Characterising therapeutic antibodies and ADCs using mass spectrometry
Since the 1970s, the advent of biotechnology has resulted in the development and commercialisation of many therapeutic proteins, including antibodies and antibody fragments, for the treatment of human diseases. Examples include antibody treatments for autoimmune diseases (for example, adalimumab [Humira®] for rheumatoid arthritis), cancers (such as trastuzumab [Herceptin®] for breast cancer), degenerative conditions (including ranibizumab [Lucentis®] for macular degeneration), viral and bacterial infections, as well as others. Additionally, antibodies are used as imaging reagents, linked to radionuclides or non-radioactive rare earth metals, and quite extensively employed as reagents in biomedical research, commercial medical tests and clinical laboratory assays (for example, enzyme-linked immunosorbent assay [ELISA]).
Therapeutic antibodies are typically monoclonal antibodies (MABs) of the immunoglobulin G (IgG) isotype, with affinities against specific antigens and which bind monospecifically to particular cells or proteins. They work either by stimulating the patient’s immune system, thus priming or enhancing the immune response, or by binding to specific molecules and thus modulating or inhibiting certain biochemical pathways involved in disease. Monoclonal antibodies have also been developed with affinities towards specific tumour antigens to deliver radionuclides to them, thereby killing tumour cells with minimal deleterious effects on non-tumour cells. Such radiommunotherapeutic antibodies are modified with a chelator that binds a radioactive metal ion that is thus transported to the tumour.
Antibody-drug conjugates (ADCs), used in cancer chemotherapy, have small-molecule chemotherapeutics covalently attached via a linker that is cleaved at the tumour site, thereby releasing a cytotoxic small molecule in the immediate vicinity of a tumour. Although only a few ADCs have received regulatory approval so far, they comprise a very active area of research and development in many biopharmaceutical companies.
Structure of therapeutic MABs and ADCs
An IgG therapeutic antibody comprises two identical longer sequences (heavy chains) and two, also identical, shorter protein sequences (light chains). IgG light chains are typically 211-217 amino acids long, whilst IgG heavy chains comprise approximately 450 amino acids. The two heavy chains are linked covalently with disulfide bonds between cysteines roughly in the middle of the respective sequences, in a Y-shaped configuration (Figure 1). The number of disulfides linking the two heavy chains depends on the IgG subclass (IgG1, IgG2, IgG3 and IgG4 isotypes). Each light chain is covalently attached to a heavy chain with a single disulfide bond between cysteine residues. Along with interchain disulfide bonds, each heavy and light chain also has several intra-chain disulfide bonds between its cysteine residues.