Rational protein engineering to enhance MHC-independent T cell receptors.

Chimeric antigen receptor (CAR)-based therapies have pioneered synthetic cellular immunity but remain limited in their long-term efficacy. Emerging data suggest that dysregulated CAR-driven T cell activation causes T cell dysfunction and therapeutic failure. To re-engage the precision of the endogenous T cell response, we designed MHC-independent T cell receptors (miTCRs) by linking antibody variable domains to TCR constant chains. Using predictive modeling, we observed that this standard "cut and paste" approach to synthetic protein design resulted in myriad biochemical conflicts at the hybrid variable-constant domain interface. Through iterative modeling and sequence modifications we developed structure-enhanced miTCRs which significantly improved receptor-driven T cell function across multiple tumor models. We found that 41BB costimulation specifically prolonged miTCR T cell persistence and enabled improved leukemic control in vivo compared to classic CAR T cells. Collectively, we have identified core features of hybrid receptor structure responsible for regulating function.

Identification of core techniques that enhance genome editing of human T cells expressing synthetic antigen receptors.

Genome editing technologies have seen remarkable progress in recent years, enabling precise regulation of exogenous and endogenous genes. These advances have been extensively applied to the engineering of human T lymphocytes, leading to the development of practice changing therapies for patients with cancer and the promise of synthetic immune cell therapies for a variety of non-malignant diseases. Many distinct conceptual and technical approaches have been used to edit T-cell genomes, however targeted assessments of which techniques are most effective for manufacturing, gene editing and transgene expression are rarely reported. Through extensive comparative evaluation, we identified methods that most effectively enhance engineering of research-scale and pre-clinical T-cell products at critical stages of manufacturing.

Costimulatory domains direct distinct fates of CAR-driven T-cell dysfunction.

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B-cell cancers yet fail to induce durable remissions for nearly half of all patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven antitumor T-cell function. We developed and validated an in vitro model that drives T-cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T-cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T-cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation using CAR T cells from a patient with progressive lymphoma confirmed the activation of this novel program in a failing clinical product. Furthermore, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T-cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T-cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.

Costimulatory domains direct distinct fates of CAR-driven T cell dysfunction.

Chimeric antigen receptor (CAR) engineered T cells often fail to enact effector functions after infusion into patients. Understanding the biological pathways that lead CAR T cells to failure is of critical importance in the design of more effective therapies. We developed and validated an in vitro model that drives T cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains contribute to T cell failure. We found that dysfunctional CD28-based CARs targeting CD19 bear hallmarks of classical T cell exhaustion while dysfunctional 41BB-based CARs are phenotypically, transcriptionally and epigenetically distinct. We confirmed activation of this unique transcriptional program in CAR T cells that failed to control clinical disease. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is a significant contributor to this dysfunction and disruption of FOXO3 improves CAR T cell function. These findings identify that chronic activation of 41BB leads to novel state of T cell dysfunction that can be alleviated by genetic modification of FOXO3. Summary: Chronic stimulation of CARs containing the 41BB costimulatory domain leads to a novel state of T cell dysfunction that is distinct from T cell exhaustion.

Inducing T cell dysfunction by chronic stimulation of CAR-engineered T cells targeting cancer cells in suspension cultures.

Several pre-clinical models reveal that chronic chimeric antigen receptor (CAR) stimulation drives a dysfunctional state that mimics in vivo failure. In this protocol, we describe steps to induce T cell dysfunction by persistent and long-term stimulation of CAR-engineered T cells using antigen-expressing cancer cells in suspension cultures. We first described a validated method for manufacturing of CAR T cells, followed by a detailed method for chronic stimulation of CAR T cells and a strategy to evaluate these cells during the process of chronic stimulation. For complete details on the use and execution of this protocol, please refer to Singh et al. (2020).1.

Antigen glycosylation regulates efficacy of CAR T cells targeting CD19.

While chimeric antigen receptor (CAR) T cells targeting CD19 can cure a subset of patients with B cell malignancies, most patients treated will not achieve durable remission. Identification of the mechanisms leading to failure is essential to broadening the efficacy of this promising platform. Several studies have demonstrated that disruption of CD19 genes and transcripts can lead to disease relapse after initial response; however, few other tumor-intrinsic drivers of CAR T cell failure have been reported. Here we identify expression of the Golgi-resident intramembrane protease Signal peptide peptidase-like 3 (SPPL3) in malignant B cells as a potent regulator of resistance to CAR therapy. Loss of SPPL3 results in hyperglycosylation of CD19, an alteration that directly inhibits CAR T cell effector function and suppresses anti-tumor cytotoxicity. Alternatively, over-expression of SPPL3 drives loss of CD19 protein, also enabling resistance. In this pre-clinical model these findings identify post-translational modification of CD19 as a mechanism of antigen escape from CAR T cell therapy.