title: Site specific discrete PEGylation of (124)I-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-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 ((124)I) 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.
authors.affiliation: Department of Radiology, The Wright Center for Innovation in Biomedical Imaging, ‡Department of Surgery, and ⊥Department of Pharmacology, The Ohio State University , Columbus, Ohio 43210, United States.
authors.forename: Haiming
authors.initials: H
authors.lastname: Ding
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Carlton
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Povoski
authors.forename: Keisha
authors.initials: K
authors.lastname: Milum
authors.forename: Krishan
authors.initials: K
authors.lastname: Kumar
authors.forename: Shankaran
authors.initials: S
authors.lastname: Kothandaraman
authors.forename: George H
authors.initials: GH
authors.lastname: Hinkle
authors.forename: David
authors.initials: D
authors.lastname: Colcher
authors.forename: Rich
authors.initials: R
authors.lastname: Brody
authors.forename: Paul D
authors.initials: PD
authors.lastname: Davis
authors.forename: Alex
authors.initials: A
authors.lastname: Pokora
authors.forename: Mitchell
authors.initials: M
authors.lastname: Phelps
authors.forename: Edward W
authors.initials: EW
authors.lastname: Martin
authors.forename: Michael F
authors.initials: MF
authors.lastname: Tweedle
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology; D003110:Colonic Neoplasms / Q000175:diagnosis*; D005260:Female; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology; D007457:Iodine Radioisotopes / Q000737:chemistry; D051379:Mice; D008819:Mice, Nude; D015394:Molecular Structure; D064847:Multimodal Imaging / Q000379:methods; D009374:Neoplasms, Experimental / Q000175:diagnosis*; D011092:Polyethylene Glycols / Q000737:chemistry*; D049268:Positron-Emission Tomography / Q000379:methods*; D014057:Tomography, X-Ray Computed / Q000379:methods*
chemical_list: D000951:Antigens, Neoplasm; D007140:Immunoglobulin Fab Fragments; D007457:Iodine Radioisotopes; D011092:Polyethylene Glycols