Predictive Molecular Signatures of Tumor Reactivity from Antigen Specific Tumor Infiltrating Lymphocytes (tils) in Human Papillomavirus (Hpv)-Positive Oropharyngeal Carcinoma (OPC)

Abstract Background Accurate identification of tumor-reactive T cells in the tumor microenvironment (TME) is critical to understand mechanisms of response and resistance to immunotherapy and to engineer effective cellular therapies. In contrast to patient-specific, mutation associated neoantigens (MANA), viral proteins are expressed in all HPV-positive cancers, thus providing a unique opportunity to identify and characterize tumor-reactive T cells across patients. Methods We performed the Functional Expansion of Specific T cell (FEST) assay using 176 CMV-EBV-Flu virus (CEFX) peptides and 587 overlapping 15 amino acid peptide pools representing the entire HPV16 peptidome on 30 newly-diagnosed, HPV16-positive OPC patients before and during induction CTLA-4 and PD-1 immune checkpoint blockade (iICB). In a subset of 19 patients, overlapping peptides for expressed MANA identified by whole exome sequencing and RNASeq of tumor were included in the assay. T cell receptors (TCRs) for antigen-experienced T cells that significantly expand in vitro in peptide vs. no-peptide control were identified by Immunoseq. The antigen-specific TCR clonotypes were used to identify and phenotype tumor-reactive CD8+TILs in the TME in paired tumor biopsy samples (28 patients) with single-cell profiling by scRNA-seq and scTCR-seq on the 10X platform. Results The FEST assay identified a total of 9748 HPV-, 2373 MANA-, and 2639 CEFX-reactive TCRs in the blood of 30 patients. When tracked into the TME, HPV-specific CD8+TILS were more frequent and more clonally expanded when compared to CEFX-and MANA-specific TILs. HPV early proteins, such as oncogenic E5-E7, elicited more specific TILs than late proteins. Compared to “bystander” CEFX-specific CD8+ TIL, gene expression signatures (GES) for a tissue-resident memory (TRM) phenotype, cytotoxicity and immune checkpoint were higher in HPV-specific CD8+ TIL. HPV-specific CD8+ TIL in tumors with a major histological response (≤10% residual tumor viability) to iICB exhibited higher cytotoxic and lower checkpoint phenotypes than nonMHRs. Some HPV16-specific TCRs were validated to be tumor specific (n=40) and capable of killing. A tumor-reactive GES score was then calculated via AUCell software and recursive feature elimination from genes upregulated in validated HPV-specific vs “bystander” CEFX-specific CD8+TILs. The score achieved an area under curve (AUC) at 0.94 in a logistic regression model for tumor-reactive TIL prediction, higher than signatures from other groups. More tumor-reactive TILs were predicted using the score and their proportion was correlated with histological response. Conclusions In HPV16-positive OPC, the phenotype of HPV-specific CD8+TILs is associated with pathological response to iICB. A tumor-reactive GES score in scRNAseq data has high accuracy for identifying of tumor-reactive CD8+TIL, thus facilitating the quantification of in the TME and associations with treatment response. These findings enhance the development of personalized cellular therapies by enabling efficient identification of TILs and TCRs. Citation Format: Xianli Jiang, Khaled Sanber, Nils-Petter Rudqvist, Bo Jiang, Joe Dan D., Keiko Akagi, Weihong Xiao, Kelli N. Smith, Ken Chen, Maura L. Gillison. Predictive Molecular Signatures of Tumor Reactivity from Antigen Specific Tumor Infiltrating Lymphocytes (TILs) in Human Papillomavirus (HPV)-Positive Oropharyngeal Carcinoma (OPC) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B047.