D-amino acid peptide residualizing agents bearing N-hydroxysuccinimido- and maleimido-functional groups and their application for trastuzumab radioiodination

Abstract

Introduction Proteins that undergo receptor-mediated endocytosis are subject to lysosomal degradation, requiring radioiodination methods that minimize loss of radioactivity from tumor cells after this process occurs. To accomplish this, we developed the residualizing radioiodination agent N∊-(3-[*I]iodobenzoyl)-Lys5-Nα-maleimido-Gly1-d-GEEEK (Mal-d-GEEEK-[*I]IB), which enhanced tumor uptake but also increased kidney activity and necessitates generation of sulfhydryl moieties on the protein. The purpose of the current study was to synthesize and evaluate a new d-amino acid based agent that might avoid these potential problems. Methods Nα-(3-iodobenzoyl)-(5-succinimidyloxycarbonyl)-d-EEEG (NHS-IB-d-EEEG), which contains 3 d-glutamates to provide negative charge and a N-hydroxysuccinimide function to permit conjugation to unmodified proteins, and the corresponding tin precursor were produced by solid phase peptide synthesis and subsequent conjugation with appropriate reagents. Radioiodination of the anti-HER2 antibody trastuzumab using NHS-IB-d-EEEG and Mal-d-GEEEK-IB were compared. Paired-label internalization assays on BT474 breast carcinoma cells and biodistribution studies in athymic mice bearing BT474M1 xenografts were performed to evaluate the two radioiodinated d-peptide trastuzumab conjugates. Results NHS-[131I]IB-d-EEEG was produced in 53.8 ± 13.4 % and conjugated to trastuzumab in 39.5 ± 7.6 % yield. Paired-label internalization assays with trastuzumab-NHS-[131I]IB-d-EEEG and trastuzumab-Mal-d-GEEEK-[125I]IB demonstrated similar intracellular trapping for both conjugates at 1 h (131I, 84.4 ± 6.1%; 125I, 88.6 ± 5.2%) through 24 h (131I, 60.7 ± 6.8%; 125I, 64.9 ± 6.9 %). In the biodistribution experiment, tumor uptake peaked at 48 h (trastuzumab-NHS-[131I]IB-d-EEEG, 29.8 ± 3.6 %ID/g; trastuzumab-Mal-d-GEEEK-[125I]IB, 45.3 ± 5.3 %ID/g) and was significantly higher for 125I at all time points. In general, normal tissue levels were lower for trastuzumab-NHS-[131I]IB-d-EEEG, with the differences being greatest in kidneys (131I, 2.2 ± 0.4 %ID/g; 125I, 16.9 ± 2.8 %ID/g at 144 h). Conclusion NHS-[131I]IB-d-EEEG warrants further evaluation as a residualizing radioiodination agent for labeling internalizing antibodies/fragments, particularly for applications where excessive renal accumulation could be problematic.

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

“…These results are similar to those obtained for [ 131 I]IB-Mal- d -GEEEK in the current study: radioiodination yields of 76.5 ± 10.9% ( n = 6), a radiochemical purity of 97.5 ± 1.1%, and 5F7 conjugation yields of 55.4 ± 10.1% ( n = 6). The conjugation efficiency for [ 125 I]IB-Mal- d -GDDDK also was comparable to the results obtained previously for the coupling of [ * I]IB-Mal- d -GEEEK to larger proteins [ 14 , 15 , 22 ], sdAbs [ 17 , 18 ], and those reported for other maleimide-containing agents used for radiolabeling biomolecules such as affibodies [ 30 ]. The molar activity for [ 125 I]IB-Mal- d -GDDDK-5F7 and [ 131 I]IB-Mal- d -GEEEK-5F7 was in the range of 0.4–7.1 MBq/nmol and 2.6–6.4 MBq/nmol, respectively, which largely reflected the activity level of the prosthetic agent used for conjugation.…”

“…This provided motivation for the current study investigating whether replacing d -glutamate (E) in the [ * I]IB-Mal- d -GEEEK template with d -aspartate (D) would reduce kidney accumulation of activity from radioiodinated internalizing proteins without affecting tumor uptake. A 5F7 sdAb was chosen for this comparison, because kidney activity is much more of an issue for sdAbs than mAbs [ 17 , 18 , 22 ]. In this study, [ * I]IB-Mal- d -GDDDK was synthesized, and 5F7 sdAb was radiolabeled using this prosthetic agent.…”

Understanding the in vivo fate of radioimmunoconjugates for nuclear imaging

“…While 89 Zr's multiday half-life allows for the collection of imaging data as late as 10 days after the administration of the radioimmunoconjugate, preclinical images taken at later time points often reveal relatively high activity concentrations in the bones of mice. It is well known that Zr 4+ is an osteophilic (bone-seeking) cation, as illustrated in several prior reports that used 93 Zr and 95 Zr. 128,129 This accumulation in bone suggests that DFO (Figure 9)-the most widely used chelator for 89 Zrmay allow for the release of the radiometal into the bloodstream or its transchelation to plasma proteins.…”

“…However, this phenomenon translates into far lower activity concentrations in tumor tissue compared with those created by radioimmunoconjugates labeled with residualizing radionuclides such as 90 To circumvent this limitation, several residualizing prosthetic groups have been developed to help trap the nuclide within the lysosomes of target cells. These include nonmetabolizable carbohydrates, [82][83][84][85][86] positively or negatively charged aromatic compounds, [87][88][89][90] bulky ionic polyhedral boron clusters, 77 and short D-amino acid peptides that are resistant to proteolytic degradation [91][92][93][94] ( Figure 6). Diethylenetriaminepentaacetic acid (DTPA) has also been added to some short D-amino acid peptides owing to its own residualizing properties.…”

“…95,96 In murine models of cancer, these strategies have been shown to increase the retention of radioiodine in tumor tissue as well as reduce the uptake of radioiodine in the thyroid. [82][83][84][85][86][87][88][89][90][91][92][93][95][96][97] Unfortunately, however, radiolabeling antibodies using these prosthetic groups is often less efficient than direct radioiodination. In addition, these residualizing prosthetic groups can also slow the clearance of radioactivity from nontarget tissues.…”