Duke University

The United States of America, As Represented by the Secretary, Department of Health and Human Services

Charles Pegram

Darell D Bigner

Ira H Pastan

Chien-Tsun Kuan

Vidyalakshmi Chandramohan

D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a recombinantexotoxin A-based immunotoxin (IT), targeting both wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFR variant III (EGFRvIII) proteins overexpressed in glioblastomas. A good laboratory practice (GLP) manufacturing process was developed to produce sufficient material for a Phase I/II clinical trial. D2C7-IT was expressed under the control of the T7 promoter inBLR (&#x3bb; DE31). D2C7-IT was produced by a 10 L batch fermentation process and was then purified from inclusion bodies using anion exchange, size exclusion, and an endotoxin removal process that achieved a yield of over 300 mg of purified protein.

Production of immunotoxin D2C7&#x2014;(scdsFv)&#x2014;PE38KDEL

We tested the in vitro and in vivo efficacy of a recombinant bispecific immunotoxin that recognizes both EGFRwt and tumor-specific EGFRvIII receptors. A single chain antibody was cloned from a hybridoma and fused to toxin, carrying a C-terminal peptide which increases retention within cells. The binding affinity and specificity of the recombinant bispecific immunotoxin for the EGFRwt and the EGFRvIII proteins was measured. In vitro cytotoxicity was measured. In vivo activity of the recombinant bispecific immunotoxin was evaluated in subcutaneous models and compared to that of an established monospecific immunotoxin. In our preclinical studies, the bispecific recombinant immunotoxin, exhibited significant potential for treating brain tumors.

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Durham, NC, United States of America

Charles Pegram