preclinical evaluation of medi0641, a ...antibody–drug conjugates (adc) are used to selectively...

Large Molecule Therapeutics

Preclinical Evaluation of MEDI0641, aPyrrolobenzodiazepine-ConjugatedAntibody–Drug Conjugate Targeting 5T4Jay Harper1, Christopher Lloyd2, Nazzareno Dimasi3, Dorin Toader3,Rose Marwood2, Leeanne Lewis2, David Bannister2, Jelena Jovanovic2,Ryan Fleming3, Francois D'Hooge4, Shenlan Mao1, Allison M. Marrero1,Martin Korade III1, Patrick Strout1, Linda Xu3, Cui Chen1, Leslie Wetzel1,Shannon Breen1, Lilian van Vlerken-Ysla1, Sanjoo Jalla5, Marlon Rebelatto6,Haihong Zhong1, Elaine M. Hurt1, Mary Jane Hinrichs7, Keven Huang1,Philip W. Howard4, David A. Tice1, Robert E. Hollingsworth1, Ronald Herbst1,and Adeela Kamal1,8

Abstract

Antibody–drug conjugates (ADC) are used to selectivelydeliver cytotoxic agents to tumors and have the potential forincreased clinical benefit to cancer patients. 5T4 is an oncofetalantigen overexpressed on the cell surface in many carcinomason both bulk tumor cells as well as cancer stem cells (CSC), hasvery limited normal tissue expression, and can internalize whenbound by an antibody. An anti-5T4 antibody was identifiedand optimized for efficient binding and internalization in atarget-specific manner, and engineered cysteines were incorpo-rated into the molecule for site-specific conjugation. ADCstargeting 5T4 were constructed by site-specifically conjugatingthe antibody with payloads that possess different mechanismsof action, either a DNA cross-linking pyrrolobenzodiazepine(PBD) dimer or a microtubule-destabilizing tubulysin, so thateach ADC had a drug:antibody ratio of 2. The resulting ADCs

demonstrated significant target-dependent activity in vitro andin vivo; however, the ADC conjugated with a PBD payload(5T4-PBD) elicited more durable antitumor responses in vivothan the tubulysin conjugate in xenograft models. Likewise, the5T4-PBD more potently inhibited the growth of 5T4-positiveCSCs in vivo, which likely contributed to its superior antitumoractivity. Given that the 5T4-PBD possessed both potent anti-tumor activity as well as anti-CSC activity, and thus couldpotentially target bulk tumor cells and CSCs in target-positiveindications, it was further evaluated in non-GLP rat toxicologystudies that demonstrated excellent in vivo stability with anacceptable safety profile. Taken together, these preclinical datasupport further development of 5T4-PBD, also known asMEDI0641, against 5T4þ cancer indications. Mol Cancer Ther;16(8); 1576–87. �2017 AACR.

IntroductionAntibody–drug conjugates (ADC) represent a promising ther-

apeutic approach to effectively treat cancer while reducing drug-related toxicities by combining the specificity of an antibodywith the potency of cytotoxic agents (reviewed in refs. 1–3). AnADC consists of an antibody that is conjugated with a potent

warhead, typically a cytotoxic small molecule, which has beenchemically modified with a linker used to conjugate the drug tothe antibody. The ADC binds to an antigen on the surface of acancer cell, and the ADC/antigen complex is internalized andtrafficked to the lysosome, where the cytotoxic agent is releasedthrough cleavage of a linker or through proteolytic degradationof the antibody itself if a noncleavable linker is used to con-jugate the warhead to the antibody. The warhead exits thelysosome where it can then bind to its target, typically eithermicrotubules or DNA, depending on its mechanism of action.The binding of these cytotoxic agents to their targets results incell-cycle arrest that subsequently leads to cell death. Given thegenerally high potency of cytotoxics most commonly used inADCs, it is important to identify targets exhibiting both goodexpression in tumors and limited normal tissue expression toreduce the possibility of on-target toxicity.

The oncofetal antigen 5T4, also known as trophoblast gly-coprotein (TPBG), is a 72-kDa glycoprotein that is typicallyonly expressed during embryonic development, whereasexpression in normal adult tissues is very limited (4, 5).Expression of 5T4, however, is reported to be significantlyupregulated in many types of carcinomas, including, but not

1Oncology Research, MedImmune, LLC, Gaithersburg, Maryland. 2AntibodyDiscovery and Protein Engineering, MedImmune, Ltd, Cambridge, United King-dom. 3Antibody Discovery and Protein Engineering, MedImmune, LLC, Gaithers-burg, Maryland. 4Spirogen, London, United Kingdom. 5Project Management,MedImmune, LLC, Gaithersburg, Maryland. 6Pathology, MedImmune, LLC,Gaithersburg, Maryland. 7Biologics Safety Assessment, MedImmune, LLC,Gaithersburg, Maryland. 8Ferring Pharmaceuticals, San Diego, California.

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

Corresponding Author: Jay Harper, MedImmune, LLC, One MedImmune Way,Gaithersburg, MD 20878. Phone: 301-398-5209; Fax: 301-398-8958; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-16-0825

�2017 American Association for Cancer Research.

MolecularCancerTherapeutics

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limited to, cancers of the lung, breast, stomach, prostate,colon, and ovaries, and its expression has been correlated withpoor prognosis in multiple indications (6–13).

The biology of 5T4 in normal and pathologic tissues is notwell understood. The extracellular domain of 5T4 containsseveral leucine-rich repeats that have been implicated in pro-tein binding that can promote cell–matrix and/or cell–cellinteractions (14). However, no ligand or coreceptor has beenidentified for 5T4 to date. During embryonic development, 5T4appears to regulate CXCR4-mediated chemotaxis (15) andmodulates dendritic branching of granule cells in the devel-oping olfactory bulb (16). The extracellular domain is criticalfor 5T4-mediated inhibition of Wnt/b-catenin signaling (17).5T4 has a short cytoplasmic domain consisting of 44 aminoacids and aside from a serine–asparagine–valine motif that isimplicated in binding PDZ-containing molecules, there are noclassical signal transduction motifs contained in the sequence(14, 18). Studies with overexpression or knockdown of 5T4have demonstrated that it likely plays a role in adhesion,cytoskeletal organization and motility, and epithelial-to-mes-enchymal transition associated with metastasis (18–20). 5T4knockout mice are viable, although adults have a number ofstructural defects in the brain as a result of changes that occurduring brain development in utero likely caused by the role of5T4 in dendritic branching (15).

5T4 is also expressed on cancer stem cells (CSC), which arehypothesized to be responsible for chemotherapeutic resis-tance and recurrence of cancer (21, 22). Targeting both 5T4-expressing bulk tumor cells and CSCs could, theoretically,produce a more durable clinical response. Previous reportshave separately shown that 5T4-targeted ADCs armed witheither a microtubule-targeting payload or a DNA cross-linkingpyrrolobenzodiazepine (PBD) payload can inhibit both bulkand CSC tumor cell populations (22–25). Given the differencesin mechanisms of action between these payloads (26–28),we wanted to compare the activities of these two drugs side-by-side.

We report data from in vitro and in vivopreclinical tumormodelscomparing the activity of the site-specifically conjugated ADCs5T4-PBD and 5T4-tubulysin (5T4-Tub). The 5T4-PBD, subse-quently labeled asMEDI0641, elicited superior antitumor activitythan 5T4-Tub and reduced the CSC population in vivo, unlikeADCs conjugated with either tubulysin or auristatin payloads.Following these observations, the preclinical safety and pharma-cokinetic profile ofMEDI0641was evaluated in rats to assess its invivo stability and off-target toxicity profile.

Materials and MethodsGeneration of the lead anti-5T4 antibody, 5T4_0108

The parental antibody, A07, was derived by immunization ofVelocImmune II mice (Regeneron; ref. 29) with recombinanthuman 5T4 using a 48-day rest/boost immunization protocol(30) and was identified from a biochemical assay of hybridomasupernatants for binding to 5T4. This antibody underwent affinityoptimization by CDR-directed mutagenesis (31) and phage dis-play selection (32, 33). The lead antibody, 5T4_0108, was iden-tified in a biochemical assay, where it demonstrated improvedbinding to 5T4 over the parental A07. This antibody was thenexpressed and purified as a full-length, human IgG1 antibodycontaining the mutations L234F to significantly reduce FcgR

binding, and S239C to provide engineered cysteines for site-specific conjugation of payloads, in the CH2 domain (34) basedon EU numbering (35).

Site-specific conjugation of warheadsSite-specific conjugates were generated as described previously

(34), and see Supplementary Data for a detailed protocol.

Cell linesAll cell lines (MDA-MB-361, HCC1937, SUM159, and T47D

breast carcinoma cells; DU 145 prostate cancer cells, NCI-N87gastric cancer cells, DMS114 lung carcinoma cells; Panc-1 andHPAC pancreatic carcinoma cells; HCT-116 colorectal carcinomacells; HepG2 hepatocellular carcinoma cells) were obtained fromATCC in the following years: MDA-MB-361, November 2011;HCC1937, November 2008; SUM159, January 2012; T47D, Sep-tember 2012; DU 145, October 2011; NCI-N87, August 2012;DMS114, July 2008; Panc-1, January 2012; HPAC, April 2008;HCT-116, January 2013; and HepG2, February 2012. Uponarrival, stock vials undergo authenticity and IMPACT testing (formurine viruses) by IDEXX Bioresearch using real-time PCR anal-yses. Cells are also designated mycoplasma negative after under-going internal PCR assays. Master cell banks are made from thesestock vials, and each time a working cell bank is created fromthese master banks, the cells are retested at IDEXX and internally.Cells used for all described experiments were derived from theseworking cell banks and were used within 10 passages followingthawing. After 10 passages (typically within 2–3 months follow-ing thawing), cells were discarded and new cell vials were thawedas needed. All cell lines were cultured using ATCC-recommendedmedia at 37�C in a humidified incubator with 5% CO2 except forthe MDA-MB-361 breast carcinoma cells that were cultured inatmospheric air at 37�C.

In vitro cytotoxicityThe cells were plated in culture media at a density of 2,000 to

5,000 per well (depending on the growth kinetics of each cellline) of tissue culture-treated 96-well plates in a volume of80 mL and allowed to adhere overnight. A 5� stock of eachconcentration of antibody or ADC to be tested was prepared bydiluting the test articles in culture medium. Twenty microlitersof each test article was added to cells in duplicate or triplicatesuch that the final dose curve ranged from 4 mg/mL down to 61pg/mL in a stepwise 1:4 serial dilution series. The treated cellswere cultured at 37�C/5% CO2 for 72 to 144 hours (dependingon the growth kinetics of each particular cell line). TheCellTiter-Glo (CTG) Luminescent Viability Assay (Promega)was used to determine relative cytotoxicity. Briefly, 100 mL ofCTG reagent was added to each well, allowed to incubate for10 minutes at room temperature with mild shaking, and thenthe absorbance of each sample at 560 nmol/L was read usingan EnVision luminometer (PerkinElmer). The percent cell via-bility was calculated by the following formula: (averageluminescence of treated samples/average luminescence ofcontrol samples) � 100. IC50 values were determined usinglogistic nonlinear regression analysis with GraphPad Prismsoftware.

In vivo efficacy studiesAll in vivo studies were carried out in compliance with the

guidelines published by the Association for Assessment and

MEDI0641: A Novel PBD-Based Anti-5T4 ADC

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Accreditation of Laboratory Care. The MedImmune InstitutionalAnimal Care and Use Committee approved all study protocols.For cell line–derived xenograftmodels, tumor cellswere harvestedfrom culture flasks, mixed with Matrigel in a 1:1 ratio, andimplanted subcutaneously or orthotopically into 5- to 6-week-old athymic nude mice (Envigo). Resulting tumors were mea-sured twice aweek throughout the course of the experiments, withtumor volume calculated using the following formula: tumorvolume (mm3) ¼ (length � width2)/2. Test articles were admin-istered intravenously. See Supplementary Data for additionalinformation.

Evaluating effects on CSCs in tumorsMice bearing NCI-N87 and MDA-MB-361 xenografts were

treated with the indicated ADC, and upon initial signs of tumorregression, tumors were excised, cut into 4-mm pieces and cryo-preserved using Cryostor. Frozen tumor pieces were thawed at37�C, washed twice in Hank's balanced salt solution (HBSS), andfurther minced using sterile scalpel blades. To obtain single-cellsuspensions, the tumor pieces were thenmixed with 200U/mL ofultrapure collagenase III in DMEM/F12 medium. The tumorsuspension was incubated at 37�C for approximately 1 hour,with mechanical disruption every 30 minutes by pipetting with a5-mL pipette. At the end of the incubation, cells were filteredthrough a 70-mm nylon mesh and centrifuged at 1,200 rpm for 5minutes. Cells were washed twice with HBSS. Following the lastwash, cells were put through a 40-mm cell strainer and countedusing a Vi-Cell XR Cell Viability Analyzer. Cells were assayed foraldehyde dehydrogenase activity as a measure of CSCs using theStemcell Technologies Aldefluor Kit following themanufacturer'sinstructions. The cells were run on an LSRII flow cytometer andanalyzed with FlowJo software. Statistical significance of differ-ences between groups was based on P values determined throughunpaired t tests.

The methods used to confirm the CSC phenotype of Aldefluorcells from NCI-N87 and MDA-MB-361 tumors can be found inSupplementary Data.

Rat toxicology studies with MEDI0641Male Sprague Dawley rats (12 per group) were administered a

single intravenous injection (day 1) of 1 or 2 mg/kg anti-5T4antibody conjugated to SG3249. Control rats (12/group) wereadministered a single intravenous injection of vehicle control onday 1. Animals were necropsied on days 8 and 29 (6 per timepoint/group) to evaluate acute and delayed effects, respectively.All main study animals were evaluated for clinical signs, changesin body weight, clinical pathology, gross pathology with organweights, and microscopic observations. In addition, groups oftoxicokinetic satellite animals (12/group) were included in eachtreatment arm tomeasure plasma concentration of total antibodyand ADC. All toxicokinetic satellite animals were evaluated forclinical signs, changes in body weight, and pharmacokineticanalysis.

Hematology and serum chemistry samples were collected andanalyzed on days 8, 14, and 29. Blood samples for pharmacoki-netic analysis were collected in K2 EDTA tubes at multiple timepoints on days 1, 2, 3, 8, 15, 16, 22, and 29.

A gross necropsy was performed on all main study animalsand a standard list of organs, including brain, lung, liver,kidney, spleen, thymus, testes, heart, and bone, were embeddedin paraffin, sectioned, stained with hematoxylin and eosin, and

examined microscopically by a board-certified veterinarypathologist.

Additional Materials and Methods can be found in the Sup-plementary Data.

ResultsExpression of 5T4 in clinical tumor samples

Previous reports have indicated that 5T4 is overexpressed in avariety of human carcinomas (6–13). To confirm these findingsand to focus on cancer types with the highest incidence of 5T4expression, extensive IHC of tumor microarrays and tumorsamples as well as normal tissue arrays was conducted. 5T4was broadly expressed in tumor microarrays and clinical tumorsamples representing a number of cancer types, with the highestincidence in gastric cancer, non–small cell lung cancer (NSCLC),and head and neck squamous cell carcinoma (HNSCC; Fig. 1A).At least 90% of tumors analyzed in each of these cancers haddetectable levels of 5T4 expression by IHC, and in NSCLCsamples, the prevalence of 5T4 was slightly higher in squamousNSCLC compared with adenocarcinoma. Greater than 60% ofbreast cancer specimens, including triple-negative samples, were5T4 positive. Clear membrane localization of 5T4 immunos-taining was prevalent in various types of cancers (Fig. 1B).Although gastric cancers had the highest incidence of staining,only about 20% of samples could be classified as high/positivewith a pathology score of 2þ or 3þ observed in greater than50% of the tumor cells. In contrast, roughly 70% of all NSCLCand HNSCC samples exhibited high/positive staining.

Similar to previous findings (6, 9), 5T4 expression in normaltissues was extremely limited with minimal expression observedin epithelial cells of the renal tubules, gastric glands, skin, adrenalgland, urinary bladder, gall bladder, uterus, and cervix. Collec-tively, these data confirmed 5T4 would be a good tumor-associ-ated antigen for a targeted approach such as anADCand identifiedpotential indications to target in the clinic due to prevalentexpression of 5T4.

Generation and in vitro characterization of 5T4_0108 antibodyA 5T4-specific antibody, A07, was isolated from transgenic

mice expressing human antibody variable genes immunizedwith recombinant human 5T4. The KD of this antibody wasmeasured to be approximately 100 nmol/L as determined viaBiacore analysis; therefore, in vitro affinity optimization usingphage display was conducted and led to the generation of5T4_0108. 5T4_0108 is a human IgG1 with the followingmutations engineered into the heavy chain CH2 domain basedon EU numbering (35): S239C to introduce a free cysteine forsite-specific conjugation of payloads, and L234F to reduceengagement of FcgR and activation of immune effector cellsin conjunction with conjugation of payload at S239C (34). The5T4_0108 antibody has a KD of 6.5 nmol/L for 5T4 with on-and off-rates of 3.3 � 105 (1/Ms) and 2.1 � 103 (1/s) respec-tively. It binds to and is internalized by 5T4-expressing cells(Fig. 2A; Supplementary Fig. S1), and conjugation of a payloadat the S239C site did not affect binding or the internalizationkinetics (Supplementary Fig. S1).

The improved affinity translated into superior efficacy.5T4-Tub,which consists of 5T4_0108 conjugatedwith a tubulysinpayload, was 14-fold more potent than an ADC consisting of asite-specific variant of the parental antibody (ssA07) conjugated

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with the same payload (ssA07-Tub; Supplementary Fig. S2;Supplementary Table S1).

In vitro efficacy of anti-5T4 ADCs conjugatedwith tubulysin andPBD payloads

As described above, the initial studies to evaluate a candidateanti-5T4 ADC were conducted with 5T4-Tub. However, the activ-ity of cytotoxics can be context dependent, where certain classes ofdrugs are more effective than others depending on the type ofcancer. Therefore, studies were conducted to determine the rela-tive activity of 5T4-Tub and 5T4-PBD, an ADC composed of5T4_0108 conjugated with SG3249, a DNA cross-linking PBDpayload (27).

Comprehensive in vitro cytotoxicity assays were then con-ducted as an initial screen to compare relative activities of5T4-PBD and 5T4-Tub. To determine whether 5T4 expressionlevels impact ADC activity, these ADCs were tested againstMDA-MB-361 breast cancer cells representing high 5T4 expres-sion (�65,000 5T4 molecules/cell), DU 145 prostate cancercells representing moderate 5T4 expression (�30,000 5T4molecules/cell) and NCI-N87 gastric carcinoma cells represent-

ing low 5T4 expression levels (�4,000 5T4 molecules per cell;Supplementary Table S2).

Although the relative activities of 5T4-PBD and 5T4-Tub werecomparable in each cell line, there was a clear correlation betweenthe level of 5T4 expression and in vitro potency for both 5T4-ADCsin general (Fig. 2). 5T4-PBD and 5T4-Tub were most potentagainst 5T4-high MDA-MB-361 cells generating IC50 values inthe pg/mL range based on antibody concentration (Table 1). Inaddition, a greater maximum cytotoxicity was achieved at dosesthat are logs lower than what was needed in the other two celllines. The 5T4-ADCs induced potent cytotoxicity (IC50 values inthe single-digit ng/mL range) of DU 145 cells with moderate5T4 expression and had theweakest activity againstNCI-N87 cellswith very low levels of 5T4 in vitro.

Both the PBD and tubulysin warheads should elicit apopto-sis, and this was confirmed using companion in vitro assays. Inone assay, the percent viability was assessed, and in a secondassay, cleaved caspase activity was used as an indicator ofapoptosis (36). Following treatment with 5T4-PBD or 5T4-Tub(Supplementary Fig. S3), decreased viability is directly corre-lated with increased caspase-3/7 activity in various cell lines,

Figure 1.

Relative prevalence of 5T4 expression in clinical tumorsamples. A, IHC for 5T4 expression was conducted todetermine the prevalence of 5T4 expression in tumorsamples from patients with various cancers. Thevalues represent the percentage of tumor samplesthat have detectable levels of 5T4 immunostaining.TNBC, triple-negative breast cancer; NSCLC, non-small cell lung cancer. The number in parenthesesrepresents the number of tumor samples tested foreach cancer type. B, Representative images of 5T4immunostaining in various cancer types.






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confirming that both 5T4-PBD and 5T4-Tub induce an apo-ptotic cell death pathway.

In vivo antitumor activity of 5T4-PBD and 5T4-TubCollectively, the in vitro data suggest that both 5T4-PBD and

5T4-Tubwere able to induce potent cytotoxicity via apoptosis in atarget-dependent manner. To confirm that the in vitro activity of5T4-PBDand5T4-Tub could translate topotent antitumor activityin vivo, these ADCs were tested against tumors with varying levelsof 5T4 expression as determined by IHC: NCI-N87 xenograftswith low/moderate in vivo 5T4 expression, DU 145 xenograftswith moderate levels of 5T4 expression and MDA-MB-361 xeno-grafts with high 5T4 levels.

In theNCI-N87model with low/moderate 5T4 levels, 5T4-PBDwas able to achieve durable regression with a single administra-tion of a 1 mg/kg dose, whereas 5T4-Tub achieved a period ofregression with a 5mg/kg dose administered once a week for fourweeks, but then the tumors began to regrow shortly after cessationof treatment (Fig. 3A). Initially, tumor regression was observed inthe DU 145model following five doses administered once a weekof either a 1mg/kg dose of 5T4-PBD or a 5mg/kg dose of 5T4-Tub(Fig. 3B). However, the 5T4-PBD–treated tumors remainedregressed for approximately 70 days following cessation of ther-apy, whereas tumor growth in the 5T4-Tub–treated group appearsto resume even before completion of the full course of therapy. Infact, the duration of the antitumor response of the 0.3mg/kg doseof 5T4-PBD was superior to the 5 mg/kg dose of 5T4-Tub. The 1mg/kg dose of 5T4-Tub only resulted in slight tumor growthinhibition. In the MDA-MB-361 xenograft model expressing highlevels of 5T4, both 5T4-Tub and 5T4-PBD achieved durableregressions, although a lower dose and less frequent dosing of5T4-PBD was required to achieve such antitumor activity (Fig.3C). Statistical significance (P < 0.05 based on two-way ANOVA)was demonstrated for the 5T4-ADCs compared with controls. Itshould also be noted that no overt signs of toxicity, such assignificant body weight loss, were observed with either ADC atthe doses administered in these studies.

Anti-CSC activity of 5T4-PBDTo determine whether the enhanced in vivo efficacy with the

PBD-conjugated 5T4-PBD compared with the tubulysin-conju-

gated 5T4-Tub could be due to differential effects of theirwarheads on the CSC populations, studies were carried out todetermine 5T4 expression on CSCs in cultured tumor cell linesand the effects of these ADCs and their respective warheads onCSCs in vitro and in vivo. Cell lines of various cancer types weretested for 5T4 expression on both total tumor cells as well asCSCs. CSCs were defined as Aldefluorþ or CD44þCD24� cellsin breast cancer (37, 38), CD44þCD24� cells in prostate cancer(39), CD90þ cells in hepatocellular carcinoma (40), Aldefluorþ

cells in gastric cancer (41), and CD24þCD44þ cells in pancre-atic cancer (42). In all cases, 5T4 expression on the CSCpopulation was equal to or higher than that on non-CSCs(Fig. 4A), even when the cell line had little to no 5T4 expressionas a whole (Supplementary Fig. S4).

We first determined the effects of the various warheads onCSCs in vitro. CSC-enriched spheroids derived from several ofthese cell lines were treated with PBD, tubulysin, or auristatinwarheads to determine whether these warheads could inhibitthe growth of CSCs in vitro. The auristatin MMAE was includedas an auristatin-conjugated 5T4-ADC has been reported tohave activity against CSCs (23). In our studies, only the PBDdimer warhead potently inhibited CSC sphere formation in alllines tested with an IC50 range of 0.18 to 0.78 nmol/L, whereasthe tubulysin or auristatin warheads had little to no effect(Supplementary Fig. S5). These data suggest that the DNAcross-linking PBD dimer warhead could potently and specif-ically inhibit CSC proliferation, whereas tubulin inhibitorsmay not.

Next, we addressed whether 5T4-ADCs conjugated withvarious payloads had differential effects on CSCs in vivo fromMDA-MB-361 breast carcinoma xenografts and NCI-N87gastric carcinoma xenografts. We first confirmed the CSC phe-notype in each of the cell lines by determining tumorigenicpotential of each of the putative CSC populations by perform-ing limiting dilution assays. In this manner, we confirmed thatAldefluorþ cells represented the tumorigenic population inboth the MDA-MB-361 and NCI-N87 cell lines (SupplementaryTable S3), and effects of these ADCs on in vivo CSCs wereevaluated. Mice were inoculated with either MDA-MB-361 orNCI-N87 cells, and when the average tumor volume reachedapproximately 500 mm3, tumor-bearing mice were treated with

Table 1. Relative activity of 5T4-ADCs against cell lines with varying levels of cell surface 5T4 expression

MDA-MB-361 (�65K 5T4/cell) DU 145 (�30K 5T4/cell) NCI-N87 (�4K 5T4/cell)ADC IC50 (ng/mL) Max kill (%) IC50 (ng/mL) Max kill (%) IC50 (ng/mL) Max kill (%)

5T4-Tub 0.85 78.8 2.67 80.5 47.3 77.05T4-PBD 0.39 90.7 2.47 77.0 111.1 91.1

NOTE: The table represents average IC50 values and the maximum cytotoxicity (max kill percentages) from multiple assays run for each ADC against eachcell line.

Figure 2.In vitro characterization of 5T4_0108 and ADCs comprised of this antibody. A, Target-specific binding of 5T4_0108 to human breast cancer (MDA-MB-361),prostate cancer (DU 145), and gastric cancer (NCI-N87) cell lines that express 5T4 or to 5T4-negative DMS 114 lung carcinoma cell lines wasdetermined by flow cytometry. B, Structures of 5T4-PBD consisting of the 5T4_0108 antibody site-specifically conjugated to the PBD payload SG3249,and 5T4-Tub consisting of the 5T4_0108 antibody conjugated to the tubulysin payload mc-Lys-MMETA. C, The 5T4-PBD and 5T4-Tub ADCs weretested against tumor cells with different levels of 5T4 expression on the cell surface. A correlation between target expression and cytotoxicity wasobserved by testing the ADCs against MDA-MB-361 breast carcinoma cells with high levels of 5T4 cell surface expression, DU 145 prostate cancer cellswith moderate levels of 5T4 cell surface expression, and NCI-N87 gastric carcinoma cells with low levels of 5T4 cell surface expression. Representativeexperiments are shown, and the values indicate the mean � SEM. Control ADCs consisting of the site-specific variant of the R347 IgG1 isotype controlantibody conjugated to either PBD (Ctrl-PBD) or tubulysin (Ctrl-Tub) were also included to determine target-specific binding.






a single dose of 5T4-PBD, 5T4-Tub, or 5T4-mcMMAF (an anti-5T4 antibody site-specifically conjugated with the auristatinmcMMAF with a DAR of 2). Once tumors began to regress (�5–7 days in this model), the tumors were harvested and the CSCpopulations were quantified. The PBD-conjugated 5T4-PBDsignificantly reduced the number of CSCs by approximately50% in both models and was more effective than either thetubulysin-conjugated 5T4-Tub or the auristatin-conjugated5T4-mcMMAF (Fig. 4B).

The data suggest that the superior antitumor activity of5T4-PBD could be due to the ability of this ADC to target5T4þ CSCs in addition to the 5T4þ bulk tumor cells, whereasADCs with tubulin inhibitors, such as 5T4-Tub, are ineffectiveat inhibiting CSCs. As 5T4 is a target that is expressed on bothCSCs as well as the non-CSCs within a tumor, it is criticalto develop a 5T4-ADC conjugated with a payload capableof effectively targeting CSCs to produce the most durableresponse.

Figure 3.

5T4-PBD produces more durableresponses in xenograft tumor modelswith varying levels 5T4 expression.5T4-PBD and 5T4-Tub were tested inathymic nudemice bearing subcutaneousNCI-N87 (A), DU 145 (B), or MDA-MB-361(C) xenografts. Following tumor cellinoculation, mice were size-matched intotreatment groups, and therapeuticadministration was initiated when theaverage tumor volume reachedapproximately 200 mm3. Theadministration of therapeutics in thestudy is indicated by the arrowheads, andthe dotted lines represent average tumorvolume at the time of treatment initiation;tumor volumes below this line areindicative of tumor regression. Values,mean � SEM. Representativeexperiments are shown and demonstratethat while 5T4-PBD and 5T4-Tub are ableto achieve tumor regressions in theMDA-MB-361 model with high 5T4expression, 5T4-PBD produces moredurable in vivo tumor regression than5T4-Tub in the NCI-N87 and DU 145models. Statistical significance of5T4-ADC treatment groups (P < 0.05)was demonstrated via two-way ANOVAanalysis.

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Tolerability of MEDI0641 in ratsGiven the greater in vivo activity and anti-CSC activity

observed with 5T4-PBD, this ADC was selected for furtherdevelopment and was designated as MEDI0641. The safetyprofile of the ADC was subsequently evaluated in a non-GLPrat toxicology study. Although the 5T4 antibody does notcrossreact with rodent 5T4, the rat was chosen as the initialtoxicity species to evaluate the off-target toxicity profile andassess the in vivo stability of the molecule. Male rats wereadministered a single intravenous dose of 1 or 2 mg/kg ofMEDI0641 on day 1 and necropsied on days 8 and 29 toevaluate acute and delayed toxicities. All animals surviveduntil scheduled necropsy. Major findings were dose-dependentdecrease in body weight gain (up to 10%) and dose-dependentbone marrow suppression, with major effects on reticulocytes,platelets, and white blood cells (Fig. 5). All findings werereversible or trending toward reversible by day 29 and are

consistent with the known off-target effects of PBD-conjugatedADCs in rats (43).

DiscussionMEDI0641 is a 5T4-targeting ADC conjugated with a PBD

payload that is capable of inducing significant antitumor activ-ity, including durable regressions in tumor models with varyingintensity and heterogeneity of 5T4 expression. Although5T4-Tub also demonstrated significant in vivo antitumor activ-ity, MEDI0641 produced a more durable response possiblydue to the fact that it had a more pronounced effect on CSCsthan 5T4-Tub. We and others have shown that 5T4 is expressedon CSCs that are hypothesized to be responsible for chemo-therapeutic resistance and recurrence of cancer (21, 22). Usingmultiple HNSCC patient-derived xenograft (PDX) models,Kerk and colleagues report that MEDI0641 was able to reduce,

Figure 4.

5T4-PBD effectively targets CSCs in vivo.A, 5T4 is expressed on CSC populationsof various cancer cell lines. CSC markersare as follows: MDA-MB-361 breastcancer: Aldefluorþ; NCI-N87 gastriccancer: Aldefluorþ; DU 145 prostatecancer: CD44þCD24�. B, ADC-treatedtumors were harvested and theCD44þCD24� breast cancer CSCs andAldefluorþ gastric carcinoma CSCs werecounted. The number of CSCs found inthe vehicle-treated control tumors wasnormalized to 100%, and the data forother treatment groups are reported as apercent of the CSC population found incontrol tumors. 5T4-PBD decreased theCSC population by approximately 50% inboth models. 5T4-Tub had no effect onthe MDA-MB-361 CSCs but had someeffect on NCI-N87 CSCs, although thiseffect was not as potent as that with5T4-PBD. Neither 5T4-mcMMAF norcontrols had any effect on CSCs in eithermodel. The 5T4-PBD–mediateddecrease in CSCs was statisticallysignificant compared with all groupsin both models except the 5T4-Tub–treated group in theNCI-N87model(P < 0.05 based on unpaired t tests).






and in one model ablate, CSCs in these models, corroboratingand extending the anti-CSC properties of MEDI0641 that wedescribe here. Like us, they also observed that 5T4 expressionwas increased in the CSC population of these PDX tumorscompared with 5T4 expression in non-CSC tumor cells. Here,we extend these findings into additional tumor types withsignificant prevalence and intensity of 5T4 expression in clinicalsamples, including breast cancer, gastric cancer, pancreaticcancer, and prostate cancer. Collectively, the strong expressionand/or upregulation of 5T4 on CSCs suggest that perhaps 5T4may have some functional relevance to CSC biology. Furtherstudies are warranted to determine such a role for 5T4 in CSCs.

It has been hypothesized that tubulin inhibitors, such as thetubulysins, auristatins, and maytansinoids, are most effectiveagainst tumor cells with high proliferative rates and are lesseffective against relatively quiescent cells, such as CSCs(23, 44). DNA-targeting agents on the other hand, particularlythose that form cross-links or otherDNA lesions that are not easilyrepaired, could more effectively target CSCs. The data presentedhere support this hypothesis. 5T4-mcMMAF conjugated with an

auristatin payload had no effect on CSC populations in vivo ineither xenograft model, and 5T4-Tub was either ineffective or lesseffective against CSCs compared with MEDI0641 in theMDA-MB-361 and NCI-N87 xenograft models, respectively.Previously, an anti-5T4 ADC conjugated with the auristatin pay-load mcMMAF (A1-mcMMAF; PF-06263507) was reported tohave potent antitumor activity in vivo and also was reported toinhibit CSC-like tumor-initiating cells in a NSCLC PDX model(23). There could be several explanations for the conflicting datawith the MMAF-conjugated ADCs: (i) The 5T4-mcMMAF ADCused in the current study was a site-specific conjugate with a DARof 2, as opposed to the classically conjugated A1-mcMMAF thathas a DAR of approximately 4; (ii) differences in dosing schedule(single administration here, every four days for four total dosesreported); (iii) different CSCþ tumormodels were used in each setof studies and each could respond to auristatins differently; and(iv) different methodologies were used to assess anti-CSC activityin vivo in each report. Testing our 5T4-ADCs in models and assayssimilar to those reported where A1-mcMMAF demonstrated anti-CSC activity, and vice versa, could help resolve the contrary

Figure 5.

Safety profile of MEDI0641 in rats. Rats (n¼ 12) were treated with a singleintravenous injection of 1 or 2 mg/kgMEDI0641. Control animals were treatedwith a single intravenous injection ofvehicle control. A, Tolerability wasassessed by measuring body weightchanges over a 29-day period. Datapoints represent the mean value of eachgroup at individual time points; errorbars, mean � SEM. Tx, treatment. B–D,MEDI0641 is associated with dose-dependent bone marrow suppression.Hematologic analyses were conductedon peripheral blood samples collected ondays 8, 14, and 29 posttreatment toenumerate platelet counts (B),reticulocytes (C), and white blood cells(D). Bars represent the mean value ofeach group; error bars, mean � SEM.

Harper et al.





observations. Regardless, the current data clearly demonstrate thatthe PBD-conjugated MEDI0641 had superior anti-CSC effectsover either tubulysin- or auristatin-conjugated ADCs.

The different anti-CSC activities observed between MEDI0641and 5T4-Tub could potentially account for the different antitu-mor responses elicited by these ADCs. MEDI0641 significantlydecreased the CSC population in both the MDA-MB-361 andthe NCI-N87 models, and in each model, durable regressionswere observed following MEDI0641 treatment. In the NCI-N87model, even though higher doses and frequency were utilized,5T4-Tub had inferior anti-CSC activity and less durable antitu-mor activity compared with MEDI0641. Interestingly, each ADCwas capable of eliciting regressions in the MDA-MB-361 modeleven though only MEDI0641 was able to inhibit the in vivoCSC population. This could be attributed to the fact that 5T4expression was significantly higher in both bulk tumor cells andCSCs in the MDA-MB-361 model compared with NCI-N87,suggesting that, hypothetically, more of each ADC would bedelivered to MDA-MB-361 tumors compared with NCI-N87tumors. It could also be due to dosing: a single administrationin the CSC experiment, while 5T4-Tub was administered once aweek for a total of four doses in the efficacy study. Takentogether, these data suggest that greater success of a 5T4-ADCin tumors with lower levels of 5T4 expression may be dependenton the ability of that ADC to deliver a payload that effectivelytargets the 5T4-positive CSC population.

Evaluating ADCs that target the same surface antigen butdeliver warheads with different mechanisms of action helped topotentially identify which ADC could produce a more durableresponse. To our knowledge, this is the first report where a head-to-head comparison was made between two ADCs comprised ofthe same antibody but conjugated with payloads from differentdrug classes with different mechanisms of action: PBDs thatform interstrand DNA cross-links and tubulysins that depoly-merize microtubules. Others have reported comparing cleav-able versus noncleavable drug linkers, but typically, thesecomparisons have included the same warhead class; forexample, comparing conjugates with either cleavablemc-VC-PABC-MMAE or noncleavable mc-MMAF (45, 46), orcleavable SPDB-DM4 versus noncleavable SMCC-DM1(47, 48). There have also been reports of comparing conjugateswith auristatins and maytansinoids (49), but these are bothinherently two classes of drugs with a similar mechanism ofaction, namely microtubule inhibition.

Prior to developing A1-mcMMAF, the same research grouphad evaluated a calicheamicin-conjugated 5T4-ADC, however,composed of a different antibody against 5T4. They found thatthe ADC bearing the calicheamicin payload was not tolerated atefficacious exposure levels in cynomolgus monkeys (23, 50).Although also a DNA-targeting agent, the mechanism of actionof calicheamicin is different from that of PBDs, and the driversof the toxicity with the 5T4-calicheamicin were not reported, soit is not clear whether they could be attributed to the differentantibody and/or the different payload. Non-GLP rat toxicologystudies with MEDI0641 demonstrated a reasonable safety pro-file. Regardless, close attention to the toxicologic findings with

MEDI0641, particularly in a species where our anti-5T4 anti-body cross-reacts, is warranted as safety studies with this ADCproceed.

In conclusion, our generation and testing of the PBD-con-jugated MEDI0641 and 5T4-Tub permitted a direct comparisonof payloads with different mechanisms of action conjugated tothe same antibody against 5T4. Our results demonstratedMEDI0641 had overall superior activity and, in particular, ismore effective at targeting 5T4-positive CSCs, which may pre-vent or reduce tumor recurrence. These data provide motivationfor further development of this promising ADC.

Disclosure of Potential Conflicts of InterestF. D'Hooge is the head of the Bioconjugation Unit at Novasep.

L. van Vlerken-Ysla is a scientist at MedImmune. R.E. Hollingsworth is thesenior director (Oncology) at MedImmune. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: J. Harper, C. Lloyd, D. Toader, R. Fleming, C. Chen,H. Zhong, E.M. Hurt, P.W. Howard, R.E. Hollingsworth, A. KamalDevelopment of methodology: J. Harper, C. Lloyd, R. Marwood, L. Lewis,D. Bannister, J. Jovanovic, R. Fleming, S. Mao, L. Xu, M. Rebelatto, E.M. Hurt,P.W. Howard, R.E. HollingsworthAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Harper, C. Lloyd, N. Dimasi, R. Marwood, L. Lewis,J. Jovanovic, R. Fleming, F. D'Hooge, S. Mao, A.M. Marrero, M. Korade III,C. Chen, L. Wetzel, S. Breen, L. van Vlerken-Ysla, M. Rebelatto, E.M. HurtAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Harper, C. Lloyd, N. Dimasi, R. Marwood,L. Lewis, J. Jovanovic, R. Fleming, F. D'Hooge, S. Mao, P. Strout, C. Chen,S. Breen, L. van Vlerken-Ysla, S. Jalla, M. Rebelatto, H. Zhong, E.M. Hurt,D.A. Tice, R.E. HollingsworthWriting, review, and/or revision of the manuscript: J. Harper, C. Lloyd,N. Dimasi, D. Toader, R. Marwood, L. Lewis, J. Jovanovic, R. Fleming, S. Mao,A.M. Marrero, M. Korade III, P. Strout, C. Chen, L. Wetzel, M. Rebelatto,H. Zhong, E.M.Hurt,M.J. Hinrichs, P.W.Howard, D.A. Tice, R. Herbst, A. KamalAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Harper, N. Dimasi, R. Marwood, L. Lewis,K. HuangStudy supervision: D. Toader, H. Zhong, E.M. Hurt, R.E. Hollingsworth,A. KamalOther (made the 5T4 protein to enable antibody generation and helpedwith biophysical analysis of the ADC candidates): D. BannisterOther (designing and performing the experiments): S. JallaOther (design and synthesis and supply of drug-linker portion of antibody–drug conjugate): P.W. Howard

AcknowledgmentsThe authors wish to thank Jean-Noel Levy for his work on synthesizing the

PBD payloads used during the course of these studies. In addition, we thank theBiological Expression Team at MedImmune, Ltd. (Cambridge, United King-dom) for their work in synthesizing the antibodies for use in these studies.

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

ReceivedDecember 1, 2016; revisedMarch 28, 2017; accepted April 28, 2017;published OnlineFirst May 18, 2017.

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2017;16:1576-1587. Published OnlineFirst May 18, 2017.Mol Cancer Ther Jay Harper, Christopher Lloyd, Nazzareno Dimasi, et al. Targeting 5T4

Drug Conjugate−Pyrrolobenzodiazepine-Conjugated Antibody Preclinical Evaluation of MEDI0641, a

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