Tumor Uptake of Anti-CD20 Fabs Depends on Tumor Perfusion

Antibodies have become an established treatment modality in cancer therapy during the last decade. However, these treatments often suffer from an insufficient and heterogeneous response despite validated antigen or target receptor expression in the tumor. In fact, therapeutic success depends on both the presence of the tumor antigen and its accessibility by the antibody. In search of a suitable preclinical animal model to evaluate the mechanisms of tumor heterogeneity and hemodynamics, we characterized two exemplary non-Hodgkin lymphoma subtypes with comparable CD20 expression and metabolism, SUDHL-4 and Granta-519, using multimodal imaging techniques.

To investigate in vivo biodistribution, two differently modified αCD20 antigen-binding fragments (Fab), prepared by PASylation with a 200-residue polypeptide tag comprising Pro, Ala, and Ser (PAS₂₀₀) and by fusion with an albumin-binding domain (ABD), were radiolabeled with ¹²⁵I and intravenously injected into immunocompromised mice bearing corresponding xenografts.

Validation with ¹⁸F-FDG revealed a similar distribution in vital tumor tissue 1 h after injection. However, large differences in tumor uptake were observed when the CD20-specific radiotracers ¹²⁵I-Fab-ABD and ¹²⁵I-Fab-PAS₂₀₀ were applied (respective percentages injected dose per gram at 24 h after injection: 12.3 and 2.4 for Granta-519 vs. 5.8 and 1.2 for SUDHL-4). Three-dimensional light-sheet fluorescence microscopy with Cy5-Fab-PAS₂₀₀ confirmed better tracer extravasation in the Granta-519 tumors. Moreover, dynamic contrast-enhanced (DCE) MRI revealed significantly reduced perfusion in the SUDHL-4 tumors.

Tracer uptake was highly dependent on local tumor perfusion and Fab permeation in the SUDHL-4 and Granta-519 tumors. Thus, the SUDHL-4 xenograft offers an excellent model for investigating the influence of therapies affecting tumor angiogenesis.