CAR-specific monoclonal FMC63 antibodies for CD19 CAR expression evaluation

In CAR-T therapy, CD19 is now the most extensively used target. It has been confirmed to be effective and safe for the treatment of CLL, B-ALL and B cell lymphoma. FMC63, an IgG2a mouse monoclonal antibody, targets CD19.

To date, a majority of the reported CART19 trials include the anti-CD19 scFv extracted from FMC63, such as both the FDA-approved CARs Kymriah and Yescarta.

Although the therapeutic results for CD19-specific CAR-T cells have been impressive, the quality control at the time of therapy manufacture has been an area where more progress is still needed. The high-affinity CD19 proteins from ACROBiosystems exhibit the potential to effectively detect the anti-CD19 CAR expression on the surface of transduced T cells. These proteins have been recognized widely for their superior sensitivity and specificity.

The ACROBiosystems high-affinity CD19 proteins have found extensive use for the quality control release testing and pharmacokinetic (PK) analysis of anti-CD19 CAR-T cells in clinical trials.

With positive feedback related to its high-affinity CD19 proteins available in the market, ACROBiosystems has now created an anti-idiotypic antibody against FMC63 scFv, known as Monoclonal Anti-FMC63 scFv Antibody. This antibody has the potential to specifically recognize the antigen-recognition domain of FMC63 derived anti-CD19 CARs with superior sensitivity and specificity.

The anti-FMC63 scFv monoclonal antibody’s performance was verified in house through flow cytometry (FCM), and it was found to be ideal for the detection of FMC63-derived anti-CD19 CARs in clinical trials.

Product list

Table 1. Source: ACROBiosystems

Product features

No background staining when detecting FMC63 derived Anti-CD19 CARs in clinical trials

Detection of anti-CD19 (FMC63) 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). Image Credit: ACROBiosystems

Assessment of non-specific binding to 293 cells

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. Image Credit: ACROBiosystems

Assessment of non-specific binding to PBMCs

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. Image Credit: ACROBiosystems

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

Binding activity and specificity measured by ELISA

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). Image Credit: ACROBiosystems

Neutralizing activity measured by ELISA

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). Image Credit: ACROBiosystems

High affinity determined by SPR

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 an SPR assay. Image Credit: ACROBiosystems

Product list of high affinity CD19 proteins

Table 2. Source: ACROBiosystems

Molecule	Cat. No.	Source	Product Description	Structure
CD19	CD9-HP2H3	HEK293	PE-Labeled Human CD19 (20-291) Protein, His Tag (Site-specific conjugation)Hot
	CD9-H5251	HEK293	Human CD19 (20-291) Protein, Fc Tag, low endotoxin (Super affinity, MALS verified) DMF FiledHot
	CD9-H52H2	HEK293	Human CD19 (20-291) Protein, His Tag DMF FiledHot
	CD9-H5259	HEK293	Human CD19 (20-291) Protein, Fc Tag, low endotoxin
	CD9-H5250	HEK293	Human CD19 (20-291) Protein, Llama IgG2b Fc Tag, low endotoxin
	CD9-H5258	HEK293	Human CD19 (20-291) Protein, Mouse IgG2a Fc Tag
	CD9-HF2H2	HEK293	FITC-Labeled Human CD19 (20-291) Protein, His Tag DMF FiledHot
	CD9-HF251	HEK293	FITC-Labeled Human CD19 (20-291) Protein, Fc Tag DMF FiledHot
	CD9-HF2H3	HEK293	FITC-Labeled Human CD19 (20-291) Protein, His Tag Star Staining
	CD9-H8259	HEK293	Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling) DMF Filed
	CD9-H82F7	HEK293	Biotinylated Human CD19 (20-291) Protein, Fc, Avitag™Hot
	CD9-H82E9	HEK293	Biotinylated Human CD19 (20-291) Protein, His, Avitag™ (SPR verified)Hot
	MBS-K005		Human CD19-coupled Magnetic Beads
	CD9-C5251	HEK293	Cynomolgus / Rhesus macaque CD19 (20-292) Protein, Fc Tag
	CD9-C5221	HEK293	Cynomolgus / Rhesus macaque CD19 (20-292) Protein, His Tag