Progress and Remaining Opportunities to Increase the Use of Animal-free Antibodies in the USA.

The scientific and ethical issues associated with the use of animal-derived antibodies in research can be overcome by the use of animal-free, sequence-defined recombinant antibodies, whose benefits are well documented. Here, we describe progress made following a 2019 expert meeting focused on improving the quality and reproducibility of biomedical research by accelerating the production and use of animal-free recombinant antibodies in the USA. In the five intervening years since the meeting, participants have established multifaceted initiatives to tackle the next steps outlined during the meeting. These initiatives include: prioritising the replacement of ascites-derived and polyclonal antibodies; distributing educational materials describing recombinant antibodies; fostering public-private partnerships to increase access to recombinant antibodies; and increasing the availability of funding for recombinant antibody development. Given the widescale use of antibodies across scientific disciplines, a transition to modern antibody production methods relies on a commitment from government agencies, universities, industry and funding organisations, to initiatives such as those outlined here.

In vitro and integrated in vivo strategies to reduce animal use in genotoxicity testing.

Scientific, financial, and ethical drivers have led to unprecedented interest in implementing human-relevant, mechanistic in vitro and in silico testing approaches. Further, as non-animal approaches are being developed and validated, researchers are interested in strategies that can immediately reduce the use of animals in toxicology testing. Here, we aim to outline a testing strategy for assessing genotoxicity beginning with standard in vitro methods, such as the bacterial reverse mutation test and the in vitro micronucleus test, followed by a second tier of in vitro assays including those using advanced 3D tissue models. Where regulatory agencies require in vivo testing, one demonstrated strategy is to combine genotoxicity studies traditionally conducted separately into a single test or to integrate genotoxicity studies into other toxicity studies. Standard setting organisations and regulatory agencies have encouraged such strategies, and examples of their use can be found in the scientific literature. Employing approaches outlined here will reduce animal use as well as study time and costs.

Itching for change: Embracing modern flea and tick product development.

The development and regulatory approval of ectoparasiticides, including flea and tick control products, involves decades-old methods and the use of large numbers of animals to evaluate toxicity and efficacy. Animals also are used to rear (breed and feed) fleas and ticks for later use in testing. Non-animal methods for regulatory-required testing and rearing currently exist and, with further development, others could soon become available. Here we provide an overview of the state-of-the-science of non-animal methods for rearing and regulatory-required efficacy testing of flea and tick control products. Several remaining challenges as well as recommendations on the steps needed to replace animals in the evaluation of these products are discussed.

Animal metrics: Tracking contributions of new approach methods to reduced animal use.

Many companies and global regulatory programs have expressed the intent to move away from in vivo animal testing to new approach methods (NAMs) as part of product safety assessments. NAMs, which include non-animal approaches for testing and assessment ­ from computer-based modeling to in chemico or in vitro models ­ allow faster data gener­ation with potentially greater relevance to humans while avoiding animal use. To monitor progress implementing NAMs, each organization first must define what is in scope, starting with the definition of "animal" (e.g., mammals, vertebrates) and applicable studies (e.g., animals used for "in-house" experiments, at contract research organizations, as part of envi­ronmental monitoring). Next, organizations must establish baseline animal use, including defined rules for inclusion/ exclusion of animals that ensure consistency in future assessments. Lastly, organizations must establish metrics for animal savings based on the utility of NAM data. This paper presents one approach to establish "animal use" metrics in a toxi­cology program at The Dow Chemical Company. The premise of our program is that most NAM information has value for animal savings, but the value depends on how data are used (e.g., research and development, screening, or regulatory requirements) and the level of certainty for internal decision-making. This manuscript provides metrics on the impact of NAMs, allowing a quantitative assessment of animal use numbers over time, accountability for resources spent on NAM development, and identification of areas where NAM development is still needed. This approach can be refined for use at other organizations.

Maximizing the relevance and reproducibility of A549 cell culture using FBS-free media.

Scientists are using in vitro methods to answer important research questions and implementing strategies to maximize the reliability and human relevance of these methods. One strategy is to replace the use of fetal bovine serum (FBS)-an undefined and variable mixture of biomolecules-in cell culture media with chemically defined or xeno-free medium. In this study, A549 cells, a human lung alveolar-like cell line commonly used in respiratory research, were transitioned from a culture medium containing FBS to media without FBS. A successful transition was determined based on analysis of cell morphology and functionality. Following transition to commercially available CnT-Prime Airway (CELLnTEC) or X-VIVO™ 10 (Lonza) medium, the cells were characterized by microscopic evaluation and calculation of doubling time. Their genotype, morphology, and functionality were assessed by monitoring the expression of gene markers for lung cell types, surfactant production, cytokine release, the presence of multilamellar bodies, and cell viability following sodium dodecyl sulphate exposure. Our results showed that A549 cells successfully transitioned to FBS-free media under submerged and air-liquid-interface conditions. Cells grown in X-VIVO™ 10 medium mimicked cellular characteristics of FBS-supplemented media while those grown in CnT-Prime Airway medium demonstrated characteristics possibly more reflective of normal human alveolar epithelial cells.

Increasing the use of animal-free recombinant antibodies.

Antibodies are used in a range of research, diagnostic, and regulatory applications. Traditional methods for producing such reagents involve the immunization of animals, which introduces variability into the methods that use them and is not aligned with efforts to replace and reduce animal use. Experts from academia, biotechnology, government, and animal protection organizations met December 3, 2019, at the National Institutes of Health in Bethesda, MD, USA to discuss the status of development and use of animal-free recombinant antibodies and their potential to replace antibodies derived from animals. This paper summarizes the discussion and the actions that resulted to facilitate increased production and use of animal-free recombinant antibodies.

Modern affinity reagents: Recombinant antibodies and aptamers.

Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research.