Site Specific Discrete PEGylation of ¹²⁴I-Labeled mCC49 Fab′ Fragments Improves Tumor MicroPET/CT Imaging in Mice

Abstract

The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2–4 kDa discrete, branched PEGylation reagents on mCC49 Fab′ (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab′ (Fab′-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 (124I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab′ with Mal-dPEG-A (Fab′-A) reduced the binding affinity of the non PEGylated Fab′ by 30%; however, in vivo, Fab′-A significantly lengthened the blood retention vs Fab′-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p < 0.01), showed excellent tumor to background, better microPET/CT images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p < 0.05). Despite the strong similarity of the three PEGylation reagents, PEGylation with Mal-dPEG-B or -C reduced the in vitro binding affinity of Fab′-NEM by 70%, blood retention, microPET/CT imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A.

Mucins and associated glycan signatures in colon adenoma–carcinoma sequence: Prospective pathological implication(s) for early diagnosis of colon cancer

“…It is overexpressed in several malignancies, including CRC, and is considered as an early marker for CRC and other dysplastic colonic diseases [42, 43]. The present results regarding TAG72 expression are consistent with those previously reported studies.…”

A 3E8.scFv.Cys-IR800 Conjugate Targeting TAG-72 in an Orthotopic Colorectal Cancer Model

“…This is generally consistent with the behavior of radiolabeled antibodies and fragments, where lower molecular weight correlates with more rapid blood clearance [49]. The scFv-sized fragments are desirable when more rapid blood clearance and renal excretion is useful for the clinical indication, and some kidney retention is tolerable.…”

“…An in vitro test of the biopsy specimen [41], followed by TAG-72 specific PET/CT would be used to select surgical candidates [49]. Then, simultaneously applied TAG-72 OSN plus RADS would guide potentially curative surgery on this well-selected population, avoiding the common finding of unexpected extensive disease that requires immediate termination of the surgery [9].…”

Solid-Phase Bioconjugation of Heterobifunctional Adaptors for Versatile Assembly of Bispecific Targeting Ligands

“…6,7 Toward this end, many strategies have been exploited, such as Dock-and-Lock, 8,9 chemical cross-linking, 10,11 peptide nucleic acid conjugation via unnatural amino acids, 12 hybrid–hybridoma, 13 assembly via short synthetic peptides, 14 and genetic engineering. 15,16 However, current methods suffer from high complexity and cost of engineering of individual constructs, hampering high-throughput production of bispecific molecules.…”