Monodispersed DOTA-PEG–Conjugated Anti-TAG-72 Diabody Has Low Kidney Uptake and High Tumor-to-Blood Ratios Resulting in Improved⁶⁴Cu PET

Abstract

Diabodies are non-covalent dimers of single chain antibody fragments (scFvs) that retain the avidity of intact IgG but have more favorable blood clearance than intact IgGs. Radiometals offer a wide range of half lives and emissions for matching imaging and therapy requirements and provide facile labeling of chelate-antibody conjugates. However, due to their high retention and metabolism in the kidney, use of radiometal labeled diabodies can be problematic for both imaging and therapy. Methods Having previously shown that 111In-DOTA-PEG3400-anti-CEA-diabody has similarly high tumor uptake and retention and less than 50% as much kidney uptake and retention as non-PEGylated diabody, we synthesized a similar derivative for an anti-TAG-72-diabody. We also reduced the molecular size of the polydispersed PEG3400 to monodispersed PEG27 and PEG12 (nominal masses of 1188 and 528, respectively). We performed biodistributions of their DOTA conjugates radiolabeled with 125I, 111In, or 64Cu in tumor bearing athymic mice. Results Addition of PEG3400 to the diabody reduced kidney uptake to a level (≈10 %ID/g) comparable to that obtained with radiometal labeled intact IgG. The PEG27 and PEG12 diabody conjugates also demonstrated low kidney uptake without reduction of tumor uptake or tumor to blood ratios. When radiolabeled with 64Cu, the DOTA-PEG12- and PEG27-diabody conjugates gave high contrast PET images of colon cancer xenografts in athymic mice. Conclusion PEGylated diabodies may be a valuable platform for delivery of radionuclides and other agents to tumors.

Diabody Pretargeting with Click Chemistry In Vivo

“…1B) (24), the synthesis of the axial isomer of TCO-oxymethylbenzamide-N-hydroxysuccinimide (26), and AVP04-07 production have been described elsewhere. AVP04-07, a stable, approximately 55-kDa diabody format of monoclonal antibody CC49, was produced by dimerization of single-chain Fv fragments with short (5-residue) linkers in a bacterial periplasm fermentation system (21,26).…”

“…1A). The results of the diabody pretargeting approach were compared with previously published data for a conventional radiometal-labeled TAG72-binding diabody analog that showed high tumor targeting and fast blood clearance but high renal retention (21).…”

“…One example is the conjugation of polyethylene glycol (PEG) to antibody fragments to increase the molecular weight beyond the renal molecular weight cutoff of 60 kDa, resulting in an impressive reduction in kidney uptake (21,22). However, because of the reduction in glomerular filtration, these PEGylated antibody fragments have a relatively long circulation time.…”

“…In comparison with our previous pretargeting work on antibodies with a long circulation time (23)(24)(25), the relatively fast clearance of low-molecular-weight agents is expected to result in high tumor-to-blood ratios without the need for clearing agents. Among antibody fragments, diabodies (;55 kDa) are of particular interest because they provide greater avidity than other agents, including monovalent single-chain Fv fragments, and therefore typically show higher tumor uptake (2,21,22). In the present study, we explored the use of a TAG72-binding diabody, AVP04-07 (21), as a model construct for a pretargeting approach in LS174T tumorbearing mice (Fig.…”

“…In recent years, several strategies that reduce the renal uptake of radiometal-labeled low-molecular-weight agents have been developed (6,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). One example is the conjugation of polyethylene glycol (PEG) to antibody fragments to increase the molecular weight beyond the renal molecular weight cutoff of 60 kDa, resulting in an impressive reduction in kidney uptake (21,22).…”