3Rs Prize: Replacing animal-derived antibodies and in vivo antivenom tests

The International 3Rs Prize, awarded by the NC3Rs and sponsored by GSK, has recognised two decades of outstanding 3Rs advances from scientists around the world. In the 20^th 3Rs Prize ceremony last week, this years’ winning and highly commended researchers shared details of their approaches to replace animal use in antibody and antivenom production. 

"GSK proudly celebrates the achievements of the NC3Rs International Prize winners, whose pioneering research drives the replacement, reduction, and refinement of animal use in science. Over two decades of partnership, we have witnessed remarkable progress in non-animal technologies and a steadfast commitment to animal welfare, all of which contribute to advancing transformative healthcare. Through our continued partnership, we look forward to inspiring the next generation of scientific leaders dedicated to the 3Rs and to shaping the future of medicine through innovative, responsible research."

– GSK, 3Rs Prize sponsor

Dr Peter McPherson presented on behalf of the winning team from McGill University in Montreal, Canada, which also include Dr Riham Ayoubi and Dr Carl Laflamme. Their paper presenting a systematic and scalable approach to assess antibody performance has already been viewed 4,448 times and cited 59 times since it was published in eLife in February 2024. Millions of animals are used every year to produce commercial antibodies for scientific research, including mice, rabbits and rats alongside larger animals such as goats and donkeys. The team demonstrated that non-animal derived recombinant antibodies performed better than traditional animal-derived monoclonal and polyclonal antibodies, in terms of target specificity and reproducibility in common laboratory applications. Their large-scale performance dataset evidences the significant benefits non-animal derived affinity reagents can offer, including coverage of human proteins and greater sensitivity and specificity for their targets.

“This year is a special occasion as it marks the 20^th 3Rs Prize the NC3Rs has awarded. The Panel were incredibly impressed by the high quality interdisciplinary nature of the entries and had a truly difficult decision in selecting this year’s winner. The methods reported in both the winning and highly commended papers have remarkable replacement potential and I look forward to seeing their full 3Rs impact being realised in the near future. Huge thanks to GSK who have generously supported this international prize since its inception.”

– Professor Dame Julia Buckingham Chair of the NC3Rs Board and 3Rs Prize Panel

We were also joined by Dr Renata N.C Nundes from the Oswaldo Cruz Foundation in Rio de Janeiro, Brazil, who received a highly commended award for a paper describing an in vitro alternative to the lethality neutralisation assay for snakebite antivenom testing. The currently used in vivo assay for quality control and batch release testing of antivenom causes severe suffering for mice. The paper details a cell-based cytotoxicity assay for potency testing of antivenoms and demonstrates its reproducibility, compared to historical in vivo data. The work demonstrates the potential of this approach to replace the current mouse lethality neutralisation assay that is recommended by the WHO.

An estimated $1BN in research funding is wasted each year in the US alone on ineffective commercial antibodies. Many fail to recognise the intended target protein in some or all laboratory applications or recognise additional targets and generate non-specific results. While it is difficult to estimate the total number of animals used in antibody production globally, one million animals per year are used for this purpose in the EU alone. To tackle the wastage of animals and resources in unreproducible research caused by poor antibody performance, the winning team of scientists created partnerships with academics, research funders and ten commercial antibody manufacturers, representing an estimated 27% of global antibody production. They agreed on optimised protocols to assess antibody specificity in the three most common uses of antibodies in biomedical research laboratories – Western blot, immunoprecipitation and immunofluorescence. Peter, Riham and Carl tested 614 commercially available antibodies provided by manufacturers to 65 target proteins involved in neurological diseases. The majority of target proteins were chosen by research funders and focused on neuroscience research, including those linked to Alzheimer's disease, Parkinson’s disease and amyotrophic lateral sclerosis. 

Their scalable screening strategy involved a panel of cells expressing each target protein at sufficient levels to be detected by an antibody with typical binding affinity (1–50 nM). Multiple antibodies were tested against each target protein (an average of nine to ten per protein), including both animal-derived and non-animal derived reagents. The team could not identify a high-performance antibody for approximately a third of the target proteins, demonstrating the prevalence of poor binding affinity and target specificity among commercially available antibodies. For two thirds of the antibodies tested (409 out of 614), the team's characterisation data conflicted with the antibody supplier’s recommendations for use. As a result, the participating companies have withdrawn 73 poorly performing antibodies from the market and changed recommendations for 153 antibodies. Notably, recombinant non-animal derived antibodies performed better than traditional animal-derived reagents. Only one of the 65 proteins tested was not recognised by any of the non-animal derived antibodies tested, with the majority (49 proteins) identified by at least three different recombinant antibodies. 

A lack of large-scale performance data comparing different antibody generation technologies has contributed to the slow uptake of replacement alternatives, despite the well-established reproducibility issues of traditional animal-derived antibodies. The authors characterisation dataset is available on ZENODO, a freely available data-sharing website operated by CERN. Based on their findings that recombinant antibodies performed better than monoclonal or polyclonal antibodies the authors encourage the increased generation of recombinant antibodies to replace the use of animal-derived antibodies. 

To detect whether a primary antibody has bound to a target protein a secondary detection system is used. This secondary detection system (e.g. secondary antibody) is bound to a detectable molecule such as a fluorophore which allows for the quantification of the target protein. Currently standard secondary detection systems are not always compatible with the animal free protein binders used in recombinant antibodies. The team will use the £28K Prize grant to develop protocols for the optimisation of secondary detection systems that are compatible with recombinant antibodies. The team, who have been approached by manufacturers of animal-free protein binders, will conduct an independent comparison of their performance compared to animal-derived antibodies. The development of reliable secondary detection systems for recombinant antibodies aims to remove another barrier to their adoption to replace animal-derived reagents.

"We’re honoured to receive this recognition from the NC3Rs. YCharOS is a public good initiative committed to improving research reproducibility and accelerating the adoption of selective, recombinant antibodies. These ethical reagents offer scientific advantages over traditional animal-derived antibodies and support the 3Rs by reducing the need for animals in antibody production. This award highlights the meaningful impact that open, community-driven efforts can have on both scientific progress and ethical practice."

– Dr Carl Laflamme

Currently, the only available treatment for snakebite envenomation is the administration of specific antivenom. Antivenoms are purified antibodies harvested from an envenomed animal – the potency varies between each batch and must be assessed as part of quality control testing. Potency determination relies on the murine lethality neutralisation assay as recommended by the WHO. In this test, antivenom therapies are mixed with lethal doses of snakebite venom and administered intravenously to mice. This can lead to severe suffering including paralysis, seizures, haemorrhage and death. Snakes of the Bothrops genus were responsible for 69% of snakebite events reported in Brazil in 2019 (almost 21,000 cases). Bothrops snakebites often cause permanent tissue damage, leading to limb amputation, disability and in some instances, life threatening complications such as tetanus and sepsis. In 2017, around 2,000 mice were used for Bothrops antivenom efficacy and batch release testing at the National Institute for Quality Control in Health in Brazil where this work was conducted.

Renata developed a cell-based cytotoxicity assay as an alternative to the murine lethality neutralisation assay specifically for Brazilian Bothrops antivenom potency testing. The in vitro method is based on the ability of Bothrops venom to induce cytotoxic effects in an immortalised cell line and assesses the capacity of the antivenom to inhibit this cytotoxicity. The in vitro assay provides a concentration-based measure of antivenom potency that allows direct comparison of their efficacy. This allows clinicians to make better informed treatment decisions, compared to the in vivo assay where the volume of antivenom that protects 50% of mice from death is measured and then used to calculate potency for clinical use. 

The cytotoxicity-based in vitro method exhibits very good inter-assay and intra-assay reproducibility, as demonstrated through comparison with existing in vivo data in collaboration with three Brazilian antivenom manufacturers and the National Control Laboratory. Further work to define the regulatory acceptance criteria for these biological assays will allow for their wider uptake to replace the murine lethality neutralisation assay in antivenom production.

“This recognition is the validation of years of passion and dedication to research, and symbolises the fulfilment of my commitment to advancing science in an ethical and innovative way. It is a great incentive for research that seeks reproducibility, efficiency and sustainability, showing that it is possible to transform the knowledge acquired into a new methodology that not only saves human lives but also those of animals.”

– Dr Renata Norbert Costa Nundes 

The 3Rs Prize highlights outstanding and original work within the 3Rs and consists of a £28k prize grant and a £2k personal award. A £20k contribution is provided by GSK with all remaining funds, including the personal award made by the NC3Rs. The prize grant is intended to maximise the 3Rs impacts of the published research. This includes purchasing new equipment and consumables to aid further development, characterisation and qualification studies or supporting dissemination and training opportunities to increase awareness and uptake of the 3Rs approach.

The criteria assessed by the 3Rs Prize selection Panel include:

• Actual or potential impact on the 3Rs.
• Dissemination of the scientific and 3Rs impacts to date.
• Strength of plans for the Prize grant.
• Potential impacts for the research, the 3Rs and development of the individual.