Background: Despite impressive responses to checkpoint inhibitors in advanced melanoma, post-treatment relapse and primary non-response indicate that some patients require additional strategies to produce clinically-effective immune responses. Here we describe the immunogenic potential and expression profile of ropporin, a tissue-restricted and highly expressed antigen in malignant melanoma.
Methods: Expression of ropporin (ROPN1) was determined in tumour tissue (various types), melanoma metastases and a large panel of melanoma cell lines by RT-qPCR. ROPN1 and ROPN1B gene expression data was also assessed by whole-genome microarray for the panel of melanoma cell lines. Protein expression was confirmed in melanoma cell lines by immunohistochemistry. Seromic screening of patient sera to identify in vivo-generated antibodies against ROPN1 was performed using a custom phage-display protein array. The minimal Ropporin epitope recognised by specific T cells was defined by in vitro peptide stimulation of peripheral blood mononuclear cells, followed by flow cytometric analysis for cytokine production.
Results: Ropporin isoforms are minimally expressed in normal tissues other than testis. Tumour samples from various sites show a range of ROPN1 expression from very low (colon, kidney, prostate, testis) to mixed (breast, glioma, lung). Melanoma tumours and cell lines derived from metastatic lesions show high levels of both ROPN1 gene and protein expression. Sera obtained from patients with advanced melanoma revealed frequent presence of ROPN1 and ROPN1B antibodies. We identified ropporin-specific T cell responses using in vitro stimulation of patient-derived peripheral blood mononuclear cells with a series of overlapping ropporin peptides. Both CD4+ T cell and CD8+ T cell responses were identified.
Conclusion: Ropporin is a highly immunogenic and tumour-specific protein commonly found in melanomas. The identification of cognate T cell responses to ropporin in melanoma patient-derived lymphocytes indicates that ropporin represents a promising immunotherapeutic target in advanced melanoma and may be a useful adjunct to checkpoint inhibition.