The objective of this project is to perform preclinical developmental studies of a novel immunoconjugate that may be useful in modifying the biological responses to Hodgkin's disease and T-cell lymphomas. Recent cytochemical studies of tissues involved by these two malignancies indicate that there is extensive extracellular deposition within the tumors of eosinophil peroxidase (EPO), a 78kD cytophilic heme-enzyme that is cytotoxic in the presence of its substrates, hydrogen peroxide and iodide. Cellular systems and animal models will, therefore, be employed to test the properties of a peroxide-generating immunoconjugate that was synthesized by using a heterobifunctional cross-linking agent to bind reduced SF25.5 (a murine monoclonal antibody directed to human EPO) to glucose oxidase (an enzymatic generator of hydrogen peroxide). The glucose oxidase-EPO (GO-EPO) system, in the presence of the appropriate substrates (glucose, iodide, and oxygen), generates a continuous flux of highly cytotoxic hypoiodous acid. In the first phase of the project, optimal affinity-chromatographic procedures for purifying active immunoconjugate will be developed, and an enzyme-linked immunoassay for defining the potency of the immunoconjugate will be validated. Flow cytometric measurement of propidium iodide exclusion will then be employed to assay the cytotoxicity of the immunoconjugate against viable, EPO-coated cells obtained from fresh human tissues involved by Hodgkin's disease and T-cell lymphomas. To define the cytotoxic properties of the GO-EPO system in greater detail, in-vitro clonogenic assays will also be performed with tumor cells coated with EPO and incubated in immunoconjugate with substrates. The toxicity of the immunoconjugate will be studies in a series of acute and potentiated toxicity experiments in mice. A rat model that utilized spectrophotometric quantification of glucose oxidase activity in tissue homogenates will be employed to define the in-vivo localization and biodistribution (pharmacokinetics) of the immunoconjugate. To determine the ability of unconjugated SF25.5 to localize at sites of Hodgkin's disease in humans, a radiolocalization pilot study, using indium-111-labeled antibody, will be performed on three patients, in collaboration with the Divisions of Nuclear Medicine and Hematology/Oncology. This study will generate important information about the properties of a novel, peroxide-generating immunoconjugate and will provide justification for clinical trials of immunoconjugate therapy in patients with Hodgkin's disease and T-cell lymphomas.
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