Heavy chain and light chain variable regions of anti-GPC3 monoclonal antibody and application

Abstract

The embodiment of the invention discloses a heavy chain variable region and a light chain variable region of an anti-GPC3 monoclonal antibody and application of the heavy chain variable region and the light chain variable region. The high-affinity anti-GPC3 sequence is obtained through screening, the binding specificity of the high-affinity anti-GPC3 sequence and the GPC3 antigen is high, and dissociation is slow. The sequence is used as an extracellular targeting domain to construct GPC3-CAR, after macrophages are transduced through lentivirus, CAR-M can specifically recognize GPC3 positive target cells, activate intracellular signal channels and remarkably improve in-vitro phagocytosis and killing activity, the hepatoma cell lysis effect is better, and technical support is provided for anti-hepatoma application.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the heavy and light chain variable regions of an anti-GPC3 monoclonal antibody and its applications. Background Technology

[0002] Primary liver cancer is one of the malignant tumors with the highest incidence and mortality rates worldwide. According to statistics from the World Health Organization (WHO) in 2024, there are more than 1.2 million new cases and more than 850,000 deaths annually. Hepatocellular carcinoma (HCC) accounts for more than 90% of these cases, making it the fourth most common malignant tumor and the second leading cause of cancer death in my country. The clinical diagnosis and treatment of liver cancer faces many intractable challenges: On the one hand, its onset is insidious, with no specific symptoms in the early stages, and only about 20%-30% of patients meet the criteria for radical surgery at the time of diagnosis, while the remaining 70%-80% of patients have progressed to the middle or late stages and lost the opportunity for surgery; on the other hand, liver cancer has an extremely poor prognosis. Even if early-stage patients undergo surgical resection or liver transplantation, the 5-year recurrence rate is still as high as 50%-70%, and the median survival of late-stage patients without effective treatment is only 3-4 months, while the median survival after receiving standard treatment (such as sorafenib combined with PD-1 antibody) is only 12-15 months. Moreover, the tumor is highly heterogeneous, and the objective response rate (ORR) of the same treatment regimen can fluctuate by 10%-40% in different patients. Some patients develop primary or secondary drug resistance due to tumor genotype or solid tumor microenvironment (TME) characteristics.

[0003] Current treatments for liver cancer all have significant limitations: local treatments (such as radiofrequency ablation and TACE) are only suitable for patients with ≤3 lesions, a maximum diameter ≤5cm, and no vascular invasion / extrahepatic metastasis. Even if treatment is successful, 40%-60% of patients will relapse within 2 years due to tumor micrometastasis, and the risk is higher for patients with severe cirrhosis. Anti-angiogenic drugs, such as sorafenib and lenvatinib, have a single mechanism of action and are prone to developing resistance due to "bypass activation" or "tumor vascular remodeling," with a median progression-free survival of only 3-6 months. Subsequent second-line drug therapy... The efficacy further declined and adverse reactions were significant; the ORR of PD-1 / PD-L1 antibody monotherapy was only 15%-20%, and even when combined with anti-angiogenic drugs, the ORR increased to 30%-35%, but more than 60% of patients still did not respond due to insufficient T cell infiltration in the TME or low PD-L1 expression, and the risk of bleeding increased; CAR-T cell therapy has significant efficacy in hematologic malignancies, but in liver cancer, due to problems such as the physical barrier of the TME, short cell survival and off-target toxicity, the ORR is only 25%-30%, and the complete response rate is less than 5%, which is difficult to meet clinical needs.

[0004] Among targeted therapies for liver cancer, phosphatidylinositol proteoglycan 3 (GPC3) exhibits unique advantages. GPC3 is a heparan sulfate glycoprotein anchored to the cell membrane surface. By binding to the Wnt signaling pathway, it promotes β-catenin nuclear translocation and inhibits the TGF-β / Smad tumor suppressor pathway, directly regulating the proliferation and malignant phenotype of liver cancer cells. Its tissue expression is highly specific, with a positive rate of <5% in normal adult liver tissue, while it is highly expressed in 70%-80% of liver cancer tissues. Moreover, its expression level is negatively correlated with the degree of tumor differentiation, with a GPC3 positive rate of >90% in poorly differentiated liver cancer. At the same time, the median survival of GPC3-positive liver cancer patients (18 months) is significantly shorter than that of negative patients (32 months), making it an independent risk factor for poor prognosis in liver cancer. Furthermore, it is highly expressed on the surface of liver cancer stem cells. Targeting GPC3 can simultaneously kill mature liver cancer cells and liver cancer stem cells, reducing the risk of recurrence from the source. Compared with other targets such as AFP and EpCAM, GPC3 is hardly expressed in normal tissues, has a higher safety profile, and is expressed independently of other targets, making it a potential target for joint development and an ideal target for targeted therapy of liver cancer.

[0005] However, existing GPC3-targeted therapies still face core technological bottlenecks: GPC3 antibody-drug conjugates (ADCs) such as TJ107 and LM-305 use antibodies (such as GC33) with a dissociation constant (KD) of approximately 100 pM for GPC3, resulting in weak binding ability to liver cancer cells with low GPC3 expression. Furthermore, the linkers suffer from an imbalance between stability and release efficiency, easily leading to toxicity or insufficient killing. GPC3-CAR-T projects such as LCAR-G33 and CT011 are in Phase I / II, with an ORR of only 25%-30%. Approximately 30% of patients experience grade 3 or higher cytokine release syndrome, and some patients experience liver damage due to off-target effects of CAR-T cells on bile duct epithelial cells. Although GPC3-targeted macrophage therapy has advantages in solid tumor infiltration, the antibodies used in current technologies have low activity, with macrophage phagocytosis rates below 20%. Moreover, the CAR structure lacks co-stimulatory signals, resulting in insufficient activation and inability to effectively activate adaptive immune responses. In addition, the in vitro expansion efficiency of macrophages is low, making it difficult to meet clinical dosage requirements. Summary of the Invention

[0006] Addressing the pain points of treating advanced / relapsed and drug-resistant liver cancer patients, such as the lack of long-acting and low-toxicity regimens, the ease with which low-expressing GPC3 liver cancer cells can escape, the difficulty in overcoming the tumor microenvironment barrier, and the high safety risks of immunotherapy, as well as the shortcomings of existing GPC3 targeting technologies, this invention aims to systematically solve the key problems of low targeting precision, difficulty in solid tumor invasion, weak immune activation, and high safety risks by developing high-affinity GPC3 targeting molecules and functionally enhanced CAR-M cells adapted to the liver cancer TME.

[0007] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0008] According to a first aspect of the present invention, the present invention provides a heavy chain and light chain variable region of an anti-GPC3 monoclonal antibody.

[0009] The heavy chain variable region comprises one or more of the following sequences:

[0010] (1) An amino acid sequence as shown in any one of SEQ ID NO.1-3;

[0011] (2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO. 1-3, and retaining the activity of the amino acid sequence shown in any one of SEQ ID NO. 1-3;

[0012] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in any of the amino acid sequences shown in any of SEQ ID NO. 1-3, and which retains the activity of the amino acid sequence shown in any of SEQ ID NO. 1-3;

[0013] The amino acid sequence of the light chain variable region comprises one or more of the following sequences:

[0014] (1) An amino acid sequence as shown in any one of SEQ ID NO. 4-6;

[0015] (2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO. 4-6, and retaining the activity of the amino acid sequence shown in any one of SEQ ID NO. 8-10;

[0016] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in any of the amino acid sequences shown in any of SEQ ID NO.4-6, and retains the activity of the amino acid sequence shown in any of SEQ ID NO.4-6.

[0017] According to a second aspect of the present invention, the present invention provides an anti-GPC3 monoclonal antibody, comprising the heavy chain variable region and the light chain variable region as described above.

[0018] According to a third aspect of the present invention, the present invention provides a chimeric antigen receptor targeting GPC3, the chimeric antigen receptor comprising: a single-chain variable fragment, a hinge region, a transmembrane domain and an intracellular domain, wherein the amino acid sequence of the single-chain variable fragment is shown in SEQ ID No. 8 to SEQ ID No. 10.

[0019] Furthermore, the hinge region is the IgG4 hinge region of the anti-degradation immunoglobulin family;

[0020] The transmembrane domain is a CD14 transmembrane domain;

[0021] The intracellular domains are the 4-1BB co-stimulatory domain and the FcγRIIIa-derived activation domain.

[0022] According to a fourth aspect of the present invention, the present invention provides a chimeric antigen receptor macrophage targeting GPC3, which expresses the chimeric antigen receptor as described above.

[0023] According to a fifth aspect of the present invention, the present invention provides the use of the heavy chain and light chain variable regions as described above, the anti-GPC3 monoclonal antibody as described above, the chimeric antigen receptor as described above, or the chimeric antigen receptor macrophage as described above in the preparation of a therapeutic tumor drug.

[0024] Furthermore, the tumor is liver cancer.

[0025] Furthermore, the liver cancer includes unresectable locally advanced liver cancer, advanced liver cancer with vascular invasion or extrahepatic metastasis, liver cancer that has recurred after surgery, or liver cancer that is resistant to targeted / immunotherapy.

[0026] The embodiments of the present invention have the following advantages:

[0027] This invention screened and obtained high-affinity anti-GPC3 sequences (such as SEQ ID No. 8~SEQ ID No. 10), which exhibit strong specificity in binding to the GPC3 antigen and slow dissociation. Using this sequence as the extracellular targeting domain, a GPC3-CAR was constructed. After transduction into macrophages via lentivirus, the CAR-M specifically recognized GPC3-positive target cells, activated intracellular signaling pathways, significantly enhanced in vitro phagocytic and killing activities, and showed superior lysis of hepatocellular carcinoma cells, providing technical support for its application in treating liver cancer. Attached Figure Description

[0028] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0029] Figure 1 The affinity of the GPC3 monoclonal antibody provided by this invention for CHOK1-GPC3;

[0030] Figure 2 The structure of CAR-M provided by this invention;

[0031] Figure 3 The expression and positive rate detection results of GPC3-CAR in macrophages provided by this invention;

[0032] Figure 4 The killing detection results of GPC3-CAR-M against Hep3B provided by this invention;

[0033] Figure 5 The results of GPC3-CAR-M killing HepG2 provided by this invention. Detailed Implementation

[0034] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1: Anti-GPC3 antibody sequence obtained by DNA and cell-mediated immunization of BALB / c mice

[0036] Two immunogens, a full-length human GPC3 protein and a truncated C-terminal polypeptide, were first prepared, and their coating titers were verified by indirect ELISA. BALB / c and SJL mice were then immunized in groups, and high-responding mice were selected by ELISA and FACS. Subsequently, spleen cells from these mice were fused with SP2 / 0 myeloma cells at a 5:1 ratio. After screening in HAT medium, initial screening by ELISA, secondary screening by FACS, and limiting dilution monocloning, 20 clones were selected for sequencing. Finally, total RNA was extracted from these monoclonal cells, and RT-PCR reverse transcription, VH / VL gene amplification, and Sanger sequencing were performed to obtain mutation-free candidate sequences with intact CDR regions. Antibody activity was confirmed by eukaryotic expression, Protein A purification, and SPR detection. Figure 1 As shown, its highly active antibodies are SYQN-2025-B1-hIgG1, SYQN-2025-B2-hIgG1 and SYQN-2025-B3-hIgG1.

[0037] The amino acid sequence of the heavy chain variable region of SYQN-2025-B1-hIgG1 is shown in SEQ ID NO.1:

[0038] EVQLQQSGPELVRPRASMKISCKASGFLFTAYTMNWMKQSHGKNLEWIGLIYPYNGDTDYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGDYGLAWFSYWGQGTLVTVSA.

[0039] The amino acid sequence of the light chain variable region of SYQN-2025-B1-hIgG1 is shown in SEQ ID NO.4:

[0040] DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYKASNLHTGVPSRFTGSGSGTGFTLTISSLQPEDIATYYCQQGQIYPFTFGGGTKLEIK.

[0041] The amino acid sequence of the heavy chain variable region of SYQN-2025-B2-hIgG1 is shown in SEQ ID NO.2:

[0042] QVQLQQSGAELVRPGASVTLSKASGYTFSDYEIHWVKQTPVHGLEWIGAIDPEAGNTAYNQRFKDKATLTADKSSSTAYMELRSLTSEDSAVYYCTRYFSFAYWGQGTLVTVSA.

[0043] The amino acid sequence of the light chain variable region of SYQN-2025-B2-hIgG1 is shown in SEQ ID NO.5:

[0044] DVLMTQTPLSLPVSLGDQASISCRSSQTFVHSNGNTYLQWYLQKPGQSPKLLIYKVSNRFSGVPDRFRGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK.

[0045] The amino acid sequence of the heavy chain variable region of SYQN-2025-B3-hIgG1 is shown in SEQ ID NO.3:

[0046] QVQLQQSGAELVRPGASVTLSKASGYTFTDYEMHWVKETPVHGLEWIGGIDPETGGTAYNQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCTRYFSFAYWGQGTLVTVSA.

[0047] The amino acid sequence of the light chain variable region of SYQN-2025-B3-hIgG1 is shown in SEQ ID NO.6:

[0048] HALMTQSPLSLPVSLGDQASISCRSSQSIVHSNGNTYLQWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCSQVSHVPYTFGGGTKLEIK.

[0049] SYQN-2025-B1-hIgG1, SYQN-2025-B2-hIgG1, and SYQN-2025-B3-hIgG1 are composed of heavy and light chains linked by a linker peptide, with the linker peptide sequence SEQ ID NO.7: GGGGSGGGGSGGGGS. The amino acid sequences of each antibody are shown in SEQ ID NO.8-10.

[0050] EVQLQQSGPELVRPRASMKISCKASGFLFTAYTMNWMKQSHGKNLEWIGLIYPYNGDTDYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGDYGLAWFSYWGQGTLVTVSAGGG GSGGGGSGGGGSDIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYKASNLHTGVPSRFTGSGSGTGFTLTISSLQPEDIATYYCQQGQIYPFTFGGGTKLEIK (SEQ ID NO.8);

[0051] QVQLQQSGAELVRPGASVTLSCKASGYTFSDYEIHWVKQTPVHGLEWIGAIDPEAGNTAYNQRFKDKATLTADKSSSTAYMELRSLTSEDSAVYYCTRYFSFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDVLMTQTPLSLPVSLGDQASISCRSSQTFVHSNGNTYLQWYLQKPGQSPKLLIYKVSNRFSGVPDRFRGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK (SEQ ID NO.9);

[0052] QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKETPVHGLEWIGGIDPETGGTAYNQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCTRYFSFAYWGQGTLVTVSAGGGGSGGGGSGGGGSHALMTQSPLSLPVSLGDQASISCRSSQSIVHSNGNTYLQWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCSQVSHVPYTFGGGTKLEIK (SEQ ID NO.10).

[0053] Example 2: Detection of the Affinity between Anti-GPC3 Monoclonal Antibody and Cell Surface GPC3 Protein

[0054] The affinity between the antibody and the antigen was determined using surface plasmon resonance (SPR) technology. The antibody was immobilized on protein A on the sensor chip as a ligand, while the antibody was passed through the sensor chip surface as an analytical stream. Using a Cytiva Biacore 8K instrument and Series S Sensor Chip Protein A, with HBS-EP+buffer (pH 7.4) as the running buffer, the target antibody and positive control antibody Codrituzumab (GC33) were captured and immobilized on the chip at a flow rate of 10 μL / min for 30–90 s. Then, gradient concentrations (200, 100, 50, 25, 12.5, 6.25 nM) of Human GPC3 Protein and Cyno GPC3 Protein were used as analytes and flowed through the chip at a flow rate of 30–75 μL / min. After binding for 120 s and dissociation for 360 s, the chip was regenerated with 10 mM Glycine-HCl (pH 1.5) at a flow rate of 30 μL / min for 120–180 s. Finally, ka, kd, and KD were calculated by fitting the data using Biacore 8K Evaluation Software 4.0.

[0055] The results are shown in Table 1: Classified by antigen type, SYQN-2025-B3-hIgG1 showed the best performance in binding to the full-length human GPC3 protein. The KD of SYQN-2025-B1-hIgG1 was lower than that of the positive control Codrituzumab (GC33). SYQN-2025-B2-hIgG1 had the largest ka, but its higher KD resulted in a slightly higher KD. In binding to cynomolgus monkey GPC3 protein, SYQN-2025-B3-hIgG1 remained the best, significantly superior to GC33. SYQN-2025-B1-hIgG1 and SYQN-2025-B2-hIgG1 showed similar affinity to GC33 and stable cross-reactivity. In binding to human GPC3… Among C-terminal truncated peptides, SYQN-2025-B1-hIgG1 still showed the best affinity, while SYQN-2025-B1-hIgG1 and SYQN-2025-B2-hIgG1 were both superior to GC33.

[0056] Table 1. Affinity data between antigen and antibody

[0057]

[0058] Example 3: Detection of viral infection positivity rate in GPC3-targeted CAR (GPC3-CAR)

[0059] The construction process of GPC3-CAR is as follows: First, CAR sequence design and plasmid construction are performed. The obtained anti-GPC3scFv (as shown in SEQ ID No. 8~SEQ ID No. 10) is used as the extracellular targeting domain, and the human IgG4 hinge region, CD14 transmembrane domain, 4-1BB co-stimulatory domain and FcγRIIIa activation domain are tandemly linked to form the full GPC3-CAR sequence. Then, it is inserted into a lentiviral vector. After sequencing verification, the plasmid is amplified using an animal-free high-density fermentation process. It is purified by gel filtration chromatography, affinity chromatography, and anion exchange chromatography to ensure that the supercoil ratio is ≥90% and the endotoxin is <10EU / mg. Subsequently, lentiviral packaging is performed, and the CAR plasmid and helper plasmids (gag / pol, rev, VSV-G) are co-transfected into Lenti-X. 293T cells were transfected, and viral supernatant was collected 30-48 hours later. The supernatant was clarified and filtered, nuclease incubation was performed to degrade residual DNA, ultrafiltration concentration was performed, impurities were removed by SEC chromatography, and 0.22μm sterile filtration was performed. Finally, the viral titer was confirmed to be ≥10 by FACS detection. 7 The concentration of TU / mL was measured, and residual impurities such as host cell proteins (HCP <10 μg / mL) and endotoxins (<10 EU / mL) were also detected. After passing the test, the cells were used for subsequent macrophage transduction. The structure of GPC3-CAR is shown below. Figure 2 As shown. During CAR transfection, activated macrophages were first centrifuged, resuspended, and counted at a rate of 1 × 10⁻⁶. 6 Cells were seeded into 6-well plates, and 1 ml of culture medium was added. The plates were incubated at 37°C with 5% CO2 for 1 hour to allow the cells to settle. The required viral volume was calculated based on an MOI of 20, and the concentrated lentiviral particles were then added to the 6-well plates. Polybrene (5 μg / mL) was added simultaneously to promote viral adsorption to cells. The plates were incubated at 37°C with 5% CO2 for 24 hours, then replaced with fresh culture medium. Incubation continued for 48-72 hours, at which point CAR gene expression began. The positivity rate was analyzed by flow cytometry after transfection. The results are shown below. Figure 3 As shown, the positive rates of SYQN-2025-B1, SYQN-2025-B2, SYQN-2025-B3, PC (anti-GC33 CAR-M), and NC were 98.65%, 92.11%, 99.2%, 99.25%, and 97.04%, respectively.

[0060] Example 4: Killing of liver cancer cells by GPC3-CAR

[0061] The killing effect of GPC3-CAR on target Hep3B and HepG2 cells was detected using the IncuCyte imaging platform. On day 1, macrophages infected with GPC3 CAR lentivirus were counted, and Hep3B and HepG2 target cells were seeded into 96-well plates at a ratio of 5E3 / well. 24 hours later, effector cells with consistent positivity were added to the target cells at effector-target ratios of 4:1, 2:1, and 1:1, and then the plates were placed on the IncuCyte imaging platform for imaging.

[0062] The results are as follows Figure 4-5 As shown, in Hep3B and HepG2 cells, the killing effect of SYQN-2025-B3 was better than that of PC (anti-GC33 CAR-M), while the killing effects of SYQN-2025-B1 and SYQN-2025-B2 were significantly better than those of NC (negative control), but slightly worse than those of SYQN-2025-B3.

[0063] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A heavy chain and a light chain variable region of an anti-GPC3 monoclonal antibody, characterized in that, the heavy chain variable region comprises one or more of the following sequences: (1) an amino acid sequence as shown in any one of SEQ ID NO. 1-3; (2) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence as shown in any one of SEQ ID NO. 1-3, and which retains the activity of the amino acid sequence as shown in any one of SEQ ID NO. 1-3; (3) an amino acid sequence with 1 or more amino acid residues added to, substituted for, deleted from, or inserted into the amino acid sequence as shown in any one of SEQ ID NO. 1-3, and which retains the activity of the amino acid sequence as shown in any one of SEQ ID NO. 1-3; the amino acid sequence of the light chain variable region comprises one or more of the following sequences: (1) an amino acid sequence as shown in any one of SEQ ID NO. 4-6; (2) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence as shown in any one of SEQ ID NO. 4-6, and which retains the activity of the amino acid sequence as shown in any one of SEQ ID NO. 8-10; (3) an amino acid sequence with 1 or more amino acid residues added to, substituted for, deleted from, or inserted into the amino acid sequence as shown in any one of SEQ ID NO. 4-6, and which retains the activity of the amino acid sequence as shown in any one of SEQ ID NO. 4-6.

2. An anti-GPC3 monoclonal antibody, characterized in that, The chimeric antigen receptor comprises a single chain variable fragment, a hinge region, a transmembrane domain, and an intracellular domain, wherein the amino acid sequence of the single chain variable fragment is as shown in SEQ ID No. 8-SEQ ID No.

10.

3. A chimeric antigen receptor targeting GPC3, characterized in that, 4.The GPC3-targeted chimeric antigen receptor of claim 3, characterized in that, the hinge region is an anti-degradation immunoglobulin family IgG4 hinge region; the transmembrane domain is a CD14 transmembrane domain; the intracellular domain is a 4-1BB co-stimulatory domain and a domain derived from FcγRIIIa activation domain. The chimeric antigen receptor expressed in claim 4.

5. A chimeric antigen receptor macrophage targeting GPC3, characterized in that, 6.The heavy chain and light chain variable region of claim 1, the anti-GPC3 monoclonal antibody of claim 2, the chimeric antigen receptor of claim 3, or the chimeric antigen receptor macrophage of claim 5 for use in the preparation of a medicament for treating tumors. The tumor is liver cancer.

7. Use according to claim 6, characterized in that, The liver cancer includes unresectable locally advanced liver cancer, advanced liver cancer with vascular invasion or extrahepatic metastasis, liver cancer recurrence after surgery, or liver cancer resistant to targeted / immunotherapy.

8. Use according to claim 7, characterized in that, ​

Images