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

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.

Visualizing cell-cell communication using synthetic notch activated MRI.

Cell-cell communication plays a fundamental role in multicellular organisms. Cell-based cancer immunotherapies rely on the ability of innate or engineered receptors on immune cells to engage specific antigens on cancer cells to induce tumor kill. To improve the development and translation of these therapies, imaging tools capable of noninvasively and spatiotemporally visualizing immune-cancer cell interactions would be highly valuable. Using the synthetic Notch (SynNotch) system, we engineered T cells that upon interaction with a chosen antigen (CD19) on neighboring cancer cells induce the expression of optical reporter genes and the human-derived, magnetic resonance imaging (MRI) reporter gene organic anion transporting polypeptide 1B3 (OATP1B3). Administration of engineered T cells induced the antigen-dependent expression of all our reporter genes in mice bearing CD19-positive tumors but not CD19-negative tumors. Notably, due to the high spatial resolution and tomographic nature of MRI, contrast-enhanced foci within CD19-positive tumors representing OATP1B3-expressing T cells were clearly visible and their distribution was readily mapped. We then extended this technology onto human natural killer-92 (NK-92) cells, observing similar CD19-dependent reporter activity in tumor-bearing mice. Furthermore, we show that when delivered intravenously, engineered NK-92 cells can be detected via bioluminescence imaging in a systemic cancer model. With continued work, this highly modular imaging strategy could aid in the monitoring of cell therapies in patients and, beyond this, augment our understanding of how different cell populations interact within the body during normal physiology or disease.

Molecular Imaging Reveals a High Degree of Cross-Seeding of Spontaneous Metastases in a Novel Mouse Model of Synchronous Bilateral Breast Cancer.

PURPOSE: Synchronous bilateral breast cancer (SBBC) patients present with cancer in both breasts at the time of diagnosis or within a short time interval. They show higher rates of metastasis and lower overall survival compared to women with unilateral breast cancer. Here we established the first preclinical SBBC model and used molecular imaging to visualize the patterns of metastasis from each primary tumor. PROCEDURES: We engineered human breast cancer cells to express either Akaluc or Antares2 for bioluminescence imaging (BLI) and tdTomato or zsGreen for ex vivo fluorescence microscopy. Both cell populations were implanted into contralateral mammary fat pads of mice (n=10), and dual-BLI was performed weekly for up to day 29 (n=3), 38 (n=4), or 42 (n=3). Primary tumors and lungs were fixed, and ex vivo fluorescence microscopy was used to analyze the cellular makeup of micrometastases. RESULTS: Signal from both Antares2 and Akaluc was first detected in the lungs on day 28 and was present in 9 of 10 mice at endpoint. Ex vivo fluorescence microscopy of the lungs revealed that for mice sacrificed on day 38, a significant percentage of micrometastases were composed of cancer cells from both primary tumors (mean 37%; range 27 to 45%), while two mice sacrificed on day 42 showed percentages of 51% and 70%. CONCLUSIONS: A high degree of metastatic cross-seeding of cancer cells derived from bilateral tumors may contribute to faster metastatic growth and intratumoral heterogeneity. We posit that our work will help understand treatment resistance and optimal planning of SBBC treatment.