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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.

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.

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.

Abstract Introduction/Objective The U.S. Bureau of Labor Statistics projects 24,000 openings for clinical laboratory jobs each year until 2031 (BLS, 2022). The American Society for Clinical Pathology (ASCP, 2023) reported a 14.8% vacancy rate for medical laboratory scientists in the South-Central Atlantic region; this region includes South Carolina. There are not enough MLT or MLS certified laboratorians to keep up with current or future demands. On average, 3779 new certifications were awarded annually between 2019-2023. Pathology and Laboratory Medicine at the Medical University of South Carolina is taking short- and long-term approaches to address workforce challenges. Methods/Case Report Since 2018, Pathology and Lab Medicine has increased the number of clinical affiliates from 2 to 9 and have trained a total of 39 MLS students and 37 MLT students. In 2023, through collaboration with the MLT program at the local technical college, we began providing didactic education for 15 current lab team members with bachelor’s degrees but no clinical laboratory background. Employing youth apprentices is a long-term strategy being initiated in the 24-25 academic year. Juniors and seniors in high school are eligible for a two-year paid apprenticeship. Training includes medical lab assistant and phlebotomy certification. Through recruitment activities, we can increase awareness of the role of laboratory professionals in medicine. Long-term, the apprenticeship training provides an academic pathway to MLT and MLS certification. Results (if a Case Study enter NA) N/A Conclusion The short-term strategies have met with varying success and course corrections have been made. Successes and barriers will be discussed. Strategies for recruiting and selecting youth apprentices will be outlined. Training plans for youth apprentices will be presented.

Intratumoral injections of macromolecules, such as biologics and immunotherapeutics, show promise in overcoming dose-limiting side effects associated with systemic injections and improve treatment efficacy. However, the retention of injectates in the tumor microenvironment is a major underappreciated challenge. High interstitial pressures and dense tumor architectures create shear forces that rapidly expel low-viscosity solutions post-injection. Injectable hydrogels may address these concerns by providing a viscoelastic delivery vehicle that shields loaded therapies from rapid expulsion from the tumor. A chitosan–glycerol hydrogel was thus developed and characterized with the goal of improving the injection retention of loaded therapeutics. The gelation parameters and mechanical properties of the hydrogel were explored to reveal a shear-thinning gel that is injectable through a 27-gauge needle. Biocompatibility studies demonstrated that the chitosan–glycerol hydrogel was nontoxic. Retention studies revealed significant improvements in the retention of model therapeutics when formulated with the chitosan–glycerol hydrogel compared to less-viscous solutions. Finally, release studies showed that there was a sustained release of model therapeutics of various molecular sizes from the hydrogel. Overall, the chitosan–glycerol hydrogel demonstrated injectability, enhanced retention, biocompatibility, and sustained release of macromolecules, indicating its potential for future clinical use in intratumoral macromolecule delivery.

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.

BackgroundCancer immunotherapies, although they can confer potent and durable antitumor effects, suffer from dose-limiting toxicities when administered systemically. Localized delivery via intratumoral (i.t.) injection can mitigate this, however, low viscosity injectates can often leak out due to high intratumoral pressure. To improve/sustain local retention of IT-injected immunotherapy, we have previously developed a novel, chitosan-based injectable hydrogel called XCSgel. Prior work has shown that XCSgel is shear-thinning, self-healing, biocompatible, imageable and capable of providing sustained release of biologic. These characteristics make XCSgel a great candidate to better treat multiple tumor types by maximizing delivery to the tumor microenvironment while limiting systemic exposure.MethodsIn vitro studies quantified the leakage of XCSgel compared to low and high viscosity solutions following injections of tumor phantoms. For in vitro studies, mice bearing established MOC1 squamous cell carcinomas or B16F10 melanomas were treated with a single injection of interleukin-12 (IL-12) co-formulated with XCSgel. Mice were monitored for injection site reactions, body composition, and weight loss. Serum cytokine levels were quantified as a function of time after i.t. XCSgel-IL12 immunotherapy. A comparison of XCSgel with other published hydrogels was performed.ResultsCo-formulations with XCSgel significantly enhanced retention in tumor phantoms. Nearly all low viscosity injectates leaked out of injected phantoms which XCSgel was completely retained. I.t. XCSgel-IL12 immunotherapy eliminated all treated MOC1 tumors XCSgel-IL12 also eliminated MOC1 tumors and significantly delayed the growth of B16F10 tumors (figure 1). Treated mice exhibited no signs of weight loss or distress indicated that treatments were well tolerated.ConclusionsXCSgel is a promising injectable hydrogel for the co-delivery of potent immunomodulators such as IL12. XCSgel’s viscoelastic properties allow it to resist expulsion from injected tumors. Localized immunotherapy with i.t. XCSgel-IL12 continues to demonstrate robust antitumor activity with minimal toxicity concerns.AcknowledgementsThis research was supported by the Chancellor’s Innovation Fund at NC State University.Ethics ApprovalStudies involving animals were approved by the IACUC at NC State University (23-126).Abstract 884 Figure 1Individual tumor growth curves following treatment with XCSgel-IL12[Image Omitted. See PDF.]

Two previously healthy males presented to the emergency symptoms with signs of pericarditis/myocarditis after being vaccinated with an mRNA vaccine for COVID-19.Two previously healthy males presented to the emergency symptoms with signs of pericarditis/myocarditis after being vaccinated with an mRNA vaccine for COVID-19.

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 StatementThe authors have declared no competing interest.

This clinician's workbook is designed to accompany and supplement the main volume \"Ending Spouse/Partner Abuse.\" It includes forms, questionnaires, and various handouts.