simultaneous targeting of fcgrs and fcari …...2015/09/30 · research article simultaneous...

Research Article

Simultaneous Targeting of FcgRs and FcaRIEnhances Tumor Cell KillingArianne M. Brandsma1, Toine ten Broeke1, Maaike Nederend1, Laura A.P.M. Meulenbroek1,Geert van Tetering1, Saskia Meyer1, J.H. Marco Jansen1, M. Alejandra Beltr�an Buitrago1,Sietse Q. Nagelkerke2, Istv�an N�emeth3, Ruud Ubink4, Gerard Rouwendal4, Stefan Lohse5,Thomas Valerius5, Jeanette H.W. Leusen1, and Peter Boross1

Abstract

Efficacy of anticancer monoclonal antibodies (mAb) is limitedby the exhaustion of effector mechanisms. IgG mAbs mediatecellular effector functions through FcgRs expressed on effectorcells. IgAmAbs can also induce efficient tumor killing both in vitroand in vivo. IgA mAbs recruit FcaRI-expressing effector cells andtherefore initiate different effector mechanisms in vivo comparedwith IgG. Here, we studied killing of tumor cells coexpressingEGFR and HER2 by the IgG mAbs cetuximab and trastuzumaband their IgA variants. In the presence of a heterogeneous pop-ulation of effector cells (leukocytes), the combination of IgG andIgA mAbs to two different tumor targets (EGFR and HER2) led toenhanced cytotoxicity compared with each isotype alone. Com-bination of two IgGs or two IgAs or IgG and IgA against the same

target did not enhance cytotoxicity. Increased cytotoxicity reliedon the presence of both the peripheral blood mononuclear celland the polymorphonuclear (PMN) fraction. Purified naturalkiller cells were only cytotoxic with IgG, whereas cytotoxicityinduced by PMNs was strong with IgA and poor with IgG.Monocytes, which coexpress FcgRs and FcaRI, also displayedincreased cytotoxicity by the combination of IgG and IgA in anovernight killing assay. Coinjection of cetuximab and IgA2-HER2resulted in increased antitumor effects compared with eithermAbalone in a xenograft model with A431-luc2-HER2 cells. Thus, thecombination of IgG and IgA isotypes optimallymobilizes cellulareffectors for cytotoxicity, representing a promising novel strategyto improve mAb therapy. Cancer Immunol Res; 1–9. �2015 AACR.

IntroductionAntibody therapy with IgG monoclonal antibodies (mAb)

represents part of the standard treatment in the clinic for variousforms of cancer. The ErbB family members EGFR and HER2 arevalidated clinical targets for mAb therapy (1). Cetuximab (anti-EGFR) is approved for colon and head and neck cancers, whereastrastuzumab (anti-HER2) is used for breast and gastric cancertreatment. EGFR mAbs have a dual mechanism of action: BothFab- and Fc-mediated antitumor effects were described (2). Thedirect Fab-mediated effects include occlusion of the ligand-bind-ing site, leading to hampering of receptor dimerization, andinternalization of EGFR, leading to inhibition of downstreamsignaling (3). The antitumor mechanisms of HER2 mAbs are less

well understood, but also involve Fab-mediated direct effects, suchas induction of receptor internalization anddegradation, or induc-tion of cell-cycle arrest by inhibiting downstream signaling (4, 5).Next to direct Fab-mediated effects, the mechanisms of action ofboth mAbs involve Fc gamma receptor (FcgR)–dependentmechanisms. The importance of Fc–FcgR interaction is supportedby clinical association studies and preclinical animal studies forboth EGFR (6–8) and HER2 mAb therapy (9, 10). However, IgGmAb therapy is rarely curative, and this has in part been attributedto exhaustion of cellular effector mechanisms (11).

Cetuximab and trastuzumab are, like most therapeutic mAbs,of the IgG1 subclass. IgG1 mAbs engage FcgRs on effector cells toinitiate antibody-dependent cell-mediated cytotoxicity (ADCC).Each immune effector cell type has a unique expression of FcgRs.For example, in humans, natural killer (NK) cells express onlyFcgRIIIa (and in some individuals FcgRIIc), polymorphonuclearcells (PMN) express FcgRIIa and FcgRIIIb, whereas monocytesexpress FcgRI, FcgRIIa, and FcgRIIIa.

Coengagement of the activating FcgRs with the inhibitoryFcgRIIb by IgG1 on monocytes results in inhibitory signalingand limits the cytotoxic activity of IgG1. PMNs abundantlyexpress a GPI-linked nonsignaling FcgRIIIb. The interaction ofIgG1 with FcgRIIIb limits IgG1-mediated PMN cytotoxicity(12, 13). Moreover, polymorphisms in several FcgRs influenceIgG1 activity: Genotypes that confer to stronger IgG binding areassociated with better response to mAb therapy (14).

Previously,wehave shown that IgA antitumormAbs can induceefficient cytotoxicity in vitro and in vivo (15, 16). IgA induces morecytotoxicity than IgG by purified PMNs and unfractionatedhuman leukocytes (15) and induces comparable cytotoxicity with

1Immunotherapy Laboratory, Laboratory for Translational Immunolo-gy, UMC Utrecht, the Netherlands. 2Department of Blood CellResearch, Sanquin Research and Landsteiner Laboratory, AcademicMedical Center, University of Amsterdam, the Netherlands. 3Develop-mental Drug Metabolism and Pharmacokinetics, Gedeon Richter Plc,Budapest, Hungary. 4Synthon Biopharmaceuticals B.V., Nijmegen,the Netherlands. 5Division of Stem Cell Transplantation and Immuno-therapy, Department of Internal Medicine II, Christian-Albrechts-Uni-versity, Kiel, Germany.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Jeanette H.W. Leusen, University Medical CenterUtrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands. Phone:31-88-755-4268; Fax: 31-88-755-4305; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-15-0099-T

�2015 American Association for Cancer Research.

CancerImmunologyResearch

www.aacrjournals.org OF1

Research. on August 15, 2020. © 2015 American Association for Cancercancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst September 25, 2015; DOI: 10.1158/2326-6066.CIR-15-0099-T

http://cancerimmunolres.aacrjournals.org/

IgG by macrophages (16, 17). The cytotoxic activity of IgA ismediated by FcaRI, and therefore IgA induces different effectorfunctions than IgG, for example, more efficient activation ofPMNs compared with IgG (18).

EGFR and HER2 coexpression is found in a number of cancers,including pancreatic cancer, non–small cell lung cancer, andbreast cancer (19–21). Moreover, EGFR and HER2 coexpressionis associatedwith themost aggressive tumors (21). To increase IgGactivity, the combination of two antitumor IgG mAbs againstthese two targets has been investigated before (22, 23). Thesestudies have revealed increased antitumor effects by the combi-nation of two IgG mAbs, which were mainly due to the dual Fab-mediated effector functions. At saturating target opsonization,this approach also increases the available Fc moieties for FcgRbinding; however, tumor cell killing is still limited by the numberof available FcgRs on effector cells and/or their cytotoxic capacity.

We reasoned that the combination of IgG and IgA wouldinduce enhanced tumor cell killing through the recruitmentand/or activation of both FcgR-bearing NK cells and FcaRI-bear-ing PMNs.Coengagement of FcgRs and FcaRI on the same effectorcell (monocytes/macrophages) might also result in stronger FcRsignaling and tumor cell killing. To this end, we studied cytotox-icity induced by the combination of IgG and IgA (cetuximab ortrastuzumab, as well as their IgA variants carrying the samevariable region) toward tumor cells coexpressing EGFRandHER2.

Materials and MethodsCell lines and cell culture

The human epidermoid carcinoma cell line A431 and thehuman breast cancer cell line SK-BR-3 (ATCC) were authenticatedbySTRprofiling.No reauthenticationassaywasperformedbecausecell lines were directly obtained from cell banks and used at earlypassages after receipt or resuscitation. All cell lines were tested andvalidated to beMycoplasma free. Cell lines were cultured in RPMI-1640 (Gibco) supplemented with 10% FCS and penicillinand streptomycin (Gibco). A431-HER2 cells were generated byretroviral transduction of A431 cells with human HER2 (pMX-puro-HER2), and positive clones were selected using puromycin.A431-luc2 cells were generated as previously described (16) andtransducedwithHER2.HER2 expression onA431-luc2-HER2 cellswas comparablewith that inA431-HER2 cells.HER2expressiononthe transduced cell lines was regularly tested by cytofluorimetryandwas found tobe stable in time. Thenumber of EGFRandHER2molecules on the cells was determined using the Qifikit (DAKO).ThenumberofHER2molecules onA431-HER2 cellswas similar tothe number in SK-BR-3 cells, whereas the number of EGFR mole-cules was comparable with the number of A431 and A1207 cells(Supplementary Table S1).

AntibodiesThe chimeric anti-human EGFR mAb cetuximab and the

humanized anti-human HER2mAb trastuzumab were purchasedfrom the pharmacy of UMC Utrecht. Both cetuximab and trastu-zumab are of IgG1 isotype. The generation and characterization ofIgA2-EGFR have been previously described (15, 24). The gener-ation and characterization of IgA2-HER2mAbwill be described indetail elsewhere (S.Meyer, submitted for publication). Briefly, thevariable regions of trastuzumab were inserted into an expressionvector containing the constant regions of the heavy and lightchains of human IgA2 (24). IgA2 was produced by transient

transfection of HEK 293F cells (FreeStyle 293 Expression System;Invitrogen) and purified by affinity and size-exclusionchromatography.

Flow cytometryExpression of HER2 and EGFR on tumor cells was determined

using different concentrations of trastuzumab and cetuximab,respectively, andmeasuredwith flow cytometry using a PE-labeledanti-human IgGAb (The JacksonLaboratory). The followingmAbswere used for flow cytometry: CD3 (UCHT1; Biolegend), CD56(HCD56; Biolegend), CD14 (HCD14; Biolegend) and CD16(3G8; Biolegend) for isolated effector cells or CD45 (HI30; Bio-legend), CD3 (OKT3; Biolegend), CD56 (HCD56; Biolegend),CD14 (G1D3; eBioscience), and CD16 (3G8; Biolegend) Abs forleukocytes. Control mouse IgG2b and mouse IgG1 mAbs did notshow any background binding. BD Trucount tubes were used todetermine the absolute amount of effector cells in 50 mL leukocytesuspension reconstituted to the original volume of the peripheralblood sample. During the measurement, leukocytes were identi-fied to be CD45þ. Flow-cytometry measurements were performedon FACS CantoII (BD Biosciences). Flow-cytometry data wereanalyzed by FACS Diva software.

ADCCADCC with 51Cr-labeled target cells was described previously

(25). Briefly, 1 � 106 target cells were labeled with 100 mCi(3.7 MBq) 51Cr for 2 hours. After extensive washing, cells wereadjusted to 105/mL. Blood was obtained from healthy donors atthe UMC Utrecht. In leukocyte ADCC, erythrocytes were lysed byincubation inwater for 30 seconds, and then total leukocytes wereresuspended in medium. The number of leukocytes used per wellcorresponded to the number of leukocytes present in 50 mL ofblood before lysis. Effector cells, sensitizing mAbs at variousconcentrations, medium, and tumor cells were added toround-bottom microtiter plates (Corning Incorporated). After 4or 20 hours (overnight) of incubation at 37�C, 51Cr release wasmeasured in counts per minute (cpm). The percentage of specificlysis was calculated using the following formula:

½ðexperimental cpm�basal cpmÞ/ðmaximal cpm�basal cpmÞ� � 100;

with maximal lysis determined in the presence of 3% triton andbasal lysis in the absence of Abs and effector cells. We includedonly those donors in our analysis that induced a minimum of5% and maximum of 80% lysis with both single mAbs (Supple-mentary Table S2).

PMNand peripheral bloodmononuclear cell (PBMC) fractionswere isolated from blood by Ficoll/Histopaque separation (GEHealthcare; Sigma-Aldrich). Monocytes were isolated from thePBMC fraction usingCD14magnetic beads (Miltenyi Bioscience).NK cells were isolated from the CD14-negative fraction by neg-ative selection using the EasySep Human NK Cell Enrichment Kit(Stemcell Technologies). Purity of effector cells was analyzed byflow cytometry and was approximately 85% for NK cells, 70%formonocytes, and close to 100% for PMNs. The effector-to-target(E:T) ratios for purified fractions were 70:1 (PBMC), 40:1 (PMN),15-25:1 (monocytes), or 10:1 (NK cells). The E:T ratio in theleukocyte ADCC was 1:1 for both NK cells and monocytes and12:1 (with a range of 6–18:1) for PMNs. IgG and IgA mAbs werecombined at a 1:1 ratio.

In certain experiments, A431-HER2 or SK-BR-3 cells wereincubated for 6 hours with both PMNs (E:T ¼ 40:1) and PBMCs

Brandsma et al.

Cancer Immunol Res; 2015 Cancer Immunology ResearchOF2




(E:T ¼ 70:1), and specific lysis was compared with conditionsusing either PMNs (E:T ¼ 80:1) or PBMCs (E:T ¼ 140:1) alone.

For the experiments using sequential incubation with effectorcells, tumor cellswerefirst incubatedwithPMNs (E:T¼40:1) for 3hours, then PBMCs (E:T¼ 70:1) were added, and incubated withthe target cells for another 3 hours. As controls, tumor cells wereincubated either with PMNs (E:T¼ 80:1) or with PBMCs (140:1)twice for 3 hours. Cetuximab and IgA2-HER2 were added at 1:1ratio at the indicated concentrations.

A431-luc2-HER2 xenograft tumor modelThe in vivo xenograft experiment was performed as described

previously (16). FcaRI Tg or nontransgenic littermate SCID micewere injected i.p. with 5 � 105 A431-luc2-HER2 cells in 100 mLPBS. Tumor outgrowth was monitored by serial bioluminescentimaging (BLI; PhotonImager; Biospace Lab). BLI images wereprocessed using M3Vision software (Biospace Lab) and edited inAdobe Photoshop. Mice with tumor outgrowth were randomizedin different treatment groups on day 13. Cetuximab was injectedonce on day 14 (10 mg), whereas 10 mg IgA2-HER2 was injecteddaily between days 14 and 23 (in total 10 times) to compensatefor its shorter half-life. The combination group was injected withboth cetuximab and IgA2-HER2. Tumor growth is expressed aspercentage signal compared with the beginning of the treatment.Circulating serum mAb levels were monitored in serial bloodsamples taken from alternating mice (2 mice/time point). Serumlevels of human IgG and IgA were measured by ELISA as previ-ously described (16). The experiment was approved by the localAnimal Ethical Committee.

Data processing and statistical analysesStatistical analyses were performed in R (3.1.0) or inGraph Pad

6.0 software. Data are represented as mean � SEM, unless indi-cated otherwise. Dose–response curves for the ADCC experimentswere calculated via nonlinear regression [log(agonist) vs.response] using Graph Pad 6.0 software.

Maximal specific lysis was analyzed by using the triplets of aconcentration level on which maximal average specific lysis wasattained in an individual ADCCexperiment. Treatment effectswereanalyzedbyusing two-way full factorial linearmixed-effectsmodel-ing. The model consisted of two indicator variables for drugsapplied (yes or no) as fixed-effects terms, and subject and tripletsnested within subject as random-effect terms. The least-squaremean differences in the maximal lysis among the full factorialinteraction groups were estimated with the Tukey adjustment.

Inferential statistical analysis regarding the effect of treatmentson the time course of tumor growth in vivowas carried out by usinglinear mixed-effects modeling. The log-transformed tumor sizewas described by a model consisted of fixed-effect terms, such astime since xenograft (days) as a continuous covariate, and itssecond- and third-order interactions with the cetuximab andIgA2-HER2 treatments as factors, and two random-effect termsallowing individual differences in tumor size at day 1 and intumor growth time course.

ResultsIgG and IgA mAbs to both EGFR and HER2 induce cytotoxicityof A431-HER2 cells

To test cytotoxicity induced by the combinations of two dif-ferent isotypes, we used IgG and IgA specific for EGFR and HER2.

IgA variants of the chimeric IgG1-EGFR mAb cetuximab werepreviously generated (15, 24). Following a similar approach, wegenerated IgA variants of the human HER2 mAb trastuzumab(S.Meyer, submitted for publication). The IgA2 subclass was usedin our experiments because it induces stronger cytotoxicity thanthe IgA1 subclass (15, 16).

IgA2-EGFR and IgA2-HER2 induced significantly highercytotoxicity than cetuximab and trastuzumab, respectively,by leukocytes in a 4-hour killing assay with tumor cell linesexpressing high levels of endogenous targets (SupplementaryFig. S1A and S1B). To study the effects of different mAbcombinations against EGFR and HER2, we established atarget cell line with high expression of both EGFR and HER2(A431-HER2 cells), because HER2 expression on A431 cellswas too low for efficient cytotoxicity by trastuzumab (Sup-plementary Fig. S2; Supplementary Table S1 and unpublishedresults).

Both IgG- and IgA-mediated targeting of either EGFR orHER2 induced specific lysis of A431-HER2 cells using leuko-cytes as effector cells and, as expected, maximal lysis by IgA washigher than by IgG for both targets (Fig. 1A). No specific lysiswas observed in the absence of mAbs or effector cells, nor wasthe lysis induced by tumor-specific mAbs, influenced by thepresence of nonspecific antibodies. At low mAb concentra-tions, cetuximab was more effective than trastuzumab, mostlikely due to the higher expression of EGFR than HER2 byA431-HER2 cells.

Cytotoxicity by IgG and IgA ismediated by distinct populationsof effector cells within the leukocytes

Leukocytes contain a heterogeneous population of effectorcells (NK cells, monocytes, and PMNs) that are all capable ofinducing cytotoxicity. We therefore purified the different effec-tor cells to determine their individual cytotoxic capacity. PMNsand monocytes expressed both FcgRs and FcaRI, whereas NKcells only expressed FcgRIIIa (Supplementary Fig. S3). Inaccordance with their FcR expression, NK cells only inducedcytotoxicity with IgG (Fig. 1B). PMNs were more efficient withIgA2-EGFR than with cetuximab, and they induced tumor cellkilling with IgA2-HER2 but not with trastuzumab (Fig. 1C).Monocytes did not induce cytotoxicity after 4 hours with any ofthe mAbs. However, monocytes induced potent cytotoxicity inan overnight killing assay with all four mAbs, which is in linewith previous findings showing that monocytes require alonger incubation time for efficient cytotoxicity (Fig. 1D;refs. 16, 17).

In conclusion, IgG-mediated cytotoxicity is mediated mainlyby NK cells, whereas IgA-mediated cytotoxicity is mediated pre-dominantly via PMNs. Monocytes are unlikely to directly con-tribute to tumor cell killing in a 4-hour assay, but mediatecytotoxicity with both IgG and IgA in an overnight assay.

Increased cytotoxicity by the combination of IgG- andIgA-targeting EGFR and HER2

We first investigated the effect of combining two IgGs (ce-tuximab and trastuzumab) or two IgAs (IgA2-EGFR and IgA2-HER2) on the specific lysis of A431-HER2 cells by leukocytes.These combinations did not decrease EC50 values or increasemaximal cytotoxicity compared with the best mAb alone(Fig. 2A–D), likely because the Fc parts of both mAbs competedfor the same set of FcRs.

Improved Cytotoxicity by the Combination of IgG and IgA

www.aacrjournals.org Cancer Immunol Res; 2015 OF3




Given that IgG and IgA recruit predominantly different cellulareffectors (NK cells and PMNs), we hypothesized that the combi-nation of IgG and IgA would result in increased cytotoxicity byleukocytes containing both NK cells and PMNs. The combinationof IgG and IgA directed against the same target (cetuximab andIgA2-EGFR or trastuzumab and IgA2-HER2) did not increasemaximal tumor cell killing compared with the best mAb alone(Fig. 2E–H). This is most likely due to competition for the targetby both IgG and IgA. IgG might decrease IgA-mediated recruit-ment and activation of PMNs from the leukocytes. Indeed, IgA2-EGFR–induced cytotoxicity by purified PMNs was inhibited bythe addition of cetuximab (Supplementary Fig. S4).

Next, we investigated the combination of IgG and IgA direct-ed against two different targets. Cetuximab and IgA2-HER2were mixed at varying ratios while keeping the total mAbconcentration constant. The combination of cetuximab andIgA2-HER2 resulted in increased lysis of A431-HER2 cellscompared with cetuximab or IgA2-HER2 alone (Fig. 3A). Usingbroad mAb concentrations with multiple donors, the maximallysis induced by the combination cetuximab of and IgA2-HER2was significantly increased compared with the maximal lysis bythe best single mAb (Fig. 3B and C).

The combination of trastuzumab and IgA2-EGFR did notincrease maximal lysis compared with IgA2-EGFR alone(Fig. 3D and E). This might be due to the relatively high cyto-toxicity induced by IgA2-EGFR alone as a result of the high EGFRexpression by A431-HER2 cells.

These data show that the combination of IgG and IgA againsttwo different tumor targets can result in increased cytotoxicity byleukocytes.

Increased cytotoxicity by the combination of IgG and IgArequires multiple effector cell types

Whereas NK cells express only FcgRIIIa, human PMNs mayexpress both FcgRs (FcgRIa, FcgRIIa, and FcgRIIIb) and FcaRI,and are capable of tumor cell killing with both IgG andIgA (Supplementary Fig. S3). Because trastuzumab alone didnot induce cytotoxicity of A431-HER2 cells with PMNs(Fig. 1C), we studied only the combination of cetuximab andIgA2-HER2 on PMN-induced cytotoxicity. Cetuximab aloneinduced 5% to 20% maximal lysis depending on the donor.The combination of cetuximab and IgA2-HER2 did notincrease cytotoxicity by purified PMNs (SupplementaryFig. S5A and S5B).

The combination of cetuximab and IgA2-HER2 resulted inincreased maximal cytotoxicity of A431-HER2 cells whenPBMCs and PMNs were combined compared with either frac-tion individually (Fig. 4A). Similar effects were observed usingSK-BR-3 cells expressing both EGFR and HER2 endogenously(Fig. 4B). In addition, the sequential incubation of A431-HER2or SK-BR-3 cells with PBMCs and PMNs in the presence ofcetuximab and IgA2-HER2 resulted in increased cytotoxicitycompared with incubation with either PBMCs or PMNs alone(Fig. 4C and D).

Monocytes express high levels of both FcgRs and FcaRI (Sup-plementary Fig. S3) and induced comparable cytotoxicity by bothIgG and IgA (Fig. 1D). Both combinations of IgG and IgA againstdifferent targets increased maximal cytotoxicity induced bymonocytes in an overnight killing assay (Fig. 5A–D).

These results show that increased cytotoxicity induced by thecombination of IgG and IgA by leukocytes in a 4-hour assay

Figure 1.Cytotoxicity by IgG and IgA is mediatedby distinct populations of effector cells.A, leukocytes corresponding to 50 mLwhole blood and mAb-opsonized A431-HER2 cells weremixed and incubated for4 hours at 37�C before 51Cr-release wasmeasured to assess specific lysis.Specific lysis of A431-HER2 cells by B,purified NK cells (E:T = 10:1); C, PMNs(E:T = 10:1) in a 4-hour assay; or D, bymonocytes (E:T = 10:1) in a 20-hourkilling assay. One graph eachrepresenting 9 (A), 3 (B), 5 (C), or4 (D) independent experiments isshown, for a total of 16 (A), 3 (B),6 (C), or 7 (D) donors.

Brandsma et al.





requires the presence of at least two effector cell types, mostlikely NK cells and PMNs. Furthermore, the combination of IgGand IgA can also increase cytotoxicity induced by monocytesalone.

Enhanced antitumor effect by the combination of IgG and IgAin an in vivo xenograft tumor model

Using an in vivo xenograft tumor model in human FcaRItransgenic SCID mice, we have earlier shown that administrationof cetuximab and IgA2-EGFR was able to decrease intraperitonealoutgrowth of A431-luc2-HER2 cells (16). PMNs and macro-phages expressing mouse FcgRs as well as human FcaRI wereboth recruited to the peritoneal cavity and could participate intumor killing (16). We now show that treatment of establishedtumors with either cetuximab or IgA2-HER2 alone reduced tumorgrowth compared with the control group. Mice that received bothcetuximab and IgA2-HER2 displayed a reduced tumor volumecompared with those treated with single mAbs (Fig. 6A and B).

The concentrationof circulating cetuximab(�3mg/mL) in serumduring thefirst 10days after treatmentwas higher than that of IgA2-HER2 (�1 mg/mL; Fig. 6C). At thesemAb concentrations, maximal

Figure 2.Leukocyte-mediated cytotoxicity by the combination of two IgGs or two IgAsagainst different targets or the combination of IgG and IgA against the sametargets. Leukocytes and mAb-opsonized A431-HER2 cells were mixed andincubated for 4 hours at 37�C before 51Cr-release was measured to assessspecific lysis. Dose–response curves are calculated from pooled data frommultiple experiments obtained using several donors. MAbs were mixed atthe 1:1 ratio and each single mAb was used at the indicated concentration(mean � SEM). Right, the maximal specific lysis from all donors is plotted. Aand B, cetuximab and trastuzumab (5 independent experiments, with a totalof 10 donors), C and D, IgA2-EGFR and IgA2-HER2 (4 independentexperiments, with a total of 7 donors), E and F, cetuximab and IgA2-EGFR(3 independent experiments, with a total of 6 donors), G and H, trastuzumaband IgA2-HER2 (5 independent experiments, with a total of 9 donors).(median, box extends from the 25th to 75th percentiles, whiskers tominimumand maximum; ANOVA and Tukey multiple comparison test, � , P < 0.05;�� , P < 0.01; �� , P < 0.001). AB, antibody; ns, not statistically significant.

Figure 3.Increased leukocyte-mediated cytotoxicity by the combination of IgG andIgA against two targets. A, specific lysis of A431-HER2 cells by leukocytes(2 independent experiments, with a total of 3 donors). Cetuximab and IgA-HER2 were combined at different ratios while keeping the total amount ofmAb constant at 1 mg/mL. B and D, pooled leukocyte ADCC data withcetuximab and IgA2-HER2 (7 independent experiments, with a total of 11donors) and trastuzumab and IgA2-EGFR (5 independent experiments, with atotal of 9 donors). MAbs were combined at 1:1 ratio, and each single mAbwasused at the indicated concentration (mean� SEM). C and E, maximal specificlysis (median, box extends from the 25th to 75th percentiles, whiskers tominimum and maximum; ANOVA, Tukey multiple comparison test,�� , P < 0.001). Ab, antibody.






lysiswas achieved in vitrowithbothmousePMNs andmacrophageswith both mAbs (unpublished results; ref. 16).

These data demonstrate that the combination of IgG and IgAenhances antitumor effects of mAbs in vivo.

DiscussionIntense efforts are being undertaken to increase efficacy of

antitumor IgG mAbs. However, in vivo activity of IgG is limitedby the number of available FcgRs, their biologic distribution, andthe cytotoxic capacity of the recruited effector cells. Here, wepresent evidence that the combination of IgG and IgA isotypescan increase mAb-induced tumor cell killing by more optimallyharnessing the cytotoxic potential of a heterogeneous populationof immune effector cells.

Increased maximal killing in the leukocyte ADCC assay resultsfrom the recruitment of bothNK cells and PMNs that—as purifiedeffector cells—only, or preferentially, mediate cytotoxicity byeither IgG or IgA. The different antitumor effector mechanismsused by NK cells and PMNs could also contribute to the increasedkilling. NK cells induce apoptosis in tumor cells through therelease of granzymes and perforin. Cytotoxicity mediated byPMNs is less well understood, but it was reported that PMNs canmediate frustrated phagocytosis or induce autophagy in tumorcells by IgA (26).

Purified PMNs preferentially induced cytotoxicity by IgA andonly poorly with IgG due to the high expression of the nonsignal-ing FcgRIIIB, which scavenges IgG away from FcgRIIa, and there-fore decreases IgG activity (12, 13, 27). The combination of IgGand IgA did not increase cytotoxicity using purified PMNs. In

contrast, purified monocytes induced comparable cytotoxicity byboth IgG and IgA and mediated increased cytotoxicity by thecombination of IgG and IgA. As both FcgRs and FcaRI signalthrough the same FcRg-chain ITAM, the combination of IgG andIgA could result in stronger cellular signaling in monocytes,resulting in increased cytotoxicity.

IgG-opsonized tumor cells in leukocyte ADCC recruit NK cells,eliciting efficient cytotoxicity, but also recruit PMNs and mono-cytes, leading to less efficient cytotoxicity in a 4-hour assay.Although monocytes alone did not induce cytotoxicity within4 hours, they could have indirectly contributed to tumor cellkilling, for example, through stimulation of NK cells (28). Trig-gering of FcRs on monocytes could result in the release ofcytokines or other mediators, which may enhance FcaRI- orFcgRI-mediated killing by PMNs (or by monocytes) throughactivation via inside-out signaling. Cross-talk between effectorcells is suggested by the fact that when purified effector cells wereused at E:T ratios present in leukocyte ADCC, they were onlypoorly cytotoxic.

The combination of IgG and IgA led to increased cytotoxicityonly when directed against two different tumor targets. Thecombination of IgG and IgA against the same target did notincrease tumor cell killing possibly due to competition for tumortarget binding by IgG and IgA. The combination of two IgGs ortwo IgAs against two different targets also did not increase tumorcell killing, which could be due to saturation of the correspondingreceptors.

In our in vitro leukocyte ADCC assays, we have found that IgA-EGFR alone already induced relatively high maximal tumor celllysis, and this was not further enhanced by adding trastuzumab.

Figure 4.Increased leukocyte-mediatedcytotoxicity by the combination of IgGand IgA requires the presence ofmultiple effector cell types. Specificlysis of A431-HER2 cells (A) or SK-BR-3cells (B) in a 6-hour ADCC assayinduced by the combination of PMNs(E:T ¼ 40:1) and PBMCs (E:T ¼ 70:1),or by PMNs (E:T ¼ 80:1), or PBMCs(E:T¼ 140:1) alone. A431-HER2 cells (C)or SK-BR-3 cells (D) were firstincubated with PBMCs (E:T ¼ 70:1) for3 hours and subsequently incubatedwith PMNs (E:T ¼ 40:1) for another 3hours (mean � SD). Representativegraphs from 5 (A and C) or 3 (B and D)independent experiments, with a totalof 6 (A and C) or 3 (B and D) donors areshown. Ab, antibody.

Brandsma et al.





The combination of trastuzumab and IgA-EGFR, however,enhanced cytotoxicity by monocytes, a major cellular effectorpopulation of both IgG and IgA, suggesting that this combinationcan also have a beneficial effect in vivo.

Previously, the combinationof EGFR andHER2 IgG1mAbs hasbeen described to lead to a synergistic increase of antitumoreffects, which was mainly attributed to increased direct Fab-mediated antitumor effects (22, 23). Although Fab-mediatedantitumor effects are readily induced by both IgG and IgA, inthis study, we focused on Fc-mediated effector functions becausewe do not expect differences mediated by the Fab portion of themAbs within the time frame of our in vitro experiments. However,we expect that combination therapy with IgG and IgA againstEGFR and HER2 in patients might also result in enhanced Fab-mediated antitumor effects. Despite the fact that maximal specifickilling by the combination of IgG and IgA in a 4-hour in vitro assaydoes not reach 100%, the measured increase (�20%) might stillhave profound consequences in vivo and may lead to therapeuticsynergy.

At the site of the tumor, the magnitude of tumor lysis will bedetermined by the number of available Fc moieties (influencedby the target expression, orientation of the Fc part, clustering of

target in the cell membrane), the available FcRs (influenced bythe numbers and types of effector cells present), and the killingcapacity of the effector cells. Studies using engineered mAbsthat have a higher affinity for activating FcRs suggest thatincreasing cellular signaling above a certain threshold does notfurther increase cytotoxicity (29). Cytotoxicity can thus belimited by both saturation of the target and the saturation ofthe FcRs. This could explain why cytotoxicity by leukocytes wasenhanced only by the combination of IgG and IgA against twotargets and not by the combination of two IgGs, or two IgAsagainst the same target or by the combination of IgG and IgAagainst the same target.

Monocytes express high levels of FcaRI and have a favorableexpression pattern of FcgRs (high expression of FcgRI and noexpression of FcgRIIIB). This may explain why cytotoxicity wasalso enhanced by the combination of IgG and IgA against twotargets when monocytes alone were used as effector cells.

Individual donors showed variability in the extend by whichthe combination of IgG and IgA increased cytotoxicity, whichcouldbe a result of polymorphisms in FcgRs. Analysis of a numberof SNP and CNV polymorphisms as tested bymultiplex ligation–dependent probe amplification (30) in various FcgR genes did not

Figure 5.Increased monocyte-mediatedcytotoxicity by the combination of IgGand IgA. Dose–response curves from arepresentative experiment in anovernight cytotoxicity assay usingA431-HER2 cells and purifiedmonocytes (E:T¼ 25:1) with cetuximaband IgA2-HER2 (A) and trastuzumaband IgA2-EGFR (C). IgG and IgA mAbswere combined at a 1:1 ratio. B and D,maximal lysis was determined andplotted from 5 independentexperiments, with a total of 7 donorsperformed with E:T ¼ 15:1 and 25:1(median, box extends from the 25th to75th percentiles, whiskers to minimumand maximum; ANOVA, Tukey multiplecomparison test, ��, P < 0.01;�� , P < 0.001). Ab, antibody.






reveal a clear correlation with increased cytotoxicity, although thenumbers analyzed were small (data not shown).

Intimate contact between effector and target cells is required forefficient cytotoxicity (31). Our preliminary data suggest that thecombination of trastuzumab and IgA2-EGFR, but not cetuximaband IgA2-EGFR or cetuximab and trastuzumab, increased theformation of conjugates between A431-HER2 cells and mono-cytes or PMNs. Thus, it appears that for PMNs increased bindingdoes not directly translate to more efficient killing, most likelyreflecting a contribution of the nonsignaling FcgRIIIB to theformation of conjugates.

In the xenograft tumor model, both PMNs and macrophageswere recruited to the tumor site (16). In FcaRI Tgmice, bothPMNsand macrophages express FcgR and FcaRI. In preclinical studies,macrophages aswell as PMNswere found tomediate effects of IgGin vivo (25, 32, 33), whereas in a different model, we previouslyidentifiedmacrophages as primary effectors of IgA (16).However,because PMNs as well as macrophages are effective in vitro, it isreasonable to assume that both effector cell types contribute tocytotoxicity in vivo. In our mouse model, the increased tumorkilling by the combination of IgG and IgA likely results from therecruitment of PMNs and macrophages and increased FcR trig-gering on both cell types.

Compared with IgG mAbs, the biology of IgA is less wellunderstood, and the production and purification processes arenot well established. However, replacing IgA with a bispecificmAb targeting both a tumor target and FcaRI might be analternative strategy to overcome this.

In conclusion, we have presented evidence that the simulta-neous targeting of both FcgRs and FcaRI appears to more opti-mally mobilize the cytotoxic resources of effector cells and there-fore represents a conceptually new approach to improve theefficacy of mAb therapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A.M. Brandsma, T. ten Broeke, L.A.P.M. Meulenbroek,G. van Tetering, R. Ubink, G. Rouwendal, S. Lohse, J.H.W. Leusen, P. BorossDevelopment ofmethodology:A.M. Brandsma, T. ten Broeke, S.Q. Nagelkerke,J.H.W. Leusen, P. BorossAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.M. Brandsma, T. ten Broeke, L.A.P.M. Meulenbroek,S. Meyer, J.H.M. Jansen, M.A. Beltr�an Buitrago, J.H.W. LeusenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.M. Brandsma, T. ten Broeke, M. Nederend,S.Q. Nagelkerke, I. N�emeth, R. Ubink, S. Lohse, J.H.W. Leusen, P. BorossWriting, review, and/or revision of the manuscript: A.M. Brandsma, T. tenBroeke, L.A.P.M. Meulenbroek, G. van Tetering, J.H.M. Jansen, S.Q. Nagelkerke,R. Ubink, S. Lohse, T. Valerius, J.H.W. Leusen, P. BorossAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. ten Broeke, M. Nederend, S. Lohse, T. ValeriusStudy supervision: J.H.W. Leusen, P. Boross

AcknowledgmentsThe authors thank Dr. Timo K. van den Berg and Prof. Dr. Taco W. Kuijpers

(Sanquin) for their help with the genotyping of the donors, Dr. Tom Vink(Genmab) for providing the p33-HER2 vector, Prof. Linde Meyaard andDr. Tomek Rygiel for critically reading the manuscript, the minidonors of theUMC Utrecht, and the animal caretakers of GDL for the excellent animal care.

Grant SupportThis project was supported by the AICR (grant number 11-0120).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 10, 2015; revised July 1, 2015; accepted July 3, 2015;published OnlineFirst September 29, 2015.

References1. Kim R. Cetuximab and panitumumab: are they interchangeable? Lancet

Oncol 2009;10:1140–1.2. BleekerWK, Lammerts van Bueren JJ, vanOjikHH,Gerritsen AF, PluyterM,

Houtkamp M, et al. Dual mode of action of a human anti-epidermal

Figure 6.Increasedantitumor effect by thecombinationof IgGand IgAmAbs ina xenograft tumormodelwithA431-luc2-HER2 cells invivo. FcaRITgorwild-typeSCIDmicewereinjected i.p. with 1� 105 A431-luc2-HER2 cells. On day 13, mice with tumor outgrowth were randomized in the following treatment groups: PBS, cetuximab (1� 10 mg),IgA2-HER2 (10 � 10 mg), or combination treatment cetuximab (1 � 10 mg) and IgA2-HER2 (10 � 10 mg). Repeated injections were necessary to compensatefor the shorter half-life of IgA2-HER2 compared with IgG1. Tumor growth was monitored by serial bioluminescent imaging. A, representative bioluminescent imagestaken on day 29. B, tumor volume as monitored by bioluminescent imaging. Signal for each individual mouse is expressed as a percentage compared with thesignal measured on day 14 (median � range). Arrows indicate mAb treatment (1 x IgG, 10 x IgA). C, serum concentration of cetuximab and IgA2-HER2 during thefirst 10 days of treatment (days 15–24) in mice receiving treatment with only one type of mAb (single) or both IgG and IgA (combination; 6–7 mice/group,mean � SD).

Brandsma et al.





growth factor receptormonoclonal antibody for cancer therapy. J Immunol2004;173:4699–707.

3. Seshacharyulu P, Ponnusamy MP, Haridas D, Jain M, Ganti AK, Batra SK.Targeting the EGFR signaling pathway in cancer therapy. Expert Opin TherTargets 2012;16:15–31.

4. Klapper LN, Waterman H, Sela M, Yarden Y. Tumor-inhibitory antibodiestoHER-2/ErbB-2may act by recruiting c-Cbl and enhancing ubiquitinationof HER-2. Cancer Res 2000;60:3384–8.

5. Junttila TT, Akita RW, Parsons K, Fields C, Lewis Phillips GD, Friedman LS,et al. Ligand-independent HER2/HER3/PI3K complex is disrupted bytrastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Cancer Cell 2009;15:429–40.

6. Galizia G, Lieto E, De VF, Orditura M, Castellano P, Troiani T, et al.Cetuximab, a chimeric human mouse anti-epidermal growth factor recep-tor monoclonal antibody, in the treatment of human colorectal cancer.Oncogene 2007;26:3654–60.

7. Peipp M, Schneider-Merck T, Dechant M, Beyer T, van Bueren JJ, BleekerWK, et al. Tumor cell killing mechanisms of epidermal growth factorreceptor (EGFR) antibodies are not affected by lung cancer-associatedEGFR kinase mutations. J Immunol 2008;180:4338–45.

8. Overdijk MB, Verploegen S, van den Brakel JH, Lammerts van Bueren JJ,Vink T, van de Winkel JG, et al. Epidermal growth factor receptor (EGFR)antibody-induced antibody-dependent cellular cytotoxicity plays a prom-inent role in inhibiting tumorigenesis, even of tumor cells insensitive toEGFR signaling inhibition. J Immunol 2011;187:3383–90.

9. Arnould L, Gelly M, Penault-Llorca F, Benoit L, Bonnetain F, Migeon C,et al. Trastuzumab-based treatment of HER2-positive breast cancer: anantibody-dependent cellular cytotoxicity mechanism? Br J Cancer 2006;94:259–67.

10. Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptorsmodulate in vivo cytoxicity against tumor targets. Nat Med 2000;6:443–6.

11. Beurskens FJ, Lindorfer MA, Farooqui M, Beum PV, Engelberts P, MackusWJ, et al. Exhaustion of cytotoxic effector systems may limit monoclonalantibody-based immunotherapy in cancer patients. J Immunol 2012;188:3532–41.

12. PeippM, Lammerts van Bueren JJ, Schneider-Merck T, Bleeker WW, DechantM, Beyer T, et al. Antibody fucosylation differentially impacts cytotoxicitymediated by NK and PMN effector cells. Blood 2008;112:2390–9.

13. Derer S, Glorius P, Schlaeth M, Lohse S, Klausz K, Muchhal U, et al.Increasing FcgammaRIIa affinity of an FcgammaRIII-optimized anti-EGFRantibody restores neutrophil-mediated cytotoxicity.MAbs 2014;6:409–21.

14. Bruhns P, Iannascoli B, England P, Mancardi DA, Fernandez N, Jorieux S,et al. Specificity and affinity of human Fcgamma receptors and theirpolymorphic variants for human IgG subclasses. Blood 2009;113:3716–25.

15. Dechant M, Beyer T, Schneider-Merck T, Weisner W, Peipp M, van deWinkel JG, et al. Effector mechanisms of recombinant IgA antibodiesagainst epidermal growth factor receptor. J Immunol 2007;179:2936–43.

16. Boross P, Lohse S,NederendM, Jansen JH, van TeteringG,DechantM, et al.IgA EGFR antibodies mediate tumour killing in vivo. EMBO Mol Med2013;5:1213–26.

17. Lohse S, Brunke C, Derer S, Peipp M, Boross P, Kellner C, et al. Charac-terization of a mutated IgA2 antibody of the m(1) allotype against theepidermal growth factor receptor for the recruitment of monocytes andmacrophages. J Biol Chem 2012;287:25139–50.

18. OttenMA, Rudolph E, Dechant M, Tuk CW, Reijmers RM, Beelen RH, et al.Immature neutrophils mediate tumor cell killing via IgA but not IgG Fcreceptors. J Immunol 2005;174:5472–80.

19. Kimple RJ, Vaseva AV, Cox AD, Baerman KM, Calvo BF, Tepper JE, et al.Radiosensitization of epidermal growth factor receptor/HER2-positivepancreatic cancer is mediated by inhibition of Akt independent of rasmutational status. Clin Cancer Res 2010;16:912–23.

20. Hirsch FR, Varella-Garcia M, Cappuzzo F. Predictive value of EGFR andHER2 overexpression in advanced non–small cell lung cancer. Oncogene2009;28 Suppl 1:S32–S37.

21. Suo Z, Risberg B, Kalsson MG, Willman K, Tierens A, Skovlund E, et al.EGFR family expression in breast carcinomas. c-erbB-2 and c-erbB-4receptors have different effects on survival. J Pathol 2002;196:17–25.

22. Larbouret C, Robert B, Navarro-Teulon I, Thezenas S, Ladjemi MZ, Mor-isseau S, et al. In vivo therapeutic synergism of anti-epidermal growth factorreceptor and anti-HER2 monoclonal antibodies against pancreatic carci-nomas. Clin Cancer Res 2007;13:3356–62.

23. Maron R, Schechter B, Mancini M, Mahlknecht G, Yarden Y, Sela M.Inhibition of pancreatic carcinoma by homo- and heterocombinations ofantibodies against EGF-receptor and its kin HER2/ErbB-2. Proc Natl AcadSci U S A 2013;110:15389–94.

24. Beyer T, Lohse S, Berger S, Peipp M, Valerius T, Dechant M. Serum-freeproduction and purification of chimeric IgA antibodies. J Immunol Meth-ods 2009;346:26–37.

25. Hamaguchi Y, Xiu Y, Komura K, Nimmerjahn F, Tedder TF. Antibodyisotype-specific engagement of Fcgamma receptors regulates B lymphocytedepletion during CD20 immunotherapy. J Exp Med 2006;203:743–53.

26. Bakema JE, Ganzevles SH, Fluitsma DM, Schilham MW, Beelen RH,Valerius T, et al. Targeting FcalphaRI on polymorphonuclear cells inducestumor cell killing through autophagy. J Immunol 2011;187:726–32.

27. Flinsenberg TW, JanssenWJ,Herczenik E, Boross P,NederendM, JongeneelLH, et al. A novel FcgammaRIIa Q27W gene variant is associated withcommon variable immune deficiency through defective FcgammaRIIadownstream signaling. Clin Immunol 2014;155:108–17.

28. Bhatnagar N, Ahmad F, Hong HS, Eberhard J, Lu IN, Ballmaier M, et al.FcgammaRIII (CD16)-mediated ADCC by NK cells is regulated by mono-cytes and FcgammaRII (CD32). Eur J Immunol 2014;44:3368–79.

29. KellnerC,Derer S, Valerius T, PeippM.BoostingADCCandCDCactivity byFc engineering and evaluation of antibody effector functions. Methods2014;65:105–13.

30. van der Heijden J, Nagelkerke S, Zhao X, Geissler J, Rispens T, van den BergTK, et al. Haplotypes of FcgammaRIIa and FcgammaRIIIb polymorphicvariants influence IgG-mediated responses in neutrophils. J Immunol2014;192:2715–21.

31. Horner H, Frank C, Dechant C, Repp R, Glennie M, Herrmann M, et al.Intimate cell conjugate formation and exchange of membrane lipidsprecede apoptosis induction in target cells during antibody-dependent,granulocyte-mediated cytotoxicity. J Immunol 2007;179:337–45.

32. Richards JO, Karki S, Lazar GA, Chen H, Dang W, Desjarlais JR. Optimi-zation of antibody binding to FcgammaRIIa enhances macrophage phago-cytosis of tumor cells. Mol Cancer Ther 2008;7:2517–27.

33. Albanesi M, Mancardi DA, Jonsson F, Iannascoli B, Fiette L, Di Santo JP,et al. Neutrophils mediate antibody-induced antitumor effects in mice.Blood 2013;122:3160–4.






Published OnlineFirst September 25, 2015.Cancer Immunol Res Arianne M. Brandsma, Toine ten Broeke, Maaike Nederend, et al. Tumor Cell Killing

RI EnhancesαRs and FcγSimultaneous Targeting of Fc

Updated version

10.1158/2326-6066.CIR-15-0099-Tdoi:

Access the most recent version of this article at:

Material

Supplementary

-T.DC1

http://cancerimmunolres.aacrjournals.org/content/suppl/2015/10/23/2326-6066.CIR-15-0099Access the most recent supplemental material at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.Thttp://cancerimmunolres.aacrjournals.org/content/early/2015/09/30/2326-6066.CIR-15-0099-To request permission to re-use all or part of this article, use this link



http://cancerimmunolres.aacrjournals.org/lookup/doi/10.1158/2326-6066.CIR-15-0099-T

http://cancerimmunolres.aacrjournals.org/content/suppl/2015/10/23/2326-6066.CIR-15-0099-T.DC1

http://cancerimmunolres.aacrjournals.org/content/suppl/2015/10/23/2326-6066.CIR-15-0099-T.DC1

http://cancerimmunolres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerimmunolres.aacrjournals.org/content/early/2015/09/30/2326-6066.CIR-15-0099-T

http://cancerimmunolres.aacrjournals.org/content/early/2015/09/30/2326-6066.CIR-15-0099-T


simultaneous targeting of fcgrs and fcari …...2015/09/30 · research article simultaneous...

Documents