Defining human CAR T cell exhaustion: discovery, characterization, prevention and reversal.
CAR-T cell exhaustion is a phenomenon wherein chronic CAR signaling induces a loss of effector function and represents a major barrier to progress for CAR T cell therapy. Our lab recently identified underlying transcriptional and epigenetic mechanisms that promote CAR T cell exhaustion, which enabled rational design of novel cell engineering strategies to prevent or even reverse exhaustion in human CAR T cells. Ongoing studies employing state-of-the-art surfaceomic, transcriptomic, and epigenomic profiling assays, gene-editing, and synthetic biology will enhance our understanding of exhaustion and inform methods to mitigate it.
Defining novel immunotherapy targets for pediatic cancer
Pediatric malignancies manifest with one of the lowest mutational burdens of all cancer, leading to a scarcity of somatic mutations that could serve as neoantigens for checkpoint-based immunotherapies. Adoptive cell therapy (ACT) using CAR-T-cells or engineered TCRs have the potential to mediate potent anti-tumor effects despite low immunogenicity. The discovery of cancer-restricted differentially expressed antigens with absent or low expression on vital tissues is crutial for the development of novel ACTs and holds the potential to uncover new insights into the underlying biology of these tumors. We characterize the antigen landscape of pediatric cancer utilizing transcriptome, surfaceome, ligandome and proteome data.
Developing mouse models of pediatic brain tumors
Pediatric brain tumors are the leading cause of cancer-related deaths in children. Moreover, treatment related toxicities of current standard therapies cause long term sequelae. Thus, there is a significant need for more effective and less toxic strategies to treat those cancers. CAR T cell Immunotherapies have shown great promise for hematologic disease and are now an FDA approved drug. We are working on identifying targets for immunotherapies for pediatric brain tumors, modify CAR design to improve CAR T cell function, evaluate toxicity and investigate immune cell trafficking in disease relevant mouse models of pediatric brain tumors.
Computational analysis of clinical samples from patients undergoing CAR T cell therapy
Multimodal single-cell data analyses and data integration enable us to investigate how chimeric antigen receptor (CAR)-expressing T lymphocytes succeed or fail in patients. The insights from this work then guide the design of the next generation of engineered cell therapies. Here, we leverage mass cytometry (CyTOF) and single-cell RNA-sequencing (scRNA-seq) data to identify CAR-T cell populations associated with clinical outcome or toxicity. With single-cell sequencing, in addition to transcriptome we also analyze surface protein expression with CITE-seq and TCR clonotype with scTCR-seq. These data enable us to trace individual CAR T-cell clones through time to define properties of the most successful CAR T-cell clones at the time of infusion into the patient.
Mapping the trajectories of dysfunctional T cell states
Recent genetic and epigenetic studies have tried to characterize the exhaustion phenotype and described T cell exhaustion (Tex) as a distinct T cell lineage. There is increasing evidence to suggest that Tex cell lineage exists in multiple, rather than a single activation and differentiation state. A better understanding of transition of dysfunctional cells could inform cell engineering efforts and improve therapy. Analysis of multimodal high-throughput single cell data present an opportunity to characterize these states and their transitions. We analyze single cell protein, transcriptomic and epigenomic data from our engineered T cell (CAR-T) model of exhaustion to define and reconstruct trajectories of T cell dysfunctional states.
Immunotherapy diagnostics with circulating DNA
Next generation sequencing (NGS) of clonal B and T cell receptors enables earlier prediction of B-ALL relapse than current standard clinical methods. In addition to detecting Minimal Residual Disease (MRD) with NGS, we utilize NGS to track circulating tumor DNA (ctDNA) with the aim of identifying tumor features associated with response and resistance to CAR T cell therapeutics. The ability to detect and monitor multiple genetic variants simultaneously enables the dynamic assessment of tumor heterogeneity to understand the underlying biologic mechanisms of effective cancer immunotherapies.
CAR T cell manufacturing research: implementation of preclinical findings into clinical production.
Manufacturing CAR T cells involves individually preparing each patient’s T cells under specially regulated conditions for infusion. Vector creation is specific to the type of virus – whether a retrovirus or a lentivirus – each requiring its own rigorous protocol. Currently, Stanford’s cell and vector manufacturing facility, the Laboratory for Cell and Gene Medicine (LCGM), is retooling to ramp up efficacy and capacity to meet the growing CAR T cell production demand. Plans are underway to create a dedicated facility for manufacturing cancer cell therapies at Stanford. This facility will be necessary to meet the growing clinical demand and the breadth of novel therapeutics that will emanate from the Center for Cancer Cell Therapy.
Synthetic biology tools for Next Generation CAR-T cells
We are developing genetically-encoded switches that allow for control over T cell activity using exogenously-delivered agents. In preclinical studies, T cells engineered with these control modules are able to circumvent CAR-associated toxicities while at the same time outperforming conventional CAR-T cells in terms of antitumor efficacy. We are also engineering NextGen “smart” CAR-T cells that integrate endogenous signals about T cell state to dynamically modulate effector activity and T cell differentiation. Through these strategies, we aim to improve the persistence of CAR-T cells and render them resistant to exhaustion, particularly in the solid tumor setting.
Clonal analysis and single cell characterization of TILs from lung cancer patients
Immunotherapies targeting T cells have revolutionized the treatment for cancer, but only a fraction of patients respond and some patients experience autoimmune toxicities. There is much to be learned about what targets the T cells in tumors are recognizing and how they recognize them. In collaboration with the Mark Davis lab, we are investigating novel T cell specificities in human lung cancer through single-cell T cell receptor and transcriptome sequencing, computational analysis, antigen discovery, and experimental validation. We aim to understand how T cell-mediated responses to tumor are impacted by their specificities and cell states.
Modulation of metabolic pathways to enhance CAR T cell function
T cell activation and differentiation is accompanied by metabolic reprograming to ensure adequate energy and substrates necessary for clonal expansion and effector function. Metabolically active tumor cells can suppress CAR T cell anti-tumor immune responses through competition for nutrients and accumulation of suppressive metabolites. Therefore, our laboratory is using multi-omics approaches to characterize the key regulators of CAR T cell metabolism and design new strategies to modulate T cell function.