are natural markers on the cell surface. The hinge region is the CD8 hinge region (CD8-hinge), and the hinge region is the CD28 hinge region (CD28-hinge).

[0036] When used in the present invention, "transmembrane region" refers to the region of the CAR across the plasma membrane. The transmembrane regions of various human proteins can be used, in particular the transmembrane regions of various different antigen receptors. The transmembrane region preferably used is the transmembrane region of the CD molecule. In one embodiment, the transmembrane region can be selected from the transmembrane region of proteins such as CD8 or CD28. In a specific embodiment, the transmembrane region is the CD8 transmembrane region (CD8-TM), and in a specific embodiment, the transmembrane region is the CD28 transmembrane region (CD28-TM).

[0037] As used herein, the term "encoding" when applied to a nucleic acid sequence means that a polynucleotide "encoding" a polypeptide, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA for the polypeptide and / or its fragment.

[0038] As used herein, the term "signal peptide" refers to a short peptide chain (5-30 amino acids in length) that directs newly synthesized proteins into the secretory pathway. Signal peptides from various proteins in the human body can be used herein, such as secreted cytokine proteins, colony-stimulating factor 2 receptor α signal peptide (CSF2Rα), and signal peptides from leukocyte differentiation antigens (CD molecules).

[0039] The term "vector" used in the present invention refers to a recombinant vector that retains the ability to infect and transduce non-dividing and / or slowly dividing cells and integrate into the genome of the target cell. In some aspects, the vector is derived from or based on a wild-type virus. In a further aspect, the vector is derived from or based on a wild-type lentivirus. Such examples include, but are not limited to, human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV). Alternatively, it should be understood that other retroviruses can be used as the basis of the vector backbone, such as murine leukemia virus (MLV).

[0040] The structure of a CAR typically consists of an extracellular antigen-binding domain, a hinge region, a transmembrane region, and an intracellular signaling domain (Grossetal., Proc. Natl. Acad. Sci. USA 86: 10024, 1989; Eshhar et al., Proc. Natl. Acad. Sci. USA 90: 720, 1993). The intracellular signaling domain of first-generation CARs contained only a single CD3ζ chain. This resulted in poor CAR-T cell activity and short in vivo survival. Second-generation CARs added intracellular costimulatory domains, such as CD27, CD28, CD134 (OX40), or CD137 (4-1BB), which can promote sustained CAR-T cell proliferation and cytokine release, enhancing anti-tumor activity (Imai et al., Leukemia 18: 676, 2004; Zhao et al., Cancer Cell 28: 415, 2015). Third-generation CARs utilize two costimulatory domains (Zhong et al., Mol. Ther. 18: 413, 2010) to enhance CAR-T activation. Fourth-generation CARs, in addition to chimeric antigen receptor genes, incorporate the expression of immune factors, costimulatory ligands, chemokines, and suicide switches to enhance CAR-T cell activation, expansion, and killing; increase tumor-localized chemotaxis and recruitment; and activate other immune cells.

[0041] In humans, the GPC3 protein expressed by the GPC3 gene exhibits significant differences in expression during different developmental stages and in different tissues. For example, it is low or absent in gastric, breast, and ovarian cancers, while often overexpressed in hepatocellular carcinoma. Therefore, developing an immune cell based on the GPC3 protein that can phagocytose and digest hepatocellular carcinoma tumor cells is of great significance for the treatment of hepatocellular carcinoma.

[0042] The macrophages modified with a GPC3-targeted chimeric antigen receptor disclosed in the present application express the GPC3-targeted chimeric antigen receptor, wherein the chimeric antigen receptor comprises an antigen-binding domain, a hinge region, a transmembrane domain, and a signaling domain. The antigen-binding domain is an anti-GPC3 single-chain antibody, wherein the antigen-binding domain is used for temporally targeted recognition of cells expressing GPC3, thereby further achieving the purpose of phagocytosis and digestion of hepatocellular carcinoma tumor cells.

[0043] Preferably, the hinge region of the chimeric antigen receptor comprises CD8.

[0044] Preferably, the transmembrane domain of the chimeric antigen receptor includes the CD8 transmembrane region.

[0045] Preferably, the signaling domain of the chimeric antigen receptor comprises CD3ζ.

[0046] Preferably, the signaling domain of the chimeric antigen receptor further comprises any one or a combination of at least two of 4-1BB, CD28 intracellular region, DAP10 or OX40.

[0047] Preferably, the chimeric antigen receptor comprises an anti-GPC3 single-chain antibody, a CD8 hinge region, a CD8 transmembrane region, and CD3ζ.

[0048] Preferably, the antigen-binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 2.

[0049] Preferably, the hinge region of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 1.

[0050] Preferably, the transmembrane domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 3.

[0051] Preferably, the signaling domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:4.

[0052] Furthermore, the present application also discloses a method for preparing macrophages modified with a GPC3-targeting chimeric antigen receptor, wherein the method for preparing macrophages modified with a GPC3-targeting chimeric antigen receptor specifically comprises: Synthesizing the nucleic acid sequences of each structural domain of the transmembrane expression protein or polypeptide macromolecule targeting the GPC3 chimeric antigen receptor to obtain the nucleic acid molecule of the target fragment targeting the GPC3 chimeric antigen receptor; Connecting the target fragment to the overexpression vector to obtain a recombinant plasmid vector; amplifying the recombinant plasmid vector and the helper plasmid vector; Obtaining a packaged virus containing the target fragment based on the recombinant plasmid vector and the helper plasmid vector; Macrophages are infected with the virus to obtain modified macrophages expressing a chimeric antigen receptor targeting GPC3.

[0053] Furthermore, the overexpression vector is an adenovirus or lentivirus overexpression vector.

[0054] The present application also discloses a recombinant lentivirus or adenovirus, which is prepared by mammalian cells transfected with the expression vector and helper plasmid.

[0055] Preferably, the nucleic acid molecule is integrated into the genome of the chimeric antigen receptor macrophage.

[0056] Preferably, the chimeric antigen receptor macrophage comprises the expression vector and / or the recombinant lentivirus and / or adenovirus.

[0057] The present application also discloses a pharmaceutical composition, one advantage of which is that the pharmaceutical composition can achieve phagocytosis and digestion of tumor cells in liver cancer patients.

[0058] Furthermore, the pharmaceutical composition includes macrophages modified with the GPC3-targeting chimeric antigen receptor disclosed in the present application.

[0059] Furthermore, the pharmaceutical composition disclosed in the present application comprises isolated modified immune response cells expressing the chimeric antigen receptor specifically targeting GPC3 and a pharmaceutically acceptable carrier.

[0060] More specifically, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

[0061] The chimeric antigen receptor nucleic acid molecule, the expression vector, the recombinant lentivirus and / or adenovirus, the chimeric antigen receptor macrophage or the pharmaceutical composition are used in the preparation of a drug for treating GPC3-positive tumors.

[0062] Preferably, the GPC3-positive tumor is liver cancer.

[0063] Through creative work, the inventors continuously carried out amino acid sequence design, sequence permutation, combination and screening, and conducted random screening tests and targeted function verification on the sequences of more than 100 CAR molecules (for example, constructing viral vectors, and further infecting macrophages to obtain modified macrophage cells, and detecting the in vitro killing activity of the modified macrophages). After that, based on the comparison of the results of multiple random combinations, the sequence was adjusted, and finally a sequence with better effect was screened out, obtaining a high-titer scFv amino acid sequence targeting GPC3 and its functional variants of the present invention.

[0064] Administration of the pharmaceutical composition can be autologous or non-autologous. For example, immune response cells expressing the chimeric antigen receptor specifically targeting GPC3 and compositions comprising the same can be obtained from one subject and administered to the same subject or a different compatible subject. Peripheral blood-derived macrophages or their progeny (e.g., derived in vivo, ex vivo, or in vitro) of the presently disclosed subject matter can be administered by methods including catheter administration, intravenous injection, or parenteral administration. When administering a pharmaceutical composition of the presently disclosed subject matter (e.g., a pharmaceutical composition comprising immune response cells expressing the chimeric antigen receptor specifically targeting GPC3), it is typically formulated into a unit dose injectable form (solution, suspension, emulsion).

[0065] The composition of the present application can be a preparation. The immune response cells of the chimeric antigen receptor (CAR) of the specific targeting GPC3 disclosed in the present application and the composition comprising it can be conveniently provided as a sterile liquid preparation, such as an isotonic aqueous solution, a suspension, an emulsion, a dispersion or a viscous composition, which can be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions and solid compositions. In addition, liquid compositions are more convenient to apply, particularly by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact time with a specific tissue. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium comprising, for example, water, normal saline, phosphate buffered saline, a polyol (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.) and a suitable mixture thereof.

[0066] Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents.

[0067] According to the present application, any carrier, diluent or additive used must be compatible with the immune response cells expressing the chimeric antigen receptor (CAR) specifically targeting GPC3 of the presently disclosed subject matter.

[0068] If necessary, the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent. The choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, such as a liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel or another liquid form, such as a time-release form or a liquid-filled form).

[0069] A kit for treating or preventing a disease, comprising the immune response cell or the nucleic acid of the present invention.

[0070] Use of the protein targeting and binding to GPC3 or its functional variant, the chimeric antigen receptor specifically targeting GPC3, the recombinant vector or expression plasmid, the recombinant virus, the isolated modified immune response cell, and the kit in products for treating or preventing diseases, discomfort or health disorders.

[0071] In some embodiments, the disease to be treated or prevented comprises liver cancer, gastric cancer, pancreatic cancer, brain cancer, prostate cancer, lymphoma, leukemia, intestinal cancer, lung cancer, ovarian cancer or breast cancer.

[0072] The macrophages modified with a chimeric antigen receptor specifically targeting GPC3 of the present invention transmit activation signals and activate the immune system by recognizing GPC3 produced on the surface of tumor cells, thereby exerting a killing effect on tumor cells.

[0073] The specific preparation steps are as follows: Construction of anti-GPC3 CAR viral vector.

[0074] In this example, an anti-GPC3 single-chain antibody was used as the antigen binding domain of the CAR molecule, which binds to the CD8α hinge region, CD8α transmembrane region, and CD3ζ to design an anti-GPC3 CAR. The amino acid sequence is shown in SEQ ID NO: 5, the nucleic acid sequence is shown in SEQ ID NO: 10, and the schematic diagram is shown in FIG. Figure 1 shown.

[0075] The nucleic acid sequence of the CD8 hinge region is shown in SEQ ID NO:6.

[0076] The nucleic acid sequence of the anti-GPC3 single-chain antibody is shown in SEQ ID NO: 7.

[0077] The nucleic acid sequence of the CD8 transmembrane region is shown in SEQ ID NO:8.

[0078] The nucleic acid sequence of CD3ζ is shown in SEQ ID NO:9.

[0079] The anti-GPC3 CAR encoding gene was fully synthesized. The synthesized CAR molecule encoding gene was cloned into an adenovirus vector through PCR, enzyme digestion, recombination and other steps, introduced into the host bacteria, and stored in glycerol. Take 100 μL of glycerol bacteria in a 50 mL centrifuge tube, add 25 mL of fresh LB medium, and culture in a shaker at 37°C and 220 rpm for 16 hours. Then, use a plasmid mini-extraction kit to extract the plasmid. Take a small amount of plasmid for sequencing to check whether its sequence matches the constructed target map. Figure 2 The remaining plasmids were stored at -20°C.

[0080] Construction of a stable cell line overexpressing GPC3.

[0081] V5 and HA tags were added to the CDS sequence of GPC3 at the C-terminus (W:14). The expression vector was transfected into 293T cells, and the target protein expression was detected by WB. The virus was packaged in 293T cells, and the crude virus was collected to infect Huh7 cells. The target protein expression was detected by WB. The verified lentivirus was used to infect Huh7 cells. Drug screening was performed for 5 days. After the screening, a monoclonal clone was constructed, and the target protein expression was detected by WB.

[0082] Day 0: When Huh7 cells grow to 70% in a 6-well plate, replace the culture medium: 1 mL DMEM + 1 mL lentivirus (pCDH-CMV-MCS-GPC3-V5-HA-puro, plasmid structure as shown in Figure 3 shown).

[0083] Day 1: Change the medium to 2 mL of DMEM complete medium.

[0084] Day 2: Cell passaging.

[0085] Day 3: Add 2 μg / mL of puro for screening.

[0086] Day 5: Change the medium (containing 2μg / mL puro).

[0087] Day 9: Passaging (containing 2 μg / mL puro).

[0088] Day 12: Part of the cells were passaged and the remaining cells were used for protein extraction and Western blotting.

[0089] Day 13: Cryopreservation of cells.

[0090] Example 2 Packaging of anti-GPC3 CAR virus and titer detection.

[0091] 1. Packaging of anti-GPC3 CAR adenovirus and titer detection.

[0092] The vector was linearized by enzyme digestion and recovered by ethanol precipitation, and then packaged into adenovirus using 293A.

[0093] 1. Preparation of chimeric antigen receptor macrophages specifically targeting GPC3.

[0094] Macrophages (RAW264.7 / miBMDM / hPBMC / THP-1) were infected with anti-GPC3 CAR adenovirus.

[0095] 2. Detect the expression of CAR protein by flow cytometry analysis.

[0096] Example 4 Anti-GPC3 CAR-M cell killing evaluation experiment in vivo and in vitro.

[0097] like Figure 4-5 As shown in the figure, the in vitro phagocytosis experiment of different GPC3 positive and negative cells by adenovirus-transduced RAW264.7 was used to verify the targeted phagocytosis ability of CAR. Huh7 / HepG2 / C3A were screened as GPC3 positive target cells by WB verification. The WB verification results are shown in the figure. Figure 4As shown, 293FT is a GPC3 negative target cell, and subsequent in vitro phagocytosis experiments were performed, and the results are shown in Figure 5 As shown in Figure 3 . After 6 hours of co-culture of adenoviral-transduced RAW264.7 cells with C3A / Huh7 / HepG2 / 293FT, it was found that when the target cells were GPC3-positive, the phagocytic efficiency of GPC3-CAR was significantly increased compared to Empty and UTD cells, whereas when the target cells were 293FT, there was no significant difference. Therefore, we demonstrated the ability of anti-GPC3-CAR to mediate antigen-specific phagocytosis.

[0098] 1. The specific steps for in vitro killing assessment are as follows:

[0099] Construction of GPC3 expression vector: The GPC3 sequence was constructed on the pCDH-CMV-MCS-EF1-Puro backbone vector.

[0100] (1) Phagocytosis of GPC3-positive macrophages.

[0101] Immunofluorescence detection.

[0102] Flow cytometry.

[0103] Detection of cytokine secretion.

[0104] Prime CD8+ T cell detection.

[0105] like Figure 6 To further validate the effect of GPC3-CAR, we evaluated the ability of adenoviral-transduced RAW264.7 cells to kill various GPC3-positive cells in vitro. In the Fluc-based killing assay, E:T ratios of 1:1 and 2:1 were used. The results demonstrated that GPC3-CAR demonstrated significantly higher killing activity than other groups, regardless of cell line type. In the LDH-based killing assay, an E:T ratio of 1:1 was used in C3A and Huh7 cells. Similarly, the results showed that GPC3-CAR demonstrated significantly higher killing activity than other groups. Therefore, we further demonstrated that adenoviral-transduced macrophages have a significant anti-tumor effect.

[0106] Compared with the prior art, the present invention has the following beneficial effects.

[0107] The present invention utilizes chimeric antigen receptor-modified macrophage technology to prepare engineered immune cells specifically targeting GPC3. This method is simple, and the resulting engineered immune cells can specifically recognize tumor cells, more effectively targeting and attacking them, with a high tumor killing rate. These cells can be used to prepare anti-tumor products, particularly drugs for treating GPC3-positive tumors. The present invention is expected to be used to prepare anti-tumor products, particularly drugs for treating liver cancer, lung adenocarcinoma, gastric cancer, pancreatic cancer, brain cancer, prostate cancer, lymphoma, leukemia, intestinal cancer, lung cancer, ovarian cancer, or breast cancer, and has promising industrial application prospects.

Claims

1. A macrophage modified with a chimeric antigen receptor targeting GPC3, characterized in that: The macrophages are used to treat GPC3-positive diseases. The chimeric antigen receptor comprises an antigen binding domain, a hinge region, a transmembrane domain and a signal transduction domain. The antigen binding domain is an anti-GPC3 single-chain antibody.

2. The macrophage modified with a chimeric antigen receptor targeting GPC3 according to claim 1, wherein The hinge region includes CD8.

3. The macrophage modified with a chimeric antigen receptor targeting GPC3 according to claim 1, wherein The transmembrane domain includes the CD8 transmembrane region.

4. The macrophage modified with a chimeric antigen receptor targeting GPC3 according to claim 1, wherein The signaling domain includes CD3ζ.

5. The macrophage modified with a chimeric antigen receptor targeting GPC3 according to claim 1, wherein The signaling domain further includes any one or a combination of at least two of 4-1BB, CD28 intracellular region, DAP10 or OX40.

6. The macrophage modified with a chimeric antigen receptor targeting GPC3 according to claim 1, wherein The chimeric antigen receptor comprises an anti-GPC3 single-chain antibody, a CD8 hinge region, a CD8 transmembrane region, and CD3ζ.

7. The macrophage modified with a chimeric antigen receptor targeting GPC3 according to claim 1, wherein The antigen-binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:

2.

8. The macrophage modified with a chimeric antigen receptor targeting GPC3 according to claim 1, wherein The hinge region includes the amino acid sequence shown in SEQ ID NO:

1.

9. The macrophage modified with a chimeric antigen receptor targeting GPC3 according to claim 1, wherein The transmembrane domain includes the amino acid sequence shown in SEQ ID NO:

3.

10. The macrophage modified with a chimeric antigen receptor targeting GPC3 according to claim 1, wherein The signaling domain includes the amino acid sequence shown in SEQ ID NO:

4.

11. The macrophage modified with a chimeric antigen receptor targeting GPC3 according to claim 1, wherein The GPC3-positive disease is specifically a GPC3-positive tumor, wherein the GPC3-positive tumor is specifically liver cancer.

12. The macrophage modified with a chimeric antigen receptor targeting GPC3 according to claim 1, wherein The method for preparing macrophages modified with a GPC3-targeted chimeric antigen receptor specifically comprises the following steps: (1) Synthesize nucleic acid sequences for various domains of transmembrane expression proteins or polypeptide macromolecules; (2) Connecting the target fragment to a lentiviral or adenoviral overexpression vector to obtain a recombinant lentiviral vector or a recombinant adenoviral vector; (3) Extraction and preparation of recombinant plasmid vectors and auxiliary plasmid vectors; (4) Virus packaging; (5) Construction of functional cell lines.

13. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the chimeric antigen receptor macrophage according to claims 1-12.

14. The pharmaceutical composition according to claim 13, wherein Also included are any one or a combination of at least two of pharmaceutically acceptable carriers, excipients or diluents.

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