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Solutions for Evaluation of CAR Expression

Fluorescent-Labeled Proteins

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Biotinylated Proteins

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Unconjugated Proteins

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Chimeric antigen receptor T (CAR-T) cell therapy is a cancer immunotherapy based on genetically modified T cells that express specific receptor fragments recognizing tumor surface antigens. The modified T cells are infused into patients and can directly target cancer cells in the body without the help of antigen-presenting cells (APCs) and exert immune killing effects.

For CAR-T cells, the effective component for tumor killing is CAR-positive T cells. The packaging specifications and clinical dosage of CAR-T cell products are expressed by the number of CAR-positive cells. Therefore, CAR transduction positive rate is a mandatory test item for CAR-T quality control. Regulatory authorities recommend using flow cytometry to detect CAR transduction positive rate. Currently, there are detection methods for different structural regions of CAR, including those targeting CAR antigen binding sites, such as CD19 antigen, or anti-Fab antibodies or Protein L proteins targeting light or heavy chain regions. Among them, the CAR positive rate detection method targeting antigen binding sites has been widely used due to its better specificity.

ACROBiosystems, as a protein supplier focused on pharmaceutical R&D, has developed a series of CAR-T target proteins in various forms including unlabeled, biotin-labeled, and fluorescent-labeled using professional protein development platform, protein labeling platform, stable cell line development platform and flow cytometry analysis platform, as well as supporting flow cytometry protocols for detecting CAR positive rates, to assist CAR-T R&D and accelerate the progress of CAR-T research. Currently, products cover more than 80 popular CAR-T targets including CD19, BCMA, CD22, MSLN and EGFR.

Direct method —Fluorescent-Labeled Proteins

KEY FEATURES

Target antigens are pre-labeled with a fluorescent dye.
Processing time can be reduced by the use of direct-labeled proteins.
Non-specific reaction of a secondary antibody is eliminated.

> PE-labeled （Click molecule view product details）

> FITC-labeled （Click molecule view product details）

> Case Study

Evaluation of Anti-CD19 CAR Expression with FITC-labeled CD19
: 293 cells were transfected with anti-CD19-scFv and RFP tag. 2e5 of the cells were stained with B. FITC-Labeled Human CD19 (20-291) (Cat. No. CD9-HF2H2, 10 µg/ml) and C. FITC-labeled protein control. A. Non-transfected 293 cells and C. FITC-labeled protein control were used as negative control. RFP was used to evaluate CAR (anti-CD19-scFv) expression and FITC was used to evaluate the binding activity of FITC-labeled Human CD19 (20-291) (Cat. No. CD9-HF2H2).; Request Protocol

Biotin-streptavidin based detection using biotinylated proteins

KEY FEATURES

Target antigens are pre-labeled with biotin and detected by labeled streptavidin (the biotin-avidin complex).
Streptavidin labeled with fluorochromes can bind biotinylated proteins with a high degree of affinity and specificity, amplifying the signal and improving the detection sensitivity and specificity.

> Biotinylated proteins -> Specially designed

BCMA CD19 CD30 EGF R Her2 Mesothelin PSMA ROR1

> Case Study

Evaluation of Anti-BCMA CAR Expression with Biotinylated BCMA
: Human T cells were transfected with anti-BCMA CAR and cultured for 3 days. Three days post-transfection, 1e6 cells were first incubated with 50 µl biotinylated human BCMA protein (Cat. No. BC7-H82F0, 8 µg/ml), washed and then stained with PE Streptavidin and analyzed by flow cytometry. (Data are kindly provided by PREGENE Biopharma); Request Protocol

Indirect detection using unconjugated proteins

KEY FEATURES

Target antigens are designed to carry a specific tag and detected using a secondary antibody (anti-epitope tag antibody) labeled with a fluorophore.
High sensitivity detection of CAR positive expression rate.
Non-specific reaction of a secondary antibody may occur.

> Unconjugated proteins -> Specially designed

> Case Study

Evaluation of Anti-CD19 CAR Expression with Fc-fusion CD19
: 293 cells were transfected with FMC63-scFv and RFP tag. 2e5 of the cells were first stained with B. Human CD19 (20-291) Protein, Fc Tag, low endotoxin (Super affinity) (Cat. No. CD9-H5251, 3 µg/ml) and C. Human Fc Tag Protein Control, followed by FITC-conjugated Anti-human IgG Fc Antibody. A. Non-transfected 293 cells and C. Human Fc Tag Protein Control were used as negative control. RFP was used to evaluate CAR (anti-CD19-scFv) expression and FITC was used to evaluate the binding activity of Human CD19 (20-291) Protein, Fc Tag, low endotoxin (Super affinity) (Cat. No. CD9-H5251).; Request Protocol

More CAR-T related products

> Click here to learn more about mAb for anti-CD19 (FMC63) CAR detection-flow cytometry validated > Click here to learn more about high affinity CD19 protein-specifically designed for detecting CD19-CAR-T

FAQ

What are the key advantages of CAR-T therapy over conventional chemotherapy and targeted therapies?

As a cell-based immunotherapy, CAR-T offers several unique advantages over conventional chemotherapy and targeted therapies. First, CAR-T cells recognize tumor antigens in an MHC-independent manner, enabling direct tumor cell killing without the need for antigen presentation and helping overcome immune escape caused by MHC downregulation. Second, CAR-T cells can expand and persist in vivo after infusion, providing continuous immune surveillance and elimination of residual tumor cells, which may result in durable remission or even long-term disease control beyond the transient effects of conventional drugs. Finally, CAR-T therapy offers highly specific tumor targeting, selectively eliminating antigen-expressing tumor cells while minimizing damage to normal tissues, and has demonstrated remarkable efficacy in patients with relapsed or refractory malignancies. The precision of CAR recognition depends heavily on high-quality target antigens with native conformations. ACROBiosystems provides a comprehensive portfolio of more than 80 CAR-T target proteins covering both hematologic and solid tumors. Featuring high biological activity, native conformation, and excellent batch-to-batch consistency, these proteins support CAR affinity optimization, construct screening, and functional validation to maximize the efficacy and safety of CAR-T therapies.

What are the major CAR-T technology platforms, and what are their key characteristics?

As CAR-T technology continues to evolve, multiple mature platforms have emerged to address diverse clinical needs. Based on cell source, CAR-T therapies are generally classified as autologous or allogeneic ("off-the-shelf") CAR-T. Autologous CAR-T therapies are manufactured from a patient's own T cells, minimizing the risk of immune rejection and representing the current clinical standard. Allogeneic CAR-T therapies leverage gene-editing technologies to enable large-scale manufacturing and ready-to-use products, addressing challenges associated with lengthy production timelines and variable product quality. Based on CAR design, current platforms include single-target CARs, dual CARs, tandem CARs (TanCARs), and logic-gated CARs, each designed for specific applications such as routine treatment, overcoming antigen escape, or improving treatment specificity and safety. In addition, next-generation platforms such as armored CAR-T and cytokine-engineered CAR-T therapies further enhance antitumor activity by remodeling the tumor microenvironment and improving CAR-T persistence and functionality.

What is in vivo CAR-T, and how does it differ from conventional ex vivo CAR-T therapy?

In vivo CAR-T represents a next-generation cell therapy approach that enables direct delivery and expression of CAR genes in T cells within the patient's body, eliminating the need for ex vivo T-cell isolation, expansion, and genetic engineering. Conventional CAR-T therapy requires collection of peripheral blood, ex vivo modification and expansion of T cells, followed by reinfusion—a complex, time-consuming, and costly manufacturing process. In contrast, in vivo CAR-T utilizes delivery systems such as lipid nanoparticles (LNPs) or viral vectors to genetically program T cells directly in vivo, substantially simplifying manufacturing, shortening treatment timelines, reducing production costs, and minimizing cell exhaustion associated with ex vivo culture. As a promising next-generation CAR-T platform, in vivo CAR-T requires highly sensitive tools for CAR expression analysis, pharmacodynamic evaluation, and delivery system characterization. ACROBiosystems provides a comprehensive in vivo CAR solution, including recombinant CAR target proteins, LNP characterization and internalization assays, and quality control kits to support translational research and clinical development.

What are the major CAR-T targets currently being pursued for hematologic and solid tumors?

The current CAR-T target landscape includes a well-established portfolio for hematologic malignancies and a rapidly expanding pipeline for solid tumors, collectively covering most major oncology indications. In hematologic cancers, CD19, BCMA, and CD22 are the leading targets for B-cell malignancies, while CD33, CD123, and CLL-1 are widely pursued in acute myeloid leukemia (AML), and CD7 is the primary target for T-cell malignancies. These targets form the foundation of most approved and investigational hematologic CAR-T therapies. For solid tumors, HER2, EGFRvIII, GPC3, CLDN18.2, and MSLN represent some of the most promising targets currently under active clinical investigation. To support the full spectrum of CAR-T research, ACROBiosystems has developed a comprehensive portfolio of more than 80 recombinant CAR-T target proteins covering both hematologic and solid tumors. With high biological activity, native conformations, and exceptional batch consistency, these products support antibody discovery, CAR construct validation, specificity analysis, pharmacological evaluation, and commercial quality control, providing an integrated solution for CAR-T development across diverse indications.

What are the applications of unlabeled, biotinylated, and fluorescently labeled CAR target proteins, and how should researchers choose between them?

Different labeling formats are designed to support distinct stages of the CAR-T development workflow. Unlabeled proteins are ideal for early-stage target validation, scFv screening, SPR/BLI affinity analysis, and in vitro functional assays. Biotinylated proteins can be used with fluorescent streptavidin for flexible applications such as multicolor flow cytometry, ELISA, and high-throughput binding assays. Fluorescently labeled proteins (Star Staining™) enable one-step flow cytometry and are well suited for CAR-positive cell detection, in vitro and in vivo CAR-T tracking, and longitudinal clinical monitoring, offering simplified workflows, low background, and excellent reproducibility. Selection should be based on the intended application. Unlabeled proteins are recommended for early discovery and affinity characterization, biotinylated proteins for flexible assay development and multiplex applications, and Star Staining™ fluorescent proteins for standardized flow cytometry, clinical sample analysis, and long-term CAR-T monitoring. ACROBiosystems offers unlabeled, biotinylated, and Star Staining™ fluorescent versions of the same CAR target proteins, providing a comprehensive solution that supports every stage of CAR-T development—from target discovery and CAR screening to clinical testing and quality control.