Abstract
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 (alphaCD20). The constructs differed in affinity or avidity towards CD20 (dimeric alphaCD20-alphaCD20 and alphaCD20 fused to a non-targeting control sdAb, referred to as alphaCD20-ctrl) and blood half-lives (alphaCD20 fused to an albumin-targeting sdAb (alphaAlb)=alphaCD20-alphaAlb). 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-alphaCD20 decreased from 4.82+/-1.80 to 0.13+/-0.05% IA/g over 72h. Due to its rapid blood clearance, tumor- to-blood (T/B) ratios of >100 were obtained within 24h. Although in vitro internalization indicated that dimeric (177)Lu-DTPA-alphaCD20-alphaCD20 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 alphaCD20-alphaAlb. Blood levels of (177)Lu-DTPA-alphaCD20-alphaAlb decreased from 68.30+/-10.53 to 3.58+/-0.66% IA/g over 120h, 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 alphaCD20-alphaCD20 or the fusion of monomeric alphaCD20 to an albumin- targeting moiety (alphaAlb) counterbalance their improved tumor targeting capacity compared to monomeric alphaCD20.
