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Ask any major pharmaceutical company whatconstitutes an ideal drug and the answerwould probably include the words ‘specificity,affinity, solubility, stability and safety’ alongwith the phrases ‘cheap to manufacture, easyto formulate, simple to deliver and the rightpharmacokinetic profile.’ Ironically, manydrugs on the market fail to deliver in one ormore of these areas because of their sub-opti-mal biophysical makeup. Even blockbusterbiologics, such as therapeutic antibodies1,suffer from drawbacks, such as the require-ment for an expensive mammalian cell pro-duction system and the need for intravenous,intramuscular or subcutaneous injection(with molecular weights of around 150,000,they are too large to be administered by anyother route). Clearly, there is room forimprovement. In this issue, Binz et al.2

describe a natural scaffold, ankyrin repeatprotein, that has promising biophysical prop-erties for therapeutic application. Ankyrinrepeats are one of several new types of scaf-fold being developed for a new generation ofprotein therapies.

An ideal drug would have the followingqualities: it would have very high affinity andexquisite specificity for its target; it could be manufactured by the bucket-load in bacte-ria or yeast; it would be both incredibly solu-ble and remarkably stable; it could bedelivered to any part of the human body byany route of administration; and, once there,it would hang around long enough to havethe desired therapeutic effect. Achieving allthese goals has been particularly difficult forprotein drugs.

Currently, protein drugs come in all shapesand sizes: some are recombinant human pro-teins (for instance, insulin, growth hormone

and erythropoietin), others are monoclonalantibodies (for instance, Remicade (inflix-imab; Johnson & Johnson, Kenilworth, NJ,USA), Rituxan (rituximab; Genentech; S. SanFrancisco, CA, USA) and Erbitux (cetuximab;ImClone, New York, NY, USA) and still othersare viral or bacterial proteins used as vaccinesto elicit a specific immune response. Naturedid not evolve proteins for manufacture exvivo. For this reason, many human proteinsproduced in recombinant form are difficult tomanufacture and some cannot be expressedat all in microbial cell culture. Furthermore,the serum half-life and tissue distribution ofendogenously expressed proteins is carefullycontrolled in vivo to optimize their biologicalactivity. Most human proteins are not

designed to be administered from outside thebody. Recombinant proteins therefore tend tobe rapidly cleared and thus require frequentinjection (thus, the growing interest inextending the serum half-life by, for example,polyethylene glycol conjugation).

Antibodies have proved useful as humanprotein therapeutics because they exhibit afavorable pharmacokinetic profile. After asingle injection, they can persist for a longtime in the bloodstream, maintaining theirbiological activity for several weeks. However,antibodies have also evolved to be secretedfrom mammalian cells and, for a variety ofreasons, cannot be expressed in yeast or bac-terial cell culture.

Given the limitations of current proteintherapies, scientists are starting to developmore tailored approaches to drug designwhereby you first assemble a list of the vari-ous properties you want the drug to have andthen engineer a drug with precisely thoseproperties. Over the past three years, severalnew biotech companies have been set up toexploit the use of ‘well-behaved’ human pro-teins as scaffolds to create a range of designerprotein drugs that have improved therapeuticproperties (see Table 1). This approach pro-ceeds through the following steps: first, chosea human protein that is well expressed in bac-teria and/or yeast and has good biophysicalproperties (solubility, stability and others);second, create a repertoire by introducingdiversity into the loop regions of the givenscaffold, preferably in a way that does not dis-rupt the overall structure of the protein; third,

Ian M. Tomlinson is Chief Scientific Officer ofDomantis Limited, 315 Cambridge SciencePark, Cambridge CB4 0WG, UK.e-mail: ian.tomlinson@domantis.com

Next-generation protein drugsIan M Tomlinson

Ankyrin repeats generate high-affinity protein binders with biophysical properties that may favor therapeutic applications.

NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 5 MAY 2004 521

Ribosome display selection forhigh-affinity binders tomaltose-binding protein

Ankyrin repeat protein

Maltose-bindingprotein

Ankyrinrepeatprotein

Figure 1 All in a bind. Binz et al. randomized 6of the 33 amino acids (red side chains) in threeankyrin repeats (dark blue) and, using ribosomedisplay, isolated a range of nanomolar binders tomannose-binding protein. The co-crystal structureconfirms the predicted binding of the engineeredankyrin repeat protein to the mannose-bindingprotein target.

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use a genotype-phenotype display system(such as phage3 or ribosome display4) toselect a range of binders to a given therapeutictarget; and fourth, use some form of screen toidentify those leads that have the desired bio-logical activity. If all goes according to plan,the outcome should be a protein that has allthe desirable biophysical properties of theparental scaffold and the required potency forthe therapeutic target.

Of course, it’s not always that straightfor-ward. In many cases, the mutations intro-duced to enable binding to the targetcompromise the biophysical propertiesand/or the three-dimensional structure of theparental scaffold. In some cases, it may noteven be possible to achieve the desiredpotency using such a small binding footprintto the given target. And of course each start-ing scaffold has its unique pros and cons—intracellular or extracellular expression, diff-erent binding sites for purification, immuno-genicity and so on. Some scaffolds may haveintrinsically long serum half-lives5, whereasothers may show unusual properties, such asthe ability to refold reversibly after heatdenaturation6.

In the present paper, Binz et al. focus on theuse of one particular scaffold, based onankyrin repeats, to generate binders with bio-physical properties designed for therapeuticapplication. Ankyrins are proteins, first iso-lated in mammalian erythrocytes, involved inthe targeting, mechanical stabilization andorientation of membrane proteins to special-ized compartments within the plasma mem-brane and endoplasmic reticulum. Naturalankyrin repeat proteins consist of many 33-amino-acid modules, each comprising a β-turn and two anti-parallel α-helices7. They donot contain any disulfide bonds and thereforecan be expressed at very high yields in thebacterial cytoplasm. They also seem to beboth highly soluble and stable.

The approach used by Binz et al. ran-domizes 6 of the 33 amino acids in each of

two or three ankyrin repeats. The diversitythereby generated (12 or 18 randomizedresidues) is sufficient to isolate a range ofnanomolar binders to mannose-binding protein using ribosome display, all of whichhave the desirable biophysical properties of the parental ankyrin scaffold. Import-antly, the authors also showed, by co-crystal-lization, that the selected binders have thesame structural fold as the parental scaffold.Although the authors have yet to demon-

strate in vivo efficacy with an engineeredankyrin repeat protein, the libraries they havecreated should be a valuable resource for the isolation of therapeutically relevant leadsthat are both well expressed and highly stable.

Undoubtedly, there is a big drive for thedrugs of the future to be much easier to man-ufacture and administer to patients. Theymust also be highly efficacious with few, ifany, side effects. By wiping the slate clean anddesigning potent drugs based on human pro-tein scaffolds with good biophysical proper-ties, we may find that the ideal drug is closerthan ever before.

1. Reichert, J.M. Nat. Biotechnol. 19, 819–822 (2001).2. Binz, H.K. et al. Nat. Biotechnol. 22, 575–582

(2004).3. Scott, J.K. & Smith, G.P. Science 249, 386–390

(1990).4. Mattheakis, L.C., Bhatt, R.R. & Dower W.J. Proc. Natl.

Acad. Sci. USA 91, 9022–9026 (1994).5. Ali, S.A., Joao, H.C., Hammerschmid, F., Eder, J. &

Steinkasserer, A. J. Biol. Chem. 274, 24066–24073(1999).

6. Jespers, L., Schon, O., James, L.C., Veprintsev, D. &Winter, G. J. Mol. Biol. 337, 893–903 (2004).

7. Sedgwick, S.G. & Smerdon, S.J. Trends Biochem. Sci.24, 311–316 (1999).

522 VOLUME 22 NUMBER 5 MAY 2004 NATURE BIOTECHNOLOGY

Table 1 Selected companies using human proteins as scaffolds to create next-generation drugs

Company Protein scaffold

BioRexis (King of Prussia, PA, USA) Transferrin

Borean Pharma (Aarhus, Denmark) C-type lectins

Compound Therapeuticsa (Waltham, MA, USA) Trinectins

Domantis (Cambridge, UK) Domain antibodies

Dyax (Cambridge, MA, USA) Kunitz domains

Pieris ProteoLab (Freising-Weihenstephan, Germany) Lipocalins

aOn 9 March 2004, Compound Therapeutics announced the acquisition of the intellectual property estate of Phylos(Lexington, MA, USA).

Why is it so hard to find new drugs? A signifi-cant amount of time goes into finding andvalidating new drug targets for the develop-ment of small-molecule or biotherapeuticleads. Why not create in vivo disease modelsand directly screen for compounds that canameliorate the disease state? In this issue,Peterson et al.1 describe an effort to do justthat, using a zebrafish mutant with ananatomical defect that resembles a malforma-tion in the human heart.

In theory, whole-organism screeningshould circumvent the need to identify spe-cific drug targets, allowing the entire genometo be screened in a single, unbiased assay. This

approach is equivalent to a classical geneticmutant suppressor screen, in which onesearches for secondary mutations that revertthe abnormal phenotype to wild type.Sensitized cell-based screens have previouslybeen used to identify chemical suppressors ofa disease process—for example, drug leadsthat block the proliferation of carcinomacells2. Peterson et al. extend this strategy to avertebrate organism. They describe com-pounds that rescue the abnormal vasculardevelopment of a zebrafish mutant and sug-gest that this could provide a rapid path todrug leads for diseases whose underlying biol-ogy is not well understood.

The gene encoding the gridlock transcrip-tion factor is a classic developmental selectorcontrolling the choice of angioblasts betweenvenous and arterial fates in the developingfish heart (Fig. 1). With a partial reduction ofgridlock activity, the bifurcation of the lateral

Overcoming the gridlock indiscovery researchJonathan Margolis & Greg D Plowman

Chemical screening in a zebrafish mutant has turned up two compoundsthat rescue a heart defect, but will this yield new drugs?

Jonathan Margolis and Greg D. Plowman are at Exelixis, 170 Harbor Way, P.O. Box 511,South San Francisco, CA, USA.e-mail: gplowman@exelixis.com

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