>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.
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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).
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.
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.
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).
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).
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.
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