ANIMAL IMMUNIZATION FOR ANTIBODY PRODUCTIONOVERLOOKED …..

….. AND OBSOLETE
Dr Alison Gray

WHY SHOULD WE BE CONCERNED ABOUT ANIMALBASED ANTIBODY PRODUCTION?
• Animal based antibody production requires immunization• Includes hybridoma (monoclonal), polyclonal and (immunized)
recombinant methods• Subsequently appear as in vitro tests
We continue to use immunized animals where…
• Welfare issues: 100’s of thousands of animals, mistreatment and undesirableprocedure• 3Rs: Replacement methods exist• A myriad of misconceptions and unawareness about antibody production…..Lack of enforcement of Directive 2010/63/EU on theprotection of animals used for scientific purposes

WHY SHOULD WE BE CONCERNED ABOUT ANIMALBASED ANTIBODY PRODUCTION?

We continue to use immunized animals where…
• Welfare issues: 100’s of thousands of animals, mistreatment and undesirable
procedureWELFARE ISSUES: STATISTICS• Multibillion dollar global industry• Only two Member State countries published this information (up to 2013 - nowceased)

ESTIMATES•

Not taking into account academic use , import , repeat production  or rebranding EU : from 833 thousand to 1.25 million animals per year
World wide : 5 fold increase and limitless new possibilities…..

WELFARE ISSUES: MISTREATMENT, UNDESIRABLE PROCEDURE

WHY SHOULD WE BE CONCERNED ABOUT ANIMALBASED ANTIBODY PRODUCTION?

We continue to use immunized animals where…

• 3Rs: Replacement methods exist

AFAs : ANIMAL FRIENDLY AFFINITY REAGENTS
Antigen• Antibodies

Antibody• Non-antibody scaffolds or mimetics• Instead of a constant supply of animals, we create a library of AFAs in atube: equivalent to a life time’s supply of animals• All AFAs formats have the characteristic of having
an affinity for / binding to a unique target / antigen• Produced in micro-organisms• No animal use, including immunization• More than 40 AFA solutions available, various stages of development,
some commercialized

WHY SHOULD WE BE CONCERNED ABOUT ANIMALBASED ANTIBODY PRODUCTION?

We continue to use immunized animals where…

• A myriad of misconceptions and unawareness about antibody productionMISCONCEPTIONS / UNAWARENESSLACK OF AWARENESS AND THEREFORE ENFORCEMENT AT
MEMBER STATE /LEC LEVELLACK OF AWARENESS AND THEREFORE ENFORCEMENT AT
MEMBER STATE /LEC LEVELEXTRACTS FROM 2013 NON-TECHNICAL SUMMARIES (UK)“…..Antibody production without the use of animals has not been shown to produce the range ofantibody specificities and affinities required in this project…..” (200 mice and 30 rats over 5 years )“..…whilst antibodies can be made without immunisation, antibodies that are generated as a result ofimmunisation have a higher specificity and higher affinity…..” (4000 mice and 500 rabbits over 5 years)“…..although antibodies can be synthesized in vitro, these are of low affinity and are unable to performthe highly demanding requirements we need from our antibodies…..” (900 mice)“…..Antibodies are produced in the laboratory animals. There are no convenient alternatives for theproduction of antibodies. A known procedure of production of antibodies in bacterial cells still requires
the use of laboratory animals in order to obtain the gene that encodes this antibody from spleens of theanimal. This technique is too labour intensive, prone to errors, time consuming and costly. …..”“….. Mouse is by far the best model for producing antibodies. Alternative approaches are being
developed, but none of them has become as reliable as the mouse model. We have tried one of the bestalternatives, but could not make any antibody……”“…. Animals produce antibodies with the highest affinity. New technologies are available which produce
antibodies in-vitro, however, these are low affinity antibodies and although we have tried to use them inour work, none have been successful…… “LACK OF AWARENESS AND THEREFORE ENFORCEMENT AT
MEMBER STATE /LEC LEVELEXTRACTS FROM 2016 NON-TECHNICAL SUMMARIES (UK)
“Antibodies are produced by a living immune system involving activation of specific cells inresponse to antigens …. This means that laboratory animals of excellent health status and known
genetic background are required …”“We are investigating more modern methods of producing antibodies in the laboratory, using
bacteria, but research in these areas is still at an early stage and is not yet ready for use”
“There are still applications for which animal derived antibodies are essential, e.g. antivenomproduction, where antibodies to all of the epitopes of the toxins and peptides are required: 10s to100s…”“…Donkeys need to be used to produce the secondary reagents…”“…Ig has an on-going research programme with partners to develop methods for …phage display
antibodies through purification of peripheral blood lymphocytes from hyper-immunised sheep….
(with) the potential to reduce…”“only the whole animal immune response is adequate … to provide cells for in-vitro selection andantibody production”

WHY IS DIRECTIVE 2010/63/EU NOT BEING ENFORCED?
• No EU led expert guidance / recommendation report available• Project license applications not adequately interrogated by LECs – massive genericapplications granted: 5 year duration, 100’s-1000’s animals• The assumption that the technology can not be ‘ready’ if scientists are not using it• Lack of expertise / motivation by users of traditional methods and a propensity toward
familiar methods due to economics /time factor / implementation• Accessibility to commercial Abs limited (but improving) –focus on pharma, high prices,royalties, few catalogue AFAs.
• Scientific misconceptions block uptake of AFAs including that technology still in its infancy,
poor affinity, immunization required etc ….
• Criticisms come from scientists who are expert in animal derived antibody production, NOT
phage display

WHY SHOULD WE BE CONCERNED ABOUT ANIMALBASED ANTIBODY PRODUCTION?

We continue to use immunized animals where…

…..Lack of enforcement of Directive 2010/63/EU on theprotection of animals used for scientific purposes

ANIMAL FRIENDLY AFFINITY REAGENTS (AFAS)
Current Status• AFAs do not necessitate the use of animals at any stage of production.
• Many AFAs are mature and available to replace ALL animal use:
• As a method to adopt in the lab• As a commercialised reagent from a supplier (catalogue or custom)• AFAs are structurally equivalent, wide range of applications, equal or greater specificity andaffinity to a huge repertoire of antigens and offer greater control over their properties,
generation time and cost
• Even the production method is similar: from combinatorial diversification to selection• This justifies the replacement of animal immunization methods in accordance withDirective 2010/63/EU.

MissionAFABILITY is striving to replace animal derived antibody (ADA) production methods withAnimal Friendly Affinity-reagents (AFAs) that do not require the use of animals, thereby
significantly reducing the numbers and suffering of animals in the biomedical sciences.
AFABILITY challenges the enforcement of Directive 2010/63/EU, improves accessibility ofreplacement methods and creates awareness of the use of animal derived antibody
production methods by all scientific disciplines

ACTIVITY HIGHLIGHTS• AFABILITY (2013) publicly launched in July2016• Website www.afability.com, facebook pageand LinkedIn discussion forum• Papers released July 2016 : open access• Winner of Beagle Freedom Science prize2016• Winner of Lush public awareness prize 2017
• Ongoing program of antibody (AFA)
development at University Nottingham• EU/ECVAM invited expert and advisor,
member of ECVAM stakeholder forum(ESTAF)• Collaborations with FRAME and others

RECOMMENDATIONS• Antibody production methods that use animal immunization should be replaced in EU
Member States• Manufacturers outside the European Union should be required to adhere to Europeanstandards to qualify for import to Member States• An expert working group should be established to set up a roadmap for replacement.
• Programs should be implemented to ensure that animal-friendly antibody producers are
fully supported• EU Statistics on the number of animals used for experimental and other scientificpurposes should include data on the use of animals for antibody production as anindependent category• These actions should be reinforced through international cooperation and national
agencies that can execute government regulation and prevent outsourcing to regions
where animal welfare is less well regulated.

FOCUS AREAS• Commitment by all relevant EU authorities (medicines, IVD, food and environment,microbiology, agriculture, pharma, cosmetics, chemicals…), including regulation ofimported commercial products• Prior to a project authorization for animal derived antibody production, clear
guidelines must be applied : Project license applicants should demonstrate steps taken to produce AFAs
 Independent centres of excellence should be part of the application process Where animal derived Ab production should be last resort and demonstrated
on case by case basis• Working toward cosmetics style ban on EU production and importPROGRESS…

GREAT NEWS FOR THE TECHNOLOGY THAT HAS
REVOLUTIONIZED ANTIBODY PRODUCTION WITHOUT THE
NEED FOR ANIMALS!

Thankyou!

With thanks and gratitude to the supporters of the AFABILITY mission including the Lush prize team, Carl Borrebaeck, Sachdev Sidhu,Coenraad Hendriksen and Charu Chandrasekera, to my colleagues Kevin Gough, Ben Maddison and Jon Owen at the School of Veterinary
Sciences, University of Nottingham, the staff and the trustees at FRAME, the Beagle freedom prize and LFVALTF