Comparison of Two Triple Therapy Regimens for Enhancing the Abscopal Effect in Mice

Abstract

Localized radiotherapy (RT) can cause a T cell-mediated abscopal effect on non-irradiated tumor lesions, especially in combination with immune checkpoint blockade (ICB). However, this effect is still clinically rare and improvements are highly desirable. We here compared two triple therapy regimens (RT+⍺PD1+⍺CTLA4 vs. RT+⍺PD1+IL2/⍺IL2 complexes (IL2c)) for their efficacy to improve systemic (abscopal) anti-tumor effects. In mice bearing bilateral subcutaneous C51 colon carcinoma tumors, the primary tumor was irradiated with two fractions of 8 Gy. ⍺PD1 and ⍺CTLA4 were given concomitantly and weekly thereafter; IL2c was given i.p. for 3 consecutive days. Besides tumor size and survival, tumor-specific CD8+ T cells were determined flow cytometrically using MHC-I tetramers and various antibodies. In addition, isolated TILs were cultured with PMA, ionomycin, and Brefeldin A in vitro to assess polyfunctionality (based on cytokine production) of CD8+ and CD4+ TILs. The abscopal effect was significantly stronger in mice treated with the ⍺CTLA4-containing triple combination (n = 13) than in mice treated with the IL2c-containing triple combination (n = 9) (p<0,05), and compared to mice treated with RT+⍺PD1 (n = 8) (p<0,05) or RT+⍺CTLA4 (n = 10) (p<0,05), respectively. The IL2c triple combination induced the abscopal effect better than RT+⍺PD1 (p<0,05). The ⍺CTLA4 triple therapy improved survival and resulted in complete cures of 8/13 mice. In mice treated with ⍺CTLA4-containing triple therapy, control of the irradiated tumor was partially dependent and that of the non-irradiated tumor was strongly dependent on T cells, primarily CD8 T cells but also CD4 T cells. With ⍺CTLA4 triple treatment (n = 9), the frequency and absolute numbers of polyfunctional TNF-⍺+IFN-γ+ AH-1 tumor-specific CD8+ T cells, IFN-γ+IL2+ AH-1 tumor-specific CD8+ T cells, TNF-⍺+IFN-γ+ CD4+ and IFN-γ+IL2+ CD4+ effector T cells were higher than with the IL2c triple combination (n = 9) (p<0,05), particularly in the non-irradiated tumors. RT+⍺PD1+⍺CTLA4 triple treatment induced more cytotoxic effector TILs than RT+⍺PD1+IL2c triple treatment, enhancing the abscopal effect and inducing a high abscopal cure rate. More experiments underway to reveal mechanisms.