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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.

CART-LP-1.png

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.

Full list of CAR-T Targets

Recommended Products

TargetsDiseasesRecommended Products
BCMAMultiple myeloma, leukemia, B-Cell lymphomaBC7-H82F0 (Biotinylated Human BCMA)
BCA-H522Y (Human BCMA)
ROR1Leukemia, breast cancerRO1-H821y (Biotinylated Human ROR1)
RO1-H522y (Human ROR1)
CD33Acute myeloid leukemiaCD3-H82E7 (Biotinylated Human CD33)
CD3-H5226 (Human CD33)
EGFRVIIIGlioblastomaEGR-H82E0 (Biotinylated Human EGFRVIII)
EGI-H52H4 (Human EGFRvIII)
CD30Leukemia, B-Cell lymphomaCD0-H82E6 (Biotinylated Human CD30)
CD0-H5229 (Human CD30)
EGFRNSCLC, epithelial carcinoma, gliomaEGR-H82E3 (Biotinylated Human EGFR)
EGR-H5222 (Human EGFR)
FOLR1Ovarian cancerFO1-M82E9 (Biotinylated Mouse FOLR1)
FO1-H5229 (Human FOLR1)
HER2Ovarian cancer, breast cancer, glioblastoma, osteosarcomaHE2-H822R (Biotinylated Human Her2)
HE2-H5225 (Human Her2)
c-MET/HGFRMalignant melanoma, breast cancerMET-H82E1 (Biotinylated Human HGF R)
MET-H5227 (Human HGF R)
CAIXRenal cell carcinoma (RCC)CA9-H5226 (Human CA9 (38-414))
CD19Acute leukemia, B-Cell lymphomaCD9-H8259 (Biotinylated Human CD19)
CD20Leukemia, B-Cell lymphomaCD0-H5268 (Human MS4A1 / CD20, Fc Tag)
CD22Leukemia, B-Cell lymphomaSI2-H5228 (Human Siglec-2 / CD22)
EpCAMLiver neoplasms, stomach neoplasmsEPM-H82E8 (Biotinylated Human EpCAM)
EPM-H5223 (Human EpCAM)
GPC3Hepatocellular carcinomaGP3-H5223 (Human Glypican 3)
IL13Rα2GliomaIL2-H5257 (Human IL13Rα2, Fc Tag)
MSLNMesothelioma, ovarian cancerMSN-H82E9 (Biotinylated Human MSLN)
MSN-H5223 (Human MSLN)
MUC1Seminal vesicle cancerMU1-H5252 (Human MUC-1, Fc Tag)
CD138Multiple myelomaSD1-H5228 (Human Syndecan-1 / CD138)
CD38B-cell MalignanciesCD8-H82E7 (Biotinylated Human CD38)
CD8-H5224 (Human CD38)

Case study: evaluation of CD19 CAR expression

  1. 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 atMacLeod DT,et al., 2017, Mol Ther. 25(4):949-961.doi: 10.1016/j.ymthe.2017.02.005.

  2. Method: Flow Cytometry

  3. Equipment: BD Fortessa flow cytometer (BD Biosciences)

  4. Reagents: Biotinylated CD19-Fc (Cat. No.CD9-H8259, ACROBiosystems); streptavidin-PE and anti-CD3-BV711 (BioLegend);

  5. Samples: TRC1-2-treated, AAV:TRAC:CAR-transduced T cells

  6. 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.

  7. 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 atMacLeod DT,et al., 2017, Mol Ther. 25(4):949-961.doi: 10.1016/j.ymthe.2017.02.005.)

  8. CAR-expression.png

    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.

References

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