Restoration of p53 activity via intracellular protein delivery sensitizes triple negative breast cancer to anti-PD-1 immunotherapy

BACKGROUND: Although immune checkpoint inhibitors (ICIs) have been shown to yield promising therapeutic outcomes in a small subset of patients with triple negative breast cancer (TNBC), the majority of patients either do not respond or subsequently develop resistance. Recent studies have revealed the critical role of TP53 gene in cancer immunology. Loss or mutation of p53 in cancer cells has been found to promote their immune escape. Given the high mutation frequency of TP53 in TNBC cells, restoration of p53 function could be a potential strategy to overcome their resistance to anti-programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) therapy. Herein, we have assessed the use of Pos3Aa crystal-based platform to mediate the intracellular delivery of p53 protein to restore p53 activity in p53 null tumors and consequently augment anti-PD-1 activity. METHODS: The efficiency of Pos3Aa-p53 crystals in delivering p53 protein was evaluated using confocal imaging, immunofluorescence staining, flow cytometry and RNA-seq. The ability of Pos3Aa-p53 crystals to remodel tumor microenvironment was investigated by examining the markers of immunogenic cell death (ICD) and the expression of PD-L1, indoleamine 2,3-dioxygenase 1, tryptophan 2,3-dioxygenase 2 and type I interferon (IFN). Finally, both unilateral and bilateral 4T1 tumor mouse models were utilized to assess the efficacy of Pos3Aa-p53 crystal-mediated p53 restoration in enhancing the antitumor activity of ICIs. T cells in tumor tissues and spleens were analyzed, and the in vivo biosafety of the Pos3Aa-p53 crystal/anti-PD-1 antibody combination was also evaluated. RESULTS: Delivery of p53 protein into p53-null TNBC 4T1 cells via Pos3Aa-p53 crystals restored the p53 activity, and therefore led to the induction of ICD, activation of type I IFN signaling and upregulation of PD-L1 expression. Pos3Aa-p53 crystals significantly enhanced T cell infiltration and activation in 4T1 tumors, thereby sensitizing them to anti-PD-1 therapy. The combination of Pos3Aa-p53 crystals with anti-PD-1 antibody also induced a systemic antitumor immunity resulting in the inhibition of distal tumor growth with minimal toxicity. CONCLUSION: This study validates that p53 restoration can be an effective approach to overcome ICI resistance and demonstrates that intracellular delivery of p53 protein can be an efficient, safe and potentially universal strategy to restore p53 activity in tumors carrying TP53 mutation.