Femtosecond laser-induced pain stimulation of dorsal root ganglion neurite induces signal conduction along the nerve fiber leading to the perception of pain. CADM1 expressed on the nerve cell, when bound by high affinity antibody A, fails to conduct the pain signal, and suppresses the perception of pain. Credit: Professor Akihiko Ito from Kindai University Faculty of Medicine

Cell surface proteins play a crucial role in cell communication and in sensing changes in the extracellular environment. Their ability to respond to external stimuli by altering the internal biological activity of the host cell makes them ideal targets for therapeutic manipulation.

Cell adhesion molecule 1 (CADM1) is one such protein expressed in various cell types, including neurons, respiratory, and endometrial epithelial cells, as well as mast cells. Recently, an antibody known as 3E1, which specifically binds to the extracellular domain of CADM1, has emerged as a promising tool for targeted drug delivery to CADM1-expressing cells.

Given the high expression of CADM1 in peripheral nerves and distribution along neurites, a question that arose is whether the anti-CADM1 antibodies have an effect on the biological activities of nerves.

Professor Akihiko Ito and Dr. Fuka Takeuchi from the Department of Pathology at Kindai University Faculty of Medicine, Japan, set out to seek answers to this critical question. They investigated the impact of anti-CADM1 antibodies on neuronal activity, and their findings were made available online on 22 August 2024 and published in the journal Life Sciences.

The team injected 3E1, the anti-CADM1 ectodomain antibody, under the mouse skin to study its localization on nerve fibers. Immunohistochemical and immunofluorescence studies revealed that the injected 3E1 was exclusively localized on peripheral nerves in the dermis.

The senior author of the study, Prof. Ito says, “As CADM1 can recruit neuronal receptors to the plasma membrane, we hypothesized that this accumulation of 3E1 may blunt neuronal sensitivity, i.e., have an analgesic effect, via altering the expression of CADM1 on nerve fibers. However, to our knowledge, there have been no studies that attempted to develop drugs in terms of inhibiting CADM1 in nerves.”

Analgesic effects were tested using a formalin-induced chemical-inflammatory pain test and video-recorded behavior analysis at 6-, 12-, and 24-hours post-injection. Mice injected with 3E1 exhibited less pain-related behaviors when compared with controls, with analgesic effects lasting up to 24 hours, which is significantly longer than the duration of 5 to 8 hours reported for the local anesthetic levobupivacaine.

In a formalin-based pain elicitation test, pre-treatment of mice with antibody A, protects the mice from experiencing pain and lessens pain-related behavioral changes. Credit: Professor Akihiko Ito from Kindai University Faculty of Medicine

Further investigations involved primary cultures of mouse dorsal root ganglion cells to study neuronal stimulation. Live cell imaging confirmed that 3E1 localized to the neurites, and protein analysis revealed that 3E1 formed a complex with CADM1 and decreased CADM1 expression. A femtosecond laser pulse was used to induce mechanical stimulation, with calcium fluorescence (Fluo-8) visualizing neuronal stimulation. The investigation revealed that neural transmission was markedly suppressed in 3E1-treated cells.

Notably, in this study no paralysis or behavioral abnormalities were observed in the treated mice, suggesting that 3E1 acts preferentially on sensory nerves rather than motor nerves.

The effects of 3E1 stand out in the field of pain management, since a decade-long effort to develop effective antibody drugs for osteoarthritis and chronic pain has met with limited success. Despite the initial promise, antibodies like tanezumab, fasinumab, and fulranumab, which targeted the nerve growth factor (NGF), faced setbacks due to severe side effects like ischemia and tissue necrosis. Unlike these NGF inhibitors, 3E1 offers a new, and safer pain relieving strategy with fewer drawbacks and more therapeutic potential.

Overall, the unique feature of antibody 3E1 to cluster CADM1 on the plasma membrane aids in its biological activity, and a single injection provides pain relief for a day or more, without motor paralysis or toxicity.

Prof. Ito said, “The identification of an antibody that, when injected, spontaneously accumulates in nerves, bringing about an analgesic effect is likely to open up a new field of discovering ‘antibody anesthetics.’ We believe that the present study is unique and significant in that it presented adhesion molecules as new targets, and our attempts to humanize 3E1 and the resulting clones are expected to result in long-acting analgesics.”