TY - JOUR
T1 - Site specific discrete PEGylation of 124I-labeled mCC49 Fab′ fragments improves tumor MicroPET/CT imaging in mice
AU - Ding, Haiming
AU - Carlton, Michelle M.
AU - Povoski, Stephen P.
AU - Milum, Keisha
AU - Kumar, Krishan
AU - Kothandaraman, Shankaran
AU - Hinkle, George H.
AU - Colcher, David
AU - Brody, Rich
AU - Davis, Paul D.
AU - Pokora, Alex
AU - Phelps, Mitchell
AU - Martin, Edward W.
AU - Tweedle, Michael F.
PY - 2013/11/20
Y1 - 2013/11/20
N2 - 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-dPEG 12-(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.
AB - 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-dPEG 12-(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.
UR - http://www.scopus.com/inward/record.url?scp=84888584556&partnerID=8YFLogxK
U2 - 10.1021/bc400375f
DO - 10.1021/bc400375f
M3 - Article
C2 - 24175669
AN - SCOPUS:84888584556
SN - 1043-1802
VL - 24
SP - 1945
EP - 1954
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
IS - 11
ER -