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Focal ablation technologies are routinely used in the clinical management of inoperable solid tumors but they often result in incomplete ablations leading to high recurrence rates. Adjuvant therapies, capable of safely eliminating residual tumor cells, are therefore of great clinical interest. Interleukin-12 (IL-12) is a potent antitumor cytokine that can be localized intratumorally through coformulation with viscous biopolymers, including chitosan (CS) solutions. The objective of this research was to determine if localized immunotherapy with a CS/IL-12 formulation could prevent tumor recurrence after cryoablation (CA). Tumor recurrence and overall survival rates were assessed. Systemic immunity was evaluated in spontaneously metastatic and bilateral tumor models. Temporal bulk RNA sequencing was performed on tumor and draining lymph node (dLN) samples. In multiple murine tumor models, the addition of CS/IL-12 to CA reduced recurrence rates by 30–55%. Altogether, this cryo-immunotherapy induced complete durable regression of large tumors in 80–100% of treated animals. Additionally, CS/IL-12 prevented lung metastases when delivered as a neoadjuvant to CA. However, CA plus CS/IL-12 had minimal antitumor activity against established, untreated abscopal tumors. Adjuvant anti-PD-1 therapy delayed the growth of abscopal tumors. Transcriptome analyses revealed early immunological changes in the dLN, followed by a significant increase in gene expression associated with immune suppression and regulation. Cryo-immunotherapy with localized CS/IL-12 reduces recurrences and enhances the elimination of large primary tumors. This focal combination therapy also induces significant but limited systemic antitumor immunity.

BACKGROUND: Intravesical administration of interleukin 12 (IL-12) co-formulated with the biopolymer, chitosan (CS/IL-12), has demonstrated remarkable antitumor activity against preclinical models of bladder cancer. However, given historical concerns regarding severe toxicities associated with systemic IL-12 administration in clinical trials, it is important to evaluate the safety of intravesical CS/IL-12 prior to clinical translation. OBJECTIVE: To evaluate the pharmacokinetics as well as the local and systemic toxicities of intravesical CS/IL-12 immunotherapy in laboratory mice. METHODS: Local inflammatory responses in mouse bladders treated with intravesical IL-12 or CS/IL-12 were assessed via histopathology. Serum cytokine levels following intravesical and subcutaneous (s.c.) administrations of IL-12 or CS/IL-12 in laboratory mice were compared. Systemic toxicities were evaluated via body weight and liver enzyme levels. RESULTS: Intravesical IL-12 and CS/IL-12 treatments did not induce significant local or systemic toxicity. IL-12 dissemination and exposure from intravesical administration was significantly lower compared to s.c. injections. Weekly intravesical CS/IL-12 treatments were well-tolerated and did not result in blunted immune responses. CONCLUSIONS: Intravesical CS/IL-12 is safe and well-tolerated in mice. In particular, the lack of cystitis and acute inflammation justifies continued investigation of intravesical CS/IL-12 immunotherapy in larger animals and patients with bladder cancer.

BackgroundUnresectable solid malignancies are responsible for a major proportion of total cancer-related mortalities, making focal ablation an attractive alternative. Nevertheless, there are high rates of recurrence after ablation [1,2]. The addition of an immune agonist to ablation has the potential to prevent this recurrence and improve treatment outcomes. The goal of this study is to determine if localized interleukin-12 (IL-12) can prevent primary tumor recurrence after cryoablation in both minimal ablation and metastasis models.MethodsLLC (LL/2) (ATCC) and MC38 (NCI) were implanted in 6–9 week old C57BL/6 mice. All tumors were treated at volumes of 200–500 mm3. LLC tumors with treated with three cycles of freeze/thaw and then monitored for tumor recurrence and lung metastasis 22–25 days after implantation. MC38 tumors were treated with a minimal cryoablation protocol where the tumor undergoes one cycle of freezing at 100% intensity up to the tumor margin by visual inspection, followed by one cycle of active thaw until the cryo probe can be removed. Ablation was performed using the Argon-Helium Visual-ICETM Cryoablation System (Boston Scientific). Interleukin-12 (IL-12) in 1.5% (w/v) chitosan acetate (CS) dissolved in dPBS was injected intratumorally within an hour after cryoablation, or as indicated. The dose of IL-12 was 1 ug unless otherwise indicated.ResultsWe established a model of 100% recurrence using the minimal cryoablation protocol. Using this protocol, we demonstrated that a single intratumoral injection of CS/IL-12 within an hour after cryoablation prevents recurrence in 7/8 mice while only 3/8 mice remained tumor-free without CS/IL-12 (Figure 1). Studies evaluating the impact of CS/IL-12 on tumor-specific T cell responses following cryoablation are ongoing. In the spontaneously metastatic LLC model, delivering CS/IL-12 two days before treatment, either resection or cryoablation, reduced the number of metastatic lung nodules and furthermore prevented the recurrence of the primary tumor after resection (Figure 2).Abstract 620 Figure 1CS/IL-12 prevents tumor recurrence after minimal cryoAbstract 620 Figure 2CS/IL-12 before cryo or surgery prevents metastasisConclusionsDue to the high percentage of solid malignancies that are unresectable at diagnosis, focal ablation is an attractive alternative, yet has a high rate of recurrence. We demonstrated that intratumoral neoadjuvant CS/IL-12 protects not only against primary recurrence after cryoablation, but also protects against lung metastasis and recurrence after resection. Further studies are necessary to explore the immune populations responsible for this therapeutic effect.AcknowledgementsThis work is supported by Boston Scientific, the NC State University Provost’s Fellowship, the NSF Graduate Research Fellowship and startup funds provided by the College of Engineering at NC State University.ReferencesWeld KJ, Landman J. Comparison of cryoablation, radiofrequency ablation and high-intensity focused ultrasound for treating small renal tumours. BJU Int. 2005;96(9):1224–1229. doi:10.1111/j.1464–410X.2005.05848.x.Guenther E, Klein N, Zapf S, et al. Prostate cancer treatment with Irreversible Electroporation (IRE): Safety, efficacy and clinical experience in 471 treatments. PloS One. 2019;14(4):e0215093. doi:10.1371/journal.pone.0215093Ethics ApprovalThe Institutional Animal Care and Use Committee at North Carolina State University approved of all animal protocols (#19–795) in compliance with The Guide for Care and Use of Laboratory Animals (National Research Council).

Interleukin-12 (IL-12) is a potent, pro-inflammatory type 1 cytokine that has long been studied as a potential immunotherapy for cancer. Unfortunately, IL-12's remarkable antitumor efficacy in preclinical models has yet to be replicated in humans. Early clinical trials in the mid-1990's showed that systemic delivery of IL-12 incurred dose-limiting toxicities. Nevertheless, IL-12's pleiotropic activity, i.e., its ability to engage multiple effector mechanisms and reverse tumor-induced immunosuppression, continues to entice cancer researchers. The development of strategies which maximize IL-12 delivery to the tumor microenvironment while minimizing systemic exposure are of increasing interest. Diverse IL-12 delivery systems, from immunocytokine fusions to polymeric nanoparticles, have demonstrated robust antitumor immunity with reduced adverse events in preclinical studies. Several localized IL-12 delivery approaches have recently reached the clinical stage with several more at the precipice of translation. Taken together, localized delivery systems are supporting an IL-12 renaissance which may finally allow this potent cytokine to fulfill its considerable clinical potential. This review begins with a brief historical account of cytokine monotherapies and describes how IL-12 went from promising new cure to ostracized black sheep following multiple on-study deaths. The bulk of this comprehensive review focuses on developments in diverse localized delivery strategies for IL-12-based cancer immunotherapies. Advantages and limitations of different delivery technologies are highlighted. Finally, perspectives on how IL-12-based immunotherapies may be utilized for widespread clinical application in the very near future are offered.

Hydrogel biomaterials are pervasive in biomedical use. Applications of these soft materials range from contact lenses to drug depots to scaffolds for transplanted cells. A subset of hydrogels is prepared from physical cross-linking mediated by host-guest interactions. Host macrocycles, the most recognizable supramolecular motif, facilitate complex formation with an array of guests by inclusion in their portal. Commonly, an appended macrocycle forms a complex with appended guests on another polymer chain. The formation of poly(pseudo)rotaxanes is also demonstrated, wherein macrocycles are threaded by a polymer chain to give rise to physical cross-linking by secondary non-covalent interactions or polymer jamming. Host-guest supramolecular hydrogels lend themselves to a variety of applications resulting from their dynamic properties that arise from non-covalent supramolecular interactions, as well as engineered responsiveness to external stimuli. These are thus an exciting new class of materials.