Antibody fragmentation is the biochemical or genetic cleavage of antibodies into smaller fragments. This process is used in various laboratory and clinical applications, as fragments retain the specific targeting capabilities of full monoclonal antibodies while altering physiochemical features. This can be beneficial for developing therapeutic agents with distinct properties or to reduce the size of the antibody while retaining its antigen-binding capabilities.
For example, smaller fragments can penetrate tissues inaccessible to full-size antibodies, as well as being easier and more economical to manufacture due to the lack of glycosylation, since it allows the use of prokaryotic expression systems. However, fragmentation without Fc domains may experience rapid degradation within the human body, as the Fc domain is responsible for stabilizing full-size antibodies and enabling FcR-mediated recycling.1
There are several different types of fragments available, such as small monovalent antibody fragments (Fab, scFv) and engineered variants (diabodies, triabodies, minibodies and single-domain antibodies). Antibody fragments have been adapted for use in creating multivalent and multispecific reagents, linked to therapeutic cargos, and enhanced for therapeutic effectiveness.2,3
Common antibody fragments include:
Fab (Fragment, Antigen-Binding): The Fab fragment is generated by cleaving an antibody molecule with the enzyme papain. It consists of the variable regions of both the heavy and light chains, along with part of the constant region of the antibody. Fab fragments retain the antigen-binding capability of the original antibody and are often used in research and diagnostic applications.
Single-Chain Variable Fragment (scFv): An scFv is a smaller antibody fragment that includes both the variable regions of the heavy and light chains linked together as a single polypeptide chain. ScFvs are often used in biotechnology and medical applications due to their smaller size and potential for engineering into various formats.
VHH or Single-Domain Antibodies (sdAbs): VHHs consist of a single variable domain derived from heavy-chain antibodies found in camelids and some cartilaginous fish. Their small size and remarkable stability offer excellent tissue penetration. They maintain high specificity and affinity for their antigens, and they are amenable to genetic engineering, enabling the development of novel constructs tailored for specific applications, such as targeted drug delivery, in vivo imaging, and as probes for various biomolecular studies.
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Holliger, P., & Hudson, P. J. (2005). Engineered antibody fragments and the rise of single domains. Nature Biotechnology, 23(9), 1126-1136. https://doi.org/10.1038/nbt1142
Bates, A., & Power, C. A. (2019). David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments. Antibodies, 8(2), 28. https://doi.org/10.3390/antib8020028
Antibodies are versatile molecules that perform a range of effector functions, many of which engage different arms of the immune system. Their modes of action extend beyond simple antigen binding, enabling the activation of various immune mechanisms that lead to pathogen neutralization and clearance. These functions include blocking molecular interactions, activating the complement system, and linking the humoral immune response to cellular immune responses via Fc receptor engagement.
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In addition to isotypes and subtypes, antibodies exhibit genetic variation known as allotypes, which are polymorphic epitopes on immunoglobulins. These allotypic differences arise from allelic variations in immunoglobulin genes, causing certain antibody subtypes to differ between individuals or ethnic groups. The presence of these polymorphic forms can influence immune responses, particularly when an individual is exposed to a non-self allotype, potentially triggering an anti-allotype immune reaction.
In mammals, antibodies are classified into five major isotypes: IgA, IgD, IgE, IgG, and IgM. Each isotype is defined by the heavy chain it contains: alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM). These structural differences in the heavy chains determine the antibody's function, tissue localization, and role in the immune response. Furthermore, antibody light chains fall into two classes—kappa and lambda—with kappa being more common, though both exhibit similar functions despite differences in sequence.
