Evaluating CAR expression with biotinylated, fluorescence-labeled, and unconjugated proteins

The chimeric antigen receptor T (CAR-T) cell therapy from ACROBiosystems is a new treatment developed for a range of cancers. The concept is to take the T-cells from the patient and genetically engineer the cells to express a chimeric antigen receptor (CAR) that identifies a particular tumor-associated antigen (TAAs).

Consequently, upon being reintroduced into the patient’s body, the CAR-expressing T cells will target and reduce the TAA-expressing tumor cells.

Assessing CAR expression is an important step in generating CAR-T cells. This is frequently done by flow cytometry, with the help of protein L, anti-Fab antibodies or target antigens as detection methods. Of these regular options, target antigens are largely regarded as the best option since they provide high specificity and the least background staining.

ACROBiosystems has designed a wide collection of recombinant proteins to assist CAR-T therapy development. This expanding list of proteins consists of several fluorescent-labeled target antigens and pre-biotinylated proteins that are especially appropriate for assessing CAR expression. Besides, the company also provides hard-to-express proteins like CD19, BCMA, ROR1 and EGFRVIII.

CAR detection strategy and product design

Direct method — Fluorescent-labeled proteins

Image Credit: ACROBiosystems

Key features

• Target antigens come pre-labeled with a fluorescent dye
• Non-specific reaction of a secondary antibody is prevented
• Processing time can be minimized by using direct-labeled proteins

PE-labeled

Table 1. Source: ACROBiosystems

FITC-labeled

Table 2. Source: ACROBiosystems

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). Image Credit: ACROBiosystems

Biotin-streptavidin based detection using biotinylated proteins

Image Credit: ACROBiosystems

Key features

• Target antigens are pre-labeled using biotin and detected using labeled streptavidin (the biotin-avidin complex)
• Streptavidin labeled with fluorochromes has the ability to bind biotinylated proteins with a high degree of specificity and affinity, thereby amplifying the signal and enhancing the detection specificity and sensitivity

Biotinylated proteins

Table 3. Source: ACROBiosystems

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). Image Credit: ACROBiosystems

Indirect detection using unconjugated proteins

Image Credit: ACROBiosystems

Key features

• Target antigens are developed to carry a particular tag and are detected with the help of a secondary antibody (anti-epitope tag antibody) labeled with a fluorophore
• Non-specific reaction of a secondary antibody might take place

Unconjugated proteins

Table 4. Source: ACROBiosystems

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). Image Credit: ACROBiosystems