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Structure-Guided Design Fine-Tunes Pharmacokinetics, Tolerability, and Antitumor Profile of Multispecific Frizzled Antibodies

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Structure-Guided Design Fine-Tunes Pharmacokinetics, Tolerability, and Antitumor Profile of Multispecific Frizzled Antibodies Structure-guided design fine-tunes pharmacokinetics, tolerability, and antitumor profile of multispecific frizzled antibodies Swetha Ramana,b,1, Melissa Beilschmidtb,1, Minh Tob, Kevin Linb, Francine Luib, Yazen Jmeianb, Mark Ngb, Minerva Fernandezb,YingFub, Keith Mascallc, Alejandro Duqued, Xiaowei Wangd, Guohua Pand, Stephane Angersc,e,JasonMoffatd,f, Sachdev S. Sidhud,f, Jeanne Magramb, Angus M. Sinclairb, Johan Franssonb,2, and Jean-Philippe Juliena,e,g,2 aProgram in Molecular Medicine, Hospital for Sick Children Research Institute, Toronto, ON M5G 0A4, Canada; bOncology Group, Northern Biologics, Toronto, ON M5G 1L7, Canada; cDepartment of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON M5S 3M2, Canada; dDonnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E2, Canada; eDepartment of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada; fDepartment of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; and gDepartment of Immunology, University of Toronto, Toronto, ON M5S 1A8, Canada Edited by Dennis A. Carson, University of California, San Diego, La Jolla, CA, and approved February 25, 2019 (received for review October 6, 2018) Aberrant activation of Wnt/β-catenin signaling occurs frequently colorectal and triple negative breast cancer (14, 24, 25); FZD8 is up- in cancer. However, therapeutic targeting of this pathway is com- regulated in acute lymphoblastic leukemia and lung cancer (17, 26); plicated by the role of Wnt in stem cell maintenance and tissue and FZD4 is elevated and drives epithelial-to-mesenchymal transition homeostasis. Here, we evaluated antibodies blocking 6 of the 10 in TMRESS2–ERG fusion prostate cancer (27). Signaling through human Wnt/Frizzled (FZD) receptors as potential therapeutics. Crys- FZD4 is also essential for normal angiogenesis (28, 29). Mutations in FZD4 tal structures revealed a common binding site for these monoclonal and its alternative ligand, Norrin, are primary drivers of retinal antibodies (mAbs) on FZD, blocking the interaction with the Wnt hypovascularization in familial exudative vitreoretinopathy (30). palmitoleic acid moiety. However, these mAbs displayed gastroin- Therefore, therapeutic modulation of the Wnt pathway is an attrac- testinal toxicity or poor plasma exposure in vivo. Structure-guided tive approach to treat multiple disease indications (13, 31). BIOPHYSICS AND engineering was used to refine the binding of each mAb for FZD Several approaches have been taken to develop drugs capable of COMPUTATIONAL BIOLOGY abrogating the Wnt pathway in cancers (13, 31, 32). These include receptors, resulting in antibody variants with improved in vivo tol- inhibition of Wnt palmitoleation with PORCN (a Wnt-specific erability and developability. Importantly, the lead variant mAb sig- acyltransferase) inhibitor LGK974, pan-Wnt neutralization with nificantly inhibited tumor growth in the HPAF-II pancreatic tumor xenograft model. Taken together, our data demonstrate that anti- FZD cancer therapeutic antibodies with broad specificity can be fine- Significance tuned to navigate in vivo exposure and tolerability while driving therapeutic efficacy. The canonical Wnt/β-catenin signaling pathway is tightly reg- ulated and plays a crucial role in a broad range of biological antibody therapeutic | Frizzled receptors | Wnt signaling | X-ray processes. Perturbations in this pathway are associated with crystallography | protein engineering multiple pathologies, including different cancers. Efforts to develop Wnt-pathway inhibitors as cancer therapeutics have been met with significant challenges in balancing therapeutic nt signaling is an evolutionarily conserved signaling cas- efficacy and toxicity, due to the ubiquitous nature of the cade that plays a critical role in diverse biological pro- W pathway. Here we describe the development and character- cesses, including embryonic development, tissue differentiation, ization of broadly specific frizzled antibodies that block the organogenesis, stem cell maintenance, and normal adult tissue ho- Wnt ligand–receptor interaction. A combination of structural, meostasis (1–3). Activation of the well-characterized β–catenin- biochemical, and cellular studies allowed refining the pharmaco- dependent canonical pathway is initiated by the binding of secreted kinetics, tolerability, and antitumor profile of multispecific frizzled Wnt proteins to Frizzled (FZD) receptors and coreceptors such as antibodies. We provide a rationale for structure-based refinement LRP5 and LRP6 (4–8). To date, 19 human Wnt and 10 FZD of therapeutic antibody leads and contribute to guiding the de- receptors have been identified that mediate differential cellular velopment of anti-Wnt/FZD cancer therapeutics. functions (9, 10). FZD receptors interact with Wnt through their N-terminal extracellular cysteine-rich domain (CRD). A Wnt Xenopus Author contributions: J.F. and J.-P.J. conceived the study; S.R., M.B., J. Magram, A.M.S., residue, Ser187, (position number according to Wnt8) is J.F., and J.-P.J. designed research; S.R., M.B., M.T., K.L., F.L., Y.J., M.N., M.F., Y.F., K.M., posttranslationally modified, leading to its palmitoleation, which A.D., and X.W. performed research; G.P., J. Moffat, S.S.S., J. Magram, and A.M.S. provided mediates interactions with the FZD–CRD (site 1); a second site of critical insights; G.P., S.A., J. Moffat, and S.S.S. contributed new reagents/analytic tools; interaction between FZD and Wnt is located at the opposing end S.R., M.B., K.L., F.L., Y.J., M.N., M.F., Y.F., K.M., A.D., and X.W. analyzed data; and S.R., M.B., J.F., and J.-P.J. wrote the paper. of Wnt (11). Conflict of interest statement: The authors have filed a patent application for the anti- Wnt/FZD signaling is essential for normal cell function, but bodies described in this work. aberrations in the pathway are frequently found in cancers, fi- brosis, and degenerative diseases (12, 13). Abnormal activation This article is a PNAS Direct Submission. of the Wnt pathway is an essential driver of primary tumor for- Published under the PNAS license. mation and metastasis in multiple cancer types (14–18). Inacti- Data deposition: Structural data have been deposited in the Protein Data Bank (PDB), vating mutations in E3 ubiquitin ligase RNF43 inhibit the down- www.wwpdb.org (PDB ID codes 6O39, 6O3A, and 6O3B). modulation of FZD expression on the cell surface and sensitize 1S.R. and M.B. contributed equally to this work. tumor cells to Wnt-dependent growth. These mutations have 2To whom correspondence may be addressed. Email: [email protected] or been identified in pancreatic, biliary duct, and colorectal cancers [email protected]. (19–21). FZD5 expression is up-regulated in renal cell carcinoma This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (22), prostate cancer (23), and pancreatic tumors (16); aberrant 1073/pnas.1817246116/-/DCSupplemental. FZD7 expression is observed in hepatocellular carcinoma and www.pnas.org/cgi/doi/10.1073/pnas.1817246116 PNAS Latest Articles | 1of6 Downloaded by guest on September 24, 2021 decoy receptor FZD8-Fc OMP54-28 (31–33), interference in down- AB stream signaling components such as tankyrase inhibitor XAV939, or inhibitors that disrupt β-catenin/cotranscriptional inhibitor interac- tions (e.g., ICG-001) (34). However, therapeutically targeting the WNT/β-catenin pathway is challenging due to the critical involvement of the pathway in normal tissue homeostasis, and complete abroga- tion may have severe adverse effects. Gastrointestinal toxicity has been a frequent dose-limiting toxicity observed in clinical trials, among other toxicities (35). Therapeutic antibodies potentially offer advantages in that they can target specific FZD receptors rather than broadly inhibiting the Wnt pathway. For example, OMP-18R5 (vantictumab), a monoclonal antibody (mAb) that interacts with the CRD of FZD1, -2, -5, -7, and -8, has been shown to inhibit tumor growth in several tumor types, including breast, colon, lung, and pancreatic cancer and did not reportedly demonstrate gastrointes- tinal toxicity (31, 32). Targeting FZD4 in addition to FZD1, -2, -5, -7, and -8 may have a broader impact on the efficacy of FZD-targeting antibodies. We have recently shown that antibodies specific to these six FZD re- ceptors inhibit endothelial tube formation in vitro, whereas anti- bodies lacking FZD4 specificity did not (36). Here we expand on these findings and present the structural definition, functional char- acterization, and refinement of FZD antibodies that target FZD1, -2, -4, -5, -7, and -8 to potently inhibit Wnt signaling, while balancing their tolerability. Indeed, despite similar epitopes, these antibodies showed a wide range of selectivity, potency, tolerability, and devel- opability. Here, we demonstrate how structure-guided activity rela- tionships were critical to fine-tune antibody-binding profiles, which led to the development of a tolerable anti-FZD therapeutic antibody with broad FZD-binding specificity and effective antitumor activity. Results Fig. 1. Binding and functional characteristics of lead FZD mAbs. (A) Binding Unique Binding and Potency Profiles of FZD Antibodies. In vitro cell- curves for mAbs F2.I, F7.B, and F6 to 32D cells overexpressing FZD4, -5, or -7. binding experiments were performed to assess the specificity and MFI indicates median fluorescence intensity. (B) Dose–response
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