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Glycoengineering of Therapeutic Enhances /Macrophage-Mediated and Cytotoxicity

This information is current as Sylvia Herter, Martina C. Birk, Christian Klein, Christian of September 25, 2021. Gerdes, Pablo Umana and Marina Bacac J Immunol 2014; 192:2252-2260; Prepublished online 31 January 2014; doi: 10.4049/jimmunol.1301249

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Supplementary http://www.jimmunol.org/content/suppl/2014/01/31/jimmunol.130124 Material 9.DCSupplemental http://www.jimmunol.org/ References This article cites 44 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/192/5/2252.full#ref-list-1

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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

Glycoengineering of Therapeutic Antibodies Enhances Monocyte/Macrophage-Mediated Phagocytosis and Cytotoxicity

Sylvia Herter, Martina C. Birk, Christian Klein, Christian Gerdes, Pablo Umana, and Marina Bacac

Therapeutic Abs possess several clinically relevant mechanisms of action including perturbation of tumor cell signaling, activation of complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), and induction of adaptive . In view of the important role of phagocytic lineage cells in the mechanism of action of ther- apeutic Abs, we analyzed FcgR receptor-dependent effector functions of and macrophages triggered by glycoengi-

neered (GE) Abs (having enhanced FcgRIIIa [CD16a] binding affinity) versus their wild-type (WT) counterparts under different Downloaded from experimental conditions. We first defined the precise FcgR repertoire on classical and nonclassical intermediate monocytes—M1 and M2c macrophage populations. We further show that WT and GE Abs display comparable binding and induce similar effector functions (ADCC and ADCP) in the absence of nonspecific, endogenous IgGs. However, in the presence of these IgGs (i.e., in a situation that more closely mimics physiologic conditions), GE Abs display significantly superior binding and promote stronger monocyte and macrophage activity. These data show that in addition to enhancing CD16a-dependent NK cell cytotoxicity, glycoengineering also enhances monocyte and macrophage phagocytic and cytotoxic activities through enhanced binding to http://www.jimmunol.org/ CD16a under conditions that more closely resemble the physiologic setting. The Journal of Immunology, 2014, 192: 2252–2260.

ver the past decades, several anticancer Ab therapeutic activating signals through ITAMs or inhibitory signals through agents have been developed and tested in the clinic, and ITIMs. Most Fc-dependent inhibitory signals are transduced via O 12 are currently approved for use in oncology (1–6). FcgRIIb (CD32b), whereas most stimulatory signals are trans- Many of these agents affect tumor growth by interfering with duced by FcgRI (CD64) and CD16a (10). CD64 is a high- receptor signaling. For example, trastuzumab (targeting Her2/neu) affinity receptor expressed by macrophages, DCs, , and cetuximab (targeting EGFR) counteract tumor progression by and , whereas CD16a is a low-affinity receptor expressed blocking receptor downstream signaling, whereas rituximab (tar- by NK cells, DCs, macrophages, and mast cells; it is required for by guest on September 25, 2021 geting CD20) and alemtuzumab (targeting CD52) induce direct NK cell–mediated ADCC (10). cell death or (3, 7, 8). Various engineering technologies have recently been developed In addition to direct antitumor effects, therapeutic mAbs con- to modulate the binding affinity of the Fc region of therapeutic Abs trol tumor progression through additional mechanisms, includ- to different FcgRs to enhance or suppress FcgR-dependent immune ing complement-dependent cytotoxicity, and immune cell effector effector functions. This has been achieved either by modifying the functions such as Ab-dependent cellular cytotoxicity (ADCC) and structure of the Fc-attached oligosaccharides (glycoengineering) Ab-dependent cellular phagocytosis (ADCP) (3). ADCC and ADCP or by engineering the Fc-polypeptide backbone. In particular, are triggered through interaction of the Ab fragment crystallizable glycoengineered (GE) Abs enriched in glycoforms lacking core (Fc) domain with the corresponding Fcg receptors (FcgRs) expressed fucose residue from oligosaccharides attached at Asn297 of the on NK cells, neutrophils, monocytes, macrophages, dendritic cells Fc have significantly enhanced binding affinity to CD16a and are (DCs), and eosinophils (3, 9). Once engaged, FcgRs transduce more potent and efficacious at mediating ADCC (6, 11–14) Although removing the core fucose residue from Fc-oligosaccharides increases the affinity of all IgG subclasses to human activating CD16a Discovery Oncology, Pharma Research and Early Development (pRED), Roche Gly- (and to its mouse ortholog FcgRIV) by 10–50-fold, binding to cart AG, 8952 Schlieren, Zurich, Switzerland other human activating and inhibitory receptors, including CD32a Received for publication May 10, 2013. Accepted for publication December 21, 2013. or CD32b, remains largely unchanged. Despite the overall simi- larity in protein structure, individual FcgRs have unique binding Address correspondence and reprint requests to: Dr. Marina Bacac, Discovery Oncology, Pharma Research and Early Development (pRED), Roche Glycart patterns. In the case of the CD16a-IgG Fc interaction, the car- AG, Wagistrasse 18, 8952 Schlieren, Zurich, Switzerland. E-mail address: marina. bohydrate attached to CD16a at its Fc binding interface, a struc- [email protected] tural element unique to CD16a among FcgRs, is required for the The online version of this article contains supplemental material. increased binding affinity to afucosylated, GE Abs (10, 15–17). Abbreviations used in this article: ABC, Ab-binding capability; ADCC, Ab-dependent Glycoengineering confers two clinically relevant properties: 1) cellular cytotoxicity; ADCP, Ab-dependent cellular phagocytosis; CLL, chronic lympho- cytic leukemia; DC, ; LDH, lactate dehydrogenase; M1, type I macrophage; the ability to achieve high levels of ADCC, even in individuals M2, type II macrophage; M-CSF, macrophage CSF; MDM, monocyte-derived macro- harboring the low-affinity CD16a (158Phe), potentially phage; WT, wild type. overcoming the problem of individual heterogeneity in CD16a This article is distributed under The American Association of Immunologists, Inc., polymorphism and therapy response and 2) the ability to preserve Reuse Terms and Conditions for Author Choice articles. activity in the presence of high concentrations of nonspecific serum Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 IgGs (.10 mg/ml) that compete with conventional therapeutic www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301249 The Journal of Immunology 2253

Abs for FcgRs and impair their activity. In line with these prop- 10% FCS and N-acetyl-L-alanyl-L-glutamine (2 mM). 1833-PPOP233 cells erties, a number of preclinical studies demonstrated that Ab Fc- (provided by Thomas Weber, Roche Diagnostics, Penzberg, Germany) were afucosylation translates into significantly enhanced activity in vivo cultivated in DMEM containing 10% FCS and N-acetyl-L-alanyl-L-glutamine (2 mM) plus 200 mg/ml G418. (6, 9, 17), and this has led to the approval of new therapeutic GE Abs. GA101 (obinutuzumab) is a GE, type II anti-CD20 mAb that Monocyte isolation and generation of polarized macrophage has recently been approved by the U.S. Food and Drug Admin- subsets istration for the first line treatment of patients with chronic lym- PBMCs were isolated from buffy coats obtained from the Blutspende- phocytic leukemia (CLL) in combination with chlorambucil (18– zentrale Zurich€ (Switzerland). Depending on the assay requirements, mono- 20). In Japan, the GE CCR4 Ab mogamulizumab has been ap- cytes were isolated using the Pan Monocyte Isolation Kit (for pan monocyte proved for treatment of patients with relapsed or refractory CCR4+ isolation), the Monocyte-Isolation Kit II (for classical, CD16 negative monocyte isolation) or CD16+ Monocyte Isolation Kit (for intermediate, leukemia-lymphoma (21). nonclassical, CD16+ monocyte isolation), all from Miltenyi Biotech. For The effector cells contributing to the efficacy of therapeutic monocyte-derived macrophage (MDM) generation, pan monocytes or Abs and their relative contribution in vivo have not been clearly classical monocytes were incubated inRPMI1640supplementedwithhuman defined. NK cells are considered as one of the players because macrophage CSF (M-CSF) (30 ng/ml), 10% FCS, 2 mM glutamine for 6–7 d. they rapidly elicit potent ADCC in vitro in comparison with After removal of M-CSF, M1 macrophage subtype was generated by polarizing MDMs with human IFN-g (100 ng/ml) and LPS (100 ng/ml) for freshly isolated monocytes or (6, 17, 22, 23) and an additional 24 h; M2c was generated by polarizing MDMs with human infiltrate tumors in trastuzumab-treated patients (24). On the IL-10 (10 ng/ml) for an additional 48 h; M2a was generated by polar- other hand, several preclinical studies in murine models con- izing MDMs with IL-4 (20 ng/ml) for an additional 24 h; and M2b was vincingly demonstrated that macrophage subsets are primarily generated by polarizing MDMs with freshly prepared immune complexes Downloaded from (10 mg/ml) and LPS (100 ng/ml) for an additional 24 h. All responsible for eliminating circulating and tissue B cells upon used were from Peprotech. Immune complexes have been prepared anti-CD20 mAb therapy and essentially excluded a role for NK using OVA (Sigma Aldrich, #A5503) and anti–chicken egg white an- cell-, T cell- or perforin-dependent mechanisms in vivo in those tiserum (Sigma Aldrich, #C6534) (31). LPS was obtained from Sigma particular models (25–29). In addition, it has been shown re- Aldrich (#L5629). cently that glycoengineering through its enhanced affinity to Quantification of FcgRs on monocytes and macrophages

CD16b on neutrophils results in enhanced mediated http://www.jimmunol.org/ ADCPandneutrophilactivationascomparedwiththerespec- Freshly isolated human classical and intermediate, nonclassical monocytes or polarized macrophages were stained with anti-human CD16-FITC (BD tive wild type (WT) Abs (30). Bioscience; clone 3G8, slightly higher affinity for CD16a compared with In view of the potential important role of phagocytic lineage cells CD16b) or anti-human CD32-FITC (BD Bioscience; clone FLI8.26, higher in the mechanism of action of therapeutic Abs, we investigated affinity and avidity for CD32a compared with CD32b) or anti-human CD64- whether Ab glycoengineering enhances their cytotoxic (ADCC) FITC (BD Bioscience, clone 10.1) for 30 min at 4˚C, washed and measured immediately by flow cytometry (BD FACSCanto II) in parallel with cali- and phagocytic (ADCP) activity in comparison with conventional bration beads consisting of Ab-coated Quantum Simply Cellular micro- Abs. To reflect the plasticity of myeloid lineage cells better we spheres (Bang Laboratories) preincubated with the same Abs according to 2 evaluated the activities on classical (CD14++/CD16 ) monocytes, the manufacturer’s instructions. The Ab-binding capability (ABC) for each nonclassical/intermediate (CD14+/CD16+) monocytes, classically Ab was calculated for monocytes and M1 and M2c macrophage subsets by guest on September 25, 2021 activated type I macrophages (M1) and alternatively activated type according to the manufacturer’s instructions. II macrophages (M2). In the current study, we report a quantitative Labeling of Abs assessment of FcgRs expression on different monocyte and mac- Labeling with Alexa Fluor 647 (Alexa Fluor 647 carboxylic acid, succinimidyl rophage subsets and provide evidence that GE, afucosylated Abs ester [Invitrogen], 5 mg, A20106, dissolved in DMSO with concentration 10 + display enhanced binding to CD16 monocytes and macrophages mg/ml) was performed in PBS (Life Technologies DPBS13, Ref no 14190- and induce significantly higher levels of ADCC and ADCP than 136) at a molar ratio of 7.5 (dye to protein), with final 0.1 M NaHCO3 and the respective WT Abs do under conditions that more closely reflect a final protein concentration of 4 mg/ml. After 1 h incubation at room tem- the natural ones. perature (end-over-end shaking), the free dye was removed on a Zeba-Spin 0.5-ml column (40 kDa cutoff; Thermo scientific, No 87766). Absorption A280 and A650 was determined and concentration was calculated: Materials and Methods Abs and reagents Concentration ðMÞ¼ðA280-ðA650p0:03ÞÞ pdilution factor=extension coefficient GA101 GE and WT, GA201 GE and WT, trastuzumab GE and WT, and rituximab were obtained from F. Hoffmann-La Roche AG. Ofatumumab Determination of labeling efficiency was calculated: was obtained from a local pharmacy. Cetuximab (commercial grade) was obtained from Merck, and the nonspecific total human IgG Redimune was Moles dye=mole protein ¼ A650pdilution factor=239000 obtained from CSL Behring. PKH67 (PKH67GL), PKH26 (PKH26GL) and p ð Þ CFSE (#21888) dyes were obtained from Sigma-Aldrich. The character- protein concentration M istics of the WT and GE variants of the therapeutic Abs evaluated in the current study were as follows: GA101 (Gazyva [obinutuzumab]; GE type Binding of therapeutic Abs to monocytes and macrophages II anti-CD20 Ab), MabThera (rituximab; type I anti-CD20 Ab approved in non-Hodgkin’s lymphoma, CLL, and ), Arzerra (ofatu- Fluorescently labeled Abs were generated in house using AlexaFluor647 mumab; type I anti-CD20 Ab approved in CLL), GA201 (GE Ab targeting fluorochrome (see Materials and Methods). AlexaFluor647-labeled GE EGFR, clinical trial phase II in colorectal carcinoma and head and neck (GA101 and GA201) and wild-type Abs (GA101 WT, GA201 WT, and carcinoma (currently on clinical hold), Erbitux (cetuximab; targeting EGFR in ofatumumab; dye/protein ratio 5.3–7.6) were incubated with human colorectal carcinoma and squamous cell carcinoma), Herceptin (trastuzumab; monocytes or polarized macrophage subsets for 30 min at 4˚C in the targeting Her2 in breast carcinoma). presence or absence of 10 mg/ml Redimune. For the assessment of the role of CD16, M1 and M2c macrophages were preincubated with sat- urating concentration of the blocking CD16 Ab (BD; FITC-labeled Raji and WIL2S cells (Ref: 85011429 and 90112121, respectively; Euro- clone 3G8) for 45 min at 37˚C. pean Collection of Cell Cultures [ECACC]) as well as A549 (Ref: ADCP 86012804, ECACC) and KPL-4 (Kawasaki Medical School) were cultivated in DMEM containing 10% FCS and N-acetyl-L-alanyl-L-glutamine (2 mM). CFSE-labeled target cells (Raji or KPL4) were incubated with monocytes, MKN45 (DSMZ Ref: ACC 409) were cultivated in RPMI 1640 containing M1 or M2c macrophages (E:T 3:1), and increasing concentrations of anti- 2254 GLYCOENGINEERING ENHANCES MONOCYTE AND MACROPHAGE ACTIVITY

CD20 (Raji) or anti-Her2 Abs (KPL-4) for 1–24 h at 37˚C in the presence or control GA101 GE at 1 mg/ml) and 10 mg/ml competing IgGs absence of competing unspecific human IgGs (10 mg/ml Redimune). Typical (Redimune). Cell culture medium was exchanged before image acquisition IgG concentrations in human plasma cover 8–18 mg/ml. Cells were subse- to Leibovitz L-15 without phenol red supplemented with 10% FBS. Cells quently stained with anti-CD206–FITC or anti-CD206–PE and anti-human were imaged using a 1003 oil immersion objective, 1.3 numerical aperture CD22-APC (Raji) or anti-human Epcam-APC (KPL-4) Abs (all from on an Axio Observer inverted microscope with stage-top incubator with BioLegend) and subject for FACS analysis. ADCP by monocytes was deter- support for 35 mm dish with CO2 (5%) and temperature (37˚C) controllers minedbygatingtotaltarget cells and determining the percentages of CFSE+/ (CTI Controller 3700 and Temp Control 37-2, respectively). Fluorescence CD206+ double-positive target cells in a child gate. ADCP by macrophages was time-lapse images were acquired using a CoolSNAPHQ2 camera every determined by gating total target cells and determining the percentages of CFSE+/ 5 min for 3 h and 14 h (overnight) on multiple positions per dish. Ex- CD206+ double-positive target cells in a child gate, creating another child gate of posure times were 30 ms for PKH67, 100 ms for PKH26, and 10 ms for this to distinguish phagocytosed targets from effector attached targets by deter- bright field. mining the percentages of Epcam-negative, CFSE+/CD206+ targets. ADCC Results Target cells (A549 or MKN45) were incubated with human monocytes Isolation of monocytes and generation of macrophage subsets (E:T ratio, 4:1) or macrophages (E:T ratio, 3:1) for 24 h in the presence of Classical and intermediate, nonclassical monocytes were isolated increasing concentrations of anti-EGFR Abs (GA201 GE, GA201 WT, from buffy coats derived from healthy donors (see Materials and cetuximab). Lactate dehydrogenase (LDH) release was measured using the LDH Cytotoxicity Detection Kit (Roche Applied Science). Target cell Methods). Macrophages were generated by culturing pan mono- killing was calculated using the following formula: cytes in the presence of M-CSF for 6–7 d before polarization to M1, M2a, M2b, and M2c macrophage subsets (see Materials and % killing ¼ððsample release 2 spontaneous releaseÞ= Methods) (34–36). The purity of isolated monocytes and the ef- Downloaded from ðmaximal release 2 spontaneous releaseÞÞp100 ficiency of were confirmed by analysis of Spontaneous release, corresponding to LDH released by target cells without cell surface markers and cytokines known to be expressed or se- Ab and effectors, was defined as 0% cytotoxicity. Maximal release (cor- creted by the above-mentioned subsets (Supplemental Fig. 1). In responding to LDH released by target cells lysed with 2% Triton X-100) was agreement with literature data, classical monocytes were highly defined as 100% cytotoxicity. In addition to LDH release, ADCC was determined by luminescence in an positive for CD14 and negative for CD16, whereas intermediate and nonclassical monocytes express CD14 and CD16. M1 mac- assay based on the assessment of active caspase-3/7 pathway in tumor target http://www.jimmunol.org/ cells using 1833-POPP233 cells stably transfected with plasmid coding for rophages expressed high levels of CD80, MHC class II and se- split luciferase (C-Luc and N-Luc domain fused with intervening caspase-3/7 creted IL-12, TNF-a, IL-6, and IP10. M2a expressed CD206 and cleavage motif [DEVD]). The cleavage of the reporter molecule by activated caspase-3/7 enables interaction of C-Luc and N-Luc and reconstitutes lu- MHC class II and secreted predominantly CCL-22. M2b expressed ciferase activity (32, 33). 1833-POPP233 target and effector cells (human M1 MHC class II and secreted predominantly TNF-a,IL-10,andIL-6, or M2c macrophages of CD16+ monocytes) were incubated for 8 or 24 h in whereas M2c were MHC class II negative, expressed CD163 and IL- the presence or absence of WT or GE GA201 (1 mg/ml), and luminescence 1RII, and secreted high levels of IL-10, confirming the validity of was quantified by the addition of D-Luciferin (Biosynth). polarization methods applied in the current study (7, 35–37). Overall monocyte and macrophage activity (cell ELISA) Expression of CD16, CD32, and CD64 and binding of by guest on September 25, 2021 MKN45 or KPL4 cells were incubated with human macrophages (E:T ratio, therapeutic Abs 3:1) and 1 mg/ml anti-EGFR (GA201 versus cetuximab) or anti-Her2 Abs (trastuzumab GE versus WT) in the presence or absence of competing IgGs The quantitative assessment of the expression of CD16a,CD16b, (10 mg/ml Redimune) for 48 h. Raji cells were incubated with human iso- CD32a, CD32b, and CD64 on monocytes and M1 and M2c lated monocytes of monocyte-derived macrophages (E:T ratio, 1:1) and macrophages was performed using quantum beads FACS-based 1 mg/ml of anti-CD20 Abs (GA101, rituximab, ofatumumab) in the presence method (see Materials and Methods). Despite the variability or absence of competing IgGs (10 mg/ml Redimune) for 24 h. At the end of between donors, CD16a and CD16b displayed a high level of the incubation time, coculture was washed and fixed with PFA. Intact target cells, which escaped macrophage ADCC and ADCP, were quantified using expression on intermediate, nonclassical monocytes and, in anti-human Epcam (MKN45, KPL-4) or anti-human CD19 (Raji) Abs fol- agreement with literature, were absent on classical monocytes. lowed by detection using HRP-labeled secondary Ab (all Abs derived from CD32a and CD32b had similar expression on both monocyte BioLegend). The OD of 540–570 nm was determined after incubation with subsets, whereas CD64 was higher on classical monocytes TMB (3,39,5,59 tetramethylbenzidine from BioLegend). The reaction was (Fig. 1A). Interestingly, all three FcgRs displayed higher levels on stopped using H2SO4 2N (Sigma-Aldrich). Ab-dependent killing of targets by macrophages was calculated using the following formula: M2c macrophages than on M1 (Fig. 1B). CD32a and CD32b were the most abundantly expressed Fcg receptors on both macrophage ¼ ðð 2 Þ= % -dependent killing sample release spontaneous release subsets, followed by CD64, CD16a, and CD16b (Fig. 1B). When ð 2 ÞÞ p maximal release spontaneous release 100 comparing monocytes with macrophages, we found that the ex- Spontaneous release, corresponding to target cells incubated with effector pression of CD16a and CD16b was comparable between inter- cells without Ab was defined as 0% cytotoxicity, with maximal release mediate, nonclassical monocytes and macrophages, whereas the (target cells lysed with 2% Triton X-100) defined as 100% cytotoxicity. The levels of CD32a, CD32b, and CD64 were 5–10-fold higher on mean and SD of quadruplicates of each experiment were calculated. macrophages (Fig. 1A, 1B). NO release The binding of therapeutic mAbs to monocytes and macrophages was assessed using WT and GE Fc variants of therapeutic Abs target- Supernatants were collected from different ADCC and cell ELISA assays described above, corresponding to cocultures of human monocytes or ing CD20 (GA101 and ofatumumab) and EGFR (GA201; Fig. 2). To macrophages with Raji or WIL2S target cells (E:T ratio, 1:1) in the presence more accurately reproduce physiologic conditions, Ab binding was of anti-CD20 Abs (GA101 GE or WT, ofatumumab or rituximab). NO also assessed in the presence of physiologic concentrations of release was detected in cell supernatants using the Griess Reagent (Mo- normal human endogenous IgGs (10 mg/ml Redimune), which in lecular Probes). The amount of NO in the supernatants was calculated based on the standard curve generated by the supplier. general caused a 10-fold reduction in Ab binding (compare mean fluo- rescence in Fig. 2A and 2B to Fig. 2C and 2D and Fig. 2E to Fig. 2F). Wide-field fluorescence microscopy WT and GE Abs displayed comparable binding patternonclassical 2 PKH26-labeled MKN45 cells were incubated with PKH67-labeled M2c (CD16 ) monocytes independently of the presence of competing macrophages in the presence of anti-EGFR Abs (GA201 GE, cetuximab or IgGs (Fig. 2A, 2C). Interestingly, CD16+ intermediate, nonclassical The Journal of Immunology 2255

FIGURE 1. Quantitative assessment of FcgRs surface expression on human monocytes and M1 and M2c macrophages. (A) Freshly isolated monocytes or (B) M1- and M2c-polarized macrophages were analyzed with flow cytometry to quantify the surface expression of CD64, CD32, and CD16. Ordinate denotes the ABC obtained for each receptor. Data represent the mean ABC fluorescence 6SD derived from analysis of four different donors. Statistical analysis, unpaired t test: *p , 0.05, **p , 0.01, ***p , 0.001. monocytes bound significantly higher amounts of GE Abs in the was assessed in the presence of blocking CD16 Abs and com- absence and presence of competing IgGs, underscoring the key peting endogenous IgGs (Fig. 2G, 2H). Blocking of CD16 abol- role of CD16 in binding of GE Abs (Fig. 2B, 2D). ished binding differences between GE and WT Abs, confirming its The binding of GE and WT Abs to M1 and M2c macrophages central role in mediating the binding superiority of GE Abs, as was undistinguishable in the absence of competing IgGs (Fig. 2E). also supported by findings on CD16+ monocytes. Downloaded from However, in the presence of competing IgGs, GE Abs displayed a clearly superior binding to both macrophage subsets (Fig. 2F). ADCP This difference was even more accentuated on M2c macrophages The ADCP of monocytes and the four different macrophage compared with M1, most likely because of their higher CD16 subsets (M1, M2a, M2b, and M2c) was initially assessed in a 1-h expression (Fig. 1B). To confirm that CD16 is the Fcg receptor coculture assay with tumor cells and GA101 (Supplemental Fig. 2).

responsible for the superior binding of GE Abs compared with Overall, monocytes mediated the highest maximal ADCP (87%), http://www.jimmunol.org/ their WT counterparts, the binding to M1 and M2c macrophages followed by M2c, M2a (62–70%), and M1 (42%) macrophages. by guest on September 25, 2021

FIGURE 2. Binding of therapeutic Abs to human monocytes and M1 and M2c macrophages. The binding of AlexaFluor647 directly labeled WT and GE Abs was assessed using classical (CD162) monocytes (A, C), nonclassical intermediate (CD16+) monocytes (B, D) and M1 and M2c macrophages (E, F). The binding of each Ab was assessed in absence (A, B, E)orpresence(C, D, F)ofcompeting endogenous human IgGs (10 mg/ml Redimune). Each symbol in (A)–(D) corresponds to median fluorescence intensity of single data points derived from three independent experiments (donors). Donors are identified by the following symbols: donor 1 = black circles; donor 2 = black squares; donor 3 = black triangles. Two GE Abs were used (GA101 and GA201) as duplicates or triplicates, therefore resulting in four to six symbols per donor per condition. Three WT Abs were used as duplicates or triplicates (GA101 WT, GA201 WT, and ofatumumab), resulting in six to nine symbols per donor per condition. (G and H) Binding of directly labeled WT and GE Abs in the presence of saturating concentrations of blocking CD16 Ab and 10 mg/ml Redimune to M1 (G) and M2c (H) macrophages. Data in (E)–(H) correspond to median fluorescence intensity of duplicates derived from one representative experiment out of a total of three independent ones using GA101 GE, GA201 GE, GA101 WT, and ofatumumab. Two of these experiments included the blocking anti- CD16 Ab. Statistical analysis, unpaired t test: *p , 0.05, **p , 0.01, ***p , 0.001. 2256 GLYCOENGINEERING ENHANCES MONOCYTE AND MACROPHAGE ACTIVITY

M2b macrophages displayed poor ADCP activity (21%) most likely to killing in absence of therapeutic Abs, to “WT” or “GE” sam- because of occupancy of their FcgRs by immune complexes used for ples, corresponding to killing obtained in the presence of 1 mg/ml their generation. In general, the addition of therapeutic Ab led to Abs). In agreement with binding data, the intermediate, nonclassical a dose-dependent enhancement of monocyte and macrophage (CD16+) monocytes induced a significantly higher level of tumor phagocytic activity of all myeloid subsets that were tested. cell killing when triggered with GE than with WT Abs (Fig. 4A). To investigate differences in the activity of WT and GE Abs, As expected, the classical (CD162) monocytes were devoid of ADCP was assessed in the absence and presence of competing any cytotoxic activity (data not shown). endogenous IgGs (10 mg/ml Redimune). In agreement with binding Interestingly, M1 macrophages displayed superior cytotoxicity data, the activity of WT and GE Abs was comparable using classical compared with M2c macrophages (Fig. 4B). As expected, the (CD162) monocytes under all experimental conditions (Fig. 3A). The addition of therapeutic Abs enhanced macrophage cytotoxicity activity of GE and WT Abs was also comparable on intermediate, (ADCC) from 22% (no Ab) to 29% (M1) and 13% (no Ab) to 19% nonclassical (CD16+) monocytes in the absence of competing IgGs (M2c; Fig. 4B). In line with phagocytosis, ADCC mediated by GE (Fig. 3B; 0 mg/ml Redimune), despite higher binding of GE Abs and WT Abs in the absence of competing human IgGs was undis- under the same experimental conditions (Fig. 2B). Notably, the supe- tinguishable (Fig. 4B). In addition to LDH, ADCC was assessed by riority of GE Abs using intermediate, nonclassical (CD16+) monocytes detection of caspase-3/7 activation in tumor cells (split luciferase became clearly detectable upon the addition of competing IgGs assay). In line with LDH data, CD16+ monocytes promptly induced (Fig. 3B; 10 mg/ml Redimune). higher levels of tumor cell killing when triggered with GE than with Similar studies were performed using M1 and M2c macrophages WT Abs (Fig. 4C). In line with LDH, the activity of GE and WT Abs

(Fig. 3C, 3D). As for binding, the presence of competing IgGs sig- was undistinguishable in the absence of competing human IgGs on Downloaded from nificantly reduced the overall ADCP, particularly on M1 macro- the macrophage subsets (Fig. 4D, 4E). As before, M1 macrophages phages on which we could not detect significant differences between mediated higher levels of ADCC when compared with M2c mac- Abs (Fig. 3C; compare ADCP at 0 mg/ml to 10 mg/ml Redimune). rophages (Fig. 4E). Taken together, CD16+ monocytes triggered with On the other hand, the phagocytic activity of M2c macrophages was GE Abs mediate higher levels of ADCC than when triggered found to be significantly higher than that of M1 macrophages and with WT Abs. M1 macrophages are more potent in inducing

was better preserved in the presence of competing IgG, allowing target cell cytotoxicity than M2c macrophages; the cytotoxic http://www.jimmunol.org/ solid differentiation of GE and WTAb-mediated ADCP (Fig. 3D). In activity of both macrophage subsets is enhanced by the addi- the presence of competing IgGs, GE Abs display superior binding to tion of therapeutic Abs. CD16+ monocytes and M2c macrophages, and they mediate higher NO release by human monocytes, M1 and M2c macrophages levels of ADCP compared with their WT counterparts. NO release was measured in supernatants recovered from the dif- ADCC ferent macrophage activity assays (as above). The level of NO se- The cytotoxic activity of monocytes and M1 and M2c macrophages creted after GE Ab-mediated ADCC/ADCP was significantly higher was detected by quantifying LDH released in cell culture super- than that secreted after WT treatment on both M1 and M2c mac- natants (Fig. 4). In contrast to phagocytosis, which occurs rapidly rophage subsets already in the absence of competing IgGs (Fig. 5A). by guest on September 25, 2021 and is efficiently quantified after 1–4 h, ADCC was found to be In addition, there was more NO release in the presence of inter- a less efficacious process exerted by monocytes and macrophages, mediate, nonclassical (CD16+) monocytes compared with CD162 which required longer incubation times of 24 h to be detected. In monocytes, particularly when triggered with GE Abs (Fig. 5B). addition, whereas ADCP (despite being significantly reduced) was still measurable in the presence of competing IgGs, the weak cyto- Assessment of the overall Ab-mediated activity using toxicity of monocytes and macrophages was completely abolished. monocytes and macrophages For that reason, data related to monocyte and macrophage cytotoxicity The antitumor activity of Abs in vivo is a combination of direct cell are reported only in the absence of competing IgGs. death induction and Fc-related phagocytic and cytotoxic activities Similar to ADCP, the addition of therapeutic Abs enhanced mediated by engagement effector cells. We therefore set up an monocyte and macrophage cytotoxicity as seen by a slight increase in vitro assay that takes into account all mechanisms of action, thus in the percent killing (compare the “no Ab” control, corresponding enabling the assessment of the overall antitumor activity (direct cell

FIGURE 3. Ab-dependent phagocytosis. CFSE-labeled Raji cells were cocultured for 4 h with isolated human classical monocytes (A)or nonclassical, intermediate monocytes (B) (E:T ratio, 3:1) in pres- ence of GA101 GE or WT Abs and in the absence or presence of 10 mg/ml Redimune. KPL-4 cells were cocultured for 4 h with M1 (C) or M2c macrophages (D) (E:T ratio, 3:1) in the presence of GE or WT trastuzumab and 10 mg/ml Redimune. The percentage of phagocytized target cells was determined with flow cytometry as described in Materials and Methods. Triplicates of representative experiments (performed using different targets and GE/WT Ab pairs (GA101, trastuzumab) are shown. Statistical analysis, unpaired t test: **p , 0.01. The Journal of Immunology 2257 Downloaded from http://www.jimmunol.org/

FIGURE 4. Macrophage-mediated cytotoxicity. (A) A549 cells were incubated with isolated human intermediate, nonclassical (CD16+) monocytes (E:T ratio, 4:1) for 24 h in the presence of 1 mg/ml GA201 GE or WT Abs. (B) MKN45 cells were incubated with M1 or M2c macrophages (E:T ratio, 3:1) for 24 h in the presence of 1 mg/ml GA201 GE or cetuximab Abs. The percent killing was determined by setting the LDH released from target cells to 0% and LDH released by target cells lysed with 2% Triton X-100 to 100%. Triplicates and quadruplicates referring to one representative experiment out of three are shown. (C) 1833-PPOP233 cells were incubated with isolated human intermediate, nonclassical (CD16+) monocytes (E:T ratio, 30:1) for 8 h in the presence of 1 mg/ml GA201 GE or WT Abs. (D) 1833-PPOP233 cells were incubated with polarized human M1 macrophages (E:T ratio, 30:1) for 24 h in the presence of 1 mg/ml GA201 GE or WT Abs. (E) 1833-PPOP233 cells were incubated with polarized human M1 or M2c macrophages (E:T ratio, 10:1 or m 50:1) for 24 h in the presence of 1 g/ml GA201 GE. Triplicates and quadruplicates referring to one representative experiment out of three are shown. by guest on September 25, 2021 Statistical analysis, unpaired t test: *p , 0.05, **p , 0.01, ***p , 0.001. death induction plus ADCC and ADCP). The assay is based on the ing. As third set of WT and GE Abs, we compared the activity of detection of tumor cells (by ELISA) that escaped to Ab-mediated type I and type II anti-CD20 Abs, which notably differ signifi- monocyte or macrophage-induced killing or phagocytosis after 24 cantly in their capacity to induce direct cell death (5). Type I, WT or 48 h of coculture of tumor cells with monocytes or macrophages anti-CD20 Abs (rituximab and ofatumumab) were therefore in the presence of WTor GE therapeutic Abs and competing human compared with the type II, GE, anti-C20 Ab GA101 using M1 and IgG (Fig. 6). In agreement with previous findings, GE Abs (GA201 M2c macrophages and CD16+ and CD162 monocyte subsets from and GE trastuzumab) were significantly more potent than WT three different donors (Fig. 6C, 6D). As before, GA101 mediated Abs (cetuximab and trastuzumab), particularly in the presence of significantly stronger elimination of CD20-expressing tumor cells competing human IgGs and M2c macrophages (Fig. 6A, 6B). As in all experimental conditions, further underscoring the relevance GA201/cetuximab and GE/WT trastuzumab are comparable in of combination of glycoengineering and direct cell death induc- direct-cell death induction, we conclude that the difference in their tion for the monocyte- and macrophage-mediated killing of tumor overall tumor-killing activity is solely due to Ab glycoengineer- cells.

FIGURE 5. NO release. (A) Raji or WIL2S cells incubated with human M1 or M2c macrophages (E:T ratio, 1:1) for 24 or 48 h in the presence of 1 mg/ ml GA101 GE or WT Abs and ofatumumab. NO release in culture supernatants was assessed with Griess reagent and corresponds to the average of pooled triplicates derived from three independent experiments (two using Raji and one using WIL2S as targets). (B) Raji cells incubated with human classical or CD16+ monocytes (E:T ratio, 10:1) for 22 h in the presence of 1 mg/ml GA101 GE or WT Abs. NO release in culture supernatants was assessed by Griess reagent using pooled quadruplicates derived from one experiment. Statistical analysis, unpaired t test: *p , 0.05, **p , 0.01, ***p , 0.001. 2258 GLYCOENGINEERING ENHANCES MONOCYTE AND MACROPHAGE ACTIVITY Downloaded from

FIGURE 6. Assessment of the overall macrophage activity. (A) The overall macrophage activity was assessed in a 48-h coculture assay of MKN45 tumor cells with human M1 or M2c macrophages (E:T ratio, 3:1) in the presence of GA201 or cetuximab and Redimune (10 mg/ml). (B) The overall macrophage

activity assessed in a 48-h coculture assay of KPL-4 tumor cells with human M1 or M2c macrophages (E:T ratio, 4:1) in the presence of trastuzumab GE http://www.jimmunol.org/ and WT Abs and Redimune (10 mg/ml). Quadruplicates of two representative experiments out of a total of four with different Abs and tumor target cells are shown. Statistical analysis, unpaired t test: *p , 0.05, **p , 0.01, ***p , 0.001. The overall macrophage (C) or monocyte (D) activity in combination with direct Ab effects was assessed in a 24-h coculture assay of Raji tumor cells with human isolated CD16+ or CD162 monocytes or monocyte-derived M1 or M2c macrophages (E:T ratio, 3:1) in the presence of GA101, rituximab and ofatumumab, and Redimune (10 mg/ml). Quadruplicates of two representative experiments out of a total of six (three with monocytes and three with macrophages as effectors) from different donors are shown. Statistical analysis, unpaired t test: *p , 0.05, **p , 0.01, ***p , 0.001, comparison of GA101 to rituximab/comparison GA101 to ofatumumab indicated on top of the graphs.

Live-cell imaging of macrophage phagocytic and cytotoxic populations because of CSF-1 deficiency (38) significantly impaired by guest on September 25, 2021 activity elimination of circulating and B cells by anti-CD20 Ab treatment (25). On the contrary, athymic nude and LAT2/2 mice The ADCP activity was further monitored by live-cell imaging of 2/2 cocultures consisting of MKN45 tumor cells (red), M2c macrophages lacking functional T cells, beige and perforin mice with defective (green), WT or GE Abs (cetuximab and GA201, respectively), and NK cell function (39), or mice depleted in NK cells or neutrophils competingIgGs(10mg/ml).Imageswereacquiredevery5minfor (28) maintained the capacity to eliminate most of and spleen a total of 3 h (Supplemental Videos 1–3) or 14 h (Supplemental Videos B cells upon Ab treatment. Therefore, macrophages have been 4, 5). Under control conditions, performed using GA101 that cannot proposed as primary effector cells involved in the activity of mAbs bind to MKN45 cells and thus cannot crosslink macrophages to tumor in murine models in vivo (25, 29). cells, macrophages migrated nearby tumor cells and sensed them In view of the important role of phagocytic lineage cells in the several times but were unable (or weakly able) to phagocytize them, mechanism of action of therapeutic Abs, we analyzed the FcgR- even after overnight incubation (Supplemental Videos 1, 4). Interest- dependent effector functions mediated by human monocytes and ingly, phagocytosis was readily induced by the addition of therapeutic macrophages upon triggering with WT and GE. Our focus was to Abs, which bound to MKN45 cells and thus cross-linked macro- assess the contribution of different phagocytic lineage cells and to phages to tumor cells (cetuximab [Supplemental Video 2] and GA201 investigate whether glycoengineering of therapeutic Abs enhances [Supplemental Videos 3, 5]). In addition to increasing ADCP, several the phagocytic and cytotoxic activity of monocyte and macro- interesting differences could be detected between WT and GE Abs: 1) phages subsets, similarly to what has been shown for NK cells. the degree of phagocytized cells was higher with GE than with WT (in Given the important role of the FcgR expression for the Ab Fc- line with quantitative FACS data), 2) GE Ab-treated macrophages mediated effector functions, we first performed a detailed quan- phagocytized tumor cells faster than those treated with cetuximab titative analysis of the expression of Fcg receptors on classical and (several cells were readily phagocytized within 30 min, rarely seen nonclassical, intermediate monocytes, in addition to M1 and M2c with cetuximab), and 3) GE Ab-treated macrophages performed macrophage populations derived from four different healthy hu- serial phagocytosis of more than one tumor cell and frequently man donors. Overall, macrophages expressed 5–10-fold higher lead to macrophages bearing two or three engulfed tumor cells (a levels of CD64, CD32a, and CD32b than monocytes, whereas phenomenon rarely seen with cetuximab; Supplemental Video 5). the level of CD16a and CD16b was comparable to intermediate, nonclassical monocytes. As expected, CD16a and CD16b were Discussion not detected on classical monocytes. Between macrophages, M2c FcgR-expressing macrophages have recently been identified as expressed overall higher levels of FcgRs than M1 with expression important immune effectors contributing to the activity of mAbs pattern being CD32a,b . CD64 . CD16a,b. in vivo (25–28). Macrophage depletion by treatment with - The Fc-dependent binding of Abs to phagocytic lineage cells was encapsulated clodronate or tissue-specific loss of macrophage sub- in line with the FcgR expression pattern: M2c macrophages bound The Journal of Immunology 2259 the highest amounts of therapeutic Abs (∼10-fold higher than phagocytized cells is higher in the presence of GE Abs (in line with monocytes), followed by M1 macrophages and monocytes. In the quantitative FACS data) and that GE Ab-treated M2c macrophages presence of nonspecific competing IgGs (i.e., under conditions phagocytize tumor cells faster than those treated with WT Abs and that more closely resemble the natural setting), the binding of GE perform serial phagocytosis resulting in engulfment of two to three Abs was significantly higher than that of their WT counterparts, tumor cells. indicating that glycoengineering also enhances the binding of Our data show that in the presence of nonspecific competing IgGs therapeutic Abs to myeloid lineage cells. We also provided evi- (i.e., as it occurs in patients) GE Abs have an advantage over the dence that the superiority acquired by glycoengineering is attrib- WT ones because they display superior binding to CD16+ mono- utable to CD16 as: 1) the binding of GE Abs was reduced to that cytes and M2c macrophages and induce them to eliminate tumor of WT ones by CD16 blocking, 2) the binding of GE Abs was cells more efficiently. The current findings potentially shed new comparable to WT ones on CD162 monocytes, and 3) GE Abs light on the activity of M2c macrophages and the traditional way were bound in significantly higher amounts to CD16+ monocytes these are viewed within tumors. The conclusions from our in vitro even in absence of nonspecific competing IgGs. Overall, the monocyte data indicate that, in the context of therapeutic IgG1 mAbs, the and macrophage binding data corroborate previous studies that presence of M2c macrophages might not necessarily be associated elucidated the structural features underlying enhanced Ab affinity with immunosuppression and poor patient outcome as suggested to CD16 given by glycoengineering (14). As reported, glyco- previously (43–45). On the contrary, our in vitro data suggest that engineering specifically enhances Ab affinity to CD16, whereas M2c macrophages can readily be engaged by therapeutic Abs (es- the affinity to CD32 and CD64 remains unaltered. In line with this, pecially when GE), and can be induced to phagocytize tumor cells

WT and GE Abs perform equally on monocyte and macrophage more efficiently than M1. Recent clinical reports further support our Downloaded from effector cells expressing low levels (or not expressing) CD16. findings indicating that a high tumor-associated macrophage score Upon Ab binding, monocytes and macrophages exert both cy- is associated with favorable prognosis of patients with follicular totoxic (ADCC) and phagocytic (ADCP) activity. Interestingly, lymphoma treated with rituximab and (44), and that we found that, under our experimental in vitro conditions, both rituximab is able to circumvent the unfavorable outcome associ- monocytes and macrophages preferentially eliminate tumor cells ated high intratumor macrophage count patients with follicular

via ADCP as quantitative assessments of both biological processes lymphoma (45). Ultimately, the study shows that therapeutic http://www.jimmunol.org/ and live-cell imaging indicated that ADCP occurs rapidly after Ab mAbs can engage circulating monocytes and different types of addition (15–30 min) and results in 40–85% phagocytized tumor macrophages present within tumors, with M1 displaying more cells within 1 h (as quantified by FACS). On the contrary, ADCC cytotoxic and M2c displaying more phagocytic activity. The Ab- appeared as a less efficient mechanism exploited by macrophages mediated tumor-cell eradication therefore results from a joint in the presence of therapeutic Abs as only ∼5–30% of tumor cells contribution of NK cells and different myeloid effector subsets were eliminated via ADCC after 24 h. Interestingly, M2 macro- in contact with tumor cells. phages displayed the highest ADCP activity, followed by mono- cytes and M1 macrophages. On the other hand, M1 macrophages Acknowledgments were superior to M2c macrophages and monocytes in mediating We thank Dr. Al Rehemtulla, Dr. Stefanie Galba´n(CenterforMolec- by guest on September 25, 2021 ADCC. ular Imaging, University of Michigan, Ann Arbor, MI), and Thomas A side-by-side comparison of different sets of WT and GE Abs Weber (Roche Diagnostics) for providing the split luciferase cells (1833- targeting Her2, EGFR, and CD20 revealed that GE Abs triggered PPOP233 cells) and Dr. Mark S. Cragg for sharing data on CD32a more potent ADCP and overall ADCC and ADCP activity (as and CD32b on different monocyte subsets, MDMs, and M1 and M2 assessed by cell ELISA) when CD16+ monocytes and M2c mac- macrophages. rophages are used, whereas their activity was comparable using CD162 monocytes and M1 macrophages under the same experi- Disclosures mental conditions. A direct comparison of the three anti-CD20 The authors have no financial conflicts of interest. Abs (GA101, rituximab, and ofatumumab) in the cell ELISA as- say further indicated the superiority of GA101 over both rituximab References and ofatumumab resulting from the combination of glycoengin- 1. Shuptrine, C. W., R. Surana, and L. M. Weiner. 2012. Monoclonal antibodies for eering and its strong direct-cell death induction. These findings the treatment of cancer. Semin. Cancer Biol. 22: 3–13. differ from the ones of Rafiq et al. (40), who reported that ofa- 2. Weiner, L. M., J. C. Murray, and C. W. Shuptrine. 2012. Antibody-based im- tumumab elicited superior ADCP when using MDMs and that munotherapy of cancer. Cell 148: 1081–1084. 3. Weiner, L. M., R. Surana, and S. Wang. 2010. Monoclonal antibodies: versatile GA101 treatment results in less potent activation of monocytes platforms for cancer . Nat. Rev. Immunol. 10: 317–327. with diminished pERK, TNF-a release, and FcgRIIa recruitment 4. Reichert, J. M. 2011. Antibody-based therapeutics to watch in 2011. MAbs 3: to lipid rafts when exposing monocytes and MDMs to surface- 76–99. 5. Herter, S., F. Herting, O. Mundigl, I. Waldhauer, T. Weinzierl, T. Fauti, G. Muth, immobilized Abs. However, the different experimental conditions D. Ziegler-Landesberger, E. Van Puijenbroek, S. Lang, et al. 2013. Preclinical used in the two studies (30 min ADCP, E:T ratio of 1:5 in Rafiq activity of the type II CD20 antibody GA101 (obinutuzumab) compared with et al. versus 4 h ADCP, E:T ratio of 3:1 in the current study) and rituximab and ofatumumab in vitro and in xenograft models. Mol. Cancer Ther. 12: 2031–2042. differences in phagocytic effector cells (monocytes and MDMs € € + 2 6. Mo¨ssner, E., P. Brunker, S. Moser, U. Puntener, C. Schmidt, S. Herter, R. Grau, derived from patients with CLL in Rafiq et al. versus CD16 /CD16 C. Gerdes, A. Nopora, E. van Puijenbroek, et al. 2010. Increasing the efficacy of monocytes and M1/M2c macrophages in the current study) might CD20 antibody therapy through the engineering of a new type II anti-CD20 have led to different results. Importantly, the elimination of leu- antibody with enhanced direct and immune effector cell-mediated B-cell cyto- toxicity. Blood 115: 4393–4402. kemic B cells in whole blood from patients with CLL is consis- 7. Adams, G. P., and L. M. Weiner. 2005. therapy of cancer. tently superior with GA101 (41, 42). Nat. Biotechnol. 23: 1147–1157. The functional superiority of GE Abs was further confirmed by 8. Jaglowski, S. M., L. Alinari, R. Lapalombella, N. Muthusamy, and J. C. Byrd. live-cell imaging of cocultures of tumor cells and M2c macro- 2010. The clinical application of monoclonal antibodies in chronic lymphocytic leukemia. Blood 116: 3705–3714. phages treated with WT or GE Abs in the presence of competing 9. Nimmerjahn, F., and J. V. Ravetch. 2005. Divergent subclass IgGs. Data collected by imaging indicated that the degree of activity through selective binding. Science 310: 1510–1512. 2260 GLYCOENGINEERING ENHANCES MONOCYTE AND MACROPHAGE ACTIVITY

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