A WNT mimetic with broad spectrum FZD-specificity decreases fibrosis and improves function in a pulmonary damage model.

BACKGROUND: Wnt/ß-catenin signaling is critical for lung development and AT2 stem cell maintenance in adults, but excessive pathway activation has been associated with pulmonary fibrosis, both in animal models and human diseases such as idiopathic pulmonary fibrosis (IPF). IPF is a detrimental interstitial lung disease, and although two approved drugs limit functional decline, transplantation is the only treatment that extends survival, highlighting the need for regenerative therapies. METHODS: Using our antibody-based platform of Wnt/ß-catenin modulators, we investigated the ability of a pathway antagonist and pathway activators to reduce pulmonary fibrosis in the acute bleomycin model, and we tested the ability of a WNT mimetic to affect alveolar organoid cultures. RESULTS: A WNT mimetic agonist with broad FZD-binding specificity (FZD1,2,5,7,8) potently expanded alveolar organoids. Upon therapeutic dosing, a broad FZD-binding specific Wnt mimetic decreased pulmonary inflammation and fibrosis and increased lung function in the bleomycin model, and it impacted multiple lung cell types in vivo. CONCLUSIONS: Our results highlight the unexpected capacity of a WNT mimetic to effect tissue repair after lung damage and support the continued development of Wnt/ß-catenin pathway modulation for the treatment of pulmonary fibrosis.

BRAIDing receptors for cell-specific targeting.

Systemic toxicity is a major challenge in the development of therapeutics. Consequently, cell-type-specific targeting is needed to improve on-target efficacy while reducing off-target toxicity. Here, we describe a cell-targeting system we have termed BRAID (BRidged Activation by Intra/intermolecular Division) whereby an active molecule is divided into two inactive or less active parts that are subsequently brought together via a so-called 'bridging receptor' on the target cell. This concept was validated using the WNT/ß-catenin signaling system, demonstrating that a multivalent WNT agonist molecule divided into two inactive components assembled from different epitopes via the hepatocyte receptor ßKlotho induces signaling specifically on hepatocytes. These data provide proof of concept for this cell-specific targeting strategy, and in principle, this may also allow activation of multiple signaling pathways where desirable. This approach has broad application potential for other receptor systems.

Novel Frizzled-specific antibody-based Wnt mimetics and Wnt superagonists selectively activate WNT/ß-catenin signaling in target tissues.

WNTs are essential factors for stem cell biology, embryonic development, and for maintaining homeostasis and tissue repair in adults. Difficulties in purifying WNTs and their lack of receptor selectivity have hampered research and regenerative medicine development. While breakthroughs in WNT mimetic development have overcome some of these difficulties, the tools developed so far are incomplete and mimetics alone are often not sufficient. Here, we developed a complete set of WNT mimetic molecules that cover all WNT/ß-catenin-activating Frizzleds (FZDs). We show that FZD1,2,7 stimulate salivary gland expansion in vivo and salivary gland organoid expansion. We further describe the discovery of a novel WNT-modulating platform that combines WNT and RSPO mimetics' effects into one molecule. This set of molecules supports better organoid expansion in various tissues. These WNT-activating platforms can be broadly applied to organoids, pluripotent stem cells, and in vivo research, and serve as bases for future therapeutic development.

Therapeutic blood-brain barrier modulation and stroke treatment by a bioengineered FZD4-selective WNT surrogate in mice.

Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer's disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/ß-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we develop L6-F4-2, a non-lipidated, FZD4-specific surrogate which significantly improves subpicomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reverses neonatal retinal angiogenesis deficits, but also restores BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduces BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with potential applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.

SZN-413, a FZD4 Agonist, as a Potential Novel Therapeutic for the Treatment of Diabetic Retinopathy.

Purpose: There remains a high unmet need for therapies with new mechanisms of action to achieve reperfusion of ischemic retina in diabetic retinopathy. We examined whether a novel frizzled class receptor 4 (FZD4) agonist could promote regeneration of functional blood vessels in animal models of retinopathy. Methods: We developed a novel Norrin mimetic (SZN-413-p) targeting FZD4 and low-density lipoprotein receptor-related protein 5 (LRP5) and examined its effect on retinal and brain endothelial cells in vitro. SZN-413-p was subsequently humanized, resulting in the therapeutic candidate SZN-413, and was examined in animal models of retinopathy. In an oxygen-induced retinopathy mouse model, avascular and neovascularization areas were measured. Furthermore, in a vascular endothelial growth factor (VEGF)-induced retinal vascular leakage rabbit model, the impact on vascular leakage by SZN-413 was examined by measuring fluorescein leakage. Results: SZN-413-p induced Wnt/ß-catenin signaling and upregulated blood-brain barrier/blood-retina barrier gene expressions in endothelial cells. In the oxygen-induced retinopathy mouse model, SZN-413-p and SZN-413 significantly reduced the neovascularization area size (P < 0.001) to a level comparable to, or better than the positive control aflibercept. Both agonists also showed a reduction in avascular area size compared to vehicle (P < 0.001) and aflibercept groups (P < 0.05 and P < 0.01 for SZN-413-p and SZN-413, respectively). In the VEGF-induced retinal vascular leakage rabbit model, SZN-413 reduced retinal vascular leakage by ∼80%, compared to the vehicle-treated group (P < 0.01). Conclusions: Reduction of neovascular tufts and avascular areas and of VEGF-driven retinal vascular leakage suggests that SZN-413 can simultaneously address retinal non-perfusion and vascular leakage. Translational Relevance: FZD4 signaling modulation by SZN-413 is a novel mechanism of action that can offer a new therapeutic strategy for diabetic retinopathy.

Robust Colonic Epithelial Regeneration and Amelioration of Colitis via FZD-Specific Activation of Wnt Signaling.

BACKGROUND AND AIMS: Current management of inflammatory bowel disease leaves a clear unmet need to treat the severe epithelial damage. Modulation of Wnt signaling might present an opportunity to achieve histological remission and mucosal healing when treating IBD. Exogenous R-spondin, which amplifies Wnt signals by maintaining cell surface expression of Frizzled (Fzd) and low-density lipoprotein receptor-related protein receptors, not only helps repair intestine epithelial damage, but also induces hyperplasia of normal epithelium. Wnt signaling may also be modulated with the recently developed Wnt mimetics, recombinant antibody-based molecules mimicking endogenous Wnts. METHODS: We first compared the epithelial healing effects of RSPO2 and a Wnt mimetic with broad Fzd specificity in an acute dextran sulfate sodium mouse colitis model. Guided by Fzd expression patterns in the colon epithelium, we also examined the effects of Wnt mimetics with subfamily Fzd specificities. RESULTS: In the DSS model, Wnt mimetics repaired damaged colon epithelium and reduced disease activity and inflammation and had no apparent effect on uninjured tissue. We further identified that the FZD5/8 and LRP6 receptor-specific Wnt mimetic, SZN-1326-p, was associated with the robust repair effect. Through a range of approaches including single-cell transcriptome analyses, we demonstrated that SZN-1326-p directly impacted epithelial cells, driving transient expansion of stem and progenitor cells, promoting differentiation of epithelial cells, histologically restoring the damaged epithelium, and secondarily to epithelial repair, reducing inflammation. CONCLUSIONS: It is feasible to design Wnt mimetics such as SZN-1326-p that impact damaged intestine epithelium specifically and restore its physiological functions, an approach that holds promise for treating epithelial damage in inflammatory bowel disease.

Development of Potent, Selective Surrogate WNT Molecules and Their Application in Defining Frizzled Requirements.

WNTs regulate myriad biological processes during embryonic development and are key regulators of stem cell function, tissue homeostasis, and injury repair in adults. The creation of WNT-based therapies has been hampered by challenges in developing soluble, potent, and selective WNT molecules. Soluble WNT surrogates have been reported, but they demonstrate relatively weak WNT signaling activity. Here, we describe a platform for potent, selective WNT surrogate generation. We identify multivalent binding to Frizzleds (FZDs) and low-density lipoprotein receptor-related proteins (LRPs) to be a requirement for maximal WNT/ß-catenin activation. Furthermore, we show that recruitment of two different FZDs together with LRP causes efficient signaling. Surrogate WNT targeting either FZD1,2,7 or FZD5,8 induces expansive growth of intestinal organoids. This flexible WNT surrogate platform yields potent agonists with any desired receptor specificity and will be useful for research and therapeutic applications for tissue regeneration.