PROJECT SUMMARY Human papillomavirus (HPV) is the causative agent of a growing proportion of incident cases of head and neck squamous cell carcinomas (HNSCCs). HPV type 16 is responsible for over 90% of HPV+ HNSCCs diagnosed in the United States. The HPV16 E7 antigen is an attractive immunotherapeutic target for HPV-associated cancers since it is a unique virus-specific oncoprotein constitutively expressed by all cancer cells. T cells that are genetically engineered to express an HLA-A*0201-restricted T cell receptor (TCR) that recognizes the HPV16 E711-19 epitope can mediate regression of HPV16-associated cancers in a preclinical model.1 A phase 1, first-in-human (FIH) multi-center study demonstrated the safety and efficacy of an autologous HPV TCR- engineered T cell (TCR-T) therapy which targets the HPV16 E7 binding epitope in HLA-A*0201-expressing HPV+ cancer patients (NCT03912831).2 However, the realized challenges with adoptive cell therapy (ACT) are: i) the high costs associated with manufacturability and monitoring and managing the toxicities from the supra- physiological bolus of ACT products which are combined with systemic IL-2 administration, and ii) the limited in vivo persistence due to the lack of appropriate antigen-specific stimulation due to poor intra-tumoral T cell recruitment into solid tumors. Herein, we propose innovative proof-of-concept (PoC) experiments to determine whether a low dose of adoptively transferred HPV16 E711-19 TCR-T cells can be exponentially expanded in situ by administering a novel biologic fusion protein, CUE-101, which delivers the HPV16 E7 antigen-specific stimulation with localized delivery of high doses of IL-2.3-5 Furthermore, we determine whether HPV16 E7 TCR- T cell persistence translates into an enhanced anti-tumor effect in preclinical HPV+ tumor models (Aim 1). Lastly, we build upon our team’s discovery of the role of the CXCR6:CXCL16 axis in the recruitment of peripheral circulating CD8+ T cells to perivascular niches of the tumor populated by CXCL16-expressing CCR7+ DC3s, which can sustain the survival of tumor-specific CD8+ T cells.6 Thus, we assess whether the efficacy of HPV16 E7 TCR-T cells can be improved through engineering strategies that introduce CXCR6 to the TCR-T cells, which can facilitate intra-tumoral recruitment, proliferation, and survival in situ (Aim 2). The completion of these aims will generate the supporting rationale for a future clinical trial administering HPV16 E7 TCR-T cells and CUE- 101 in HPV+ cancer patients. These efforts can potentially transform our current approaches to adoptive cell therapy.