For complex diseases in which multiple mediators contribute to overall disease pathogenesis by distinct or redundant mechanisms, simultaneous blockade of multiple targets may yield better therapeutic efficacy than inhibition of a single target. However, developing two separate monoclonal antibodies for clinical use as combination therapy is impractical, owing to regulatory hurdles and cost. Multi-specific, antibody-based molecules have been investigated; however, their therapeutic use has been hampered by poor pharmacokinetics, stability and manufacturing feasibility. Here, we describe a generally applicable model of a dual-specific, tetravalent immunoglobulin G (IgG)-like molecule--termed dual-variable-domain immunoglobulin (DVD-Ig)--that can be engineered from any two monoclonal antibodies while preserving activities of the parental antibodies. This molecule can be efficiently produced from mammalian cells and exhibits good physicochemical and pharmacokinetic properties. Preclinical studies of a DVD-Ig protein in an animal disease model demonstrate its potential for therapeutic application in human diseases.

A B S T R A C T Although recent studies suggest that interferons can increase the number of IgG Fc receptor (FcR'y) sites on mouse macrophages, direct assessment of similar effects on human mononuclear phagocytes is lacking. We therefore measured the specific binding of 125.-and fluorescein-labeled IgGl to human monocytes and leukemic cell lines after culture in vitro with highly purified human interferons. We report that natural and recombinant human 'y-interferon causes a dramatic (nearly 10-fold) increase in the number of FcR-y on normal human monocytes and on the human cell lines HL-60 and U-937. Alpha and ,B-interferons cause a modest but significant increase in these receptors. This report demonstrates that 'v-interferon acts directly on human mononuclear phagocytes to increase FcR-y sites, it identifies a qualitative difference in the physiologic actions of human type I and type II interferons, and it suggests that HL-60 and U-937 cells will be important models for further study of the molecular mechanisms of interferon action. The results reported here could also be the basis for a bioassay to assess the pharmacokinetics and variability of '-interferon action on monocytes of individual patients during treatment in vitro and in vivo.

Signal transduction through the interleukin-1 receptor (IL-1R) pathway mediates a strong pro-inflammatory response, which contributes to a number of human diseases such as rheumatoid arthritis. Within the IL-1 family, IL-1alpha and IL-1beta are both agonistic ligands for IL-1R, whereas IL-1 receptor antagonist (IL-1ra) is an endogenous antagonist that binds to IL-R, but does not signal. Therefore, the ideal therapeutic strategy would be blocking both IL-1alpha and IL-1beta, but not IL-1ra. However, due to low sequence homology between the three members of the family, it has been exceedingly difficult to identify potent therapeutic agents, e.g., monoclonal antibodies (mAbs), that selectively recognize both IL-1alpha and IL-1beta, but not IL-1ra. Currently, several anti-IL-1 therapeutic agents in clinical development either inhibit only IL-1beta (i.e., anti-IL-1beta mAb), or recognize all three ligands (i.e., anti-IL-1R mAb or IL-1R Trap). We have recently developed a novel dual variable domain immunoglobulin (or DVD-Ig) technology that enables engineering the distinct specificities of two mAbs into a single functional, dual-specific, tetravalent IgG-like molecule. Based on this approach, we have developed anti-human IL-1alpha/beta DVD-Ig molecules using several pairs of monoclonal antibodies with therapeutic potential, and present a case study for optimal design of a DVD-Ig agent for a specific target pair combination.

IL-18 is a pleiotropic proinflammatory cytokine that is involved in induction of inflammatory mediators, regulation of the cytotoxic activity of NK cells and T cells, and differentiation and activation of both Th1 and Th2 cells. IL-18 signals through its specific cell surface receptor IL-18R, which comprises two subunits: IL-18Rα and IL-18Rβ. IL-18Rα alone has a weak affinity for IL-18 binding, while the IL-18Rα/β complex has a high affinity. By using several anti-IL-18 mAbs and IL-18 binding protein, we have examined whether these site-specific inhibitors could block the binding of IL-18 to IL-18Rα and to the IL-18Rα/β complex. Here we show that IL-18 binding to IL-18Rα was inhibited by a neutralizing mAb, 125-2H, while binding of IL-18 to the α/β receptor complex was not. This suggests that IL-18Rβ-induced conformational changes may occur in IL-18Rα upon dimerization, leading to changes in the presentation of IL-18 binding sites. Epitope mapping of 125-2H using human-mouse IL-18 chimeras identified a region in IL-18 that was required for 125-2H recognition. This region, as examined by IL-18R binding and functional analysis, appeared to be critical for triggering signal transduction through the heterodimeric receptor.