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¹ Genencor International, Palo Alto, California and ² Department of Medicine and Endocrinology, University Campus Bio-Medicio, Rome, Italy

Requests for reprints: Volker Schellenberger, Genencor International, 925 Page Mill Road, Palo Alto, CA 94304. Phone: 650-846-7646. E-mail: vschellenberger{at}genencor.com

Antibody-directed enzyme prodrug therapy (ADEPT) delivers chemotherapeutic agents in high concentration to tumor tissue while minimizing systemic drug exposure. ß-Lactamases are particularly useful enzymes for ADEPT systems due to their unique substrate specificity that allows the activation of a variety of lactam-based prodrugs with minimal interference from mammalian enzymes. We evaluated the amino acid sequence of ß-lactamase from Enterobacter cloacae for the presence of human T-cell epitopes using a cell-based proliferation assay using samples from 65 community donors. We observed a low background response that is consistent with a lack of preexposure to this enzyme. ß-Lactamase was found to contain four CD4⁺ T-cell epitopes. For two of these epitopes, we identified single amino acid changes that result in significantly reduced proliferative responses while retaining stability and activity of the enzyme. The ß-lactamase variant containing both changes induces significantly less proliferation in human and mouse cell assays, and 5-fold lower levels of IgG1 in mice were observed after repeat administration of ß-lactamase variant with adjuvant. The ß-lactamase variant should be very suitable for the construction of ADEPT fusion proteins, as it combines high activity toward lactam prodrugs, high plasma stability, a monomeric architecture, and a relatively low risk of eliciting an immune response in patients.

Note: F.A. Harding is currently at Protein Design Labs, 34801 Campus Drive, Fremont, CA 94555.

Received 6/ 9/05; revised 7/27/05; accepted 8/30/05.

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