Thousands of new antigens are recognized in mice via endogenous antibodies after being cured of a B78 melanoma via immunotherapy

Abstract

Abstract Antibodies can play an important role in innate and adaptive immune responses against cancer. Using a high-density peptide array, we assessed potential protein-targets for antibodies activated in mice cured of their melanoma through a combined immunotherapy regimen. Our goal was to determine the linear peptide sequences recognized by anti-tumor antibodies produced in mice cured of their melanoma with immunotherapy. Mice with GD2-expressing syngeneic B78 melanoma were treated with a combination immunotherapy capable of inducing an “in situ vaccine” effect (ISV), enabling mice to be cured of their tumors with long-term immune memory. Naïve and immune sera were collected from these mice. Using flow cytometry, immune sera showed strong antibody-binding against B16 (parental cell line of B78 without GD2 expression). These sera were then used on a Nimble Therapeutics’ peptide-array (either whole proteome or a curated list of ~650 proteins) to determine specific antibody-binding sites, and data were analyzed using a probabilistic model. Using the “curated list” peptide array, proteins were selected if the protein was bound in immune sera but not bound in the sera from naïve or non-responding tumor-bearing mice. When focusing on the whole mouse proteome data, thousands of peptides were targeted by 2 or more mice and exhibited strong antibody binding only by immune sera. We are continuing to refine our analytical methods and are further investigating all identified proteins. These peptides may be new targets for antibody-based or cellular therapies and some of the tumor-specific endogenous antibodies that we have identified may be used as biomarkers to predict response to our ISV regimen and potentially other immunotherapy treatments.