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Rösner et al: Effector functions of IgG2 1 Immune effector functions of human IgG2 antibodies against EGFR Thies Rösner 1* , Steffen Kahle 1* , Francesca Montenegro 1 , Hanke L. Matlung 2 , J. H. Marco Jansen 3 , Mitchell Evers 3 , Frank Beurskens 4 , Jeanette H.W. Leusen 3 , Timo K. van den Berg 25 , Thomas Valerius 1# 1 Section for Stem Cell Transplantation and Immunotherapy, Department of Medicine II, Christian Albrechts University and University Hospital Schleswig-Holstein, Campus Kiel 2 Sanquin Research, Landsteiner Laboratory and Department of Molecular Cell Biology and Immunology, VU Medical Center, Amsterdam, The Netherlands 3 Laboratory of Translational Immunology, University Medical Center, Utrecht, The Netherlands 4 Genmab BV, Utrecht, The Netherlands 5 Dept. Molecular Cell Biology and Immunology, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam Infection and Immunity Institute, Amsterdam, The Netherlands * these authors contributed equally # Corresponding author: Thomas Valerius, MD; Section for Stem Cell Transplantation and Immunotherapy, Department of Medicine II, Christian Albrechts University and University Hospital Schleswig-Holstein, Campus Kiel, Arnold Heller Str. 3, 24105 Kiel, Germany; phone: 0049-431-500-22710; e-mail: [email protected] Disclosure of Potential Conflicts of Interest F.B. is an employee of Genmab, the other authors declare that they have no conflict of interest. Research. on December 15, 2020. © 2018 American Association for Cancer mct.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 3, 2018; DOI: 10.1158/1535-7163.MCT-18-0341

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Rösner et al: Effector functions of IgG2

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Immune effector functions of human IgG2 antibodies against EGFR

Thies Rösner1*, Steffen Kahle1*, Francesca Montenegro1, Hanke L. Matlung2, J. H. Marco

Jansen3, Mitchell Evers3, Frank Beurskens4, Jeanette H.W. Leusen3, Timo K. van den Berg25,

Thomas Valerius1#

1 Section for Stem Cell Transplantation and Immunotherapy, Department of Medicine II, Christian Albrechts University and University Hospital Schleswig-Holstein, Campus Kiel 2 Sanquin Research, Landsteiner Laboratory and Department of Molecular Cell Biology and Immunology, VU Medical Center, Amsterdam, The Netherlands 3 Laboratory of Translational Immunology, University Medical Center, Utrecht, The Netherlands

4 Genmab BV, Utrecht, The Netherlands 5 Dept. Molecular Cell Biology and Immunology, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam Infection and Immunity Institute, Amsterdam, The Netherlands

* these authors contributed equally

# Corresponding author: Thomas Valerius, MD; Section for Stem Cell Transplantation and

Immunotherapy, Department of Medicine II, Christian Albrechts University and University

Hospital Schleswig-Holstein, Campus Kiel, Arnold Heller Str. 3, 24105 Kiel, Germany; phone:

0049-431-500-22710; e-mail: [email protected]

Disclosure of Potential Conflicts of Interest

F.B. is an employee of Genmab, the other authors declare that they have no conflict of

interest.

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Abstract

Three FDA approved epidermal growth factor receptor (EGFR) antibodies (Cetuximab,

Panitumumab, Necitumumab) are clinically available to treat patients with different types of

cancer. Interestingly, Panitumumab is of human IgG2 isotype, which is often considered to

have limited immune effector functions. Unexpectedly, our studies unraveled that human

IgG2 antibodies against EGFR mediated effective CDC when combined with another non

cross-blocking EGFR antibody. This second antibody could be of human IgG1 or IgG2 isotype.

Furthermore, EGFR antibodies of human IgG2 isotype were highly potent in recruiting

myeloid effector cells such as M1 macrophages and PMN for tumor cell killing by ADCC.

Tumor cell killing by PMN was more effective with IgG2 than with IgG1 antibodies if tumor

cells expressed lower levels of EGFR. Additionally, lower expression levels of the “don’t eat

me” molecule CD47 on tumor cells enabeled ADCC also by M2 macrophages, and improved

PMN and macrophage mediated ADCC. A TCGA enquiry revealed broadly varying CD47

expression levels across different solid tumor types. Together, these results demonstrate

that human IgG2 antibodies against EGFR can promote significant Fc mediated effector

functions, which may contribute to their clinical efficacy. The future challenge will be to

identify clinical situations in which myeloid effector cells can optimally contribute to

antibody efficacy.

abstract word count: 206

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Introduction

Monoclonal antibodies against checkpoint controlling molecules of the adaptive immune

system – such as CTLA-4, PD-1, PD-L1 and others – currently revolutionize cancer therapy (1).

The scientific and economic interest to identify biomarkers to predict response or resistance

to these novel therapies has encouraged studies to closer investigate the mutational status

of different tumor types and their immune cell infiltrate (2). The latter studies benefit from

recently developed technologies, which enable more detailed analyses of the different

tumor infiltrating cell types as well as their functional states (3). Across many different

cancer types, myeloid cells – including monocytes/macrophages, granulocytes and dendritic

cells – were found to make a numerically significant contribution to the tumor cell infiltrate

(3). Depending on their state of differentiation, these infiltrating myeloid cells have been

divided into anti-tumorigenic macrophages and neutrophils (M1 and N1, respectively), which

can be differentiated from tumor-promoting M2/N2 phenotypes (4, 5). Similar to, but less

well investigated than in T cells, also the tumoricidal activity of innate myeloid cells is

controlled by activating and inhibitory receptors (6, 7). Among the identified inhibitory

receptors, SIRPα appears to have a prominent function on myeloid cells, which

predominantly interacts with CD47 expressed on malignant and non-malignant cells to

provide a “don’t eat me signal”. In the clinical setting of antibody-based immunotherapy,

activating signals to myeloid effector cells are predominantly provided by antibody binding

Fcγ receptors.

Human myeloid cells express a panel of molecularly and functionally distinct receptors for

IgG (8, 9). These receptors e.g. differ in their affinity for different antibody isotypes (10).

Peripheral blood monocytes typically express low levels of FcγRI (CD64), which is up-

regulated upon exposure to interferon-γ, as well as FcγRIIa (CD32a) and the inhibitory

FcγRIIb (CD32b) isoform. Monocyte-derived macrophages can additionally express FcγRIIIa

(CD16a), but usually do not express FcγRI. Human, but not murine PMN abundantly express

the GPI-linked FcγRIIIb isoform, which does not trigger antibody-dependent cell-mediated

cytotoxicity (ADCC), but rather functions as a “decoy receptor” for antibodies with increased

FcγRIII binding affinity (11). For FcγRIIa and FcγRIIIa functionally relevant polymorphisms

have been identified (12, 13), which were correlated to clinical benefit from tumor-directed

antibodies of human IgG1 isotype such as rituximab, herceptin or cetuximab (14). Although

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controversial, these studies support the concept that these approved antibodies can

mediate their therapeutic efficacy, at least in part, by effector cell recruitment. Studies in

mice have suggested that myeloid cells are particularly relevant for the therapeutic efficacy

of several antibodies (15-17), but at present it is unclear how these results translate to

clinical situations in patients.

IgG2 is the second most prevalent antibody isotype in human serum, which is predominantly

produced in response to bacterial polysaccharide antigens (18). Differences in the antibody

isotype repertoire are increasingly recognized to contribute to the protective immune

response in infectious diseases (19). At present, human IgG1 is by far the most commonly

used isotype in tumor immunotherapy, but also four human IgG2 antibodies are FDA

approved, and many more are in clinical development (20). Since human IgG2 only weakly

binds to FcγRIIIa (10), this isotype does not effectively recruit NK cells for ADCC (21, 22).

These results led to the conclusion that human IgG2 is an isotype with limited immune

effector functions. However, myeloid effector cells such as monocytes and PMN mediated

ADCC with human IgG2 antibodies against the epidermal growth factor receptor (EGFR) as

effectively as with human IgG1 antibodies (22). In comparison to human IgG1, human IgG2

antibodies poorly activated complement-dependent cytotoxicity (CDC) (21). However, at

higher target antigen densities and increased serum and antibody concentrations, IgG2

induced CDC by the classical and the alternative complement pathways (23, 24).

Nevertheless, the approved IgG2 antibody against EGFR (Panitumumab) did not trigger CDC

as a single agent, even under optimal experimental conditions (25).

EGFR is a tyrosine kinase receptor, which is over-expressed on many solid tumor types and

constitues a promising target antigen for small molecule and antibody-based approaches

(26). Thus, three generations of tyrosine kinase inhibitors are currently available for the

treatment of patients with lung cancer, whose tumors habor charateristic activating

mutations in the ATP-binding domain (27). Additionally, EGFR antibodies have been

approved for patients with colo-rectal cancer lacking KRAS mutations (Cetuximab and

Panitumumab), head and neck cancer (Cetuximab), and recently also for lung cancer

(Necitumumab). However, several other EGFR antibodies have also failed during clinical

development (e.g. Matuzumab, Zalutumumab), and many more clinical indications could be

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envisioned with respect to EGFR’s broad expression profile. A better understanding of the

required mechanisms of action in particular tumor indications and respective biomarker

identifcation may increase the chances of successful drug development (27).

Here, we investigated immune effector functions of human IgG2 antibodies against EGFR in

more detail. With the exception of NK cell mediated ADCC, the human IgG2 isotype proved

at least as effective in triggering antibody mediated effector functions as human IgG1. Thus,

tumor cell types or stages of tumor development with a myeloid rather than NK cell

predominated immune cell infiltrate may particularly benefit from the application of human

IgG2 antibodies against EGFR. Since myeloid cell mediated tumor cell killing is effectively

regulated by CD47/SIRPα interactions, CD47 low expressing tumor types could be promising

candidates for this approach.

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Materials and Methods

Experiments with human material were approved by the Ethical Committees of the

participating institutions in accordance with the Declaration of Helsinki.

Antibodies

The approved EGFR antibodies Panitumumab (human IgG2; E7.6.3, VectibixR) and Cetuximab

(chimeric human IgG1; 225, ErbituxR) were from Amgen (Thousand Oaks, CA) and Merck

(Darmstadt, Germany), respectively. Human IgG1 and IgG2 variants of the EGFR antibodies

Zalutumumab (sequence in patent WO 002004056847A2), 003, 005, 018 (sequences in

patent WO 2009/030239A1) and Matuzumab (sequence in patent WO 002009043490A1)

were produced at Genmab (Utrecht, The Netherlands) and in Southampton (Cancer and

Vaccine Group, Cancer Sciences, University of Southampton, UK), respectively. Antibodies

were purified by Protein A affinity chromatography (rProtein A FF; GE Healthcare), dialyzed

overnight to PBS, and filter-sterilized over 0.2-µM dead-end filters. The concentration of

purified IgGs was determined by absorbance at 280 nm. Quality assessment of purified

antibodies was performed by SDS/PAGE (> 90% intact IgG, > 95% HC + LC under reducing

conditions), ESI-TOF mass spectrometry (identity confirmation), and HP-SEC (aggregate level

< 5%), as described previously (28).

Cell lines

Cell lines were obtained from DSMZ (German Collection of Microorganisms and Cell

Cultures, Braunschweig, Germany), ECACC (European Collection of Authenticated Cell

Culture, Salisbury, UK) or kindly provided by Dr. M. Binder (University Hospital Eppendorf,

Hamburg, Germany). All cells were grown in media as recommended by the suppliers and

were obtained between 2011 and 2016.

C1q ELISA

C1q binding was measured by ELISA. Microtiter stripes (Thermo Fisher Scientific, Waltham,

MS, USA) were coated with serial dilutions (0 - 50 µg/ml) of IgG1 or IgG2 at 4°C before 25%

NHS was added as source of human C1q. Bound C1q was quantified by 10 µg/ml monoclonal

mouse-anti-human C1q antibody (Quidel, San Diego, CA, USA) visualized by 1:000 diluted

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HRP-conjungated goat-anti-mouse antibody (West Grove, PA, USA). Absorbance was

measured by a SunRise microplate reader (TECAN, Männedorf, Schweiz).

Generation of CD47 knock-out A431 cells

gRNA directed against the coding sequence of CD47 were designed using the Zhang

laboratory online tool (http://crispr.mit.edu/). As a control oligo,

5’GCACTACCAGAGCTAACTCA3’ from pCas-Scramble CrispR vector (Origene) was used. The

control oligo or CD47 coding sequences were cloned into pLentiCrispR-v2, a second

generation SIN lentiviral plasmid. Lentiviral particles were produced by transient transfection

of pLentiCrispR-v2–CD47ko or -control and co-constructs psPAX2 and pCMV-VSVg in 293T

cells. On days 2 and 3 after transfection, supernatants of transfected cells were filtered

through a 0.45µm filter and added to A431 cells. Transduced cells were selected by 1 ug/ml

puromycin. Single cell clones were obtained by limiting dilution.

Growth inhibition

Growth inhibition of DiFi cells was analyzed using the 3-(3,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay (Promega,

Madison, WI). DiFi cells were seeded at a density of 5 × 103 cells/well and treated with

antibodies at the indicated concentrations. After incubation for 72 h at 37°C, MTS was added

and absorption at 490 nm/670 nm was measured. Percentage of growth inhibition was

calculated using the formula: % growth inhibition = absorption with antibody /absorption

without (w/o) antibody × 100.

Flow cytometric analyses

EGFR and CD47 expression were quantified using the murine antibodies m425 (EGFR) and

B6H12 (CD47, BioXCell, West Lebanon, NH, USA). Binding of murine antibodies was

determined by FITC-conjugated goat anti-mouse Fcγ fragment-specific F(ab)2 staining. A

murine CD20 antibody served as a control.

PE-labeled antibodies against CD16 (FcγRIII), CD32 (FcγRII), CD64 (FcγRI) (Beckman Coulter,

Brea, CA, USA) or against CD80, CD163, CD47 or SIRPα (Miltenyi BioTec, Bergisch-Gladbach,

Germany) were used for direct immunofluorescence analyses. Fcγ receptor blockade was

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achieved by IVIG (Privigen, CSL Behring, Marburg, Germany). Immunofluorescence was

analyzed on flow cytometers (Epics Profile or Navios; Beckman Coulter, Fullerton, CA, USA).

Complement deposition

Deposition of complement components on target cells was determined by incubating 2.5 ×

105 cells with control or single EGFR antibodies (all at 10 µg/ml) alone or in combination with

a non–cross-blocking EGFR antibody (anti–EGFR-IgG1 003 at 5 µg/ml) for 15 min at 4°C.

Subsequently, 25% v/v human serum was added in the presence of a C5-blocking antibody

(Eculizumab, Alexion Pharma; New Haven, CT, USA; 100 µg/ml) to avoid CDC. Samples were

either stained with polyclonal FITC-conjugated C1q or C4b/c antibodies (both from Agilent,

Santa Clara, CA, USA) or polyclonal FITC-conjugated anti-C3c serum (Abcam, Cambridge, UK)

and analyzed on flow cytometers (Epics Profile or Navios; Beckman Coulter).

CD47 siRNA-induced knockdown experiments

Target cells were seeded at a density of 2.5 × 105/well in 6-well plates. Four hours later,

transfection of siRNA was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA,

USA). Synthetic siRNA targeting CD47 (ID Hs.446414) or a control siRNA (Low GC Duplex #1

from Invitrogen) were used. Efficacy of siRNA-induced knockdown of CD47 was analyzed

using a PE-conjugated mouse anti-human CD47 antibody and an irrelevant PE-conjugated

antibody (both Miltenyi BioTech).

CDC assays

CDC assays were performed as described (25). Briefly, target cells were preincubated with

200 μCi [51Cr] for 2 hours. As the source of complement, 25% v/v freshly drawn human

serum was used in the presence of the indicated antibodies. For combination assays, non-

cross blocking EGFR antibodies of IgG1 isotype (003, 005, 018 all from Genmab) were used at

a final concentration of 5 µg/ml. Percentage of [51Cr] release was calculated using the

formula: % lysis = (experimental cpm − basal cpm)/(maximal cpm − basal cpm) × 100.

Isolation of human effector cells

Human effector cells (MNC, PMN) were isolated from peripheral blood of healthy

volunteers, as previously described (11) using Polymorphprep (Progen, Heidelberg,

Germany). Macrophages were generated by adherence of MNC using monocyte-attachment

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medium (PromoCell, Heidelberg, Germany). After attachment, cells were incubated for 24 h

in serum-free X-Vivo medium (Lonza, Basel, Switzerland). Subsequently, cytokines for

macrophage generation and polarization were added. M1-like macrophages were generated

by incubation with 10 ng/ml GM-CSF for 6 days followed by 10 ng/ml interferon-γ and 100

ng/ml LPS for 2 additional days. To generate M2-like macrophages, attached MNC were

incubated with 25 ng/ml M-CSF for 6 days. Subsequently, IL-4 at 10 ng/ml was added for 2

additional days to complete M2-like polarization. All cytokines were from Peprotech

(Hamburg, Germany), LPS from Sigma-Aldrich (St. Louis, MO, USA).

ADCC assays

ADCC was analyzed in [51Cr] release assays as described (11). Briefly, isolated effector cells

(MNC, PMN, monocytes or macrophages), antibodies at various concentrations, and medium

were added to round-bottom microtiter plates (Nunc, Rochester, NY). Assays were started

by adding effector and target cells at indicated E:T ratios. After 3 h (MNC and PMN) or 16 h

(macrophages) at 37°C, [51Cr] release from triplicate samples was measured. The percentage

of cellular cytotoxicity was calculated using the formula: percentage of specific lysis =

(experimental cpm − basal cpm)/(maximal cpm − basal cpm) × 100; maximal [51Cr] release

was determined by adding Triton-X (2% final concentration) to target cells, and basal release

measured in the absence of sensitizing antibodies and effector cells.

Trogocytosis assay

The transfer of membrane from tumor cells to neutrophils was measured by flow cytometry

as described (29). Briefly, lamin-B-TRQ transfected A431 cells were labelled at 37⁰C with the

lipophilic membrane dye DiO (5 µM, Invitrogen, CA, USA). Labelled target cells were then

incubated with GM-CSF stimulated neutrophils at an effector:target cell ratio of 5:1 in the

absence or presence of either 5 µg/ml Cetuximab (IgG1 (225)) or Panitumumab (IgG2

(E7.6.3)). After 60 minutes, samples were fixed and measured on a flow cytometer. After

gating for the TRQ negative neutrophil population, the percentage of DiO positive cells and

their mean fluorescent intensity (MFI) were evaluated.

TCGA data analysis

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RNA sequencing data sets (RNAseqV2) from 17 different human cancer types (ovary, thyroid,

lung, cervix, head and neck, prostate, glioma, pancreas, colon, stomach, breast,

endometrium, urothelium, melanoma, testis, kidney, liver) were obtained from The Cancer

Genome Atlas (TCGA) (https://tcga-data.nci.nih.gov/tcga/) via The Human Protein Atlas

(https://www.proteinatlas.org/ENSG00000196776-CD47/pathology). Expression in

fragments per kilobase per million (FPKM values) was compared via box-whisker plots

(whiskers indicating the 10th and 90th percentile).

Data processing and statistical analyses

Data were generated from at least three independent experiments with blood from different

healthy donors. Graphical and statistical analyses were performed using GraphPad Prism 6.0

(GraphPad Software). Group data are reported as mean ± SEM. Significance was determined

by two-way ANOVA repeated measures test with Bonferroni's post hoc correction. EC50

values were calculated from dose–response curves, reported as means ± SEM.

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Results

EGFR-directed IgG1 and IgG2 antibodies do not differ in Fab-mediated effector

functions

Although IgG1 and IgG2 share approx. 90 % sequence homology, both isotypes differ in

structural aspects, especially in their heavy and light chain disulfide linkage (Figure 1A).

While IgG1 antibodies contain disulfide bonds between the C-terminus of the light chains

and the upper hinge region of the heavy chains, IgG2 antibodies are linked between the light

chains’ C-terminus and the N-terminal regions of the heavy chains’ CH1 domains. Thus, we

analyzed whether these structural differences influenced Fab-mediated effector functions

induced by EGFR-directed IgG1 and IgG2 antibodies. First, the ability of EGFR-directed IgG1

as well as IgG2 antibodies for target antigen binding was compared. Both clinically approved

EGFR antibodies Cetuximab (IgG1, 225) and Panitumumab (IgG2, E7.6.3) displayed similar

binding to EGFR-expressing A431 cells, as indicated by similar EC50 values (1.6 ± 1.3 µg/ml

and 1.5 ± 1.3 µg/ml for Cetuximab and Panitumumab, respectively). Cetuximab and

Panitumumab contain different variable regions and bind to different, although overlapping

EGFR epitopes (25). Thus, we next compared IgG1 and IgG2 isotype switch variants of

Zalutumumab (2F8) in binding to EGFR-expressing cells (Figure 1B). Again, the binding

characteristics of both IgG variants were similar (EC50 = 3.2 ± 1.4 µg/ml and 2.0 ± 1.4 µg/ml

for IgG1 and IgG2, respectively), and no significant differences in the number of EGFR

binding sites were observed between IgG1 and IgG2 antibodies (Figure 1B and Figure S1A).

Next, we used MTS assays to investigate growth inhibition of DiFi cells in the presence of

increasing concentrations of EGFR antibodies of IgG1 or IgG2 isotypes. All three EGFR

antibodies (225, E7.6.3, 2F8) mediated similar maximal growth inhibition of DiFi cells (Figure

1C). No differences between IgG1 (225) and IgG2 (E7.6.3) were observed, while the IgG2

variant of 2F8 was less efficient at lower antibody concentrations than 2F8-IgG1. These

results demonstrated that Fab-mediated effector functions of EGFR antibodies were

marginally affected by selecting IgG1 or IgG2 isotypes.

EGFR-directed IgG2 antibodies induced efficient CDC in combination with non-epitope

overlapping IgG1 or IgG2 antibodies

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Combinations of IgG1 antibodies against non-overlapping EGFR epitopes were shown to

efficiently induce complement deposition and CDC (25). Results from ELISA experiments

confirmed the previously reported low C1q binding capacity of human IgG2 compared to

IgG1 antibodies under these experimental conditions (Figure 2A). Next, we analyzed

whether IgG2 antibodies will induce complement deposition on tumor cells and CDC in

combination with IgG1 antibodies to non-overlapping EGFR epitopes. As a combination

partner the EGFR antibody 003 was chosen, which reacts with a non-ligand binding EGFR

epitope and does not interfere with Cetuximab or Panitumumab binding (25). As expected,

all individual EGFR antibodies did not induce complement binding or deposition on A431

cells as single molecules, as previously shown (25). Interestingly, however, the IgG1/IgG2

combination of antibody 003 and Panitumumab was as efficient in inducing C1q binding

(Figure 2B), C4b deposition (Figure 2C) and C3b deposition (Figure 2D) as the IgG1/IgG1

combination of antibody 003 and Cetuximab. Subsequently, the CDC activity of the

respective IgG1/IgG2 (mAb 003 and Panitumumab) and IgG1/IgG1 (mAb 003 and Cetuximab)

combinations was analyzed in 51Cr release assays against A431 cells. Importantly, the EGFR-

directed IgG1/IgG2 antibody combination was as effective as the IgG1/IgG1 combination in

triggering CDC, while the individual antibodies were ineffective (Figure 2E). Control

experiments with heat-inactivated human serum or addition of the C5 blocking antibody

Eculizumab confirmed that the observed tumor cell killing was indeed complement mediated

(Figure 2E). In addition, the CDC activity of IgG1/IgG2 and IgG1/IgG1 combinations was also

determined using a fixed 003 antibody concentration and increasing concentrations of

Panitumumab or Cetuximab. Both EGFR-directed IgG combinations displayed similar dose-

dependent CDC efficacy (Figure 2F, left panel). Next, the complement activation kinetics of

IgG1/IgG2 or IgG1/IgG1 combinations were analyzed by stopping the assays at different time

points. Interestingly, both IgG1/IgG2 and IgG1/IgG1 combinations showed similarly fast

kinetics in complement activation (Figure 2F, right panel). Together, these results indicate

that both EGFR-directed IgG1/IgG2 and IgG1/IgG1 combinations triggered CDC

predominantly via the classical complement pathway. Furthermore, the ability of the

IgG1/IgG2 combination to trigger CDC of other target cell lines was tested. Also against

A1207 and DiFi cells, both IgG1/IgG2 and IgG1/IgG1 combinations lead to similar CDC activity

(Figure 2G). In addition, IgG2 variants of other EGFR antibodies against different target

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antigen epitopes were tested. In combination with 003-IgG1, 2F8-IgG2 triggered similar CDC

(max. lysis = 30.5 ± 4.7 %) as Panitumumab (max. lysis = 28.7 ± 8.4 %), while 425-IgG2 was

less potent in combination with 003-IgG1 (max. lysis = 7.8 ± 2.3 %) (Figure 2H). In addition,

Cetuximab and Panitumumab were combined with other non-epitope overlapping EGFR-

directed IgG1 combinatorial partners (003, 005 or 018). While IgG1/IgG2 and IgG1/IgG1

combinations utilizing 005 (225/005 max. lysis = 30.9 ± 8.3 %; E7.6.3/005 max. lysis = 25.8 ±

7.4 %) led to similar CDC as 003 combinations (225/003 max. lysis = 30.1 ± 7.7%; E7.6.3/003

= 25.8 ± 6.9 %), 018 was a more potent combination partner (225/018 max. lysis = 45.9 ± 3.4

%; E7.6.3/018 max. lysis = 40.3 ± 6.3 %) (Figure 2I). Next, we analyzed the capacity of two

human IgG2 antibodies against non-overlapping EGFR epitopes to mediate complement

activation. Thus, IgG1 and IgG2 isotype variants of EGFR antibody 018 were combined with

either Cetuximab (IgG1) or Panitumumab (IgG2), resulting in IgG1/IgG1, IgG1/IgG2 and

IgG2/IgG2 combinations, respectively. Interestingly, the IgG2/IgG2 combination displayed

similar efficiency in C1q binding, C4b/c and C3b deposition (Figure S1B) and triggered similar

levels of CDC (Figure 2J) as the respective IgG1/IgG1 and IgG1/IgG2 combinations. These

results clearly show that human IgG2 EGFR antibodies can induce efficient CDC in

combination with another EGFR antibody of either human IgG1 or IgG2 isotype.

EGFR-directed IgG2 antibodies activate M1-like macrophages for ADCC

Numerous studies suggested the importance of macrophages in antibody-based

immunotherapy (reviewed in (7)). Thus, we analyzed the ability of human IgG2 antibodies to

activate macrophages for tumor cell killing. Monocyte-derived macrophages were generated

by adhesion, and were either stimulated with GM-CSF/interferon-γ/LPS for M1-like

polarization, or with M-CSF/IL-4 for M2-like differentation. As expected, M1-like

macrophages displayed strong expression of CD80, while M2-like macrophages expressed

lower levels. In contrast, CD163 expression was higher on M2- than on M1-like macrophages

(Figure 3A). Next, Fcγ receptor expression of M1- or M2-like macrophages was determined

by direct immunofluorescence. FcγRIII (CD16) was only weakly expressed by both types of

macrophages. Interestingly, M2-like macrophages displayed higher CD32 expression, while

M1-like macrophages showed a higher expression of CD64 (Figure 3B). Next, we analyzed

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the ability of EGFR-directed IgG1 or IgG2 antibodies to mediate ADCC by macrophages of

either M1- or M2-like differentiation. A431, SAT and Kyse-150 cells with high, intermediate

or low EGFR expression levels, respectively, were used as target cells (Figure 3C). While

EGFR-directed IgG1 was able to induce efficient ADCC by M1-like macrophages against all

tested target cell lines, EGFR-directed IgG2 antibodies only induced ADCC by M1-like

macrophages against high and moderate EGFR-expressing cells (A431 and SAT) (Figure 3D,

upper panel). Interestingly, neither EGFR-directed IgG1 nor IgG2 antibodies induced

significant ADCC by M2-like macrophages (Figure 3D, lower panel).

EGFR antibodies of human IgG2 isotype are more effective in PMN mediated ADCC

than their IgG1 variants

Human IgG2 antibodies effectively activated myeloid cells for ADCC, which was triggered by

FcγRIIa (CD32a) and affected by the FcγRIIa-131H/R polymorphism of donors (22). Here, we

extended these observations and investigated PMN recruitment by IgG2 antibodies in more

detail. First, Fcγ receptor profiling showed GM-CSF-primed granulocytes to express high

levels of CD16, moderate levels of CD32 and low levels of FcγRI (CD64) (Figure 4A). Next, we

studied to a broader spectrum of target cell lines expressing different levels of EGFR (Figure

4B), since target antigen expression levels were correlated to PMN mediated ADCC (30).

These experiments confirmed that EGFR antibodies of IgG1 and IgG2 isotypes were similarly

effective against A431 and A1207 cells, expressing very high EGFR levels. Interestingly, IgG2

antibodies became significantly more effective than IgG1 against target cell lines expressing

moderate (Kyse-30, SAT) or low (Kyse-150, SCC-25) EGFR levels (Figure 4B). Correlation

analyses between EGFR expression levels and the amount of PMN-mediated killing

demonstrated a linear positive correlation for both IgG1 and IgG2 antibodies (Figure 4C).

Next, we used Kyse-30 cells, which display moderate EGFR expression, as target cells for

concentration dependent induction of PMN- mediated ADCC by EGFR-directed IgG1 or IgG2

antibodies. In comparison to IgG1, IgG2 was significantly more efficient in triggering PMN-

mediated ADCC in a concentration-dependent manner (Figure 4D, left panel). Furthermore,

IgG2 mediated significantly more efficient ADCC at lower effector to target cell ratios than

IgG1 (Figure 4D, right panel). Additionally, three IgG2 antibodies targeting different EGFR

epitopes were tested for their capacity to induce PMN-mediated ADCC. All three human

IgG2 antibodies were effective in inducing PMN-mediated ADCC against Kyse-30 cells, with

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Panitumumab being the most efficient at lower antibody concentrations (Figure 4E). Next,

we were interested to analyse the mechanism of IgG2 mediated tumor cell killing by PMN,

which was recently reported to be induced by trogocytosis (29). Incubation of PMN with

DiO-membrane labelled A431 cells resulted in the up-take of tumor cell membranes by PMN

in the presence of EGFR antibodies (Figure 4F/I), but not in the absence of antibodies. EGFR

antibodies of both IgG1 (225) or IgG2 (E7.6.) isotypes were similarly effective in triggering

trogocytosis – measured as increase in “MFI of all neutrophils” (Figure 4F/II) or as “% of

neutrophils staining positive for DiO” (Figure 4F/III). Together, these novel data

demonstrated superior activity of human IgG2 antibodies to induce PMN-mediated ADCC,

especially against target cells displaying moderate to low EGFR expression.

Inhibition of CD47/SIRPα interactions improves PMN mediated ADCC by EGFR

antibodies

Interactions between CD47 on tumor cells and SIRPα on PMN were shown to affect PMN

mediated ADCC by the HER-2/neu antibody trastuzumab (31). Here, we characterized the

impact of CD47/SIRPα interactions on PMN mediated ADCC by EGFR antibodies of IgG1 or

IgG2 isotype. To address this issue, a CD47-negative variant and a control cell line of EGFR-

positive A431 cells was generated by CRISPER/Cas9 knock-out of the CD47 gene (A431 CD47

Ko) or a control vector (A431 crtl) (see Material & Methods). While EGFR expression was not

altered (Figure S1C), both cell lines differed in their CD47 expression, as displayed in Figure

5A. In addition, expression of SIRPα on GM-CSF stimulated granulocytes as well as M1 and

M2 macrophages was measured (Figure 5B). Next, we analyzed macrophage mediated

ADCC. Using A431 CD47 Ko and A431 control cells, M1 macrophage mediated ADCC by IgG1

or IgG2 was significantly improved in the absence of CD47 (Figure 5C/left panel).

Furthermore, inhibition of CD47/SIRPα interactions rescued the cytotoxic potential of M2

macrophages in the presence of IgG1 or IgG2 EGFR targeting antibodies (Figure 5C/right

panel). Taken together, these results show that macrophage mediated ADCC by EGFR-

directed IgG2 antibodies was regulated by CD47/SIRPα interactions. Furthermore, we

analyzed PMN mediated ADCC by EGFR-directed IgG1 or IgG2 antibodies in the presence or

absence of CD47 using unstimulated granulocytes as effector cells. In these assays, both

EGFR antibodies were unable to induce efficient PMN mediated ADCC against wildtype A431

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cells. Interestingly, in the absence of CD47 only the EGFR-directed IgG2 antibody was

capable of inducing significant ADCC by PMN (max. lysis = 25.7 ± 6.8%), while the EGFR-

directed IgG1 antibody did not (Figure 5D/I.). Subsequently, the efficacy of EGFR-directed

IgG1 and IgG2 mediated ADCC by PMN in the presence or absence of CD47 expression was

measured using GM-CSF stimulated granulocytes. The ability of EGFR-directed IgG1 (A431

wt: max. lysis = 28.1 ± 6.8% / A431 CD47 ko: max. lysis = 58.6 ± 8.9%) and IgG2 (A431 wt:

max. lysis =24.6 ± 8.8% / A431 CD47 ko: max. lysis = 69.6 ± 10.6%) to mediate ADCC by GM-

CSF-stimulated PMN was strongly improved in the absence of CD47 expression (Figure

5D/II). In addition, EGFR-directed antibodies mediated significantly higher ADCC by PMN at

lower effector to target ratios in the absence of CD47 (Figure 5D/III). Next, siRNA mediated

down-regulation of CD47 expression on SAT (moderate EGFR expression levels) and Kyse-

150 (low EGFR expression levels) was employed to investigate the impact of CD47 expression

on other target cell lines. Again, CD47 knock-down did not affect EGFR expression (Figure

S1C), while CD47 expression was reduced by approx. 50% (Figure 5E, left panels). In ADCC

assays, target cells with reduced CD47 expression were significantly more susceptible to

IgG1 and IgG2 induced cytotoxicity by GM-CSF stimulated PMN compared to control target

cells. While both EGFR antibodies were similarly effective in PMN mediated ADCC against

CD47-siRNA treated SAT cells, the EGFR-directed IgG2 antibody was more potent than IgG1

against CD47-siRNA treated Kyse-150 cells (Figure 5E, right panels). Next, three IgG2

antibodies, targeting different EGFR epitopes (E.6.3, 425, 2F8), were tested for their ADCC

activity against A431 control and CD47 ko cells, respectively, using GM-CSF stimulated PMN

as effector cells. In the presence of CD47 all three antibodies mediated low levels of ADCC,

which was improved for all three IgG2 antibodies in the absence of CD47 expression (Figure

5F). While 2F8 and Panitumumab were similarly effective, ADCC by 425-IgG2 was less

potent. These data demonstrate that similar to IgG1- also IgG2-mediated ADCC by PMN is

regulated by the interaction of CD47 and SIRPα.

CD47 is differentially expressed across different human solid cancer types

CD47 is highly expressed on many hematological malignancies, where overexpression of

CD47 often correlated with worse clinical outcomes. Here, we showed that EGFR-directed

IgG2 antibodies could induce efficient tumor cell killing by PMN, particularly against tumor

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cells with lower CD47 expression. Therefore, we were interested in analyzing CD47

expression across different solid tumor entities. For this purpose, we made use of RNAseq

data obtained from TCGA. Interestingly, CD47 expression varied considerably between the

analyzed cancer types (Figure 6). While ovarian cancer displayed the highest CD47

expression, CD47 expression in liver cancer was particularly low. EGFR antibodies are

currently approved for colo-rectal, head & neck and lung cancer. Among these three entities,

lung and head & neck cancer displayed higher CD47 expression levels compared to

colorectal cancer. Additional studies would be interesting to investigate the impact of CD47

expression on clinical outcomes during EGFR antibody therapy, particularly for

Panitumumab.

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Discussion

Human IgG2 has traditionally been selected as antibody isotype in immunotherapy when Fc

mediated effector functions were undesired (10, 20). The interest in human IgG2 was

recently fostered by studies confirming older observations that human IgG2 can exist in two

different isoforms, IgG2A and IgG2B. These two isoforms differ in the conformation of their

disulfide bonds in the hinge region and their Fab arm linkage, leading to differences in the

rigidity of the hinge (32). The IgG2B isoform with the more rigid hinge region was found to

be particularly effective in mediating immune cell activation via CD40 antibodies of human

IgG2 isotype (33). Currently available IgG2 antibodies can switch between the IgG2A and

IgG2B isoforms, depending e.g. on redox conditions. Thus, efforts are in progress to better

characterize the IgG2 hinge region and to generate IgG2 variants with well defined and

stable disulfide profiles. Here, we did not observe differences in Fab mediated effector

functions between IgG1 and IgG2 antibodies against EGFR (Figure 1C). Furthermore, also

isoform skewed IgG2A and IgG2B variants of Matuzumab did not differ in inhibiting growth

of EGFR-positive DiFi cells (Figure S1D). Others have reported that human IgG2 variants of

CD20 antibodies were associated with more effective direct tumor cell killing than their

respective IgG1 variants (34). Together, these results suggest that the impact of the isotype

and the rigidity of the hinge on Fab mediated effector functions may critically depend on the

selected target antigen and its epitope, as recently elegantly demonstrated for CD40

antibodies (35).

EGFR epitopes have been identified from crystal structures for Cetuximab (C225),

Matuzumab (H425) and Panitumumab (E7.6.3) (36-38) and by mutational studies for

Zalutumumab (2F8) (39), which were all mapped to the ligand-binding domain III. The

epitopes for the non-ligand blocking antibodies 003, 005 and 018 were defined by cross-

blocking experiments (25). Studies with hexamerization enhanced variants of EGFR

antibodies have demonstrated that Matuzumab is particularly efficient in triggering CDC,

which may be related to its particular orientation of binding (37). Recently reported results

have demonstrated that characteristics of target antigen epitopes – such as their distance to

the tumor cell membrane – differently affect the efficacy of particular effector mechanisms

of therapeutic antibodies (40, 41).

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Fc mediated effector functions such as ADCP, ADCC and CDC can contribute to the

therapeutic efficacy of EGFR antibodies (27). Frustrated phagocytosis leading to tumor cell

death (trogocytosis/trogoptosis) is a documented mechanism of tumor cell killing by

macrophages (42) and has recently been described as PMN’s major tumor cell killing

mechanism (29). Individual EGFR antibodies do not trigger CDC against human tumor cells,

unless they are specifically Fc engineered (43, 44). However, combinations of two non cross-

blocking EGFR antibodies of human IgG1 isotype were demonstrated to trigger significant

CDC (25). Results from clinical studies with EGFR antibody combinations may reveal whether

CDC contributes to their clinical efficacy (45-47). Here, we demonstrate that unexpectedly

similar levels of CDC were observed when the second EGFR antibody was of human IgG2

instead of human IgG1 isotype (Figure 2). Importantly, also two IgG2 antibodies against non-

overlapping EGFR epitopes triggered CDC (Figure 2J). Although human IgG2 has low affinity

for C1q in ELISA, these observations suggest that C1q binding by human IgG2 is sufficient for

effective CDC. Recent studies have demonstrated that effective CDC requires hexamerisation

of antibodies’ Fc parts on the tumor cell surface, which can be modified by Fc engineering

(48, 49). Our results suggest that multimerisation is also facilitated by EGFR antibody

combinations, and that IgG2’s low affinity for C1q sufficiently supports this initial step of

complement activation.

IgG1 and IgG2 isotype variants of the 2F8 EGFR antibody have demonstrated similar activity

in immunodeficient mice (22). Furthermore, the in vivo activity of the human IgG2 EGFR

antibody Panitumumab was enhanced, when CD47/SIRPα interactions were abolished (50).

However, translation of these results to humans is complicated by the lack of a FcγRIIa

homologue in mice, which is functionally the predominant Fcγ receptor for human IgG2 in

man. Furthermore, mice do not express a homologue of human FcγRIIIb, which can inhibit

ADCC by human IgG1 antibodies (11), but does not bind human IgG2 with relevant affinity

(10). Thus, studies in conventional mice may significantly underestimate the

immunotherapeutic potential of human IgG2 antibodies when these antibodies can recruit

PMN as effector cells.

Many EGFR expressing tumor entities demonstrate an infiltration by myeloid cells, which is

often associated with a worse clinical prognosis for patients (3). Myeloid cells can directly

promote tumor growth by different mechanisms, e.g. by providing growth factors, increasing

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tumor cell angiogenesis or by mediating immunosuppressive functions on tumor-directed T

cells – all leading to increased tumor progression. On the other hand, myeloid cells, and

macrophages in particular, were identified as the predominant effector cell population for

many tumor-directed monoclonal antibodies (15-17). Additionally, also PMN contributed to

tumor control in several murine tumor models (51). Thus, optimal activation and

recruitment of myeloid cells may improve the efficacy of antibody therapy. Previous studies

demonstrated that PMN as the most numerous myeloid cell population were particularly

effective in tumor cell killing when activated by tumor directed antibodies of human IgA

isotype (52, 53). However, the introduction of human IgA antibodies into clinical studies is

still in progress (54), while experience in the clinical development of human IgG2 antibodies

is broadly available. Thus, human IgG2 may be selected as antibody isotype for clinical

situations were recruitment of myeloid rather than NK effector cells appears promising.

For example, early lung cancer appears to actively exclude NK cell infiltration, while myeloid

cells are prominently present (55, 56). In addition to the tumor stage also underlying genetic

alterations may critically effect the composition of the immune cell infiltrate. For example,

Myc activation was demonstrated to actively increase myeloid cell infiltration in lung cancer

models, while lymphoid cell numbers were significantly reduced (57). At present, it is unclear

why Necitumumab was successful in prolonging survival in lung cancer patients (58), while

Cetuximab demonstrated therapeutic efficacy only in patients with high EGFR expressing

lung cancer (59). Both antibodies are of human IgG1 isotype, recognize similar/overlapping

epitopes and thus block EGFR signalling similarly. Interestingly, addition of Panitutumab to

chemotherapy did not improve therapeutic outcome in two phase II lung cancer studies, but

was associated with increased toxicity (60, 61).

The impact of Fc mediated effector functions on clinical outcomes after Cetuximab

treatment are still controversial, although individual studies have suggested that Fcγ

receptor poylmorphims are associated with Cetuximab’s efficacy in colo-rectal cancer

patients (14). Human IgG2 has moderate affinity for FcγRIIa, but very low affinity for FcγRIIIa

and FcγRIIIb (10), which is in line with its ADCC activity by different effector cell populations

(11, 22). Preclinical studies employing human IgG2 antibodies have demonstrated that the

131-H alloform of the FcγRIIa receptor interacts more effectively with human IgG2 than the

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131-R alloform (12), leading to increased PMN mediated ADCC by EGFR antibodies of human

IgG2 isotype (22). However, we are not aware of studies addressing the impact of Fcγ

receptor polymorphisms on the clinical efficacy of human IgG2 antibodies. Furthermore, no

direct comparisons between isotype variants of individual antibodies have been performed

in patients. Nevertheless, there are results from a head-to-head comparison between the

two EGFR antibodies Cetuximab (IgG1) and Panitumumab (IgG2) in patients with metastatic

colo-rectal cancer. Interestingly, both antibodies demonstrated similar clinical activity (62).

Subgroup analyses of this informative study could assist to identify biomarkers to predict

clinical responses to EGFR antibodies of human IgG2 isotype. A similar scenario is developing

for CTLA-4 antibodies, where an IgG1 antibody (Ipilimumab) is approved, while an IgG2

antibody (Tremelimumab) is in late stage clinical development. Myeloid effector cell

mediated depletion of regulatory T (Treg) cells by Fc-mediated mechanisms contributes to

the therapeutic efficacy of Ipilimumab, and was recently also demonstrated for

Tremelimumab (63-65).

The identification of appropriate biomarkers has been demonstrated to increase the chances

of patients to respond to novel therapies and to pave the way for accelerated approval of

innovative drugs (27). Our in vitro studies suggest the clinical evaluation of several potential

biomarkers for antibodies of human IgG2 isotype. For example, the amount of myeloid cells

within tumors would be expected to be relevant. Additionally, myeloid cells require rather

high EGFR expression levels for effective ADCC (30), a biomarker which is actively

investigated in NSCLC therapy (59). Furthermore, patients expressing the 131-H alloform of

FcγRIIa should have higher chances to respond than patients homozygous for the FcγRIIa-

131R alloform.

Following the therapeutic success of immune checkpoint blocking antibodies (1), novel

approaches aim to improve myeloid cell recruitment for tumor immunotherapy. Prominent

examples include antibodies blocking CD47/SIPRα interactions (6) as well as e.g. antibodies

against CD27 (66). To develop their full therapeutic activity, these novel antibodies probably

need to be combined with antibodies against classical “tumor antigens” like CD20 or EGFR.

Our studies presented here suggest that the human IgG1 isotype is expected to be

suboptimal for these approaches, and that human IgG2 antibodies may improve myeloid

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effector cell activation. Thus, additional studies are required “to harvest the high hanging

fruits” in EGFR-directed antibody therapy (67).

word count: 5.823

Acknowledgement

The authors thank C. Wildgrube and Y. Claussen for their excellent technical assistance, and

Prof. M. Cragg and the Antibody and Vaccine Group (Cancer Sciences Unit, University of

Southampton, UK) for providing antibodies. We gratefully acknowledge Dr. J. Lammerts van

Bueren (Genmab, Utrecht, NL) for assistance with the TCGA enquiry.

These studies were supported by the German Research Organization (DFG Va124/9-1),

granted to T. Valerius, and by the Dutch Cancer Society (grant: #10300), granted to T. K. van

den Berg.

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Figure Legends

Figure 1: EGFR antibodies of IgG1 and IgG2 isotype induce similar F(ab)-mediated effector

functions. (A) Cartoon of human IgG1 and IgG2 with disulfide bonds indicated. (B) Target

antigen binding of different EGFR antibodies (225, E7.6.3, 2F8) was analyzed by indirect

immunofluorescence and was not affected by their isotype (IgG1 vs. IgG2). (C) Growth

inhibition of DiFi cells was analyzed by MTS assays in the presence of increasing

concentrations of EGFR antibodies. No differences between IgG1 (225) and IgG2 (E7.6.3)

isotypes were observed, while the IgG2 variant of 2F8 was less efficient at lower antibody

concentrations than 2F8-IgG1. Mean values ± SEM of at least three independent

experiments are displayed. * p ≤ 0.05 for IgG1 vs IgG2 (two-way ANOVA).

Figure 2: Complement activation by EGFR antibodies of human IgG1 and IgG2 isotypes. (A)

IgG1 and IgG2 antibodies significantly differed in C1q binding in ELISA. Combinations of

EGFR-IgG1 and -IgG2 antibodies (5 µg/ml each) induced C1q binding (B), C4b/c deposition (C)

and C3b/c deposition (D) on tumor cells, while single agents did not result in complement

activation. Complement binding as well as deposition was analyzed by indirect

immunofluorescence analyses. (E) CDC by EGFR-directed IgG1/IgG2 and IgG1/IgG1

combinations was analyzed in 51Cr release assays. Fresh or heat-inactivated human serum

(NHS and HI-NHS, respectively) served as complement source. To inhibit MAC formation, NHS

was supplemented with 100 µg/ml of the C5 blocking antibody Eculizumab (ECU). (F) CDC

induced by EGFR-directed IgG1/IgG1 and IgG1/IgG2 combinations was similarly dependent on

antibody concentrations (left panel) or time course (right panel). (G) EGFR-directed IgG1/IgG1

and IgG1/IgG2 combinations (5 µg/ml each) triggered CDC against different cell lines (left

panel: DiFi, right panel: A1207). (H) IgG2 antibodies (5 µg/mL) against different EGFR

epitopes (E7.6.3, 425, 2F8) induced CDC in combination with a non-epitope-overlapping IgG1

antibody (003, 5 µg/ml). (I) EGFR-directed IgG1/IgG1 and IgG1/IgG2 combinations induced

similar CDC using different IgG1 combination partners (antibodies 003, 005 or 018). (J) EGFR

antibody combinations of IgG1/IgG1, IgG1/IgG2 or IgG2/IgG2 isotypes (5 µg/ml each)

triggered efficient CDC. CDC was analysed by 51Cr-release assays using 25% NHS as a source

of complement. Mean values ± SEM of at least three independent experiments are

displayed. Data were analyzed by ANOVA, and significant differences (p ≤ 0.05) are depicted

by *.

Figure 3: EGFR-directed IgG2 antibodies induced ADCC by M1, but not M2 macrophages.

(A) M1 (A/I) and M2 macrophages (A/II) were characterized by CD80 and CD163 expression

and further analyzed for Fcγ receptor expression (CD16/CD32/CD64) (B). EGFR expression of

A431, SAT and Kyse-150 cells was analyzed by indirect immunofluorescence (C). Macrophage-

mediated ADCC by EGFR-directed antibodies of human IgG1 and IgG2 isotypes (5 µg/ml) was

analyzed in 51Cr-release assays (D). A431, SAT and Kyse-150 cells with high, moderate and low

EGFR expression, respectively, served as target cells. M1 (GM-CSF/IFN-γ/LPS stimulated) or

M2 (M-CSF/IL-4 stimulated) macrophages were utilized as effector cells at an E:T-ratio of

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10:1. Mean values ± SEM of at least three independent experiments are displayed. Data

were analyzed by ANOVA, and significant differences (p ≤ 0.05) are depicted by *.

Figure 4: Human IgG2 antibodies against EGFR trigger effective PMN mediated ADCC. (A)

Fc receptor expression of GM-CSF stimulated PMN was measured by direct

immunofluorescence analyses. (B) ADCC by GM-CSF stimulated PMN and EGFR antibodies of

human IgG1 or IgG2 isotypes (5 µg/mL) was analyzed by 51Cr-release assays against different

solid tumor cell lines (E:T ratio 80:1) expressing different amounts of EGFR (indicated as MFI).

IgG2 was significantly more effective than IgG1 against tumor cell lines expressing

intermediate or low EGFR levels. (C) EGFR expression levels positively correlated with PMN-

mediated ADCC for antibodies of both IgG1 and IgG2 isotypes (r-values 0.92 and 0.94,

respectively). (D) Against Kyse-30 target cells, IgG2 mediated more effective ADCC by GM-CSF

stimulated PMN than IgG1, requiring lower antibody concentrations (left panel, E:T ratio

80:1) and lower E:T ratios (right panel, mAb concentration 5 µg/ml). (E) IgG2 antibodies

against different EGFR epitopes (E7.6.3, 2F8, 425) mediated significant ADCC by GM-CSF

stimulated PMN against Kyse-30 target cells. Mean values ± SEM of at least three

independent experiments are displayed as “% cytotoxicity”. Data were analyzed by ANOVA,

and significant differences (p ≤ 0.05) are depicted by *. (F) Induction of trogocytosis by IgG1

(225) or IgG2 (E7.6.3) EGFR antibodies was analyzed by flow cytometry. DiO labelled A431-

lamin B-TRQ cells and neutrophils were gated as shown in F/I. After incubation with EGFR

antibodies of IgG1 or IgG2 isotype for 60 minutes, PMN became DiO positive. Trogocytosis

was quantified as “MFI of the total neutrophil population” (F/II), or as the “% of neutrophils

that became DiO positive” (F/III). Results are shown as averages ± SEM of 3 independent

experiments with neutrophils from at least 5 individual donors. Data were analyzed by

ANOVA with statistical significance as depicted (* p ≤ 0.05).

Figure 5: Myeloid cell mediated ADCC by IgG2 antibodies is regulated by CD47/SIRPα

interactions. CD47 expression by CD47 knock-out (CD47 Ko) and A431 control (crtl) cells (A),

as well as SIRPα expression by PMN, M1 and M2 macrophages (B) were determined by direct

immunofluorescence analyses. (C) In the absence of CD47 expression, ADCC by M1

macrophages was significantly improved for IgG1 or IgG2 antibodies, and also M2

macrophages aquired the capacity for ADCC by both isoytpes. Effector to target ratios were

10:1, and antibody concentrations were 10 µg/ml. (D/I) Unstimulated PMN mediated

significant ADCC only with IgG2 against A431 CD47 Ko target cells. In the presence of GM-

CSF, EGFR anibodies of both IgG1 and IgG2 isotypes triggered PMN-mediated ADCC, which

was significantly higher against CD47 Ko than against control A431 cells. Killing was

dependent on antibody concentrations (D/II) and E:T cell ratios (D/III). (E) ADCC by PMN

(E:T-ratio = 80:1) induced by EGFR-directed IgG1 or IgG2 antibodies (5 µg/ml) was improved

by siRNA-mediated down-regulation of CD47 expression on SAT (moderate EGFR expression)

and Kyse-150 (low EGFR expression). (F) Also IgG2 antibodies against other EGFR epitopes

(2F8, 425) mediated improved ADCC against CD47 Ko vs control A431 cells. ADCC by human

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IgG1 or IgG2 EGFR antibodies was analyzed in 51Cr-release assays. Mean values ± SEM of at

least three independent experiments are displayed. Data were analyzed by ANOVA, and

significant differences (p ≤ 0.05) are depicted by *.

Figure 6: CD47 expression across different tumor types. Total mRNA scores across 17

cancer types were obtained from TCGA datasets and are displayed as boxplots. Highest CD47

expression were found in ovarian carcinoma, while liver cancer displayed the lowest

expression of CD47. Presented results are means ± SD of the indicated number of analyzed

samples.

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