highly potent, anthracycline-based antibody drug ... · nikolas stefan, remy gebleux, lorenz...

Large Molecule Therapeutics

Highly Potent, Anthracycline-basedAntibody–Drug Conjugates Generated byEnzymatic, Site-specific ConjugationNikolas Stefan, R�emy G�ebleux, Lorenz Waldmeier, Tamara Hell, Marie Escher,Fabian I.Wolter, Ulf Grawunder, and Roger R. Beerli

Abstract

Antibody–drug conjugates (ADC) are highly potent and spe-cific antitumor drugs, combining the specific targeting of mAbswith the potency of small-molecule toxic payloads. ADCs gener-ated by conventional chemical conjugation yield heterogeneousmixtures with variable pharmacokinetics, stability, safety, andefficacy profiles. To address these issues, numerous site-specificconjugation technologies are currently being developed allow-ing the manufacturing of homogeneous ADCs with predeter-mined drug-to-antibody ratios. Here, we used sortase-mediatedantibody conjugation (SMAC) technology to generate homo-geneous ADCs based on a derivative of the highly potent

anthracycline toxin PNU-159682 and a noncleavable peptidelinker, using the anti-HER2 antibody trastuzumab (part ofKadcyla) and the anti-CD30 antibody cAC10 (part of Adcetris).Characterization of the resulting ADCs in vitro and in vivoshowed that they were highly stable and exhibited potenciesexceeding those of ADCs based on conventional tubulin-target-ing payloads, such as Kadcyla and Adcetris. The data presentedhere suggest that such novel and highly potent ADC formatsmayhelp to increase the number of targets available to ADCapproaches, by reducing the threshold levels of target expressionrequired. Mol Cancer Ther; 16(5); 879–92. �2017 AACR.

IntroductionAntibody–drug conjugates (ADC) take advantage of the spec-

ificity of mAbs for the targeted delivery of toxic payloads to cancercells (1, 2). Because of the specific targeting of ADCs to the tumor,this class of drugs is expected to be associated with less side-effectsand a higher therapeutic index than standard chemotherapy.Although highly plausible, this straightforward concept has onlyslowly translated into the clinic and the development of ADCs hasbeen associated with difficulties and setbacks (1, 3).

The first ADC to be approved was Mylotarg (4), an anti-CD33ADC for the treatment of acute myeloid leukemia (AML). Thisfirst-generation ADC suffered from unstable covalent coupling ofthe toxin to the antibody, leading to significant release of toxinmolecules in circulation, which resulted in an unfavorable ther-apeutic index of this first-generation ADC. Eventually, Mylotarg,which had been FDA approved in 2000, was withdrawn from theU.S. market in 2010. Because of the safety concerns of this ADC,Mylotarg was never approved for cancer treatment by the Euro-pean Medicines Agency (EMA; London, United Kingdom). Morerecently, also the anti-CD22ADC inotuzumab–ozogamicin, com-prising the same linker-payload as Mylotarg, yielded disappoint-

ing clinical data and did not show statistically significant benefitfor the treatment of non-Hodgkin lymphoma (NHL; ref. 5).

As a result of the difficulties associated with such first-generation ADCs, the quality and functionality of chemicallinkers for coupling toxins to antibodies has been improvedsubstantially in recent years (6). Various new linker-payloadshave been developed for incorporation into "second-genera-tion" ADCs, including lysine-reactive noncleavable and cleav-able derivatives of the maytansinoids, DM1 and DM4 (7), andcysteine-reactive derivatives of the auristatins monomethylaur-istatin F (MMAF) and E (MMAE; refs. 8, 9). Consequently, twoADCs, Adcetris and Kadcyla, have been approved for cancertherapy. Adcetris (brentuximab vedotin) is an ADC for thetreatment of CD30-positive lymphomas (e.g., Hodgkin lym-phoma), and employs a protease-cleavable linker based on thedipeptide valine–citrulline and a self-immolative p-aminoben-zylcarbamate (PAB) group connected to the N-terminus ofMMAE (8). Kadcyla (trastuzumab-emtansine or T-DM1) is anADC approved for second-line therapy of HER2-positive breastcancer based on Herceptin, and employs a noncleavable SMCCthioether linker connected to DM1 (10).

Despite these highly encouraging developments, severalchallenges are still encountered even with second-generationADCs. One challenge is related to the inherent instability of theutilized maleimide-containing linkers in serum. The thio–suc-cinimid linkage present in these linkers can be reversed via aretro-Michael reaction involving free thiol groups present inhigh concentrations in human blood in the form of cysteine-34of human serum albumin (11). This problem has been recog-nized and was recently addressed with the design of novellinker variants with maleimides displaying improved serumstability (12, 13).

NBE-Therapeutics AG, Basel, Switzerland.

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

Corresponding Author: Roger R. Beerli, NBE-Therapeutics AG, Hochberger-strasse 60C, 4057 Basel, Switzerland. Phone: þ41-61-633-2233; Fax: þ41-61-633-2231. E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-16-0688

�2017 American Association for Cancer Research.

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The second challenge encountered by second-generation ADCsis related to their inherent heterogeneity. Conjugation of payloadsto lysine or cysteine residues occurs randomly and only allows alimited control over the site of conjugation or the number oftoxins coupled to the antibody (the drug-to-antibody ratio, orDAR; ref. 14). For the maytansine- and auristatin-based linker-payloads mentioned above, which are quite hydrophobic, it hasbeen empirically determined that the "sweet-spot" for the averageDAR is between 3 and 4, which is also reflected by theDARs of thetwo commercial products, Adcetris and Kadcyla, which have beenreported to be 4.0 and 3.5, respectively (15, 16). However, it isimportant to note that evenwith an averageDAR in this range, theADC preparation consists of a mixture of molecules comprisingindividual components with DARs ranging from 0 to 8 (14), eachbehaving differently with respect to their pharmacokinetic, effi-cacy, and safety profiles (17).

It is possible that the abovementioned issues of current ADCsare, at least in part, responsible for disappointing results obtainedrecently in late-stage clinical trials, including the failure of Roche'sKadcyla in gastric cancer and first-line therapy of breast cancer(18), and the discontinuation of Sanofi's SAR3419 in diffuse largeB-cell lymphoma (19). Thus, there is currently intense researchongoing to develop strategies for the manufacturing of site-spe-cifically conjugated, homogeneous ADCs. These strategies notonly include the introduction of unique handles for chemicalconjugation, such as by incorporation of unnatural amino acids(20) or by site-directed mutagenesis (21), but also various enzy-matic approaches, including the use of bacterial transglutaminase(22), formylglycine-generating enzyme (FGE; ref. 23), and S.aureus sortase A (24). Using such novel approaches, highly homo-geneous, site-specifically conjugated "next-generation" ADCs cannow be prepared (1). As maleimide-containing linkers are oftenavoided, site-specific ADCs circumvent the stability issuesdescribed above which are observed with conventional ADCs.

Anadditional limitationof conventional ADCs is their potency.By far, the most abundant payloads employed in clinical-stageADCs, as well as those used in Adcetris and Kadcyla, are based onauristatin and maytansine toxins, respectively, both of whichinterfere with microtubule reorganization (2). While these pay-loads work very well with certain tumor-specific antigens (TSA), itis becoming increasingly clear that their efficacy is limited to TSAsthat are highly expressed and/or show rapid internalization andtrafficking of the ADC into late endosomal or lysosomal compart-ments. This is particularly well documented in the case of Kadcyla,where there is ample clinical data to support this notion (25, 26).To address this limitation, numerous approaches are currentlybeing taken. The most obvious approach is to use ADCs withhigher DARs, which in the case of auristatins and maytansines islimited by the hydrophobicity of these payloads, leading to ADCswith a tendency to aggregate and with poor pharmacokineticprofiles (14). To circumvent this problem, high DAR ADCs weredeveloped, for example, by using so-called hydrophilic fleximersfor attachment of the payloads to the mAb. Because of the highlyhydrophilic nature of fleximers, ADCs with a DAR of 20 can beprepared that still exhibit favorable pharmacokinetic propertiesand substantially improved efficacy (27). Another approachreported recently is based on induction of receptor clustering bytargeting two independent epitopes on a TSA. As a result, suchbiparatopic ADCs show enhanced internalization and lysosomaltrafficking and, consequently, superior potency when comparedwith a conventional, monospecific ADC (28). Finally, alternative

payloads with substantially higher potency than microtubule-disrupting agents are being investigated, including pyrroloben-zodiazepine (PBD) dimers (29), enedyenes such as calicheamicin(30), indilino-benzodiazepine (IBD) pseudodimers (termedIGNs; ref. 31), as well as novel anthracylines (32, 33).

In this study, we describe ADCs that address all the above-mentioned shortcomings of conventional ADCs. Site-specificallyconjugated and homogeneous ADCs based on a derivative of theanthracycline PNU-159682 (34) were generated using SMACtechnology (24), and their in vitro and in vivo activities wereinvestigated.

Materials and MethodsCell lines

The breast cancer cell lines, SKBR3 and T47D, received as giftfrom Nancy E. Hynes at the Friedrich Miescher Institute (Basel,Switzerland) in July-2013, were grown in DMEM/10% FCS.Karpas-299 NHL cells, received as a gift from Alfred Zippelius atthe University Hospital Basel (Basel, Switzerland) in September-2013, and REH ALL cells (purchased from ATCC, order numberACRL-8286), were passaged in RPMI/10% FCS. Cell lines werealways used within less than 10 passages, after which a fresh vialwas thawed. The expression level of HER2 and CD30 on each cellline was evaluated by FACS analysis. No additional authentica-tion was done by the authors. The cells used in the Karpas-299xenograft study in CB17.SCID mice, performed by ProQinase,were purchased from the DSMZ in February 2007. They were lastauthenticated on October 23, 2013 by STR profiling (performedby LGC Standards). The cells used in the study were <10 passagesaway from the authenticated sample tube. The cells used in theJIMT-1 xenograft study, performedbyCharles Rivers Laboratories,were purchased from theDSMZ in August 2011 (ACC589, lot#5).A frozenMaster Cell bank was created from the original source onAugust 24, 2011. From that initial bank, aWorking Cell Bank wasgenerated on September 29, 2011. A vial from this working bankwas thawed and used for the JIMT-1 study. All cell banks weretested and authenticated on March 30, 2016 by STR profiling(performed by Genetica DNA laboratories). The results wereentered into the DSMZ's database for confirmation.

Generation of recombinant antibodiesCloning, expression in CHO cells, and purification of

HER2-specific trastuzumab and CD30-specific cAC10/brentux-imab, with their heavy and light chains C-terminally taggedwith a sortase A recognition motif and a Strep II-tag, was de-scribed previously (24). The tag sequences appended were: IgHchain, LPETG-G-WSHPQFEK; IgL chain, GGGGS-LPETG-G-WSHPQFEK. Expression in 293T cells and purification ofTwinStrep-tagged trastuzumab was done as described previous-ly (35). The tag sequences appended were: IgH chain, LPETG-G-WSHPQFEKGGGSGGGSGGSAWSHPQFEK-GS; IgL chain,GGGGS-LPETG-G-WSHPQFEKGGGSGGGSGGSAWSHPQFEK-GS. TwinStrep-tagged trastuzumab was only used in the experi-ments described in Figs. 2 and 4A and B; all other experimentswere performed with Strep II-tagged mAbs.

Synthesis of oligo-glycine linker-payloadsSynthesis of Gly5-EDA-Dox, Gly5-EDA-Nemo, Gly5-EDA-PNU

and Gly3-vcPAB-PNU-159682 was performed by Levena Bio-pharma and the glycine-modified payloads were provided in

Stefan et al.

Mol Cancer Ther; 16(5) May 2017 Molecular Cancer Therapeutics880




>90% purity according to high-performance liquid chromato-graphy (HPLC) analysis.

Production of recombinant Sortase A variants fromStaphylococcus aureus

Highly active evolved Sortase A (eSrtA) was produced asdescribed previously (24).

Sortase-mediated toxin conjugationSMAC was performed essentially as described (24). Briefly,

tagged mAbs [10 mmol/L] were incubated with oligo-glycine–modified toxin [200 mmol/L] in the presence of 3 mmol/L SortaseA in 50mmol/L HEPES, 150mmol/L NaCl, 1 mmol/L CaCl2, pH7.5 for 3.5 hours at 25�C. The reaction was stopped by passing itthrough an rProtein A GraviTrap column (GE Healthcare) equil-ibrated with 25 mmol/L HEPES, 150 mmol/L NaCl, pH 7.5,followed by washing with 20 column volumes (CV) of buffer.Bound conjugatewas elutedwith5CVsof elutionbuffer (0.1mol/L glycine, 50 mmol/L NaCl, pH 2.5) with 0.5 CV fractionscollected into tubes containing 25% v/v 1 mol/L HEPES pH 8to neutralize the acid. Protein-containing fractions were pooledand formulated using ZebaSpin desalting columns (ThermoFisher Scientific) according to the manufacturer's instructions.cAC10 ADCs were formulated in PBS without Ca2þ and Mg2þ

(Amimed-Bioconcept) and Trastuzumab ADCs in Kadcyla-buffer(10 mmol/L sodium succinate pH 5.0, 175 mmol/L sucrose,0.02% w/v polysorbate 20). ADCs used in in vivo studies weresubjected to an additional polishing step prior to formulation byusing 0.5 mL Pierce High-Capacity Endotoxin Removal Spincolumns (Thermo Fisher Scientific) according to the manufac-turer's instructions. For subsequent DAR 4.0 enrichment (Fig. 2)and the cytotoxicity assay described in Fig. 4A and B, TwinStrep-tagged trastuzumab was used for conjugation. Trastuzumab con-jugates for DAR 4.0 enrichment were formulated in PBS.

DAR 4.0 enrichmentTras-Gly5-May and Tras-Gly5-PNU (1.1 mg) produced as

described above was passed over a 0.5-mL StrepTactin Superflowhigh-capacity gravity flow column (IBA) and washed with PBSwithout Ca2þ andMg2þ. Flow through andwashwere collected infractions. Peak fractions as determined by Bradford assay werepooled and concentrated by ultrafiltration.

ADC analyticsThe drug loading was assessed by reverse-phase chromatogra-

phy performed on a PLRP-S 2.1 mm � 5 cm, 5 mm column(Agilent) run at 1 mL/min/80�C with a linear gradient between0.05% TFA/H2O and 0.04% TFA/CH3CN. Prior to analysis, sam-ples were reduced with DTT at 37�C for 15 minutes.

Cell killing assaysCytotoxicity of ADCs was investigated in cell killing assays

essentially as described previously (24). Briefly, cells were platedon 96-well plates in 75mL growthmediumand grown at 37�C in ahumidified incubator in a 7.5% CO2 atmosphere. The cell den-sities varied, depending on the experiment and cell lines (SKBR3and T47D, 5,000 or 10,000 cells per well; Karpas-299, 2,500 or5,000 cells per well; REH, 10,000 cells per well). After one dayincubation, 25 mL of 3.5-fold serial dilutions of each ADC ingrowth medium were added, typically resulting in final ADCconcentrations from 20 mg/mL to 0.02 ng/mL. Each dilution was

done in duplicate. For competition, ADC serial dilutions weredone in growth medium containing unconjugated trastuzumabat a concentration of 200 mg/mL. After 4 additional days, plateswere removed from the incubator and equilibrated to roomtemperature. After approximately 30 minutes, 50 mL mediumwas removed carefully from each well and replaced with 50 mLCellTiter-Glo Luminescent Solution (Promega, catalog no.G7570). After shaking the plates at 450 rpm for 5 minutesfollowed by 10 minutes incubation in the dark without shaking,luminescence was measured on a Tecan Spark with an integra-tion time of 1 second per well.

In vitro serum stabilityTo evaluate serum stability, cAC10-Gly5-PNU was diluted to

a concentration of 100 mg/mL in freshly prepared serum fromBalb/c mice, or in commercially available sera from rat (Sigma),cynomolgus monkey (Abcam), and human (Sigma), and incu-bated at 37�C. Samples were removed and snap-frozen in liquidnitrogen on days 0, 3, 7, and 14 and stored at �80�C until ELISAanalysis. Serum concentrations of mAb backbone and PNU-conjugated mAb were evaluated using an ELISA-based assay.For rodent sera, dilution series of ADC serum samples werecaptured on ELISA plates coated with 2 mg/mL of a mouse anti-PNU mAb (generated in-house by immunization of mice withGly5-EDA-PNU-conjugated ADC) to bind PNU-conjugated mAb,or with goat anti-human Fc F(ab0)2 (Jackson Immunoresearch)to bind the ADC's mAb backbone, and detected with a 1:2,500dilution of an HRP-conjugated anti-human IgG (Jackson). Forprimate sera, dilution series of ADC serum samples were capturedon ELISA plates coated with 2 mg/mL of recombinant extracellulardomain of human CD30 (Sino Biological) and detected with a1:2,500 dilution of HRP-conjugated donkey anti-human IgG(Jackson) to measure the concentration of the ADC's mAb back-bone. To measure PNU-conjugated mAb, 2 mg/mL of mouseanti-PNU mAb followed by a 1:5,000 diluted HRP-conjugatedgoat anti-mouse Fcg F(ab0)2 (Jackson) was used for detection.Serum concentrations of PNU-conjugated mAb and total mAbwere calculated from the half-maximal OD values of the sampletitrations, by comparison with serial dilutions of a known con-centration of the same ADC.

Pharmacokinetic studyPharmacokinetics of cAC10 and cAC10-Gly5-PNU was eval-

uated in female CD-1 mice. The choice of an immunocompe-tent mouse strain was based on our prior observation that lowdoses of antibody-based therapeutics in mice lacking endoge-nous antibodies suffer from a severely impaired half-life (Sup-plementary Fig. S1), possibly by rapid adsorption to unoccu-pied Fc receptors. Animals were treated with 1 mg/kg ofunconjugated antibody or ADC by tail vein injection. Bloodsamples were collected at 1 hour, 24 hours, 72 hours, 7 days,and 14 days after injection. For each time point, three animalswere bled. Individual animals were used for a maximum of twoblood draws, at least one week apart. Blood was collected intotubes containing K2-EDTA and plasma was isolated from bloodby centrifugation at 1,500 � g for 10 minutes. Plasma was thentransferred to sterile cryovials, aliquoted, and stored at �80�Cuntil analysis. Plasma concentrations of mAb backbone andPNU-conjugated mAb was determined by ELISA, as describedabove. Noncompartmental pharmacokinetic parameters werecalculated with Kinetica (Thermo Fisher Scientific).

Potent Anthracycline ADCs

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In vivo efficacy studiesFor the JIMT-1 xenograft model, 107 cells in 100 mL PBS/

Matrigel (1:1) were implanted subcutaneously into the left flanksof CB17.SCID mice (Charles River Laboratories). For the Karpas-299 xenograft model, 5 � 106 cells in 100 mL PBS/Matrigel (1:1)were implanted subcutaneously into the left flanks of femaleNOD-SCID common-gamma chain-deficient (NSG) or CB17.SCIDmice (Charles River Laboratories). In the following, primarytumor volumes were measured by a caliper. After mean tumorvolumes had reached approximately 100 to 150 mm3, tumor-bearing animals were block-randomized into treatment groupseach according to tumor sizes. Group sizes were 5–6 animals inthe case of NSG mice and 8 animals in the case of CB17.SCIDmice. The next day (day 1), ADC therapy was initiated by intra-veneous injection of the indicated ADC. Treatment was repeatedtwice, in weekly intervals (i.e., on days 8 and 15). One day beforeeach ADC administration (i.e., on days 0, 7 and 14), animals werepretreated by intraveneous injection of 30mg/kg IVIG (Privigen).Preconditioning of NSG and CB17.SCID mice with exogenousIgG was done because it was previously found to improve the invivohalf-life ofmAbs andADCs used at the low dose of 1mg/kg inanimals lacking endogenous antibodies (Supplementary Fig. S1).Animals that reached predetermined abortion criteria (tumordiameter 2 cm or 20% weight loss) during the course of thestudies were sacrificed for ethical reasons. Tumor sizes and bodyweights were monitored for 43 days (JIMT-1), 69 days (Karpas-299 in NSG), or 57 days (Karpas-299 in CB17.SCID) afterrandomization.

Ethics statementAnimal experiments have been approved by the local Ethics

Committee for Animal Experimentation.During thewhole courseof animal experiments, all efforts were made to minimizesuffering.

ResultsGeneration of anthracycline-based linker-payloads for sortaseA-mediated conjugation

The anthracyclines doxorubicin, nemorubicin, and PNU-159682 were evaluated for their suitability as ADC payloads.Doxorubicin is an anthracycline that is widely used as a chemo-therapeutic agent for the treatment of various hematologic andsolid tumors (36) and acts via DNA intercalation, although theprecise mechanism of action remains controversial (37). Nemor-ubicin, a morpholinyl analogue of doxorubicin, is significantlymore potent than doxorubicin, also acts on multidrug-resistantcells and is not cardiotoxic at therapeutic doses (38, 39). PNU-159682 is a liver metabolite of nemorubicin and is about threeorders of magnitude more potent than its parent molecule oncultured human tumor cells (34).

To allow for sortase-mediated conjugation, anthracyclinederivatives were generated by adding a pentaglycine peptide tothe tetracyclic aglycone structure, common to all anthracyclines.For this, the 14C and the attached hydroxyl group were removedand the glycine stretch was added directly to the carbonyl groupof 13C via an amide bond using an ethylenediamine (EDA)linker, generating the linker-drugs Gly5-EDA-Dox, Gly5-EDA-Nemo and Gly5-EDA-PNU, respectively (Figs. 1A–C). In thecase of PNU-159682, a cleavable linker-payload based on theprotease-sensitive dipeptide valine-citrulline (vc) and a self-

immolative spacer, PAB was also evaluated. For this, Gly3-vcPAB was attached to the primary alcohol of PNU-159682through a N-formyl-N,N0-dimethylethylendiamine spacer gene-rating Gly3-vcPAB-PNU-159682 (Fig. 1D). A similar linker-payload for maleimide-based conjugation to thiols, referredto as NMS249, has recently been described and was evaluatedin the context of a CD22-specific ADC (33).

Manufacturing of noncleavable, anthracycline-based ADCstargeting HER2

To allow for Sortase A-mediated conjugation, a variant of theHER2-specific therapeutic antibody trastuzumab was produced,with each of its C-termini tagged with an LPETG Sortase Arecognition motif followed by a Strep-tag. In case of the heavychain, the sequence was directly appended to the C-terminus. Incase of the light chain, a five amino acid spacer peptide with thesequence GGGGS was placed between C-terminus and tag. Thisspacer was previously shown to facilitate conjugation to the lightchain, presumably by improving accessibility of the sortase Arecognition motif for the enzyme (24). Functionally enhancedevolved sortase A (eSrtA; ref. 40) was then used to conjugate therespective Gly5-modified anthracycline-based linker-payload tothe antibody. The transpeptidation reaction catalyzed by sortase Aleads to the formation of a new peptide bond between thethreonine residue of the LPETG sortase A recognition motif andthe amino terminus of the linker-payload, therefore all resultingADCs comprise the sequence LPETGGGGG in their linker. Wepreviously showed that conjugation reactions with maytansine-and auristatin-based linker-payloads occuredwith an efficiency ofapproximately 80%–90%, yielding ADCs with DARs in the rangeof 3.1 to 3.5, which is similar to the DARs of the currentlymarketed ADCs, Kadcyla and Adcetris (24). Surprisingly, conju-gation efficiencies with anthracycline-based linker-payloads wereconsistently and significantly higher and theADCs Tras-Gly5-Dox,Tras-Gly5-Nemo, and Tras-Gly5-PNU manufactured here eachhad a DAR ranging between 3.7 and 3.9.

Enrichment of DAR 4.0 speciesWe next tested the feasibility of enriching DAR 4.0 conjugates,

by taking advantage of the fact that sequences C-terminallyappended to the sortase pentapeptide recognition motif arereleased upon successful conjugation. This unique feature of thesortase conjugation process should in principle allow the use ofthe Strep-tag as an affinity handle for the selective removal ofunder-conjugated ADC species by a simple StrepTactin affinitychromatography step. Thus, a trastuzumab variant with a Twin-Strep-tag was produced and evaluated. Conjugation of Gly5-EDA-PNU to TwinStrep-tagged trastuzumab occurred nearly quantita-tively on heavy and light chain and yielded a DAR of approx. 3.8,as shown by reverse-phase chromatography (Fig. 2A, left). Sig-nificantly, a Tras-Gly5-PNU conjugate polished by a single passover a StrepTactin affinity column did no longer show any tracesof unconjugated heavy or light chain and was completely homo-geneous with a DAR of 4.0 (Fig. 2A, right).

Selective removal of under-conjugated ADC species is of par-ticular interest when using linker-payloads, which are less effi-ciently conjugated. Thus, the same strategy was used with atrastuzumab–maytansine conjugate. Whereas the initial ADCpreparation was comparable with previously described maytan-sine-based conjugates (24) and exhibited a DAR of approx. 3.4(Fig. 2B, left), a single pass over a StrepTactin column removed the

Stefan et al.





majority of under-conjugatedmolecules to yield an almost homo-geneous ADC preparation (Fig. 2B, right).

Taken together, these data demonstrate that SMAC techno-logy allows efficient enrichment of DAR 4.0 ADCs. Neverthe-less, given the very high conjugation efficiencies achievedconsistently, especially with Gly5-anthracycline linker-pay-loads, all ADCs used in this study were evaluated withoutfurther purification.

In vitro cell killing activity of noncleavable, anthracycline-basedADCs targeting HER2

Next, the activity of SMAC technology–manufactured tras-tuzumab–anthracyline ADCs was evaluated in in vitro cellkilling assays using the HER2-overexpressing breast cancer cellline SKBR3 (HER2high) and the HER2-negative acute lympho-

blastic leukemia (ALL) cell line REH, as a negative control(Supplementary Fig. S2). HER2 expression levels in SKBR3cells were determined experimentally using a bead-based assay(Biocytex) and found to be approximately 694,000 moleculesper cell (Supplementary Fig. S3). Significantly, ADCs based ondoxorubicin and nemorubicin showed hardly any toxicity andwere only able to kill a minor fraction of HER2-overexpressingSKBR3 cells at high concentrations (Supplementary Fig. S2A).This low level of toxicity appeared to be specifically mediatedby HER2, as no sign of toxicity was observed on HER2-negativecells (Supplementary Fig. S2B). In line with the dramaticallyincreased cytotoxicity of the anthracycline PNU-159682 com-pared with other anthracylines (34), the Tras-Gly5-PNU ADCdisplayed a significantly enhanced cytotoxicity compared withthe Tras-Gly5-Dox and Tras-Gly5-Nemo ADCs and killed SKBR3

Figure 1.

Structures of oligoglycine-modifiedanthracycline linker-payloads used inthis study. A, Gly5-EDA-Dox; B, Gly5-EDA-Nemo; C, Gly5-EDA-PNU; D,Gly3-vcPAB-PNU-159682.






cells with an IC50 of 2.8 ng/mL (Supplementary Fig. S2A). AlsoTras-Gly5-PNU–mediated cytotoxicity was highly specific, asonly very high concentrations of the ADC had an effect onHER2-negative REH cells (Supplementary Fig. S2B).

In light of its high potency, the Tras-Gly5-PNU conjugate wasfurther investigated and compared with SMAC technology–manufactured trastuzumab-maytansine ADC (Tras-Gly5-May),using HER2-overexpressing SKBR3 cells and the breast cancercell line T47D, expressing low levels of HER2 (Figs. 3A and B).T47D cells were determined to express approximately 32,000HER2 molecules per cell (Supplementary Fig. S3), that is, about20 times less than SKBR3. Adcetris, which was used as a negativecontrol, only had a modest effect on the growth of either cellline. As we had observed previously (24), Tras-Gly5-May washighly effective on HER2high SKBR3 cells (Fig. 3A), with IC50

of 18 ng/mL, but failed to have an appreciable cytotoxic effecton HER2low T47D cells (Fig. 3B). In striking contrast, Tras-Gly5-PNU was effective on both cell lines, and killed SKBR3 aswell as T47D cells with an IC50 of 2.7 ng/mL and 14.7 ng/mL,respectively (Fig. 3A and B).

Generation and evaluation of a noncleavable, PNU-basedADC targeting CD30

To further validate SMAC technology–manufactured, PNU-based ADCs, the Gly5-EDA-PNU linker-payload (Fig. 1C) wasalso conjugated to a sortase motif-modified version of theCD30-specific mAb cAC10, which is the basis of commerciallyavailable ADC brentuximab vedotin (Adcetris; ref. 8). Activity ofcAC10-Gly5-PNU was evaluated and compared with Adcetrisand to a maytansine-conjugated ADC, cAC10-Gly5-May, incytotoxicity assays, using the CD30high non-Hodgkin lympho-ma (NHL) cell line Karpas-299 and the CD30-negative ALL cellline REH (Fig. 3C and D). Tras-Gly5-May, which was used as anegative control, had no detectable growth-inhibitory effect oneither cell line even at very high concentrations. In contrast,cAC10-Gly5-PNU potently killed CD30high Karpas-299 cells,with an IC50 of 1.8 ng/mL, and thus was more potent thanAdcetris, which had an IC50 of 4.8 ng/mL (Fig. 3C). cAC10-Gly5-

May showed a similar potency as Adcetris (IC50 ¼ 4.7 ng/mL).Significantly, none of the cAC10-based ADCs had any detectableeffect on the growth of CD30-negative REH cells (Fig. 3D). Thus,these data demonstrate that our novel, noncleavable ADCformat, based on sortase-mediated conjugation of a Gly5-modi-fied PNU-derivative, consistently delivers highly potent andspecific drugs.

Evaluation of PNU-based ADCs with a cleavable linkerThe noncleavable PNU ADC described above is likely to

require lysosomal trafficking and degradation in late lyso-somes for release of the payload to occur. We next evaluatedwhether the utilization of a cleavable linker would yield aneven more potent ADC by increasing efficiency of payloadrelease. Thus, a PNU-based linker-payload, incorporating thecathepsin B-cleavable dipeptide valine-citrulline (vc) and aself-immolative spacer, PAB (Fig. 1D), was conjugated totrastuzumab using SMAC technology. The cytotoxic activityof the resulting ADC, Tras-Gly3-vcPAB-PNU, was then com-pared with that of the noncleavable variant, Tras-Gly5-PNU,again using the breast cancer cell lines SKBR3 (HER2high) andT47D (HER2low), and the NHL cell line Karpas-299 (Figs. 4Aand B; Supplementary Fig. S4). The CD30-specific ADC cAC10-Gly5-PNU, which was used as a negative control, only had amodest effect on the growth of the CD30-negative breast cancercells whereas it efficiently killed CD30-positive Karpas-299cells (IC50 ¼ 3.7 ng/mL). Kadcyla, which was used as an addi-tional control, as expected was highly effective on HER2high

SKBR3 cells (IC50 ¼ 3.6 ng/mL), but did hardly exhibit acytotoxic effect on HER2low T47D cells or on HER2-negativeKarpas-299 cells. Importantly, use of a cleavable linker didnot substantially increase ADC potency, and Tras-Gly5-PNUand Tras-Gly3-vcPAB-PNU exhibited cytotoxicity on SKBR3cells with IC50 values of 1.9 ng/mL and 0.24 ng/mL, respec-tively (Fig. 4A). The cytotoxic activity on HER2low T47Dcells differed even less between Tras-Gly5-PNU and Tras-Gly3-vcPAB-PNU, with IC50 values of 16.8 ng/mL and12.0 ng/mL, respectively (Fig. 4B). Addition of a large excess

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Analytical characterization of site-specific ADCs trastuzumab–PNU (A)and trastuzumab–maytansine (B)by RP-HPLC. ADCs were generatedby enzymatic conjugation ofGly5-modified payloads to sortase Atag-modified trastuzumab. TheTwinStrep tag added to theC-terminus of the sortase A tag is lostupon conjugation, allowing theselective removal of under-conjugated species by StrepTactinchromatography. Red traces,unconjugated mAb; black traces,conjugated mAb; HC, heavy chain;LC, light chain. Arrow indicates minorspecies of unconjugated HC or LC.

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In vitro cytotoxicity assays with Sortase A-conjugated noncleavable anti-HER2 and anti-CD30ADCs. HER2-overexpressing SKBR3 breast cancercells (A), HER2-low T47D breast cancer cells (B),CD30-high Karpas-299 NHL cells (C) and CD30-negative REH ALL cells (D) were grown in thepresence of serial dilutions of the indicated ADCs.Viable cells were quantified using a luminescent cellviability assay. Datapoints represent mean of tworeplicates and error bars represent SD.






of unconjugated trastuzumab efficiently competed awaymost of the cytotoxic activity of both ADCs, indicating that theeffect is specifically mediated via binding of the ADCs to HER2.However, these competition experiments also revealed thatTras-Gly3-vcPAB-PNU displayed a higher level of nonspecificcytotoxicity in comparison to its noncleavable counterpart,Tras-Gly5-PNU (Figs. 4A and B). Nonspecific cell killing activity

of the cleavable ADC was also revealed on HER2-negativeKarpas-299 cells, where Tras-Gly3-vcPAB-PNU was approxi-mately 37 times more toxic than Tras-Gly5-PNU (Supplemen-tary Fig. S4). As expected, addition of the large excess of tras-tuzumab (200 mg/mL at all ADC concentrations tested) per sehad a noticeable effect on the proliferation of HER2-overex-pressing SKBR3 cells but not T47D cells (Figs. 4A and B).

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Comparison of in vitro cell killingactivity of sortase A-conjugated anti-HER2 and anti-CD30 ADCs withcleavable and noncleavable linkers.HER2-overexpressing SKBR3 breastcancer cells (A), HER2-low T47Dbreast cancer cells (B), CD30-highKarpas-299 NHL cells (C) and CD30-negativeREHALL cells (D)weregrownin the presence of serial dilutions of theindicated ADCs. Dotted lines, cellkilling activity in the presence of anexcess of unconjugated mAb. Viablecells were quantified using aluminescent cell viability assay.Datapoints represent mean of tworeplicates and error bars represent SD.

Stefan et al.





The cleavable Gly3-vcPAB-PNU-159682 linker-payload wasalso evaluated in the context of the CD30-specific mAb cAC10.The cytotoxic activity of the resulting ADC, cAC10-Gly3-vcPAB-PNU, was compared with that of the noncleavablevariant, cAC10-Gly5-PNU, using the CD30high NHL cell lineKarpas-299 and the CD30-negative ALL cell line REH (Figs. 4Cand D). Tras-Gly5-PNU, which was used as a negative control,only had a modest effect on the growth of both cell lines. Incontrast, cAC10-Gly5-PNU efficiently killed CD30-positiveKarpas-299 cells (IC50 ¼ 1.1 ng/mL), while having no appre-ciable effect on CD30-negative REH cells. Significantly, thecleavable cAC10-Gly3-vcPAB-PNU ADC did not show enhanc-ed cytotoxicity on Karpas-299 cells (IC50 ¼ 1.1 ng/mL).However, similar to the anti-HER2 ADCs, nonspecific cyto-toxicity of the cleavable ADC on antigen-negative cells wasincreased significantly compared with its noncleavable coun-terpart (550-fold; Fig. 4D).

In summary, the results demonstrate that PNU-based ADCsmanufactured by SMAC technology work efficiently in theabsence of a cleavable linker and display enhanced nonspecificcytotoxicity in the presence of a cleavable linker.

In vitro serum stability and pharmacokinetics of CD30-specificPNU conjugate

Considering the high potency of noncleavable PNU-ADCsin vitro, and due to concerns about the stability and potentialtoxicity of cleavable PNU-ADCs in vivo, the focus of all addi-tional experiments was on noncleavable PNU-ADCs. Thus, wenext investigated the stability of the SMAC technology–manu-factured cAC10-Gly5-PNU ADC in human serum and in serumfrom species relevant for preclinical efficacy and safety testing(Figs. 5A–D). For this, cAC10-Gly5-PNU was diluted in mouse,rat, cynomologus monkey, or human serum and incubated at37�C for two weeks. Aliquots were removed at different timesand the concentrations of total cAC10 antibody and intact ADCwere determined by ELISA. The concentration of total cAC10antibody remained essentially constant for the duration of theexperiment. Significantly, also the concentration of intact ADCremained unchanged over time and payload loss could not bedetected by toxin ELISA at any significant level. Thus, the novelnoncleavable ADC format described herein, based on sortase-conjugation of aGly5-modifiedPNU-derivative, is highly stable inserum from all species analyzed.

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A pharmacokinetic study was carried out, to analyze in vivohalf-life and stability of the cAC10-Gly5-PNU ADC in compar-ison to its parental mAb (Fig. 5E). For this, nontumor-bearingCD-1 mice were intravenously injected with cAC10-Gly5-PNUor naked mAb cAC10 as a control. Plasma was collected fromthe mice after various times and analyzed by ELISA to quantifyintact ADC and total IgG. The pharmacokinetic parameterswere determined by noncompartmental analysis. The time/concentration curves of cAC10 and cAC10-Gly5-PNU appearedto follow biexponential declines (Fig. 5E). The terminal half-lives of cAC10, cAC10-Gly5-PNU (total IgG detected by anti-FcELISA) and cAC10-Gly5-PNU (intact ADC detected by anti-PNU-toxin ELISA) were 13.1, 11.5, and 11.3 days, respectively.The half-life of nonconjugated cAC10 measured here thuscorrelates quite well with the 16.7 days reported previously(17). The half-life of cAC10-Gly5-PNU ADC was slightlyreduced compared with the nonconjugated mAb cAC10. Thisis expected, because increased hydrophobicity of ADCs incomparison to nonconjugated antibodies is known lead to afaster turnover of the conjugate (17). However, comparison ofintact ADC versus total IgG of the ADC revealed that there washardly any detectable loss of PNU payload during the time ofthe experiment (14 days). Therefore, these data demonstratethat SMAC technology–manufactured PNU-ADCs are not onlyhighly stable in vitro, but also in vivo.

In vivo antitumor activity of HER2-specific PNU conjugateWe next evaluated the in vivo efficacy of our novel SMAC

technology–manufactured Tras-Gly5-PNU ADC in a trastuzu-mab-resistant JIMT-1 breast cancer xenograft model. JIMT-1cells are known to express moderate HER2 levels despite geneamplification (41). On the basis of FACS analysis, we foundthat these cells express 4-fold lower levels of HER2 than SKBR3cells, that is, around 170,000 molecules per cell. We comparedthe activity of Tras-Gly5-PNU with Kadcyla, as a benchmarkcontrol, and cAC10-Gly5-PNU ADC, as a negative control(Fig. 6A). For this, JIMT-1 cells were transplanted subcutane-ously into CB17.SCID mice and tumors were allowed to growto a volume of 100–150 mm3. Mice were then treated intra-venously 3 times weekly with the different ADC preparations.Tumors grew relatively slowly and reached 800 mm3 within 3–4 weeks post randomization in vehicle control mice. Signif-icantly, Kadcyla treatment in JIMT-1 xenotransplanted miceshowed only limited efficacy even at the high dose of 15 mg/kgand tumor growth was delayed by only about a week. Instriking contrast, Tras-Gly5-PNU was highly effective at themuch lower dose of 1 mg/kg and led to complete tumor regres-sion. The negative control cAC10-Gly5-PNU did not inducetumor regression, demonstrating that the strong antitumoreffect seen with Tras-Gly5-PNU was mediated by target bind-ing. The mild target-independent effect on tumor growth hasalready been observed with other highly potent ADCs, forexample, using PBD-based payloads (29), and may be exac-erbated by the slow growth of the JIMT-1 xenografts. All ADCswere well tolerated, as shown by the absence of weight loss ineach of the treatment groups compared with the vehiclecontrol group (Supplementary Fig. S5A). Taken together, thesedata demonstrate that PNU-based ADCs are highly potent notonly in vitro, but also in vivo, allowing to address targetsexpressed at levels not accessible to ADCs with conventionalpayloads.

In vivo antitumor activity of CD30-specific PNU conjugateWe then evaluated the in vivo efficacy of the SMAC technol-

ogy–manufactured cAC10-Gly5-PNU ADC in two differentCD30-positive NHL mouse xenograft models in comparisonto: (i) Adcetris, as a benchmark control; (ii) SMAC technology–manufactured Tras-Gly5-PNU ADC, as a negative control; and(iii) SMAC technology–manufactured cAC10-Gly5-MaytansineADC, as an additional control (Figs. 6B and C). For this,CD30high Karpas-299 cells were either transplanted subcutane-ously into NSG, or into CB17.SCID mice. In both models,tumors grew very aggressively in vehicle control groups andreached sizes of around 1,000 mm3 within 10 or 14 days postrandomization, respectively. Mice were treated intravenously 3times weekly with different ADC preparations, beginning oneday post randomization, when the tumors had reached a sizeranging between 100 and 150 mm3. Interestingly, Adcetristreatment in Karpas-299 xeno-transplanted NSG mice showedonly limited efficacy at the standard concentration of 1 mg/kgand tumor growth was only delayed by about a week (Fig. 6B).In line with the previously reported high efficiency of Adcetris ina Karpas-299 model in SCID mice (8), Adcetris was highlyeffective in the CB17.SCID model at a dose of 1 mg/kg, leadingto tumor regression in all treated animals (Fig. 6C). In contrastto Adcetris, SMAC technology–manufactured anti-CD30 ADCs(either with Gly5-PNU at 1 mg/kg or with Gly5-Maytansinepayload at 10 mg/kg) were found to be highly effective for thetreatment of Karpas-299 tumors in both models, although alsothese ADCs were slightly less efficacious in NSG mice (Figs. 6Band C). As expected, animals treated with Tras-Gly5-PNU ADCas a negative control developed tumors with similar kinetics asin the vehicle control groups in both models, showing that theeffect of the cAC10-Gly5-PNU and cAC10-Gly5-May ADCs washighly antibody and target specific.

Significant differences in the efficacies of the different ADCpreparations become apparent when antitumor effects are ana-lyzed in individual mice (Supplementary Fig. S6). In the NSGmodel, all mice treated with cAC10-Gly5-May, which initiallyresponded well to the 10 mg/kg treatment, eventually relapsed.Thus, in this model antitumor activity of the maytansine conju-gate appears to require the continuous administration of the ADCto permanently suppress tumor growth. In striking contrast, evenin NSGmice, treatment with cAC10-Gly5-PNU at themuch lowerdose of 1mg/kg led to a long lasting tumor regression until day 69in all but one of the animals.

These data demonstrate that SMAC technology–manufacturedanti-CD30 ADCs exhibit comparable or, depending on the pre-clinical animal model employed, even better potency than theapproved ADC Adcetris. Most notably, only the SMAC technol-ogy–manufactured cAC10-Gly5-PNU ADC lead to long-lastingtumor eradication in a majority of animals in both models, evenwithout continuous ADC administration.

Significantly, all ADCs were well tolerated, as shown by theabsence of weight loss in each of the treatment groups com-pared with the vehicle control group (Supplementary Fig. S5Band S5C).

DiscussionNumerous strategies to improve properties of conventional

ADCs have been described in recent years, aiming at increasingthe degree of homogeneity, level of serum stability and/or cell

Stefan et al.





killing potency (1, 2). In this study, we describe an ADCplatform based on SMAC technology (24), which addressesall these aspects. The ADCs produced are homogeneous by wayof their predefined attachment sites for the payload on the C-termini of the antibody's heavy and light chains. The use of anoncleavable, entirely peptide-based linker confers exquisiteserum stability, whereas a highly potent payload derived fromthe anthracycline PNU-159682 provides superior potency.

We have previously described the use of SMAC technology forthe generation of maytansine- and auristatin-based ADCs (24).Enzymatic conjugation using an engineered sortase A enzymefrom S. aureus allowed site-specific attachment of pentaglycine-modified payloads with an efficacy of 80% to 90%. This cor-relates well with the conjugation efficiency described in thisstudy, where a trastuzumab–maytansine conjugate with a DARof approx. 3.4 was generated (Fig. 2B), corresponding to a

conjugation efficiency of 85%. As shown in this study, the useof a StrepTactin affinity chromatography step allows for deple-tion of underconjugated ADC species and manufacturing ofhomogeneous DAR4.0 ADCs. Surprisingly, we found that theGly5-PNU linker-payload allowed for significantly better conju-gation efficiencies than other payloads, with efficiencies oftenexceeding 95% and DARs close to 4.0 routinely achieved with-out any additional enrichment step (Fig. 2A). It is conceivablethat accessibility of the oligo-glycine peptide for the enzyme'sactive site differs between various Gly5-modified payloads,leading to different conjugation efficiencies. The high degree ofcompatibility of the Gly5-PNU linker-payload with the sortaseenzyme allowed us to generate and analyze high-quality ADCswithout further purification.

Anthracyclines such as doxorubicin, epirubicin, and daunoru-bicin are frequently used chemotherapeutic agents for the

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In vivo evaluation of HER-2 and CD30-specific ADCs in mouse xenograftmodels. JIMT-1 breast cancer cells weregrown subcutaneously in CB17.SCIDmice (A). Karpas-299 non-Hodgkinlymphoma cells were grownsubcutaneously in NSG (B) or CB17.SCIDmice (C). When tumors reached avolume of 100–150 mm3, animals wererandomized into different treatmentgroups and treated via the tail vein asindicated 1, 8, and, 15 days later (arrows).Tumor volumes were monitored. Plotsare terminated when the first animalreached abortion criteria and needed tobe sacrificed for ethical reasons. Datapoints represent mean and barsrepresent SEM.






treatment of hematologic and solid cancers (42). Accordingly,a doxorubicin-based ADC has already been evaluated for can-cer-cell targeting many years ago (43). While this ADC, likeother early day ADCs based on chemotherapeutic agents, suf-fered from a number of issues related to immunogenicity andlinker instability, also the potency of the standard chemother-apeutic agents used as payloads was simply too low for targeteddelivery (2). Indeed, a subsequently evaluated ADC calledBR96-doxorubicin, targeting the Lewis Y antigen, successfullycircumvented the hypersensitivity reactions by using a mouse/human chimeric mAb but did not progress beyond phase II dueto insufficient efficacy (44). It will be interesting to see howImmunomedic's anti-CD74 conjugate milatuzumab–doxorubi-cin for the treatment of multiple myeloma performs in theclinic (http://www.immunomedics.com/adc.shtml). In linewith clinical results available to date, the ADCs utilizing link-er-payloads based on doxorubicin or nemorubicin analyzedin this study did not display significant cell killing activity(Supplemenary Fig. S2A).

In light of the limited potency of doxorubicin- and nemoru-bicin-based ADCs, the Gly5-PNU linker payload, based on aderivative of the nemorubicin metabolite PNU-159682, was alsoinvestigated in the context of SMAC technology. PNU-159682was described to be several orders ofmagnitudemore potent thandoxorubicin (34) andhas recently shownpromise as apayload forADCs in the context of conventional chemical conjugation (32,33). Indeed, when conjugated to trastuzumab, a significantlyincreased cytotoxic activity was achieved in comparison withmaytansine conjugates and even cells with moderate HER2expression levels, that were not sensitive to Kadcyla, could effi-ciently be killed (Figs. 3A and B). This observation is reminiscentof other HER2-targeting ADCs currently in development that alsodisplay cell killing activities surpassing that of Kadcyla, includingSynthon's SYD985, a novel trastuzumab–duocarmycin conjugate(45), and Mersana's XMT-1522, a high-DAR ADC based on anovel anti-HER2 mAb and an auristatin payload (http://www.mersana.com/pipeline). The fact that numerous highly potentanti-HER2 ADCs are currently being developed underlines theattractiveness of HER2 as a cancer target. The ability to address notonly cancer cells displaying extremely high levels of HER2 (IHCstatus 3þ) but also those expressing moderate levels (IHC status1þ or 2þ) has the potential to allow treatment of a significantlylarger fraction of metastatic breast cancers, as well as of severaladditional types of cancer. Considering that HER2 is widelyexpressed in numerous healthy tissues (46), it will be interestingto see the results of the clinical evaluation of such high potencyanti-HER2 ADCs.

An additional advantage of highly potent ADC formats such asthe one described here is that they may be effective at relativelylow target receptor densities, thereby increasing the number oftargets accessible to ADC strategies. The current paradigmdemands that the ideal ADC target be not only tumor-specific,but also highly expressed and/or highly internalizing (2, 3). It isreasonable to expect that there are numerous potential ADCtargets that fulfill the requirement for tumor-selective expression,but fail to show sufficient expression levels and/or sufficientinternalization rates for conventional ADCs. Time will tell howthe opportunity provided by high potency ADCs, as described inthis study, will translate into the clinic.

In light of the highly potent PNU payload evaluated in thisstudy, we decided to use a noncleavable pentaglycine linker in

our initial trastuzumab- and cAC10-based ADCs. Noncleavablelinkers provide the highest degree of in vivo stability (6) andtherefore have the potential to reduce nonspecific toxicity ofhighly potent ADCs. Significantly, we demonstrated that non-cleavable ADCs based on the PNU payload were not onlyhighly potent, but also effective on cells expressing only mod-erate target levels (Figs. 3C and D). Nevertheless, we wonderedwhether the use of a cleavable linker would further enhancethe potency of PNU-based ADCs. Efficient delivery of a toxicpayload is commonly believed to be improved by using cleav-able linkers. Whereas early hydrazone-based linkers sufferedfrom insufficient stability, more recent cleavable linkers, suchas reducible disulfide-based or enzymatically cleavable di-pep-tide linkers (7, 8), achieve a high level of in vivo stability andare by far the most frequently used linkers in clinical-stageADCs (2). Surprisingly, only a moderate improvement of thecytotoxic activity of trastuzumab-based ADCs was achievedwhen using a cleavable vcPAB linker, especially on target cellsexpressing low target levels (Figs. 4A and B). In the case ofcAC10-based ADCs, use of a cleavable linker had no appreci-able effect at all (Fig. 4C), indicating that efficient release ofthe PNU payload inside cells occurs even in the absence of acleavable linker. In addition, the nonspecific cell killing activ-ity of cleavable PNU-based ADCs was found to be signi-ficantly higher, as shown by competition experiments withunconjugated mAb (Figs. 4A and B), and by evaluation oftarget-negative cells (Supplementary Fig. S4 and Fig. 4D). Thus,at least in the context of SMAC technology, a noncleavablelinker appears to be the preferred choice for a PNU-basedADC, by combining high efficacy with increased selectivity.

The ADC platform described here is universally applicable. Weshow that in addition to maytansines and auristatins (24), alsoanthracyclines can be modified with oligo-glycine linkers andefficiently conjugated using SMAC technology. Furthermore, theGly5-PNU payload described here can be attached to differentmAbs and consistently delivers highly potent ADCs. For instance,conjugation of Gly5-PNU to the CD30-specific mAb cAC10,basis of the marketed ADC Adcetris, yielded an ADC that exceed-ed the potency of Adcetris by almost 3-fold (Fig. 3C). Finally, inline with our previous studies (24), PNU-based ADCs generatedusing SMAC technology display potent antitumor activities notonly in vitro, but also in vivo (Fig. 6).

However, we found a striking difference in the antitumorefficacy of the clinically approved benchmark ADC Adcetris inKarpas-299 xenograft models, depending on the mouse strainused. Whereas antitumor activity was limited in NSG mice,Adcetris was very potent on Karpas-299 tumors in CB17.SCIDmice (Fig. 6). Similar strain-specific differences in the efficacyof cAC10-MMAE conjugates have been observed recently andwere attributed to significantly faster clearance of the ADCs inNSG versus SCIDmice (13). Interestingly, this observation wasnot limited to specific ADCs or ADC formats, but rather ageneral feature of mAb-based drugs including naked mAbs.Although we have not performed a pharmacokinetic study inNSG mice with our SMAC technology–conjugated ADCs, thesemight also be subject to faster clearance in this mouse strain.This is supported by the observation that the cAC10-Gly5-MayADCs lead to permanent eradication in CB17.SCID, but not inNSG host mice, where only a transient antitumor effect wasobserved. The fact that our SMAC technology–manufacturedcAC10-Gly5-PNU-ADC maintains a strong antitumor activity

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http://www.immunomedics.com/adc.shtml

http://www.mersana.com/pipeline

http://www.mersana.com/pipeline


even in NSG mice is an impressive demonstration of the highpotency of PNU-based, SMAC technology–conjugated ADCs.

The site of conjugation has previously been shown to have asignificant impact on in vivo stability, thereby influencing thetoxicity profile and antitumor efficacy of an ADC (23, 47, 48).With respect to the placement of payloads at the C-termini of anantibody's heavy or light chains, there have been conflictingresults. Suboptimal serum stability has been observedwhen usingcleavable vcPAB linkers (47). However, this observation seems tobe specific to this type of cleavable linker and can be mitigated bychanges in the linker design (49). Accordingly, no stability issueshave been observed upon C-terminal conjugation using nonclea-vable linkers (23, 50). In agreement with these observations, theC-terminally conjugated ADCs described here, employing non-cleavable peptide linkers, are highly stable in serum, both in vitro(Figs. 5A–D) and in vivo (Fig. 5E). The avoidance of maleimide-based linkers, shown to be associated with a certain degree ofserum instability of conventional ADCs (11), is likely to add to thestability of ADCs generated using sortase.

In summary, our results show that SMAC technology is capableof generatinghighlyhomogeneous, stable andpotentADCsbasedon a derivative of the anthracycline PNU-159682, that can resultin long-lasting and complete tumor eradication with only 3treatment cycles in xenotransplantation models. It will be inter-esting to see how these early preclinical data will translate into theclinic. First clinical evaluation of this novel ADC format isexpected in 2019.

Disclosure of Potential Conflicts of InterestR.R. Beerli andU.Grawunder hold stocks ofNBE-Therapeutics AG. Thiswork

has been included in apatent applicationbyNBE-Therapeutics AG.Nopotentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: N. Stefan, L. Waldmeier, U. Grawunder, R.R. BeerliDevelopment of methodology: N. Stefan, U. Grawunder, R.R. BeerliAcquisition of data (provided animals, acquired and managed pati-ents, provided facilities, etc.): N. Stefan, R. G�ebleux, L. Waldmeier, T. Hell,M. Escher, F.I. Wolter,Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N. Stefan, L. Waldmeier, R. R. BeerliWriting, review, and/or revision of the manuscript: N. Stefan, R. G�ebleux,U. Grawunder, R.R. BeerliAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N. StefanStudy supervision: N. Stefan, U. Grawunder, R.R. Beerli

AcknowledgmentsWe would like to thank Dr. Birgit Dreier for her critical reading of the

manuscript.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Revised October 14, 2016; received October 18, 2016; accepted February 2,2017; published OnlineFirst March 3, 2017.

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Stefan et al.




2017;16:879-892. Published OnlineFirst March 3, 2017.Mol Cancer Ther Nikolas Stefan, Rémy Gébleux, Lorenz Waldmeier, et al. Generated by Enzymatic, Site-specific Conjugation

Drug Conjugates−Highly Potent, Anthracycline-based Antibody

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