Use of antibody as carrier of oligomers of peptidomimetic α_vβ₃ antagonist to target tumor-induced neovasculature

Sulfhydryl selective reactions were explored to conjugate oligomers of a peptidomimetic integrin α_vα₃ 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 α_vβ₃ 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 α_vβ₃. 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 α_vβ₃ 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.