>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.
Despite the early excitement, the actual path to a clinical success is not an easy one. Both Juno's and Kite Pharma's clinical trials ended up with unexpected and unexplained deaths caused by cerebral edema, which cast a dark shadow over the entire field.
The long-awaited breakthrough finally came this year. Kymriah, Novartis's CAR-T therapy, was approved by FDA this August for the treatment of patients up to 25 years of age with B-cell precursor ALL that is refractory or in second or later relapse. This success marks a milestone in the development of targeted cell therapies, and 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.
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, ROR1, and EGFRVIII.
|BCMA||Multiple myeloma, leukemia, B-Cell lymphoma||BC7-H82F0 (Biotinylated Human BCMA)|
BCA-HF254 (FITC-Labeled Human BCMA)
|CD19||Acute leukemia, B-Cell lymphoma||CD9-H8259 (Biotinylated Human CD19)|
CD9-H5259 (Human CD19)
|ROR1||Leukemia, breast cancer||RO1-H821y (Biotinylated Human ROR1)|
RO1-H82F4 (Biotinylated Human ROR1)
|CD33||Acute myeloid leukemia||CD3-H82E7 (Biotinylated Human CD33)|
CD3-H5257 (Human CD33)
|EGFRVIII||Glioblastoma||EGR-H82E0 (Biotinylated Human EGFRVIII)|
EGI-H52H4 (Human EGFRvIII)
|CD30||Leukemia, B-Cell lymphoma||CD0-H82E6 (Biotinylated Human CD30)|
CD0-H5250 (Human CD30)
|EGFR||NSCLC, epithelial carcinoma, glioma||EGR-H82E3 (Biotinylated Human EGFR)|
EGR-H5252 (Human EGFR)
|FOLR1||Ovarian cancer||FO1-H5229 (Human FOLR1)|
|HER2||Ovarian cancer, breast cancer, glioblastoma, osteosarcoma||HE2-H822R (Biotinylated Human Her2)|
HE2-H5225 (Human Her2)
|HGFR||Malignant melanoma, breast cancer||MET-H82E1 (Biotinylated Human HGF R)|
MET-H5256 (Human HGF R)
|CAIX||Renal cell carcinoma (RCC)||CA9-H5226 (Human CA9 (38-414))|
|CD20||Leukemia, B-Cell lymphoma||CD0-H5268 (Human MS4A1 / CD20, Fc Tag)|
|CD22||Leukemia, B-Cell lymphoma||SI2-H5228 (Human Siglec-2 / CD22)|
|EpCAM||Liver neoplasms, stomach neoplasms||EPM-H82E8 (Biotinylated Human EpCAM)|
EPM-H8254 (Biotinylated Human EpCAM)
|GPC3||Hepatocellular carcinoma||GP3-H5258 (Human Glypican 3)|
|IL13Rα2||Glioma||IL2-H5257 (Human IL13Rα2, Fc Tag)|
|MSLN||Mesothelioma, ovarian cancer||MSN-H82E9 (Biotinylated Human MSLN)|
MSN-H826x (Biotinylated Human MSLN)
|MUC1||Seminal vesicle cancer||MU1-H5252 (Human MUC-1, Fc Tag)|
|CD138||Multiple myeloma||SD1-H5228 (Human Syndecan-1 / CD138)|
|CD38||B-cell Malignancies||CD8-H82E7 (Biotinylated Human CD38)|
CD8-H5255 (Human CD38)
The following case was described in a recent paper by MacLeod DT, et al. published on Jounal of Molecular therapy. The detailed infomation can be found at MacLeod DT, et al., 2017, Mol Ther. 25(4):949-961.doi: 10.1016/j.ymthe.2017.02.005.
Method: Flow Cytometry
Equipment: BD Fortessa flow cytometer (BD Biosciences)
Reagents: Biotinylated CD19-Fc (Cat. No. CD9-H8259, ACROBiosystems); streptavidin-PE and anti-CD3-BV711 (BioLegend);
Samples: TRC1-2-treated, AAV:TRAC:CAR-transduced T cells
Brief protocol: For evaluation of CAR expression, cells were stained with biotinylated CD19-Fc (Cat. No. CD9-H8259, ACROBiosystems) for 15 min at room temperature. Cells were thoroughly washed before staining with antibodies for additional surface markers. Streptavidin-PE and anti-CD3-BV711 were used to stain surface antigens. Cell Trace Violet (Thermo Fisher Scientific) was used at a concentration of 1 mM to label cells for 10 min. All FACS data was analyzed using FlowJo software.
Results: Analysis of CD3 and CAR expression by flow cytometry showed a high frequency of CD19 CAR+ cells in the CD3－ population. (More details can be found at MacLeod DT, et al., 2017, Mol Ther. 25(4):949-961.doi: 10.1016/j.ymthe.2017.02.005.)
Figure A. 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 abiotinylated 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.
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