Acro-Evaluation of CAR Expression

Acro-Evaluation of CAR Expression

2018.03.31

 

Evaluation of CAR Expression

 

 

 

Evaluating CAR expression is an essential step in the production of CAR-T cells. This is often done by flow cytometry, using protein L, anti-Fab antibodies or target antigens as detection antibodies. Among these common choices, target antigens are widely considered to be the best option, because it offers high specificity and minimal background staining.

 

Reagents 

Mechanism 

Pros 

Cons 

Target Antigens 

Specifically bind to the antigen-binding domains of CARs. 

High specificity;
Minimal background staining. 

High cost;
Each unique CAR has to be stained with corresponding antigens. 

Protein L 

Binds to the kappa light chain of immunoglobulin. 

Universal and low cost.

High background staining;
Cannot detect the anti-lambda light chain CAR. 

Anti-Fab antibody 

Binds to the Fab portion of immunoglobulin. 

Universal and low cost.

High background staining. 

 

ACROBiosystems has developed an extensive collection of recombinant proteins to support CAR-T therapy development. This growing list of proteins includes many pre-biotinylated proteins that are uniquely suitable for screening CAR-expressing cells. In addition, we also supply hard-to-make proteins such as BCMA, CD19, ROR1, and EGFRVIII.

 

 

Case Studies

 

  • Evaluation of anti-BCMA CAR expression using biotinylated BCMA

 

Human T cells were transfected with anti-BCMA CAR and cultured for 3 days. Three days post-transfection, 1×10 6 cells were first incubated with 50ul biotinylated human BCMA protein (Cat.No. BC7-H82F0, 8ug/ml ), washed and then stained with PE Streptavidin. The results showed that the expression of anti-BCMA CARs on transduced T cell surface from donor 1 and donor 2 were 52.72% and 73.49%, respectively. (Data are kindly provided by PREGENE Biopharma)

 

  • Evaluation of anti-CD19 CAR expression using biotinylated CD19

 

293 cells were transfected with FCM63-scFv and RFP tag. 2×105 of the cells were first incubated with A. Biotinylated protein control. B. Recombinant biotinylated human CD19 (Cat. No. CD9-H8259, 10ug/ml). C. Recombinant biotinylated human CD19 (Cat. No. CD9-H8259, 10ug/ml) and FMC63 (Mouse anti-CD19 antibody), followed by FITC Streptavidin, and then analyzed using NovoCyteTM Flow Cytometer. RFP was used to evaluate CAR (FMC63-scFv) expression and FITC was used to evaluate the binding activity  of recombinant biotinylated human CD19 (Cat. No. CD9-H8259).

 

 

Full list of CAR-T Targets

 

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