A New Class of Dimeric α-Interferons (IFN-α) Made by the Dock and Lock (DNL) Method for Improved Cancer Therapy.

Abstract

Background: IFN-α2 is indicated for the therapy of hairy cell leukemia, chronic myelogenous leukemia, follicular lymphoma, and malignant melanoma. As is the case for most cytokines, the pharmacokinetic (PK) properties of IFN-α2 are critical for dosing and efficacy. In vivo, the protein is quickly degraded, diffuses widely throughout the body, and has a rapid rate of renal clearance. Pegylation of IFN-α2 significantly increases the serum half-life and reduces renal clearance, thus enhancing its efficacy. However, established pegylation of IFN-α2 results in a mixture of positional isomers and reduced in vitro activity, as known for PEGASYS (Roche) and PEG-INTRON (Schering-Plough).Methods: To improve the PK properties and the potency, the DNL method (Rossi et al. Proc. Natl. Acad. Sci. USA, 2006,103:6841) was used to generate novel agents having two copies of IFN-α2b conjugated to polyethylene glycol (PEG).Results: A fusion protein (DDD2-IFN-α2b) composed of IFN-α2b with a dimerization-and-docking domain (DDD2) and a six-His tag was expressed both in myeloma cells and in E. coli. Two PEG-based modules, each composed of a fluorescent molecule, an anchor domain (AD) and either a 20-kDa PEG (IMP362) or a 30-kDa PEG (IMP413), were synthesized. Combining DDD2-IFN-α2b and IMP362 or IMP413 under redox conditions resulted in the desirable DNL conjugates consisting of two copies of IFN-α2b and one PEG linked site-specifically via the DDD and AD interaction. The cytotoxic activity of DDD2-IFN-α2b on Daudi lymphoma cells was similar to that of commercially available recombinant IFN-α2 (rhIFN-α2). The purity and identity of the two DNL conjugates (α2b-413 and α2b-362) were demonstrated by SDS-PAGE, immunoblotting, and fluorescence imaging. Both also showed potent cytotoxic activity on Daudi cells in vitro and superior PK properties to PEG-INTRON. For example, the mean blood residence times for α2b-362 (10.3 h) and α2b-413 (21.7 h) were significantly longer than those of rhIFN-α2 (0.7 h) and PEG-INTRON (5.1 h). Initial studies in mice bearing Daudi xenografts showed a significant therapeutic advantage over PEG-INTRON for both α2b-362 and α2b-413. Animals given 14,000 IU of PEG-INTRON had a median survival (MS) of 32 days compared to 21 days for saline control, whereas those receiving α2b-362 at 14,000 IU, 7,000 IU and 3,500 IU resulted in MS of 45, 41 and 32 days, respectively. More remarkably, α2b-413 was the most effective, achieving MS of 46, >53, and >53 days with 3,500 IU, 7,000 IU and 14,000 IU, respectively, all statistically significant improvements (P< 0.0028) compared to PEG-INTRON given at each equivalent activity.Conclusions: The DNL method provides a novel pegylation strategy for generating a dimeric IFN-α2b that is linked site-specifically to a single PEG at the predetermined location. Since the resulting conjugates exhibit improved PK and efficacy in a Burkitt lymphoma model, they may represent a new class of interferons for use in cancer and infectious disease therapy.