Dissecting the molecular basis of CAR-driven T cell dysfunction

Similar to native T cells responding to chronic infections, CAR-engineered T cells also become dysfunctional when they are persistently stimulated. We have found, however, that the phenotypes of these cells, as well as their transcriptional and epigenetic circuitry, is distinct from classical TCR-driven T cell exhaustion. From these observations, we aim to understand how CARs and TCRs direct distinct states of T cell dysfunction. Using next-generation and single-cell sequencing, CRISPR-based editing, high-resolution mass cytometry and evaluation of samples from patients receiving CAR therapies, this project is dissecting how these two antigen receptors differ in instructing T cell responses against cancer. Our ultimate goal is to develop advanced protein and genome engineering strategies to bypass dysfunctional CAR-driven molecular circuits and make “dysfunction-resistant” CAR T cells.

Identify how tonic CAR signaling impacts T cell functional state

While CARs are designed to signal only when they encounter their target antigens, nearly all CARs activate intracellular signaling in the absence of antigen. Our lab and other labs have found that this phenomenon, referred to as “tonic” signaling, has a distinct impact on T cell functional potential that is dependent on the CAR co-stimulatory domain (Lynn R., Nature 2019; Singh N., Nature Medicine 2021). This project integrates high-resolution imaging, novel proteomics techniques and design of controllable transcriptional circuits to understand how CAR co-stimulation can direct T cell lineage commitment.

Understanding how cancer cells are resistant to T cell killing

While many patients will demonstrate initial responses to CAR T cell therapy, many of these patients will relapse. Loss of the CAR target antigen has proven to be a main cause of CAR T cell failure after initial response, however the biology that permits relapse without loss of the target antigen remains unclear. Furthermore, the etiologies that prevent even initial responses are unknown. Based on observations from in vitro studies, we are evaluating clinical tumor samples to identify features that associate with response to CAR therapy.

Protein engineering to improve translational cell therapies

Chimeric antigen receptors are a prime example of the power of synthetic biology. Integrating functional domains from several distinct native proteins to create a novel molecule with multiple functions opens endless possibilities for regulation of cellular circuits. We are using this approach to address the most relevant barriers to the success of translational cellular therapies – aberrant receptor function, promiscuous signaling protein activity, and delivery of gene therapy vectors. While new for our lab, we have developed expertise in protein engineering, purification and evaluation to build several new molecules that we hope will open new possibilities for synthetic receptor-based therapies.