Project Abstract/Summary Despite their success, immunotherapies fail to engage T cells on an antigen-dependent basis rather opting to reactivate a broad spectrum of T cells of unknown specificity based on their location or phenotype. Markers such as PD-1 have been posited to delineate anti-tumor T cells but are also expressed on those recognizing non- tumor antigens. Likewise, tumor-resident T cells are surmised to exhibit tumor antigen recognition though they have been shown by us and others to also recognize viral and bacterial antigens (ie. bystander T cells). MHC multimers present a potent alternative to identify anti-tumor T cells but are limited by a need for anterior identification of the antigen(s) of interest. This limitation is compounded by a lack of understanding of the antigens that are immunogenic and the T cells that recognize them. Recent discoveries into the impact of tumor mutational burden (TMB) have bolstered our grasp of the origins of tumor antigens, despite TMB failing to consistently predict clinical outcomes. This is perhaps best epitomized by the dismal antigen validation rates which hover at ~1%. Hence, our understanding of which T cells exhibit therapeutic anti-tumor potential in solid tumors remains problematic. Therefore, there is a critical need for unbiased approaches capable of identifying anti- tumor T cell responses, a lack of which will substantially impede the progress of immunotherapies in solid tumors. We previously developed ATTACH (Assessment of T cells Tethered to Antigen Class I/II Histocompatibility), a powerful microfluidics assay which allows direct isolation of anti-tumor T cells by leveraging HLA/peptide binding avidity of T cell receptors (TCR) to matched tumor cells serving as de facto “tetramer pools”. Here, we propose the development of an ATTACHER (ATTACH via Electrothermal flow-enhanced Recovery), a microfluidic device capable of streamlining this assay while increasing sensitivity, specificity, and antigen-specific T cell recovery. Our central hypothesis is that T cells recognizing multiple tumor antigens can be directly isolated in vitro using a T cell ATTACHER, and that these cells harbor increased anti-tumor cytotoxic potential compared to bulk T cells. We have formulated this hypothesis on the basis of preliminary studies outlining the ability of ATTACH to enrich for tumor-infiltrating lymphocytes (TILs) with increased cytotoxic potential. The rationale for the proposed research is that applying electrothermal flow to ATTACH via development of a T cell ATTACHER will promote T cell/tumor cell contact and allow T cells to scan multiple MHC/antigen complexes in order to encounter their cognate antigen. In Aim 1, we will design and construct the T cell ATTACHER and evaluate its ability to recover antigen-specific T cells across a set of 20 different human antigens and TCRs of different affinities developed in our lab. In Aim 2, we will characterize TILs isolated using the T cell ATTACHER both phenotypically and functionally using paired human patient-derived xenografts and tumor-infiltrating lymphocytes from the ICON cohort. We anticipate our work will allow us to demonstrate the versatility of the T cell ATTACHER and its ability to improve anti-tumor responses in vitro. Overall, our work will allow direct, accurate and unbiased identification of anti-tumor T cells in solid tumors within hours rather than months as is currently required, laying the foundation for subsequent studies into the predictive and therapeutic potential of this method in solid tumors.

Project Narrative T cells are highly effective at destroying cancer cells based on the antigens they display but our understanding of which T cells destroy tumors and which antigens enable them to do so is rudimentary. Current approaches to decipher these questions rely on challenging experimental procedures which are costly, labor-intensive, exhibit a low success rate and can take months to perform, impacting their clinical applicability. Our novel device overcomes these hurdles, allowing the direct identification and isolation of anti-tumor T cells within only hours thereby improving potential applicability to the treatment of solid tumors.