Pharmacokinetics of radiolabeled dimeric sdAbs constructs targeting human CD20.

Single-domain antibody fragments (sdAbs) are the smallest functional antigen-binding fragments, derived from heavy chain-only camelid antibodies. When designed as radiolabeled monomeric probes for imaging and therapy of cancer, their fast and specific targeting results in high tumor-to-background ratios early after injection. However, their moderate absolute uptake into tumors might not always be sufficient to treat cancerous lesions. We have evaluated the pharmacokinetics of seven constructs derived from a CD20-targeting monomeric sdAb (αCD20). The constructs differed in affinity or avidity towards CD20 (dimeric αCD20-αCD20 and αCD20 fused to a non-targeting control sdAb, referred to as αCD20-ctrl) and blood half-lives (αCD20 fused to an albumin-targeting sdAb (αAlb) = αCD20-αAlb). The constructs were radiolabeled with 111 In (imaging) and 177 Lu (therapy) using the bifunctional chelator CHX-A"-DTPA and evaluated in vitro and in vivo. In mice, tumor uptake of 177 Lu-DTPA-αCD20 decreased from 4.82 ± 1.80 to 0.13 ± 0.05% IA/g over 72 h. Due to its rapid blood clearance, tumor-to-blood (T/B) ratios of >100 were obtained within 24 h. Although in vitro internalization indicated that dimeric 177 Lu-DTPA-αCD20-αCD20 was superior in terms of total cell-associated radioactivity, this was not confirmed in vivo. Blood clearance was slower and absolute tumor uptake became significantly higher for αCD20-αAlb. Blood levels of 177 Lu-DTPA-αCD20-αAlb decreased from 68.30 ± 10.53 to 3.58 ± 0.66% IA/g over 120 h, while tumor uptake increased from 6.21 ± 0.94 to 24.90 ± 2.83% IA/g, resulting in lower T/B ratios. Taken together, these results indicate that the increased size of dimeric αCD20-αCD20 or the fusion of monomeric αCD20 to an albumin-targeting moiety (αAlb) counterbalance their improved tumor targeting capacity compared to monomeric αCD20.