title: Use of antibody as carrier of oligomers of peptidomimetic alphavbeta3 antagonist to target tumor-induced neovasculature.
abstract: Sulfhydryl selective reactions were explored to conjugate oligomers of a peptidomimetic integrin alphavbeta3 antagonist, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonylamino-beta-alanine (IA) to monoclonal antibody (MoAb) to increase integrin alphavbeta3 receptor-binding avidity. To generate sulfhydryl groups, N-succinimidyl-S-acetylthioacetate (SATA) was conjugated to both MoAb and IA. Sulfhydryl groups were then generated upon the deacetylation of the protecting acetyl group from the S-acetylthioacetato (ATA) moiety of MoAb-(ATA)n or IA-ATA with 0.02 M hydroxylamine in the presence of 1 mM EDTA at pH 7.2. The major focus was on optimizing the reaction concentrations, molar ratios, and reaction pH to conjugate high levels of IA-(A-SH) to MoAb-(A-SH)n without causing the inter- and intramolecular cross-linking of MoAb. Stepwise reactions of MoAb-(A-SH)n (15 microM MoAb) with a homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol (BM[PEO]2) at a >50x molar excess to the -SH, followed by the reaction of the purified product MoAb-(A-S-succinimidomaleimido-[PEO]2)n with IA-(A-SH) at pH 7.2 afforded monomeric MoAb-(A-S-succinimido-[PEO]2-succinimido-S-A-IA)n with <10% high molecular weight oligomeric MoAb. Monomeric MoAb-(A-S-S-[PEO]2-S-S-A-IA)10 (MoAb-IA10) radiolabeled with 111In using 2-(p-isothiocyanatobenzyl)cyclohexyl-DTPA and with 125I using the Iodogen method showed >70% bindability to 0.4 microM alphavbeta3. When injected iv to nude mice with the receptor-positive M21 tumor, MoAb-IA10 radiolabeled with both 111In and 125I accumulated rapidly and was retained in the tumor for a 44 h period while the radioactivity cleared rapidly from the blood, thereby resulting in increasing tumor-to-blood ratios over time. The tumor uptake was similar between the 125I label and the 111In label for a 44 h period. In contrast, the blood radioactivity was lower, but liver and other organ uptakes were much higher for the 111In label than for the 125I. The 111In label produced higher tumor-to-blood ratios but much lower tumor-to-organ ratios than the 125I. The rapid blood clearance, a short peak tumor uptake time, and a low peak tumor uptake value with prolonged tumor retention of this macromolecule appear to support a hypothesis that MoAb-IA10 primarily binds to alphavbeta3 receptors on angiogenic vessels, but not on the tumor. This hypothesis was substantiated by the fluorescence microscopic analysis of FITC-MoAb-IA10, which showed that FITC-MoAb-IA10 outlined neovasculatures but not tumor cells at 4 and 21 h ex vivo. Additional proof was observed when blood vessels outlined with rhodamine-lectin, which specifically binds to blood vessels, were superimposable on neovasculatures outlined with FITC-MoAb-IA10.
authors: Department of Nuclear Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors: In Soo
authors: IS
authors: Shin
authors: Beom-Su
authors: BS
authors: Jang
authors: S Narasimhan
authors: SN
authors: Danthi
authors: Jianwu
authors: J
authors: Xie
authors: Sarah
authors: S
authors: Yu
authors: Nhat
authors: N
authors: Le
authors: Jin-Soo
authors: JS
authors: Maeng
authors: In Sook
authors: IS
authors: Hwang
authors: King C P
authors: KC
authors: Li
authors: Jorge A
authors: JA
authors: Carrasquillo
authors: Chang H
authors: CH
authors: Paik
mesh_terms: D020533:Angiogenesis Inhibitors / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics*; D040761:Biomimetic Materials / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics; D045744:Cell Line, Tumor; D004337:Drug Carriers / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D006801:Humans; D007205:Indium Radioisotopes / Q000737:chemistry; D039302:Integrin alphaVbeta3 / Q000037:antagonists & inhibitors*; D007457:Iodine Radioisotopes / Q000737:chemistry; D051379:Mice; D008815:Mice, Inbred Strains; D009369:Neoplasms / Q000098:blood supply / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000276:immunology / Q000378:metabolism*; D013388:Succinimides / Q000737:chemistry; D013440:Sulfides / Q000737:chemistry; D013449:Sulfonamides / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics*; D014018:Tissue Distribution; D023041:Xenograft Model Antitumor Assays; D015091:beta-Alanine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000737:chemistry / Q000493:pharmacokinetics
chemical_list: C521824:4-(2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)ethyloxy)benzoyl-2-aminoethylsulfonylamino-beta-alanine; D020533:Angiogenesis Inhibitors; D000911:Antibodies, Monoclonal; D004337:Drug Carriers; D007205:Indium Radioisotopes; D039302:Integrin alphaVbeta3; D007457:Iodine Radioisotopes; D013388:Succinimides; D013440:Sulfides; D013449:Sulfonamides; D015091:beta-Alanine; C039011:N-hydroxysuccinimide S-acetylthioacetate; D016650:Fluorescein-5-isothiocyanate