Antibody Drug Conjugates – The Importance of an Emerging Modality

by Michael Caves, Thursday, 25th July 2024

What are Antibody Drug Conjugates (ADCs)?

Antibody Drug Conjugates are playing an increasingly important role in the Biotech revolution, combining the specificity of antibody-antigen binding interactions with high-toxicity payloads (API) that could not be safely administered systemically on its own. Following the first release in the early-2000s, seven ADCs received FDA-approval in the 2010s, a number already surpassed in the first half of the current decade.

Why now?

Conjugation to antibodies allows highly cytotoxic agents to be more effectively delivered to targets, expanding the therapeutic window. The risk of side-effects due to the payloads reaching healthy tissues is minimized, whilst the effect of the drug on the diseased tissue is maximized. Monoclonal antibodies have been on the market for nearly 40 years, and research on toxic agents used in ADCs, including DNA and tubulin inhibitors, also have a long history of research behind them. It is improvements to the linker technology in the current millennium that has led to the steep increase in ADCs reaching the market. Early linkers had issues with chemical instability leading to non-specific release of payload narrowing the therapeutic window, as well as limits to the amount of linker (and therefore quantity of drug) that could be conjugated with the antibody. Over the last 10-15 years, more linkers have been designed to release the API inside the target cells through cleavage and pH sensitivity.

How much?

ADCs have proven to be a highly effective means of treating cancer, a disease for which there are ~ 20 million new cases per year. Enhertu gave its developers (AstraZeneca and Daiichi Sankyo) combined sales of 2.5 M USD in 2023, and is one of several blockbuster ADCs currently on the market. In 2023, the ADC market surpassed 10 M USD for the first time. Such returns are necessary for such a research-intensive modality for which characterization is key to reducing the risks of costly failure.

Quality requirements

In addition to the nature of the linker, a key factor in the success of ADC development is the structure and stability of the antibody, given the high-hydrophobicity of most cytotoxic agents. To give an example, Lysine-based conjugation is one of the most widely used conjugation strategies. Lysine residues are widely dispersed across the surface of IgG molecules, with ~20 being solvent accessible acting as potential conjugation sites. Some of these surface lysine residues are in the variable regions of the antibody that are responsible for antigen binding, with the conjugation potentially affecting the effectiveness of the antibody vector and the efficacy of the ADC.

It is vital, therefore, that we characterize the binding of the ADC with antigen after conjugation, and this is something that Malvern Panalytical helps pharmaceutical companies across the world with. The WAVEsystem, a Creoptix GCI technology, uses no-clog microfluidic cartridges to screen the binding kinetics of the widest range of sample types and sizes. Microcal PEAQ ITC, on the other hand, is a highly sensitive, low volume Isothermal Titration Calorimeter that gives comprehensive label-free characterization of binding under physiological conditions. The impact of conjugation on the mechanism, not just the strength, of binding is explored using PEAQ ITC across the biotechnology industry.

Stability concerns

In addition to binding efficacy, conjugation also impacts the structural stability of the antibody-vectors, generally reducing it. To confirm the processability of the antibody, and an optimal shelf-life if successful, the nature of this reduction must be characterised.

Microcal PEAQ DSC is the gold standard method for structural stability analysis of biotherapeutics in solution, whether it be for protein engineering, formulation, and batch-to-batch comparability studies. PEAQ DSC is fully automatable, both in terms of hardware and software, and 21 CFR Part 11 compliant, designed with the requirements of the biopharmaceutical industry in mind. Whatever impact the conjugation of an API has on stability, PEAQ DSC characterizes and gives information required to either help mitigate the change (through excipient selection, structural engineering, etc.) or halt development early.

Aggregation and submicron particle development

The negative impact of conjugation on ADC stability leads to an increased propensity for aggregation, in part due to shifts induced in the Isoelectric Point (pI) and changes in surface charge heterogeneity, in addition to hydrophobic interactions between the API and the core of the protein.

The Zetasizer Advance range is used to predict and detect the earliest stages of aggregation, Dynamic Light Scattering (DLS) being highly sensitive to the presence of large particles in solution. A simple cuvette-based technique, a 5-minute assay can be used to assess the presence of aggregates in an ADC sample. Zeta Potential measurements on the same system, meanwhile, allow assessment of the charge status of ADCs, a critical factor in the propensity for these biomolecules to aggregate. Oligomer content is another predictor of aggregation, and our Size Exclusion Chromatography product (OMNISEC) includes light scattering detectors to characetrize better detect and quantify the earliest stages of aggregation. In addition to monomeric purity, OMNISEC system reports on Drug Antibody Ratio (DAR) which is another CQA for ADCs.

To further characterize submicron particles within ADCs, Malvern Panalytical also offers the NanoSight Pro system, a Nanoparticle Tracking Analysis (NTA) system designed for the requirements of the industry. The NanoSight Pro allows automated, high resolution sizing and concentration analysis for submicron particles, helping ADC developers control the levels of potentially immunogenic larger particles in their drug, an area of increasing concern for regulatory authorities.

Enabling ADC development across the development process

With our technology and expertise, both in terms of cutting-edge research and innovation, Malvern Panalytical is a key part of the development of successful ADCs. From assessment of binding-site structure and effectiveness to stability and particle mitigation, our products and knowledge are used to optimize efficiency and quality whilst minimizing the risk of costly failure.