@article{mantooth_hancock_thompson_varghese_meritet_vrabel_hu_zaharoff_2024, title={Characterization of an Injectable Chitosan Hydrogel for the Tunable, Localized Delivery of Immunotherapeutics}, volume={10}, ISSN={["2373-9878"]}, url={https://doi.org/10.1021/acsbiomaterials.3c01580}, DOI={10.1021/acsbiomaterials.3c01580}, abstractNote={Localized delivery of immunotherapeutics within a tumor has the potential to reduce systemic toxicities and improve treatment outcomes in cancer patients. Unfortunately, local retention of therapeutics following intratumoral injection is problematic and is insufficiently considered. Dense tumor architectures and high interstitial pressures rapidly exclude injections of saline and other low-viscosity solutions. Hydrogel-based delivery systems, on the other hand, can resist shear forces that cause tumor leakage and thus stand to improve the local retention of coformulated therapeutics. The goal of the present work was to construct a novel, injectable hydrogel that could be tuned for localized immunotherapy delivery. A chitosan-based hydrogel, called XCSgel, was developed and subsequently characterized. Nuclear magnetic resonance studies were performed to describe the chemical properties of the new entity, while cryo-scanning electron microscopy allowed for visualization of the hydrogel's cross-linked network. Rheology experiments demonstrated that XCSgel was shear-thinning and self-healing. Biocompatibility studies, both in vitro and in vivo, showed that XCSgel was nontoxic and induced transient mild-to-moderate inflammation. Release studies revealed that coformulated immunotherapeutics were released over days to weeks in a charge-dependent manner. Overall, XCSgel displayed several clinically important features, including injectability, biocompatibility, and imageability. Furthermore, the properties of XCSgel could also be controlled to tune the release of coformulated immunotherapeutics.}, number={2}, journal={ACS BIOMATERIALS SCIENCE & ENGINEERING}, author={Mantooth, Siena M. and Hancock, Asher M. and Thompson, Peter M. and Varghese, P. J. George and Meritet, Danielle M. and Vrabel, Maura R. and Hu, Jingjie and Zaharoff, David A.}, year={2024}, month={Jan}, pages={905–920} }
 @misc{mantooth_abdou_saez-ibanez_upadhaya_zaharoff_2024, title={Intratumoral delivery of immunotherapy to treat breast cancer: current development in clinical and preclinical studies}, volume={15}, ISSN={["1664-3224"]}, DOI={10.3389/fimmu.2024.1385484}, abstractNote={Breast cancer poses one of the largest threats to women’s health. Treatment continues to improve for all the subtypes of breast cancer, but some subtypes, such as triple negative breast cancer, still present a significant treatment challenge. Additionally, metastasis and local recurrence are two prevalent problems in breast cancer treatment. A newer type of therapy, immunotherapy, may offer alternatives to traditional treatments for difficult-to-treat subtypes. Immunotherapy engages the host’s immune system to eradicate disease, with the potential to induce long-lasting, durable responses. However, systemic immunotherapy is only approved in a limited number of indications, and it benefits only a minority of patients. Furthermore, immune related toxicities following systemic administration of potent immunomodulators limit dosing and, consequently, efficacy. To address these safety considerations and improve treatment efficacy, interest in local delivery at the site of the tumor has increased. Numerous intratumorally delivered immunotherapeutics have been and are being explored clinically and preclinically, including monoclonal antibodies, cellular therapies, viruses, nucleic acids, cytokines, innate immune agonists, and bacteria. This review summarizes the current and past intratumoral immunotherapy clinical landscape in breast cancer as well as current progress that has been made in preclinical studies, with a focus on delivery parameters and considerations.}, journal={FRONTIERS IN IMMUNOLOGY}, author={Mantooth, Siena M. and Abdou, Yara and Saez-Ibanez, Ana Rosa and Upadhaya, Samik and Zaharoff, David A.}, year={2024}, month={May} }
 @article{vrabel_schulman_gillam_mantooth_nguyen_zaharoff_2023, title={Focal Cryo-Immunotherapy with Intratumoral IL-12 Prevents Recurrence of Large Murine Tumors}, volume={15}, ISSN={["2072-6694"]}, DOI={10.3390/cancers15082210}, abstractNote={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.}, number={8}, journal={CANCERS}, author={Vrabel, Maura R. and Schulman, Jacob A. and Gillam, Francis B. and Mantooth, Siena M. and Nguyen, Khue G. and Zaharoff, David A.}, year={2023}, month={Apr} }
 @article{nguyen_mantooth_vrabel_zaharoff_2022, title={Intranasal Delivery of Thermostable Subunit Vaccine for Cross-Reactive Mucosal and Systemic Antibody Responses Against SARS-CoV-2}, volume={13}, ISSN={["1664-3224"]}, DOI={10.3389/fimmu.2022.858904}, abstractNote={Despite the remarkable efficacy of currently approved COVID-19 vaccines, there are several opportunities for continued vaccine development against SARS-CoV-2 and future lethal respiratory viruses. In particular, restricted vaccine access and hesitancy have limited immunization rates. In addition, current vaccines are unable to prevent breakthrough infections, leading to prolonged virus circulation. To improve access, a subunit vaccine with enhanced thermostability was designed to eliminate the need for an ultra-cold chain. The exclusion of infectious and genetic materials from this vaccine may also help reduce vaccine hesitancy. In an effort to prevent breakthrough infections, intranasal immunization to induce mucosal immunity was explored. A prototype vaccine comprised of receptor-binding domain (RBD) polypeptides formulated with additional immunoadjuvants in a chitosan (CS) solution induced high levels of RBD-specific antibodies in laboratory mice after 1 or 2 immunizations. Antibody responses were durable with high titers persisting for at least five months following subcutaneous vaccination. Serum anti-RBD antibodies contained both IgG1 and IgG2a isotypes suggesting that the vaccine induced a mixed Th1/Th2 response. RBD vaccination without CS formulation resulted in minimal anti-RBD responses. The addition of CpG oligonucleotides to the CS plus RBD vaccine formulation increased antibody titers more effectively than interleukin-12 (IL-12). Importantly, generated antibodies were cross-reactive against RBD mutants associated with SARS-CoV-2 variants of concern, including alpha, beta and delta variants, and inhibited binding of RBD to its cognate receptor angiotensin converting enzyme 2 (ACE2). With respect to stability, vaccines did not lose activity when stored at either room temperature (21-22°C) or 4°C for at least one month. When delivered intranasally, vaccines induced RBD-specific mucosal IgA antibodies, which may protect against breakthrough infections in the upper respiratory tract. Altogether, data indicate that the designed vaccine platform is versatile, adaptable and capable of overcoming key constraints of current COVID-19 vaccines.}, journal={FRONTIERS IN IMMUNOLOGY}, author={Nguyen, Khue G. and Mantooth, Siena M. and Vrabel, Maura R. and Zaharoff, David A.}, year={2022}, month={May} }
 @article{mantooth_zaharoff_mantooth_2021, title={INJECTABLE CHITOSAN HYDROGEL FOR LOCALIZED DELIVERY OF IMMUNE CHECKPOINT INHIBITORS}, volume={9}, ISSN={["2051-1426"]}, DOI={10.1136/jitc-2021-SITC2021.259}, abstractNote={BackgroundSystemic delivery of checkpoint inhibitors risks the development of immune-related adverse events (irAEs) in up to 85% of patients.1 Localized delivery methods with slow-release kinetics have the potential to avoid systemic exposure and reduce irAEs. Direct tumor injection is extremely difficult, as saline-based solutions are rapidly excluded from the high-pressure tumor environment. Utilizing hydrogels as a delivery medium and local depot can address this shortcoming. To this end, we developed an injectable chitosan-based hydrogel for intratumoral delivery of checkpoint antibodies.MethodsHydrogelLow-viscosity, 80% deacetylated chitosan (Heppe Medical Chitosan; Halle, Germany) was reacted with 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in the presence of β-glycerophosphate at room temperature for 48 hours. The mixture was then washed with ethanol and dried at 60°C. The resulting solid was dissolved in phosphate buffered saline (PBS) at concentrations from 35–70 mg/mL.In vitro release. 300 μg/mL bovine serum albumin (BSA) labeled with fluorescein-5-isothiocyanate (FITC) as a model protein drug was loaded into the hydrogel. The hydrogel was injected through a 28g needle and incubated with PBS. Samples were taken over a week period. Release kinetics were analyzed by fitting fluorescence data to zero-order, first-order, and Korsmeyer-Peppas models. To visualize retention after injection, dye-loaded hydrogels or dye in PBS alone were injected into a 0.6 wt% agar tissue phantom.In vivo imaging and tumor treatment. Flank MC38 tumors will be established in C57BL/6 mice. At tumor volumes of 50–100 mm3, 200 ug of fluorescently labeled aCTLA-4 and aPD-L1 included in the chitosan hydrogel will be delivered intratumorally. Images will be captured using an In Vivo Imaging System (IVIS). Antitumor activity will be assessed in a separate cohort using unlabeled antibodies.ResultsThe chitosan hydrogel was found to be injectable in needles as thin as 28g. After exiting the needle, the hydrogel reformed (figure 1A). Upon injection into the tissue phantom, dyed PBS immediately leaked out, primarily through the needle track, while the dyed hydrogel was retained (figure 1B). In vitro release studies demonstrated long-term, nearly zero-order, week-long sustained release (figure 1C). In vivo retention and tumor treatment studies are ongoing.Abstract 259 Figure 1Injectable chitosan hydrogel. (A) Re-formed BSA-FITC hydrogel in 1x PBS; (B) (i) Retained hydrogel in agar tissue phantom, (ii) Excluded 1x PBS in agar tissue phantom; (C) Release kinetics in 1x PBS.ConclusionsA novel injectable chitosan hydrogel was found to provide sustained release of a large model protein over a 1–2 week period with favorable in vitro kinetics. Importantly, this hydrogel can be engineered to provide faster or slower release as needed. Ongoing studies in vivo will quantify intratumoral retention, systemic dissemination, and antitumor activity.AcknowledgementsThis work is supported by the National Science Foundation Graduate Research Fellowship.ReferenceHommes J, Verheijden R, Suijkerbuijk K, Hamann D. Biomarkers of checkpoint inhibitor induced immune-related adverse events—a comprehensive review. Front Oncol 2021;10:1–16.Ethics ApprovalAnimal use was in compliance with the Public Health Service Policy on Human Care and Use of Laboratory Animals. All experiments involving laboratory animals were approved by the Institutional Animal Care and Use Committee at North Carolina State University (Protocol #19–795).}, journal={JOURNAL FOR IMMUNOTHERAPY OF CANCER}, author={Mantooth, Siena and Zaharoff, David and Mantooth, Siena}, year={2021}, month={Nov}, pages={A281–A281} }
 @article{vrabel_zaharoff_mantooth_2021, title={ONCO-IMMUNOLOGICAL MECHANISMS OF FOCAL ABLATION AND LOCALIZED IL-12 IMMUNOTHERAPY}, volume={9}, ISSN={["2051-1426"]}, DOI={10.1136/jitc-2021-SITC2021.620}, abstractNote={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).}, journal={JOURNAL FOR IMMUNOTHERAPY OF CANCER}, author={Vrabel, Maura and Zaharoff, David and Mantooth, Siena}, year={2021}, month={Nov}, pages={A650–A650} }
 @article{nguyen_wagner_vrabel_mantooth_meritet_zaharoff_2021, title={Safety and Pharmacokinetics of Intravesical Chitosan/Interleukin-12 Immunotherapy in Murine Bladders}, volume={7}, ISSN={["2352-3735"]}, DOI={10.3233/BLC-211542}, abstractNote={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.}, number={4}, journal={BLADDER CANCER}, author={Nguyen, Khue G. and Wagner, Ethan S. and Vrabel, Maura R. and Mantooth, Siena M. and Meritet, Danielle M. and Zaharoff, David A.}, year={2021}, pages={427–437} }