CD133(PROM1) Chimeric Antigen Receptor (CAR): A Comprehensive Guide and Our Service & Product Introduction

Our CD133(PROM1) CAR Expression Plasmid Vector Products and Custom Services

Product Features

1) Multiple Generations of CD133 CAR: We offer 1st to 4th generation CD133 CAR expression plasmids to meet different research requirements.
a) 1st Generation: Contains only the CD3ζ intracellular signaling domain, suitable for basic research on CD133 recognition and T cell activation.
b) 2nd Generation: Adds one co-stimulatory domain (CD28 or 4-1BB), enhancing the proliferation and survival of CAR-T cells.
c) 3rd Generation: Contains two co-stimulatory domains (CD28+4-1BB, CD28+OX40, etc.), further improving the anti-tumor efficacy and persistence of CAR-T cells.
d) 4th Generation: Integrates cytokine secretion elements (such as IL-12, IL-15) or immune checkpoint blocking molecules (such as PD-1 scFv), which can remodel the tumor microenvironment and enhance the anti-tumor immune response.

2) Diverse Vector Backbones: Our CD133 CAR plasmids are available in various vector backbones, including non-viral vectors, lentiviral vectors, retroviral vectors, and adeno-associated virus (AAV) vectors. Customers can choose the appropriate vector backbone according to their experimental system and delivery method.

3) Optimized Promoters: We use high-efficiency promoters to ensure strong and stable expression of CD133 CAR, including CMV (cytomegalovirus promoter), EF1α (elongation factor 1α promoter), and PGK (phosphoglycerate kinase promoter). EF1α and PGK promoters are preferred for long-term stable expression in primary cells and animal models, while CMV promoter is suitable for high-efficiency transient expression.

4) Fluorescent Markers for Visualization: Some of our CD133 CAR plasmids are equipped with fluorescent markers for easy detection and sorting of CAR-expressing cells, such as GFP (green fluorescent protein) and RFP (red fluorescent protein). The fluorescent markers are linked to the CAR gene or the antibiotic marker gene via an a 2A peptide, ensuring co-expression of CAR and fluorescent protein.

5) Antibiotic Selection Markers: To facilitate the screening of positive clones, our plasmids contain common antibiotic selection markers, such as Ampicillin (Amp) or Kanamycin (Kan) for bacterial screening, and Puromycin (Puro), Neomycin (Neo), or Hygromycin (Hyg) for eukaryotic cell screening. Customers can choose the appropriate selection marker according to their cell lines and experimental needs.

Product Advantages

1) High Expression Efficiency: The CD133 CAR gene sequence is optimized to improve the expression level of CAR on the cell surface, enhancing the recognition and killing ability of CAR-T cells against CD133⁺ tumor cells.

2) Strong Targeting Specificity: The scFv of CD133 CAR is specifically designed to recognize the CD133 antigen, ensuring high specificity and reducing off-target effects.

3) Good Compatibility: Our plasmids are compatible with various cell lines and delivery methods, meeting the needs of different experimental designs.

4) Strict Quality Control: Each batch of plasmids undergoes Sanger sequencing to confirm that the CD133 CAR gene sequence are correct, with no mutations or deletions.

5) Cost-Effective: We provide high-quality products at competitive prices, and offer bulk purchase discounts to reduce the research cost of customers.

6) Comprehensive Technical Support: Our professional technical team provides full-range technical support to help customers smoothly carry out their research.

Product Applications

Our CD133 CAR expression plasmid vectors are widely used in scientific research and preclinical studies, including but not limited to:

1) Basic research on CD133 CAR: Study the mechanism of CD133 CAR-mediated immune cell activation, proliferation, and tumor killing.

2) In vitro anti-tumor activity detection: Construct CD133 CAR-T/NK cells in vitro, and detect their killing ability against CD133⁺ tumor cell lines (such as U87MG, A375, HepG2).

3) In vivo animal model studies: Evaluate the anti-tumor efficacy and safety of CD133 CAR-T cells in mouse tumor models (such as glioblastoma, melanoma, colon cancer models).

4) Optimization of CD133 CAR structure: Screen and optimize the scFv, co-stimulatory domains, and promoters of CD133 CAR to improve its anti-tumor efficacy and safety.

5) Research on combination therapy: Explore the combined effect of CD133 CAR-T therapy with radiotherapy, chemotherapy, immunotherapy, or heat therapy in preclinical models.

Customized CD133 CAR Plasmid Vector Construction Services

In addition to our standard products, we also provide customized CD133 CAR plasmid vector construction services to meet the personalized needs of customers.
1) Customized CD133 CAR structure design: Design and construct CD133 CAR with specific scFv (targeting CD133/1 or CD133/2 epitopes), co-stimulatory domains, and cytokine secretion elements according to customers' research needs.
2) Customized vector backbone selection: Choose the appropriate vector backbone (non-viral, lentiviral, retroviral, AAV) according to customers' experimental system and delivery method.
3) Customized markers: Replace or add fluorescent markers (such as GFP, mCherry) or selection markers according to customers' needs.
4) Codon optimization: Optimize the CD133 CAR gene sequence according to the target cell species (human, mouse, rat) to improve the expression efficiency.
Our customized services have the advantages of fast delivery, high accuracy, and strict quality control. Our professional technical team will communicate with customers in detail to ensure that the customized products meet their research needs.

Introduction of CD133(PROM1)

CD133, also known as Prominin-1 (PROM1), is a cell surface glycoprotein that is widely used as a key marker in stem cell and cancer research. It is primarily recognized for its role in identifying cancer stem cells (CSCs), a subpopulation of tumor cells with stem cell-like characteristics that contribute to tumor initiation, progression, recurrence, and resistance to chemotherapy and radiotherapy. The expression of CD133 is regulated by both intrinsic cellular mechanisms and external microenvironmental factors, and its dysregulation is closely associated with the development and progression of various malignancies.

The human CD133 gene (PROM1) is located on chromosome 4p16.12-p16.1 and chromosome 2. Its official gene symbol is PROM1, and the NCBI Gene ID is 8842. The CD133 gene encodes multiple transcript variants due to alternative splicing, resulting in different protein isoforms. The reference sequence (mRNA) of human CD133 can be accessed via ENA Browser with the accession number X59131.1. The open reading frame (ORF) of CD133 encodes a protein with approximately 513 amino acids, and its expression is tightly regulated in a tissue- and cell type-specific manner.

CD133 is a pentaspan transmembrane glycoprotein with a relative molecular mass of approximately 120 kDa. Its structure consists of five transmembrane domains, an N-terminal extracellular domain, and a C-terminal intracellular domain. The extracellular domain contains several N-glycosylation sites, which are crucial for its function and antigenic properties. Notably, different epitopes of CD133 have been identified, including CD133/1 (recognized by clone AC133, a glycosylated epitope associated with stem cell phenotype) and CD133/2 (recognized by clones 293C3 and AC141, non-glycosylated epitopes used for flow cytometry staining). These epitope differences have important implications for the design of CD133-targeting therapies such as CAR-T.

CD133 plays a critical role in various key cellular processes, particularly in the regulation of stem cell properties and cancer progression. It is involved in the activation of signaling pathways that are frequently dysregulated in cancer, including the PI3K/Akt and Wnt/β-catenin pathways. The expression of CD133 enhances cell self-renewal, migration, invasion, and survival under cancer stress conditions, thereby conferring an adaptive advantage to tumor cells. Additionally, CD133 is associated with autophagy regulation in cancer stem cells; for example, α-1,2-mannosylation of CD133 enhances the self-renewal and tumorigenesis of intrahepatic cholangiocarcinoma-initiating cells by upregulating the autophagy-related gene FIP200. It also participates in the formation of resistance to immunotherapies, such as macrophage-based therapy, by activating the TGF-β signaling pathway and remodeling the tumor microenvironment.

CD133 is not exclusively expressed in tumor cells; it is also present in various normal tissues, primarily in stem/progenitor cells and differentiated cells. Normal tissues expressing CD133 include the hematopoietic system (hematopoietic stem cells in peripheral blood, bone marrow, and umbilical cord blood), nervous system (neural stem cells), gastrointestinal tract, liver, and kidney. In tumors, CD133 is highly expressed in a variety of solid tumors, including glioblastoma, melanoma, intrahepatic cholangiocarcinoma, non-small cell lung cancer, hepatocellular carcinoma, gastric cancer, colorectal cancer, breast cancer, and prostate cancer. Notably, the positive expression rate of CD133 in non-small cell lung cancer tissues is approximately 48.9%, and its expression is closely associated with poor tumor differentiation, lymph node metastasis, and advanced clinical stage.

Introdution of CD133(PROM1) Chimeric Antigen Receptor (CAR)

CD133 Chimeric Antigen Receptor (CAR) is a genetically engineered receptor expressed on the surface of immune cells (primarily T cells, known as CD133 CAR-T cells) that specifically recognizes and binds to the CD133 antigen on the surface of tumor cells, particularly cancer stem cells. The structure of CD133 CAR typically includes an extracellular antigen-binding domain (scFv) that specifically recognizes CD133, a transmembrane domain, and an intracellular signaling domain. Different generations of CD133 CAR differ in the composition of their intracellular signaling domains, which affects the activation, proliferation, and anti-tumor efficacy of CAR-T cells.

Research Achievements of CD133 CAR

In recent years, significant progress has been made in the research and development of CD133 CAR, particularly in the treatment of solid tumors.

1) Dual-Target CD133/PD-L1 CAR-T Therapy: A novel dual-target CD133/PD-L1 CAR-T strategy has been developed, which shows stronger activation phenotype, cytokine secretion ability, and in vitro and in vivo killing effect in various CD133⁺ solid tumor models. The combination of this dual-target CAR-T with low-dose radiotherapy and PD-1 inhibitors (a "sandwich" model) can significantly enhance the anti-tumor efficacy by inducing the expansion of CXCR6⁺CD103⁺ tissue-resident memory-like (Trm) CAR-T cells and remodeling the tumor microenvironment.

2) CD133/CD44 Dual-Target CAR-T for Glioblastoma: P134 cell injection, an autologous CAR-T product targeting CD133 and CD44, has obtained clinical trial approval in China for the treatment of recurrent glioblastoma. This product targets glioblastoma stem cells (the root cause of treatment resistance and recurrence), with a target coverage rate of over 80%. Clinical trial results show that it can significantly slow tumor growth, with a disease control rate of 80%, and no systemic cytokine release syndrome or central nervous system toxicity has been observed.

3) Combination Therapy Strategies: Heat therapy has been shown to reverse the resistance of CD133⁺PD-L1⁺ cancer cells to macrophage-based therapy by reducing ECM density, inhibiting the TGF-β pathway, and enhancing immune responses. In a mouse melanoma model, the combination of heat therapy and engineered macrophage transfer significantly prolonged the median survival time of mice, and even achieved complete tumor cure in a mouse colon cancer model when combined with NIR-II fluorescence image-guided therapy.

4) Mechanism Research: Studies have revealed that CD133⁺ cancer cells confer resistance to immunotherapy by activating the TGF-β signaling pathway and inducing tumor-associated fibroblasts (CAFs) to enhance ECM stiffness, thereby blocking the migration and infiltration of immune cells. Targeting the TGF-β signaling pathway or tumor microenvironment components can enhance the efficacy of CD133-targeted therapies.

Approved Drugs of CD133 CAR

To date, there are no CD133 CAR products officially approved for marketing globally. However, several CD133-targeted CAR-T products are in clinical development stages, showing promising efficacy and safety. The most advanced product is P134 cell injection (developed by Tasly Pharmaceutical and Beijing Neurosurgical Institute), which obtained clinical trial approval from China's National Medical Products Administration (NMPA) in April 2025 for the treatment of recurrent glioblastoma. This is the first CD133-targeted CAR-T product in China to enter clinical trials, marking an important breakthrough in the clinical transformation of CD133 CAR therapy. Other CD133 CAR products are in preclinical or early clinical stages, focusing on various solid tumors such as melanoma, intrahepatic cholangiocarcinoma, and non-small cell lung cancer.

Research Hotspots of CD133 CAR

The current research hotspots of CD133 CAR mainly focus on overcoming the bottlenecks of CAR-T therapy in solid tumors and improving its efficacy and safety.

1) Dual-Target or Multi-Target CAR Design: Developing dual-target CARs (such as CD133/PD-L1, CD133/CD44) to improve the specificity of tumor targeting, reduce off-target effects, and overcome tumor heterogeneity. This strategy can simultaneously target cancer stem cells and immunosuppressive molecules in the tumor microenvironment, enhancing anti-tumor efficacy.

2) Combination Therapy Strategies: Exploring the combination of CD133 CAR-T therapy with radiotherapy, chemotherapy, immunotherapy (such as PD-1/PD-L1 inhibitors), heat therapy, or targeted therapy to reverse tumor resistance, remodel the tumor microenvironment, and enhance the infiltration and persistence of CAR-T cells.

3) Fourth-Generation CAR (CAR-Treg, CAR-NK, CAR-Mφ): Developing fourth-generation CD133 CARs that can secrete cytokines (such as IL-12, IL-15) to enhance the anti-tumor immune response, or engineering other immune cells (such as NK cells, macrophages) to express CD133 CAR, expanding the application scope of CD133-targeted immunotherapy.

4) Optimization of CD133 Epitope Targeting: Studying the differences between CD133 epitopes (such as CD133/1 and CD133/2) and their effects on CAR-T efficacy, and designing CARs targeting specific epitopes to improve the recognition and killing of cancer stem cells.

5) Regulation of CD133 Glycosylation: Exploring the role of CD133 glycosylation (such as α-1,2-mannosylation) in cancer stem cell properties and CAR-T recognition, and developing strategies to regulate CD133 glycosylation to enhance the efficacy of CD133-targeted therapies.

Research Challenges of CD133 CAR

1) Tumor Heterogeneity: CD133 expression is not uniform in tumors, and some tumor cells may lose CD133 expression, leading to CAR-T therapy escape. Additionally, the presence of multiple cancer stem cell subpopulations further increases the difficulty of targeting.

2) Immunosuppressive Tumor Microenvironment (TME): The solid tumor microenvironment is rich in immunosuppressive cells (such as Tregs, M2 macrophages) and molecules (such as TGF-β, PD-L1), which inhibit the activation, proliferation, and infiltration of CD133 CAR-T cells, reducing their anti-tumor efficacy.

3) Off-Target Effects: CD133 is also expressed in normal stem/progenitor cells, which may lead to CD133 CAR-T cells attacking normal tissues, causing severe side effects. How to improve the tumor-specific targeting of CD133 CAR is a key challenge.

4) Limited Infiltration of CAR-T Cells into Solid Tumors: Solid tumors have a dense extracellular matrix and high interstitial pressure, which hinder the infiltration of CAR-T cells into the tumor core, resulting in insufficient killing of tumor cells, especially cancer stem cells in the tumor microenvironment.

5) Resistance of Cancer Stem Cells: CD133⁺ cancer stem cells have strong self-renewal ability and drug resistance, and can escape the killing of CAR-T cells through various mechanisms (such as activating the TGF-β pathway, upregulating PD-L1 expression), leading to tumor recurrence.

References

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