Background Human papillomavirus (HPV) infection causes at least 650,000 anogenital and oropharyngeal cancers (OPC) worldwide annually. Despite the viral immunological target, immune checkpoint inhibitors produce responses only in a minority of patients, and cancer therapies that directly target HPV-antigens represent an attractive alternative therapeutic approach. With this work, we introduce a high-throughput and epitope-agnostic pipeline for HPV16-reactive T cell discovery and validation.
Methods Using the FEST assay , T cell responses against HPV16 were assessed in 3 patients with HPV16-positive OPC that participated in an open-label phase I clinical trial studying the safety and immunogenicity of vaccination with a DNA-based HPV16-E7 vaccine. T cell receptor (TCR) motifs were constructed using GLIPH2 . Enrichment of motifs in tumors and HLA alleles among patients was determined using Wilcoxon signed-rank test and Fisher Exact test, respectively. Association between TCR motifs and survival was assessed using a log-rank test. Single cell sequencing (sc-seq) was performed using the 10x platform. Expression of TCRs in effector cells was achieved using electroporation. All studies were approved by MD Anderson IRB (PA17-0149, PA19-0470, and 2019-1059).
Results Sixteen HPV16-reactive TCRs were identified in 3 patients with HPV16-positive OPC, and responses were found against most HPV16 proteins. Five clones were associated with commonly shared TCR motifs identified in our combined patient cohort. A TCR motif against HPV16-E6 (E6-TCR-motif) was found enriched in tumors (p=0.007; figure 1) of patients who expressed a common HLA allele (p=0.0019; figure 2). Strikingly, no patients that naturally harbor the E6-TCR-motif died (p=0.04) nor progressed (p=0.023; figure 3). Recognition of a region within E6 by a T cell belonging to the E6-TCR-motif was validated via transgenic TCR expression in Jurkat cells co-cultured with HLA-matched peptide-pulsed target cells, and eventually the minimal epitope was determined. Impressively, an interaction between TCR and epitope was still detectable at 10-15 M peptide concentration, a factor 1000 lower compared to our positive control TCR  against HPV16-E6 (figure 4).
Conclusions We have identified a common HPV16-E6 specific TCR motif shared among patients with HPV16-positive OPC cancer that is associated with survival. We are currently working to translate this finding to the clinic as an adoptive cellular therapy. Given the success of this approach, we are working to map the antigenic/TCR landscape in patients with HPV16-positive tumors, with the goal of developing a suite of TCR-based therapies restricted to common HLA alleles and to construct a therapeutic vaccine against persistent HPV16 infection.
Acknowledgements This research was supported by The University MD Anderson HPV-Related Cancers Moon Shot, the MD Anderson Oropharynx Cancer Program, and Intramural Startup funds. FEST assays were performed by the FEST and TCR Immunogenomics core at Johns Hopkins University. We would also like to thank Drs. Alexandre Reuben and Minying Zhang for sharing reagents.
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