Tumor-responsive, multifunctional CAR-NK cells cooperate with impaired autophagy to infiltrate and target glioblastoma

Tumor antigen heterogeneity, a severely immunosuppressive tumor microenvironment (TME) and lymphopenia resulting in inadequate immune intratumoral trafficking have rendered glioblastoma (GBM) highly resistant to therapy. As a result, GBM immunotherapies have failed to demonstrate sustained clinical improvements in patient overall survival (OS). To overcome these obstacles, here we describe a novel, sophisticated combinatorial platform for GBM: the first multifunctional immunotherapy based on genetically-engineered, human NK cells bearing multiple anti-tumor functions, including local tumor responsiveness, that addresses key drivers of GBM resistance to therapy: antigen escape, poor immune cell homing, and immunometabolic reprogramming of immune responses. We engineered dual-specific CAR-NK cells to bear a third functional moiety that is activated in the GBM TME and addresses immunometabolic suppression of NK cell function: a tumor-specific, locally-released antibody fragment which can inhibit the activity of CD73 independently of CAR signaling and decrease the local concentration of adenosine. The multifunctional human NK cells targeted patient-derived GBM xenografts, demonstrated local tumor site specific activity in the tissue and potently suppressed adenosine production. We also unveil a complex reorganization of the immunological profile of GBM induced by inhibiting autophagy. Pharmacologic impairment of the autophagic process not only sensitized GBM to antigenic targeting by NK cells, but promoted a chemotactic profile favorable to NK infiltration. Taken together, our study demonstrates a promising new NK cell-based combinatorial strategy that can target multiple clinically-recognized mechanisms of GBM progression simultaneously

Countermeasures for Cyanide: Organometallics as Novel Cyanide Scavengers

Exposure to cyanide occurs more commonly in everyday life than many would believe, although most exposure is rather innocuous. However, the main concern is the misuse of cyanide in mass casualty events. Misuse of cyanide as a poison has been documented since before World War II. 1 The potential for malicious use still pervades in our society. Current scavenging treatments center on cobalt ions to bind cyanide in the blood. 2,3 Unfortunately, these treatments require high doses of cobalt that can be associated with some significant toxic side effects when used in higher doses, possibly resulting in a dose limiting toxicity. 4–6 When administered shortly after cyanide exposure, cobalt-based scavengers only improves survival by 50-70%, thus adding concern over the therapeutic options.6–10 In addition, the delivery of the FDA approved scavenger, hydroxocobalamin requires intravenous infusion over several minutes, adding delays to treatment time. Slow delivery has been demonstrated to reduce the chance of survival in patients suffering from cyanide exposure.5Therefore, there is an ever-pressing need for the rapid delivery of an effective scavenger to mitigate the morbidity and mortality associated with high levels of exposure. Chapter 1 is a literature review for topics related to cyanide poisoning, current scavengers, and emerging solutions in development for treating cyanide in.Previous work with platinum demonstrated the capacity to mitigate cyanide-associated toxicity in both zebrafish and mice when exposed to lethal levels.11 The resulting Pt-sulfide complexes might have been acting as a cis or trans-directing ligand that was later hypothesized to have contributed to the efficacious response. Mechanistically, Pt-S interactions may activate platinum, improving the rate of cyanide substitution onto the platinum center. 12–15 A possible advantage of platinum complexes over cobalt lies in the fact that little evidence supports strong interactions between sulfur and cobalt, which would limit the usefulness of the trans effect in these complexes. Interactions of cobalt with amino acids would occur primarily through carboxylate and amines, which have stronger bonds and would limit cyanide reactivity.16,17 Chapter 2 focuses on identifying suitable Pt-sulfide complexes using a combination of in vitro and in vivotesting.The overall project aims to identify suitable formulation conditions which maintain efficacy by intramuscular injection. Associative properties of ligands (e.g. amine and hydroxide) on platinum might be influenced by hydrogen ion concentration (pH) in solution.18,19 Regarding the trans effect, a conversion to related isomers may change the reactivity between platinum and cyanide. Chapter 3 begins identifying the relationship between formulation pH, cyanide scavenging, and efficacy. One of the leading adverse reactions with platinum drugs is acute kidney injury (AKI), which leads to renal disfunction.20 Sulfides have been found to modulate renal injury in vivo, suggesting sulfide complexed to platinum might reduce nephrotoxicity from platinum.21 One such example, thioethers (e.g. methionine) co-administered with cisplatin almost completely removed symptoms of renal injury in rats.22,23Chapter 3 also describes our investigation of the risk of AKI and lingering renal injury for the most efficacious platinum complexes identified in chapter 2. Furthermore, we highlight a potential strategy to mitigate AKI with the complexes

Intramuscular administration of glyoxylate rescues swine from lethal cyanide poisoning and ameliorates the biochemical sequalae of cyanide intoxication

Cyanide-a fast-acting poison-is easy to obtain given its widespread use in manufacturing industries. It is a high-threat chemical agent that poses a risk of occupational exposure in addition to being a terrorist agent. FDA-approved cyanide antidotes must be given intravenously, which is not practical in a mass casualty setting due to the time and skill required to obtain intravenous access. Glyoxylate is an endogenous metabolite that binds cyanide and reverses cyanide-induced redox imbalances independent of chelation. Efficacy and biochemical mechanistic studies in an FDA-approved preclinical animal model have not been reported. Therefore, in a swine model of cyanide poisoning, we evaluated the efficacy of intramuscular glyoxylate on clinical, metabolic, and biochemical endpoints. Animals were instrumented for continuous hemodynamic monitoring and infused with potassium cyanide. Following cyanide-induced apnea, saline control or glyoxylate was administered intramuscularly. Throughout the study, serial blood samples were collected for pharmacokinetic, metabolite, and biochemical studies, in addition, vital signs, hemodynamic parameters, and laboratory values were measured. Survival in glyoxylate-treated animals was 83% compared with 12% in saline-treated control animals (p < .01). Glyoxylate treatment improved physiological parameters including pulse oximetry, arterial oxygenation, respiration, and pH. In addition, levels of citric acid cycle metabolites returned to baseline levels by the end of the study. Moreover, glyoxylate exerted distinct effects on redox balance as compared with a cyanide-chelating countermeasure. In our preclinical swine model of lethal cyanide poisoning, intramuscular administration of the endogenous metabolite glyoxylate improved survival and clinical outcomes, and ameliorated the biochemical effects of cyanide

Identification of Platinum(II) Sulfide Complexes Suitable as Intramuscular Cyanide Countermeasures

The development of rapidly acting cyanide countermeasures using intramuscular injection (IM) represents an unmet medical need to mitigate toxicant exposures in mass casualty settings. Previous work established that cisplatin and other platinum­(II) or platinum­(IV)-based agents effectively mitigate cyanide toxicity in zebrafish. Cyanide’s in vivo reaction with platinum-containing materials was proposed to reduce the risk of acute toxicities. However, cyanide antidote activity depended on a formulation of platinum-chloride salts with dimethyl sulfoxide (DMSO) followed by dilution in phosphate-buffered saline (PBS). A working hypothesis to explain the DMSO requirement is that the formation of platinum–sulfoxide complexes activates the cyanide scavenging properties of platinum. Preparations of isolated NaPtCl5–DMSO and Na (NH3)2PtCl–DMSO complexes in the absence of excess DMSO provided agents with enhanced reactivity toward cyanide in vitro and fully recapitulated in vivo cyanide rescue in zebrafish and mouse models. The enhancement of the cyanide scavenging effects of the DMSO ligand could be attributed to the activation of platinum­(IV) and (II) with a sulfur ligand. Unfortunately, the efficacy of DMSO complexes was not robust when administered IM. Alternative Pt­(II) materials containing sulfide and amine ligands in bidentate complexes show enhanced reactivity toward cyanide addition. The cyanide addition products yielded tetracyanoplatinate­(II), translating to a stoichiometry of 1:4 Pt to each cyanide scavenger. These new agents demonstrate a robust and enhanced potency over the DMSO-containing complexes using IM administration in mouse and rabbit models of cyanide toxicity. Using the zebrafish model with these Pt­(II) complexes, no acute cardiotoxicity was detected, and dose levels required to reach lethality exceeded 100 times the effective dose. Data are presented to support a general chemical design approach that can expand a new lead candidate series for developing next-generation cyanide countermeasures