Molecular construction and optimization of anti-human IL-1alpha/beta dual variable domain immunoglobulin (DVD-Ig) molecules

Abstract

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.

Figure 1

Schematic diagram of Dual Variable Domain (DVD)-Ig. (A) Generation DVD-Ig from two parental antibodies. The VH and VL of 13F5.G5 was directly fused to the N-terminus (without signal sequences) of the HC and LC of 3D12.E3, respectively, to generate HC and LC of DVD-Igs that were subsequently co-expressed in mammalian cells. (B) Specific constructs (1^st set) generated include DVD1-Ig, DVD2-Ig and two chimeric mAbs from hybridoma clones 3D12.E3 (anti-IL-1α) and 13F5. G5 (anti-IL-1β).

Figure 2

(A) SDS-PAGE analysis of DVD-Ig molecules. Purified chimeric mAbs 3D12.E3 and 13F5.G5, DVD1-Ig and DVD2-Ig were run on 10% SDS tris-glycine gel in non-reduced (left) and reduced (right) conditions. (B) Analysis of mAbs/DVD-Igs by size exclusion chromatography (SEC). Std: protein standards ranging from 1.35 to 670 kDa.

Figure 3

Binding mode analysis of DVD-Ig by Biacore. DVD2-Ig was immobilized by a goat anti-human Fc antibody on the sensor chip, and hIL-1β was first injected and a binding signal observed, followed by the injection of hIL-1α, which exhibited the second binding signal (A). In the reverse order, hIL-1α was first injected followed by the injection of hIL-1β (B), showing a similar result of dual-specific binding.

Figure 4

Inhibition of both IL-1α and IL-1β biological activities by DVD-Ig. DVD1-Ig (○) and DVD2-Ig (▪) were tested in MRC-5 bioassay to determine their neutralization potency against hIL-1α (A) and hIL-1β (B), in comparison to 3D12.E3 (▵) and 13F5.G5 (▿) chimeric mAbs. DVD1-Ig and DVD2-Ig were also able to inhibit IL-8 production in the presence of both IL-1α and IL-1β in the same cell culture system (C).

Figure 5

Monomeric profiling of the second set of mAbs/DVD-Igs by size exclusion chromatography. Std: protein standards ranging from 1.35 to 670 kDa.