Abstract C21: Generation of Wnt- and mitogenic receptor binding bispecific antibodies to target cancer stem cells
Authors: Eppink Berina , Roovers Rob , Herpers Bram , de Lau Wim , Clements Carina , Zondag van der Zande Vanessa , Basmeleh Abdul , Bartelink Willem , van de Wetering Marc , Vries Robert , Price Leo , De Kruif John , Throsby Mark 
Published: May 2016

Abstract
Background: In colorectal cancer (CRC) and other solid tumors, cancer stem cells (CSC) contribute to tumor progression and resistance to standard chemotherapies. The continuous regeneration of the colon is dependent on strict control of developmental (e.g. Wnt) and mitogenic (e.g. EGF) pathway signaling; dysregulation results in uncontrolled proliferation forming the basis of aggressive tumors with metastatic potential. Here we describe the generation of novel bispecific antibodies designed to target CSC through Wnt signaling receptors and block growth factor signaling. The Wnt targets LGR5, LGR4, ZNRF3 and RNF43 were selected since their expression is modulated in CSC populations. The GPCR family members LGR4/LGR5 are positive Wnt regulators and the transmembrane E3 ligases ZNRF3/RNF43 are negative Wnt regulators. The growth factor receptor EGFR is frequently (&gt;70%) overexpressed in CRC and its blockade has demonstrated clinical benefit in a subgroup of patients. More recently, HER3 pathway activation has been implicated in resistance to EGFR-targeted therapies.
Experimental procedures and results: Two parallel strategies were applied to generate panels of common light chain (cLC) Fab against LGR4, LGR5, ZNRF3 and RNF43. Humanized cLC mice (MeMo®) were immunized with recombinant protein or DNA, and materials harvested from these mice used to generate Fab regions against these antigens. The second approach utilized large and diverse synthetic cLC Fab-phagemid libraries. Combined, these methods resulted in ∼1500 unique antigen-specific Fab from which ∼300 were selected for further testing.
Bispecific antibodies were produced in a human cLC IgG1 format using substitutions in the IgG Fc regions for coexpression of two different heavy chains resulting in the generation of large panels of pure and stable bispecific IgG suitable for screening. The Wnt target specific Fab were combined with a Tetanus toxoid-specific control Fab arm allowing for stringent ranking of these Wnt-specific panels in a monovalent format for specificity, affinity, stability, and ligand (R-Spondin3) blocking potency. Based on this characterization the 54 most promising Wnt targeting arms were combined with a panel of previously characterized EGFR and HER-3 specific Fab arms resulting in ∼ 500 different cLC bispecific IgG for functional testing. All bispecific IgG were screened for potency of growth inhibition of CSC using novel 3D high content imaging readouts on patient-derived CRC organoids. The organoids are cultured using growth factors that allow for the maintenance and proliferation of healthy and diseased stem cells and their offspring. Functional analysis revealed several bispecific antibodies that inhibited CRC organoid growth much more potently than comparator drugs such as cetuximab or erlotinib.
Conclusion: Bispecific antibodies present a biological modality that result in unexpected functional activities by mechanisms possible unique to the architecture of these molecules. Identifying these unique properties requires the rapid generation and screening of large panels of bispecific IgG directly in the therapeutic format in relevant functional assays. Initial screening results of the bispecific antibodies targeting Wnt and HER family members supports the concept of CSC targeting and several leads are currently undergoing more extensive characterization.
Citation Format: Berina Eppink, Rob Roovers, Bram Herpers, Wim de Lau, Carina Clements, Vanessa Zondag van der Zande, Abdul Basmeleh, Willem Bartelink, Marc van de Wetering, Robert Vries, Leo Price, John De Kruif, Mark Throsby. Generation of Wnt- and mitogenic receptor binding bispecific antibodies to target cancer stem cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C21.