746 Checkpoint inhibitor sensitization and systemic anti-tumor immunity generated by local gene delivery nanoparticles

<h3>Background</h3> Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, but novel treatment strategies are needed for resistant or recurrent cases. We previously demonstrated that polymeric nanoparticles (NPs) delivering plasmids encoding 4-1BBL and IL-12 elicit productive immune responses in vivo.^1–3 Here, we assess 4-1BBL/IL-12 NPs in two additional murine cancer models with varying degrees of sensitivity to ICI to investigate their ability to synergize with ICI and generate systemic immunity. <h3>Methods</h3> MC38 colorectal cancer was established on the flank or E0771 breast carcinoma was established on the mammary fat pad in C57BL/6J mice. Animals were untreated or received intratumoral injections of control NPs or 4-1BBL/IL-12 NPs on days 9, 11, 16, and 18. A subset of animals also received systemic anti-PD1 on the same days and twice a week for four additional weeks. Mice were sacrificed when surface area reached 200 mm² or for moribund states. In E0771, all animals with fully regressed primary tumors were rechallenged with a second tumor on the opposite flank. Significance was assessed via log-rank (Mantel-Cox) test with Bonferroni correction, or two-way ANOVA comparing conditions to no treatment or anti-PD1 only. <h3>Results</h3> In MC38, 4-1BBL/IL-12 NPs alone or with anti-PD1 led to modest decreases in tumor size (figure 1A-B), though survival was not improved with NPs alone (figure 2A). Systemic anti-PD1 led to a slight increase in median survival compared to no treatment (37 versus 29 days, figure 2A-B), but combined with 4-1BBL/IL-12 NPs led to 4/7 animals with fully regressed tumors (figure 2B). In E0771, 4-1BBL/IL-12 NPs alone and with anti-PD1 slowed tumor growth significantly (figure 3A-B). 4-1BBL/IL-12 NPs alone led to significantly improved survival compared to untreated animals (p=0.0006) and 6/8 long-term tumor-free survivors (figure 4A), and anti-PD1 led to 4/8 long-term tumor-free survivors (figure 4B). Notably, 4-1BBL/IL-12 NPs with anti-PD1 led to 100% tumor-free survival (figure 4B). On day 83, all animals with fully regressed primary E0771 tumors were rechallenged with a tumor on the opposite flank. In all cases, the primary tumor did not recur, the flank tumor was rejected, and all mice remained tumor-free through day 151. <h3>Conclusions</h3> Here, we demonstrate that 4-1BBL/IL-12 NPs can sensitize resistant cancer to ICI. We further show that locally administered 4-1BBL/IL-12 NPs, with and without systemic ICI, can generate systemic immunity preventing establishment of distant tumor. Together, these results highlight the utility of these immunostimulatory NPs in generating potent anti-cancer responses <i>in vivo</i>. <h3>References</h3> Tzeng SY, Patel KK, Wilson DR, Meyer RA, Rhodes KR, Green JJ. In situ genetic engineering of tumors for long-lasting and systemic immunotherapy. <i>Proc Natl Acad Sci U S A</i>. 2020;<b>117</b>(8):4043–52. Luly KM, Green JJ, Sunshine JC, Tzeng SY. Biomaterial-mediated genetic reprogramming of merkel cell carcinoma and melanoma leads to targeted cancer cell killing in vitro and in vivo. <i>ACS Biomater Sci Eng [Internet]</i>. 2023 Nov 13;<b>9</b>(11):6438–50. Luly K, Green J, Tzeng S, Sunshine J. 222 Genetic reprogramming of merkel cell carcinoma and melanoma leads to increased MHC-I expression and antitumor immune activation in vitro and in vivo. <i>J Immunother Cancer [Internet]</i>. 2021 Nov;<b>9</b>(Suppl 2):A235–6. <h3>Ethics Approval</h3> All animal work was conducted within the guidelines of the Johns Hopkins Animal Care and Use Committee under approved protocol numbers MO18M388/MO21M384.