https://phaidra.vetmeduni.ac.at/o:2223 Rabbit derived VL single-domains as promising scaffolds to generate antibody-drug conjugates André, Ana S. Ana S. André University of Lisbon / Associate Laboratory for Animal and Veterinary Sciences Aires-da-Silva, Frederico Frederico Aires-da-Silva University of Lisbon / Associate Laboratory for Animal and Veterinary Sciences Tavares, Luís Luís Tavares University of Lisbon / Associate Laboratory for Animal and Veterinary Sciences Gil, Solange Solange Gil University of Lisbon / Associate Laboratory for Animal and Veterinary Sciences Gois, Pedro M. P. Pedro M. P. Gois Universidade de Lisboa Goncalves, Joao Joao Goncalves Universidade de Lisboa Castanho, Miguel Miguel Castanho Universidade de Lisboa Correia, João D. G. João D. G. Correia Universidade de Lisboa Gano, Lurdes Lurdes Gano Universidade de Lisboa Rütgen, Barbara Barbara Rütgen 0000-0001-9080-0164 University of Veterinary Medicine Vienna Braz, Berta São Berta São Braz University of Lisbon / Associate Laboratory for Animal and Veterinary Sciences Carrapiço, Belmira Belmira Carrapiço University of Lisbon / Associate Laboratory for Animal and Veterinary Sciences Vicente, Gonçalo Gonçalo Vicente Universidade de Lisboa Oliveira, Soraia Soraia Oliveira Technophage SA Neves, Vera Vera Neves Universidade de Lisboa Cavaco, Marco Marco Cavaco Universidade de Lisboa António, João P M João P M António Universidade de Lisboa Carvalho, Joana Inês Joana Inês Carvalho University of Lisbon Bule, Pedro Pedro Bule University of Lisbon / Associate Laboratory for Animal and Veterinary Sciences Nogueira, Sara Sara Nogueira University of Lisbon / Associate Laboratory for Animal and Veterinary Sciences Aguiar, Sandra Sandra Aguiar University of Lisbon / Associate Laboratory for Animal and Veterinary Sciences Dias, Joana N. R. Joana N. R. Dias University of Lisbon / Associate Laboratory for Animal and Veterinary Sciences Nature Portfolio text eng Antibody-drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer. Therapeutic Activity; Next-Generation; Strategies; Selection; Peptide http://creativecommons.org/licenses/by/4.0/ https://phaidra.vetmeduni.ac.at/api/object/o:2223/download 10.1038/s41598-023-31568-x https://phaidra.vetmeduni.ac.at/o:605 application/pdf Scientific Reports 13(1) (2023) Scientific Reports 13 1 863.16 kB 2023