Identifying and dissecting the anti-tumor regulation networks of CAR T cells in Pancreatic Cancer and Chronic Lymphoblastic Leukemia using CRISPR-based genetic approaches

To develop truly novel approaches for immunotherapy to reverse immune dysfunction, it is critical to understand the basic molecular mechanisms of this process.

Goals:

• Determine how best to deliver Cas9 and sgRNAs to primary human T cells
• Map the various T cell effector functions and determine the general suppressive nature of CLL cells and pancreatic cancer cells using cultures of T cells transduced with CARs and transgenic TCRs
  • Phenotype and function (antigen-specific stimulations)
• Identification of potency enhancing measures: The gene knockout landscape of cells will be assessed prior to culture with control, non-suppressive target cells and the suppressive CLL and PDAC tumor cells
• Retrospective sgRNA screen with CLL patient T cells
• CRISPR/Cas9-mediated correction of hypofunctional patient T cells

Accomplishments:

• Established a biobank of 22 CLL patient  and 5 healthy donor PBMC samples for experiments
• Carried out deep immunophenotyping of tumor and T cell populations for samples in the biobank
• Established an in vitro CAR T cell suppression assay based on the stimulation with primary CLL cells
• Tested various sgRNA delivery systems for primary T cells
• Developed an sgRNA/CAR lentiviral delivery method for primary human T cells

PICI Investigators:  Carl June, Jos Melenhorst, Junwei Shi,  McKensie Collins, and Emily Duffner