After decades of investment, antibody-drug conjugates (ADCs) are finally realizing their potential, evidenced by a growing number of clinical approvals, application in earlier lines of therapy, and integration into drug combinations including immunotherapies. This progress has spurred investment in novel ADC development and expanded the clinical use of approved ADCs. ADC design is complex, involving multiple molecular components that interact with the tumor and host tissue microenvironments. This article explores the molecular and immunological factors influencing ADC efficacy and toxicity. It describes how the molecular components of an ADC determine its systemic, tissue, and cellular distribution, which ultimately dictates therapeutic effect. These interactions also define the toxicity profile and set the maximum tolerated dose. Finally, the impact of ADC treatment on immune cells is discussed, highlighting distinct yet interconnected roles for immunogenic cell death, activation of immune cells such as dendritic cells, and antibody-Fc interactions. These mechanisms are crucial for enhancing efficacy beyond the direct cytotoxic effects of the payload. By unveiling the intricate interplay of ADCs, this review aims to provide insights for the rational design of combination therapies and guide the development of next-generation, clinically effective ADCs.

Development of Antibody-Drug Conjugates (ADCs) 

After decades of investment and effort, ADCs are beginning to realize their full clinical potential in cancer therapy. ADCs are having a broad impact on both clinical practice and drug development, reshaping the oncology landscape. For instance, an ADC targeting human epidermal growth factor receptor 2 (HER2) conjugated to a topoisomerase I inhibitor, trastuzumab deruxtecan, has shown remarkable efficacy across multiple tumor types and in patients with varying HER2 expression levels, demonstrating its broad impact in cancer treatment. Similarly, an ADC targeting nectin-4 conjugated to a microtubule inhibitor, enfortumab vedotin, has demonstrated deep and durable responses when combined with immune checkpoint inhibitors. These and other recent successes have driven substantial investment in the field and reshaped the pharmaceutical and clinical landscape. Although the concept of using monoclonal antibodies to selectively deliver chemotherapeutic agents to cancer cells is straightforward, its application is exceedingly complex. This article aims to cover the multifaceted molecular interactions and mechanisms involved in the in vivo pharmacokinetics, distribution, and efficacy of ADCs, thereby providing insights to inform the design of the next wave of effective therapeutics.

Structural Features of Antibody-Drug Conjugates (ADCs)

ADCs typically consist of a monoclonal antibody, primarily in the IgG format, linked to a small molecule drug, typically a cytotoxic payload, via a linker (Figure 1). However, these components do not function in isolation; the complex interactions between the ADC and cells within various human tissues, including the tumor, ultimately determine its efficacy and toxicity.

Figure 1: Structural analysis of antibody-drug conjugates

ADC Conjugation Methods and Systemic Pharmacokinetics

As systemically administered therapies, ADCs are well-suited for treating metastatic disease and have shown efficacy even in patients with brain metastases. Following intravenous administration, ADCs rapidly distribute in the plasma and are taken up by tissues throughout the body. Due to the size difference between the antibody (approximately 150 kDa) and the payload (typically approximately 0.5 kDa), ADCs generally distribute in a manner similar to monoclonal antibodies. However, it was recognized early on that modifying antibodies with multiple payloads could significantly alter their distribution and clearance. Conjugating multiple, often lipophilic, payloads to the antibody surface can increase non-specific interactions with cells, accelerating systemic clearance. Conversely, using too few payloads may fail to deliver a sufficient amount for effective cell killing. This balance historically led to the common drug-to-antibody ratio (DAR) for many ADCs, aiming to optimize efficacy while minimizing undesirable pharmacokinetic effects.

Early ADCs primarily utilized non-specific conjugation methods, such as attaching payloads to free amines on lysines or through partial reduction of disulfide bonds in the hinge region. Most current ADCs employ hinge region disulfide bond conjugation, which provides semi-specific conjugation in the hinge region, avoiding interference with antibody functional domains (Figure 1). In recent years, advances in site-specific labeling and conjugation chemistry have significantly improved ADC conjugation technologies. These innovations allow conjugation at nearly any site on the antibody, although only a few sites are suitable for stable drug attachment with minimal pharmacokinetic impact. Using hydrophilic linkers can also enhance linker stability and improve ADC circulation, enabling fine-tuning of the DAR and optimization of the dosing of both antibody and payload portions for better therapeutic outcomes. 

Once released from the ADC, the free payload distributes similarly to small molecule drugs, undergoing processes like hepatic metabolism and excretion. Separate tracking of the ADC, unconjugated antibody, and free payload provides a more detailed picture of systemic processing. Free payload in systemic circulation originates primarily from two sources: de-conjugation occurring in circulation, particularly for ADCs with less stable linkers; or ADC uptake into tissues followed by intracellular payload release and subsequent efflux back into the blood, which is more common for ADCs with more stable linkers (Figure 2). The correlation between systemic payload levels and overall antitumor efficacy of an ADC is debated; the magnitude of this contribution likely depends on specific features of each ADC, such as linker stability and payload potency, as well as key tumor-related factors like target expression and the sensitivity of the treated tumor to the payload.

Figure 2: Systemic pharmacokinetics of antibody-drug conjugates.

ADC Distribution in Tumors

ADC distribution within tumors is a critical consideration for the efficacy of these agents. It is well-established that antibody distribution in tumors is heterogeneous, and recent advances using fluorescent antibodies have enhanced our ability to visualize these distribution gradients with cellular resolution (Figure 3a, b). In some cases, such as pancreatic cancer, physical barriers can impede antibody penetration into tissue. However, pharmacokinetic barriers commonly limit distribution because antibodies and ADCs bind to their targets much faster than they diffuse into the tumor, effectively immobilizing the drug perivascularly (Figure 3b). Increasing the antibody dose can saturate these perivascular regions, enabling deeper tissue penetration. As many antibodies are well-tolerated even at high doses (for example, margetuximab (anti-HER2 monoclonal antibody) at doses >10 mg/kg, atezolizumab (anti-PDL1 monoclonal antibody) doses, and intravenous immunoglobulin (IVIG) therapy at gram/kg doses), this approach can overcome the diffusion limitations of biologics. However, for ADCs, drug toxicity primarily dictates the maximum tolerated dose. Therefore, a high DAR or an ultra-potent payload limits the administrable ADC dose, consequently reducing tumor tissue penetration and diminishing efficacy.

Figure 3: Tissue and cellular pharmacokinetics of antibody-drug conjugates.

Cellular Processing of ADCs

Once an ADC distributes within the tumor, it is processed by cells to release its payload. In most cases, this process begins with ADC binding to its receptor on the cancer cell surface, followed by entry into the endosome-lysosome pathway. Within the lysosome, the payload is released and subsequently escapes via transporters or passive diffusion. Once free, the payload targets specific subcellular compartments, such as DNA or topoisomerase in the nucleus, or microtubules in the cytoplasm. However, overall ADC efficacy is influenced by multiple interconnected and sequential steps along this pathway (Figure 3c).

ADC Toxicity: On-Target vs. Off-Target Toxicity 

It is estimated that the majority of systemically administered ADC molecules, >99% of the total dose, are catabolized within healthy cells. Nevertheless, compared to small molecule chemotherapy, ADCs more effectively deliver and maintain payload concentrations within tumors. This uptake and catabolism likely represent a key driver of ADC-related toxicity, potentially mediated by two main mechanisms: binding of the ADC to its target receptor on healthy cells, termed on-target toxicity; or internalization of intact ADC via mechanisms not involving the target receptor, such as non-specific endocytosis. Additionally, cellular uptake of de-conjugated payload may contribute to both ADC toxicity and efficacy, referred to as the bystander effect (Figure 4).

Figure 4: Mechanisms contributing to antibody-drug conjugate toxicity

The nature and severity of on-target toxicity depend primarily on the expression level of the target receptor on healthy cells and its internalization rate. To minimize on-target toxicity risk, ADC development efforts typically focus on cell surface proteins, such as CD33, that have minimal or no expression on healthy cells susceptible to chemotherapy toxicity (e.g., rapidly dividing cells like hematopoietic stem cells). Due to this focus on "clean" targets, ADC-related toxicity is often associated with non-target uptake of intact ADC or released payload. However, toxicity from some ADCs may be driven by on-target mechanisms. For instance, cardiotoxicity observed in patients treated with trastuzumab and trastuzumab-based ADCs is a rare but serious side effect, suggesting that interaction of the antibody or ADC with HER2-positive cardiomyocytes is a primary driver of this toxicity. Another example is dysgeusia, reported by approximately 40% of patients in clinical trials receiving enfortumab vedotin. This side effect may be linked to nectin-4 expression in salivary glands. Notably, dysgeusia and rash are not common adverse events in patients treated with other ADCs carrying the same payload, monomethyl auristatin E (MMAE), further supporting an on-target mechanism. ADCs with more stable linkers and more potent payloads may exacerbate on-target toxicity, highlighting the importance of carefully considering target expression in healthy tissues during ADC design and development.

Immunomodulatory Effects of ADCs

Although the primary mechanism of action for ADCs is inducing cancer cell death via the payload, these therapies can also exert potent anti-tumor immune responses due to their unique design, bridging immuno-oncology with ADC development. The immune system continuously surveys and eliminates malignant cells to prevent cancer development. Cytotoxic CD8+ T cells specifically recognize tumor antigens and establish durable immune memory, a key advantage of the adaptive immune system. However, robust and sustained anti-tumor immunity also relies on the innate immune system, including neutrophils, natural killer (NK) cells, and antigen-presenting cells such as macrophages and dendritic cells (DCs).

Cytotoxic Payloads and Immunogenic Cell Death

ADCs carrying cytotoxic payloads have demonstrated significant immunomodulatory potential. The efficacy of these ADCs is significantly reduced in mouse models lacking CD8+ T cells or in genetically immunodeficient mice, suggesting that immune modulation may indeed be a primary mechanism of action. The extent of the immunomodulatory contribution to ADC potency is still being explored, but ongoing research continues to uncover novel mechanisms by which ADCs interact with immune pathways (Figure 5).

Figure 5: Immune interactions of antibody-drug conjugates in the tumor microenvironment

ADCs are a complex therapeutic modality combining the targeting precision of antibodies with the potent activity of small molecules to concentrate payload delivery within the tumor microenvironment (TME). While their design complexity presents challenges, it also offers opportunities to finely tune their molecular properties for deep and durable clinical responses. Insights gleaned from antibody therapy, small molecule drug development, and immunology, when applied judiciously, can guide the design of next-generation ADCs. Although the numerous interactions between ADCs and the immune system may seem overwhelming, not all mechanisms are equally important in every context. By prioritizing the most impactful mechanisms, such as optimizing ADC uptake and tumor distribution at tolerable doses, or enhancing specific immune pathways, more effective therapies can be designed. The potential of this approach is already evident in clinical outcomes, demonstrating the transformative power of ADCs in cancer treatment.

Tek Biotech (Tianjin) Co., Ltd. specializes in phage display and yeast display antibody development services as its core business. We are dedicated to providing high-quality targeted antibody discovery, antibody humanization, ADC design and conjugation, cell killing assays, and animal model experiments to scientists worldwide, offering robust support for customer research projects and the development of monoclonal antibody therapeutics for refractory diseases.

References

[1] Zippelius, A., Tolaney, S.M., Tarantino, P. et al. Unveiling the molecular and immunological drivers of antibody-drug conjugates in cancer treatment. Nat Rev Cancer (2025).