A Novel Glycoengineered Bispecific Antibody Format for Targeted Inhibition of Epidermal Growth Factor Receptor (EGFR) and Insulin-like Growth Factor Receptor Type I (IGF-1R) Demonstrating Unique Molecular Properties

Juergen M Schanzer,Katharina Wartha,Rebecca Croasdale,Samuel Moser,Klaus-Peter Künkele,Carola Ries,Werner Scheuer,Harald Duerr,Sandra Pompiati,Jan Pollman,Jan Stracke,Wilma Lau,Stefan Ries,Ulrich Brinkmann,Christian Klein,Pablo Umana

doi:10.1074/jbc.m113.528109

Abstract

In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the "knob-into-hole" technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats.

Highlights

Bispecific antibodies are currently emerging as a promising new class of cancer therapeutics
Design of a Heterodimeric One-arm singlechain Fab fragment (scFab) Bispecific Antibody Targeting epidermal growth factor receptor (EGFR) and IGF-1R (XGFR)—The bispecific antibodies generated in this study are based on a human IgG1 isotype with heavy chains composed of a variable VH domain and three constant domains CH1, CH2, and CH3
XGFR bispecific antibodies were assembled with an EGFR binding arm composed of the GA201 light and heavy chain

Summary

Background

Bispecific antibodies are currently emerging as a promising new class of cancer therapeutics. We have developed an IGF-1R antibody termed R1507 that has been evaluated in combination with erlotinib in phase II clinical trials for advanced stage non-small cell lung cancer [19] Both receptor tyrosine kinases contribute to tumor growth via activation of the PI3K-AKT and RAS-RAF-MAPK signaling pathways, and cross-talk between EGFR and IGF-1R signaling has been reported. We rationally designed a bispecific heterodimeric and bivalent onearm scFab IgG (OAscFab-IgG) antibody format based on the “knob-into-hole” technology [34] targeting EGFR and IGF-1R with distinct binding arms derived from the parental antibodies GA201 (EGFR) and R1507 (IGF-1R) [18, 20] This novel antibody format combines robust expression and overcomes the light chain association issue in bispecific heterodimeric IgG antibodies [35]. In several ADCC-competent mouse xenograft models, antibody XGFR demonstrated highly effective antitumoral activity

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION