Therapeutic antibody drugs are playing an increasing role in the field of disease diagnosis and treatment, placing greater demands on antibody development. In particular, most antibody-drug conjugate (ADC) drugs rely on the endocytosis of antibodies. Whether an antibody can be internalized is primarily determined by the target, while the efficiency of antibody internalization is closely related to antigen density, antibody affinity, and the antibody-antigen binding epitope. Different antibodies targeting the same antigen will also show varying internalization efficiencies. Therefore, screening antibodies with high affinity and efficient internalization becomes crucial to ensuring the targeting and safety of ADC drugs. Based on our rich field experience and advanced phage display platform, Creative Biolabs provides comprehensive services for internalizing antibody discovery to support therapeutic antibody development.

Internalizing Antibody

Antibodies or peptide fragments can specifically bind to receptors on the surface of mammalian cells and enter the cell through receptor-mediated endocytosis. Such antibodies or antibody fragments are known as internalized antibodies. The antibody internalization assay is a commonly used method to study the cellular uptake and internalization process of specific antibodies, using techniques like phage display and others. Internalized antibodies find a wide-ranging applications as carriers for the targeted delivery of drugs, toxins, enzymes, or DNA, particularly in ADC drugs.

ADCs are composed of monoclonal antibodies targeting specific antigens and small-molecule cytotoxic drugs linked by a linker. They combine the potent killing effect of traditional small molecule chemotherapy with the tumor-targeting abilities of antibody drugs. After administration to a patient, ADCs enter the patient's bloodstream. When the antibody portion of the ADC binds specifically to the surface of a tumor cell, the cell membrane of the tumor cell curves inward, forming an endosomal vesicle, a process known as internalization. ADCs are designed to act through this internalization mechanism. Once the antibody binds to the target antigen on the cancer cell, the ADC is endocytosed and degraded in the lysosome, releasing the cytotoxic payload. If the payload is appropriately membrane permeable, it can diffuse into surrounding "bystander" cancer cells, whether or not they express the target antigen, a phenomenon known as the bystander effect.

There are three common internalization pathways for ADCs: lattice protein-mediated endocytosis, cytosolic caveolae-mediated endocytosis, and cytosolic caveolae-mediated cytosolic drinking. The first two pathways are antigen-mediated, while the last one is antigen-independent. Lattice protein-mediated endocytosis involves several sequential and partially overlapping steps.

Clathrin-mediated endocytosis can be initiated by certain receptors on the plasma membrane or requires ligand and/or by antibody binding. It starts when endocytosed coat proteins in the cytoplasm begin to accumulate on the inner leaflets of the plasma membrane. Clathrin continues to assemble and grow by recruiting proteins from the cytoplasm and interacting with additional protein adapters. Key bridging proteins bend the membrane, concentrating the internalized receptor/ligand into "clathrin coated pits". As the invagination of these pits increases, the neck narrows and separates from the plasma membrane through a process of rupture. Actin polymerization helps pull the pits inward into the cytoplasm until the rupture is complete. The "clathrin-coated pits" are released and become clathrin-coated vesicles. Finally, the outer shell of the vesicle disassembles, and the vesicle fuses with an endosome for transport to a specific subcellular location or can be recycled back to the cell surface.

The development of most ADCs depends on endocytosis by antibodies. However, not every target can be endocytosed, and the efficiency of endocytosis varies for each antibody-antigen pair. Different antibodies against the same immunogen can exhibit different rates of internalization. The efficiency of endocytosis between ADCs and naked antibodies is subject to change, affecting the uptake and release of the drug into tumor and normal cells. In some cases, both are internalized at the same rate, while in other cases, ADCs have a higher internalization efficiency. Rapid internalization enhances the efficacy and safety of ADCs, reducing the chance of off-target release.

Creative Biolabs possesses extensive knowledge and experience in antibody discovery. We would be happy to share our knowledge and experience in internalizing antibody discovery with you.

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