From the publishers of - Donutsdocshare01.docshare.tips/files/29550/295502902.pdfthe findings of the latest Review on Antimicrobial Resistance report published by O’Neill and colleagues:

www.drugtargetreview.com Winter 2015

From the publishers of

IN THIS ISSUE:

Mass SpectrometryFocusWith articles from Michelle M. Hillfrom the University of Queensland,and Nicola Burgess-Brown andcolleagues from the StructuralGenomics Consortium

Target ValidationScientists from MRC Technology’sCentre for Therapeutics Discoverydiscuss the challenges in identifyingand working with early-stagetherapeutic targets

Protein ExpressionQiang Chen and Huafang Lai fromArizona State University explainhow plants like tobacco are ideal forproducing monoclonal antibodies

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Last year, the UK government bestowed on renownedeconomist Jim O’Neill the task of finding solutions to theincreasing global threat of antibiotic resistance; so he tolddelegates at the annual FT Global Pharmaceutical and Biotechnology conference that I attended recently.His keynote talk sparked a lively discussion amongst apanel of healthcare stakeholders, all of whom agreed withthe findings of the latest Review on AntimicrobialResistance report published by O’Neill and colleagues: weneed to both encourage the uptake of diagnostics andimprove innovation in antimicrobials drugs.

But will efforts be rewarded in time? The Reviewforecasts an extra 10 million people across the world eachyear dying by 2050 if drug-resistant infections fail to betackled. In China, the world’s largest pork consumer, astrain of bacteria that can survive the antibiotic colistin –used as a last resort in humans but fed routinely to pigs – has just been found both in pigs and humans.Worryingly, the resistance gene, MCR-1, can readily spreadbetween different bacteria. We really do seem to belooking at a future in which the elderly and infirm start todie, as their great-grandparents did, from conditions thatcan currently be treated.

Many blame the pharmaceutical industry for notdoing enough to invest in new anti-infectives. Thissituation was surely highlighted by the recent West AfricanEbola crisis that claimed over 10,000 lives, during whichtime, no matter how well-meaning governments were,there were simply no late-stage pipeline candidates todraw upon. Industry has long argued that paymentsystems need to be overhauled in order to stimulate thisvital research, and stronger commercial incentivesoffered. Perhaps the ‘global innovation fund’ O’Neillproposes setting up to support early-stage research willhelp to address this.

Despite these antimicrobial challenges that lie ahead, exciting developments in the drug disc-overy world continue, as our Winter issue of Drug Target Review demonstrates. You can subscribe to Drug Target Review, free, on our website(www.drugtargetreview.com/subscribe) and feel free tocontact me at [email protected] or on+44(0)1959 563331. We are also on LinkedIn and Twitter – details are opposite.

No responsibility can be accepted by Russell Publishing Limited, the editor, staff or any contributors for action taken as a result of the information and other materials contained inour publications. Readers should take specific advice when dealing with specific situations. In addition, the views expressed in our publi cations by any contributor are not necessarilythose of the editor, staff or Russell Publishing Ltd. As such, our publications are not intended to amount to advice on which reliance should be placed. We therefore disclaim all liabilityand responsibility arising from any reliance placed on such materials by any reader, or by anyone who may be informed of any of its contents. Published November 2015

Drug Target Review is published quarterly (four issues per annum) and circulated on a free-of-charge subscription membership. Drug Target Review is avialable for drug discovery andpharmaceutical industry professionals and you cansubscribe now by visiting www.drugtargetreview.com.Please send subscription enquiries [email protected] or telephone +44 (0)1959 563311.

Drug Target Review is published by Russell Publishing Ltd, Court Lodge,

Hogtrough Hill, Brasted, Kent, TN16 1NU, UK. Telephone: +44 (0)1959 563311 Fax: +44 (0)1959 563123

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ISSN 2059-1349Copyright rests with the publishers.

All rights reserved.©2015 Russell Publishing LtdABC application approved – April 2014

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registered as a Limited Companyin England, Number 2709148VAT Number GB 577 8978 47Follow us on Twitter:

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INTRODUCTION

Russell Publishing Ltd

Editorial Board

James AdjayeChair of Stem Cell Researchand Regenerative Medicine,Heinrich Heine University

Michelle ArkinAssociate Professor,Pharmaceutical Chemistry,Associate Director, Biology,Small Molecule DiscoveryCenter, University of California

Mohamed BoudjelalHead of Medical ResearchCore Facility and Platforms,King Abdullah InternationalMedical Research Center

Justin BryansDirector of Drug Discovery, Centre forTherapeutics Discovery MRC Technology

Hakim DjaballhDirector, HTS Core Facility,Memorial Sloan-KetteringCancer Center

Horst FlotowGroup Leader, HTS, Head,Singapore Screening Centre,Experimental TherapeuticsCentre, A*STAR

Sheraz GulVice President and Head of Biology, Fraunhofer-IME SP

Kamyar HadianHead of Assay Developmentand Screening Platform,Helmholtz Zentrum München

Steven A. HaneySenior Research Advisor andGroup Leader, Oncology DrugDiscovery, Eli Lilly

Namir J. HassanDirector of TranslationalResearch, Immunocore Ltd

Rudi MarquezIan Sword Reader of Organicand Bioorganic Chemistry,EPSRC Leadership Fellow,School of Chemistry,University of Glasgow

Andrew PopeDirector, Platform Technology& Science, GlaxoSmithKline

Ulrich SchopferDirector, Integrated LeadDiscovery Head, Novartis

Anton SimeonovActing Branch Chief atNational Institutes of Healthand Group Leader, NationalInstitutes of Health

David E. ThurstonProfessor of Drug Discovery,Kings College London

Giovanna ZinzallaAssistant Professor inChemical Biology, Centre forAdvanced Cancer Therapies(ACT), Microbiology, Tumourand Cell Biology (MTC) Dept.,Karolinska Institutet

Back to thedark ages?

Caroline RichardsEditor

[email protected]

VOLUME 2 ISSUE 4 2015 Drug Target Review 1Front Cover Artwork: © Promotive / Shutterstock.com

SUBSCRIBEON-LINE: www.drugtargetreview.com

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1 | INTRODUCTIONBack to the dark ages?Caroline Richards, Editor, Drug Target Review

5 | FOREWORDAvoid falling off a ladder and into the dockJacob Gifford Head, Thomas More Chambers

6 | NEWS

9 | EVENTS

10 | BIOMARKERSBiomarkers as molecular footprints for cancer therapy: current perspectivesand promisesMahfoozur Rahman and Vikas Kumar, Sam Higginbottom Institute of Agriculture, Technology & Sciences, Sarwar Beg, Panjab University, and Firoz Anwar, King Abdulaziz University

24 | TARGET VALIDATIONThe challenges associated with ‘de-risking’ early-stage therapeutic targetsAhmad Kamal, Debbie Taylor, Tim Chapman and CatherineKettleborough, MRC Technology’s Centre for Therapeutics Discovery

29 | DRUG DEVELOPMENTEnabling new targets by integrated lead discoveryUlrich Schopfer and Sylvain Cottens, Novartis

33 | PERSONALISED MEDICINES3D printing: A new era for personalised medicines?Dr Simon Gaisford, University College London School of Pharmacy

36 | NEXT-GENERATION SEQUENCINGNext-generation transcriptomic analysis in cancer vascular researchJoseph W. Wragg and Roy Bicknell, University of Birmingham

40 | WEBINAR REVIEWGenomic support for target selection and validation in drug development: design of a new genotyping arraySponsored by Illumina

41 | PROTEIN EXPRESSIONThe growing potential of plant-made monoclonal antibodiesQiang “Shawn” Chen and Huafang Lai, Arizona State University

46 | COMPANION DIAGNOSTICSChallenges in the development ofpharmacist-based point-of-care testsAllison M. Dering-Anderson and Meagan Doyle, University of Nebraska College of Pharmacy

VOLUME 2 ISSUE 4 2015 Drug Target Review 3

IN-DEPTH FOCUS:

MASSSPECTROMETRY15

With articles from Michelle M. Hill, University of Queensland, and Nicola Burgess-Brown, Rod Chalk, Claire Strain-Damerell and Pravin Mahajan, Structural Genomics Consortium.Turn to page 23 for a mass spectrometry Q&A withThermo Fisher Scientific’s Eric E. Niederkofler

Volume 2, Issue 4, 2015

■ Drug Targets In-Depth Focus■ Assays In-Depth Focus

■ Antibodies ■ Oncology

Published March 2016. Don’t miss out on your copy – subscribe for free today by visiting www.drugtargetreview.com

COMING UP IN THE NEXT ISSUE:

DutiesIn many jurisdictions, as with other regulatory offences, the legislationcovering Health and Safety prosecutions is not new, but there is agrowing appetite amongst regulatory authorities to seek criminalsanctions for non-compliance. For example, in England and Wales, thelegislation governing health and safety law dates back to 19741 butprovides for a wide-ranging set of duties at section 2. These includeduties towards, as far as is reasonably practicable:(a) the provision and maintenance of plant and systems of work that

are safe and without risks to health;(b) arrangements for ensuring safety and absence of risks to health in

connection with the use, handling, storage and transport of articlesand substances;

(c) the provision of such information, instruction, training andsupervision as is necessary to ensure the health and safety at workof his employees;

(d) as regards any place of work under the employer’s control, themaintenance of it in a condition that is safe and without risks tohealth (including the ability to safely enter and exit it); and

(e) the provision and maintenance of a working environment for hisemployees that is without risks to health, and adequate as regardsfacilities and arrangements for their welfare at work.

Less obvious risksAlthough it is likely that most pharmaceutical companies and otherorganisations engaged in drug development work have a goodunderstanding of the risks associated with working with potentiallytoxic materials (which may, indeed, be covered by separate regulation) or dangerous laboratory equipment, often the dangers maybe more prosaic. For example, I have been involved in a prosecutionarising out of the cleaning of a company’s roof – something entirelyunrelated to the company’s business – whilst the Health and Safety Executive’s guidance on the pharmaceutical industry is mostly concerned with laboratory ergonomics, such as the height of lab stools2. It is therefore clear that a wide view should be taken to all risks, including ones incidental to the ‘core business’ and mostobvious risks.

Prosecutions and director’s personal liabilityIn addition to civil enforcement action (such as the service of notices

requiring a company to stop certain work or improve it), for companydirectors there is a further danger in the act, under section 37, whichmakes breach of any of these duties a criminal offence. Liability extendsto any company director or manager with ultimate authority whoconsents to or connives (i.e., turns an eye away) from the breach, or isnegligent in his or her duties. Punishments can include an unlimitedfine and up to two years’ imprisonment (in addition to any fine imposedon the company, itself).

Smaller drug development companies may be particularly at risksince they will have fewer resources to be able to devote to the subject.Furthermore, anecdotal experience suggests that prosecutingauthorities seem more willing to prosecute directors of smallercompanies personally, perhaps since it is easier to show knowledge ofthe problems when there are fewer management layers.

Avoid prosecutionsThere are various ways for a company to protect itself againstprosecution. The most important is to engage, positively, with Healthand Safety so that in the event of an investigation following an accident,it can produce a substantial body of material demonstrating that it hasconsidered the risks, assessed them, and attempted to mitigate them.Openness with regulators, in many jurisdictions, can also help.

InsuranceFurthermore, whilst all drug development companies should haveinsurance in place to cover themselves against civil actions caused bytheir work, it is worth considering the scope of this coverage in respectof prosecutions. Whilst insurance is unlikely to be available to cover anyfinancial penalty or fine, it may cover the costs of criminal proceedingswhich can be lengthy and expensive. For directors, it may be worthconsidering if the company’s insurance will also cover individual liabilityand, if not, whether separate Director’s and Officer’s Insurance shouldbe taken out for protection.

www.giffordhead.co.ukwww.thomasmore.co.uk

FOREWORD


Jacob Gifford HeadThomas More Chambers

1. Health and Safety at Work etc Act 1974

2. http://www.hse.gov.uk/pharmaceuticals/index.htm

References

Avoid falling off a ladderand into the dockHealth and Safety Law rarely arouses interest, either amongst companies or lawyers, yet its importance is growingand it has become the area in which companies and their directors are likely to find themselves in the dock, facinga criminal prosecution.

NEWS

6 Drug Target Review VOLUME 2 ISSUE 4 2015 Get daily news updates at www.drugtargetreview.com @DrugTargetReview

UNIVERSITY OF ADELAIDE

Researchers identify protein that drives multiple sclerosisUniversity of Adelaide scientists have identified that the chemokine receptor CCR2 is involved ina ‘super-inflammatory’ immune response that drives the progression of multiple sclerosis (MS)and other autoimmune diseases, and not the CCR6 receptor as previously assumed.

CCR2 is involved in moving T-cells around the body when they are in the super-inflammatorymode and needed to fight persistent infections or conversely, as in the case of autoimmune diseaseslike MS, attacking the body’s own tissues.

“Blocking CCR6 makes the disease worse. If we can find an antagonist to block the CCR2receptor specifically on these T-cells, we should be able to control the progression of MS,” saidproject leader Professor Shaun McColl, Director of the Centre for Molecular Pathology at theUniversity of Adelaide. Another potential benefit of the research is making improved vaccines tofight infection.

UNIVERSITY OF BONN

Scientists decodesignal cascadeassociated withepileptic seizuresUniversity of Bonn and the Hebrew Uni -versity of Jerusalem researchers have foundthat blocking a central switch known asmetal-regulatory transcription factor 1(MTF1) in epileptic mice decreases thefrequency and severity of seizures.

The concentration of zinc ions in thehippocampus increases following transientsevere brain injury and prior to a spontaneousepileptic seizure and the team found thatthese ions dock in greater numbers ontoMTF1. This leads to a large increase in theamount of a special calcium ion channel inthe nerve cells and overall, this significantlyboosts the risk of epileptic seizures. Thescientists demonstrated the fact that the transcription factor MTF1 plays a centralrole in this connection in an experiment onmice suffering from epilepsy. When MTF1was inhibited in these mice, seizures becamerarer and weaker.

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Gene could hold key to treatingParkinson’s diseaseResearchers have identified a new genelinked to nerve function, which could providea treatment target for ‘switching off’ the genein people with neurodegenerative diseasessuch as Parkinson’s disease.

Previous research suggests that defectsin mitochondria play an important role in anumber of diseases that affect the nervoussystem, including Parkinson’s. However,until now the neuronal processes under-lying the development of these conditionswere unknown.

The study, conducted by researchers atKing’s College London, discovered thatdamaged mitochondria in fruit flies producea signal which stops nerve cells fromworking. A gene called HIFalpha was found to regulate the nerve signals fromdamaged mitochondria and, when this genewas ‘switched off’ by the research team,nerve function in flies with Parkinson’sdisease was restored. By deactivating the HIFalpha gene, the early failure of nervecells caused by mitochondrial damage was prevented.

Since the HIFalpha gene is also found inhumans, this new finding could pave the wayfor new treatments in the future.

BILL AND MELINDA GATES FOUNDATION

£1 billion fund will focus on malaria and other infectious diseasesA new £1 billion fund has been set up to support the global fight against malaria and other infectiousdiseases. The Ross Fund, named after the Nobel Laureate who discovered that mosquitoes transmitmalaria, Sir Ronald Ross, has been set up in a partnership between the UK government and the Billand Melinda Gates foundation.

The £1 billion will include a £300 million package focused on malaria and other infectiousdiseases. This will include £115 million to develop new drugs, diagnostics and insecticides for malaria,tuberculosis and other infectious disease resistance. Research and development into products forinfectious diseases will receive £100 million.

Commenting on the announcement, Chancellor George Osborne said: “Eradicating malaria wouldsave 11 million lives so today’s announcement of the £1 billion Ross Fund is an important step to helptackle this global disease.”

Get daily news updates at www.drugtargetreview.com @DrugTargetReview VOLUME 2 ISSUE 4 2015 Drug Target Review 7

NEWS

BOEHRINGER INGELHEIM

Boehringer Ingelheim to plough€11 billion into R&DBoehringer Ingelheim has pledged to invest a total of €11 billion in a new research anddevelopment programme spanning five years. Of the total investment, €5 billion will go towardspreclinical R&D with €1.5 billion thereofplanned for collaborations with external partners.

The firm recently announced newcollaborations with four major scientificpartners to enrich R&D with novel therapeuticapproaches for patients suffering frominflammatory bowel disease. It has alsorecently signed exclusive agreements with Hanmi Pharmaceuticals in Korea todevelop a third generation EGFR-targeted

therapy for lung cancer and with CircuitTherapeutics, California, to apply thetechnique of optogenetics to find newtreatments for psychiatric disorders andcardiometabolic diseases.

Other ways the company is engaging inearly-stage research is through public-privatepartnerships, such as with the StructuralGenomics Consortium (SGC), InnovativeMedicines Initiative (IMI) and the G-proteincoupled receptor (GPCR) Consortium, and viacrowdsourcing initiatives with organisationssuch as InnoCentive and the BioMed XInnovation Centre.

CANCER RESEARCH UK

Chemical probehighlights potential cancer-causing proteinsScientists have created a highly specific andwell-characterised chemical probe which canswitch off two important protein kinases –CDK8 and CDK19 – implicated in cancer,shedding new light on the role these proteinsplay in driving cancer cell proliferation.

This probe (CCT251545) will allow moreprecise analysis than ever before of thebiological the roles of CDK8 and CDK19 incancer and other cells. It was discovered byscreening a large collection of chemicals againstthe WNT signalling pathway in cancer cells. Inthis new study, the team describe the discoverythat CDK8 and CDK19 are the biochemicaltargets of CCT251545. The researchers used arange of biological and biophysical techniques toshow that CCT251545 potently and selectivelybinds to CDK8 and CDK19. They go on toexplain how the probe binds to CDK8 andCDK19 and how this in turn blocks the WNTsignalling pathway, a known driver of manybowel cancers.

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UNIVERSITY OF WASHINGTON

Stem cell triumph with mini kidneysMini-kidney organoids containing all cell types found in the human kidney have been grown frompluripotent stem cells in petri dishes. The work paves the way for personalised drug discovery forkidney disease.

When treated with a chemical cocktail, the stem cells matured into structures that resembleminiature kidneys, containing tubules, filtering cells and blood vessel cells. They were able totransport chemicals and respond to toxic injury in ways that are similar to kidney tubules in people.

To re-create human disease, Benjamin Freedman and colleagues at Brigham and Women’sHospital in Boston used the CRISPR gene-editing technique. They engineered mini-kidneys withgenetic changes linked to two common kidney diseases, polycystic kidney disease andglomerulonephritis. The organoids developed characteristics of these diseases. Those withmutations in polycystic kidney disease genes formed balloon-like, fluid filled sacks, called cysts,from kidney tubules. The organoids with mutations in podocalyxin, a gene linked toglomerulonephritis, lost connections between filtering cells.

The organoids will be used in disease modelling and cell therapy and are a step towardsconstructing human organs in a lab.

UNIVERSITY COLLEGE LONDON

UCL launches genetherapy company University College London (UCL) hasannounced the formation of Athena Vision, abiopharmaceutical company focused ondeveloping gene therapies for eye diseases.

Athena Vision has entered into a globalpartnership with MeiraGTx to develop and commercialise Athena’s ocular genetherapy programmes.

The partnership will pursue four initialclinical programmes in inherited retinalconditions. These include Leber congenitalamaurosis type 2 (LCA2) caused bydeficiencies in RPE65, achromatopsiacaused by mutations in CNGB3 or CNGA3and X-linked retinitis pigmentosa caused bymutations in RPGR. A phase I/II dose-escalation clinical trial in LCA2 is expectedto start in the first quarter of 2016. Thedevelopment costs for all four of theseprogrammes are supported by an undisclosedupfront payment by MeiraGTx.

The companies stated that they will haveunparalleled access to resources due to their affiliation with the UCL Institute ofOphthalmology and its partner MoorfieldsEye Hospital, which together form one of the world’s largest vision research centres, with access to a large and diversepatient population.

UNIVERSITY OF MICHIGAN MEDICAL SCHOOL

BanLec: the banana-derived antiviral agentScientists have created a new form of the bananalectin protein, BanLec, which can fight virusesthat cause AIDS, hepatitis C and influenza intests in tissue, blood samples and mice, but doesnot cause irritation or unwanted inflammation.

BanLec can read the sugars on the outside ofboth viruses and cells. Five years ago, scientistsshowed it could keep the virus that causes AIDSfrom getting into cells – but it also caused sideeffects that limited its potential use. The team hasnow succeeded in peeling apart these twofunctions by pinpointing the part of the moleculethat triggered side effects. By altering a gene,

they engineered a new version of BanLec, calledH84T. The researchers also showed that H84TBanLec protected mice from getting infected by ‘flu virus.

The investigators hope that BanLec could bedeveloped into a broad spectrum antiviral agent.In addition, the new version of BanLec has oneless ‘Greek key’ site on its surface for sugars to attach. This makes it impossible for T cells toattach in multiple spots at once and triggerinflammation. But it still allows BanLec to grabon to sugars on the surface of viruses and keepthem from getting into cells.

SANOFI PASTEUR

Sanofi Pasteur unveils universal ‘flu vaccine researchSanofi Pasteur revealed that it will shift itsresearch onto developing broader-spectrumcross-reactive antigens against seasonal and pandemic influenza, at the recent WorldVaccine Congress.

The company has an existing R&Dcollaboration agreement with the University ofGeorgia on a method it believes could yield a novel, synthetic vaccine based on thehaemagglutinin protein, designed to protect

against seasonal influenza strains spanningseveral years, including drifted strains not yet in existence. Traditional influenza vaccinemanufacturers are directed by, and provided with,candidate vaccine viruses from public-healthauthorities, determined through active surveill -ance of influenza viruses circulating each year.

Sanofi’s experimental vaccine is a novelsynthetic vaccine generated from key geneticsequences of many flu viruses, and is termed

“computationally optimised broadly reactiveantigen” or COBRA, and is designed to protectagainst many strains over several years, due tothe common sequences many flu viruses share.The key advantage is broader coverage againstseveral seasonal flu strains, which is importantwhen there is a mismatch to the vaccine strain.An additional advantage of this approach is notrelying upon annual strain selection, allowingyear-round manufacturing.

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8 Drug Target Review VOLUME 2 ISSUE 4 2015 Get daily news updates at www.drugtargetreview.com @DrugTargetReview

NEWS

UNIVERSITY OF COPENHAGEN

Malaria vaccineprovides hope for a general cure for cancerScientists from the University of Copenhagenand the University of British Columbia havefound that the carbohydrate that the malariaparasite attaches itself to in the placenta inpregnant women is identical to a carbo -hydrate found in cancer cells.

In the laboratory, scientists have createdthe protein that the malaria parasite uses toadhere to the placenta and added a toxin. Thiscombination of malaria protein and toxinseeks out the cancer cells, is absorbed, thetoxin released inside, and then the cancer cellsdie. This process has been witnessed in cellcultures and in mice with cancer.

“For decades, scientists have beensearching for similarities between the growth of a placenta and a tumour,” said Ali Salanti from the Department of Imm -unology and Microbiology at the Universityof Copenhagen.

The two university research groups havetested thousands of samples and it seems themalaria protein is able attack more than 90%of all types of tumours. The drug has beentested on mice that were implanted with three types of human tumours. With non-Hodgkin’s lymphoma, the treated mice’stumours were about a quarter of the size of the tumours in the control group. Withprostate cancer, the tumours disappeared intwo of the six treated mice a month afterreceiving the first dose. And in metastaticbone cancer, five out of six of the treated mice were alive after almost eight weeks,compared to none of the mice in a controlgroup. The scientists hope the results willprovide the basis for a drug against cancer.

In collaboration with the scientistsbehind the discovery, the University ofCopenhagen has created the biotechcompany, VAR2pharmaceuticals, which willdrive the clinical development forward.

Get daily news updates at www.drugtargetreview.com @DrugTargetReview VOLUME 2 ISSUE 4 2015 Drug Target Review 9

NEWS

EVENTSJANUARY 2016

Festival of GenomicsDate: 19 – 21 January Location: London, UKe: [email protected]: www.festivalofgenomicslondon.com

SLAS2016Date: 23 – 27 January Location: San Diego, CA, USAw: www.slas.org

FEBRUARY 2016

Targeted Drug DeliveryDate: 11 – 12 February Location: Basel, Switzerlande: [email protected]: www.pharma.flemingeurope.com

Cell Culture World CongressDate: 23 – 24 February Location: Munich, Germanye: [email protected]: www.bit.ly/1WRRbmk

11th Annual BiomarkersCongressDate: 25 – 26 FebruaryLocation: Manchester, UKe: [email protected]: www.biomarkers-congress.com

MARCH 2016Molecular Med Tri-ConferenceDate: 6 – 11 MarchLocation: San Francisco, CA, USAe: [email protected]: www.triconference.com

APRIL 20164th Annual Biosimilars &Biobetters CongressDate: 18 – 19 AprilLocation: London, UKe: [email protected]: www.biosimilars-congress.com/download-agenda-marketing

MAY 20162nd Annual Formulation andDrug Delivery Congress 2016Date: 18 – 19 MayLocation: London, UKe: [email protected]: www.formulation-congress.com

GeneOne Life Science has announced thefiling of an Investigational New Drug Applica-tion with the United States Food and DrugAdministration for its collaborative vaccinefor Middle East Respiratory Syndrome(MERS), GLS-5300.

GeneOne is partnered with Inovio Pharma -ceuticals on the development of the vaccineand the firms expect to move the MERSvaccine into a phase I clinical trial in healthyvolunteers before the end of the year. Earlierthis year, Inovio’s MERS vaccine induced100% protection from a live virus challenge ina preclinical study. Inovio and its collaboratorsevaluated its MERS vaccine in mice, camelsand monkeys, and non-human primates. As pub lished in Science TranslationalMedicine, the vaccine induced robust immuneresponses capable of preventing the virus from

infecting cells in all three species. In monkeys,all vaccinated animals in the study wereprotected from symptoms of MERS whenchallenged with a live MERS virus.

MERS is caused by a coronavirus that isrelated to severe acute respiratory syndrome.There is no vaccine or effective treatmentagainst the illness, which spreads from humanto human. Since 2012, MERS has infected over1,500 people and killed almost 600 (40%).Recently, the largest outbreak outside of Saudi Arabia of this emergent global healthconcern infected 186 people with 36 fatalities inSouth Korea. Much about the behaviour of theMERS virus remains shrouded in scientificuncertainty, though evidence is mounting thatcamels are likely to be a major reservoir host forMERS-CoV and an animal source of MERSinfection in humans.

UNIVERSITY OF NEW MEXICO

Cholesterol-lowering vaccineshows promise inpreclinical studiesAn experimental cholesterol-loweringvaccine led to reductions in low-densitylipoprotein (LDL) cholesterol in mice andmacaques after just one dose, and could havethe potential to be a more powerful treatmentthan statins alone, scientists at the Universityof New Mexico believe.

The vaccine targets the protein PCSK9,which regulates the cholesterol in the blood.The protein works by encouraging the bodyto break down receptors that cholesterolbinds to when it is flushed out of the body.People who have a mutation in the proteinoften suffer from an increased risk of heartdisease, and people who do not produce theprotein have a decreased risk. By targetingthis protein, the vaccine can stop it fromfunctioning, lowering the amount of chol -esterol in the blood.

Several drug companies have beendeveloping high cholesterol treatments thattarget PCSK9, for example, Praluent(alirocumab, Sanofi and Regeneron) andRepatha (evolocumab, Amgen), which theFDA recently approved. Results have beenpositive, but their treatments, which usemonoclonal antibodies, cost upwards of$10,000 per year. The researchers now plan to expand their studies in macaques and find commercial partners to move thetechnology forward.

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GENEONE LIFE SCIENCES

Inovio and partner advance MERS vaccine

Increased knowledge of cancer biology, combined with rapidadvancements in molecular technology, have led to the recognition ofumpteen numbers of biomarkers in cancer. To that end, a paper ispublished almost on a daily basis in this realm1,2. The availability ofabundant literature makes the task easier for clinicians and scientists tocritically understand the biomarkers, which helps in understanding how they can be targeted and incorporated into routine medicalpractice3,4. The National Cancer Institute defines biomarkers as footprintmolecules found in blood, other body fluids and tissues, which flag up

normal or abnormal process of any disease condition5. Every cell has itsown unique molecular signature, which gives rise to certain activities by the response of genes and proteins5,6. Therefore, evaluation ofbiomarkers is an important approach for understanding the biologicaland pathogenic processes or pharmacological responses fortherapeutic intervention7.

Every year more than 11 million people are diagnosed with cancerand it is estimated that the number will reach up to 16 million by 20207.A complex disease, cancer involves multistep processes involving

Knowledge about cancer biomarkers has increased tremendously and provides clinicians and researchers withvalued opportunities to understand the molecular mechanism of cancer, thus applying it to effective, early-stagediagnosis and treatment. Cancer biomarkers include a vast range of biochemical molecules such as nucleic acids,proteins, sugars, lipids, small metabolites, cytogenetic and cytokinetic parameters, as well as whole tumour cellsinside the body. A comprehensive understanding of each biomarker enables the perfect diagnosis of the disease andmakes it easy to choose from multiple different therapeutics, although it is important to note that technologycannot currently identify all biomarkers that play a key role in cancer. This article provides an account of severalbiomarkers used in the diagnosis, prognosis and treatment of cancer, discussing the uses of some of the markersthat are both already in clinical practice, and in trials.

BIOMARKERS

Biomarkers as molecular footprints forcancer therapy: currentperspectives and promises

Mahfoozur Rahman and Vikas Kumar Sam Higginbottom Institute of Agriculture, Technology & SciencesSarwar Beg Panjab University � Firoz Anwar King Abdulaziz University

10 Drug Target Review VOLUME 2 ISSUE 4 2015

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Enabled by SOMAmer® Technology: SOMAmers® (Slow Off-rate Modified Aptamers) are short DNA sequences thathave been modified with “protein-like” side chains to combine the specificity and affinity of antibodies with the manufacturing consistency of DNA.

conversion of proto-oncogenes tooncogenes, which may lead to thedevelopment of unmatured group of cells(tumours)8. There are three main categoriesof genes involved in the development ofcancer: proto-oncogenes, tumour-suppressor genes and DNA repair genes8.Epigenetic changes such as DNA methylationand changes in pattern of histonemodification can also lead to cancer, andsuch changes may result in further alterationin chromatins, leading to abnormal geneexpression9. Modern technology can be usedto understand the footprints of cells whichbecome cancerous, and their recognition isimportant for early cancer diagnosis,improved patient prognosis and therapyselection9. Using biomarkers, it is easy tolocate the tumour and determine its stageand subtypes for undertaking necessarytherapeutic interventions.

Genetic cell alteration, which includes gene rearrangements, pointmutations and gene amplifications, is profound in cancer cells. Thesemay alter several molecular pathways, regulating cell growth andmetastasis10-11. To date, genetics, genomics, proteomics and many non-invasive imaging techniques allow several biomarkers to be measured11,and scientists now have a broad understanding of disease pathways,corresponding protein targets and pharmacotherapy for variousdisorders beyond cancer. Nowadays, the major challenges in cancer arethe difficulties in establishing the exact relationship between cancerbiomarkers and the clinical pathology, and non-invasively detecting thetumour at an early stage11. Similarly, recognition of small changes in

genomics and proteomics can highlight molecular targets fordeveloping cancer therapeutics.

Diagnostic and prognostics biomarkers are useful for clinicaloncologists, since they aid in identifying the suspected patients who areat a high risk for the disease and in diagnosing them at an early stage12.These biomarkers are available in various forms, such as traditionalbiomarkers, which can be easily assessed with radiological techniquessuch as mammography, and detection of tumour-specific antigens (e.g., prostate-specific antigen; PSA)12. The availability of the completehuman genome and advancement in technologies such as high-throughput DNA sequencing, microarray and mass spectrometry, have,in this regard, made the task tremendously easier8,12. The list of cancerbiomarkers have dramatically expanded to include the sequence andexpression level of DNA, RNA and protein, as well as metabolites12.Cancer biomarkers are depicted in Figure 1.

Examples of of biomarkers in cancerCells as biomarkersIn the advanced stage of cancer, cells start appearing in the bloodstream. Circulating tumour cells (CTCs) are recognised as power-ful biomarkers in oncology and have been predicted in metastatic breast cancer. They provide an early, reliable indication of diseaseprogression and survival for patients on systemic therapy for metastaticbreast cancer. Therefore, elimination of CTCs could be a betterapproach in breast cancer therapy13. This is summarised in Table 1 for different diseases.

Cytogenetic and cyto-kinetic markers Any changes in chromosomes are classical markers of cancer, since theassociation between chromosomal aberration and neoplastictransformation has been well established. For example, aneuploidy hasfrequently been found in malignant tumours. Somatic mutations, whichare associated with malignant transformation, are promisingbiomarkers for cancer risk. Meanwhile, genome-based biomarkers have


BIOMARKERS

Table 1: Biomarkers in cancer

Cancer biomarker Types of tumour Reference

Metabolic biomarkers

Glucose metabolism All types of cancer covered [4, 5]

Cells as biomarkers

Circulating tumour cells Metastatic breast cancer [8-10]

Cancer stem cell Brain tumour, breast cancer [8-10]and prostate cancer

Biomolecules as biomarkers

Prostate specific antigen Prostate cancer [12, 13]

Alpha-fetoprotein Hepatocellular carcinoma [7, 10]

BRCA-1, BRCA-2 Colorectal cancer [11, 12]

Heat shock proteins Cervical, uterine, and [13, 14](Hsp27 and Hsp70) prostate carcinoma

Throglobulin Papillary and thyroid cancer [8, 15]

Cancer antigen 125 Ovarian cancer [11-13]

Human chorionic- Ovarian and testicular cancer [6, 11]gonadotrophin (hCG)

Genetic biomarkers

Philadelphia chromosomes Burkitt lymphoma [9, 11]and Bcl-2 gene

APC gene Adeno-carcinoma and [10-13]squamous cell carcinoma

Figure 1: Pictorial depiction of various cancer biomarkers

been identified in certain epithelial tumours such as mRNA ofcytokeratin-19, EGFR and MU-1, etc10,13. Recently, exon-3 has beenobserved as a novel transcriptome marker, which is primarily involved inprostate cancer cell proliferation and endocrine cancer. Elsewhere,protein encoded by the mini-chromosome maintenance gene hasemerged as useful marker for cell proliferation10,13.

Viral biomarkers Hepatocellular carcinoma (HCC) is one of the most common cancersworldwide and a major cause of death in developing countries.Infection with either hepatitis B virus or hepatitis C virus, both of whichpromote carcinogenesis of HCC, is known to be a major cause of HCC14.Another cancer in which viral biomarkers can be implicated is cervicalcancer. As the second most widespread gynecological cancer in women,persistent infection by human papillomaviruses (HR-HPVs) is known tobe the major cause of the main types of cervical cancer – squamous cellcancer and adenocarcinoma, with other risk factors such as smokingand use of the pill also acting as risk factors. HPVs are also profound inoral, oesophageal and vaginal, vulvar and penile cancer14-15.

Genetic biomarkersCancer is also mediated by alterations in oncogene and tumoursuppressor genes, which regulates cell proliferation and otherhomeostatic functions7. Non-random mutations, translocation, andrearrangement in the regulatory region of chromosomes and genes arealso known to be associated with particular types of malignancy. Forexample, the ‘Philadelphia chromosome’ associated with chronic

myelogenous leukaemia results from translocation betweenchromosomes 9 and 22, leading to cancer-causing gene BCR-ABL7,16,17.Meanwhile, several tumour suppressor genes, such as p53, Rb, DCC,Brush-1, BRCA-1, and BRCA-2, have been implicated in breast tumours7,16,17.

Conclusion Cancer is a complex disease; molecular heterogeneity and adaptiveresistance makes it challengeable, thus its exact pathomechanismneeds be understood for patients to receive effective chemotherapy.Cancer biomarkers have paved the way for the development of novelpharmaceuticals and, therefore, it is vital that clinical, translational andlaboratory-based researchers become aware of the issues related tobiomarker development.

BIOMARKERS


Mr. Mahfoozur Rahman* is a young Research Scientist.Presently, he works as a research fellow at the Department ofPharmaceutical Sciences, Faculty of Health Sciences, SamHigginbottom Institute of Agriculture, Technology &Sciences, in Allahabad, India, under the supervision of Dr. Vikas Kumar. He is intensively involved in thedevelopment of nano-sized drug delivery systems for

the treatment of inflammatory disorders. He has been selected in UGCnetworking for his research work in the liposome research laboratory, UIPS,at Panjab University in Chandigarh, India. He is a member of IPGA andeditorial board member of various journals. He has an international bookchapter in CRC press (Taylor and Francis), Springer and Elsevier andpublished his research findings in various peer-reviewed journals ofinternational repute. *E-mail: [email protected]

Mr Sarwar Beg is a UGC-Doctoral Meritorious ResearchFellow in Science at University Institute of PharmaceuticalSciences, Panjab University, in Chandigarh, India. He completed his Masters in Pharmaceutics from HamdardUniversity in New Delhi. His major areas of researchinterest include DoE/QbD-based development andcharacterisation of controlled-release drug delivery systems,

and bioenhanced drug delivery systems like self-nanoemulsifying systems,lipidic nanoparticles, liposomes, microspheres, nanoparticles, dendrimers,carbon nanotubes and nanocomposites. To date he has authored more than30 publications in various high impact peer-reviewed journals, 10 bookchapters, two books and has one Indian patent to his credit.

Dr. Vikas Kumar*, Assistant Professor (Pharmacognosy)at the, Department of Pharmaceutical Sciences, SamHigginbottom Institute of Agriculture, Technology &Sciences, Allahabad, India, has been working on traditionalmedicine in drug discovery leading to the development oftherapeutic leads from natural resources. His research workis highlighted on screening, evaluation, formulation and

standardisation of herbal drugs with their validation to ensure quality,efficacy and safety. He has made innovative, outstanding and originalcontributions both in research and education in the area of natural products.He has to his credit more than 30 publications in peer-reviewed impactjournals, three books and three book chapters. *E-mail: [email protected]

Prof Firoz Anwar is working as Professor at the Departmentof Biochemistry, Faculty of Science, King AbdulazizUniversity in Jeddah, Saudi Arabia. He has been working onscreening, evaluation, formulation and standardisation ofherbal drugs as well as synthetic drugs. He was the chairmanand Ex Dean of Siddhartha Institute of Pharmacy, Dehradun,India. Dr. Firoz Anwar is a pharmacist and completed his

Master’s and PhD from Jadavpur University. His research career has beenoutstanding, including globally acclaimed contributions to developmentfrom natural resources including Ayurveda, ethnopharmacology, herbal drugtechnology and synthetic drugs. His pioneering work has led to manyimportant national and international projects in the field of pharmacologyand clinical pharmacology. Based on these works, he has to his credit over 100 publications in peer-reviewed journals, several patents, books and chapters.

1. Srinivas PR, Kramer BS, Srivastava S. Trends in biomarker 1. Research for cancerdetection. Lancet Oncol 2001; 2: 698- 704

2. Cho WSC. Contribution of onco-proteomics to cancer 2. Biomarker discovery. MolCancer 2007; 6: 25

3. Hanahan D, Weinberg RA. The hallmarks of cancer.3. Cell 2000; 100: 57-70

4. Bayli SB, Ohm JE. Epigenetic gene silencing in cancer-a. Mechanism for early oncogenicpathway addiction? Nature Rev Cancer 2006; 6: 107-17

5. Ludwig JA, John N. Weinstein biomarkers in cancer staging, prognosis and treatmentselection. Nat Rev Cancer 2005; 5: 845-56

6. Sidransky BD. Emerging molecular markers of cancer. Nat Rev Cancer 2002; 2: 210-9

7. Vogelstein CB, Kinzler KW. Cancer genes and the pathways they control. Nat Med 2004; 10: 789-99

8. Rahman M, Ahmad MZ, Kazmi I, et al.Advancement in multifunctional nanoparticles forthe effective treatment of cancer. Expert Opin Drug Deliv. 2012; 9 (4): 367-81

9. Sawyers CL. The cancer biomarker problem. Nature 2008; 452: 548-52

10. Whitfield ML, George LK, Grant GD, Perou CM. Common markers of proliferation. NatRev Cancer 2006; 6: 99-106

11. Toru H, Masaharu Y, Shinji T, Kazuaki C. Genetic polymorphisms and head and neckcancer risk. Int J Oncol 2008; 32: 945-73

12. Shinjo K, Kondo Y. Targeting Cancer Epigenetics: Linking Basic Biology to ClinicalMedicine. Adv Drug Deliv Rev. 2015

13. Frantzi M, Latosinska A, Merseburger AS, Mischak H. Recent progress in urinaryproteome analysis for prostate cancer diagnosis and management. Expert Rev Mol Diagn.2015; 22:1-16

14. Middleton FK, Patterson MJ, Elstob CJ. Common cancer-associated imbalances in theDNA damage response confer sensitivity to single agent ATR inhibition. Oncotarget.2015; 6(32): 32396-409

15. Rahman M, Akhter S, Ahmad MZ, Ahmad J, Addo RT, Ahmad FJ, Pichon C. Emergingadvances in cancer nanotheranostics with graphene nanocomposites: opportunities andchallenges. Nanomedicine (Lond). 2015; 10 (15): 2405-22

16. Rahman M, Ahmad MZ, Kazmi I, Akhter S, Afzal M, Gupta G, Sinha VR. Emergence ofnanomedicine as cancer targeted magic bullets: recent development and need to addressthe toxicity. Curr Drug Discov Technol. 2012; 9(4): 319-29

17. Ahmad MZ, Akhter S, Jain GK, Rahman M, Pathan SA, Ahmad FJ, Khar RK. Metallicnanoparticles: technology overview & drug delivery applications in oncology. ExpertOpin Drug Deliv. 2010; 7(8): 927-42

References


MassSpectrometry

IN-DEPTH FOCUS

© Sebastian Tomus / Shutterstock.com

16 Mass spectrometric andsystems biology approachesin drug target discovery Michelle M. Hill, University of Queensland and the Translational Research Institute

19 Analysing recombinantproteins by massspectrometryNicola Burgess-Brown, Rod Chalk, Claire Strain-Damerell & Pravin Mahajan, Structural Genomics Consortium

23 Q&AWith Eric E. Niederkofler PhD, R&D Manager at Thermo Fisher Scientific

SPONSORS:

To use an analogy of a motor vehicle; the catalogue of available parts islike the genetic code (genome) which dictates the final product. Not allparts will be used in the manufacturing of a particular vehicle; a race carwill use different parts to a van. The list of actual parts to be used is akinto the mRNA (transcriptome), while the actual parts used are similar tothe proteins (proteome). Once the vehicles are assembled correctly,fuel and waste products are analogous to the metabolites required andproduced by cells (metabolome). With completed sequencing ofgenomes and advancements in bio-analytical technologies, we can nowefficiently measure all of the omics data, however, as with the motorvehicles, what is essential is the blue-print of how all parts fit togetherwith respect to each other (the system). Successful application ofsystems biology and systems pharmacology approaches require multi-disciplinary collaborations between computational biology, engineeringand bio-molecular analysts.

Mass spectrometry is a key enabling technology for the high-throughput measurement of the proteome and metabolome, the mainactive ‘parts’ and ‘outputs’ of the biological system. This article will

review recent successes in the application of mass spectrometry andsystems biology approaches in improving drug target identification and drug performance.

Systems biology approach for drug target discovery Traditional drug target identification workflows and drug developmentpipelines focus on a single target, or class of targets, based on knownbiology. As examples, kinases are common targets for cancer drugssince they are commonly mutated in cancers and neurotransmitterreceptors are targets for neurodegenerative disease and psychiatricdisorders such as depression. While this approach has some successes,it is not applicable to the majority of pathologies which do not have asingle, underlying biological fault (e.g., a gene mutation). Furthermore,complex pathologies such as neurological disorders are not readilyresponsive to single-target therapies1,2. And finally, mono-targetedtherapies often lead to drug resistance, since the biological system re-wires to bypass the blocked drug target.

How can systems biology and mass spectrometry help to find better

Cells, organs and organisms are complex biological systems consisting of inter-related components that co-operatively work to maintain function and respond to change. Malfunction of any component can lead topathologies and disease, leading to system-wide changes and subsequent adaptation. Hence, the ability to studyand predict system behaviour will considerably improve the selection of appropriate drug targets, and enable theprediction of any catastrophic side-effects prior to the actual drug development process.

IN-DEPTH FOCUS: MASS SPECTROMETRY

Michelle M. HillUniversity of Queensland


© style-photography / Shutterstock.com

Mass spectrometricand systems biologyapproaches in drugtarget discovery

drug targets? Firstly, a blue-print of howthe system is wired can be developed.Sequencing of individual genomes is nowfeasible using next-generation sequencingtechnologies. The genome provides acatalogue of all possible systems (celltypes), while the actual transcriptome and proteome determines the finalphenotype (neuron versus liver cell). Massspectrometry is a key technology for thecharacterisation of proteomes and metab -olomes. In addition to high-throughputprotein and metabolite identification andquantitation, mass spectrometry-basedprotein-protein interactome analysisprovides critical information for developinga map of how each protein componentinteracts within specific cell systems3.Furthermore, high resolution tandem massspectrometry enables the high throughput detection and monitoring ofpost-translational protein modifications, which affects protein stability,localisation and function. Innovative proteomics and massspectrometry methods for post-translational modification arecontinually being developed4. Since post-translational modificationsare ‘biological switches’ in the system, the ability to measure andinclude this information is critical in a systems biology approach.

After acquisition of proteomics and metabolomics data using massspectrometry, computational and mathe matical models need to bedeveloped to capture protein-protein interactions, post-translationalmodifications to proteins, and to organise the proteomics data into acorrelated system. The metabolomics datawhich represent substrates and outputs ofdifferent proteins/pathways should be overlaidon the proteomics pathways. This map of thesystem then becomes the blueprint to under -stand the pathways altered in pathologies,which can lead to the identification of keynodes as drug targets. Importantly, in silico analysis can be performedto first understand the system-wide consequences of inhibitingselected drug targets, before any funds are spent on developing actualdrugs and assays.

Recent works on anti-angiogenesis strategies have furtherincorporated multi-cell and multi-compartment models due to theinvolvement of several cell types and the importance of the tissuemicro-environment in angiogenesis5. The systems biology approachrequires up-front investment in characterising the system andestablishing a robust computational model, in the hope of identifyingthe best drug targets from a thorough systems perspective, andreducing failures during later stages of drug development.

Mass spectrometry to evaluate drug effects Traditionally, drug effects are evaluated for their on-target effect,general cytotoxicity, and a limited selection of off-target effects. Forexample, Gleevec® (imatinib) was developed as an inhibitor for thePDGF receptor, and then discovered to be a potent inhibitor of two

additional tyrosine kinases, ABL and c-Kit6. The additional high potency‘off-targets’ for imatinib has led to additional approved uses of thedrug. In most other cases, drugs with undesired off-target effects, whendetected, will be dropped from further development. However, if onlyspecific molecular types are monitored, in this example, tyrosinekinases, off-target effects may not be discovered during the initialdevelopment. The delayed realisation of wanted off-target effectsduring the drug development process can translate to highexpenditures for failed leads.

Systems biology and mass spectrometry can, respectively, help topredict and enable high-throughput analysis of potential off-target

effects. The systems model can be used togenerate in silico predictions of any unwantedsystem-wide effects in inhibiting the drugtarget. Targeted mass spectrometry methods,such as multiple reaction monitoring (MRM),can then be devel oped to measure a spectrumof parameters to ensure specificity and lack of

off-target effects. Bioinformatic predictions of potential additionaltargets due to structural similarity can also be evaluated in silicoand with targeted mass spectrometry assays. In addition, massspectrometry can be used to experimentally evaluate system-wideeffects including off-target. A recent study used quantitativeproteomics and phosphoproteomics to evaluate the effect of MEKinhibitors on three tissues following administration of the drugs tomice7, and identified potential mechanisms of the unwanted side effect of oedema.

Understanding drug resistance Drug resistance often develops against single-target drugs due tovarious escape mechanisms which may be difficult to decipher usingtraditional approaches. High-throughput proteomics and metab -olomics in a systems analysis can be used to identify resistancemechanisms. Several recent studies illustrate this application. Canutoand colleagues utilised mass spectrometry coupled to three differentseparation methods to compare the metabolome of miltefosine-


Mass spectrometry is a key enablingtechnology for the high-throughputmeasurement of the proteome and

metabolome, the main active ‘parts’ and‘outputs’ of the biological system

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resistant with miltefosine-sensitive Leishmania . The study identified anincrease in the levels of amino acids as a possible adaptationmechanism. Analysis of the proteomic and pathways adaptations mayprovide additional clues on miltefosine-resistance. Parker andcolleagues evaluated the mechanism of resistance to BRAF inhibitor inmelanoma using phosphoproteomics and proteomics . BRAF inhibitorssuch as vemurafenib and dabrafenib cause dramatic regression ofmetastatic melanoma, only for relapses to occur after several months .Re-activation of the MAP Kinase pathway was detected using traditionalwestern blotting approaches. This phosphoproteomics study identifiedadditional pathways including IGF signaling, which could be consideredfor combination therapy.

As an example of targeted mass spectrometry evaluation, Rebeccaand colleagues examined adaptive resistance mechanisms to inhibitorsof HSP90 and MEK in melanoma cells, using multiplexed MRM assays toquantify the expression of >80 key signaling proteins . An advantage oftargeted MRM assays over tandem mass spectrometry assays is theincreased sensitivity, which was demonstrated by Rebecca andcolleagues by successfully measuring signalling proteins in needleaspirates from melanoma lesions .

Mass spectrometry in personalised systems medicineThe sensitivity of multiplex targeted mass spectrometry assays enable the translation of personalised medicine to the clinic, throughmeasurement of biomarkers which indicate or predict response totherapies. The goal of personalised medicine is to deliver the rightdrugs to the right patients at the right time. This approach requires theidentification of clinically useful biomarkers which can be easilymeasured. These biomarkers could be proteins within tumour tissues,as in the example of the HSP90 inhibitor response study by Rebecca andcolleagues , or surrogate biomarkers that can be easily sampled inbiofluids such as blood, urine and saliva. Although discoveryproteomics in serum/plasma is highly challenging, due to the presenceof high abundance proteins, recent studies have reported promisingprotein biomarker panels for predicting response to bevacizumab inovarian cancer , and to thalidomide in multiple myeloma .

Metabolite profiles may also be useful surrogate systemsbiomarkers. Shah and colleagues found a panel of plasma metabolitesto be predictive of adverse events following coronary artery bypasssurgery. Kamleh and colleagues found elevated plasma free amino acid

levels to correlate with severity of psoriasis, which was ameliorated byanti-tumour necrosis factor (TNF)α therapy . This data not onlysuggested potential biomarkers for diagnosis, but also for monitoringtherapeutic response in psoriasis.

In summary, mass spectrometry is a key enabling technology forsystems biology studies, allowing the simultaneous measurement of alarge number of analytes. Movement to a systems biology approach indrug development heralds a change in focus, from single molecule orpathway analysis, to evaluation of the entire cellular or organismalsystem. Deployment of this global approach brings the hope of reducingoff-target effects and increasing the success of drug development.



obtained her PhD, BSc (Hon I) and BA fromthe University of Queensland (UQ) in Brisbane, Australia.Following postdoctoral positions in Switzerland, Ireland andAustralia, she established the Cancer Proteomics Group atthe UQ Diamantina Institute (UQDI) in 2009. Her multi-disciplinary research programme aims to translate researchoutcomes to patient benefits. She currently holds an

Australian Research Council Future Fellowship, and is the academic leaderof the Proteomics & Mass Spectrometry Core Facility of the TranslationalResearch Institute (TRI), Brisbane, in which UQDI is housed.

1. Geerts H, Kennis L. Multitarget drug discovery projects in CNS diseases: quantitativesystems pharmacology as a possible path forward. Future medicinal chemistry.2014;6(16):1757-69

2. Kotelnikova E, Bernardo-Faura M, Silberberg G, Kiani NA, Messinis D, Melas IN, et al.Signaling networks in MS: a systems-based approach to developing new pharmacologicaltherapies. Multiple sclerosis. 2015;21(2):138-46

3. Ngounou Wetie AG, Sokolowska I, Woods AG, Roy U, Deinhardt K, Darie CC. Protein-protein interactions: switch from classical methods to proteomics and bioinformatics-based approaches. Cellular and molecular life sciences : CMLS. 2014;71(2):205-28

4. Doll S, Burlingame AL. Mass spectrometry-based detection and assignment of proteinposttranslational modifications. ACS chemical biology. 2015;10(1):63-71

5. Finley SD, Chu LH, Popel AS. Computational systems biology approaches to anti-angiogenic cancer therapeutics. Drug discovery today. 2015;20(2):187-97

6. Buchdunger E, Cioffi CL, Law N, Stover D, Ohno-Jones S, Druker BJ, et al. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit andplatelet-derived growth factor receptors. The Journal of pharmacology and experimentaltherapeutics. 2000;295(1):139-45

7. Paulo JA, McAllister FE, Everley RA, Beausoleil SA, Banks AS, Gygi SP. Effects ofMEK inhibitors GSK1120212 and PD0325901 in vivo using 10-plex quantitativeproteomics and phosphoproteomics. Proteomics. 2015;15(2-3):462-73

8. Canuto GA, Castilho-Martins EA, Tavares MF, Rivas L, Barbas C, Lopez-Gonzalvez A.Multi-analytical platform metabolomic approach to study miltefosine mechanism ofaction and resistance in Leishmania. Analytical and bioanalytical chemistry.2014;406(14):3459-76

9. Parker R, Vella LJ, Xavier D, Amirkhani A, Parker J, Cebon J, et al. PhosphoproteomicAnalysis of Cell-Based Resistance to BRAF Inhibitor Therapy in Melanoma. Frontiers inoncology. 2015;5:95

10. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, et al. Improvedsurvival with vemurafenib in melanoma with BRAF V600E mutation. The New Englandjournal of medicine. 2011;364(26):2507-16

11. Rebecca VW, Wood E, Fedorenko IV, Paraiso KH, Haarberg HE, Chen Y, et al.Evaluating melanoma drug response and therapeutic escape with quantitative proteomics.Molecular & cellular proteomics : MCP. 2014;13(7):1844-54

12. Collinson F, Hutchinson M, Craven RA, Cairns DA, Zougman A, Wind TC, et al.Predicting response to bevacizumab in ovarian cancer: a panel of potential biomarkersinforming treatment selection. Clinical cancer research : an official journal of theAmerican Association for Cancer Research. 2013;19(18):5227-39

13. Rajpal R, Dowling P, Meiller J, Clarke C, Murphy WG, O’Connor R, et al. A novel panelof protein biomarkers for predicting response to thalidomide-based therapy in newlydiagnosed multiple myeloma patients. Proteomics. 2011;11(8):1391-402

14. Shah AA, Craig DM, Sebek JK, Haynes C, Stevens RC, Muehlbauer MJ, et al. Metabolicprofiles predict adverse events after coronary artery bypass grafting. The Journal ofthoracic and cardiovascular surgery. 2012;143(4):873-8

15. Kamleh MA, Snowden SG, Grapov D, Blackburn GJ, Watson DG, Xu N, et al. LC-MSmetabolomics of psoriasis patients reveals disease severity-dependent increases incirculating amino acids that are ameliorated by anti-TNFalpha treatment. Journal ofproteome research. 2015;14(1):557-66

References

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The process of recombinant protein production involves quality checksthroughout; on the expression constructs and also on their products.Verification of expression constructs by sequencing confirms identity atthe DNA level but in addition to standard SDS-PAGE and gel filtrationanalyses, MS adds another dimension to protein analysis. Intact massmeasurement, as well as confirming protein molecular weight, providesa quick indication of post-translational modifications (PTMs) such asphosphorylation, acetylation, glycosylation etc. Moreover, identificationof proteins by tandem mass spectrometry (MS/MS) not only providesassurance on the protein of interest, but is also a valuable tool to

identify host proteins that co-purify with recombinant proteins thatcould be mistaken as target proteins of interest.

Structural genomics laboratories apply MS as part of their proteinproduction pipelines to provide information and guidance throughoutthe process1. Our process at the SGC begins at construct design; toincrease the likelihood of obtaining soluble proteins and well-diffracting crystals, the multi-construct approach has been employed2,3.In order to implement this approach on multiple protein families,development of a high-throughput (HTP) protein expression andpurification pipeline is required. This requirement has necessitated

The drug discovery landscape is changing: no longer limited to big pharma, it is now within reach of academics andsmall consortia alike. Regardless of the setting, drug discovery requirements are always the same: strong biologicaltheory, good chemical starting material and high quality protein samples from which to determine the binding andinhibition of the lead compounds. Production of such high calibre protein is facilitated by a strong pipeline process(Figure 1; page 20), such as that established at the Structural Genomics Consortium (SGC), but ultimately there hasto be a means to assess the quality of the protein before progressing to drug development. Traditionally this was limited to an approximate measure of mass and purity from SDS-PAGE and gel filtration profiles, however,advancements in mass spectrometry (MS) equipment and methodologies have greatly enhanced the breadth ofinformation that is available from both pure and complex protein samples. MS can now be used to ask questionsabout protein folding, composition and modification state, to determine interactions between lead compounds orother proteins, and can even be used to predict the propensity for crystallisation. Here we discuss the application of MS at the SGC to aid production of proteins for structural and functional studies.



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Analysing recombinant proteins by mass spectrometry

Nicola Burgess-Brown, Rod Chalk, Claire Strain-Damerell and Pravin MahajanStructural Genomics Consortium

development of HTP expression methods utilising prokaryotic andeukaryotic systems3-6. Also, where possible, we apply HTP methods toour MS process to enable target protein identification for structural andfunctional studies as described below7.

Target protein identityUse of SDS-PAGE alone as a measure of protein expressionconfirmation, monodispersity and identity based on mobility of proteinbands is insufficient for several reasons: (i) poorly expressed proteins may not stand out clearly against theback ground of host proteins; (ii) target proteinsmay have anomalous gel mobility – for examplemembrane proteins and glycoproteins usuallyrun faster than predicted and stain diffusely;(iii) the target protein may not actually have theexpected sequence length – truncation or degradation are commonresulting in faster gel mobility, while read-through to the next stopcodon results in a larger than predicted protein bearing extraneousvector sequence; (iv) over-expression of host proteins such as heatshock proteins may appear due to the expression system itself and; (v) affinity capture can enrich for host proteins bearing sequencesimilarity to the affinity tag (Figure 2; page 21).

For all the above reasons, MS is rightly regarded as the ‘goldstandard’ for protein identification, and this can be readily applied toHTP test expression8. Tryptic digest MS/MS analysis of SDS-PAGE gel

bands is available in most laboratories and is sensitive to femtomolesof protein. Full sequence coverage is not required and confidentidentification of the target is possible from fragmentation data of asingle peptide. Target bands can be precisely excised using gel-cuttingtips, and the process of reduction-alkylation and tryptic digestion canbe performed on a large number of samples in parallel using 96-wellplates and multichannel pipettes. Fast LC-MS/MS analysis protocolsand automated database searching means that MS/MS identification of all gel bands from a 96-well test expression is possible in under

72 hours7. In a HTP environment and in theabsence of MS, the risk of misidentification of expressed soluble proteins is in the region of10%, whereas for membrane proteins the risk is as high as 50%. Incorporation of tryptic digest MS/MS into a HTP pipeline enables

selection of constructs for scale-up with confidence that the target is indeed present.

Target protein structureWhile tryptic digest MS/MS can confirm target identity, it cannotconfirm covalent structure because, for technical reasons, 100%peptide coverage is never achieved. Truncated and full lengthconstructs may generate fragment spectra from the same subset oftryptic peptides and therefore yield identical Mascot results. PTMs suchas terminal methionine loss and acetylation may be naturally present,

and others may have been artificially introduced, suchas biotinylation. These PTMs will not come to light ifthey are absent from the database being searched,which is normally the case. In general, results fortryptic digest MS/MS of cut gel bands are not availablein less than 24 hours. Techniques exist for rapidtrypsinisation9 and top-down fragmentation10 butthese are not immediately available to the scientistperforming protein purification at the bench. It is wellknown that speed and success go together forpurification of native proteins. Having purified aprotein, the preparation cannot wait days for theresults of MS analysis to become available.

Protein intact mass analysis is an extremely fastmethod for determining covalent structure and is alsosimple enough to be performed by bench scientists



Figure 1: Overview of the protein production pipeline operated at the SGC

MS can now be used to ask questions about protein folding,

composition and modification state

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with minimal MS training on an open access basis. Typically amicrogram of purified protein is diluted in an acidic buffer andundergoes LC-MS analysis using a guard column as a protein trap and areversed-phase elution over less than two minutes7. Data analysisinvolves summing together of mass to charge (m/z) spectra over theretention time of the relevant peak and mathematical transformation ofthe multiple charge states of the electrospray spectrum to a singleneutral peak representing the observed proteinmass. This can be fully automated, leaving the experimenter to compare the observedmass with that expected from the constructsequence and to interpret any mass deviations.In principle, MALDI MS can be used in this way,but in practice the necessary sensitivity and mass accuracy of less than1 Da is only achievable using electrospray. Where observed andexpected masses are within 1 Da, this is sufficient to confirm both theidentity and structure of the target protein. A decreasing series ofsodium adduct peaks is characteristically observed which has nofunctional effect upon the protein.

Large negative mass shifts can often be accounted for by truncationof the target protein. These will match a C-terminal or N-terminalsequence string to within 1 Da and will be inclusive of any MS/MSpeptide data. Most PTMs involve mass additions. Serial additions, asobserved in phosphorylation and glycosylation, are simple to interpret.While there are just under a thousand PTMs in the Unimod database(http://www.unimod.org)11, the vast majority are extremely rare, such asmutation, or involve chemical derivitisation or heavy isotopes.Excepting glycans, there are actually fewer than a dozen modifications

routinely observed in E. coli, insect and mammalian expressionsystems. Parallel mass additions involving more than one modificationcan be more difficult to interpret, but knowledge of PTMs whichcommonly occur together, such as methionine loss and acetylation, stillmakes this possible.

Like soluble proteins, integral membrane proteins are alsoamenable to intact mass analysis, though different methods are

required. The main difficulty involves separa -tion of the protein from detergent, which is apowerful ion suppressant. This may be doneeither off-line using size exclusion or proteinprecipitation, or on-line using reversed phasecolumn separation12. Techniques also exist for

gas-phase separation of the protein-detergent complex in the massspectrometer itself by collision-induced dissociation13.

The same high-throughput methods which can be applied to trypticdigest MS/MS may also be applied to intact mass analysis at the small-scale test expression stage. Proteins from 96-well test purification maybe analysed overnight with results available the following day. Unlikethe former technique, intact mass analysis is insufficiently sensitive fortargets expressing with low to medium yield, yet this need not be ofconcern since often highly expressing constructs are preferred for scale-up. When intact mass data can be obtained, this is availablefaster and is more informative during the protein purification processthan tryptic digest MS/MS.

Target protein functionFunctionally-active protein is the endpoint goal of most recombinant



Figure 2: Illustration of the purification process, demonstrating the applications of mass spectrometry

MS is rightly regarded as the ‘gold standard’ for protein

identification

protein expression. Functional activity of an enzyme can be measuredindirectly by quantitative mass spectrometric analysis of the conversion of substrate to product14. Where biological activity involvesspecific binding of a small molecule ligand, interaction with another protein, or formation of a multimeric protein complex, this canbe measured directly using native MS15. Even when the activity of aprotein is unknown, the proportion of correctly folded native proteinmay be determined and hence its functional activity inferred. The methods discussed earlier involve loss of all activity either by proteolysis or by denaturing HPLC. Native MS involves achieving ionisation whilst allowing the protein to retain its funct-ional conformation.

Efficient desalting is performed off-line and proteins are introducedinto the mass spectrometer by direct infusion. Natively folded proteinsacquire two-to-three-fold fewer charges upon ionisation, hence them/z ratio (the parameter which mass spectrometry actually measures)will be higher. For small proteins m/z will fall within the normal range ofmost instruments of up to m/z 3500. Larger proteins and proteincomplexes with a higher native m/z will require a modern massspectrometer which can operate to m/z 20,000 and above. The transmission and detection efficiency falls away as m/z increases,meaning that some optimisation of the instrument is needed for native MS. In spite of this, the resolution and mass accuracy obtainable by native MS can equal or even surpass that seen usingconventional methods.

At the SGC, all of these analyses are used together to build acomplete picture of what our protein samples consist of and how theybehave. This allows independent purifications to be compared andnormalised for consistency, giving weight and reliability to thedownstream uses in structural biology, as well as assay and chemicalprobe development.

AcknowledgementsThis work was supported by the SGC which is a registered charity(number 1097737) that receives funds from AbbVie, Bayer Pharma AG,Boehringer Ingelheim, Canada Foundation for Innovation, EshelmanInstitute for Innovation, Genome Canada, Innovative MedicinesInitiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck & Co.,

Novartis Pharma AG, Ontario Ministry of Economic Development andInnovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and the Wellcome Trust [092809/Z/10/Z].



1. Jeon WB, Aceti DJ, Bingman CA, Vojtik FC, Olson AC, Ellefson JM, et al. High-throughputpurification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structuralgenomics. Journal of structural and functional genomics. 2005;6(2-3):143-7

2. Graslund S, Sagemark J, Berglund H, Dahlgren LG, Flores A, Hammarstrom M, et al.The use of systematic N- and C-terminal deletions to promote production and structuralstudies of recombinant proteins. Protein Expr Purif. 2008;58(2):210-21

3. Savitsky P, Bray J, Cooper CD, Marsden BD, Mahajan P, Burgess-Brown NA, et al. High-throughput production of human proteins for crystallization: the SGC experience. J StructBiol. 2010;172(1):3-13

4. Strain-Damerell C, Mahajan P, Gileadi O, Burgess-Brown NA. Medium-throughputproduction of recombinant human proteins: ligation-independent cloning. Methods MolBiol. 2014;1091:55-72

5. Burgess-Brown NA, Mahajan P, Strain-Damerell C, Gileadi O, Graslund S. Medium-throughput production of recombinant human proteins: protein production in E. coli.Methods Mol Biol. 2014;1091:73-94

6. Mahajan P, Strain-Damerell C, Gileadi O, Burgess-Brown NA. Medium-throughputproduction of recombinant human proteins: protein production in insect cells. Methods MolBiol. 2014;1091:95-121

7. Chalk R, Berridge G, Shrestha L, Strain-Damerell C, Mahajan P, Yue W, et al. High-Throughput Mass Spectrometry Applied to Structural Genomics. Chromatography.2014;1(4):159-75

8. Cohen SL, Chait BT. Mass spectrometry as a tool for protein crystallography. Annual reviewof biophysics and biomolecular structure. 2001;30(1):67-85

9. Sebela M, Stosova T, Havlis J, Wielsch N, Thomas H, Zdrahal Z, et al. Thermostable trypsinconjugates for high-throughput proteomics: synthesis and performance evaluation.Proteomics. 2006;6(10):2959-63

10. Brunner AM, Lossl P, Liu F, Huguet R, Mullen C, Yamashita M, et al. Benchmarkingmultiple fragmentation methods on an orbitrap fusion for top-down phospho-proteoformcharacterization. Analytical chemistry. 2015;87(8):4152-8

11. Creasy DM, Cottrell, JS. Unimod: Protein modifications for mass spectrometry. Proteomics.2004;4:1534–6

12. Berridge G, Chalk R, D’Avanzo N, Dong L, Doyle D, Kim JI, et al. High-performance liquidchromatography separation and intact mass analysis of detergent-solubilized integralmembrane proteins. Analytical biochemistry. 2011;410(2):272-80

13. Barrera NP, Isaacson SC, Zhou M, Bavro VN, Welch A, Schaedler TA, et al. Massspectrometry of membrane transporters reveals subunit stoichiometry and interactions.Nature methods. 2009;6(8):585-7

14. Forbes CD, Toth JG, Ozbal CC, Lamarr WA, Pendleton JA, Rocks S, et al. High-throughputmass spectrometry screening for inhibitors of phosphatidylserine decarboxylase. Journal ofbiomolecular screening. 2007;12(5):628-34

15. Heck AJ. Native mass spectrometry: a bridge between interactomics and structural biology.Nature methods. 2008;5(11):927-33

References

Nicola Burgess-Brown is the Principal Investigator of theBiotech Group at the SGC, responsible for molecularbiology, cell culture, protein production and massspectrometry analysis of the targets of interest at the Oxfordsite. Working closely with other SGC teams, the groupdevelops methods for increasing protein expression anddriving throughput. Following her degree in Applied

Biochemical Sciences in 1997, Nicola worked as a Molecular Biologist forSmithKline Beecham. She received her PhD in Molecular Microbiology atthe University of Nottingham in 2001 then returned to industry focusing onhigh-throughput cloning and validation of therapeutic cancer antigens forOxford Glycosciences.

Rod Chalk is a post doc running the mass spectrometryfacility at the SGC, where he has worked since 2008. Rodgained a PhD at the Liverpool School of Tropical Medicinein 1992 and has worked in mass spectrometry in industryand academia for 19 years in a variety of roles. Industrialpositions include Oxford Glycosciences (proteomics),Comet Analytics (cryodetector mass spectrometry)

and Lonza (biologics). He held academic posts in proteomics at QUB,Oxford and Reading. His current interests include high-throughput proteinanalysis, integral membrane proteins and native mass spectrometry and theirapplication to drug discovery.

After studying Molecular Genetics in Biotechnology at theUniversity of Sussex, Dr Claire Strain-Damerell

continued on to obtain her DPhil on the role of the redoxsensitive transcriptional repressor; Rex, in Streptomycescoelicolor. After completing her PDhil, Dr Strain-Damerellthen progressed on to a postdoctoral position at theStructural Genomics Consortium in Oxford, focusing

on the optimisa tion of the cloning and crystallographic pipelines to improvethe success rates for structural determination.

Pravin Mahajan obtained his PhD from De MontfortUniversity, Leicester, where he studied genetic engineeringof drug-metabolising human cytochrome P450 enzymes andtheir interaction with NADPH-cytochrome P450 reductase.Prior to his PhD, he worked in the area of cancer drugdiscovery for five years. Pravin joined the SGC in 2008 andhas been working on high-throughput expression of human

proteins in mammalian cells, insect cells and E. coli. He developed a high-throughput method for protein expression in mammalian cells usingBacMam and contributed significantly towards method development andimprovement of the baculovirus expression system.

Given the prevalence and ease ofgenomics and transcriptomics, whatrole and value do you place on massspectrometry as a biomolecularcharacterisation technique on itsown, and applied together with other–omics’ in a systems approach? For bimolecular characterisation, massspectrometry has significant value in theability to provide detailed molecularinformation, enabling users to potentiallyidentify a range of biomolecular hetero -geneity. However, selectivity and sensitivitycan be improved by utilising this technique inconjunction with immunoprecipitation/capture. This offers the ability to target andcharacterise biomolecules from complexmatrices. Such hybrid LC-MS approaches havecontinued to grow within scientific research,and could eventually lead to a cheaper andmore reusable testing system.

Mass spectrometry is one of thetechnology pillars for The HumanProteome Project (HPP). What do youthink is the most important outputfor this technology for the HPP? For translational research?Mass spectrometry is an important biomarkerdiscovery and validation tool for the HumanProteome Project, especially due to its abilityto provide accurate selectivity and sensitiveanalysis. Once validated, this information can

become the basis for translational research,and then be used to develop products toimprove human health and wellbeing. Thesesame benefits provided by hybrid LC-MSmethodologies also apply to translationalresearch, thus enabling the development ofimproved biotherapeutics and diagnostics.

With multiplex targeted massspectrometry assays such as multiple reaction monitoringbecoming more common in theresearch setting, do you think massspectrometry assays can become amodality for clinical tests to measuremultiple proteins? What are thehurdles that need to be overcome?Yes, I believe that mass spectrometry assayscan become a modality for clinical tests tomeasure multiple proteins. In doing so,relevant multiplexes need to be overcome –for example, the number and nature of teststo run on a disease panel, as well as a suitablereimbursement structure. Production capacityand stability could also become an issue forantibody components that are more complexand multiplicative.

What are your thoughts on massspectrometry data sharing throughopen source repositories? What is thevalue and what are the obstacles?The main challenge with mass spectrometry

data sharing is when details of how the datawas generated is withheld. These specificsoften contain critical and important infor -mation that could provide great benefit if itwere able to be shared efficiently andeffectively. In addition to this, there needs tobe an incentive to scientists to share theircutting-edge data.

Mass spectrometry can seem to be a highly specialised technique to many biomedicalresearchers, and perhaps not asaccessible as, say, genomics ortranscriptomics. How do you thinkthis barrier can be overcome toincrease its application? Can massspectrometry for the masses become a reality?The use of mass spectrometry has come a long way in the past decade, and whatonce seemed complicated is now commonpractice. Yet one of the main barriers to using this technique is that it lacks auto -mation. If the instrumentation and softwarecan be improved to incorporate moreautomated means of optimising MS methods,generating and interpreting mass spectraldata, then the use of this technique willbecome more prevalent. Automated samplepreparation is another way to truly push the technology forward into a more acc-essible area.



Eric E. Niederkofler Thermo Fisher Scientific

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Michelle M. Hill, PhD, invites Eric E. Niederkofler, PhD, to discuss his views on the benefits and importance of massspectrometry as a biomolecular discovery and validation tool. Eric is an R&D Manager at Thermo Fisher Scientific.Michelle is Head of the Cancer Proteomics Group at The University of Queensland Diamantina Institute, Faculty ofMedicine and Biomedical Sciences, The University of Queensland, Brisbane, Australia. She is also Academic Lead atthe Proteomics & Mass Spectrometry Core Facility at the Translational Research Institute in Brisbane.

Early-stage innovative drug targets The identification of protein targets that have genuine therapeuticpotential is a major challenge. Although there is wide variation, fromour experience over the past 10 years, most new targets that areproposed by academics to translational groups have limited disease-association and target validation data to progress them as candidatesfor drug discovery. These data are not limited to, but may typicallyinclude, a combination of evidence including: (i) in vivo or cellular genesilencing or knock-out/knock-in data to indicate a significant change inphenotype; (ii) evidence that gain- or loss-of-function mutations orchanges in gene expression are observed in patient populations; (iii) data from the use of non-specific tool compounds that cellsignalling is altered or a functional effect or change in phenotype is

observed in a cellular or in vivo model; (iv) evidence to show interactionwith specific receptors or protein complexes and involvement in cellularprocesses or signalling events.

Often the data have not yet been published or reproduced by anyother research groups. It is also common for the target recombinantprotein, essential for the development of screening assays, structuralstudies and immunisations, to be unavailable, available in only limitedamounts or of inadequate quality. In addition, it is unusual for robusttarget-specific biochemical, biophysical or phenotypic cellular assaysamenable to pharmacological or high-throughput screening to beavailable, further adding to the challenges of progression into a drugdiscovery program.

The main challenge as we see it and one of the most common

The pharmaceutical industry is searching for novel drug targets that could produce the next generation of ‘first-in-class’ therapeutic agents. The challenge for academia is to identify and translate such targets and to providestarting points and confidence in the underlying science. The aim should be to generate evidence that modulationwith chemical or biological modulators is likely to result in clinical benefit. As a ‘not-for profit’ drug discoveryorganisation, MRCT is just one of a number of groups in the UK that conducts translational and drug discovery workin collaboration with academics (http://ukddc.org/). Here, we review some of the issues related to working oninnovative drug targets, reflect on our own experiences of working in the early-stage drug discovery arena anddiscuss approaches that allow target ‘de-risking’ and validation for progression into the drug discovery pipeline.

TARGET VALIDATION

The challenges associatedwith ‘de-risking’ early-stage therapeutic targets

Ahmad Kamal, Debbie Taylor, Tim Chapman and Catherine KettleboroughMRC Technology, Centre for Therapeutics Discovery


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reasons for failure of projects is target validation and linked to this isthe availability of tool molecules that can be used for conducting suchstudies, as well as the availability of reagents and assays to enable theidentification of such tools. We have learnt that at the beginning of anydrug discovery project, it is important that a thorough review isconducted around the potential new target and that key data is bothindependently reproduced and expanded upon prior to embarking onexpensive and time-consuming work to identify tools and/or potentialdrugs. The target review should encompass a comprehensive review ofthe scientific, patent and clinical literature, as well as bioinformaticdatabases to establish a robust target rationale and freedom tooperate, as well as determining the availability of reagents, toolcompounds/antibodies, biochemical and cellular assays, and in vivomodels. It is also vital at this stage that a clear strategy for targetvalidation, identification of modulators, and the generation of reagents and assays should be established, by asking some keyquestions (see Table 1).

Addressing the challengesGeneration of proteins and reagents for assay developmentTo date, our focus at MRCT has very much been target-based drugdiscovery and as such the availability of high quality target protein for the development of assays, as an antigen for immunisation, forstructural work, or for target engagement studies, has been critical.Although cellular assays, where the target protein is eitherrecombinantly or endogenously expressed, can be used as a means toscreen for modulators, ideally a source of purified target protein isrequired for biochemical and biophysical assays. Unfortunately, and

particularly when working on more novel and unprecedented targets,protein availability, i.e., expression, purification and stability, can be a challenge.

While in some instances commercial sources may be available, thiscan prove expensive, particularly if the project progresses into a fulldrug discovery programme. In addition, careful consideration should begiven to protein from commercial sources; which expression system hasbeen used? Is it full length or truncated? How has it been purified andwhat is the level of purity? Is there evidence it is functional? How hasthis activity been assayed? In prosecuting early-stage projects this is aproblem that we have encountered on numerous occasions andsignificant commitment of resource is required to generateactive/functional protein in the required amounts.

In some instances, protein stability has been an issue and freshbatches of protein have been required on a regular basis . In order toaddress this problem, we invest early to ensure target proteins andantibodies are “fit for purpose” for target validation studies and thenonward into a drug discovery programme. As well as establishing in-house protein expression (prokaryotic and eukaryotic), we endeav -our to achieve high levels of purity and routinely assess aggregation,solubility and stability.

Target validation Ultimately, target validation can only be achieved in the clinic, however,robust disease-linkage and modulation of cellular phenotypes inphysiologically-relevant assays can contribute to mitigating attrition. To conduct thorough target validation studies, access to bioinformaticsand good quality selective tool molecules/antibodies are important.Equally vital is the ability to confirm engagement of the modulator withthe target, especially in a cellular format. Unfortunately, such tools or information may not be readily available, requiring a combination ofalternative approaches to achieve confidence in the target. In ourexperi ence it is vital to start by asking key target-related andphenotype-related questions (see Table 1) and put together a plan of the required studies. We should not be afraid to “fearlesslyevaluate the science” .

Target validation can involve a combination of many differenttechniques including amongst others: various methods for gene


Techniques that can be used for target validation

Target validation questions that should be considered prior to working onnew targets

Target-related

Is the target expressed in relevant human cells/tissues?

Is there a disease/diseases associated with the target based on clinical observations?

If the association is genetic, what evidence is there that there is equivalentmodification of protein levels in response to disease status and outcome?

Should the target be blocked or activated?

Are there any existing modulators used clinically or available commercially oridentified by originating lab?

If target validation is based on knockdown/deletion studies, how well controlled arethey?

Are they complimented by small molecule or antibody studies that show the samephenotype?

Is there a relevant in vivo model of the disease(s)?

How well does it ‘model’ or predict human disease?

Can the target be assayed in a relevant cell-based or in vivo system? How robust isthis assay?

Can we access primary human material to investigate the biology of the target(expression, function)?

Is there a phamacodynamic biomarker directly linked to target activity?

How easy is it to assay?

Phenotype-related

What is the cellular/physiological process being modulated?

Is it relevant to disease and supported by clinical observations?

Can it be modelled in vitro? In what cellular system?

Can it be modelled in vivo? In what system?

TARGET VALIDATION

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editing/silencing, the use of various forms of biological and chemicaltools and the use of multiple types of animal models (see Figure 1; page 25). Where possible target validation studies can be supported bythe use of patient-derived or primary human cells/tissues orphysiologically relevant cell lines, such that species differences andartificial systems do not impact on the results obtained and effects canbe translated into the clinic. The use of phenotypic and high-contentassays, and the use of cells generated from human induced pluripotentstem cells also have a role.

It is essential to have confidence in the authenticity of any tools,reagents, antibodies and cell lines used for target validation work andensure that they are ‘fit for purpose’. In-house generation of proteinand characterisation will address some of these issues, however, otherthings to consider include: authentication and mycoplasma-testing ofany cell lines used; whether antibodies from commercial or externalsources are being used as part of target validation studies; how theywere generated and, depending on the studies they will be used for, arethere preservatives?

Another question to ask is: are they endotoxin-free? Since targetvalidation studies can involve complex experiments, in our experienceinvesting in the characterisation of reagents and tools will save timeand resources, as well as give confidence in the results obtained fromsuch studies. We consider it important that any published or reporteddata is robustly reproducible for “landmark experiments”, butunfortunately in our experience, and as others have reported, not allpublished data stands up to scrutiny2,3,4.

At MRCT we have prosecuted more than 83 projects over the past 10 years and an analysis of the data indicates that 25% of theseprograms were closed early due to lack of target validation. In asignificant number of these programs, the data we generated,particularly where we could access pharmacological modulators, did infact help to ‘de-validate’ targets. Based on this experience, we are now putting greater emphasis and resource into early targetvalidation work and are establishing powerful new technologies ‘inhouse’ that can be used for such studies including: CRISPR gene silencing/editing and cellular thermal shift assays. Overall, we aim to fulfil three criteria during the project lifecycle, encomp-assing: (i) evidence that the target is associated with disease; (ii) that the modulator interacts with the target; and (iii) that the

modulator results in a relevant phenotype, ideally in relevant human cells.

MRCT also recognises the advantage of working in collaborationwith other groups, particularly in relation to sharing of target valida-tion data. We have numerous collaborations with pharmaceuticalcompanies worldwide on specific targets and play a key role intherapeutically-focussed initiatives with charitable organisations (e.g., Alzheimer’s Research UK Dementia Consortium)5.

Generation of toolsIt has become increasing clear that the availability of tools, includingboth antibodies and selective small molecules, has a significant impacton the ability to validate a drug target and at MRCT we recognise theimportance of generating such tools during the early stages of aprogramme. We do this via a combination of ‘in-house’ synthesis,commercially-available compounds or expression of antibodiesdescribed in the scientific or patent literature, as well as via screeningprograms to identify novel modulators.

AntibodiesIn addition to the traditional approach of generating mono-clonal antibodies via the immunisation of rodents and screening of hybridomas, we are increasingly using phage library generation and screening from immunised sources (mouse, rabbit, llama) togenerate scFvs or nanobodies (single heavy chain variable regions) as tools for target validation. If required these formats can readily be converted to whole IgGs or other isotypes if applicable. The ideal criteria for an antibody tool are shown in Table 2, but key is that it demonstrates selective nanmolar binding affinity and can be used to obtain functional effects when used at ~1μg/ml(~7nM). For target validation studies it needs to be soluble,endotoxin–free, and of a relevant isotype. Nanobodies are generally easy to express and purify and give high yields from eithermammalian cells or E.coli. They have proved to be useful tools for

TARGET VALIDATION


Table 2: Ideal criteria for an antibody tool

Criteria

Affinity Min 1-10 nM

Functional activity Observed at concentrations <1ug/ml(this is the key criteria)

Solubility Better than 10 mg/mL in PBS

Biophysical properties No aggregation, not sticky, purifies understandard conditions

Stability Stable at 4°C for more than 1 month

Cross-reactivity Project dependent: needs to bind to speciesrequired for assays

For a therapeutic: human and cynomologusbinding with preferred rodent binding

Alternatively, an orthologue rodent antibodyalso developed

Known epitope/sequence Not necessary for a tool

Effector function Formatted as appropriate

Table 3: Ideal criteria for an antibody tool

Criteria

Structure Well characterised; no reactive groups unless aselective mechanistic requirement

Stability High chemical stability in aqueous media

Biochemical activity <100nM

Cellular activity <1µM; concentration-dependent effect observed

SAR Closely related structures with similar activity

Selectivity >100-fold versus anti-target/s;polypharmacology undesirable

Permeability High

Solubility High (>100 µM) in low % DMSO aqueousbuffered solutions, no aggregation effects

Negative control compound Inactive compound with similar structure showsno activity in cells

Orthogonal probes Different chemotypes with similar levels ofactivity/selectivity

Mechanism of action Well-defined quantitative relationship betweenbiochemical and cellular effects consistent withtarget-dependent action

Pharmacokinetics Good pharmacokinetics not essential for in vitroand cellular work, but required for animal work

Toxicity No undesired effects

target validation, for affinity purification of proteins, as well as forprotein stabilisation to aid structural studies.

Small molecules The ideal criteria for small molecule tools or probes are shown in Table 3 (page 27). With the unprecedented nature of the targets that we prosecute at MRCT, typically there are no existing probesavailable. Where compounds are available, even if they meet potencycriteria, they are often not sufficiently characterised or do not meet thedesired quality criteria in other areas, such as selectivity orphysicochemical properties and sometimes they contain knownfrequent hitter motifs. High-quality probes are essential for performingtarget validation work and therefore these have to be generated. Wefind that it is important to have a range of compound sets available forscreening as potential start points, so the hit generation approach canbe tailored depending on the project. These sets include annotatedknown bioactives, target-class based sets and diversity-based sets spanning fragment, lead-like and drug-like chemical spaces, as well as pathway-focused sets to support phenotypic approaches.Active curation of these is important and we have recently expandedour fragment set, renovated our diversity set and are currently renew-ing our kinase-focused set.

As noted elsewhere6,7,8, the requirements for a chemical probe aresomewhat different from those of a drug. Some parameters are lessrigorous for a probe, for example, tools for cellular studies will notrequire optimisation of pharmacokinetic (PK) parameters. However,other requirements such as selectivity are more stringent for a probethan a drug, since the polypharmacology that may be advantageous forthe efficacy of a drug is undesirable in a probe, because it does notallow the target to be confidently linked with the phenotype. It isrecommended that different chemotypes with similar levels of activityare used for validation work, as well as control compounds that areinactive, although in practice for novel targets at MRCT it can be difficultto generate multiple active series with the right profiles. Importantly,the impact of a probe in elucidating the biology will partly depend on itsready and widespread availability6, and at MRCT we endeavour to maketools available to academics.

An evaluation of projects conducted over the past 10 years indicatesthat tool compounds were generated and made available for 38% ofsmall molecule and 57% of antibody projects. Notably, a new resourceto share information around probes has just been launched(http://www.chemicalprobes.org/). We are also making increased useof bespoke tagged probes alongside the development of SAR in achemical series, to increase confidence in the relationship between

engagement of the target and the observed phenotype. These includebiotinylated probes to allow identification or confirmation of the compound interaction partners in the cell; we typically conduct theproteomics analysis work in collaboration with academic partners9,10

and intend to build our capabilities in this area to examine targetengagement in intact cells as well as lysates.

ConclusionOur experiences of working as a translational unit at the interfacebetween academia and pharma over the past 10 years has taught us that early investment in validation of targets is essential forsuccessful management of a portfolio of novel and unprece-dented targets. Historically, a significant number of our projects havefailed due to lack of target validation and on some occasions projects have stalled because of issues with protein generation or aninability to identify validated hits. To address this we are putting agreater emphasis on generation of reagents and tools fit for purpose,increased the use of new technologies and are repeating key studies to confirm the proposed therapeutic hypothesis. Coupled witha rigorous target selection procedure we envisage a greater success rate going forward.


TARGET VALIDATION

References

Ahmad Kamal is a Team Leader in MRCT’s CellularPharmacology group, establishing cell-based assays fortarget validation and profiling of both small molecules andantibodies. He has worked in the pharmaceutical industry as well as academia, having graduated with a PhD inPharmacology from Imperial College.

Debra Taylor is a Drug Discovery Manager at MRCT’sCentre for Therapeutics Discovery. She is responsible formanaging the team responsible for target validation andconducting cell-based assays to support small molecule and antibody programs. She obtained her PhD from Universityof London and previously worked in antiviral drug discovery.

Tim Chapman is a Team Leader in chemistry at MRCTechnology’s Centre for Therapeutics Discovery, where hehas worked on discovery projects across a range of disease areas including malaria, TB, oncology andinflammation. He obtained a DPhil in chemistry at OxfordUniversity, followed by a postdoctoral fellowship at the University of Toronto.

Catherine Kettleborough is the Associate Director ofBiology at MRC Technology’s Centre for TherapeuticsDiscovery (CTD). CTD’s Biology group is responsible forconducting target validation, assay development, screeningand cell-based assays to support prosecution of early drugdiscovery projects for novel therapeutic targets sourcedfrom academic research groups worldwide.

1. McNeil, EM, Ritchie, AM, Astell, KR, Shave, S; Houston, DR, Bakrania, P., Jones, HM,Khurana, P, Wallace, C, Chapman, T, Wear, MA, Walkinshaw, MD, Saxty, B and Melton,DW. Inhibition of the ERCC1-XPF structure-specific endonuclease to overcome cancerchemoresistance. 2015; DNA Repair 31: 19-28

2. Dahlin, JL, Inglese, J and Walters, MA. Mitigating risk in academic preclinical drugdiscovery. Nat. Rev. Drug Discov. 2015; 14:279-294

3. Prinz, F, Schlange, T and Asadullah, K. Believe it or not: how much can we rely on publisheddata on potential drug targets? Nat. Rev Drug Discov. 2011; 10: Correspondence

4. Mullard A. Reliability of ‘new drug target’ claims called into question. Nature Rev DrugDiscov. 2011; 10: 643

5. Bryans, J. ( 2015). A new dawn in academic and pharma collaboration – a UK perspective.Drug Target Review, 2: 12-14

6. Frye, SV. The art of the chemical probe. Nat. Chem. Biol. 2010; 6: 159-161

7. Workman, P and Collins, I. Probing the probes: Fitness Factors for Small Molecule Tools.Chemistry & Biology. 2010; 17: 561-577

8. Bunnage, ME, Piatnitski Checkler, EL and Jones, LH. Target validation using chemicalprobes. Nature Chem Biol. 2013; 9: 195-199

9. Tsaytler, P, Harding, HP, Ron, D and Bertolotti, A. Selective inhibition of a regulatorysubunit of protein phosphatase 1 restores proteostasis. Science. 2011; 332: 91-94.

10. Green, J, Moon, R, Whalley, D, Bowyer, P, Wallace, C, Rochani, A, Kumar, R, Howell, S,Jones, H, Ansell, K, Chapman, T, Taylor, D, Osborne, S, Baker, D, Tatu, U and Holder, A.Imidazopyridazine inhibitors of Plasmodium falciparum calcium dependent protein kinase 1also target cGMP-dependent protein kinase and heat shock protein 90 to kill the parasite atdifferent stages of intracellular development. Antimicrob. Ag. Chemother. 2015. Submitted

The advances that have been made in biomedical understanding ofdisease need to be translated into treatments to improve the lives of patients. Lead discovery is a key step in this translation, aiming atfinding molecules that modulate a target or phenotype to affect theirrole in disease. However, despite successes, there remains a need to further improve lead discovery, and in particular to addressunprecedented and difficult target classes. High-throughput screening

(HTS) was widely adopted in the 1990s to tackle this problem. Especiallyin combination with combinatorial chemistry, HTS was considered a wayto turn drug discovery from a game of chances into a rational,predictable industrial process.

Later, HTS was rather seen as a contributor to the escalating researchand development (R&D) costs in the pharmaceutical industry withoutsignificantly adding to pipelines1,2. It is only recently that a more balanced

Progress in the understanding of disease mechanisms provides new opportunities to discover molecules thatmodulate disease. To capitalise on these opportunities, successful lead discovery strategies need to build oninsights into how a cellular phenotype or a target contributes to disease biology. Recent advances in inducedpluripotent stem cells (iPSCs), gene editing and imaging technologies enable unprecedented access to relevantcellular models of disease, while methods such as fragment-based and DNA-encoded library screening haveexpanded our ability to find ligands, even for difficult and unusual targets. Realising that only a tight integration ofapproaches can overcome the challenges of these novel targets, we introduce the concept of ‘integrated leaddiscovery’ and highlight the scientific, technological and organisational implications of the concept.

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Enabling new targets byintegrated lead discovery

Ulrich Schopfer and Sylvain CottensNovartis

assessment of the role and contribution of HTSto drug discovery has become possible andtoday’s R&D pipelines contain many drugcandidates that originate from HTS.3 A closerlook, however, shows that a drug was rarelydiscovered by a single technology, and fre -quently it is difficult to retrospectively trace therelative contributions of screening, design,structural information, in silico modelling orother technologies in the complex and highlyiterative path of drug discovery.

Meanwhile, the recent renaissance ofphenotypic drug discovery4 has been accomp -anied by advances in cell biology and imagingtechnologies that allow the development ofmore disease-relevant cellular models. Work- ing with these more complex models meansthat lead discovery becomes even less of astandardised process. This led us to re-thinkour approach to lead discovery. We saw the need to place the disease-relevant pathway or target at the centre of our thinking. Based on thebiological understanding of disease pathology, we then brought the breadth of our capabilities to bear on the scientific hypothesis, withan emphasis on the integration of technologies. We termed thisconcept “integrated lead discovery (ILD)” (Figure 1).

A new concept for lead discoveryILD has its foundation in the biological understanding of the role of atarget or pathway in disease. A thorough understanding of how a cellular phenotype or a signalling pathway contributes to disease canbe used to design lead discovery strategies thatcapture essential elements of biology. Primarycells or iPSC-derived cellular systems play anincreasing role here, since they are more reflec -tive of the disease tissue than transformed celllines. Live or fixed cell imaging with advanced image analysis arepowerful methods to interrogate cellular systems. In silico methodshelp to select focused compound sets and to examine results in thecontext of pathway knowledge. Similarly, a detailed understanding of

the role of a protein complex, a protein, or a specific mechanism ofaction of a protein domain can be incorporated into strategies thatcapitalise on a unique insight into protein function, and which are alsoopen to non-canonical findings. Strong protein biochemistrycapabilities, in silico modelling and X-ray crystallography combined withbiochemical and biophysical assays that detect binding or enzymeactivity can build on the strength of each technology.

An essential element of the ILD concept is to also pursue lowaffinity/low activity hits. To differentiate these hits from assay noise andfalse positives, biophysical methods that show true compound bindingto the target protein play a critical role. Also, fragment-based discovery

efforts that use small, low-affinity molecules tointerrogate binding sites on proteins are a coreelement of ILD, because fragments have ahigher likelihood of binding to a protein pocket.

Importantly, the concept implies that weaim to develop the most biologically-relevant assay even when it does notoffer the throughput to test millions or even hundreds of thousands of compounds. In most projects, HTS has been replaced by iterations of low- or medium-throughput assays, testing between 1000 to 100,000

compounds at each run. Hits from these runsprovide tool compounds that can be used todevelop follow-up assays or even to challengethe underlying project hypothesis. The ILDapproach requires a tight interaction and closecommunication across disciplines. As no twoprojects follow the same process, it is vital that project teams learn from each other’sexperi ences and accumulate a body of knowl -edge across projects. These requirementsrepresent significant changes to earlier appr -oaches. To realise the full potential of ILD, a shiftin culture and a new organisation were required.

A new cultureAs in most other companies, lead discovery at


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Figure 1: Integrated lead discovery: a project perspective. Synergy between lead discovery approaches

Figure 2: A new lead discovery organisation. A matrix with focus on projects and on scientific disciplines

Lead discovery aims at finding molecules that modulate a target or phenotype to affect their

role in disease

Novartis started in a central department thathad established the technologies andprocesses required to test large numbers ofcompounds in high-throughput assays. Also forstructural biology, a process flow had beendeveloped and partially automated thatoptimised efficiency at each step to maximisethe number of structures that could be solved.This had created a culture of technologyexperts who took pride in the processes theyhad developed. This mind-set had to be shiftedin favour of emphasising biological relevance.Numerical performance indicators werereplaced by assessing how contributions hadenabled lead discovery. While performance andinnovation were still core values of theorganisation, they were complemented by astrong emphasis on collaboration.

Integrating knowledge across disciplines and sharing credit withcolleagues, while being easy to work with for partners in disease areasand medicinal chemistry, required training in self-awareness andcommunication. It was also designed toquestion the leadership model5 of theorganisation. Leaders took the role of mentors,who help with problem-solving and connectingpeople. They establish the direction but refrain from prescribing a solution. In this way they enable individuals, teams and theentire organisation to learn. As we found out, the standard hierarchicalorganisational model, with technology-centric groups at its core, wasnot flexible enough to enable project-centric, fluid teams to form anddisassemble over time.

A new organisationA new matrix organisation was built along two axes: lead discoveryprojects and technologies (Figure 2; page 30). Projects were grouped

according to disease area to facilitate interactions with disease areasand project portfolio management. Technologies were grouped to buildstrong expertise cores. Mid-level group leaders were replaced by astrengthened project leader role. These leaders have the responsibility

of developing and executing the project leaddiscovery strategy, together with the coreproject team from the disease area and fromchemistry. They could come from any disciplinebut were expected to have sufficient under -standing of the other disciplines to be able toguide strategy development.

ILD groups united projects into a portfolio that partnered with thesame disease area. The leader of this ILD group became a member ofthe disease area management board under the agreement that thisboard would be the single governance board for that project. In thatrole, he or she mentors project leaders and ensures alignment with the disease area with regard to scientific strategy and projectprioritisation. Along the second axes, Core Science Groups were formed to ensure the continued evolution of capabilities and skills.

The leaders of these groups provide theinfrastructure and resources for the projects, aswell as guiding innovation projects thatadvance the technology in their area. Theyensure flexibility of resource assignment acrossthe ILD groups as the size and nature of theproject portfolio of any disease area changesover time.

The organisation was designed to maxi-mise synergies across technologies andapproaches, as well as across project port -folios. Project teams retain a high degree ofscientific autonomy to empower them to fromstrong collaborations with partners in diseaseareas and chemistry and quickly react toscientific discoveries.

Integrated lead discovery in actionThe experience with this model has been

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Figure 3: Ligands identified by fragment-based screening and in silico docking were merged and guided by co-crystal structures and biochemical assays to yield potent inhibitors

The recent renaissance ofphenotypic drug discovery has been

accompanied by advances in cell biologyand imaging technologies that allow thedevelopment of more disease-relevant

cellular models

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overwhelmingly positive. While in the past thelead discovery group was often viewed as slow, bureaucratic and process-driven, theperception has changed and the group has nowbecome a strong, flexible and science-drivenpartner for disease areas and other plat-forms. Project teams address key questions in aproject by applying relevant expertise andtechnologies rather than executing a projectplan. Rapid iterations between small pilotscreens, early hit confirmation, input by earlystructure and biophysical information havebecome standard practice. These activities can generate tool compounds to validateflowcharts all the way to providing in vivoconcept validation and thus bridge betweenphenotypic- and target-based approaches.

For targets that were previously deemed‘undruggable’, activity-agnostic binding assaysare used to find ligands, identify binding sitesand elucidate mechanisms of action. Fragment-based drug discovery using biophysicalmethods such as nuclear magnetic resonance,differential scanning fluorimetry or others has proven a powerfulapproach. Also, new selection-based screening technologies, such asDNA-encoded libraries, are used to gain an understanding of pocketsthat can be used to modulate target activity. In combination with in silico methods, ligands can rapidly be improved to become higher inaffinity and enable a proof of principle in cells (Figure 3; page 31).

Initially, a concern was that the project focus in the neworganisation would lead to reduced efforts in technology developmentand that it would become more difficult to dedicate resources totechnology innovation. This has not been the case. On the contrary,activities in technological innovation have become more deliberate andmore strategic, with a focus on keeping the technology base of thedepartment at the forefront of the discipline.

However, the adaptation to working in a matrix organisation is notwithout difficulties. Since there is a high degree of co-dependencyacross the matrix, the organisation needs a high level of trust andcommunication to function well. Decision-making can be more complexthan in a hierarchical organisation. Initially, scientists were not surewho to approach to obtain decisions, because depending on the topic, they interacted with different leadership team members. The closer collaboration with a disease-area partner in the ILD groupstherefore has two sides. On balance, however, the ILD approach hastransformed the department to become more flexible and moreinnovative. Delayering and focused leadership roles strengthened both the project and the technology focus of the organisation andempowered associates to take ownership of the scientific challenge oflead discovery.

ConclusionRethinking our approach to lead discovery and the understanding ofdisease mechanisms has been placed at the centre of lead discoverystrategies. Key scientific questions are addressed in an iterative,

hypothesis-driven manner. We believe that this flexible, science-drivenapproach will give rise to the next generation of drug targets for which the reliance on standard flowcharts and processes will no longerbe suitable.


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1. Peter Landers, Human Element: drug industry's big push into technology falls short –testing machines were built to streamline research – but may be stifling it – officials seepayoff after 2010. The Wall Street Journal (24 Feb 2004)

2. Roger Lahana, Who wants to be irrational? Drug Discov. Today. 2003; 8, 655–656

3. Ricardo Macarron, Martyn N. Banks, Dejan Bojanic, David J. Burns, Dragan A. Cirovic,Tina Garyantes, Darren V. S. Green, Robert P. Hertzberg, William P. Janzen, Jeff W.Paslay, Ulrich Schopfer, G. Sitta Sittampalam, Impact of high-throughput screening inbiomedical research. Nature Reviews Drug Discovery. 2011; 10, 188-195

4. David C. Swinney, Jason Anthony, How were new medicines discovered? NatureReviews Drug Discovery. 2011; 10, 507-519

5. Andreas Schneider, Zeynep Erden, Hans Widmer, Guido Koch, Christine Billy, Georgvon Krogh. Rethinking leadership in drug discovery projects. Drug Discovery Today.2012; 23–24, 1258-1262

References

Ulrich Schopfer is Executive Director and Integrated LeadDiscovery Head at the Center of Proteomic Chemistry of theNovartis Institutes for BioMedical Research (NIBR). He isoverseeing lead discovery projects in the oncology andmuscoskeletal disease area as well as lead discovery projectspartnered with the disease molecular pathways group.Previously he led a biochemical screening group and

was responsible for global compound management at NIBR. UlrichSchopfer holds a PhD in chemistry and started his career at Novartis asMedicinal Chemist and Project Team Leader of different discoveryprograms in Neuroscience.

Sylvain Cottens is the Global Head of the Center forProteomic Chemistry at the Novartis Institutes for Bio -medical Research (NIBR). Previously he led the NovartisExpertise Platform Proteases. Sylvain Cottens holds a PhDin chemistry and started his career at Novartis as a medicinal chemist, first in the Neurosciences area andthen in the Immunology department, before becoming aMedicinal Chemistry Unit Head. From 2000 to 2002, Sylvain Cottens wasthe acting head of the Novartis Transplantation Research department.

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3D printing technologyPrinting a pharmaceutical product requires the design to be createdwith a computer-aided-design (CAD) software package; it must then bedigitally sliced in order for the printer to fabricate it. The most widelyused file format for encoding the individual layers is stereolithographic.Once encoded, the file is transferred to the printer’s driving software sothe printer can fabricate the object.

The first 3DP technology was essentially a form of ink-jet printing,wherein a liquid adhesive is jetted onto a powder bed to create eachsolid layer. After forming each layer, a new powder bed is laid on top of

the build plate and the process repeats. Once the printing is finished,excess powder is blown away and the object may be furtherstrengthened by heat treatment. This approach is used to manufactureSpritam® tablets (Zipdose® technology) and one of the mainadvantages it confers on the product is rapid disintegration in themouth (since the powder particles are only loosely bound). Powder-bedprinting also offers the familiarity of using powders and so there ispotential for reformulating current tablets made from compressedpowders. One limitation is that it cannot be used to make hollowobjects, since unbound powder will be trapped in any cavities during

3D printing (3DP) is attracting increasing interest as a new method of fabricating pharmaceutical products,especially with the recent United States Food and Drug Administration (FDA) approval of the first three-dimensional(3D) printed tablet Spritam® (levetiracetam). 3DP is considered to be an additive manufacturing technique, becauseregardless of their principles of action, all 3D printers create objects layer by layer on a build plate. Additivemanufacturing technology has a number of advantages compared with large-scale processes (such as powdercompaction) including the ability to locate the active drug at specific locations in the product, to vary size and shape,to incorporate layers with different release properties or containing different actives and, possibly mostimportantly, to fabricate as few as a single dosage form. If developed correctly, 3DP technology thus offers thepotential to herald a new era of personalised medication, where doses or dose combinations can be tailored to the specific requirements of the patient. In this article, some of the latest types of 3D printing technology will bediscussed, and their potential to pharmaceutical formulation highlighted.

PERSONALISED MEDICINE

3D printing: A new era forpersonalised medicines?

Dr Simon GaisfordUniversity College London School of Pharmacy


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fabrication, which may limit the shapes of unit doses that canbe manufactured.

More recently, fused-filament fabrication (FFF) – alsoknown as fused deposition modelling (FDM) – 3D printershave become available. Their increasing popularity is down totheir low cost and simplicity, which lends itself to homeoperation, and numerous desktop systems are available. The basic principle of operation is simple. The feedstock,which is a filament of extruded polymer, is fed through aheated tip. The polymer softens and is deposited on a buildplate, which is kept cold enough to solidify the polymer. Thetip is moved in a raster pattern across the build plate tocreate a layer of the object being fabricated. The printer thendeposits polymer in the same fashion layer by layer until thefinal object is created. Although the commercially-availablefeedstock polymers are usually hard and non-ingestible (suchas polyvinyl alcohol or acrylonitrile butadiene styrene), it ispossible to extrude pharmaceutical grade polymers withsuitable melting and solidifying temperatures for printing,and so the number of applications of FDM printing isincreasing. From a pharmaceutical perspective, preparationof polymer filaments is convenient because it can beachieved with hot-melt extrusion (HME), and so the infrastructure andunderstanding of polymer properties exists. Furthermore, it is relativelyeasy to incorporate actives into the polymer filaments during extrusion.

FDM printers can easily fabricate dosage forms, such as tablets andcaplets. Dosage forms will comprise individual strands that adheretogether (see Figure 1) and the diameter of the strands will beapproximately the size of the print tip. It takes ca. five-to-10 minutes toprint each unit, although this is partly becausethe printers used are designed to print high-resolution objects, and so the printer tip istypically 30-100 microns in diameter. Largerdiameter tips would reduce the printing time.The first report of an FDM printer being used tofabricate tablets was in 20141 and since then thenumber and complexity of dosage forms being printed has increased.One immediate benefit of FDM printing is that the density of the objectis easily varied by altering the ‘infill percentage’ (essentially, how muchpolymer is deposited inside the printed object), and density is usuallyfound to affect the rate of dissolution of drug. Another advantage is theease with which objects with complex or unusual geometries can beprinted, which would be impossible to achieve with powdercompaction2. While tablets of unusual shapes might not ultimately

make commercial production, they do allow exploration and validationof dissolution kinetics models.

Complex or multilayer tablets are also easily fabricated with FDMprinting3 (see Figure 2). This is because FDM printers are easilyconstructed with multiple print heads, and so it is possible to printobjects from different filaments. The filaments can be comprised ofdifferent polymers, for fabrication of modified or sustained-release

devices, or may contain different drugs,allowing for the manufacture of multi-drugpolypills. For instance, Figure 3 (page 35) showsRaman images of a polypill containing caffeineand paracetamol3. Each drug was encapsulatedin a separate polymer filament, and the tabletwas printed with a dual-head FDM printer.

The Raman images are colour-coded to show the locations of the twodrugs and it is clearly evident that the drugs remain in separate layersafter printing.

One drawback of FDM printers is that the printing temperature isdependent on the softening or melting temperature of the polymerfrom which the filament is constructed. If the drug to be printed isphotolabile, there is a risk of significant thermal degradation as thematerial passes through the print head. This issue may be ameliorated

to a degree by careful blending of thepolymers used to make the filament, or byadding plasticisers, to lower the printingtemperature. It may also be possible toincrease the speed of printing, to reduce thetime the drug is exposed to heat. However,careful thought is needed when consideringusing FDM printing to formulate thermallylabile drugs.

It is also possible to use 3D printers todeposit gels and semi-solids. In this case, the



Figure 1: Cross-section of a printed dosage form, showing the individual polymer strands thatadhere to form the final object

Figure 2: 3D representations of heterogeneous printed solid dosage forms: (A) sectioned multilayer device and (B) sectioned DuoCaplet (caplet in caplet)

3DP technology offers the potentialto herald a new era of personalisedmedication, where doses or dose

combinations can be tailored to thespecific requirements of the patient

print head is essentially a syringe which is driven by a motor.The semi-solid material is extruded from the syringe by themotor, and moving the print head in a raster pattern across abuild plate builds up the object, in a similar fashion to theFDM printers just described. Printing a semi-solid materialrequires careful formulation, because the material must beviscous enough to flow, but must remain in place on the buildplate after deposition. This may be accomplished bystrategies such as developing thixotropic systems or using aheated build plate to dehydrate or ‘set’ the mixture.

The final type of 3D printer is stereolithographic (SLA). Inthis case, the feedstock is a resin monomer and the printhead is a laser. The laser is focussed to a specific depth in theresin tank and the wavelength is selected so that excitation ofthe resin monomers with laser light causes photo-polymerisation.Then, as in the cases described, the laser is moved in a raster pattern tocreate one layer of the object, before the build plate is moved and theprocess repeats. If the excitation wavelength of the laser is not exactlymatched to the resin, a photo-initiator can be added.

SLA printers have some unique advantages that favour productionof pharmaceuticals. Firstly, they do not require elevated printingtemperatures (although there can be local heating as the polymerpolymerises), and so have more potential for thermally labile drugs.Also, drugs with poor aqueous solubility maynaturally dissolve to a higher concentration inthe resins, and so be easier to formulate at highdose (in cases where drug solubility in the resinis poor, drug particles may be suspended in theresin). Where the resin used is miscible orsoluble in water, it is possible to encapsulate water in the printedobject, and so print drug-loaded hydrogels directly.

One drawback of SLA printing is that the resin monomer (which istypically comprised of polyethylene glycol diacrylates) and photo-initiator may have toxicity issues and so work is needed to developcurable pharmaceutical materials. The technique does, however, lookvery promising as a manufacturing technology for making implantable,drug-loaded devices.

The role of 3DP in pharmaceutical manufacturingThe current applications of 3DP to pharmaceuticals are at an earlystage, and all the types of printer described herein need properlydeveloped feedstock, comprised of pharmaceutical-grade materials.The powder-bed system has clear potential in light of the recent firstFDA-approved 3DP tablet and naturally lends itself to agglomeratedpowder systems. Feedstock filaments for FDM printers are beingdeveloped and it is surely only a matter of time before filaments ofpharmaceutical-grade polymers are developed. Polymeric printeddosage forms lend themselves to targeted delivery, either throughcareful control of shape, selection of pH-sensitive enteric polymers orfabrication of multi-layered polypills. SLA printers have potential in theareas of hydrogels and drug-loaded implantable devices.

Selection of the drugs to be formulated will remain a critical issue.There has not, as of yet, been a drive to replace commercial batchprocesses, such as powder compaction or capsule filling, with 3DPtechnology. And so the immediate areas of application lie, for instance,

in drugs that (i) are highly-potent and so have a low dose and/or narrowtherapeutic index, (ii) can be formulated as an oro-dispersible tablet, inwhich case 3DP technology might be economically favoured overmanufacturing routes involving freeze-drying, (iii) need to beincorporated into implantable devices manufactured to contour-specific patients and (iv) are biopharmaceuticals, and so have a limitednumber of patients.

It is relatively easy to envisage how 3DP may alter the pharma -ceutical manufacturing landscape, if appropriately developed over

the next few decades. In the short term, moreprinted dosage forms will be approved,probably designed as fast-dissolving orcontrolled-release. Over the longer term, thebasic technology will reduce further in priceand will be optimised for pharmaceutical

manufacture, reducing the fabrication time of unit doses to a fewseconds. The printers could be situated in pharmacies, while thefeedstock materials could be commercially produced. The pharmacistcould then extemporaneously print a supply of tablets, with the doseand/or dose combination tailored to the patient. For biopharma -ceutical products, it can be imagined that a biopharmaceutical mightbe prepared from a patient’s blood sample, and formulated into aprinted form in a continuous process – truly ‘printing a dose of one’sown medicine’.



Dr. Gaisford is a Reader and Head of Department ofPharmaceutics at UCL School of Pharmacy, UniversityCollege London. With Professor Abdul Basit and Drs. SteveHilton and Alvaro Goyanes, he leads a team pioneering theuse of ink-jet and 3D printing in fabricating pharmaceuticaldosage forms and has established a company, FabRx Ltd, tocommercialise the work. He has published 80 papers, three

books and six book chapters. Contact Dr. Gaisford at: [email protected](Tel: +44(0)20 7753 5863. Fax: +44(0)20 7753 5942).

1. Goyanes A, Buanz ABM, Basit AW, Gaisford S. Fused-filament 3D printing (3DP) forfabrication of tablets. Int. J. Pharm. 2014; DOI:10.1016/j.ijpharm.2014.09.044

2. Goyanes A, Robles Martinez P, Buanz ABM, Basit A, Gaisford S. Effect of geometry on drug release from 3D printed tablets. Int. J. Pharm., 2015, DOI:10.1016/j.ijpharm.2015.04.069

3. Goyanes A, Wang J, Buanz A, Martinez-Pacheco R, Telford R, Gaisford S, Basit A. 3Dprinting of medicines: Engineering novel oral devices with unique design and drugrelease characteristics. Mol. Pharm., 2015, DOI: 10.1021/acs.molpharmaceut.5b00510

References

Figure 3: Raman microscopy images of a polypill printed with an FDM printer. The image on theleft is the original image from the microscope (inset shows the tablet itself) and the image on the right is colour coded to show the location of the drugs (caffeine in green, paracetamol in red)

The powder-bed system has clear potential in light of the recent first

FDA-approved 3DP tablet

NGS is an emerging technology for genomic, epigenomic andtranscriptomic profiling, which can be performed more rapidly thantraditional Sanger sequencing and in far greater depth than microarraytechnologies1. NGS technologies have been used in a wide variety offields from sequencing of bacterial and viral genomes2,3, searching forpatient-specific genetic variations4 and profiling of DNA-bindingproteins by ChIP-Seq5, to characterising the transcriptomes of cells,tissues and organisms by RNA-Seq6. NGS covers a wide variety oftechnologies but primarily operates by sequencing DNA or RNA samplesin a massively multiplexed manner, generating images, from whichshort sequence reads can be determined, aligned to a referencegenome and converted to genomic data7.

Tumours are highly dependant on their vasculature to support theirgrowth, being unable to expand beyond 2mm in size or metastasise inthe absence of vessels to supply nutrients, remove waste products and

propagate tumour cells around the body8,9. Considerable effort has beenexpended to investigate the mechanisms by which tumour vasculaturedevelops, as well as examining its distinctiveness from the vessels ofhealthy tissues, with the aim of depriving the tumour of its blood supply.The vast majority of transcriptomic analysis involved in this work hasbeen performed using microarray technologies, though SAGE and cDNAlibrary analysis have also been used (reviewed in10). The advent of high-throughput next-generation analysis offers the opportunity for newdiscoveries to be made in this field, discoveries which are not possiblewith older technologies, and this will contribute to refinement andimprovement of vessel-targeted anti-cancer therapies.

RNA-Seq to study antiangiogenic therapiesCancer vessel-targeted therapies broadly divide into two categories:antiangiogenic therapies, which aim to block the processes by which

Over the past decade significant advances have been made in the fields of genomic and transcriptomic profiling,inspired by the advent of next-generation sequencing (NGS). Yet despite the considerable promise of these newtechnologies, uptake has been slow. The focus of this review is the use of next-generation transcriptomic analysis inthe field of cancer endothelial biology, highlighting its advantages and a few of the disadvantages compared withcurrent-generation technologies.

NEXT-GENERATION SEQUENCING

Next-generationtranscriptomic analysis incancer vascular research

Joseph W. Wragg and Roy BicknellUniversity of Birmingham


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cancers vascularise11 and vascular disrupting agents, which aim todestroy existing vessels in the tumour12. Antiangiogenics are the better-established therapeutic intervention, contributing several therapies tothe clinic, including Sutent® (sunitinib), Nexavar® (sorafenib), cediranib(in development by AstraZeneca) and Avastin® (bevacizumab), which have contributed considerably to survival in several cancer types (reviewed in10).

However, the efficacy of this approach has been curtailed bydifficulties of acquired and innate resistance (reviewed in10,13).Transcriptomic analysis techniques have been utilised in the search forthe molecular mechanisms behind resistance and for markers ofresponse to these therapies, to better tailor the use of these drugs tothe most profitable indication (reviewed in 14).

The majority of this work has been performed using present-generation technologies. Next-generation technologies do offer someadvantages, however. For example, Illumina RNA-Seq NGS was used togenetically profile the tumour from a patient exhibiting an unusuallypositive response to sunitinib in a clinical trial of testicular cancer. This study identified the amplification of several candidate markers ofresponse to sunitinib including RET, EGFR and KRAS15. The study waspart of a wider program at the MD Anderson Cancer Center in Texas,profiling abnormal responders to therapeutics using next-generationgenomic technologies with the aim of identifying novel markers of drugresponse or resistance16.

RNA-Seq differs from traditional transcriptomic analyses such asprobe-based microarray systems in that rather than providing a relative

expression level of a candidate gene via comparative fluorescencesignal analysis, NGS provides an absolute expression level. This isundertaken by sequencing the prepared genetic material from thesample and determining the number of copies of transcriptscorresponding to candidate genes. In the example given, the use of NGStechnologies allowed the identification of genes that were not justdifferentially expressed but also present in sufficient quantities to beclinically relevant for further investigation15. Thus RNA-Seq has

overcome one of the weaknesses of microarray-based analysis, which identify a huge number ofdifferentially expressed genes, most of which arepresent in such minuscule quantities that they are irrelevant for further investigation.

RNA-Seq based analyses offer considerablepower in investigating tumour and stromal responsesto anti-angiogenic therapies with human tumourxenograph models, improving understanding of the effect these therapies have on the tumourenvironment. Widely used array-based systemsprofile the transcriptome expression through the useof probes complementary to transcripts from anyknown gene in the genome. Thus being a semi-targeted approach it suffers from biased transcriptcoverage17, but also is often unable to differentiatebetween transcripts derived from homologous genes in different species. Bradford et al. (2013)17

demonstrated how RNA-Seq can be used to dissecttranscripts derived from the host (mouse) tissue andthe tumour (human) tissue through a species-specific mapping approach, which allowed them toindependently investigate tumour and stromalresponses to AstraZeneca’s cediranib (Figure 1).

Vascular-targeted therapiesVascular disrupting therapies target differences in the vasculature supplying the tumour and healthytissues. These are either structural differences, such



Figure 1: Dissection of human tumour and mouse stromal responses in a xenograph model by RNA-Seq. The human Calu-6 non-small cell lung cancer cell line was grown in mouse. The tumour was excised, theRNA extracted and subjected to RNA-Seq. The sequence read fragments were aligned to a human and mousereference genome and reads that mapped uniquely to either genome were used for gene expression profilingof both the mouse- and human-derived tumour compartments

Figure 2: Vascular-targeted therapies induce tumour necrosis. Gross appearance of asubcutaneous neuroblastoma tumour treated with a MHC class II targetedimmunotoxin. At day 0 the tumours appear highly vascular. Two days after treatment,the tumour is blackened (indicating massive intratumoural haemorrhage). At day 7 the tumour has collapsed into a scab-like plug and by day 10 there is no visibleliving tumour (figure adapted from19)

as combretastatin-based therapies, which destabilise the vessels18, ormolecular changes that have occurred in the endothelial cells lining thevessels, such as vascular-targeted therapies (VTAs), which aim tospecifically kill these cells in a targeted manner12. For VTAs to be effective,targets must be found that are uniquely over expressed on tumourvessels, so that therapeutics can be engineered to target thesemolecules, localise to the tumour and deliver a toxic payload, killing the vessels. Burrows and Thorpe 199319 were the first to demonstrate thepower of this approach. They engineered a neuroblastoma tumour to express interferon gamma and this induced the expression of MHC-class II on the surface of the vessels supplying the tumour. Theytreated the tumour with the intravenous inoculation of an antibodytargeted against MHC class II and conjugated to ricin. This resulted inrapid haemorrhagic necrosis within the tumour, which becamecompletely necrotic over a period of nine days19 (Figure 2, page 37).

The search for specific tumour vascular targets is an active area ofinvestigation with many groups using transcriptomic analysis to probefor molecular differences between the vessels supplying the tumourand healthy tissues20-23 (reviewed in10). Again, the majority of this workhas been performed using current-generation technologies, howeverNGS is starting to be used to provide a more in-depth analysis of thetumour vessel transcriptome.

Zhuang et al. (2013)24 used a combined approach of both microarrayand RNA-Seq NGS transcriptomic profiling of isolated endothelial cellsfrom lung cancer. It has been reported that there is only moderateconcordance between differentially expressed genes identified by RNA-Seq and by microarray, probably due to the considerable inherentdifferences between the approaches1. Both approaches suffer from high

false positive rates25,26. By using the current- and next-generationtechnologies concurrently, the selection process for candidate genes inlung cancer was streamlined, since the true differences between thehealthy and cancer vessels should be identified by both approaches24.

One approach used to identify novel markers of tumourendothelium is to model the environment to which the tumour vesselsare exposed. The tumour often grows at a rate exceeding the capacityof the vessel bed to expand and supply it sufficiently27. Because of this, the tumour microenvironment is generally highly hypoxic,hypoglycaemic and acidic with poor blood flow. The vessels areadditionally often disordered and poorly structured, leading tobreakages within the vessel wall and opening a thrombogenic surfaceon which clots can form28. This environment has a considerable impact on the expression profile of tumour vessels, creating distinctchanges that can be targeted therapeutically.

Zhang et al. (2012)29 modelled the impact that exposure to thrombinhas on gene expression in pulmonary microvessels, in cancer and otherdiseases by RNA-Seq transcriptomic profiling. This analysis identified150 known genes and 480 known isoforms which were upregulated and2,190 known genes and 3,574 known isoforms downregulated byexposure to thrombin by at least two-fold. Of note however, is that 1.775 previously unknown isoforms were found to be upregulated and12.202 downregulated29, highlighting another considerable advantageprovided by the next-generation approach. As discussed, microarray-based systems rely on a finite number of predesigned probes to assessthe transcript content of a sample, meaning that it is only ever possibleto compare the expression of those genes and isoforms for whichprobes have been generated. On the other hand, by sequencing the

entire sample and providing a readout for eachtranscript encountered, RNA-Seq provides virtuallyunlimited genomic analysis capacity, allowing theidentification of previously unknown genes, isoformsand splice variants1. NGS therefore offers a hugepotential for the identification of previously unknowncandidates for tumour vascular-specific targeting.

Complexity of analysisThe generation of such in-depth genomic data is notwithout its disadvantages. Gigabytes of data areproduced for each sample analysed, making thehandling of large data sets from multiple samples aconsiderable and costly challenge. Additionally,bioinformatic tools to assure sequence quality,conduct sequence alignment and assembly andbiologically interpret the data are in their infancy, and require considerable development in order toallow NGS to fully achieve its potential for genomicand transcriptomic discovery. The use of these toolsto analyse NGS data is costly due to its requirementfor considerable investment in infrastructure, but alsodue to its need for specialist bioinformaticians. Theanalysis process is also labour- and time-intensive.Firstly, the images from the NGS sequencers must beanalysed and converted into sequence reads. Thereads are then quality assessed and aligned to a



Figure 3: A typical workflow for the analysis of RNA-Seq data. Consists of the conversion of raw dataimages to short reads, the quality assessment of the reads, alignment to a reference genome (if available) andproduction of useful data from uniquely mapped reads

reference genome, if one exists. Finally, mapped and unmapped readsare assessed through a process of gene expression profiling1 (Figure 3;page 38). This process of data analysis is considerably more complex andcostly than for array-based systems30.

Therefore, despite the rapidly falling costs of performing NGS, thedifficulties of analysing the data make it an unattractive option for allbut the best-funded research groups, compared with cheap and easilyanalysed array-based systems. This issue must be addressed withinstitutional investment in infrastructure, improvement in the qualityand usability of analysis software and education in how to use it. The most intelligently designed experiments and the best analysis is achieved when researchers themselves can perform it rather thanrelying on specialist bioinformaticians unfamiliar with the research areafor which NGS is being used. Until the usability of NGS analysis softwareis improved to the level at which microarray analysis operates, theutility of NGS to enable scientific discovery will be curtailed.

ConclusionIn conclusion, NGS is an extremely promising approach to enablescientific discovery in the field of tumour vascular biology and in thefuture could transform our ability to both research and treat cancer. The MD Anderson’s genetic profiling of abnormal responders is an earlyexample of the potential of NGS to permit personalised medical care.With time, databases for disease-specific mutations and alterations willbecome more comprehensive and better characterised, allowing NGS of

patient material to become a viable option for clinical as well asscientific evaluation, superseding traditional diagnostic methods,which are limited by their narrow focus and usually only useful for oneintended purpose.

AcknowledgementsThe authors would like to thank Cancer Research UK for financial supportand members of the Bicknell/Heath group for intellectual support.

This article was previously published in European PharmaceuticalReview, Issue 4, 2015.



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5. Kharchenko PV, Tolstorukov MY, Park PJ. Design and analysis of ChIP-seq experiments forDNA-binding proteins. Nature Biotechnology. Nature Publishing Group; 2008 Dec1;26(12):1351–9

6. Hiller D, Jiang H, Xu W, Wong WH. Identifiability of isoform deconvolution from junctionarrays and RNA-Seq. – PubMed – NCBI. Bioinformatics. Oxford University Press; 2009Nov 17;25(23):3056–9

7. Voelkerding KV, Dames SA, Durtschi JD. Next-generation sequencing: from basic researchto diagnostics. – PubMed – NCBI. Clinical Chemistry. American Association for ClinicalChemistry; 2009 Mar 26;55(4):641–58

8. Folkman J, Cole P, Zimmerman S. Tumor behavior in isolated perfused organs: in vitrogrowth and metastases of biopsy material in rabbit thyroid and canine intestinal segment.Annals of Surgery. Lippincott, Williams, and Wilkins; 1966 Sep;164(3):491–502

9. Folkman J. Tumor Angiogenesis: Therapeutic Implications. N Engl J Med. 1971 Nov18;285(21):1182–6

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11. Folkman J. Anti-angiogenesis: new concept for therapy of solid tumors. Annals of Surgery.Lippincott, Williams, and Wilkins; 1972 Mar;175(3):409–16

12. Thorpe PE. Vascular targeting agents as cancer therapeutics. Clin Cancer Res. AmericanAssociation for Cancer Research; 2004 Jan 15;10(2):415–27

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14. Bergers G, Hanahan D. Modes of resistance to anti-angiogenic therapy. Nature PublishingGroup. 2008 Aug;8(8):592–603

15. Subbiah V, Meric-Bernstam F, Mills GB, Shaw KRM, Bailey A, Rao P, et al. Next generationsequencing analysis of platinum refractory advanced germ cell tumor sensitive to Sunitinib(Sutent®) a VEGFR2/PDGFRβ/c-kit/ FLT3/RE... – PubMed – NCBI. J Hematol Oncol.BioMed Central Ltd; 2014;7(1):52

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medicine infrastructure at a major cancer center. – PubMed – NCBI. Journal of ClinicalOncology. American Society of Clinical Oncology; 2013 May 17;31(15):1849–57

17. Bradford JR, Farren M, Powell SJ, Runswick S, Weston SL, Brown H, et al. RNA-SeqDifferentiates Tumour and Host mRNA Expression Changes Induced by Treatment ofHuman Tumour Xenografts with the VEGFR Tyrosine Kinase Inhibitor Cediranib. MüllerR, editor. PLoS ONE. 2013 Jun 19;8(6):e66003

18. Nihei Y, Suzuki M, Okano A, Tsuji T, Akiyama Y, Tsuruo T, et al. Evaluation of antivascularand antimitotic effects of tubulin binding agents in solid tumor therapy. Jpn J Cancer Res.1999 Dec;90(12):1387–95

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20. Herbert JM, Stekel D, Sanderson S, Heath VL, Bicknell R. A novel method of differentialgene expression analysis using multiple cDNA libraries applied to the identification oftumour endothelial genes. BMC Genomics. 2008;9(1):153

21. Masiero M, Simões FC, Han HD, Snell C, Peterkin T, Bridges E, et al. A core human primarytumor angiogenesis signature identifies the endothelial orphan receptor ELTD1 as a keyregulator of angiogenesis. Cancer Cell. 2013 Aug;24(2):229–41

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References

Joseph W. Wragg carried out his undergraduate studies atThe University of York and is currently studying for his PhDat the University of Birmingham. He has published three peer-reviewed articles in the fields of cancer micro environmentand cancer vascular targeting. He has a particular interest inthe use of high-throughput gene expression profiling tech -niques to identify specific genes for cancer targeting.

Roy Bicknell carried out his undergraduate and doctoralstudies at Oxford University and his postdoctoral training atHarvard Medical School. He was formerly Professor ofCancer Cell Biology at Oxford and is currently Professor of Cancer Biology and Genomics at the University ofBirmingham. He has published over 200 peer reviewedarticles in the fields of endothelium and angiogenesis and a particular interest is the difference between tumours and healthy tissue endothelium.

WEBINAR REVIEW: ILLUMINA


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Our keynote speakers were Prof. Aroon Hingorani, Chair of GeneticEpidemiology, and Prof. John Overington, Visiting Professor ofComputational Chemical Biology, both at University College London’sInstitute of Cardiovascular Science. Dr. Anna Gaulton, Senior DataIntegration and Development Officer for ChEMBL at the EuropeanBioinformatics Institute in Hinxton, also participated.

Firstly, Aroon Hingorani guided the listener through the processof drug development: the motivations behind the process, thepotential role for genetic evidence and benefits of genomic supportfor drug target selection and validation. He summarised the series oftasks that must be completed when developing a new drug, includingdisease hypothesis, target selection and molecule development. It iswhen the drug enters the testing phases (proof of concept) thatthings can begin to go wrong, he explained – something that worked perfectly in vitro can fail in vivo. Aroon also discussed why late-stage failure is a major issue in drug development in thecontext of randomised controlled trials and Mendilian randomisationtrials, the latter enabling researchers to distinguish on- from off-target effects.

Next, John Overington moved on to the druggable genome. Hebegan by explaining the different types of drugs that have beenapproved, moving on to discuss how ChEMBL, the world’s largestprimary database of publically-available medicinal chemistry data, is

a valuable tool. John also revealed the finding, from some of his ownresearch, that only ~1% of the genome was found to be a drug target.This discovery is striking since it brings us to question whether thereis something special about current drug targets that predicts theproperties and features of future drug targets. John also delved intothe topic of drug targets across model organisms, highlighting thesimilarities and differences between species, although he warnedthat data are not always correct, since other organisms’ genomeshave not been as well investigated as the human genome.

In the last presentation, Dr. Anna Gaulton introduced a newgenotyping array design created by Illumina. The company found thatexisting arrays either provided sparse coverage of a whole genome, ordense coverage of a few genes; this design offers a single array whichprovides a dense coverage of known and likely drug targets across alldisease areas. This allows for identification of tractable targets anddrug repurposing opportunities. Anna explained the three tiers oftargets for this array and how it provides coverage of the druggablegenome by genotyping platforms. She finished by suggesting thetypes of people who would benefit from the new array, and discussingpotential applications.

To see these presentations, you can go to the Drug Target Reviewwebsite to watch the webinar now.

Genomic support for targetselection and validation indrug development: design of a new genotyping array

This webinar, hosted by Drug Target Review and sponsored by Illumina, provided an insight into the drugdevelopment process. It was broadcast live on the 12th November 2015.

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Since the commercialisation of the first therapeutic mAb in 1986, theyhave represented the largest and fastest-growing class of bio -pharmaceutical products. The development of mAbs has revolutionisedthe pharmaceutical industry, created a multibillion dollar market, andprovided vast opportunities to treat a wide range of diseases fromautoimmune disorders to cancer to viral or bacterial infections. MAbs produced in mammalian cell culture have achieved remarkablepharmaceutical and financial success leading to several blockbustertherapeutic products. However, they are prohibitively expensive,making them unaffordable for the majority (>75% population) ofcountries around the globe1. MAbs produced in mammalian cellcultures require capital-intensive facilities, fermenters, expensivedownstream processing, cold storage and transportation, and sterile

delivery methods2-4. The high cost of this system has encouraged thedevelopment of alternative production systems including yeast, insectcells and plants. While there is much progress being made with thesesystems, the general public has very little knowledge about them andtheir utility in producing mAb drugs for human use.

ZMapp’s full potential revealed in LiberiaEarly in 2014, a new strain of the Zaire species of Ebola virus, one of theworld’s most deadly pathogens, emerged in Guinea, West Africa, andrapidly spread to Liberia, Sierra Leone and Nigeria. It was the largestEbola outbreak in history, infecting 28,512 people with 11,313 fatalities todate5. Out of this total devastation, a dramatic chapter came from thesurvival of two American health aid workers, Dr. Kent Brantly and Nancy

The success story of Mapp Pharmaceuticals’ experimental drug, ZMapp, during last year’s Ebola outbreak highlights thepotential of plant-made monoclonal antibodies (mAbs) as life-saving treatments. Current plant expression systemsoffer features beyond the traditional advantages of eukaryotic protein modification, such as low cost, high scalabilityand increased safety. The development of novel transient expression vectors has allowed mAbs to be produced atunprecedented speed to mitigate potential pandemics. Glycoengineering allows plants to make mAbs with uniquemammalian glycoforms with differential binding to various Fc receptors (FcγRs), providing new utility to makebiobetters with superior potency or safety. Along with ZMapp, several human clinical trials have been conducted.Ultimately, obtaining regulatory approval is key to fulfilling the vast commercial potential of this technology.

PROTEIN EXPRESSION


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The growing potential of plant-mademonoclonal antibodies

Qiang “Shawn” Chen and Huafang LaiArizona State University

Writebol, who contracted Ebola at the Liberian hospital. Brantly’scondition had deteriorated to the point where he thought that he wasdying and called his wife to say goodbye. At this critical moment, he was given an intravenous dose of a little-known, experimental drugcalled ZMapp. What happened next was described as ‘miraculous’ by hisdoctors: Brantly’s breathing became easier, and he was able to take ashower without any assistance by the next morning. Similarly, NancyWritebol’s condition also dramatically improved after receiving twodoses of ZMapp6.

The story of Brantly and Writebol became a media sensation andpeople became intrigued about the nature of ZMapp. The drug is acocktail of three chimeric mAbs made in Nicotiana benthamianaplants (Figure 1). The fact that it is produced in plants is both fascinatingand surprising for most people – how can a drug that might savepeople’s lives come from tobacco, a plant has done so much harm to people’s health?

But plants, especially tobacco and its related Nicotiana plants, havebeen used to produce mAbs for decades, with the first mAb made intobacco in 19897. Despite the complexity of mAb production requiringthe assembly of four polypeptides into a heterotetramer and complexglycosylation, a variety of mAbs and their derivatives have beenproduced in many plant species, such as secretory IgAs, tetravalentmAbs, immune-complex, single-domain fragments, single-chainvariable fragments (scFv), and diabodies. An increasing number anddiversity of MAbs are being produced every year and several of themhave now reached human clinical trials4.

However, until now, these efforts were largely unknown beyond theclose-knit field of plant-made pharmaceuticals (PMPs). The intense

media interest in ZMapp has given the public unprecedented exposureto the fact that PMPs can be life-saving drugs, and an internationalstage that we, who have worked in the PMP field for many years, couldnever have anticipated. We are repeatedly asked by the media andgeneral public – why plants? Why tobacco and how can this work?

Why use plants?The traditional advantages of utilising plants to produce mAbs are theirdemonstrated low cost, high scalability and increased safety2,8, while amajor advantage of plant-production systems is their low-costupstream protein expression9. In contrast to mammalian cell culturesystems, which require capital-prohibitive bioreactors and expensiveculture media, plant mAb production can be easily scaled in

PROTEIN EXPRESSION

Figure 1: Production of ZMapp in tobacco plants

inexpensive green houses. Tobacco plants can be grown in soil orhydroponically in simple mineral solutions to produce a large amountof biomass and millions of seeds per plant for a facile scale-up ofproduction, making high levels of mAbs efficiently and sustainably4.

Plants rarely carry human or animal pathogens, reducing the risk ofcontamination. In addition, plants’ cellular protein machinery can easilyassemble multiple subunits required for the production of mAbs andefficiently perform critical post-translational modifications4.

New innovations in viral vector development provide additionalbenefits for plants to make mAbs. For example, the development of the‘deconstructed’ viral vector system has successfully addressed the challenges of sufficient protein expression levels, consistency andspeed of mAb production in plants10-12.

In contrast to using stable transgenic plants, transient expressionwith deconstructed viral vectors avoids the months-long process of generating and selecting transgenic plants. It can produce up to 5mg mAb per gram of fresh leaf weight (LFW) within two weeks due to the robust transcriptional and translational activities of the plant virus components in the vector11,13. These deconstructed viralvectors are non-competitive, allowing mAbs and other proteins withmore than one hetero-subunit to be expressed and assembled in oneindividual cell11,13,14.

Agrobacterium tumefaciens, a bacterium that naturally transfers a portion of its plasmid into plant cells, is used to deliver thedeconstructed viral vectors to plant cells15-17. Thus, the deconstructedviral vector system provides the flexibility ofnuclear gene expression with the speed andhigh-yield of viral vectors. The rapid and highlevel mAb production capability of the transientexpression systems make them the system ofchoice for obtaining the milligram and gramlevels of mAbs needed for preclinical studies. These vectors have alsobeen adapted for scale-up manufacturing of mAbs in stable transgenicplants, while retaining consistent and high mAbs expression level andtransgene stability2. Therefore, a rapid evaluation of mAb candidatesand transition to a large-scale commercial production can beaccomplished by the combinatorial use of both transient and stableplant expression based on deconstructed viral vectors.

N-glycosylation in plants is similar to human cells yet has minordifferences. These differences were once a cause for concern, since they

may produce plant glycan-specific antibodies that could reducetherapeutic efficacy or cause adverse effects. But glycoengineering ofhost plants via knocking out/down plant-specific glycan genes or/andinserting mammalian glycosylation genes has overcome thischallenge18,19. Extensive studies have shown that mAbs produced in theglycoengineered plants do not contain any plant-specific xylose or 1,3-frucose, but have authentic mammalian N-glycans18,19. Astonishingly,

these mAbs also have a remarkably high degreeof glycan uniformity that cannot be producedby mammalian cells or achieved by in vitrotreatments20-23. A portfolio of plant lines hasbeen developed, with each producing mAbswith a unique mammalian N-glycoform and

providing a potential system for producing mAbs with defined, uniformcarbohydrate moieties based on their functional needs.

Leading candidates for plant-derived mAbsThe early work of plant-made mAbs against Ebola that contributed to the eventual development of ZMapp started at Arizona StateUniversity (ASU). ASU had received several large grants from US federalagencies to develop mAbs or vaccines against Ebola virus24. Due to thedeadly nature of Ebola virus, the use of plant-based mAb therapeuticsand vaccines meets a critical public health challenge and builds animportant biodefense stockpile. Three mAbs: 6D8, 13C6 and 12F6, wereinitially expressed in N. benthamiana plants. Our characterisationshowed that fully assembled mAbs can be detected just two daysfollowing infiltration (DPI) of viral vectors and reached the peakaccumulation at 4 DPI up to 0.5mg/g LFW14 (Figure 2).

Our results also indicate that these mAbs can be produced in lettuce with a comparable accumulation level and rapidity as in N. benthamiana25. These mAbs can be effectively purified to greaterthan 95% purity by simple chromatographic steps14,25,26. The successfuluse of commercially-produced lettuce demonstrated a viable option ofsupplying an almost unlimited amount of cost-effective material forlarge-scale production of anti-Ebola mAb therapeutics. Both tobaccoand lettuce-produced mAbs retain their specific affinity for Ebola GPprotein, suggesting they are functionally active14,25 (Figure 3).

PROTEIN EXPRESSION


Figure 2: Expression of anti-Ebola mAb in N. benthamiana plants with geminiviraldual- or single-vector system. Levels of mAb accumulation in leaves days post vectorinfiltration (DPI) was measured by ELISA

Figure 3: Lettuce (L) or tobacco (T)-produced 6D8 mAb shows specific binding toirradiated Ebola virus

The traditional advantages ofutilising plants to produce mAbs are theirdemonstrated low cost, high scalability

and increased safety

Our colleagues at Mapp Biopharmaceutical demonstrated the in vivo efficacy of these mAbs and in concert with our efforts,demonstrated that a vaccine candidate based on these mAbs alsoprotected mice from a lethal challenge of Ebola . The efficacy of acocktail of the three plant-made mAbs (6D8, 13C6 and 12F6), called MB-003, was subsequently established in nonhuman primates .Remarkably, the mouse and macaque experiments also revealed thatplant-derived individual anti-Ebola mAb and MB-003 have a superiorpotency to their mammalian-produced counterparts, most likely due tothe fact that they carry the homogenous GnGn mammalian glycans, incontrast to the heterogeneous glycan populations of the mammaliancell-derived mAbs . This paved the way for combining one mAb fromMB-003 with two other mAbs to formulate ZMapp and its experimentaluse in Brantly, Writebol and five other human patients during the Ebola outbreak. A subsequent report proved that ZMapp is able torescue 100% of rhesus macaques even when given five days followinglethal Ebola challenge . A clinical trial with the drug is now beingconducted in Liberia by the US Institutes of Health (NIH) to examine itssafety and efficacy.

The rapid nature of plant transient expression can also facilitatenew treatments for cancer. This includes the development of atreatment for non-Hodgkin’s lymphoma (NHL), a group of blood cancersthat have a significant impact on global health. The extremely variablenature of NHL calls for the development of a patient-specificimmunotherapeutic approach through vaccination with the patient’sown idiotype immunoglobulin (Ig). This requires a production platformthat offers the flexibility to rapidly produce tailor-made, patient- andtumour-specific antigens at low cost. These requirements present adifficult obstacle for mammalian cell cultures, but can be readily met byplant transient expression.

In fact, idiotype-specific scFvs of the Ig from the 38C13 mouse B celllymphoma can be expressed in plants within two weeks of vectorinoculation, which protect 90% of mice against a lethal challenge of thesyngeneic 38C13 tumour . Subsequently, 44 human tumour-specificscFvs were rapidly produced, and were shown to recognise the Ig onhuman tumour cells and induce idiotype responses with minimal crossreactivity to irrelevant Igs . The safety and immunogenicity of thesescFv-based personalised vaccines were tested in a phase I clinical trial .The results indicated that all individualised scFv vaccines were well-tolerated by all patients . Greater than 70% of the patients developedhumoral or cellular immune responses . Thirteen of 16 patientsremained alive with a median follow-up of 78 months. These findingssuggest that plant-produced idiotype vaccines are feasible to produce,safe to administer, and provide a viable option for idiotype-specificimmune therapy in follicular lymphoma patients.


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18. M Schahs, R Strasser, J Stadlmann, R Kunert, T Rademacher and H Steinkellner. Productionof a monoclonal antibody in plants with a humanized N-glycosylation pattern. PlantBiotechnol J. 2007; vol. 5, no. 5, pp. 657-663

19. R Strasser, J Stadlmann, M Schahs, G Stiegler, H Quendler, L Mach, J Glossl, K Weterings,M Pabst and H Steinkellner. Generation of glyco-engineered Nicotiana benthamiana for theproduction of monoclonal antibodies with a homogeneous human-like N-glycan structure.Plant Biotechnology Journal, 2008; vol. 6, no. 4, pp. 392-402

20. J He, H Lai, M Engle, S Gorlatov, C Gruber, H Steinkellner, MS Diamond and Q Chen.Generation and Analysis of Novel Plant-Derived Antibody-Based Therapeutic Moleculesagainst West Nile Virus. PLoS ONE, vol. 9, no. 3, pp. e93541 DOI:93510.91371/journal.pone.0093541, 2014

21. H Lai, J He, J Hurtado, J Stahnke, A Fuchs, E Mehlhop, S Gorlatov, A Loos, MS Diamondand Q Chen. Structural and functional characterization of an anti-West Nile virus monoclonalantibody and its single-chain variant produced in glycoengineered plants. PlantBiotechnology Journal, 2014; vol. 12, no. 8, pp. 1098-1107, 2014

22. J He, H Lai, C Brock and Q Chen. A Novel System for Rapid and Cost-Effective Productionof Detection and Diagnostic Reagents of West Nile Virus in Plants. Journal of Biomedicineand Biotechnology, 2012; vol. 2012, no. 10.1155/2012/106783, pp. 1-10

References

Plant-produced anti-WNV E16 mAb glycovariants (pE16 and scFv-FcpE16) have no or greatly reduced risk of ADE compared with mammalian cell-produced E16 mAb (mE16)

The availability of a collection of plant lines that produce mAbs withuniform and distinct mammalian glycoforms also offers opportunitiesto address the safety issue of mAb therapeutics against flaviviruses,which are prone to the development of so-called antibody-dependentenhancement of infection (ADE). ADE may render anti-flavivirus mAb-treated subjects more susceptible to infection. ADE occursbecause sub-neutralising concentrations of antibodies (includingtherapeutic mAbs) form complexes with the infecting flavivirus thatbind to FcγR-bearing cells, resulting in increased virus uptake andinfection35. To address this major impediment, our laboratory hasdeveloped a plant-derived mAb (E16) against West Nile virus (WNV) withvarious unique N-glycoforms20-23. Our results indicate that a single dose of plant-made E16 protected mice from lethal infection of WNV,even given four days post infection23. Furthermore, in contrast to mammalian cell-made E16, all plant E16 glycovariants lost ADE activity on CD32A-expressing K562 cells (Figure 4; page 44). Thus,mammalian forms of glycans carried by plant-produced E16 mAb do notinduce the development of ADE, consistent with their in vitro bindingpattern as shown by SPR20. These results demonstrate the potential of plant-made mAbs and their variants to be used as therapeuticsagainst ADE-prone viruses.

ConclusionsPlant expression systems not only offer the traditional advantages ofproper eukaryotic protein modification, low cost, high scalability andincreased safety, but they also allow the production of mAbs atunprecedented speed to control potential pandemics or with specificglycoforms for superior potency or safety. These advantages offer plantsas a superior alternative production system for mAb production. Plants can be used to produce mAb biosimilars because of their large

capacity to rapidly generate mAb at low cost, but can also be exploredto produce safer and more effective mAb biobetters due to theflexibility of producing mAbs with specific and unique mammalianglycoforms that preferentially bind to various FcγRs.

The approval of the first PMP by the US FDA for treating Gaucher’sdisease has established the regulatory pathway and warmed up theinterests of large pharmaceutical companies in the PMPs, fueling newhopes for the field36,37. The successful story of ZMapp has sparked new interests and promoted several large government investments toexpand the capacity of producing mAbs from plants under cGMPregulations38-40. We speculate that we should see the regulatory approvalof the first few plant-made mAb-based therapeutics or vaccines withinthe next decade.

AcknowledgementThe authors thank J. Caspermeyer for the critical reading of themanuscript.

PROTEIN EXPRESSION


23. H Lai, M Engle, A Fuchs, T Keller, S Johnson, S Gorlatov, MS Diamond and Q Chen.Monoclonal antibody produced in plants efficiently treats West Nile virus infection in mice.Proceedings of the National Academy of Sciences of the United States of America, 2010; vol.107, no. 6, pp. 2419-2424

24. C Arntzen. Plant-made pharmaceuticals: from ‘Edible Vaccines’ to Ebola therapeutics. Plant Biotechnology Journal. 2015; vol. 13, no. 8, pp. 1013-1016

25. H Lai, J He, M Engle, MS Diamond & Q Chen. Robust production of virus-like particles andmonoclonal antibodies with geminiviral replicon vectors in lettuce. Plant BiotechnologyJournal, 2012; vol. 10, no. 1, pp. 95-104

26. A Fulton, H Lai, Q Chen and C Zhang. Purification of monoclonal antibody against EbolaGP1 protein expressed in Nicotiana benthamiana. Journal of Chromatography A. 2015; vol.1389, no. 0, pp. 128-132

27. L Zeitlin, J Pettitt, C Scully, N Bohorova, D Kim, M Pauly, A Hiatt, L Ngo, H Steinkellner, KJWhaley & GG Olinger. Enhanced potency of a fucose-free monoclonal antibody beingdeveloped as an Ebola virus immunoprotectant. Proceedings of the National Academy ofSciences of the United States of America. 2011; vol. 108, no. 51, pp. 20690-20694

28. W Phoolcharoen, JM Dye, J Kilbourne, K Piensook, WD Pratt, CJ Arntzen, Q Chen, HSMason and MM Herbst-Kralovetz. A nonreplicating subunit vaccine protects mice againstlethal Ebola virus challenge. Proceedings of the National Academy of Sciences of the UnitedStates of America. 2011; vol. 108, no. 51, pp. 20695-20700

29. W Phoolcharoen, SH Bhoo, H Lai, J Ma, CJ Arntzen, Q Chen and HS Mason. Expression ofan immunogenic Ebola immune complex in Nicotiana benthamiana. Plant BiotechnologyJournal. 2011; vol. 9, no. 7, pp. 807-816

30. GG Olinger, J Pettitt, D Kim, C Working, O Bohorov, B Bratcher, E Hiatt, SD Hume, A K Johnson, J Morton, M Pauly, KJ Whaley, CM Lear, JE Biggins, C Scully, L Hensley & LZeitlin. Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodiesprovides protection in rhesus macaques. Proceedings of the National Academy of Sciences ofthe United States of America, 2012

31. X Qiu, G Wong, J Audet, A Bello, L Fernando, JB Alimonti, H Fausther-Bovendo, H Wei, J Aviles, E Hiatt, A Johnson, J Morton, K Swope, O Bohorov, N Bohorova, C Goodman, D Kim, MH Pauly, J Velasco, J Pettitt, GG Olinger, K Whaley, B Xu, JE Strong, L Zeitlin andGP Kobinger. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp.Nature, 2014; vol. 514, no. 7520, pp. 47-53

32. AA McCormick, MH Kumagai, K Hanley, TH Turpen, I Hakim, LK Grill, D Tuse, S Levyand R Levy. Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants. Proceedings of the National Academy ofSciences of the United States of America, 1999, vol. 96, no. 2, pp. 703-708

33. AA McCormick, SJ Reinl, TI Cameron, F Vojdani, M Fronefield, R Levy and D Tuse.Individualized human scFv vaccines produced in plants: humoral anti-idiotype responses invaccinated mice confirm relevance to the tumor Ig. Journal of Immunological Methods. 2003;vol. 278, no. 1-2, pp. 95-104

34. AA McCormick, S Reddy, SJ Reinl, TI Cameron, DK Czerwinkski, F Vojdani, KM Hanley,SJ Garger, EL White, J Novak, J Barrett, RB Holtz, D TusÃ© & R Levy. Plant-producedidiotype vaccines for the treatment of non-Hodgkin's lymphoma: Safety and immunogenicityin a phase I clinical study. Proceedings of the National Academy of Sciences of the UnitedStates of America. 2008; vol. 105, no. 29, pp. 10131-10136

35. DM Morens. Antibody-dependent of enhancement of infection and the pathogenesis of viraldisease. Clin Inf Dis. 1994; vol. 19, pp. 500-512

36. Q Chen & H Lai. Plant-derived virus-like particles as vaccines," Human Vaccines &Immunotherapeutics, 2013; vol. 9, no. 1, pp. 26-49

37. K Traynor. Taliglucerase alfa approved for Gaucher disease. Am J Health Syst Pharm. 2012;vol. 69, no. 12, pp. 1009

38. BR Holtz, BR Berquist, LD Bennett, VJM Kommineni, RK Munigunti, EL White, DCWilkerson, K-YI Wong, LH Ly & S. Marcel. Commercial-scale biotherapeuticsmanufacturing facility for plant-made pharmaceuticals. Plant Biotechnology Journal. 2015;vol. 13, no. 8, pp. 1180-1190

39. JKC Ma, J Drossard, D Lewis, F Altmann, J Boyle, P Christou, T Cole, P Dale, CJ vanDolleweerd, V Isitt, D Katinger, M Lobedan, H Mertens, MJ Paul, T Rademacher, M Sack,PAC Hundleby, G Stiegler, E Stoger, RM Twyman, B Vcelar & R Fischer. Regulatoryapproval and a first-in-human phase I clinical trial of a monoclonal antibody produced intransgenic tobacco plants. Plant Biotechnology Journal. 2015; vol. 13, no. 8, pp. 1106-1120

40. H Lai & Q Chen. Bioprocessing of plant-derived virus-like particles of Norwalk virus capsidprotein under current Good Manufacture Practice regulations. Plant Cell Reports. 2012; vol.31, no. 3, pp. 573-584

Dr. Qiang ‘‘Shawn’’ Chen, PhD, is a Professor in theCenter for Infectious Diseases at Arizona State University.Dr. Chen spent more than ten years in the biotechnology andpharmaceutical industry directing research in therapeuticprotein development in both plant and mammalian cellculture systems. Prior to joining ASU, Dr. Chen was theAssociate Director of the Division of Protein Chemistry at

Cardinal Health and a Sr. Scientist at Monsanto. Email Dr. Chen at:[email protected]

Huafang Lai is a research scientist in Dr. Chen’s laboratory.Their lab focuses on optimising the expression and assemblyof monoclonal antibodies and designing novel mAb fusion proteins to enhance their targeting and efficacy. Email Huafang at: huafang.lai@ asu.edu

In an industry of discovery and development, federal regulation from avariety of United States’ (US) departments and agencies is expected.Innovators in health care products and companion diagnostic testsexpect a degree of regulatory oversight, but rarely expect that everystate and jurisdiction may manage that oversight differently. Productregulation is most often a task assigned to the federal government,because these products enter into interstate commerce. The regulationof people, in this case health care providers, is reserved to the states

and jurisdictions under the 10th Amendment to the US Constitution.Navigating the pharmacist regulations for all 50 states and additionaljurisdictions can seem nearly impossible. This article will review thecommonalities of professional regulation amongst all states andjurisdictions, as well as offer areas of difference to be considered bydrug and point-of-care testing developers and manufacturers.Projected changes in regulation and opportunities for industrycollaboration will be highlighted.

A 2010 overview article on companion diagnostics, compiled by authors Stephen Naylor and Toby Cole, commentedon the need for the companion diagnostic to fit into the logistics of the testing laboratory1. The intervening five yearshave shown that patients are demanding more rapid and more convenient testing for diagnosis, screening andpharmacotherapy optimisation. Patients are expecting to receive these home-approved tests and screens in thecommunity pharmacy. Home-use tests and Clinical Laboratory Improvement Amendment (CLIA)-waived tests arenot synonymous2, but both have been considered point-of-care tests (POC) by various authors3. For the purposes ofthis article, POC is defined as: performing a test outside of a traditional laboratory that produces a reliable resultrapidly to aid in disease screening, diagnosis and/or patient monitoring4. Unlike tests approved for ‘home use’, CLIA-waived tests are intended to be used by individuals trained in properly conducting and interpreting the tests despitetheir classification as ‘simple to use’.

COMPANION DIAGNOSTICS

Challenges in the development of pharmacist-based point-of-care tests

Allison M. Dering-Anderson and Meagan DoyleUniversity of Nebraska College of Pharmacy


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Each state and jurisdiction (Washington DC and US Territories) willhave a definition of the practice of pharmacy called a ‘scope of practice’and codified by state law. Each scope of practice will list thoseprofessional tasks and functions requiring licensure as a pharmacist.Most states have a process whereby any professional act not expresslylisted in the law will require a ruling by a governing body – a Board ofPharmacy, a Board of Public Health, a Department of ProfessionalLicensing, etc. In a few states, the law is absolute and any task notspecifically listed in law is disallowed.

Pharmacists’ responsibilities varyAll pharmacists, regardless of state and jurisdiction, have theprofessional ability to dispense drugs, to counsel patients, and toprovide comprehensive care with regard to pharmacotherapy. Likewise,pharmacists in each state are allowed to administer vaccines, butlimitations on this service vary greatly. By wayof example, in Nebraska, pharmacists areallowed to administer all vaccines to patients of any age; in New York pharmacists areprohibited from vaccinating children. There are similar variances among the state regula -tions that allow for pharmacists to conduct POC in communitypharmacies. Many states’ laws and regulations are moot regard-ing pharmacist-based POC; others are progressive on the issue, writinginto the pharmacists’ scope of practice legislation the authority toorder and conduct lab tests and to make pharmacotherapy decisionsbased on the results.5 Kansas not only recognises this process as apharmacist’s professional function, but has declared it to beunprofessional conduct to order or run unnecessary tests6.

A final element to the functionality of companion diagnostics incommunity pharmacies, where the pharmacist or technician willconduct the test and the pharmacist will professionally act upon the result, is the necessity for the result to be actionable. It is theprofessional action taken by the pharmacist that is regulated differentlyin nearly every state and jurisdiction. This action, the seamless use ofthe test results, is accomplished in a variety of ways depending uponthe state or jurisdiction. In some states, the pharmacist will work underprotocol or collaborative agreement with a single diagnostician orprescriber. In others, there will need to be collaboration with multiplediagnosticians or prescribers. Advanced practice pharmacists in yetother states will be able to make these decisions independently7.

If a true ‘companion’ diagnostic is developed, pharmacists willreceive a single prescription that includes both the diagnostic test andthe drug. This combination will by-pass the collaboration requirementsin many states. There will likely be pharmacotherapeutic agents wherea test will be required prior to dispensing a refill. Examples in practicetoday include a pregnancy test for an isotretinoin product or a whiteblood cell analysis for clozapine-containing drugs. Currently, patientscannot undergo these tests in a community pharmacy. Adherence toprescribed therapy could be improved if CLIA-waived tests, appropri-ate for this monitoring, were available. Pharmacists are currently testing for and treating influenza and group A streptococcus8,9,10. For many years, they have tested international normalised ratio andadjusted anticoagulant doses, tested haemoglobin A1c and adjustedhypoglycaemics and insulin doses11,12,13. The pharmacies offering these

services have an existing CLIA certificate of waiver. Over 1,350pharmacists and pharmacist interns have completed a certificate-training program in Community Pharmacy Based Point of Care Testing14,indicating that community pharmacy is well positioned to participate inthis process for an expanding number of companion diagnostics.

In addition to the scope of practice laws and differences inauthority, via collaboration or independent authority, there will be astate agency or department charged with the regulation of the CLIA andcertificates of waiver. Research by Sallach shows that these agenciesand departments are also varied15. In the vast majority of statesreporting, the community pharmacy needs only to follow the federallaw governing CLIA and certificates of waiver. Four (8%) of the reportingstates and jurisdictions (51/57) note having state laws in addition tofederal requirements: California reported more stringent staterequirements, Maine reported expanded requirements, Nevada

reported having additional personnel restric -tions, New York expressly limits those peoplewho may perform CLIA-waived tests andcurrently excludes pharmacists. Most USTerritory’s laboratory regulations are managedby state agencies: American Samoa uses the

Hawaii State Agency; Guam and Saipan use the regional office in SanFrancisco, California; and the Virgin Islands use the New York regionaloffice. Interestingly, the Boards of Pharmacy do not always agree withthe information provided by the state or jurisdiction laboratory division.With this confusion, it is often difficult for the innovators anddevelopers of POC to know when to approach community pharmacistsas collaborators.

The decision to develop tests and companion diagnosticsappropriate for use in a community pharmacy will be based on several variables16: 1. Is the patient likely to be receiving the prescription or over-the-

counter drug being monitored at a community pharmacy? If thedrug is used only during surgery, for instance, the communitypharmacist will not be a reasonable resource. If, however, the drugbeing monitored treats a chronic condition, the patient is likelygetting those prescriptions filled in the community pharmacy.

2. What is the analyte for the test and what is the volume? Many statesand jurisdictions require separate licensure or credentialing as aphlebotomist, therefore large volume blood draws, even for a CLIA-waived tests, will not be possible in the pharmacy. There are somesamples that cannot be easily collected in the communitypharmacy – faecal testing would be difficult, for instance. There areCLIA-waived tests that can be performed on patient self-collectedsamples, but the collection has to be easy and straightforward.

3. The test has to meet the standard of rapid; meaning results in lessthan 60 minutes. Results in 10 minutes would be optimal.

4. The test and any machinery needed to read the test must have asmall footprint and not require specialised storage conditions. Incommunity pharmacies, professional space is limited. The optimal‘reader’ would read multiple tests and be safe to leave in thepatient counseling space unattended while running. This meansthe patient cannot interfere with the test and cannot be exposed to the analyte nor the reagent used.

5. If there is no mechanical assistance with reading a test, the results



If a true ‘companion’ diagnostic isdeveloped, pharmacists will receive a

single prescription that includes both thediagnostic test and the drug

must be visible and accurate for a length of time to allow thepharmacist or technician to walk away from the test without a needto return at exactly the right moment for an accurate reading.

There will be instances where a CLIA-waived test is not available.Considering the community pharmacist may still be an option. Today, pharmacies are screening for Hepatitis C and HIV infections17,18.These are not diagnostic tests, but the pharmacists are serving to get the patients with reactive tests into thehealth care system. Community pharmacies arealso reasonable sample collection sites.Rebecca Chater, RPh, MPH, FAPhA, an executivehealthcare strategist at Ateb Inc, notes thatpharmacogenomic testing services offer an opportunity for communitypharmacies to improve patient care and avoid drug problems whengenomic variances could compromise a drug’s efficacy and/or safety19.

Transitioning timesThe laws and regulations around pharmacist-led POC are changingrapidly. This is due to: patients demanding accessible and convenientcare; an expanding shortage of primary care providers20,21; and thetraining necessary for pharmacists to order, conduct and evaluate testsbecoming a mandatory skill in colleges and schools of pharmacy in theUS22. Many of today’s frustrations with variations in regulation will startto resolve as the regulators embrace this professional service providedby pharmacists. This transition is very similar to the changes inregulation seen when pharmacists became vaccinators, however therecognition of POC is moving even more quickly23.

The creating of companion diagnostics suitable for CLIA-waived POCwill hasten this transition and will improve patient convenience andultimately patient adherence to pharmacotherapy. Moving requiredmonitoring tests and pre-dose tests to the pharmacy will enable thepatient to receive needed pharmacotherapy in a much more timelyfashion. The American Pharmacists Association, the national US

association representing all pharmacists, will consider new policy on POC during their annual meeting in March 201624. This, too, willincrease the recognition that pharmacists can serve a pivotal role inpatient care using companion diagnostics. Laws and regulations willmirror this recognition.

It is essential that the pharmaceutical industry considers POC earlyin the drug development and safety monitoring processes. Whencompanion diagnostics and new drugs are presented to federal

regulators as a ‘package deal’, the recognitionwill expand from state regulation of pro -fessionals to the federal government regulationof product, where this industry has much moreexperience and comfort. States and Territories

will still have the ultimate authority to regulate their professionals, butthey are unlikely to craft or maintain laws and regulations that willdecrease patient access to care or patient convenience. Working withcommunity pharmacists on the front line of drug dispensing and therecognised pharmacotherapy experts on the health care team makessense for all involved.



Dr. Ally Dering-Anderson, Clinical Assistant Professor atthe Department of Pharmacy Practice, University ofNebraska College of Pharmacy, is a community pharmacist.Ally was the 2015 College of Pharmacy Teacher of the Year.She has spoken nationally and internationally on communitypharmacy topics. Her interests include point-of-care testing,excellence in education, medical marijuana and public

health policy. Her correspondence details are: Dr. Ally Dering-Anderson;University of Nebraska College of Pharmacy; 986045 Medical Center;Omaha, NE 68198-6045

Meagan Doyle is a senior-level pharmacy student at theUniversity of Nebraska College of Pharmacy. She is the past-president of the Student Alliance for Global Health at UNMC and a coordinator for the Bridge to Carementoring program which she presented at the GlobalHealth Conference Midwest 2015. Her career interestsinclude cardiology.

1. Overview of Companion Diagnostics in the Pharmaceutical Industry; Stephen Naylor andToby Cole; Drug Discovery World; Spring 2010

2. Title 42 CFR Chapter IV Subchapter G Part 493; Public Health, Laboratory Requirements

3. CLIA Waived Tests; CDC; https://wwwn.cdc.gov/clia/Resources/WaivedTests/; accessed 20October 2015

4. Gubbins PO, et al. Point-of-care testing for infectious diseases: Opportunities, barriers, andconsiderations in community pharmacy J Am Pharm Assn. 2014;54:163-71

5. 2012 Minnesota Statutes 1501.01 Subdivision 27

6. 2006 Kansas Code – 65 – 1626 (hh)(10)

7. 2015 California Statue: Section 4016.5

8. Klepser D, et al. Time and motion study of influenza diagnostic testing in a communitypharmacy. Innovations in Pharmacy. 2014;5:1-8

9. Klepser DG, et al. Health Care Resource Utilization and Cost for Influenza-like Illness Among Midwestern Health Plan Members. J Manag Care Spec Pharm.2015;21(7):568-73

10. Collins S. Point-of-Care testing: Emergency market, opportunity for pharmacists; PharmacyToday; APhA; https://www.pharmacist.com/point-care-testing-emerging-market-opportunity-pharmacists; accessed October 2015

11. Witt DM, et al. Effect of a Centralized Clinical Pharmacy Anticoagulation Service on theOutcomes of Anticoagulation Therapy; Chest. 2005;127(5):1515-1522doi:10.1378/chest.127.5.1515

12. Developing Trends in Delivery and Reimbursement of Pharmacist Services; Avalere HealthLLC; November 2015; pp 3

13. Bunting BA. Training Pharmacists for Disease Management Roles; Pharmacy Times; 1 October, 2007 (online)

14. Sara Roszak, Director of Research programs, National Association of Chain Drug StoresFoundation; personal correspondence; October 2015

15. Rory Sallach Ruma, PhD, PharmD. Point-of-Care in Community Pharmacies; podiumpresentation; Asheville, NC; January 2015

16. Kenneth Hohmeier. University of Tennessee; Preliminary research data; November 2015

17. Darin KM, et al. Pharmacist-provided rapid HIV testing in two community pharmacies.J Am Pharm Assn. 2014:e7-e14. doi:10.1331/JAPhA.2015.14070

18. Heath D, et al. Screening for human immunodeficiency virus and hepatitis C virus incommunities: an emerging opportunity. Michigan Pharmacist. 2012;50:40-3

19. Hesselgrave B. Pharmacogenomics: Right med, right dose, every time; Drug Topics;October 2015; vol 159; No 10: pp22

20. Projecting the Supply and Demand for Primary Care Practitioners Through 2020;http://bhpr.hrsa.gov/healthworkforce/supplydemand/usworkforce/primarycare/; accessedSeptember 2015

21. Prognosis E-New; Wayne State University School of Medicine; Michigan Academy ofFamily Physicians elects residency director Pierre Morris, MD, to leadership role; 20 August 2014

22. Accreditation Council for Pharmacy Education Accreditation Standards and Guidelines for2016

23. Higginbotham S, Stewart A, Pfalzgraf A. Impact of a pharmacist immunizer on adultimmunization rates; J Am Pharm Assoc (2003) 2012;52:367-371. doi:10.1331/JAPhA.2012.10083

24. Theresa Tolle, BSPharm, FAPhA; Speaker of the APhA House of Delegates 2015-2016;personal correspondence; July 2015

References

Pharmacists can serve a pivotal role in patient care using

companion diagnostics

InformationPodium presentations, case studies, posters, tutorials, exhibits, shortcourses, user perspectives, career services, panel discussions and off-site facility tours make SLAS2016 five days of high-volume, high-impactinformation catered to the needs of today’s life science professional.

InnovationWhat’s new in scientific innovation is at SLAS2016; from breakthroughproducts to breakthrough applications of established technology,SLAS2016 will showcase what is coming – and what has arrived – andhow that innovation can drive value.

InspirationBe it in a hallway conversation with a fellow attendee or hearing a casestudy from a podium, SLAS2016 will be serving up a full dose of ‘aha’moments. As a community united by pioneering new scientific horizonsusing technology, and with a programme curated by a committee ofpracticing researchers, SLAS2016 will leave attendees inspired.

Scientific programmeThe scientific programme is the cornerstone of SLAS2016. Compiled by ateam of practicing scientists and scientific technology users, theprogramme represents original research, case studies and innovativetechnology advancement that will provide inspiration on a year-roundbasis. In-depth educational tracks include: � Advances in Bioanalytics, Biomarkers and Diagnostics;� Assay Development and Screening;� Automation and High Throughput Technologies;� Cellular Technologies;� Drug Target Strategies;� Informatics; and� Micro/Nano Technologies.

See complete details on the scientific programme, includingpresentation and session titles, abstracts and presenter information onthe website.

The exhibitionThe SLAS2016 exhibition is an all-in-one venue allowing you to see thelatest technologies and to visit with product experts and developersfrom more than 300 leading multinational providers of scientifictechnologies. From robotics to reagents, the SLAS2016 is a unique

opportunity to see, hear, touch and feel breakthrough innovation.Nowhere else will you have such convenient access to a wealth of newtechnology, product expertise and user experiences.

Visit SLAS2016.org for the latest list of exhibiting companies,product descriptions, an exhibition floorplan and a schedule of eventsin the exhibition.

NetworkingA hallmark of the SLAS experience is intelligent network building. AttendSLAS2016 and reap the benefits of being a part of the SLAS community,not only during the conference, but for the other 360 days of the year.Networking activities at SLAS2016 include daily meals and receptions inthe exhibition, evening functions and programming, a fun run withfellow attendees, Special Interest Groups (SIGs), special programmingfor students, early career professionals and international guests, andmuch more.

SLAS2016 last night celebrationThe final evening of SLAS2016 will be one to remember as conversationscontinue aboard the USS Midway – one of America’s longest-serving andmost impressive aircraft carriers. From stem to stern, the entire ship willbelong exclusively to the SLAS community. Attendees will explore the 20 stories high, 1,000 ft. long, 64,000 ton, 212,000 horsepower aircraftcarrier while meeting old friends and making new professional contacts.

Career connectionsSLAS2016 provides a host of unique resources to help you accelerateyour career and distinguish yourself in a competitive job market. Careercoaching, career workshops, one-on-one mentoring and individual anddiscreet resume reviews are among the complimentary servicesprovided to SLAS2016 attendees.

Registration SLAS2016 is pleased to offer significant registration discounts toadvance registrants, for groups of registrants from the same industryorganisation, for professionals from academic institutions andgovernment agencies, and for students. Advance discounts are availableuntil the 18th December 2015.

SHOW PREVIEW

Date: 23-27 January 2016 · Location: San Diego, California, USAMore information: www.SLAS2016.org

SLAS is proud to present the Society’s Fifth Annual Conference and Exhibition. Bringing together the best and brightest minds vested in developing and using technology for life science R&D, SLAS2016 will serve as the epicenter of science and technology for five days this January.

Michael M. Gottesman (left), M.D.,

National Cancer Institute and

Adam Diedrich Steltzner (above), NASA

Jet Propulsion Laboratory, will be hosting

keynote presentations at SLAS 2016

Visit Drug Target Review at Booth 337

German engineering at its best.

The New Gold Standard HTS Reader

PHERAstar® FSX

Find out more at SLAS 2016. www.bmglabtech.com

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Documents

From the publishers of - Donutsdocshare01.docshare.tips/files/29550/295502902.pdfthe findings of the latest Review on Antimicrobial Resistance report published by O’Neill and colleagues: