Foreword
In recent years, antibody-drug conjugates (ADCs) have emerged as one of the most promising breakthroughs in targeted cancer therapy. Often described as “biological missiles”, ADCs combine the high specificity of monoclonal antibodies with the potent killing ability of cytotoxic small-molecule drugs, enabling precise tumor cell elimination with reduced systemic toxicity.
This article focuses on the mechanism of action of ADC drugs, key factors influencing their therapeutic efficacy, and—most importantly—endocytosis detection strategies, which play a decisive role in ADC development and optimization.
ADC Drugs: Mechanism Analysis and Multiple Actions
ADC drugs consist of three core components: a monoclonal antibody that specifically targets tumor antigens, a highly effective cytotoxic small molecule drug (payload), and a linker connecting the two. This ingenious design allows ADCs to precisely identify and eliminate cancer cells while minimizing damage to normal tissues. The mechanism of action of ADCs can be divided into the following key steps:
Targeted binding: ADCs specifically recognize and bind to target antigens on the surface of tumor cells through their antibody components.
Endocytosis: The antibody-antigen complex is endocytosed by tumor cells, entering the cell interior.
Payload release: Under specific intracellular conditions (such as a low pH environment or protease action), the linker breaks, releasing the cytotoxic drug. The cytotoxic drug damages cellular structures such as DNA or microtubules, leading to tumor cell death.
Schematic diagram of the mechanism of action of ADC drugs (Data source: Micro-source detection, Clinical bioanalysis case of antibody-drug conjugates ADC 2024)
The killing effect of ADC drugs is not limited to direct action, but also includes:
Bystander effect: Some cytotoxic drugs can penetrate the cell membrane and kill neighboring tumor cells, even if these cells do not express the target antigen.
Reduction of membrane proteins: After ADC binding, the internalization of the target antigen-antibody complex leads to a reduction in the number of target proteins on the cell surface.
Impact on membrane protein structure: The antigen-antibody complex causes conformational changes in cell surface membrane proteins.
Immune effects: The antibody portion of ADCs can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP), mobilizing the immune system to attack tumor cells.
Diagram illustrating the mechanisms by which ADCs kill cancer cells through different methods (Data source: DOI: 10.1038/s41392-022-00947-7)
Key Factors Affecting ADC Efficacy
The efficacy of ADC drugs is influenced by a variety of factors, among which several key elements include:
Target Selection: Targets should be highly expressed on tumor cells and low-expressed or restricted in normal tissues; the target antigen should possess endocytic properties, effectively internalizing the ADC-antigen complex into cells.
Antibody Properties: The affinity, specificity, stability, and immunogenicity of antibodies all affect the efficacy and safety of ADCs. High-affinity antibodies can improve tumor targeting efficiency but may also lead to a "binding site barrier" effect, limiting drug penetration and distribution in solid tumors.
Linkers and Small Molecule Drugs: The stability of linkers directly affects the blood circulation time and tumor-selective release of ADCs. The potency, mechanism of action, and physicochemical properties of small molecule drugs determine the final therapeutic effect.
Endocytosis Efficiency: A Decisive Factor in ADC Efficacy Among all influencing factors, the endocytosis efficiency of the target antigen is particularly critical. Efficient endocytosis ensures that sufficient ADCs are delivered into cells, releasing the effective payload and exerting their killing effect. Accurately assessing the endocytosis properties of the target antigen is a crucial step in the ADC development process.
Endocytosis Detection: Methods and Development Stage Adaptation Strategies
Endocytosis detection is a crucial method for evaluating the efficacy of ADC candidate drugs. As ADC drug development progresses from early discovery to clinical trials, endocytosis detection methods need corresponding adjustments.
Common Endocytosis Detection Methods:
Flow Cytometry: Assessing endocytosis efficiency by detecting the reduction of target antigens on the cell surface or the internalization of fluorescently labeled ADCs. This method is suitable for high-throughput screening and is used in the early antibody screening stage to evaluate antibody endocytic activity. However, it is less intuitive and accurate than imaging and killing methods.
Confocal Microscopy: Allows direct observation of the localization and distribution of ADCs within cells, distinguishing between surface-bound and internalized portions. However, it requires sophisticated equipment and has a lower throughput than flow cytometry. It is suitable for in vitro pharmacological evaluation after molecular confirmation.
pH-Sensitive Fluorescent Probes: Utilizing pH changes after endocytosis, secondary antibody fluorescent probes are designed and co-incubated with primary antibodies before being endocytosed by cells. The subsequent pH change triggers a specific fluorescent signal within the cell, which is used to evaluate antibody endocytic activity. This method is suitable for evaluating the endocytic activity of candidate molecules.
pH-sensitive killing agents: Secondary antibody toxin molecules designed based on pH changes after endocytosis are co-incubated with primary antibodies and then endocytosed by cells. The toxin molecules are then released due to pH changes to kill cells, thus evaluating the endocytic activity of the antibody. This method is suitable for evaluating the endocytic activity of candidate molecules.
AlpVHHs: Advanced Nano-pH-Sensitive Probes for Endocytosis Detection
AlpVHHs is an industry-leading nanobody company specializing in VHH antibodies, recombinant antibodies, and recombinant monoclonal antibody development, providing discovery, engineering, expression, and large-scale production services for nanobodies and antibody fragments, supporting both research and diagnostic product development.
AlpVHHs offers a new generation of Nano-pH-sensitive probe coupled to pH-dependent fluorescent probes for endocytosis detection. pH-sensitive dyes have no fluorescence excitation outside the cell, but emit bright red or green fluorescence in some acidic environments inside the cell (including phagosomes). Unlike traditional labeling methods using amino or thiol-reactive markers, Nano-pH-sensitive probes, which are site-specifically coupled to pH-dependent fluorescent probes by specific nanobodies, are very stable and compatible with BSA and other stable proteins.
Code | Description | Application |
023-101-013 | Internalization Test | |
023-102-012 | Internalization Test | |
023-102-014 | Internalization Test | |
001-101-012 | Internalization Test | |
001-101-014 | Internalization Test |
Learn More
Visit www.alpvhhs.com to explore advanced nanobody-based solutions for ADC research and development.
