Use of antibody as carrier of oligomers of peptidomimetic αvβ3 antagonist to target tumor-induced neovasculature

Soo Shin In, Beom Su Jang, S. Narasimhan Danthi, Jianwu Xie, Sarah Yu, Nhat Le, Jin Soo Maeng, Sook Hwang In, King C.P. Li, Jorge A. Carrasquillo, Chang H. Paik

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Sulfhydryl selective reactions were explored to conjugate oligomers of a peptidomimetic integrin αvα3 antagonist, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S) -aminoethylsulfonylamino-β-alanine (IA) to monoclonal antibody (MoAb) to increase integrin αvβ3 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 μM 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 monomelic MoAb-(A-S-succinimido-[PEO]2-succinimido-S- A-IA)n with <10% high molecular weight oligomeric MoAb. Monomelic 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 μM αvβ3. 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 αvβ3 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.

Original languageEnglish (US)
Pages (from-to)821-828
Number of pages8
JournalBioconjugate Chemistry
Issue number3
StatePublished - 2007
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry


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