Target discovery for immunotherapy of cervical cancer and high-risk human papillomavirus infection

Human papillomaviruses (HPVs) are small, double-stranded DNA viruses, and more than 170 types, including 15 types that are designated “high risk” (HR) or oncogenic, have been described. HR types are present in more than 99% of cervical cancers and each year, over 570,000 women are diagnosed with the disease causing 311,000 death per year worldwide. The emergence of cancer immunotherapy has provided new possibilities for the treatment of cancer. For this strategy, it is important to understand which proteins are presented on human leukocyte antigens (HLA) by the tumour and, therefore, could be recognised by T-cells and used as antigens in vaccination approaches or adoptive and recombinant T cell therapies.
High-performance mass-spectrometric techniques have provided the ability to globally investigate which peptides are presented by HLA in cells and tissues. In this study, these technological advances were applied for the identification of HLA class I peptidomes from cervical cancer cell lines and primary cervical cancer resections. We explored how to integrate whole exome sequencing data for the identification of neoantigens spanning tumour-specific mutation events, RNA sequencing data for definition of viral open reading frames and identification of viral HLA-presented peptides, and finally, the use of LC-MS de novo sequencing approaches that combine a in silico binding and peptide retention time prediction for identification of non-canonical ligands (MAR-S linear model tool).
In our human cervical cancer line panel, 55,474 peptides (48,278 unique peptides） were identified from 15,129 (10,920 unique proteins) proteins. More than 84% of the peptides had the typical length of HLA-I peptides (8 to 14 amino acids), with the majority (60%) being nine amino acids long. In ten human primary cervical tumour resections, 78,618 peptides (52,382 unique peptides) from 9,765 proteins (9,215 unique proteins). More than 88% of these peptides had the typical length of HLA-I peptides (8 to 14 amino acids), and the majority (60%) were nine amino acids long. KDM5B and H1.2 were identified as shared cancer-testis, tumour-associated antigens which were presented by all 5 cell lines and 10 tumour resections. Using DNA exome sequencing-based protein databases, 6 peptides were identified to originate from 5 different neoantigens in C33, DoTc2 and SiHa cell lines and the presentation of E1, and cryptic peptide epitopes originating from viral transcripts in cervical cancer cells is reported for the first time. RNA sequencing and de novo assembly supported the discovery of 5 viral peptides in CaSki and HeLa cells. Immunogenicity of the viral peptides was demonstrated by ELIspot in HPV-positive women with and without cervical intraepithelial neoplasia (CIN). The importance of HPV-E1 as a tumour-specific antigen in cervical cancer is highlighted further by demonstration of consistently high expression levels in primary human tumour resections relative to E6 and E7. We finally identified 12 lncRNA-related HLA peptide candidates in the primary cervical tumour samples, which warrant further validation.
Together, we conclude that E1 is an important target to consider for future immunotherapeutic approaches to cervical tumour treatment, and suggest further antigen candidates that are suitable for the consideration as molecular targets in next-generation immunotherapeutic development.

Keywords: Human papillomaviruses
antigens
cancer immunotherapy
cervical cancer
Advisor: Ternette, N
Dorrell, L