Robust generation of IgG bispecific antibodies has been a long-standing challenge. Existing methods require extensive engineering of each individual antibody, discovery of common light chains, or complex and laborious biochemical processing. Here we combine computational and rational design approaches with experimental structural validation to generate antibody heavy and light chains with orthogonal Fab interfaces. Parental monoclonal antibodies incorporating these interfaces, when simultaneously co-expressed, assemble into bispecific IgG with improved heavy chain-light chain pairing. Bispecific IgGs generated with this approach exhibit pharmacokinetic and other desirable properties of native IgG, but bind target antigens monovalently. As such, these bispecific reagents may be useful in many biotechnological applications.

(2011) 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, mAbs, 3:3, 273-288,

Therapeutic antibodies directed against the type 1 insulinlike growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting downstream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 M) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy. The type I insulin-like growth factor receptor (IGF-1R)2 is a large transmembrane receptor tyrosine kinase expressed on most somatic cells. IGF-1R is activated by the binding of its constitutive ligands, IGF-1 and IGF-2 (and at a much lower affinity, insulin). Ligand binding to the IGF-1R extracellular domains leads to activation of its cytoplasmic tyrosine kinase domain, receptor autophosphorylation, and phosphorylation of downstream targets such as insulin receptor substrate-1 (IRS-1), the Src homology and collagen domain protein (Shc), and others (1, 2). Phosphorylation of IRS-1 activates the phosphoinositol kinase 3/AKT cellular growth and survival pathways, and Shc phosphorylation leads to the activation of other signal cascades, including the extracellular signal-regulated kinase(Erk)/mitogen-activated protein kinase (MAPK) cellular growth and proliferation pathways (3).Human IGF-1R is synthesized as a 1368-amino acid polypeptide whose primary and tertiary structures have been reviewed (4,5). The N-terminal region (consis...

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

The insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor tyrosine kinase that mediates cell survival signaling and supports tumor progression in multiple tumor types. We identified a spectrum of inhibitory IGF-IR antibodies with diverse binding epitopes and ligand-blocking properties. By binding distinct inhibitory epitopes, two of these antibodies, BIIB4 and BIIB5, block both IGF-I and IGF-II binding to IGF-IR using competitive and allosteric mechanisms, respectively. Here, we explored the inhibitory effects of combining BIIB4 and BIIB5. In biochemical assays, the combination of BIIB4 and BIIB5 improved both the potency and extent of IGF-I and IGF-II blockade compared with either antibody alone. In tumor cells, the combination of BIIB4 and BIIB5 accelerated IGF-IR downregulation and more efficiently inhibited IGF-IR activation as well as downstream signaling, particularly AKT phosphorylation. In several carcinoma cell lines, the antibody combination more effectively inhibited ligand-driven cell growth than either BIIB4 or BIIB5 alone. Notably, the enhanced tumor growth-inhibitory activity of the BIIB4 and BIIB5 combination was much more pronounced at high ligand concentrations, where the individual antibodies exhibited substantially reduced activity. Compared with single antibodies, the BIIB4 and BIIB5 combination also significantly further enhanced the antitumor activity of the epidermal growth factor receptor inhibitor erlotinib and the mTOR inhibitor rapamycin. Moreover, in osteosarcoma and hepatocellular carcinoma xenograft models, the BIIB4 and BIIB5 combination significantly reduced tumor growth to a greater degree than each single antibody. Taken together, our results suggest that targeting multiple distinct inhibitory epitopes on IGF-IR may be a more effective strategy of affecting the IGF-IR pathway in cancer. Mol Cancer Ther; 9(9); 2593-604. ©2010 AACR.