An Injectable Chitosan Hydrogel Localizes and Tunably Releases Immunotherapeutics Intratumorally Eliminating Both Treated and Abscopal Murine Triple Negative Breast Tumors

Systemic delivery of immunotherapy is dose-limited and often causes serious immune-related adverse events. Intratumoral injections can reduce systemic immunotoxicities and increase immunotherapy concentrations within a tumor. However, high pressures associated with direct tumor injection limits injectate retention, as low viscosity, saline-based solutions rapidly leak out of tumors. Viscoelastic solids, such as hydrogels, can improve local retention of co-formulated immunotherapies and provide sustained delivery. Prior work demonstrated that a chitosan-based hydrogel, XCSgel, was shear-thinning, self-healing, injectable, biocompatible, and clinically imageable. Here, we investigated XCSgel as a localized intratumoral delivery platform in the context of murine models of orthotopic triple-negative breast cancer. The intratumoral retention of immunotherapeutics co-formulated in XCSgel was characterized both ex vivo and in vivo via fluorescence imaging. Histopathological responses to intratumoral injections of XCSgel alone were scored by a veterinary pathologist. Initial antitumor studies evaluated a range of antitumor cytokines co-formulated with XCSgel. Subsequent antitumor and rechallenge studies evaluated the efficacy of a single intratumoral injection of interleukin-12 (IL-12) co-formulated in XCSgel (XCSgel-IL12) to control the growth of primary and abscopal tumors while inducing protective immunity. Pharmacokinetics studies quantified the systemic dissemination of IL-12 and consequent production of interferon-gamma following intratumoral injection with XCSgel co-formulation. Spectral flow cytometry was used to document changes in the tumor-immune microenvironment (TIME). XCSgel resisted tumor leakage and slowly released three model cytokines. XCSgel could be tuned for faster or slower release of embedded therapeutics. XCSgel-IL12 outperformed XCSgel formulations with other commonly used antitumor cytokines. A single injection of XCSgel-IL12 eliminated 86% E0771 and 20% mWnt orthotopic primary TNBC tumors. Mice rendered tumor-free resisted a live tumor challenge. XCSgel-IL12 also eliminated 67% untreated abscopal E0771 tumors. XCSgel-IL12 induced profound changes to the TIME, including a 3-fold reduction in the frequency of exhausted CD8+ T cells and a 3.2-fold increase in activated, proliferating CD8+ T cells. XCSgel is a promising localized delivery platform well-suited to enhance the retention and antitumor activity of potent immunotherapeutics. A single injection of XCSgel-IL12 can eliminate both primary and abscopal solid tumors, indicating that systemic immunotherapy may not be required for systemic control of cancer. ### Competing Interest Statement The authors have declared no competing interest.