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 stimulatory conditions in a model system using myeloid cells and defined tumor antigen- in 50 ␮l HBSS. Stimulations included 1 ␮g/ml PMA (Sigma positive tumor cell lines. Aldrich, Zwijndrecht, The Netherlands), 0.1 mg/ml opsonized zymosan (Sigma Aldrich) and 0.1 ␮g/ml in-house protein A-pu- MATERIAL AND METHODS rified mouse MAbs either alone or in combination with secondary cross-linking human antimouse Ig antibodies. Antibody-FcR–me- Cells diated myeloid cell activation was performed in the presence of GLC-1 (EGP-2-negative) and GLC-8 (EGP-2-positive) are both 2.5 ϫ 105 GLC-1 or GLC-8 cells. Superoxide generation was small cell lung carcinoma–derived cell lines.23 EGP-2 is a 38 kDa detected using 30 ␮M lucigenin (Sigma Aldrich), which was added transmembrane antigen, also known as 17-1A or Ep-CAM, which just prior to the first of a series of measurements to analyze is widely used for targeted immunotherapy of carcinomas.3,24–27 superoxide production kinetically for up to 1 hr. Chemilumines- MonoMac-6 (ATCC, Rockville, MD) is a monoblastic leukemia- cence was measured using a Lucy-1 luminescence reader (Phar- derived cell line with all of the phenotypic and biologic charac- macia, Uppsala, Sweden). Data were collected using Biolise soft- teristics of mature monocytes.28 MonoMac-6 cells were primed by ware. culturing for 48 hr in the presence of 200 U/ml IFN-␥ (Boehringer- Ingelheim, Ridgefield, CT). All cell lines were cultured according Flow cytometry to routine procedures in RPMI-1640 culture medium (GIBCO All incubations and wash steps were performed at 4°C. Anti- BRL, Grand Island, NY) supplemented with 10% heat-inactivated body incubations (30 min) were carried out in PBS supplemented FBS (GIBCO BRL), 2 mM L-glutamine (GIBCO BRL), 60 ␮g/ml with 5% human pooled serum and 5% normal rabbit serum. gentamicin (GIBCO BRL), 0.05 mM ␤-mercaptoethanol (Merck, Control stainings (conjugate and isotype-matched control antibod- Darmstadt, Germany) and 1 mM sodium pyruvate (GIBCO BRL) ies) were routinely included to rule out the possibility of detecting at 37°C in a humidified atmosphere containing 5% CO2. a positive signal due to nonspecific binding of the antibodies. HUVECs were obtained from the Endothelial Cell Facility, Immediately after staining, cells were analyzed on a Coulter (Hi- Faculty of Medical Sciences and University Hospital Groningen aleah, FL) Elite Cytometer using Winlist (version 3.1) (Verity (Dr. G. Molema, Mr. H. Moorlag), isolated and cultured as de- Software House, Topsham, ME). scribed.29 By routine flow-cytometric phenotyping for CD31, cells were found to be uniformly positive. HUVEC passages 2–4 were TNF-␣ ELISA used for the adhesion/transendothelial migration studies presented. An antibody-based ELISA (British Biotechnology, Oxford, UK) was used according to the manufacturer’s instructions to assess Antibodies TNF-␣ in the culture supernatant of activated MonoMac-6 cells. Antibodies used to activate myeloid cells included the EGP-2- Cell suspensions were centrifuged at 500g at 4°C to remove cells. specific mouse MAbs MOC31 (IgG1) and MOC181 (IgG2a),24 Supernatants were stored at –20°C until analysis. which recognize the same epitope on EGP-2. As isotype-matched control antibodies with irrelevant specificity MAbs R73 (mouse Complement lysis antirat TcR, IgG1) and WT32 (mouse antihuman CD3, IgG2a) Cell lysis by rabbit complement was assessed as follows. Prior were used. FcR binding experiments were performed with to starting the assay, 5 ϫ 106 GLC-1 and GLC-8 cells were labeled MOC31, MOC181, T1 (mouse antihuman CD5, IgG2b) and RIV-9 51 ␮ with 3.7 MBq Na2 CrO4 (Amersham, Aylesbury, UK) in 100 l (mouse antihuman CD3, IgG3). The amounts of antibody added in 51 medium at 37°C for 1 hr. Unbound Na2 CrO4 was removed by the experiments were in all cases saturating, as determined by flow washing cells 3 times with medium. Cells were then incubated cytometry in separate experiments (data not shown). with the antibodies described above for 30 min at 4°C, washed For flow cytometry, the MAbs MAb22, mouse IgG1 anti-CD64 once with medium, counted and diluted in culture medium at 5 ϫ (Medarex, Annandale, NJ), H18/7 (anti-E-selectin, a kind gift of 104 cells/ml. Aliquots of 100 ␮l were seeded into a 96-well plate Dr. M.A. Gimbrone, Jr., Brigham and Women’s Hospital and at 5 ϫ 103 cells/well. Complement (Low-Tox-R rabbit comple- TUMOR-TARGETED IMMUNE COMPLEX FORMATION 859 ment; Cedarlane, Ontario, Canada) was added in a volume of 100 ␮linafinal dilution of 1:40. Plates were incubated for 2.5 hr at 37°C, 5% CO2. After incubation, plates were centrifuged for 5 min at 200g and 100 ␮l samples collected and counted in a ␥-counter (LKB, Uppsala, Sweden). Cell lysis, indicative of complement activation, was calculated from the percentage 51Cr released ac- cording to the following formula: (experimental release – sponta- neous release)/(maximal release – spontaneous release) ϫ 100%, in which maximal release was determined from a sample to which 1% Triton X-100 was added and spontaneous release was deter- mined from a sample to which only medium was added. Cell lysis by human complement was assessed as follows. Aliquots of 2.5 ϫ 105 cells were incubated with the antibodies described above for 30 min at 4°C, washed once with medium and suspended in 300 ␮l medium to which 100 ␮l freshly isolated human serum were added. Samples were incubated for 2.5 hr at 37°C, 5% CO2. After incubation, cells were centrifuged for 5 min at 200g and 150 ␮l fresh medium containing 2 ␮g/ml propidium iodide (Molecular Probes, Leiden, The Netherlands) were added. Cell lysis was determined flow cytometrically on the Coulter Elite flow cytometer by determining the percentage propidium iodide- positive cells. Determination of lymphocyte adhesion/transendothelial migration FIGURE 1 – Superoxide production by MonoMac-6 cells. IFN-␥– Experiments to investigate the effects of myeloid cell activation 5 on PBMC interactions with endothelium were performed essen- primed MonoMac-6 cells (2.5 ϫ 10 ) were incubated with 1 ␮g/ml tially as described.31,32 Briefly, HUVECs were seeded into 24-well PMA (solid circles) or 0.1 mg/ml opsonized zymosan (open circles) or without stimulus (solid triangles) in HBSS containing 30 ␮M lucige- plates (Costar) on Vitrogen 100 collagen layers (Collagen Corpo- nin. MonoMac-6 cells had been prestimulated for 48 hr with 200 IU/ml ration, Palo Alto, CA) at confluent cell density and cultured for 48 IFN-␥. After adding lucigenin, chemiluminescence (CL) was mea- hr. Tumor cell opsonization with EGP-2–directed antibodies sured repeatedly for up to 1.5 hr. Values represent means Ϯ SEM, MOC31 or MOC181 was done for 45 min at 4°C. Subsequently, n ϭ 3. antibody-opsonized tumor cells and IFN-␥–primed MonoMac-6 cells or human peripheral blood monocytes (both 2.5 ϫ 105 in 0.5 ml) were added to HUVECs in the presence or absence of human antimouse Ig antibodies. After incubation for 4 hr, wells were washed with RPMI-1640 medium and freshly isolated PBMCs ϫ 6 (1 10 ) were added. After 3 hr incubation at 37°C, 5% CO2, nonadherent cells were removed by washing 3 times with HBSS. The remaining cells were treated with 0.05% trypsin (ICN Bio- medicals, Zoetermeer, The Netherlands) in 0.5 mM EDTA solu- tion and collagenase (2 mg/ml freshly prepared in PBS; collage- nase was a gift from Dr. Knoll, Ludwigshafen, Germany) for 15–30 min at 37°C to release adherent and migrated lymphocytes, respectively and put on ice. Flow-cytometric determination of absolute cell numbers recovered was performed using fluorosphere reagent (Flow Count Fluorospheres, Coulter).

FIGURE 2 – Expression of CD64 (Fc␥RI) and binding of the various RESULTS subclasses of mouse IgG to IFN-␥–primed or untreated MonoMac-6 Antibody binding and activation of MonoMac-6 cells cells. MonoMac-6 cells (0.5 ϫ 106) were incubated with the antibodies MonoMac-6 cells could be stimulated to produce easily detect- (10% hybridoma culture supernatant) shown in the absence of human able levels of ROS in response to triggering with immune com- pooled serum. Anti-CD64 (MAb22, reactive with an epitope outside via the ligand-binding site) was applied as purified IgG in the presence of plexes serum-opsonized zymosan and PMA (Fig. 1). A pre- 5% human pooled serum. Antibody binding was performed using requisite for activation appeared to be treatment of cells with Ј FITC-labeled F(ab )2 goat antimouse Ig antibody fragments, followed IFN-␥ prior to zymosan/PMA stimulation (data not shown). Ad- by flow-cytometric analysis. Representative experiment of 3 indepen- dition of superoxide dismutase completely blocked superoxide- dent experiments, performed in duplicate, is shown. Values represent induced chemiluminescence (data not shown), which is in line with means Ϯ SEM. earlier reports on the strict specificity of lucigenin as a substrate for oxygen radicals. Direct binding of specific mouse antibody isotype classes to (GLC-1) tumor cells coated with anti-EGP-2–directed IgG1 MonoMac-6 cells was assessed flow-cytometrically using mouse (MOC31) and IgG2a (MOC181) antibodies. Coincubation of IFN- IgG1 (MOC31), IgG2a (MOC181), IgG2b (T1) and IgG3 (RIV-9) ␥–treated MonoMac-6 cells with GLC-8 tumor cells in the pres- MAbs. As shown in Figure 2, preferential binding of mouse IgG2a ence of MOC181 antibodies resulted in direct and specific activa- antibodies to MonoMac-6 cells was observed. Binding depended tion of MonoMac-6 cells, as assessed by superoxide generation largely on prior treatment of cells with IFN-␥ and appeared to be (Fig. 3a) and TNF-␣ production (Fig. 3c). No activation of Mono- proportional to the amount of Fc␥RI (CD64) expressed. Mac-6 cells was noted when GLC-1 tumor cells (Fig. 3b), anti- EGP-2–directed IgG1 antibodies (MOC31) or irrelevant mouse Functional binding of tumor antigen-specific MAb IgG2a antibodies were used. This showed the physiologic potential Functional binding of tumor-directed MAbs by MonoMac-6 of tumor antigen-specific mouse IgG2a MAbs to trigger myeloid cells was assessed using EGP-2-positive (GLC-8) and -negative cell activation when presented properly, i.e., cross-linked by rel- 860 KROESEN ET AL.

FIGURE 4 – Flow-cytometric analysis of E-selectin and ICAM-1 expression by HUVECs. IFN-␥–primed MonoMac-6 cells were acti- vated overnight with antibody-opsonized tumor cells as shown and coincubated with HUVECs for 4 hr (for E-selectin expression) or 24 hr (for ICAM-1 expression). TNF-␣ (0.1 ␮g/ml) was included as a positive control. Values represent means Ϯ SEM, n ϭ 3.

MOC181 to MonoMac-6 cells as shown by flow-cytometric anal- ysis. Accordingly, activation of MonoMac-6 cells by MOC181- opsonized tumor cells, assayed by the production of ROS, also decreased in the presence of human serum (Fig. 5b). Cross-linking of mouse anti-EGP-2 MAbs using human antimouse Ig To overcome the inhibitory effect of serum on antibody–FcR- induced myeloid cell activation, we included an additional anti- body cross-linking step using human antimouse Ig in combination FIGURE 3 – Superoxide production (a,b) and TNF-␣ production by MonoMac-6 cells after activation with mouse IgG-opsonized tumor with a first tumor binding step of mouse anti-EGP-2 MAbs cells. IFN-␥–primed MonoMac-6 cells (2.5 ϫ 105) and MOC31- (open (MOC181 or MOC31). Such a 2-step cross-linking approach pro- circles) or MOC181- (solid circles) opsonized GLC-8 (a,c) or GLC-1 vides a means to specifically deposit human Ig at the site of the (b) cells (2.5 ϫ 105) were coincubated in the presence of 30 ␮M tumor, which is likely to be favorable for effective binding of lucigenin. WT32 (solid triangles, mIgG2a) was used as an isotype- FcR-positive cells. In addition, a 2-step antibody binding strategy matched control for MOC181. Superoxide-induced chemilumines- potentially increases the extent of Ig complexation and, as a result, cence (CL) was measured for up to 1 hr after adding lucigenin to cells. will be more efficient in activating myeloid cells or the comple- Ϯ ϭ Values represent means SEM, n 3. ment system. Human antimouse Ig antibody-opsonized tumor cells preincubated with MOC181 proved to be an excellent substrate for tumor cell–directed myeloid cell activation (Fig. 6). Moreover, evant tumor cells. Similar results were obtained using U937 cells myeloid cell activation was unaffected by the presence of normal instead of MonoMac-6 cells (data not shown). Prior treatment of human serum. Interestingly, MOC31 (IgG1), in addition to U937 or MonoMac-6 cells with IFN-␥ was found to be a prereq- MOC181 (IgG2a), was able to elicit myeloid cell activation when uisite for activation since neither superoxide generation nor TNF-␣ used in combination with human antimouse Ig antibodies, whereas production could be measured when omitting preactivation with MOC31 by itself was unable to induce any form of myeloid cell IFN-␥ (data not shown). activation, even in the absence of normal human serum (Fig. 3). Functional activation of endothelial cells Besides Ig–FcR-induced myeloid cell activation, cross-linking an- Functional activation of endothelial cells was induced by Mono- tibodies are effective activators of the complement system. As Mac-6 cells after activation via Fc␥R cross-linking. We first ana- shown in Figure 7, MOC181-, but not MOC31-, opsonized tumor lyzed E-selectin and ICAM-1 expression by HUVECs in response cells were able to activate complement to the extent that cell lysis to coincubation with MonoMac-6 cells, activated via MOC181- could be observed using both rabbit or human complement. How- opsonized tumor cells. As shown in Figure 4, both E-selectin and ever, using human antimouse Ig antibodies as a second cross- ICAM-1 adhesion molecules became upregulated on HUVECs in linking agent, specific lysis of EGP-2-positive cells was observed the presence of MonoMac-6 cells activated via MOC181-opso- using both MOC181 and MOC31 MAbs (Fig. 7). No complement- nized tumor cells. Maximal E-selectin expression was noted 4 hr mediated cell lysis was observed against EGP-2-negative (GLC-1) after coincubation, whereas ICAM-1 became maximally expressed target cells. 24 hr after coincubation. In the absence of MOC181 or IFN-␥– To extend the myeloid cell–activating properties of this dual- primed MonoMac-6 cells, significantly less E-selectin and antibody cross-linking approach, we next studied lymphocyte ICAM-1 were expressed by HUVECs (p Ͻ 0.002, t-test). transmigration mediated by the antibody–complex-induced in- flammatory response. To this end, we employed a flow-cytometric Inhibition of antibody-induced myeloid cell activation transmigration assay to quantitate lymphocyte transmigration over The interaction of tumor EGP-2-bound antibodies with FcR a HUVEC monolayer. Endothelial cells were preincubated over- expressed by myeloid cells may be blocked in the presence of night with a mixture of IFN-␥–activated MonoMac-6 cells and excess human Ig, as is present in normal human serum. Such MOC181-opsonized tumor cells. This allowed a significant num- interference could result in less efficient FcR-triggered myeloid ber of freshly added lymphocytes to migrate over the endothelial cell activation. Indeed, binding of MOC181 to MonoMac-6 cells cell layer (data not shown). Myeloid cell activation appeared to be was blocked in the presence of serum (Fig. 5a). Incubation of a prerequisite for the observed transmigration induction since MonoMac-6 cells with MOC181 in the presence of an increasing omitting any component that allowed ROS or TNF-␣ production concentration of serum resulted in a decreased binding of by MonoMac-6 cells resulted in significantly reduced lymphocyte TUMOR-TARGETED IMMUNE COMPLEX FORMATION 861

FIGURE 6 – Superoxide production by IFN-␥–primed MonoMac-6 cells by combined mouse tumor antigen-specific and human antimouse Ig antibody-opsonized tumor cells. GLC-8 cells were sequentially incubated with MOC181 (solid circles), MOC31 (open circles) or WT32 (solid triangles) and human antimouse Ig. MonoMac-6 cells were added to antibody-opsonized tumor cells in a ratio of 1:1 in the presence of 30 ␮M lucigenin and superoxide-induced chemilumines- cence (CL) was measured up to 1 hr thereafter. Values represent means Ϯ SEM, n ϭ 3.

FIGURE 7 – Complement-mediated lysis of antibody-opsonized tu- mor cells. GLC-8 or GLC-1 tumor cells were incubated with MOC31 or MOC181 in the presence or absence of human antimouse Ig. IGURE 51 ϫ 3 F 5 – Effects of human serum on the binding of MOC181 to Antibody-opsonized Na2 CrO4-labeled tumor cells (5 10 ) were and superoxide production by MonoMac-6 cells. (a) IFN-␥–primed incubated with 1:40 diluted rabbit serum (RCЈ) or 1:4 diluted freshly MonoMac-6 cells were incubated with MOC181 (10% hybridoma derived human serum (HCЈ) for 2.5 hr at 37°C. Values represent culture supernatant) in the absence or presence of an increasing means Ϯ SEM, n ϭ 3. amount of human pooled serum and analyzed by flow cytometry for binding of MOC181 (representative experiment of 3 independent experiments, performed in duplicate is shown). Values represent nized tumor cells. However, as shown in Figure 8, when human means Ϯ SEM. (b) MonoMac-6 cells were incubated with MOC181- opsonized GLC-8 tumor cells in the absence or presence of an increas- antimouse Ig antibodies were added to MOC181- or MOC31- ing amount of human pooled serum and assayed by chemilumines- preincubated tumor cells, lymphocyte transmigration was restored cence (CL) for superoxide production. Values represent means Ϯ in agreement with the previously observed restoration of myeloid SEM, n ϭ 3. cell activation using this antibody cross-linking approach.

DISCUSSION transmigration. In line with the data presented in Figure 5, normal human serum completely abrogated lymphocyte migration induced We have studied tumor-directed myeloid cell activation using by myeloid cells that had been preincubated with MOC181-opso- tumor-specific antibodies to induce tumor-directed immune effec- 862 KROESEN ET AL.

induce (tumor cell) specific activation of immune effector cells and to enhance the specific recognition and killing of tumor cells. Limited studies have focused on the development of ways to improve tumor-directed immune effector-cell migration. However, migration of immune effector cells to the tumor site is an important prerequisite for effective cell-mediated immunotherapy and con- trolled regulation of such a process holds significant potential for the further development of diverse immunotherapeutic approaches.33 Indeed, previous in vivo studies by us and others have indicated that immunotherapeutic approaches using bispecific antibody–re- targeted T cells are effective only in when micrometastases are treated.34,35 Treatment of large tumor nodules was not effective, most probably due to insufficient T-cell infiltration into the tumor mass. These observations implicate the necessity to combine treat- ments, as mentioned above, with strategies aimed at specific in- FIGURE 8 – Transendothelial migration of lymphocytes by antibody- duction of immune effector-cell migration toward the tumor site. opsonized, tumor cell–mediated MonoMac-6 activation. A monolayer of HUVECs was incubated with a 1:1 mixture of IFN-␥–primed Under physiologic conditions, the humoral response becomes a MonoMac-6 cells and GLC-8 tumor cells in the absence or presence of polyclonal antibody response. This allows for maximal binding of MOC31 or MOC181 and/or human antimouse Ig. After 4 hr, the diverse classes of antibodies to often a number of antigens ex- MonoMac-6/GLC-8 cell suspension was washed off and freshly iso- pressed by the pathogen or target cell, which subsequently in- lated PBMCs were added to the HUVEC monolayer. After an addi- creases the efficiency of complement activation and FcR cross- tional incubation period of 3 hr, mononuclear cells that had migrated linking. In addition, one of the Fc␥Rs (CD32) has been described were quantitated flow-cytometrically, as described in Material and to preferably bind extensively cross-linking IgG.17,18 The other Ϯ ϭ Methods. Values represent means SEM, n 3. Fc␥Rs, CD64 and CD16, bind monomeric IgG with high and intermediate affinity, respectively.17,18 Because MAbs by defini- tion are monospecific, they result in only limited IgG cross-linking tor cell migration. We describe myeloid cell activation, as assessed and, thus, a reduced efficiency of myeloid cell activation. Due to by ROS production and the expression of typical myeloid activa- the abundant presence of serum IgG, the FcR-binding monomeric tion markers by MonoMac-6 cells using tumor antigen-specific IgG is normally occupied by irrelevant human serum IgG and, mouse IgG2a, but not IgG1, MAbs. The observed antibody-medi- thus, unavailable for binding of any therapeutic, exogenously ated activation of MonoMac-6 cells most probably involved CD64 applied MAb. Nevertheless, a number of reports have documented since preincubation with IFN-␥ increased both membrane CD64 potent tumor antigen-specific responses in both experimental ani- expression and responsiveness to FcR-bound antibodies. In addi- mal models and humans using MAbs.22,27 In some cases, immune tion, human serum strongly inhibited IgG2a-induced MonoMac-6 activation was achieved by combining the application of antibod- cell activation, suggesting displacement of mouse tumor antigen- ies with immune-stimulating cytokines such as TNF-␣.22 Priming specific antibodies by human monomeric serum IgG, which is of polymorphonuclear granulocytes with cytokines such as TNF-␣ present in vast excess. To increase the extent of antibody cross- or compounds such as formyl-methionyl-leucyl-phenylalanine linking and make the antibody Fc domains presented more com- (fMLP) have been reported to considerably increase the biologic patible with the human FcRs expressed by MonoMac-6 cells, we effects of FcR cross-linking. We also observed substantially in- added human antimouse Ig as a second antibody cross-linking step creased sensitivity to the administered antibodies when Mono- to mouse IgG-opsonized tumor cells. This incubation step com- Mac-6 cells were precultured in the presence of IFN-␥. This effect pletely restored the activation signal induced by mouse IgG2a was not just due to the marked upregulation of CD64, allowing a antibody-opsonized tumor cells, which was inhibited in the pres- direct increase in the binding of the antibodies, because ROS ence of human serum. Moreover, MonoMac-6 cell activation was production by PMA, a direct activator of protein kinase C and ROS observed using mouse IgG1-opsonized tumor cells in combination production, also depended on priming with IFN-␥. This is in line with the added human antimouse Ig. This latter observation in with numerous reports on the effects of IFN-␥ on the activation of particular opens up possibilities to exploit this concept clinically in myeloid cells, which involve the translocation of transcription a 2-phase treatment setting. The high-affinity anti-EGP-2–directed factors such as NF␬B and signal transducer and activator of mouse IgG1 antibody MOC3124,26 could thus be used to attain transcription (STAT), inducing the expression of several (pro)in- optimal tumor localization without premature binding of the anti- flammatory cytokines.36–38 In addition, ROS augment Fc␥R sig- body to circulating FcR-positive cells in the blood. Subsequently naling via a catalase-dependent pathway.39 However, a direct applied human antimouse Ig will deposit specifically on MOC31- correlation between the level of Fc␥R expression and the severity opsonized tumor tissue, thereby providing optimal conditions for of immune complex–induced synovial inflammation has been local activation of myeloid cells. This could initiate a natural tumor shown.40 antigen-specific immune response or help to direct immune effec- The same dependence on IFN-␥ was observed using extensively tor-cell migration, which could be used in adoptive immunother- cross-linking antibodies as these provided, via opsonized zymosan apeutic approaches. and human antimouse Ig, cross-linking mouse IgG-opsonized tu- The clinical setting proposed here differs from the situation mor cells. In the latter situation, it did not matter whether human where mouse antibodies are administered to a patient positive for serum was present. This may indicate the involvement of FcRs human antimouse Ig antibodies due to previous treatments with other than CD64, such as CD32 and CD16, which have been mouse antibodies. Whereas preexisting human antimouse Ig anti- implemented in the functional binding of complexed Ig.40,41 We bodies could potentially neutralize the administered MAb, sequen- did not observe upregulation of CD32 or CD16 after priming with tial use of tumor-targeted MAbs and human antimouse Ig antibod- IFN-␥. This may underscore the intracellular effects of IFN-␥ ies would result in the induction of antibody complexes other than CD64 upregulation. In addition, FcRs may cooperate in specifically at the site of the tumor, as described here. In the the transduction of signals upon cross-linking such that the CD64- present study, we confined ourselves to in vitro proof of principle. associated ␥ chains may participate in the signal transduction of Ig The in vivo implications of our results are currently being inves- complexes bound to CD89, CD32 or CD16 via coclustering of the tigated. different FcRs, as described.39,41–43 Future studies may elucidate Thus far, the development of immunotherapeutic strategies for the relative contribution of the various specific FcRs that resulted the treatment of cancer has mainly concentrated on studies to in myeloid cell activation, as described here. TUMOR-TARGETED IMMUNE COMPLEX FORMATION 863

Also, further dissection of the relative contribution of FcR contribution of complement activation in a human setting to the cross-linking vs. activation of the complement system should be phenomena described here is highly relevant and the subject of investigated. In our experimental set-up, we used rabbit com- current investigation. plement and cell lysis as a read-out for complement activation. In conclusion, we have described a potential role for the use of Susceptibility of cells to complement-mediated lysis is a func- human antimouse Ig antibodies to attain mouse antibody-directed, tion of the rate by which complement-regulating factors, which tumor site-specific activation of myeloid cells. Myeloid cell acti- act in a syngeneically restricted fashion, degrade activated vation was dependent on pretreatment with IFN-␥, was not inhib- complement components. In addition, biologically active inter- ited by human serum and resulted in the production of inflamma- mediates such as C3a, C4a and C5a could be involved in tory intermediates that could activate endothelial cells to facilitate attracting and activating myeloid cells. Therefore, the in vivo leukocyte recruitment.

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