As opioid overdose rates continue to climb, one drug class has emerged as a deadly and pervasive culprit. Synthetic opioids such as fentanyl and its analog carfentanil (originally used as an elephant tranquilizer) are orders of magnitude more potent than morphine and even heroin, making them far more likely to cause respiratory depression. Fentanyl and carfentanil are also frequently added to illegal recreational substances such as cocaine, giving rise to unexpected and potentially life-threatening drug effects and interactions. In 2021, two out of five fentanyl-laced pills contained a possibly deadly dose of the drug, and synthetic opioids were responsible for almost 88 percent of all deaths due to opioid overdose (1,2).

Synthetic opioids are also resistant to current overdose treatment options. For example, bystanders can easily administer intranasal naloxone to quickly reverse respiratory depression due to opioid overdose (3). But with a half-life of approximately one hour, naloxone cannot compete with the three to seven hour half-life of fentanyl (4,5). “These synthetic opioids sit in your fat and slowly leak out. And when they leak out, then you can relapse again,” said Kim Janda, a chemist at Scripps Research Institute. Therefore, multiple doses of short-acting naloxone are required to prevent renarcotization over longer time periods (6). 

To overcome this limitation, Janda and his team developed a novel human antibody that binds to fentanyl and carfentanil. In a recent study in ACS Chemical Neuroscience, the researchers demonstrated that the antibody effectively reverses respiratory depression in an animal model of overdose and remains in the bloodstream for up to almost seven days (7). They hope that their work will provide a longer lasting treatment for overdose from dangerous synthetic opioids. 

To engineer their antibody, Janda’s team injected a chemically modified form of carfentanil into transgenic rats that express human antibodies. By using fully human antibodies, they aimed to eliminate the potential for cross-species reactivity and immune responses when the antibody is administered to humans. After injecting carfentanil multiple times for a period of 16 weeks, the researchers collected the rats’ spleens and lymph nodes.

The team then sorted B cells from the spleens and lymph nodes based on the cells’ reactivity with both carfentanil and fentanyl. They collected antibody-encoding mRNA from the B cells, combined it with sequences for human antibody backbones, and expressed this genetic material in cell culture. From this, the researchers produced 146 unique antibodies and characterized their ability to bind carfentanil, fentanyl, and other synthetic opioids. They identified C10-S66K as the top candidate due to its high affinity and specificity for this class of drugs.

The team then adapted C10-S66K from a full-sized antibody to a single-chain variable fragment to enable additional administration routes. While intravenous administration offers the fastest onset of activity, setting up an IV is not always feasible in settings of opioid overdose. Compared to a full antibody, a smaller fragment may be more quickly and easily circulated via an intramuscular route. 

The team then reengineered the surface of the antibody, introducing specific mutations to enhance its thermostability and pharmaceutical properties. Finally, they added a protein domain that binds to albumin, which is produced by hepatocytes and has a plasma half-life of over three weeks (8). In this way, the researchers increased blood absorption of the antibody and extended its half-life from 0.9 to 47 hours.

With the candidate ready for testing, the researchers administered a dose of carfentanil that causes respiratory depression in mice and, after 15 minutes, treated the mice with the C10-S66K antibody, naloxone, or saline. Mice that received the antibody showed significantly greater recovery from respiratory depression compared to saline-treated mice. The naloxone-treated group showed some initial recovery from respiratory depression, but the effect was outlived by the slow-leaching opioid.

We’re looking to see if we can attack this poly-substance abuse problem using multifunctional antibodies. 
– Kim Janda, Scripps Research Institute

In addition to its longevity, another advantage of C10-S66K is that it binds to and sequesters the opioid rather than blocking specific receptors. “People think that a lot of the overdose issues with fentanyl and carfentanil are due to actions other than actions at opiate receptors,” said John Traynor, a neuropharmacologist at University of Michigan who was not involved in the study. “Traditional treatments don’t work at those [other] receptors, but by just simply taking the drug away, this would work.” 

Despite efforts to optimize its circulation, C10-S66K may still not be fast-acting enough to reverse overdose in humans. “It’s got a fairly slow onset of action. So, people would be dead before it was working, I think,” Traynor said. He envisions that C10-S66K could be used in tandem with naloxone as the two drugs do not cross react. A bystander could administer intranasal naloxone to immediately reverse the overdose state, and once paramedics arrive onto the scene, they could provide an intramuscular injection of the antibody for long-term protection against renarcotization.

Janda’s team has licensed C10-S66K to the biotechnology company Cessation Therapeutics for further studies. Given the high cost of production for monoclonal antibodies, early collaboration with a biotechnology company may provide better opportunities for commercializing the antibody affordably for at-risk communities. The researchers also plan to adapt their approach to target multiple overdose-causing drugs at once, which better reflects how drugs are used. “We’re looking to see if we can attack this poly-substance abuse problem using multifunctional antibodies,” Janda said. “That would be our long-term goal.”  

References

1. Centers for Disease Control and Prevention. Drug overdose deaths in the United States, 2001 – 2021. 
2. Drug Enforcement Administration. DEA issues public safety alert on sharp increase in fake prescription pills containing fentanyl and meth. 
3. Zuckerman, M., Weisberg, S., & Boyer, E. Pitfalls of intranasal naloxone. Prehosp Emerg Care  18, 550-554 (2014).
4. Lynn, R.R. & Galinkin, J.L. Naloxone dosage for opioid reversal: current evidence and clinical implications. Ther Adv Drug Saf  9, 63-68 (2018).
5. Ramos-Matos, C.F., Bistas, K.G., & Lopez-Ojeda, W. Fentanyl. StatPearls (2023). 
6. Moss, R. & Carlo, D. Higher doses of naloxone are needed in the synthetic opioid era. Subst Abuse Treat Prev Policy  14, 6 (2019).
7. Eubanks, L.M. et al. An engineered human-antibody fragment with fentanyl pan-specificity that reverses carfentanil-induced respiratory depression. ACS Chemical Neuroscience  14, 2849-2856 (2023). 
8. Dennis, M.S. et al. Albumin binding as a general strategy for improving the pharmacokinetics of proteins. Journal of Biological Chemistry  277, 35035-35043 (2002).