Abstract
Immunotoxin (ITX)-mediated cell targeting enables selective elimination of neuronal types of interest from a complex neural network. In this technology, human interleukin-2 receptor α-subunit or CD25 (hCD25) is expressed in specific cell types in transgenic rodents, and then the animals are treated with a recombinant ITX composed of monoclonal antibody variable regions for hCD25 fused to a Pseudomonas exotoxin fragment (PE38), resulting in the ablation of hCD25-expressing cells. However, there is a critical issue on the cross-reactivity of the recombinant ITX for endogenous CD25 in non-human primates (NHPs), leading to off-target effects. Here we generated a mouse CD25 (mCD25)-specific recombinant ITX, termed anti-mCD25-PE38, based on variable regions of a rabbit monoclonal antibody that specifically reacts to mCD25, but not to hCD25. Anti-mCD25-PE38 showed high-affinity binding to mCD25 and cytotoxic activity toward mCD25-expressing cells. Injection of anti-mCD25-PE38 into the ventral midbrain of common marmosets, in which the mCD25 transgene was expressed in dopamine neurons by a lentiviral vector for retrograde gene transfer, induced a significant loss of midbrain dopamine neurons. Therefore, anti-mCD25-PE38 provides a useful strategy for selective targeting of neuronal types to study the behavioral and neurological functions of these neurons in the NHP brain.
Data availability
The datasets generated and/or analyzed during the current study are available in the Mendeley Data [https://data.mendeley.com/datasets/9zz9x8b3rs/2].
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Acknowledgements
We are grateful to Dr. Ira Pastan for providing the expression plasmid pRTK749K; to Minako Kikuchi, Yoko Nakasato, and Hiromi Hashimoto for their technical support in animal and histological experiments; and to Drs. Takeshi Machida and Hideharu Sekine for their help with plasmid DNA purification using an automated DNA extraction system.
Funding
This work was supported by grants-in-aid for Brain/MINDS 2.0 under Grant Number 25wm0625103h0002 (K.K.) and for Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research: BINDS) under Grant Number JP21am0101077 (T.O.) from the Japan Agency for Medical Research and Development; and a grant-in-aid for Scientific Research on Transformative Research Areas (A) Adaptive Circuit Census (21H05244) (K.K.) from the Ministry of Education, Science, Sports, and Culture of Japan.
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T. K., M.T., and K.K. conceived the study, designed the experiments, and directed the project. T.O., K.I., and K.K. designed the antibody screening, and T.O. performed ISAAC and recombinant antibody production. M.O., I.P., and T.O. generated the recombinant ITX. S.K., S.K., M.S., and K.I. performed the intracranial injections and histological/biochemical examinations. T.O., M.T., and K.K. wrote the manuscript. All authors discussed the results and their implications and commented on the manuscript at all stages.
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Kobayashi, T., Kato, S., Kimura, S. et al. Targeting of specific neuronal types in the non-human primate brain by using a murine CD25-specific recombinant immunotoxin. Sci Rep (2026). https://doi.org/10.1038/s41598-026-39662-6
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DOI: https://doi.org/10.1038/s41598-026-39662-6
