A synthetic notch (synNotch) system linking intratumoral immune-cancer cell communication to a synthetic blood biomarker assay.

Abstract

Introduction: Cellular immunotherapy has greatly improved cancer treatment in recent years. For instance, chimeric antigen receptor (CAR) T cell therapy has been proven highly effective in treating hematological malignancies, and many CAR cell designs are being explored for solid tumors. However, many questions remain why responses differ across patients and some tumor types are resistant. Improved and relatively inexpensive ways to monitor these cells could provide some answers. Clinically, blood tests are regularly used to monitor these therapies, but blood signals often do not reflect the activity of immune cells within the tumor(s). Here, using the synthetic Notch (synNotch) receptor that tethers antigen binding to customized transgene expression, we linked intratumoral immune-cancer cell communication to a simple secreted reporter blood test. Specifically, we engineered immune cells with a CD19-targeted synNotch receptor and demonstrated that binding to CD19 on cancer cells in vivo resulted in the production of secreted embryonic alkaline phosphatase (SEAP) at levels that are readily detected in the blood. Methods and Results: Jurkat T cells were engineered via sequential lentiviral transduction of two components: an anti-CD19 synNotch receptor and a synNotch response element encoding SEAP. Co-culture of engineered cells with CD19+, but not CD19-, Nalm6 cells, resulted in significantly elevated SEAP in media. Nod-scid-gamma (NSG) mice were subcutaneously injected with either CD19+ or CD19- Nalm6 cells. Intratumoral injection of engineered T cells (1x107) resulted in significantly elevated blood SEAP activity in mice bearing CD19+ tumors (n = 7), but not CD19- tumors (n = 5). Discussion: Our synNotch reporter system allows for the monitoring of antigen-dependent intratumoral immune-cancer cell interactions through a simple and convenient blood test. Continued development of this system for different target antigens of interest should provide a broadly applicable platform for improved monitoring of many cell-based immunotherapies during their initial development and clinical translation, ultimately improving our understanding of design considerations and patient-specific responses.