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Ideal Timing of Immunotherapy With Radiation in Murine Tumor Models
S58
International Journal of Radiation Oncology Biology Physics
218 mm2 (p Z 0.028). Similarly, anti-PD-1 therapy with RT resulted in significantly longer survival than PD-1 blockade therapy alone (29 days vs 19 days; p Z 0.021). The mean tumor size on day 19 at the primary site was 103 mm2 (anti-PD-1) and 26.2 mm2 (anti-PD-1 + RT) (p Z 0.043). Secondary tumor growth was 197.8 mm2 and 88.4 mm2 for PD-1 therapy alone or with RT (p Z 0.037). RT alone to the primary tumor had no suppressive effect on the distant tumor growth. Flow analysis confirmed the presence of anti-melanoma PD-1+ CD8+ effector T cells in both primary and distant tumor sites. Conclusions: In our preclinical models, the addition of RT to PD-1 blockade (PD-1 KO and anti-PD-1 antibody) suppressed tumor growth at both radiated and non-radiated sites, and successfully recapitulated the clinical abscopal effect of RT. Two mice developed anti-melanoma immunity and did not exhibit melanoma growth upon rechallenge. These results provide a rationale for testing RT and PD-1 blockade combination therapy in a clinical setting. Author Disclosure: S.S. Park: A. Employee; Mayo Clinic. E. Research Grant; RSNA Research Scholar Grant Recipient. H. Dong: A. Employee; Mayo Clinic. W. Zhao: A. Employee; Mayo Clinic. M.P. Grams: A. Employee; Mayo Clinic. X. Liu: A. Employee; Mayo Clinic. S.M. Harrington: A. Employee; Mayo Clinic. K.M. Furutani: A. Employee; Mayo Clinic. C.J. Krco: A. Employee; Mayo Clinic. K.R. Olivier: A. Employee; Mayo Clinic. S.N. Markovic: A. Employee; Mayo Clinic. E.D. Kwon: A. Employee; Mayo Clinic.
tumor (p < 0.05). However, in each case, all mice that were cured of their tumors utilizing immunotherapy in combination with radiation resulted in long-term tumor memory, as mice were resistant to tumor rechallenge. Conclusions: These data demonstrate that the combination of immunotherapy and hypofractionated radiation results in improved therapeutic efficacy; and that the ideal timing of administration with radiation is dependent on the type of immunotherapy utilized with aCTLA optimally delivered prior to radiation and aOX40 optimally delivered following radiation. Author Disclosure: K. Young: None. B. Cottam: None. J.R. Baird: None. M.J. Gough: None. M. Crittenden: None.
120 Ideal Timing of Immunotherapy With Radiation in Murine Tumor Models K. Young,1 B. Cottam,2 J.R. Baird,2 M.J. Gough,2 and M. Crittenden2,3; 1 Oregon Health & Science University, Portland, OR, 2Earle A. Chiles Research Institute, Providence Portland Medical Center, Portland, OR, 3 The Oregon Clinic, Portland, OR Purpose/Objective(s): Multimodality therapy has become the mainstay of oncologic treatment, including surgery, chemotherapy, and radiation therapy with the goal of tumor debulking and cytotoxicity. Recently, a fourth modality has been increasingly utilized: immunotherapy. Immune evasion has long been recognized as a critical step in tumorigenesis. Immunotherapy aims to target pathways of immune evasion, and activate an individual’s immune response to their cancer. Traditional cytotoxic therapies aim to target proliferating tumor cells, inducing cellular damage rendering them incompetent for replication. However, an under-recognized benefit to radiation is its ability to expose tumor antigen and create a focal inflammatory response. This study was designed to test the hypothesis that the optimal timing of immunotherapy in conjunction with hypofractionated radiation depends on the immunotherapy agent being utilized. Materials/Methods: The aCTLA4 antibody or aOX40 agonist antibody (250 ug) were administered either before or after a single dose of 20 Gy radiation to a subcutaneous CT26 colorectal adenocarcinoma. Endpoints included tumor growth, survival, immune infiltrate, and drug permeability. Results: We demonstrate that aCTLA4 antibody administration results in decreased T regulatory, increased CD4 T, and decreased myeloid cell tumor infiltrate in a mouse colorectal cancer model (p < 0.05). Hypofractionated radiation therapy delivered to this altered tumor environment it results in 100% tumor cure rather than tumor growth delay followed by outgrowth in control animals treated with radiation alone (Median survival not reached versus 47 days, p < 0.01). Administration of aCTLA4 following hypofractionated radiation resulted in only 50% tumor clearance, which remains a significant improvement over radiation alone and antibody alone (Median survival 91 days vs 47 days vs 32 days, p < 0.01). However, the optimum timing is dependent on the immunotherapy reagent delivered. Administration of an OX40 agonist antibody resulted in an increase in activated CD8 T cells (p < 0.05) and was optimal when delivered one day following hypofractionated radiation (Median survival not reached versus 50 days with RT alone, p < 0.05). The differing mechanism may be due to aOX40 pretreatment resulting in IFNg dependent decreased drug penetrance and increased tumor hypoxia as a result of inflammation in the
121 Radiation Therapy Sensitizes a Poorly Immunogenic Breast Cancer to PD-1 Blockade K.A. Pilones,1 A. Joseph,1 R. Vatner,2 S. Formenti,1 and S. Demaria1; 1 New York University School of Medicine, New York, NY, 2New York University Medical Center, New York, NY Purpose/Objective(s): We have shown that radiation therapy (RT) converts poorly immunogenic tumors resistant to antibodies (mAbs) against CTLA-4 into susceptible ones (Demaria et al). In part, the synergy between RT and anti-CTLA-4 was due to RT-induced upregulation on cancer cells of molecules that facilitate the formation of an immune synapse between target and effector CD8 T cells activated by treatment (Ruocco et al). Programmed death-1 (PD-1) is a checkpoint receptor upregulated on T cells shortly after activation and expressed at high levels on exhausted T cells. Anti-PD-1 mAbs have shown marked clinical activity in some cancer patients but the majority of patients do not respond. Here we tested the hypothesis that RT can sensitize poorly immunogenic tumors to immunotherapy targeting PD-1. Materials/Methods: BALB/c mice were inoculated s.c. with the syngeneic poorly immunogenic TSA breast cancer cells. When tumors became palpable mice were randomly assigned to one of 4 treatment groups: control, RT, anti-PD-1 mAb and RT + anti-PD-1 mAb. RT was delivered exclusively to the primary tumor in 8 Gy fractions on days 13, 14, and 15 post-tumor inoculation. PD-1 blocking mAb RMP1-14 was given on day 15 and every 4 days thereafter, and mice were followed for tumor growth. In a separate experiment, mice were euthanized on day 20 to characterize tumor-infiltrating lymphocytes and development of CD8+ T cells specific for the tumor epitope AH1 by pentamer analysis. Results: TSA tumors were resistant to PD-1 blockade. RT significantly delayed tumor growth (p < 0.01), but tumor regression was seen only in 1 of 6 mice. In contrast, all mice given RT + RMP1-14 completely rejected tumors by day 25. Splenic AH1-specific CD8+ T-cells were markedly increased (4.6%) with RT + RMP1-14 compared to control (1.7%) or RMP1-14 (1.8%) or RT (2.9%) treated mice (p < 0.05). Compared to controls, RT-treated mice showed a significant increase in CD8+ T cells expressing high levels of PD-1 (CD8+ PD-1hi) (67% vs 36%, p < 0.01). PD-1 ligands PDL-1 and PDL-2 were also upregulated by RT on TSA cells and tumor-infiltrating myeloid cells, suggesting that PD-1 interaction with its ligands may limit activity of anti-tumor T cells. Anti-PD-1 also significantly decreased PD-1hi exhausted CD8+ T cells (14% RMP1-14 versus 36% control, p < 0.01; and 30% RT + RMP1-14 versus 67% RT, p < 0.01). In contrast, CD8+ T cells expressing activation markers CD69 and CD137 and low levels of PD-1 were increased significantly in tumors of mice treated with RT (64%) compared to control (42%) (p < 0.001) irrespective of the treatment with RMP1-14. Conclusions: Results suggest that anti-PD-1 mAb selectively decreases exhausted CD8+PD-1hi TILs, while enhancing the RT-elicited priming and activation of tumor-specific CD8+ T cells and support testing this combination in the clinic. Acknowledgment: This research was supported by the Breast Cancer Alliance (Exceptional Project Award). Author Disclosure: K.A. Pilones: None. A. Joseph: None. R. Vatner: None. S. Formenti: None. S. Demaria: None.
International Journal of Radiation Oncology Biology Physics
218 mm2 (p Z 0.028). Similarly, anti-PD-1 therapy with RT resulted in significantly longer survival than PD-1 blockade therapy alone (29 days vs 19 days; p Z 0.021). The mean tumor size on day 19 at the primary site was 103 mm2 (anti-PD-1) and 26.2 mm2 (anti-PD-1 + RT) (p Z 0.043). Secondary tumor growth was 197.8 mm2 and 88.4 mm2 for PD-1 therapy alone or with RT (p Z 0.037). RT alone to the primary tumor had no suppressive effect on the distant tumor growth. Flow analysis confirmed the presence of anti-melanoma PD-1+ CD8+ effector T cells in both primary and distant tumor sites. Conclusions: In our preclinical models, the addition of RT to PD-1 blockade (PD-1 KO and anti-PD-1 antibody) suppressed tumor growth at both radiated and non-radiated sites, and successfully recapitulated the clinical abscopal effect of RT. Two mice developed anti-melanoma immunity and did not exhibit melanoma growth upon rechallenge. These results provide a rationale for testing RT and PD-1 blockade combination therapy in a clinical setting. Author Disclosure: S.S. Park: A. Employee; Mayo Clinic. E. Research Grant; RSNA Research Scholar Grant Recipient. H. Dong: A. Employee; Mayo Clinic. W. Zhao: A. Employee; Mayo Clinic. M.P. Grams: A. Employee; Mayo Clinic. X. Liu: A. Employee; Mayo Clinic. S.M. Harrington: A. Employee; Mayo Clinic. K.M. Furutani: A. Employee; Mayo Clinic. C.J. Krco: A. Employee; Mayo Clinic. K.R. Olivier: A. Employee; Mayo Clinic. S.N. Markovic: A. Employee; Mayo Clinic. E.D. Kwon: A. Employee; Mayo Clinic.
tumor (p < 0.05). However, in each case, all mice that were cured of their tumors utilizing immunotherapy in combination with radiation resulted in long-term tumor memory, as mice were resistant to tumor rechallenge. Conclusions: These data demonstrate that the combination of immunotherapy and hypofractionated radiation results in improved therapeutic efficacy; and that the ideal timing of administration with radiation is dependent on the type of immunotherapy utilized with aCTLA optimally delivered prior to radiation and aOX40 optimally delivered following radiation. Author Disclosure: K. Young: None. B. Cottam: None. J.R. Baird: None. M.J. Gough: None. M. Crittenden: None.
120 Ideal Timing of Immunotherapy With Radiation in Murine Tumor Models K. Young,1 B. Cottam,2 J.R. Baird,2 M.J. Gough,2 and M. Crittenden2,3; 1 Oregon Health & Science University, Portland, OR, 2Earle A. Chiles Research Institute, Providence Portland Medical Center, Portland, OR, 3 The Oregon Clinic, Portland, OR Purpose/Objective(s): Multimodality therapy has become the mainstay of oncologic treatment, including surgery, chemotherapy, and radiation therapy with the goal of tumor debulking and cytotoxicity. Recently, a fourth modality has been increasingly utilized: immunotherapy. Immune evasion has long been recognized as a critical step in tumorigenesis. Immunotherapy aims to target pathways of immune evasion, and activate an individual’s immune response to their cancer. Traditional cytotoxic therapies aim to target proliferating tumor cells, inducing cellular damage rendering them incompetent for replication. However, an under-recognized benefit to radiation is its ability to expose tumor antigen and create a focal inflammatory response. This study was designed to test the hypothesis that the optimal timing of immunotherapy in conjunction with hypofractionated radiation depends on the immunotherapy agent being utilized. Materials/Methods: The aCTLA4 antibody or aOX40 agonist antibody (250 ug) were administered either before or after a single dose of 20 Gy radiation to a subcutaneous CT26 colorectal adenocarcinoma. Endpoints included tumor growth, survival, immune infiltrate, and drug permeability. Results: We demonstrate that aCTLA4 antibody administration results in decreased T regulatory, increased CD4 T, and decreased myeloid cell tumor infiltrate in a mouse colorectal cancer model (p < 0.05). Hypofractionated radiation therapy delivered to this altered tumor environment it results in 100% tumor cure rather than tumor growth delay followed by outgrowth in control animals treated with radiation alone (Median survival not reached versus 47 days, p < 0.01). Administration of aCTLA4 following hypofractionated radiation resulted in only 50% tumor clearance, which remains a significant improvement over radiation alone and antibody alone (Median survival 91 days vs 47 days vs 32 days, p < 0.01). However, the optimum timing is dependent on the immunotherapy reagent delivered. Administration of an OX40 agonist antibody resulted in an increase in activated CD8 T cells (p < 0.05) and was optimal when delivered one day following hypofractionated radiation (Median survival not reached versus 50 days with RT alone, p < 0.05). The differing mechanism may be due to aOX40 pretreatment resulting in IFNg dependent decreased drug penetrance and increased tumor hypoxia as a result of inflammation in the
121 Radiation Therapy Sensitizes a Poorly Immunogenic Breast Cancer to PD-1 Blockade K.A. Pilones,1 A. Joseph,1 R. Vatner,2 S. Formenti,1 and S. Demaria1; 1 New York University School of Medicine, New York, NY, 2New York University Medical Center, New York, NY Purpose/Objective(s): We have shown that radiation therapy (RT) converts poorly immunogenic tumors resistant to antibodies (mAbs) against CTLA-4 into susceptible ones (Demaria et al). In part, the synergy between RT and anti-CTLA-4 was due to RT-induced upregulation on cancer cells of molecules that facilitate the formation of an immune synapse between target and effector CD8 T cells activated by treatment (Ruocco et al). Programmed death-1 (PD-1) is a checkpoint receptor upregulated on T cells shortly after activation and expressed at high levels on exhausted T cells. Anti-PD-1 mAbs have shown marked clinical activity in some cancer patients but the majority of patients do not respond. Here we tested the hypothesis that RT can sensitize poorly immunogenic tumors to immunotherapy targeting PD-1. Materials/Methods: BALB/c mice were inoculated s.c. with the syngeneic poorly immunogenic TSA breast cancer cells. When tumors became palpable mice were randomly assigned to one of 4 treatment groups: control, RT, anti-PD-1 mAb and RT + anti-PD-1 mAb. RT was delivered exclusively to the primary tumor in 8 Gy fractions on days 13, 14, and 15 post-tumor inoculation. PD-1 blocking mAb RMP1-14 was given on day 15 and every 4 days thereafter, and mice were followed for tumor growth. In a separate experiment, mice were euthanized on day 20 to characterize tumor-infiltrating lymphocytes and development of CD8+ T cells specific for the tumor epitope AH1 by pentamer analysis. Results: TSA tumors were resistant to PD-1 blockade. RT significantly delayed tumor growth (p < 0.01), but tumor regression was seen only in 1 of 6 mice. In contrast, all mice given RT + RMP1-14 completely rejected tumors by day 25. Splenic AH1-specific CD8+ T-cells were markedly increased (4.6%) with RT + RMP1-14 compared to control (1.7%) or RMP1-14 (1.8%) or RT (2.9%) treated mice (p < 0.05). Compared to controls, RT-treated mice showed a significant increase in CD8+ T cells expressing high levels of PD-1 (CD8+ PD-1hi) (67% vs 36%, p < 0.01). PD-1 ligands PDL-1 and PDL-2 were also upregulated by RT on TSA cells and tumor-infiltrating myeloid cells, suggesting that PD-1 interaction with its ligands may limit activity of anti-tumor T cells. Anti-PD-1 also significantly decreased PD-1hi exhausted CD8+ T cells (14% RMP1-14 versus 36% control, p < 0.01; and 30% RT + RMP1-14 versus 67% RT, p < 0.01). In contrast, CD8+ T cells expressing activation markers CD69 and CD137 and low levels of PD-1 were increased significantly in tumors of mice treated with RT (64%) compared to control (42%) (p < 0.001) irrespective of the treatment with RMP1-14. Conclusions: Results suggest that anti-PD-1 mAb selectively decreases exhausted CD8+PD-1hi TILs, while enhancing the RT-elicited priming and activation of tumor-specific CD8+ T cells and support testing this combination in the clinic. Acknowledgment: This research was supported by the Breast Cancer Alliance (Exceptional Project Award). Author Disclosure: K.A. Pilones: None. A. Joseph: None. R. Vatner: None. S. Formenti: None. S. Demaria: None.