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Development of nanobody-based PROTAC (NbTAC) for ROR1 degradation by RNF149
Yue Zhao1, Hongyang Wang1, Ping Wu2
1School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
Abstract:
Receptor tyrosine kinase-like orphan receptor 1 (ROR1) plays a key role in noncanonical Wnt signaling. It is frequently overexpressed in tumors, and high ROR1 level is associated with poor patient survival. ROR1 has emerged as a potential therapeutic target for cancer therapy. Here, we report the first nanobody-based proteolysis-targeting chimeras (NbTAC) to degrade ROR1, by bringing the membrane-anchored E3 ubiquitin ligase RING finger protein 149 (RNF149) and membrane-bound ROR1 to proximity. We used phage display approach, discovered specific nanobodies for the extracellular domains (ECD) of RNF149 and ROR1, respectively. A number of bispecific NbTACs, formed by fusing an anti-RNF149 nanobody with an anti-ROR1 nanobody, were tested for their activities to degrade ROR1 in the cell. Among the NbTACs, N16-R14 exhibited specific and efficient elimination of ROR1 and proliferation inhibition of triple-negative breast cancer (TNBC) cell lines at nanomolar concentration. This ROR1 NbTAC may serve as a potential therapeutic agent to block ROR1-associated cancer progress in various indications. This RNF149-induced target degradation strategy may also be adapted to other membrane target proteins for broad applications.
Insights
Researchers developed novel nanobody-based proteolysis-targeting chimeras (NbTACs) to degrade Receptor tyrosine kinase-like orphan receptor 1 (ROR1). This targeted ROR1 degradation effectively inhibited triple-negative breast cancer cell proliferation.
Area of Science:
- Oncology
- Molecular Biology
- Biotechnology
Background:
- Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is implicated in noncanonical Wnt signaling and frequently overexpressed in various cancers.
- Elevated ROR1 levels correlate with reduced patient survival, establishing ROR1 as a significant therapeutic target in oncology.
- Current therapeutic strategies targeting ROR1 are limited, necessitating novel approaches for its effective inhibition.
Purpose of the Study:
- To develop and characterize the first nanobody-based proteolysis-targeting chimeras (NbTACs) for the targeted degradation of ROR1.
- To investigate the efficacy of NbTACs in degrading ROR1 and inhibiting cancer cell proliferation, particularly in triple-negative breast cancer (TNBC).
- To explore the potential of RNF149-induced target degradation as a versatile platform for targeting membrane-bound proteins.
Main Methods:
- Phage display was employed to identify specific nanobodies targeting the extracellular domains of RNF149 (an E3 ubiquitin ligase) and ROR1.
- Bispecific NbTACs were constructed by fusing anti-RNF149 and anti-ROR1 nanobodies.
- The ROR1-degrading efficacy and anti-proliferative effects of NbTACs were evaluated in TNBC cell lines.
Main Results:
- Specific nanobodies targeting RNF149 and ROR1 were successfully isolated.
- Constructed NbTACs demonstrated the ability to induce proximity between RNF149 and ROR1, leading to ROR1 degradation.
- The NbTAC N16-R14 exhibited potent and specific ROR1 elimination and significant inhibition of TNBC cell proliferation at nanomolar concentrations.
Conclusions:
- Nanobody-based proteolysis-targeting chimeras (NbTACs) represent a promising new strategy for targeted ROR1 degradation.
- NbTAC N16-R14 shows therapeutic potential for treating ROR1-driven cancers, including TNBC.
- The RNF149-induced target degradation approach is adaptable for targeting other membrane proteins, offering broad therapeutic applications.
