Hidden Animals in Science: A Call for an Antibody Revolution

Ladies and gentlemen, let me introduce you to The Antibody. Seldom does he traverse the thoughts or the tongues of those outside the scientific community – but you can be sure this Y-shaped workhorse is toiling away behind the scenes of every medical breakthrough and scientific discovery that has shaped our world over the past sixty or so years.

In biomedical research, questions of animal ethics usually pertain to the use of whole animals to model disease and test pharmaceutical efficacy. In truth, the problem extends far beyond this. Many of us are oblivious to the mass of the iceberg concealed beneath the glamorous ocean of science.  It is easy to turn a blind eye to these lesser known cruelties – the use of animals for science beyond direct testing – and some will say it is acceptable to do so. If we need to use animals here and here, and if it’s helping cure diseases and create a better world, and if there’s no alternative technology that gives the same result, maybe it’s okay to brush the dust under the carpet and go about our lives. Basically, when it comes to medicine and there are human lives on the line, if there is no alternative technology that gives the same result as an animal-based test, it’s very hard to make an argument for change. I will not try to do so.

Antibodies – what are they, and why do we use them?

The antibody is one of the most indispensable tools used in biomedical experiments today. Many essential techniques routinely performed in biomedical laboratories are dependent on their use – Western blotting, enzyme linked immunosorbent assay (ELISA), immunohistochemistry – basically all techniques which rely on identifying or isolating some protein target. In fact, every time a scientist reports some new discovery about a disease or biological process, chances are that somewhere down the line an experiment was done using antibodies.

In essence, antibodies are protective proteins produced by our immune system in response to foreign substances. They help us to fight off infections caused by bacteria and viruses by binding strongly and specifically to molecules – so-called antigens – on the surface of these pathogens. During the immune response the antibody latches on to whichever unique antigen has triggered its production, thereby helping to eliminate some invading substance from the body which might otherwise provoke disease (1). This remarkable specificity for binding to its target substance is exactly what makes the antibody such a treasure in the molecular laboratory. The unique specificity of the antibody-antigen interaction underlies its value as a research tool in molecular biology, as well as its use in diagnosing and producing medicines for diseases such as cancer (2).

The global antibody industry, which today relies heavily on animals, is worth $80 billion, and continues to expand each year. Though the actual number of animals used to generate antibodies is not known, as statistics in this field are scarcely reported, it is estimated that about 2.6 million antibodies are in production worldwide, each requiring 2-5 animals at a minimum (3).

The Low-down on Conventional Antibody Production

Traditionally, production of antibodies requires repeated injections of the substance of interest – lets call it Antigen X – into the animal. This stimulates the animal’s immune response so that it produces antibodies which recognise and bind specifically to Antigen X. Later, these antibodies are extracted from the animal and then used in experiments that require detection or quantification of Antigen X.  When scientists think about antibody-based tests, the thought is generally far removed from animal experimentation because no animals are directly tested on. But animals buried in the deeper layers of the production process are still animals. These are usually mice, rabbits and goats, and after their use, just like those animals used directly in experiments, they will ultimately be killed by euthanasia.

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I am not here to propose that we stop using antibodies. Doing so would arrest medical progress. Rather I am here to question whether antibodies can be produced without animal suffering. Is there even such a thing as a cruelty free antibody? The answer is yes –  so-called Animal Friendly Affinity reagents (AFAs) have actually been around for quite some time.

It is nearly impossible to live a life abstaining from any product or service which somewhere down the line caused harm to an animal. But replacing animal-derived antibodies is not impossible – it is entirely feasible and it is tremendously urgent.

The Case for a Transition to Animal-Friendly Affinity Reagents

AFAs are most commonly created using a technology called Phage Display. Phage Display technologies are independent of any living immune system, instead relying on a type of virus called bacteriophage. These techniques are accessible, cost-effective and robust, and in fact are considered to produce more specific and better functioning antibodies than traditional animal based methods. A shift towards Phage Display technology could spare thousands of unnecessary animal deaths while at the same time improving the quality and reproducibility of scientific research and reducing associated costs of repeat experiments (4).

Poor quality antibodies are polluting the market today, posing serious problems in science. When an antibody doesn’t stick to what it’s supposed to, or sticks to other things when it’s not supposed to, any experimental findings garnered from it’s use are unknowingly destroyed. Moreover, batch-to-batch variability of identically labelled antibodies is too often observed (5). False findings, inconsistency and confusion ensue, requiring experiments to be repeated again and again, often at colossal, and otherwise avoidable costs.  A large assessment of some 20,000 commercial antibodies conducted by Swedish researchers at The Human Protein Atlas reported bleakly that up to 50% of commercially available antibodies are unreliable (6). That scientists can no longer trust the label on the vial – usually having spent a few hundred to a thousand dollars to procure its contents – is truly unacceptable. Evidently, regardless of whether animal ethics are on your agenda, the need for an antibody revolution is undeniable, and this is an issue that AFAs might help to address. AFAs can be designed to have higher affinity and binding specificity than is possible for their animal-derived counterparts, meaning that they can bind more strongly to their target, without having undesirable off target effects (7).

AFAs can also be produced more quickly, and unlike those from animal sources, they can be used to generate antibodies against non-immunogenic antigens as well as toxic ones. And these recombinant antibodies can be generated synthetically in the lab without using animals at all.

Another advantage of AFAs is that they are much safer for therapeutic applications where animal antibodies may elicit unwanted immune responses in a patient. When animal antibodies are made for clinical purposes, they must first be “humanised” or altered in such a way as to avoid unwanted immune reactions before being introduced into the human body. Scientists have known for quite some time – yet seem hesitant to admit – that mice are in fact not human. Nor are their antibodies, and our clever immune systems are well designed to fight off anything that it perceives as foreign.

How do current strategies align with EU Legislation?

Whenever we address the use of animals for science, the Three R’s – Replacement, Reduction and Refinement – must be considered in a strict hierarchy. This means replacing animal use where an alternative is available, reducing animal use where no alternative is available, and as a very last resort, refining conditions of animal use so as to minimise suffering. Researchers today seem to have flipped this hierarchy on its head, focusing their efforts on Refinement but neglecting the possibility for Replacement where it exists (8)

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In article 4 and 13 of the Directive 2010/63/EU (9), which legislates for the protection of animals used for scientific purposes, states:

‘Wherever possible, a scientifically satisfactory method or testing strategy, not entailing the use of live animals, shall be used instead.’

‘An animal procedure should not carried out if another method or testing strategy for obtaining the result sought, not entailing the use of a live animal, is recognised.’

It seems then that in the case of antibodies, given this hierarchy, the factors of Reduction and Refinement should not even need to be considered. Is it not clearly essential, and in accordance with EU law, that animal antibodies are completely eradicated from the system and replaced by AFAs?

It is important to recognise that AFAs are not merely “antibody lookalikes” – they are true antibodies, of at least equal – if not superior – scientific quality as animal-derived ones, as has been demonstrated in several large scale studies (4). AFAs are functionally and structurally indistinguishable from those produced in vivo. AFAs are not “fake meat” – they are truly beef without the cow. And without the cow – or in our case, without the mouse, the rabbit and the goat –  you get the added advantages of time, money, and scientific progress – probably the top three priorities for anyone in the research community. So what’s standing in our way?

They’re just as sticky, but they still won’t stick

Despite high quality AFAs being available for the past two decades, and despite having enormous potential to reduce animal use in medical research, the technology has not been widely adopted by the scientific community. Why the resistance? Supposedly, a marriage of complacency and misconceptions regarding costs and scientific quality has left scientists eyes-deep in animal antibodies, and flailing about in a sea of Bad Science. Not to mention that all of the leading muli-million dollar antibody companies which have already well established large-scale facilities dedicated to traditional production methods, are not ready or willing to face reform. A transition to Phage Display technology, while it would likely save money in the long run, would call for a complete revamping of these facilities.

I will finish with an excerpt from a revealing article in Cell Press’s Trends in Biotechnology, authored by Immunotechnology Professor Carl Borrebaek from Lund University, Sweden, and founder of AFABILITY Dr Allison Gray, from Nottingham University (4).

“We have at our disposal a mature and widely used technology that is set to have an enormous impact on animal use owing to the fact that the reliance on antibodies by biomedical scientists, health-care professionals, and consumers impacts all areas of research, development, and safety testing. For that reason, it is bewildering to consider that, despite the readiness of new molecular methodologies and the wealth of literature to support implementation, and despite the growing availability of companies offering AFAs, antibodies are continuing to be produced using animal immunisation techniques. It is even more perplexing that we are not already deeply committed to a programme of replacement of animal-derived antibody production techniques.”

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References:

  1. Forthal DN. Functions of antibodies. Antibodies for Infectious Diseases. 2015 May 30:23-48.
  2. Chames P, Van Regenmortel M, Weiss E, Baty D. Therapeutic antibodies: successes, limitations and hopes for the future. British journal of pharmacology. 2009 May;157(2):220-33.
  3. Biocompare: https://www.biocompare.com/Editorial-Articles/177815-2015-Antibody-Market-Report/
  4. Gray AC, Sidhu SS, Chandrasekera PC, Hendriksen CF, Borrebaeck CA. Animal-friendly affinity reagents: Replacing the needless in the haystack. Trends in biotechnology. 2016 Dec 1;34(12):960-9.
  5. Baker M. Blame it on the antibodies. Nature. 2015 May 21;521(7552):274.
  6. Berglund L, Björling E, Oksvold P, Fagerberg L, Asplund A, Szigyarto CA, Persson A, Ottosson J, Wernérus H, Nilsson P, Lundberg E. A genecentric Human Protein Atlas for expression profiles based on antibodies. Molecular & cellular proteomics. 2008 Oct 1;7(10):2019-27.
  7. Bradbury AR, Sidhu S, Dübel S, McCafferty J. Beyond natural antibodies: the power of in vitro display technologies. Nature biotechnology. 2011 Mar;29(3):245.
  8. Franco NH, Sandøe P, Olsson IA. Researchers’ attitudes to the 3Rs—An upturned hierarchy?. PloS one. 2018;13(8)
  9. Directive 2010/66/EU: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=celex%3A32010L0063

 

2 Comments

  1. Really interesting post. I’m currently finishing my bachelor thesis in bioinformatics, when talking to my friend at lunch time someone’s problem with an antibody was brought up almost daily. I can’t understand why I haven’t learnt about AFAs during the career

    Liked by 1 person

    1. Thanks Barbara! Yes, I remember during my bachelors studies in biomedicine, we were always told that the most important thing for our experiments is that we have a good antibody – but I also never heard anything about AFAs until doing my own reading recently. I agree with you, it is mind-boggling.

      Like

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