1121 Injectable hydrogel for intratumoral delivery localizes cytokine immunotherapy and eliminates murine breast tumors with systemic immune activity

<h3>Background</h3> Cytokine immunotherapies represent powerful approaches for manipulating the immune response in cancer, but their systemic toxicity limits their clinical application. Clinical interest in intratumoral delivery of immunotherapy to minimize systemic exposure while maximizing cytokine concentrations has increased in the recent decade. However, dense tumor architectures and high interstitial pressures rapidly exclude saline-based solutions via the needle track following intratumoral injection. To this end, we have developed an injectable delivery medium to effectively localize cytokines at the tumor. <h3>Methods</h3> XCSgel is a shear-thinning, self-healing, biocompatible, imageable hydrogel synthesized by cross-linking chitosan. To monitor local retention of injected immunotherapies, cytokines were fluorescently labeled, co-formulated in XCSgel, delivered subcutaneously in mice, and imaged over four weeks. To identify the lead cytokine candidate, cytokines IL-12, IL-15, IFNα2, and GM-CSF were co-formulated in XCSgel and delivered intratumorally in murine E0771 breast tumors. To evaluate the systemic uptake of cytokines following intratumoral injection, blood samples were taken over three days after the intratumoral delivery of IL-12 in XCSgel (XCSgel-IL-12). Anti-tumor efficacy of intratumorally delivered XCSgel-IL-12 was evaluated in four murine breast cancer models: (1) orthotopic E0771 tumor, (2) treated orthotopic E0771 tumor with an untreated abscopal E0771 tumor, (3) orthotopic mWnt tumor, and (4) orthotopic 4T1 tumor as a neoadjuvant to surgery. <h3>Results</h3> Imaging data revealed that XCSgel could maintain cytokine immunotherapy for over a month period <i>in vivo</i> (figure 1). The lead anti-tumor cytokine co-formulated in XCSgel was IL-12 (figure 2). Pharmacokinetic studies revealed tunable release of IL-12 contingent on XCSgel character after intratumoral delivery. IL-12 systemic circulation was significantly reduced using XCSgel as a delivery system. In the first E0771 study, a single injection with XCSgel-IL-12 eliminated 86% of tumors, with tumor rejection upon rechallenge. In the E0771 abscopal study, XCSgel-IL-12 eliminated both the treated orthotopic and untreated abscopal tumor in 67% of the mice (figure 3). XCSgel-IL-12 also eliminated 80% of the mWnt orthotopic tumors. Finally, neoadjuvant treatment with XCSgel-IL-12 prevented primary tumor recurrence in 4T1 tumors to date, with survival and metastasis studies ongoing. <h3>Conclusions</h3> XCSgel effectively localized and slowly released cytokine immunotherapies. XCSgel-IL-12 reduced systemic exposure to IL-12 following intratumoral delivery. A single injection of XCSgel-IL-12 eliminated both primary and abscopal E0771 tumors and mWnt tumors, with induced immune memory. It prevented 4T1 tumor recurrence. Given its ability to induce systemic antitumor immunity, XCSgel deserves further consideration as a neoadjuvant treatment prior to breast surgery. <h3>Acknowledgements</h3> This work is supported by the National Science Foundation Graduate Research Fellowship. <h3>Ethics Approval</h3> All experiments involving laboratory animals were approved by Institutional Animal Care and Use Committee at North Carolina State University (protocol numbers 19-795, 22-414, and 23-126).