Development of a robust reporter-based ADCC assay with frozen, thaw-and-use cells to measure Fc effector function of therapeutic antibodies

Journal of Immunological Methods xxx (2014) xxx–xxx

JIM-11903; No of Pages 13

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Journal of Immunological Methods

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Development of a robust reporter-based ADCC assay withfrozen, thaw-and-use cells to measure Fc effector functionof therapeutic antibodies

Zhijie Jey Cheng⁎, Denise Garvin, Aileen Paguio, Richard Moravec, Laurie Engel,Frank Fan, Teresa SurowyDepartment of Research, Promega Corporation, Madison, WI 53711, United States

a r t i c l e i n f o

Abbreviations: ADCC, antibody-dependent cell-medicomplement-dependent cytotoxicity; PBMC, peripheracell; NK cells, natural killer cells; LDH, lactate dehglyceraldehyde 3-phosphate dehydrogenase; EC50, meacy; maximum FI, maximum fold of induction; DOE, Desiratio, Effector cell:Target cell ratio; CV, coefficient ofrelative luminescence units; IgG1, immunoglobulin Gactivated cell sorting; FBS, fetal bovine serum; HPLC, hichromatography; TCR, T cell receptor.⁎ Corresponding author. Tel.: +1 6082744330.

E-mail address: [email protected] (Z.J. Cheng

http://dx.doi.org/10.1016/j.jim.2014.07.0100022-1759/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Cheng, Z.J., et al., Dmeasure Fc effector function of therapeutic

a b s t r a c t

Article history:Received 3 May 2014Received in revised form 18 July 2014Accepted 21 July 2014Available online xxxx

Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the main mechanisms of actionfor many therapeutic antibodies. Classic ADCC assays measure antibody-dependent target cellcytotoxicity induced by primary effector cells that are isolated from human blood. They sufferfrom high assay variability due to the genetic and immune-status-mediated variation from blooddonors. Here we report the development of a robust reporter-based ADCC assay that uses anengineered Jurkat stable cell line as the source of effector cells. These engineered effector cellswere further developed as frozen, thaw-and-use format that can be plated for assay immediatelyafter thaw. We demonstrate that frozen, thaw-and-use Jurkat effector cells showed appropriateFc effector function similar to fresh cells from continuous culture, with added benefits ofconvenience and consistency. This robust assay is able to measure antibody potency for severaltherapeutic antibodies targeted to hematopoietic or solid tumors. The assay can distinguisheffector functions for different antibody IgG isotypes in two antibody model systems: anti-CD20and anti-EGFR. It is able to detect changes in ADCC biological activity for heat-stressed rituximaband trastuzumab, demonstrating that it possesses proper stability-indicting property. Whencompared with a classic PBMC-based ADCC assay, the ADCC reporter assay showed better assayprecision and similar correlation of antibody glycosylation with ADCC biological activity for apanel of glyco-modified trastuzumab mixtures. Together these data demonstrate that this robustADCC reporter assay meets the requirement of a potency bioassay that can quantify antibody Fceffector function in ADCC mechanism of action during drug discovery and development.

© 2014 Elsevier B.V. All rights reserved.

Keywords:Antibody-dependent cell-mediatedcytotoxicityADCC reporter assayFc effector functionFcγRIIIaMonoclonal therapeutic antibody

ated cytotoxicity; CDC,l blood mononuclearydrogenase; GAPDH,sured antibody poten-gn of Experiments; E:Tvariation; RLUs, raw1; FACS, fluorescencegh performance liquid

).

evelopment of a robustantibodies, J. Immunol.

1. Introduction

Therapeutic antibodies are used in targeted therapy formany human diseases. To date, more than 30 recombinanttherapeutic antibodies are approved for human therapy, andseveral hundred candidates are in clinical development world-wide (Chan and Carter, 2010; Jiang et al., 2011; Leader et al.,2008). There are two classes of clinical applications oftherapeutic antibodies that are governed by antibody structuresand their binding partners: blocking or stimulation of receptorsignaling by target-specific binding via antibody Fab domain;and stimulation of immune-mediated effector function via the

reporter-based ADCC assaywith frozen, thaw-and-use cells toMethods (2014), http://dx.doi.org/10.1016/j.jim.2014.07.010

http://dx.doi.org/10.1016/j.jim.2014.07.010

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2 Z.J. Cheng et al. / Journal of Immunological Methods xxx (2014) xxx–xxx

antibody Fc domain, such as via complement-dependentcytotoxicity (CDC) and antibody-dependent cell-mediatedcytotoxicity (ADCC) (Chan and Carter, 2010; Jiang et al., 2011;Nimmerjahn and Ravetch, 2008). ADCC is an importantmechanism of action for many therapeutic antibodies inoncology and autoimmune disease, including rituximab,trastuzumab and cetuximab (Reff et al., 1994; Carter et al.,1992; Vermorken et al., 2008; Prang et al., 2005). In ADCC, theantibody recognizes the antigen receptors on the target cells viaits Fab domain, while the Fc portion of the antibody binds to theFc receptors on immune cells such as natural killer (NK) cells ormonocytes. This is followed by the crosslinking and activation ofthe Fc receptors, the release of cytokines, and the formation ofcytotoxic granules containing perforin and granzyme whichultimately leads to the lysis of the target cells (Leibson, 1997;Azzoni et al., 1992; Lyubchenko et al., 2001). The extent of ADCCresponse depends on many factors, including the effector celltypes (Fanger et al., 1989a,b; Graziano et al., 1989), different Fcreceptors and polymorphism of Fc receptors on effector cells(Nimmerjahn and Ravetch, 2008; Koene et al., 1997; Dall'Ozzoet al., 2004; Siberil et al., 2007), antibody IgG isotypes(Nimmerjahn and Ravetch, 2008; Siberil et al., 2007; Patelet al., 2010), and antigen expression level on target cells (Pranget al., 2005; Patel et al., 2010).

Despite the tremendous interest in the development oftherapeutic antibodies, it has been highly challenging tomeasure the Fc effector function in ADCC for therapeuticantibodies in a reproducible and quantitative manner. ClassicADCC assaysmeasure the short-term cytotoxicity of target cellsafter exposure to antibody-bound primary effector cells such asPBMCs or the subset NK cells. The target cells are pre-loadedwith radioactive chromium (51Cr) in the earliest version of theADCC assay, or loaded with the lanthanide Europium, or afluorescent dye, typically calcein-AM, in later modified ADCCassays (Brunner et al., 1968; Roden et al., 1999; Patel and Boyd,1995; von Zons et al., 1997). Alternatively, the enzyme releaseof lactate dehydrogenase (LDH), glyceraldehyde 3-phosphatedehydrogenase (GAPDH), or a specific dead cell protease intarget cells can be measured to quantify cytotoxicity withoutthe need of a labeling process (Korzeniewski and Callewaert,1983; Stewart et al., 2011; Sergeeva et al., 2011). These assaysbring the benefit of convenience by eliminating the handling ofradioactive isotopes and the variability of cell labeling. Still, theneed for primary effector cells isolated from fresh donor bloodhas made classic ADCC assays vulnerable to high variability.This variability has multiple sources, including tedious stepsneeded for the isolation of primary cells, and the inherentdonor-to-donor variation due to differences in genetic back-ground and the immune status of blood donors (Brunner et al.,1968; Roden et al., 1999; Patel and Boyd, 1995; von Zons et al.,1997). To overcome this, NK cell lines expressing endogenousFcγRIIIa or engineered NK cell lines expressing exogenousFcγRIIIa have been developed and used to replace primaryPBMCs as effector cells in ADCCassays (Schnueriger et al., 2011;Binyamin et al., 2008). These NK cell-line based ADCC assaysshowed improved precision and reproducibility. However, NKcell lines are challenging to use in potency bioassays due totheir tendency to form large cell aggregates, being difficult tohandle, and genetic instability of endogenous or engineered Fcreceptors (Gong et al., 1994; Robertson et al., 1996). As analternative, the Jurkat T-cell line has been used as a model

Please cite this article as: Cheng, Z.J., et al., Development of a robustmeasure Fc effector function of therapeutic antibodies, J. Immunol.

system to study FcγRIIIa receptor function in ADCC since thecells are easy to handle in cell culture and engineering whilesharing similar immune cell background with NK cells (Vivieret al., 1992; Wirthmueller et al., 1992). Recently, Parekh et al.reported the development of a reporter gene assay as asurrogate ADCC assay, by building a Jurkat stable cell lineexpressing human FcγRIIIa, human CD3γ, and an NFAT-response element regulating a luciferase reporter (Parekhet al., 2012). The assay was successfully validated as a potencybioassay in an anti-CD20 antibodymodel systemwith CD20+ Bcell line WIL2-S cells. Since other target cells and non-CD20antibodies were not evaluated in that study, it would be ofgreat value if a reporter-based surrogate ADCC assay can bedemonstrated to broadly apply to many other target systems.

Low CV and high precision are considered critical require-ments of a cell-based potency bioassay. Reproducibility isextremely important during assay transfer. Cells are consideredas critical reagents in cell-based bioassays. Many factors duringcell culture and preparation can cause run-to-run assayvariation, and make assay transfer very challenging (Buttner,2006). Therefore, a well-controlled, consistent source of cellreagents should significantly decrease assay variability, im-prove assay consistency, and be highly desired for bioassaydevelopment. Frozen, single-use cells are now widely used inthe high-throughput screening community for small moleculedrug discovery because they offer convenience, uniformity andefficiency (Fursov et al., 2005; Zaman et al., 2007). In the case ofbiologic drug discovery, there was an early report on frozenprimary PBMCs in ADCC assay (Strong et al., 1982). In recentyears, frozen cells have been applied in a few quality controllaboratories for biologic drug development (TerWee et al.,2011), and in detection of neutralizing antibodies againstbiologic drugs in clinical research (Lallemand et al., 2011).However, the use of frozen cells is still limited and not welladopted for potency bioassays for biologics, despite the benefitsit could bring. For previously reported ADCC bioassays that useeffector cell lines that exogenously express FcγRIIIa (Azzoniet al., 1992; Schnueriger et al., 2011; Binyamin et al., 2008;Parekh et al., 2012), the assays are performed using fresh cellsfrom culture. Thus, like many other cell-based bioassays, theyhave stringent training requirements for laboratory analysts onhow to consistently handle fresh cells from culture in assay.Therefore, fresh cells in culture share potential challenges toachieve high assay precision especially in order to maintaingood assay reproducibility during assay transfer to differentsites in house or to contract laboratories.

In the current study, we describe construction of a quanti-tative surrogate ADCC reporter bioassay, by applying a similarassay design as that used by Parekh et al. (2012), through theestablishment of an engineered Jurkat reporter cell line andtheir use as effector cells in assay. We further developed theengineered effector cells in frozen, thaw-and-use formatwithout the need of daily cell culture and cell washing afterthaw. We demonstrated that the assay using these thaw-and-use cells showed specific Fc effector function, antibody IgGisotype specificity, good precision and sensitivity to antibodyactivity changes due to heat stress, when tested in multipletherapeutic antibody model systems. Using a panel of glyco-modified antibodies in studies comparing this assay to a classicPBMC-based ADCC assay, we showed the biological relevance ofthe ADCC reporter assay response, and similar correlation of



3Z.J. Cheng et al. / Journal of Immunological Methods xxx (2014) xxx–xxx

biological activity with the degree of antibody glycosylation byboth types of assays. These results demonstrate that, the ADCCreporter assay using frozen, thaw-and-use Jurkat effector cellscan serve as a quantitative surrogate ADCC potency bioassay fortherapeutic antibodies. This is the first report on the applicationof thaw-and-use effector cells in anADCCbioassay. The assay hashigh potential for antibody characterization, lot release, andstability studies during therapeutic antibody drug development.

2. Results

2.1. Establishment of reporter-based ADCC assay specificity

In order to build a quantitative cell line-basedADCC assay fora range of antibody therapeutics, we generated a stable Jurkatcell line for use as effector cells in ADCC assay. This Jurkat cellline, similar to an engineered effector cell line previouslyreported (Parekh et al., 2012), exogenously expresses the higheraffinity variant of human FcγRIIIa, V158 variant, and a NFAT-REdriven luciferase reporter. We chose to use a new generation,codon-optimized luciferase reporter, luc2, to replace the nativeluciferase gene, because luc2 provides a more specific andenhanced reporter response (Cheng et al., 2010). We chose notto introduce exogenous CD3γ as previously described (Parekhet al., 2012). Instead, we selected Jurkat cell line, clone E6-1,which is reported to endogenously express CD3ζ, a CD3 chainsupporting FcγRIIIa surface expression and signaling (Vivieret al., 1992; Moingeon et al., 1992). We confirmed CD3ζexpression in the Jurkat clone E6-1 using FACS analysis (datanot shown). In the following studies, we refer to the engineeredJurkat cell line as “Jurkat effector cells” and the reporter-basedsurrogate ADCC assay using these Jurkat effector cells as “ADCCreporter assay”.

Previously, anti-CD20 model system was evaluated in cellline-based ADCC assays, which demonstrated appropriateassay specificity and precision (Schnueriger et al., 2011;Parekh et al., 2012). To evaluate assay specificity for ADCCreporter assay that we established, we started with a similarCD20 target system that includes two anti-CD20 antibodies

Fig. 1. Assay specificity. ADCC reporter assay using fresh Jurkat effector cells was car(A) Luciferase reporter responses were measured in the presence or in the absence(B) Luciferase reporter response from Jurkat NFAT-RE reporter cell line expressing FcγWIL2-S cells and rituximab. (C) Dose-dependent inhibition of ADCC reporter respoantibodies, mouse anti-human CD16 antibody or mouse IgG1 control, were then addecontrol was determined as described in “Materials and methods”. Results shown were


rituximab and B1, and a human CD20+ B lymphoma cell lineWIL2-S as target cells. A robust, dose-dependent reporter signalwas producedwhen the Jurkat effector cells were co-incubatedwith WIL2-S and rituximab (Fig. 1A) or WIL2-S and B1 (datanot shown). No signal was observed in the absence of antibodyor when the non-CD20 antibody, trastuzumab, was added,confirming that a specific antibody is required to recognize theantigen on target cells. Adding rituximab (Fig. 1A) ortrastuzumab (data not shown) alone to Jurkat effector cells,without target cells, did not produce any reporter signal,demonstrating that soluble antibody is not sufficient to induceFc receptor activation and ADCC (Wirthmueller et al., 1992).

To demonstrate that the reporter signal is mediated byFcγRIIIa exogenously introduced into Jurkat effector cells, wegenerated a Jurkat single stable reporter cell line expressingNFAT-RE-luc2 reporter alone. Both Jurkat effector cells andJurkat single stable NFAT-RE reporter cells responded well toanti-CD3 antibody to stimulate endogenous TCR activation(data not shown). After the incubation with WIL2-S boundrituximab, only Jurkat effector cells showed robust reporterresponse while Jurkat single stable NFAT-RE reporter cellsshowed no reporter activity with the same treatment (Fig. 1B).In addition, the ADCC reporter response induced by anti-CD20antibody B1, using WIL2-S target cells, was almost completelyblocked by pre-incubation of Jurkat effector cells with anti-CD16 antibody in a dose-dependent manner (Fig. 1C). Thesedata demonstrated that the reporter signal in ADCC reporterassay is specifically dependent on 1) target cells expressingspecific antigens, 2) a specific antibody that recognizes theantigen on target cells, and 3) expression of FcγRIIIa in Jurkateffector cells.

2.2 . Development of Jurkat effector cells in a frozen, thaw-and-useformat

Having established a specific ADCC reporter assay, wesought to minimize assay variability further by using frozen,thaw-and-use effector cells in the assay. To develop this format,we first explored cell culture and cryopreservation conditions,

ried out as described in “Materials and methods”, unless otherwise indicated.of Jurkat effector cells, WIL2-S cells, rituximab or trastuzumab, as indicated.RIIIa (Jurkat effector cells) or without FcγRIIIa, after overnight incubation withnse by anti-CD16 antibody. Jurkat effector cells pre-incubated with blockingd to assay plates containing WIL2-S and anti-CD20 antibody B1. Percentage ofrepresentative data from at least two independent experiments.




in order to generate frozen cells that could provide robust andconsistent assay response upon thaw and immediate use. Wechose to use the anti-CD20/CD20+ B cell linemodel systemwehad already used to develop the assay using continuouslycultured effector cells. We investigated several cell culture andfreezing conditions, including cell density at harvest, carryoverof DMSO from cell freezing medium, and cell freezing density.Antibody potency (EC50) and maximum fold of induction(maximum FI) induced by test antibodywere used as the assayoutputs to compare conditions.

To evaluate the impact of cell culture density on frozeneffector cell performance in the ADCC reporter assay, we testeda range of cell densities at harvest from continuous culture. Asshown in Fig. 2A, when frozen Jurkat effector cells wereprepared from cell culture with cell densities of 1.2 × 106 and1.8 × 106 viable cells/ml, the assay using these cells showed arelatively consistent and robust reporter response, uponinduction by WIL2-S bound rituximab. Similar results wereobserved when a second CD20+ B cell line, Raji, or anti-CD20antibody B1 was tested (data not shown). In contrast, frozenJurkat effector cells prepared from cell densities outside therange of 1.2 to 1.8 × 106 cells/ml showed decreased maximumFI or a right shifted EC50. All the cells tested gave similar post-thaw cell viability in a range of 75%–85%, indicating that theimpact on assay performance by cell culture density was notdue to the changes in the numbers of viable effector cellsavailable in the assay. Thus, the range of 1.2–1.8 × 106 cells/mlwas chosen as the acceptable range of cell density for cellharvest in the following studies.

Washing and centrifugation to remove cryoprotectantssuch as DMSO, after thaw, are routine practices to initiate cellculture from freezing. Here, we aimed to avoid such washingprocedures by using thawed cells immediately in assay, thusobviatingmishandling that could potentially lead to cell loss ordamage and increase assay variability run-to-run. This decisionnecessitated our prior evaluation of any impact of DMSOcarryover in the assay reaction. Fig. 2B shows that in an initialexperiment, Jurkat effector cells used in an ADCC reporter assaydirectly without washing after thaw, gave assay performancesimilar to that obtained with washed cells. This indicated

Fig. 2. Optimization of cell culture and freezing conditions for frozen, thaw-and-use Ju“Materials and methods”, unless otherwise indicated. WIL2-S cells are used as target ccell culturewith different cell densities at harvest, (B) frozen Jurkat effector cells, with 2washed with PBS and centrifuged before being re-suspended in assay buffer, (C) Jurkaassay buffer after thaw to generate equivalent cell densities for assay. EC50 and maximand methods”. Results shown were representative data from at least two independent


that the DMSO carryover had minimal impact on the assay,under the conditions we tested in which cells used were at 2 ×107 cells/ml when frozen. In the following study, Jurkat effectorcells that were plated for assay immediately after thaw, withoutany washing, are defined as thaw-and-use.

Assay response of cells used immediately after thaw isdependent on both conditions at cell cryopreservation and atthaw. We next evaluated cell freezing density for its impact oncell viability and cell performance in the ADCC reporter assayafter freeze/thaw, as well as any potential side effect due tovarious concentrations of DMSO carryover without washingafter cell thaw (Fig. 2C). The cells frozen at three differentfreezing densities, ranging from 1 × 107/ml to 2 × 107/ml,performed similarly in ADCC reporter assay in response torituximab stimulation, using WIL2-S (Fig. 2C) or Raji (data notshown) target cells. In contrast, the cells frozen at the lowestfreezing density tested, 5 × 106/ml, showed over 50% decreaseof reporter response (maximum FI) when tested under thesame condition. The post-thaw cell viability is equivalent forfrozen cells from different freezing densities (data not shown).Therefore, this decrease of reporter response from the cells at 5×106/ml freezing density is not due to the change of viable effectorcell number initially plated for assay. Since the concentration ofDMSO carryover in the cell suspension prepared from cryovialswith 5 × 106/ml cell freezing density is 2–4 fold higher thanthose in cell suspensions from vials with higher freezingdensities, it is likely that the decrease of reporter response iscaused by the cytotoxicity induced by extended incubation ofboth effector and target cells in high concentration DMSO-containing assay reactions. We tested this hypothesis byspiking aliquots of the same effector cells at high freezingdensity of 2 × 107/ml upon thaw into different concentrationsof DMSO-containing assay buffer and compared effectorresponses side-by-side in the ADCC reporter bioassay usingrituximab and WIL2-S target cells. The range of final DMSOconcentrations in assay reaction is the same as in theexperiment depicted in Fig. 2C. Our results showed that theeffector cells spiked into 3% total DMSO-containing cellsuspension showed 30% of decrease in reporter signal com-pared with the cell suspension with DMSO carryover from

rkat effector cells. The frozen Jurkat effector cells were prepared as described inells. ADCC reporter response from (A) frozen Jurkat effector cells prepared from× 107 cells/ml freezing density, directly suspended in assay buffer after thaw, ort effector cells frozen at different freezing densities then immediately diluted inum fold of induction (maximum FI) were determined as described in “Materialsexperiments.




freezingmediumonly at 0.75%DMSO final concentration in cellsuspension (data not shown). Thus the equivalent 3% DMSOconcentration in effector cell suspension made from frozencells at 5 × 106 cells/ml (Fig. 2C) could partially contribute tothe N50% decrease of reporter signal observed in the initialexperiment. Additional factors such as cell freezing density incombination with final DMSO concentration during assay mayalso contribute. Based on these results,we decided to use a highcell freezing density of 2 × 107/ml effector cells for thaw-and-use format cells in all future studies. This minimized thecytotoxicity of DMSO carryover and at the same time providedsufficient effector cells from one single cryovial for onecomplete 96-well assay, a convenient single-use size.

2.3. Comparison of assay performance using fresh from culture orfrozen, thaw-and-use Jurkat effector cells in ADCC reporter assay

To assess whether frozen Jurkat effector cells in thaw-and-use, assay-ready format retain appropriate effector cell functionin ADCC reporter assay, we compared the assay performance offresh from continuous culture and frozen, thaw-and-use Jurkateffector cells using the rituximab/WIL2-S target cell modelsystem (Fig. 3A and B).

Assay fold-induction responses using both fresh fromculture or frozen, thaw-and-use Jurkat effector cells, showedtime-dependent increase in the first 7 h of induction byWIL2-Sbound rituximab. For each time point in the first 7 h we tested,assay response from frozen cells was higher than that fromfresh cells. The response with thaw-and-use cells decreased, by15–20%, after overnight induction (Fig. 3A). In contrast, theassay signal from fresh cells continued to increase and reachedits peak after overnight induction (Fig. 3B) matching that seenfor the thaw-and-use cells at the same time point of induction.A similar trend in assay dynamics, between frozen and freshJurkat effector cells, was also observed for other antibodies andtarget cell systems we tested (data not shown). These dataindicate that frozen Jurkat effector cells aremore sensitive than

Fig. 3. Comparison of assay performance using frozen or fresh Jurkat effector cells. ADCeffector cells was carried out as described in “Materials andmethods”. ADCC reporter refor a series of time points as indicated. EC50 and maximum fold of induction (maximumwere representative data from at least two independent experiments.


fresh cells to stimulation by target cell-bound antibody duringthe first few hours after thaw. The exact cause of the differentassay dynamics between frozen and fresh Jurkat effector cells isnot clear and will need further investigation.

The measured antibody potency (EC50) stabilized andremained consistent across later time points for assays usingfrozen, thaw-and-use or fresh from culture Jurkat effector cells.The stabilization occurred earlier for the thaw-and-use cellsand thus was more robust across time. The EC50 valuesobtained from fresh and frozen cell assays are in the ng/mlrange for rituximab. This is in the same range as EC50 valuespreviously reported in classic ADCC assays (Lewis et al., 2011;Idusogie et al., 2000). These data indicated that frozen Jurkateffector cells have appropriate effector cell function, similar tothat of fresh cells, and are suitable for measuring ADCCbiological activity for therapeutic antibodies. Use of frozen,thaw-and-use Jurkat effector cells in ADCC reporter assayprovides advantages over fresh effector cells: shorter assaytime for a better reporter response, more robust assay, andconvenience and consistency.

2.4 . Evaluation of ADCC reporter bioassay with multipletherapeutic antibodies and target cell lines derived fromhematopoietic or solid tumors

Wenext evaluatedwhether this robust ADCC reporter assayusing frozen, thaw-and-use Jurkat effector cells could bebroadly applied to therapeutic antibodies targeted to bothhematopoietic and solid tumors (Fig. 4). Three therapeuticantibodies, anti-CD20 rituximab, anti-HER2 trastuzumab andanti-EGFR cetuximab, were each tested with multiple targetcell lines that are known to differ in antigen expressionlevel and to have different ADCC effects. Assay optimizationincludingDesign of Experiments (DOE)was performed. Severalkey assay parameters, including Effector cell:Target cell ratio(E:T ratio), assay buffer components and induction time, wereidentified to affect assay performance in terms of antibody EC50

C reporter assay using frozen, thaw-and-use (A) or fresh from culture (B) Jurkatsponseswere determined by Bio-Glo™ Luciferase Assay System, after inductionFI) were determined as described in “Materials and methods”. Results shown



Fig. 4. ADCC reporter assay response to therapeutic antibodies. Frozen, thaw-and-use Jurkat effector cells were induced with target cell-bound rituximab(A), trastuzumab (B), or cetuximab (C). In testing each therapeutic antibody the same E:T ratio was used with different target cell lines, as described in “Materials andmethods”. Results shown were representative data from at least three independent experiments.

Table 1EC50 and maximum fold of induction (maximum FI) values (mean ± SD,N ≥ 3) obtained for therapeutic antibodies each tested with multiple targetcells in the ADCC reporter assay using frozen Jurkat effector cells.

Antibody Target cells Maximum FI EC50, g/ml

Rituximab WIL2-S 45 ± 14 4.9 × 10−9 ± 1.7 × 10−9

Raji 31 ± 8 1.9 × 10−8 ± 7.0 × 10−9

Daudi 18 ± 5 2.7 × 10−8 ± 1.1 × 10−8

Ramos 15 ± 8 1.9 × 10−8 ± 5.4 × 10−9

Trastuzumab SK-BR-3 95 ± 31 1.1 × 10−8 ± 9.0 × 10−10

SK-OV-3 31 ± 11 7.7 × 10−9 ± 9.0 × 10−10

MCF7 17 ± 5 5.9 × 10−9 ± 5.0 × 10−10

Cetuximab A431 22 ± 2 1.7 × 10−8 ± 4.1 × 10−9

PC-3 10 ± 3 5.3 × 10−9 ± 1.3 × 10−9


and maximum FI. In direct comparison of response usingsame therapeutic antibody with different target cells, we usedidentical assay conditions.

To test rituximab, two low CD20 expression B-cell lines,Daudi and Ramos, along with two high CD20 expression B-celllines, WIL2-S and Raji, were tested using an E:T ratio of 6:1(Fig. 4A). The assay with each target cell showed a dose-dependent ADCC reporter response induced by rituximab. Therank order of maximum FI wasWIL2-S N Raji N Daudi N Ramos,which correlates with the CD20 expression levels and thereported target cell sensitivity to ADCC measured in classicADCC assay as percentage ofmaximum lysis (Lewis et al., 2011;Idusogie et al., 2000; Cardarelli et al., 2002).

We tested the ADCC reporter assay using several solid tumorcell lines as targets. These have not, to the best of ourknowledge, been formally reported in other cell line-basedADCC assays (Schnueriger et al., 2011; Parekh et al., 2012). An E:T ratio of 15:1 was used and shown to work for all the adherenttarget cells tested. Two breast cancer cell lines, SK-BR-3 (highHER2 expression) and MCF7 (low HER2 expression), and anovarian cancer cell line, SK-OV-3 (medium HER2 expression)(Prang et al., 2005; Suzuki et al., 2007; Reim et al., 2009), weretested with the anti-HER2 antibody trastuzumab (Fig. 4B). Therank order of maximum FI observed was SK-BR-3 N SK-OV-3 N

MCF7, which correlates with the HER2 expression levels andreported sensitivity to ADCC effects in classic ADCC assay. TwoEGFR+ cell lines, the human epithelial carcinoma cell line A431and the human prostate cell line PC-3, were used to testcetuximab (Dhupkar et al., 2010; Kimura et al., 2007). Weobserved good ADCC reporter response in both EGFR+ A431cells and PC-3 cells (Fig. 4C).

As shown in Table 1, the EC50 values for these therapeuticantibodies in ADCC reporter assay are similar to those reportedby others where classic PBMC-based ADCC assays were used(Prang et al., 2005; Dall'Ozzo et al., 2004; Lewis et al., 2011;Kimura et al., 2007). These data suggest that ADCC reporterassay using frozen, thaw-and-use effector cells providesappropriate Fc effector function quantification for therapeuticantibodies targeted to both hematopoietic and solid tumors.

2.5. Assessment of assay precision of the ADCC reporter assay usingfrozen, thaw-and-use Jurkat effector cells

In traditional cell-based assays, cell banks are generated toprovide a shared starting point for production of fresh cells incontinuous culture, for a relatively consistent cell source in


independent bioassay runs within specific periods of time. Forassays using frozen cells, we were able to further control thecell source by generating hundred-vial batches of thaw-and-use effector cells. Since thaw-and-use cells from the samebatchare used immediately after thaw in assay without furthermanipulation, they provide a consistent source of critical cellreagent that can be used in independent bioassay runs acrossspecific periods of time, for up to several months.

Good assay precision was reported in an ADCC reportergene assay validation using fresh effector cells, performedaccording to ICH-QR2 guidelines (Parekh et al., 2012), andtested using an anti-CD20 model system with suspensiontarget cells. We have demonstrated broad applicability of theADCC reporter assay using thaw-and-use effector cells; there-fore, we chose to evaluate intra-assay precision and inter-assayprecision using an adherent target cell system representing thebroad target base of solid tumors. We performed an assayqualification study with trastuzumab and HER2+ SK-BR-3target cells (Tables 2, 3).

Intra-assay precision, or repeatability, refers to plate-to-plate variability under the same assay condition on the sameday. The inter-assay precision refers to day-to-day, or analyst-to-analyst assay variability that reflects variation resulting fromroutine laboratory practices. We assayed trastuzumab withHER2+ SK-BR-3 target cells over three days with four plateseach day. As shown in Tables 2 and 3, the plate-to-platevariation (%CV) we observed for measured antibody potency(EC50) is in the range of 3.9%–6.3% over the three days, and theday-to-day variation (%CV) is 7.7%. Maximum FI obtained fromplate-to-plate, or day-to-day runs were also compared. The



Table 2Plate-to-plate assay variability of the ADCC reporter assay response. Trastuzumab was tested over three days with four plates each day, with SK-BR-3 target cells usingsame batch of frozen, thaw-and-use Jurkat effector cells.

Day 1 Day 2 Day 3

Maximum FI EC50, g/ml Maximum FI EC50, g/ml Maximum FI EC50, g/ml

Plate 1 152 1.3 × 10−8 168 1.2 × 10−8 163 1.4 × 10−8

Plate 2 151 1.3 × 10−8 163 1.1 × 10−8 164 1.6 × 10−8

Plate 3 151 1.2 × 10−8 164 1.1 × 10−8 169 1.6 × 10−8

Plate 4 152 1.3 × 10−8 163 1.1 × 10−8 174 1.5 × 10−8

Mean 152 1.3 × 10−8 165 1.1 × 10−8 168 1.5 × 10−8

SD 0.55 5.0 × 10−10 2.38 5.0 × 10−10 5.07 9.6 × 10−10

%CV 0.4% 3.9% 1.4% 4.4% 3.0% 6.3%


plate-to-plate assay variation (%CV) is in the range of 0.4%–3.0%for maximum FI obtained in the same day over the three days,and 5.0% for day-to-day variation. Raw relative luminescenceunits (RLUs) are not suitable to be used to monitor assayprecision, as we found that the assay variations for RLUs aremuch higher than those seen for EC50 or maximum FI (data notshown). This is due to the fact that raw RLUs are not onlyaffected by reporter gene expression, but also dependent on thereal-time luciferase enzymatic reaction, which is sensitive toambient temperature, instrument set-up, incubation condi-tions of luciferase reagent, and other factors.

Overall, the ADCC reporter assay described here usingfrozen, thaw-and-use Jurkat effector cells is robust, providesgood intra-assay precision and inter-assay precision with lowassay variability, in terms of antibody potency and maximumADCC reporter response. It meets the main requirements forpotency bioassays: high precision and low CV.

2.6. Assessment of antibody IgG isotype — specific ADCC reporterresponse

The binding of the antibody Fc portion to FcγRIIIa is a criticalstep in the initiation and control of the antibody's effectorfunction. Each subclass of human IgG (IgG1–IgG4) exhibitsdistinct effector function in ADCC, which is dictated by its Fcbinding affinity to human FcγRIIIa. Human IgG1 and IgG3 haverelatively high binding affinities; human IgG2 and IgG4 haverelatively low binding affinities (Jiang et al., 2011; Siberil et al.,2007). Human IgG2 and IgG4, therefore, are often chosen toserve as the backbone for a therapeutic antibody when Fceffector function is not desired, whereas the human IgG1isotype is most often chosen for antibody drug developmentwhen Fc effector function is required.

Table 3Day-to-day assay variability of the ADCC reporter assay response. Trastuzumabwas tested on three different dayswith SK-BR-3 target cells using same batch offrozen, thaw-and-use Jurkat effector cells.

Maximum FI EC50, g/ml

Day 1 152 1.3 × 10−8

Day 2 168 1.2 × 10−8

Day 3 163 1.4 × 10−8

Mean 161 1.3 × 10−8

SD 8 1.0 × 10−9

%CV 5.0% 7.7%


To evaluate whether the ADCC reporter assay with thaw-and-use effector cells would exhibit the appropriate correlationof antibody IgG isotypeswith their Fc effector function in ADCC,we tested different antibody IgG isotypes using two therapeuticantibody model systems: anti-CD20 and anti-EGFR (Fig. 5).When frozen, thaw-and-use Jurkat effector cells were incubat-ed with WIL2-S target cells and various rituximab variabledomain containing IgG isotypes, the rank order of maximum FIobserved was: human IgG1, human IgG3, mouse/human IgG1chimera Nmouse IgG2a⋙ human IgG2 and IgG4, mouse IgG1(Fig. 5A). We also compared two anti-EGFR therapeuticantibodies, cetuximab (mouse/human IgG1 chimera) andpanitumumab (human IgG2), using EGFR+ A431 target cells(Fig. 5B). Cetuximab, but not panitumumab, is reported toefficiently bind to FcγRIIIa and initiate ADCC (Patel et al., 2010).Our data showed that cetuximab induced a robust, dose-dependent, ADCC reporter signal. In contrast, panitumumabdid not induce any reporter signal under equivalent assayconditions. Altogether, these data are in complete agreementwith the reported binding affinities of IgG isotypes to humanFcγRIIIa (Jiang et al., 2011; Patel et al., 2010), and support thehypothesis that frozen Jurkat effector cells have the appropriateIgG isotype-dependent effector cell function in ADCC pathwayactivation.

2.7. Evaluation of stability-indicating property of the ADCCreporter assay

We evaluated whether the ADCC reporter assay usingfrozen, thaw-and-use effector cells provides sufficient assaysensitivity to detect potency changes due to heat stress (Fig. 6).Rituximab and trastuzumab were incubated at 65 °C fordifferent periods of time up to five days, before being testedin the assay. Extended heat-treatment significantly impactedantibody stability and decreased antibody potencies forrituximab, as indicated by protein precipitation from visualobservation, and a time-dependent right shift of EC50 anddecrease of maximum FI as shown in ADCC reporter assay(Fig. 6A). A time-dependent, moderate but consistent increaseof EC50 and decrease of maximum FI were also seen fortrastuzumab during heat treatment (Fig. 6B). For bothantibodies, time-dependent degradation or aggregation ofantibody was confirmed using size exclusion HPLC (data notshown). These results indicate that the ADCC reporter assay issensitive to loss of antibody activity due to heat stress, and thuscan potentially be used in stability studies for therapeuticantibody drug development.



Fig. 5.Antibody IgG isotype-specificity. ADCC reporter assay using frozen, thaw-and-use Jurkat effector cellswas carried out as described in “Materials andmethods” forsuspension or adherent target cells. (A) ADCC reporter assay response to various IgG isotypes of anti-CD20 rituximab using CD20+ WIL2-S target cells. The therapeuticbiologic, mouse/human IgG1 rituximab (Trade name: Rituxan) was included as positive control. (B) ADCC reporter response to anti-EGFR antibody cetuximab orpanitumumab using EGFR+ A431 target cells. Results shown were representative data from at least two independent experiments.


2.8. Correlation of antibody biological activity with antibodyglycosylation in ADCC reporter assay and a PBMC-based ADCCassay

Antibody N-glycosylation at amino acid 297 is wellknown as a critical requirement for antibody binding to Fcγreceptors to elicit effector functions including ADCC (Becket al., 2008; Chung et al., 2012). Evaluation of the impact ofantibody N-glycosylation on ADCC was performed using theADCC reporter assay with thaw-and-use effector cells and aPBMC-based classic ADCC assay using LDH release as read-out (Fig. 7). N-glycosylated trastuzumab was treated withPNGase F to generate deglycosylated trastuzumab, whichwas confirmed by size shift in SDS-PAGE (data not shown).Untreated and deglycosylated trastuzumab showed equiva-lent binding affinity to HER2+ SK-BR-3 by FACS analysis(Fig. 7A), indicating that deglycosylation by PNGase Ftreatment did not affect Fab binding ability of trastuzumabto the HER2 antigen receptors on SK-BR-3 cells. When testedin ADCC reporter assay and a PBMC-based ADCC assay,untreated trastuzumab gave a robust ADCC response andshowed similar antibody potency in both assays (EC50 = 9.8 ×10−9 g/ml in ADCC reporter assay, EC50 = 7.5 × 10−9 g/mlin PBMC-based ADCC assay). In contrast, deglycosylatedtrastuzumab almost completely lost its Fc effector function in

Fig. 6. Evaluation of stability-indicating properties of the ADCC reporter assay. The ADC“Materials and methods” for suspension or adherent target cells. (A) ADCC reporter asvarious durations as indicated; target cells wereWIL2-S. (B) ADCC reporter assay respodurations as indicated; target cells were SK-BR-3. Results shown were representative


ADCC, as shown in both ADCC assays (Fig. 7B, C), confirmingthe critical role of antibody glycosylation in an antibody's ADCCeffector function. This result also demonstrated that the ADCCreporter assay possess the capability to detect loss of functionthat is biologically relevant.

A panel of antibody samples with a defined percentagerange of N-glycosylated trastuzumab, from 10% to 50%, wasprepared by mixing untreated trastuzumab (N-glycosylated)with fully deglycosylated trastuzumab. Each antibody samplewas tested in the ADCC reporter assay and classic PBMC-basedADCC assay, with untreated trastuzumab included in each plateas reference. The ratio of EC50 between the reference andeach antibody test sample was calculated to represent thepercentage of ADCC biological activity for that antibodysample relative to trastuzumab reference. A linear correla-tion between percentage of ADCC biological activity andpercentage of N-glycosylated antibody in a mixture wasobserved in the ADCC reporter assay with good assayprecision obtained from multiple assay runs using samebatch of frozen Jurkat effector cells (Fig. 7D). A similarcorrelation was also observed in the PBMC-based ADCCassay, but with large run-to-run variation due to the use ofprimary effector cells isolated from different donors for eachrun. These data showed that both ADCC assays exhibitedsimilar correlation of antibody N-glycosylation with ADCC

C reporter assay using frozen Jurkat effector cells was carried out as described insay response to a panel of heat-stressed rituximab samples treated at 65 °C fornse to a panel of heat-stressed trastuzumab samples treated at 65 °C for variousdata from at least two independent experiments.



Fig. 7. Analysis of glycosylation-dependent ADCC Fc effector function of trastuzumab. Deglycosylated trastuzumab was generated by treating trastuzumab(N-glycosylated) with PNGase F, as described in “Materials and methods”. (A) FACS analysis of antibody binding to antigen HER2 on SK-BR-3 cells for untreated(control) or PNGase F-treated trastuzumab. (B, C) Untreated and deglycosylated trastuzumab were tested in ADCC reporter assay using frozen Jurkat effector cells orPBMC-based ADCC assay, as described in “Materials andmethods”. SK-BR-3 cells were used as target cells in both assays. Results shownwere representative data fromat least two independent experiments. (D) Correlation of percentage of ADCC biological activity with percentage of N-glycosylated trastuzumab in antibody blendedmixtures, by both ADCC reporter assay and PBMC-basedADCC assay. Blended trastuzumab sampleswith various percentage of trastuzumab (N-glycosylated) from10%to 50%, were prepared as described in “Materials andmethods”. For the ADCC reporter assay, results aremean± SD of three independent experiments using the samebatch of frozen, thaw-and-use Jurkat effector cells. For PBMC-based ADCC assay, results are mean ± SD of three independent experiments using PBMCs isolated fromdifferent donors. Percentage of ADCC biological activity is defined as the ratio of the EC50 of untreated trastuzumab (N-glycosylated) to the EC50 of the blendedtrastuzumab mixture placed in same assay plate.


biological activity, and that ADCC reporter assay showedgreat run-to-run assay precision.

3. Discussion

In recent years especially with tighter regulatory guidance,a variety of new strategies and improvements have beenadopted by biotherapeutic drug developers as to how cells arehandled as critical reagents in cell-based bioassays. Cell lineshave been substituted over primary cells, often stably express-ing recombinant receptors or reporters and using sensitivepathway readouts (Schnueriger et al., 2011; Parekh et al.,2012). Specially engineered cell lines have been developed foreasier manageability, such as mutants that exhibit lessclumping (Gazzano-Santoro et al., 1999). Pre-primed cellshave been cryopreserved in readiness for brief propagation forbioassay, such as those prepared for a nerve growth factorbioassay (Rukenstein and Greene, 1983). Frozen, ready-to-plate cells have been produced as cell banks for plating inassays after centrifugation and washing for both cell prolifer-ation and reporter gene bioassays (TerWee et al., 2011). In drug


development, banks of cell vials are frequently developed andpropagated for bioassay from well-characterized working andmaster cell banks. All these approaches can improve bioassayprecision and greater assay consistency.

For cell-based ADCC assays, classic PBMC or subset NK cell-based ADCC assays are technically challenging. Several reportson modified ADCC assays using engineered cell lines to replaceprimary PBMCs as effector cells (Schnueriger et al., 2011;Binyamin et al., 2008; Parekh et al., 2012) demonstrate bothimproved assay precision and overall equivalence to classicADCC assay in detection of ADCC activity changes due todifferences in antibody fucosylation level when evaluated inanti-CD20 model systems (Schnueriger et al., 2011; Binyaminet al., 2008; Parekh et al., 2012). In our approach reported here,we implemented several additional strategies to minimize andcontrol effector cell variability and generate a robust reporter-based surrogate ADCC assay. These included developing anengineered Jurkat effector cell line to replace primary effectorcells, creating banks of frozen thaw-and-use effector cells forimmediate use in assay without the need of cell propagation,and eliminating cell washing or centrifugation before each




assay run. The frozen, thaw-and-use Jurkat effector cells do notclump and are easy to handle for passage and assay. Weestablished standard protocols for cell propagation, harvest,and cryopreservation to make large batches of cell banks withconsistent cell viability and assay performance. We alsooptimized cell handling procedures for thawing cells andpreparing cell suspensions for assay. Additionally, we devel-oped a simple, optimized assay protocol for the bioassay itselfin order to obtain robust and consistent assay response. Byperforming all of the above, we have taken control of theeffector cells as a critical reagent, to provide precision andconvenience to an ADCC bioassay.

We show that the ADCC reporter assay with thaw-and-useeffector cells can be used for potency measurement formultiple therapeutic antibodies targeted to both hematopoieticand solid tumors. The fact that antibody potencies obtained inthe assay are in close alignment with those reported in classicPBMC-based ADCC assays demonstrates that the ADCC reporterassay provides appropriate ADCC biological activities fortherapeutic antibodies. We believe that this robust ADCCassay can be used for drug development for a wide range ofbiosimilars, and new generation therapeutic antibodies.

We also demonstrate that the freezing process did notimpact the effector cell function for Jurkat effector cells. Theobservation that the thaw-and-use cells gave similar antibodyEC50 values as cells from continuous culture demonstrates thatthere is no shift in the sensitivity of the cells when used directlyfrom the newly-thawed state. The ADCC reporter assay usingfrozen, thaw-and-use effector cells, has the appropriate IgGisotype selectivity and high specificity. One differencewe notedfrom assays using frozen effector cells was that it gave fasterand higher ADCC reporter response than assay using fresheffector cells. This difference may be due to an NFAT pathway-specific response in Jurkat cells to cryo-reagent components, orto the freezing process, or a combination of both, as we did notobserve the same enhanced reporter response in frozen Jurkatcells for reporter assays using other response elements(unpublished results). The exact cause of the faster ADCCreporter response in frozen Jurkat effector cells is not clear, andremains to be further investigated.

Using frozen, thaw-and-use Jurkat effector cells providesabundant benefits when building quantitative potency bioas-says. Cell passage number and cell growth conditions are well-known variants in traditional cell-based assays when usingcells from continuous culture. Through our manufacturingprocess control, frozen effector cells are produced in largebatches and at controlled passage numbers. This eliminatesassay variation caused by cell culture and cell passage number.The availability of large banks of cells provides researchers theconvenience of running an assay without pre-assay cellplanning or cell staging. Availability of same batches of cellreagents facilitates assay transfer between different laboratorysites. Bulk production of the cells as reagents and stored frozenas assay banks reduces development costs in the long-term,saving time and labor otherwise required for daily cell cultureto supply fresh cells for assays. The reporter-based format andsimple homogeneous nature of the ADCC reporter assaysimplifies procedure and reduces assay variability. The conve-nience and simplicitymakes this assay automation friendly andhas potential for use in high-throughput antibody screeningand assays for neutralizing antibodies. We have in fact


successfully developed the assay in 384-well format (data notshown). An additional advantage is assay response andsensitivity due to the inherent broad dynamic range ofluminescent readout from the effector cells. This should allowfor the detection of antibody Fc effector function in ADCC forthose target cells that are more resistant to cell lysis and thusgive poor assay response in classic ADCC assays. We believethat this is the first report of an ADCC bioassay using frozen,thaw-and-use cells that are simply thawed immediately priorfor assay use, and that there is great potential for use of frozen,thaw-and-use cells in many other cell-based bioassays. Usingsimilar cell freezing and handling strategies, we were able togenerate two B-cell lines in frozen, thaw-and-use format, andthey showequivalent target cell activity in ADCC reporter assaywhen compared with cells from continuous culture (data notshown). We have applied the same assay concept to build aJurkat effector cell line expressing F158 variant of FcγRIIIa inorder to better evaluate the impact of FcγRIIIa polymorphismon therapeutic antibody's ADCC response. We are also able todevelop the F variant Jurkat effector cells in frozen, thaw-and-use format (work in progress).

Antibody development and manufacture has many stagesrequiring a quantitative ADCC assay with accuracy, precision,and good sensitivity. One important application of a quantitativeADCC assay is in antibody stability studies. Regulatory agenciesrequire data to support claims of storage conditions and tojustify process changes for therapeutic antibodies, but theclassic PBMC-based ADCC assays are not able to serve thisfunction effectively due to high assay variability. We demon-strate here that the ADCC reporter assay using frozen, thaw-and-use effector cells provides good intra-assay precision andinter-assay precision and shows low assay variability. The assayalso has sufficient sensitivity in detecting loss of ADCC biologicalactivity due to heat stress for two therapeutic antibodies,rituximab and trastuzumab. This provides an assay with highprecision that is also stability-indicating, demonstrating poten-tial suitability for use in antibody stability studies.

Another important application of a quantitative ADCC assayis its ability to appropriately detect changes in ADCC biologicalactivity due to antibody structure changes such as differences inantibody glycosylation or amino acid sequence (Jiang et al.,2011; Beck et al., 2008; Chung et al., 2012). It is well establishedthat the precise structure of N-linked oligosaccharides at N297in therapeutic antibodies determines the binding affinity of theIgG to Fcγ receptor, and thus plays a critical role in the Fceffector function of the antibodies. Many glyco-engineered oramino acid-engineered therapeutic antibodies are currently indevelopment in an effort to increase ADCC activity over existingantibody drugs. It is clearly important to have a quantitative andprecise ADCC potency assay to determine improvements inADCC activity for next-generation antibody drugs. For existingantibody therapeutics, the level of antibody glycosylation needsbe tightly controlled and monitored during antibody manufac-ture to ensure optimal drug efficacy and safety. However, batch-to-batch variations using mammalian cell systems in glycanprofiles are common issues in antibodymanufacture. One of thereasons is a lack of a sensitive and quantitative ADCC potencyassay to monitor and better understand the impact ofmanufacturing processes on antibody glycosylation profiles.Recently, Chung et al. compared various in vitro ADCC assaysusing different effector cells: primary effector cells (PBMCs or




NKs), engineered NK cell lines and the engineered Jurkatreporter cell line that is described here. The article reported onlinear correlation between the reporter cell line assay responsesand the amount of afucosylated antibody in the test samples andthat this correlation was similar to that observed in a previousstudy of theirs using PBMCs as effectors (Chung et al., 2014). Inour current study, when we tested a panel of glyco-modifiedtrastuzumab samples whichwere differentmixtures of untreat-ed and deglycosylated trastuzumab, we were able to showlinear correlation of antibody N-glycosylation with ADCCbiological activity in the ADCC reporter assay using the samebatch of frozen, thaw-and-use Jurkat effector cells providedconsistent run-to-run results. We also obtained a similarcorrelationwhenwe tested the same panel of antibody samplesin a classic PBMC-based ADCC assay. However, the results werehighly variable from run-to-run because of the need to use freshPBMCs from different donors; the PBMCs from some donorsshowed little ADCC activity and the results from thoseexperiments were not included in Fig. 7D. Thus, the ADCCreporter assay using frozen, thaw-and-use effector cells pro-vides much better day-to-day assay precision in identifying thebiological impact of antibody glycosylation than classic PBMC-based ADCC assays. In our glycosylation studies we alsoobserved decreased signal (efficacy) in addition to EC50 changeswith increased percentage representation of non-glycosylatedtrastuzumab. Since Fc-deficient deglycosylated trastuzumab canstill efficiently bind to HER2 antigen on target cells, thedecreased assay response in the blended antibody samplesmay be also due to competitive binding to HER2 antigen ontarget cells between deglycosylated samples and untreatedantibody samples, in addition to the impact of the attenuated Fcbinding affinity to Fc receptors on effector cells.

Altogether, our results demonstrate that the ADCC reporterassay using frozen, thaw-and-use effector cells has potential tofulfill gaps in therapeutic antibody drug development where aquantitative and precise ADCC potency bioassay is needed,which the classic PBMC-based ADCC assays are not able tomeet. It can be used for antibody characterization, stabilitystudies and potentially as a starting point for development ofQC lot release assays.

4. Materials and methods

4.1. Reagents

Anti-CD20 antibody B1 was from Beckman Coulter. Ritux-imab and trastuzumab were from Genentech, cetuximab fromEli Lilly, and panitumumab from Amgen. Various rituximab IgGisotypes were obtained from InvivoGen. Anti-CD16 3G8antibody and goat anti-human IgG (H + L) were obtainedfrom Thermo Scientific. Low IgG FBS (≤5 μg/ml IgG) was fromHyclone. PBMCs from different donors were from AllCells.PNGase F, Magne™ Protein G Beads, Bio-Glo™ Luciferase AssaySystem and CytoTox 96® Non-Radioactive Cytotoxicity Assaywere from Promega.

4.2. Cell lines and cell culture

Jurkat (clone E6-1), WIL2-S, Raji, Ramos, SK-BR-3, MCF-7,A431, and PC-3 cells were obtained from ATCC. Jurkat cellswere cultured in RPMI 1640 with 10% FBS supplemented with


sodiumpyruvate andMEMNon-Essential Amino Acids.WIL2-Sand Raji were maintained in RPMI 1640 with 10% FBS; Ramosin RPMI 1640 with 10% heat inactivated newborn calf serum;SK-BR-3 cells inMcCoy's 5AMediumwith 10% FBS;MCF-7 cellsin EMEMwith 10% FBS supplemented with 0.01mg/ml humanrecombinant insulin; A431 in DMEM with 10% FBS; PC-3 inF-12 K with 10% FBS.

4.3. Plasmid construction

The DNA sequence of human FcγRIIIa, 158 V allotype(accession number: NM_000569) was synthesized by DNA2.0,and was inserted into pF9A CMV hRluc-neo Flexi® Vector(Promega). The resulting construct was named as pF9AFcγRIIIa-V158. The DNA sequence of three copies of the NFATresponse element was synthesized and inserted into pGL4.14[luc2/Hygro] (Promega). The resulting construct was named aspGL4 [luc2/NFAT-RE/Hygro].

4.4. Generation of stable Jurkat effector cell line

Jurkat cells, clone E6-1,were co-transfectedwith pGL4 [luc2/NFAT-RE/Hygro] and pF9A FcγRIIIa-V158 by Nucleofection(Lonza). Stable clones were generated by hygromycin andG418 double selection and limiting dilution. The chosen stablecell clone was maintained with complete cell culture mediumsupplemented with hygromycin and G418.

4.5. Generation of frozen, thaw-and-use Jurkat effector cells

Jurkat effector cells grown from continuous culture wereharvested, and frozen in freezing medium containing RPMI1640, 5% DMSO, and 10% low IgG FBS at a density of 2 × 107/ml,unless otherwise indicated. Cells were then frozen in acontrolled rate freezer and stored in the vapor phase of liquidnitrogen before being used in ADCC reporter assay. Low IgG FBSwas used to minimize non-specific IgG binding to FcγRIIIaduring ADCC reporter assay.

4.6. Generation of heat-stressed antibodies

Rituximab and trastuzumab at 10 mg/ml were incubated at65 °C for 1, 3, and 5 days. The treated antibody samples werethen stored at 4 °C before assay. Antibody samples that werestored at 4 °C at all times, according to the manufacturer'srecommendation, were used as untreated control.

4.7 . Generation of deglycosylated trastuzumab

In brief, trastuzumab stock at 10 mg/ml was treated with0.1 mg/ml PNGase F at 37 °C overnight. The antibody in thereaction was then purified and concentrated with Magne™Protein G Beads according to the manufacturer's instructions.The protein concentration of treated trastuzumab was quanti-fied by spectrophotometer at A280.

4.8. FACS analysis

SK-BR-3 cells were incubated with 1 μg/mL untreated ordeglycosylated trastuzumab for 60 min at 4 °C in FACS buffer(PBS with 0.5% BSA). The cells were then incubated with




fluorescein-conjugated goat anti-human IgG (H+L) secondaryantibody before being analyzed on a Guava easyCyte 8HT FlowCytometer (Millipore). The relative fluorescence was deter-mined by excitation at 488 nm using 525/30 nm band passfilters.

4.9 . Preparation of antibody blended mixture with untreated anddeglycosylated trastuzumab

Untreated and PNGase F-treated deglycosylated trastuzumabwere prepared in ddH2O at 0.5 mg/ml. The antibody blendedmixture containing 10%, 20%, 30%, 40%, or 50% of untreatedtrastuzumab (fully glycosylated) were then prepared by mixingthe untreated and PNGase F-treated trastuzumab in theappropriate proportions.

4.10. ADCC reporter assay

All assays were run in 96-well flat, white bottom plates. Alltarget cells used in the assays are freshly harvested fromculture. Suspension target cells were plated on the day of assayin assay buffer (RPMI 1640 with 4% low IgG FBS). Serialdilutions of antibodies were then added to the plates. Adherenttarget cells were plated the day before the assay in culturemedium. The medium was then replaced with assay buffer onthe morning of assay, and followed by the addition of serialdilutions of antibodies. The E:T ratio is 6:1 for suspension targetcells, and 15:1 for adherent target cells based on the target cellnumber at the time of plating. After induction at 37 °C asindicated time, Bio-Glo™ Luciferase Assay Reagent was added,and luminescence was determined using a GloMax®-MultiDetection System (Promega).

For ADCC reporter assay using fresh Jurkat effector cells, theJurkat effector cells from continuous culture were harvested,and resuspended in assay buffer (RPMI 1640 with 0.5% low IgGFBS) on the day of assay. Cells were then added for 150,000cells per well to assay plates already containing target cells andantibodies. The assay plates were incubated in a CO2 incubatorat 37 °C overnight or as otherwise indicated before addition ofBio-Glo™ Luciferase Assay Reagent. For the competitionexperiment using anti-CD16 antibody, fresh Jurkat effectorcells were pre-incubated with a titration series of blockingantibodies, mouse IgG1 as control or mouse anti-CD16antibody (3G8) before being added to assay plates alreadycontaining WIL2-S target cells and 5 × 10−8 g/ml anti-CD20antibody B1. Percentage of control was calculated as: % ofcontrol = 100 × (RLUblocking antibody + B1 − RLUbackground) /(RLUB1 alone − RLUbackground).

For ADCC reporter assay using frozen, thaw-and-use Jurkateffector cells, frozen Jurkat effector cells were thawed on theday of assay, and immediately resuspended in assay buffer(RPMI 1640 with 4% low IgG FBS). Cells were then added for75,000 cells per well to assay plates already containing targetcells and antibodies. The assay plates were incubated in a CO2

incubator at 37 °C for 6 h or as otherwise indicated, beforeaddition of Bio-Glo™ Luciferase Assay Reagent.

For data analysis, fold of induction was calculated asfollows: fold of induction = (RLUinduced − RLUbackground) /(RLUuninduced − RLUbackground). RLU or fold of induction wasplotted against Log10 [antibody concentration]. The doseresponse curve was fitted with a 4-parameter model using


GraphPad Prism® software. EC50 and maximum fold ofinduction (maximum FI) were determined after curve fitting.

4.11. ADCC assay

ADCC assay was performed using PBMCs as effector cells in96-well flat, clear bottom plates. Briefly, Fresh SK-BR-3 targetcells harvested from culture were plated in culture medium at10,000 cells per well on the day before assay and incubatedovernight in a CO2 incubator at 37 °C. On the day of assay, themedium was replaced with assay buffer (RPMI 1640 with 4%low IgG FBS) followed by addition of serial dilutions oftrastuzumab samples. PBMCs were then added to assay plates,at a density of 500,000 cells perwell. After overnight incubationin a CO2 incubator at 37 °C, target cell lysis was measured bydetecting the release of lactate dehydrogenase (LDH) fromlysed SK-BR-3 cells using CytoTox 96® Non-RadioactiveCytotoxicity Assay according to the manufacturer's instruc-tions. The plates were read on a Spectra Max (MolecularDevices) plate reader for absorbance at 490 nM. SpontaneousLDH release was measured in wells containing target andeffector cells without antibody. Maximal LDH release wasmeasured in wells containing target cells completely lysed bylysis buffer supplied in the assay kit.

For data analysis, the extent of specific ADCCwas calculatedas follows: % lysis= (experimental LDH release− spontaneousLDH release) / (maximal LDH release − spontaneous LDHrelease) × 100. The % lysis was plotted against Log10 [antibodyconcentration]. The dose response curve was fitted with a4-parameter model using GraphPad Prism® software. EC50

was determined after curve fitting.

Acknowledgments

The authors would like to thank Tom Lubben for establishingmethods and performing the size-exclusion HPLC confirmationof antibody degradation and/or aggregation upon heat treat-ment, and Neal Cosby for critical review and comments on themanuscript.

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Documents

Development of a robust reporter-based ADCC assay with frozen, thaw-and-use cells to measure Fc effector function of therapeutic antibodies