Targeting the cell-killing effects of drugs or radiation with the specificity of antibodies is not a new idea. Early attempts at producing antibody-drug conjugates (ADCs), the most advanced embodiment of that idea, focused chiefly on finding antibodies with the appropriate target-seeking activity, which were not immunogenic and could be produced at a reasonable cost. Spacers and toxic payload considerations were considered “engineering problems” that would be easily overcome. But by the 1990s, with robust monoclonal antibody discovery and manufacturing methodology in place, it became apparent that antibodies were not the only piece of the ADC puzzle holding back these promising therapeutics.
Interconnected Issues
Today, nearly a quarter-century after the first U.S. ADC drug approval, developers still grapple with drug resistance, suboptimal therapeutic windows, and molecular heterogeneity—interconnected issues for which any of an ADC’s physical components may be responsible.
For example, improving tumor penetration by altering the charge or hydrophilicity of the linker may adversely affect pharmacokinetics or target affinity, thereby solving one problem while introducing another.
“Resistance to ADCs is a complex issue that significantly limits their effectiveness,” says Namrata Jayanth, Ph.D., Research Leader for Biotherapeutics at Charles River. The main mechanisms of ADC resistance involve loss or downregulation of the target antigen, changes in ADC processing, and “payload resistance” involving increased drug efflux, altered drug targets, and enhanced DNA repair mechanisms.
To address resistance mechanisms, researchers are exploring variations on the classical drug-plus-spacer-plus-payload theme, including:
- Bispecific ADCs targeting two different epitopes or antigens
- Combining ADCs with other targeted therapies to overcome resistance
- Novel linkers that are more stable in circulation but are readily cleaved within the tumor
- Exploring new cytotoxic agents or dual-payload ADCs to combat payload resistance
- Immunomodulatory ADCs that stimulate the immune system to enhance the anti-tumor response
The therapeutic window (or therapeutic index) of a drug is the range of dosages a typical patient tolerates without severe toxicity. Since many ADC payloads are even more toxic than standard chemotherapy agents, the potential for serious side effects is great and the therapeutic index for ADCs tends to be narrow.
Strategies for improving ADCs’ therapeutic window include incorporating payloads with improved potency and selectivity, enhancing tumor penetration by replacing intact antibodies with antibody fragments or bispecific antibodies, improving linker technology to reduce off-target effects and improve safety, and exploring alternative targets to improve selectivity and reduce off-target toxicity.
Here again, developers walk a thin line between effectiveness and toxicity.
“By addressing resistance and narrow therapeutic windows, researchers and clinicians can work toward creating more effective ADC therapies with improved durability of response and better patient outcomes,” Jayanth tells Biocompare.
Heterogeneity, a project-killer?
Molecular homogeneity—having just one active species in a drug formulation—is a given for small molecule drugs but challenging for biologicals and even more of an issue for large, multicomponent ADCs.
“ADCs exhibit significant molecular heterogeneity, which stems primarily from the complexities involved in their manufacturing process and the stochastic nature of drug conjugation,” says Claes Gustafsson, Co-founder at ATUM. Because many proteins carry multiple potential attachment sites, ADC conjugation methods often result in variable drug-to-antibody ratios (DARs), leading to heterogeneous mixtures of ADC species that affect pharmacokinetics, effectiveness, and toxicity. “Furthermore, the conjugation sites on the antibody can differ, influencing stability and activity due to variations in structural orientation and linker accessibility.”
The chemical linker also contributes to heterogeneity through differences in stability and susceptibility to enzymatic cleavage or hydrolysis. Linker design and attachment chemistry play critical roles in ensuring controlled drug release while minimizing premature detachment during circulation.
“Manufacturing-related factors like process conditions, buffer compositions, and purification techniques further amplify heterogeneity,” Gustafsson says. Additionally, post-production modifications, such as aggregation or degradation, can alter ADC characteristics over time.”
Efforts to reduce heterogeneity focus on site-specific conjugation technologies, which enable precise control over drug attachment sites and DARs. These approaches enhance uniformity, improving therapeutic performance and safety. Addressing molecular heterogeneity remains a pivotal aspect of ADC development to ensure consistent and effective cancer treatment outcomes.
Analytical challenges
Although ADCs work on disease targets through their antibody component, linker and payload choices also affect the efficacy-safety equation. Each component and each combination presents unique analytical challenges, particularly in determining payload-antibody stoichiometry.
For antibody oligonucleotide conjugates, where the payload is a gene-modifying oligo, characterization methods must be capable of independently assessing gene and protein components, both bound and unbound. According to Salma Jalal, Market Development Manager at Refeyn, “Platform methods developed for monoclonal antibodies often fail to accommodate RNA’s unique chemical properties, which can slow development timelines.”
The diverse chemistries of antibody, linker, and payload complicate ADC analytics because each may provide an independent signal, especially in optical methods like UV absorbance—a modality typically used to monitor size exclusion chromatography. Similarly, post-translational modification analysis via mass spectrometry becomes complicated by RNA’s high negative charge in ADCs.
“Further challenges with column-based approaches, such as size exclusion, include column-sample interactions that alter retention times and sample properties,” Jalal adds. “Column clogging by heavily glycosylated and other aggregation-prone antibodies is also challenging because it adversely affects the assessment of antibody stability. These issues all necessitate tailored analytics for each combination of targeting protein, linker, and payload, and so can extend development timelines.”
Conventional analytical method development, which is already quite slow for relatively homogeneous therapeutics like antibodies, slows to a crawl for ADCs, hindering rapid decision-making for candidate selection. “In biopharma, typical turnarounds for aggregation analysis by SEC is around a day, and ultracentrifugation can take up to a week,” Jalal says.
In some cases, such as in-use studies of IV-delivered drugs, samples at nanomolar concentrations must be concentrated to micromolar levels for SEC analysis. In addition to being time- and resource-intensive, the concentration step risks altering a sample’s properties.
“That’s why next-generation treatments require next-generation analytical technologies,” Jalal tells Biocompare.
By directly measuring molecular masses in solution in the analyte’s native state, column-free mass photometry, Refeyn’s lead technology, offers a potential solution, according to the company.
“Mass photometry makes the assessment of antibody aggregation, fragmentation, and target binding possible,” Jalal says. “It eliminates potential column interactions and the need for method development as it only requires simple dilution optimization. Mass photometry accommodates measurements of samples loaded at a wide range of concentrations, including nanomolar, which are inaccessible by other aggregation assessment techniques. Mass photometry is also very easy to use as anyone can run it after just half a day of training.”
Key Takeaways
Challenges with ADC resistance: ADCs face significant hurdles due to resistance mechanisms, including loss of target antigens, changes in ADC processing, and increased drug efflux. Researchers are addressing these challenges through novel strategies such as bispecific ADCs, combining ADCs with other therapies, and exploring new cytotoxic agents or dual-payload ADCs.
Narrow therapeutic window: ADCs have a narrow therapeutic window due to their potent cytotoxic payloads, which can lead to severe toxicity. Developers are working on strategies to improve this window, such as enhancing payload potency and selectivity, improving linker technology, and using alternative targets to minimize off-target effects and improve safety.
Molecular heterogeneity: ADCs often exhibit molecular heterogeneity, which complicates their development. Variations in drug-to-antibody ratios (DARs), differences in conjugation sites, and variations in linker stability all contribute to this issue, affecting pharmacokinetics, effectiveness, and toxicity. Efforts to reduce heterogeneity focus on site-specific conjugation techniques.
Complexity in ADC analytics: ADCs present unique analytical challenges due to the complex interplay between the antibody, linker, and payload. These challenges include difficulties in assessing payload-antibody stoichiometry and post-translational modifications. Traditional analytical methods are slow and may not be sufficient, highlighting the need for next-generation technologies like mass photometry.
Manufacturing and development challenges: ADCs are complex to manufacture due to factors like variability in conjugation, stability, and post-production modifications (e.g., degradation and aggregation). This molecular heterogeneity, combined with challenges in ensuring consistent drug release and minimizing premature detachment, makes developing ADCs a highly intricate process that requires precise control over each component.