d-Amino Acid Peptide Residualizing Agents for Protein Radioiodination: Effect of Aspartate for Glutamate Substitution

Abstract

The residualizing prosthetic agent Nε-(3-[*I]iodobenzoyl)-Lys5-Nα-maleimido-Gly1-d-GEEEK ([*I]IB-Mal-d-GEEEK) showed promise for the radioiodination of monoclonal antibodies (mAbs) that bind to internalizing molecular targets. Although enhanced tumor uptake was achieved in these studies, elevated kidney accumulation also was observed, particularly with low-molecular-weight, single-domain antibody fragments (sdAbs). Here, we developed an analogous agent (IB-Mal-d-GDDDK), in which glutamate residues (E) were replaced with aspartates (D) to determine whether this modification could decrease renal uptake. [125I]IB-Mal-d-GDDDK and [131I]IB-Mal-d-GEEEK were synthesized with similar radiochemical yields (60–80%) and coupled to the anti-HER2 sdAb 5F7 at 50–60% efficiency. Paired-label internalization assays in vitro indicated similar levels of intracellular activity residualization in HER2-expressing BT474M1 cells for [125I]IB-Mal-d-GDDDK-5F7 and [131I]IB-Mal-d-GEEEK-5F7. A paired-label biodistribution comparison of the two labeled conjugates was performed in mice with HER2-expressing SKOV-3 xenografts, and the results of this study indicated that renal uptake at 1 h was 127.5 ± 18.7% ID/g and 271.4 ± 66.6% ID/g for [125I]IB-Mal-d-GDDDK-5F7 and [131I]IB-Mal-d-GEEEK-5F7, respectively. The tumor uptake of the two radioconjugates was not significantly different. These results demonstrate that substitution of E with D in the IB-Mal-d-GEEEK construct reduced kidney accumulation of the sdAb. However, renal activity levels need to be reduced further if d-amino acid derived prosthetic agents are to be of practical value for labeling low molecular weight biomolecules such as sdAbs.

Immuno-PET Imaging and Pharmacokinetics of an Anti-CEA scFv-based Trimerbody and Its Monomeric Counterpart in Human Gastric Carcinoma-Bearing Mice

“…With that aim, we decided to radiolabel the protein scaffold with 131 I and perform dissection and gamma counting experiments. For the radioiodination, different approaches both applied to intact IgG antibodies , and antibody fragments have been reported, showing a method-dependent accumulation of radioiodinated antibody fragments in tumor xenografts. However, none of the labeling methods has shown clear advantages in terms of the subsequent evaluation in vivo , and direct radioiodination has shown preserved immunoreactivity in anti-CEA scFv-Fc variants .…”

Imaging using radiolabelled targeted proteins: radioimmunodetection and beyond

“…Such labels are called residualizing labels. Strong residualizing properties are typical for radiometal-chelator complexes, but could also be observed when a radiohalogen is coupled to a polar or charged molecular moiety, which is non-degradable by proteolytic enzymes (Stein et al 1995;Pruszynski et al 2018). If radiometabolites are lipophilic, they can diffuse through lysosomal and cellular membranes and leak from cells to be further redistributed through the blood and excreted in the urine (Tolmachev et al 2003).…”

Synthesis and Applications of Synthetic Peptides

“…Selective prosthetic group conjugation is provided to the thiols with the help of pre-radioiodinated maleimides, and prelabeling of the corresponding peptide is carried out by the use of stannylated vinyl alkylating agents. In this way, tissue deiodinase and unfavorable structural conditions for radioiodination can be overcome [90]. [91].…”