Background 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 detected in mice cured of 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 melanoma following immunotherapy.

Methods Mice with GD2-expressing syngeneic B78 melanoma were treated with a combination immunotherapy (local radiation therapy + intratumoral anti-GD2 mAb linked to IL2) capable of inducing an ‘in situ vaccine’ effect (ISV), enabling mice to be cured of their tumors with long-term immune memory.¹ 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 dynamic programming method that scans adjacent peptides to determine whether a peptide is bound by antibodies. Proteins were selected if peptides were bound using immune sera but not bound with the sera from naïve or non-responding tumor-bearing mice.

Results Multiple proteins were selectively identified by immune sera that were not detected by 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 also identified a few proteins that elicited an immune response in the naïve mouse sera that showed a significantly stronger signal in the immune sera of the same mice indicating that the cancer and/or the received therapy strengthened the immune response to these proteins.

Conclusions We are able to detect selective antibody binding to immune sera. However, we are continuing to refine our analytical methods and are further investigating the identified proteins. These peptides may potentially serve as targets for antibody-based or cellular therapies. In addition, we are examining whether some of the identified tumor-specific endogenous antibodies might be used as biomarkers to predict response to our ISV regimen and potentially other immunotherapy treatments.

Reference

1. Morris ZS, et al. In Situ Tumor Vaccination by Combining Local Radiation and Tumor-Specific Antibody or Immunocytokine Treatments. Can Res. 2016; 76:3929–3941