Site‐Specific Radioiodination of HER2‐Targeting Affibody Molecules using 4‐Iodophenethylmaleimide Decreases Renal Uptake of Radioactivity

Abstract

Affibody molecules are small scaffold-based affinity proteins with promising properties as probes for radionuclide-based molecular imaging. However, a high reabsorption of radiolabeled Affibody molecules in kidneys is an issue. We have shown that the use of 125I-3-iodo-((4-hydroxyphenyl)ethyl)maleimide (IHPEM) for site-specific labeling of cysteine-containing Affibody molecules provides high tumor uptake but low radioactivity retention in kidneys. We hypothesized that the use of 4-iodophenethylmaleimide (IPEM) would further reduce renal retention of radioactivity because of higher lipophilicity of radiometabolites. An anti-human epidermal growth factor receptor type 2 (HER2) Affibody molecule (ZHER2:2395) was labeled using 125I-IPEM with an overall yield of 45±3 %. 125I-IPEM-ZHER2:2395 bound specifically to HER2-expressing human ovarian carcinoma cells (SKOV-3 cell line). In NMRI mice, the renal uptake of 125I-IPEM-ZHER2:2395 (24±2 and 5.7±0.3 % IA g−1at 1 and 4 h after injection, respectively) was significantly lower than uptake of 125I-IHPEM-ZHER2:2395 (50±8 and 12±2 % IA g−1at 1 and 4 h after injection, respectively). In conclusion, the use of a more lipophilic linker for the radioiodination of Affibody molecules reduces renal radioactivity.

Al18F-Labeling Of Heat-Sensitive Biomolecules for Positron Emission Tomography Imaging

“…Affibody molecules are non-immunoglobulin-based scaffold proteins characterized by high target specificity and binding affinity (Kd in the low-nanomolar to picomolar range) 52 , 53 . These small proteins have been generated against different cancer-associated molecular targets and have been labeled with different radionuclides ( 68 Ga, 18 F, 64 Cu, 99m Tc, and 111 In) suitable for immuno-PET and SPECT 54 - 59 .…”

Influence of Residualizing Properties of the Radiolabel on Radionuclide Molecular Imaging of HER3 Using Affibody Molecules

“…[ 125 I]I-para-iodobenzoate is only one of the potential pendant groups providing a non-residualizing radioiodine label. Alternatively, labeled iodo-hydroxyphenylethyl-maleimide (I-HPEM) [55,56] or 4-iodophenethylmaleimide (I-PEM) [57] can be used for indirect radioiodination of affibody molecules. These alternative pendant groups have different rates of excretion from normal tissues compared to PIB, which opens an opportunity for finding the most suitable variant.…”

Affibody Molecules as Targeting Vectors for PET Imaging

“…The release of activity from kidneys depends on the radioiodine-bearing prosthetic group and the position of its coupling to an affibody molecule. Therefore, the clearance of activity from kidneys is quicker when using 3-iodo-((4-hydroxyphenyl) ethyl)maleimide [55] and 3-bromo-((4-hydroxyphenyl)ethyl)maleimide [18] (Figure 2) than using 4-iodobenzoate and 4-bromobenzoate. The use of iodophenetylmaleimide ( Figure 2) as a precursor resulted in an even more rapid clearance of activity from kidneys [55].…”

“…Therefore, the clearance of activity from kidneys is quicker when using 3-iodo-((4-hydroxyphenyl) ethyl)maleimide [55] and 3-bromo-((4-hydroxyphenyl)ethyl)maleimide [18] (Figure 2) than using 4-iodobenzoate and 4-bromobenzoate. The use of iodophenetylmaleimide ( Figure 2) as a precursor resulted in an even more rapid clearance of activity from kidneys [55]. Similarly, low renal uptake was associated with the use of non-residualizing radiofluorine labels N-2-(4-[ 18 [59] (Figure 3).…”

“…Cobalt-55 (T 1 2 = 17.5 h, positron yield 76%) might be a better alternative from a dosimetry point of view. This radionuclide can be produced using low-energy cyclotrons [116], and the broad availability of a long-lived surrogate 57 Co (T 1 2 = 275 d) simplifies the preclinical development of 55 Co-labeled probes [117]. It has been demonstrated that NOTA-and DOTA-conjugated recombinant HER2-, EGFR-, and HER3-binding affibody molecules can be quantitatively labeled with 55/57 Co by heating at 60 • C in 0.2 M ammonium acetate buffer at pH 5.5 for 30 min [51,101,118].…”