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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).

Background Clinically, cryoablation is used to treat certain early stage prostate, liver, and kidney tumors in addition to bone and soft tissue sarcomas. However, for late-stage cancers, ablation is only an auxiliary step before complete resection. This leaves a gap of patients with advanced and inoperable tumors, where up to 90% of all pancreatic, and 80–85% of all prostate cancers are unresectable at diagnosis.1 2 Because cryoablation can release large amounts of antigen, it is uniquely capable of not only treating advanced, unresectable tumors, but also may induce an in situ vaccination response when combined with the appropriate immunotherapy. Previously, our results in single primary tumor models indicated that the addition of interleukin-12 (IL-12) to cryoablation (cryo) improved tumor burden and survival We hypothesized that intratumoral injection of IL-12 after cryo would activate a strong T cell response and induce systemic immunity in bilateral tumor models. Methods Panc02 cells were purchased from ATCC; MC38 and MB49 cells were acquired from the NIH. Female C57BL/6 mice were purchased from Jackson Laboratory. For primary tumor implantation, 1 × 106 Panc02 cells and 3 × 105 MC38 cells were injected subcutaneously (s.c.) in the right flank. For rechallenge, the same dose of cells was implanted on the left flank of cured mice. For bilateral models, in both the MB49 and MC38 models, 3 × 105 and 1.5 × 105 cells were injected s.c. in the right and left flanks respectively on the same day. For the Panc02 model, 1 × 106 cells were implanted s.c. on both the right and left flanks on the same day. Tumor volume was calculated as 0.5*a*b2 given the perpendicular long (a) and short (b) dimensions. Tumors measuring between 150–300 mm3 were cryoablated with three cycles of freeze/thaw using the Visual-ICETM Cryoablation System (Boston Scientific). The dose of IL-12 was 1 µg/mouse in 1.5% (w/v) chitosan acetate (CS) dissolved in DPBS, and then injected intratumorally within an hour after cryoablation. For the anti-PD-1 and isotype antibodies (BioXCell, clone: RMP1.14). 300µg was injected intraperitoneally every 3 days starting on the day of cryoablation for a total of 4 doses. Results In the bilateral MB49 mouse bladder cancer model, the median survival for the cryo alone group was 20 days post treatment (p.t.) compared to 23 days p.t. for cryo + CS/IL-12, which was not significant, and 12 days for the untreated control group. In the bilateral Panc02 model, the median survival for both the cryo alone and cryo + CS/IL-12 groups was the same at 20.5 days p.t., compared to 10 days p.t. for the untreated control. In the bilateral MC38 model, the addition of anti-PD-1 to cryo + CS/IL-12 did not significantly improve survival compared to isotype + cryo + CS/IL-12, with a median survival of 24 days p.t. and 16 days p.t. respectively (p=0.53, Log-rank test) (figure 1). However, addition of anti-PD-1 did significantly delay abscopal tumor growth up to 500 mm3 when compared to the isotype + cryo + CS/IL-12 (p=0.0398, Unpaired t test) (figure 2). Finally, the addition of IL-12 worsens memory in the MC38 model, where 100% of rechallenged cryo alone mice were protected (5/5) compared to only 43% protected of the cryo + CS/IL-12 group (3/7). Conclusions Conclusions: While cryoablation in combination with immunotherapy has the potential to treat advanced, unresectable primary tumors and distant untreated tumors, the addition of a single injection of IL-12 is not enough to induce a strong abscopal effect. Furthermore, it may actually worsen the establishment of effector memory cells. The addition of anti-PD-1 only slows abscopal tumor growth. Future work is needed to understand the mechanism of T cell priming in the context of the post-ablative tumor. Acknowledgements This 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. References van der Geest LGM. et al. Trends in treatment and survival of patients with nonresected, nonmetastatic pancreatic cancer: a population-based study. Cancer Med 2018; 7, 4943–4951. Takaki H. et al. Thermal ablation and immunomodulation: from preclinical experiments to clinical trials. Diagn. Interv. Imaging 2017; 98:651–659.

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.

Current methods for detection and diagnosis of allergies do not provide epitope specific immunogenic information and hence lack critical information that could aid in the prediction of clinical responses. To address this issue, we developed a nanoparticle based platform, called nanoallergens that enable multivalent display of potential allergy epitopes for determining the immunogenicity of each IgE binding epitope. By synthesizing nanoallergens that present various epitopes from the major peanut allergen, Ara h2, we directly determined the immunogenicity of each epitope, alone and in combination with other epitopes, using patient sera. This information provided insights on which epitopes are most critical for physiological responses to Ara h2 and revealed the importance of both high and low affinity epitopes for allergic responses. We anticipate the nanoallergen platform to be used to provide information regarding allergic reactions and therefore potentially aid in more accurate diagnosis and design of personalized treatment options.

Degranulation caused by type I hypersensitivity (allergies) is a complex biophysical process, and available experimental models for studying relevant immunoglobulin E binding epitopes on allergen proteins lack the ability to adequately evaluate, rank, and associate these epitopes individually and with each other. In this study, we propose a new allergy model system for studying potential allergen epitopes using nanoallergens, liposomes modified to effectively display IgE binding epitopes/haptens. By utilizing the covalently conjugated lipid tails on two hapten molecules (dinitrophenol and dansyl), hapten molecules were successfully incorporated into liposomes with high precision to form nanoallergens. Nanoallergens, with precisely controlled high-particle valency, can trigger degranulation with much greater sensitivity than commonly used bovine serum albumin conjugates. In rat basophil leukemia cell experiments, nanoallergens with only 2% hapten loading were able to trigger degranulation in vitro at concentrations as low as 10 pM. Additionally, unlike bovine serum albumin-hapten conjugates, nanoallergens allow exact control over particle size and valency. By varying the nanoallergen parameters such as size, valency, monovalent affinity of hapten, and specific IgE ratios, we exposed the importance of these variables on degranulation intensity while demonstrating nanoallergens’ potential for evaluating both high- and low-affinity epitopes. The data presented in this article establish nanoallergen platform as a reliable and versatile allergy model to study and evaluate allergen epitopes in mast cell degranulation.