Introduction How does this affect assay development?

A cost-effective solution to unfit monoclonal antibodies in assay development Jan Voskuil, CSO at Everest Biotech Ltd, Cherwell Innovation Centre, 77 Heyford Park, Upper Heyford, Bicester, OX25 5HD, United Kingdom

The many different types of polyclonal antibodies Entire protein as antigen

Summary One of the many reasons why biomarkers do not make it to the clinic is related to poorly characterized or

Specific region as antigen

poorly reported antibodies. In addition, assay developers get regularly stuck with unfit monoclonal antibodies without having access to budgets to generate new ones. However, there is a cost-effective alternative. Peptide-generated polyclonal antibodies also benefit of mono-specificity and batch-to-batch consistency, but they are highly cost-effective to generate and the epitope is determined beforehand thus preventing

Peptide as antigen Peptide-generated antibodies approximate the mono-specificity of monoclonal antibodies

cross-reactivity to other closely related proteins.

Introduction Currently, an estimated 85% of research resources are wasted1

The advantages of peptide polyclonal antibodies

Faked peer reviews prompt 64 retractions2

•Cheap and quick in manufacturing

Sluggish data sharing hampers reproducibility effort3 Irreproducible biology research costs put at $28 billion per year4 Reproducibility crisis: Blame it on the antibodies5 Reproducibility: Standardize antibodies used in research6 Integrity of protein biomarkers may depend on well-reported antibodies7

•Sequence of epitope designed upfront •Avoidance of cross-reactivity at design •Limited batch-to-batch variations due to peptide size •Scaled up production feasible by using multiple animals

What is the problem?

•Large animals enable long-lasting batches •Potential to immortilize well-established reagents (recMabs)

Most biomarkers do not make 8 it to the clinic. Why ? • • • • • • • • •

How does Everest Biotech contribute to the

Preparations of biospecimen Choosing the right assays Choosing the right reagents Using the right criteria for positive/negative signals Using the right statistics Comparing with external data Coming to the right conclusions Verifying bias/unbias enforced by commercial setting Putting the results and conclusions in public domain

progress in biomedical science? •Catalogue of thousands self-manufactured goat peptide antibodies •Antibodies available in bulk to assay developers •Decades worth of relevant knowledge and experience •Offering of custom antibody service •Offering to markets of LDT and CDx

How does this affect assay development? • Unreliable science makes poor biomarkers • Waste of investment on resources with the wrong biomarker • Danger of using unfit antibodies in immunoassays • Unnecessary high false positives due to unexpected crossreactivity

•Quality guarantees in place

Example of bad diagnostic monoclonal antibodies Cross reactivity of clinically validated anti-HER2 antibodies10 HER2 antibodies

• Lack of faith in commercial research antibodies • Unnecessary project failure due to not finding the right reagents

HER1

HER2

HER3

HER4

Herceptest™ (Dako)

v

PATHWAY® (Vantana)

v

v

Oracle™ (Leica)

v

v

Conclusion

Are there solutions ? 9

• Use only antibodies with known epitope (see example below) • Buy antibodies in bulk preferably straight from the original manufacturer in order to avoid variations between purchases • Make sure new antibody batches are validated as if an entire new antibody • Specify each reagent by its catalogue number and by its formulation

Avoid antibodies with unknown epitopes Literature 1. Macleod MR et al, Biomedical research: increasing value, reducing waste. Lancet. 2014 Jan 11;383(9912):101-4. 2. Callaway E, Faked peer reviews prompts 64 retractions. Nature. 2015 Aug 18; doi:10.1038/nature.2015.18202 3. Van Noorden R, Sluggish data sharing hampers reproducibility effort. Nature 2015 Jun 03; doi:10.1038/nature.2015.17694 4. Baker M, Irreproducible biology research costs put at $28 billion per year. Nature 2015 Jun 09; doi:10.1038/nature.2015.17711 5. Baker M, Reproducibility crisis: Blame it on the antibodies. Nature 2015 May 19; 521(7552):274-6 6. Bradbury A and Plückthun A, Reproducibility: Standardize antibodies used in research. Nature. 2015 Feb 5;518(7537):27-9. 7. Voskuil J. Biomarkers and their dependence on well-reported antibodies. Per. Med. 2015 Nov; doi:10.2217/pme.15.24 8. Voskuil J. How difficult is the validation of clinical biomarkers? F1000Res. 2015 Apr 28;4:101. 9. Voskuil J. Commercial antibodies and their validation. Version 2. F1000Res. 2014 Oct 2 [revised 2014 Oct 15];3:232. 10. Schrohl A et al, Human epidermal growth factor receptor 2 (HER2) immunoreactivity: specificity of three pharmacodiagnostic antibodies. Histopathology. 2011 Nov;59(5):975-83.