to placebo, 100 µg ZYC101a or 200 µgZYC101a. Treatment was administeredthree times at three-week intervals asintramuscular injections. After sixmonths, the patients underwent Loopelectrosurgical excision procedure(LEEP) surgery and the cervical tissuewas evaluated.

At the DNA Vaccines 2002 conferencein Edinburgh, UK (23–25 October 2002),Hedley presented preliminary resultsthat showed that, in the studypopulation as a whole, lesions wereresolved in 43% versus 27% of women(ZYC101a versus placebo; p = 0.12),whereas in women <25 years of age,the lesions resolved in 70% versus 23%of women (ZYC101a versus placebo,p <0.01). The results are encouraging,says Tyring. He believes that ‘thesuccess of a therapeutic vaccine willstimulate other people to work on thisarea with more intensity’.

There is a great need for a medicalalternative to currently available

treatment options. At present, womenwith high-grade cervical dysplasiaundergo surgery to remove thediseased tissue. However, surgicalmethods, such as LEEP, do not alwaysclear the disease, and they can lead tocomplications during pregnancy. This is a crucial problem because moreand more women between 12 and25 years of age present with thedisease, relates Hedley.

Future workZycos now wants to confirm these data in younger women and find out why the regression of lesions wasnot significant in women over 25 yearsof age. ‘There should not be a magic cut-off at 25’, says Hedley.‘Perhaps we can see some efficacy in those older women as well,potentially by changing the route of injection.’

Hedley and colleagues also want toexplore the efficacy of the drug in other

indications. Although cervical dysplasiais mainly associated with HPV-16 andHPV-18, 20 different HPV subtypeswere present at baseline in the studypopulation. ZYC101a cross-reacted withmost of these subtypes.

This result suggests that the agent, ora similar formulation, could be beneficialin treating genital warts, anal dysplasiaand even cervical cancer. ‘I think whatwe are seeing is the tip of the iceberg’,says Hedley. ‘HPV causes so many[conditions] that once you have thisinitial efficacy, this really opens up alarge possibility for you.’

References1 American Social Health Association (1998)

Sexually Transmitted Diseases in America: How Many Cases and at What Cost? Kaiser Family Foundation

2 Sheets, E.E. et al. Immunotherapy of humancervical high-grade cervical intraepithelialneoplasia with microparticle delivered HPV16 E7 plasmid DNA. Am. J. Obstet. Gynecol.(in press)

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New immunotherapy triple strategy for AMLKathryn Senior, freelance writer

A patient’s own blood cells could bemanipulated to ‘mop up’ residualmalignant cells that might remain afterstandard treatments for acute myeloidleukaemia (AML), according to a studypublished in late October 2002 [1].Edward Ball’s group at the UCSDCancer Center (University of California at San Diego, CA, USA;http://cancer.ucsd.edu/) treated theAML cells of patients to turn them intoantigen-presenting cells and expanded

T cells with anti-AML specificity duringin vitro culture. ‘The basic idea was tomake the AML cells more visible to thepatient’s immune system, and to boostspecific immunity to AML cells at thesame time’, explains Ball.

Immune response important inAML recoveryAML is a difficult disease to treat.Standard therapy is usually high-dosechemotherapy, with haematopoietic

stem cell transplantation (HSCT). It hasbeen known for some time that theimmune system is important in theeradication of residual leukaemia afterHSCT. Ironically, the removal of T cellsfrom the allogeneic graft, done toreduce graft-versus-host-disease (GVHD),is associated with an increased incidenceof leukaemia recurrence, althoughpatients who experience GVHD tend tohave a reduced risk of relapse.‘Attempts have been made to enhance

the anti-leukaemic effect without riskingmassive GVHD, but this has proved verydifficult’, says Ball. The group at UCSDreasoned that it must be possible to usethe patient’s own blood cells to bringabout a therapeutic immune response.‘Theoretically, the blood of a patient atpresentation contains all the elementsnecessary to induce that immuneresponse’, adds Ball.

Manipulating the blood cells ofAML patientsRui-Kun Zhong, Ball and colleaguesobtained blood from 12 AML patientsand cultured the white cells from eachsample. Typically, the samplescontained 92.3% AML blasts and 3.4%CD3+ T cells. These were culturedin vitro with interleukin-4 (IL-4) andrecombinant granuloctye–monocytecolony-stimulating factor (Fig. 1).Recombinant IL-2 was added on day 8.On day 21, the culture conditions werechanged to anti-CD3–anti-CD28monoclonal antibodies and IL-2. Byday 42, the amount of T cells in theculture had expanded 354-fold. CytotoxicT-lymphocyte assays demonstrated thatthe T cells produced had a significantkilling effect against autologousleukaemia cells and AML cell lines, butdid not kill cells of other lineages [1].

‘We originally started this study toinvestigate the possibility that T-cellswith anti-AML specificity could beexpanded from the blood of patientswith active disease, but we have alsomanaged to develop a strategy of3-stage sequential modulation of growthfactors to induce the patients ownT cells to attack AML cells’, explains Ball.The first phase is the initiation of AMLblast differentiation into dendritic cells,the second is the priming of T cells, andthe third stage is the expansion of theprimed T cells, ready for transfer backinto the patient. However, thisimportant next step has yet to be done.Work is under way to refine and scale upthe procedure for clinical application.

‘This may require substitution of someof the culture ingredients with productsthat can be used clinically’, commentsBall. This is expected to take up to ayear, because of the need to complywith complex regulatory issues.

Further studiesStan Riddell (Fred Hutchinson CancerResearch Center, Seattle WA, USA;http://www.fhcrc.org/) is also cautiousabout the time schedule, as he thinksthat further preclinical work is stillnecessary. ‘Others have shown thatinducing differentiation of AML blaststo a dendritic cell like phenotype canprovide stimulator cells that eliciteffector T cells that are cytotoxic forAML cells. Ball and colleagues havestreamlined the approach to culturingthe cells by adding steps that promoteT cell expansion but importantquestions remain about the nature ofthe effector cells that are generated andwhat antigens are being recognised’,comments Riddell. He points out thatthe responses seen cannot be entirelyclassic major histocompatibility complex(MHC)-restricted responses, becauseMHC mismatched leukemia lines arekilled very efficiently. ‘The authorsprovide some data that the responserequires class I antigens on the targetcell based on blocking with an anti classI antibody but this blocking occurs evenif the target is not class I matched’, henotes. It will be interesting, addsRiddell, to see whether it is possible togenerate T cell clones that can be morecarefully and adequately characterized.‘This could prove very interestingbecause the approach could provideT cells for molecular identification ofthe target antigens’, he continues.

T cell therapy is still in its infancyT-cell therapy for AML and chromicmyeloid leukaemia (CML) is a new fieldthat is currently generating excitingresults. Riddell reports that several othergroups are looking at defined tumor

associated antigens. In November 2002,Jeff Molldrem (Department of BoneMarrow Transplantation, MD AndersonCancer Center, University of Texas,Houston, TX, USA; http://www.mdanderson.org/) published a study inwhich his group identified proteinase-3as a potential target in AML and CML[2]. Hans Staus (Department ofImmunology, Hammersmith HospitalImperial College School of Medicine,London, UK; http://www.med.ic.ac.uk)claims to have exciting data showingthat T cells specific for the antigen WT-1 can eliminate human AML inimmunodeficient mice. Edus Warren,an immunologist colleague of Riddell’sat the Fred Hutchinson Cancer Research Center, and Els Goulmy(Leiden University Medical Centre, theNetherlands; http://www.lumc.nl/) have identified T cell responses tominor histocompatibility antigens thatare expressed on leukaemic cells buthave limited expression onnonhematopoietic cells in allogeneicstem cell transplant recipients [3,4].Riddell considers that identification ofsuch antigens might make it possible toaugment the graft versus leukemiaeffect without GVHD, and data fromboth groups in a few patients treatedfor post-transplant relapse in Phase I

DDT Vol. 8, No. 1 January 2003 updatenews

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Figure 1. Acute myeloid leukaemia cellsshowing dendritic morphology after8–10 days culture with interleukin-4and recombinant granuloctye–monocytecolony-stimulating factor. Figurecourtesy of Edward Ball at the UCSDCancer Centre (http://www.cancer.ucsd.edu)

trials of T cell therapy suggest that this is feasible. But he stresses the neednot to forget the preliminary stage of all of this research: ‘A much largernumber of candidate minor antigensmust be identified to allow thisapproach to be tested more broadly’,he concludes.

References1 Zhong, R.K. et al. (2002) Sequential

modulation of growth factors: a novelstrategy for adoptive immunotherapy ofacute myeloid leukaemia. Biol. Blood MarrowTransplant. 8, 557–568

2 Molldrem, J.J. et al. [2002] Overexpresseddifferentiation antigens as targets of graft-versus-leukemia reactions. Curr. Opin.Hematol. 6, 503–508

3 Warren, E.H. et al. (2002) Feasibility of usinggenetic linkage analysis to identify the genesencoding T cell-defined minor histocompa-tibility antigens. Tissue Antigens 4, 293–303

4 Mutis, T. et al. (2002) Generation of minorhistocompatibility antigen HA-1-specificcytotoxic T cells restricted by nonself HLAmolecules: a potential strategy to treatrelapsed leukemia after HLA-mismatchedstem cell transplantation. Blood 100, 547–552

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Targeting cell migrationStephani Sutherland, freelance writer

There are many ways in which cellsmigrate around the body to carry outessential functions, such as targetedimmune reactions, metastatic invasionand angiogenesis. In spite of thediversity of cell type involved, they alluse the same basic machinery formotility: the actin cytoskeleton and itsassociated proteins. Scientists arebeginning to develop new ways toidentify and study these proteins.Researchers at the University of Illinoisin Chicago (http://www.uic.edu/index.html/), for example, have recentlyidentified a compound that stops cells intheir tracks in a wound-healing assay [1].

Gabriel Fenteany, lead author of thestudy, says that the work represents ashift toward therapeutic drugs thattarget cell motility. But finding aninhibitory compound is only the firststep in elucidating the still-mysteriousworkings of cell movement, and in theeven more distant process ofdeveloping drugs to exploit it.

Screening for mobility-affectingcompoundsThe researchers used a screening assaythat they report is inexpensive, easy touse, and gives unambiguous results. Theassay uses standard 384-well tissueculture plates; a ‘wound’ is drawn across

the confluent cells, and the effects ofvarious compounds on the wound sizeare then examined. By using Madin-Darby Canine Kidney (MDCK)cells, which maintain a smooth woundedge and migrate as a sheet, Fenteanysays that they have eliminated theambiguity that comes with individuallymotile cells. The assay enables thescreening of 1000 compounds per day,which Fenteany says is ‘getting into therealm of respectable high-throughput’.Although that might be highthroughput for an individual in anacademic lab, there are also commercial operations that screenmotility-affecting compounds, such asAutomated Cell in Pittsburgh, PA, USA(http://www.automatedcell.com).

Instead of starting with an individualprotein, manipulating it, and thenmeasuring its effects on the actin systemand motility, Fenteany has taken adifferent approach: to find compoundsthat affect motility and work backwardto the proteins involved. The newlyidentified compound, dubbed UIC-1005(Fig. 1), is similar in structure to a classof antibiotics but has no antibacterialactivity. Although both types ofcompound contain an oxazolidinone ring,says Fenteany, ‘it’s what’s attached to it,and where, that makes all the difference’.

Compound identificationJustin Yarrow, working in the lab ofTim Mitchison at Harvard UniversityMedical School, MA, USA(http://www.hms.harvard.edu/), hasused a similar assay to find smallmolecules that affect motility [2].Although he agrees that Fenteany’sassay is unambiguous, he suggests thatit might be an oversimplification to callUIC-1005 a specific inhibitor of cellmovement. ‘When you’re asking for abinary read-out, you have to be askinga very specific question.’ One caveatthat Yarrow says comes with the assayis that there is a multitude ofmolecules, from transcription factorsto membrane trafficking proteins, thatcould be affected upstream of actinproteins. ‘There are probably billions ofcompounds that can inhibit wound

Figure 1. Structure of the compound UIC-1005. Figure provided by Gabriel Fenteany (University of Illinois in Chicago; http://www.uic.edu).

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