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Peritumoral IL-15 Potentiates Radiation-Induced Anti-Tumor Immunity
Oral Scientific Sessions S129
Volume 96 Number 2S Supplement 2016 Author Disclosure: M. Hettich: None. F. Braun: None. G. Niedermann: None.
289 Unique Changes in the TCR Repertoire of Tumor-Infiltrating Lymphocytes Underlie the Synergy of Radiation Therapy With CTLA-4 Blockade K.A. Pilones,1 R. Emerson,2 S. Formenti,3 and S. Demaria1; 1Weill Cornell Medical College, New York, NY, 2Adaptive Biotechnologies, Seattle, WA, 3 Weill Cornell Medicine, New York, NY Purpose/Objective(s): Monoclonal antibodies (mAb) that target CTLA-4, a negative regulator of T-cell activation, have significantly extended survival of patients with solid malignancies. However, the number of responders remains low. We have previously shown in the 4T1 mouse breast tumor model that resistance to anti-CTLA-4 therapy can be overcome by concurrent local radiotherapy (RT) (Demaria et al Clin Cancer Res 2005). Multiple effects of RT on the tumor microenvironment contributed to its ability to sensitize tumors to anti-CTLA-4 treatment, including enhanced homing of effector T cells to the tumor and formation of stable immune synapses between CTL and tumor cells (Matsumura et al. J Immunol 2008; Ruocco et al. J Clin Invest 2012). Importantly, CD8 T cell responses to the endogenous tumor antigen gp70 were seen only in mice treated with the combination, consistent with the hypothesis that RT generates an in situ vaccine and CTLA-4 expands primed T cells. Materials/Methods: BALB/c mice were inoculated s.c. with 4T1 cells and thirteen days later, when tumors became palpable, randomly assigned to one of 4 treatment groups (N Z 5 mice/group): control, RT alone, antiCTLA-4 alone or RT + anti-CTLA-4. RT was given in 2 fractions of 12 Gy on days 13 and 14 post-tumor inoculation. Anti-CTLA-4 mAb (Clone 9H10) was given i.p. on days 15, 18, and 21. Tumors and sorted CD4+ and CD8+ T-cells were harvested on day 22 for high throughput sequencing of TCRb CDR3 regions performed using the ImmunoSEQ platform. Results: We observed distinct and non-overlapping effects of RT and immunotherapy on TIL repertoire. CTLA-4 blockade increased clonality and significantly expanded the top 5 most frequent clonotypes. On the other hand, RT increased TIL numbers and broadened their repertoire by selectively expanding the top 6-20 clonotypes. By comparing the exact nucleotide sequences from sorted TILs with those from paired tumor samples, oligoclonal expansion driven by RT + anti-CTLA-4 mAb was essentially driven by CD8+ T-cells. Significantly reduced diversity of the TCRb profile seen only with combination therapy is consistent with antigen-driven TIL clonal expansion. Conclusion: Overall, tumor rejection induced by RT + anti-CTLA-4 is associated with both quantitative and qualitative changes in TIL repertoire. Data suggests that a broader repertoire of tumor-specific T cells may be critical for tumor rejection, and is achieved by coupling the effects of RT, which promotes priming of T cells specific for endogenous tumor antigens, and CTLA-4 blockade, which drives expansion of selected clones. These results have important implications for the therapeutic synergy of radiation and CTLA-4 blockade. Author Disclosure: K.A. Pilones: None. R. Emerson: None. S. Formenti: Advisory committee; BMS, Smith Kline, Astra Zeneca, EISAI. Research Grant; Regeneron, Janssen Biotech, Eli Lilly. Speaker’s Bureau; Varian. S. Demaria: Consultant; Sanofi US Inc., Regeneron Pharmaceuticals, Inc.
290 Peritumoral IL-15 Potentiates Radiation-Induced Anti-Tumor Immunity K.A. Pilones,1 S. Formenti,2 and S. Demaria1; 1Weill Cornell Medical College, New York, NY, 2NewYork-Presbyterian Hospital/Weill Cornell Medicine, New York, NY Purpose/Objective(s): Radiotherapy (RT) can induce T cell-mediated anti-tumor immune responses by multiple mechanisms but is often unable to overcome immunosuppression in the tumor microenvironment. The common gamma-chain cytokines interleukin (IL)-2 and IL-15 promote the
proliferation of activated T cells and, therefore, are prime agents for immunotherapy strategies aimed at sustaining anti-tumor T cell responses. The benefits of high dose IL-2, however, are undermined by serious toxicity and by regulatory T cell (Treg) stimulation. In contrast, IL-15 is well-tolerated and lacks Treg stimulatory activity, making it an attractive candidate for testing in combination with RT. Here we tested the hypothesis that IL-15 strengthens the pro-immunogenic effect of local RT to potentiate a durable anti-tumor immune response. Materials/Methods: The poorly immunogenic mouse TSA breast cancer cells were implanted s.c. in syngeneic BALB/c mice and randomly assigned to one of 4 treatment groups when tumors reached 5mm average diameters: control, RT, IL-15, or RT + IL-15. RT was delivered locally in 8 Gy fractions on days 13, 14, and 15. IL-15 (2 mg/mouse) was administered s.c. peritumorally daily for 10 days starting on day 12. Mice were followed for tumor growth. A parallel experiment was done to characterize tumorinfiltrating lymphocytes (TILs) at the end of treatment (day 22). Results: Low dose IL-15 given peritumorally as a monotherapy induced marginal tumor growth control and had no effect on survival (median survival Z 45 days compared to 76 days for control). Local RT significantly delayed tumor growth (P < 0.05 compared to control) and improved survival (median Z 76 days, P < 0.05). However, highest survival advantage was seen in mice given IL-15 + RT (median Z 102 days, P < 0.05 compared to all groups) with 1 of 6 mice showing complete tumor rejection and development of anamnestic response against tumor re-challenge. Analysis of TILs showed marked infiltration of CD8+ T cells expressing the activation marker CD137 (35.3% in RT + IL-15 versus 5.90% In control, P < 0.05) while the increase was modest with each monotherapy (18.8% in RT, 20.7% in IL-15, P < 0.05 compared to control). In addition, we found a significant increase in the ratio of effector CD4+ T-cells to Tregs (2.5 in RT + IL-15 versus 0.78 in control, P < 0.05) whereas monotherapy had no effect (1.14 in RT, 0.96 in IL-15). Additionally, IL-15 treatment, regardless of RT, led to significant intratumoral enrichment of NKp46+ NK cells. Conclusion: Overall these results support the rational combination of low dose intratumoral IL-15 with local RT. We are currently elucidating the mechanisms involved in pre-clinical models in preparation for future testing in patients. Author Disclosure: K.A. Pilones: None. S. Formenti: Research Grant; Regeneron, Janssen, Eli Lilly. Speaker’s Bureau; Varian. Advisory Board; BMS, Smith Kline, Astra Zeneca. S. Demaria: None.
291 Impact of Incidental Cardiac Radiation on Cardiopulmonary Toxicity and Survival for Locally Advanced Non-Small Cell Lung Cancer: Reanalysis of NRG Oncology/RTOG 0617 With Centrally Contoured Cardiac Structures E.M. Gore,1 C. Hu,2 V. Bar Ad,3 C.G. Robinson,4 M.D. Wheatley,5 J.A. Bogart,6 Y. Garces,7 V.S. Kavadi,8 S. Narayan,9 P. Iyengar,10 J.S. Witt,11 J.W. Welsh,12 C.D. Koprowski,13 J.M. Larner,14 and J.D. Bradley15; 1Medical College of Wisconsin and Clement J. Zablocki VA Medical Center Department of Radiation Oncology, Milwaukee, WI, 2NRG Oncology, Philadelphia, PA, 3Sidney Kimmel Medical College of Thomas Jefferson University, Philadelphia, PA, 4 Washington University in St. Louis, Department of Radiation Oncology, St. Louis, MO, 5Mercy Medical Group, Sacramento, CA, United States, 6 SUNY Upstate Medical University, Syracuse, NY, 7Mayo Clinic, Rochester, MN, 8Texas Oncology, Fort Worth, TX, United States, 9St. Joseph Mercy Hospital, Ypsilanti, MI, 10University of Texas Southwestern Medical Center, Dallas, TX, 11Washinginton University School of Medicine, St Louis, MO, 12Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 13 Christiana Care Health System, Newark, DE, United States, 14University of Virginia, Department of Radiation Oncology, Charlottesville, VA, 15 Washington University School of Medicine, St. Louis, MO Purpose/Objective(s): RTOG 0617 showed high dose RT had a greater risk of death than standard dose. Higher heart dose was associated with
Volume 96 Number 2S Supplement 2016 Author Disclosure: M. Hettich: None. F. Braun: None. G. Niedermann: None.
289 Unique Changes in the TCR Repertoire of Tumor-Infiltrating Lymphocytes Underlie the Synergy of Radiation Therapy With CTLA-4 Blockade K.A. Pilones,1 R. Emerson,2 S. Formenti,3 and S. Demaria1; 1Weill Cornell Medical College, New York, NY, 2Adaptive Biotechnologies, Seattle, WA, 3 Weill Cornell Medicine, New York, NY Purpose/Objective(s): Monoclonal antibodies (mAb) that target CTLA-4, a negative regulator of T-cell activation, have significantly extended survival of patients with solid malignancies. However, the number of responders remains low. We have previously shown in the 4T1 mouse breast tumor model that resistance to anti-CTLA-4 therapy can be overcome by concurrent local radiotherapy (RT) (Demaria et al Clin Cancer Res 2005). Multiple effects of RT on the tumor microenvironment contributed to its ability to sensitize tumors to anti-CTLA-4 treatment, including enhanced homing of effector T cells to the tumor and formation of stable immune synapses between CTL and tumor cells (Matsumura et al. J Immunol 2008; Ruocco et al. J Clin Invest 2012). Importantly, CD8 T cell responses to the endogenous tumor antigen gp70 were seen only in mice treated with the combination, consistent with the hypothesis that RT generates an in situ vaccine and CTLA-4 expands primed T cells. Materials/Methods: BALB/c mice were inoculated s.c. with 4T1 cells and thirteen days later, when tumors became palpable, randomly assigned to one of 4 treatment groups (N Z 5 mice/group): control, RT alone, antiCTLA-4 alone or RT + anti-CTLA-4. RT was given in 2 fractions of 12 Gy on days 13 and 14 post-tumor inoculation. Anti-CTLA-4 mAb (Clone 9H10) was given i.p. on days 15, 18, and 21. Tumors and sorted CD4+ and CD8+ T-cells were harvested on day 22 for high throughput sequencing of TCRb CDR3 regions performed using the ImmunoSEQ platform. Results: We observed distinct and non-overlapping effects of RT and immunotherapy on TIL repertoire. CTLA-4 blockade increased clonality and significantly expanded the top 5 most frequent clonotypes. On the other hand, RT increased TIL numbers and broadened their repertoire by selectively expanding the top 6-20 clonotypes. By comparing the exact nucleotide sequences from sorted TILs with those from paired tumor samples, oligoclonal expansion driven by RT + anti-CTLA-4 mAb was essentially driven by CD8+ T-cells. Significantly reduced diversity of the TCRb profile seen only with combination therapy is consistent with antigen-driven TIL clonal expansion. Conclusion: Overall, tumor rejection induced by RT + anti-CTLA-4 is associated with both quantitative and qualitative changes in TIL repertoire. Data suggests that a broader repertoire of tumor-specific T cells may be critical for tumor rejection, and is achieved by coupling the effects of RT, which promotes priming of T cells specific for endogenous tumor antigens, and CTLA-4 blockade, which drives expansion of selected clones. These results have important implications for the therapeutic synergy of radiation and CTLA-4 blockade. Author Disclosure: K.A. Pilones: None. R. Emerson: None. S. Formenti: Advisory committee; BMS, Smith Kline, Astra Zeneca, EISAI. Research Grant; Regeneron, Janssen Biotech, Eli Lilly. Speaker’s Bureau; Varian. S. Demaria: Consultant; Sanofi US Inc., Regeneron Pharmaceuticals, Inc.
290 Peritumoral IL-15 Potentiates Radiation-Induced Anti-Tumor Immunity K.A. Pilones,1 S. Formenti,2 and S. Demaria1; 1Weill Cornell Medical College, New York, NY, 2NewYork-Presbyterian Hospital/Weill Cornell Medicine, New York, NY Purpose/Objective(s): Radiotherapy (RT) can induce T cell-mediated anti-tumor immune responses by multiple mechanisms but is often unable to overcome immunosuppression in the tumor microenvironment. The common gamma-chain cytokines interleukin (IL)-2 and IL-15 promote the
proliferation of activated T cells and, therefore, are prime agents for immunotherapy strategies aimed at sustaining anti-tumor T cell responses. The benefits of high dose IL-2, however, are undermined by serious toxicity and by regulatory T cell (Treg) stimulation. In contrast, IL-15 is well-tolerated and lacks Treg stimulatory activity, making it an attractive candidate for testing in combination with RT. Here we tested the hypothesis that IL-15 strengthens the pro-immunogenic effect of local RT to potentiate a durable anti-tumor immune response. Materials/Methods: The poorly immunogenic mouse TSA breast cancer cells were implanted s.c. in syngeneic BALB/c mice and randomly assigned to one of 4 treatment groups when tumors reached 5mm average diameters: control, RT, IL-15, or RT + IL-15. RT was delivered locally in 8 Gy fractions on days 13, 14, and 15. IL-15 (2 mg/mouse) was administered s.c. peritumorally daily for 10 days starting on day 12. Mice were followed for tumor growth. A parallel experiment was done to characterize tumorinfiltrating lymphocytes (TILs) at the end of treatment (day 22). Results: Low dose IL-15 given peritumorally as a monotherapy induced marginal tumor growth control and had no effect on survival (median survival Z 45 days compared to 76 days for control). Local RT significantly delayed tumor growth (P < 0.05 compared to control) and improved survival (median Z 76 days, P < 0.05). However, highest survival advantage was seen in mice given IL-15 + RT (median Z 102 days, P < 0.05 compared to all groups) with 1 of 6 mice showing complete tumor rejection and development of anamnestic response against tumor re-challenge. Analysis of TILs showed marked infiltration of CD8+ T cells expressing the activation marker CD137 (35.3% in RT + IL-15 versus 5.90% In control, P < 0.05) while the increase was modest with each monotherapy (18.8% in RT, 20.7% in IL-15, P < 0.05 compared to control). In addition, we found a significant increase in the ratio of effector CD4+ T-cells to Tregs (2.5 in RT + IL-15 versus 0.78 in control, P < 0.05) whereas monotherapy had no effect (1.14 in RT, 0.96 in IL-15). Additionally, IL-15 treatment, regardless of RT, led to significant intratumoral enrichment of NKp46+ NK cells. Conclusion: Overall these results support the rational combination of low dose intratumoral IL-15 with local RT. We are currently elucidating the mechanisms involved in pre-clinical models in preparation for future testing in patients. Author Disclosure: K.A. Pilones: None. S. Formenti: Research Grant; Regeneron, Janssen, Eli Lilly. Speaker’s Bureau; Varian. Advisory Board; BMS, Smith Kline, Astra Zeneca. S. Demaria: None.
291 Impact of Incidental Cardiac Radiation on Cardiopulmonary Toxicity and Survival for Locally Advanced Non-Small Cell Lung Cancer: Reanalysis of NRG Oncology/RTOG 0617 With Centrally Contoured Cardiac Structures E.M. Gore,1 C. Hu,2 V. Bar Ad,3 C.G. Robinson,4 M.D. Wheatley,5 J.A. Bogart,6 Y. Garces,7 V.S. Kavadi,8 S. Narayan,9 P. Iyengar,10 J.S. Witt,11 J.W. Welsh,12 C.D. Koprowski,13 J.M. Larner,14 and J.D. Bradley15; 1Medical College of Wisconsin and Clement J. Zablocki VA Medical Center Department of Radiation Oncology, Milwaukee, WI, 2NRG Oncology, Philadelphia, PA, 3Sidney Kimmel Medical College of Thomas Jefferson University, Philadelphia, PA, 4 Washington University in St. Louis, Department of Radiation Oncology, St. Louis, MO, 5Mercy Medical Group, Sacramento, CA, United States, 6 SUNY Upstate Medical University, Syracuse, NY, 7Mayo Clinic, Rochester, MN, 8Texas Oncology, Fort Worth, TX, United States, 9St. Joseph Mercy Hospital, Ypsilanti, MI, 10University of Texas Southwestern Medical Center, Dallas, TX, 11Washinginton University School of Medicine, St Louis, MO, 12Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 13 Christiana Care Health System, Newark, DE, United States, 14University of Virginia, Department of Radiation Oncology, Charlottesville, VA, 15 Washington University School of Medicine, St. Louis, MO Purpose/Objective(s): RTOG 0617 showed high dose RT had a greater risk of death than standard dose. Higher heart dose was associated with