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mAb for Anti-CD19 CAR Detection-Flow Cytometry Validated

CD19 is currently the most widely used target in CAR-T therapy, which has been validated to be effective and safe to treat B-ALL, CLL, and B cell lymphoma. FMC63 is an IgG2a mouse monoclonal antibody targeting CD19. So far, most of reported CART19 trials contain the anti-CD19 scFv derived from FMC63, including the two FDA-approved CARs Kymriah and Yescarta.

While the therapeutic outcomes for CD19-specific CAR-T cells have been impressive, the quality control during therapy manufacture remains an area in which more progress is required. Our high-affinity CD19 proteins can effectively detect the expression of anti-CD19 CAR on the surface of transduced T cells and have been widely recognized for their high sensitivity and specificity. These high-affinity CD19 proteins have been widely used for the quality control release testing and pharmacokinetic (PK) study of anti-CD19 CAR-T cells in clinical trials.

After receiving positive feedback on high-affinity CD19 proteins in market, ACROBiosystems now developed an anti-idiotypic antibody against FMC63 scFv, named Monoclonal Anti-FMC63 scFv Antibody, which can specifically recognize the antigen-recognition domain of FMC63 derived anti-CD19 CARs with high sensitivity and high specificity. The performance of anti-FMC63 scFv monoclonal antibody was validated by flow cytometry (FCM) in house and it is suitable for detecting FMC63 derived anti-CD19 CARs in clinical trials.

About Premium grade Anti-FMC63 Antibody

It is designed for cell isolation and cell culture applications, and produced under animal-free manufacturing conditions. No animal derived components are used throughout the production process. It is produced under our rigorous quality control system that incorporates a comprehensive set of tests including sterility and endotoxin tests. Product performance is carefully validated and tested for compatibility for cell culture use or any other applications in the early preclinical stage.

Product Features

Highly Specific Binding to CAR-Positive Cells

Highly Specific Binding to CAR-Positive Cells

After Fc receptor blockade, whole blood only control (Figure A) and whole blood spiked with CAR-T cells (Figure B) were first stained with PE-Labeled Monoclonal Anti-FMC63 Antibody, Mouse IgG1 (Y45) (Site-specific conjugation) (Preservative free) (Cat. No. FM3-HPY53) , and then stained with 7-AAD. After 100-fold dilution, acquire data using a CytoFLEX S and then analyze the data with FlowJo. CAR-T cells were identified as PE-positive events within the live CD45+CD3+ (or CD45+) lymphocyte population. The proportion of CD45+CAR+ cells is around 20%, which is largely consistent with the theoretical spike-in value.

Highly Specific Binding to CAR-Positive Cells

After Fc receptor blockade, whole blood only control (Figure A) and whole blood spiked with CAR-T cells (Figure B) were first stained with PE-Labeled Monoclonal Anti-FMC63 Antibody, Rabbit IgG (1G10) (Cat. NO. FM3-PFM721), and then stained with 7-AAD. After 100-fold dilution, acquire data using a CytoFLEX S and then analyze the data with FlowJo. CAR-T cells were identified as PE-positive events within the live CD45+CD3+ (or CD45+) lymphocyte population. The proportion of CD45+CAR+ cells is around 20%, which is largely consistent with the theoretical spike-in value.

Detection of Anti-CD19 (FMC63) CAR Expression
CAR-specific Monoclonal Antibody to Detect Anti-CD19 CAR Expression

2e5 of FMC63 scFv-based anti-CD19 CAR-293 cells were stained with 100 µL of the working solution of Monoclonal AntiFMC63 scFv Antibody, Mouse IgG1 (Cat. No. FM3-Y45) and isotype control respectively, washed and then followed by PE anti-mouse IgG1 Antibody and analyzed with flow cytometry (QC tested).

Assessment of Non-specific Binding to 293 Cells
CAR-specific Monoclonal Antibody to Detect Anti-CD19 CAR Expression

Non-specific binding of Monoclonal Anti-FMC63 scFv Antibody (Cat. No. FM3-Y45) to non-transfected 293 cells was determined by flow cytometry. The data showed that Anti-FMC63 scFv Antibody didn’t bind to non-transfected 293 cells.


Assessment of Non-specific Binding to PBMCs
CAR-specific Monoclonal Antibody to Detect Anti-CD19 CAR Expression

Non-specific binding of Monoclonal Anti-FMC63 scFv Antibody (Cat. No. FM3-Y45) to non-transfected human PBMCs was determined by flow cytometry. The data showed that Anti-FMC63 scFv Antibody didn’t bind to non-transfected human PBMCs.

Specifically recognize the antigen-binding site of FMC63 derived CARs with high affinity

Binding Activity and Specificity Measured by ELISA
CAR-specific Monoclonal Antibody to Detect Anti-CD19 CAR Expression

Immobilized FMC63 scFv, His Tag at 2 μg/mL (100 μL/well) can bind Monoclonal Anti-FMC63 scFv Antibody, Mouse IgG1 (Clone Y45) with a linear range of 1-19 ng/mL. Anti-DNP antibody, mouse IgG1 (Cat. No. DNP-M1) was used as an isotype control (QC tested).


Neutralizing Activity Measured by ELISA
CAR-specific Monoclonal Antibody to Detect Anti-CD19 CAR Expression

ELISA analysis shows that the binding of Human CD19, Fc Tag (Cat. No. CD9-H5251) to FMC63 scFv, His Tag was inhibited by increasing concentration of Monoclonal Anti-FMC63 scFv Antibody, Mouse IgG1 (Clone Y45). The concentration of Human CD19, Fc Tag used is 5 μg/mL (100 μL/well). The IC50 is 0.084 μg/mL (Routinely tested).

High Affinity Determined by SPR
CAR-specific Monoclonal Antibody to Detect Anti-CD19 CAR Expression

Monoclonal Anti-FMC63 scFv Antibody, Mouse IgG1 (Cat. No. FM3-Y45) captured on CM5 chip via anti-mouse antibodies surface can bind FMC63 scFv with an affinity constant of 1.08 nM as determined in a SPR assay.

Product List

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FAQ

Q

What is the relationship between the FMC63 clone and modern FDA-approved CAR-T therapies?

FMC63 is a mouse monoclonal IgG2a antibody that targets human CD19. It is the most widely utilized target in current CAR-T research because the antigen-recognition domains (scFv) of the two leading FDA-approved therapies, Kymriah and Yescarta, are both derived from the FMC63 clone. Because of this clinical validation, FMC63-based constructs have become the gold standard for treating B-cell malignancies.
Q

Why is an anti-idiotypic antibody, like Anti-FMC63, essential for monitoring CAR-T cells in patients?

Monitoring the persistence and expansion of CAR-T cells (Pharmacokinetics) is vital for clinical success. While target proteins like CD19 can be used for detection, an anti-idiotypic antibody specifically recognizes the unique antigen-binding site of the CAR. This allows researchers to accurately distinguish and quantify the engineered T cells even in complex biological environments, such as patient blood samples, with high sensitivity.
Q

How can I overcome the challenge of high background noise when detecting CAR expression in whole blood?

Detecting CAR-T cells in whole blood for PK studies is often difficult due to non-specific binding. ACROBiosystems addresses this by providing monoclonal anti-idiotypic antibodies validated for flow cytometry in whole blood samples. Our data shows that after Fc receptor blockade, our antibodies (such as clone Y45) demonstrate negligible non-specific binding to non-transfected PBMCs or 293 cells, providing a clear signal even at low spike-in concentrations.
Q

What are the strategies for evaluating the immunogenicity of a CD19 CAR‑T therapeutic candidate?

A major safety concern in cell therapy is the development of Anti-Drug Antibodies (ADA) against the scFv of the CAR. ACRO supports this by offering a specialized Anti-CD19 (FMC63) CAR Immunogenicity ELISA Kit. Additionally, we provide Carrier-free and Rabbit Anti-Mouse Polyclonal versions specifically recommended as positive controls or reference standards for ADA assays to help researchers satisfy regulatory safety requirements.
Q

What are the unique advantages of using ACRO’s "Premium Grade" Anti-FMC63 antibodies in cell culture?

For applications involving live cells, safety and purity are paramount. ACRO’s Premium Grade antibodies are produced under animal-free manufacturing conditions. They undergo rigorous quality control, including sterility and endotoxin testing, ensuring they are fully compatible with cell culture use and early preclinical research without introducing exogenous contaminants.
Q

Can ACRO’s Anti-FMC63 tools be used to quantify the density of CAR expression on the cell surface?

Yes. Since our antibodies can identify CAR‑positive cells, they are also used for quality control release testing to assess the quality of the manufactured therapy. By using our high-affinity monoclonal antibodies (linear range of 1-19 ng/mL in ELISA), researchers can more accurately characterize the antigen-recognition domain density across different production batches.
Q

Why is site-specific conjugation a superior standard for fluorescently labeled Anti-FMC63 antibodies?

Traditional random labeling can interfere with the antibody's binding site or lead to inconsistent signals. ACRO utilizes a site-specific labeling platform for many of its fluorescent Anti-FMC63 products. This "Star Staining" Anti-FMC63 antibody ensures that every antibody molecule is labeled in a way that preserves its native conformation, thereby ensuring high affinity, and provides maximum sensitivity for flow cytometry detection.
Q

How should I choose between Mouse and Rabbit host species for my Anti-FMC63 detection reagents?

The choice typically depends on your existing secondary reagents and the species of your sample: Mouse IgG1 (Clone Y45): This is our most widely used clone, available in diverse formats including Biotinylated, PE, FITC, and Alexa Fluor labels. Rabbit IgG (Clone 1G10 or M4H08): These provide an excellent alternative for researchers who need to avoid anti-mouse cross-reactivity or who prefer the high affinity often associated with rabbit monoclonals.
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