Project Summary / Abstract Immunotherapy has become a rapidly growing segment of cancer treatment with impressive success across a spectrum of malignancies including melanoma, colorectal, and non-small cell lung cancers. Notwithstanding these advances, a significant fraction of patients fails to respond to immunotherapy and suffers from serious adverse side effects. While peptide-loaded major histocompatibility complex (pMHC) tetrameric structures allow the detection of antigen-specific T cells, a few antigen specificities can be detected in parallel because of limitations on the number of available fluorescent or metal labels. On the other hand, thousands of cells are needed for the workflow. Improved prognostic methods to monitor the specificity and functional behavior of tumor antigen-specific cytotoxic T cells is greatly needed to enhance the overall effectiveness of a range of immunotherapies, especially adoptive cell therapy (ACT), and provide better outcomes for cancer patients. This project develops a first-of-its-kind biosensor platform that enables the rapid and parallel detection of antigen- specific T cells. The proposed lab-on-chip technology allows detection and characterization at a single-cell level without requiring the use of labeling, complicated operational controls, or expensive equipment. As a result, the technology can be implemented in point-of-care settings and rapidly provide medical professionals with critical information, such as the ideal timing of future injected doses and any off-target effects. The key innovations behind the proposed technology include its high-throughput biosensor architecture, the ability to scale-up manufacturing using existing silicon foundries, label-free cell detection, simple operation and product design, and the implementation of novel algorithms of robust, real-time data analysis. Moreover, the commercialization of the proposed technology is facilitated by a mature semiconductor industry to achieve this high level of multiplexing in a small form factor. The proposed project focuses on engineering and optimization of the proposed biosensor platform and iterative development using six well-characterized tumor-antigens that are frequently recognized in melanoma patients. Peripheral blood mononuclear cells (PBMCs) from melanoma patients and from healthy donors will be used for analysis and technology validation. Successful completion of the project will provide a laboratory proof-of- concept, allowing the technology to move forward to a clinical setting where it can be used to monitor patients’ ongoing responses to immunotherapy, in specific checkpoint blockade and/or adoptive cell therapy. The tumor profiling market is projected to grow to about $12B by 2024, with the largest sector being immunoassays. If successful, the proposed technology will be a groundbreaking development in the cancer immunology toolbox, especially for early ex vivo identification of resistant tumor cell subpopulations,...