Antibody fragmentation is a process in which antibodies are selectively cleaved into smaller fragments via biochemical or genetic methods. This fragmentation is done to study or utilize specific parts of the antibody molecule without interference from other regions.
The main approach to fragmentation is through enzymatic digestion, typically with papain or pepsin, which can cleave a whole antibody into antigen binding fragment (Fab) or bivalent Fab, named F(ab’)2, respectively. This removes the crystallizable fragment (Fc) region while keeping both the variable regions of the heavy and light immunoglobulin chains linked together with disulfide bonds, thus allowing the remaining fragment to continue to bind their target antigen without directing an immune response.
Enzymatic Fragmentation Methods: Papain and Pepsin Digestion
The most common approach to antibody fragmentation is enzymatic digestion using specific proteolytic enzymes. Papain and pepsin are widely used to cleave antibodies at precise locations, generating functional fragments without compromising antigen-binding capability.
Papain Digestion: Papain, a proteolytic enzyme derived from papaya, cleaves antibodies above the hinge region, producing two identical Fab fragments and an Fc fragment. Fab fragments retain their variable domains, allowing them to bind to specific antigens. However, since the Fc region is removed, these fragments do not engage immune effector functions, such as complement activation or antibody-dependent cellular cytotoxicity (ADCC). Fab fragments are useful in applications where the immune system’s involvement is undesirable, such as in diagnostic assays or therapeutic delivery to specific tissue sites.
Pepsin Digestion: Pepsin, another protease, cleaves antibodies below the hinge region, resulting in a bivalent F(ab’)2 fragment and degraded Fc fragments. The F(ab’)2 fragment retains both antigen-binding sites, making it capable of cross-linking antigens, much like a full-length antibody. However, like the Fab fragment, the F(ab’)2 fragment lacks the Fc region and, consequently, the ability to recruit immune cells or activate complement. This makes F(ab’)2 fragments advantageous in therapeutic applications where antibody-mediated immune responses could lead to adverse side effects.
Applications of Antibody Fragments in Research and Therapy
Antibody fragments, including Fab and F(ab’)2 fragments, have found widespread use in both research and therapeutic settings. Their smaller size, lack of immune effector functions, and ability to bind antigens with high specificity make them particularly valuable in situations where full-length antibodies may not be ideal.
Research Applications: In research, antibody fragments are frequently used in diagnostic assays, such as ELISAs and immunohistochemistry (IHC), where the antigen-binding capability is needed, but the Fc region’s immune-activating properties may introduce unwanted background noise. For example, using Fab fragments in IHC reduces nonspecific binding to Fc receptors on tissue cells, improving the clarity and specificity of staining.
Therapeutic Applications: Antibody fragments are also gaining traction in therapeutic settings, particularly in cases where full-length antibodies may elicit an unwanted immune response. Fab fragments are used in therapies like the antivenom CroFab®, where neutralizing snake venom is the primary goal without activating the immune system.1 Similarly, F(ab’)2 fragments have been explored for treating autoimmune diseases, where their ability to bind antigens without triggering inflammation is highly desirable.
Tissue Penetration: Due to their smaller size, antibody fragments penetrate tissues more efficiently than full-length antibodies, making them ideal for targeting antigens in dense tissues, such as tumors. Fab fragments have a molecular weight of approximately 50 kDa, while scFv fragments are typically in the range of 30 kDa.2
Genetically Engineered Antibody Fragments
In addition to enzymatic fragmentation, genetic engineering can develop fragments tailored for particular applications, such as improved stability, affinity, or tissue targeting.
Single-Chain Variable Fragments (scFv): These fragments can be created by genetically linking the variable regions of the heavy (VH) and light (VL) chains of an antibody with a short peptide linker. scFvs retain antigen-binding specificity and are much smaller than Fab or F(ab’)2 fragments, making them even more suitable for applications that require deep tissue penetration, such as imaging or therapeutic targeting of solid tumors. Furthermore, scFvs can be produced using recombinant DNA technology in various expression systems, allowing for large-scale production and optimization.
Diabodies and Triabodies: These are multivalent forms of scFvs that increase the avidity of binding by bringing together multiple antigen-binding sites. Diabodies (with two binding sites) and triabodies (with three binding sites) are engineered by modifying the length of the linker between the variable regions, allowing the fragments to form stable, multivalent structures. These engineered fragments are particularly useful in therapeutic applications that require strong antigen binding, such as in bispecific antibodies or targeted cancer therapies.
VHH Antibodies: Derived from camelid heavy-chain antibodies, these consist of only a single variable domain (VHH). These fragments are highly stable, small, and retain full antigen-binding capabilities, making them excellent candidates for applications requiring high tissue penetration or for delivery into intracellular compartments.
References:
Dart, R. C., Seifert, S. A., Boyer, L. V., Clark, R. F., Hall, E., McKinney, P., McNally, J., Kitchens, C. S., Curry, S. C., Bogdan, G. M., Ward, S. B., & Porter, R. S. (2001). A randomized multicenter trial of crotalinae polyvalent immune Fab (ovine) antivenom for the treatment for crotaline snakebite in the United States. Archives of internal medicine, 161(16), 2030–2036. https://doi.org/10.1001/archinte.161.16.2030
Xenaki, K. T., & Oliveira, S. (2017). Antibody or Antibody Fragments: Implications for Molecular Imaging and Targeted Therapy of Solid Tumors. Frontiers in Immunology, 8. https://doi.org/10.3389/fimmu.2017.01287
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