Use of Antibody as Carrier of Oligomers of Peptidomimetic v
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- 作者：In Soo Shin ; Beom-Su Jang ; S. Narasimhan Danthi ; Jianwu Xie ; Sarah Yu ; Nhat Le ; Jin-Soo Maeng ; In Sook Hwang ; King C. P. Li ; Jorge A. Carrasquillo ; Chang H. Paik
- 刊名：Bioconjugate Chemistry
- 出版年：2007
- 出版时间：May 2007
- 年：2007
- 卷：18
- 期：3
- 页码：821 - 828
- 全文大小：448K
- 年卷期：v.18,no.3(May 2007)
- ISSN：1520-4812
文摘
Sulfhydryl selective reactions were explored to conjugate oligomers of a peptidomimetic integrin 
v
3 antagonist,4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonylamino--alanine (IA) tomonoclonal antibody (MoAb) to increase integrin v3 receptor-binding avidity. To generate sulfhydryl groups,N-succinimidyl-S-acetylthioacetate (SATA) was conjugated to both MoAb and IA. Sulfhydryl groups were thengenerated upon the deacetylation of the protecting acetyl group from the S-acetylthioacetato (ATA) moiety ofMoAb-(ATA)n or IA-ATA with 0.02 M hydroxylamine in the presence of 1 mM EDTA at pH 7.2. The majorfocus was on optimizing the reaction concentrations, molar ratios, and reaction pH to conjugate high levels ofIA-(A-SH) to MoAb-(A-SH)n without causing the inter- and intramolecular cross-linking of MoAb. Stepwisereactions of MoAb-(A-SH)n (15 
M MoAb) with a homobifunctional cross-linker, 1,8-bis(maleimido)diethyleneglycol (BM[PEO]2) at a >50× 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-DTPAand with 125I using the Iodogen method showed >70% bindability to 0.4 M v3. When injected iv to nudemice with the receptor-positive M21 tumor, MoAb-IA10 radiolabeled with both 111In and 125I accumulated rapidlyand was retained in the tumor for a 44 h period while the radioactivity cleared rapidly from the blood, therebyresulting in increasing tumor-to-blood ratios over time. The tumor uptake was similar between the 125I label andthe 111In label for a 44 h period. In contrast, the blood radioactivity was lower, but liver and other organ uptakeswere much higher for the 111In label than for the 125I. The 111In label produced higher tumor-to-blood ratios butmuch lower tumor-to-organ ratios than the 125I. The rapid blood clearance, a short peak tumor uptake time, anda low peak tumor uptake value with prolonged tumor retention of this macromolecule appear to support a hypothesisthat MoAb-IA10 primarily binds to v3 receptors on angiogenic vessels, but not on the tumor. This hypothesiswas 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 observedwhen blood vessels outlined with rhodamine-lectin, which specifically binds to blood vessels, were superimposableon neovasculatures outlined with FITC-MoAb-IA10.
v3 antagonist,4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonylamino--alanine (IA) tomonoclonal antibody (MoAb) to increase integrin v3 receptor-binding avidity. To generate sulfhydryl groups,N-succinimidyl-S-acetylthioacetate (SATA) was conjugated to both MoAb and IA. Sulfhydryl groups were thengenerated upon the deacetylation of the protecting acetyl group from the S-acetylthioacetato (ATA) moiety ofMoAb-(ATA)n or IA-ATA with 0.02 M hydroxylamine in the presence of 1 mM EDTA at pH 7.2. The majorfocus was on optimizing the reaction concentrations, molar ratios, and reaction pH to conjugate high levels ofIA-(A-SH) to MoAb-(A-SH)n without causing the inter- and intramolecular cross-linking of MoAb. Stepwisereactions of MoAb-(A-SH)n (15 M MoAb) with a homobifunctional cross-linker, 1,8-bis(maleimido)diethyleneglycol (BM[PEO]2) at a >50× 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-DTPAand with 125I using the Iodogen method showed >70% bindability to 0.4 M v3. When injected iv to nudemice with the receptor-positive M21 tumor, MoAb-IA10 radiolabeled with both 111In and 125I accumulated rapidlyand was retained in the tumor for a 44 h period while the radioactivity cleared rapidly from the blood, therebyresulting in increasing tumor-to-blood ratios over time. The tumor uptake was similar between the 125I label andthe 111In label for a 44 h period. In contrast, the blood radioactivity was lower, but liver and other organ uptakeswere much higher for the 111In label than for the 125I. The 111In label produced higher tumor-to-blood ratios butmuch lower tumor-to-organ ratios than the 125I. The rapid blood clearance, a short peak tumor uptake time, anda low peak tumor uptake value with prolonged tumor retention of this macromolecule appear to support a hypothesisthat MoAb-IA10 primarily binds to v3 receptors on angiogenic vessels, but not on the tumor. This hypothesiswas 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 observedwhen blood vessels outlined with rhodamine-lectin, which specifically binds to blood vessels, were superimposableon neovasculatures outlined with FITC-MoAb-IA10.