B-Cell Depletion In Vitro and In Vivo with an Afucosylated Anti-CD19 Antibody

Ronald Herbst, Yue Wang, Sandra Gallagher, Nanette Mittereder, Ellen Kuta,Melissa Damschroder, Rob Woods, Daniel C. Rowe, Li Cheng, Kim Cook, Krista Evans,Gary P. Sims, David S. Pfarr, Michael A. Bowen, William Dall’Acqua, Mark Shlomchik,1

Thomas F. Tedder,2 Peter Kiener, Bahija Jallal, Herren Wu, and Anthony J. CoyleDepartment of Research, MedImmune, LLC, Gaithersburg, Maryland (R.H., Y.W., S.G., N.M., E.K., M.D., R.W., D.C.R., L.C.,K.C., K.E., G.P.S., D.S.P., M.A.B., W.D., B.J., H.W., A.J.C.); Department of Immunology, Duke University Medical Center,Durham, North Carolina (T.F.T.); and Department of Immunology, Yale University School of Medicine, New Haven,Connecticut (M.S.)

Received March 16, 2010; accepted July 2, 2010

ABSTRACTThe pan B-cell surface antigen CD19 is an attractive target fortherapeutic monoclonal antibody (mAb) approaches. We havegenerated a new afucosylated anti-human (hu)CD19 mAb,MEDI-551, with increased affinity to human Fc�RIIIA andmouse Fc�RIV and enhanced antibody-dependent cellular cy-totoxicity (ADCC). During in vitro ADCC assays with B-cell lines,MEDI-551 is effective at much lower mAb concentrations thanthe fucosylated parental mAb anti-CD19-2. Furthermore, theafucosylated CD19 mAb MEDI-551 depleted B cells from nor-mal donor peripheral blood mononuclear cell samples in anautologous ADCC assay, as well as blood and tissue B cells inhuman CD19/CD20 double transgenic (Tg) mice at lower con-

centrations than that of the positive control mAb rituximab. InhuCD19/CD20 Tg mice, both macrophage-mediated phagocy-tosis and complement-dependent cytotoxicity contribute to de-pletion with rituximab; MEDI-551 did not require complementfor maximal B-cell depletion. Furthermore, extended B-cell de-pletion from the blood and spleen was achieved with MEDI-551, which is probably explained by bone marrow B-cell de-pletion in huCD19/CD20 Tg mice relative to the control mAbrituximab. In summary, MEDI-551 has potent B-cell-depletingactivity in vitro and in vivo and may be a promising new ap-proach for the treatment of B-cell malignancies and autoim-mune diseases.

IntroductionB lymphocytes are an important component of the immune

system and are the source of humoral immunity. However, Bcells can also be pathogenic and the origin of disease. Dereg-ulated B-cell function has been implicated in several autoim-mune diseases, including systemic sclerosis, systemic lupuserythematosus, and rheumatoid arthritis (Edwards andCambridge, 2006; Gabrielli et al., 2009). B cells contribute topathological immune responses through the secretion of cy-tokines, costimulation of T cells, antigen presentation, andthe production of autoantibodies (Browning, 2006; Walkerand Fritzler, 2007; Yanaba et al., 2008). Furthermore, the

majority of human leukemias and lymphomas are of B-cellorigin (Kuppers, 2005). Despite recent advances in the clinic,B-cell malignancies, such as acute lymphoblastic leukemia,chronic lymphocytic leukemia, follicular lymphoma, and dif-fuse large B-cell lymphoma (DLBCL), still present a signifi-cant challenge, and curative treatment is not possible in mostcases (Coffier et al., 2005a; Coffier et al., 2005b).

B-cell depletion by targeting B-cell-restricted cell surfaceantigens with monoclonal antibodies (mAbs) has gained sig-nificant attention in recent years. In particular, the CD20mAb rituximab, which is already approved for the treatmentof non-Hodgkin’s lymphoma and DLBCL, has shown prom-ising results in the treatment of B-cell malignancies. Al-though the combination of rituximab with chemotherapy hasled to significant improvements in patient survival in follic-ular lymphoma and other malignancies, many patients re-lapse after treatment (Maloney, 2005). In addition, low CD20expression has been associated with poor response to ritux-

1 Current affiliation: Yale University, New Haven, Connecticut.2 Current affiliation: Department of Immunology, Duke University, Durham,

North Carolina.Article, publication date, and citation information can be found at

http://jpet.aspetjournals.org.doi:10.1124/jpet.110.168062.

ABBREVIATIONS: DLBCL, diffuse large B-cell lymphoma; mAb, monoclonal antibody; ADCC, antibody-dependent cellular cytotoxicity; Tg,transgenic; CDC, complement-dependent cytotoxicity; NK, natural killer; PBMC, peripheral blood mononuclear cell; PBS, phosphate-bufferedsaline; LDH, lactate dehydrogenase; FACS, fluorescence-activated cell sorting; CoVF, cobra venom factor; BM, bone marrow.

0022-3565/10/3351-213–222$20.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 335, No. 1Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 168062/3623104JPET 335:213–222, 2010 Printed in U.S.A.

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DC1.html http://jpet.aspetjournals.org/content/suppl/2010/11/15/jpet.110.168062.Supplemental Material can be found at:

http://jpet.aspetjournals.org/content/336/1/294.full.pdfAn erratum has been published:

imab in DLBCL, and rituximab treatment can lead to down-regulation of CD20 or the selection of CD20-deficient tumorclones (Davis et al., 1999; Kennedy et al., 2004; Johnson etal., 2009). Rituximab has also shown activity in autoimmunediseases, in particular in RA, but reductions in the levels ofcirculating autoantibodies, which may play an important rolein systemic lupus erythematosus and other indications, arenot consistent (Levesque and St Clair, 2008; Tedder, 2009).

CD19 is a B-cell-specific surface antigen that is expressed byearly pre-B cells from the time of heavy chain rearrangementuntil the molecule is eventually down-regulated during termi-nal differentiation into plasma cells (Nadler et al., 1983). CD19belongs to the immunoglobulin domain-containing superfamilyof transmembrane receptors. As a component of the B-cell re-ceptor complex, CD19 regulates the threshold for B-cell activa-tion (Engel et al., 1995). Relatively small changes in CD19surface expression can lead to loss of tolerance and autoanti-body production (Sato et al., 2004).

In hematopoietic malignancies of B-cell origin, expression ofCD19 is maintained after malignant transformation of B cells,and the majority of B-cell-derived leukemias and lymphomasconsistently express CD19 (D’Arena et al., 2000). It is importantto note that malignant B cells that have lost CD20 expressionduring the course of rituximab therapy maintain expression ofCD19 (Johnson et al., 2009). Thus, CD19 is an attractive targetfor antibody-mediated B-cell depletion therapy in multiple he-matologic malignancies. Due to the expression of CD19 on plas-mablasts, which are CD20 negative (Levesque and St. Clair,2008), a depleting CD19 mAb may also have a more significantimpact than CD20-targeted therapies on the levels of patho-genic autoantibodies in autoimmune diseases (Tedder, 2009).

Here, we report the generation of a new anti-human-CD19mAb in development for the therapeutic depletion of B cells.The humanized and affinity-optimized MEDI-551 mAb hasbinding characteristics that are favorable for an ADCC-depen-dent mechanism. MEDI-551 was generated by removal of thefucose carbohydrate modification from the Fc portion of thehumanized CD19 mAb anti-CD19-2, which resulted in a selec-tive increase in the affinity for human Fc�RIIIA and mouseFc�RIV. The anti-CD19-2 mAb was developed by humanizationand affinity optimization of the HB12b mAb, which has potentB-cell-depleting activity in transgenic (Tg) mice expressing hu-man CD19 (huCD19) (Kansas and Tedder, 1991; Yazawa et al.,2005). During in vitro ADCC assays, MEDI-551 depleted pri-mary human B cells at lower mAb concentrations than theCD20 mAb rituximab. Furthermore, at low mAb doses, MEDI-551 is more effective than rituximab in depleting blood andtissue B cells in huCD19/CD20 double Tg mice, despite theabsence of complement-dependent cytotoxicity (CDC), whichcontributes to in vivo depletion with rituximab. In the Tg mousemodel, macrophages are important for in vivo B-cell depletionwith the afucosylated human IgG1, consistent with the in-creased binding to mouse Fc�RIV. In summary, MEDI-551 isa new anti-CD19 mAb optimized for enhanced ADCC ef-fector function and may be found to be a useful new re-agent for the treatment of B-cell malignancies and B-cell-dependent autoimmune disease.

Materials and MethodsCells and Reagents. The Burkitt’s lymphoma cell lines Raji,

Daudi, and Ramos and the human monocyte line THP-1 were ob-

tained from the American Type Culture Collection (Manassas, VA).The KC1333 NK cell line expressing human CD16 was obtained fromBioWa Potelligent Technology (Princeton, NJ). All cell lines weremaintained in RPMI 1640 medium, supplemented with 10% fetalbovine serum. Blood samples [peripheral blood mononuclear cells(PBMCs) and serum] were obtained from healthy donors after ob-taining informed consent. PBMCs were isolated by Ficoll densitygradient centrifugation using Histopaque-1077 (Sigma-Aldrich, St.Louis, MO) according to the manufacturer’s instructions, and B cellswere purified by negative selection using the Naive B Cell IsolationKit II according to the manufacturer’s instructions (Miltenyi Biotec,Auburn, CA). The mAbs used were anti-CD22 clone S-HCL-1 (Im-munocytometry Systems, San Jose, CA) and rituximab (Biogen Idec,Inc., Cambridge, MA) (Anderson et al., 1997). The human IgG1 mAbR347 (MedImmune, LLC, Gaithersburg, MD) was used as isotypecontrol. Mouse specific mAbs used were B220 (CD45R; Invitrogen,Carlsbad, CA) and 1D3 (CD19; BD Biosciences Pharmingen, SanDiego, CA).

Antibody Engineering. To generate a CD19 mAb with enhancedADCC effector function, the mouse IgG1 mAb HB12b (Kansas andTedder, 1991), which recognizes human CD19, was humanized andaffinity-optimized, resulting in mAb anti-CD19-2. To generate ahomogenously afucosylated antibody, the humanized IgG1 mAb anti-CD19-2 was expressed in a fucosyltransferase-deficient producerChinese hamster ovary cell line (BioWa Potelligent Technology) togenerate MEDI-551.

Cell Proliferation Assay. To test the effect of immobilized(cross-linked) mAb on B-cell function, 100 ng of anti-CD19-2 mAb orisotype control mAb in phosphate-buffered saline (PBS) was used tocoat triplicate wells of a flat-bottomed high-binding 96-well plate(Costar 3361; Corning Life Sciences, Lowell, MA) overnight at roomtemperature. Next, the wells were washed and the cells were seededat a density of 10,000 cells/well. Cells were assayed on day 3 usingthe CellTiter-Glo luminescent cell viability assay according to themanufacturer’s instructions (Promega; Madison, WI).

BIAcore Affinity Measurements. The affinity (KD) for the bind-ing of human and murine Fc�Rs to IgG mAbs was measured on aBIAcore 3000 instrument (BIAcore AB, Uppsala, Sweden). In brief,the fucosylated and afucosylated forms of the IgG1-humanized CD19mAb were immobilized onto separate flow cells on a CM5 sensorchips using a standard amino coupling chemistry as outlined by theinstrument’s manufacturer. A reference flow cell without mAb wasalso prepared on each sensor chip. Stock solutions of Fc�Rs (Ogane-syan et al., 2008) were serially diluted using instrument buffer(HBS-EP buffer containing 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3mM EDTA, and 0.005% P-20 detergent). Fc�Rs were injected overboth the IgG and reference cell surfaces at a flow rate of 5 �l/min.Binding data were collected for 50 min, followed by a 60-se pulse of5 mM HCl between injections to remove bound Fc�R from the IgGsurfaces. After all binding data were collected, individual data setswere averaged for binding (response at equilibrium) at each concen-tration, and then fit to a 1:1 binding isotherm (response at equilib-rium versus concentration) plot. From this, the KD values werederived. Such calculations were performed with BIAevaluation soft-ware, version 4.1 (BIAcore AB).

ADCC Assays. ADCC assays were performed with a set effector-target cell ratio of 2.5:1. In brief, Daudi target cells were washed withPBS, resuspended in RPMI 1640 phenol-free media at a cell densityof 0.4 � 106/ml. KC1333 NK cells were washed once in PBS andresuspended in RPMI 1640 phenol-free media at a cell density 1 �106/ml. Assays were performed in U-bottomed 96-well plates. Exper-imental wells were set up by combining 50 �l of the appropriate mAbdilution, 50 �l of target cell suspension, and 50 �l of effector cellsuspension. Reactions were set up in triplicates. After setup, plateswere spun at 120g for 3 min to pellet the cells. Plates were incubatedat 37°C, 5% CO2 for 4 h. Target cell lysis was measured by detectingthe release of lactate dehydrogenase (LDH) from the cytoplasm oflysed cells using the CytoTox 96 nonradioactive cytotoxicity assay

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(Promega) performed according to the manufacturer’s directions.Forty-five minutes before the end of incubation, 15 �l of manufac-turer-provided lysis buffer was added to the target cell maximalrelease control wells and detergent background wells. After incuba-tion the plate was centrifuged at 120g for 3 min. Then, 50 �l of thesupernatant from each well was transferred to a new flat-bottomed96-well plate. Next, 50 �l of reconstituted substrate mix (assembledfrom manufacturer provided components) was added, and the platewas incubated at room temperature for 10 to 20 min, protected fromlight. Finally, 50 �l of manufacturer-provided stop buffer was added,and absorbance at 490 nM was measured in a plate reader: %cytotoxicity � (experimental LDH release � spontaneous LDH re-lease)/(maximal LDH release � detergent background) � 100. Beforecalculating the percentage of cytotoxicity, all values were reduced bythe media background.

Multiparameter flow cytometry was used to quantify in vitroADCC activity using PBMCs from healthy adult volunteer donors. Inthis assay, the PBMCs are the source of both the natural killereffector cells and the B-cell targets. The PBMCs were resuspended at106 cells/ml in culture media (RPMI 1640 medium, 10% heat in-activated fetal bovine serum, and 2 mM L-glutamine), supplementedwith 200 ng/ml recombinant human interleukin-2 (PeproTech, RockyHill, NJ). In total, 2 � 105 PBMCs were added to each well inU96-well round-bottomed microwell plates (Nalge Nunc Interna-tional, Rochester, NY). Serial dilutions of MEDI-551 or rituximabwere added in 10 �l to wells in triplicate and incubated for 20 h at37°C with 5% CO2. Cells were then washed once in stain buffer (PBScontaining 0.5% bovine serum albumin) and resuspended in 100 �l ofa cocktail of fluorescently labeled antibodies in stain buffer contain-ing anti-CD19 phycoerythrin-Cy7, anti-CD20 Pacific Blue, anti-CD22 allophycocyanin, or anti-CD22 phycoerythrin, and anti-FcεR1� fluorescein isothiocyanate. The cells were stained for 30 minon ice, washed once in stain buffer, and then resuspended in a finalvolume of 100 �l of stain buffer containing 5 �l of the viabilitysolution, 7-amino-actinomycin D. As a counting standard, 20 �l/wellCountBright absolute counting beads was added to determine cellconcentration of cell subsets. Samples were acquired on an LSR IIflow cytometer (BD Biosciences, Franklin Lakes, NJ) equipped witha high-throughput capability for running 96-well microwell plates.

The fluorescence-activated cell sorting (FACS) data were analyzedwith FlowJo software, version 7.2.2 for PC (TreeStar, Inc., Ashland,OR). The gating strategy used to quantify B-cell depletion was asfollows. Initially, dead 7-amino-actinomycin D-positive cells wereexcluded, and then live lymphocytes were gated using their forwardand light scatter characteristics. Basophils, eosinophils, and mastcells within the lymphocyte gate were excluded on the basis of theirexpression of FcεR1�. With rituximab, it was evident that the anti-body treatment reduced CD19 expression. Consequently, for thisantibody treatment, the total number of surviving B cells was de-fined by a single gate of all CD22� cells. For MEDI-551 treatment,the number of surviving B cells in the CD20�CD22� gate wasdetermined. The IgG1 afucosylated antibody R347-aFuc was used asa nondepleting treatment control and was used to define these gates.The number of absolute counting beads in each sample was quanti-fied in a forward scatter gate. The numbers of surviving B cells in thetotal CD22� or CD20�CD22� gates were converted to cell concen-trations using the standard counting beads according to the manu-facturer’s instructions.

The B-cell depletion (percentage of cytotoxicity) was then calcu-lated according to the following formulae: for rituximab, {1 – [ritux-imab CD22� (cells/milliliter)]/(R347-aFuc CD19�CD22� (cells/mil-liliter)} � 100; and or MEDI-551, and {1� [MEDI-551 CD20�CD22�(cells/milliliter)]/(R347-aFuc CD20�CD22� (cells/milliliter)} � 100.

The half-maximal effective concentration (EC50) of B-cell depletionwas calculated using a four-variable curve-fit equation in Prism,version 5.01 software (GraphPad Software Inc., San Diego, CA). Theresults are shown in Table 2 and include the 95% confidence intervalas well as the R2 goodness-of-fit value.

Complement-Dependent Cytotoxicity Assay. The CDC assaywas performed using the same LDH readout assay as the ADCCassay. In brief, human donor serum was collected and divided intotwo separate aliquots. One aliquot was treated at 56°C for 30 min toinactivate complement followed by centrifugation for 10 min at3000g, whereas the other aliquot remained untreated. Media con-taining phenol-free RPMI 1640 medium and 10% of either heat-inactivated or nonheat-inactivated serum were prepared. Targetcells were washed with PBS, resuspended in either RPMI 1640medium containing heat-inactivated or nonheat-inactivated serum,at a cell density of 0.4 � 106/ml. Experimental wells were set up bycombining 50 �l of the appropriate antibody dilution (in either heat-inactivated or nonheat-inactivated media), 50 �l of target cell sus-pension (in either heat-inactivated or nonheat-inactivated media),and 50 �l of media (either heat-inactivated or nonheat-inactivated).Reactions were set up in triplicates, and plates were incubated at37°C, 5% CO2 for 4 h. Forty five minutes before the end of incubation,15 �l of manufacturer-provided lysis buffer was added to the targetcell maximal release control well and detergent background wells.After incubation, the plate was centrifuged at 120g for 3 min. Then,50 �l of the supernatant from each well was transferred to a newflat-bottomed 96-well plate. Next, 50 �l of reconstituted substratemix (assembled from manufacturer-provided components) wasadded, and the plate was incubated at room temperature for 10 to 20min, protected from light. Finally, 50 �l of manufacturer-providedstop buffer was added, and absorbance at 490 or 492 nm was mea-sured in a plate reader. Percentage of cytotoxicity was calculated asdescribed above for ADCC.

B-Cell Depletion in huCD19/CD20 Transgenic Mice. Allmouse experiments were carried out in a pathogen-free environmentat the MedImmune, LCC, animal facility in accordance with Insti-tutional Animal Care and Use Committee-approved protocols. Invivo depletion of B cells by CD19 and CD20 mAbs was evaluated inhuCD19/CD20 double Tg mice, which were generated by crossinghuCD19 Tg with huCD20 Tg animals (Zhou et al., 1994; Ahuja et al.,2007). Tg mice (10–15 weeks old; five to eight animals/group) wererandomized and treated with MEDI-551 or rituximab by tail veininjection. PBS-treated animals were used as controls (three to fiveanimals/group). Blood, spleen, and bone marrow samples were col-lected for further analysis using flow cytometry. B-cell numbers weredetermined in each sample by staining with allophycocyanin-Cy5.5-conjugated B220 (CD45R) and PerCP-Cy5.5-conjugated muCD19 an-tibodies. Single-cell bone marrow and spleen cell suspensions wereloaded at 1.5 million cells/well for flow cytometry analysis.

Mechanism of In Vivo B-Cell Depletion. Tg mice were treatedwith MEDI-551 or rituximab in the absence of complement or of oneof the potential effector cell populations.

To study the contribution of complement for B-cell depletion,hCD19 Tg mice (three mice/group; 8–12 weeks old) were treated with30 �g/mouse cobra venom factor (CoVF, Sigma-Aldrich) or PBS everyother day for 5 days (Minard-Colin et al., 2008). Twenty-four hoursafter the first intraperitoneal dose of CoVF, mice were treated witheither MEDI-551 or rituximab (10 mg/kg i.v.) or PBS. Serum C3levels were detected before mAb injection and at the study endpointby enzyme-linked immunosorbent assay (Immunology ConsultantsLaboratory, Newberg, OR; data not shown).

To deplete NK cells and neutrophils, hCD19 Tg mice were treatedwith intraperitoneal injections of 0.25 mg/mouse anti-NK1.1 (clonePK136) or anti-GR1 (clone RB6-8C5) (Biolegend, San Diego, CA),respectively (Gong et al., 2005). Anti-NK1.1 treatment occurred ev-ery other day for 5 days (days �1, 1, and 3). Anti-GR1 was given asa single injection (day �1). Treatment with MEDI-551 or rituximab(10 mg/kg mAb) followed 24 h after the first injection (day 0). Thedepletion of NK cells and neutrophils was verified with FACS anal-ysis before treatment with MEDI-551 or rituximab and throughoutthe duration of B-cell depletion. NK cells were detected asCD49b�CD335� in the blood and CD49b�Ly49� in the spleen and

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bone marrow. Neutrophils were detected as CD11b�SSChigh (datanot shown).

To assess the role of macrophages, hCD19 Tg mice were treatedwith injections of liposome-encapsulated clodronate (Encapsula,Nashville, TN; five to seven animals/group). PBS-loaded liposomeswere used as control (three to four animals/group). Loaded liposomeswere intraperitoneally injected at 0.2 ml/mouse every other day for 5days before treatment with 10 mg/kg MEDI-551 or rituximab. Anadditional 0.2 ml/mouse was injected 2 days after mAb treatment.The depletion of macrophages was verified by FACS and immuno-fluorescence histology. The number of macrophages in spleen wasdetermined by FACS staining of F4/80� cells before the treatmentwith MEDI-551 and again at the endpoint. The spleens of mice werealso harvested for histology. Slides of spleen sections, prepared fromoptimal cutting temperature blocks, were stained for B cells (AlexaFluor 555 or Pacific Blue-conjugated B220), T cells (Alexa Fluor647-conjugated CD90), red pulp macrophages (Alexa Fluor 488-con-jugated F4/80), marginal zone metaphilic macrophages (biotinylatedMOMA-1 followed by Pacific Blue-conjugated streptavidin), and/ormarginal zone macrophages (Alexa Fluor 488-conjugated ERTR9)(data not shown).

Statistical Analysis. The data were analyzed and graphed usingPrism software (GraphPad Software Inc.). Mean values and S.D.were calculated for experiments as indicated in the figure legends.Statistics were performed using a two-tailed, two-sample t test inPrism and are determined from triplicate wells of each donor tested.Significant differences are noted by p value: �, p � 0.05; ��, p � 0.01;and ���, p � 0.005.

ResultsRemoval of Fucose Enhances the ADCC Effector

Function of the Anti-CD19-2 mAb. ADCC is an importantmechanism by which therapeutic mAbs mediate target celldepletion (Glennie et al., 2007). Engagement of effector cellsrequires the interaction of the mAb Fc with Fc� receptors onthe surface of NK cells, monocytes/macrophages, or neutro-phils. It has been demonstrated that the removal of fucosefrom the carbohydrate modification of the mAb Fc preferen-tially increases the affinity to Fc�RIIIA, resulting in in-creased ADCC effector function (Shields et al., 2002; Nim-merjahn and Ravetch, 2008).

To generate a CD19 mAb with enhanced effector function,the humanized and affinity-optimized CD19 mAb anti-CD19-2 was expressed in a fucosyltransferease-deficient Chi-nese hamster ovary cell line, which generates homogenouslyafucosylated mAbs (BioWa Potelligent Technology). The afu-cosylated form of anti-CD19-2 is termed MEDI-551. We firsttested the effect of fucosylation on the binding of the hu-manized IgG1 anti-CD19 mAb to various human andmouse Fc� receptors by measuring the equilibrium bindingconstant (KD) on a BIAcore instrument. The removal offucose had no significant effect on the binding to humanFc�RI and Fc�RIIa (Table 1). However, the afucosylatedmAb has approximately 9-fold increased affinity to theactivating Fc�RIIIA. Likewise, removal of fucose resultedin a 10-fold increase in binding affinity to mouse Fc�RIV,the mouse ortholog of human Fc�RIIIA. The binding tomouse Fc�RIIb and Fc�RIII was unaffected by the removalof fucose. These results are consistent with previouslypublished data and further demonstrate a selective in-crease in the binding of afucosylated IgG1 mAbs to humanFc�RIIIA and mouse Fc�RIV (Shields et al., 2002; Nim-merjahn and Ravetch, 2008).

Although the relative contribution of different effector cellpopulations in various tissues is still not fully established forhuman subjects, NK cells probably play an important role. InADCC assays with human PBMCs as the source of the effec-tor cells, almost all activity is provided by the NK cells(Desjarlais et al., 2007). Therefore, we tested the effectorfunction of MEDI-551 and the fucosylated version the mAb(anti-CD19-2) in assays with NK cells as the effector cellpopulation. The increased affinity of MEDI-551 for Fc�RIIIAresulted in a substantial enhancement of its ADCC activityin vitro (Fig. 1A). This increase in ADCC effector functionwas reflected in a lower EC50 value for cytotoxicity (3.1 ng/mlfor MEDI-551 versus 57.6 ng/ml for the fucosylated mAb) aswell as an increase in overall cytotoxicity (50% for MEDI-551versus 17% for mAb anti-CD19-2). In repeated assays withDaudi cells as targets, MEDI-551 was also more effectiveat low mAb concentrations than the rituximab CD20 mAb(EC50 � 41.0 ng/ml), which was included as a positivecontrol (Fig. 1A).

To further characterize the anti-CD19 mAb, we tested mAbanti-CD19-2 for additional Fc-dependent and independenteffects on B cells. In addition to ADCC, CDC is an Fc-depen-dent mechanism by which mAbs against cell surface antigenscan mediate target cell killing and is well characterized forrituximab (Glennie et al., 2007). When tested in vitro withDaudi cells and human serum as a source of complement, theanti-CD19-2 mAb was not active in a CDC assay, whereasthe rituximab CD20 mAb showed significant cytotoxicity(Fig. 1B).

Antibodies against cell surface antigens can also affectproliferation and/or viability of the target cell. Previous re-ports demonstrated that some, but not all, mAbs againstCD19 can inhibit proliferation of B leukemia or lymphomacells (Ghetie et al., 1990). Therefore we tested the effect ofanti-CD19-2 mAb on cell proliferation of Raji, Daudi, andRamos B lymphoma cell lines, using a luminescent viabilityassay. As a control, we also included the monocytic cell lineTHP-1. As shown in Fig. 1C, incubation with anti-CD19-2mAb resulted in a significant decrease in the number ofviable B cells compared with cells treated with the isotypecontrol mAb. Proliferation of THP-1 cells, which do not ex-press CD19, was not affected by anti-CD19-2 mAb. Whenadded in solution, anti-CD19-2 mAb also inhibited the pro-liferation of primary human B cells stimulated with anti-IgMand CpG-B oligonucleotide (data not shown).

Depletion of Primary Human B Cells Ex Vivo withMEDI-551. Having demonstrated potent activity in anADCC assay with a B-cell line and NK cells, we asked

Table 1BIAcore affinity measurements of fucosylated and afucosylated anti-CD19 antibodies to mouse and human Fc� receptors

Fc� Receptor

Fucosylated Anti-CD19 IgG1 KD

aAfucosylated

Anti-CD19 IgG1 KD

Mouse Human Mouse Human

nM

I N.T. 20 N.A. 26IIa N.A. 3030 N.A. 3490IIb 1230 14,500 1200 N.T.IIIA (V158) N.A. 656 N.A. 77III 3440 N.A. 3560 N.A.IV 306 N.A. 31 N.A.

N.A., not applicable; N.T., not tested.

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whether DI-551 was also effective in depleting primary hu-man B cell in an autologous assay in which donor PBMCs arethe source of both the target and effector cell populations.Figure 2 shows the results from three representative assays(of six individual PBMC samples) in which the activities ofMEDI-551 and rituximab were compared. The calculatedEC50 values for B-cell depletion with MEDI-551 mAb andrituximab for all PBMC samples are summarized in Table 2.In all assays, MEDI-551 demonstrated potent ADCC activityagainst primary human B cells. The EC50 values determined

ranged from 3 to 29 pM for MEDI-551 and from 18 to 431 pMfor rituximab. The results demonstrate that MEDI-551 is ac-tive against primary human B cells in autologous ADCC assays.

MEDI-551 Depletes Blood and Tissue B Cells In Vivo.The HB12b and anti-CD19-2 mAbs are specific for humanCD19 and do not bind rodent CD19 or CD19 from nonhumanprimates (Yazawa et al., 2005; data not shown). In addition,rituximab does not cross-react with rodent CD19 (Gong et al.,2005). Therefore, we made use of a Tg mouse model to com-pare the in vivo B-cell depletion activity of the two mAbs.Double Tg animals were generated by crossing huCD19 Tgmice with huCD20 Tg mice. Both strains have been well

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∗∗∗

Raji Daudi Ramos THP1Cell Lines

Fig. 1. Effector function of fucosylated and afucosylated anti-CD19 mAbs.A, afucosylated anti-CD19 mAb MEDI-551 has increased ADCC effectorfunction against Daudi Burkitt lymphoma cells compared with the fuco-sylated mAb anti-CD19-2. ADCC activity was determined using an LDHrelease assay and KC1333 NK cells as effectors (effector cell/target cellratio � 2.5:1). Rituximab was included for comparison. All ADCC assayswere done in triplicate. Results are mean S.D. and representative ofthree independent experiments. B, anti-CD19 mAb anti-CD19-2 is devoidof CDC activity. Daudi cells were incubated with mAb anti-CD19-2 orrituximab as a positive control in the presence of human serum as asource of complement. In contrast to rituximab, mAb anti-CD19-2 has nodetectable CDC activity. Cells incubated with mAb in the presence ofheat-inactivated (hi) serum served as a negative control. All measure-ments were done in duplicate. The results are representative of twoindependent experiments. C, anti-CD19 mAb anti-CD19-2 inhibits theproliferation of B-lymphoma cells. The Burkitt lymphoma lines Raji,Daudi, and Ramos and the monocytic cell line THP-1 were cultured for 3days in multiwell plates coated with anti-CD19-2 mAb or the control mAbR347. The effect of mAb treatment on cell proliferation was determinedusing the CellTiter-Glo assay. The results shown are mean S.D. oftriplicate samples.

-3 -2 -1 0 1 2-25

0

25

50

75

100rituximab

MEDI-551

Donor 343 (158 V/V)

Log Antibody concentration [nM]

% C

ytot

oxic

ity-3 -2 -1 0 1 2

-25

0

25

50

75

100rituximab

MEDI-551

Donor 374 (158 V/F)

Log Antibody concentration [nM]

% C

ytot

oxic

ity

-3 -2 -1 0 1 2-25

0

25

50

75

100rituximab

MEDI-551

Donor 310 (158 V/V)

Log Antibody concentration [nM]

% C

ytot

oxic

ity

Fig. 2. MEDI-551 has potent ADCC activity in an autologous ADCCassays with normal donor PBMCs. PBMC samples from healthy donorswere incubated with MEDI-551 and rituximab as indicated. Cytotoxicitywas measured after overnight incubation using an FACS-based method(see Materials and Methods). Shown are representative results withPBMCs from three individual donors (of six donors tested). Donor 374(middle) is heterozygous for the high-affinity allotype at amino acidposition 158 of Fc�RIIIA (158 V/F), whereas all other samples tested werefrom donors homozygous for the high-affinity allotype (158 v/v). Theresults shown are mean S.D. of triplicate samples.

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characterized previously, express the transgene in a B-cellrestricted manner, and have been used successfully to studyB-cell depletion with CD19 and CD20 mAbs, respectively(Zhou et al., 1994; Yazawa et al., 2005; Ahuja et al., 2007). Asshown in Fig. 3A, the B-cell-restricted expression of bothtransgenes is also maintained in the huCD19/CD20 doubleTg animals. To further characterize the expression ofhuCD19 and huCD20 in this model, purified spleen B cellswere incubated with serial dilutions of anti-CD19-2 mAb,

rituximab, or the R347 isotype control mAb and mAb bindingwas determined by FACS. The results demonstrate that max-imal surface binding to huCD19/CD20 Tg B cells is compa-rable for the anti-CD19-2 and rituximab mAbs. The anti-CD19 mAb, however, seems to have stronger binding at lowmAb concentrations, suggesting a higher affinity than ritux-imab (Fig. 3B).

To compare in vivo B-cell depletion with MEDI-551 andrituximab, huCD19/CD20 Tg mice received a single dose ofeach mAb at 0.5, 2, or 10 mg/kg, and remaining blood andspleen B-cell numbers were determined 4 days later by flowcytometry. At the highest dose of 10 mg/kg, both mAbs werevery effective in depleting B cells from blood and spleen(Fig. 4, A and B). At the lower doses, however, B-celldepletion with rituximab was incomplete, whereas deple-tion with MEDI-551 was as effective at 0.5 mg/kg as at 10mg/kg mAb. For example, a single dose of 0.5 mg/kg ritux-imab reduced splenic B cells on average by 42%, whereasMEDI-551 mAb used at this dose eliminated 85% of B cellsfrom treated mice (Fig. 4B).

Further analysis showed that B-cell depletion in blood andspleen was maintained for more that 2 wk after a single 10mg/kg administration of MEDI-551 (Fig. 4, C and D). In micetreated with rituximab, significant recovery of blood andspleen B-cell numbers was observed by day 14, despite al-most complete depletion of blood and spleen B cells duringthe first days after dosing. In clinical studies with rituximab,immature B cells are often the first B cells detectable inperipheral blood during B-cell recovery, suggesting repopu-lation from the bone marrow (BM) (Roll et al., 2008). To testwhether differences in the depletion of BM B cells betweenCD19 and CD20 mAbs could account for the differences inthe kinetics of B-cell recovery in the periphery and secondarylymphoid organs, we analyzed the extent of B-cell depletionin this compartment with the two mAbs. As shown in Fig. 4E,a single dose of MEDI-551 mAb resulted in a substantialreduction (on average by 91.4% by day 3) in BMB220�muCD19� B cells, whereas rituximab depleted fewerthan 50% of the cells. Thus, the shorter time to B-cell recov-ery in mice treated with rituximab is probably caused by thepartial depletion of BM B cells.

Macrophages Are Required for Efficient In VivoB-CellDepletion with MEDI-551. Several cell types, including NKcells, neutrophils, and macrophages, can potentially contributeto antibody-mediated target cell depletion in vivo (Glennie etal., 2007; Nimmerjahn and Ravetch, 2008). Previous studieshave emphasized the importance of monocytes/macrophages forB-cell depletion in mice with mouse IgG2a mAbs (Uchida et al.,2004; Gong et al., 2005; Yazawa et al., 2005). However, themechanism by which an afucosylted human IgG1 mediatesits effector function has not yet been determined in a

Table 2Depletion of B cells from normal donor PBMC samples with the afucosylated anti-CD19 mAb MEDI-551 and the anti-CD20 mAb rituximab

Donor MEDI-551 EC50 (95% CI) R2 Rituximab EC50 (95% CI) R2

nM nM

343 0.016 (0.0096–0.0264) 0.991 0.431 (0.2158–0.8625) 0.975310 0.014 (0.0073–0.0251) 0.970 0.252 (0.2166–0.2947) 0.998374 0.029 (0.0132–0.0645) 0.968 0.063 (0.0500–0.0802) 0.989421 0.0006 (0.0003–0.0011) 0.896 0.032 (0.0240–0.0416) 0.989442 0.003 (0.0005–0.0183) 0.989 0.018 (0.0157–0.0208) 0.825467 0.0007 (0.0001–0.0055) 0.957 0.039 (0.0348–0.0442) 0.992

B

Antibody concentration (M)

0

500

1000

1500isotype controlanti-CD19-2rituximab

Mea

nflu

ores

cenc

ein

tens

ity

10-11 10-10 10-9 10-8 10-7

A Spleen

muCD19

Blood

muCD19

huC

D19

huC

D20

isot

ype

cont

rol

Fig. 3. Cell surface expression of huCD19 and huCD20 in huCD19/20double Tg mice. A, live blood mononuclear cells and splenocytes fromhuCD19/CD20 Tg mice were analyzed for expression of human CD19 andCD20, along with murine CD19, a marker for B cells. B, purified mousespleen B cells from huCD19/CD20 Tg mice were incubated with serialdiluted anti-huCD19 mAb anti-CD19-2, anti-huCD20 mAb rituximab, orisotype control mAb for 1 h at 37°C. The cell surface-bound mAbs werethen detected with a mouse anti-human IgG1 secondary antibody andmeasured by FACS. The graph shows one of two experiments with similarresults.

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mouse model. To explore the mechanism of in vivo B-celldepletion with MEDI-551, huCD19/CD20 Tg mice weretreated with the CD19 mAb in the absence of one of thepotential effector cell populations or after complementneutralization.

Elimination of NK cells or neutrophils with anti-NK1.1 oranti-GR-1 mAbs, respectively (Gong et al., 2005), did notaffect the efficiency of B-cell depletion (data not shown).However, elimination of monocytes/macrophages, by treat-ment of mice with liposome-encapsulated clodronate (Uchidaet al., 2004), almost completely prevented depletion of bloodand spleen B cells with MEDI-551 (Fig. 5, A and B). Elimi-nation of monocytes/macrophages had a similar effect onblood B-cell depletion with rituximab. Depletion of splenic Bcells with rituximab, however, was less affected. Therefore,we also tested for a potential contribution of complement tothe depletion of spleen B cells with CD19 and CD20 mAbs.Treatment of Tg mice with CoVF, which almost eliminatedserum C3 levels as determined by enzyme-linked immu-nosorbent assay (data not shown) (Minard-Colin et al., 2008),significantly reduced spleen B-cell depletion by rituximab(Fig. 5C). Depletion of spleen B cells by MEDI-551 was muchless affected by the elimination of complement with CoVF. Tofurther confirm the contribution of macrophages for deple-tion of mouse B cells, we tested MEDI-551 and rituximab ina phagocytosis assay with mouse peritoneal macrophages. Asnegative control, we also included a Fc-mutated version of

mAb anti-CD19-2, mAb anti-CD19-2-TM, which is devoid ofeffector function (Oganesyan et al., 2008). As shown in Fig.5D, the MEDI-551 mAb efficiently mediated phagocytosis ofmouse B cells, whereas the effector-less anti-CD19-2-TMmAb was inactive. Although rituximab was also active in thephagocytosis assay as expected, the overall activity was lessthan with MEDI-551.

In summary, macrophages play an important role for invivo B-cell depletion in mice with the afucosylated humanIgG1 mAb MEDI-551. Although the results do not rule outsome contribution of NK cells and neutrophils, especiallyin the absence of macrophages, these cells seem to play aminor role and are not required for efficient depletionwhen functional macrophages are present. Furthermore,consistent with the in vitro data, the anti-CD19 mAb doesnot mediate CDC in vivo. For the depletion of blood B cells,rituximab also largely depends on macrophages. For thedepletion of splenic B cells with rituximab, however, CDCseems to also play a role, in accordance with the resultsfrom Gong et al. (2005).

DiscussionB-cell depletion with targeted mAbs has proven successful

for the treatment of hematologic malignancies of B-cell originas well as for autoimmune diseases. Given the success ofrituximab, a variety of new agents targeting CD20 are now in

A B

600

800

ber p

er µ

L

rituximabMEDI-551

1.0´107

1.5´107

ll nu

mbe

r

rituximabMEDI-551

∗∗∗

0

200

400

B c

ell n

umb

∗∗∗

∗∗

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ll num

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0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Days after treatment

∗

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 150

1.0×106

2.0×106

Days after treatment

Bone

mar

row

B ce ∗

∗∗∗

∗∗

y

Fig. 4. Depletion and recovery of B cells afterinjection of a single dose of MEDI-551 or ritux-imab. HuCD19/CD20 Tg mice were treated withMEDI-551 or rituximab at 0.5, 2, or 10 mg/kg.Control animals were treated with PBS only.Mice (six to seven animals/dose group and timepoint) were sacrificed for analysis of blood andtissue B-cell depletion. The dose-response for B-cell depletion in blood (A) and spleen B cells (B)was analyzed on day 3 after mAb administra-tion. To compare the duration of B-cell depletionbetween MEDI-551 and rituximab, additionalgroups of mice treated with 10 mg/kg mAb weresacrificed and analyzed on days 7 and 14 aftermAb administration. Shown are depletion ofblood (C), spleen (D), and BM (cell number perfemur; E) over time. Statistical significance atindividual time points between groups treatedwith MEDI-55 and rituximab is indicated.

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preclinical and clinical development (Cheson and Leonard,2008). Targeting CD20, however, also has limitations, andthe antigen can be lost from malignant cells through down-regulation, selection, or both (Davis et al., 1999; Kennedy etal., 2004; Takei et al., 2006). Recent data from clinical trialsof autoimmune patients treated with rituximab suggest thatthe depletion of memory B cells from lymphoid tissues is notalways complete. It is important to note that the presence ofmemory cells, rather than naive immature and mature Bcells, at the time of repopulation was generally associatedwith a poor response to rituximab therapy (Leandro et al.,2006; Pers et al., 2008; Roll et al., 2008). The broader expres-sion along the B-cell lineage and presence on most malignantB cells make CD19 an attractive alternative to CD20 fortherapeutic mAb approaches.

Several different mechanisms, including ADCC, CDC, andinduction of apoptosis, can lead to target cell elimination bytherapeutic mAbs, such as rituximab (Glennie et al., 2007).Although CDC may play a role in B-cell depletion with rit-uximab, the contribution of direct apoptosis to in vivo B-celldepletion is controversial (Glennie et al., 2007).

In contrast to CDC and apoptosis, the relevance of ADCCand antibody-mediated phagocytosis for in vivo activity isnow well established (Desjarlais et al., 2007; Glennie et al.,2007). The results from this study demonstrate that MEDI-551 has potent ADCC activity but does not mediate CDC.Although potentially contributing to overall activity of CDC-competent mAbs, such as rituximab, activation of comple-ment may also be involved in infusion-related reactions, aside effect often observed with therapeutic mAbs. Using dif-ferent forms of a mAb against HLA-DR in preclinical animalmodels, Tawara et al. (2008) demonstrated that infusionreactions were correlated to the ability of the mAbs to medi-ate CDC. It will have to be determined in the clinic whether

the absence of CDC in MEDI-551 results in fewer infusionreactions compared with other CDC-competent mAbs.

The ADCC activity of therapeutic mAbs can be enhancedby increasing the affinity of the mAb Fc for activating Fc�receptors, in particular Fc�RIIIA (Desjarlais et al., 2007).This can be achieved by the introduction of point mutationsin the Fc or by modification of the Fc carbohydrate. Theremoval of fucose from the Fc of the anti-CD19 mAb anti-CD19-2 resulted in 9-fold increased affinity to Fc�RIIIA. Itis interesting that the increase in ADCC activity was muchgreater than the fold affinity increase to Fc�RIIIA. Not onlydid the EC50 drop by approximately 20-fold but also themaximal level of cytotoxicity was significantly greater for theafucosylated mAb MEDI-551 compared with the fucosylatedmAb anti-CD19-2 (Fig. 1A; data not shown). Because theactivity and potency of the anti-CD20 mAb rituximab havebeen well characterized, we included this mAb as control inthis study. The results from ADCC assays with B-cell lines aswell as primary B cells demonstrate that MEDI-551 is moreeffective at low mAb concentrations than rituximab (Figs. 1Aand 2; data not shown). In addition, in vivo, using huCD19/CD20 double Tg mice as a model system, MEDI-551 effec-tively depleted blood and tissue B cells. At high doses MEDI-551 and rituximab achieved comparable depletion in bloodand secondary lymphoid organs. However, MEDI-551 wasmore effective in eliminating B cells at lower mAb doses. Thelonger duration of B-cell depletion with MEDI-551 is proba-bly a result of the greater impact MEDI-551 has on BM Bcells. CD20 is expressed by immature and mature BM B cells,but, in contrast to CD19, it is not yet present in earlier stagesof B-cell development (Levesque and St Clair, 2008). Giventhat rituximab was almost as effective as MEDI-551 in de-pleting B cells from blood and spleen when used at highdoses, the relatively incomplete depletion of B cells from the

1.0´103

1.0´104

PBSMEDI-551rituximab

L b

lood

1.0×107

1.0×108

PBSMEDI-551rituximab

een

B c

ells

A B

* *

1.0´101

1.0´102

PBS Clod. PBS Clod. PBS Clod.

B c

ells

/µL

1.0×105

1.0×106

PBS Clod. PBS Clod. PBS Clod.

num

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rituximab

C D

107

PBSMEDI-551rituximab *

0

20

40

60

80 MEDI-551anti-CD19-2-TM

% d

eple

tion

106

107

cell n

umbe

r

*

-20

Antibdy concentration [M]10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4

105

PBS PBS CoVF PBS CoVF

Fig. 5. MEDI-551 leads to depletion of Bcell by mouse macrophages in vivo andphagocytosis of murine B cells ex vivo.A, HuCD19/CD20 Tg mice were pre-treated with clodronate liposome or con-trol liposome and subsequently treatedwith either MEDI-551 or rituximab at10 mg/kg. Numbers of remainingB220�muCD19� blood (A) spleen (B) Bcells were determined on day 4 byFACS. Depletion of spleen macrophagesand liver Kupffer cells was confirmed byFACS and histology analysis of F4/80�cells (data not shown). C, effect of comple-ment neutralization with CoVF (30 �g/mouse on days �1, 1, and 3) on spleenB-cell depletion with MEDI-551 and rit-uximab. D, B cells from huCD19/CD20 Tgmice and B6 wild-type mice were stainedwith fluorescence dyes CFSE and PKH-26, respectively. The cells were mixed ata 1:1 ratio and cultured with mouse peri-toneal macrophages, in the presence ofserially diluted MEDI-551 or anti-CD19-2-TM mAbs, or with rituximab. After24 h, cells were recovered, and the num-bers of CFSE� or PKH26� B cells weredetermined by FACS. Percentage of B-cell depletion was calculated as describedunder Materials and Methods. The graphshows one of two experiments with simi-lar results.

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BM of Tg mice is probably a result of the differential expres-sion of the two antigens along the B-cell lineage. It has beennoted that in RA patients treated with rituximab clinicalbenefit often lasts for the duration of B-cell depletion (Dorneret al., 2010). Thus, more complete depletion of early B cellsfrom the BM could provide additional benefit in this indica-tion and possibly other autoimmune diseases.

Consistent with previous reports, the removal of fucoseselectively increased the affinity of the IgG1 anti-CD19mAbs to human Fc�RIIIA, which is expressed on NK cells,monocytes/macrophages, and neutrophils (Shields et al.,2002; Nimmerjahn and Ravetch, 2008). Among mouse Fc�receptors tested, the affinity increase was limited to Fc�RIV.In contrast to human Fc�RIIIA, however, mouse Fc�RIV isnot expressed on NK cells. This has potential consequencesfor the in vivo activity when testing the afucosylated mAb ina murine model. Analysis of the effector mechanisms en-gaged in the mouse model by MEDI-551 clearly demonstratesan important contribution by macrophages for in vivo B-celldepletion. Elimination of neutrophils or NK cells on theirown had no effect on the ability of MEDI-551 to deplete Bcells from blood and lymphoid tissues (data not shown).MEDI-551 also efficiently depleted splenic B cells in theabsence of complement, although a minor, but statisticallysignificant difference was observed between CoVF-treatedand untreated animals (Fig. 5C). In previous studies carriedout in huCD19 Tg animals, elimination of complement byCoVF had no noticeable effect on depletion of blood or spleenB cells by MEDI-551 (data not shown). Overall, the findingsare consistent with the results from the in vitro CDC assay(Fig. 1B). The absence of CDC activity is probably the resultof the inability of the anti-CD19 mAb to mobilize the targetantigen into lipid rafts, a phenomenon required for anti-CD20 mAbs to mediate CDC (data not shown; Cragg et al.,2003). Complement, however, was required to achieve max-imal depletion of spleen B cell with rituximab (Fig. 5C). Inaddition, using a huCD20 Tg mouse model, Gong et al. (2005)investigated the mechanisms of B-cell depletion with anti-huCD20 mAbs. Consistent with the findings presented here,neutralization of complement reduced the efficiency of B-celldepletion in the spleen. It is interesting that the marginalzone B cells were selectively dependent on complement foranti-CD20-mediated depletion (Gong et al., 2005). Using wild-type mice and anti-mouse-CD20 mAbs, however, Minard-Colinet al. (2008) did not observe a significant contribution ofcomplement to depletion of spleen B cells. It is possible thatthese different observations reflect differences in potency ofthe mAbs used. In the huCD19/CD20 Tg model used here,marginal zone and follicular B cells were equally susceptibleto depletion with MEDI-551, despite the lack of CDC activity(data not shown). Thus, the combined in vivo data demon-strate that the afucosylated human IgG1 mAb behaves mostsimilarly to a mouse IgG2a mAb, which has the highestaffinity to FcgRIV and largely relies on macrophages fortarget cell depletion in mouse models (Uchida et al., 2004;Gong et al., 2005; Yazawa et al., 2005).

As mentioned, the affinity of mAbs for Fc� receptors can beincreased by point mutations in the Fc (Desjarlais et al.,2007). Although a selective increase in Fc�RIIIA bindingseems difficult, several mAbs with significantly enhancedADCC have been reported previously (Bowles et al., 2006;Lazar et al., 2006). One such mAb is XmAb5574, an anti-

CD19 mAb that has been engineered for enhanced ADCC byintroducing two point mutations in the Fc (Horton et al.,2008). These mutations result in a significant increase inaffinity not only to Fc�RIIIA but also to Fc�RIIa and theinhibitory receptor Fc�RIIb. It is, however, difficult to com-pare the in vitro activity of XmAb5574 with the activity ofMEDI-551, due to differences in the assay systems used.Using rituximab as a template, Masuda et al. (2007) com-pared the effect of removal of fucose to Fc mutations on Fc�receptor binding and ADCC activity. In their study, the afu-cosylated mAb had 10-fold better binding to Fc�RIIIA,whereas the affinity of the Fc mutant versions of rituximabwas improved up to 90-fold. When tested in vitro for ADCCactivity, however, the afucosylated and Fc mutant mAbswere equipotent over a range of effector cell/target cell ratios(Masuda et al., 2007). The results suggest the existence of athreshold effect with regard to the affinity of the mAb Fc toFc�RIIIA. Furthermore, point mutations in the Fc can poten-tially result in undesired effects, such as increased immuno-genicity or decreased stability. Removal of fucose, however,does not affect mAb stability and is unlikely to change theimmunogenicity in vivo.

While this manuscript was in preparation, Cardarelli et al.(2009) reported another afucosylated anti-CD19 mAb, MDX-1342. Although the same approach for ADCC enhancementwas used in the generation of MEDI-551 and MDX-1342, it isdifficult to compare the activity in vitro and in vivo from thepublished data. MDX-1342 cross-reacts with CD19 fromcynomolgus monkeys and was compared with rituximab forin vivo B-cell depletion. When given as a single dose, extentand duration of blood B-cell depletion with the two mAbs wascomparable, although the onset of depletion with rituximabappeared more rapid (Cardarelli et al., 2009). In addition toFc modifications, the ADCC potency of mAbs is also deter-mined by other factors. The anti-CD19 mAb anti-CD19-2 wasgenerated by humanization and affinity maturation, whichimproved the binding characteristics with decreased inter-nalization rate, and prolonged residence time on the cellsurface, features favorable for an ADCC-dependent mecha-nism (D. C. Rowe, G. P. Sims, and R. Herbst, unpublished).Ghetie et al. (1990) have shown that some, but not all, CD19mAbs can inhibit B-cell proliferation, suggesting that bind-ing properties and/or the particular epitope recognized playan important role for this effect. Here, we show that theCD19 mAb anti-CD19-2 (the fucosylated version of MEDI-551) also reduces proliferation of B cells; thus, over time, themAb could also affect B cells in vivo, independently of ADCCeffector function. To test this, we also generated an Fc-mu-tated version of anti-CD19-2, anti-CD19-2-TM, that is unableto bind to Fc� receptors (Oganesyan et al., 2008) and com-pared the effect to MEDI-551 in SCID mouse human lym-phoma xenograft models. Although less potent than MEDI-551, anti-CD19-2-TM resulted in noticeable tumor growthinhibition, which is probably the result of the antiprolifera-tive effect of the mAb (E. Ward and R. Herbst, unpublisheddata).

In summary, MEDI-551 is a new glycoengineered anti-CD19 antibody, optimized for ADCC effector function. Theremoval of fucose resulted in a selective increase in affinity tohuman Fc�RIIIA and mouse Fc�RIV and enhanced potencyin vitro and in vivo. Furthermore, the in vitro activity ofMEDI-551 with lymphoma cells and primary human B cells

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compared favorably to the anti-CD20 mAb rituximab, whichwas included as positive control in our experiments. Giventhe broad expression of CD19 in B-cell malignancies andcontinued expression on late stage memory B cells and plas-mablasts, B-cell depletion with MEDI-551 has therapeuticpotential in the treatment of B-cell malignancies as well as inautoimmune disease. Clinical studies with MEDI-551 havealready been initiated.

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Address correspondence to: Dr. Ronald Herbst, MedImmune, LCC, De-partment of Research, Respiratory, Inflammation, and Autoimmunity,One MedImmune Way, Gaithersburg, MD 20787. E-mail: herbstr@medimmune.com

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Correction to “B-Cell Depletion In Vitro and In Vivo with anAfucosylated Anti-CD19 Antibody”

In this article [Herbst R, Wang Y, Gallagher S, Mittereder N, Kuta E, Damschroder M,Woods R, Rowe DC, Cheng L, Cook K, Evans K, Sims GP, Pfarr DS, Bowen MA, Dall’AquaW, Shlomchik M, Tedder TF, Kiener P, Jallal B, Wu H, and Coyle AJ (2010) J PharmacolExp Ther 335:213–222], one author’s name was spelled incorrectly. The correct spelling isWilliam Dall’Acqua.

The online version of this article has been corrected in departure from the print version.

The authors regret this error and apologize for any confusion or inconvenience it may havecaused.

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