Poster J1 - Antibody-Mediated Degradation of Intracellular Protein Targets

Thursday, October 24, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Primary Investigator(s)

Andrew Tsourkas, PhD

Primary Investigator
University of Pennsylvania
Philadelphia, Pennsylvania, United States

Presenting Author(s)

Chiara Cline

Undergraduate Researcher
University of Pennsylvania
Menlo Park, California, United States

Co-Author(s)

SM

Shruthi Murali, MSE

PhD Candidate
University of Pennsylvania, United States

Introduction: Targeted protein degradation is an emerging field, as it enables the removal of specific disease-related proteins that may not respond to small-molecule targeted therapy. One method involves biological Proteolysis Targeting Chimeras (bioPROTACs), which have a protein-targeting domain fused to an E3 ligase to facilitate targeted ubiquitination (1). This process requires significant resources, as a new protein-targeting domain is required for each new target protein. By fusing full-length antibodies to an E3 ligase, this project aims to establish a universal platform for intracellular protein ubiquitination where any targeting antibody can be used to degrade any intracellular target. The E3 ligase used will be IpaH 9.8, and the E3 ligase will be linked to an antibody via a photoreactive antibody-binding domain (pAbBD), which has been shown to form a covalent bond with IgG Fc under ultraviolet light (2). A glycine-glycine-serine (GGS) linker of various lengths – 43 amino acids, 56 amino acids, or 77 amino acids will be used to connect IpaH 9.8 and pAbBD. By modifying the linker lengths between IpaH 9.8 and pAbBD, this project seeks to minimize the spatial distance between the E3 ligase and the target substrate, since ubiquitination is proximity-based. At the C-terminus of IpaH-pAbBD, there is a 25 amino acid-long chain of negatively-charged aspartic acid (D25), which complexes with a cationic lipid (Lipofectamine 2000) to facilitate intracellular delivery and endosomal escape of the protein-antibody complex (3). The goal of this project is to create a modular intracellular degradation platform that can be adapted to various proteins.

Materials and

Methods: The 77AA linker modification was introduced as a gene fragment via InFusion Snap Assembly into the IpaH-pAbBD vector backbone. Following Sanger sequencing to confirm insertion of the linker, a restriction digest was performed to produce the shorter linkers (56AA and 43AA). The products were expressed at 25C for 48 hours in E. Coli Shuffle T7 Express competent cells and purified using Sortase-Tagged Expressed Protein Ligation (STEPL), a purification technique previously developed by our lab, based on immobilized metal affinity chromatography via a His tag and ligation of triglycine (GGG) via SortaseA (4). Following purification, IpaH-GGS(77AA)-pAbBD was crosslinked to a panel of antibodies for two hours under UV light. The clinical anti-EGFR antibody Cetuximab was used as a negative control and a rabbit polyclonal anti-GFP antibody was used as the targeting domain for the model substrate eGFP. Crosslinking efficiency was analyzed by reducing SDS-PAGE (180V, 30 minutes). The ubiquitination activity of IpaH 9.8 was tested using an in vitro Ubiquitination Assay (R&D Systems) and Western Blot. To confirm cytosolic delivery of the cells, flow cytometry was performed on the IpaH protein alone and both crosslinked products, where endosomal escape was confirmed by a split-GFP reporter system.

Results, Conclusions, and Discussions: The protein with the 77 amino acid GGS linker (IpaH-GGS(77AA)-pAbBD) expressed the highest, at around 3.1mg/mL, after STEPL purification. A crosslinking pilot determined that a 3:1 IpaH:antibody ratio achieved the highest crosslink efficiency, 66%. The in vitro ubiquitination Western Blot demonstrated polyubiquitination of eGFP in the presence of ATP catalyzed by free IpaH and IpaH crosslinked to an anti-GFP antibody. Cetuximab, which does not target GFP, showed polyubiquitination products to a lesser degree. The eGFP ubiquitination products in the Cetuximab and uncrosslinked IpaH lanes indicate that nonspecific, proximity-based ubiquitination occurs due to an excess of free IpaH in the crosslinked product. When the molar ratio of IpaH:eGFP in the in vitro ubiquitination assay was reduced from 1:10 to 0.05:10, there was no eGFP ubiquitination, indicating that in order to eliminate non-specific degradation, 95% of free IpaH should be removed from the crosslinked product. To remove free IpaH, magnetic beads coated in rabbit IgG were used (AlphaThera), relying on the C-terminal pAbBD domain’s affinity for Rabbit IgG Fc. These beads removed 90.7%-96.2% of free IpaH from the crosslinked product with some loss of product. Benchtop anion exchange chromatography (AEX) was also used to remove free IpaH. Since the IpaH alone is negatively charged at pH 4, it can be separated from the positively charged crosslinked IpaH by using a positively charged column bed. A silver stain of the AEX washes and elutions showed successful separation of free antibody from the solution, but the crosslinked product remained attached to the column. In future experiments, a weaker positive column should be used. The eGFP ubiquitination to a higher degree in the anti-GFP crosslinked lane is promising, but the purification of the crosslinked product using IgG beads or AEX needs optimization. Flow cytometry confirmed via split-GFP fluorescence that IpaH alone delivered with 43.7% efficiency, IpaH crosslinked to Cetuximab delivered with 48.5% efficiency, and IpaH crosslinked to a rabbit anti-GFP delivered with 17.1% efficiency. A future experiment will involve delivery of anti-GFP crosslinked to IpaH to degrade eGFP in GFP-expressing cells.

Acknowledgements (Optional):

References:
1. Yao T, Xiao H, Wang H, Xu X. Recent Advances in PROTACs for Drug Targeted Protein Research. Int J Mol Sci. 2022 Sep 7;23(18):10328. doi: 10.3390/ijms231810328. PMID: 36142231; PMCID: PMC9499226.
2. Zappala F, Tsourkas A. Site-Specific Photocrosslinking to Immunoglobulin G Using Photoreactive Antibody-Binding Domains. Methods Mol Biol. 2019;2033:275-286. doi: 10.1007/978-1-4939-9654-4_18. PMID: 31332760.
3. Wang HH, Tsourkas A. Cytosolic delivery of inhibitory antibodies with cationic lipids. Proc Natl Acad Sci U S A. 2019 Oct 29;116(44):22132-22139. doi: 10.1073/pnas.1913973116. Epub 2019 Oct 14. PMID: 31611380; PMCID: PMC6825285.
4. Warden-Rothman R, Caturegli I, Popik V, Tsourkas A. Sortase-tag expressed protein ligation: combining protein purification and site-specific bioconjugation into a single step. Anal Chem. 2013 Nov 19;85(22):11090-7. doi: 10.1021/ac402871k. Epub 2013 Oct 28. PMID: 24111659; PMCID: PMC3843242.