Cancer immunotherapy has revolutionized cancer treatment and has helped thousands of patients (Couzin-Frankel, 2013; Patel & Minn, 2018). However, most patients are still not showing positive responses to current cancer immunotherapy treatment regimens (Patel & Minn, 2018). Using radiation therapy (RT) and intratumoral injections of immunocytokine (IC), our lab has developed a local in situ vaccine regimen capable of curing mice bearing B78 melanoma tumors with protective immune memory (Morris et al., 2016). Our in situ vaccine (RT+IC) cures 70% of treated mice bearing a single large B78 tumor (which expresses GD2) and creates strong immunologic memory to reject a second challenge of B78 melanoma. We have also demonstrated that our in situ vaccine causes epitope spread; 75% of cured mice reject a challenge with B16 melanoma cells (which do not express the GD2 antigen), and we observed strong antibody-binding to B16 cells using serum from cured as compared to naïve mice (Baniel, Heinze, et al., 2020; Morris et al., 2016). Although we observed epitope spread, the exact antigen targets of these endogenous antibodies were unknown. Knowledge of these additional targets could help to identify biomarkers of positive responses, as well as identify possible new therapeutic targets. In this thesis we utilized a peptide array approach to probe every mouse protein (broken into 16-mer peptides in a stepwise overlapping fashion) to identify antibody targets, using serum from cured mice vs. their matched naïve sample to identify immunodominant tumor antigens on cold murine tumors. We were able to develop a robust analysis method (HERON) to identify a number of proteins recognized by multiple mice that we are further investigating (explained in detail in Chapter 2). We furthermore saw that within the top epitopes (measured by signal strength and detection in multiple mice) peptides containing a specific four amino acid long sequence were highly overrepresented. The presence of this sequence seemed to be associated with binding, although not being the sole source recognized for antibody binding. Multiple analysis approaches have led us to know more about this motif while yet not being able to elucidate it fully (described in chapter 3). Further investigation is needed into top proteins co-recognized by multiple immune mice as well as the four amino acid motif and possible implications and uses as biomarkers or treatment alternatives. We are pursuing experimental as well as bioinformatic approaches to enhance our knowledge of the identified antibody targets. The methods generated will provide useful tools to identify the immunodominant tumor-specific antigens which may be translatable to mechanisms of resistance (and how to overcome such resistance) in human cancers and find potential uses as biomarkers or new treatment targets.