Structure reveals function of the dual variable domain immunoglobulin (DVD-Ig™) molecule

Abstract

Several bispecific antibody-based formats have been developed over the past 25 years in an effort to produce a new generation of immunotherapeutics that target two or more disease mechanisms simultaneously. One such format, the dual-variable domain immunoglobulin (DVD-Ig™), combines the target binding domains of two monoclonal antibodies via flexible naturally occurring linkers, which yields a tetravalent IgG - like molecule. We report the structure of an interleukin (IL)12-IL18 DVD-Ig™ Fab (DFab) fragment with IL18 bound to the inner variable domain (VD) that reveals the remarkable flexibility of the DVD-Ig™ molecule and how the DVD-Ig™ format can function to bind four antigens simultaneously. An understanding of how the inner variable domain retains function is of critical importance for designing DVD-Ig™ molecules, and for better understanding of the flexibility of immunoglobulin variable domains and linkers, which may aid in the design of improved bi- and multi-specific biologics in general.

Keywords: DFab; DVD-Ig™; Dual Specific Antibody; IL12; IL18.

Figure 1. DVD-Ig™ Technology Overview. (A) A DVD-Ig™ binding protein is constructed from two parent antibodies by addition of the first variable domain to the second via a flexible linker sequence (in this case antiIL12 antiIL18 and SS linkers). (B) Solid surface representation of IL12-IL18 DVD-Ig™ DFab with IL18 shown as ribbon. VD1 is shown in teal and VD2 and constant domains are shown in blue. Heavy and light chains are dark and light shades respectively. Short-short linkers are shown in green. IL18 is shown in magenta. (C) View of b from the top.

Figure 2. Structure of IL12-IL18 DVD-Ig™ DFab fragment with IL18 bound. (A) Ribbon and solid surface representation of the structure with heavy and light chains in respective shades of gray, linkers in green, and IL18 in magenta. The epitope of IL18 (dark blue sticks) interacts mainly with heavy chain CDRs 1 (lt. cyan) and 3 (lt. orange) with minor contributions from heavy and light chain CDR 2 (lt. yellow). (B) The VD2 CDR plane as viewed from above and in ribbon with IL18 removed for viewing clarity (C) Ribbon representation viewed from below the outer VD. Heavy chain is shown in dark blue and light chain in teal. (D) Stereo diagram of a representative portion of the electron density map at the DFab IL18 VD/IL18 interface.

Figure 3. Linker Length Affects Inner Antigen Affinity. (A) Illustration of outer VD position as a function of linker length, relative to the inner VD. Arrows indicate likely reorientation of the outer VD. (B) The VEGF and AgA binding kinetics are shown in plots of off-rate (k_d) vs. on-rate (k_a). Symbols are defined in Table 2; briefly, data points are colored by linker set (red, green, dark blue and brown correspond to linker sets SS, LS, SL and LL, respectively) and shaped by antiAgA sequence (circle, triangle, square and diamond are sequences 1, 2, 3 and 4, respectively). Binding curves with fits, for both VEGF and AgA, as well as the detailed methodology, can be found in supplemental materials.