Optimizing Timing of Immunotherapy Improves Control of Tumors by Hypofractionated Radiation Therapy

Abstract

The anecdotal reports of promising results seen with immunotherapy and radiation in advanced malignancies have prompted several trials combining immunotherapy and radiation. However, the ideal timing of immunotherapy with radiation has not been clarified. Tumor bearing mice were treated with 20Gy radiation delivered only to the tumor combined with either anti-CTLA4 antibody or anti-OX40 agonist antibody. Immunotherapy was delivered at a single timepoint around radiation. Surprisingly, the optimal timing of these therapies varied. Anti-CTLA4 was most effective when given prior to radiation therapy, in part due to regulatory T cell depletion. Administration of anti-OX40 agonist antibody was optimal when delivered one day following radiation during the post-radiation window of increased antigen presentation. Combination treatment of anti-CTLA4, radiation, and anti-OX40 using the ideal timing in a transplanted spontaneous mammary tumor model demonstrated tumor cures. These data demonstrate that the combination of immunotherapy and radiation results in improved therapeutic efficacy, and that the ideal timing of administration with radiation is dependent on the mechanism of action of the immunotherapy utilized.

Fig 1. Anti-CTLA4 pretreatment optimizes tumor control by radiation therapy.

(a) Immunocompetent BALB/c mice bearing CT26 tumors were left untreated (NT) or treated with 250μg anti-CTLA4 on d7, d15 or d19. Select groups were additionally treated with 20Gy focal radiation (RT) on d14. (b) Individual tumor measurements from mice in these groups i) untreated or treated with ii) anti-CTLA4 d7; iii) RT 20Gy d14; iv) anti-CTLA4 d7+RT 20Gy d14; v) anti-CTLA4 d15+RT 20Gy d14; vi) anti-CTLA4 d19+RT 20Gy d14. (c) i) average tumor size and ii) overall survival. Representative experiment shown with n = 6 mice per group. Experiment replicated a minimum of two times.

Fig 2. Timing of immunotherapy is dependent on the mechanism of action.

(a) Immunocompetent BALB/c mice were challenged with CT26 in the flank and randomized to no further treatment (NT), 20Gy focal radiation therapy (RT) alone or along with administration of 250μg anti-OX40 on d7, d15 or d19. (b) Overall survival of mice following treatment. Data combined from 3 experiments, total n = 12–18 mice per group. (c) Immunocompetent C56BL/6 mice were left untreated (NT) or vaccinated with DEC205ova plus anti-CD40 (Vaccine) on d0. Matched groups of vaccinated mice were additionally treated with 250μg anti-OX40 on d1 or d7, or 250μg anti-CTLA4 on d1 or d7. Graph shows the percent of CD8 T cells that are specific for the immunodominant SIINFEKL peptide of Ova on (c) d7, (d) d14 or (e) over time. Key: NS = not significant; * = p<0.05; ** = p< 0.01. Each symbol represents one animal.

Fig 3

Effect of Treg depetion on tumor control by radiation therapy (a) i) Immunocompetent BALB/c mice bearing CT26 tumors were left untreated (NT) or treated with 250μg anti-CTLA4 clone 9D9 or UC10 on d7. Select groups were additionally treated with 20Gy focal radiation (RT) on d14. Graphs show ii) average tumor size and iii) overall survival. (b) Immunocompetent BALB/c mice bearing CT26 tumors were left i+iii) untreated (control) or treated with ii) 100μg anti-CD4 depleting antibody or iv) anti-CD25 depleting antibody. Representative flow cytometry plots show whole blood 1 day following control or antibody-depletion i+ii) gated on lymphocytes and stained for CD8 and CD4 or iii) and iv) gated on CD4⁺ lymphocytes and stained for CD4 and CD25. (c) i) Immunocompetent BALB/c mice bearing CT26 tumors were left untreated (NT) or treated with 100μg anti-CD4 or anti-CD25 on d7. Select groups were additionally treated with 20Gy focal radiation (RT) on d14. ii) Overall survival in all groups.

Fig 4. Combination immunotherapy and radiation therapy of spontaneous mammary tumors in immune competent mice.

(a) MMTV-PyMT tumors were harvested from approximately 100 day old female MMTV-PyMT⁺ mice, the tumor disrupted ex vivo and 1x10⁶ viable cells injected orthotopically into immunocompetent syngeneic FVB mice. (b) Mice with d14-d17 tumors underwent CT-guided radiation therapy (RT) using a Small Animal Radiation Research Platform and i) images used to place isocenters within individual mammary tumors and collimeters and beam angles designed to deliver focal radiation to the tumor and minimal dose to radiosensitive organs. ii) CT images were segmented by tissue density and this information used to predict dose delivery. Mice were also randomized to receive 250μg anti-CTLA4 immunotherapy 7d prior to RT, and 250μg anti-OX40 immunotherapy d1 and d4 following radiation. (c) Graphs show i) average tumor growth and ii) overall survival.