Abstract 1377: Endogenous antibodies recognize multiple proteins on B78 melanoma in mice cured via immunotherapy
Authors: Hoefges Anna , McIlwain Sean J. , Erbe Amy K. , Le Trang Q. , Tetreault Kaitlin , Mathers Nicholas , Rakhmilevich Alexander L. , Hank Jacquelyn A. , Patel Jigar , Garcia Brad , Morris Zachary S. , Ong Irene M. , Sondel Paul M. 
Published: June 2022

Abstract
Introduction: Antibodies can play an important role in innate and adaptive immune responses against cancer. By using a high-density whole mouse proteome peptide array, we were able to assess potential protein-targets for antibodies present in mice cured of their melanoma by a combination immunotherapy regimen of 12Gy local radiation and intratumoral administration of a tumor-specific immunocytokine (anti-GD2 antibody linked to IL-2). This regimen can induce an “in situ vaccine” effect (ISV) enabling mice to be cured of their tumors with long-term immune memory.
Methods: Immunocompetent C57/BL6 mice bearing large B78 (GD2-expressing) melanoma tumors were treated with ISV. Sera were collected at different timepoints (before tumor implantation, after implantation before treatment, after completion of treatment, after the mouse was rendered tumor-free and after a rejected rechallenge). Tumor cell surface binding of these serum samples was assessed via flow cytometry and showed a strong increase in binding of sample obtained following immunotherapy, and after rejection of a tumor rechallenge. Binding was assessed against the original tumor line, B78, as well as B16 (parental to B78 without GD2 expression).We used the naïve and immune sera on a Nimble Therapeutics whole mouse proteome peptide array to determine specific antibody-binding sites. Whole proteome data was analyzed using a newly developed algorithm which scans adjacent peptides to detect differential binding between naïve and immune samples.
Results: Multiple proteins were selectively identified by immune sera which were not well detected by sera from naïve mice. Hundreds of proteins were targeted by 3 or more of 6 mice and exhibited strong antibody binding only by immune sera. Some proteins were recognized via the same binding epitopes, others via different epitopes in separate mice. Some of the whole protein peptide array results were validated via JPT multi-well peptide array and some via independent peptide ELISA. Results from Nimble peptide array and ELISA validated well.
Conclusions: We were able to detect antibody in immune sera binding selectively to specific linear epitopes and identify proteins of interest for further investigation as potential targets for antibody-based or cellular therapies. Results from our whole proteome peptide array were validated in separate assays via peptide ELISA. We are also exploring if some of the identified tumor-specific endogenous antibodies could be used as biomarkers to predict response to our ISV regimen and potentially other immunotherapy treatments.
Citation Format: Anna Hoefges, Sean J. McIlwain, Amy K. Erbe, Trang Q. Le, Kaitlin Tetreault, Nicholas Mathers, Alexander L. Rakhmilevich, Jacquelyn A. Hank, Jigar Patel, Brad Garcia, Zachary S. Morris, Irene M. Ong, Paul M. Sondel. Endogenous antibodies recognize multiple proteins on B78 melanoma in mice cured via immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1377.