ADCT-301, a Pyrrolobenzodiazepine (PBD) Dimer Containing … · benzodiazepine (PBD) dimer warhead with a drug–antibody ratio (DAR) of 2.3. ADCT-301 binds human CD25 with pico-molar

Large Molecule Therapeutics

ADCT-301, a Pyrrolobenzodiazepine (PBD)Dimer–Containing Antibody–Drug Conjugate(ADC) Targeting CD25-Expressing HematologicalMalignanciesMichael J. Flynn1,2, Francesca Zammarchi3, Peter C. Tyrer2, Ayse U. Akarca4,Narinder Janghra4, Charles E. Britten3, Carin E.G. Havenith3, Jean-Noel Levy2,Arnaud Tiberghien2, Luke A. Masterson2, Conor Barry2, Francois D'Hooge2,Teresa Marafioti4, Paul W.H.I. Parren5,6, David G.Williams2, Philip W. Howard2,Patrick H. van Berkel3, and John A. Hartley1,2

Abstract

Despite the many advances in the treatment of hematologicmalignancies over the past decade, outcomes in refractorylymphomas remain poor. One potential strategy in this patientpopulation is the specific targeting of IL2R-a (CD25), which isoverexpressed on many lymphoma and leukemic cells, usingantibody–drug conjugates (ADC). ADCT-301 is an ADC com-posed of human IgG1 HuMax-TAC against CD25, stochasticallyconjugated through a dipeptide cleavable linker to a pyrrolo-benzodiazepine (PBD) dimer warhead with a drug–antibodyratio (DAR) of 2.3. ADCT-301 binds human CD25 with pico-molar affinity. ADCT-301 has highly potent and selective cyto-toxicity against a panel of CD25-expressing human lymphomacell lines. Once internalized, the released warhead binds in theDNA minor groove and exerts its potent cytotoxic action via theformation of DNA interstrand cross-links. A strong correlation

between loss of viability and DNA cross-link formation isdemonstrated. DNA damage persists, resulting in phosphory-lation of histoneH2AX, cell-cycle arrest in G2–M, and apoptosis.Bystander killing of CD25-negative cells by ADCT-301 is alsoobserved. In vivo, a single dose of ADCT-301 results in dose-dependent and targeted antitumor activity against both subcu-taneous and disseminated CD25-positive lymphoma models.In xenografts of Karpas 299, which expressed both CD25 andCD30, marked superiority over brentuximab vedotin (Adce-tris) is observed. Dose-dependent increases in DNA cross-linking, g-H2AX, and PBD payload staining were observedin tumors in vivo indicating a role as relevant pharmacody-namic assays. Together, these data support the clinical testingof this novel ADC in patients with CD25-expressing tumors.Mol Cancer Ther; 15(11); 2709–21. �2016 AACR.

IntroductionIn 2015 in the United States, there were an estimated 80,900

and 54,270 new diagnoses of all-type lymphoma and leukemia,and 20,490 and 24,450 deaths, respectively (1). Both Hodgkinlymphoma and various T- and B-cell non-Hodgkin lymphomatumor cells express IL2R-a (CD25) (2, 3). At least two leukemicdiseases, adult T-cell leukemia lymphoma (ATLL; ref. 4) and hairy

cell leukemia (5), show nearly 100% expression of CD25 ontheir circulating tumor cells. In the refractory setting, there isevidence of maintenance of CD25 expression, and in acutemyeloid leukemia (AML), its upregulation (6). Although notunique to hematologic malignancy, increased CD25 expressioncan be detected via shedding of this receptor (7, 8). Indeed, highsoluble IL2R-a serum levels have helped identify patients withpoorer prognoses in diffuse large B-cell lymphoma, cutaneouslymphomas, follicular lymphoma, AML, acute lymphoblasticleukemia, Hodgkin lymphoma (9–14), and are used as a part ofa prognostic index in ATLL (15).

A number of clinical trials have demonstrated the utilityof targeting this receptor in oncology. Phase I clinical trials ofthree radioimmunoconjugates, 90Yttrium-labeled anti-Tac (16),131Iodine-labeled basiliximab (17), and 90Yttrium-labeled dacli-zumab (18) have demonstrated complete responses in patientswith CD25-expressing lymphomas. The delayed myelosuppres-sion seenwith each of these anti-CD25 radionuclideswas thoughtto be due to each b-emitting radionuclide having an effect on redmarrow rather than an anti-CD25 antibody–specific effect (16–18). Two anti-CD25 immunotoxin conjugates have been tested ina number of human trials. LMB-2, a pseudomonal exotoxinconjugated to an anti-CD25 single-chain variable fragment

1Cancer Research UK Drug DNA Interactions Research Group, UCLCancer Institute, London, United Kingdom. 2Spirogen Ltd, QMB Inno-vation Centre, London, United Kingdom. 3ADC Therapeutics (UK)Limited, QMB Innovation Centre, London, United Kingdom. 4Depart-ment of Pathology, University College London, London, United King-dom. 5Genmab, Utrecht, the Netherlands. 6Department of Immuno-hematology and Blood Transfusion, Leiden University Medical Center,Leiden, the Netherlands.

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

CorrespondingAuthor: John A. Hartley, UCL Cancer Institute, 72 Huntley Street,London WC1E 6BT, United Kingdom. Phone: 4420-7679-6055; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-16-0233

�2016 American Association for Cancer Research.

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(19) has shown objective responses in CD25-expressing lympho-mas including ATLL (20) and denileukin–diftitox (Ontak), adiphtheria exotoxin conjugated to an IL2 fragment (21), islicensed in CD25-positive cutaneous T-cell lymphoma. Despiteimmunogenicity andmanufacturing obstacles (22), immunotox-ins remain apromisingfield of targeted therapies (23). Anti-CD25radioimmunoconjugates and immunotoxins have both providedproof of concept for the feasibility of targeting the CD25 receptorwith few toxicity concerns and have provided a platform for thepreclinical development of new anti-CD25 targeting drugs.

The development of antibody–drug conjugates (ADC), how-ever, has arguably been the immunoconjugate class most pro-pelled by the commercial successes of therapeutic mAb technol-ogies (24). This is at least partly because the synthesis of purifiedADCs can now be carried out in a controlled way using optimizedlinker technologies (24). The use of ADCs in oncology indicationshas risen to prominence in the past decade, highlighted by FDAlicensing of Adcetris in 2011 and trastuzumab emtansine (Kad-cyla) in 2013 (24). More recently, novel ADCs delivering pyrro-lobenzodiazepine (PBD) dimers as the cytotoxic warhead havebeen developed. PBDdimers are a class of exquisitely potentDNAminor groove interstrand cross-linking agents, one of which,SG2000 (SJG-136), has shown activity against both solid tumorsand hematologic malignancies (25, 26). Advantages over ADCstargeting other warheads including tubulin inhibitors (e.g., aur-istatins andmaytansinoids), DNA damaging drugs (e.g., calichea-micin), and classical chemotherapeutics include the ability totarget low copy number antigens, and to exploit low drug–antibody ratios (DAR). Because of their novel mechanism ofaction, PBD-containing ADCs are active in tumors inherentlyresistant to other warhead types and against multidrug-resistanttumors (27).

Here we report the characterization, mechanism of action, andpreclinical evaluation of a novel anti-CD25 ADC, ADCT-301.

Materials and MethodsSynthesis of ADCT-301

HuMax-TAC antibody was first buffer exchanged into a pro-prietary histidine buffer at pH 6 containing various carbohydrate-based stabilizing additives using tangential flow filtration (TFF)on Pellicon 30-kDa cassettes using 10 diavolumes at constantTMP of 1.0 bar. Antibody was concentrated to approximately 10mg/mL and the pHwas adjusted to 7.5 using a TRIS/EDTA pH 8.5buffer. TCEP reductant (10 mmol/L) was added to the batch as a1.25 excess with respect to HuMax-TAC mAb and the reductionreactionwas allowed to proceed for 60minutes at 20�C.Dimethy-lacetamide (DMA) and 10 mmol/L SG3249 (28) were added tothe solution to a final 5% DMA v/v and 3-fold excess relative toantibody. The conjugation reaction was incubated at 20�C for 60minutes, then quenched with 3-fold molar excess of N-acetylcysteine and incubated at 20�C for 20 minutes. The pH was thendecreased to 6.0 using histidine hydrochloride solution and theADC purified by TFF using the same conditions as the first bufferexchange step, this time with 12 diavolumes of the histidine/carbohydrate additive buffer. The solution was filtered (0.22 mm)and stored at �70�C. Final yield was estimated from startingantibody by UV/Vis.

Human cell linesThe anaplastic large cell lymphoma (ALCL) cell lines Karpas

299 (2012) and Su-DHL-1 (2012) and the Hodgkin lymphoma

cell lines L-540 (2014) and HDLM-2 (2014) were obtained fromthe Deutsche Sammlung von Mikroorganismen und Zellkuturen(DSMZ). The Burkitt lymphoma cell lines Daudi (2012) andRamos (2012) and the cutaneous T-cell lymphoma line HuT-78 (2013) were obtained from the ATCC supplier LGC Standards.The myeloid leukemia cell lines, EoL-1 and KG-1, were obtainedfrom DSMZ and LGC Standards, respectively (2015). Character-ization of thesemycoplasma-negative cell lines was performed bythe cell bank supplier and included the determination and theverification of tissue-specific antibodies, karyotyping, and expres-sion of aberrantly expressed genes. As cell lines were passaged forfewer than 6 months after receipt or resuscitation, they were notauthenticated or tested in our laboratory. Cells were cultured inRPMI1640 media (Sigma-Aldrich) or Iscove's modified Dulbec-co's medium (LGC Standards) with added L-glutamine (Sigma-Aldrich) or in L-glutamine–containing media (Life Technologies)each of which was supplemented with heat-inactivated FBS as perthe manufacturer's instructions.

In vitro binding and internalization assaysBinding of HuMax-TAC or ADCT-301 to cell lines was detected

with an Alexa Fluor 488 (AF488)-labeled anti-human IgG (LifeTechnologies). For internalization studies, Karpas 299 cells wereexposed (1 hour at 4�C) to ADCT-301 in blocking buffer (PBS,10% (v/v) normal goat serum, 0.1% sodium azide (w/v)). Cellswere washed and incubated for various periods inmedium over atime course at 37�C after which time they were centrifuged,washed, and fixed (neutral buffered formalin). Once all timepoints were collected, plates were centrifuged (400 � g), washed,and incubated with either a saturating antibody concentration ofmouse IgG1 CD25 antibody (BD Biosciences) or a mouse non-targeted (isotype control) mAb MOPC (BD Biosciences), inblocking buffer for 1 hour at 4�C. The potential for competitionof binding to the same IL2R-a epitope by ADCT-301 and mouseanti-human CD25 was measured in a competition assay bycoincubation of cells with increasing concentrations of ADCT-301 and a saturating concentration of mouse anti-human CD25.After washing and resuspension, the Qifikit protocol (ref. 29;DAKO) was followed, labeling cells with an anti-mouse FITC-labeled antibody (DAKO). For immunofluorescent imaging ofADCT-301–treated CD25-positive cell lines, cells were fixed (4%paraformaldehyde) at each time point, resuspended in permea-bilization/wash buffer (BDBiosciences), and then incubatedwithAPC-labeled mouse anti-human LAMP-1 (BD Biosciences) andAF488-labeled anti-human antibody (Life Technologies), whilenuclear counterstaining was performed with Hoechst 33342 (LifeTechnologies; 1 hour at 4�C). After cell cytospins were generated,slides were mounted with ProLong gold antifade (Life Technol-ogies) and slide edges sealed with clear nail polish. Images fromdried slides kept at 4�C were captured on a TCS SPE confocalsystem utilizing a DM2500 microscope (Leica; 40 or 63� oilimmersion objective) using three solid-state excitation lasers(405, 488, and 635 nm). Alternatively, Karpas 299 cells wereincubated with ADCT-301, washed, and then either kept at 4�C inPBS, 0.1% azide or resuspended in medium and incubated at37�C over a time course to 4 hours. After each time point, cellswere washed and incubated with an AF488-labeled anti-humanIgG (1 hour at 4�C). After washing and resuspension of all labeledcells in PBS, 0.1% sodium azide, cells were analyzed on an AccuriC6 flow cytometer (BD Biosciences). Median fluorescent intensity(MFI) of the live cell populationwas determined using FlowJo 7.0

Flynn et al.

Mol Cancer Ther; 15(11) November 2016 Molecular Cancer Therapeutics2710




(Tree Star Inc.). GraphPadwas used to calculate an EC50 or a linearregressionmodel for calculatingCD25molecule numbers per cell.The Qifikit protocol (29) was also used to determine CD30 copynumber with a mouse IgG1 CD30 antibody (GeneTex).

Surface plasmon resonanceHuman soluble CD25-streptavidin fusion protein transiently

produced in CHO cells (Evitria) was immobilized by aminecoupling to a Biacore CM3 sensor chip (GE Healthcare). Aconcentration range (0.05–5 nmol/L) of HuMax-TAC andADCT-301 was injected over the chip for 120 seconds as per theBiacore multi-cycle kinetics method and allowed to dissociate byrunning HBS-EP buffer (Hepes-buffered saline containing EDTAand surfactant P20) over the chip for 60minutes. The equilibriumdissociation constant (KD) was calculated with BIAevaluationsoftware (GE Healthcare).

Cell survival determination by MTS assayCell viability was measured following either 96-hour incuba-

tion at 37�C toADC,nonbindingADC, orwarheaddilution series,or a 2-hour exposure and wash, followed by further 94-hourincubation. For themeasurement of bystander killing, Karpas 299cells were incubated with serial dilutions of ADCT-301 or non-binding ADC in 96-well round-bottom plates for 48 hours.Centrifuged Karpas 299 conditioned medium (50 mL) was addedto 50 mL of Ramos cell culture in 96-well flat-bottom plates andincubated for 96 hours. For all experiments, cell survival wasdetermined by absorbance measurements on a Varioskan FlashMultimode Reader (Thermo Fisher Scientific) according to astandard MTS protocol (30). Percentage growth inhibition wascalculated from the mean absorbance in the triplicate ADC-treated wells, compared with the mean absorbance in the controlwells (100%). Dose–response curves were generated from themean data of each independent experiment. The GI50 was deter-mined by fitting a sigmoidal dose–response curve to the datausing GraphPad.

Coculture and viability flow cytometry assaysUnlabeled Karpas 299 cells and PKH-26–labeled (Sigma-

Aldrich) Ramos cells (20 mmol/L) were diluted (1.5 � 105

cells/mL) in medium and dispensed into round-bottom, ultra-low-attachment 96-wellmicroplates (Corning) as follows:mono-culture: 50 mL cells plus 50 mL growth medium; cocultures: 50 mLKarpas 299 plus 50 mL labeled Ramos. Serial dilutions of ADCT-301 and nonbinding ADC, containing 20 mmol/L TGF-b inhib-itor, SB431542 (Sigma-Aldrich) were added (100 mL/well) tosingle and cocultures. Cells were mixed on an IKA Vibrax shaker(10 seconds) and incubated at 37�C for 96 hours. After washingand resuspension in PBS, Fixable Dead Cell Stain (Life Techno-logies) was added. Cells were resuspended and incubated in thedark for 30 minutes. Alternatively, in apoptosis experiments,Karpas 299 cells (1.5 � 105/mL) were dispensed with ADCT-301 or nonbinding ADC as described above. Etoposide was usedas a positive apoptosis control. Cells were incubated at 37�C overa time course. At each time point, cells were centrifuged, washed,resuspended in Fixable Dead Cell Stain, and FITC-labeledAnnexin-V antibody (BD Biosciences) diluted in Annexin-V Cal-cium-free buffer and incubated for 30 minutes at room temper-ature. Annexin-V buffer (250 mL) was added to each well beforeanalysis on an Accuri C6 flow cytometer. Data were analyzed inFlowJo. Total, live, and dead cell counts from three replicate

experiments were obtained from quadrant plots after compensa-tion was carried out as necessary. Counts were normalized to thetotal cell count per well for each condition, to reflect the percent-age of cells that remained viable or were apoptosed.

In vitro and in vivo cross-linking determination by single-cell gelelectrophoresis (comet) assay

Cell lines were incubated with ADCT-301 or nonbinding ADC(20 ng/mL) for 2 hours at 37�C, washed, resuspended inmediumand incubated at 37�C over a further time course (2–36 hours).After each time point, cells were centrifuged, resuspended infreezing medium (FBS with 10% DMSO), and frozen at �80�C.An equimolar concentration of SG3199 was dispensed into cellsuspension, incubated for 2 hours, and the same procedure forharvesting ADC-exposed cells followed. To determine percentagecross-linking of increasing doses of ADCT-301 or nonbindingADConexpressing cell lines, eachADCdilutionwas added to cellsfor 2 hours at 37�C. Cells were washed, resuspended in culturemedium, dispensed into 24-well plates, incubated for 24 hours at37�C and harvested, as above. For analysis of cross-linking onKarpas 299 subcutaneously implanted into SCID mice, cell sus-pensions were prepared from the excised tumors removed 24hours after dosing with 0.2 or 0.6 mg/kg ADCT-301 or leftuntreated and stored in freezing medium at �80�C. Peripheralbloodmononuclear cells (PBMC) from eachmouse were isolatedfrom whole blood using a Ficoll gradient and stored in freezingmedium at�80�C. Frozen cells were thawed on ice, resuspendedin cold RPMI (Sigma), and counted. Cell suspensions (2–3� 104

cells/mL), except unirradiated controls, were irradiated (15 Gy)and kept on ice. The Comet assay was performed as describedpreviously (31). Slideswere reviewed under a 20�objective on anepifluorescence microscope equipped with Hg arc lamp, 580 nmdichroic mirror, and 535 nm excitation and 645 nm emissionfilters suitable for visualizing propidium iodide staining with aminimum of 50 Comet images acquired per treatment condition.The olive tail moment (OTM) was determined as the product ofthe tail length and the fraction of total DNA in the tail as recordedby Komet 6 software (Andor Technology) and the percentagereduction in OTM calculated according to the formula:

%Decrease in tailmoment

¼ 1� TMdi� TMcuð Þ= TMci� TMcuð Þ½ � � 100

where TMdi ¼ tail moment drug irradiated; TMcu ¼ TM controlunirradiated; TMci ¼ TM control irradiated.

Analysis was performed on Microsoft Excel and GraphPad.

g-H2AX foci quantificationKarpas 299 cells treated with serial dilutions of ADCT-301

concentrations and frozen at �80�C were thawed, washed inPBS, fixed with 4% paraformaldehyde for 5 minutes at roomtemperature, and washed in PBS. Cell cytospins were generatedfor each treatment condition and cells were permeabilized withPBS/0.2% Triton-X100, washed in PBS, and blocked in 10% FBS,5% BSA in PBS. After incubation overnight (4�C) with goat anti-mouse g-H2AX (JBW301;Millipore) in 1%FBS in PBS, slideswerewashed and incubated (1 hour at room temperature) with AF488-labeled goat anti-mouse IgG antibody (Life Technologies) andthen counterstained with Hoecsht 33342 (Life Technologies).Slides were washed in PBS and distilled water, mounted with

ADCT-301 Targets CD25-Expressing Hematological Malignancies

www.aacrjournals.org Mol Cancer Ther; 15(11) November 2016 2711




ProLong gold antifade (Life Technologies), and the coverslipedges sealed with clear nail polish. Images captured on a confocalsystem as described above were compressed to a maximumintensity projection image of z-stack acquired images to allowmaximal detectionof foci and analyzed in ImageJ (NIH, Bethesda,MD). Cell Profiler was used to quantify g-H2AX foci per cell in atleast 20 cells per treatment.

Cell-cycle analysisKarpas 299 cells were treated with ADCT-301, SG3199, non-

binding ADC, or left untreated. Ethanol-fixed nuclei were stainedwith 0.5 mg/mL propidium iodide (PI; Sigma) in the presence of100 mg/mL RNAase (Sigma; 1 hour at 37�C). Cells were analyzedby flow cytometry (FACSVerse, BD Biosciences). Histograms ofthe singlet live cell population in the PI emission spectrum weredrawn using the cell-cycle function in FlowJo and analyses of cell-cycle percentages performed in GraphPad.

Assessment of ADCT-301 efficacy in subcutaneous in vivomodels

Karpas 299 xenografts were established in 6- to 8-week-oldfemale Fox Chase SCID (C.B-17/Icr-Prkdcscid, Charles River Lab-oratories) by implanting 107 cells subcutaneously into theirflanks. Su-DHL-1 xenografts were established by injection of107 cells (50% Matrigel) into 10-week-old nude mice [Nu(NCr)-Fox1nu, Charles River Laboratories]. When tumorsreached approximately 100–155 mm3 mice were randomly allo-cated into groups to receive ADCT-301, nonbinding ADC, PBS(vehicle), or Adcetris intravenously. Clinical grade Adcetris waspurchased from Seattle Genetics. Mice were in the body weightrange of 15.5 to 27.6 g on day 1 of the studies. Each animal waseuthanizedwhen its tumor reached the endpoint volumeof 2,000mm3 or at study end. The time-to-endpoint (TTE) for analysis wascalculated for each mouse by the following equation:

TTE daysð Þ ¼ log10 endpoint volume � intercept�ð �=slope�½ Þð

where � of the line was obtained by linear regression of a log-transformed tumor growth dataset

The log-rank test was used to analyze the significance ofdifferences between the TTE values of two groups. The setup ofexperimental procedures used in Karpas 299 and Ramos dissem-inated models are described in Supplementary Methods andTables.

Assessment of ADCT-301 pharmacokinetics and toxicity innon–tumor-bearing SCID mice

ADCT-301 was administered intravenously as a single dose(1–12mg/kg) to non–tumor-bearingCB.17 SCIDmice, followingwhich mice were observed for weight changes and toxicities.Alternatively, to measure the pharmacokinetic profile of nakedantibody, HuMax-Tac or ADCT-301, 1 mg/kg of each drug wasadministered intravenously as a single dose to non–tumor–bear-ingmice. Blood collection was performed at specified time points(25 mL of blood frommice tail veins diluted in 225-mL PBS in 6%EDTA). The sampleswere spun down at 400� g for 5minutes, thesupernatant was aspirated and stored at �80�C, and measure-ment of samples' total antibody and ADC (DAR � 1) concentra-tions performed by an optimized ELISA assay. To measure anti-body in each sample, Immuno MaxiSorp plates (Thermo Fisher

Scientific) were first coated with sCD25 Strepavidin (Evitria) inPBS and incubated overnight (4�C). Alternatively, to measureconjugated ADC, DAR � 1, identical plates were coated withantiPBD antibody in PBS and incubated overnight (4�C). Plateswere then washed three times (0.05% Tween) and blocked (3%BSA/PBS) for 1 hour. Reference, QC, and test samples wereprepared and added to polypropylene plates (Thermo FisherScientific; 2 hours at room temperature). They were then washedthree times and mouse antihuman IgG1-HRP conjugate (San-quin) added andplates incubated for 1hour at room temperature.Plates were washed three times, Ultra TMB (Thermo Fisher Sci-entific) added, and incubated for 10 minutes at room tempera-ture. Stop solution of 1 mol/L HCL was added and absorbancewas read at 450 nm on an Envision plate reader. To quantifyabsorbance, calibration curves were established in assay buffer(0.5� Blotto þ 0.05% Tween in PBS) containing 2.5% or 0.5%pooledmouse serum. The pharmacokinetic analysis of ADCT-301and HuMax-TAC concentrations was performed using PhoenixWinNonlin Version 6.2 with noncompartmental analysis.

Animal care complianceThe Institutional AnimalCare andUseProgramatCharles River

Discovery Services are accredited by the Association for Assess-ment and Accreditation of Laboratory Animal Care International(AAALAC), which assures compliance with accepted standards forthe care and use of laboratory animals.

IHC of tumor xenograft sectionsMurine xenograft tumors untreatedor treatedwith 0.2mg/kgor

0.6mg/kg ADCT-301were fixed in buffered formalin and embed-ded in paraffin according to a conventional protocol. Two to 5-mmsections were cut and transferred on electrically charged slides tobe subjected to IHC. Testing of an anti-SG3249 payload antibody(14B3-B7; ADC Therapeutics) and an antibody to detect phos-phorylation of Histone H2A.X at position Ser139 (New EnglandBiolabs) was carried out with staining conditions (i.e., antibodydilution, incubation time, and antigen retrieval method) opti-mized on ADCT-301–treated or irradiated Karpas 299 cytospins.Single immunostaining was performed using a BOND-III Auto-stainer (Leica Microsystems) according to protocols describedelsewhere (32, 33). Stained slides were scanned with the Nano-Zoomer Digital Pathology System (Hamamatsu) and imagesreviewed with NDP viewer.

ResultsProperties of ADCT-301

ADCT-301 is an ADC composed of HuMax-TAC, a humanIgG1 anti-CD25 antibody, maleimide-conjugated at reducedinterchain cysteines via a cathepsin-cleavable valine-alanine(val-ala) linker to PBD dimer warhead SG3199 (Fig. 1A). TheADC was 97% monodisperse as determined by size-exclusionchromatography (Supplementary Fig. S1A; SupplementaryTable S1). Drug antibody ratio (DAR) was determined byreverse-phase liquid chromatography to be 2.25 (Supplemen-tary Fig. S1B; Supplementary Table S2) and by hydrophobicinteraction chromatography to be 2.19 (SupplementaryFig. S1C; Supplementary Table S3).

HuMax-TAC and ADCT-301 showed strong binding affinity toCD25-positive human anaplastic large cell lymphoma–derivedcell lines Karpas 299 and Su-DHL-1 (Fig. 1B). Estimated EC50s

Flynn et al.





were in the subnanomolar range and were marginally lower forHuMax-TAC than ADCT-301 in both lines. Binding saturated ataround1mg/mL for both cell lines, but theMFIwas approximately3-fold higher in the Su-DHL-1 compared with the Karpas 299 cellline reflecting the difference in copynumber (Table 1). In contrast,a nonbinding ADC containing the same drug linker payloadshowed no binding to either cell line at 30 mg/mL. Using solubleCD25 ectodomain, equilibrium dissociation constant (KD) mea-surements of HuMax-TAC or ADCT-301 determined by surfaceplasmon resonance (SPR) were 15 pmol/L and 20 pmol/L,respectively. Representative SPR sensorgrams forHuMax-TAC andADCT-301 are shown in Fig. 1C and D, respectively.

Selective cytotoxicity of ADCT-301 in vitroThe in vitro cytotoxicity of ADCT-301 was determined in a

panel of human lymphoma CD25-expressing (Su-DHL-1,HDLM-2, L540, Karpas 299) and nonexpressing (Ramos,HuT-78, Daudi) cell lines. These values were derived fromgraphs shown in Supplementary Fig. S2A. In addition, cyto-toxicity in myeloid leukemia lines, EoL-1 (CD25-expressing)and KG-1 (CD25 non-expressing) was determined. As shown

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Figure 1.

A, structure of ADCT-301. B, flow cytometry measurement of median fluorescent intensity showing binding of ADCT-301, HuMax-TAC, and nonbinding ADCbinding to Karpas 299 and Su-DHL-1 CD25–positive human cell lines. C, Biacore sensogram depicting injections of naked antibody, HuMax-TAC over a BiacoreCM3 chip that had soluble CD25 ectodomain immobilized onto its surface. Arrow indicates the end of the ADC injection and the beginning of the dissociationrun. D, corresponding sensorgram for ADCT-301.

Table 1. CD25-positive and -negative cell lines were incubated with increasingconcentrations of ADCT-301 or free warhead (SG3199) for 96 hours beforeprocessing by the MTS assay

Mean CD25 GI50

Cell lineTumortype

molecules per cellsurface (thousands)

ADCT-301 ng/mL(pmol/L)

SG3199pmol/L

Su-DHL-1 ALCL 310 0.74 (4.96) 12.11HDLM-2 HL 167 2.94 (19.60) 158.60L-540 HL 96 1.11 (7.49) 14.80Karpas 299 ALCL 77 2.56 (17.07) 18.49Ramos Burkitt <1a >1,000 (>6,667) 5.83Daudi Burkitt <1a >1,000 (>6,667) 18.11HuT-78 CTCL <1a >1,000 (>6,667) 12.98EoL-1 AML 17 0.04 (0.26) 0.79KG-1 AML <1a >1,000 (>6,667) NDb

NOTE: Data for each cell line are presented as the GI50, that is, 50% of the nettetrazolium reduction product increase in untreated cells. The mean number ofCD25 molecules per cell line was determined by flow cytometric analysis usingQifikit (DAKO).Abbreviation: ALCL, anaplastic large cell lymphoma; AML, acute myeloid leu-kemia; CTCL, cutaneous T cell lymphoma; HL, Hodgkin lymphoma.a1,000 reflects the limits of sensitivity of this assay below which cell lines wereconsidered CD25 negative.bNot determined.






in Table 1, the GI50 values for ADCT-301 in the CD25-positivelines ranged from 0.04–2.94 ng/mL. GI50 values in the CD25-negative lines were >1,000 ng/mL. The nonbinding ADC gaveGI50 values >1,000 ng/mL in both antigen-expressing andnonexpressing lines (Supplementary Fig. S2B). No clearrelationship between GI50 value and mean CD25 copy numberwas observed for ADCT-301 in the five CD25-positive cell lines(r ¼ �0.07; P ¼ 0.91). In contrast to the ADC, the nakedwarhead, SG3199, was cytotoxic against all the lines tested anddid not discriminate between CD25-expressing and nonexpres-sing cells (Table 1). HuMax-TAC antibody up to 1,000 ng/mLwas not cytotoxic in CD25-positive or CD25-negative cell lines(Supplementary Fig. S2C).

After exposure to ADCT-301, reduction of CD25-negativeRamos cell viability was shown when cocultured (50:50) withCD25-expressing Karpas 299 cells, indicating a bystander killing

effect (Fig. 2A, top). ADCT-301 at both 1 and 10 ng/mL induced ahighly significant difference (P � 0.001) in Ramos viability inmonoculture compared with Ramos þ Karpas 299 coculture. Asignificant difference (P � 0.05) between GI50 values for Ramostreated for 96 hours with themedium taken from 48-hour ADCT-301–treated Karpas 299 and Ramos treated for 96 hours withADCT-301 in freshmediumwas also demonstrated (Fig. 2B, top).In contrast, therewas no significant effect onRamos survival whentreated with 1 or 10 ng/mL of nonbinding ADC (Fig. 2A, bottom)or conditioned media taken from nonbinding ADC-treated Kar-pas 299 (Fig. 2B, bottom).

ADCT-301 internalizationCD25-expressing cells exposed to ADCT-301 (1 hour at 4�C)

showed prominent cell surface binding (T ¼ 0 hour; Fig. 2C).On incubation at 37�C, ADCT-301 was beginning to be

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A, histograms depicting percentageviable CD25-negative Ramos cells inmonoculture (black bar) and RamosþKarpas 299 (white bar) in coculture(50:50) treated with 1 or 10 ng/mL ofeither ADCT-301 (top; �� , P � 0.001)or nonbinding ADC (bottom) for 96hours (ns, not significant). B, viabilitycurves showing percentage Ramosviability compared with untreatedcontrol wells after a 96-hour exposureeither to ADC (black square) or tomedia transferred from ADC-treatedKarpas 299 (white triangle). Theoriginal ADCT-301 concentrationtitration series is plotted in the toppanel (P � 0.05) and that of thenonbinding ADC in the bottom panel(not significant). C, mergedimmunofluorescence images of CD25-positive L-540 cells treated withADCT-301 (1 mg/mL) for 1 hour,washed and fixed after T ¼ 0, 1, or4 hours in medium at 37�C andstained with labeled anti-human IgG(green), anti-LAMP-1 (red), andHoechst 33342 (blue) nuclear stain.Yellow indicates colocalization.Nonbinding ADC-treated L-540 cellsare shown at T ¼ 0. D, line graphshowing themeanCD25moleculespercell present on the surface of ADCT-301–treated (1 mg/mL) Karpas 299cells over a time course to 16 hours as apercentage of the mean CD25molecules per cell present onuntreated Karpas 299. E, line graphsdepicting median fluorescence of cellsincubated at 37�C in medium versuscells kept at 4�C in 0.1% sodium azideat 1, 2, and 4 hours after ADCT-301treatment.

Flynn et al.





internalized by 1 hour with cell membrane labeling still evidentand colocalization with lysosomes observed by 4 hours. Incontrast, no cell surface binding was observed with a nonbind-ing ADC at T ¼ 0 hours. For Karpas 299, there was a reduction

of CD25 molecules on the cell surface, as measured by areduction in MFI of cells labeled with a FITC-conjugatednoncompetitive anti-CD25 antibody, over the first three hoursafter treatment. The change in surface CD25 molecules after

Figure 3.

A, the single-cell gel electrophoresis (comet) assay was carried out on untreated irradiated or unirradiated Karpas 299 and 1 or 15 ng/mL ADCT-301 or nonbindingADC-treated irradiated cells, with image panels showing DNA labeled with propidium iodide for each treatment condition. B, line graph showing thepercentage reduction in OTM compared with untreated control in Karpas 299 cells treated with 300 pmol/L naked warhead, 20 ng/mL (133 pmol/L; ADCT-301or nonbinding ADC). C, percentage reduction in OTM in Su-DHL-1 cells treated with 300 pmol/L naked warhead or 20 ng/mL (133 pmol/L) ADCT-301. D, percentageviability and percentage reduction in OTM are plotted versus concentration of equivalent warhead in ADCT-301–treated Karpas 299. E, the equivalentcurves are plotted for ADCT-301–treated Su-DHL-1.






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Flynn et al.





ADCT-301 exposure was significant (P � 0.05) compared withthat observed after nonbinding ADC exposure. A return topretreatment CD25 MFI occurred by 16 hours (Fig. 2D). Inaddition to a change in CD25 receptor number, the MFI of cellsincubated with ADCT-301 and stained with an AF488-labeledanti-human IgG antibody was reduced over a time course up to4 hours after incubation at 37�C (Fig. 2E).

DNA interstrand cross-link formationDNA interstrand cross-linking was measured using a modifi-

cation of the single-cell gel electrophoresis (comet) assay. Rep-resentative comet images are shown in Fig. 3A. Cross-linking wasexpressed as the percentage decrease in the OTM compared withcontrol irradiated cells. Following a 2-hour exposure of Karpas299 cells to 20 ng/mL of ADCT-301, the time course of cross-linkformation is shown in Fig. 3B. Cross-links formed after an initialdelay, reaching a peak between 4 and 8 hours. In contrast, the freewarhead at an equimolar equivalent warhead dose reached thepeak of cross-linking during the 2-hour exposure. In both cases,cross-links persisted over at least 36 hours. The nonbinding ADCat 20 ng/mL did not show a significant reduction inOTMover thesame time course. An equivalent experiment in Su-DHL-1 cellsgave the same kinetics of cross-link formation resulting in aslightly higher peak (Fig. 3C), possibly reflecting the higher CD25copy number and sensitivity of this line to ADCT-301.

At maximum cross-linking (24 hours) in both Karpas 299 (Fig.3D) and Su-DHL-1 (Fig. 3E), a correlation between loss ofviability and cross-link formation (r ¼ 0.9745 and r ¼ 0.9485,respectively; P � 0.05) was established. No evidence of cross-linking after treatment with 1 mg/mL ADCT-301 could be dem-onstrated in CD25-negative Daudi cells (Supplementary Fig. S3).However, cross-linking by naked warhead in CD25-negativeDaudi was similar to that in CD25-positive cell lines.

DNA damage response, cell-cycle arrest, and apoptosisWe have previously shown that PBD-induced DNA interstrand

cross-links induce a robust, but delayed g-H2AX response (34). InKarpas 299 cells, g-H2AX foci were observed 24 hours after a 2-hour exposure to sub-GI50 concentrations of ADCT-301 (Fig. 4A).After treatment with 60 pg/mL or 1,000 pg/mL ADCT-301,g-H2AX foci increased by 17- or 34-fold relative to untreatedcells, respectively (P � 0.05 and P � 0.01). In these cells, con-tinuous exposure to 3 or 10 ng/mL ADCT-301 resulted in a dose-dependent G2–M arrest reaching a maximum at 48 hours asevidenced by a decreased percentage of cells in G0–G1 and anincreased percentage in G2–M compared with untreated control(Fig. 4B). A GI50 warhead concentration (15 pmol/L) inducedG2–M cell-cycle arrest in a similar fashion to a GI50 concentrationof ADCT-301 (3 ng/mL, containing the equivalent of 46 pmol/Lof payload in this cell line), although this occurred earlier peak-

ing at 24 hours (Fig. 4C). After continuous exposure to 10 ng/mLADCT-301, the peak of the early apoptosis marker Annexin-Von the cell surface of Karpas 299 cells was observed between60 and 72 hours (Fig. 4D). Maximal loss of viability was at96 hours. Nonbinding ADC (10 ng/mL) did not induce cell-cycleG2–M arrest (Fig. 4B) or increased Annexin-V and loss of viability(Fig. 4D).

In vivo efficacy of ADCT-301In vivo, ADCT-301 demonstrated dose-dependent antitumor

activity against both subcutaneous and disseminated lymphomamodels. In the subcutaneous CD25-expressing Karpas 299 xeno-graft, a single dose of ADCT-301 delayed tumor growth in a dose-dependent fashion with the 0.6 mg/kg cohort demonstrating 10of 10 tumor-free survivors at day 60 (Fig. 5A). TTE survivalanalyses compared with vehicle control were superior at 0.1mg/kg (log-rank test P � 0.05) and at 0.2, 0.4, and 0.6 mg/kg(P�0.001; Supplementary Fig. S4A). Similarly a single dose of 0.5mg/kg ADCT-301 produced 10 of 10 tumor-free survivors main-tained at day 75, whereas a single dose of 0.5 mg/kg Adcetris(targeting CD30) only delayed tumor growth and even four 0.5mg/kg doses (q4d � 4) failed to cure mice (Fig. 5B). CD30expression on Karpas 299 is two to three times higher than CD25(inset to Fig. 5B). Kaplan–Meier analyses showed ADCT-301significantly superior (P � 0.01) to Adcetris single-dose admin-istration (Supplementary Fig. S4B). In a disseminated Karpas 299model, single-dose ADCT-301 also showed significant dose-dependent extensions of survival in comparison with vehiclecontrol at all four doses tested (P � 0.001; Supplementary Fig.S5A). Single-doseADCT-301 at 0.6mg/kg showed significant (P�0.05) improvement in survival compared with single-dose Adce-tris administration (Supplementary Fig. S5B). In both subcuta-neous and disseminated models (0.5 mg/kg and 0.6 mg/kg,respectively), nonbinding ADC failed to show significant tumorregression or survival benefit compared with vehicle controls(Fig. 5B and Supplementary Figs. S4B and S5A). In the subcuta-neous Su-DHL-1 xenograft, a single dose of ADCT-301 delayedtumor growth in a dose-dependent fashion with the 0.6 mg/kgcohort demonstrating 6 of 8 tumor-free survivors at day 74(Fig. 5C). In contrast, single-dose (0.5 mg/kg) ADCT-301 indisseminated CD25-negative Ramos xenografts failed to showa survival benefit (Supplementary Fig. S6). In non–tumor-bearingSCIDmice the MTD, as defined by less than 10%weight loss, was9 mg/kg ADCT-301. The half-life of naked antibody, HuMax-Tac,was 7.3 days. Similarly, the half-life of ADCT-301 by analysis ofthe conjugated antibody with DAR component� 1 was 7.3 days.

Pharmacodynamic assessment of ADCT-301 in vivoIn SCID mice with Karpas 299 subcutaneous tumors, a single

dose of ADCT-301 was administered at 0.2 or 0.6 mg/kg. Twenty-

Figure 4.A, image panels depict Karpas 299 cells untreated or treatedwith 60pg/mL or 1 ng/mLADCT-301 and then stainedwith Hoechst 33342 nuclear stain (first column) orAF-488–labeled anti-g-H2AX (middle column). The third column shows the image overlays in color. Scale bars, 20 mm. The bar chart (right) shows the mean foldincrease in foci per cell in three independent experiments at each concentration comparedwith foci per untreated cell. (� , P�0.05; �� , P�0.01).B, cell histograms ofKarpas 299 either untreated, or treated with 10 ng/mL of nonbinding ADC, 3 or 10 ng/mL of ADCT-301 for 48 hours, in each cell-cycle phase (G1, S, orG2–M) as determined by intensity of propidium iodide staining in one representative experiment. The bar chart (below) shows the percentage of cells in each cell-cycle phase in three independent experiments for each of these treatment conditions. (� , P � 0.05). C, bar chart showing mean fold increase of cells in G2 phasecompared with untreated control for cells treated with 3 ng/mL (20 pmol/L) ADCT-301 or 15 pmol/L naked warhead at 16, 24, or 48 hours. D, line graph showingpercentage of Karpas 299 cells per well with surface Annexin V exposure or nonviable cells as indicated by permeability to viability dye over a time course (24, 48,60, 72, and 96 hours) after treatment with 10 ng/mL ADCT-301 or nonbinding ADC.






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Figure 5.

A, in vivo antitumor efficacy of ADCT-301 in subcutaneously implanted Karpas 299 xenograft model. Compared with injection of vehicle (PBS), ADCT-301administered intravenously (i.v.) at a mean tumor volume of 160 mm3 as a single dose at 0.1, 0.2, 0.4, and 0.6 mg/kg showed dose-dependent antitumor activity.B, in vivo antitumor efficacy of ADCT-301 in comparison to Adcetris and nonbinding ADC. Vehicle (PBS), nonbinding ADC (DAR 2.1), ADCT-301 (DAR 2.2), or Adcetris(DAR 4) were administered intravenously at a mean Karpas 299 tumor volume of 130 mm3 as single doses at 0.5 mg/kg. Adcetris was also tested q4d � 4 at0.5mg/kg. Themean number of CD25 andCD30molecules perKarpas 299 cell as determined by theflowcytometricQifikit (DAKO) is shown in the inset to the graph.C, in vivo antitumor efficacy of ADCT-301 in subcutaneously implanted Su-DHL-1 xenograft model. ADCT-301 was administered intravenously at mean tumorvolumeof 155mm3at single doses of 0.3mg/kgor0.6mg/kg and tumor growth comparedwith that observed after injection of vehicle (PBS).D, representative scansof formalin-fixed paraffin-embedded Karpas 299 tumor sections, obtained from untreated control SCID mice or mice treated with 0.2 and 0.6 mg/kg ADCT-301 andstained with an anti-PBD linker antibody (top) or an anti-g-H2AX antibody (bottom). Scale bars, 100 mm. A magnified area of the anti-PBD linker antibodystaining of tumors excised from animals dosed at 0.6mg/kg is shown in the inset to this figure. E, histogramdepictingmeanOTM in irradiated tumor cell suspensionstaken from Karpas 299 xenograft models untreated or 24 hours after injection with 0.2 mg/kg or 0.6 mg/kg ADCT-301 (��, P � 0.01; �� , P � 0.001).

Flynn et al.





four hours after treatment, excised tumors showed a dose-relatedincrease in intensity of staining by an anti-PBD payload antibodyand a g-H2AX antibody (Fig. 5D). A magnified section of theADCT-301 0.6 mg/kg anti-PBD payload antibody panel indicatesthat the tumor cells demonstrate both membrane and cytosolicstaining. Reduction inOTMwas 23%(0.2mg/kg) versus 49%(0.6mg/kg; P� 0.01) using the comet assay (Fig. 5E). No cross-linkingwas observed in peripheral blood lymphocyte samples from thesame mice (Supplementary Fig. S7). The selective targeting ofADCT-301 to CD25-expressing tumor cells and resulting forma-tion of DNA interstrand cross-links and associated DNA damageresponse are therefore confirmed in vivo.

DiscussionADCs delivering PBD dimer payloads represent a novel mode

of action in the ADC arena. They exploit a completely differentcellular mechanism to the widely used auristatin and maytansi-noid tubulin inhibitors and a different mode of action to otherDNA-interacting warheads such as calicheamicin (27). SeveralPBD-containing ADCs are now in phase I clinical trials includingSGN-33A (27), SGN-70A (35), SC16LD6.5 (36), and ADCT-301.In the synthesis of ADCT-301, the warhead SG3199 is connectedvia its N10 position to a maleimidocaproyl val-ala dipeptidelinker (as in SC16LD6.5). The maleimide then covalently andstochastically reacts with a limited number of reduced inter-chaindisulphide bonds inHuMax-TAC. In contrast, SGN-33AandSGN-70A are conjugated via a val-ala linker to the C2 position on thePBD dimer SGD-1882. As the N10 position imine is involved incovalent binding toDNA, linker attachment at this point producesa prodrug and an ADC payload as well as potentially adding afurther level of safety. As a result, attachment at the N10 iminerequires a self-immolative linker that becomes completely trace-less following cleavage.

Binding of ADCT-301 and HuMax-TAC to CD25-positive celllines (flow cytometry) and to soluble CD25 (SPR) was compa-rable, indicating that conjugation to PBD linker did not affectantigen binding. Our in vitromodels show that HuMax-TAC is notitself directly cytotoxic. All cell lines studied were sensitive toSG3199 warhead. The CD25-positive cell line least sensitive toADCT-301 was also least sensitive to SG3199 warhead. In all fourCD25-positive lymphoma cell lines tested, picomolar potency toADCT-301 is demonstrated but there was no clear relationbetween sensitivity to ADCT-301 and CD25 expression level. Allthese cells lines, however, had high levels of CD25 expression(77,000–310,000) and cell lines with much lower CD25 expres-sion may be required to determine the threshold for cytotoxicsensitivity. An eosinophilic leukemia cell line, EoL-1, with asurface CD25 expression of 17,000 demonstrated femtomolar invitro sensitivity to ADCT-301 and free SG3199, suggesting thattumor type sensitivity to free warhead varies considerablybetween tumor types and may predict targeted ADC cytotoxicityindependently of CD25 expression level above a certain thresholdexpression level.

If ADCT-301 is to be efficacious in lymphomas with hetero-geneous CD25 expression (2), the existence of bystander toxicityon target-negative tumor cells is important. PBDwarhead releasedfrom the target-positive cell would be expected to be diffusible incontrast to less permeable payloads such as MMAF (37). Initialcoculture experiments of CD25-negative Ramos with CD25-pos-itive Karpas 299 resulted in loss of Ramos cells, possibly due to

the Treg-suppressive phenotype of Karpas 299 (38). Therefore,Ramos and Karpas 299 were cocultured in the presence of aTGF-b signaling antagonist to ablate the Treg-suppressive effect(39). In bothmedium transfer and coculture experiments, Ramoscells were killed by a soluble factor released from ADCT-301–treated Karpas 299 cells into the medium. We hypothesize thatthis soluble factor is SG3199 warhead, released by lysosomalcleavage of the val-ala dipeptide linker in ADCT-301 and theself-immolative cleavage of the residual linker stub on the PBDN10 imine, as we have shown that any transfer of intact ADCwould be inactive against the CD25-negative Ramos cells.

Evidence for internalization of ADCT-301 is provided both bythe decline of detected ADC on the cell surface of Karpas 299 andthe reduction in intensity of membrane immunofluorescentstaining on L-540 cells. Furthermore, a reduction of CD25 surfaceexpression over the first 3 hours after ADCT-301 treatment isevident, after which expression returns to pretreatment levels by16 hours. There is no binding competition between the two anti-CD25 antibodies used in this latter assay suggesting that thedecline in the CD25 copy number represents internalization anddownmodulation. Immunofluorescent colocalization imagesshow that processing of ADCT-301 is at least, in part, lysosomal.

An important feature of the highly cytotoxic interstrand cross-linking produced by PBD dimer warheads is the minimal DNAdistortion which may be responsible for a lack of repair of thesecross-links and their persistence (40, 41). The time lag between thepeak of cross-linking by ADCT-301 and by equimolar warheadlikely reflects the slower time taken for ADC internalization andcellular processing compared with the diffusible active warhead.We detected no cross-link repair over 36 hours. The strongcorrelation between the reduction in OTM and the loss of cellsurvival indicates that DNA cross-linking is an importantmode ofaction of ADCT-301.

A DNA damage response (increased g-H2AX foci) is evident inCD25-positive cells 24 hours after a 2-hour exposure to ADCT-301 at concentrations as low as 60 pg/mL. This supports previousobservations that g-H2AX is highly sensitive to cellular eventsinduced by DNA cross-linking agents including PBD dimers (34,41). DNA cross-linking leads to G2–M cell-cycle arrest and apo-ptotic cell death (40, 42). We have shown that ADCT-301 causedG2–M arrest peaking at 48 hours and apoptotic cell death. Thus,we confirm thatDNAcross-linking peakedby4 to 8hours andwasfollowed by a g-H2AX response by 24 hours. G2–M arrest ensuedat 48 hours, phosphatidylserine and phosphatidyltyramine expo-sure at 72 hours, and loss of cell viability at 96 hours.

The MTD, pharmacokinetics, and stability of ADCT-301 wereexplored in non–tumor-bearing SCIDmice. These mice toleratedADCT-301 up to 9 mg/kg. Measured half-lives of naked antibodyand ADCT-301 in mice serum were identical. CD25 is constitu-tively expressed on Treg and on activated cytotoxic T lymphocytes,B lymphocytes (43),macrophages (44, 45), eosinophils (46), andbasophils (47). Anti-CD25 immunoconjugatesmay contribute tothe depletion of intratumoral CD25-expressing T cells includingTregs as has been demonstrated for 90Yttrium-labeled daclizumab(18) and LMB-2 (48). Although the safety and efficacy of admin-istering anti-CD25 radiommunoconjugates (16–18) and immu-notoxins (19, 20) has been established in previous phase I trials,the consequences of prolonged ablation of Tregs remains to bedetermined.

Mice xenografted with Karpas 299 and treated with ADCT-301show remarkable dose–response improvements in survival.






Single-dose ADCT-301 shows superiority to single-dose Adcetrisadministration in TTE statistical analyses despite the 3-fold highercell surface target expression for Adcetris and the higher DAR ofAdcetris versus ADCT-301. Another CD25-expressing xenograftedcell line, Su-DHL-1, showed significant dose-dependent delays intumor growth and tumor-free survival after treatmentwith ADCT-301. The targeted in vivo efficacy of ADCT-301 is illustrated in thelack of efficacy in both nontargeted ADC treatment of Karpas 299xenografts and inADCT-301 treatment of aCD25-negative Ramosmodel.

Using amonoclonal anti-PBD antibody against a PBD payloadand anti-g-H2AX staining has confirmed the dose-dependentdelivery of ADC to the tumor xenograft. The comet assay andg-H2AX immunofluorescence have been used as pharmacody-namic assays in previous clinical trials of PBD dimer, SG2000(25, 26) and the comet assay was used to quantitatively assessDNA damage in excised tumor samples in the current study.Extrapolating from in vivo data, the mean reduction in OTM in0.6 mg/kg-treated tumor cells compared with the 0.2 mg/kgcohort, suggests that a threshold level of DNA cross-linking maybe required for in vivo efficacy. The absence of significant DNAcross-linking in SCID mouse PBMCs (CD25-negative) supportsthe CD25 specificity of ADCT-301 in vivo. These comet data thusprovide a relevant pharmacodynamic assay for use in the currentclinical development of PBD-based ADCs including ADCT-301.

Disclosure of Potential Conflicts of InterestP.C. Tyrer has ownership interest (including patents) in Medimmune.

F. D'Hooge is the Head of the Bioconjugation Unit at Novasep. P.W.H.I. Parrenhas ownership interest (including patents) in Genmab. P.W. Howard hasownership interest (including patents) in ADC Therapeutics. P.H.C. van Berkelhas ownership interest (including patents) in ADC Therapeutics. J.A. Hartleyreports receiving a commercial research grant from, has ownership interest(including patents) in, and is a consultant/advisory board member for ADCTherapeutics. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design:M. Flynn, C.E.G. Havenith, J.-N. Levy, A. Tiberghien,L.A. Masterson, P.W.H.I. Parren, D.G. Williams, P.W. Howard, P.H.C. vanBerkel, J.A. Hartley

Development of methodology: M. Flynn, P.C. Tyrer, C.E. Britten, C.E.G.Havenith, L.A. Masterson, T. Marafioti, P.W. Howard, J.A. HartleyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Flynn, P.C. Tyrer, N. Janghra, C.E. Britten,J.-N. Levy, A. Tiberghien, C. Barry, F. D'HoogeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Flynn, F. Zammarchi, N. Janghra, C.E. Britten,C.E.G. Havenith, J.-N. Levy, A. Tiberghien, L.A.Masterson, C. Barry, F. D'Hooge,T. Marafioti, P.H.C. van Berkel, J.A. HartleyWriting, review, and/or revision of the manuscript: M. Flynn, F. Zammarchi,C.E.G. Havenith, F. D'Hooge, P.W.H.I. Parren, D.G. Williams, P.W. Howard,J.A. HartleyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Flynn, F. Zammarchi, A.U. Akarca,C.E. Britten, C.E.G. Havenith, F. D'HoogeStudy supervision: F. Zammarchi, P.H.C. van Berkel, J.A. HartleyOther (performed IHC-related experiments and provided images via scan-ning): A.U. AkarcaOther (development of pbd chemistry): L.A. Masterson

AcknowledgmentsThe authors thank Charles River Laboratory Services for conducting the

majority of the in vivo efficacy studies. Much appreciation is extended tomembers of other departments at UCL including GCLP staff, Janet Hartley,and Victoria Spanswick for sharing their expertise (comet assay and g-H2AXimmunofluorescence), staff of the Biological Services Unit, Mathew Robsonand Barbara Pedley for their assistance and guidance in conducting addi-tional in vivo experiments, and Vishvesh Shende and Joseph Linares from thePathology Department for their help in setting up IHC assays. Otherlaboratory staff at Spirogen and ADCT in both chemistry and biologydepartments are acknowledged for their guidance in setting up assays usedin this project.

Grant SupportJ.A. Hartley acknowledges Programme Grant support from Cancer Research

UK (C2559/A16569).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.

Received April 15, 2016; revised July 7, 2016; accepted July 13, 2016;published OnlineFirst August 17, 2016.

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2016;15:2709-2721. Published OnlineFirst August 17, 2016.Mol Cancer Ther Michael J. Flynn, Francesca Zammarchi, Peter C. Tyrer, et al. Hematological Malignancies

Drug Conjugate (ADC) Targeting CD25-Expressing−Antibody Containing−ADCT-301, a Pyrrolobenzodiazepine (PBD) Dimer

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ADCT-301, a Pyrrolobenzodiazepine (PBD) Dimer Containing … · benzodiazepine (PBD) dimer warhead with a drug–antibody ratio (DAR) of 2.3. ADCT-301 binds human CD25 with pico-molar