Epidermal Growth Factor Receptor Targeting IgG3 Triggers Complement-Mediated Lysis of Decay-Accelerating Factor Expressing This information is current as Tumor Cells through the Alternative of September 25, 2021. Pathway Amplification Loop Thies Rösner, Stefan Lohse, Matthias Peipp, Thomas Valerius and Stefanie Derer Downloaded from J Immunol 2014; 193:1485-1495; Prepublished online 27 June 2014; doi: 10.4049/jimmunol.1400329 http://www.jimmunol.org/content/193/3/1485 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Epidermal Growth Factor Receptor Targeting IgG3 Triggers Complement-Mediated Lysis of Decay-Accelerating Factor Expressing Tumor Cells through the Alternative Pathway Amplification Loop

Thies Ro¨sner, Stefan Lohse, Matthias Peipp, Thomas Valerius, and Stefanie Derer

Binding of C1q to target-bound IgG initiates complement-mediated lysis (CML) of pathogens, as well as of malignant or apoptotic cells, and thus constitutes an integral part of the innate immune system. Despite its prominent molecular flexibility and higher C1q binding affinity compared with human IgG1, IgG3 does not consistently promote superior CML. Hence the aim of this study was to investigate underlying molecular mechanisms of IgG1- and IgG3-driven complement activation using isotype variants of the ther- apeutic epidermal growth factor receptor (EGFR) Ab cetuximab. Both IgG1 and IgG3 Abs demonstrated similar EGFR binding Downloaded from and similar efficiency in Fab-mediated effector mechanisms. Whereas anti–EGFR-IgG1 did not promote CML of investigated target cells, anti–EGFR-IgG3 triggered significant CML of some, but not all tested cell lines. CML triggered by anti–EGFR-IgG3 negatively correlated with expression levels of the membrane-bound complement regulatory CD55 and CD59, but not CD46. Notably, anti–EGFR-IgG3 promoted strong C1q and , but relatively low C4b and C5b-9 deposition on analyzed cell lines. Furthermore, anti–EGFR-IgG3 triggered release on all cells but failed to induce and C5a release on CD55/CD59 http://www.jimmunol.org/ highly expressing cells. RNA interference-induced knockdown or overexpression of membrane-bound complement regulatory proteins revealed CD55 expression to be a pivotal determinant of anti–EGFR-IgG3–triggered CML and to force a switch from classical complement pathway activation to C1q-dependent alternative pathway amplification. Together, these data suggest human anti–EGFR-IgG3, although highly reactive with C1q, to weakly promote assembly of the classical C3 convertase that is further suppressed in the presence of CD55, forcing human IgG3 to act mainly through the alternative pathway. The Journal of Immunology, 2014, 193: 1485–1495.

ctivation of the human by cell- new and accelerates the decay of preformed C3 and C5 con- surface ligated Igs leads to opsonization and destruc- vertases by associating with C4b or C3b to force dissociation of by guest on September 25, 2021 A tion of pathogens or malignant cells and constitutes an C2b (generally designated as C2a) from classical convertases, integral part of the innate immune system (1). Thus, complement- as well as of factor Bb from alternative convertases. CD59 blocks dependent cytotoxicity (CDC) has been supposed to constitute the assembly of the membrane attack complex by displacement a pivotal mechanism of action of different therapeutic Abs in tu- of C9 (3). Hence mCRPs are often found to be highly upregulated mor therapy (2). However, membrane-bound complement regu- on tumor cells and, therefore, confer resistance against CDC. In- latory proteins (mCRPs), such as CD46, CD55, and CD59, are terestingly, small interfering RNA (siRNA)-induced knockdown ubiquitously expressed in human cells to tightly regulate local of CD55 in lymphoma cells has been demonstrated to overcome complement activation and protect against uncontrolled complement- resistance against rituximab-triggered CDC (4). Similar results mediated cell destruction. CD46 (membrane cofactor ) acts have been recently reported for the therapeutic Her2/neu-directed as a cofactor of the serine protease “factor I” to degrade C3b or Abs trastuzumab and pertuzumab (5). C4b. CD55 (decay-accelerating factor) prevents the formation of CDC against target cells is evoked by the efficient fixation of the initial complement component C1q on at least two Ig-Fc portions in the vicinity to the cell membrane. However, based on the finding Division of Stem Cell Transplantation and Immunotherapy, 2nd Department of Med- that complement activation by Ig does not solely depend on C1q icine, Christian-Albrechts-University and University Hospital Schleswig-Holstein, fixation (6), it might be hypothesized that other essential molec- 24105 Kiel, Germany ular features are required besides strong C1q binding for efficient Received for publication February 4, 2014. Accepted for publication May 29, 2014. induction of CDC, such as Ag cell-surface expression levels, the This work was supported by the German Research Foundation (Grant DE 1874/1-1) binding epitope of the Ab, Ags’ mobility within the cell mem- and intramural funding from Christian-Albrechts-University Kiel. brane, as well as Abs’ isotype (7–9). This suggestion has been Address correspondence and reprint requests to Dr. Stefanie Derer, Division of Stem Cell Transplantation and Immunotherapy, 2nd Department of Medicine, Christian- strengthened by results received from functional characterization Albrechts-University, Schittenhelmstraße 12, 24105 Kiel, Germany. E-mail address: of the therapeutic epidermal growth factor receptor (EGFR)–tar- [email protected] geting Ab cetuximab, a mouse-human chimeric IgG1 molecule. Abbreviations used in this article: AP, alternative pathway; BHK-21, baby hamster Unexpectedly, cetuximab does not trigger CDC against solid tu- kidney 21; CDC, complement-dependent cytotoxicity; CHO-K1, Chinese hamster ovarian K1; CML, complement-mediated lysis; CRIg, of the mor cells as a single agent while being effective in combination Ig-superfamily; EGFR, epidermal growth factor receptor; mCRP, membrane-bound with a second noncompeting EGFR Ab (10, 11). Besides human complement regulatory protein; MFI, mean fluorescence intensity; RFI, relative fluo- IgG1, human IgG3 has been demonstrated to be highly reactive rescence intensity; SABC, specific Ag-binding sites per cell; w/o, without. with complement (12, 13). Although both human IgG1 and IgG3 Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 efficiently initiate CDC against target cells expressing high Ag www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400329 1486 CD55 CONTROLS IgG3-DRIVEN COMPLEMENT ACTIVATION levels, human IgG3 gains advantage over human IgG1 against amounts of L and H chain vectors using Lipofectamine 2000 (Invitrogen) target cells expressing low Ag levels (14, 15). This difference in according to the manufacturer’s instructions for CHO cells. For selection, CDC activity has been ascribed to the 4-fold longer hinge region, 25 mM methionine sulfoximine (Sigma-Aldrich, St. Louis, MO) was added for inhibition of the endogenous glutamine synthetase expression of CHO- as well as to the higher flexibility of human IgG3 compared with K1 cells. The IgG1 and IgG3 mAbs were affinity purified using Capture human IgG1, enabling IgG3 to better span widely spaced Ag Select Fab anti-human k-L chain chromatography matrices (Capture Se- molecules (13, 16, 17). Referring to these structural features, an lect, Naarden, the Netherlands). For analytical size exclusion chromatog- IgG3 isotype switch variant of the therapeutic IgG1 Ab rituximab raphy, a Superdex 200 26/600 column (GE Healthcare) was used. All purification steps were run on an A¨ KTAprime liquid chromatography has been demonstrated to possess superior complement-activating system (GE Healthcare). UV absorbance at 280 nm, pH, and conductivity capacity against CD20 low-expressing cells such as chronic of the effluent stream were continuously recorded and analyzed using lymphocytic leukemia cells (18). However, contrasting results also Unicorn 4.11 software (GE Healthcare). Ab concentrations were deter- have been reported regarding complement activation by human mined by capillary electrophoresis using the Experion system (Bio-Rad IgG1 and IgG3, demonstrating lower complement-activating Laboratories GmbH, Munich, Germany). capacities for IgG3 than for IgG1 (12). Referring to these stud- Growth inhibition assay ies, it might be hypothesized that structural differences between human IgG1 and IgG3 may result in distinct complement- Growth inhibition of DiFi cells was investigated using the MTS assay according to the manufacturer’s instructions (Promega, Madison, WI). activating mechanisms. However, in-depth analyses elucidating Cells were seeded in a 96-well microtiter plate at a density of 5 3 103 these modes of action have not been reported. cells/well and treated with Abs at indicated concentrations. After 72 h of Hence the aim of this study was to gain novel insights into the incubation at 37˚C, MTS was added as a substrate, and absorption at 490 mode of action and to decipher main determinants of human IgG3- nm was measured. Percentage of growth inhibition was calculated using Downloaded from following formula: % growth inhibition = absorption (EGFR Ab)/absorption driven complement activation. For this purpose, we systematically (without [w/o] Ab) 3 100. investigated underlying molecular mechanisms of IgG3-triggered complement activation in the background of the therapeutic SDS-PAGE and immunoblotting EGFR Ab cetuximab. Thus, we found anti–EGFR-IgG3 to mediate Purified Abs were separated by denaturing NaDodSO4 PAGE (SDS-PAGE) potent C1q fixation and to promote the classical pathway exclu- on gradient Bis-Tris gels (4–12%; Invitrogen) under reducing or nonre- 2 sively on CD55 target cells. When target cells express CD55, anti– ducing conditions and either stained with Coomassie brilliant blue simple http://www.jimmunol.org/ EGFR-IgG3 mainly triggers CDC through the alternative pathway Safe stain (Invitrogen) or transferred onto polyvinylidene fluoride mem- (AP) amplification route after initial classical pathway activation. branes (Millipore, Billerica, MA) for immunoblotting experiments. Immunoblots were performed using HRP-conjugated goat anti-human k-L chain–specific or HRP-conjugated goat anti-human IgG–specific Ab. Proteins were visualized by chemiluminescence (ECL; Thermo Fisher Materials and Methods Scientific, IL). Study population and consent Experiments reported in this article were approved by the Ethics Committee Flow cytometric analyses of the Christian-Albrechts-University, Kiel, Germany, in accordance with EGFR binding experiments were carried out by indirect immunofluores- the Declaration of Helsinki. Blood donors were randomly selected from cence as described previously (21). Relative fluorescence intensity (RFI) by guest on September 25, 2021 healthy volunteers, who gave written informed consent before analyses. was determined by the following formula: mean fluorescence intensity (MFI) EGFR Ab/MFI control Ab. EGFR, CD46, CD55, and CD59 ex- Cell lines pression were quantified using the following murine Abs: EGFR Ab m425, Human epidermoid carcinoma cell line A431 (German Collection of CD46 Ab 0846 (Immunotech Laboratories, Monrovia, CA), CD55 Ab Microorganisms and Cell Cultures, Braunschweig, Germany) and baby S031 (BD, Franklin Lakes, NJ) or the CD59 Ab mem43, and the QIFIKIT hamster kidney 21 (BHK-21) cells transfected with the human EGFR (9) (DAKO, Glostrup, Denmark), according to the manufacturer’s instructions. were kept in RPMI 1640. The human colon carcinoma cell line DiFi and A murine CD20 Ab served as a control. Immunofluorescence was analyzed glioblastoma cell line A1207 (European Collection of Cell Cultures, Sal- on a flow cytometer (Epics Profile; Beckman Coulter, Fullerton, CA). isbury, U.K.) were kept in DMEM, and Chinese hamster ovarian K1 Complement deposition (CHO-K1) cells were kept in DMEM select according to manufacturer’s instructions. Culture media were supplemented with 10% heat-inactivated Deposition of complement components on the cell surface of target cells FCS, except DMEM select, which was supplemented with dialyzed heat- was determined by incubation of 1.5 3 105 cells with EGFR-directed or inactivated FCS. In addition, all culture media were supplemented with control Abs (all at 66.67 nM), as well as with a combination of two non– 100 U/ml penicillin and 100 mg/ml streptomycin (all from Invitrogen, cross-blocking EGFR Abs (anti–EGFR-IgG1 and 003, each at 33.34 Carlsbad, CA). Selection pressure for BHK-EGFR+ transfectants 1–5 was nM), serving as a positive control (10), for 15 min at 4˚C. Subsequently, maintained by adding 1 mg/ml geneticin (PAA, Pasching, Austria). 25% v/v human serum was added, followed by incubation for 10 min at 37˚C. After washing, samples were either stained with polyclonal FITC- Generation and production of mAbs conjugated C1q or C4(b, c) Abs (both from DAKO) or with mouse anti- human (i)C3b mAb (Thermo Fisher Scientific), as well as with mouse Anti–EGFR-IgG1, as well as anti–EGFR-IgG3, was generated based on anti-human Factor Bb or C5b-9 mAb (both from Quidel, San Diego, the variable regions of the 225 (cetuximab) Ab, whereas control mAbs CA), followed by staining with FITC-conjugated goat anti-mouse Fcg were derived from the variable regions of C2B8 (rituximab) Ab. In brief, fragment-specific F(ab)2 (Jackson Immunoresearch Laboratories, West 225-k-L chain and 225-variable heavy genes were each cloned Grove, PA). Samples were analyzed on a flow cytometer (Epics Profile; into pEE14.4 GS-vector (Lonza Biologics, Slough, U.K.), resulting in Beckman Coulter). pEE14.4-225-VL-k-vector and pEE14.4-225-VH-vector (19). The cDNA sequence encoding the variable k-L chain and variable H chain region of Anaphylatoxin release C2B8 (patent no. US006399061B1) was de novo synthesized (Entelechon, Regensburg, Germany) and cloned in the pEE14.4 GS-vector, resulting in Fresh human plasma (25% v/v), supplemented with 25 mg/ml lepirudin pEE14.4-C2B8-VL-k-vector and pEE14.4-C2B8-VH-vector. Next, the (Refludan; Pharmion, Hamburg, Germany), sensitizing Abs (66.67 nM) or g1- or g3-genes, synthesized de novo, were subcloned into either pEE14.4- Ab combination (33.34 nM each mAb), and RPMI 1640 (10% FCS) were 225-VH-vector or pEE14.4-C2B8-VH-vector, resulting in pEE14.4-225- added to a 96-round-well plate. As a control, target cells were incubated VH-IgG1, pEE14.4-225-VH-IgG3, pEE14.4-C2B8-IgG1, or pEE14.4- only with RPMI 1640 (10% FCS). The assay was started by adding 5 3 C2B8-IgG3. The g3- was a Gm3 allotype of the Ig G3 family, in 104 target cells in 50 ml culture medium, therefore resulting in a final which a point mutation at position 435 was inserted, resulting in an amino volume of 200 ml/well. After 3 h at 37˚C, anaphylatoxin release from the acid exchange from arginine to histidine. This mutation has been shown to supernatants was determined by calibrated flow cytometry using the Hu- confer binding to FcRn and, therefore, to increase the in vivo half-life of an man Anaphylatoxin Kit (BD Biosciences, San Diego, CA) according to the IgG3 molecule (20). CHO-K1 cells were stably cotransfected with equal manufacturer’s instructions. The Journal of Immunology 1487

CDC assays AUAAUUUGAACCA-39; CD59 siRNA #1 (ID HSS101611) sense 59-

51 CCAAAGCUGGGUAUCAAGUGUAUAA-39 and antisense 59UUAUA- CDC assays were performed by preincubating target cells with 200 mCi [ Cr] CACUUGUAACCCAGCUUUGG-39; CD59 siRNA #2 (ID HSS10613) for 2 h. Afterward, cells were resuspended in PBS (1X) supplemented sense 59-AGUUCUUCUGCUGGUGACUCCAUUU-39 and antisense with 0.11 mM Mg2+ (PBS (1X) supplemented with 0.5 mM Mg2+ and 2+ 59AAAUGGAGUCACCAGCAGAAGAACU-39. As control, siRNA the 0.15 mM Ca in the case of experiments with factor B–depleted serum) Low GC Duplex #1 (Invitrogen) was used. Efficacy of siRNA-induced or cell culture medium in the case of BHK-EGFR transfectants. To block knockdown of CD46, CD55, or CD59 was determined by direct immuno- complement regulatory activity of CD55, we used mouse anti-human fluorescence using Pacific blue–conjugated mouse anti-human CD46 mAb CD55 (66.67 nM, BRIC216, mouse IgG1; Bio-Rad) blocking mAb in (Exbio, Vestec, Czech Republic), PE-conjugated mouse anti-human CD55 CDC experiments at saturating concentrations. To inhibit the AP of mAb (Beckman Coulter), or FITC-conjugated mouse anti-human CD59 complement activation, we used a fusion protein of the human AP inhibitor mAb (Exbio). Irrelevant Pacific blue/PE/FITC-conjugated Abs served as complement receptor of the Ig-superfamily (CRIg) and human IgG1-Fc controls. (Genentech, San Diego, CA) in CDC experiments at 10 mg/ml. As the source of complement, 25% v/v freshly drawn human serum, 25% v/v Overexpression of CD55 C1q-depleted serum, or 12.5% v/v factor B–depleted serum (Com- + 3 6 plement Technology) was used. C1q-depleted serum was reconstituted BHK-EGFR #5 cells were seeded at a density of 1 10 /well in a 10-cm with 100 mg/ml C1q (Complement Technology), whereas factor B–depleted plate. Next day, cells were transfected with pCMV6-Entry vector (catalog serum was reconstituted with 200 mg/ml factor B (Complement Tech- no. PS100001; OriGene Technologies, Rockville, MD) or pCMV6-Entry- nology). Percentage of [51Cr] release was calculated using following CD55 vector (catalog no. RC201615; OriGene Technologies) by lip- formula: % lysis = (experimental cpm 2 basal cpm)/(maximal cpm 2 ofection using Lipofectamine 2000 (Invitrogen) according to the manu- basal cpm) 3 100. facturer’s instructions. Expression of CD55 was analyzed by flow cytometry using PE-conjugated mouse anti-human CD55 mAb (Beckman siRNA-induced knockdown experiments Coulter). Downloaded from 6 A431 cells were seeded at a density of 1 3 10 /well in 10-cm plates. Data processing and statistical analyses Twenty-four hours later, transfection of siRNA was performed using Lip- ofectamine 2000 (Invitrogen). For all siRNA transfection experiments, Graphical and statistical data analyses were carried out using GraphPad standard protocols were used according to the manufacturer’s instructions. Prism 5.0. Curves were adjusted by using a nonlinear regression model with Synthetic siRNAs targeting CD46, CD55, or CD59 were purchased from a sigmoidal dose response. Statistical significance was determined by Invitrogen. Target sequences were as follows: CD46 siRNA #1 (ID the two-way ANOVA repeated-measures test with Bonferroni’s posttest. HSS142895) sense 59-CAUGUCCAUAUAUACGGGAUCCUUU-39 and Results are presented as mean 6 SEM of at least three independent antisense 59-AAAGGAUCCCGUAUAUAUGGACAUG-39; CD46 siRNA experiments. Correlations between CD46, CD55, or CD59 molecules/cell http://www.jimmunol.org/ #2 (ID HSS181049) sense 59-GGUGAACGAGUAGAUUAUAAGUGUA- and CDC activity were determined by the Pearson correlation test. The p 39 and antisense 59-UACACUUAUAAUCUACUCGUUCACC-39; CD46 values were calculated and null hypotheses were rejected when p # 0.05. siRNA #3 (ID HSS181050) sense 59-CAAAUGGGACUUAGGAGUUU- GGUUA-39 and antisense 59-UAACCAAACUCGUAAGUCCCAUUUG- 39; CD55 siRNA #1 (ID HSS102621) sense 59-ACAGUCUGUAACGU- Results AUGCAUGUAAU-39 and antisense 59AUUACAUGCAUACGUUACA- Anti–EGFR-IgG1 and anti–EGFR-IgG3 do not differ in GACUGU-39; CD55 siRNA #2 (ID HSS175910) sense 59-CACAGUAAAU- Fab-mediated effector mechanisms GUUCCAACUACAGAA-39 and antisense 59UUCUGUAGUUGGAAC- AUUUACUGUG-39; CD55 siRNA #3 (ID HSS175911) sense 59-GGGUA- Human IgG3 in comparison with human IgG1 displays a longer

CAAAUUAUUUGGCUCGACUU-39 and antisense 59AAGUCGAGCCAA- hinge region, 62 versus 14 aa, respectively, and therefore a higher by guest on September 25, 2021

FIGURE 1. Anti–EGFR-IgG1 and anti–EGFR-IgG3 display similar Ag binding and Fab-mediated effector mechanisms. (A) Structural model of IgG1 and IgG3 molecules. Arrows highlight differences between the hinge regions of IgG1 and IgG3. (B) Monomeric anti–EGFR-IgG1 and anti–EGFR-IgG3 were separated under reducing or nonreducing conditions by SDS-PAGE and were either stained using Coomassie blue (left panel) or immunoblotted (right panel) against k L chain or human IgG. (C) Size-exclusion chromatography of anti–EGFR-IgG1 and anti–EGFR-IgG3 were performed to separate ag- gregated from monomeric molecules. (D) Binding of anti–EGFR-IgG1 and anti–EGFR-IgG3 to EGFR expressing DiFi cells were analyzed by indirect immunofluorescence. Means 6 SEM of three independent experiments are presented. (E) Inhibition of DiFi cell growth was analyzed by MTS assay. Means 6 SEM of three independent experiments are presented. *p # 0.05, specific mAb versus respective control Ab. 1488 CD55 CONTROLS IgG3-DRIVEN COMPLEMENT ACTIVATION molecular mass, 170 versus 150 kDa, respectively, as demonstrated dependent manner when compared with the respective control Abs by biochemical analyses (Fig. 1A, 1B). Purity of monomeric anti– (Fig. 1E). EGFR-IgG1 and anti–EGFR-IgG3, as well as of control-IgG1 and control-IgG3 molecules, was investigated by size exclusion chro- CDC is induced by anti–EGFR-IgG3, but not by matography. No contamination with aggregated immune complexes, anti–EGFR-IgG1 known to encompass stronger complement activation capacities, To analyze the effect of different EGFR expression levels on anti– was observed (Fig. 1C). EGFR-IgG1– or anti–EGFR-IgG3–mediated CDC, we used BHK- Monomeric anti–EGFR-IgG1 and anti–EGFR-IgG3 were com- EGFR+ #1–#5 cells, displaying varying expression levels of hu- pared regarding their ability to bind to EGFR on DiFi colorectal man EGFR (#1 , #2 , #3 , #4 , #5), in CDC assays (9). To carcinoma cells. No differences in EGFR binding were detected exclude different EGFR binding capacities of anti–EGFR-IgG1 between anti–EGFR-IgG1 (EC50:1.73nM)andanti–EGFR- and anti–EGFR-IgG3 on these transfectants, we generated dose– IgG3 (EC50: 1.26 nM; Fig 1D). Similar results were obtained response curves for both Abs, as well as for the respective control from experiments using A431 cells as a second EGFR+ tumor Abs by flow cytometry analyses (Fig. 2A). No significant differ- cell line (data not shown). Furthermore, anti–EGFR-IgG1 (EC50: ences were observed between anti–EGFR-IgG1 and anti–EGFR- 0.41 6 0.18 nM) and anti–EGFR-IgG3 (EC50: 0.58 6 0.15 nM) IgG3, except for significantly different binding of anti–EGFR- similarly induced growth inhibition of DiFi cells (anti–EGFR-IgG1: IgG1 (MFI = 13.4 6 0.2) and anti–EGFR-IgG3 (MFI = 7.55 6 66.9 6 6.3%; anti–EGFR-IgG3: 68.7 6 6.4%) in a concentration- 2.75) on BHK-EGFR+ #3 cells at 0.53 nM (Fig. 2A). Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Anti–EGFR-IgG3 in contrast with anti–EGFR-IgG1 triggers CDC. (A) Binding of anti–EGFR-IgG1 or anti–EGFR-IgG3, as well as of respective control Abs, to BHK cells transfected to express increasing levels of EGFR (BHK-EGFR+ cell lines #1–#5) was analyzed by indirect flow cytometry at indicated Ab concentrations. (B)In[51Cr] release assays, BHK-EGFR+ cell lines #1–#5 were incubated for 3 h in the presence of 25% v/v normal human serum and anti–EGFR-IgG1, anti–EGFR-IgG3, or control Abs at increasing concentrations. Means 6 SEM of three independent experi- ments with different blood donors are presented. (C) CDC against three different tumor cell lines was analyzed by 3-h [51Cr] release assay in the presence of 25% v/v human serum and anti–EGFR-IgG1, anti–EGFR-IgG3, or the respective control Abs at increasing concentrations. Means 6 SEM of at least three independent experiments are presented. *p # 0.05 anti–EGFR-IgG1/anti–EGFR-IgG3 versus respective control Abs, #p # 0.05 anti–EGFR-IgG3 versus anti–EGFR-IgG1. (D) Surface expression levels of EGFR were quantified by calibrated flow cytometry. Means 6 SEM of at least three independent experiments are presented. (E) Correlation between EGFR expression levels and anti–EGFR-IgG3–mediated CDC was calculated for the four cell lines. The Journal of Immunology 1489 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Anti–EGFR-IgG3 fails to initiate the formation of C5b-9 on the cell surface of A431 cells. (A) Schematic models of the classical pathway and the AP of complement activation triggered by IgG molecules. (B–F) Deposition of complement components was analyzed on cell lines that were either resistant (A431) or susceptible (BHK-EGFR+ #5 and DiFi) to anti–EGFR-IgG3–triggered CML. Cells were incubated in the (Figure legend continues) 1490 CD55 CONTROLS IgG3-DRIVEN COMPLEMENT ACTIVATION

Notably, when compared with the respective control Abs, anti– To investigate functional consequences of complement depo- EGFR-IgG3, but not anti–EGFR-IgG1, triggered significant CDC sition on analyzed cell lines, we analyzed the release of C4a, C3a, in a concentration-dependent manner (0.11–66.67 nM) against and C5a anaphylatoxins by calibrated flow cytometry (Table I). BHK-EGFR+ cell lines #3, #4, and #5, whereas no CDC was Significant release of C4a, C3a, and C5a was triggered by anti– detected against BHK-EGFR+ cell lines #1 and #2 (Fig. 2B). EGFR-IgG3 on DiFi and BHK-EGFR+ #5 cells. Notably, on A431 To investigate whether results received from BHK-EGFR+ #1– cells, the positive control induced significant C4a, C3a, and C5a #5 cell lines could be transferred to human tumor cell lines, we release, whereas anti–EGFR-IgG3 evoked only significant C4a, analyzed CDC activity evoked by anti–EGFR-IgG1 or anti–EGFR- but not C3a, as well as C5a release. In addition, significant C3a IgG3 against different tumor cell lines by [51Cr] release assays. release was measured in the case of anti–EGFR-IgG1 on A431 Anti–EGFR-IgG3 triggered strongest CDC activity against DiFi and DiFi, but not on BHK-EGFR+ #5 cells. However, no signifi- cells (35.2 6 3.1% maximum lysis), followed by A1207 cells cant augmentation of C4a and C5a levels was detected on all three (11.8 6 1.8% maximum lysis). However, no complement-mediated cell lines by anti–EGFR-IgG1. lysis (CML) of A431 cells was induced by anti–EGFR-IgG3. In The extent of CML evoked by anti–EGFR-IgG3 negatively addition, anti–EGFR-IgG1, as well as the respective control Abs, correlates with CD55 and CD59 expression levels did not mediate CDC activity against any of the analyzed tumor cell lines (Fig. 2C). CDC triggered by IgG1 Abs directed against Besides the impact of Ag expression levels on the extent of CDC, EGFR has been recently demonstrated to depend on cell-surface cell-surface expression levels of mCRPs such as CD46, CD55, or expression levels of the Ag (9). To analyze whether the extent of CD59 have been demonstrated to control CDC activity triggered by

CDC activity induced by anti–EGFR-IgG3 is controlled by EGFR Her2/neu-directed Abs (5). To investigate whether the extent of Downloaded from expression levels, we quantified EGFR cell-surface expression CDC activity induced by anti–EGFR-IgG3 is controlled by mCRP levels by calibrated flow cytometry and correlated them to the expression levels, we performed calibrated flow cytometry to extent of anti–EGFR-IgG3–triggered CML of the respective cell quantify CD46, CD55, or CD59 cell-surface expression levels on + line. However, no correlation was observed because of similar A431, DiFi, A1207, and BHK-EGFR #5 cells (Fig. 4A). As ex- EGFR-Ab binding sites per cell between all four analyzed cell pected, no expression of human CD46, CD55, or CD59 was 6 6 + + lines (A431 = 1.2 3 10 6 0.1 3 10 ; BHK-EGFR #5 = 1.1 3 detected on BHK-EGFR #5 cells, whereas significant differences http://www.jimmunol.org/ 106 6 0.1 3 106; DiFi = 1.1 3 106 6 0.1 3 106; A1207 = 1.0 3 were observed for A431, A1207, and DiFi cells. Highest CD46 6 106 6 0.2 3 105; Fig. 2D, 2E). expression levels were identified on DiFi cells (0.4 3 10 6 0.02 3 106 specific Ag-binding sites per cell [SABC]), followed by A431 Anti–EGFR-IgG3 fails to efficiently initiate generation of and A1207 cells. However, highest expression levels of human functional C3 convertases on A431 cells CD55 (0.9 3 105 6 0.1 3 105 SABC) and human CD59 (0.5 3 Because anti–EGFR-IgG3 triggered CDC against DiFi and BHK- 106 6 0.1 3 106 SABC) were measured on A431 cells, followed EGFR+ #5 cells, but not against A431 cells, the ability of anti– by A1207 and DiFi cells. Furthermore, correlation coefficients (R) EGFR-IgG3 to deposit complement components on A431, DiFi, between mCRPs (CD46, CD55, and CD59) cell-surface expression + or BHK-EGFR #5 cells was analyzed by flow cytometry. In these levels and relative anti–EGFR-IgG3–induced cytotoxic activity by guest on September 25, 2021 experiments, anti–EGFR-IgG3 was compared with anti–EGFR- were calculated for the analyzed cell lines (Fig. 4B). Correlation IgG1, as well as with a combination of two noncompeting was calculated at saturating anti–EGFR-IgG3 Ab concentration EGFR-directed IgG1 Abs, serving as a positive control (Fig. 3). (2 mg/ml) by equating cytotoxic activity against BHK-EGFR+ Interestingly, despite stronger C1q binding by anti–EGFR-IgG3 #5 cells with 100% (maximum lysis), which was related to the compared with the positive control on A431, DiFi, and BHK- cytotoxic activity measured for all other cell lines. A negative EGFR+ #5 cells (Fig. 3B), lower amounts of C4(b/c) deposition on correlation between CD55 and/or CD59 cell-surface expression A431, DiFi, or BHK-EGFR+ #5 cells were observed for anti– levels and cytotoxic activity triggered by anti–EGFR-IgG3 was EGFR-IgG3 in comparison with the positive control (Fig. 3C). observed (R = 20.9, p 5 0.06 for CD55, R = 20.9, p 5 0.06 for However, in contrast with C4(b/c) deposition, anti–EGFR-IgG3 CD59), whereas no correlation was found for CD46 cell-surface displayed similar C3b deposition as detected for the positive expression levels. control on A431, DiFi, and BHK-EGFR+ #5 cells (Fig. 3D). Because of results received from correlation analyses, the Notably, although significantly higher factor Bb deposition was impact of CD46, CD55, or CD59 on CML of A431 cells was obtained for anti–EGFR-IgG3 compared with the positive control analyzed in more detail by siRNA-induced knockdown experi- on DiFi and A431 cells, significantly lower factor Bb deposition ments. Knockdown of CD46, CD55, or CD59 was tested using three was found on BHK-EGFR+ #5 cells, pointing to stronger activa- distinct siRNAs specific either for CD46, CD55, or CD59, whereas tion of the AP of complement on DiFi and A431 cells than on a nonspecific siRNA served as a control siRNA (data not shown). BHK-EGFR+ #5 cells (Fig. 3E). Furthermore, anti–EGFR-IgG3 Based on these data, siRNAs displaying strongest knockdown induced significant C5b-9 levels on DiFi and BHK-EGFR+ #5 effects were chosen for further experiments. Significant siRNA- cells, but not on A431 cells, compared with the respective control induced knockdown was achieved for CD46, CD55, or CD59: IgG3 Ab (Fig. 3F). In contrast with anti–EGFR-IgG3, anti–EGFR- CD46-specific siRNA (∼73% knockdown), CD55-specific siRNA IgG1 or used control Abs did not trigger significant deposition of (∼67% knockdown), or CD59-specific siRNA (∼67% knockdown). C1q, C4(b/c), C3b, and C5b-9 on all three analyzed cell lines A combination of CD46-, CD55-, and CD59-specific siRNAs also (Fig. 3B–F). induced a significant knockdown of CD46, CD55, and CD59,

presence of 25% v/v human serum and indicated EGFR-Abs or the respective control Abs (66.67 nM for individual mAbs and 33.34 nM each for the positive control). Deposition of (B) C1q, (C) C4(b/c), (D) (i)C3(b), (E) factor Bb, and (F) C5b-9 were analyzed by direct or indirect immunofluorescence. Relative deposition levels were calculated by equating RFI measured in the presence of the positive control with 100% for each cell line. Means 6 SEM of at least three independent experiments are presented. *p # 0.05 anti–EGFR-IgG1/anti–EGFR-IgG3/positive control versus respective control Abs; #p # 0.05 indicated mAb versus anti–EGFR-IgG3. The Journal of Immunology 1491

which was comparable with the extent of knockdown induced

a a a by single siRNAs (CD46: ∼77% knockdown; CD55: ∼55% 5.8 2.1 3.1 knockdown; CD59: ∼58% knockdown; Fig. 4C). 6 6 6 51 C5a Subsequently, [ Cr] release assays with increasing concen- 46

15.8 trations of anti–EGFR-IgG1, anti–EGFR-IgG3, or the respective control Abs in the presence of human serum were performed with a

a control siRNA or with mCRP-specific siRNA-transfected A431

4.9 4443.2 14.1 cells (Fig. 4D–G). In the case of anti–EGFR-IgG3, siRNA- C3a 6 6 6 induced knockdown of CD46 (Fig. 4D, lower panel) did not af-

592 509 473 fect CDC activity, whereas siRNA-induced knockdown of CD55 Positive Control (10.4 6 4.4% lysis; Fig. 4E, lower panel) or CD59 (14.0 6 4.3% a

a a lysis; Fig. 4F, lower panel) significantly improved CML of A431 49.4 23 57 cells. This improvement was further enhanced by a combined 6 6 6 C4a siRNA-induced knockdown of CD46, CD55, and CD59 (36.5 6

447 358 2.1% lysis; Fig. 4G, lower panel). Anti–EGFR-IgG1 and respec- tive control Abs did not trigger CDC neither against control a a 1.9 446 9.4 3.2 siRNA-transfected nor against mCRP-specific siRNA-transfected 6 6 6 A431 cells. Results received from RNA interference-induced C5a

knockdown experiments with CD55-specific siRNA were addi- Downloaded from 40.2 15.1 tionally verified by CDC experiments against A431 cells in the

a a presence of a CD55 blocking Ab at a saturating concentration of

17.2 51.2 43.6 8.8 66.67 nM. In these blocking experiments, anti–EGFR-IgG3 6 C3a 6 6 6 (9.6 2.6% lysis), but not anti–EGFR-IgG1, triggered CDC

EGFR-IgG3 against A431 in the presence of the CD55 Ab (Fig. 4H, 4I). Based 602 555 410

on its earlier inhibitory function in the complement cascade in http://www.jimmunol.org/

a a comparison with CD59, these data point to a crucial role of CD55 a 50 51.2 39.8 in anti–EGFR-IgG3–evoked complement-dependent cytotoxic C4a 6 6 6 activity. CD55 constitutes the main regulator of anti–EGFR-IgG3–driven CML 1.7 424 3.4 431 2.2 393 6 6 6 To further analyze the influence of CD55 on anti–EGFR-IgG3– C5a 2

25 11 dependent CML, we transiently transfected human CD55 BHK- EGFR+ #5 cells with a plasmid encoding human CD55, leading to by guest on September 25, 2021

a strong overexpression of CD55 (control vector RFI = 1.2 6 0.2 a 21.6 67 50 8.4 versus CD55-vector RFI = 380.8 6 39.7) on the cell surface C3a 6 6 6 (Fig. 5A). Accordingly, dose-dependent CDC activity against +

EGFR-IgG1 control vector or CD55 vector–transfected BHK-EGFR #5 cells triggered by anti–EGFR-IgG3 was analyzed by [51Cr] release assays in the presence of human serum (Fig. 5B). Compared with 43.2 584 45.4 452 50.8 471 control vector–transfected BHK-EGFR+ #5 cells, overexpression 6 6 6 C4a of CD55 led to a significant decrease (∼60%) in anti–EGFR- IgG3–induced complement-dependent cytotoxic activity. No CML of analyzed transfectants was observed in the presence of a 3.7 291 2.1 379 1.6 323 control IgG3 Ab (Fig. 5B). 6 6 6

C5a Activation of the classical pathway and the AP of complement can be distinguished by analyzing the kinetics of CML, with the AP being slower than the classical pathway (22, 23). Hence CDC assays using control vector or CD55 vector–transfected BHK- 17.2 18.6 53.6 9.7 49.8 8.1 EGFR+ #5 cells were performed at different time points (0–240 6 6 6 C3a w/o min) at saturating Ab concentrations (anti–EGFR-IgG3 and control-IgG3, both at 13.3 nM). Notably, significantly slower anti– EGFR-IgG3–promoted CDC kinetics were observed on CD55 6 61.4 519 55.8 384 55 474 transfectants (EC50 = 85.00 1.95 min) compared with control 6 6 6 6 C4a vector transfectants (EC50 = 32.41 2.71 min; Fig. 5C), pointing to the predominant activation of the alternative complement 255 326 322 pathway on CD55 transfectants. The contribution of the alterna- tive complement pathway in anti–EGFR-IgG3–induced CDC + #5 against CD55- or control vector–transfected BHK-EGFR #5 cells + was additionally examined by CDC assays using different serum concentrations (0–25% v/v). As presented in Fig. 5D, compared 0.05 anti–EGFR-IgG1/anti–EGFR-IgG3/positive control versus w/o Ab.

Anaphylatoxin with control vector–transfected cells, overexpression of CD55 on Release (ng/ml) # + p DiFi BHK-EGFR A431

a BHK-EGFR #5 cells was accompanied by a statistically signifi-

Table I. EGFR Ab–triggered anaphylatoxin release cant decline of anti–EGFR-IgG3–driven CML efficiency at lower 1492 CD55 CONTROLS IgG3-DRIVEN COMPLEMENT ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. CML triggered by anti–EGFR-IgG3 negatively correlates with CD55 and CD59 expression levels. (A) Surface expression levels of CD46, CD55, and CD59 on analyzed cell lines were quantified by calibrated flow cytometry. Means 6 SEM of at least three independent experiments are presented. (B) Correlations between CD46, CD55, or CD59 and anti–EGFR-IgG3–mediated CDC were calculated for all four cell lines. CDC results at 2 mg/ml Ab concentration were taken from experiments presented in Fig. 2. (C) A431 cells were seeded into 10-cm plates and grown overnight. On the following day, cells were transfected with 50 nM control siRNA or with single siRNAs specific for CD46, CD55, CD59, or with a combination of all three mCRP-specific siRNAs for 72 h. Efficiency of siRNA-induced knockdown was analyzed by direct flow cytometry using fluorochrome-labeled, mCRP- specific Abs (CD46-Pacific blue, CD55-PE, CD59-FITC), or respective control Abs. (D–G) CDC against control siRNA or mCRP-specific, siRNA- transfected A431 cells was analyzed by 3-h [51Cr] release assays in the presence of 25% v/v human serum and anti–EGFR-IgG1 (upper panels), anti– EGFR-IgG3 (lower panels), as well as the respective control Abs at increasing concentrations. (H) Concentration-dependent binding of CD55-Ab (BRIC216, mouse IgG1) to A431 cells was analyzed by indirect immunofluorescence. Results from one representative experiment are presented. (I) CDC triggered by anti–EGFR-IgG1, anti–EGFR-IgG3, or respective control Abs (all at 66.67 nM) against A431 cells in the presence of saturating concentrations of CD55-Ab or a control Ab (both at 66.67 nM) was analyzed by 3-h [51Cr] release assays in the presence of 25% v/v human (Figure legend continues) The Journal of Immunology 1493 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. CD55 dampens anti–EGFR-IgG3–triggered CML and promotes C1q-dependent induction of AP amplification. BHK-EGFR+ #5 cells were transiently transfected with a control vector or a CD55 vector for 48 h. (A) Cell-surface expression of CD55 was analyzed by direct flow cytometry using PE-conjugated CD55-specific or control Abs. (B–D) The influence of CD55 overexpression on anti–EGFR-IgG3–mediated CDC was investigated by [51Cr] release assays either (B) in an Ab concentration–response curve, (C) in a time-dependent manner, or (D) in serum titration experiments. (E–H) The influence of the alternative complement pathway inhibitor CRIg (E and G), the presence of C1q in serum (F; at 66.67 nM Ab concentration; mean 6 SEM of triplicates), as well as of factor B (H; 13.33 nM Ab concentration, 12.5% v/v factor B–depleted serum, 200 mg/ml factor B), on anti–EGFR-IgG3–mediated CDC was analyzed using either (E and F) control vector–transfected or CD55 vector–transfected BHK-EGFR+ #5 cells or (G and H) DiFi cells (66.67 nM Ab concentration). (I) Deposition of factor Bb on control vector– or CD55 vector–transfected BHK-EGFR+ #5 cells was analyzed by flow cytometry. Relative deposition levels were calculated by equating RFI measured in the absence of Ab with 100%. Results are presented as mean 6 SEM of at least three independent experiments with different blood donors. *p # 0.05 anti–EGFR-IgG3 versus respective control Ab; (B–D, I) #p # 0.05 control vector versus CD55 vector; (E and G) #p # 0.05 without CRIg-Fc versus CRIg-Fc; (H) #p # 0.05 w/o factor B versus with factor B. serum concentrations. To more directly analyze activation of the In addition, CDC experiments using DiFi cells that endogenously AP by anti–EGFR-IgG3 on CD55 overexpressing BHK-EGFR+ #5 express CD55 were performed in the absence and presence of cells, we performed CDC assays in the presence of the alternative CRIg-Fc (Fig. 5G). As presented in Fig. 5G, CRIg-Fc also sig- complement pathway inhibitor CRIg-Fc (Fig. 5E) (24). In line nificantly inhibited anti–EGFR-IgG3–induced CML of DiFi cells with CDC experiments presented in Fig. 5C and 5D, no inhibition (∼80% inhibition) compared with untreated control experiments. of anti–EGFR-IgG3–induced CML of control vector–transfected Similar results were received from CDC experiments using factor BHK-EGFR+ #5 cells was observed in the presence of CRIg-Fc. B–depleted serum (Fig. 5H). Although no CDC was induced However, CRIg-Fc strongly inhibited (∼50% inhibition) anti– against DiFi cells by anti–EGFR-IgG3 in the absence of factor B, EGFR-IgG3–triggered CML of CD55-transfected BHK-EGFR+ #5 significant cytotoxicity was triggered when factor B–depleted cells. For both transfectants, control vector– or CD55-transfected serum was reconstituted with factor B. Results received from CDC BHK-EGFR+ #5 cells, C1q-dependent initiation of CDC was experiments were further supported by flow-cytometry analyses observed using C1q-depleted serum in cytotoxic assays (Fig. 5F). demonstrating anti–EGFR-IgG3 to promote significantly more

serum. Results are presented as mean 6 SEM of at least three independent experiments with different blood donors. (D–I)*p # 0.05 anti–EGFR-IgG1/anti– EGFR-IgG3 versus respective control Abs; (D–G) #p # 0.05 specific siRNA versus control siRNA. 1494 CD55 CONTROLS IgG3-DRIVEN COMPLEMENT ACTIVATION factor Bb deposition on CD55-transfected than on control vector– human IgG1 and IgG3 molecules significantly differ in C4b bind- transfected BHK-EGFR+ #5 cells (Fig. 5I, Fig. 6). ing. In this study, human IgG3 against EGFR was also found to display low C4b in relation to high C1q deposition levels and, Discussion therefore, to mainly recruit the AP after initial C1q-dependent CDC constitutes a powerful effector mechanism of the immune complement activation on CD55 high-expressing cells (Fig. 6). system and is suggested to contribute to the clinical efficacy of Hence although human IgG3 has been described to be an activator some tumor-directed therapeutic Abs (3). Unexpectedly, human of the classical pathway of complement activation (34), recent data IgG1 Abs targeting EGFR lack the capacity to activate the com- also demonstrated that human IgG3 directed against plement cascade on solid tumor cells as single agents, whereas binding protein expressed by meningococcal strains mainly acti- being potent in activating CDC as noncompeting Ab combinations vates the AP of complement at low Ag expression levels (35). (10, 25). Because efficient C1q fixation to the CH2 domain of Ig Furthermore, this study shows that CD55 controls anti–EGFR- molecules depends on the close proximity of at least two Ig-Fc IgG3–driven complement activation on human cells by interfering parts (26, 27), it can be assumed that a combination of non- with the assembly of classical C3 convertase and forces anti–EGFR- competing EGFR Abs, in contrast with single EGFR Abs, more IgG3 to more efficiently activate the AP. In line with these findings, potently enables clustering of closely spaced Ig-Fc parts. a previous study reported superior human IgG3-induced CML of To enhance the CDC capacity of single EGFR-targeting Abs, we tumor cells expressing high CD55 and CD59 but low CD46 levels generated an IgG3 isotype variant of cetuximab to take advantage by neutralization of CD46, CD55, and CD59 compared with sole of human IgG3-related structural features. Notably, despite strong neutralization of CD59 on these cells (36). Moreover, recent data

C1q binding capacity, single EGFR-specific IgG3 Abs triggered revealed human IgG3 specific for CD20 to more potently trigger Downloaded from significant CDC against CD552 or low-expressing target cells, but CML of CD55-expressing CLL cells than the human IgG1 coun- not against CD55 high-expressing cells. This discrepancy between terpart, potentially pointing to different mechanisms of action of strong human IgG3 triggered C1q deposition on target cells, but human IgG3 Abs displaying distinct target Ag specificities (18). relatively low lytic activity has been also previously reported (12) CD55, the decay-accelerating factor of C3 (C4b2b/C4b2a or and further related to inefficient deposition of C4b and C3b on C3bBb) and C5 (C4b2b3b/C4b2a3b or C3bBb3b) convertases, is

target cells (28). Furthermore, lower C4b deposition, although not a GPI-anchored protein that is widely expressed in human cells. http://www.jimmunol.org/ as much as demonstrated by Bindon et al. (28), but stronger CDC However, dysregulated CD55 expression was identified by many activity by IgG3 compared with IgG1 has been reported in a recent studies in distinct tumor types, with some revealing an upregula- study analyzing CD20-directed Abs on lymphoma cells (29). tion and some a downregulation (37). The importance of CD55 in Notably, in two additional studies, using enzyme immunoassays, tumorigenesis has been underlined by studies revealing high CD55 either similar (30) or even stronger (31) C4 binding was observed expression levels as a poor prognosis marker in carcinomas of the for IgG3 compared with IgG1. Because C4b has been reported to breast, the gallbladder, or the colon (38–40). Furthermore, pan- covalently bind to Ag-bound Abs in the Fd region, comprising the creatic and breast carcinomas were identified to display stronger VH and the CH1 domains (32, 33), it can be hypothesized that CD55 cell-surface expression levels compared with oral squamous by guest on September 25, 2021

FIGURE 6. Overview of complement activation by human anti–EGFR-IgG3 in the context of CD55 expression. On CD55-deficient target cells (left panel), anti–EGFR-IgG3 mediates strong C3b but low C4b deposition and induces assembly of classical and alternative C3 convertases, predominantly resulting in the induction of fast and efficient CDC via the classical pathway of complement activation. In contrast, on CD55-expressing target cells (right panel), CD55 mainly accelerates the decay of low amounts of classical C3 convertases, leading to amplification of the AP and finally to slow and inefficient CDC induction. The Journal of Immunology 1495 cell or colorectal carcinomas, as well as melanomas (41). In ac- 16. Brekke, O. H., T. E. Michaelsen, and I. Sandlie. 1995. The structural require- ments for complement activation by IgG: does it hinge on the hinge? Immunol. cordance with these results, lowest CD55 expression and, there- Today 16: 85–90. fore, strong CDC activity by anti–EGFR-IgG3 was detected on the 17. Roux, K. H., L. Strelets, and T. E. Michaelsen. 1997. Flexibility of human IgG colorectal carcinoma cell line DiFi in this study. subclasses. J. Immunol. 159: 3372–3382. 18. Ro¨sner, T., S. Derer, C. Kellner, M. Dechant, S. Lohse, G. Vidarsson, M. Peipp, In conclusion, an isotype switch from human IgG1 to human IgG3 and T. Valerius. 2013. An IgG3 switch variant of rituximab mediates enhanced is accompanied by improvement of complement-activating capaci- complement-dependent cytotoxicity against tumour cells with low CD20 ex- ties of EGFR targeting Abs. However, despite its strong C1q fixing pression levels. Br. J. Haematol. 161: 282–286. 19. Beyer, T., S. Lohse, S. Berger, M. Peipp, T. Valerius, and M. Dechant. 2009. capacity, human anti–EGFR-IgG3 lacks the ability to efficiently Serum-free production and purification of chimeric IgA antibodies. J. Immunol. deposit C4b on targets’ cell surfaces, and thus to initiate the com- Methods 346: 26–37. plement cascade on CD55 high-expressing tumor cells. These novel 20. Stapleton, N. M., J. T. Andersen, A. M. Stemerding, S. P. Bjarnarson, R. C. Verheul, J. Gerritsen, Y. Zhao, M. Kleijer, I. Sandlie, M. de Haas, et al. findings revealed human IgG3 directed against EGFR to encompass 2011. Competition for FcRn-mediated transport gives rise to short half-life of improved cytotoxic potential, and hence to display a promising human IgG3 and offers therapeutic potential. Nat. Commun. 2: 599. strategy in Ab-based therapy of CD55 low-expressing tumors. 21. Schlaeth, M., S. Berger, S. Derer, K. Klausz, S. Lohse, M. Dechant, G. A. Lazar, T. Schneider-Merck, M. Peipp, and T. Valerius. 2010. Fc-engineered EGF-R antibodies mediate improved antibody-dependent cellular cytotoxicity (ADCC) Acknowledgments against KRAS-mutated tumor cells. Cancer Sci. 101: 1080–1088. 22. Oganesyan, L. P., G. M. Mkrtchyan, S. H. Sukiasyan, and A. S. Boyajyan. 2009. We thank Christyn Wildgrube, Yasmin Brodtmann, and Kathinka Tuxen€ for Classic and alternative complement cascades in post-traumatic stress disorder. excellent technical assistance. CRIg-Fc was kindly provided by Dr. M. van Bull. Exp. Biol. Med. 148: 859–861. Lookeren Campagne (Genentech, South San Francisco, CA). We thank 23. Harboe, M., and T. E. Mollnes. 2008. The alternative complement pathway revisited. J. Cell. Mol. Med. 12: 1074–1084. Dr. F. Beurskens (Genmab, Utrecht, the Netherlands) for critically reading Downloaded from 24. Wiesmann, C., K. J. Katschke, J. Yin, K. Y. Helmy, M. Steffek, W. J. Fairbrother, the manuscript. S. A. McCallum, L. Embuscado, L. DeForge, P. E. Hass, and M. van Lookeren Campagne. 2006. 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