For decades, traditional polyclonal and monoclonal antibodies have driven progress in many different fields of research. More recently, they have been complemented by recombinant antibodies that offer opportunities for modification and engineering. Here, we recap the benefits of recombinant antibodies and comment on the reasons for their increased use. We also highlight some novel discoveries made possible by recombinant antibody technology.
Addressing the reproducibility crisis
Recombinant antibodies are produced by cloning antibody-encoding genes into high-yielding expression vectors such as E. coli or Chinese Hamster Ovary (CHO) cells for in vitro production. Critically, this approach overcomes the problem of genetic drift that is known to limit the lifespan and batch-consistency of hybridoma-derived monoclonals and is a contributing factor in the ongoing reproducibility crisis.
“Poor standardization and reproducibility of antibodies is an issue the life sciences industry has long struggled with,” reports Andy Lane, Ph.D., Commercial Director at The Native Antigen Company. “For example, a recent study shows traditional monoclonal antibodies frequently express additional functional variable regions that negatively impact specificity and signal. By heterologously expressing antibodies in vitro, high batch-to-batch consistency can be achieved to ensure reproducible results and long-term supply.” Other advantages of recombinant antibodies include high purity, low endotoxin levels, and the capacity for engineering into different formats.
Tight control over antibody properties
According to Dr. Michael Fiebig, Chief Scientific Officer at Absolute Antibody, recombinant technology gives antibody manufacturers complete control over the products they are making—right down to the sequence level. “As well as helping safeguard antibody identity during production, this aids in troubleshooting when there are unexpected results,” he says. “Moreover, recombinant antibody manufacturers can engineer antibodies to suit specific requirements, essentially tailoring antibodies to the assay in question rather than compromising on experimental design with sub-optimal antibodies.”
Engineering can include changing the species of an antibody to improve compatibility with different detection reagents in multiplexed studies or reduce immunogenicity in vivo, and silencing Fc gamma receptor binding to minimize Fc-receptor mediated background staining in methods like flow cytometry. Fiebig does, however, caution that many antibodies are still unavailable in recombinant form, meaning recombinant antibody products are not always an option. Considerable efforts are being made within the antibody manufacturing industry to address this.
Applicability to IVD testing
A main reason for using antibody engineering to perform species-switching is that it avoids human anti-mouse antibody (HAMA) effects, which can especially be problematic during in vitro diagnostic (IVD) testing. If an individual has produced HAMA following an encounter with mouse proteins, these can bind to mouse antibodies used as immunoassay components, yielding false positive or false negative results by bridging a matched antibody pair or blocking antibody binding to the target analyte, respectively.
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“At Medix Biochemica, we have developed chimeric antibodies containing variable domains from mouse or chicken immunoglobulins and the constant region from human IgG to decrease HAMA interference in IVD assays,” notes Sari Tiitinen, R&D Director for Immunodiagnostic Reagents. “Recombinant technology has also allowed us to rescue monoclonal antibodies from unstable cell lines, thereby securing raw material supply, and has provided opportunities to modify existing antibodies or develop new antibodies for analytes that are toxic or non-immunogenic in animals.”
Importantly, Tiitinen stresses the value of using both traditional and recombinant technologies to enable antibody selection based on performance—namely, specificity, affinity, and suitability for application. “The production method or technology used is not an ultimate determining factor when selecting antibodies for IVD tests,” she says. “By ensuring our in vitro hybridoma manufacturing processes produce highly pure mouse monoclonals with excellent batch-to-batch consistency and applying state-of-the-art recombinant technologies to complement our toolbox, we offer our customers the best of both worlds.”
High rates of target specificity
Despite gaining momentum in popularity, recombinant antibodies have been described as relatively underutilized reagents. “Our discussions with the life science community suggest that this is predominantly due to lack of awareness of the benefits recombinant antibody reagents offer or a historic misplaced perception that recombinant antibodies don’t cover as many targets as traditional antibody reagents,” explains Dr. Alejandra Solache, Senior Vice President for R&D at Abcam.
“Indeed, the switch to recombinant antibodies is often driven by past frustrations with antibodies that don’t work reliably. Yet, researchers are increasingly mindful of the fact that recombinant monoclonals often have the highest rate of target specificity when compared to polyclonals and hybridoma monoclonals, which is driving uptake. To meet demand, we have developed over 25,000 recombinant antibody reagents to date, combining the high affinity and specificity of our rabbit monoclonal antibodies with recombinant technologies that include a recombinant synthetic DNA method for higher reproducibility and consistency from batch to batch.”
Reduced animal use
Last year, a committee from the European Commission’s EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) issued a recommendation urging end users and other stakeholders to recognize the scientific validity of non-animal-derived antibodies. The review focused on non-animal-derived antibodies generated by phage display, a mature technology that allows the enrichment of binding proteins from large libraries.
Amanda Turner, Product Manager for Custom Antibody Products at Bio-Rad, notes that antibody phage display has been used by Bio-Rad’s custom antibody team since 2004 to generate highly specific antibodies in eight weeks for research and diagnostic use. “Using an in vitro guided selection method rapidly provides reagents with a level of specificity that is difficult to achieve using conventional animal immunization methods and, importantly, removes animal use from the equation,” she says.
“In a recent innovation we incorporated SpyTag technology into the process. This adds completely new versatility to recombinant antibodies, giving the user easy access to multiple formats of an antibody for use in different applications.” An antibody with a SpyTag can be coupled to a range of SpyCatcher proteins in a reaction that takes under an hour and enables site-specific conjugation and switching between monovalent, bivalent, and Ig-like formats with isotypes of different species, to suit the experimental set-up.
Driving novel research
As the availability of recombinant antibodies has increased, their use has been cited in a growing number of publications. For example, Abcam’s anti-ATG16L1 (phospho S278) antibody was recently used to analyze autophagy induction, something that has historically been challenging due to a lack of suitable tools, while its HMGB1, SMARCA4, and H3K27ac antibodies have helped reveal host factors involved in SARS-CoV-2 infection.
Recombinant antibodies have also opened up new avenues of drug development; at the end of last year, two T cell-engaging murine bispecific antibodies engineered by Absolute Antibody were used to recruit virus-specific T cells and induce anti-tumor activity in vivo, providing hope to patients with solid tumors where drug efficacy is often limited by insufficient numbers of infiltrating T cells.
Additionally, recombinant antibody use has seen rapid uptake in the diagnostic industry, where Fiebig suggests there is huge potential for designing better controls and capture/detection reagents. “With the COVID-19 pandemic, we have seen a dramatic need for reliable and accurate diagnostic testing,” he says. “Recombinant engineering allows you to generate even unusual controls quickly, such as anti-SARS-CoV2 human IgG1, IgG3, IgA, IgM, and IgE panels, and we expect this market to continue to grow.”
Aside from these developments, Lane remarks that traditional polyclonal and monoclonal antibodies will likely continue to be used for a long time, especially in research and smaller scale applications. “As it stands, traditional antibody generation methods are still the go-to for antibody discovery, given their comparative simplicity,” he says. “However, as phage libraries and validation technologies continue to improve, recombinant antibody production will become more powerful and accessible for many users. It is clear that recombinant antibodies are promising tools with bright futures in both research and clinical use, but I suspect traditional antibodies will be around for some time yet.”
Image: Bispecific antibody formats include heterodimeric knob-into-hole, where one arm of the antibody targets one antigen and the other targets a second, and homodimeric IgG-dAb, where each antigen is targeted by two antigen binding sites. Image provided by Absolute Antibody.