Abstract 7016: PROTiNb: A novel platform for developing biodegraders targeting intracellular proteins

Abstract Since the concept of targeted protein degradation (TPD) was first introduced in the 1990s, innovative technologies such as PROTACs and molecular glues have rapidly emerged and advanced, leveraging pathways including the ubiquitin-proteasome system, lysosome-mediated degradation, and autophagy. A notable limitation of PROTAC technology is its dependence on small molecule ligands to engage the target protein or the E3 ligase. These small molecules are often vulnerable to target mutations, which can interfere with binding and are considered a major cause of drug resistance. Furthermore, most existing TPD technologies rely on a limited set of endogenous E3 ligases, primarily CRBN and VHL.Nanobodies, on the other hand, provide larger binding surfaces and greater structural flexibility, allowing for a broader range of target proteins and potentially mitigating resistance risks. Recognizing these advantages, we established PROTiNb (Proteolysis Targeting intra-Nanobody) platform. This platform integrates engineered E3 ligases with VHH nanobodies in RNA form to be translated in the cells, enabling highly efficient and specific protein degradation. For instance, in GFP-overexpressing cell lines, LaG16-SPOPm (modified SPOP) and GFP enhancer-RNF4m (modified RNF4) demonstrated higher degradation efficiency compared to their wild-type E3 ligase counterparts. Similarly, when fused with KRAS-specific VHH RT22, RT22-SPOPm showed superior degradation activity over RT22-SPOPwt, leading to enhanced inhibition of tumor cell proliferation. Furthermore, the fusion of RNF4m with the PCNA-specific VHH efficiently degraded PCNA in cancer cells, inducing cell cycle arrest and significantly suppression of tumor cell growth in vitro. We inoculated Nude mice with HCT116 cells overexpressing doxycycline-induced KRAS PROTiNb and induced PROTiNb expression by feeding doxycycline. The induction of KRAS PROTiNb significantly inhibited tumor growth. To translate this platform into a therapeutic application, we used a tumor-targeted lipid nanoparticle (LNP) for in vivo delivery. In vitro studies revealed that tumor-targeted LNPs enhanced the delivery specificity and increased the tumor-suppressive potency. Most notably, these nanobody-based degraders exhibited robust activity in vivo, effectively suppressing tumor growth. In summary, our study demonstrates the versatility of the PROTiNb platform in rapidly generating degraders against intracellular targets once considered undruggable, with promising potential for clinical therapy for cancers. Citation Format: Hongzhuan Gu, Lijun Ye, Zexiu Huang, Dan Xu, Lizhen Zhan, Tian Wang, Liang Xiao, Yue Huang, Baiguang Ren, Chang Zhou, Ke Chen, Xiaodong Wu, Di Lu. PROTiNb: A novel platform for developing biodegraders targeting intracellular proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7016.