Creation and Exploration of Single and Multi Antigen-Targeting Synthetic T Cell Immuno-Stimulating Treatments

Abstract

The human body has developed many mechanisms and layers of defense against cancer, requiring multiple systems to fail for a malignancy to escape the immune system and pose a threat to the body. When cancers do develop, immunotherapy has emerged as a new potent way to harness these defense layers: using the body’s own immune system, reprogramming a patient’s immune cells using synthetically designed proteins that can stimulate T cells to attach to and kill cancerous cells. Here, I have explored the development and results of two distinct technological approaches for creating synthetic T cell stimulants: (1) Bi-specific T cell engagers (bTCEs); fusion proteins that combine multiple antibody-binding domains to simultaneously engage both T-cells and cancer cells in the body; and (2) Chimeric antigen receptors (CARs), fusion proteins that combine antigen targeting and immune cell stimulating domains that remain tethered to patient T cells, resulting in a living cell that can directly detect and respond to cancer cells. I created two interoperable platforms to quickly and efficiently identify promising therapeutic TCE or CAR molecules. For TCEs, I established a high throughput function-first screening platform, allowing identification of effective TCE molecules without the need for protein purification. The second platform looked to expand a pre-existing CAR platform towards exploration of multi-antigen targeting CAR therapeutics. Specifically, two high-throughput molecular platforms were created to allow screening of multi-antigen CARs in either co-expression or tandem-CAR formats. I found that regardless of molecular strategy, multi antigen CARs maintained the ability to engage both targeted antigens and successfully suppressed tumor growth in vitro. In contrast to in vitro findings, co-expression of CARs showed superior responses in a murine xenograft model of human lung cancer, relative to tandem CAR. Despite this clear superiority of co-expression in our experiments, I do not expect this finding to generalize to all multi-antigen targeting CARs, rather these results underline the need to approach multi-antigen CARs as unique entities. In order to expand our screening methods for CAR-T, we developed two novel assays for CAR assessment. The first assessed the avidity of CAR-Jurkat cells using flow cytometry by assessing doublet formation of CAR-target pairs. When tested using CD22 CARs, we observed unique binding maximums for each CAR which appear to be enforce by an equilibrium in a biologically driven interaction. The second is an ongoing study using cell surface proteomics, phosphoproteomics and transcriptomics to better understand why the CD22 lead 1ug36 was superior to the other CD22 CARs tested along side it. Although still ongoing, this study has revealed potentially relevant differences in signaling through LCK, LAT, CD28 and the MAPK pathways. As a whole, this work presents novel methods and findings related to the creation and assessment of synthetic T cell immuno-stimulating treatments.