Innovative therapies to complement current treatments are needed to curb the growing incidence of fatal overdoses related to synthetic opioids. Murine and chimeric monoclonal antibodies (mAb) specific for fentanyl and its analogs have demonstrated pre-clinical efficacy in preventing and reversing drug-induced toxicity in rodent models. However, mAb-based therapeutics require extensive engineering as well as in vitro and in vivo characterization to advance to first-in-human clinical trials. Here, novel murine anti-fentanyl mAbs were selected for development based on affinity for fentanyl, and efficacy in counteracting the pharmacological effects of fentanyl in mice. Humanization and evaluation of mutations designed to eliminate predicted post-translational modifications resulted in two humanized mAbs that were effective at preventing fentanyl-induced pharmacological effects in rats. These humanized mAbs showed favorable biophysical properties with respect to aggregation and hydrophobicity by chromatography-based assays, and thermostability by dynamic scanning fluorimetry. These results collectively support that the humanized anti-fentanyl mAbs developed herein warrant further clinical development for treatment of fentanyl toxicity.
ID 53762 Poster Board 315Strychnine is an extremely toxic compound that can cause seizures, respiratory failure, and death with no available treatment. To determine if strychnine-specific antibodies could protect against strychnine-related effects and alter strychnine distribution in serum and brain, mice were vaccinated once every two weeks with a strychnine conjugate vaccine (Strychnine-sKLH adsorbed to aluminum) or aluminum alone. One week later, mice received a 0.9 mg/kg IP strychnine dose and blood and brain were collected 30 minutes. To quantitively determine the strychnine in mouse serum and brain, a sensitive method has been developed and validated on liquid chromatography-tandem mass spectrometry (LC-MS/MS) in Electrospray ionization (ESI) inlet with dynamic multiple reaction monitoring (DMRM) mode. The method consists of a protein precipitation spiked with internal standard followed by solid phase extraction (SPE) using a Bond Elut PCX 96 well plate. Serum and brain supernatant samples of 200 mL were pretreated via protein precipitation and internal standard. Chromatographic separation was carried out on a Poroshell EC C18 2.1 x 50mm 2.7u column. The method is linear in the range of 2.5-500 ng/mL and a relative standard error of 4.8%. Maximum detected strychnine in vaccinated mouse serum ranged from 79 to 471 ng/mL, 19 to 124 ng/mL in unvaccinated mice. Strychnine concentrations in vaccinated mouse brains ranged from 10 to 34 ng/gm, 46 to 97 ng/gm in unvaccinated mice. The results of this study showed that strychnine-sKLH reduced brain strychnine concentrations and demonstrate that a strychnine-immunotherapy could be developed to protect against strychnine-related effects.
OBJECTIVES/GOALS: The innate immune responses to Multisystem Inflammatory Syndrome in Children (MIS-C) are not fully known. Using samples from MIS-C, we will assess the cellular responses and develop a novel Tri-Specific Killer Engager (TRiKE) that engages innate immune cells to improve those responses. METHODS/STUDY POPULATION: We collected blood samples from 60 pediatric patients from which we isolated plasma and peripheral blood mononuclear cells. We received blood samples from 13 MIS-C, 32 severe acute COVID, 5 COVID-19 asymptomatic, and 15 COVID-19 negative patients. Using plasma, we then performed ELISAs to determine IgG antibody levels against SARS-CoV-2 and plaque reduction neutralization tests to determine neutralizing antibody functions. We isolated DNA to look at Fc receptor genetics. We also utilized utilize flow cytometry assays determine the phagocytosis and killing abilities of the innate cells from these patients. This data will be correlated with clinical outcomes. Additionally, we have developed a novel SARS-CoV-2 TRiKE which directs natural killer (NK) cell killing specifically to of COVID-19 infected cells. RESULTS/ANTICIPATED RESULTS: MIS-C patients had higher IgG antibody titers against SARS-CoV-2 compared to children with symptomatic or asymptomatic COVID. MIS-C patients also neutralized SARS-CoV-2 more effectively than children with acute symptomatic or asymptomatic COVID-19. We found natural killer cells and monocytes are dysfunctional in MIS-C patients and do not kill SARS-CoV-2 infected cells as well. Specifically, NK cells do not kill COVID-19 infected cells as well. To combat this, we have successfully generated and are now testing a Tri-Specific Killer engager (TRiKE) which binds one ends to NK cells, one end to the Spike protein on COVID-19 infected cells and contains IL-15 to improve NK cell function. We anticipate that we can improve NK cell killing of COVID-19 infected cells with this TRiKE. DISCUSSION/SIGNIFICANCE: We found that MIS-C patients have antibodies that can neutralize SARS-CoV-2 but that that innate immune cells that engage antibodies are dysfunctional. We are have successfully developed and are targeting this response with a TRiKE to improve innate immune cell functional; this may serve as an adjunctive therapeutic if proven successful.
Strychnine poisoning induces seizures that result in loss of control of airway muscles, leading to asphyxiation and subsequent death. Current treatment options are limited, requiring hands-on medical care and isolation to low-stimulus environments. Anticonvulsants and muscle relaxants have shown limited success in cases of severe toxicity. Furthermore, nonfatal strychnine poisoning is likely to result in long-term muscular and cognitive damage. Due to its potency, accessibility, and lack of effective antidotes, strychnine poses a unique threat for mass casualty incidents. As a first step toward developing an anti-strychnine immunotherapy to reduce or prevent strychnine-induced seizures, a strychnine vaccine was synthesized using subunit keyhole limpet hemocyanin. Mice were vaccinated with the strychnine immunoconjugate and then given a 0.75 mg/kg IP challenge of strychnine and observed for seizures for 30 min. Vaccination reduced strychnine-induced events, and serum strychnine levels were increased while brain strychnine levels were decreased in vaccinated animals compared to the control. These data demonstrate that strychninespecific antibodies can block the seizure-inducing effects of strychnine and could be used to develop a therapeutic for strychnine poisoning.
Synthetic opioids including fentanyl are the lead cause of opioid-related deaths in the United States. Current treatments for opioid overdose include the opioid receptor antagonist naloxone; however, naloxone is not as effective against highly potent opioids, has a relatively short half-life, and precipitates withdrawal symptoms. Monoclonal antibody (mAb) therapy offers a promising strategy to combat opioid-related overdoses. Opioid-specific mAb bind to target molecules in the bloodstream and inhibit them from crossing the blood-brain barrier. We produced a lead fentanyl-specific mAb that protects against fentanyl toxicity in rats. The efficacy of mAbs is reliant on maintaining a high concentration in the bloodstream to ensure sufficient binding to the target drug. The neonatal Fc receptor (FcRn) is responsible for the recycling of IgG in circulation, thus increasing their effective half-life. Therefore, to increase the duration of efficacy of a lead chimeric fentanyl-specific mAb, we introduced three different mutations to the Fc region of the mAb. Biolayer interferometry paired with recombinant FcRn showed a 10-fold increase in affinity of the recombinant mAb compared to wild-type. Efficacy of the mutated and WT mAb against fentanyl-induced behavior and toxicity was also tested in mice. Both the mutated and WT mAb reduced fentanyl-induced antinociception, respiratory depression, and bradycardia. Finally, the half-life and long-term efficacy of these mutated mAbs was assessed in transgenic mice expressing human FcRn. Extending the half-life of these mAbs will facilitate more successful clinical approaches in preventing opioid-related overdoses. This work was funded by NIDA under CounterACT grant U01-DA051658
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