Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design

Abstract

The DVD-Ig (TM) protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.

Figure 1

The schematic depicts the general structure of a DVD-Ig protein where CH and VH refer to constant and variable heavy chain regions, respectively, and CL and VL refer to constant and variable light chain regions, respectively. CD and VD refer to constant domain and variable domain, respectively. The variable regions form tandem inner and outer variable domains, connected by both light and heavy chain linkers, on each arm of the DVD.

Figure 2

Cleavage of DVD-03, DVD-04 and DVD-05 (referred to simply as 03, 04 and 05) with entorokinase or thrombin visualized by reducing SDS-PAGE. (A) The DVD-Ig proteins before (−) and after (+) cleavage with either enterokinase or thrombin. (B) The cleaved DVD-Ig proteins after purification on Mab Select SuRe columns. HC and LC refer to the heavy and light chains, respectively. Frag 1 refers to the cleavage product of the light chain consisting of the constant region and the inner variable region; Frag 2 refers to the cleavage product of the light chain consisting of the outer variable region.

Figure 3

Papain cleavage can be used to generate functional fragments from DVD-Ig proteins. (A) Papain can cleave a DVD-Ig protein between CH₁ and CH₂ resulting in a product denoted as a DFab that contains intact outer and inner variable domains. (B) Papain cleavage of DVD-02 resulted in an additional secondary product, denoted DFab(48k) where papain cleavage also occurred in both heavy and light chain linkers between the inner and outer variable domains.

Figure 4

k_a vs. k_d plot derived from data presented in Table 2A for TNFα binding kinetics of the intact reference mAb and DVD-Ig proteins. Diagonals show affinities (K_D); the vertical and horizontal dashed lines intersect at the reference mAb kinetic constants.

Figure 5

k_a vs. k_d plot derived from data presented in Table 2A for TNFα binding kinetics of the intact reference mAb and DVD-03, DVD-04 and DVD-05 before (solid circles) and after (empty circles) cleavage of the light chain linkers with either enterokinase or thrombin proteases. Diagonals show affinities (K_D); the vertical and horizontal dashed lines intersect at the reference mAb kinetic constants.

Figure 6

k_a vs. k_d plot derived from data presented in Table 2B for TNFα binding kinetics (anti-human(H+L) assay format). Solid circles are the intact reference mAb, DVD-01 and DVD-02; empty circles are the reference Fab, DVD-02-DFab and the DVD-02-DFab(48k). The red arrow highlights the difference in binding kinetics for the DVD-02-DFab and the DVD-02-DFab(48k). Diagonals show affinities (K_D); the vertical and horizontal dashed lines intersect at the reference mAb kinetic constants when assayed via anti-human (Fc).