Structure-guided design fine-tunes pharmacokinetics, tolerability, and antitumor profile of multispecific frizzled antibodies

Abstract

Aberrant activation of Wnt/β-catenin signaling occurs frequently in cancer. However, therapeutic targeting of this pathway is complicated by the role of Wnt in stem cell maintenance and tissue homeostasis. Here, we evaluated antibodies blocking 6 of the 10 human Wnt/Frizzled (FZD) receptors as potential therapeutics. Crystal structures revealed a common binding site for these monoclonal antibodies (mAbs) on FZD, blocking the interaction with the Wnt palmitoleic acid moiety. However, these mAbs displayed gastrointestinal toxicity or poor plasma exposure in vivo. Structure-guided engineering was used to refine the binding of each mAb for FZD receptors, resulting in antibody variants with improved in vivo tolerability and developability. Importantly, the lead variant mAb significantly 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 finetuned to navigate in vivo exposure and tolerability while driving therapeutic efficacy.antibody therapeutic | Frizzled receptors | Wnt signaling | X-ray crystallography | protein engineering W nt signaling is an evolutionarily conserved signaling cascade that plays a critical role in diverse biological processes, including embryonic development, tissue differentiation, organogenesis, stem cell maintenance, and normal adult tissue homeostasis (1-3). Activation of the well-characterized β-catenindependent canonical pathway is initiated by the binding of secreted Wnt proteins to Frizzled (FZD) receptors and coreceptors such as LRP5 and LRP6 (4-8). To date, 19 human Wnt and 10 FZD receptors have been identified that mediate differential cellular functions (9, 10). FZD receptors interact with Wnt through their N-terminal extracellular cysteine-rich domain (CRD). A Wnt residue, Ser187, (position number according to Xenopus Wnt8) is posttranslationally modified, leading to its palmitoleation, which mediates interactions with the FZD-CRD (site 1); a second site of interaction between FZD and Wnt is located at the opposing end of Wnt (11).Wnt/FZD signaling is essential for normal cell function, but aberrations in the pathway are frequently found in cancers, fibrosis, and degenerative diseases (12,13). Abnormal activation of the Wnt pathway is an essential driver of primary tumor formation and metastasis in multiple cancer types (14-18). Inactivating mutations in E3 ubiquitin ligase RNF43 inhibit the downmodulation of FZD expression on the cell surface and sensitize tumor cells to Wnt-dependent growth. These mutations have been identified in pancreatic, biliary duct, and colorectal cancers (19-21). FZD5 expression is up-regulated in renal cell carcinoma (22), prostate cancer (23), and pancreatic tumors (16); aberrant FZD7 expression is observed in hepatocellular carcinoma and colorectal and triple negative breast cancer (14,24,25); FZD8 is upregulated in acute lymphoblastic leukemia and lung cancer (17, 26); and FZD4 is elevated and drives epithelial-to-mesenchy...

Long Non-Coding RNA Landscape in Prostate Cancer Molecular Subtypes: A Feature Selection Approach

“…Our evidence on LINC00688 is in contrast with those reported in gastric, breast and colon cancers [ 48 ]. Therefore, further investigation is needed to verify the role of this lncRNA in ERG-positive prostate tumors, in which it is reported that the expression of cell cycle-related genes is negatively regulated by ERG [ 53 ]. Likewise, the lnc-SAYSD1-1 also bioinformatically allows to distinguish the ERG-positive from all the other subclasses of prostate cancer with a Gleason score ≤ 3+4.…”

Aberrant WNT/CTNNB1 Signaling as a Therapeutic Target in Human Breast Cancer: Weighing the Evidence

“…A phase Ib clinical trial in HER2 − breast cancer patients identified a four-gene signature (FBXW2, CCND2, CTBP2, and WIF1) as a potential predictive biomarker for the response to combined treatment with paclitaxel and vantictumab (Zhang et al, 2018). Structure guided design will likely help in generating more specific antibodies that target individual FZD receptors (Raman et al, 2019). Based on the available data, FZD6 and FZD7 seem obvious candidates for therapeutic intervention (Figure 1 and Table 1).…”

Review Article: Is Wnt Signaling an Attractive Target for the Treatment of Osteoarthritis?

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