Making the Switch to Non-Animal Derived Antibodies

Is it now the responsibility of the scientific community to take a major leap towards the replacement of animals for antibody generation? A recent report by the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) concluded that non-animal derived antibodies should be recommended for future antibody development (Barroso et al. 2020). This blog discusses the reasons why making the switch to non-animal derived recombinant antibodies is something that both antibody suppliers and researchers should consider.

Could Recombinant Antibodies Be Key to Solving the Reproducibility Crisis?

The reproducibility of scientific results is something that scientists are deeply concerned about. Not only must results be repeatable between experiments, but also findings should be verifiable by different research groups.

Antibodies are a common research tool used in many different assays, and results are heavily influenced by the quality of the antibodies used and the robustness of the experimental procedure. Polyclonal antibodies are often a desirable choice due to their low cost, but they can suffer from variability in performance between batches, even when the same animal is used repeatedly for antibody production, leading to inconsistent results (Baker 2015). While monoclonal antibodies are in theory less susceptible to performance issues due to much better batch-to-batch consistency, a recent study has found that almost a third of hybridoma cell lines tested contained additional productive heavy or light chains (Bradbury et al. 2018). Furthermore, hybridoma cell lines are sometimes susceptible to genetic drift over time leading to loss of antibody binding or even complete loss of antibody expression. However, there is a way to have a secure supply of antibodies that can perform consistently time-after-time.

Recombinant antibodies offer a way to produce antibodies with high consistency in a range of formats with an unlimited supply. While it is possible to make recombinant antibodies from sequencing hybridomas, there is also a way of producing recombinant antibodies that eliminates the requirement of animals and associated ethical dilemmas. Non-animal derived antibodies utilize for instance, universal phage display libraries which do not rely on the immune response of an animal to generate antibodies, and result in the generation of recombinant antibodies in vitro with a defined sequence, which can be well-characterized and reproduced indefinitely through production in E. coli or a mammalian cell line. These libraries cover an enormous immune repertoire, meaning that antibodies can be generated targeting almost any conceivable antigen.

In vitro guided selection methods enable isolation of antibodies targeted to defined epitopes. It is possible to select antibodies from a library that bind a very specific region like a phosphorylated residue or a point mutation, enabling very precise detection or inhibition of target molecules.

In a Correspondence published in Nature, scientists urge the use of non-animal derived recombinant antibodies to improve scientific reproducibility (Gray et al. 2020). A central finding of the EURL ECVAM report is that non-animal derived antibodies are equivalent to those generated using animal immunization methods, meaning the requirement for animals to generate new antibodies is obsolete. The report concluded that there are no detrimental changes in shelf life, stability, specificity, or affinity (Barroso et al. 2020). Moreover, non-animal derived antibodies can be used in place of traditional antibodies in all commonly used applications, so there should be no limits to their experimental potential. It was for all of these reasons that the report made the following recommendation:

“Therefore, taking into consideration the ESAC Opinion on the scientific validity of replacements for animal-derived antibodies, EURL ECVAM recommends that animals should no longer be used for the development and production of antibodies for research, regulatory, diagnostic, and therapeutic applications. The provisions of Directive 2010/63/EU should be respected and EU countries should no longer authorise the development and production of antibodies through animal immunisation, where robust, legitimate scientific justification is lacking.”

Time to Prioritize Ethical Arguments for Antibody Generation

Scientists are being actively encouraged to think about how they can minimize the involvement of animals in their research. Choosing to generate new antibodies using methods that do not involve the immunization of animals is one way of achieving this, and for many people, making an ethical choice is a top priority.

In the UK, the government set up the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) in 1994 to provide guidance on how to perform more humane animal research. Their recommendations have become embedded in the guidelines for many funding bodies like the Wellcome Trust and the  Medical Research Council (MRC). Furthermore, the EU Directive 2010/63/EU, adopted in September 2010, embeds the protection of animals used for scientific purposes into legislation in all member states and calls for stringent ethical and welfare standards. In line with this legal requirement, where a non-animal alternative exists that produces results to the same or higher standards, it should be used preferentially. Despite these initiatives, it is estimated that close to 1 million animals continue to be used yearly for antibody development and production in the EU alone. Non-animal derived antibodies are endorsed and recommended by EURL ECVAM scientific advisory committee (ESAC) as a suitable replacement for animal-derived antibodies, along with a recommendation to focus on eliminating animals from antibody production, and instead utilize alternative technology for future antibody production (Barroso et al. 2020). The committee focused its review on the mature and proven method of monoclonal antibody production from universal phage display libraries, the technology for which the 2018 Nobel Prize in Chemistry was awarded.

Overcoming the Limitations of Animal Immunization

In vitro methods of antibody generation can be used to solve problems that a traditional approach is unable to address, such as selecting antibodies that bind to a toxin, which couldn’t be used to immunize an animal, or that bind to a low immunogenic protein, or to overcome self-tolerance, for example through a highly specific antibody in the case of a cardioprotective MASP-2 antibody (Clark et al. 2017). The challenge of raising antibodies against a protein complex has also been solved using phage display libraries and guided selection strategies. Antibodies that recognize a drug-target complex can be used to improve analysis of biotherapeutics during pre-clinical and clinical development, and in routine drug monitoring in clinical practice.

Antibody phage display libraries can be developed by investing in expertise, resources, and equipment. Once in place, the selection of antibodies can be done in as little as four weeks with dedicated personnel. This is considerably faster than the standard 6-9 months for the generation of mouse monoclonal antibodies using immunization followed by hybridoma cell line development. After the initial investment to create the synthetic library, the cost of generating non-animal derived antibodies is comparable to monoclonal generation using traditional immunization methods, with the added significant advantage of being able to move a project forward by several months.

What Do the EURL ECVAM Report Findings Mean for the Antibody Field?

The report highlighted the importance of investing in alternatives to animal-derived antibodies, not only to reduce the use of animals in research but also to improve the reproducibility of experimental data. There are compelling arguments for the research community to adapt their current common practices and move towards non-animal derived antibodies for new antibody generation, and recombinant antibodies where possible. Antibody manufacturers are called upon to cease producing antibodies by the ascites method, work towards phasing out antibody production using animals, and increase the availability of non-animal derived antibodies. Where possible, antibody suppliers, end users, and publishers should switch to methods that do not require the use of animals.