Evading immune evasion via reprograming of the tumor microenvironment by radiotherapy and simultaneous targeting of TGF-β/PD-L1

Tumor immunogenicity enhanced by radiotherapy (RT), is often counterbalanced by immune evasive mechanisms and tissue remodeling effects via upregulation of transforming growth factor-β (TGF-β) and programmed cell death-ligand 1 (PD-L1). We report that bintrafusp alfa (BA), a bifunctional fusion protein that simultaneously inhibits TGF-β and PD-L1, is a favorable combination partner for RT in eradicating multiple treatment-resistant tumor models. Beneficial effects of BA+RT (BART) are partly attributed to counterbalancing undesired RT effects and local tumor microenvironment reprograming, leading to reconstitution of tumor immunity and regression of spontaneous lung metastases. Intriguingly, BA, but neither TGF-β trap nor anti–PD-L1 alone, attenuates late-stage RT-induced lung fibrosis. Single cell transcriptome show TGF-β expression is confined to PD-L1+ endothelium and M2/lipofibroblast-like cells. Hence, superior effects of BA could be attributed to its ability to trap TGF-β to relevant PD-L1+ compartments. Here, we provide rationales that BART resensitizes tumors and broadens the therapeutic window. 4T1 cells, 0.5 × 105, were inoculated im in the thigh of BALB/c mice on day -6, mice were treated on day 0 (n = 9 mice/group) and euthanized on day 6. To assess the combination of radiation with Bintrafusp alfa (BA), mice were randomized into the following treatment groups: isotype control (400 μg), RT (exact dose and schedule provided in the figure legends) + isotype control, BA (492 μg), or BA + RT (BART). For transcriptome analysis of 4T1 tumors, total RNA was extracted and RNA sequencing (RNAseq) analyses were performed using a targeted mouse Immuno-Oncology panel (RMM-009Z, Qiagen) and a Qiaseq targeted RNA custom panel (Qiagen) with RT2 Profiler PCR arrays containing murine genes related to TGF-β signaling targets (PAMM-235Z), DNA damage signaling pathway (PAMM-029Z), DNA repair (PAMM-042Z), and fibrosis (PAMM-120Z).