The ABCs of ADCs: Armed Antibodies on the Frontlines of Cancer Wars

Antibody-drug conjugates (ADCs) are one of the fastest growing classes of biologics with a market size that is estimated to reach $22.9 billion by 2030, according to a Grand View Research report. A key driver of this trend is the global rise in cancers, which are the main targets of ADCs. This blog post will highlight the analytical technologies that are helping biopharmaceutical companies bring these life-saving drugs to patients.  

^{This article is posted on our Science Snippets Blog}

Downside of conventional chemotherapy

The goal of therapy is to treat the disease, while sparing healthy tissue. But this is not always possible. For example, traditional chemotherapies used to treat rapidly-dividing cancer cells also target other rapidly-dividing cells, leading to hair loss, fatigue and other adverse reactions. Advancements in monoclonal antibody (mAb) technology and alternate antibody modalities has opened the door to a new generation of potent anticancer drugs with minimal cytotoxicity to healthy tissue.

What are ADCs?

ADCs are modified antibodies consisting of three components: a mAb, a covalent linker, and a cytotoxic payload. By taking advantage of the specificity of mAbs, ADCs deliver their cytotoxic payloads directly to cancer cells that express a specific cell-surface protein marker. The mechanism of action of ADCs involves two stages: 

• Binding of the mAb to the cell-specific surface protein
• Internalization of the entire ADC, releasing the cytotoxic drug and causing targeted cell death

This precision targeting of cells has made ADCs one of the fastest growing anticancer drugs, with successes like Kadcyla^®, Mylotarg^®, Blenrep^® and Zynlonta™, which have been used to treat breast, leukemia, multiple myeloma and lymphoma cancers.

Characterizing ADCs

ADCs are structurally complex molecules that require thorough characterization during the development process. Commonly used analytical methods for characterizing ADCs, like liquid chromatography, electrophoresis, and mass spectrometry, are useful, but need to be paired with methods that can add biophysical and functional information. 

Biophysical characterization of binding to target

One of the biggest challenges in developing ADCs is choosing the linker that connects the cytotoxic drug to the mAb. The linker chemistry can influence the overall structure of the ADC and, therefore, affects stability, specificity, binding and other important ADC properties. Detailed kinetic studies are needed to evaluate and optimize the linker.

Label-free detection offers many advantages for measuring interaction kinetics during ADC characterization. The method relies on optics-based biosensors to convert binding responses into signals without using a detection label. This helps to avoid experimental artifacts resulting from labeling and produces more biologically-relevant data. 

One popular label-free technology is available on the Octet^® Bio-layer Interferometry (BLI) Platform. You can use it to study the binding kinetics between ADCs and target proteins like the HER2 receptor, in real time. The Octet^® uses a dip-and-read assay format; either ADC or antigen is immobilized on a biosensor and dipped into increasing concentrations of the appropriate binding partner to get fast dose-response curves. 

Antibody-dependent cellular cytotoxicity (ADCC) studies

Several mechanisms of action of therapeutic mAbs are initiated via binding to Fc gamma receptors (FcγRs) expressed on the surface of immune effector cells. The ability of therapeutic mAbs and derivatives like ADCs to bind FcγRs can greatly impact their safety and efficacy, so analyzing interactions with FcγRs and understanding the functional consequence is integral to biotherapeutic development.

Antibody-dependent cellular cytotoxicity (ADCC) is a natural immune response that is triggered when an antibody bound to its target cell engages a natural killer (NK) cell through the FcγRs, resulting in cell lysis. With the iQue^® Advanced Flow Cytometry Platform, you can assess ADCC function much faster than traditional flow cytometers. In addition to its unique speed, the iQue^® enables easier evaluation of cell health, function and cytokine release in one assay, using validated kits for profiling NK cells and powerful tools for analyzing complex datasets.
 

Real-time internalization and cytotoxicity in target cells

Internalization and release of cytotoxic payload is the last stage of the ADC action. Popular end-point techniques, like FACS, ELISA and microscopy are not amenable to detailed kinetic analysis of time-dependent processes such as internalization. 

For industrial scale workflows, the Incucyte^® Live-Cell Analysis System is a well-established platform for visualizing and quantifying cell behavior over time, and in a completely non-perturbing way. Using a validated pH-sensitive dye, you can watch in real time as the ADC is internalized causing the dye to fluoresce in the low-pH environment of the lysosome. 

Biophysical and functional assessment is a core part of biologics development, including ADCs. Advanced high-throughput systems for cell and protein analysis can cut down the number of steps and speed up the time to results in biopharmaceutical development. To learn more, read about how these technologies were used to characterize the Kadcyla^®, and ADC used to treat some types of breast cancers.