Decoding Wnt Signalling Challenges in Cancer Therapy

The Wnt signalling pathway is a critical player in cancer progression, implicated in tumour growth, resistance mechanisms, and stem-cell renewal. However, therapeutics targeting this pathway have faced numerous challenges, from lack of specificity to poor tissue penetration in dense tumour environments.

This case study delves into the development of anti-LRP6 VHH inhibitors that overcome these challenges.

The Problem: Challenges in Targeting the Wnt Pathway

The Wnt pathway is integral to cell signalling, embryonic development, and tissue homeostasis. Dysregulation of this pathway is closely associated with cancers, including liver, lung, pancreatic, colorectal and triple-negative breast cancer.

A key player in Wnt signalling is LRP6 (Figure 1), a co-receptor critical for activating oncogenic signalling. However, traditional antibody approaches targeting LRP6 have encountered several hurdles. One significant issue is limited specificity, as these antibodies often cross-react with other receptors, causing off-target effects and subsequent toxicity. Additionally, their large size restricts tissue penetration, making it difficult to reach dense tumour microenvironments effectively.  Compounding these challenges is the production of therapeutic monoclonal antibodies, which involves high costs and lengthy development timelines.

Figure 1. Wnt signalling is driven by the binding of Wnt simultaneously to LRP5/6 and Frizzled. Both Frizzled and either LRP5 or LRP6 are needed for Wnt signaling.

Despite the promise of Wnt pathway modulation in oncology, monoclonal antibodies have struggled to achieve the necessary specificity and efficacy. Overcoming these challenges demands a shift toward innovative molecular designs that prioritise precision and robust activity.

Innovative VHH Strategies to Overcome the Issue

In partnership with the Oncode Institute (at University Medical Center Utrecht ), anti-LRP6 VHH inhibitors were discovered from Isogenica’s libraries, leveraging the unique structural properties of VHH antibodies and the strength of Isogenica’s CIS Display technology.

These antibodies exhibited exceptional target specificity. They selectively bind to LRP6 while minimizing off-target effects. Other therapies often struggle with specificity, leading to toxicity in healthy tissues. Their small size also enhances tissue penetration, which allows them to access dense tumour regions more effectively and to improve therapeutic efficacy.

Our experimental findings, published here in Nature Communications, demonstrate the therapeutic potential of these LRP6-specific VHHs. The following data will illustrate how these antibodies achieve potent Wnt signaling inhibition in vitro, resulting in significant tumour growth reduction in Wnt-dependent cancers.

Real-World Insights: Anti-LRP6 VHHs in Action

Isogenica’s LlamdA® VHH library, was used in the study by The Oncode Institute, to identify potent VHH antibodies targeting the LRP6 Wnt3-binding domain.

A standout achievement of the anti-LRP6 VHH project was its application in tumour models dependent on aberrant Wnt signalling. Using colorectal cancer cell lines with overactive Wnt signalling, VHH inhibitors effectively disrupted the pathway, leading to a marked reduction in cell proliferation.

Figure 2 shows the target region of these VHHs, concretely the LRP6P3E3P4E4 region.

Figure 2. VHHs targeting the LRP6 P3E3P4E4 module block cellular responses to Wnt3a. This module, derived from the extracellular domain of LRP6, was produced using HEK 293 cells.

Key results:

• Binding Data: Anti-LRP6 VHHs exhibited exceptional specificity with dissociation constants (Kds) in the low nanomolar range; <40nM (Figure 3).

• Functional Impact: WNT pathway activity was significantly reduced in VHH-treated cells (Figure 4).

• Therapeutic Potential: Tumour models treated with anti-LRP6 VHHs showed effective disruption of tumour growth. (Figure 5).

From an initial selection and screening process, 33 unique anti-LRP6 VHH clones were identified. Wnt luciferase reporter assays revealed that the majority effectively inhibited Wnt3α-mediated responses. Among them, the three most potent candidates – LP2-B10, L-P2-D07, and L-P2-H07, were advanced for further investigation.

The activity and specificity of these lead VHHs were validated through cellular titration assays. Notably, anti-LRP6 VHHs fully blocked Wnt3α-mediated pathway activation and cellular responses to LRP5 overexpression in LRP6-/- cells, demonstrating their ability to effectively disrupt the Wnt/β-catenin signalling pathway.

Isothermal titration calorimetry (ITC) showed that all three VHHs had low nanomolar binding affinities (<40 nM) and a 1:1 binding ratio with the LRP6P3E3P4E4 module (Figure 3)

Figure 3. ITC experiment. DP: differential power, kD: dissociation constant, N: stoichiometry. Credits: Nature Communications

To identify the best clone, a characterization of anti-LRP5/6P3 VHH with highest potency was carried out (Figure 4).

Figure 4. IC50 values were calculated for Wnt3α-induced cellular response inhibition using luciferase reporter assays, with and without the Wnt pathway agonist Rspo1, showing consistent results across experiments. Credits: Nature Communications

To understand the tumour-killing potential of these VHHs, a treatment was tested on organoids with R/Z mutations, which involve RNF43 and ZNRF3 – genes that regulate WNT signalling and whose mutations lead to excessive pathway activation. Tumourigenic organoids treated with L-P2-B10 or L-P2-H07 showed extensive cell death, similar to the effects of IWP-2, a strong WNT inhibitor (Figure 5).

Further analysis using qRT-PCR and confocal microscopy revealed that anti-LRP5/6P3 VHHs effectively blocked the growth of WNT-hypersensitive organoids by disrupting a pathway critical for stem-like tumour cell renewal.

Figure 5.  Anti-LRP5/6 VHHs promote the differentiation of R/Z-mutant tumour organoids and effectively block their growth. When treated with 10 μM of anti-LRP5/6P3 VHHs for 4 days, the organoids exhibited significant cell death, structural changes, and features of differentiation. Credits: Nature Communications

Supporting Insight

Currently, there are no LRP6-targeted therapies approved for clinical use, despite a critical unmet need in Wnt-driven cancers like colorectal and pancreatic cancer triple-negative breast cancer, where Wnt signaling drives tumour progression and drug resistance. These results underline the potential of anti-LRP6 VHHs to transform the therapeutic landscape of Wnt-related cancers.

The success of Isogenica’s anti-LRP6 VHH aligns with broader industry trends emphasising the need for smaller, more precise therapeutic molecules. Tumour penetration remains a major challenge in solid tumours due to dense stroma and high interstitial pressure. VHHs’ compact size and superior penetration enable deeper tumour access, while their low immunogenicity and high specificity make them ideal for targeting complex oncogenic pathways like Wnt.

Conclusion

The development of Anti-LRP6 VHH antibodies provides high specificity and enhanced tissue penetration. These antibodies precisely target the Wnt pathway by selectively binding to LRP6 and minimising off-target effects. This advancement tackles important obstacles in cancer therapy and overcomes limitations of traditional approaches.

Furthermore, they leverage scalable production methods, reducing costs and timelines compared to traditional antibodies. By combining these advantages, this results in a transformative solution for targeting the complex Wnt pathway in cancer.

Application note: 

Despite the critical need for therapies targeting LRP6 in Wnt-driven cancers no approved treatments exist. Isogenica’s anti-LRP6 VHHs offer significant promise, with the potential to transform the treatment landscape of these cancers.

Aligning with industry trends, Isogenica’s VHHs antibodies compact size, low immunogenicity, and high specificity make them ideal for targeting complex oncogenic pathways like Wnt, showcasing the strength of Isogenica’s vhh libraries in advancing cancer therapies.

Read the full application note to explore the groundbreaking science behind our approach and its potential in cancer therapy.