Imaging-guided optimization of biodistribution and antitumor efficacy of L19-based immunocytokines

Antibody-based targeted delivery of pharmaceuticals is an attractive approach to preferentially localize anti-cancer payloads to neoplastic lesions. The L19 antibody, specific for the extra domain B of fibronectin, is used in several antibody-cytokine fusion proteins investigated in clinical trials involving different tumor types. However, improving the efficacy of L19-based immunotherapies requires a detailed understanding of how delivery strategies influence intratumoral distribution and therapeutic outcomes. In this study, we investigate the biodistribution of the L19 antibody in murine models of primary Eμ-myc lymphoma and metastatic MC38 colon carcinoma. Using high-resolution in vivo and ex vivo microscopy, we compared subcutaneous (s.c.) and intravenous (i.v.) administration of L19, revealing rapid accumulation in tumor invaded lymph nodes within 10-30 min post injection. While both routes enabled initial tumor targeting, i.v. injection led to longer retention (up to 72 h) and greater selectivity for tumor associated blood vasculature. In contrast, s.c. delivery favored transient accumulation near lymphatic vessels and exhibited reduced tumor residence. These distribution patterns directly influenced the therapeutic efficacy of the L19-IL2 immunocytokine, which showed superior tumor control following i.v. administration in the MC38 model, consistent with enhanced blood vascularization in this model. Our findings demonstrate that L19 binds both blood and lymphatic vasculature in primary and metastatic disease, underscoring the critical impact of the administration route on antibody biodistribution, microanatomical localization, and therapeutic outcome. Moreover, this work highlights the utility of microscopy guided analysis in optimizing delivery strategies and supports the rationale for tailoring administration routes based on tumor type and vascular context in antibody-based theranostics.