Preview

PDF, English Download (7MB) | Terms of use

Abstract

High-risk human Papilloma viruses (HR-HPVs) are the main causative agents of cervical, anal, vulvar, vaginal and penile cancer as well as head and neck cancer. The most prevalent HR-HPV types are HPV16 and HPV18. HPV16 is responsible for about 50% of HPV-related cancers and thus it is a preferred type to study. The HPV16 E7 (11-19) peptide is a well-characterized epitope presented by HLA-A*02:01 molecules on HPV16+ tumor cells. Therefore, it represents a tumor-specific antigen and is an ideal target for Adoptive T cell therapy (ATC) due to its viral source and being absent in healthy tissues. Recently, a proof-of-concept study in metastatic cervical cancer patients has shown that administration of HPV16-specific tumor infiltrating lymphocytes (TILs) expanded ex vivo and infused back to the patient induced tumor regression. However, selection and expansion of TILs has some important disadvantages compared to genetically modified T cells. This project was aimed at identifying HPV16 E7-specific TCRs that could be applied as an effective immunotherapy to cancers caused by this virus. We believe that tumor samples from patients at developing stages of HPV+ cancers might not be optimal for finding HPV-specific TCRs, since their TILs react weakly against the E6/E7 oncoproteins. Furthermore, HPV16- specific cytotoxic T cells can be frequently found in peripheral blood of healthy donors. Therefore, in vitro stimulation of CD8+ T cells derived from healthy individuals and subsequent identification of their TCR repertoire could be a better way to obtain efficient TCR candidates for adoptive T cell therapy of HPV16+ cancers. For an efficient in vitro stimulation of T cells, we used a previously constructed and purified recombinant fusion protein composed of an N-terminal fragment of E7 (amino acids 1-30, E71- 30) linked to the N terminus of Flt3L. Immature dendritic cells isolated from PBMCs of healthy donors were pre-incubated with the fusion protein and then co-cultured with autologous T cells. E7-reactive CD8+ T cells (IFN- γ + , CD137+ and HLA-A2-E711-19-Tetramer+ ) were isolated. The exact TCR profile of the E7-reactive T cells was determined by single-cell V(D)J sequencing using the 10X Genomics platform. Our results showed that the E7-Flt3L is a functional protein that can efficiently increase the frequency of CD8+ T cells targeting the HLA-A*02:01-restricted iv HPV16 E7(11-19) epitope among peripheral lymphocytes of healthy donors. Moreover, our workflow combined tetramer binding and activation of T cells to improve the selection of truly reactive T cells. Three TCR candidates were screened in vitro, which showed different patterns of specificity, avidity, and reactivity. Further, we were able to identify several E7(11-19)-reactive motifs in the CDR3β region through in silico characterization of 22 selected TCRs. Importantly, most of these motifs were enriched in patients who cleared HPV16 infection compared to patients with cervical intraepithelial neoplasia grade 3 (CIN3) or higher. Taken together, our study has established an efficient workflow for the identification of TCRs targeting tumor viral antigens which can be an important step toward TCR discovery.