Int.J.Cancer:98,857–863(2002) PublicationoftheInternationalUnionAgainstCancer ©2002Wiley-Liss,Inc. DOI10.1002/ijc.10245 TUMOR-TARGETEDIMMUNECOMPLEXFORMATION:EFFECTSONMYELOID CELLACTIVATIONANDTUMOR-DIRECTEDIMMUNECELLMIGRATION 1 1 2,3 1 1 Bart-JanKROESEN ,*PamelaM.J.MCLAUGHLIN ,PetraH.L.SCHUILENGA-HUT ,SusanC.JACOBS ,GrietjeMOLEMA , 1 1 WijnandHELFRICH andLouF.M.H.DELEIJ 1DepartmentofPathologyandLaboratoryMedicine,UniversityHospitalGroningen,Groningen,TheNetherlands 2DepartmentofMedicalGenetics,UniversityofGroningen,Groningen,TheNetherlands 3GroningenUniversityInstituteforDrugExploration,Groningen,TheNetherlands Theeffectivenessofcellularimmunotherapyofsolidtu- inflammatoryreactionisbeinginitiated.TypeIIandtypeIII morsisoftenhamperedbythelackofspecificinfiltrationof inflammatoryreactionsareinducedbycytotoxicandcomplexed immuneeffectorcellsintothetumormass.Therefore,we antibodies,respectively,which,afterspecificornonspecificdep- studiedthepotentialoftumorantigen-specificantibodiesto ositionatcertainsites,activatemyeloidcellspresentinsituvia elicittumor-specificmyeloidcellactivation,toinduceor enhancetumorinfiltrationbyimmunecells.Tothisend,we Fc–FcRinteraction.Myeloidcellactivationcanbeinitiatedsolely developedaninvitromodelsystemusingthehumanmyeloid byFc–FcRinteraction,butothersignals,suchasthoseprovidedby celllineMonoMac-6.IncubationofIFN-␥–primedMono- componentsoflocallyactivatedcomplementand/orbycytokines Mac-6cellswithserum-opsonizedzymosanorEGP-2–di- suchasTNF-␣andIFN-␥,cangreatlyaugmentmyeloidcell rected,mouseIgG2a-opsonized,EGP-2-positivetumorcells activation.Ingeneral,thetypeIIIinflammatoryreaction,modeled resultedintheproductionofROSandTNF-␣ andinduced intheArthusreaction,appearstobestrongerthanthetypeII E-selectinandICAM-1expressiononHUVECs.FcR-medi- inflammatoryreaction.Thismaybeduetothefactthatcomplexed atedMonoMac-6cellactivationwasstrictlydependentonthe 17,18 activationofMonoMac-6cellswithIFN-␥.Inaddition,no IgbindswithhigheraviditytoFcRsthannoncomplexedIg. In myeloidcellactivationwasobservedinthepresenceofhu- addition,thedegreetowhichcomplementisactivatedbycell- manserumorusingtumorantigen-specificmouseantibody boundIgisdeterminedinpartbytheextentofIgcomplexation.In subclassesotherthanIgG2a,suggestingthecrucialinvolve- responsetoimmunecomplexbinding,macrophagesarestimulated mentofCD64(Fc␥R1)intheeffectsobserved.However, togenerateandreleaseoxygenradicalsandvariousinflammatory serum-inhibitedmyeloidcellactivationwascompletelyre- cytokines,suchasIL-1,TNF-␣,colony-stimulatingfactorsand storedemployinga2-steptargetingapproachinwhichtumor chemoattractants,whichactivateendotheliumandareinvolvedin cellopsonizationwithmouseanti-EGP-2antibodieswasfol- lowedbyincubationwithhumanantimouseIgantibodies. thesubsequentrecruitmentofmononuclearimmuneeffector Moreover,usingthis2-stepapproach,notonlyanti-EGP-2– cells.19 Vasculitidesandrheumatoidarthritisrepresentclinical directedmouseIgG2abutalsomouseIgG1antibodieseffec- pathologicmanifestationsofthistypeofinflammatoryresponse, tivelyinducedtumor-specificmyeloidcellactivation.Incon- exemplifyingitspathophysiologicpotential. clusion,wedescribeamethodtoinduceefficientandtumor- Sincethedevelopmentofhybridomatechnology,MAbsdi- specificactivationofmyeloidcellsbasedonthesequential useofmousetumorantigen-specificandhumanantimouse rectedagainsthumantumor-associatedantigenshavebeenusedin Igantibodies.Targetedmyeloidcellactivationmayprovidea variousimmunotherapeuticstrategies.20 Disappointingclinicalre- meanstoaidintheinductionofatumor-directedimmune sults,obtainedforthegreaterpartwithmouseMAbs,havebeen responseandassuch,themethoddescribedherecouldbeof ascribedtoaninabilityofmouseIgmoleculestobindtohuman clinicalsignificance. Fc␥Randactivatethecomplementcascade.20,21 Studiesusing ©2002Wiley-Liss,Inc. mouseantibodiesthatappeartoactivatethecomplementsystem,at leasttoacertainextent,suggestthattheseantibodiescanbe Keywords:immunecomplex;Fcreceptor;epithelialglycoprotein-2; inflammation;targeting effectiveasasoletherapeuticentity.Thecombinationofonesuch antibodywithTNF-␣resultedinconsiderablyenhancedimmune activity,leadingtolocalcomplementandmyeloidcellactiva- Manystudieshaveindicatedapotentialrolefortheimmune tion.22 Antibodyhumanizationpartlyresolvesthedrawbacksen- systeminthemanagementofcancer.1–6 Immunecells,especially CTLs,harborhighcytolyticactivityandareinvolvedinanatural responseagainstcertaintumors.7,8 TheeffectivenessofCTL-based Abbreviations:CTL,cytotoxicT-lymphocyte;EGP-2,epithelialglyco- protein-2;HBSS,Hanksbalancedsaltsolution;HUVEC,humanumbilical immunotherapeutictumorantigen-specificresponsesdependson3 veinendothelialcell;ICAM,intracellularadhesionmolecule;IFN,inter- prerequisites.Firstly,specificimmuneeffectorcellsneedtobe- feron;MAb,monoclonalantibody;PBMC,peripheralbloodmononuclear comeactivatedtogaincytolyticpotential.Secondly,immune cell;PMA,phorbolmyristateacetate;ROS,reactiveoxygenspecies;TNF, effectorcellsneedtomigratetowardthesiteofthetumor.Thirdly, tumornecrosisfactor. themigratedimmunecellsshouldexerteffectiveantitumoractiv- ity.Tumorsareabletoevadethedestructiveimpactofanimmu- Grantsponsor:DutchCancerSociety;Grantnumber:RUG98-1669; nologictumorantigen-specificresponsebyunderminingoneor Grantsponsor:Nijbakker-MorraFoundation. moreoftheseprerequisites.9–14 Theimmune-modulatingactivity ofthetumorcanpotentiallyimpairtheefficacyofimmunothera- peuticmodalities.Therefore,manystudiesaimtoshiftthebalance *Correspondenceto:DivisionofMedicalBiology,TumorImmunology ofthismodulatoryactivityofthetumor.15,16 Inthisrespect,many Branch,DepartmentofPathologyandLaboratoryMedicine,University HospitalGroningen,Hanzeplein1,9713GZ,Groningen,TheNetherlands. studieshavefocusedontheactivationand(directed)cytolytic Fax:ϩ31-50-3619911orϩ31-50-3633113. potentialofcytotoxicTcells,whereasrelativelylittleattentionhas E-mail:[email protected] beengiventothedevelopmentofstrategiestospecificallyincrease immuneeffectorcellmigrationintothetumor. Received13June2001;Revised17August,7November2001;Ac- Normally,immuneeffectorcellsareefficientlyattractedtoward cepted16November2001 inflammatorysites,wheretheyinteractwithpathogensaspartof thecellularhostdefense.Differentcategoriesofinflammation induction(typesI–IV)canbedistinguishedbasedonthewaythe Publishedonline6February2002 858 KROESEN ET AL. countered with the therapeutic use of mouse MAbs; however, Harvard Medical School, Boston, MA), CD54 (anti-ICAM-1; technical problems are associated with the generation, affinity and Beckman Coulter, Mijdrecht, The Netherlands) and FITC-labeled Ј large-scale production of such recombinant molecules. F(ab )2 rabbit antimouse Ig conjugate (Dako, Glostrup, Denmark) In our present study, we combined the predefined specificity of were used. mouse MAbs with human antimouse Ig antibodies in a 2-step Human antimouse Ig antibodies were isolated from the serum of approach to induce extensive immune complex formation at the a patient (patient 925) previously treated with repeated doses of the site of the tumor. Such strategy holds potential advantages over mouse bispecific antibody BIS-1. The total human Ig fraction single mouse or humanized antibody-mediated immunotherapy. containing antimouse Ig antibodies was isolated according to stan- No direct immune effector function needs to be associated with the dard protein A column chromatography, as described.25 Protein A tumor antigen-specific mouse antibody, which can thus be selected eluate was dialyzed against PBS and stored at a concentration of 5 merely on the basis of specificity and affinity for the tumor. mg/ml in PBS, 0.5% human serum albumin (CLB, Amsterdam, Moreover, the monomeric mouse Ig is not likely to bind exten- The Netherlands). The antimouse Ig recognizing a fraction of the sively to circulating myeloid cells, allowing good initial localiza- isolated human Ig was analyzed by capture ELISA using MOC31 tion at the site of the tumor. Then, after localization of the mouse immobilized to a 96-well medium-binding ELISA plate (Costar, antitumor antibodies and clearance of circulating mouse Ig, sub- Cambridge, MA) as a catching antigen and peroxidase-labeled sequently applied human antimouse Ig will specifically bind at the goat antihuman IgG and goat antihuman IgM as a detecting con- site of the tumor, thus optimizing local Fc–FcR interaction and jugate. Using a standard curve of known amounts of human IgG complement activation. The resulting inflammatory reaction could and IgM, the IgG antimouse Ig antibody fraction was determined be exploited in numerous immunotherapeutic treatment modali- to be approximately 13 ␮g/ml and the IgM antimouse Ig antibody ties. Naturally occurring antitumor activity may be induced or fraction was 31 ␮g/ml of the 5 mg/ml total human Ig. A 10ϫ enhanced and adoptive immunotherapeutic approaches could ben- dilution of the 5 mg/ml human antimouse Ig antibody preparation efit from such targeted inflammation induction strategies by en- was used. hancement of immune effector-cell migration specifically toward the site of the tumor. In addition, the local inflammatory reaction Superoxide analysis could establish a favorable environment for cellular immune ef- Superoxide production was measured essentially as described fector functions, such as enhanced cytolytic activity, antigen pre- by Ginsburg et al.30 In brief, 2.5 ϫ 105 myeloid cells suspended in sentation, costimulation and immune cell survival. Here, we de- 50 ␮l HBSS were added to each well of a sterile solid white scribe the immunobiologic effects of the 2-step targeting approach 96-well plate (Costar) containing different