Evaluating CAR Expression for CAR-T Cell Therapy

Chimeric antigen receptor T, or CAR-T, cell therapy is a new therapy developed for a wide range of cancers. The main idea is to harvest T-cells from the patient and genetically engineer the cells in such a way that they express a chimeric receptor (CAR) which detects specific tumor-associated antigens (TAAs). Consequently, when the CAR-expressing T cells are reintroduced into the patient's body, they will target and remove the TAA-expressing tumor cells.

An important step in the production of CAR-T cells is the evaluation of CAR expression. This is usually achieved via flow cytometry, using protein L, anti-Fab antibodies, or target antigens as detection antibodies.

Among these common choices, target antigens are generally believed to be the best option as they provide minimal background staining and high specificity.

ACROBiosystems has produced a comprehensive collection of recombinant proteins to support the development of CAR-T therapy. This increasing list of proteins includes a number of pre-biotinylated proteins and fluorescent-labeled target antigens that are exclusively suitable for evaluating CAR expression. The company also supplies difficult-to-express proteins, such as EGFRVIII, ROR1, CD19, and BCMA.

CAR Detection Strategy and Product Design

Direct Method—Fluorescent-Labeled Proteins

Key Features

  • Processing time can be decreased using direct-labeled proteins
  • A fluorescent dye is used to pre-label target antigens
  • Non-specific reaction of a secondary antibody is completely removed

FITC-Labeled -> Specially Designed

BCMA CD19 CD22 GPC3 MSLN Protein L

 

PE- Labeled -> Specially Designed

MSLN

 

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).

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).

Biotin-Streptavidin Based Detection Using Biotinylated Proteins

Key Features

  • Biotin is used to pre-label target antigens, which are detected by labeled streptavidin (the biotin-avidin complex)
  • Streptavidin labeled with fluorochromes is capable of binding biotinylated proteins with a high degree of specificity and affinity, amplifying the signal, and enhancing the detection specificity and sensitivity

Avitag TM Biotinylated Proteins -> Specially Designed

BCMA CD22 CD30 CD33 CD38 CD70
EGF R EGFRVIII EpCAM FOLR1 GPC3 HER2
HGFR MSLN ROR1      

 

Chemically Biotinylated Proteins -> Specially Designed

CD19 EpCAM HER2 MSLN Protein L

 

Case Study

Evaluation of Anti-BCMA CAR Expression with Biotinylated BCMA

Human T cells were transfected with anti-BCMA CAR and cultured for three 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)

Human T cells were transfected with anti-BCMA CAR and cultured for three 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)

Indirect Detection Using Unconjugated Proteins

Key Features

  • Target antigens are designed to perform a particular tag and are identified using a secondary antibody (anti-epitope tag antibody) labeled with a fluorophore
  • Non-specific reaction of a secondary antibody may take place

Fc-Tag Fusion Proteins -> Specially Designed

BCMA CD19 CD30 CD33 CD38 CD70
EGFR FOLR1 GPC3 HER2 HGFR IL13RA2
MSLN MUC1 ROR1 EpCAM    

 

His-Tag Fusion Proteins -> Specially Designed

. . . . . .
BCMA CD19 CD123 CD138 CD22 CD30
CD33 CD38 CD70 CAIX EGFR EGFRVIII
EpCAM FOLR1 GPC3 HER2 HGFR MSLN
ROR1          

 

All Fusion Proteins -> Specially Designed

. . . . . .
BCMA CD19 CD123 CD138 CD22 CD30
CD33 CD38 CD70 CAIX EGFR EGFRVIII
EpCAM FOLR1 GPC3 HER2 HGFR IL13RA2
MSLN MUCI ROR1      

 

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).

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).

ACROBiosystems

ACROBiosystems is an internationally recognized manufacturer of recombinant proteins committed to supporting cancer immunotherapy. We specialize in mammalian cell-based recombinant protein production and process development.

Our goal is to support professionals from pharmaceutical companies, CROs and research institutes who are working on the cancer immunotherapy area by providing high-quality proteins, antibodies and assay kits.

We have multiple offices and branches in North America, Europe, and Asia, and we are proud of serving customers from over 50 countries.


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Last updated: Nov 21, 2018 at 9:37 AM

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