Multivalent Anti-CD20/Anti-CD22 Bispecific Antibody Fusion Proteins Made by the DNL Method Show Potent Lymphoma Cytotoxicity.

Abstract

Purpose: Multivalent and either monospecific or bispecific antibodies have shown the potential for improved efficacy in a number of preclinical studies. A panel of such agents based on hLL2 (epratuzumab, anti-CD22) and hA20 (humanized anti-CD20) was made and evaluated for cytotoxicity in vitro and anti-tumor activity in vivo.Methods: The Dock and Lock (DNL) method (Rossi et al., Proc. Natl. Acad. Sci. USA, 2006,103:6841) is a novel platform technology for the site-specific and covalent assembly of modular components. DNL was utilized to generate two hexavalent monospecific (hex-hA20 and hex-hLL2), and two hexavalent bispecific (DNL-1 and DNL-2) stably tethered complexes, each composed of four Fab fragments conjugated to an IgG at the latter's carboxyl termini of the heavy chain. DNL-1 consists of four hA20 Fab fragments tethered to an hLL2 IgG. DNL-2 consists of four hLL2 Fab fragments tethered to an hA20 IgG.Results: For each construct, the DNL method produced a single, defined, homogeneous structure that is stable in serum. All of the constituent Fab fragments are functional, with binding affinities similar to the parental MAbs. In vitro analyses using human Burkitt lymphoma cell lines demonstrate that both the monospecific and bispecific constructs induce strong cell adhesion and potently inhibit cell growth without the requirements for further cross-linking. DNL-1, DNL-2 and hex-hA20 each completely inhibited the growth of cultured Daudi cells at concentrations as low as 1 nM and exhibited two to three logs greater potency than that of hA20 IgG or rituximab under the same experimental conditions. Anti-tumor efficacy was demonstrated in SCID mice bearing disseminated Daudi lymphoma. Treatment with 4 μg of hex-hA20 or DNL-2 at 1, 4 and 7 days post intravenous injection of 1 × 107 Daudi cells resulted in significantly increased survival of tumor-bearing mice compared to controls treated with saline.Conclusion: These findings suggest that the DNL method can be used to make multivalent proteins with potent anti-tumor activity, as shown here with anti-CD20/anti-CD22 bispecific antibody fusion proteins.