Faculty Bibliography

The incidence of Clostridioides difficile infection (CDI) and associated mortality have increased rapidly worldwide in recent years. Therefore, it is critical to develop new therapies for CDI. Here we report on the development of mRNA-LNPs encoding camelid-derived V_HH-based neutralizing agents (VNAs) targeting toxins A and/or B of C. difficile. In preclinical models, intravenous administration of the mRNA-LNPs provided serum VNA levels sufficient to confer protection of mice against severe disease progression following toxin challenge. Furthermore, we employed an mRNA-LNP encoded effector antibody, a molecular tool designed to specifically bind an epitopic tag linked to the VNAs, to prolong VNA serum half-life. Co-administration of VNA-encoding mRNA-LNPs and an effector antibody, either provided as recombinant protein or encoded by mRNA-LNP, increased serum VNA half-life in mice and in gnotobiotic piglets. Prolonged serum half-life was associated with higher concentrations of serum VNA and enhanced prophylactic protection of mice in challenge models.

While covalent drug discovery is reemerging as an important route to small-molecule therapeutic leads, strategies for the discovery and engineering of protein-based irreversible binding agents remain limited. Here, we describe the use of yeast display in combination with noncanonical amino acids (ncAAs) to identify irreversible variants of single-domain antibodies (sdAbs), also called VHHs and nanobodies, targeting botulinum neurotoxin light chain A (LC/A). Starting from a series of previously described, structurally characterized sdAbs, we evaluated the properties of antibodies substituted with reactive ncAAs capable of forming covalent bonds with nearby groups after UV irradiation (when using 4-azido-l-phenylalanine) or spontaneously (when using O-(2-bromoethyl)-l-tyrosine). Systematic evaluations in yeast display format of more than 40 ncAA-substituted variants revealed numerous clones that retain binding function while gaining either UV-mediated or spontaneous crosslinking capabilities. Solution-based analyses indicate that ncAA-substituted clones exhibit site-dependent target specificity and crosslinking capabilities uniquely conferred by ncAAs. Interestingly, not all ncAA substitution sites resulted in crosslinking events, and our data showed no apparent correlation between detected crosslinking levels and distances between sdAbs and LC/A residues. Our findings highlight the power of yeast display in combination with genetic code expansion in the discovery of binding agents that covalently engage their targets. This platform streamlines the discovery and characterization of antibodies with therapeutically relevant properties that cannot be accessed in the conventional genetic code.

Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.

Somatodendritic dopamine (DA) release from midbrain DA neurons activates D2 autoreceptors on these cells to regulate their activity. However, the source of autoregulatory DA remains controversial. Here, we test the hypothesis that D2 autoreceptors on a given DA neuron in the substantia nigra pars compacta (SNc) are activated primarily by DA released from that same cell, rather than from its neighbors. Voltage-clamp recording allows monitoring of evoked D2-receptor-mediated inhibitory currents (D2ICs) in SNc DA neurons as an index of DA release. Single-cell application of antibodies to Na⁺ channels via the recording pipette decreases spontaneous activity of recorded neurons and attenuates evoked D2ICs; antibodies to SNAP-25, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, also decrease D2IC amplitude. Evoked D2ICs are nearly abolished by the light chain of botulinum neurotoxin A, which cleaves SNAP-25, whereas synaptically activated GABA_B-receptor-mediated currents are unaffected. Thus, somatodendritic DA release in the SNc autoinhibits the neuron that releases it.

Botulism is caused by a potent neurotoxin that blocks neuromuscular transmission, resulting in death by asphyxiation. Currently, the therapeutic options are limited and there is no antidote. Here, we harness the structural and trafficking properties of an atoxic derivative of botulinum neurotoxin (BoNT) to transport a function-blocking single-domain antibody into the neuronal cytosol where it can inhibit BoNT serotype A (BoNT/A1) molecular toxicity. Post-symptomatic treatment relieved toxic signs of botulism and rescued mice, guinea pigs, and nonhuman primates after lethal BoNT/A1 challenge. These data demonstrate that atoxic BoNT derivatives can be harnessed to deliver therapeutic protein moieties to the neuronal cytoplasm where they bind and neutralize intracellular targets in experimental models. The generalizability of this platform might enable delivery of antibodies and other protein-based therapeutics to previously inaccessible intraneuronal targets.

Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E). The 19 VHHs which bind to BoNT/E were characterized for their subunit specificity and 8 VHHs displayed the ability to neutralize BoNT/E intoxication of neurons. Heterodimer antitoxins consisting of two BoNT/E-neutralizing VHHs, including one heterodimer designed using structural information for simultaneous binding, were shown to protect mice against co-administered toxin challenges of up to 500 MIPLD₅₀. The 22 unique VHHs which bind to LC/E were characterized for their binding properties and 9 displayed the ability to inhibit LC/E protease activity. Surprisingly, VHHs selected on plastic-coated LC/E were virtually unable to recognize soluble or captured LC/E while VHHs selected on captured LC/E were poorly able to recognize LC/E coated to a plastic surface. This panel of anti-LC/E VHHs offer insight into BoNT/E function, and some may have value as components of therapeutic antidotes that reverse paralysis following BoNT/E exposures.

Background: Botulinum neurotoxins (BoNTs) pose serious military and civilian mass casualty threats with potential to rapidly overwhelm medical resources. Currently, the only FDA-approved treatment for botulism is infusion of antibody-based antitoxins to neutralize BoNT that circulates in the bloodstream. Presently, there is no treatment to inactivate the protease activity of BoNTs within the neuron. Here, we describe a first-in-kind treatment approach to block the protease activity of BoNT at the site of intoxication. The technology is based on "atoxic" derivatives of botulinum neurotoxins (serotypes A and C) that can maintain their natural biological trafficking properties and deliver single-chain antibodies to inhibit the protease activity of BoNTs. This report focuses on in vitro and in vivo studies of a treatment candidate for BoNT/A, termed Cyto-111. Method(s): In vitro studies were performed using E18 rat cortical neurons. In vivo studies were performed in: (1) CD-1 female mice (22 to 27 g); (2) Hartley guinea pigs (250 to 350 g); and (3) adult Rhesus macaques (5 to10 kg). Result(s): In vitro, Cyto-111 binds and inhibits the light chain of BoNT/A. When administered intraperitoneally (ip), Cyto-111 traffics to the neuromuscular junction of the diaphragm, where it localizes with presynaptic markers. In a murine postsymptomatic model of BoNT/A toxemia, treatment ip or intravenously with Cyto-111 prevented death at stages of disease that are completely refractory to antitoxin treatment. In an FDA clinical surrogate model of lethal respiratory botulism in guinea pigs, treatment with Cyto-111 provided 55% survival. Currently, we are evaluating the safety and efficacy of Cyto-111 in a Rhesus macaque model of botulism. Conclusion(s): Collectively, these results demonstrate that Cyto-111 reverses clinical symptoms of botulism and prevents mortality following exposure to lethal doses of BoNT/A. Cyto-111 represents a first-in-class, novel therapeutic approach that allows the precise delivery of single-domain function-blocking antibodies to the presynaptic compartment of BoNT/A-intoxicated neurons. Disclaimer: The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the US Government. The experimental protocol was approved by the Animal Care and Use Committee at the United States Army Medical Research Institute of Chemical Defense, and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act of 1966 (P.L. 89-544), as amended. Funding(s): (1) Defense Threat Reduction Agency (DTRA), (2) NIH; R01-5R01AI093504, (3) ORISE, and (4) Geneva.

Botulinum neurotoxin (BoNT) binds to and internalizes its light chain into presynaptic compartments with exquisite specificity. While the native toxin is extremely lethal, bioengineering of BoNT has the potential to eliminate toxicity without disrupting neuron-specific targeting, thereby creating a molecular vehicle capable of delivering therapeutic cargo into the neuronal cytosol. Building upon previous work, we have developed an atoxic derivative (ad) of BoNT/C1 through rationally designed amino acid substitutions in the metalloprotease domain of wild type (wt) BoNT/C1. To test if BoNT/C1 ad retains neuron-specific targeting without concomitant toxic host responses, we evaluated the localization, activity, and toxicity of BoNT/C1 ad in vitro and in vivo. In neuronal cultures, BoNT/C1 ad light chain is rapidly internalized into presynaptic compartments, but does not cleave SNARE proteins nor impair spontaneous neurotransmitter release. In mice, systemic administration resulted in the specific co-localization of BoNT/C1 ad with diaphragmatic motor nerve terminals. The mouse LD50 of BoNT/C1 ad is 5 mg/kg, with transient neurological symptoms emerging at sub-lethal doses. Given the low toxicity and highly specific neuron-targeting properties of BoNT/C1 ad, these data suggest that BoNT/C1 ad can be useful as a molecular vehicle for drug delivery to the neuronal cytoplasm.

Cyto-012 is a recombinant derivative of Botulinum neurotoxin Type A (BoNT/A). It primarily differs from wild type (wt) BoNT/A1 in that it incorporates two amino acid substitutions in the catalytic domain of the light chain (LC) metalloprotease (E224 > A and Y366 > A), designed to provide a safer clinical profile. Cyto-012 is specifically internalized into rat cortical and hippocampal neurons, and cleaves Synaptosomal-Associated Protein 25 (SNAP-25), the substrate of wt BoNT/A, but exhibits slower cleavage kinetics and therefore requires a higher absolute dose to exhibit pharmacologic activity. The pharmacodynamics of Cyto-012 and wt BoNT/A have similar onset and duration of action using the Digital Abduction Assay (DAS). Intramuscular LD50 values for Cyto-012 and wt BoNT/A respectively, were 0.63 ug (95% CI = 0.61, 0.66) and 6.22 pg (95% CI = 5.42, 7.02). ED50 values for Cyto-012 and wt BoNT/A were respectively, 0.030 ug (95% CI = 0.026, 0.034) and 0.592 pg (95% CI = 0.488, 0.696). The safety margin (intramuscular LD50/ED50 ratio) for Cyto-012 was found to be improved 2-fold relative to wt BoNT/A (p < 0.001). The DAS response to Cyto-012 was diminished when a second injection was administered 32 days after the first. These data suggest that the safety margin of BoNT/A can be improved by modulating their activity towards SNAP-25.

Introduction: The Ichtchenko laboratory has developed methods that enable facile production of purified recombinant derivatives of botulinum neurotoxins (BoNTs) that retain the structural and trafficking properties of wt BoNTs. Surprisingly, and unlike previously described recombinant BoNT derivatives, they remain physiologically active despite inactivating mutations to the light chain (LC) protease. As such, they are referred to as atoxic derivatives rather than nontoxic derivatives. In this study, we compared the biological activities of BoNT/A ad0 (E224 > A, Y336 > A) with more extensively modified BoNT/A ad derivatives. Methods: Recombinant BoNT/A derivatives and primary neuronal cultures derived from E19 rat hippocampus were prepared as previously described, and their toxicity determined using a modified murine LD50 assay. Mouse embryonic stem cell–derived neuron (ESN) cultures were tested at 34 d after differentiation. After 24 and 48 hours exposure to 25 nM BoNT/A derivative, monosynaptic miniature postsynaptic currents were measured. Intrinsic electrical characteristics of treated ESNs were measured to evaluate any potential cytotoxic activity. Results: BoNT/A ad0 is internalized into hippocampal neurons. It co-localizes with and cleaves SNAP-25. In ESN cultures, BoNT/A ad0 reduced synaptic activity by 88% at 24 hours and by 99% at 48 hours. The introduction of more extensive amino acid substitutions disabled in vitro detection of BoNT/A ad derivatives, such that they had no effect on synaptic activity in ESN cultures for up to 72 hours. None of the BoNT/A ad derivatives tested evoked evidence of cytotoxicity. Despite the effect of amino acid substitutions on physiological activities measured in vitro, all BoNT ad derivatives tested retained some level of toxicity in vivo, with LD50 values 100,000-fold to 500,00-fold greater than wt BoNT/A. Conclusion: To design atoxic BoNT derivatives as molecular vehicles for delivering drugs to the neuronal cytoplasm, metalloprotease and substrate-binding activity need to be carefully balanced.