>Targets of CAR-T Cell Therapy
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.
|Target Antigens||Specifically bind to the antigen-binding domains of CARs.||
|Protein L||Binds to the kappa light chain of immunoglobulin.||
|Anti-Fab antibody||Binds to the Fab portion of immunoglobulin.||
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.
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.
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)
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.
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).
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.
|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)
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)
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)
FO1-H82E2 (Biotinylated Human FOLR1, His & Avi Tag)
FO1-H5229 (Human FOLR1, His Tag)
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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