The Fundamentals of ADCs: Which Assay is Right for Your Work?

Antibody–drug conjugates (ADCs) represent a promising frontier in targeted cancer therapy, marrying the specificity of monoclonal antibodies with the potent cell-killing capabilities of cytotoxic drugs. As complex bioconjugates, ADCs use multifaceted mechanisms of action (MOA) to deliver their therapeutic payloads precisely to tumor cells. In recent years, the clinical landscape has witnessed accelerated development and approval of ADCs, reflecting both their innovative design and the expanding understanding of their molecular dynamics. Approved ADCs deploy a variety of MOAs—including direct cytotoxicity, bystander killing, and receptor blockade—while also engaging immune-mediated processes such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) (1). Looking ahead, next-generation payloads aim to broaden this therapeutic arsenal by incorporating immune modulation, altering protein expression, and directly targeting specific oncogenic mutations.

At its core, an ADC is composed of three critical components: the antibody, the linker, and the payload. The antibody confers target specificity, binding with high affinity to tumor-associated antigens, thereby ensuring that the cytotoxic drug is delivered selectively to cancer cells. The linker, which bridges the antibody and the payload, is engineered to be stable in circulation yet labile enough to release the drug once internalized by the target cell. This balance in linker design is pivotal for optimizing therapeutic efficacy while minimizing off-target toxicity. Finally, the payload itself is typically a highly potent cytotoxic agent, capable of inducing cell death even at very low concentrations, a necessity given the limited number of molecules delivered per antibody (Figure 1).

Beyond acting as delivery vehicles, the antibodies in ADCs play an active role in modulating immune responses through their Fc regions. The Fc domain can engage immune effector cells by binding to Fc receptors on natural killer cells, macrophages, and other components of the immune system. The majority of ADCs use IgG1 antibodies, due to their long serum half-life and robust Fc effector functions. However, some ADCs leverage differing affinities of IgG isotypes for FcyR to help shape the immune response. Using immunoassay approaches, such as the Lumit® FcγR Binding Immunoassays, researchers can quickly measure antibody and receptor interactions. Fc effector functions can include ADCC, ADCP, and CDC, thereby contributing an additional layer of antitumor activity.

The immune-mediated functions not only assist in the direct killing of tumor cells but also help in shaping the tumor microenvironment to favor an immune response against cancer. As such, modifications in the Fc region are being explored to fine-tune these interactions and enhance overall therapeutic outcomes (2). Just like unconjugated monoclonal antibody drugs, regulators will likely require the Fc function of ADCs to be evaluated. Published studies of some drugs have shown that payload conjugation has little impact on Fc function (3), but as conjugation chemistry evolves, sometimes enabling very large drug-antibody-ratios (DAR), it is essential to quantify to what extent Fc function has been preserved in the final drug substance. Functional reporter assays, such as the ADCC and ADCP Reporter Bioassays can provide ADC developers the measurements required for ranking their effector functions.  

For an ADC to exert its direct cytotoxic effects, it must be internalized by the tumor cell. This process begins with the binding of the ADC to its target antigen on the cell surface, triggering receptor-mediated endocytosis. Once inside the cell, the ADC is trafficked through endosomal compartments where the linker is cleaved—either enzymatically or via environmental triggers such as pH changes—releasing the cytotoxic payload into the intracellular space. Solutions like the pHAb Reactive dyes or the NanoLuc® Internalization Assay provide two methods for measuring internalization. The pH reactive dyes use the endosomal trafficking pH changes to drive fluorescence, while the Internalization Assay takes advantage of a luminescent readout. Antigen expression, antibody affinity, binding site, and the intrinsic internalization dynamics of the antigen itself can affect the rate and degree of ADC internalization (4). Ongoing research is focused on optimizing these parameters to enhance the selective killing of tumor cells while sparing healthy tissues.

Once the ADC payload—a highly potent cytotoxic agent designed to kill cancer cells – is released, there are several common targets. Common payloads include microtubule inhibitors such as auristatins and maytansinoids, which disrupt cell division by inhibiting tubulin polymerization, ultimately leading to cell cycle arrest and apoptosis. DNA-damaging agents like calicheamicin and duocarmycins operate through a different mechanism, inducing double-strand DNA breaks that trigger cell death pathways. In addition, newer payloads are being explored that target specific oncogenic pathways or modulate the immune system, further diversifying the arsenal against cancer. The potency of these agents is balanced by the need for precise delivery—ensuring that even a small amount of the drug can exert cytotoxic effects while minimizing collateral damage to healthy tissues. Determining the cytotoxic potency of the ADC payloads may require a suite of real-time assay tools including cell viability assays, apoptosis assays and for the newer and more complex payloads, tools for understanding targeted protein degradation or oligo-targeted MoAs.

In addition to the direct cytotoxic effects of ADC payloads, one of the most intriguing features of certain ADCs is their ability to induce a bystander killing effect. In this context, the released payload is not confined to the target cell alone but can diffuse into the surrounding tumor microenvironment, affecting neighboring cells that may not express the target antigen (5). This phenomenon is particularly beneficial in heterogeneous tumors where antigen expression can vary widely among cancer cells. The clinical importance of bystander killing can be readily seen in the dramatic response to T-DXd, which has bystander activity, relative to T-DM1, which does not (6).  Using co-culture of cell lines, ADC developers can specifically measure bystander killing that broader cytotoxicity screens miss. By broadening the spectrum of cell killing, bystander effects help eradicate tumor cells that might otherwise escape direct targeting, thus enhancing the overall therapeutic impact of the ADC. 

Antibody–drug conjugates stand at the nexus of targeted therapy and precision medicine, offering a versatile platform for the treatment of cancer. With their intricate design, encompassing specific antibodies, carefully engineered linkers, and potent payloads, ADCs employ a diverse range of mechanisms—from immune effector engagement to bystander killing—to combat tumor cells effectively. As research continues to unveil new molecular insights and harness advanced technologies, the next generation of ADCs promises to expand therapeutic possibilities even further, paving the way for more personalized and effective cancer treatments.