Structural biology and drug discovery

Affinity Maturation of Monoclonal Antibodies with Radical Footprinting

Hetal Marble, Ph.D.

Head of Commercial, Immuto Scientific

Monoclonal antibodies (mAbs) are frequently used to treat a wide range of conditions, including cancer, autoimmune disorders, and infectious diseases. However, not all mAbs are equally effective in treating these diseases, and the affinity maturation process is critical in improving binding affinity and therapeutic efficacy. Affinity maturation of monoclonal antibodies (mAbs) is a crucial process in the development of therapeutic antibodies. It involves iteratively mutating the antibodies to increase their binding affinity to the target antigen, resulting in high-affinity mAbs that are more effective in treating diseases and have improved pharmacokinetic properties compared to low-affinity mAbs.

Traditionally, affinity maturation has been achieved using conventional techniques such as cryo-electron microscopy (cryoEM) and surface plasmon resonance (SPR). These procedures, however, are time-consuming, complicated, and costly. Furthermore, they necessitate large amounts of purified protein, which can be difficult to obtain. In addition, these approaches have resolution limitations and are unsuitable for high-throughput analysis of large numbers of mAbs.

Hydroxyl radical footprinting (HRF) is a new technology that has the potential to significantly improve and simplify the affinity maturation process. HRF is based on the reactivity of hydroxyl radicals with proteins, which results in peptide bond cleavage. Researchers can assess the position of the antigen-antibody interface and identify key amino acids that contribute to binding affinity by measuring the level of cleavage.

HRF offers various advantages over typical affinity maturation procedures. For starters, HRF may be performed with modest amounts of protein, making it more accessible and affordable. Second, HRF can assess many mAbs in parallel, making it a more efficient and high-throughput approach. Finally, HRF provides a high-resolution map of the antigen-antibody interface that can be used to guide the rational design of mAbs with higher binding affinity.

A study used HRF to discover the epitope of the anti-TNF-alpha monoclonal antibody adalimumab. The researchers employed mass spectrometry to determine the cleavage sites after obtaining hydroxyl radicals via the Fenton reaction. They then employed molecular modeling to predict the orientation of adalimumab relative to TNF-alpha, and their findings agreed with prior research that used other approaches.

While HRF has several advantages for affinity maturation, this method has several drawbacks. For starters, HRF necessitates specialized equipment and experience, which may limit its broad use. Second, HRF can be challenging to understand, especially in the case of complicated protein-protein interactions. Immuto's PLIMB platform was developed to circumvent these exact issues - results delivered for each project are always easy to understand, immediately actionable, and reproducible, and every project is staffed with highly trained expert scientists who reduce HRF to practice on a daily basis, guaranteeing success.

HRF has been used in several real-world applications, demonstrating its potential in mAb development. For example, a recent work employed HRF to identify key amino acids involved in the interaction of the SARS-CoV-2 spike protein and neutralizing mAbs. The researchers discovered that HRF could detect small changes in the interaction of different mAbs with the spike protein, which might be utilized to guide the development of more effective neutralizing mAbs. Another study employed HRF to identify key amino acids involved in the interaction between the HIV gp120 protein and mAbs that target it. The researchers discovered that HRF could be utilized to identify crucial binding affinity residues that might be targeted for modification to improve the efficacy of the mAbs.

Affinity maturation of monoclonal antibodies is an important step in generating therapeutic antibodies, and hydroxyl radical footprinting can enhance and simplify the process. HRF generates a high-resolution map of the antigen-antibody interface, making it a valuable tool for rationally designing mAbs with higher binding affinity. However, more research is required to address the difficulties associated with this method and determine its optimal conditions.

AUTHOR BIO

Hetal Marble, Ph.D.

Head of Commercial, Immuto Scientific

Dr. Hetal Marble is Head of Commercial for Immuto Scientific, a boutique CRO focused on protein structural characterization and drug discovery. Dr. Marble has had varied and extensive experience leading life science businesses. She led diagnostic product management at Quanterix Corporation, where she launched Quanterix’s first diagnostic test for Alzheimer’s disease and directed product strategy and go-to-market for all clinical diagnostic and research services products. She also led companion diagnostics and biomarker development for the Center for Integrated Diagnostics at MGH where she grew the division’s in-house CRO into a multi-million dollar enterprise by translating novel technologies into clinical practice and leading precision biomarker strategies for clinical trials. Prior to her work at MGH, Hetal worked in business development, alliance management, and strategic product marketing for both Promega Corporation and Pall Biotech (a Danaher company), managing and growing burgeoning commercial territories into millions of dollars in revenue. Hetal is foundationally an experienced research scientist – she received her Ph.D. at Brown University, where her graduate work focused on development of biomarkers for lineage-specific stem cell differentiation - she is the holder of two U.S. patents emerging from that work. Hetal is active in the scientific community; she is the elected Chair of Diversity, Equity, and Inclusion for the Association for Molecular Pathology (AMP); she is a member of the 2022 Women in Bio (WiB) cohort of Emerging Executives; she served on the Pathology Informatics Collaborative Community (PIcc), a collaborative community of the US Food and Drug Administration; and she is a contributing author on precision medicine for the book Taking Care of You, a book on women’s health being published by the Mayo Clinic Press in October 2022.

Related articles

Immuto Technology and Platform

Common Techniques for Protein Footprinting Analysis: A Quick Guide

Immuto Technology and Platform

What is Protein Footprinting, and How Can it Be Used in Drug Discovery?

Structural biology and drug discovery

Future-Proofing Drug Discovery: The Power of Rational Drug Design