GPC3 Chimeric Antigen Receptor (CAR): A Comprehensive Guide and Our Service & Product Introduction

Glypican-3 (GPC3) is a specific tumor antigen highly expressed in hepatocellular carcinoma and other solid tumors. It is a crucial tumor-associated antigen that has become a research hotspot in the field of targeted cancer therapy, especially in chimeric antigen receptor (CAR) technology.

RGBiotech offers the high-quality GPC3 CAR expression plasmid vector products as well as professional customized construction services, aiming to provide comprehensive support for researchers and enterprises engaged in cancer immunotherapy research and development. We are committed to becoming a reliable partner in the field of GPC3 CAR research, providing high-quality products and professional services for global researchers and enterprises.

Our GPC3 CAR Expression Plasmid Vector Products & Custom Services

To meet the needs of researchers and enterprises in GPC3 CAR research and development, RGBiotech provides a full range of GPC3 CAR expression plasmid vector products and professional customized plasmid vector construction services. Our products cover different generations of GPC3 CAR, with diverse carrier backbones and complete quality control systems, providing reliable support for GPC3 CAR-related research.

Our GPC3 CAR expression plasmid vector products are designed for the efficient expression of GPC3 CAR molecules in immune cells (T cells, NK cells, etc.), covering first-generation, second-generation, third-generation, and fourth-generation GPC3 CAR, and can meet different research needs. The vector backbones are diverse, including non-viral vectors (plasmid vectors) and viral vectors (lentiviral vectors, retroviral vectors, AAV vectors), providing flexible choices for different cell transfection and transduction methods.

1) First-generation GPC3 CAR: Only contains the CD3ζ signal transduction domain, suitable for basic research on GPC3 recognition and immune cell activation.

2) Second-generation GPC3 CAR: Contains CD3ζ + one co-stimulatory domain (CD28 or 4-1BB), which can enhance the proliferation and survival of CAR-modified cells, and is the most commonly used type in preclinical research.

3) Third-generation GPC3 CAR: Contains CD3ζ + two co-stimulatory domains (CD28+4-1BB, CD28+OX40, etc.), which further improves the anti-tumor activity and persistence of CAR cells.

4) Fourth-generation GPC3 CAR (armored CAR): On the basis of the third generation, it is modified to express cytokines (IL-7, IL-15) or immune checkpoint inhibitors (PD-1 scFv), which can resist the immunosuppressive effect of the tumor microenvironment and improve the in vivo anti-tumor effect.

Product Features

1) Diverse Vector Backbones: We provide a variety of vector backbones to adapt to different experimental scenarios: (A) Non-viral plasmid vectors: Simple operation, low cost, suitable for transient transfection and stable cell line construction. (B) Lentiviral vectors: Production of lentiviral particles, high transduction efficiency, can infect both dividing and non-dividing cells, and can integrate the CAR gene into the host genome to achieve long-term stable expression. (C) Retroviral vectors: Production of retroviral particles, suitable for the transduction of dividing cells, with high integration efficiency, suitable for the construction of stable CAR-T cell lines. (D) AAV vectors: Production of AAV particles, Low immunogenicity, high safety, suitable for in vivo delivery of CAR genes, especially in the research of in vivo CAR therapy.

2) Optimized Promoters: The vectors are equipped with high-efficiency promoters to ensure the high-level expression of GPC3 CAR molecules, including CMV promoter, EF1α promoter, and Tet/on inducible promoter etc., which can be selected according to experimental needs.

3) Multiple Fluorescent Labels: The vectors can be equipped with fluorescent labels such as GFP, mCherry and so on, which are convenient for monitoring the transfection efficiency and expression level of CAR molecules, and for sorting and tracing CAR-modified cells .

4) Diverse Antibiotic Selection Markers: We provide vectors with different antibiotic selection markers, including puromycin (Puro), neomycin (Neo), hygromycin (Hygro) and Blasticidin (Bla), which are convenient for the screening of positive clones and stable cell lines.

Product Advantages

1) High Expression Efficiency: The optimized promoter and vector structure ensure that the GPC3 CAR gene is highly expressed in target cells, improving the anti-tumor activity of CAR-modified cells.

2) Good Compatibility: The vectors are compatible with a variety of immune cells (human T cells, mouse T cells, NK cells, etc.) and delivery methods (electroporation, liposome transfection, viral production and transduction), with strong versatility.

3) High Stability: The vector backbone is optimized to reduce the probability of gene recombination and deletion, ensuring the stable expression of the CAR gene in long-term culture of cells.

4) Strict Quality Control: Each batch of products undergoes strict quality inspection to ensure the correctness of the sequence, providing reliable products for experiments.

5) Cost-Effective: We provide high-quality products at competitive prices, and provide preferential policies for long-term cooperative customers, reducing the research cost of customers.

Product Applications

1) Basic Research: Used for the study of GPC3 CAR structure and function, the mechanism of CAR-modified immune cell activation and killing, and the interaction between CAR-T cells and tumor cells.

2) Preclinical Research: Used for the construction of GPC3 CAR-T/NK cells, in vitro anti-tumor activity detection, animal model experiments (xenograft tumor model, syngeneic tumor model), and the evaluation of the safety and efficacy of CAR therapy.

3) Clinical Transformation: Provide high-quality vector tools for the development of GPC3 CAR-T cell therapy products, supporting the research and development of clinical-grade CAR-T cells.

4) Teaching and Training: Used for teaching experiments in the field of immunology and genetic engineering, helping students understand the principle and application of CAR technology.

Customized GPC3 CAR Plasmid Vector Construction Services

In addition to standard products, we also provide customized GPC3 CAR plasmid vector construction services to meet the personalized needs of customers. Our professional R&D team has rich experience in vector design and construction, and can provide one-stop customized services from vector design to product delivery.

1) Customized Design: According to the customer's research needs, design GPC3 CAR vectors of different generations, select appropriate promoters, fluorescent labels, antibiotic selection markers, and vector backbones; design and insert specific GPC3 scFv sequences, co-stimulatory domains, cytokines, or other functional genes.

2) Vector Construction: Complete the construction of the recombinant plasmid through gene synthesis, enzyme digestion, ligation, transformation, and clone screening, ensuring the correctness and efficiency of the vector.

3) Quality Inspection and Verification: Conduct strict quality inspection on the customized vector through sequence verification.

4) Fast Delivery: With efficient R&D and production processes, we can complete the customized construction of vectors in the shortest time and deliver them to customers on time.

Introduction of GPC3

GPC3, also known as glypican 3, is a member of the glypican family of heparan sulfate proteoglycans (HSPGs). The human GPC3 gene is located on the X chromosome (Xq26), with the official gene symbol GPC3 and HGNC ID HGNC:4410. It is also aliased as DGSX, GTR2-2, MXR7, and OCI-5. The GPC3 gene encodes a precursor core protein with a molecular weight of approximately 70 kDa, consisting of 580 amino acids in humans. This gene is highly conserved among species, and its abnormal expression is closely related to the occurrence and development of various tumors, making it an important molecular target for tumor diagnosis and treatment.

The GPC3 protein is a cell surface proteoglycan that is anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor. Its structure mainly includes three parts: an N-terminal signal peptide, an extracellular domain, and a C-terminal GPI anchor region. The extracellular domain contains two conserved cysteine-rich regions and can be modified by heparan sulfate (HS) chains, which are crucial for its biological functions. The mature GPC3 protein is formed by cleavage of the precursor protein into a 40 kDa N-terminal fragment and a C-terminal fragment that can be recognized by monoclonal antibodies. The crystal structure of GPC3 (PDB: 7zaw) has been resolved, providing a structural basis for the design of targeted drugs and CAR molecules.

GPC3 plays a key regulatory role in cell growth, differentiation, adhesion, and migration by interacting with various growth factors and signaling pathways. It can regulate cell morphogenesis and growth through insulin-like growth factor (IGF), bone morphogenetic protein (BMP), fibroblast growth factor (FGF), or Hedgehog signaling pathways. In normal embryonic development, GPC3 is highly expressed and participates in the regulation of embryonic growth and organ formation; however, its expression is significantly downregulated after birth. Abnormal overexpression of GPC3 in tumor cells can promote cell proliferation, inhibit apoptosis, and enhance tumor invasion and metastasis, thereby accelerating tumor progression.

GPC3 shows a strictly tissue-specific expression pattern. In normal adult tissues, GPC3 is only weakly expressed in the heart, lungs, kidneys, and ovaries, and is barely expressed in skeletal muscle, pancreas, small intestine, colon, and other tissues. In contrast, it is highly expressed in a variety of malignant tumors, especially in hepatocellular carcinoma (HCC), where the expression rate reaches 70%-90%. In addition, GPC3 is also highly expressed in testicular and ovarian yolk sac tumors, malignant melanoma, ovarian clear cell carcinoma, special-type gastric cancer, esophageal squamous cell carcinoma, testicular germ cell tumors, colon cancer, and renal rhabdoid tumors. This specific expression pattern makes GPC3 an ideal target for tumor-specific therapy.

The abnormal expression of GPC3 is closely associated with the occurrence and development of multiple diseases, mainly including tumors and genetic diseases. In terms of tumors, GPC3 is most closely related to HCC, which is the fourth leading cause of cancer-related death worldwide. GPC3 can be used as a reliable biomarker for HCC, helping to confirm the nature of lesions in the early stage of HCC and improve the accuracy of diagnosis. In addition, GPC3 is also involved in the progression of testicular cancer, ovarian cancer, melanoma, and other tumors, and its high expression is often associated with poor prognosis of patients. In terms of genetic diseases, mutations in the GPC3 gene can cause Simpson-Golabi-Behmel syndrome (SGBS), a rare X-linked recessive genetic disease characterized by gigantism, visceral hypertrophy, and an increased risk of childhood tumors.

Introduction of GPC3 Chimeric Antigen Receptor (CAR)

Chimeric antigen receptor (CAR) technology is a revolutionary immunotherapeutic strategy that modifies immune cells (mainly T cells and NK cells) through genetic engineering to express CAR molecules, enabling them to specifically recognize and kill tumor cells expressing target antigens. GPC3 CAR is a CAR molecule targeting the GPC3 antigen, which has become one of the most promising directions in the field of solid tumor immunotherapy due to the specific high expression of GPC3 in tumors.

The basic structure of GPC3 CAR is composed of four parts: an extracellular antigen-binding domain, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signal transduction domain. The extracellular antigen-binding domain is usually a single-chain variable fragment (scFv) derived from a GPC3-specific monoclonal antibody, which can specifically bind to the extracellular domain of GPC3 on the surface of tumor cells. The transmembrane domain is derived from CD4, CD8α, or other membrane proteins, which anchors the CAR molecule to the cell membrane. The intracellular co-stimulatory domain (such as CD28, 4-1BB, OX40) enhances the activation, proliferation, and survival of CAR-modified immune cells. The intracellular signal transduction domain (usually CD3ζ) transmits activation signals to trigger the immune cell killing response.

In recent years, GPC3 CAR research has made remarkable progress, with a large number of preclinical and clinical studies verifying its safety and effectiveness, especially in the treatment of HCC.

1) Preclinical Research Achievements: Preclinical studies have shown that GPC3 CAR-T cells can specifically recognize and kill GPC3-positive tumor cells in vitro, and exhibit significant anti-tumor activity in animal models. For example, GPC3 CAR-T cells can effectively inhibit the growth of HCC xenografts in nude mice and prolong the survival time of mice. In addition, researchers have optimized GPC3 CAR molecules, such as introducing armored negative dominant TGF-β type II receptors to improve the anti-tumor activity of CAR-T cells. Preclinical data of GPC3×4-1BB bispecific antibodies also show that they can specifically target GPC3-positive tumor cells and locally activate T cells, with a tumor cell inhibition rate of up to 65% in vitro.

2) Clinical Research Achievements: A number of clinical trials of GPC3 CAR-T cells have been carried out worldwide, and preliminary results show promising anti-tumor effects. For example, C-CAR031, a second-generation GPC3-targeted CAR-T cell product, showed positive anti-tumor efficacy in the phase I clinical trial, with 3 subjects achieving confirmed partial remission (PR) and 2 subjects achieving stable disease. CT011, a GPC3-targeted autologous CAR-T cell candidate product, has obtained IND approval from the National Medical Products Administration for the treatment of GPC3-positive solid tumors, and some advanced HCC patients have achieved continuous disease-free survival for more than seven years . In addition, BGB-B2033, a GPC3×4-1BB bispecific antibody, has obtained FDA fast track designation and its clinical application has been accepted by CDE, entering the clinical research stage in China.

Marketed Drugs Targeting GPC3

At present, there are no GPC3 CAR-T drugs officially approved for marketing worldwide, but a number of candidate products are in the clinical research stage and have shown good development prospects. In addition to the aforementioned CT011 and C-CAR031, ABZ-706 (RYZ801), a GPC3-targeted radioactive drug, is also in phase I clinical research, and BMS has obtained its exclusive development and commercialization rights in the Greater China region. With the continuous advancement of clinical research, it is expected that GPC3-targeted CAR drugs and other related therapies will be approved for marketing in the near future, bringing new treatment options for patients with GPC3-positive tumors.

Research Hotspots of GPC3 CAR

1) Optimization of GPC3 CAR Structure: Researchers are constantly optimizing the structure of GPC3 CAR, such as screening high-affinity scFv to improve the specificity and affinity of tumor recognition, and combining different co-stimulatory domains (such as CD28+4-1BB) to enhance the anti-tumor activity and persistence of CAR-T cells.

2) Application of GPC3 CAR in Combination Therapy: Combining GPC3 CAR-T cells with immune checkpoint inhibitors (such as PD-1/PD-L1 inhibitors), radiotherapy, chemotherapy, or targeted drugs to improve the anti-tumor effect. For example, the combination of GPC3×4-1BB bispecific antibody and PD-1 inhibitor can increase the tumor cell inhibition rate from 65% to 89%.

3) Development of GPC3 CAR-NK Cells: Compared with CAR-T cells, CAR-NK cells have the advantages of no graft-versus-host disease (GVHD), fast killing effect, and wide source, so the development of GPC3 CAR-NK cells has become a new research hotspot.

4) Overcoming Tumor Microenvironment (TME) Suppression: The immunosuppressive tumor microenvironment is one of the main factors limiting the efficacy of GPC3 CAR-T cells. Current research focuses on modifying CAR-T cells to resist the immunosuppressive effect of TME, such as expressing cytokines (IL-7, IL-15) or blocking immunosuppressive molecules (PD-1, TGF-β).

5) Development of GPC3-Targeted Bispecific Antibodies and Radioactive Drugs: In addition to CAR-T cells, GPC3-targeted bispecific antibodies (such as GPC3×4-1BB) and radioactive drugs (such as ABZ-706) are also important research directions, which can complement CAR-T therapy and enrich the GPC3-targeted treatment system.

Research Challenges of GPC3 CAR

1) Tumor Antigen Heterogeneity: HCC is one of the most heterogeneous malignant tumors, and the expression level of GPC3 in different tumor cells of the same patient is inconsistent, which may lead to tumor cell escape and limit the therapeutic effect of GPC3 CAR-T cells.

2) Immunosuppression of Tumor Microenvironment: The HCC microenvironment contains a variety of immunosuppressive factors (such as TGF-β, IL-10, PD-L1) and immunosuppressive cells (such as Treg cells, M2-type macrophages), which can inhibit the activation, proliferation, and killing function of GPC3 CAR-T cells.

3) Insufficient T Cell Infiltration: The vascular structure and matrix components in solid tumors such as HCC may hinder the effective infiltration of CAR-T cells into the tumor tissue, resulting in the inability of CAR-T cells to fully contact and kill tumor cells.

4) Off-Target Effects and Toxicity: Although GPC3 is rarely expressed in normal adult tissues, low-level expression in some tissues may lead to off-target effects of GPC3 CAR-T cells, causing damage to normal tissues. In addition, CAR-T cells may trigger cytokine release syndrome (CRS) and neurotoxicity, which need to be effectively controlled.

5) Limited Persistence of CAR-T Cells: The in vivo persistence of GPC3 CAR-T cells is insufficient, which may lead to the recurrence of tumors after treatment. How to improve the survival time of CAR-T cells in vivo is an important challenge.

References

Contact Us