Preparation and Characterization of Anti-Tenascin Monoclonal Antibody-Streptavidin Conjugates for Pretargeting Applications

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• 作者：Catherine F. Foulon ; Darell D. Bigner ; and Michael R. Zalutsky
• 刊名：Bioconjugate Chemistry
• 出版年：1999
• 出版时间：September 1999
• 年：1999
• 卷：10
• 期：5
• 页码：867 - 876
• 全文大小：238K
• 年卷期：v.10,no.5(September 1999)
• ISSN：1520-4812

文摘

Radioimmunopretargeting is based on the separate injection of a modified mAb and the radionuclideand most frequently exploits the very high avidity of biotin for streptavidin (SA). Currently, we areevaluating the therapeutic potential of directly labeled monoclonal antibody (mAb) 81C6, reactivewith the extracellular matrix protein tenascin, in surgically created glioma resection cavity patients.To be able to investigate pretargeting in this setting, the synthesis of 81C6 mAb-SA conjugates wasrequired. In the current study, we have evaluated five methods for preparing both murine 81C6(m81C6) and human/mouse chimeric 81C6 (c81C6) SA conjugates with regard to yield, biotin-bindingcapacity, immunoreactivity, and molecular weight. The 81C6 mAb and SA were coupled by covalentinteraction between sulfhydryl groups generated on the mAb via N-succinimidyl-S-acetylthioacetate,dithiothreitol or 2-iminothiolane (2IT), and maleimido-derivatized SA, prepared via sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or N-succinimidyl-3-(2-pyridyldithio)-propionate. A noncovalent approach involving reaction of a biotinylated mAb, prepared using biotincaproate, and SA also was studied. The evaluation criteria were yield of mAb-SA 215 kDa monomer,as well as conjugate biotin-binding capacity and immunoreactive fraction. The optimal procedureinvolved activation of m81C6 or c81C6 with 30 equiv of 2IT and reaction of SA with 10 equiv of SMCCand yielded a conjugate with excellent biotin-binding capacity and immunoreactivity. The (¹²⁵I-labeledm81C6)-2IT-SMCC-SA was stable and did not lose biotin-binding capacity after a 72 h incubation inhuman glioma cyst fluid in vitro. Although the conjugate was stable in murine serum in vivo, itsbiotin-binding capacity declined rapidly, consistent with high endogenous biotin levels in the mouse.After injection of the radioiodinated conjugate into athymic mice with subcutaneous D-54 MG humanglioma xenografts, high tumor uptake (36.0 ± 10.7% ID/g at 3 days) and excellent tumor:normal tissueratios were observed.