Non-destructive High Content Analysis of Heterogeneity in CAR T cell Immunotherapies
Within the emerging field of cancer immunotherapy, genetically engineered human T cells expressing cancer-specific chimeric antigen receptors (CARs) represent an exciting new treatment possibility. However, in spite of notable successes in combating leukemia, CAR T cells have shown limited efficacy against solid tumors. We hypothesize that this difficulty may stem from engineering methods that rely on viruses to deliver the CAR, thus causing it to randomly integrate into the genome, and producing heterogeneous cell populations. These random integrations may lead to significant variability in expression levels and signaling activation of the CARs. As such, it is necessary to isolate functionally cytotoxic CAR T cell fractions from the transfected cells. To mitigate this issue, we designed a microscale co-culture system to study the behavior of CRISPR-edited CAR T cells in vitro. This system allows us to assay interactions between engineered T cells and cancer cells in a high-throughput manner, and to develop mathematical models that predict CAR T cell survival rates and cytotoxicity levels against cancer cells. My role in this process has been as follows:
- Cloned plasmid necessary for generating CRISPR CAR T cells
- Collaborated with other researchers on my team to optimize cell culture, microwell assay, cell imaging, immunophenotyping, and gene editing experiments
- Currently adding new imaging capabilities that will allow for the use of other nondestructive phenotypic measurements to probe the metabolic behavior of CAR T cells in an in vitro model.
*Piscopo NJ, *Mueller KP, *Das A, Hematti P, Murphy WL, Palecek SP, Capitini CM and Saha K. Bioengineering Solutions for Manufacturing Challenges in CAR T Cells. Biotechnol J. 2017 Aug 25. PMID: 28840981. * co-first authors