69 Background: In a murine melanoma (MEL) model, we reported an in situ vaccination response to combined radiation (RT) and intra-tumor (IT) injection of anti-GD2 hu14.18-IL2 immunocytokine (IC). This treatment resulted in 71% complete regression of 5-week (~ 200mm3) tumors, a memory T cell response, and augmented response to systemic anti-CTLA-4 antibody (mAb) checkpoint blockade. We hypothesized that mice rendered disease-free (DF) by RT, IT-IC, and anti-CTLA-4 mAb might also exhibit a memory B cell response. Methods: C57BL/6 mice were implanted with 2x106 syngeneic, GD2+ B78 MEL cells and tumors developed for 5 weeks. Mice were treated with 12 Gy RT to this tumor followed by 5 daily IT injections of hu14.18-IL2 d6-10 after RT and IP injection of anti-CTLA-4 d3, 6, and 9 after RT. DF mice and naïve controls were challenged by subcutaneous implantation with 2x106 B78 MEL cells. Peripheral blood was collected from mice before and after B78 challenge and serum was evaluated for presence of tumor-specific mAbs using flow cytometry and ELISA. Results: Seventy-three percent of mice were rendered DF by treatment with RT, IT-hu14.18-IL2, and anti-CTLA-4. All of these (13/13) rejected a rechallange B78 implantation > 1 year later (range d378 – 511), whereas no naïve mice rejected B78 implantation (0/66). IgG from serum of DF mice bound selectively to B78 and parental GD2- B16 MEL cells and the level of this mAb response appeared to increase modestly d14 after B78 challenge. In naïve mice, a modest increase in tumor-specific mAb was identified between non-tumor implanted mice and d35 post-implantation mice (bearing tumors > 200mm3), however this level remained ~ 5 fold below that observed in DF mice prior to B78 rechallenge. In contrast, no appreciable mAb response was observed for unrelated syngeneic GD2+ Panc02 pancreatic tumor cells in serum of DF or naïve mice. Conclusions: We report an endogenous anti-tumor IgG humoral response in DF mice > 1 year after treatment with RT, IT-IC, and anti-CTLA-4 mAb, concurrent with demonstration of long lasting immune protection from re-challenge. Studies are underway to determine whether this response is involved in the therapeutic efficacy of this in situ vaccination regimen.
SARS-CoV-2 infection severity is linked to superior humoral immunity against the spike