Characterization of dynamic changes in tumor-infiltrating lymphocytes (TIL) after neoadjuvant administration of CUE-101, an HPV16 E7-HLA-IL2 fusion protein, to patients with HPV+ locally advanced oropharyngeal squamous cell carcinoma (LA-OPSCC).

6055 Background: CUE-101 is a novel fusion protein designed to selectively engage and expand HPV16-specific T-cells to promote an anti-tumor immune response in HPV+ malignancies. CUE-101 is comprised of the HPV16 E7 11-20 epitope, HLA-A*0201, and a reduced affinity human IL2. In this single-arm Phase 2 trial (NCT04852328), CUE-101 is administered to HLA-A*0201+ patients with HPV16+ LA-OPSCC in three schedules (A, B, and C) before curative-intent surgery and adjuvant therapy or definitive chemoradiation therapy. Primary endpoints were safety of CUE-101 and changes in the frequency of HPV16 E7 11-20 -specific CD8+ T cells in blood and tumor. Correlates in TIL for Schedules A and B are presented here. Methods: CUE-101 (4 mg/kg IV) was administered 14 days (Schedule A), 14 and 7 days (Schedule B), or 7 days (Schedule C) before curative-intent treatment. Biopsies of the primary tumor were collected before CUE-101 administration and within 2 days prior to curative-intent therapy. Single-cell RNA and T-cell receptor (TCR) sequencing (scRNAseq, 10X Genomics) was performed on fresh sorted CD8 T-cells. HPV16 E7 11-20 dextramer staining and flow cytometry assessed reactivity to HPV E7. Multiplex immunofluorescent (mIF) tissue staining with a 30-marker Phenocycler panel (Akoya Biosciences) assessed tumor, myeloid, and T-cell states. Changes in T-cell clonal frequency were significant if fold change >2 and fisher exact test adjusted p <0.05. Changes in cell populations after treatment were evaluated by two-tailed paired student’s t-test. Results: Of 20 total patients enrolled, paired biopsies from 13 of sufficient quality were analyzed by scRNAseq, flow cytometry, and mIF. In a joint analysis of Schedules A (n=7) and B (n=6), we identified multiple T-cell clones that significantly expanded following CUE-101 treatment, representing 4-51% (mean 18.3%) of tumor-infiltrating CD8 cells by scRNAseq. Among these, we found significant enrichment for phenotypes of chronic T cell activation (PD1+CD39+, often associated with tumor reactivity, p =0.041). In agreement with this finding, flow cytometry of TIL showed increased CD8+CD39+ cells (mean 10.9%, p =0.026) among CD45 cells, and a small increase in NK cells (1.7%, p =0.005). Absolute cell counts by mIF confirmed these trends. Direct dextramer staining of TIL samples has not specifically identified HPV E7 11-20 reactive T cells pre- or post-treatment, therefore cloning of TCRs from expanded T-cells is being performed to enable functional testing of HPV reactivity. Conclusions: Significant clonal expansion of intra-tumoral CD8 T-cells with signatures associated with tumor reactivity were observed within 2 weeks of CUE-101 administration in HPV16+ LA OPSCC. Studies in blood and of T-cell specificities are ongoing. Clinical trial information: NCT04852328 .