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Your Position: Home > Solutions for Evaluation of CAR Expression

Solutions for Evaluation of CAR Expression

The chimeric antigen receptor T (CAR-T) cell therapy is a new treatment for a variety of cancers.The idea is to take the T-cells from the patient, and genetically engineer the cells to express a chimeric receptor (CAR) which recognizes a specific tumor-associated antigen (TAAs). As a result, the CAR-expressing T cells, when reintroduced into the patient's body, will target and eliminate the TAA-expressing tumor cells.

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.

ACROBiosystems has developed an extensive collection of recombinant proteins to support CAR-T therapy development. This growing list of proteins includes many fluorescent-labeled target antigens and pre-biotinylated proteins that are uniquely suitable for evaluation of CAR expression. In addition, we also supply difficult-to-express proteins such as BCMA, CD19, ROR1, and EGFRVIII.

CAR Detection Strategy and Product Design
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.
> FITC-labeled (Click molecule view product details)-> Specially designed
> PE-labeled (Click molecule view product details) -> Specially designed
> 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).
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> Application —Evaluation of CAR Expression

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.
> Avitag TM biotinylated proteins -> Specially designed
> Chemically biotinylated proteins -> Specially designed
> 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)
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> Application — Evaluation of CAR Expression

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.
Non-specific reaction of a secondary antibody may occur.
> Fc-tag fusion proteins -> Specially designed
> His-tag fusion proteins -> Specially designed
> All fusion 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).
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> Application — Evaluation of CAR Expression

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