ESTRO 37 Abstract book

S21

ESTRO 37

addition of the immunocytokine L19-IL2 to RT (single dose 10Gy) resulted in 75% of tumor remission in a heterotopic C51 murine colon carcinoma model. The immune system has the capacity of conferring protective immunity over a long period of time; the memory potential. Thus, we investigated whether this combination therapy also resulted in a treatment-specific long-lasting immunological memory dependent on the interleukin 7 receptor (IL7R) – IL7 axis. Material and Methods Balb/c mice were challenged with C51 tumor cells unilaterally and received different therapeutic schedules: 10Gy + L19-IL2, surgery + L19-IL2 or high single dose RT (40Gy) + vehicle. L19-IL2 was administered i.v (20 µg) 1, 3 and 5 days after irradiation or surgery. Control groups were mice without tumors receiving 10Gy on healthy tissue + L19-IL2 or not receiving any treatment. Mice who were cured after treatment were rechallenged with C51 tumor cells bilaterally after 150 days and tumor take was assessed. To investigate the role of the IL7R-IL7 axis, an in vivo blocking study of the IL7R (CD127) was performed in cured mice treated with 10Gy+L19-IL2. Intraperitoneal injections of anti-CD127 (400 ug) or IgG isotype control were given one day before tumor rechallenge and every 48 hours for 3 weeks. Blocking capacity of the antibody was confirmed in blood by flow cytometry. The presence of central memory and effector memory T cells was assessed in peripheral lymphoid organs and blood. Results After rechallenge, 8 out of 12 mice did not show tumor take after the initial 10Gy + L19-IL2 and blockade of CD127 had no influence on this protective effect. Mice cured after surgery + L19-IL2 or 40Gy + vehicle showed significant (p<0.05) less protective effects. Control groups were all able to form tumors within 13 days. Immunological endpoint analyses revealed that mice able to reject tumor cells upon rechallenge have a significant high expression of CD44 + CD127 + on CD8 + T in lymph nodes, spleens and blood. Moreover, mice able to reject tumor cells have a significantly high expression of central memory CD44 + CD62L + (7.3% [5.7-9.8] vs 2.0% [1.0-4.0], p= 0.01) and effector memory CD44 + CD62L - (36.3%, [34.5- 42.8] vs 28.4% [17.7-40.3], p=0.01) CD8 + T cells compared to other treatment groups. Conclusion To obtain long-lasting protection against C51 tumors, prior tumor cure by combining RT and the immunocytokine L19-IL2 is the main requirement. Our data suggest that the IL7R-IL7 axis is associated with long-lasting anti-tumor responses but is not causative. Due to the high association of CD44 + CD127 + CD8 + T cells with the memory effect, their cytotoxic and cytokine release capacities will be assessed ex-vivo with mixed culture assays of tumor cells and splenocytes. OC-0052 Abscopal effects with RT schedules extending to the effector phase of the antitumor T cell response X. Zhang 1 , G. Niedermann 1 1 University Clinics, Department of Radiation Oncology and German Cancer Consortium DKTK, Freiburg, Germany Purpose or Objective Hypofractionated radiotherapy (hRT) combined with immune checkpoint blockade (ICB) can induce T cell- mediated local and abscopal anti-tumor effects. We previously observed peak levels of tumor-infiltrating lymphocytes (TILs) between day 5 and day 8 after hRT; since TILs are regarded as radiosensitive, hRT schedules extending into this period might be less immunogenic, prompting us to compare clinically relevant, short and extended schedules with equivalent biologically effective dose in combination with anti-PD1 antibody treatment. Material and Methods In mice bearing two B16 melanoma tumors, the primary was irradiated with 3 × 9.18 Gy in 3 or 5 days, or with 5 ×

6.43 Gy in 10 days; anti-PD1 antibody was given weekly. Mice were followed for tumor growth and survival. T cell responses were determined on day 8 and 15 of treatment. The role of regional lymph nodes was studied by administration of FTY720, which blocks lymph node egress of activated T cells. Results Growth inhibition of irradiated primary and non- irradiated secondary tumors and overall survival were similar with all three hRT/anti-PD1 combinations, superior to hRT or anti-PD1 monotherapy, and depended strongly on CD8+ T cells. Apart from small kinetic differences, TIL infiltration and local and systemic tumor- specific CD8+ T cell responses were also similar, regardless of whether short or extended hRT was used. Simultaneous administration of FTY720 accelerated growth of both primary and secondary tumors, strongly reduced their TIL infiltration and increased tumor- specific CD8+ T cells in the lymph nodes draining the irradiated tumor. Conclusion Our data suggest that combination therapy with hRT schedules extending into the period during which treatment-induced T cells infiltrate the irradiated tumor can provoke similar local and systemic anti-tumor effects as therapy based on shorter schedules, if regional lymph nodes supply sufficient tumor-specific CD8+ T cells. This has implications for planning clinical RT/ICB trials. OC-0053 Combining hyperthermia and checkpoint inhibitors: a method of increasing tumour immunogenicity? P.B. Elming 1 , T.R. Wittenborn 1 , M.R. Horsman 1 1 Aarhus University Hospital, Dept. of Experimental Clinical Oncology, Aarhus, Denmark Purpose or Objective Checkpoint inhibitors (CI’s) are used in the clinic with success, but only affect a small percentage of patients. Methods to increase efficacy are needed. Hyperthermia treatments, using temperatures up to 45°C, can directly kill cells, and also has indirect effects through either a reduction in tumour blood flow or by inducing an immune response. We are currently doing experiments with the combination of a CI and heat treatment. The CI of interest is the monoclonal antibody CDF1 male mice were inoculated on the right rear foot with a C3H mammary carcinoma. Treatments were performed when tumours reached a volume of 200mm 3 . These included sham treated mice (controls), heat (42.5°C for 1 hour) on day 0, intra peritoneal injection with anti-CTLA-4 (10 mg/kg) on day 1, days 1 and 3, or days 1-4, or the combination of heat with anti-CTLA-4. Tumour size was measured daily, and the time to reach five times treatment volume (TGT5) was the endpoint. Mouse body weight was also recorded to assess toxicity. Results are listed as Mean (± Standard Error). One-way ANOVA comparison of group means was performed, and a P<0.05 was considered significant. Results The TGT5 for the control group was 6.6 days (+ 0.2). For the groups treated with anti-CTLA4 on day 1, days 1 and 3, or days 1-4, the TGT5 was 5.8 days (+ 0.4), 5.8 days (+ 0.4) and 6.8 days (+0.3), respectively. None of these values were significantly different from controls. In the group treated with heat the TGT5 was significantly increased to 11.1 days (+ 0.9). When combined with the anti-CTLA-4 this value was increased to 11.6 days (+ 1.2) for treatment on day 1, 12.9 days (+ 2.7) for days 1 and 3, and 15.4 days (+ 0.8) for days 1-4. The values for day 1 and 1 and 3 were not significantly different from heat alone, but when heat and anti-CTLA-4 on days 1-4 were combined we saw a significantly increase in treatment that targets CTLA-4. Material and Methods