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.
Infection is one of the most dangerous complications that can be seen when implanting bone or tendon allografts from a deceased donor. The most common germs isolated are found among the cutaneous florae, but sometimes they may be present in the bloodstream as a result of severe injuries suffered before the time of the decease. We present a case of contamination of allografts in a musculoskeletal tissue donor deceased after an accident, whose allografts were contaminated by gastrointestinal microorganisms, probably disseminated through the donor's blood.
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.
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