Cytokine-chitosan bioconjugates for localized cancer immunotherapy

A new class of biopharmaceuticals. Cytokines (shapes) and/or immune-modulators can be conjugated to chitosan (wavy lines) to form cytokine-chitosan bioconjugates. The resulting therapeutics are administered and retained locally to elicit a desired immune response

A new class of biopharmaceuticals. Cytokines (shapes) and/or immune-modulators can be conjugated to chitosan (wavy lines) to form cytokine-chitosan bioconjugates. The resulting therapeutics are administered and retained locally to elicit a desired immune response

Cytokines are master regulators of immune function. For decades, scientists have hypothesized that exogenous cytokines can be administered to treat a wide range of diseases. However, despite hundreds, if not thousands, of preclinical and clinical studies, only 2 of 40+ identified cytokines have been approved as single agents for a limited number of malignancies. Unintentional signaling and dose-limiting side effects due to systemic delivery of pleiotropic cytokines have prevented cytokine-based immunotherapies from fulfilling their clinical potential. A safer, more effective approach for the administration of cytokine therapeutics is through local, sustained delivery. Local delivery reduces cytokine-mediated toxicities and exploits the preferred paracrine mechanism of action of most cytokines.

To help cytokines reach their full potential as cancer immunotherapies, the Zaharoff lab has pioneered the use of chitosan-based delivery systems for the local, sustained delivery of recombinant cytokines. Chitosan, an abundant, natural polysaccharide derived primarily from the exoskeletons of crustaceans, has an established safety profile in humans. A key advantage of chitosan over other polymers commonly used in drug and vaccine delivery is that chitosan can be formulated in mild, aqueous conditions, and thus, maintains bioactivity of fragile co-formulated proteins. Chitosan, by itself, has no antitumor activity.

In foundational studies, the Zaharoff lab demonstrated that simple mixtures of chitosan and interleukin-12 (IL-12) can induce complete regression and durable tumor-specific immunity against colorectal, pancreatic and bladder tumors. Recently, we invented a novel class of molecules in which a cytokine is covalently attached to a polysaccharide. Naturally, our initial experiments have focused on IL-12 and chitosan. Because chitosan is large (100-500kDa) and bioadhesive, we hypothesize that chitosan anchors IL-12 to an injection site and completely eliminate its dissemination.

 

Role of heparin in interleukin-12 function

Electrostatic potential map of hIL-12. Blue regions are electronegative; red regions are electropositive. Potential heparin binding sites are circled.

Electrostatic potential map of hIL-12. Blue regions are electronegative; red regions are electropositive. Potential heparin binding sites are circled.

With our collaborators at the University of Arkansas (Prof. Suresh Kumar et al), we recently identified putative heparin-binding segments on the p40 subunit of IL-12 and exploited this specific binding to develop a protocol for single-step purification of tagless, authentic IL-12 via heparin-sepharose chromatography. In addition, we demonstrated that heparin increased the activity of IL-12. Specifically, heparin enhanced hIL-12 bioactivity by up to 661%. Comprehensive biophysical and cell-based studies indicated that heparin enhances hIL-12 bioactivity by serving as a co-receptor. Heparin’s potential to localize IL-12, for example at a site of infection, while increasing its activity may provide humans a way to limit the systemic toxicity of this potent cytokine. Interestingly, heparin was also found to restore IL-12 activity in natural killer cells in which both IL-12 receptor subunits had been functionally deleted via CRISPR/Cas9 editing. These data have potential clinical implications for immune-compromised patients with IL-12Rb1 mutations.

 

 

Development of intravesical immunotherapies for bladder cancer

Abscopal responses following intravesical immunotherapy. C57BL/6 mice were implanted with MB49 either subcutaneously (a, c) or both subcutaneously and orthotopically (b, d). Mice in groups C (n = 5) and D (n = 10) received intravesical treatments of chitosan/IL-12 (CS/IL-12) (1 μg) twice per week starting 7 days after implantation. Group B (n = 4) received intravesical PBS. Group A (n = 5) served as a negative control. Tumor volume, hematuria, and overall survival were monitored. Arrows indicate treatments.

Abscopal responses following intravesical immunotherapy. C57BL/6 mice were implanted with MB49 either subcutaneously (a, c) or both subcutaneously and orthotopically (b, d). Mice in groups C (n = 5) and D (n = 10) received intravesical treatments of chitosan/IL-12 (CS/IL-12) (1 μg) twice per week starting 7 days after implantation. Group B (n = 4) received intravesical PBS. Group A (n = 5) served as a negative control. Tumor volume, hematuria, and overall survival were monitored. Arrows indicate treatments.

Bladder cancer is the fifth most common non-cutaneous cancer diagnosis in the U.S. with an estimated 79,030 new cases and 16,870 deaths in 2017.  Although 70-80% of patients are diagnosed with early stage non-muscle invasive bladder cancer (NMIBC), bladder cancer has a recurrence rate of approximately 65%. Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been standard-of-care intravesical therapy for high risk NMIBC for more than 30 years. Yet, 20-30% of patients will fail initial BCG therapy and 30 - 50% of BCG responders will develop recurrent tumors within 5 years. The inability of BCG to induce tumor-specific immunity necessitates long-term maintenance therapy and continuous surveillance of bladder cancer patients. As a result, domestic bladder care expenditures are significant, >$4 billion per year, and bladder cancer has the highest lifetime treatment costs per patient of all cancers.

The overall goal of this research is to develop an intravesical immunotherapy capable of eliciting tumor-specific immune responses in order to limit the progressive recurrence of high-grade NMIBC. IL-12 is under investigation as a candidate to replace BCG. Our previous studies demonstrated that chitosan, a mucoadhesive, natural polysaccharide derived from the exoskeletons of crustaceans, can enhance the delivery and antitumor efficacy of intravesically administered IL-12.3,7 In fact, a co-formulation of chitosan solution and recombinant IL-12 (chitosan/IL-12) was found to eliminate established orthotopic bladder tumors in 80-100% of mice. Furthermore, recent data demonstrated that mice develop systemic tumor-specific protective immunity following intravesical chitosan/IL-12 immunotherapy. Specifically, all cured mice rejected a distant, subcutaneous challenge with the original MB49 bladder tumor but not an irrelevant B16 melanoma. Furthermore, intravesical chitosan/IL-12 immunotherapy was found to induce abscopal responses, i.e. regression of uninjected tumors, when an orthotopic bladder tumor was present. To our knowledge, these data are the first to show that an intravesical immunotherapy can induce systemic and abscopal tumor-specific immune responses.

 

Development of topical esophageal delivery systems

Esophageal diseases, including gastroesophageal reflux disease (GERD), Barrett’s esophagus (BE), eosinophilic esophagitis (EoE), and esophageal cancer, are collectively a rapidly increasing cause of morbidity. GERD causes symptoms of heartburn, regurgitation, and chest pain, and is one of the most common medical conditions, seen in up to a third of all individuals. It is increasing in incidence and prevalence and is a major driver of healthcare utilization. GERD can be complicated by erosive esophagitis, esophageal strictures, and Barrett’s esophagus (BE). BE is a risk factor for esophageal adenocarcinoma (EAC) and is seen in approximately 2-3% of the U.S. population. EoE is a chronic allergic disease of the esophagus that causes trouble swallowing due to esophageal strictures. It has rapidly emerged as an important esophageal condition, with greater than 5-10 fold increases in incidence and prevalence over the past decade. The estimated prevalence for EoE is at least 1 in 2000 Americans while EoE-associated healthcare costs are about $1.4 billion per year. EoE is now the most common cause of food impaction, responsible for >50% of impaction-related emergency room visits, and the second most common cause of esophagitis. Up to 23% of all patients undergoing endoscopy for dysphagia will have EoE. Lastly, the incidence of EAC, in contrast to the decreasing incidence rates of many malignancies, has increased faster than any other cancer. Treatments for esophageal cancer remain ineffective, and the overall 5-year survival is dismal at 18%. Despite the incredible burden of esophageal diseases, there are no FDA-approved pharmaceuticals that directly target the esophagus. The overall goal of this research is to develop an orally administered topical delivery system to target esophageal disorders.

 

Vaccination against methamphetamine, opioids and other drugs of abuse

Drug abuse and addiction place an overwhelming burden on society. Annual crime-related, health care and loss-in-productivity costs associated with illicit drug and prescription opioid abuse in the U.S. are about $309 billion. In comparison, the total annual cost of cancer, which accounts for nearly 1 in 4 American deaths, is about $217 billion. The death rate due to the current opioid epidemic has skyrocketed to 10.3 per 100,000 – a rate approximately the same as automobile accidents. In 2016, approximately 62,000 Americans died of a drug overdose. In comparison, colorectal cancer, the second leading cause of cancer-related deaths, causes less than 51,000 deaths annually.

Medical interventions for drug addiction are sorely lacking. Drug addiction is frequently miscast as a behavioral deficiency. It is commonly assumed that drug addicts can choose to stop abusing drugs at any time. While it is true that the vast majority of addiction begins with a voluntary choice, the major risk factors for becoming addicted are similar to risk factors for heart disease and cancer, e.g. genetic predisposition, age and stress. Taken together, the burden of addiction and the lack of medical interventions illuminate an urgent, unmet clinical need for novel, effective treatments for illicit drug addiction.

The concept that substance abuse immunotherapies involving antibodies to selectively bind and prevent abused drugs from crossing the blood-brain barrier was first advanced by Owens et al in the 1980s. These antibodies and antibody fragments were shown in rodent studies to reduce drug-craving and drug-seeking behaviors. Despite promising preclinical data, nicotine and cocaine vaccines failed in recent Phase III trials.

In order to improve the immunogenicity and efficacy of substance abuse vaccines, we are developing a novel vaccine platform based on conjugation of drug-like haptens to the natural polysaccharides alginate and chitosan. These polysaccharides are more versatile than traditional carrier proteins in that they can be engineered to incorporate a wider range of drug-like haptens as well as other immune response modifiers.

 

Optimizing tumor-specific immunity following tumor ablation

In 20 years, we believe that tumor resection will not be part of the standard-of-care for most solid tumors. Tumors are a rich source of patient-specific antigens that can be exploited via local immunomodulation to induce a personalized in situ vaccination. However, large or highly immunosuppressive tumors are not likely to regress following immunotherapy alone. For these tumors, it is important to explore tumor ablative strategies that can kill the tumor while leaving tumor-specific antigens behind. We are interested in exploring cryoablation, radiofrequency ablation, high-intensity ultrasound and irreversible electroporation as ablative strategies. Our goals are: 1) to determine the conditions which maximize tumor-specific immunity; and 2) to assess the potential to partner localized adjuvant immunotherapies with ablative therapies to enhance antitumor cell-mediated immune responses.