Abstract 4011: Effective in situ immunization via local radiation therapy (RT) and tumor-specific immunocytokine (IC): Suppression from distant tumor is blocked by RT or Treg-depleting CTLA-4 antibody
Authors: Morris Zachary S. , Guy Emily , Francis David , Gressett Monica M. , Armstrong Eric A. , Huang Shyhmin , Werner Lauryn R. , Gillies Stephen D. , Korman Alan , Hank Jacquelyn A. , Rakhmilevich Alexander L. , Harari Paul M. , Sondel Paul M. 
Published: July 2016

Abstract
PURPOSE: Use “off the shelf” reagents to eradicate an established tumor and induce a tumor specific T cell response that destroys distant tumor.
PROCEDURES: We have identified a cooperative interaction between local tumor RT, intratumoral (IT) injection of IC hu14.18-IL2 (anti-GD2 hu14.18 mAb linked to IL2), and checkpoint blockade with anti-CTLA4 mAb. C57Bl/6 mice were implanted with 2×106 B78 (GD2+) melanoma in one flank (1-tumor model). In the 2-tumor model, mice received 2×106 B78 3 weeks (w) later in the opposite flank. After 5w the primary (1st) tumor was ∼200mm3, and ∼500mm3 after 7w. The 2nd tumor was ∼ 50mm3 at 5w. At 5w or 7w, mice received single fraction RT (12Gy) to the 1st tumor and 6 days later received 5-daily 50μg injections of IC (IT-IC). When used, anti-CTLA4 was given i.p. on days 3, 6 and 9 after RT.
NEW UNPUBLISHED DATA: For mice bearing a single 200mm3 tumor, RT+ IT-IC results in complete response (CR) in 71% of mice and a tumor-specific memory T cell response. Mice with a single 500mm3 tumor showed slowing of tumor growth, but only 27% CR after radiation + IT-IC. Adding anti-CTLA-4 to RT + IT-IC improved tumor response (73% CR) and survival compared to doublet combinations of these 3 modalities. In contrast, in the 2-tumor model, providing RT + IT-IC to the 1st ∼200mm3 tumor, but not to the distant ∼50mm3 tumor, did not enhance 1st tumor shrinkage compared to RT alone and had no effect on the 2nd 50mm3 tumor. The presence of the 2nd B78 tumor resulted in systemic immune suppression that prevented the local RT and IT-IC from eliminating the 1st tumor. This was tumor specific, as local RT + IT-IC to the 1st ∼200mm3 B78 tumor was still effective in treating the 1st tumor if the 2nd (∼50 mm3 tumor) was the syngeneic but unrelated Panc02 tumor. Delivering RT to both the 1st + 2nd B78 tumors eliminated the inhibitory effect of the 2nd tumor, enabling IT-IC to the 200mm3 tumor to cause eradication of that tumor in 64% of mice. Preliminary PCR analyses of FoxP3 in the 1st tumor showed Tregs are depleted by 1st tumor RT only in mice with 1 tumor and not in mice with 2 tumors. In this 2 tumor model we combined RT + IT-IC of the 1st tumor with anti-CTLA-4. The IgG2b anti-CTLA-4 (which doesn't substantially deplete Tregs) had minimal effect on 1st tumor response to RT + IT-IC. In contrast the IgG2a anti-CTLA-4 (that depletes Tregs) rendered 60% of mice disease-free (durable CR of both the treated 200mm3 and the untreated 50mm3 tumors). Preliminary data, using DEREG mice that enable diphtheria toxin to deplete Tregs, show Treg depletion in the 2 tumor model also enables eradication of both 1st and 2nd tumors in 60% of mice after RT + IT-IC to only the 1st tumor.
CONCLUSIONS: Local RT+ IT-IC can result in long-term tumor eradication of macroscopic tumors via adaptive immunity to the “in situ vaccine”, provided that Treg-associated immune suppression from distant tumor is eliminated by RT or Treg-depletion.
Citation Format: Zachary S. Morris, Emily Guy, David Francis, Monica M. Gressett, Eric A. Armstrong, Shyhmin Huang, Lauryn R. Werner, Stephen D. Gillies, Alan Korman, Jacquelyn A. Hank, Alexander L. Rakhmilevich, Paul M. Harari, Paul M. Sondel. Effective in situ immunization via local radiation therapy (RT) and tumor-specific immunocytokine (IC): Suppression from distant tumor is blocked by RT or Treg-depleting CTLA-4 antibody. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4011.