Multi-faceted strategies to combatdisease by interference with thechemokine systemZoe Johnson, Matthias Schwarz, Christine A. Power, Timothy N.C. Wells and

Amanda E.I. Proudfoot

Serono Pharmaceutical Research Institute, 14 chemin des Aulx, 1228 Plan les Ouates, Geneva, Switzerland

Inappropriate cell recruitment is a hallmark of all

autoimmune, allergic and inflammatory diseases. The

prevention of inflammation by interfering with cellular

recruitment through the neutralization of cytokines and

adhesion molecules has proven to be successful in the

clinic. Chemokines are important potential targets

owing to their central role in the cell recruitment

process. Chemokines are unique among cytokines

because they signal through seven transmembrane

receptors, thus enabling the identification of small

molecule inhibitors through high throughput screening.

The object of this Review is to discuss the validity and

feasibility of targeting several points of therapeutic

intervention offered by the chemokine system and to

assess the state of play within the field to date.

Chemokines and disease

The holy grail of many biotechnology and pharmaceuticalcompanies is to find the key targets that controlpathologies and to develop appropriate therapeutics tocontrol them. Recently, there have been several successstories, through the targeting of key proteins that areinvolved in autoimmune diseases with therapeutic anti-bodies. Antibody neutralization of the cytokine tumornecrosis factor-a (TNF-a) represents one such successfultherapeutic platform, and attempts to inhibit specificadhesion molecule interactions have also proved to besuccessful, as demonstrated by Raptivae [Efalizumabfrom Genentech (http://www.gene.com/gene/index.jsp)and Serono (http://www.serono.com/index.jsp)], an anti-body that targets lymphocyte function-associated antigen(LFA-1). Efalizumab has recently been approved for thetreatment of psoriasis, and Tysabrie, formerly known asAntegren [Natalizumab from Biogen–IDEC (www.biogen.com)], which targets the a4 integrin subunit of very lateantigen-4 (VLA-4), has recently been approved in the USAfor the treatment of multiple sclerosis (MS) and is inadvanced-stage clinical trials for inflammatory boweldisease (IBD).

Chemokines are another family of proteins that func-tion as key regulators of cell migration and therefore

Corresponding author: Proudfoot, A.E.I. (amanda.proudfoot@serono.com).Available online 19 March 2005

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represent a third class of proteins that might be exploitedto therapeutic advantage. As pivotal mediators of directedcell migration, their therapeutic potential includes auto-immune diseases, such as rheumatoid arthritis (RA),multiple sclerosis, systemic lupus erythematosus andpsoriasis, as well as allergic disorders, such as allergicdermatitis and asthma, IBD, transplant rejection, cancer,endometriosis, peripheral neuropathies and infectiousdiseases, such as AIDS. The biology of the chemokinesystem has been extensively described in several recentreviews [1–4].

A frequent criticism of targeting chemokines as atherapeutic strategy is that the apparent redundancy inthe system (Figure 1), arising from the promiscuity ofmany of the ligands involved in inflammatory processes[e.g. CCL5 (RANTES) binds to and activates CCR1, CCR3and CCR5], and the lack of fidelity many receptors displayfor their ligands {e.g. CCR1 is activated by CCL5(RANTES), CCL3 [macrophage inflammatory protein-1a(MIP-1a)] and CCL4 (MIP-1b)} might lead to lack ofspecificity and side effects. However, results from the lastten years of research using knockout mice, specificmonoclonal antibodies (mAbs) and receptor antagonistshave largely allayed such fears (Box 1). The potentialtherapeutic effect of blocking individual chemokines and/or receptors has been has borne out in efficacy studies inanimal models of disease and will be discussed in moredetail later.

Modes of intervention

There are several approaches to interfering with identifiedtargets (Figure 2), which are described in more detail inlater sections. The approach taken depends on severalfactors, including whether the target is intracellular orextracellular, exemplified by the traditional division ofanti-inflammatory strategies by the biotechnology com-panies and the pharmaceutical companies as ‘outside thecell’ and ‘inside the cell’ intervention, respectively. Inter-ference with the activity of cytokines, growth factors andinterleukins has focused on biological therapeuticsbecause the interaction of these proteins with their cellsurface receptors involves large protein–protein inter-actions, which are particularly difficult to break with smallmolecule inhibitors. However, neutralizing antibodies and

Review TRENDS in Immunology Vol.26 No.5 May 2005

. doi:10.1016/j.it.2005.03.001

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CXCR4

CCR8

CXCR5

CXCR6 CCR6

CXCR1CCR10

CCR4

CCR5

CXCR3

CXCR2

CCR2

CCR1

CCR3

CCR7

CX3CR1

XCR1

CCR9

CXCL8CXCL6

CXCL8CXCL5CXCL1CXCL2CXCL3CXCL7

CXCL9CXCL10CXCL11

CCL17CCL22

CCL3CCL4CCL5CCL8

CCL5CCL3CCL7

CXCL20

CCL25

CXCL16

CXCL13

CCL27CCL28

CXCL12

CCL19CCL21

CCL1

XCL1XCL2

CX3CL1

MSRATransplantAsthmaAtherosclerosisPeripheral neuropathy

AIDSCancer

AsthmaAllergy

Asthma?Skin diseases

Skin diseasesIBD

SepsisAtherosclerosis

MSRATransplant

MSRATransplantAsthmaNephritis

Asthma?

MSRATransplantAsthmaNephritisIBDAIDS

Atherosclerosis

Constitutive: Basaltrafficking or homing

Inducible: Inflammatory

CCL2CCL8CCL7CCL13

CCL11CCL5CCL7CCL13

Figure 1. There are currently 40 known ligands and 19 known receptors that comprise the chemokine system. Receptors that are regarded as being constitutively expressed

are shown in blue and those that are induced by proinflammatory cytokines are shown in red. The receptor–ligand pairings illustrated display the apparent redundancy in the

system. It is interesting to note that constitutively expressed receptors that are generally involved in homeostatic processes tend to havemonogamous pairings (e.g. CXCL13

with CXCR5), whereas inducible receptors that are involved in inflammatory processes tend to bind multiple ligands that are themselves promiscuous (e.g. CCR5 binds to

CCL3, CCL4, CCL5 and CCL8; CCL5 binds to CCR1, CCR3 and CCR5). Receptor–ligand pairs implicated in disease processes following target validation approaches are shown

in grey.

Review TRENDS in Immunology Vol.26 No.5 May 2005 269

neutralizing binding proteins are proving to be successfulfor inhibiting the action of several cytokine ligands orreceptors.

The chemokine system lends itself to both antibody(or biological) and small molecule approaches, with onecaveat. Seven transmembrane (7TM) G protein-coupledreceptors (GPCRs) represent the most ‘drugable’ class ofreceptors for pharmaceutical intervention. However, themajority of these receptors have small molecule ligands,such as histamine, dopamine or even peptides, whereaschemokines, which are small proteins in that they are onlyw8 kDa, have relatively large 7TM receptor ligands. Inaddition, in the case where multiple chemokines areknown to bind to a single receptor, for example CXCR3,the binding site of the receptor for the different ligandsmight not necessarily be the same, demonstrating that acompetitive inhibitor might not be optimal for theinhibition for all ligands [5,6]. Thus, successful smallmolecule inhibitors are often non-competitive, binding to asite in the transmembrane helices, which is well demon-strated by the CCR5 inhibitor, TAK779 [7]. Another pointof interest for the chemokine receptor system is that

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certain receptors have shown themselves to be moreamenable to screening campaigns for the identification ofinhibitors – the best example is the number of patentssubmitted for CCR5 antagonists compared to thosesubmitted for CXCR4, both of which were identified asco-receptors for HIV infection within weeks of each other.

Small molecules

Themost widely adopted approach to block the interactionof the chemokine with its receptor is using small moleculeantagonists (Figure 3a). It has been an exciting andinstructive experience to see the field evolve, from theidentification of the first chemokine receptors in 1991 [8,9],until today, where several small molecule chemokinereceptor antagonists are being evaluated in advancedhuman clinical trials. In the pioneering days, the mainchallenge consisted of designing small molecules able tofunction as antagonists of chemokine receptors in vitro,illustrated by the scarce results obtained frommany of theearly high-throughput screening campaigns [10]. For along time, it was a matter of debate whether smallmolecule chemokine receptor antagonists would ever be

Box 1. Target validation

The chemokine system consists of a large number of ligands and

receptors. To identify the relevant target for a drug discovery

program, it is essential to delineate which ligands and receptors

are important in the disease of interest. This can be achieved by one

or more methods, outlined here:

(i) Expression of protein target in the disease

Measured directly at the protein level in patient samples (e.g. biopsy,

surgical specimen or blood) by immunohistochemistry or ELISA

techniques, or indirectly by measuring the relevant mRNA. Thus, for

MS, the inflammatory chemokines CCL5 (RANTES), CCL2 (MCP-1),

CXCL10 (IP-10) and CXCL9 (Mig) have all been identified in lesions in

autopsy samples taken from the brains of MS patients [42,43]. It is

unclear, however, if the presence of these chemokines and receptors

implies a protective or pathological role.

(ii) Neutralizing antibodies

Neutralization of CCL3 (MIP-1a) and CCL2 (MCP-1) highlights the

categorical role of these two chemokines, and by implication their

receptors, in relapsing remitting EAE, the rodent model for MS [44].

This study shows that CCL3 (MIP-1a) is involved in the onset of the

disease, whereas CCL2 (MCP-1) has a key role in the relapses. The

role of CXCR3 in the prevention of allograft rejection was elegantly

demonstrated using neutralizing antibodies in a cardiac allograft

model and corroborated using CXCR3K/K mice – an excellent

example of two approaches arriving at the same conclusion [45].

(iii) Genetic targeting of chemokines or chemokine

receptorsTargeted deletion of chemokines or receptors is an excellent

approach to study the role of specific chemokine ligand–receptor

interactions in vivo, particularly in models of inflammatory and

infectious diseases. Knockout of CCR1, CCR2 and JE [murine CCL2

(MCP-1)], support their potential as targets for therapeutic interven-

tion in MS [46–49]. The unequivocal role of CCL2 (MCP-1) and CCR2

in the pathogenesis of atherosclerosis has also been demonstrated

using knockout mice [50,51].

Although many studies with knockout mice have pointed to

exclusive roles for specific receptor–ligand pairings, in many cases,

the results obtained from disease models are not always conclusive

and indeed are sometimes in paradox with what is known about the

human disease. Eosinophils are often the predominant inflamma-

tory cell type present in allergic diseases. CCL11 (eotaxin) was first

identified as an eosinophil-specific chemoattractant in a guinea pig

model of allergic asthma [52] and the unique functional receptor for

CCL11, CCR3, is expressed at high density on both eosinophils and

basophils. CCR3 and CCL11 are highly expressed in the bronchial

biopsies and bronchoalveaolar lavage fluid of asthmatics [53] and

serum CCL11 level is considered to be amarker of disease severity in

asthma patients [54]. Yet, the mice in which these genes are deleted

do not express the predicted phenotype, that is, a resistance to

experimental allergic asthma. Whether or not this result reflects the

inadequacy of the animal model remains to be seen. Nevertheless,

the true contribution that eosinophils make to disease progression is

still hotly debated [55,56] and, in this case, the initial optimism at

finding ‘the receptor’ to control allergy that was present in the mid-

1990s has now dissolved because a much more complex picture of

chemokine involvement in this disease has emerged.

Review TRENDS in Immunology Vol.26 No.5 May 2005270

found [11], and consequently, considerable excitement wasgenerated when the first potent small molecule antagon-ists were reported in 1998 [12,13]. Once the first dam wasbroken, chemists quickly learned to decipher whichstructural determinants are required for small moleculesto act as chemokine receptor antagonists [14] and havesince developed numerous distinct chemical series ofantagonists acting specifically on a wide variety oftherapeutically relevant chemokine receptors [15].

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Having solved the problem of potency in vitro, the nextchallenge consisted of demonstrating efficacy in vivo, inappropriate animal models of disease. Again, this turnedout to be more difficult than anticipated, owing to the factthat some of the early classes of antagonists were afflictedwith sub-optimal physicochemical and pharmacokineticproperties. In addition, several highly potent antagonistsof human chemokine receptors were found to be much lessactive against the corresponding mouse or rat receptors[16], rendering an evaluation in rodent pharmacologymodels problematic because of the higher doses needed. Toovercome the problem, researchers have often chosen toperform the pharmacology studies with close analogues inthe same chemical series that show at least residualactivity on rodent receptors and/or entirely resorted tonon-rodent animal models. Bearing this in mind, the firstreports in 2001 of successful in vivo studies involvingchemokine receptor antagonists is undoubtedly a signifi-cant milestone in the annals of chemokine receptor-baseddrug discovery [17–19].

The final hurdle on the way towards the clinic thatseveral, otherwise promising, compounds have failed topass, relates to their safety profile. Arguably, the require-ments in terms of safety are particularly stringent forchemokine receptor antagonists, given that themajority ofpotential therapeutic applications are in the area ofchronic disease. From the scarcity of information on thesubject that has been publicly disclosed, one can infer thatinteraction with the cardiac potassium channel hERG(human ether-a-go-go related gene), inhibition of certaincytochrome P450 isoforms, as well as lack of selectivitytowards certain biogenic amine receptors, are among themost frequently encountered problems with chemokinereceptor antagonists. In earlier reviews, we could onlyspeculate as to whether small molecule chemokinereceptor antagonists would ever reach the stage ofentering human clinical trials. Today, 12 years after theidentification of the first chemokine receptors, we areseeing several compounds that have successfully passedPhase I clinical trials and are now hopefully poised todeliver the final proof of therapeutic efficacy in Phase IItrials. Table 1 is a summary of the current status of smallmolecule antagonists currently in development.

Modified chemokines

A second approach to block the chemokine–receptorinteraction is by using chemokines that have beenmodified in such a way that they retain high affinity fortheir receptor but have abrogated signaling properties,thereby producing a receptor antagonist (Figure 3b).Traditionally, these have been chemokines modified attheir N-terminus, the region, which in most chemokines,is responsible for signaling. The approach of producingN-terminally truncated chemokines was particularlysuitable to solid phase synthesis because a gamut ofsuccessive truncations could be produced in one syntheticprogram. Thus, a vast array of such modified chemokineshas been produced, yielding valuable structural infor-mation and, more importantly, has identified chemokinesthat retain receptor binding but do not signal. One suchantagonist, 9–76- monocyte chemoattractant protein-1

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P

P

P

PP

(d)(a)

(c)(b)

Figure 2. Points of intervention that can be used to therapeutically target the chemokine system (grey). (a) Blocking the ligand–receptor interaction with antibodies or small

molecule inhibitors. This approach is the most widely adopted for interfering directly with the chemokine system. (b) Blocking the ligand–GAG interaction through the use of

modified chemokines. This is a novel approach and might be amenable to inhibition with antibodies or small molecule inhibitors. (c) Interfering with downstream signaling

through the use of small molecule inhibitors that interfere with the phosphorylating enzyme capacity of, for example, phosphoinositide 3-kinase. (d) Inhibiting receptor

recycling through modified chemokines that prevent the recycling of the receptor to the cell surface.

Review TRENDS in Immunology Vol.26 No.5 May 2005 271

(MCP-1) is effective in preventing the arthritis thatdevelops spontaneously in MRL-lpr mice [20].

Another CCR2 antagonist that has been producedrecombinantly has a deletion of seven amino acids inthe N-terminal region (7ND-MCP-1) and shows goodefficacy in renal fibrosis using a gene therapy approach[21]. Antagonists can also be produced by extension ofthe N-terminus, demonstrated by the retention of theinitiating methionine residue in CCL5 (RANTES)during recombinant protein production in bacterialcells [22], which produces Met–RANTES, which isefficacious in a large number of models [23]. A second

(a) (b)

Figure 3. Modes of intervention that can be used to therapeutically target the chemokine s

the small molecule is a receptor antagonist but might also bind to the ligand (light grey

(b) Modified chemokines (orange indicatesmodification, compared to green indicating n

binding. (c) Antibodies (blue) might prevent chemokine function through blockade o

(d) Chemokine-binding proteins (purple) can prevent the receptor–ligand interaction an

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series of CCL5 (RANTES) variants created by thechemical extension of the N-terminus was identified bythe creation of aminooxypentane–RANTES (AOP–RANTES), which has remarkable anti-HIV infectivityproperties [24] and prevents receptor recycling, thushighlighting another point of intervention in thesystem (Figure 3d). More potent analogues weresubsequently produced based on this strategy, andthe most potent, known as PSC–RANTES, has recentlybeen reported to prevent vaginal simian HIV (SHIV)transmission in Rhesus macaques, indicating a poten-tial topical protective therapeutic approach [25].

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(c) (d)

ystem. (a) Small molecules (red) traditionally target the receptor (dark grey). That is,

) and block the receptor interaction or the GAG (green) interaction with the ligand.

omodification) can interferewith normal chemokine function of the receptor or GAG

f the receptor, neutralization of the ligand or blockade of the GAG interaction.

d/or the ligand–GAG interaction.

Table 1. Most advanced chemokine receptor antagonistsa

Target Compound Development stage Indicationsb Company

CCR1 BX-471 Phase II MS, psoriasis Berlex (www.berlex.com) and

Schering (www.sch-plough.com)

CP-481715 Phase II RA Pfizer (www.pfizer.com)

MLN-3897 Phase I completed (MS, RA, psoriasis) Sanofi-Aventis (http://en.sanofi-

aventis.com) and Millenium

(www.mlnm.com)

CCR2 INCB-3284 Phase I completed (RA) Incyte (www.incyte.com)

CCR3 766994 Phase II Asthma, allergic rhinitis GlaxoSmithKline (www.gsk.com)

DPC-168 Phase I completed (discontinued?) (Asthma, allergic rhinitis) BMS (www.bms.com)

CCR5 UK-427857 Phase III HIV Pfizer

873140 Phase II HIV GlaxoSmithKline and Ono

(www.ono.co.jp/eng/)

Sch-417690 (Sch-D) Phase II HIV Schering-Plough

Sch-351125 (Sch-C) Phase I completed (HIV) Schering-Plough

CXCR2 656933 Phase I (COPD) GlaxoSmithKline

CXCR3 T-487 Phase II Psoriasis Amgen (Tularik) (www.amgen.com)

and Chemocentryx

(www.chemocentryx.com)

CXCR4 AMD-3100 Phase III Bone marrow transplantation AnorMED (www.anormed.com)aData obtained from the Investigational Drugs Database (www.iddb.com/).bBrackets indicate that a compound is about to be used in patients with the disease given, whereas no brackets indicates that a compound has actually been tested in patients

with the given disease.

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Recently, through using specific mutants, evidence hasbeen provided that glycosaminoglycan (GAG) binding andchemokine oligomerization are essential for the in vivoactivity of chemokines [26]. Using an assay for cellularrecruitment into the peritoneal cavity, we demonstratedthat a RANTES variant deficient in GAG bindingantagonizes CCL5 (RANTES)-induced recruitment and,moreover, could also prevent the cellular recruitmentinduced by the non-specific inflammatory agent, thiogly-collate. This antagonistic property translates into an anti-inflammatory effect in a murine model of MS, experimentalautoimmune encephalomyelitis (EAE), in which itsadministration before disease onset significantly reducesclinical score from complete hind limb paralysis toweakness in the hind limbs [27]. A novel strategy forantagonizing the chemokine system through interferingwith heparin binding was thus identified (Figure 2 andFigure 3b).

Antibodies

The use of neutralizing mAbs (Figure 3c), principallyagainst the chemokines themselves, has been usedextensively in animal models of disease, although surpris-ingly, few are being developed for therapeutic use –perhaps a reflection of the worry that orally availablesmall molecule receptor inhibitors would supersede theuse of antibodies. Therefore, the use of neutralizingantibodies against specific chemokines has, for themoment, been of more use in proving that inhibition ofchemokine activity in animal models successfully blocksinflammation in a wealth of publications (Box 1). Thedevelopment of antibodies for human disease encountersthe same problem as small molecule receptor inhibitors –that of species selectivity to prove their efficacy in rodentmodels. One example is Millennium’s humanized mAbtargeting CCR2, which was tested in a model of restinosisin a non-human primate, before progression into humans;it is currently being tested in Phase IIa studies for RA. Themost advanced anti-chemokine antibody was an anti-

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CXCL8 [interleukin-8 (IL-8)] mAb, which was tested in aPhase II trial of chronic obstructive pulmonary disease(COPD), and showed an improvement in the transitiondyspnea index (TDI) but no improvement in lung functionor health status of the patients [28]. In a second trial, forpsoriasis, anti-CXCL8 (IL-8) failed to show efficacy,underlining the risks in drug development. The fact thatCXCL8 (IL-8) is present in high quantities in psoriaticskin does not support the jump to the conclusion thatblocking CXCL8 (IL-8) activity would result in a reversalof the disease state.

Binding proteins

An interesting conundrum exists in the human system:the human genome has evolved several endogenousstrategies to combat inflammation, in the guise of bindingproteins that neutralize proinflammatory cytokines(Figure 3d). Examples of these are, IL-1 receptor a (IL-1Ra)[29], TNF-binding protein-I (TBP-I) and TBP-II [30],interferon receptor a (IFNRa) [31] and IL-18-bindingprotein (IL-18BP) [32]. Similarly, viruses have evolved toproduce such proteins and their genomes encode bindingproteins that neutralize cytokines, such as IFN-g [33] andIL-18 [34], as well as chemokines [35]. Several chemokinebinding proteins have been described (reviewed inRef. [36]). An interesting general point is that poxvirusestend to produce binding proteins, whereas herpes virusesuse molecular piracy by producing viral cytokines andchemokines as well as scavenger chemokine receptors.Two poxvirus-encoded chemokine-binding proteins, myx-oma virus T1 protein (M-T1), which inhibits receptoractivation, and M-T7, believed to prevent chemokinebinding to GAGs, were tested along with the Herpes-encoded binding protein, M3, in a rat aortic allografttransplantation model. Single administrations show dose-dependent activity at surprisingly low doses, ranging from5 to 5000 pg, with remarkable efficacy in preventingintimal hyperplasia and vasculopathy, correlating withdecreased mononuclear cell recruitment [37]. These

Review TRENDS in Immunology Vol.26 No.5 May 2005 273

findings are supported by the use of an inducible M3transgenic mouse, which shows a significant reduction inintimal hyperplasia, crucial in the development of athero-sclerosis [38].

These experiments highlight the usefulness of thesemolecules for acute indications, however, immunogenicityis a potential problem with this approach in the treatmentof chronic diseases. There is as yet no published evidencefor the existence of secreted chemokine-binding proteinsin humans, however, recently, an anti-CXCL8 (IL-8)activity was described in tick saliva [39]. On reflection,this is an obvious line of defense for parasites, such asticks, which need to feed on their hosts for relatively longperiods of time without apparently eliciting an immuneresponse – a mechanism that might lead to the adoption ofthese proteins as potential anti-inflammatory moleculesby the pharmaceutical and biotechnology industry. It willalso be interesting to see if homologous molecules exist inhumans.

Perspectives

The initial discovery of chemokines led to the perhapsnaıve hope that each leukocyte subtype would express aspecific chemokine receptor, which would facilitate thera-peutic intervention in an unprecedented manner. How-ever, our knowledge of the system as it is today indicatesthat this is not the case. The chemokine system is highlycomplex. It is often described as redundant – this iscertainly evident from in vitro studies, although webelieve that there are certainly levels of control andspecificity that exist in vivo, which are difficult to predictfrom in vitro studies. It is anticipated that this conundrumwill be resolved with the advent of small molecules in theclinic – is the system really redundant or is there sufficientspecificity in vivo to offer valuable therapeutic potential?We believe that we are close to answering this question, atleast for certain chemokine–receptor pairs (Table 1).

Two small molecule inhibitors are in Phase III trialsand four are in Phase II trials, between them covering fiveof the 19 receptors, and other molecules are active in pre-clinical development, progressing towards Phase I. Inaddition, biological therapeutics will confirm that thechemokine system is a valid therapeutic target.

Those involved in these development programs shouldbe congratulated for the speed with which these moleculesare reaching the clinic. Historically in the pharmaceuticalindustry, it has taken between 10 and 15 years (or longer)from the identification of a target to the launch of aproduct on the market. For example, it has taken 20 yearsto bring an anti-adhesion molecule to the clinic, such asanti-LFA-1 (Efalizumab or Raptiva), a treatment formoderate to severe psoriasis, if one considers the initialidentification of LFA-1 in 1983 [40], to its approval by theFood and Drug Administration in October 2003. This longtimescale must, however, take into consideration the factthat the role of LFA-1 in T-cell adhesion was onlybeginning to be elucidated in 1988 [41].

The rapid progression of small molecule inhibitors ofCCR5, from the identification of CCR5 in 1996 as theprincipal co-receptor for HIV transmission, to the firsthuman trials in 2000, is extraordinary, even given the fact

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that the development time for new drugs is constantlybeing reduced by technological improvements. Finally, thechemokine system offers the pharmaceutical industrythe possibility of developing new chemical entities, thenumber of which has been declining over the past fewyears. Therefore, with the promising new therapeuticstrategies targeting the chemokine system described inthis Review, we can expect exciting new drugs to emergein the very near future.

References

1 Rot, A. et al. (2004) Chemokines in innate and adaptive host defense:basic chemokinese grammar for immune cells. Annu. Rev. Immunol.22, 891–928

2 Lau, E.K. et al. (2004) Chemokine–receptor interactions: GPCRs,glycosaminoglycans and viral chemokine binding proteins. Adv.Protein Chem. 68, 351–391

3 Moser, B. et al. (2004) Chemokines: multiple levels of leukocytemigration control. Trends Immunol. 25, 75–84

4 Balkwill, F. (2004) Cancer and the chemokine network. Nat. Rev.Cancer 4, 540–550

5 Cox, M.A. et al. (2001) Human interferon-inducible 10-kDa proteinand human interferon-inducible T cell a chemoattractant are allotopicligands for human CXCR3: differential binding to receptor states.Mol.Pharmacol. 59, 707–715

6 Xanthou, G. et al. (2003) Molecular characterization of the chemokinereceptor CXCR3: evidence for the involvement of distinct extracellulardomains in a multi-step model of ligand binding and receptoractivation. Eur. J. Immunol. 33, 2927–2936

7 Dragic, T. et al. (2000) A binding pocket for a small molecule inhibitorof HIV-1 entry within the transmembrane helices of CCR5. Proc. Natl.Acad. Sci. U. S. A. 97, 5639–5644

8 Holmes, W.E. et al. (1991) Structure and functional expression of ahuman interleukin-8 receptor. Science 253, 1278–1280

9 Murphy, P.M. et al. (1991) Cloning of complementary DNA encoding afunctional human interleukin-8 receptor. Science 253, 1280–1283

10 Mellor, G.W. et al. (1997) Searching for chemokine receptor bindingantagonists byhigh throughput screening.J.Biomol. Screen.2, 153–157

11 McKinnon, M. et al. (1996) Strategies for the discovery of cytokinereceptor antagonists. Drug News Perspect. 9, 389–398

12 White, J.R. et al. (1998) Identification of a potent, selective non-peptide CXCR2 antagonist that inhibits interleukin-8-induced neutro-phil migration. J. Biol. Chem. 273, 10095–10098

13 Hesselgesser, J. et al. (1998) Identification and characterization ofsmall molecule functional antagonists of the CCR1 chemokinereceptor. J. Biol. Chem. 273, 15687–15692

14 Schwarz, M.K. et al. (2002) New therapeutics that modulatechemokine networks. Nat. Rev. Drug Discov. 1, 347–358

15 Gao, Z. et al. (2003) Unraveling the chemistry of chemokine receptorligands. Chem. Rev. 103, 3733–3752

16 Liang, M. et al. (2000) Species selectivity of a small moleculeantagonist for the CCR1 chemokine receptor. Eur. J. Pharmacol.389, 41–49

17 Horuk, R. et al. (2001) CCR1-specific non-peptide antagonist: efficacyin a rabbit allograft rejection model. Immunol. Lett. 76, 193–201

18 Horuk, R. et al. (2001) A non-peptide functional antagonist of theCCR1 chemokine receptor is effective in rat heart transplant rejection.J. Biol. Chem. 276, 4199–4204

19 Auten, R.L. et al. (2001) Nonpeptide CXCR2 antagonist preventsneutrophil accumulation in hyperoxia-exposed newborn rats.J. Pharmacol. Exp. Ther. 299, 90–95

20 Gong, J.H. et al. (1997) An antagonist of monocyte chemoattractantprotein 1 (MCP-1) inhibits arthritis in the MRL-lpr mouse model.J. Exp. Med. 186, 131–137

21 Wada, T. et al. (2004) Gene therapy via blockade of monocytechemoattractant protein-1 for renal fibrosis. J. Am. Soc. Nephrol.15, 940–948

22 Proudfoot, A.E. et al. (1996) Extension of recombinant humanRANTES by the retention of the initiating methionine produces apotent antagonist. J. Biol. Chem. 271, 2599–2603

Review TRENDS in Immunology Vol.26 No.5 May 2005274

23 Proudfoot, A.E. (2002) Chemokine receptors: multifaceted therapeutictargets. Nat. Rev. Immunol. 2, 106–115

24 Simmons, G. et al. (1997) Potent inhibition of HIV-1 infectivity inmacrophages and lymphocytes by a novel CCR5 antagonist. Science276, 276–279

25 Lederman, M.M. et al. (2004) Prevention of vaginal SHIV trans-mission in rhesus macaques through inhibition of CCR5. Science 306,485–487

26 Proudfoot, A.E. et al. (2003) Glycosaminoglycan binding and oligo-merization are essential for the in vivo activity of certain chemokines.Proc. Natl. Acad. Sci. U. S. A. 100, 1885–1890

27 Johnson, Z. et al. (2004) Interference with heparin binding andoligomerization creates a novel anti-inflammatory strategy targetingthe chemokine system. J. Immunol. 173, 5776–5785

28 Mahler, D.A. et al. (2004) Efficacy and safety of a monoclonal antibodyrecognizing interleukin-8 in COPD: a pilot study. Chest 126, 926–934

29 Eisenberg, S.P. et al. (1990) Primary structure and functionalexpression from complementary DNA of a human interleukin-1receptor antagonist. Nature 343, 341–346

30 Seckinger, P. et al. (1988) A human inhibitor of tumor necrosis factor a.J. Exp. Med. 167, 1511–1516

31 Novick, D. et al. (1995) Soluble and membrane-anchored forms of thehuman IFN-a/b receptor. J. Leukoc. Biol. 57, 712–718

32 Novick, D. et al. (1999) Interleukin-18 binding protein: a novelmodulator of the Th1 cytokine response. Immunity 10, 127–136

33 Goodbourn, S. et al. (2000) Interferons: cell signalling, immunemodulation, antiviral response and virus countermeasures. J. Gen.Virol. 81, 2341–2364

34 Xiang, Y. et al. (1999) IL-18 binding and inhibition of interferon g

induction by human poxvirus-encoded proteins. Proc. Natl. Acad. Sci.U. S. A. 96, 11537–11542

35 van Berkel, V. et al. (2000) Identification of a g-herpesvirus selectivechemokine binding protein that inhibits chemokine action. J. Virol.74, 6741–6747

36 Alcami, A. (2003) Viral mimicry of cytokines, chemokines and theirreceptors. Nat. Rev. Immunol. 3, 36–50

37 Liu, L. et al. (2004) Viral chemokine-binding proteins inhibitinflammatory responses and aortic allograft transplant vasculopathyin rat models. Transplantation 77, 1652–1660

38 Pyo, R. et al. (2004) Inhibition of intimal hyperplasia in transgenicmice conditionally expressing the chemokine-binding protein M3. Am.J. Pathol. 164, 2289–2297

39 Kocakova, P. et al. (2003) Effect of fast protein liquid chromatographyfractionated salivary gland extracts from different ixodid tick specieson interleukin-8 binding to its cell receptors. Folia Parasitol. (Praha)50, 79–84

40 Sanchez-Madrid, F. et al. (1983) A human leukocyte differentiationantigen family with distinct a-subunits and a common b-subunit: thelymphocyte function-associated antigen (LFA-1), the C3bi comple-ment receptor (OKM1/Mac-1), and the p150,95 molecule. J. Exp. Med.158, 1785–1803

Free journals for dev

The WHO and six medical journal publishers have launched the Acc

poorest countries to gain free access to bio

The science publishers, Blackwell, Elsevier, the Harcourt Worldwide

Springer-Verlag and JohnWiley,were approachedby theWHOand th

will be available for free or at significantly reduced prices to universitie

countries. The second stage involves extending t

Gro HarlemBrundtland, director-general for theWHO, said that this in

the health information gap betw

See http://www.healthinternetw

www.sciencedirect.com

41 Prieto, J. et al. (1988) Molecules mediating adhesion of T and B cells,monocytes and granulocytes to vascular endothelial cells. Immunol-ogy 63, 631–637

42 Sorensen, T.L. et al. (1999) Expression of specific chemokines andchemokine receptors in the central nervous system of multiplesclerosis patients. J. Clin. Invest. 103, 807–815

43 Balashov, K.E. et al. (1999) CCR5C and CXCR3C T cells are increasedin multiple sclerosis and their ligandsMIP-1a and IP-10 are expressedin demyelinating brain lesions. Proc. Natl. Acad. Sci. U. S. A. 96,6873–6878

44 Kennedy, K.J. et al. (1998) Acute and relapsing experimentalautoimmune encephalomyelitis are regulated by differentialexpression of the CC chemokines macrophage inflammatory protein-1a and monocyte chemotactic protein-1. J. Neuroimmunol. 92, 98–108

45 HancockWayne,W. et al. (2000) Requirement of the chemokine receptorCXCR3 for acute allograft rejection. J. Exp. Med. 192, 1515–1520

46 Rottman, J.B. et al. (2000) Leukocyte recruitment during onset ofexperimental allergic encephalomyelitis is CCR1 dependent. Eur.J. Immunol. 30, 2372–2377

47 Fife, B.T. et al. (2000) CC chemokine receptor 2 is critical for inductionof experimental autoimmune encephalomyelitis. J. Exp. Med. 192,899–905

48 Izikson, L. et al. (2000) Resistance to experimental autoimmuneencephalomyelitis in mice lacking the CC chemokine receptor (CCR)2.J. Exp. Med. 192, 1075–1080

49 Huang, D. et al. (2001) Absence of monocyte chemoattractant protein 1in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimentalautoimmune encephalomyelitis. J. Exp. Med. 193, 713–726

50 Peters, W. et al. (2001) Involvement of chemokine receptor 2 and itsligand, monocyte chemoattractant protein-1, in the development ofatherosclerosis: lessons from knockout mice. Curr. Opin. Lipidol. 12,175–180

51 Gosling, J. et al. (1999) MCP-1 deficiency reduces susceptibility toatherosclerosis in mice that over express human apolipoprotein B.J. Clin. Invest. 103, 773–778

52 Jose, P.J. et al. (1994) Eotaxin: a potent eosinophil chemoattractantcytokine detected in a guinea pig model of allergic airwaysinflammation. J. Exp. Med. 179, 881–887

53 Ying, S. et al. (1999) Eosinophil chemotactic chemokines (eotaxin,eotaxin-2, RANTES, monocyte chemoattractant protein-3 (MCP-3),and MCP-4), and C–C chemokine receptor 3 expression in bronchialbiopsies from atopic and nonatopic (intrinsic) asthmatics. J. Immunol.163, 6321–6329

54 Nakamura, H. et al. (1999) Eotaxin and impaired lung function inasthma. Am. J. Respir. Crit. Care Med. 160, 1952–1956

55 Humbles, A.A. et al. (2004) A critical role for eosinophils in allergicairways remodeling. Science 305, 1776–1779

56 Lee, J.J. et al. (2004) Defining a link with asthma in mice congenitallydeficient in eosinophils. Science 305, 1773–1776

eloping countries

ess to Research Initiative, which enables nearly 70 of the world’s

medical literature through the Internet.

STM group, Wolters Kluwer International Health and Science,

eBritishMedical Journal in 2001. Initially,more than 1000 journals

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his initiative to institutions in other countries.

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ork.net for more information.