Monoclonal antibodies (mAbs) used in both preclinical and clinical settings have been traditionally manufactured by hybridoma technology. Recombinant antibodies, on the other hand, offer unique advantages as compared to those produced by hybridoma technology, some of which include superior reproducibility, specificity, scalability, and lot-to-lot consistency.
Antibodies. Image Credit: vitstudio/Shutterstock.com
Introduction to antibodies
In 1890, Emil von Behring and Shibasaburo Kitasato discovered that serum obtained from diptheria-infected animals could be used to treat other animals. Following this discovery, Paul Ehrlich standardized this antidiphtheritic serum production method and identified the presence of “sidechains” in cells that could link to specific toxins.
Today, we now understand that the side chains that Ehrlich was referring to are antibodies (Abs) that belong to a family of proteins known as immunoglobulins (Igs). IgG is considered to be the most common type of antibody produced by humans, which can be further distinguished by four subclasses that include IgG1, IgG2, IgG3, and IgG4. The molecules that recognize and bind to antibodies in the body are referred to as antigens.
Antibodies used in research
The first documented use of antibodies in research was reported by Albert Coons, Hugh Creech, and Norman Jones in 1941. In their work at Harvard University, Coons, Creech, and Jones tested the use of an anti-pneumococci type III rabbit antibody that was conjugated with a fluorescent dye. This fluorescent conjugation process did not impair the function of the antibody, thus leading to the first documented use of the immunofluorescence staining technique.
Since 1941, several additional technical advancements have been made through the use of antibodies in preclinical and clinical research. During the 1970s, for example, antibodies were adapted for their use in immunohistochemistry (IHC), enzyme-linked immunosorbent assays (ELISA), and sorting cells with a flow cytometer. By 1975, the world’s first immortal hybridoma cell line was created, which was soon followed by the first western blot and chromatin immunoprecipitation (ChIP) assays in the early 1980s.
Limitations of hybridoma technology
As previously mentioned, the first immortal hybridoma cell line was created in 1975 by Georges Köhler and César Milstein. To this end, these researchers fused myeloma cells, which are known to produce antibodies while simultaneously remaining immortal, with B cells obtained from an animal.
Current hybridoma antibody generation technologies begin with inoculating the animals with a target antigen to induce the generation of B cells. Hybridoma cell lines normally secrete a single species of IgG antibodies that exhibit high specificity and affinity towards the target antigen.
Despite these advantages, the antigen used to stimulate the antibody-generating immune response for a hybridoma model is quite susceptible to proteolytic degradation. This type of damage to the antigen can therefore prevent the antibodies that are produced by the hybridoma cell line to recognize the native form of the antigen when used in other animal or cell models, thus limiting their utility in both the preclinical and clinical settings.
The development of hybridoma-refractory antigens is another limitation of hybridoma technology. These antigens arise when IgG proteolysis does not accurately produce the Fragment of the antigen-binding domain (Fab). Without the ability to retain the same antigen-binding properties as the original IgG, an immune response cannot be generated against the native antigen.
Aside from these potential failures of hybridomas, there is a high level of variability that can arise in the specificity and cross-reactivity of antibodies produced through hybridoma technologies by different companies, as well as between the lots produced by the same laboratory.
Advantages of recombinant antibodies
As compared to the antibodies produced through hybridoma technology, recombinant antibodies are monoclonal antibodies (mAbs) that are produced through in vitro genetic manipulation techniques. More specifically, the genes of these mAbs are cloned into an expression vector that is then transfected into a specified host cell line that will be used to produce the recombinant antibodies.
Since recombinant antibodies are developed from a specified genetic sequence, their production is considered to be more controlled and reliable, which allows for the production of highly specific mAbs. Furthermore, this method of mAb production is associated with the highest level of consistency between batches to ensure a high level of reproducibility during their use in preclinical experiments. As compared to hybridoma-based systems that are vulnerable to genetic drift and instability that can contribute to a loss of antibody expression, recombinant antibodies are superior.
An additional advantage of recombinant antibodies is their amenability to genetic engineering. Since the peptide sequence of the recombinant antibody is known from the start, certain engineering techniques can be utilized to increase the expression of certain properties, such as the antibody’s specificity. Isotype-switching and species-switching, for example, are two common genetic engineering techniques that can be used to enhance recombinant antibodies.
Recombinant antibodies are widely used as therapeutic and imaging agents, as well as to assist in the understanding of protein-protein mediated processes. Therapeutic recombinant antibodies, for example, are often used to prevent the binding of ligands to their given receptor. Several different checkpoint inhibiting antibodies, such as those targeted against the immune checkpoint protein programmed death (PD-1) and its ligand (PD-L1) have already been approved for clinical use in several countries around the world.
Several recombinant antibodies derived from phage display have been developed against bacterial targets for diagnostic purposes. As compared to the traditional diagnostic methods used to detect the presence of bacteria, which include time-consuming cultural and microbial techniques, antibody-based diagnostic assays like ELISA and fluorescence-activated cell sorting (FACS) are simple and easy to use.
To this end, antibodies against the bacteria responsible for tuberculosis, periodontitis, and foodborne gastrointestinal infections, to name a few, have been generated by phage display for their use in diagnostic assays.
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