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Targets of CAR-T Cell Therapy

CAR-T targets

The chimeric antigen receptor T (CAR-T) cell therapy is a new treatment for a variety of cancers. The idea is to take out the T-cells from the patient, and genetically engineer the cells to make them express a chimeric receptor (CAR) recognizing 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.

2017 is a milestone year for CAR-T cell therapy. The FDA approved two CD19-targetting CAR-T therapies. Novartis's Kymriah is approved for B-cell precursor ALL treatment in children and young adults, while Kite Pharma's Yescarta is approved for the treatment of adult patients. This success fueled the efforts to develop / advance CAR-T treatment targeting other cancer-specific antigens. Beyond CD19, there is a growing list of targets being investigated for therapeutic intervention.

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.
  1. High specificity

  2. Minimal background staining

  1. High cost

  2. Each unique CAR has to be stained with corresponding antigens

Protein L Binds to the kappa light chain of immunoglobulin.
  1. Universal and low cost

  1. High background staining.

  2. Cannot detect the anti-lambda light chain CAR

Anti-Fab antibody Binds to the Fab portion of immunoglobulin.
  1. Universal and low cost

  1. High background staining

ACROBiosystems has developed an extensive collection of recombinant proteins to support these investigations. 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

The following case study was provided by PREGENE Biopharma. The data 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.

CAR-T Cell Therapy

Human T cells were transfected with anti-BCMA CAR and cultured for 3 days. Three days post-transfection, 1x106 cells were first incubated with 50ul biotinylated human BCMA protein (Cat. No. BC7-H82F0, 8ug/ml), washed, and then stained with PE Streptavidin. (Data are kindly provided by PREGENE Biopharma)

Evaluation of anti-CD19 CAR expression using biotinylated CD19

The following case study was provided by our in-house R&D team. The data showed that the binding of biotinylated human CD19 (Cat. No. CD9-H8259) to anti-CD19 scFv-modified cells was specifically mediated by anti-CD19-CAR and CD19 interaction.

CAR-T Cell Therapy products

293 cells were transfected with FCM63-scFv and RFP tag. 2x105 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).

Evaluation of anti-CD19 CAR expression using biotinylated CD19

The following case was described in a recent paper by MacLeod DT, et al. published on Jounal of Molecular therapy. The detailed information can be found at MacLeod DT, et al., 2017, Mol Ther. 25(4):949-961.doi: 10.1016/j.ymthe.2017.02.005.


Activated T cells were electroporated with TRC1-2 mRNA and transduced with AAV:TRAC:CAR at an MOI of 400,000 vg/cell and cultured for 5 days in the presence of IL-2. Five days post-transduction, cells were stained for expression of the CAR using a biotinylated CD19-Fc reagent and CD3, with TRC1-2-treated, mock-transduced cells used as a control for gating of CAR expression. CD3+ cells were then depleted. Enriched CD3- cells were cultured for 3 additional days in the presence of IL-15 and IL-21 and then analyzed again by flow cytometry for CD3 and CAR expression.

Product List

Targets Diseases Recommended Products
BCMA Multiple myeloma, leukemia, B-Cell lymphoma BC7-H82F0 (Biotinylated Human BCMA, Fc & Avi Tag)
BCA-HF254 (FITC-Labeled Human BCMA, Fc Tag)
CD19 Acute leukemia, B-Cell lymphoma CD9-H8259 (Biotinylated Human CD19, Fc Tag)
CD9-H5259 (Human CD19, Fc Tag)
CD20 Leukemia, B-Cell lymphoma CD0-H5268 (Human MS4A1 / CD20, Fc Tag)
CD22 Leukemia, B-Cell lymphoma SI2-H82F8 (BiotinylatedHuman Siglec-2 / CD22, Fc & Avi Tag)
CD2-H52H8 (Human Siglec-2 / CD22, His Tag)
CD30 Leukemia, B-Cell lymphoma CD0-H82E6 (BiotinylatedHuman CD30, Avi & His Tag)
CD0-H5250 (Human CD30, Fc Tag)
CD33 Acute myeloid leukemia CD3-H82E7 (BiotinylatedHuman CD33, Avi & His Tag)
CD3-H5257 (Human CD33, Fc Tag)
CD38 B-cell Malignancies CD8-H82E7 (BiotinylatedHuman CD38, Avi & His Tag)
CD8-H5255 (Human CD38, Fc Tag)
CD138 Multiple myeloma SD1-H5228 (Human Syndecan-1 / CD138, His Tag)
CAIX Renal cell carcinoma (RCC) CA9-H5226 (Human CA9 (38-414))
EGFR NSCLC, epithelial carcinoma, glioma EGR-H82E3 (Biotinylated Human EGFR, His & Avi Tag)
EGR-H5252( Human EGFR, Fc Tag)
EGFRVIII Glioblastoma EGR-H82E0 (Biotinylated Human EGFRVIII, Avi & His Tag)
EGI-H52H4(Human EGFRvIII, His Tag)
EpCAM Liver neoplasms, stomach neoplasms EPM-H82E8 (Biotinylated   Human EpCAM, His & Avi Tag)
EPM-H8254 (BiotinylatedHuman EpCAM, Fc Tag)
FOLR1 Ovarian cancer FO1-H82E2 (Biotinylated  Human FOLR1, His & Avi Tag)
FO1-H5229 (Human FOLR1, His Tag)
GPC3 Hepatocellular carcinoma GP3-H82E5 (Biotinylated  Human Glypican 3, His & Avi Tag)
GP3-H5258 (Human Glypican 3, Fc Tag)
HER2 Ovarian cancer, breast cancer, glioblastoma, osteosarcoma HE2-H822R( Biotinylated  Human Her2, His Tag)
HE2-H5253 (Human Her2, Fc Tag)
HGFR Malignant melanoma, breast cancer MET-H82E1 (BiotinylatedHuman HGF R, Avi & His Tag)
MET-H5256 (Human HGF R, Fc Tag)
IL13Rα2 Glioma IL2-H5257 (Human IL-13 R alpha 2, Fc Tag)
MSLN Mesothelioma, ovarian cancer MSN-H82E9(BiotinylatedHuman MSLN, His & Avi Tag)
MSN-H826x(BiotinylatedHuman MSLN, Fc Tag)
MUC1 Seminal vesicle cancer MU1-H5252 (BiotinylatedHuman MUC-1, Fc Tag)
ROR1 Leukemia, breast cancer RO1-H821y (BiotinylatedHuman ROR1, Avi Tag)
RO1-H82F4 (BiotinylatedHuman ROR1, Fc & Avi Tag)


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