CD47 Chimeric Antigen Receptor (CAR): A Comprehensive Guide and Our Service & Product Introduction

CD47 has emerged as a pivotal target in tumor immunotherapy, renowned for its role in mediating tumor immune escape. Chimeric Antigen Receptors (CARs) targeting CD47 have become a research hotspot in recent years, offering a promising strategy to overcome the "don't eat me" signal of tumor cells. RGBiotech is committed to becoming a leading supplier of CD47 CAR expression plasmid vectors and customized services, providing high-quality products and professional technical support for global researchers. We can provide you with personalized solutions to help you accelerate the research process and achieve research goals. If you are interested in our CD47 CAR expression plasmid vector products or customized construction services, please contact us at admin@rgbiotech.com for more details. We will reply to you within 24 working hours and provide you with professional consultation and services.

Our CD47 CAR Expression Plasmid Vector Products and Custom Services

RGBiotech is committed to providing high-quality CD47 CAR expression plasmid vector products and professional customized construction services, covering multiple generations of CD47 CAR and various vector backbones, to meet the diverse needs of researchers in research. With strict quality control standards and perfect technical support, we provide reliable tools for the research and development of CD47 CAR therapy.

We provide a complete series of CD47 CAR expression plasmid vectors, covering multiple generations of CD47 CAR, with diverse vector backbones, suitable for different cell modification and delivery needs. Our products are designed and optimized based on the latest research progress, ensuring high expression efficiency, strong specificity, and good safety.

1) First-generation CD47 CAR: Only contains the CD3ζ intracellular signaling domain, which can activate the cytotoxicity of T cells, suitable for preliminary research on CD47 CAR function;

2) Second-generation CD47 CAR: Adds a costimulatory signal domain (CD28 or 4-1BB) on the basis of the first generation, which can enhance the proliferation and survival ability of CAR-T cells;

3) Third-generation CD47 CAR: Contains two costimulatory signal domains (such as CD28+4-1BB or CD28+OX40), which can further improve the anti-tumor activity and long-term persistence of CAR-T cells;

4) Fourth-generation CD47 CAR (also known as armored CAR): Adds specific cytokine (IL-12, IL-15, etc.) or chemokine signal sequences on the basis of the third generation, which can reverse the immunosuppressive TME and enhance the infiltration and activity of CAR cells in solid tumors.

Product Features

1) Multiple Generations of CD47 CAR Coverage: We provide first-generation, second-generation, third-generation, and fourth-generation CD47 CAR expression plasmids, which can meet different research needs.

2) Diverse Vector Backbones: Our CD47 CAR expression plasmids cover a variety of vector backbones, including non-viral vectors (plasmid vectors, transposon vectors) and viral vectors (lentiviral vectors, retroviral vectors, AAV vectors), to meet different delivery requirements: (A) Non-viral vectors: Simple preparation, low immunogenicity, suitable for transient expression or large-scale production, especially suitable for in vivo delivery research using LNP and other carriers; (B)Lentiviral vectors: Production of lentiviral particles, high transduction efficiency, can infect both dividing and non-dividing cells, and integrate CAR genes into the host genome to achieve long-term stable expression, which is the most commonly used vector in CAR-T cell research and clinical application; (C)Retroviral vectors: Production of retroviral particles, high transduction efficiency for dividing cells, suitable for the modification of T cells and other rapidly dividing cells; (D)AAV vectors: Production of AAV particles, low immunogenicity, good safety, can achieve long-term stable expression in non-dividing cells, suitable for in vivo delivery of CD47 CAR to solid tumors and other tissues.

1) Optimized Promoters: We use high-efficiency promoters to ensure high-level expression of CD47 CAR in target cells, including CMV promoter (strong constitutive expression, suitable for most cell types), EF1α promoter (stable expression, low cell type dependence), and tissue-specific promoters (such as CD4 promoter, CD8 promoter, suitable for T cell-specific expression), which can be selected according to research needs.

1) Multiple Fluorescent Labels: The plasmid vectors are equipped with various fluorescent reporter genes, including GFP (green fluorescent protein), mCherry (red fluorescent protein), mKate2 (orange-red fluorescent protein), etc., which can be used to monitor the transfection efficiency and expression level of CD47 CAR in real time, facilitating the screening and identification of modified cells.

1) Diverse Antibiotic Selection Markers: We provide multiple antibiotic selection markers, including ampicillin (Amp), kanamycin (Kan), puromycin (Puro), neomycin (Neo), hygromycin (Hygro), blasticidin (Bsd), etc., which can be used for the screening of positive clones in prokaryotic cells (E. coli) and eukaryotic cells (T cells, macrophages), improving the efficiency of cell modification.

Product Advantages

1) High Expression Efficiency: The vector is optimized for the codon of CD47 CAR, and the high-efficiency promoter is used to ensure that CD47 CAR is highly expressed in target cells, enhancing the anti-tumor activity of modified immune cells;

2) Good Specificity: The scFv domain in the CD47 CAR is carefully selected and verified, which can specifically bind to CD47, reducing off-target effects and improving the safety of the product;

3) Easy to Use: The plasmid vector can be used for the modification of various immune cells (T cells, macrophages, NK cells);

4) Sequence Verification: Perform Sanger sequencing on the target gene (CD47 CAR) to ensure that the sequence is correct and consistent with the design, without base mutation or deletion;

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 researchers.

Product Applications

1) Basic research: Study the structure and function of CD47 CAR, explore the mechanism of CD47 CAR-modified immune cells killing tumor cells, and optimize the design of CD47 CAR.

2) Preclinical research: Construct CD47 CAR-modified immune cells (CAR-T, CAR-macrophages, CAR-NK cells), carry out in vitro cell killing experiments and in vivo animal model experiments, evaluate the anti-tumor effect and safety of CD47 CAR therapy.

3) Drug screening: Establish a CD47 CAR-based drug screening model, screen small molecules or biological agents that can enhance the anti-tumor effect of CD47 CAR, and develop combined therapy strategies.

Custom CD47 CAR Plasmid Vector Construction Services

In addition to the standard CD47 CAR expression plasmid vectors, we also provide professional customized plasmid vector construction services to meet the personalized needs of researchers. Our customized services include:

1) Customized Design of CD47 CAR: According to the research needs of customers, construct CD47 CAR with specific scFv domain, costimulatory domain, signaling domain, or cytokine sequence, and optimize the codon to improve expression efficiency;

2) Customized Vector Backbone: Select or modify the vector backbone (non-viral, lentiviral, retroviral, AAV, etc.) according to the customer's delivery method and cell type, and add specific elements (such as tissue-specific promoters, suicide genes);

3) Modification of Fluorescent Labels and Selection Markers: According to the customer's needs, replace the fluorescent labels or antibiotic selection markers in the vector, or add multiple fluorescent labels/selection markers for double screening;

4) Whole-Cycle Technical Support: Provide professional technical consultation, design schemes and construction services to ensure that the customized vector meets the customer's research needs.

Our customized service team is composed of experienced molecular biologists, who can provide one-stop customized services from scheme design to vector delivery, with fast construction cycle and high construction success rate, helping customers save research time and cost.

Introduction of CD47

CD47, also known as integrin-associated protein (IAP), is encoded by the CD47 gene located on human chromosome 3q13.31. The CD47 gene spans approximately 28 kb and consists of 8 exons, which undergo alternative splicing to generate five main isoforms with different cytoplasmic tail lengths and sequences, leading to functional diversity in different cell types. The gene is highly conserved across species, reflecting its essential role in physiological processes. As a key immune regulatory gene, CD47 mutation or abnormal expression is closely associated with the occurrence and development of various diseases, especially tumors.

CD47 is a transmembrane glycoprotein with a molecular weight of approximately 50 kDa, belonging to the immunoglobulin superfamily (IgSF). Its structure is composed of three parts: an N-terminal extracellular IgV domain, five hydrophobic transmembrane segments, and a short hydrophilic cytoplasmic tail. The extracellular IgV domain is the core functional region, which is responsible for binding to its ligands, mainly signal regulatory protein α (SIRPα), thrombospondin-1 (TSP-1), and other molecules. The cytoplasmic tail, though short, interacts with Gi proteins and other signaling molecules to mediate intracellular signal transduction, regulating cell proliferation, apoptosis, and immune response.

CD47 plays a crucial role in maintaining immune homeostasis and regulating cell-cell interactions. Its main functions include the following aspects: First, it mediates the "don't eat me" signal: CD47 binds to SIRPα on the surface of macrophages and dendritic cells, inhibiting their phagocytic activity, thereby preventing the immune system from attacking normal cells (such as red blood cells) and maintaining self-immune tolerance. Second, it regulates cell proliferation and apoptosis: CD47 interacts with TSP-1 to activate integrin signaling, further regulating Ras/ERK and PI3K/Akt pathways, which are involved in promoting tumor cell proliferation, migration, and invasion; at the same time, it can also inhibit cAMP-dependent signaling pathways or interact with BNIP3 to regulate cell apoptosis. Third, it participates in anti-angiogenesis: TSP-1 binds to CD47, leading to the dissociation of CD47 from VEGFR2, inhibiting VEGFR2 phosphorylation and thus blocking angiogenesis pathways. Fourth, it regulates immune cell function: CD47 is widely expressed on immune cells such as T cells, B cells, and macrophages, and is involved in regulating the activation, proliferation, and differentiation of immune cells.

CD47 is widely expressed in almost all normal tissues and cells of the human body, including hematopoietic cells (red blood cells, white blood cells, platelets), epithelial cells, endothelial cells, and immune cells (macrophages, dendritic cells, T cells, B cells). Among them, the expression level of CD47 on the surface of red blood cells is relatively high, which is crucial for preventing red blood cells from being phagocytosed by macrophages during their normal life cycle. In addition, CD47 is also expressed in some normal tissues such as the brain, heart, liver, and kidney, but its expression level is relatively low. In pathological states, especially in tumors, CD47 is often abnormally highly expressed, which is one of the important mechanisms for tumor cells to escape immune surveillance.

Abnormal expression of CD47 is closely related to a variety of diseases, among which tumors are the most studied. More than 15 types of tumors have been found to highly express CD47, including hematological tumors (lymphoma, leukemia, multiple myeloma) and solid tumors (breast cancer, colorectal cancer, glioblastoma, gastric cancer, lung cancer, ovarian cancer). In breast cancer, especially triple-negative breast cancer (TNBC), CD47 high expression is significantly associated with tumor clinical stage, lymph node metastasis, distant metastasis, and poor prognosis. In glioblastoma, CD47 high expression helps tumor cells escape macrophage phagocytosis, further worsening the prognosis of patients. In addition to tumors, CD47 is also involved in the occurrence and development of autoimmune diseases (such as systemic lupus erythematosus), cardiovascular diseases, and neurodegenerative diseases, but its role in these diseases is still in the preliminary research stage.

Introduction of CD47 Chimeric Antigen Receptor (CAR)

CD47 Chimeric Antigen Receptor (CD47 CAR) is a genetically engineered receptor that can specifically recognize CD47 on the surface of tumor cells. It is usually composed of three core components: an extracellular antigen-binding domain (scFv, single-chain variable fragment) that specifically binds to CD47, a transmembrane domain that anchors the receptor on the cell membrane, and an intracellular signaling domain that activates immune cells (such as T cells, macrophages) after antigen binding. By transducing CD47 CAR into immune cells, the modified immune cells can specifically recognize and kill CD47-positive tumor cells, overcoming the "don't eat me" signal mediated by CD47-SIRPα, and providing a new strategy for tumor immunotherapy. Compared with traditional CD47 blocking therapies (such as anti-CD47 monoclonal antibodies), CD47 CAR-modified immune cells have the advantages of strong specificity, long-term efficacy, and ability to continuously kill tumor cells, which has become a new direction in CD47-targeted therapy research.

Research Achievements of CD47 CAR

In recent years, research on CD47 CAR has made remarkable progress, and a large number of preclinical studies have confirmed its significant anti-tumor effect. In 2024, Zhu Mingzhao's team from the Institute of Microbiology, Chinese Academy of Sciences, and Fu Yangxin's team from Tsinghua University found that CD47-SIRPα checkpoint blockers can promote anti-tumor immune response by regulating tumor cell metabolic reprogramming through type I interferon (IFN-I) signaling, which provides a theoretical basis for the combination of CD47 CAR therapy and other therapies. In 2025, HCB101, a new generation of CD47-targeted therapy developed by Hankang Biotech, achieved breakthrough results in clinical trials: when combined with standard therapy, the objective response rate (ORR) in the middle dose group of second-line gastric cancer reached 80%, and even in the low-dose group of head and neck squamous cell carcinoma, the partial response rate reached 100% when combined with PD-1 inhibitors, which verified the potential of CD47-targeted therapy in clinical application and provided a reference for the development of CD47 CAR therapy. Preclinical studies have shown that CD47 CAR-T cells can effectively kill CD47-positive tumor cells in vitro and in vivo, and significantly inhibit tumor growth in mouse models of colorectal cancer, lymphoma, and breast cancer, extending the survival period of mice. In addition, CD47 CAR-macrophages have also attracted wide attention: modified macrophages can break through the "don't eat me" signal of tumor cells, enhance phagocytic activity, and activate adaptive immune response by presenting tumor antigens, achieving synergistic anti-tumor effect with CAR-T cells.

Marketed Drugs Related to CD47 Target

At present, there are no marketed drugs specifically targeting CD47 CAR, but the research and development of CD47-targeted therapies (including monoclonal antibodies, fusion proteins, etc.) is in full swing, and some candidates have entered late-stage clinical trials. It should be noted that the development of CD47-targeted therapies has experienced twists and turns: Gilead acquired Forty Seven, a company focusing on anti-CD47 monoclonal antibody development, for 4.9 billion US dollars, but in April 2024, it completely abandoned the anti-CD47 monoclonal antibody Magrolimab due to clinical trial failure. However, with the continuous optimization of molecular design, new CD47-targeted drugs have broken through the safety bottleneck: HCB101, developed by Hankang Biotech, did not show significant anemia, thrombocytopenia, or other toxic reactions in dose-escalation trials, and its therapeutic window reached 25mg, which rewritten the industry's cognition of CD47-targeted therapy toxicity. The progress of CD47-targeted therapies provides a good foundation for the development of CD47 CAR drugs. It is expected that with the deepening of research, CD47 CAR drugs will enter clinical trials in the next 3-5 years and bring new treatment options for patients with CD47-positive tumors.

Research Hotspots of CD47 CAR

1) Optimization of CD47 CAR structure: Improving the affinity of the extracellular scFv domain to CD47, optimizing the intracellular signaling domain, and enhancing the anti-tumor activity, persistence, and safety of CAR-modified immune cells. For example, the fourth-generation CD47 CAR adds specific cytokine signals, which can make CAR-T cells resistant to the immunosuppressive effect of the tumor microenvironment (TME) and improve their in vivo survival time.

2) Development of CD47 CAR-modified immune cells other than T cells: Focusing on CD47 CAR-macrophages, CAR-NK cells, etc. Macrophages are the main cells mediating the phagocytosis of tumor cells, and CD47 CAR-macrophages can directly break through the "don't eat me" signal, which has unique advantages in the treatment of solid tumors.

3) Combination therapy strategy: Combining CD47 CAR therapy with PD-1/PD-L1 inhibitors, STING ligands, CD39/CD73 inhibitors, or chemotherapy drugs to enhance anti-tumor effect. Studies have shown that the combination of CD47 antibody and STING ligand cGAMP can significantly inhibit tumor growth in mouse breast cancer models, and the combination of CD47 CAR therapy and PD-1 inhibitors can improve the response rate of "immune-cold tumors".

4) Development of in vivo delivery systems for CD47 CAR: Using viral vectors (lentivirus, AAV) or non-viral vectors (LNP, polymer nanoparticles) to achieve in vivo delivery of CD47 CAR, avoiding the complex process of ex vivo cell modification and improving the convenience of treatment.

5) Exploration of resistance mechanisms: Studying the mechanism of tumor cells' resistance to CD47 CAR therapy, such as the up-regulation of MHC1 expression in tumor cells, and developing corresponding solutions to improve the long-term efficacy of therapy.

Research Challenges of CD47 CAR

Despite the promising prospects of CD47 CAR, there are still many difficulties and challenges in its research and development.

1) Toxicity and side effects: CD47 is widely expressed on normal cells (such as red blood cells), so CD47 CAR-modified immune cells may attack normal cells, leading to anemia, thrombocytopenia, and other side effects. How to improve the tumor specificity of CD47 CAR and reduce off-target toxicity is a key challenge.

2) Tumor microenvironment (TME) immunosuppression: The TME of solid tumors is rich in immunosuppressive cells (such as regulatory T cells, tumor-associated macrophages) and immunosuppressive factors (such as IL-10, TGF-β), which can inhibit the activity of CD47 CAR-modified immune cells and reduce the therapeutic effect. How to reverse TME immunosuppression and enhance the infiltration and activity of CAR cells in solid tumors is another major challenge.

3) Limited efficacy in solid tumors: At present, most preclinical studies of CD47 CAR are focused on hematological tumors, and its efficacy in solid tumors is relatively limited, which is related to the poor infiltration of CAR cells into solid tumors and the strong immunosuppressive microenvironment.

4) Long-term persistence and recurrence: The long-term persistence of CD47 CAR-modified immune cells in vivo is insufficient, and tumor recurrence is easy to occur after treatment. In addition, tumor cells may develop resistance to CD47 CAR therapy through down-regulating CD47 expression or activating alternative immune escape pathways, which affects the long-term therapeutic effect.

5) Technical bottlenecks in vector delivery: The delivery efficiency of viral vectors is high, but there are risks of insertional mutation; non-viral vectors have low immunogenicity and simple preparation, but their delivery efficiency and expression persistence are poor. How to choose an appropriate delivery vector and improve the delivery efficiency and safety of CD47 CAR is a technical bottleneck that needs to be broken through.

References

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