Diabody-Ig: a novel platform for the generation of multivalent and multispecific antibody molecules

Abstract

Multivalent mono-or bispecific antibodies are of increasing interest for therapeutic applications, such as efficient receptor clustering and activation, or dual targeting approaches. Here, we present a novel platform for the generation of Ig-like molecules, designated diabody-Ig (Db-Ig). The antigen-binding site of Db-Ig is composed of a diabody in the V H-V L orientation stabilized by fusion to antibody-derived homo-or heterodimerization domains, e.g., C H 1/C L or the heavy chain domain 2 of IgE (EHD2) or IgM (MHD2), further fused to an Fc region. In this study, we applied the Db-Ig format for the generation of tetravalent bispecific antibodies (2 + 2) directed against EGFR and HER3 and utilizing different dimerization domains. These Db-Ig antibodies retained the binding properties of the parental antibodies and demonstrated unhindered simultaneous binding of both antigens. The Db-Ig antibodies could be purified by a single affinity chromatography resulting in a homogenous preparation. Furthermore, the Db-Igs were highly stable in human plasma. Importantly, only one short peptide linker (5 aa) per chain is required to generate a Db-Ig molecule, reducing the potential risk of immunogenicity. The presence of a fully functional Fc resulted in IgG-like pharmacokinetic profiles of the Db-Ig molecules. Besides tetravalent bispecific molecules, this modular platform technology further allows for the generation of other multivalent molecules of varying specificity and valency, including mono-, bi-, tri-and tetraspecific molecules, and thus should be suitable for numerous applications.

Inhibition of Tumor Cell Growth and Cancer Stem Cell Expansion by a Bispecific Antibody Targeting EGFR and HER3

“…ELISA-based binding analysis was performed as described previously (25). For flow cytometry-based binding studies, 1Â10 5 FaDu, MCF-7 or MDA-MB-468 cells were seeded per well and incubated with serial dilutions of the proteins in PBA [PBS, 2% (v/v) FBS, 0.02% (w/v) sodium azide] at 4 C for 1 hour.…”

Development of bispecific antibodies in China: overview and prospects

“…1M ). An IgG-like bsAb with two full-length and functional Fc chains can be expected to have half-life time, pharmacodynamics (PD) and pharmacokinetics (PK) behaviors similar to those of its parent monoclonal antibodies (mAb) when their charges, glycosylation, polyreactivity and binding activity to the FcRn receptors are maintained [ 79 , 80 ], while a bispecific fragment such as Blinatumomab has a shorter half-life due to its faster clearance, which provides a safety advantage to mitigate the drug-associated neurological adverse events [ 80 , 81 ].…”

A bivalent, bispecific Dab-Fc antibody molecule for dual targeting of HER2 and HER3

“…We used our Db-Ig technology, to generate a bivalent bispecific antibody composed of a C H 1/C L -stabilized diabody (Dab) fused to an Fc region. 25 In this Dab-Fc format, the V H A-V L B domains are fused to a human γ1 C H 1-C H 2-C H 3 hole chain and the V H B-V L A domains are fused to a complementary human γ1 C L -C H 2-C H 3 knob chain, respectively, resulting in a heterodimeric molecule ( Figure 1a ). This Dab-Fc 2 × 3 molecule was produced in transiently transfected HEK293–6E cells and purified by protein G affinity chromatography followed by a preparative size-exclusion chromatography (SEC) step.…”

“…Here, we have developed a bivalent, bispecific antibody targeting HER2 and HER3 by combining the antigen-binding site of IgG 3–43 with that of the HER2-neutralizing antibody trastuzumab, using our recently established Db-Ig platform technology. 25 In this novel format, a C H 1/C L -stabilized bivalent and bispecific diabody is fused to a heterodimerizing Fc region. This Dab-Fc 2 × 3 molecule retained antigen-binding activity and efficiently inhibited HRG-mediated receptor activation.…”