Improved Translational PK/PD by Using Ces1c Knockout Mice Ruud Ubink1, Eef H.C

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

Large Molecule Therapeutics Molecular Cancer Therapeutics Unraveling the Interaction between Carboxylesterase 1c and the Antibody–Drug Conjugate SYD985: Improved Translational PK/PD by Using Ces1c Knockout Mice Ruud 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-drug spectrometric studies enabled identification of the CES1c instability and poor pharmacokinetics (PK) of several anti- cleavage site on the linker-drug and the structure of the body–drug conjugates (ADC) in mice, but not in monkeys CES1c adduct. To assess the in vivo impact, CES1c / SCID or humans. Preclinical development of these ADCs could be mice were generated that showed stable PK for SYD985, improved if the PK in mice would more closely resemble comparable to that in monkeys and humans. Patient- that of humans and is not affected by an enzyme that is derived xenograft (PDX) studies in these mice showed þ irrelevant for humans. SYD985, a HER2-targeting ADC enhanced efficacy compared with PDX studies in CES1c / þ based on trastuzumab and linker-drug vc-seco-DUBA, is also mice and provided a more accurate prediction of clinical sensitive to CES1c. In the present studies, we first focused on efficacy of SYD985, hence delivering better quality data. It the interaction between CES1c and SYD985 by size- exclu- seems reasonable to assume that CES1c / SCID mice can sion chromatography, Western blotting, and LC/MS-MS increase quality in ADC development much broader for all analysis, using recombinant CES1c and plasma samples. ADCs that carry linker-drugs susceptible to CES1c, without Intriguingly, CES1c activity not only results in release of the need of chemically modifying the linker-drug to specif- the active toxin DUBA but also in formation of a covalent ically increase PK in mice. Mol Cancer Ther; 17(11); 2389–98. bond between CES1c and the linker of vc-seco-DUBA. Mass 2018 AACR.

Introduction the market (7, 8). Besides early cleavage, other possible causes of instability in plasma include maleimide exchange of the cysteine- Stability of the linker-drug in plasma has been, and still is, a conjugated linker-drugs to other thiol-containing molecules cir- major challenge in the development of antibody–drug conjugates culating in plasma, such as serum albumin (9) and enzymatic (ADC). On the one hand, linkers should be stable enough to instability by circulating enzymes in plasma (10, 11), which can prevent release of the active toxin in plasma, whereas on the other cleave functional groups like esters, amides, lactones, lactams, hand, linkers should allow for an efficient release of active toxin carbamides, sulfonamides, and peptide mimetics (12). once the ADC targets a tumor or tumor cell. The difficulty of SYD985, a HER2-targeting ADC based on trastuzumab and developing optimal linker characteristics is illustrated by the vc-seco-DUBA, a cleavable linker-duocarmycin payload, was limited amount of linker-drug technologies in ADCs that have found to be stable in human and monkey plasma, but unstable been studied in the clinic (1, 2), although novel linker-drug in mouse and rat plasma (13, 14). Despite its instability in mouse strategies are emerging (for recent reviews see 3, 4, 5, 6). For plasma, and its subsequent poor exposure in mice, SYD985 was Mylotarg, the first approved ADC based on a pH-labile hydrazone found to be very active in HER2 3þ,2þ, and 1þ breast cancer linker, chemical instability of the linker-drug in the blood circu- patient-derived xenograft (PDX) models, whereas T-DM1 only lation probably contributed to the temporary withdrawal from showed significant antitumor activity in HER2 3þ models (15). Although good antitumor activity of SYD985 was observed in

1 these mouse models, it was still hypothesized that the poor Department of Preclinical, Synthon Biopharmaceuticals BV, Nijmegen, the stability yields an underestimation of the expected anti-tumor Netherlands. 2Oncodesign, Dijon, France. activity in humans. Therefore, in parallel to the clinical develop- Note: Supplementary data for this article are available at Molecular Cancer ment of SYD985, subsequent preclinical studies aimed at (i) Therapeutics Online (http://mct.aacrjournals.org/). identifying the cause of instability of SYD985 in mouse and rat Corresponding Author: Ruud Ubink, Synthon Biopharmaceuticals, Microweg 22, plasma, (ii) testing the hypothesis that anti-tumor activity in mice þ 6503 GN Nijmegen, the Netherlands. Phone: 31-24-372-7700; E-mail: is improved when plasma stability of SYD985 improves, and (iii) [email protected] delivering a mouse model that more closely resembles the mon- doi: 10.1158/1535-7163.MCT-18-0329 key and human pharmacokinetics (PK) to allow for reliable 2018 American Association for Cancer Research. assessment of the minimal effective dose in humans. In addition,

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and most ideally, such a mouse model could be used for samples were snap-frozen in liquid nitrogen and stored at 80C other ADC programs as well and deliver more relevant until bioanalysis. pharmacokinetic/pharmacodynamic (PK/PD) data in preclinical studies right away. As reported earlier, the rodent-specific carbox- PK studies and bioanalytical assays ylesterase 1c (CES1c) was identified as the crucial enzyme that Adult female CES1c / (B6-Ces1ctm1.1Loc/J; Charles River) cleaves vc-seco-DUBA-based ADCs, leading to the poor PK mice, CES1c / SCID (B6-Ces1ctm1.1Loc/J x B6.CB17-Prkdcscid/SzJ; of SYD985 in mice and rats compared with that observed Charles River) mice, and MAXF1162 tumor-bearing CES1c / in human and monkey (14, 15). After these reports, it was pub- SCID mice, rats (Wistar; Charles River), and monkeys (Macaca lished that CES1c is also responsible for cleavage of other fascicularis; Mauritian) were dosed intravenously with 0.3, 1, 3, 8, valine-citrulline-p-aminobenzyloxycarbonyl (vc-PABC) contain- and/or 10 mg/kg SYD985. Blood samples were taken at multiple ing linker-drugs in mouse plasma and that the extent of CES1c- time points after dosing, cooled on ice water, and processed to mediated cleavagedepends on the site of conjugation (16). Several K2-EDTA plasma as soon as possible. Plasma samples were snap- additional papers report instability in mouse and rat plasma of frozen in liquid nitrogen and stored at 80C until bioanalysis. vc-PABCorcarbamate-containinglinker-drugs(10,17),indicating SYD985 plasma levels were quantified using ELISA-based meth- CES1c activity might be a more widespread cause of linker-drug ods with anti-idiotype capture and reporting for total antibody, instability in these species, hampering the development of ADCs. and anti-toxin capture and anti-idiotype reporting for conjugated In this paper, we will first present the data that led to the antibody, as previously described (15). Based on the reported identification of CES1c as the enzyme responsible for the insta- plasma levels, PK parameters were calculated in WinNonlin bility of SYD985, causing release of active toxin. In order to be version 6.3. complete and set the context right, we needed to recapture a limited amount of data that we published before, mainly as Release of DUBA in mouse and rat plasma supplementary data. We will subsequently identify the cleavage Human tumor cell line SW-620 (ATCC number: CCL-227; Lot site of CES1c on the linker-drug vc-seco-DUBA, being different number: 58483168; P: 86 received on 19SEP2012) was obtained from the cleavage site identified by (16). Furthermore, it was from and characterized by the ATCC. No further cell-line authen- found that CES1c activity not only cleaves the linker-drug, but also tication was conducted. Mycoplasma contamination in cell cul- leads to the formation of a covalent bond between CES1c and the tures was tested at Minerva Biolabs GmbH using the VenorGeM remaining linker. To circumvent the translational issues caused by Prime test. No detectable levels of mycoplasma were found in the the CES1c activity, we developed a mouse model which closely working cell bank p:86þ14 tested on the April 11, 2013. The mimics the human PK and is suitable for xenograft studies, by number of passages between collection and use in the described cross-breeding CES1c / mice with SCID mice. Using this mouse experiments is 5 and 6. SW-620 cells (90 mL/well; 4,000 cells/well) model we demonstrate that improved PK indeed leads to were plated in 96-wells plates in RPMI1640 medium (Lonza), improved efficacy in PDX studies and improved human dose containing FBS, heat-inactivated (HI; Gibco-Life Technologies) predictions for SYD985. Potentially, this model would also and 80 U/mL Pen/Strep and incubated at 37 C, 5% CO2 over- increase PK/PD relationships of other ADCs that contain link- night. After an overnight incubation 10 mL SYD985 or DUBA was er-drugs that are susceptible to CES1c cleavage. added to each well of the 96-wells plate, containing 10% mouse (BALB/c), CES1c / mouse, rat (Wistar), monkey (Macaca fascicularis), or human plasma. Serial dilutions were made in culture Materials and Methods medium with plasma, to reach a final concentration range of ADCs and related materials 200 nmol/L (10 mg/mL) to 6.3 pmol/L (0.316 ng/mL) for SYD985 SYD985 was prepared as previously described (15). [3H]- and 10 to 0.316 pmol/L for DUBA and 1% plasma. The cell SYD985 was prepared at Pharmaron, Cardiff, UK, using the same viability was measured after 6 days using the CTG Assay Kit procedure and a linker-drug in which two tritium labels were (Promega; G7572). incorporated in the hydroxybenzoyl moiety of the toxin. N-acetyl cysteine-quenched linker-drug (NAc-Cys-linker-drug) was pre- Affinity extraction of SYD985–protein complexes pared by mixing linker-drug and N-acetyl cysteine in a 1:10 molar SYD985 was spiked at 100 mg/mL in mouse (BALB/c) and ratio in an acetonitrile/water mixture for 4 hours followed by CES1c / mouse plasma and incubated up to 48 or 96 hours at purification by preparative HPLC. The head-to-head studies com- 37C, followed by storage at 80C until extraction. For the T ¼ 0 paring T-DM1 with SYD985 were conducted with two batches of samples, SYD985 was spiked in plasma and immediately frozen. T-DM1 from Roche, EU batch N0001B02 (in nude mice) and SYD985 and associated protein complexes were extracted from N1037B19 (in CES1c / SCID mice). plasma using an anti-idiotype mini-antibody (Bio-Rad; AbD15916) coupled via amino coupling to NHS-activated In vitro plasma stability and cleavage by CES1c sepharose 4 fast flow (GE Healthcare). Prior to incubation of the The antibody–drug conjugate SYD985 was spiked into pooled SYD985 plasma samples with the anti-idiotype coupled sephar- female mouse (BALB/c), rat (Sprague Dawley), monkey (Macaca ose, a 50% ammonium sulfate precipitation step was performed fascicularis), and human K2-EDTA plasma, at a concentration of to improve the purity of the extracted samples be selectively 100 mg/mL and incubated at 37 C. After 0, 1, 6, 24, 48, and 96 precipitating the IgG fraction, including SYD985. Following incu- hours of incubation, plasma samples were snap-frozen and stored bation with the anti-idiotype coupled sepharose, the SYD985 at 80 C until bioanalysis. Recombinant mouse CES1c (rCES1c; sample was transferred to a disposable column (Thermo Scien- Cusabio Biotech, CSB-MP338557MO) was spiked in human K2- tific; cat. no. 29920). After removal of the flow-through, sequen- EDTA plasma at 0, 10, 100, 200, and 400 mg/mL) together with tial washes (1 M NaCl in PBS, 10% acetonitrile in PBS and PBS) 100 mg/mL SYD985. After 96 hours of incubation at 37 C, plasma were performed to remove nonspecifically bound proteins.

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SYD985 and associated protein complexes were eluted with proteins were in-gel digested using trypsin (o/n, 10 mmol/L 10 mmol/L glycine-HCl, pH 2.5, followed by immediate neutral- Tris-HCl, pH 8.5). ization with 0.1 M Tris-HCl, pH 8.5. Incubation of rCES1c with NAc-Cys-linker-drug and SYD985 SYD985 analysis by SEC and subsequent proteolytic digestion Radiolabeled [3H]-SYD985 was incubated at 100 mg/mL in Recombinant CES1c (rCES1c) was incubated with NAc-Cys- plasma from female mouse (BALB/c), CES1c / mouse, rat linker-drug or SYD985 in PBS, at a concentration of 100 mg/mL for (Wistar), monkey (Macaca fascicularis), and human for 6 hours 96 hours at 37C under gentle shaking. The resulting sample was at 37C. The resulting plasma samples were analyzed directly by acidified to pH 3.5 using glycine-HCl and reduced and alkylated size exclusion chromatography (SEC) using a Waters BEH200 SEC using TCEP (10 mmol/L final concentration, 37C, 30 minutes) column (4.6 150 mm, 1.7 mm particles) and isocratic elution and N-ethyl maleimide (NEM; 20 mmol/L final concentration, with 100 mmol/L NaPO4, pH 6.8 þ 0.3 M NaCl. Eluting com- RT, 45 minutes in the dark), respectively. Considering that a pounds were detected using a Model 4 b-Ram Radiodetector potential (serine) ester of CES1c with NAc-Cys-linker-drug is (LabLogic). probably base-labile, all incubations and sample preparations of For "cold" (i.e., not radiolabeled) SYD985 that was incubated the resulting CES1c – NAc-Cys-linker-drug complex were per- in plasma of mouse under the conditions mentioned above, anti- formed at low pH (3). For protein digestion, pepsin was added idiotype affinity-extracted material was evaporated to dryness and and the mixture was incubated at 37C (pH 3.0, o/n). reconstituted in 100 mmol/L NaPO4, pH 6.8 þ 0.3 M NaCl before analyses by SEC using the same analytical column and solvent as LC/MS-MS analysis of proteolytic digests described for radiolabeled SYD985, but using UV absorbance Proteolytic peptide mixtures were separated by reversed phase detection at 214 and 330 nm. Eluting compounds, including the liquid chromatography (Dionex Ultimate 3000) using a Waters 0 high molecular weight (HMW) species, were collected, buffer- BEH-300 C18 column (1.0 150 mm, 1.7 mm particles) and a 40 exchanged, and concentrated into 50 mmol/L Tris-HCl pH 8.0 5%–95% linear gradient of acetonitile þ 0.1% formic acid (FA) in using 10K centrifugal filters (Amicon). 8M urea was added to Milli-Q þ 0.1% FA at a flow rate of 0.1 mL/min and a column denature the proteins, followed by reduction and alkylation temperature of 30 C. Eluting peptides were detected using with dithiothreitol (DTT; 4.5 mmol/L final concentration in electrospray ionization mass spectrometry [Thermo Orbitrap 50 mmol/L Tris-HCl, pH 8.5, 37C, 1 hour) and iodoacetamide Fusion, heated electrospray ionization (HESI) at a spray voltage (IAM; 9 mmol/L final concentration in 50 mmol/L Tris-HCl, of 3.8 kV] with MS detection in the Orbitrap at 120K resolution pH 8.5, 37C, 1 hour in the dark), respectively. Following over the scan range m/z 400 to 1,600 and further analyzed an overnight (o/n) precipitation in EtOH, the resulting pellets using data-dependent tandem mass spectrometry (MS-MS) in were digested using trypsin (4 hours, 100 mmol/L Tris-HCl, the quadrupole, using HCD (30%), on the top-3 most intense pH 8.5). precursor ions detected per survey scan to enable protein identification. Fragments were detected in the ion trap. Proteins Coomassie blue staining and Western blot analysis were identified against the Swissprot database using Mascot Samples obtained by affinity extraction were evaporated to search engine. dryness, reconstituted in Milli-Q water and electrophoretically In addition, MS and MS-MS fragmentation data were inter- separated under nonreducing conditions on a 3% to 8% Tris- preted manually using Qualbrowser (Thermo XCalibur 3.0.63) by Acetate gel (Thermo scientific) for total protein staining, or on a comparing proteolytic digests of rCES1c with and without 4% to 12% Bis-Tris gel (Thermo Scientific) for Western blot NAc-Cys-linker-drug and proteolytic digests of rCES1c and analysis. Total protein staining was performed with Bio-safe SYD985 individually to that of the incubated mixtures to inves- Coomassie stain (Bio-Rad) according to manufacturer's instruc- tigate and characterize cross-linked peptide candidates. tions. For Western blot analysis, proteins were transferred to a PVDF membrane (GE Healthcare) with a Mini Trans-Blot Elec- Patient-derived xenograft studies trophoretic Transfer Cell (Bio-Rad). The PDVF membrane was The in vivo antitumor activity of SYD985 versus T-DM1 was blocked with Western blocker solution (Sigma-Aldrich). SYD985 tested as single-dose therapy in MAXF1162 breast cancer PDX þ þ and associated protein complexes were detected with an HRP- model (Charles River) in both CES1c / NMRI-Foxn1nu mice labeled anti-idiotype mini-antibody (Bio-Rad). CES1c was as well as CES1c / SCID mice. Initial tumor volumes at the detected with a biotinylated polyclonal rabbit anti-CES1c anti- day of randomization and treatment ranged from 46 to 252 body (Abcam) in combination with streptavidin-HRP (R&D mm3. The HER2 FISH and IHC status of the tumor from the systems). SYD985 and associated protein complexes and CES1c PDX model as determined by the CROs was independently were visualized by using the Metal Enhanced DAB Substrate Kit confirmed. Studies were conducted as previously described (Thermo Scientific). (15). All in vivo studies and protocols were approved by the local animal care and use committees according to estab- In-gel protein digestion lished guidelines. Protein bands of interest were excised from the Coomassie- stained SDS-PAGE and washed with Milli-Q water and acetonitril, to remove Coomassie and SDS before proceeding to the next step. Results Following in-gel reduction and alkylation with DTT (10 mmol/L CES1c expressed in rodent plasma cleaves vc-seco-DUBA final concentration in 10 mmol/L Tris-HCl, pH 8.5, 37C, 30 In vitro plasma stability studies, as well as in vivo PK studies, minutes) and IAM (20 mmol/L final concentration in 10 mmol/L showed that SYD985 conjugated antibody levels rapidly Tris-HCl, pH 8.5, RT, 45 minutes in the dark), respectively, decrease in mouse and rat plasma, but are stable in monkey

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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 37C 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 37C in human plasma with increasing concentrations of mouse rCES1c (mean SEM, n ¼ 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, n ¼ 3). E, Cytotoxic activity of released active toxin on HER2-negative SW-620 cells after 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.

and human plasma (Fig. 1A and B; Supplementary Fig. S1), Mechanism of action of CES1c-mediated vc-seco-DUBA cleavage similar to what was previously found for closely related ADCs To study if CES1c activity results in release of the toxin DUBA, (13, 14). A literature study pointed towards mouse and rat- SYD985 was incubated with HER2-negative cells in the presence specific CES1c as a potential hydrolyzing enzyme. To assess its or absence of 1% wild-type mouse, CES1c / mouse, rat, monkey, role in SYD985 instability, increasing amounts of rCES1c were or human plasma (Fig. 1E; Supplementary Table S2; ref. 15). spiked in human plasma in combination with 100 mg/mL Compared with the medium control, presence of 1% plasma SYD985 and incubated at 37 C up to 96 hours. SYD985 conju- caused a clear shift in the IC50 for rat (8.08 nmol/L) and especially gated antibody levels decreased with increasing CES1c concen- mouse (0.82 nmol/L) plasma, but not with monkey, human, or / tration (Fig. 1C, previously published as supplementary data; ref. CES1c mouse plasma, all showing an IC50 > 100 nmol/L. 15). Significant cleavage was observed as of 100 mg/mL CES1c As part of a metabolism study, radiolabeled SYD985 was which are physiologically relevant CES1c concentrations, com- analyzed by size exclusion chromatography (SEC) after incuba- parable to the in vivo CES1c concentration in mice (80 mg/mL; tion in mouse, rat, CES1c / mouse, monkey, and human plas- ref. 18). A PK study with SYD985 in CES1c homozygous / ma. In SEC profiles of mouse and rat plasma a new peak appeared þ þ mice, heterozygous CES1c , and homozygous CES1c / wild- (Fig. 2B and D), eluting at an earlier retention time (7.15 minutes) type littermates, showed that the PK in homozygous CES1c / compared with the SYD985 peak (RT ¼ 7.8–8.02 minutes). This mice was similar to the PK in monkey and human (Fig. 1D; new peak was hardly observed in samples taken from CES1c / Supplementary Table S1, previously published as supplementary mouse, monkey, and human plasma (Fig. 2A, C, and E), suggest- data in ref. 15). ing that a SYD985-containing compound with a higher molecular

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CPM CPM 16000.0 CES1c-/- mouse 6000.0 Mouse 14000.0 A B HMW 5000.0 SYD985 SYD985 12000.0

10000.0 4000.0

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14000.0 Monkey Rat C 5000.0 D SYD985 12000.0 HMW SYD985

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0.0 0.0 0.00 10.00 20.00 mins 0.00 10.00 20.00 mins CPM Mouse 14000.0 E Human 0.60 F 12000.0 SYD985 0.50

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0.00 0.0 0.00 10.00 20.00 mins 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 Minutes GH I

0 1 62496 kDa 0 1 62496 kDa 0 1 62496 kDa 250 250 250

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SDS-PAGE αId 75 αCES1c 75 75

Figure 2. A–E, SEC-HPLC radiochromatograms of [3H]-SYD985 after 6 hours incubation in plasma of indicated species at 100 mg/mL and 37C. F–I, Analysis of HMW species in affinity-extracted material obtained from BALB/c mouse plasma incubated with SYD985. F, Confirmatory SEC-HPLC UV chromatogram of non-radiolabeled SYD985 and associated material affinity-extracted after 48 hours of incubation in wild-type mouse plasma at 37C. G, Coomassie staining 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 Western blot analysis with an anti-CES1c antibody showing the presence of CES1c in the HMW species.

weight than SYD985 alone was formed in rat and mouse plasma. subsequently extracted from plasma using anti-idiotype extrac- To identify the compound(s) eluting in this mouse and rat- tion. SEC profiles of the affinity-extracted material confirmed the specific peak, mouse plasma was incubated with SYD985 up to appearance of a SYD985-containing peak, at an earlier retention 48 hours and SYD985 and SYD985-associated material was time as compared with SYD985 itself (Fig. 2F). The extract was

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also analyzed using nonreducing SDS-PAGE and Coomassie generated peptide mixture was analyzed using LC-MS/MS. This staining, which revealed a unique band pattern around 200 to dataset revealed several candidate cross-linked peptide molecules. 250 kDa (Fig. 2G). Although SYD985 has a molecular weight of These were confirmed using a combination of their accurate mass 150 kDa, bands were also observed at lower molecular weight in and the distinctive fragment ions that were also observed when the extracted samples. This is due to the fact that as a result of rCES1c was incubated with NAc-Cys-linker-drug, corresponding conjugation on interchain cysteines, used to manufacture to the rCES1c part of a cross-linked peptide. Subtracting the mass SYD985, its structural integrity (partially) relies on noncovalent of the rCES1c sequence (with part of the linker-drug on it), as well interactions that are disrupted during SDS-PAGE analysis. The as the remainder of the linker-drug part that was conjugated to the 200 to 250 kDa bands were cut out of the gel, trypsin-digested and cysteine residue originating from SYD985, from the precursor analyzed by LC/MS-MS. Next to trastuzumab, CES1c (Uniprot mass, revealed one of the cross-linked peptide candidates to entry P23953) was confidently identified based on 6 unique contain the trastuzumab sequence KSFNRGEC (Supplementary peptides (9% sequence coverage, Mascot score 337) in the lower Fig. S2), originating from a conjugated trastuzumab light chain. migrating band (around 200 kDa), whereas CES1c was also This confirmed the presence of a SYD985-rCES1c cross-link identified in the higher migrating band (around 250 kDa) based through the linker-drug side chain via the proposed mechanism. on 12 unique peptides (25% sequence coverage, Mascot score Finally, this conclusion was underscored by the identification of a 745). CES1c was also observed by LC-MS/MS analysis of a direct cross-link between a rCES1c peptide and the SYD985 hinge (i.e., in solution) digest of affinity-extracted material from wild- region-containing heavy chain peptide THTC(NEM)PPCPA- type mouse plasma following incubation of SYD985 at 37C for PELLGGPSVF (Supplementary Fig. S2), that was originally con- 96 hours. Western blot studies showed the presence of both jugated to one linker-drug. The other cysteine in the sequence was SYD985 and CES1c in the 200 to 250 kDa bands, because it found to be alkylated with N-ethylmaleimide as a result of the stained positive for SYD985 using an anti-idiotypic antibody, sample preparation. Overall, four different rCES1c sequences (all and for CES1c using an anti-CES1c antibody (Fig. 2H and I), containing the active site serine, Ser221) were found to be cross- respectively. linked to three different peptides originating from the light chain, Next, it was attempted to identify the CES1c cleavage site(s) on and to one peptide originating from the hinge region of the heavy the linker-drug in SYD985 and the position of the covalent bond chain (Table 2). between CES1c and SYD985. Covalent interactions between carbamates and esterases have been described for human CES1 Efficacy studies in CES1c knockout mice and CES2 (19). Based on that work, it was hypothesized that the Since in vivo CES1c activity could not be completely blocked by interaction potentially occurred via the active site serine (Ser221) carboxylesterase inhibitors (Supplementary Fig. S3), efficacy stud- in CES1c and one of the carbamates present in the linker-drug ies were performed in CES1c / SCID mice. Before efficacy (Fig. 3A). Due to the structural orientation of the carbamates, the studies were initiated, it was first verified whether the SYD985 PK covalent bond formation with the ADC would most likely in CES1c / SCID mice was identical to that in the original proceed via the carbamate connecting the linker to the DNA- CES1c / strain. As shown in Fig. 4A, the PK profiles indeed alkylating moiety of the toxin, resulting in concomitant release overlapped. Pilot efficacy studies with CES1c / SCID mice of the toxin. using different PDX models (15), showed tumor take and growth To investigate this hypothesis, rCES1c was first incubated with rates similar to those observed in the nude mice. In these models, N-acetylcysteine-quenched linker-drug (NAc-Cys-linker-drug), efficacy was indeed improved at 3 mg/kg SYD985 (Supplemen- for 72 hours at 37C in PBS. Following reduction, alkylation and tary Fig. S4). To confirm the data obtained at a single dose level of proteolytic digestion with pepsin (all at pH 3), LC-MS/MS 3 mg/kg, and to get an accurate estimate of the fold change in analysis of the generated peptide mixture resulted in the identi- efficacy, an experiment comprising of different dose levels was fication of five covalently modified CES1c peptides (Fig. 3B– performed in the HER2 3þ MAXF1162 PDX model. The efficacy of E; Table 1), all including the active site serine Ser221. The proposed different SYD985 dosages was compared head-to-head against covalent interaction was confirmed by the accurate masses of T-DM1, similar to a previous study performed in nude mice (15), these modified peptides and the concomitant mass increase of to control for potential strain differences between the nude and 968.1 Da with respect to the corresponding original (unmodified) SCID background. A non-binding isotype control ADC, bearing peptide mass as a result of the binding of NAc-Cys-linker-drug the same linker-drug and with the same drug-antibody ratio, was (minus the released toxin DUBA). Furthermore, the characteristic included to study the effect of improved PK on efficacy for a non- fragment ions observed in the corresponding MS/MS data, such as target-binding ADC. The efficacy of SYD985 was improved almost the neutral loss of 779.3 Da, corresponding to the majority of the threefold in CES1c / SCID mice showing tumor-regression at 3 NAc-Cys-linker-drug modification; the loss of the complete NAc- mg/kg SYD985 in these animals whereas 10 mg/kg SYD985 Cys-linker-drug modification (985.4 Da) resulting in the forma- induced tumor-stasis in CES1c þ/þ nude mice. The isotype tion of a dehydroalanine moiety from the serine residue, and control also showed efficacy at 3 mg/kg, in contrast to the lack several NAc-Cys-linker-drug specific fragment ions (Fig. 3E) sup- of efficacy in nude mice. This difference is most likely also caused ported the hypothesized structure of the CES1c-linker-drug cross- by an increased exposure of intact isotype control ADC. Extracel- link. In addition, free seco-DUBA, the pro-toxin that is released lular cleavage or pinocytosis of the isotype control could explain upon linker-drug cleavage and is stable at acidic pH, was also the efficacy, which is not uncommon for the isotype control (15). identified in the incubated digest at pH < 3, confirming the release As expected, since T-DM1 is not susceptible to CES1c cleavage, of the toxin. T-DM1 showed similar efficacy in nude mice versus CES1c / Next, SYD985 was incubated with rCES1c in PBS during 72 SCID mice, with prolonged tumor stasis at 10 mg/kg. hours at 37C. Following the same, low pH, sample work up as Recently, phase I clinical data were published describing described for the NAc-Cys-linker-drug incubated sample, the the efficacy of SYD985 at the recommended phase II dose of

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CES1c Knockout Mice for ADC Development

A C V

G G E S G S

V Exact mass: m/z 980.49 (1+) V S B S I D I G G E S G S G G E S G dhA

Exact mass: m/z 880.39 (2+) Exact mass: m/z 774.36 (1+)

681.2722 NL: 2.57E4 GESSGG E 100 Full ms2 [email protected]

50 349.0958 207.1686 588.2110 730.3232 1035.2388 1210.5594 1401.1116 0 NL: 1.49E5 GESSGGIS 881.4395 100 Full ms2 [email protected]

50 349.2315 675.3375 240.2588 987.4726 1251.5571 1390.4729 1567.6655 0 980.5031 NL: 1.36E5 GESSGGISV 100 Full ms2 [email protected]

50 349.2002 774.4719 240.2942 588.3439 1085.4099 1350.6631 1512.6050 1717.5129 0

Relave abundance 1067.4961 NL: 2.21E4 GESSGGISVS 100 Full ms2 [email protected]

50 349.1088 861.3724 240.1815 588.2286 1142.3851 1399.5941 1607.2213 1808.9518 0 1141.6213 NL: 8.60E4 IFGESSGGIS 100 Full ms2 [email protected]

50 349.1663 935.4435 240.2594 588.2765 830.3983 1226.6826 1530.7034 1717.8667 0 200 400 600 800 1000 1200 1400 1600 1800 m/z

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 resulting in 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 peptide GESSGGISV 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 carboxylesterase with 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) fromthe precursor ion is clearly visible (note: all precursors are doubly-charged), just like the presence of the dehydroalanine-containing peptide fragments (at m/z "precursor ion": 987.3 Da) and linker-specific fragments, such as those at m/z 163.1, 207.2, 240.3, and 349.1.

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Table 1. Characteristics of the peptides containing the CES1c active site serine (Ser221) that were found to be conjugated to NAc-Cys-linker-drug Theoretical Observed m/z (z) Observed m/z (z) Most predominant Peptide sequence peptide mass (Da) without conjugate with conjugate fragment ion m/z GES221SGG 492.19 493.19 (1) 730.79 (2) 681.27 GES221SGGIS 691.37 692.37 (1) 830.85 (2) 881.44 GES221SGGISV 790.37 791.37 (1) 880.39 (2) 980.50 GES221SGGISVS 877.40 878.40 (1) 923.90 (2) 1067.50 IFGES221SGGIS 951.45 952.45 (1) 960.93 (2) 1141.62

1.2 mg/kg in HER2 positive metastatic breast cancer patients PDX model very nicely correlated with that in humans at the (20, 21). The 1.2 mg/kg dose was shown to be efficacious recommended phase II dose at 1.2 mg/kg which turned out to with an overall response rate (ORR) of 33% (16 of 48 patients) be efficacious. and a progression-free survival (PFS) of 9.4 months (95% CI, Based on the in vivo PK data it was reasoned that the instability 4.5–12.4). The human PK of SYD985 at 1.2 mg/kg (AUCinf ¼ of SYD985 is most likely caused by cleavage or release of the 1,725 h.µg/mL) almost overlaps with the PK observed in linker-drug, rather than clearance of the entire ADC, because total MAXF1162 tumor bearing CES1c / SCID mice at the 1 mg/kg antibody levels were similar in all tested species. It was hypoth- dose that shows tumor stasis (AUCinf ¼ 1,332 h.µg/mL; Fig. 4D; esized that the instability is the result of hydrolysis of one of the Supplementary Table S3). carbamate moieties in the linker-drug because these are known substrates for esterase-like activity (22, 23). Preliminary studies with human esterases suggested that neither human CES1 and Discussion CES2 nor the plasma esterase BCHE seem to be able to cleave the Until recently, all xenograft studies with vc-seco-DUBA-based linker-drug in SYD985 (data not shown). Based on a literature ADCs, including SYD985, were performed in immune- study focused on species differences in esterase activity in plasma, compromised mice expressing CES1c. Despite the low exposure the mouse- and rat-specific CES1c was identified as a candidate levels for conjugated antibody in these mice, SYD985 showed hydrolyzing enzyme (18, 24, 25). Incubation of SYD985 in good efficacy, which was superior to that of T-DM1 in numerous human plasma spiked with rCES1c indeed revealed that CES1c xenograft models with different levels of HER2 target expression caused instability of SYD985. The final evidence was provided by (15). However, because the SYD985 PK profiles in the mouse were a PK study in CES1c / mice, showing a PK profile similar to the dramatically different from those in monkey and the anticipated PK in monkey and human. The identification of CES1c triggered profile in human based on in vitro plasma stability data, an additional studies to reveal its mode of action. The increase in accurate estimation of an effective human dose based on efficacy potency observed after in vitro incubation of SYD985 with HER-2 in PDX models was not possible. Based on extrapolation of negative cells and 1% mouse or rat plasma, compared with þ þ available PK data for SYD985 in CES1c / mice (either nude or medium, human, monkey and CES1c / plasma suggested active SCID) AUCs of conjugated antibody at tumor static dosages are toxin DUBA is released by CES1c activity. LC/MS-MS analysis estimated to be between 150 and 260 h.mg/mL. This is based on confirmed the presence of significant amounts of DUBA in plasma data generated in the MAXF1162 PDX model and the BT474 samples from mice and rats dosed with SYD985. model (15) and in normal (non-immunedeficient) mice. This Most surprisingly, CES1c activity not only results in release of AUC is more than tenfold below the observed AUC in the clinic at DUBA: here it is shown that it also results in the formation of the recommended phase II dose of 1.2 mg/kg that showed efficacy HMW species, as identified by SEC. Using SDS-PAGE, Western þ þ (21). Thus, using the AUC at tumor static dose in CES1c / mice blotting and LC-MS/MS, these HMW species were found to to predict the effective human dose would lead to a significant contain both trastuzumab and CES1c, which suggests that at least under-prediction of this dose. It was anticipated that the trans- one CES1c molecule is capable of covalently binding to SYD985. lational value of PDX models would improve if the PK profile in Next, attempts to identify the site of interaction between CES1c mice was more in line with the anticipated human PK profile. As and the linker-drug disclosed it to be the carbamate group con- will be discussed later, the AUC of conjugated SYD985 at a necting the linker and toxin. Other carbamates in the linker-drug tumor static dose of 1 mg/kg in CES1c / mice in the MAXF1162 seem to remain unaffected. CES1c activity results in release of

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 chain 828.04 (3þ) 2482.13 1140.6 618.28 723.25 FNRGEC 773.74 (3þ) 2319.23 979.5 618.28 723.30 FNRGEC 741.32 (3þ) 2221.98 880.4 618.28 723.30 FNRGEC 813.03 (3þ) 2437.11 880.4 618.28 938.43 KSFNRGEC 684.32 (4þ) 2734.28 880.4 618.28 1235.60 PVTKSFNRGEC Heavy chain 836.39 (4þ) 3342.55 680.3 618.28 2045.99 THTC(NEM)PPCPAPELLGGPSVF

891.18 (4þ) 3561.70 880.4 618.28 2064.02 THTC(NEM)PPCPAPELLGGPSVF þH2O 1181.89 (3þ) 3543.69 880.4 618.28 2046.01 THTC(NEM)PPCPAPELLGGPSVF a CES1c peptide fragments (uncharged): 680.3 ¼ GESSGG; 880.4 ¼ GESSGGIS; 979.5 ¼ GESSGGISV; 1140.6 ¼ IFGESSGGIS. "þH2O" refers to hydrolysis (ring opening) of the maleimide moiety in the linker drug.

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CES1c Knockout Mice for ADC Development

Figure 4. Pharmacokinetics of SYD985 in A, CES1c/ mice versus CES1c/ SCID mice after an intravenous dose of 3 mg/kg (mean SEM, n ¼ 3) and D, after dosing 0.3, 1, and 3 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 3þ MAXF1162 breast cancer PDX model in CES1cþ/þ nude mice (B, C) or in CES1c/ SCID mice (E, F; mean SEM, n ¼ 6–8).

DUBA and formation of a covalent bond between SYD985 and drug optimization specifically for the mouse, it was attempted to CES1c, most likely via the serine (Ser221) in its active site, although block mouse-specific CES1c activity in vivo. Initial experiments it cannot be excluded that the modification occurs on serine-222, with esterase inhibitors failed to completely block CES1c activity. adjacent to the active site serine. CES1c-SYD985 cross-links were Thus, immune-compromised CES1c / mice were needed to observed both in the hinge region of SYD985 heavy chain and evaluate the full impact of the absence of CES1c activity on PK SYD985 light chain. All together, these interaction studies clarified and in vivo efficacy in PDX models. Cross-breeding of CES1c / the structure of the covalent bond between CES1c and SYD985 mice with SCID and nude mice was setup in parallel. The cross- that is -unexpectedly- formed upon CES1c cleavage of the vc-seco- breeding of CES1c / mice with nude mice showed poor repro- DUBA linker-drug at a specific carbamate. ductive performance, whereas the SCID cross-breeding was suc- Clearly, CES1c activity is a more general concern in the devel- cessful. Pilot PDX studies in CES1c / SCID mice suggested opment of ADCs. In a recent publication, Dorywalska and col- improved efficacy of SYD985, compared with previous studies þ þ leagues (15) showed that CES1c also cleaves other linker-drugs. in CES1c / nude mice. The full PKPD study in the MAXF1162 Their vc-PABC containing linker-drug was postulated to be PDX model with T-DM1 to control for potential strain differences cleaved at the carbamate C-terminal to the citrulline moiety. confirmed improved efficacy of SYD985 in CES1c / SCID mice Cleavage by, or covalent interaction with, CES1c would not result showing tumor stasis at 1 mg/kg. At this dose, the PK profile and in a complex between the ADC and the esterase due to the AUC are very similar to those at the recommended phase II dose in structural orientation of the carbamate in the linker-drug (19). human. This shows that use of CES1c / mice is a good and for us Even though this linker-drug and vc-seco-DUBA share the vc-PABC the preferred alternative to unnecessary structural optimization of structure, we found no evidence for CES1c-mediated cleavage the linker-drug that might even compromise on human-relevant C-terminal to citrulline, or for hydrolysis at the other carbamate parameters. positions in vc-seco-DUBA, underlining the complexity of the – structure activity relationship of CES1c. Several other publica- Conclusions tions describe carbamate-containing linker-drugs that are unstable in studies with mice and rat and are therefore potentially Even though it is known that the rodent-specific esterase CES1c sensitive to cleavage by CES1c (17, 26). So far, modifying the is able to cleave linker-drugs on ADCs at distinct sites, the impact structure of the linker-drug is the general mitigation to enhance of CES1c activity on the predictive value of PK, efficacy and safety ADC stability in mouse plasma, even though the linker-drug is studies with ADCs in mice and rats should not be underestimated. perfectly stable in human plasma and from that perspective does Here it is shown that cleavage of vc-seco-DUBA results in the not require further optimization. In order to circumvent linker- formation of a covalent bond between CES1c and the ADC, which

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indicates that the structure-activity relationship of CES1c is more Analysis and interpretation of data (e.g., statistical analysis, biostatistics, complex than originally thought. It not only depends on the computational analysis): R. Ubink, E.H.C. Dirksen, E.S. Bos, M.M.C. van structure of the linker-drug, but also on the site of conjugation in der Lee, F. Bichat, P.G. Groothuis, W.H.A. Dokter Writing, review, and/or revision of the manuscript: R. Ubink, E.H.C. Dirksen, the ADC. Consequently, developing linker-drugs that are stable in M. Rouwette, I. Janssen, D.F. Egging, E.M. Loosveld, K. Berentsen, M.M.C. van / both mice and humans, remains challenging. The use of CES1c der Lee, M.A.J. van der Vleuten, P.G. Groothuis, W.H.A. Dokter mice widens the spectrum of linker-drugs suitable for the devel- Administrative, technical, or material support (i.e., reporting or organizing opment of ADCs, thereby increasing the chance that ADCs are data, constructing databases): F. Bichat successfully developed. Study supervision: R. Ubink, E.H.C. Dirksen, O. Raguin, W.H.A. Dokter

Disclosure of Potential Conflicts of Interest Acknowledgments No potential conflicts of interest were disclosed. Financial support for this study was provided by Synthon Biopharmaceu- ticals BV. Authors' Contributions Conception and design: R. Ubink, E.H.C. Dirksen, E.S. Bos, D.F. Egging, The costs of publication of this article were defrayed in part by the M.M.C. van der Lee, P.G. Groothuis, W.H.A. Dokter payment of page charges. This article must therefore be hereby marked Development of methodology: E.H.C. Dirksen, D.F. Egging, T.A. van advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Achterberg, M.M.C. van der Lee, F. Bichat this fact. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): E.H.C. Dirksen, M. Rouwette, I. Janssen, E.M.Loosveld,T.A.vanAchterberg,K.Berentsen,F.Bichat,O.Raguin, Received March 30, 2018; revised June 19, 2018; accepted July 27, 2018; M.A.J. van der Vleuten published first August 9, 2018.

References 1. Lambert JM. Drug-conjugated antibodies for the treatment of cancer. Br J benefit in low HER2-expressing breast cancers. Mol Cancer Ther Clin Pharmacol 2013;76:248–62. 2015;14:692–703. 2. Jain N, Smith SW, Ghone S, Tomczuk B. Current ADC linker chemistry. 16. Dorywalska M, Dushin R, Moine L, Farias SE, Zhou D, Navaratnam T, Pharm Res 2015;32:3526–40. et al. Molecular basis of valine-citrulline-PABC linker instability in site- 3. Lu J, Jiang F, Lu A, Zhang G. Linkers having a crucial role in antibody-drug specific ADCs and its mitigation by linker design. Mol Cancer Ther conjugates. Int J Mol Sci 2016;17:561. 2016;15:958–70. 4. Beck A, Goetsch L, Dumontet Cl, Corva€a N. Strategies and challenges for 17. Gupta N, Kancharla J, Kaushik S, Ansari A, Hossain S, Goyal R, et al. the next generation of antibody-drug conjugates. Nat Rev Drug Discov Development of a facile antibody-drug conjugate platform for increased 2017;16:315–37. stability and homogeneity. Chem Sci 2017;8:2387–95. 5. Drake PM, Rabuka D. Recent developments in ADC technology: preclinical 18. Li B, Sedlacek M, Manoharan I, Boopathy R, Duysen EG, Masson P, et al. studies signal future clinical trends. BioDrugs 2017;31:521–31. Butyrylcholinesterase, paraoxonase, and albumin esterase, but not carbox- 6. Frigerio M, Kyle AF. The chemical design and synthesis of linkers used in ylesterase, are present in human plasma. Biochem Pharmacol 2005;70: antibody drug conjugates. Curr Top Med Chem 2017;17:3393–424. 1673–84. 7. Ducry L, Stump B. Antibody-drug conjugates: linking cytotoxic payloads to 19. Crow JA, Bittles V, Borazjani A, Potter PM, Ross MK. Covalent inhibition of monoclonal antibodies. Bioconjug Chem 2010;21:5–13. recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase 8. van der Velden VH, te Marvelde JG, Hoogeveen PG, Bernstein ID, by the carbamates JZL184 and URB597. Biochem Pharmacol 2012;84: Houtsmuller AB, Berger MS, et al. Targeting of the CD33-calicheamicin 1215–22. immunoconjugate Mylotarg (CMA-676) in acute myeloid leukemia: in 20. Van Herpen CML, Banerji U, Mommers ECM, Koper NP, Goedings P, Lopez vivo and in vitro saturation and internalization by leukemic and normal J, et al. Phase I dose-escalation trial with the DNA-alkylating anti-HER2 myeloid cells. Blood 2001;97:3197–204. antibody-drug conjugate SYD985. ESMO 2015 poster presentation. 2015. 9. Shen BQ, Xu K, Liu L, Raab H, Bhakta S, Kenrick M, et al. Conjugation site 21. Saura C, Thristlethwaite F, Banerji U, Lord S, Moreno V, MacPherson I, et al. modulates the in vivo stability and therapeutic activity of antibody-drug A phase I expansion cohorts study of SYD985 in heavily pretreated patients conjugates. Nat Biotechnol 2012;22:184–9. with HER2-positive or HER2-low metastatic breast cancer. ASCO 2018 10. Durbin KR, Nottoli MS, Catron ND, Richwine N, Jenkins GJ. High- poster presentation. 2018. throughput, multispecies, parallelized plasma stability assay for the 22. Yang Y, Aloysius H, Inoyama D, Chen Y, Hun L. Enzyme-mediated determination and characterization of antibodydrug conjugate aggre- hydrolytic activation of prodrugs. Acta Pharmaceutica Sinica B 2011;1: gation and drug release. ACS Omega 2017;2:4207–15. 143–59. 11. Shadid M, Bowlin S, Bolleddula J. Catabolism of antibody drug conjugates 23. Laizure SC, Herring V, Hu Z, Witbrodt K, Parker RB. The role of human and characterization methods. Bioorg Med Chem 2017;25:2933–45. carboxylesterases in drug metabolism: have we overlooked their 12. Di L, Kerns EH, Hong Y, Chen H. Development and application of high importance? Pharmacotherapy 2013;33:210–22. throughput plasma stability assay for drug discovery. Int J Pharm 24. Berry LM, Wollenberg L, Zhao Z. Esterase activities in the blood, liver and 2005;297:110–9. intestine of several preclinical species and humans. Drug Met Lett 13. Dokter W, Ubink R, van der Lee M, van der Vleuten M, van Achterberg T, 2009;3:70–7. Jacobs D, et al. Preclinical profile of the HER2-targeting ADC SYD983/ 25. Duysen EG, Cashman JR, Schopfer LM, Nachon F, Masson P, Lockridge O. SYD985: introduction of a new duocarmycin-based linker-drug platform. Differential sensitivity of plasma carboxylesterase-null mice to parathion, Mol Cancer Ther 2014;13:2618–29. chlorpyrifos and chlorpyrifos oxon, but not to diazinon, dichlorvos, 14.ElgersmaRC,CoumansRG,HuijbregtsT,MengeWM,JoostenJA, diisopropylfluorophosphate, cresyl saligenin phosphate, cyclosarin thio- Spijker HJ, et al. Design, synthesis, and evaluation of linker-duocarmy- choline, tabun thiocholine, and carbofuran. Chem Biol Interact cin payloads: toward selection of HER2-targeting antibody-drug con- 2012;195:189–98. jugate SYD985. Mol Pharm 2015;12:1813–35. 26. Dorywalska M, Strop P, Melton-Witt JA, Hasa-Moreno A, Farias SE, 15. van der Lee MM, Groothuis PG, Ubink R, van der Vleuten MA, Galindo Casas M, et al. Site-dependent degradation of a non-cleavable van Achterberg TA, Loosveld EM, et al. The preclinical profile of the auristatin-based linker-payload in rodent plasma and its effect on ADC duocarmycin-based HER2-targeting ADC SYD985 predicts for clinical efficacy. PLoS One 2015;10:e0132282.

2398 Mol Cancer Ther; 17(11) November 2018 Molecular Cancer Therapeutics

Unraveling the Interaction between Carboxylesterase 1c and the Antibody−Drug Conjugate SYD985: Improved Translational PK/PD by Using Ces1c Knockout Mice

Ruud Ubink, Eef H.C. Dirksen, Myrthe Rouwette, et al.

Mol Cancer Ther 2018;17:2389-2398. Published OnlineFirst August 9, 2018.

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