Journal of Immunological Methods 389 (2013) 52–60

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Journal of Immunological Methods

j ourna l homepage: www.e lsev ie r .com/ locate / j im

Research paper

Development of an enzymatic assay for the detection of neutralizingantibodies against therapeutic angiotensin-converting enzyme 2 (ACE2)

Karen Liao, Daniel Sikkema, Catherine Wang, Thomas N. Lee⁎Clinical Immunology, GlaxoSmithKline, King Of Prussia, PA 19406, United States

a r t i c l e i n f o

⁎ Corresponding author at: GlaxoSmithKline, 709 SwE-mail address: Thomas.n.lee@gsk.com (T.N. Lee).

0022-1759/$ – see front matter © 2013 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.jim.2012.12.010

a b s t r a c t

Article history:Received 14 September 2012Received in revised form 6 December 2012Accepted 10 December 2012Available online 5 January 2013

Therapeutic proteins have the potential to elicit immune responses in animals and humans(Mire-Sluis et al., 2004; Yu et al., 2006; Shankar et al., 2008). Contributors to the responsecould include product related factors such as chemical modifications, impurities that co-purifywith product, contaminants, formulation, aggregates, and clinical factors such as doseconcentration, dosing frequency, route of drug administration, rate of administration, patientunderlying disease, concomitant medication, and genetic status among others (Patten andSchellekens, 2003). Further, an immune response triggered by a therapeutic enzyme mayneutralize the endogenous counterpart resulting in a decrease or depletion of the therapeuticand endogenous enzymes imposing safety concerns for patients. Therefore, monitoring ofanti-drug antibody (ADA) and neutralizing antibody (NAb) responses to both the recombinanttherapeutic enzyme and endogenous enzyme is important during early development andsubsequent clinical studies. Testing considerations for NAb detection against therapeuticenzymes have been published mostly for lysosomal storage diseases (Wang et al., 2008). NAbcross-reactivity to the endogenous counterpart has also been characterized (Sominanda et al.,2010). Here, we describe an enzymatic NAb assay which detects neutralizing antibodies toboth recombinant and endogenous angiotensin-converting enzyme 2 (ACE2). NAb assaysensitivity was optimized by selecting the assay incubation time as 20 min with an enzymeconcentration of 0.5 μg/mL. Four anti-ACE2 antibodies out of a commercial panel of 18 werefound to have neutralizing capabilities based upon their ability to abrogate ACE2 enzymaticactivity. We demonstrated assay specificity by small peptide inhibitors specific for ACE orACE2. DX600, an ACE2 specific inhibitor did not cross-react with ACE. Conversely, captopril, aninhibitor of ACE did not inhibit ACE2. The assay specificity for ACE2 neutralizing antibodieswas further demonstrated by the lack of reactivity of two species control antibodies and 14anti-ACE2 antibodies. Moreover, we demonstrated assay specificity to human endogenousACE2 from human epithelial cells. Three human cell lines (Calu-3, Caco-2, Huh-7) wereevaluated for the cell surface expression of ACE2 by flow cytometry and Western blot.Subsequently, whole cell lysates, cell culture supernatant, and live cells were evaluated in theassay. Results demonstrated that Calu-3 had elevated levels of ACE2 compared to Caco-2 orHuh-7. Calu-3 also demonstrated elevated ACE2 enzymatic activity in all three sources andcould be inhibited by the ACE2 specific inhibitor DX600 as well as the neutralizing antibodiesfor the recombinant ACE2. Thus, we describe here a method to detect NAb against atherapeutic enzyme and assess NAb cross-reactivity to the native endogenous enzyme. Theapproach of method development described here could be applied for the assessment of NAbresponses to other enzymatic therapeutics.

© 2013 Elsevier B.V. All rights reserved.

Keywords:ACE2Neutralizing antibodyNAb assayCalu-3ALI/ARDS

edeland Road, King Of Prussia, PA 19406, United States. Tel.: +1 610 270 4238; fax: +1 610 270 5155.

ll rights reserved.

53K. Liao et al. / Journal of Immunological Methods 389 (2013) 52–60

1. Introduction

ACE and ACE2 are part of the renin-angiotensin system(RAS) which controls blood pressure, electrolytes, and intra-vascular fluid volume. Increased ACE activity has beenassociated with acute lung injury (ALI) and acute respiratorydistress syndrome (ARDS) (Cooke et al., 2002). ACE2, ahomolog to the carboxypeptidase ACE, functions as a counter-regulatory mechanism and thus may be a potential therapeuticfor ALI/ARDS patients. ACE2 counterbalances the multiple func-tions of ACE by cleaving a single amino acid from angiotensin II(Ang II), forming Ang 1-7. Ang 1-7 has been associated withanti-inflammation and vasodilation. In the ARDS rat model, ACEactivity was enhanced along with reduced levels of Ang 1-7.Therapeutic intervention with Ang 1-7 attenuated the inflam-matory mediator response, markedly decreased lung injuryscores, and improved lung function (Wosten-van Asperen et al.,2011). Thus, ACE2 offers a promising novel treatment modalityfor patients. However, the therapeutic enzyme may elicit animmune response of anti-drug antibodies (ADA) within dosedsubjects (Mire-Sluis et al., 2004; Yu et al., 2006; Shankar et al.,2008). The presence of ADA could have multiple outcomeswhich could include: no impact, hypersensitivity/anaphylaxis,reduced bioavailability, or reduced clinical efficacy (Patten andSchellekens, 2003). In addition, an ADA response could cross-react and neutralize the native endogenous counterpart leadingto the generation of autoimmunity. Thus, vigorous immunoge-nicity evaluation plans are warranted for such a therapeuticenzyme like ACE2, including the assessment of ADA/NAb cross-reactivity to native and the recombinant enzyme (Wang et al.,2008; Sominanda et al., 2010).

Cell-based or ligand binding assays are currently acceptedmethods for detecting neutralizing antibodies (NAb) directedagainst therapeutic antibodies and proteins. We previouslypublished a cell-based method for NAb directed against amonoclonal antibody therapeutic (Liao et al., 2012). Here, wedescribe a method to determine NAb presence directed againstthe therapeutic enzyme rhACE2 and cross-reactivity with theendogenous counterpart enzyme. An enzymatic activity assaywas chosen since this format directly assesses the catalyticactivity of ACE2 and neutralization capacity of anti-ACE2antibodies. We optimized NAb sensitivity by evaluating assayincubation time and enzyme concentration. We characterizedenzyme neutralization with commercially available antibodiesand a hyperimmunized monkey serum. We determined theassay to be ACE2 specific by evaluating enzyme specific in-hibitor and also by determining that the homolog ACE was notreactive under these conditions. We further selected a humancell line with ample ACE2 expression and demonstrated thatthe positive control anti-ACE2 monkey serum was cross-reactive to human native endogenous ACE2. The assay wasvalidated according to FDA guidance (FDA, 2009) and deemedsuitable for clinical sample testing.

2. Materials and methods

2.1. Cell lines, reagents, ACE2 antibodies

Calu-3 (a human lung epithelial cell line), Caco-2 (humanepithelial colorectal adenocarcinoma cell line), and Huh-7(human hepatic cell line), were purchased from ATCC. Heat-

inactivated fetal bovine serum (FBS) was purchased from SAFCBiosciences (Lenexa, KS). Dulbecco's Modified Eagle Medium(high glucose) (DMEM), Eagle's minimal essential medium(EMEM), PBS and Trypsin-EDTA were obtained from InvitrogenCorp. (Carlsbad, CA). Recombinant ACE2 was provided byGlaxoSmithKline BioPharm unit. ACE2 substrate Mca-APK-Dnpand ACE2 specific inhibitor (DX600) were purchased fromAnaSpec (Fremont, CA). Recombinant ACE was purchased fromR&D systems (Minneapolis, MN). ACE inhibitor, captopril waspurchased from Sigma (St. Louis, MO). Normal human sera werepurchased from Bioreclamation (Hicksville, NY). The ACE2antibodies and the sources were listed in Table 1.

2.2. Measurement of cellular ACE2 by Western blotting and flowcytometry

Confluent cells were trypsonized and collected by centri-fuging at 2000 rpm for 10 min. Cell pellets were washed oncewith cold phosphate-buffered saline and lysed with Mamma-lian Protein Extraction Reagent (M-PER, Thermo Scientific)containingprotease inhibitor (Roche) on ice for 20 min. Lysateswere centrifuged at 12,000 rpm for 10 min. The supernatantwas collected and protein concentrations were determined byBCA kit (Pierce) using bovine serumalbumin as standard. 50 μgof cell lysate, 32.5 μL cell culture supernatant, and 1 ng rhACE2protein were resolved by NuPAGE 4–12% gradient Bis-Tris gelsin MOPs buffer then transferred to a nitrocellulose membranein 1× transfer buffer (Invitrogen)with 10% (v/v)methanol. Themembranewas saturatedwithOdyssey blocking buffer at roomtemperature for 30 min then incubated with anti-ACE2antibody (AF933, R&D systems) at room temperature for 1 h.IRDye680 (red, Li-Cor Bioscience) conjugated anti-goat sec-ondary was used at 1:7500 dilution and incubationwas 30 minat room temperature. The membrane was reblotted with anti-GAPDH (mAb374, Millipore) detected with IRDye800 (green,Li-Cor Bioscience) anti-mouse secondary antibody at 1:7500.Bound antibodies were detected by Odyssey.

Cell surface ACE2 was detected by flow cytometry. Briefly,cells were fixed by 4% paraformaldelhyde on ice for 20 minthen washed twice in assay buffer (1× PBS with 1% BSA and2 mM EDTA). Cells were resuspended in assay buffer con-taining 1 μg/mL anti-ACE2 antibody (AF933, R&D systems) andincubated on ice for 30 min. Cells were washed twice with assaybuffer, resuspended in assay buffer containing 0.5 μg/mL PEconjugated donkey anti-goat (Abcam, Cambridge, MA) andincubated on ice for 20 min. Cellswerewashed and resuspendedfor flow cytometry analysis.

2.3. ACE2 enzymatic activity kinetics

The catalytic activity of recombinant ACE2 and cellularACE2wasmeasuredbyhydrolysis of a highly specific quenchedfluorescent substrate, 7-methoxycomarin-4-yl acetyl-Ala-Pro-Lys-(2,4-dinitrophenyl)-OH (Mca-APK-Dnp) (AnaSpec, Fre-mont, CA). rhACE2 was serial diluted with dilution buffer(100 mM Glysine, 50 μM ZnCl, 150 mM, 1% BSA) startingwith 5 μg/mL. The serial diluted enzyme was further diluted1:5 with activity buffer (150 mM NaCl, 75 mM Tris, 10 μMZnCl2, 0.01% Triton X-100, pH 7.2). 50 μL of the dilutedenzyme was transferred to a black 96-well plate, then 50 μLof substrate at 200 μM was added to each well (final

Table1

Anti-ACE

2an

tibo

dies

andthesources.Allan

tibo

dies

weretested

foran

ti-drugan

tibo

dy(A

DA)bind

ingactivity

inava

lidated

bridging

ADAassay(d

atano

tsh

own)

.

Antibo

dy1

23

45

67

89

1011

1213

1415

1618

Ven

dor

Abc

amR&

Dsystem

sNov

usBiolog

icals

AbD

seroTe

cEn

zoLife

Scienc

eCo

vanc

e

Cat#

ab15

350

ab38

888

ab38

941

ab76

526

ab15

347

ab78

512

ab89

111

AB9

896

MAB5

676

AF9

33MAB9

33MAB9

331

NBP

1-33

275

AHP8

88AC1

8FAC3

84n/a

Form

poly

poly

poly

poly

poly

mAb

mAb

poly

mAb

poly

mAb

mAb

poly

poly

mAb

mAb

poly

Binding

activity

ina

bridging

ADAassay

No

No

No

No

No

Yes

No

Yes

Yes

Yes

No

No

No

No

No

Yes

Yes

54 K. Liao et al. / Journal of Immunological Methods 389 (2013) 52–60

substrate concentration was 100 μM). The fluorescenceresulting from substrate hydrolysis was measured on FluRex(Molecular Devices, Sunnyvale, CA) within 60 min. All reac-tions were performed at room temperature in microtiterplates with a 100 μL total volume.

2.4. Cellular ACE2 enzymatic activity

Three cellular sources of endogenous ACE2 were evaluatedfor enzymatic activity: total cell lysates, cell culture superna-tant, and live cells. To prepare total cell lysates (TCL), cells werelysedwithM-PER lysis buffer (Pierce, Rockford, IL) containing aprotease inhibitor cocktail. Total protein wasmeasured by BCAprotein assay kit (Pierce). TCL was prepared at 4 mg/mL anddiluted 1:5 with activity buffer. 50 μL of the diluted cell lysatewas incubated with 50 μL of stock 200 μM substrate at 37 °Cfor 20 min.

Cell culture supernatant was collected after a 72 h incuba-tionwith cells reaching 90% confluence. ACE2 enzymatic activityfrom cell culture supernatant was measured with the samedilution scheme except the protein concentration in the super-natant was not determined.

To measure live cell ACE2 enzymatic activity, cells wereseeded at 1×105 per well in a 96-well cell culture plate,followed by overnight incubation at 37 °C. Supernatant wasremoved. Cells were then washed with 1× PBS and 50 μL ofactivity buffer was added to each well followed by addition of50 μL substrate. Cells were incubated at 37 °C for 20 min thenthe solution was transferred to a black plate for fluorescencereading on the Envision.

2.5. Neutralizing assay with recombinant enzyme

Serum samples or positive controls (PCs) were first pre-incubated with 0.5 μg/mL rhACE2 at room temperature for 1 hon a shaker (neutralization incubation). After the neutralizationincubation, the neutralization solution was 1:5 diluted withactivity buffer (150 mM NaCl, 75 mM Tris, 10 μM ZnCl2, 0.01%Triton X-100, pH 7.2) and then added to plates with 50 μL of200 mMsubstrate. The sample/substrate solutionwas incubatedat 37 °C for 20 min. Fluorescent signals were generated on theEnvision at an excitationwavelength of 320 nmand an emissionwavelength of 400 nm. Data was exported as .csv file to a studyfolder and all calculations were performed in Microsoft Excel orequivalent software. Data was normalized to the mean relativefluorescent units (RFU) of the negative control (NC: a normalhuman serum pool) and reported as the % maximum response.

2.6. Neutralizing assay with cellular ACE2 enzyme

To measure NAb activity against endogenous ACE2, therecombinant ACE2 was replaced by cellular ACE2. 100 μL of celllysate at 4 mg/mL or same volume of cell culture supernatantwas pre-incubated with either PCs at a final concentration of10 μg/mL, equal volume control serum, DX600, ACE2 specificinhibitor at final concentration of 10 μM, or equal volume ofcontrol buffer (PBS) at room temperature for 1 h. The solutionwas then diluted 1:5with activity buffer (150 mMNaCl, 75 mMTris, 10 μM ZnCl2, 0.01% Triton X-100, pH 7.2). The dilutedsolution was then added to a plate with 50 μL of 200 mMsubstrate. The sample/substrate solutionwas incubated at 37 °C

55K. Liao et al. / Journal of Immunological Methods 389 (2013) 52–60

for 20 min. For NAb assay with live cells, cells were seeded at1×105 per well in a 96-well cell culture plate followed byovernight incubation at 37 °C. Cell culture supernatant wasremoved from the plate. 100 μL of 10 μg/mL PC, 10 μM DX600,or respective controlwas added to eachwell of the cell plate andincubated at 37 °C with 5% CO2 for 1 h. The treatment solutionwas then removed from cell plate, and 50 μL activity buffer wasadded to eachwell and followed by addition of 50 μL of 200 mMsubstrate. The cell plate was incubated at 37 °C for 20 min, thensubstrate reaction solution from the cell platewas transferred toa black plate for fluorescence reading on the Envision.

2.7. Specificity and cross-reactivity

2.7.1. Enzyme specificityTo evaluate enzyme specificity of the NAb assay, various

concentrations of an ACE2 specific inhibitor (DX600) or an ACEspecific inhibitor (captopril) were pre-incubated with enzymefor 1 h at room temperature. The treated enzyme was thendiluted and mixed with substrate as described earlier. The platewas incubated at 37 °C for 20 min and fluorescencewas read onthe Envision. The enzyme specificity of endogenous ACE2 wasalso evaluated by the ACE2 specific inhibitor. Similarly, thecellular enzyme (total cell lysates, cell culture supernatant, orlive cells)was pre-incubatedwithDX600 or diluent for 1 h, thentreated total cell lysates or cell culture supernatants werediluted 1:5 with activity buffer and then 50 μL of the dilutedsamples was mixed with 50 μL substrate. For live cell activity,treatment solution was removed, 50 μL activity buffer wasadded to the cells and then 50 μL substrate was added. Enzyme/substrate plates were incubated at 37 °C for 20 min and thenfluorescence was read on Envision.

2.7.2. Enzyme and PC cross-reactivityTo evaluate enzyme cross-reactivity in the NAb assay,

homolog enzyme ACE was tested in parallel with ACE2 asdescribed in Section 2.5. To evaluate specificity for the PC, aspecies control sera and an irrelevant PC raised from the samespecies (Covance, Denver, PA) were tested in parallel with the

Fig. 1. ACE2 enzymatic activity. (A) ACE2 enzyme kinetics. Various concentrations (μgwas determined every 2 min for 60 min. (B) ACE2 enzymatic activity dose-response c

specific PC in the NAb assay with rhACE2 as described inSection 2.5.

3. Results and discussion

3.1. ACE2 enzymatic activity kinetics

A time-dependent and dose-dependent fluorescence accu-mulation resulting from substrate hydrolysis was observed(Fig. 1A). At an enzyme concentration of 1 μg/mL, fluorescentsignal reached a plateau in less than 10 min. As enzymeconcentrations decreased, the time to reach a signal plateauincreased. At concentrations of 0.09 μg/mL or below, thesubstrate hydrolysis response remained linear within 60 min.Based on the kinetics data, we selected 20 min as the assayincubation time. At the 20 min incubation time, enzymaticactivity showed a linear dose-response up to 0.3 μg/mL (finalassay concentration) or 1.5 μg/mL before dilution (Fig. 1B). Theselected enzyme concentration of 0.5 μg/mL (before dilution)for the NAb assay was within the linear range. Thus, the de-crease of fluorescence signal at this selected enzyme concen-tration reflected the decrease of ACE2 enzymatic activity andtherefore could be attributed to neutralizing activity of ADA inthe reaction. ACE2 neutralizing antibodies may bind to theenzyme catalytic domain or induce conformational changes andconsequently prevent enzymatic cleavage of substrate andtherefore reduce the fluorescent product. The selection ofenzyme concentration within the linear range of the responsecurve enabled NAb sensitivity.

3.2. Neutralizing activity of ACE2 antibodies

To effectively detect neutralizing activity, the anti-ACE2antibody and ACE2 need to remain complexed within theenzymatic reaction. Thus, optimal binding and enzymatic ac-tivity buffer conditions were characterized. To achieveoptimal binding and enzymatic activity, various assaybuffers were evaluated for both binding and enzymaticactivity. The buffer containing 150 mM NaCl, 75 mM Tris,

/mL) of rhACE2 were diluted 1:5, then 1:1 mixed with substrate. Fluorescenceurve at 20 min incubation.

56 K. Liao et al. / Journal of Immunological Methods 389 (2013) 52–60

10 μM ZnCl2, 0.01% Triton X-100, pH 7.2 was found to havebest profile in both binding and enzymatic activity (data notshown). Thus, this buffer was selected as neutralizing assaybuffer. To test if an anti-ACE2 antibody had neutralizingactivity, the antibody was pre-incubated with recombinantenzyme and then assayed for enzymatic activity as describedin Materials and methods section 2.5. We tested 18 anti-ACE2 antibodies including 17 commercial and one hyper-immunized monkey serum (Covance, Denver, PA) (Fig. 2A andB). Among the 18 antibodies, four antibodies showed neutral-izing activity. A dose-dependent decrease of enzymatic activitywas seen with increased concentrations of anti-ACE2 anti-bodies, indicating neutralizing activity. All four neutralizingantibodies showed ADA binding activity in a validated ADAassay (listed in Table 1). The four antibodies showing neu-tralizing activity were raised from four different species (goat,rabbit, mouse and monkey). One common characteristic of thefour antibodies was that these animals were immunized withectodomain or whole protein. The monkey serum was selectedas NAb assay PC. Based on the PC response curve, a high positivecontrol (2.5 μg/mL) and low positive control (0.5 μg/mL) wereselected for assay validation along with three other assaycontrols (enzyme, substrate background control, NC maximumresponse control). The assay was validated according to FDAguidance (2009) (data not shown), with a cut point establishedwithin normal human serum. The assay demonstrated accept-able sensitivity and precision, and was deemed suitable forclinical sample testing.

3.3. Matrix interference and minimum required dilution (MRD)

Matrix interference was evaluated by two methods. Oneexamined the maximum response (enzymatic activity in thepresence of serum or buffer) and the basal enzymatic activity.As demonstrated in Fig. 3A, the enzymatic activities from 10individual sera or pooled serum were similar to the activitiesobserved in assay diluent; and the basal responses resultingfrom endogenous ACE2, or other sources, were also similarbetween serum and diluent. Secondly, PC was spiked into 10individual sera, pooled serum, or assay diluent. As shown inFig. 3B, 2.5 μg/mL and 0.5 μg/mL PC spiked serum samplesdemonstrated no differences compared with assay diluent.

Fig. 2. Neutralizing activity of various anti-ACE2 antibodies. (A) Examples of antipre-incubatedwith 0.5 μg/mL rhACE2 for 1 h, then 1:5 diluted beforemixingwith subsmaximum response=100∗(RFUsample/RFUNC). (B) Response curve of the hyperimmu

Together, data indicated that the assay had equivalent PCdetection regardless of matrix and was devoid of interference.

To optimize assay MRD, the PC was tested neat or diluted1:2, 1:5, and 1:10 within buffer. Fig. 3C illustrates how thesame concentration of PC had the most inhibition within neatsamples, indicating assay sensitivity was decreased with anincreasing dilution factor. Based on these results, the assaywas performed by incubating neat serum with 0.5 μg/mL ofACE2. The serum/enzyme mixture was 1:5 diluted withactivity buffer then 1:1 mixed with substrate. The final serumconcentration within the enzymatic reaction was 10%.

3.4. Specificity and cross-reactivity

Enzymatic specificitywas demonstrated by DX600, anACE2specific inhibitor, and captopril, a homolog ACE specific in-hibitor. DX600 demonstrated a dose-dependent inhibition ofassay activity, while the ACE inhibitor captopril did not inhibitat 100 μM(Fig. 4A). Assay specificity was also demonstrated bysubstituting ACE2with the ACE enzyme. Equivalent amounts ofACE enzyme showed no signal, indicating that the substratecould not be cleaved by the homolog (Fig. 4B). The assayspecificity was further demonstrated by two species controlantibodies which did not inhibit enzymatic activity (Fig. 4C).The non-reactivity of 14 from the 18 various anti-ACE2 anti-bodies, including two antibodies which demonstrated bindingactivity to ACE2 in a validated bridging ADA assay (binding butnon-neutralizing) provided further evidence for the assayspecificity of the ACE2 NAb assay.

3.5. NAb assay with endogenous enzyme

Since there is an endogenous counterpart for the recombi-nant ACE2 therapeutic, we needed to evaluate the possibility ofneutralizing antibodies cross-reacting with endogenous en-zyme. We evaluated three human cell lines for ACE2 ex-pression. Evaluations with total cell lysates or cell culturesupernatants were performed by Western blot analysis. Cellsurface ACE2 expression was evaluated by flow cytometry.Among the three cell lines tested (Fig. 5A), Calu-3 had elevatedlevels of ACE2 within the total cell lysates relative to Caco-2cells. ACE2was not detected inHuh-7 cell lysates. In agreement

body response within the neutralizing assay. Serial diluted antibodies weretrate. Fluorescence activitywas detected after a 20 minute incubation at 37 °C. %nized monkey sera (PC) under the same assay condition as described in A.

Fig. 3. Matrix interference and minimum required dilution (MRD) determination. (A) Matrix interference — enzymatic activity in serum and assay diluent. 25 μL ofindividual serum, serum pool, or assay diluent was incubated with 25 μL of 0.5 μg/mL rhACE2 (maximum response) or assay diluent (basal response) at roomtemperature for 1 h. The serum/enzyme mixture was 1:5 diluted with activity buffer, then 1:1 mixed with substrate, incubated at 37 °C for 20 min, and fluorescencewas determined. (B) Matrix interference — PC performance in serum and assay diluent. PC was spiked in 10 individual sera, serum pool, or assay diluent at 2.5 and0.5 μg/mL. The PC spiked samples or unspiked samples were assayed as described in A. % maximum response=100∗(RFUspiked/RFUunspiked). (C) MRD determination.Serial diluted PC curve was further diluted with assay diluent at 1:2, 1:5, and 1: 10. Neat or diluted PC curve was incubated with 25 μL of 0.5 μg/mL rhACE2 at roomtemperature for 1 h, then analyzed for enzymatic activity as described in A.

57K. Liao et al. / Journal of Immunological Methods 389 (2013) 52–60

with the total cell lysate data, ACE2was also detected in Calu-3and Caco-2 cell culture supernatants, yet levels in Calu-3 cellculture supernatant were higher compared to those in Caco-2.ACE2 was not detected in Huh-7 cell culture supernatant.Moreover, cell surface ACE2 was also detected on Calu-3 cells(Fig. 5B), but not on Caco-2 or Huh-7 cells (data not shown).

To demonstrate if endogenous ACE2 could be incorporatedinto the NAb assay, enzymatic activity was assessed from allthree cellular sources: whole cell lysates (Fig. 6A), cell culturesupernatant (Fig. 6B), and live cells (Fig. 6C). All sourcesgenerated signal within the assay, and enzyme specificity wasdemonstrated by ACE2 specific inhibitor (DX600). Also, thehyperimmunized monkey serum PC was evaluated for NAbcapabilities on these endogenous sources. As shown in Fig. 6A–C, the elevated enzymatic activity was seen with all threesources of Calu-3 and the activity could be inhibited by DX600,suggesting ACE2 specific enzyme activity. PC also demonstratedenzymatic inhibition on all Calu-3 sources similar to 10 μMDX600. However, live cells demonstrated the best inhibitoryprofile fromPC among the three sources and it also provided themost convenient way to carry out the assay. We consistentlyobserved that themaximum % inhibition by the PC ranged from50% to 80% for the endogenous enzyme compared to approx-imately 100% inhibition by PC with recombinant ACE2. Theincomplete inhibition of cellular activity, by either specific

inhibitor or PC, may have been attributable to non-specificsubstrate cleavage other than ACE2 that could not be inhibited.

3.6. PC neutralizing activity with endogenous enzyme

As demonstrated above, Calu-3 live cells were the optimaland most convenient source to measure endogenous ACE2enzymatic activity. To demonstrate NAb cross-reactivity withendogenous enzyme, the PC neutralizing potential was evaluat-ed using live Calu-3 cells and compared to NAb neutralizedrhACE2. As shown in Fig. 7, a similar neutralizing curve wasobserved with live Calu-3 cells compared to rhACE2. The PCneutralizing activity was slightly more potent against endoge-nous ACE2 as same concentrations of PC showed lower values of% maximum response against endogenous ACE2 compared torhACE2 at PC concentrations below 1250 ng/mL. The EC50 withCalu-3 live cells was 0.35 μg/mL compared to 1.08 μg/mL withrhACE2. Themaximum inhibition (10,000 ng/mL) achievedwithlive Calu-3 cells was 62% compared to 90% in rhACE2 for thereason discussed in Section 3.5.

4. Conclusion

In summary,we have developed andoptimized an enzymaticassay for the detection of neutralizing antibodies directed against

Fig. 4. Assay specificity. (A) Enzyme specificity by specific inhibitor. 25 μL of 0.5 μg/mL rhACE2 was mixed with 25 μL of 2× inhibitors and incubated at roomtemperature for 1 h. The enzyme/inhibitor mixture was 1:5 diluted with activity buffer, then 1:1 mixed with substrate, incubated at 37 °C for 20 min, andfluorescence was determined. (B) Enzyme cross-reactivity. 25 μL of 0.5 μg/mL rhACE2 or rhACE was mixed with 25 μL of serial diluted PC, incubated at roomtemperature for 1 h, then analyzed for enzymatic activity by the same dilution scheme as described in A. (C) PC cross-reactivity. Specific or control PC wasincubated with rhACE2 at room temperature for 1 h, then analyzed for enzymatic activity.

58 K. Liao et al. / Journal of Immunological Methods 389 (2013) 52–60

the ACE2 enzyme. The assay provided a direct measurement ofACE2 catalytic activity for the detection of NAb in clinicalsamples. The assay was specific in that it did not demonstrate aneutralizing response to anti-drug antibodies not directed toACE2, and the protein homolog ACE was inactive within theassay. The specificity for neutralizing antibodies was also

Fig. 5. ACE2 expression in three human cell lines. (A) Western blot with anti-ACE2 (A

demonstrated by profiling the reactivity of 18 ACE2 specificantibodies. Among the 18 antibodies, only four showed NAbactivity and all four demonstrated binding activity in a validatedADA assay. The remaining 14 antibodies showed no NAbactivity, but two did have ADA binding activity. We alsodemonstrated a method to determine if NAb cross-reactivity

F933). (B) Calu-3 cell surface ACE2 expression by flow cytometry with AF933.

Fig. 6. Enzymatic activity of various sources of endogenous ACE2. (A) Endogenous ACE2 activity in total cell lysates. 100 μL of cell lysate was pre-incubated withPBS, DX600, control serum, or PC at room temperature for 1 h. The final concentration was 10 μM for DX600 and 10 μg/mL for PC. The pre-incubated cell lysateswere 1:5 diluted with activity buffer, then analyzed for enzymatic activity. (B) Endogenous ACE2 activity in cell culture supernatant. 100 μL of cell culturesupernatants was pre-incubated with the same concentrations of DX600, PC, or controls as described in A. (C) Endogenous ACE2 activity in live cells. 100 μL ofmedium, 10 μM DX600, control serum, or 10 μg/mL PCs were added to cells in a 96 well plate, incubated at 37 °C for 1 h, then the treatment solution wasremoved. 50 μL activity buffer was then added, followed by addition of 50 μL substrate. Fluorescence was determined after a 20 minute incubation at 37 °C.

59K. Liao et al. / Journal of Immunological Methods 389 (2013) 52–60

between the recombinant enzyme and endogenous enzymeexists. The matrix interference was not observed and subse-quently, the assay was validated according to FDA guidance and

0 39 78 156

312

625

1250

2500

5000

1000

0

0

50

100

150rhACE2Calu-3

PC (ng/mL)

% M

axim

um r

espo

nse

Fig. 7. PC neutralizing activity with live Calu-3 cells. Serial diluted PC wasadded to overnight seeded Calu-3 cells, or mixed with an equal volume of0.5 μg/mL rhACE2, and incubated at 37 °C for 1 h. Treatment solution was thenremoved from cells. 50 μL of activity buffer was added to cells, followed by50 μL addition of substrate. The pre-incubated rhACE2 was 1:5 diluted withactivity buffer and then 50 μL of the diluted enzyme was mixed with 50 μLsubstrate. Fluorescence was determined after a 20 minute incubation at 37 °C.

deemed suitable for clinical sample testing. We believe that theevaluation described here could be useful for other therapeuticenzyme programs as well and should be incorporated into theimmunogenicity evaluation especially when a human endoge-nous homolog could be present.

Acknowledgments

We thank Shirisha Kanthala and Boyenoh Gaye for theirexcellent technical assistance. We also thank Susan Chiappinelliand Shahid Khan.

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