In situ vaccination with local radiation and intratumoral immunocytokine to elicit a tumor-specific memory B-cell response.

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 (~ 200mm 3 ) 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 2x10 6 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 2x10 6 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 > 200mm 3 ), 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.