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

Abstract

Radioimmunopretargeting is based on the separate injection of a modified mAb and the radionuclide and most frequently exploits the very high avidity of biotin for streptavidin (SA). Currently, we are evaluating the therapeutic potential of directly labeled monoclonal antibody (mAb) 81C6, reactive with 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 was required. 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-binding capacity, immunoreactivity, and molecular weight. The 81C6 mAb and SA were coupled by covalent interaction between sulfhydryl groups generated on the mAb via N-succinimidyl-S-acetylthioacetate, dithiothreitol or 2-iminothiolane (2IT), and maleimido-derivatized SA, prepared via sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or N-succinimidyl-3-(2-pyridyldithio)-propionate. A noncovalent approach involving reaction of a biotinylated mAb, prepared using biotin caproate, 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 procedure involved activation of m81C6 or c81C6 with 30 equiv of 2IT and reaction of SA with 10 equiv of SMCC and yielded a conjugate with excellent biotin-binding capacity and immunoreactivity. The ((125)I-labeled m81C6)-2IT-SMCC-SA was stable and did not lose biotin-binding capacity after a 72 h incubation in human glioma cyst fluid in vitro. Although the conjugate was stable in murine serum in vivo, its biotin-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 human glioma xenografts, high tumor uptake (36.0 +/- 10.7% ID/g at 3 days) and excellent tumor:normal tissue ratios were observed.

Radiation Therapy of Glioblastoma

“…This unique form of RT delivery involves the use of radiolabeled antibodies targeting malignant brain tissue. Investigators at Duke University have been studying the efficacy of a 131-I-labeled murine anti-tenascin monoclonal antibody (131-I-m81C6) in the treatment of newly diagnosed and recurrent malignant brain tumors [67][68][69][70][71][72][73][74][75][76][77][78][79]. Tenascin is an extracellular matrix glycoprotein expressed ubiquitously in multiple tumor types, including high-grade gliomas, but not in normal brain tissue.…”

Preparation, Characterization, and Biological Evaluation of a Streptavidin-Chimeric T84.66 Conjugate for Antibody Pretargeting

“…Specifically, poor imaging contrast and dose-limiting toxicity caused by conventionally radiolabeled mAbs can potentially be circumvented. 3,6,9 In the present studies, we developed a new pretargeting system, using cT84.66 for pretargeting CEA, which is highly expressed in colorectal, breast, and lung cancers. An SA conjugate of the mAb cT84.66 was synthesized, characterized, and evaluated for biotin pretargeting, using 111 In as an imaging radiometal and surrogate for pretargeted therapy.…”

“…Therefore, antibody pretargeting has been actively investigated as an alternative approach in recent years. [3][4][5][6][7][8][9] In pretargeting, a nonradioactive mAb-"receptor" construct is injected and allowed to reach maximum tumor uptake, after which a radiolabeled small molecule with high affinity and specificity for the mAb-"receptor" construct is administered. In this strategy, the mAb is decoupled from the radionuclide.…”

The present and future of cerebral tumor surgery in children

“…Chicoine and Silbergeld showed the ability of isolated cells from malignant gliomas to invade cerebral tissue in the C6 glioma model. Using fluorescent labeling of tumor cells in rodents, they demonstrated that individual neoplastic cells could be found more than 1 cm from the original tumor within 2 weeks of the initial implantation [24]. These cells retained the ability to begin new tumor nodules in soft agar, demonstrating that they continued their tumorogenicity.…”

“…Invasion is the most troublesome attribute of malignant cerebral tumors for the surgeon [24,39,62,63]. Invasion of tumor cells has been demonstrated both in animal models and in human tumors.…”