ADC Bystander Effect: Mechanism and Tumor Microenvironment

Antibody-drug conjugates (ADCs) have transformed targeted cancer therapy by delivering cytotoxic agents directly to tumor cells while minimizing systemic toxicity. However, their effects can extend beyond antigen-expressing cells through the bystander effect, where ADC payloads impact neighboring cells, including those lacking the target antigen. This phenomenon can enhance treatment efficacy but also raises concerns about unintended toxicity.

Understanding this effect is crucial for optimizing ADC design and improving clinical outcomes.

Mechanism of the Bystander Effect

The bystander effect occurs when cytotoxic payloads, initially delivered to antigen-expressing tumor cells, diffuse into the surrounding microenvironment and affect neighboring cells. This process begins with antigen-mediated internalization of ADCs, followed by lysosomal degradation, which releases the cytotoxic payload. The ability of this payload to escape the targeted cell and reach adjacent cells depends on its membrane permeability and chemical stability. Lipophilic payloads can readily cross cell membranes, enabling them to reach antigen-negative tumor cells.

Once released, the free payload can exert cytotoxic effects through passive diffusion, moving across cell membranes based on concentration gradients, or active efflux, where drug transporters like P-glycoprotein (P-gp) expel the drug into the extracellular space. In tumors with heterogeneous antigen expression, this effect extends the reach of ADC therapy.

The stability of the released payload in the tumor microenvironment further influences its impact. Some cytotoxic agents degrade rapidly or bind to extracellular components, limiting their diffusion, while others, such as DNA-damaging agents or microtubule inhibitors, remain active longer, increasing their potential to affect neighboring cells. Factors like pH and enzymatic activity also modulate the persistence and potency of the released drug.

Significance of Payload Properties

The effectiveness of the bystander effect depends on the physicochemical characteristics of the cytotoxic payload, which influence its stability, diffusion, and retention within the tumor microenvironment. Membrane permeability is a key factor—lipophilic payloads, such as certain auristatins and camptothecin derivatives, readily cross cellular membranes, allowing them to impact antigen-negative tumor cells. In contrast, hydrophilic payloads remain confined within the targeted cell, limiting the bystander effect.

The stability of the released payload determines its reach and duration. DNA alkylators like pyrrolobenzodiazepines (PBDs) form covalent bonds with DNA, leading to sustained cytotoxicity in both antigen-positive and antigen-negative cells. Conversely, payloads that degrade rapidly or are enzymatically inactivated have a more localized effect, reducing unintended toxicity but also limiting therapeutic advantage.

Payload potency also plays a role. Highly potent agents, such as duocarmycins and PBD dimers, can be effective even at low concentrations, making them useful in tumors with heterogeneous antigen expression. However, this also increases the risk of off-target toxicity if the payload escapes beyond the tumor microenvironment. Balancing potency and selectivity is essential to maximize therapeutic benefit while minimizing systemic exposure.

Intercellular Transfer of Cytotoxic Agents

Once an ADC delivers its cytotoxic payload, its transfer to neighboring cells is influenced by chemical properties, the tumor microenvironment, and cellular transport mechanisms. Free payload molecules move via passive diffusion and active efflux. Lipophilic payloads, such as auristatins and maytansinoids, cross cellular membranes easily, while disrupted vasculature and a dense extracellular matrix create localized drug concentration gradients that drive diffusion.

Active transport mechanisms also contribute to redistribution. Efflux pumps like P-glycoprotein (P-gp) and multidrug resistance proteins (MRPs) expel payload molecules into the extracellular space, where they may be taken up by nearby cells. This enhances the bystander effect but also introduces variability, as cells with high efflux activity may shield neighboring cells from sufficient cytotoxic concentrations.

Extracellular stability further determines how effectively the payload transfers between cells. Some agents degrade rapidly or bind to serum proteins, limiting diffusion, while others remain chemically active for extended periods. DNA-alkylating agents like pyrrolobenzodiazepine dimers form covalent DNA crosslinks that persist after uptake, prolonging cytotoxic impact. Tumor pH variations, extracellular enzymes, and protein-binding interactions can either enhance or restrict drug spread, affecting both tumor penetration and systemic toxicity.

Influence of Tumor Heterogeneity

Tumor heterogeneity significantly impacts the bystander effect, complicating treatment responses and drug distribution. Within the same malignancy, tumor cells can vary in antigen expression, metabolism, and drug sensitivity, creating an uneven landscape for ADC efficacy. Some populations efficiently internalize ADCs and release cytotoxic payloads, while others lack antigen expression and rely on bystander killing. This variability can lead to inconsistent drug exposure across the tumor.

The spatial distribution of antigen-positive and antigen-negative cells influences the bystander effect. In tumors where antigen-expressing cells are interspersed among antigen-negative populations, payload diffusion can improve therapeutic reach. However, if antigen-positive cells are clustered, the released drug may have limited access to distant antigen-negative regions. The extracellular matrix also plays a role—rigid or fibrotic stroma can act as a barrier, restricting drug movement and limiting bystander activity.

Interaction with the Tumor Microenvironment

The tumor microenvironment (TME) influences how cytotoxic payloads diffuse, persist, and interact with tumor cells. Composed of stromal cells, extracellular matrix components, blood vessels, and signaling molecules, the TME can enhance or restrict the bystander effect, impacting treatment efficacy and toxicity.

The extracellular matrix (ECM) can act as a physical barrier to drug diffusion. Dense fibrotic stroma, common in pancreatic and breast cancers, impedes the spread of free cytotoxic agents, while a more porous ECM facilitates broader diffusion. Enzymatic degradation of ECM components by tumor-associated fibroblasts further modulates drug distribution, either enhancing or limiting payload movement. Interstitial fluid pressure variations create heterogeneous drug concentration gradients, influencing how far the released payload travels before becoming inactive or cleared.

The metabolic state of the TME also affects drug stability and activity. Tumors often exhibit hypoxia and acidity due to abnormal vasculature and high metabolic demands. Acidic pH can alter the solubility and diffusion of certain ADC payloads, sometimes enhancing their penetration into surrounding cells. However, hypoxia-induced drug resistance mechanisms, such as increased efflux transporter expression and altered apoptotic pathways, can reduce the susceptibility of bystander cells to cytotoxic effects. These factors determine whether the bystander effect contributes to a comprehensive tumor response or is hindered by local resistance.

Role of Immune Cells

Immune cells within the TME introduce another layer of complexity to the bystander effect. Their interactions with tumor cells and free cytotoxic agents influence drug distribution, enhance antitumor responses, or contribute to unintended toxicities. In tumors with high immune infiltration, the interplay between ADC payloads and immune mechanisms affects treatment outcomes.

Macrophages, a predominant immune cell type in the TME, play dual roles in ADC therapy. Tumor-associated macrophages (TAMs) can internalize ADCs through Fc receptor-mediated phagocytosis, leading to intracellular processing and potential payload release into the extracellular space, expanding the bystander effect. However, macrophages also express drug efflux pumps that reduce local drug concentrations, limiting the impact on surrounding cells. The balance between these effects depends on macrophage phenotype—M1-like macrophages enhance antitumor responses, while M2-like macrophages contribute to drug sequestration and resistance.

Dendritic cells and cytotoxic T lymphocytes (CTLs) further influence the bystander effect by modulating immune surveillance and tumor recognition. Some ADC payloads, particularly those that induce immunogenic cell death (ICD), stimulate dendritic cell activation and antigen presentation, triggering a broader immune response. This can complement the bystander effect, particularly in tumors with partial antigen expression. However, excessive immune activation may lead to off-target toxicities, as immune cells contribute to inflammation in surrounding tissues. The interplay between ADC-derived cytotoxicity and immune mechanisms underscores the complexity of designing ADCs that maximize therapeutic benefit while minimizing unintended consequences.