Two research groups have designed chimeric fusion proteins that constitute a new “trojan horse” approach to neutralize lethal botulinum toxins.

Botulinum neurotoxins (BoNTs)—a family of bacterial toxins with seven major serotypes (BoNT/A to BoNT/G)—are classified by the United States Centers for Disease Control and Prevention (CDC) as one of the most dangerous bioterrorism agents. BoNTs infiltrate into motor neurons, block neurotransmitter release, and stop essential functions such as breathing.

Chimeric therapeutic fusion protein
Chimeric therapeutic fusion protein created by fusing a single-domain antibody against BoNT/A with the delivery platform (yellow: nanobody; blue: BoNT/X light chain and translocation domain; red: BoNT/A receptor-binding domain) [Source: Min Dong]
“It’s not killing the neurons, but it silences them,” said Konstantin Ichtchenko, PhD, assistant professor, department of biochemistry and molecular pharmacology, NYU Grossman School of Medicine.

Once paralysis sets in, the only therapeutic option is to put patients on ventilators and allow the toxin to wear off, which can be weeks or months. The failure in developing botulinum anti-toxins is primarily because the toxin sequesters within nerve terminals—a challenging target for delivery systems of therapeutic molecules.

“Currently, there are anti-toxins, but these only work before the toxins enter the motor neurons,” said Min Dong, PhD, assistant professor in the Boston Children’s Hospital department of urology and corresponding author on the paper. “What we have developed is the first therapy that can eliminate toxins after they get inside neurons.”

McNutt, Patrick
Patrick M. McNutt, PhD, associate professor at the Wake Forest Institute for Regenerative Medicine

Two independent studies have used nontoxic derivatives of BoNT to deliver therapeutic antibodies that bind and neutralize these toxins within neurons.

The study led by Shin-Ichiro Miyashita, PhD, a postdoctoral fellow in Dong’s lab and now assistant professor at Tokyo University of Agriculture, targeted BoNT/A and BoNT/B and reported therapeutic effects in mice, in an article titled, “Delivery of single-domain antibodies into neurons using a chimeric toxin–based platform is therapeutic in mouse models of botulism.”

Using a similar approach to target BoNT/A, another study titled, “Neuronal delivery of antibodies has therapeutic effects in animal models of botulism,” showed increased survival in mice, guinea pigs, and monkeys after lethal exposures, and was led by co-first authors Patrick M. McNutt, PhD, and  Edwin J. Vazquez-Cintron, PhD., of the Ichtchenko group.

Konstantin Ichtchenko
Konstantin Ichtchenko, PhD, assistant professor, department of biochemistry and molecular pharmacology, NYU Grossman School of Medicine

Both studies published in the January issue of Science and Translational Medicine can potentially be harnessed to develop a safe and effective treatment against BoNT and target other intracellular proteins in neurons.

“This is one of those serendipitous moments in science where two groups, working independently, demonstrate similar results for a long-standing problem,” McNutt said. “We are currently modifying this drug to enhance its therapeutic properties against botulism and exploring whether the same approach can be repurposed to treat other neuronal diseases.”

Dong says both teams began working on similar approaches based on inactive botulinum neurotoxins, an idea many groups have explored. “It works but has a flaw of residual toxicity that has been very difficult to get rid of,” said Dong. “The two groups then took different approaches. The [Ichtchenko] group pushed ahead to test an inactive botulinum neurotoxin-based system on large animal models and obtained successful therapeutic results, although their therapeutic dose would be capped by the concern of safety, limiting the efficacy. We explored a new avenue and utilized a new toxin recently discovered in our lab as the delivery platform, which showed no residual toxicity. This allowed us to develop a safe therapeutic protein, which achieved highly effective therapeutic effects as we can use the protein at much higher doses than before.”

Shin-Ichiro Miyashita
Shin-Ichiro Miyashita, PhD, assistant professor at Tokyo University of Agriculture

McNutt said: “Dr. Ichtchenko first proposed the idea of using an atoxic version of BoNT as a ‘trojan horse’ to deliver therapeutic anti-botulism antibodies to peripheral neurons around 2010. He developed several versions of this drug based on different types of Botulinum neurotoxins, finally settling on an inactivated version of BoNT/C around 2016. In 2018, Dr. Min Dong discovered the BoNT/X derivative. He then repurposed Dr. Ichtchenko’s approach using BoNT/X. We were not aware he was doing so. It was essentially chance that these two manuscripts were ready to be submitted at nearly the same time.”

Dong said, “We provided the first post-symptom treatment of botulism, that also potentially can be used to quickly reverse Botox-induced paralysis in clinics.” Dong’s team hopes to move the drug into clinical trials as soon as possible.

“Our goal is both to prepare treatments that accelerate recovery for sparse, natural cases of botulism while simultaneously developing options for mass-casualty scenarios. In the bigger picture, we believe that a similar Trojan Horse approach can be repurposed to deliver other therapeutic proteins and antibodies to motor and sensory nerve terminals, providing the first neuron-specific delivery platform for therapeutic development,” said McNutt.

Min Dong
Min Dong, PhD, research associate, assistant professor, Boston Children’s Hospital

Commenting on the significance of the study in the context of biological insights into botulism mediated neurotoxicity, Dong said, “The findings demonstrate that the persistent paralysis of botulism is due to toxins maintaining their activities within neurons for long period.”

Botulinum neurotoxins are made of a heavy chain that allows the toxin to enter neurons and, and an enzymatic light chain that inactivates specific neuronal proteins. “Our molecular delivery vehicle is composed of a wild-type heavy chain attached to a mutant, enzymatically inactive light chain. It was surprising that the molecular delivery vehicle still exhibits a transient toxicity at doses about 100,000-fold higher than the native toxin,” said McNutt. This indicates a non-enzymatic form of toxicity that comes into play only at very high doses.

“The lack of toxicity in the chimeric toxin produced by Dr. Dong is very interesting, and contributes to the hypotheses we are currently testing,” said McNutt. The team is currently investigating the non-enzymatic cause of the toxicity of their molecular delivery vehicle.

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