Surrozen : Pep Talk 2022 Poster

Development of Potent, Selective Surrogate WNT Molecules and their Application in Tissue Regeneration

Hui Chen*, Chenggang Lu, Brian Ouyang, Haili Zhang, Zhong Huang, Diksha Bhatia, Sung-Jin Lee, Darshini Shah, Asmiti Sura, Liqin Xie, Wen-Chen Yeh, and Yang Li Surrozen Inc., South San Francisco, CA 94080, USA; *presenting author

Abstract

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 the platforms 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/β-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. In addition, bispecific FZD5,8 WNT surrogate in IgG1 format repairs the damaged colon epithelium in DSS mouse model. The flexible WNT surrogate platforms yield potent agonists with any desired receptor specificity and will be useful for research and therapeutic applications for tissue regeneration.

Introduction

The WNT pathway is crucial for embryonic development, adult tissue homeostasis and injury repair. There are 19 mammalian WNTs that can induce WNT/β-catenin signaling through binding to receptors Frizzled (FZD) and low- density lipoprotein receptor-related protein (LRP) on the cell surface. The formation of the WNT-FZD-LRP complex inhibits the β-catenin destruction complex, leads to stabilization of the β-catenin protein and regulates target gene transcription via the T cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors. WNTs are highly hydrophobic lipoglycoproteins, difficult to express, solubilize, and purify. Due to the conserved interaction sites between WNTs and FZDs, WNTs are also highly promiscuous in their interactions with the ten FZDs (FZD1-10) and the two LRPs (LRP5 and LRP6), with each WNT able to activate multiple FZD and LRP pairs. Therefore, while WNT signaling plays a central role in tissue regeneration and holds promise for the treatment of degenerative diseases and diseases where tissue regeneration could confer a therapeutic benefit, the difficulty in manufacturing the ligands, the complexity in signaling, and lack of specificity have made the endogenous ligands intractable as drug targets.

The discovery of WNT surrogates addressed these challenges (Janda et al., 2017) by fusing FZD binding domain and LRP binding domain of the C-terminal fragment of the WNT antagonist Dickkopf-1 (DKK1c) into a bispecific molecule which agonized β-catenin dependent signaling and phenocopied WNT in vitro and in vivo (Janda et al., 2017, Yan et al., 2017). Due to the lack of the fatty acyl modification, these surrogate molecules are water soluble, opening opportunities to design therapeutic molecules for tissue regeneration. However, whether there are alternative approaches to generate soluble WNT surrogates was not explored in the original surrogate WNT report (Janda et al., 2017). In the present work, we explore the general applicability of the surrogate bispecific concept by assembling various combinations of FZD and LRP binders with different stoichiometry and FZD specificity, ultimately, test their ability to stimulate intestinal organoid growth and repair the damaged colon epithelium in acute DSS mouse model.

Results

Figure 1. Tetravalent bispecific L1/F1 tandem scFv-Fc molecules efficiently activate β-catenin dependent WNT signaling

Figure 3. Exploring different stoichiometries of FZD and LRP binders and combining binders of different receptor specificities or epitopes in the 2:2 tetravalent multispecific formats

Figure 4. The diagram of the valency	Figure 5. Surrogate WNTs stimulated proliferation and growth of
requirements for activating WNT signaling	mouse intestinal organoids

Figure 1A

F

Figure 1A. 1:1 bivalent bispecific L1/F1 tandem scFv molecules are not efficient in activating β-catenin dependent WNT signaling.

Figure 1B

A

B

C	L1: LRP6 E1E2
	domain binder in scFv
	format. F1:18R5,
	FZD1,2,7,5,8
	cysteine-rich domains
	(CRD) binder in scFv
	format

Figure 1B. Increasing the valency of L1 and F1 tandem scFv by fusing to a Fc domain significantly increased the activity in WNT signal.

Figure 6. The tetravalent bi-specific IgG1 antibody, SZN-1326 activates WNT signaling in Huh7 cells and restores the damaged colon epithelium in acute DSS model

SZN-1326 STF		SZN-1326 Treatments Repaired Damaged Colon Epithelium
		SZN-1326		Wnt signal	Epithelial	Anti-inflammatory	Functional
		dose		activation	repair		effect	improvement
	anti-Lrp6
	VHH
		No DSS			DSS + Anti-GFP		SZN-1326 10 mpk x 2

anti-FZD(5,8) IgG

SZN-1326 10 mpk x 4	SZN-1326 1 mpk x 4	Anti-TNF 25 mpk x 7

Figure 2. The 2:2 tetravalent bispecific molecules, consisting of the two F and two L binding arms, are highly potent in inducing WNT signaling

Summary

• A flexible system for generating potent and selective surrogate WNTs is developed
• Multivalent binding to FZD and LRP is a requirement for maximal activation
• Active receptor complex can contain different ratios of receptors
• Recruitment of two different FZDs together with LRP induces efficient signaling
• SZN-1326,the tetravalent bi-specific IgG1 WNT mimetic, restores the damaged colon epithelium in acute DSS model

Figure 2. L2: LRP6E3E4 domain binder in scFv format. F2: FZD1,2,7 CRD binder in scFv format. F3: FZD5,8 CRD binder in scFv format

Note: Figures 1-5 are reprinted from "Development of Potent, Selective Surrogate WNT Molecules and Their Application in Defining Frizzled Requirements," Chen et al., 2020 Cell Chemical Biology 27, 598-609, with permission from Elsevier.

Presenting author: Hui Chen

Surrozen Inc., South San Francisco, CA 94080 email: [e-mail]

www.surrozen.com