Designing high affinity therapeutic nanobodies through the incorporation of unnatural a mino acids

2018 Fiscal Year Annual Research Report

• Project/Area Number: 18F18074
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: ZHANG KAM 国立研究開発法人理化学研究所, 生命機能科学研究センター, チームリーダー (60558906)
• Co-Investigator(Kenkyū-buntansha): PADHI ADITYA 国立研究開発法人理化学研究所, 生命機能科学研究センター, 外国人特別研究員
• Project Period (FY): 2018-10-12 – 2021-03-31
• Keywords: Unnatural amino acid / Nanobodies

Outline of Annual Research Achievements

Incorporation of non-canonical amino acids (ncAA) by genetic fusion or chemical conjugation offers a promising strategy for protein engineering, drug designing and therapeutics development. One of the potential applications of ncAAs is to couple them in the production of highly potent and stable antibodies, protein scaffolds that have inhibitory competence. The single domain heavy-chain only antibodies or nanobodies, due to their small size and ease of production offer such benefits. Although experimental methods can aid in the production of ncAA-coupled macromolecules, they pose limitations while selecting suitable ncAAs from a large number of possible modifications and eventually incorporating into nanobodies. Moreover, these approaches are often time consuming and cumbersome. To bridge this gap, we propose to develop a unified computational platform integrated with state-of-the-art biomolecular energetics, quantum, machine learning, structural and in cerebro methods for the generation of hyperstable, potent ncAA-coupled inhibitory nanobodies. Preliminary results obtained from our approach yielded good correlation with experimental measurements and showed prominent affinity towards EGFR. Overall, our computational approach has the potential to generate experimentally verifiable designs and can prioritize the selection of non-natural sidechains. We believe our technology offers a powerful alternative to high-throughput ncAA screening and could pave the way towards therapeutics development.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

To demonstrate the feasibility of our computational approach, we introduced 4 types of halogenated ncAAs (3-Chloro, 3-Iodo, 3-Bromo and 3-meta Nitro) to tyrosine residues of the 7D12 nanobody that target EGFR and carried out molecular dynamics (MD) simulations 33 systems. Binding energies were calculated for each mutant and wild-type by high-throughput MM-PBSA method from 100 snapshots using g_mmpbsa tool. The Kd values determined from surface plasmon resonance (SPR) experiments yielded a high correlation of 0.76, 0.70, 0.68 and 0.57 for Chloro, Iodo, Nitro and Bromo-tyrosine mutants respectively. Notably, an order of magnitude increase in potency was obtained towards EGFR when the 7D12 nanobody was incorporated with ncAA-tyrosines at certain positions.

Strategy for Future Research Activity

i.Since the incorporation of halogenated tyrosines in 7D12 nanobody exhibited a notable increase in binding affinity towards EGFR, we planned to expand our approach to other important nanobodies such as 9G8 and EgA1 that target EGFR.
ii.In order to increase the accuracy of computational calculations and contemplate the halogenated side chains, several other in-silico features including bio- and chemo-informatics, structural modeling, machine learning and quantum calculations will be taken into consideration in addition to MD simulation and MM-PBSA based free energy calculations, eventually to be tested in vitro.