Large Molecule Therapeutics

Unraveling the Interaction betweenCarboxylesterase 1c and the Antibody–DrugConjugate SYD985: Improved TranslationalPK/PD by Using Ces1c Knockout MiceRuud Ubink1, Eef H.C. Dirksen1, Myrthe Rouwette1, Ebo S. Bos1, Ingrid Janssen1,David F. Egging1, Eline M. Loosveld1, Tanja A. van Achterberg1, Kim Berentsen1,Miranda M.C. van der Lee1, Francis Bichat2, Olivier Raguin2, Monique A.J.van der Vleuten1,Patrick G. Groothuis1, and Wim H.A. Dokter1

Abstract

Carboxylesterase 1c (CES1c) is responsible for linker-druginstability and poor pharmacokinetics (PK) of several anti-body–drug conjugates (ADC) in mice, but not in monkeysor humans. Preclinical development of these ADCs could beimproved if the PK in mice would more closely resemblethat of humans and is not affected by an enzyme that isirrelevant for humans. SYD985, a HER2-targeting ADCbased on trastuzumab and linker-drug vc-seco-DUBA, is alsosensitive to CES1c. In the present studies, we first focused onthe interaction between CES1c and SYD985 by size- exclu-sion chromatography, Western blotting, and LC/MS-MSanalysis, using recombinant CES1c and plasma samples.Intriguingly, CES1c activity not only results in release ofthe active toxin DUBA but also in formation of a covalentbond between CES1c and the linker of vc-seco-DUBA. Mass

spectrometric studies enabled identification of the CES1ccleavage site on the linker-drug and the structure of theCES1c adduct. To assess the in vivo impact, CES1c�/� SCIDmice were generated that showed stable PK for SYD985,comparable to that in monkeys and humans. Patient-derived xenograft (PDX) studies in these mice showedenhanced efficacy compared with PDX studies in CES1cþ/

þ mice and provided a more accurate prediction of clinicalefficacy of SYD985, hence delivering better quality data. Itseems reasonable to assume that CES1c�/� SCID mice canincrease quality in ADC development much broader for allADCs that carry linker-drugs susceptible to CES1c, withoutthe need of chemically modifying the linker-drug to specif-ically increase PK in mice. Mol Cancer Ther; 17(11); 2389–98.�2018 AACR.

IntroductionStability of the linker-drug in plasma has been, and still is, a

major challenge in the development of antibody–drug conjugates(ADC). On the one hand, linkers should be stable enough toprevent release of the active toxin in plasma, whereas on the otherhand, linkers should allow for an efficient release of active toxinonce the ADC targets a tumor or tumor cell. The difficulty ofdeveloping optimal linker characteristics is illustrated by thelimited amount of linker-drug technologies in ADCs that havebeen studied in the clinic (1, 2), although novel linker-drugstrategies are emerging (for recent reviews see 3, 4, 5, 6). ForMylotarg, the first approved ADCbased on a pH-labile hydrazonelinker, chemical instability of the linker-drug in the blood circu-lation probably contributed to the temporary withdrawal from

the market (7, 8). Besides early cleavage, other possible causes ofinstability in plasma includemaleimide exchange of the cysteine-conjugated linker-drugs to other thiol-containing molecules cir-culating in plasma, such as serum albumin (9) and enzymaticinstability by circulating enzymes in plasma (10, 11), which cancleave functional groups like esters, amides, lactones, lactams,carbamides, sulfonamides, and peptide mimetics (12).

SYD985, a HER2-targeting ADC based on trastuzumab andvc-seco-DUBA, a cleavable linker-duocarmycin payload, wasfound to be stable in human and monkey plasma, but unstableinmouse and rat plasma (13, 14). Despite its instability inmouseplasma, and its subsequent poor exposure in mice, SYD985 wasfound to be very active in HER2 3þ, 2þ, and 1þ breast cancerpatient-derived xenograft (PDX) models, whereas T-DM1 onlyshowed significant antitumor activity in HER2 3þ models (15).Although good antitumor activity of SYD985 was observed inthese mouse models, it was still hypothesized that the poorstability yields an underestimation of the expected anti-tumoractivity in humans. Therefore, in parallel to the clinical develop-ment of SYD985, subsequent preclinical studies aimed at (i)identifying the cause of instability of SYD985 in mouse and ratplasma, (ii) testing the hypothesis that anti-tumor activity inmiceis improved when plasma stability of SYD985 improves, and (iii)delivering a mouse model that more closely resembles the mon-key and human pharmacokinetics (PK) to allow for reliableassessment of the minimal effective dose in humans. In addition,

1Department of Preclinical, Synthon Biopharmaceuticals BV, Nijmegen, theNetherlands. 2Oncodesign, Dijon, France.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

CorrespondingAuthor:RuudUbink, SynthonBiopharmaceuticals, Microweg22,6503 GN Nijmegen, the Netherlands. Phone: þ31-24-372-7700; E-mail:ruud.ubink@synthon.com

doi: 10.1158/1535-7163.MCT-18-0329

�2018 American Association for Cancer Research.

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and most ideally, such a mouse model could be used forother ADC programs as well and deliver more relevantpharmacokinetic/pharmacodynamic (PK/PD) data in preclinicalstudies right away. As reported earlier, the rodent-specific carbox-ylesterase 1c (CES1c) was identified as the crucial enzyme thatcleaves vc-seco-DUBA-based ADCs, leading to the poor PKof SYD985 in mice and rats compared with that observedin human and monkey (14, 15). After these reports, it was pub-lished that CES1c is also responsible for cleavage of othervaline-citrulline-p-aminobenzyloxycarbonyl (vc-PABC) contain-ing linker-drugs in mouse plasma and that the extent of CES1c-mediated cleavagedependson the siteof conjugation (16). Severaladditional papers report instability in mouse and rat plasma ofvc-PABCorcarbamate-containing linker-drugs(10,17), indicatingCES1c activity might be a more widespread cause of linker-druginstability in these species, hampering the development of ADCs.

In this paper, we will first present the data that led to theidentification of CES1c as the enzyme responsible for the insta-bility of SYD985, causing release of active toxin. In order to becomplete and set the context right, we needed to recapture alimited amount of data that we published before, mainly assupplementary data. We will subsequently identify the cleavagesite of CES1c on the linker-drug vc-seco-DUBA, being differentfrom the cleavage site identified by (16). Furthermore, it wasfound thatCES1c activity not only cleaves the linker-drug, but alsoleads to the formation of a covalent bond between CES1c and theremaining linker. To circumvent the translational issues caused bythe CES1c activity, we developed a mouse model which closelymimics the human PK and is suitable for xenograft studies, bycross-breeding CES1c�/� mice with SCIDmice. Using this mousemodel we demonstrate that improved PK indeed leads toimproved efficacy in PDX studies and improved human dosepredictions for SYD985. Potentially, this model would alsoincrease PK/PD relationships of other ADCs that contain link-er-drugs that are susceptible to CES1c cleavage.

Materials and MethodsADCs and related materials

SYD985 was prepared as previously described (15). [3H]-SYD985 was prepared at Pharmaron, Cardiff, UK, using the sameprocedure and a linker-drug in which two tritium labels wereincorporated in the hydroxybenzoyl moiety of the toxin. N-acetylcysteine-quenched linker-drug (NAc-Cys-linker-drug) was pre-pared bymixing linker-drug andN-acetyl cysteine in a 1:10molarratio in an acetonitrile/water mixture for 4 hours followed bypurification by preparative HPLC. The head-to-head studies com-paring T-DM1 with SYD985 were conducted with two batches ofT-DM1 from Roche, EU batch N0001B02 (in nude mice) andN1037B19 (in CES1c�/� SCID mice).

In vitro plasma stability and cleavage by CES1cThe antibody–drug conjugate SYD985 was spiked into pooled

female mouse (BALB/c), rat (Sprague Dawley), monkey (Macacafascicularis), and human K2-EDTA plasma, at a concentration of100 mg/mL and incubated at 37�C. After 0, 1, 6, 24, 48, and 96hours of incubation, plasma sampleswere snap-frozen and storedat �80�C until bioanalysis. Recombinant mouse CES1c (rCES1c;Cusabio Biotech, CSB-MP338557MO) was spiked in human K2-EDTA plasma at 0, 10, 100, 200, and 400 mg/mL) together with100 mg/mL SYD985. After 96 hours of incubation at 37�C, plasma

samples were snap-frozen in liquid nitrogen and stored at�80�Cuntil bioanalysis.

PK studies and bioanalytical assaysAdult female CES1c�/� (B6-Ces1ctm1.1Loc/J; Charles River)

mice, CES1c�/� SCID (B6-Ces1ctm1.1Loc/J x B6.CB17-Prkdcscid/SzJ;Charles River) mice, and MAXF1162 tumor-bearing CES1c�/�

SCID mice, rats (Wistar; Charles River), and monkeys (Macacafascicularis; Mauritian) were dosed intravenously with 0.3, 1, 3, 8,and/or 10 mg/kg SYD985. Blood samples were taken at multipletime points after dosing, cooled on ice water, and processed toK2-EDTA plasma as soon as possible. Plasma samples were snap-frozen in liquid nitrogen and stored at �80�C until bioanalysis.SYD985 plasma levels were quantified using ELISA-based meth-ods with anti-idiotype capture and reporting for total antibody,and anti-toxin capture and anti-idiotype reporting for conjugatedantibody, as previously described (15). Based on the reportedplasma levels, PK parameters were calculated in WinNonlinversion 6.3.

Release of DUBA in mouse and rat plasmaHuman tumor cell line SW-620 (ATCC number: CCL-227; Lot

number: 58483168; P: 86 received on 19SEP2012) was obtainedfrom and characterized by the ATCC. No further cell-line authen-tication was conducted. Mycoplasma contamination in cell cul-tures was tested at Minerva Biolabs GmbH using the VenorGeMPrime test. No detectable levels of mycoplasma were found in theworking cell bank p:86þ14 tested on the April 11, 2013. Thenumber of passages between collection and use in the describedexperiments is 5 and6. SW-620 cells (90mL/well; 4,000 cells/well)were plated in 96-wells plates in RPMI1640 medium (Lonza),containing FBS, heat-inactivated (HI; Gibco-Life Technologies)and 80 U/mL Pen/Strep and incubated at 37�C, 5% CO2 over-night. After an overnight incubation 10 mL SYD985 or DUBA wasadded to each well of the 96-wells plate, containing 10% mouse(BALB/c), CES1c�/� mouse, rat (Wistar), monkey (Macaca fasci-cularis), or human plasma. Serial dilutions were made in culturemedium with plasma, to reach a final concentration range of200nmol/L (10mg/mL) to 6.3 pmol/L (0.316 ng/mL) for SYD985and 10 to 0.316 pmol/L for DUBA and 1% plasma. The cellviability was measured after 6 days using the CTG Assay Kit(Promega; G7572).

Affinity extraction of SYD985–protein complexesSYD985 was spiked at 100 mg/mL in mouse (BALB/c) and

CES1c�/� mouse plasma and incubated up to 48 or 96 hours at37�C, followed by storage at�80�Cuntil extraction. For the T¼ 0samples, SYD985 was spiked in plasma and immediately frozen.SYD985 and associated protein complexes were extracted fromplasma using an anti-idiotype mini-antibody (Bio-Rad;AbD15916) coupled via amino coupling to NHS-activatedsepharose 4 fast flow (GE Healthcare). Prior to incubation of theSYD985 plasma samples with the anti-idiotype coupled sephar-ose, a 50% ammonium sulfate precipitation step was performedto improve the purity of the extracted samples be selectivelyprecipitating the IgG fraction, including SYD985. Following incu-bation with the anti-idiotype coupled sepharose, the SYD985sample was transferred to a disposable column (Thermo Scien-tific; cat. no. 29920). After removal of the flow-through, sequen-tial washes (1 M NaCl in PBS, 10% acetonitrile in PBS and PBS)were performed to remove nonspecifically bound proteins.

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SYD985 and associated protein complexes were eluted with10 mmol/L glycine-HCl, pH 2.5, followed by immediate neutral-ization with 0.1 M Tris-HCl, pH 8.5.

SYD985 analysis by SECRadiolabeled [3H]-SYD985 was incubated at 100 mg/mL in

plasma from female mouse (BALB/c), CES1c�/� mouse, rat(Wistar), monkey (Macaca fascicularis), and human for 6 hoursat 37�C. The resulting plasma samples were analyzed directly bysize exclusion chromatography (SEC)using aWaters BEH200 SECcolumn (4.6 � 150 mm, 1.7 mm particles) and isocratic elutionwith 100 mmol/L NaPO4, pH 6.8 þ 0.3 M NaCl. Eluting com-pounds were detected using a Model 4 b-Ram Radiodetector(LabLogic).

For "cold" (i.e., not radiolabeled) SYD985 that was incubatedin plasma of mouse under the conditions mentioned above, anti-idiotype affinity-extractedmaterial was evaporated to dryness andreconstituted in 100mmol/LNaPO4, pH6.8þ 0.3MNaCl beforeanalyses by SEC using the same analytical column and solvent asdescribed for radiolabeled SYD985, but using UV absorbancedetection at 214 and 330 nm. Eluting compounds, including thehigh molecular weight (HMW) species, were collected, buffer-exchanged, and concentrated into 50 mmol/L Tris-HCl pH 8.0using 10K centrifugal filters (Amicon). 8M urea was added todenature the proteins, followed by reduction and alkylationwith dithiothreitol (DTT; 4.5 mmol/L final concentration in50 mmol/L Tris-HCl, pH 8.5, 37�C, 1 hour) and iodoacetamide(IAM; 9 mmol/L final concentration in 50 mmol/L Tris-HCl,pH 8.5, 37�C, 1 hour in the dark), respectively. Followingan overnight (o/n) precipitation in EtOH, the resulting pelletswere digested using trypsin (4 hours, 100 mmol/L Tris-HCl,pH 8.5).

Coomassie blue staining and Western blot analysisSamples obtained by affinity extraction were evaporated to

dryness, reconstituted in Milli-Q water and electrophoreticallyseparated under nonreducing conditions on a 3% to 8% Tris-Acetate gel (Thermo scientific) for total protein staining, or on a4% to 12% Bis-Tris gel (Thermo Scientific) for Western blotanalysis. Total protein staining was performed with Bio-safeCoomassie stain (Bio-Rad) according to manufacturer's instruc-tions. For Western blot analysis, proteins were transferred to aPVDF membrane (GE Healthcare) with a Mini Trans-Blot Elec-trophoretic Transfer Cell (Bio-Rad). The PDVF membrane wasblocked with Western blocker solution (Sigma-Aldrich). SYD985and associated protein complexes were detected with an HRP-labeled anti-idiotype mini-antibody (Bio-Rad). CES1c wasdetected with a biotinylated polyclonal rabbit anti-CES1c anti-body (Abcam) in combination with streptavidin-HRP (R&Dsystems). SYD985 and associated protein complexes and CES1cwere visualized by using the Metal Enhanced DAB Substrate Kit(Thermo Scientific).

In-gel protein digestionProtein bands of interest were excised from the Coomassie-

stained SDS-PAGE andwashedwithMilli-Qwater and acetonitril,to remove Coomassie and SDS before proceeding to the next step.Following in-gel reduction and alkylation with DTT (10 mmol/Lfinal concentration in 10 mmol/L Tris-HCl, pH 8.5, 37�C, 30minutes) and IAM (20mmol/L final concentration in 10mmol/LTris-HCl, pH 8.5, RT, 45 minutes in the dark), respectively,

proteins were in-gel digested using trypsin (o/n, 10 mmol/LTris-HCl, pH 8.5).

Incubation of rCES1c with NAc-Cys-linker-drug and SYD985and subsequent proteolytic digestion

Recombinant CES1c (rCES1c) was incubated with NAc-Cys-linker-drug or SYD985 in PBS, at a concentration of 100mg/mL for96 hours at 37�C under gentle shaking. The resulting sample wasacidified to pH 3.5 using glycine-HCl and reduced and alkylatedusing TCEP (10 mmol/L final concentration, 37�C, 30 minutes)and N-ethyl maleimide (NEM; 20 mmol/L final concentration,RT, 45 minutes in the dark), respectively. Considering that apotential (serine) ester of CES1c with NAc-Cys-linker-drug isprobably base-labile, all incubations and sample preparations ofthe resulting CES1c – NAc-Cys-linker-drug complex were per-formed at low pH (�3). For protein digestion, pepsin was addedand the mixture was incubated at 37�C (pH 3.0, o/n).

LC/MS-MS analysis of proteolytic digestsProteolytic peptide mixtures were separated by reversed phase

liquid chromatography (Dionex Ultimate 3000) using a WatersBEH-300 C18 column (1.0� 150mm, 1.7 mmparticles) and a 400

5%–95% linear gradient of acetonitileþ 0.1% formic acid (FA) inMilli-Q þ 0.1% FA at a flow rate of 0.1 mL/min and a columntemperature of 30 �C. Eluting peptides were detected usingelectrospray ionization mass spectrometry [Thermo OrbitrapFusion, heated electrospray ionization (HESI) at a spray voltageof 3.8 kV] with MS detection in the Orbitrap at 120K resolutionover the scan range m/z 400 to 1,600 and further analyzedusing data-dependent tandem mass spectrometry (MS-MS) inthe quadrupole, using HCD (30%), on the top-3 most intenseprecursor ions detected per survey scan to enable protein iden-tification. Fragments were detected in the ion trap. Proteinswere identified against the Swissprot database using Mascotsearch engine.

In addition, MS and MS-MS fragmentation data were inter-pretedmanually usingQualbrowser (ThermoXCalibur 3.0.63)bycomparing proteolytic digests of rCES1c with and withoutNAc-Cys-linker-drug and proteolytic digests of rCES1c andSYD985 individually to that of the incubated mixtures to inves-tigate and characterize cross-linked peptide candidates.

Patient-derived xenograft studiesThe in vivo antitumor activity of SYD985 versus T-DM1 was

tested as single-dose therapy in MAXF1162 breast cancer PDXmodel (Charles River) in both CES1cþ/þ NMRI-Foxn1nu miceas well as CES1c�/� SCID mice. Initial tumor volumes at theday of randomization and treatment ranged from 46 to 252mm3. The HER2 FISH and IHC status of the tumor from thePDX model as determined by the CROs was independentlyconfirmed. Studies were conducted as previously described(15). All in vivo studies and protocols were approved bythe local animal care and use committees according to estab-lished guidelines.

ResultsCES1c expressed in rodent plasma cleaves vc-seco-DUBA

In vitro plasma stability studies, as well as in vivo PK studies,showed that SYD985 conjugated antibody levels rapidlydecrease in mouse and rat plasma, but are stable in monkey

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and human plasma (Fig. 1A and B; Supplementary Fig. S1),similar to what was previously found for closely related ADCs(13, 14). A literature study pointed towards mouse and rat-specific CES1c as a potential hydrolyzing enzyme. To assess itsrole in SYD985 instability, increasing amounts of rCES1c werespiked in human plasma in combination with 100 mg/mLSYD985 and incubated at 37�C up to 96 hours. SYD985 conju-gated antibody levels decreased with increasing CES1c concen-tration (Fig. 1C, previously published as supplementary data; ref.15). Significant cleavage was observed as of 100 mg/mL CES1cwhich are physiologically relevant CES1c concentrations, com-parable to the in vivo CES1c concentration in mice (80 mg/mL;ref. 18). A PK study with SYD985 in CES1c homozygous �/�mice, heterozygous CES1c�, and homozygous CES1cþ/þ wild-type littermates, showed that the PK in homozygous CES1c�/�

mice was similar to the PK in monkey and human (Fig. 1D;Supplementary Table S1, previously published as supplementarydata in ref. 15).

Mechanismof actionofCES1c-mediated vc-seco-DUBA cleavageTo study if CES1c activity results in release of the toxin DUBA,

SYD985 was incubated with HER2-negative cells in the presenceor absence of 1%wild-typemouse, CES1c�/�mouse, rat,monkey,or human plasma (Fig. 1E; Supplementary Table S2; ref. 15).Compared with the medium control, presence of 1% plasmacaused a clear shift in the IC50 for rat (8.08 nmol/L) and especiallymouse (0.82 nmol/L) plasma, but not with monkey, human, orCES1c�/� mouse plasma, all showing an IC50 > 100 nmol/L.

As part of a metabolism study, radiolabeled SYD985 wasanalyzed by size exclusion chromatography (SEC) after incuba-tion in mouse, rat, CES1c�/� mouse, monkey, and human plas-ma. In SEC profiles ofmouse and rat plasma a new peak appeared(Fig. 2B andD), eluting at an earlier retention time (7.15minutes)compared with the SYD985 peak (RT ¼ 7.8–8.02 minutes). Thisnew peak was hardly observed in samples taken from CES1c�/�

mouse, monkey, and human plasma (Fig. 2A, C, and E), suggest-ing that a SYD985-containing compoundwith a highermolecular

Figure 1.

In vitro and in vivo kinetics of SYD985. A, Conjugated antibody concentration during a 96-hour incubation of 100 mg/mL SYD985 at 37�C in human,monkey, rat, and mouse plasma (mean � SEM, n ¼ 3). B, Total (TAb) and conjugated antibody (Conj. Ab) concentration (mean � SEM, n ¼ 2 for rat,three for mouse, and five for monkey) after single-dose IV administration of 10 mg/kg SYD985 to mice, rats (dose normalized from 8 mg/kg), or monkeys.C, Conjugated antibody concentrations of 100 mg/mL SYD985 after 96 hours in vitro incubation at 37�C in human plasma with increasing concentrations ofmouse rCES1c (mean� SEM, n¼ 2). D, Conjugated antibody concentrations in plasma in CES1c knock-out (�/�), CES1c heterozygous (þ/�), and CES1c wild-type(þ/þ) mice, after a single IV bolus injection of 5 mg/kg SYD985 (mean� SEM, n¼ 3). E, Cytotoxic activity of released active toxin on HER2-negative SW-620 cellsafter 6 days incubation of the toxin DUBA in plasma-free medium (positive control) or SYD985 in the presence of 1% mouse, CES1c�/� mouse, rat, monkey,or human plasma in the culture medium. Data show the percentage of survival � SD of two experiments performed in duplicate.

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weight than SYD985 alone was formed in rat and mouse plasma.To identify the compound(s) eluting in this mouse and rat-specific peak, mouse plasma was incubated with SYD985 up to48 hours and SYD985 and SYD985-associated material was

subsequently extracted from plasma using anti-idiotype extrac-tion. SEC profiles of the affinity-extracted material confirmed theappearance of a SYD985-containing peak, at an earlier retentiontime as compared with SYD985 itself (Fig. 2F). The extract was

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A–E, SEC-HPLC radiochromatograms of [3H]-SYD985 after 6 hours incubation in plasma of indicated species at 100 mg/mL and 37�C. F–I, Analysis ofHMW species in affinity-extracted material obtained from BALB/c mouse plasma incubated with SYD985. F, Confirmatory SEC-HPLC UV chromatogramof non-radiolabeled SYD985 and associated material affinity-extracted after 48 hours of incubation in wild-type mouse plasma at 37�C. G, Coomassiestaining of affinity-extracted samples from BALB/c mouse plasma using non-reducing SDS-PAGE revealed an increase in the formation of HMW species over time.H, A Western blot analysis with the anti-idiotype antibody directed against SYD985 showing the presence of SYD985 in the HMW species. I, A Westernblot analysis with an anti-CES1c antibody showing the presence of CES1c in the HMW species.

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also analyzed using nonreducing SDS-PAGE and Coomassiestaining, which revealed a unique band pattern around 200 to250 kDa (Fig. 2G). Although SYD985 has a molecular weight of�150 kDa, bandswere also observed at lowermolecular weight inthe extracted samples. This is due to the fact that as a result ofconjugation on interchain cysteines, used to manufactureSYD985, its structural integrity (partially) relies on noncovalentinteractions that are disrupted during SDS-PAGE analysis. The200 to 250 kDabandswere cut out of the gel, trypsin-digested andanalyzed by LC/MS-MS. Next to trastuzumab, CES1c (Uniprotentry P23953) was confidently identified based on 6 uniquepeptides (9% sequence coverage, Mascot score 337) in the lowermigrating band (around 200 kDa), whereas CES1c was alsoidentified in the higher migrating band (around 250 kDa) basedon 12 unique peptides (25% sequence coverage, Mascot score745). CES1c was also observed by LC-MS/MS analysis of a direct(i.e., in solution) digest of affinity-extracted material from wild-type mouse plasma following incubation of SYD985 at 37�C for96 hours. Western blot studies showed the presence of bothSYD985 and CES1c in the 200 to 250 kDa bands, because itstained positive for SYD985 using an anti-idiotypic antibody,and for CES1c using an anti-CES1c antibody (Fig. 2H and I),respectively.

Next, it was attempted to identify the CES1c cleavage site(s) onthe linker-drug in SYD985 and the position of the covalent bondbetween CES1c and SYD985. Covalent interactions betweencarbamates and esterases have been described for human CES1and CES2 (19). Based on that work, it was hypothesized that theinteraction potentially occurred via the active site serine (Ser221)in CES1c and one of the carbamates present in the linker-drug(Fig. 3A). Due to the structural orientation of the carbamates, thecovalent bond formation with the ADC would most likelyproceed via the carbamate connecting the linker to the DNA-alkylating moiety of the toxin, resulting in concomitant releaseof the toxin.

To investigate this hypothesis, rCES1c was first incubated withN-acetylcysteine-quenched linker-drug (NAc-Cys-linker-drug),for 72 hours at 37�C in PBS. Following reduction, alkylation andproteolytic digestion with pepsin (all at pH � 3), LC-MS/MSanalysis of the generated peptide mixture resulted in the identi-fication of five covalently modified CES1c peptides (Fig. 3B–E; Table 1), all including the active site serine Ser221. The proposedcovalent interaction was confirmed by the accurate masses ofthese modified peptides and the concomitant mass increase of968.1Dawith respect to the corresponding original (unmodified)peptide mass as a result of the binding of NAc-Cys-linker-drug(minus the released toxin DUBA). Furthermore, the characteristicfragment ions observed in the correspondingMS/MSdata, such asthe neutral loss of 779.3 Da, corresponding to the majority of theNAc-Cys-linker-drug modification; the loss of the complete NAc-Cys-linker-drug modification (985.4 Da) resulting in the forma-tion of a dehydroalanine moiety from the serine residue, andseveral NAc-Cys-linker-drug specific fragment ions (Fig. 3E) sup-ported the hypothesized structure of the CES1c-linker-drug cross-link. In addition, free seco-DUBA, the pro-toxin that is releasedupon linker-drug cleavage and is stable at acidic pH, was alsoidentified in the incubated digest at pH < 3, confirming the releaseof the toxin.

Next, SYD985 was incubated with rCES1c in PBS during 72hours at 37�C. Following the same, low pH, sample work up asdescribed for the NAc-Cys-linker-drug incubated sample, the

generated peptide mixture was analyzed using LC-MS/MS. Thisdataset revealed several candidate cross-linked peptidemolecules.These were confirmed using a combination of their accurate massand the distinctive fragment ions that were also observed whenrCES1c was incubated with NAc-Cys-linker-drug, correspondingto the rCES1c part of a cross-linked peptide. Subtracting the massof the rCES1c sequence (with part of the linker-drug on it), as wellas the remainder of the linker-drug part that was conjugated to thecysteine residue originating from SYD985, from the precursormass, revealed one of the cross-linked peptide candidates tocontain the trastuzumab sequence KSFNRGEC (SupplementaryFig. S2), originating from a conjugated trastuzumab light chain.This confirmed the presence of a SYD985-rCES1c cross-linkthrough the linker-drug side chain via the proposed mechanism.Finally, this conclusion was underscored by the identification of across-link between a rCES1c peptide and the SYD985 hingeregion-containing heavy chain peptide THTC(NEM)PPCPA-PELLGGPSVF (Supplementary Fig. S2), that was originally con-jugated to one linker-drug. The other cysteine in the sequence wasfound to be alkylated with N-ethylmaleimide as a result of thesample preparation. Overall, four different rCES1c sequences (allcontaining the active site serine, Ser221) were found to be cross-linked to three different peptides originating from the light chain,and to one peptide originating from the hinge region of the heavychain (Table 2).

Efficacy studies in CES1c knockout miceSince in vivoCES1c activity could not be completely blocked by

carboxylesterase inhibitors (Supplementary Fig. S3), efficacy stud-ies were performed in CES1c �/� SCID mice. Before efficacystudies were initiated, it was first verified whether the SYD985 PKin CES1c �/� SCID mice was identical to that in the originalCES1c �/� strain. As shown in Fig. 4A, the PK profiles indeedoverlapped. Pilot efficacy studies with CES1c �/� SCID miceusing different PDXmodels (15), showed tumor take and growthrates similar to those observed in the nude mice. In these models,efficacy was indeed improved at 3 mg/kg SYD985 (Supplemen-tary Fig. S4). To confirm the data obtained at a single dose level of3 mg/kg, and to get an accurate estimate of the fold change inefficacy, an experiment comprising of different dose levels wasperformed in theHER23þMAXF1162PDXmodel. The efficacy ofdifferent SYD985 dosages was compared head-to-head againstT-DM1, similar to a previous study performed in nude mice (15),to control for potential strain differences between the nude andSCID background. A non-binding isotype control ADC, bearingthe same linker-drug and with the same drug-antibody ratio, wasincluded to study the effect of improved PK on efficacy for a non-target-binding ADC. The efficacy of SYD985was improved almostthreefold inCES1c�/� SCIDmice showing tumor-regression at 3mg/kg SYD985 in these animals whereas 10 mg/kg SYD985induced tumor-stasis in CES1c þ/þ nude mice. The isotypecontrol also showed efficacy at 3 mg/kg, in contrast to the lackof efficacy in nude mice. This difference is most likely also causedby an increased exposure of intact isotype control ADC. Extracel-lular cleavage or pinocytosis of the isotype control could explainthe efficacy, which is not uncommon for the isotype control (15).As expected, since T-DM1 is not susceptible to CES1c cleavage,T-DM1 showed similar efficacy in nude mice versus CES1c �/�SCID mice, with prolonged tumor stasis at 10 mg/kg.

Recently, phase I clinical data were published describingthe efficacy of SYD985 at the recommended phase II dose of

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200 400 600 800 1000 1200 1400 1600 1800m/z

0

50

100

0

50

100

0

50

100

Rela

�ve

abun

danc

e

0

50

100

0

50

100681.2722

349.0958207.1686 730.3232588.2110 1035.2388 1210.5594 1401.1116

881.4395

349.2315675.3375240.2588 1251.5571987.4726 1390.4729 1567.6655

980.5031

349.2002774.4719240.2942 588.3439 1512.60501350.66311085.4099 1717.5129

1067.4961

349.1088861.3724240.1815 588.2286 1399.59411142.3851 1607.2213 1808.9518

1141.6213

349.1663935.4435240.2594 588.2765 830.3983 1530.70341226.6826 1717.8667

NL: 2.57E4Full ms2 730.7888@hcd30.00

NL: 1.49E5Full ms2 830.8510@hcd30.00

NL: 1.36E5Full ms2 880.3849@hcd30.00

NL: 2.21E4

Full ms2 923.9033@hcd30.00

NL: 8.60E4Full ms2 960.9280@hcd30.00

GESSGGIS

GESSGGISVS

GESSGGISV

IFGESSGGIS

GESSGGE

A

B

C

D

Exact mass: m/z 880.39 (2+)

G ES

S GG

I

S

V

G EdhA

S GG

I

S

V

Exact mass: m/z 774.36 (1+)

G ES

S GG

I

S

V

Exact mass: m/z 980.49 (1+)

Figure 3.

A, Structure of NAc-quenched linker-drug with carbamate moieties indicated by circles. The blue-circled group was hypothesized to be the one resultingin the covalent interaction upon binding of CES1c. B, Proposed structure of the linker-drug–modified CES1c active site-containing peptide GESSGGISV.C and D, Proposed structures of the corresponding predominant fragment ions formed from the linker-drug–modified CES1c active site-containing peptideGESSGGISV upon collision-induced dissociation: one that loses part of the linker moiety (C) and one that loses the complete side chain modification,resulting in the formation of a dehydroalanine (dhA) residue (D). Please refer to the middle spectrum in E for the MS-MS data of this modified peptide. E,Ion trap MS-MS fragmentation spectra (HCD @30 eV) of the five linker-drug–modified CES1c peptides that were found after incubation of the carboxylesterasewith the NAc-Cys-linker-drug. The predominant fragment resulting from the loss of most of the conjugated NAc-Cys-linker-drug moiety (780.3 Da) from theprecursor ion is clearly visible (note: all precursors are doubly-charged), just like thepresenceof thedehydroalanine-containingpeptide fragments (atm/z "precursorion": 987.3 Da) and linker-specific fragments, such as those at m/z 163.1, 207.2, 240.3, and 349.1.

CES1c Knockout Mice for ADC Development

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1.2 mg/kg in HER2 positive metastatic breast cancer patients(20, 21). The 1.2 mg/kg dose was shown to be efficaciouswith an overall response rate (ORR) of 33% (16 of 48 patients)and a progression-free survival (PFS) of 9.4 months (95% CI,4.5–12.4). The human PK of SYD985 at 1.2 mg/kg (AUCinf ¼1,725 h.µg/mL) almost overlaps with the PK observed inMAXF1162 tumor bearing CES1c�/� SCID mice at the 1 mg/kgdose that shows tumor stasis (AUCinf ¼ 1,332 h.µg/mL; Fig. 4D;Supplementary Table S3).

DiscussionUntil recently, all xenograft studies with vc-seco-DUBA-based

ADCs, including SYD985, were performed in immune-compromised mice expressing CES1c. Despite the low exposurelevels for conjugated antibody in these mice, SYD985 showedgood efficacy, which was superior to that of T-DM1 in numerousxenograft models with different levels of HER2 target expression(15).However, because the SYD985PKprofiles in themouseweredramatically different from those in monkey and the anticipatedprofile in human based on in vitro plasma stability data, anaccurate estimation of an effective human dose based on efficacyin PDX models was not possible. Based on extrapolation ofavailable PK data for SYD985 in CES1cþ/þ mice (either nude orSCID) AUCs of conjugated antibody at tumor static dosages areestimated to be between 150 and 260 h.mg/mL. This is based ondata generated in the MAXF1162 PDX model and the BT474model (15) and in normal (non-immunedeficient) mice. ThisAUC ismore than tenfold below the observed AUC in the clinic atthe recommended phase II dose of 1.2mg/kg that showed efficacy(21). Thus, using the AUC at tumor static dose in CES1cþ/þ miceto predict the effective human dose would lead to a significantunder-prediction of this dose. It was anticipated that the trans-lational value of PDX models would improve if the PK profile inmice was more in line with the anticipated human PK profile. Aswill be discussed later, the AUC of conjugated SYD985 at atumor static dose of 1 mg/kg in CES1c�/� mice in the MAXF1162

PDX model very nicely correlated with that in humans at therecommended phase II dose at 1.2 mg/kg which turned out tobe efficacious.

Based on the in vivo PK data it was reasoned that the instabilityof SYD985 is most likely caused by cleavage or release of thelinker-drug, rather than clearance of the entire ADC, because totalantibody levels were similar in all tested species. It was hypoth-esized that the instability is the result of hydrolysis of one of thecarbamate moieties in the linker-drug because these are knownsubstrates for esterase-like activity (22, 23). Preliminary studieswith human esterases suggested that neither human CES1 andCES2 nor the plasma esterase BCHE seem to be able to cleave thelinker-drug in SYD985 (data not shown). Based on a literaturestudy focused on species differences in esterase activity in plasma,the mouse- and rat-specific CES1c was identified as a candidatehydrolyzing enzyme (18, 24, 25). Incubation of SYD985 inhuman plasma spiked with rCES1c indeed revealed that CES1ccaused instability of SYD985. The final evidence was provided bya PK study in CES1c�/� mice, showing a PK profile similar to thePK in monkey and human. The identification of CES1c triggeredadditional studies to reveal its mode of action. The increase inpotency observed after in vitro incubation of SYD985 with HER-2negative cells and 1% mouse or rat plasma, compared withmedium, human, monkey and CES1c�/� plasma suggested activetoxin DUBA is released by CES1c activity. LC/MS-MS analysisconfirmed the presence of significant amounts ofDUBA inplasmasamples from mice and rats dosed with SYD985.

Most surprisingly, CES1c activity not only results in release ofDUBA: here it is shown that it also results in the formation ofHMW species, as identified by SEC. Using SDS-PAGE, Westernblotting and LC-MS/MS, these HMW species were found tocontain both trastuzumab and CES1c, which suggests that at leastone CES1c molecule is capable of covalently binding to SYD985.Next, attempts to identify the site of interaction between CES1cand the linker-drug disclosed it to be the carbamate group con-necting the linker and toxin. Other carbamates in the linker-drugseem to remain unaffected. CES1c activity results in release of

Table 1. Characteristics of the peptides containing the CES1c active site serine (Ser221) that were found to be conjugated to NAc-Cys-linker-drug

Peptide sequenceTheoreticalpeptide mass (Da)

Observed m/z (z)without conjugate

Observed m/z (z)with conjugate

Most predominantfragment ion m/z

GES221SGG 492.19 493.19 (1) 730.79 (2) 681.27GES221SGGIS 691.37 692.37 (1) 830.85 (2) 881.44GES221SGGISV 790.37 791.37 (1) 880.39 (2) 980.50GES221SGGISVS 877.40 878.40 (1) 923.90 (2) 1067.50IFGES221SGGIS 951.45 952.45 (1) 960.93 (2) 1141.62

Table 2. Characteristics of the peptides containing the CES1c active site serine (Ser221) that were found to be conjugated to SYD985

Precursor (1þ) CES1c fragmenta Linker mass Residual mass SYD985 sequence

Light chain828.04 (3þ) 2482.13 1140.6 618.28 723.25 FNRGEC773.74 (3þ) 2319.23 979.5 618.28 723.30 FNRGEC741.32 (3þ) 2221.98 880.4 618.28 723.30 FNRGEC813.03 (3þ) 2437.11 880.4 618.28 938.43 KSFNRGEC684.32 (4þ) 2734.28 880.4 618.28 1235.60 PVTKSFNRGECHeavy chain836.39 (4þ) 3342.55 680.3 618.28 2045.99 THTC(NEM)PPCPAPELLGGPSVF891.18 (4þ) 3561.70 880.4 618.28 2064.02 THTC(NEM)PPCPAPELLGGPSVF þH2O1181.89 (3þ) 3543.69 880.4 618.28 2046.01 THTC(NEM)PPCPAPELLGGPSVFaCES1c peptide fragments (uncharged): 680.3¼GESSGG; 880.4¼GESSGGIS; 979.5¼GESSGGISV; 1140.6¼ IFGESSGGIS. "þH2O" refers to hydrolysis (ring opening)of the maleimide moiety in the linker drug.

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DUBA and formation of a covalent bond between SYD985 andCES1c,most likely via the serine (Ser221) in its active site, althoughit cannot be excluded that the modification occurs on serine-222,adjacent to the active site serine. CES1c-SYD985 cross-links wereobserved both in the hinge region of SYD985 heavy chain andSYD985 light chain. All together, these interaction studies clarifiedthe structure of the covalent bond between CES1c and SYD985that is -unexpectedly- formed upon CES1c cleavage of the vc-seco-DUBA linker-drug at a specific carbamate.

Clearly, CES1c activity is a more general concern in the devel-opment of ADCs. In a recent publication, Dorywalska and col-leagues (15) showed that CES1c also cleaves other linker-drugs.Their vc-PABC containing linker-drug was postulated to becleaved at the carbamate C-terminal to the citrulline moiety.Cleavage by, or covalent interaction with, CES1c would not resultin a complex between the ADC and the esterase due to thestructural orientation of the carbamate in the linker-drug (19).Even though this linker-drug and vc-seco-DUBA share the vc-PABCstructure, we found no evidence for CES1c-mediated cleavageC-terminal to citrulline, or for hydrolysis at the other carbamatepositions in vc-seco-DUBA, underlining the complexity of thestructure–activity relationship of CES1c. Several other publica-tions describe carbamate-containing linker-drugs that are unsta-ble in studies with mice and rat and are therefore potentiallysensitive to cleavage by CES1c (17, 26). So far, modifying thestructure of the linker-drug is the general mitigation to enhanceADC stability in mouse plasma, even though the linker-drug isperfectly stable in human plasma and from that perspective doesnot require further optimization. In order to circumvent linker-

drug optimization specifically for the mouse, it was attempted toblock mouse-specific CES1c activity in vivo. Initial experimentswith esterase inhibitors failed to completely block CES1c activity.Thus, immune-compromised CES1c�/� mice were needed toevaluate the full impact of the absence of CES1c activity on PKand in vivo efficacy in PDX models. Cross-breeding of CES1c�/�

mice with SCID and nude mice was setup in parallel. The cross-breeding of CES1c�/� mice with nude mice showed poor repro-ductive performance, whereas the SCID cross-breeding was suc-cessful. Pilot PDX studies in CES1c�/� SCID mice suggestedimproved efficacy of SYD985, compared with previous studiesin CES1cþ/þ nude mice. The full PKPD study in the MAXF1162PDXmodel with T-DM1 to control for potential strain differencesconfirmed improved efficacy of SYD985 in CES1c�/� SCID miceshowing tumor stasis at 1 mg/kg. At this dose, the PK profile andAUCare very similar to those at the recommendedphase II dose inhuman. This shows that use of CES1c�/�mice is a good and for usthe preferred alternative to unnecessary structural optimization ofthe linker-drug that might even compromise on human-relevantparameters.

ConclusionsEven though it is known that the rodent-specific esterase CES1c

is able to cleave linker-drugs on ADCs at distinct sites, the impactof CES1c activity on the predictive value of PK, efficacy and safetystudieswithADCs inmice and rats should not be underestimated.Here it is shown that cleavage of vc-seco-DUBA results in theformation of a covalent bond betweenCES1c and the ADC,which

Figure 4.

Pharmacokinetics of SYD985 inA,CES1c�/�mice versus CES1c�/� SCIDmice after an intravenous dose of 3mg/kg (mean� SEM, n¼ 3) andD, after dosing 0.3, 1, and3 mg/kg in MAXF1162 tumor bearing CES1c�/� SCID mice. Antitumor activity of SYD985 compared with T-DM1 and isotype control ADC in the HER-2 3þMAXF1162breast cancer PDX model in CES1cþ/þ nude mice (B, C) or in CES1c�/� SCID mice (E, F; mean � SEM, n ¼ 6–8).

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CES1c Knockout Mice for ADC Development

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indicates that the structure-activity relationship of CES1c is morecomplex than originally thought. It not only depends on thestructure of the linker-drug, but also on the site of conjugation inthe ADC. Consequently, developing linker-drugs that are stable inbothmice andhumans, remains challenging. The use ofCES1c�/�

mice widens the spectrum of linker-drugs suitable for the devel-opment of ADCs, thereby increasing the chance that ADCs aresuccessfully developed.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: R. Ubink, E.H.C. Dirksen, E.S. Bos, D.F. Egging,M.M.C. van der Lee, P.G. Groothuis, W.H.A. DokterDevelopment of methodology: E.H.C. Dirksen, D.F. Egging, T.A. vanAchterberg, M.M.C. van der Lee, F. BichatAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E.H.C. Dirksen, M. Rouwette, I. Janssen,E.M. Loosveld, T.A. van Achterberg, K. Berentsen, F. Bichat, O. Raguin,M.A.J. van der Vleuten

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Ubink, E.H.C. Dirksen, E.S. Bos, M.M.C. vander Lee, F. Bichat, P.G. Groothuis, W.H.A. DokterWriting, review, and/or revision of the manuscript: R. Ubink, E.H.C. Dirksen,M. Rouwette, I. Janssen, D.F. Egging, E.M. Loosveld, K. Berentsen, M.M.C. vander Lee, M.A.J. van der Vleuten, P.G. Groothuis, W.H.A. DokterAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): F. BichatStudy supervision: R. Ubink, E.H.C. Dirksen, O. Raguin, W.H.A. Dokter

AcknowledgmentsFinancial support for this study was provided by Synthon Biopharmaceu-

ticals BV.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 30, 2018; revised June 19, 2018; accepted July 27, 2018;published first August 9, 2018.

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2018;17:2389-2398. Published OnlineFirst August 9, 2018.Mol Cancer Ther   Ruud Ubink, Eef H.C. Dirksen, Myrthe Rouwette, et al.   by Using Ces1c Knockout Mice

Drug Conjugate SYD985: Improved Translational PK/PD−Antibody Unraveling the Interaction between Carboxylesterase 1c and the

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Published OnlineFirst August 9, 2018; DOI: 10.1158/1535-7163.MCT-18-0329