SECUAH 2025

dianas | Vol 14 No 1 | marzo 2026 | e202603x003

Preclinical evaluation of a CD44v6-specific scFv antibody for targeted bladder cancer therapy

Laura García-Gómez, Martina Turon-Orra, Lucía Morales, Cristian Suárez-Cabrera, Esther Montesinos, Ignacio A. Reina, Carolina Rubio, Marika Nestor, Jesús M. Paramio, Marta Dueñas, Iris Lodewijk

Instituto de Investigación Hospital 12 de Octubre. Centro de Actividades Ambulatorias, 6ª Planta Bloque D Avda. de Córdoba, s/n 28041 - MADRID)

Laura.Garcia@externos.ciemat.es

XI Congreso de Señalización Celular, SECUAH 2026.
XX Simposio de Dianas Terapéuticas.
16 a 18 de marzo, 2026. Universidad de Alcalá. Alcalá de Henares, Madrid. España.

Keywords: Bladder cancer; CD44v6; single-chain variable fragment (scFv); tumor targeting; antibody-based therapy

Abstract

One of the major challenges in antibody-based cancer therapy is the identification of specific tumor-associated antigens that enable selective targeting of malignant cells. CD44v6, an isoform of the CD44 glycoprotein, has recently been characterized as a novel tumor-associated antigen in bladder cancer (BC). Its expression correlates with poor clinical outcomes and contributes to tumor progression by promoting proliferation, migration, and invasion, highlighting its potential as a therapeutic target (Lodewijk et al., 2025, bioRxiv). In this study, we aimed to develop and comprehensively characterize a single-chain variable fragment (scFv) antibody directed against CD44v6 (scFv-CD44v6) as a tumor-targeting tool for BC. A scFv antibody against CD44v6 (scFv-CD44v6) was generated from a full-length humanized CD44v6 antibody (AKIR001), kindly provided by Prof. Nestor (Akiram Therapeutics). The binding specificity and selectivity of scFv-CD44v6 were assessed in vitro by flow cytometry through direct binding and competition assays using CD44v6-positive BC cell lines. To assess its in vivo targeting capacity, scFv-CD44v6 was fluorescently labeled and administered intraperitoneally to mice bearing orthotopic bladder tumors, followed by longitudinal in vivo bioluminescent and fluorescent imaging to monitor biodistribution and tumor accumulation. Subsequent ex vivo fluorescence imaging and immunofluorescence analyses were performed to validate tissue-specific localization. In vitro assays demonstrated highly specific binding of scFv-CD44v6 to CD44v6-expressing BC cells, including competitive binding with a commercial anti-CD44v6 antibody. In vivo imaging revealed preferential accumulation of scFv-CD44v6 in the bladder tumor area compared with control groups, indicating its tumor-targeting capability. Ex vivo analyses of resected bladder tumors confirmed these findings, showing strong fluorescent signal and antibody localization within tumor tissues, but not in healthy bladders. Taken together, our results demonstrate that scFv-CD44v6 exhibits high specificity toward CD44v6-expressing BC cells and efficient tumor localization in vivo, supporting its suitability as a tumor-targeting candidate. Overall, these findings underscore its potential for developing new antibody-based diagnostic and therapeutic strategies in BC.