Stable IgG-like Bispecific Antibodies Directed toward the Type I Insulin-like Growth Factor Receptor Demonstrate Enhanced Ligand Blockade and Anti-tumor Activity

Abstract

Bispecific antibodies (BsAbs) target multiple epitopes on the same molecular target or different targets. Although interest in BsAbs has persisted for decades, production of stable and active BsAbs has hindered their clinical evaluation. Here, we describe the production and characterization of tetravalent IgG-like BsAbs that combine the activities of allosteric and competitive inhibitors of the type-I insulin-like growth factor receptor (IGF-1R). The BsAbs, which were engineered for thermal stability, express well, demonstrate favorable biophysical properties, and recognize both epitopes on IGF-1R. Only one BsAb with a unique geometry, denoted BIIB4-5scFv, was capable of engaging all four of its binding arms simultaneously. All the BsAbs (especially BIIB4-5scFv) demonstrated enhanced ligand blocking over the single monoclonal antibodies (mAbs), particularly at high ligand concentrations. The pharmacokinetic profiles of two IgG-like BsAbs were tested in nude mice and shown to be comparable with that of the parental mAbs. The BsAbs, especially BIIB4-5scFv, demonstrated an improved ability to reduce the growth of multiple tumor cell lines and to inhibit ligand-induced IGF-1R signaling in tumor cells over the parental mAbs. BIIB4-5scFv also led to superior tumor growth inhibition over its parental mAbs in vivo. In summary, BsAbs that bridge multiple inhibitory mechanisms against a single target may generally represent a more effective strategy for intervention in oncology or other indications compared with traditional mAb therapy.

A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity

“…The thermal stability profile of purified EI-04 as assessed by differential scanning calorimetry (DSC), shown in Figure 1D, confirms the T m of the stability-engineered BIIB5 scFv domain (~68°C) within the context of the final bispecific antibody format. 44 Stability studies of purified EI-04 BsAb showed the material to remain monomeric, homogeneous and stable at 4°C over at least 2 months at 25 and 50 mg/ml in a citrate-based pre-formulation buffer (data not shown).…”

“…C06 does not bind to rodent IGF-1R and the half-life of C06 was previously reported to be ~10 days in mice. 44 Therefore, M60-A02 and EI-04 were dosed twice weekly (BIW) to compensate for their faster clearance while C06 was dosed on a weekly schedule (QW) in the efficacy study.…”

“…41 We previously reported a new and robust BsAb platform that utilizes stability-engineered scFvs appended to full length IgGs to target two tumor necrosis factor receptor family members or two distinct epitopes of IGF-1R. [42][43][44] Both BsAbs displayed IgGlike pharmaceutical properties, dual specificity and improved anti-tumor activity. Here, we describe the development of a novel BsAb targeting EGFR and IGF-1R using this platform technology.…”

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

“…10 There are 3 broad categories of bispecific antibody design. The first category makes use of a conventional IgG [11][12][13] or Fab 14 as a platform, and introduces a second functional entity as a single variable domain (sVD), single-chain variable fragment (scFv) or single-chain Fab to the N-or C-terminus of the light chain (LC) or heavy chain (HC) via a flexible peptide linker, leading to tetravalent or hexavalent bispecific antibodies. Among the 3 formats, scFv is most popular because its compact structure of tandem linked V L and V H domains makes the molecule highly amenable to gene engineering, yet it still possesses complete antigen-binding activity.…”

Applications of immunochemistry in human health: advances in vaccinology and antibody design (IUPAC Technical Report)

“…Engineering the stability of the antibody variable region has mainly focused on scFv due to its inherently poor stability. Stability of scFv can be improved by optimizing the hydrophobic core residues, charge cluster residues, residues which determine the framework subclasses, conserved proline and glycine residues, hydrophobic surface residues, and V H /V L interface residues using either a rational mutagenesis approach or a library approach [265,[318][319][320][321].…”