Large Molecule Therapeutics

MI130004, a Novel Antibody–Drug ConjugateCombiningTrastuzumabwith aMoleculeofMarineOrigin, ShowsOutstanding InVivoActivity againstHER2-Expressing TumorsPablo Avil�es, Juan Manuel Domínguez, María Jos�e Guill�en, María Jos�e Mu~noz-Alonso,Cristina Mateo, Raquel Rodriguez-Acebes, Jos�e Manuel Molina-Guijarro,Andr�es Francesch, Juan Fernando Martínez-Leal, Simon Munt,Carlos M. Galmarini, and Carmen Cuevas

Abstract

In the search for novel payloads to design new antibody–drugconjugates (ADC), marine compounds represent an interestingopportunity given their unique chemical features. PM050489 is amarine compound that binds b-tubulin at a new site and disruptsthe microtubule network, hence leading to mitotic aberrationsand cell death. PM050489 has been conjugated to trastuzumabvia Cys residues through a noncleavable linker, and the resultingADC, namedMI130004, has been studied. Analysis of MI130004delivered data consistent with the presence of two molecules ofPM050489 per antibody molecule, likely bound to both sides ofthe intermolecular disulfide bond connecting the antibody lightand heavy chains. The antitumor activity of MI130004 wasanalyzed in vitro and in vivo in different cell lines of diverse tumororigin (breast, ovary, and gastric cancer) expressing different levels

of HER2. MI130004 showed very high in vitro potency andgood selectivity for tumor cells that overexpressed HER2. At thecellular level, MI130004 impaired tubulin polymerization,causing disorganization and disintegration of the microtubulenetwork, which ultimately led to mitotic failure, mirroring theeffect of its payload. Treatment with MI130004 in mice carryinghistologically diverse tumors expressing HER2 induced a long-lasting antitumor effect with statistically significant inhibitionof tumor growth coupled with increases in median survivaltime compared with vehicle or trastuzumab. These resultsstrongly suggest that MI130004 is endowed with remarkableanticancer activity and confirm the extraordinary potential ofmarine compounds for the design of new ADCs. Mol Cancer Ther;17(4); 786–94. �2018 AACR.

IntroductionAntibody–drug conjugates (ADC) are a relatively novel class of

therapeutic agents that have drawn the attention of the pharma-ceutical industry in the past decade. Pursuing the "magic bullet"concept coined by Paul Ehrlich in 1917 (1), it was not until 2000that the first ADC (Mylotarg, gemtuzumab ozogamicin) wasapproved by the FDA for treating acute myeloid leukemia.Although Mylotarg was withdrawn from market in 2010, twonew ADCs were launched soon thereafter: Adcetris (brentuximabvedotin) in 2011, for the treatment ofHodgkin lymphoma aswellas systemic anaplastic large cell lymphoma (2), and Kadcyla(trastuzumab emtansine) in 2013 for treating HER2-positivemetastatic breast cancer (3). These successful cases have boosted

interest in the discovery and development of new ADCs. Inter-estingly, the vast majority of ADCs in clinical development or inearly preclinical studies include cytotoxic payloads belonging tothe same chemical series that those of Mylotarg, Adcetris, andKadcyla, that is, calicheamicins, auristatins, or maytansinoids.Although new scaffolds acting through different mechanisms ofaction are also being explored, with the notable examples ofpyrrolobenzodiazepines and camptothecin analogues, they onlyaccount for a minority of cases (4, 5). Consequently, there is aneed for novel molecules to be used as payloads to increase thediversity of ADCs and thus to overcome possible resistance (6).

The wide multiplicity of life forms harbored by the sea and thedifferent physical and chemical environment that marine organ-ismsmust face account for themuch richer chemical diversity thatcan be found in the oceans as compared with that on earth, butsuch diversity still remains largely unexplored. Therefore, marinemolecules constitute an attractive source of inspiration in thesearch for novel chemical scaffolds and successful cases of marinecompounds with notable therapeutic potential are frequentlyreported. PM050489 is a good example of such success with itsdechlorinated analogue PM060184 currently undergoing phase IIclinical trials for the treatment of advanced, hormone receptor–positive, HER2-negative breast cancer (EU clinical trials databaseEudraCT Number: 2015-002395-24). The molecule, originallyisolated from the sponge Lithoplocamia lithistoides (7), is an

Research Department, PharmaMar S.A., Colmenar Viejo, Madrid, Spain.

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

P. Avil�es and J.M. Domínguez contributed equally to this article.

Corresponding Author: Juan Manuel Domínguez, PharmaMar S.A., Avenida delosReyes 1, ColmenarViejo 28770,Madrid, Spain. Phone: 34 918466000; Fax: 34918 466 001; E-mail: jmdominguez@pharmamar.com

doi: 10.1158/1535-7163.MCT-17-0795

�2018 American Association for Cancer Research.

MolecularCancerTherapeutics

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extremely potent interfacial microtubule inhibitor that binds tob-tubulin with single-digit nanomolar affinity at a new site (8, 9).On the basis of the interaction with tubulin dimers, these mole-cules inhibit microtubule assembly through a novel mechanismof action suppressing microtubule shortening and growing at asimilar extent (10) and leading cells to death with subnanomolarin vitro antiproliferative activity against cancer cell lines (7). Suchextremely high potency, an imperative requisite for ADCpayloads(11), together with the novel mechanism of microtubule dynam-ics impairment, led us to consider the opportunity to usePM050489 as warhead for novel ADCs. Given the proven successof trastuzumab-based ADCs and the extensive validation for thisantibody in the field, we decided to conjugate PM050489 withtrastuzumab, and thus the resulting ADC, called MI130004, wasprepared. This article summarizes the characterization ofMI130004 and the results it has delivered both in vitro and in vivo.

Materials and MethodsReagents

PM050489, PM120160 (the result of adding a noncleavablelinker to PM050489, see Fig. 1A), and MI130004 were preparedin PharmaMar S.A. Trastuzumab used in this study is the Euro-pean Medicines Agency–approved version and formulation.Chromatography reagents and materials were from GE Health-care. Immunoglobulin-degrading enzyme of Streptococcus pyo-genes (IdeS) was purchased from Genovis. Mouse monoclonalanti–a-tubulin and rabbit anti–g-tubulin antibodies were fromSigma-Aldrich. Alexa Fluor–conjugated goat anti-mouse or anti-rabbit antibodies were from Thermo Fisher Scientific. All otherreagents were of the highest purity available. Throughoutthis work, trastuzumab concentration was determined spectro-photometrically by monitoring its absorbance at 280 nm usinga molar extinction coefficient of 2.18E5 M�1 cm�1 and a molec-ular weight of 150 kDa.

Preparation of MI130004Trastuzumab was initially reduced with Tris(2-carboxyethyl)

phosphine hydrochloride (TCEP). Briefly, a 70 mmol/L (10.5 mg/mL) solution of the antibody in 50 mmol/L sodium phosphatepH 8.0 buffer was mixed with the appropriate amount of a 5mmol/L solution of TCEP in water to keep the reducing agent in a2.5-fold molar excess over the antibody. The mixture was incu-bated and stirred for 60 minutes at 20�C, and afterward, theappropriate volume of a 10 mmol/L PM120160 solution inDMSO was added to reach a fair molar excess of the compoundover the antibody. DMSO was added if needed to keep itsconcentration at 5% (v/v), and the mixture was incubated for30 minutes at 20�C. Afterward, N-acetyl-cysteine was added toquench the reaction, using the appropriate volume of a 10mmol/L solution in water tomatch the concentration of PM120160. Theresulting ADC was finally purified from the rest of reagents by gelfiltration in SephadexG-25 using PD-10 columns. The presence ofaggregates was checked through analytic size exclusion chroma-tography using an €Akta FPLC system equipped with a Superdex-100 10/300 column running an isocratic method with PBS at 1mL/minute: If the area of the peak corresponding to aggregatesexceeded 10% of the total peak area, monomers were purifiedusing the same chromatography system with a Superdex 200 16/600 preparative column running the same method describedabove. Final ADC concentration was determined spectrophoto-

metrically by monitoring its absorbance at 280 nm using themolar extinction coefficient of trastuzumab; if the ADC concen-tration was below 2 mg/mL, it was concentrated using Vivaspindevices fromGEHealthcare, and the new concentrationwas againdetermined as above.

HIC analysis of MI130004Analysis of MI130004 by hydrophobic interaction chromatog-

raphy (HIC) was performed on a Agilent 1100 HPLC system(Agilent) equipped with a 3.5 � 4.6 mm TSK-gel butyl-NPRcolumn with 2.5 mm particle size (Tosoh Bioscience), using 1.5mol/L ammonium sulfate in 25 mmol/L sodium phosphate, pH7.5 as mobile phase A, and 25% (v/v) isopropanol in 25mmol/Lsodium phosphate pH 7.5 asmobile phase B. Elutionwas run at aconstant 0.8mL/minutes flow rate using a 0% to 100%B gradientin12minutes, followedby a5-minute isocratic elution in100%B.

Figure 1.

In vitro activity of MI130004 against tumor cell lines. A, Structure of MI130004,PM050489, and PM120160. B, Antiproliferative assay showing the in vitropotency of MI130004. The assay was performed as described in "Materials andMethods" with SK-BR-3 (HER2 positive, solid circles), HCC-1954 (HER2 positive,solid squares), MDA-MB-231 (HER2 negative, hollow circles), and MCF7 (HER2negative, hollow squares) cell lines. C, Fluorescence microscopy analysis of cellstreatedwith 1% (v/v) DMSO ("control") orwith 0.1 mg/mLMI130004 in 1%DMSO.Cells were stained for a-tubulin (green), g-tubulin (red), and nuclei (blue).MI130004 induced notable alterations in the tubulin cytoskeleton of HER2-positive cells: An altered mitotic phenotype with multiple mitotic poles as wellas an irregular formation of microtubules can be observed.

Evaluation and Characterization of the Novel ADC MI130004

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IdeS digestion and LC-MS analysis of MI130004A total of 100 mg of trastuzumab or MI130004 was incubated

with 100 U of enzyme in 100 mL of PBS at 37�C for 30 minutes.The sample was then diluted with 200 mL of 100 mmol/L Tris-HCl pH 7.5 with 6 mol/L guanidine-HCl, and 2 mmol/L EDTA,and afterward, 2 mL of 1 mol/L DL-dithiothreitol (DTT) wasadded and the mixture incubated at 56�C for 45 minutes.Reduction was stopped by adding 1 mL of glacial acetic acid,and samples were finally subjected to LC-MS analysis followingthe same procedure described by Wagner-Rousset and collea-gues (12).

Cell lines and cell cultureThe following cell lines were obtained from ATCC: MDA-

MB-231 (HTB-26) and BT-474 (HTB-20), breast adenocarci-noma; N87 (CRL-5822) and Hs 746T (HTB-135), gastricadenocarcinoma; and SK-OV-3 (HTB-77) ovarian adenocarci-noma. JIMT-1 breast adenocarcinoma cells (ACC589) wereobtained from DSMZ. A2780cis ovarian adenocarcinoma cells(93112517) were obtained from ECACC. Gastric-008 gastricadenocarcinoma cell line was generated and generously givenby Dr. Manuel Hidalgo (Beth Israel Deaconess Medical Center,Boston, MA). All cell lines were maintained in DMEM supple-mented with 10% FBS, 2 mmol/L L-glutamine, and 100 U/mLof penicillin/streptomycin at 37�C and 5% CO2. Cell lineswere classified as HER2 positive or negative according to IHCanalysis.

Cell viability assayA colorimetric assay based on the reduction of 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)was used for quantitative measurement of cell viability asdescribed elsewhere (13). Briefly, cells were seeded in 96-welltrays. Serial dilutions of all compounds dissolved in DMSOwere prepared and added to the cells in fresh medium, intriplicates. Exposure to the drug was maintained during 72hours, and cellular viability was estimated from conversion ofMTT (Sigma) to its colored reaction product, MTT-formazan,which was dissolved in DMSO so as to measure its absorbanceat 540 nm with a POLARStar Omega Reader (BMG Labtech).Determination of IC50 values was performed by iterative non-linear regression to a 4-parameter logistic equation using thePrism 5.0 statistical software (GraphPad). The data presentedhere correspond to the geometric mean of three independentexperiments performed in triplicate.

Immunofluorescence of microtubule cytoskeletonTumor cells were treated for 24 hours with 1% (v/v) DMSO

in the presence or absence of MI130004 at 0.1 mg/mL or of 1nmol/L PM050489, then fixed with methanol for 10 minutes at�20�C and incubated with a blocking solution (5% BSA inPBS) for 30 minutes. Cells were then incubated with primarymouse anti-human a-tubulin or primary rabbit anti-humang-tubulin antibodies for 1 hour at room temperature. Afterthree washes with a 1% (w/v) BSA in PBS solution, cells wereincubated with Alexa Fluor 488–conjugated goat anti-mouseIgG or Alexa Fluor 594-conjugated goat anti-rabbit IgG sec-ondary antibodies at room temperature for 1 hour. Cells werefinally counterstained with addition of Hoechst 33258 (1 mg/mL)for 5 minutes and mounted with Mowiol mounting medium.Pictures were takenwith a Leica DM IRM fluorescencemicroscope

equippedwith a 100� oil immersion objective and a DFC 340 FXdigital camera (Leica).

Flow cytometric cell-cycle determinationFor cell-cycle experiments, cells were treated for 24 hours

with 1% (v/v) DMSO in the presence or absence of MI130004at 0.1 mg/mL and then stained with 0.4 mg/mL propidium iodide.Samples were analyzed with a BD Accuri C6 flow cytometer(BecktonDickinson) and the FlowJo7 cytometry analysis software(FlowJo).

Xenograft murine modelsAll animal protocols were reviewed and approved according

to regional Institutional Animal Care and Use Committees.Design, randomization, andmonitoring of experiments (includ-ing body weights and tumor measurements) were performedusing NewLab Software v2.25.06.00 (NewLab Oncology).Female athymic Nude-Foxn-1 nu/nu or CB17/IcrHsd-PrKdc-SCID mice (Envigo, RMS Spain S.L.) between 4 to 6 weeks ofage were subcutaneously xenografted with each cell line (JIMT-1,BT-474, N87, gastric-008, A2780Cis, and SK-OV-3 as HER2positive; MDA-MB-231 and Hs 746T as HER2 negative) intotheir right flank with, depending on the model, either ca. 5-10E6cells suspended in 0.05 mL of a solution consisting of 50%Matrigel (Corning Inc.) and 50% cell culture medium, withoutserum or antibiotics, or with tissue from serial transplanteddonor mice. Tumors were removed from donor animals deb-rided of membrane, hemorrhagic and necrotic areas cut intofragments (3 mm3), placed in Matrigel, and subcutaneouslyimplanted. When tumors reached ca. 150 mm3, mice (n ¼ 8–10 animals/group) were randomly allocated (day 0) to receiveMI130004 (1, 5, or 10mg/kg), trastuzumab (30 or 10mg/kg), orvehicle. Intravenous treatments were weekly administered for 5consecutive weeks. The control animals received an equal vol-ume of vehicle with the same schedule. Calipermeasurements ofthe tumor diameters were made three times a week, and tumorvolumes were calculated according to the following formula:volume¼ (a� b2)/2, where a and bwere the longest and shortesttumor diameters, respectively. For survival evaluation, time toendpoint was defined as the time from day 0 to death as a resultof tumor growth (larger than 2,000 mm3) or any other cause (e.g., tumor necrosis). Complete tumor regression (CR) wasdefined as tumor volume below 63 mm3 for 2 or more conse-cutive measurements, such value corresponding to the lowestmeasurable limit considering the contribution of the mass fromfibrous material, scar tissue, etc. Statistical differences, in animalsurvival, were assessed by Kaplan–Meier curves with the log-ranktest. Animals were humanely sacrificed when their tumorsreached 2,500 mm3 or if significant toxicity (e.g., severe bodyweight reduction) was observed. Differences in tumor volumesbetween treated and control group were evaluated using theMann–Whitney U test. Statistical analyses were performed byGraphPad Prism v5.03 (GraphPad Software Inc.).

IHC and immunofluorescence analysis in biopsies from animalexperiments

Tumors (or skin around the former tumor site) were dissected,formalin fixed, and paraffin embedded for histopathology eva-luations. Mitotic aberrations were revealed by nuclear stainingwith Hoechst 33258 after 24 hours of the first administration ofMI130004.

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ResultsMolecular characterization of MI130004

MI130004 is an ADC resulting from the conjugation ofPM050489 to Cys residues of trastuzumab via amaleimide-basednoncleavable linker (structure in Fig. 1A). According to sizeexclusion chromatography analysis, the ADC preparation wascomposed nearly exclusively (99.8% purity) of singlemonomericspecies (Supplementary Fig. S1A). HIC of MI130004 revealed theexistence of onemajor peak, eluting at 6.23minutes, correspond-ing to the conjugated species and accounting for 75% of the totalpeak area, together with the peak corresponding to the nakedantibody (16%) and another minor peak (7%) of higher hydro-phobicity, eluting at 8.06minutes, likely corresponding tominor-ity species with a higher drug load (Supplementary Fig. S1B).

To determine the number and position of the drug in the ADCmolecule, limited proteolysis and reduction of both ADC andantibody were performed, followed by LC-MS analysis of theresulting fragments. Molecular mass assignments to these frag-ments were done using the values reported in ref. 12 as we areusing the same source of trastuzumab; therefore, a similar glyco-sylation pattern may be expected. The UV chromatograms of thedigestion products of trastuzumab and MI130004 showed threecommonpeaks, eluting at circa 12.7, 15.2, and 19.0minuteswhoseMS analysis allowed the assignment to the Fc/2, LC, and Fdfragments of the antibody, respectively (Supplementary Figs.S1C–S1D and S2A–S2C) with different degrees of glycosylationcausing the multiplicity of masses identified in the first peak. OnlytheMI130004 chromatogramshowed twoadditionalpeaks elutingat longer times: 20.82 and 23.88minutes.MSdeconvolutionof thepeak at 20.82 minutes (Supplementary Fig. S3A) allowed theidentification of four species, the major one with a mass of24,303 Da, consistent with that of the LC fragment (23,439 Da)conjugated to one drug molecule (856 Da). Similarly, MS decon-volution of the peak at 23.88 minutes (Supplementary Fig. S3B)yielded two species, the major one corresponding to a mass of26,244 Da, consistent with that of the Fd fragment (25,384 Da)conjugated to one payload (856Da). Likewise, analysis of the peakareas in these chromatograms suggests a DAR of 1.04, whereas theresults of the HIC chromatography (Supplementary Fig. S1B)suggest aDAR of 1.8. These data, together with the aforementionedmass assignments (summarized in Supplementary Table S1), sug-gest that, in average, one molecule of MI130004 contains 1 to 1.8molecules of PM050489 conjugated to cysteines involved in one ofthe disulfide bonds connecting the light and the heavy chains.

Antiproliferative activity of MI130004The in vitro antiproliferative activity of MI130004 was deter-

mined using a panel of tumor cell lines of different histotypes(breast, gastric, and ovary) expressing or not HER2 (Table 1).MI130004 showed excellent in vitro potency with selectivity forHER2-positive tumor cells (Table 1; Fig. 1B; SupplementaryFig. S4). The HER2-positive panel IC50 geometric mean valuewas 0.44 mg/mL, whereas it was >10 mg/mL for the HER2-negativetumor cells. Of note, the potency ofMI130004 in the parental andP-gp overexpressing versions of the A2780 cell line are virtuallyidentical (2.00 vs. 3.44 mg/mL), consistent with the resultsobtained with PM050489 and its derivatives and confirming thepotential of these series to overcome resistance mechanismsrelated to drug efflux. On the other hand, PM050489 caused aclear antiproliferative effect in all the cell lines tested regardless oftheir HER2 expression (Supplementary Table S2). Trastuzumab

alone did not cause any effect on cell growth in any of these lineseven at the highest concentration tested (50 mg/mL).

Biological effects of MI130004 in vitroWe have previously described that, in tumor cell lines,

PM050489 impaired tubulin polymerization, causing mitoticaberrations with massive disorganization and disappearance ofthe microtubule mass and cell-cycle arrest in G2–M (8). Interest-ingly, and differently from PM050489 that affected both HER2-positive and HER2-negative tumor cells (Supplementary Fig. S5),MI130004 mirrored the cellular effects of the payload only inHER2-positive tumor cells. Indeed, as shown in Fig. 1C andSupplementary Fig. S6, MI130004-treated HER2-positive breasttumor cells of different histotypes exhibited a disordered micro-tubule distribution and less microtubule fibers compared withnontreated cells. Moreover, immunofluorescent analyses showedthatMI130004 induced alteredmitotic phenotypes withmultiplemitotic poles. This coincidedwith an increase in the percentage ofcells in G2–M phase (Table 2). In contrast, HER2-negative breastcancer cells didnot showalterations in tubulin cytoskeletonnor incell cycle (Fig. 1C; Supplementary Fig. S6; Table 2). Trastuzumabdid not induce any alterations on the microtubule network (14).

Animal studiesTo determine whether the in vitro antiproliferative activity of

MI130004 translated into in vivo antitumor activity, we evaluatedthe effect of the ADC inmice xenograft models. The efficacy of the

Table 1. Antiproliferative activity of MI130004 in HER2-positive and -negativecell lines

Tumor cell line Histotype HER2 status IC50 (mg/mL) GSD

HCC-1954 Breast Positive 0.04 2.1SK-BR-3 Breast Positive 0.03 2.2BT-474 Breast Positive 0.73 1.2JIMT-1 Breast Positive 2.38 1.4HGC-27 Gastric Positive 0.97 1.3A2780 Ovary Positive 2.00 1.3A2780cis Ovary Positive 3.44 1.2MDA-MB-231 Breast Negative >10 —

MCF-7 Breast Negative >10 —

Hs 746T Gastric Negative >10 —

NOTE: Cell lines were classified as HER2 positive or negative according to IHCanalysis. Values represent the geometric mean of three or more differentexperiments, each performed in triplicate.Abbreviations: GSD, geometric standard deviation; IC50, concentration thatinhibits cell growth by 50%.

Table 2. Cell-cycle analysis of MI130004 in HER2-positive and -negative celllines

Tumorcell line Histotype

HER2status

% G2–Muntreated

% G2–MMI130004

HCC-1954 Breast Positive 19 74SK-BR-3 Breast Positive 11 46JIMT-1 Breast Positive 14 35HGC-27 Gastric Positive 8 8A2780 Ovary Positive 8 13MDA-MB-231 Breast Negative 14 15MCF-7 Breast Negative 20 20Hs 746T Gastric Negative 15 21

NOTE: Cell lines were classified as HER2 positive or negative according to IHCanalysis. Values represent the mean of three or more different experiments. "%G2–M" denotes the percentage of cells arrested in G2 or mitosis as detected byflow cytometry in cells either untreated or treated with MI130004 at 0.1 mg/mL.

Evaluation and Characterization of the Novel ADC MI130004

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molecule was first tested in xenografts with breast tumor cells.HER2 expression levels in the tumors were confirmed by IHC 24hours after the first administration (Supplementary Fig. S7A–S7C). At the drug doses used in the experiment, no significanttoxicity or body weight loss was observed in the treated animals.As shown in Fig. 2A, MI130004 presented antitumor activity witha statistically significant inhibition of tumor growth and animprovement of mice survival only in the HER2-positive testedmodels but not in the HER2-negative one (i.e., MDA-MB-231),thereby confirming the selectivity of the ADC forHER2-expressingcells. For instance, in animals bearing JIMT-1 (HER2 positive)xenografts, MI130004 induced a long-lasting antitumor effectwith statistically significant reduction of tumor volume comparedwith both vehicle and trastuzumab treatments. Moreover, a dose-dependent disappearance of HER2-expressing cells was observedin JIMT-1 xenografts afterMI130004 (Fig. 2B).Mitotic aberrationsconsistent with the mechanism of action of PM050489 (revealedby nuclear staining with Hoechst-33258) were also noticeable(Fig. 2B).

In addition to breast xenografts, gastric and ovarian xenograftmodels were also used to evaluate the efficacy of MI130004, andsimilar results were obtained. In HER2-positive gastric cell lines(gastric-008 and N87), MI130004 induced a reduction in tumorvolume as well as an increase in survival compared with HER2-negative cells (Hs 746T, Fig. 3A), whereas trastuzumab did notshow any effect on tumor growth. In gastric-008 cells extractedfrom the tumor, a reduction of HER2 expression as well as anincrease inmitotic aberrations can be noticed as a consequence ofMI130004 treatment (Fig. 3B). Alike, the compound also showedactivity in HER2-positive ovarian cancer cell lines (SK-OV-3 andA2780cis; Fig. 4A). Again, in SK-OV-3,MI130004wasmore activethan trastuzumab and induced a dose-dependent reduction ofHER2 expression in tumor xenografts (Fig. 4B).

Altogether, our results indicate that MI130004, an ADC-con-jugating PM050489 to trastuzumab, demonstrated significantand selective in vivo antitumor activity in human-derivedHER2-expressing xenografts corresponding to breast, gastric, andovarian tumors.

DiscussionSince the approval of Mylotarg in 2000, a vast research effort

has been made in the ADC field, resulting in two novel entitiesbeing approved in 2011 to 2013 and a full pipeline with a largenumber of conjugates undergoing clinical studies. Despite theevident need for novel payloads that might help to overcomefuture resistance, a close study to the current pipeline shows thatthe vast majority of the novel ADCs under clinical investigationincludes cytotoxic warheads belonging to the same chemicalfamilies than those of Mylotarg, Adcetris and Kadcyla, that is,calicheamicins, auristatins, and maytansinoids, respectively.Moreover, the novel payloads not belonging to those classes actthrough similar mechanisms of action, affecting either DNAbiology (cross-linkers like pyrrolobenzodiazepines, alkylatingagents such as indolinobenzodiazepines and duocarmycins orinhibitors of topoisomerases like camptothecin derivatives ordoxorubicin) ormicrotubule dynamics (amberstatin or tubulysinanalogues; see ref. 15 for a complete review). The discovery ofnovel warheads acting on unprecedented targets is a difficult taskgiven the requisites such targets must accomplish, namely intra-cellular location and critical involvement in essential processes for

cell survival or division. Therefore, the search for novel scaffoldsacting differently on microtubules or DNA seems a more realisticapproach to face possible resistance to current ADC payloads.

PM050489 is a marine molecule with extremely high antipro-liferative potency that has inspired a chemical series leading to itsdechlorinated analogue, PM106184, with promising therapeuticprojection in oncology. Despite being a tubulin-binding agent,the molecule looks appropriate to be considered for antibodyconjugation even if it binds to the so-called maytansine siterecently described in b-tubulin and, therefore, its potentiality toovercome the hypothetical appearance of future resistance tomaytansinoids could be argued. However, there are structuraland functional results that may rebut such argument. First,although PM050489 derivatives share a common pharmaco-phore pocket with maytansine, their interaction area expandsbeyond such pocket to reach other zones within the b-tubulinsurface, and indeed, they are involved in additional steric clasheswith a-tubulin units, thus contributing further to prevent theformation of longitudinal contacts between tubulin moleculesneeded for microtubule growth (9). Second, although the mainresistance mechanism known so far to affect ADC payload con-sists on the overexpression and increased activity of drug effluxpumps (16), an otherwise classical resistance mechanism fortubulin-binding agents (17), PM050489 derivatives are highlypotent on inhibiting the in vitro growth of P-gp–overexpressingtumor cell lines and have shown activity in animal tumor modelsregardless of the P-gp status of the cell line used (10). In fact,MI130004 displays identical potencies in the ovarian cell lineA2780 and in the P-gp–overexpressing version A2780cis. In ourview, these two facts invest PM050489 derivatives with significantdifferential attributes with respect to maytansinoids that lead toconsider them as attractive warheads for overcoming possibleresistance to ADC payloads of current use.

PM050489 was conjugated to trastuzumab using a nonclea-vable linker. The stability of the linker used was demonstrated bythe complete lack of activity observed for MI130004 in HER-negative cells both in vitro and in vivo. Furthermore, the extraor-dinary potency observed in vitro with MI130004, consistent inmolar units with that of PM050489, demonstrates that thepresence of the linker is not impairing significantly the antipro-liferative potential of the original molecule and indeed the in vitrobiological effects observed in cells treated withMI130004 (mitot-ic aberrations, massive disorganization, and disappearance of themicrotubule network and cell-cycle arrest in G2–M) resemblethose obtained with PM050489.

Analytic studies performed onMI130004 reveal the presence ofone major species (75%, with naked trastuzumab being the mostabundant species of the remaining 30%) likely bearing twomolecules of payload bound per molecule of MI130004. Tointerpret the mass spectra data obtained, we have consideredthat, since we have used in this study the European MedicinesAgency–approved version and formulation of trastuzumab, theglycosylation pattern of the antibody should be identical to thatpublished for this version of the molecule; hence, the molecularmass assignations of the fragments obtained with the nakedantibody should readily match those previously published, forexample, in ref. 12. Our results are consistent with that assump-tion and enable us to propose that the two cytotoxicmolecules arebound to both sides of the disulfide bond connecting one lightand one heavy chain of the antibody as it has been possible toidentify fragments whose masses correspond to the addition of

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Figure 2.

In vivo activity of MI130004 on breast tumor xenografts developed in mice. A, Left, evolution of tumor volumes during treatment; right, Kaplan–Meier survivalcurves. Animals (8–10/group) were treated weekly for 5 consecutive weeks with either MI130004 at 1 (hollow triangles), 5 (pink inverted triangles), and 10(red triangles) mg/kg, trastuzumab at 30 mg/kg (blue circles), or vehicle (hollow circles) following the procedure described in the Materials and Methods section.B, IHCof samples,withdrawn24hours after thefirst dose ofMI130004, frommice xenograftedwith JIMT-1 tumor cells and treatedwithMI130004at different doses asshown in the panels. Top row shows HER2 staining with anti–c-erb2 antibody (magnification,�20), and figures show percentages of HER2 expression. Bottom rowshows DNA staining with Hoechst 33258, and bottom figures denote mitotic catastrophe nuclei per five high-power fields with magnification being �40.

Evaluation and Characterization of the Novel ADC MI130004

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only one payload of each chain (LC and Fd) located at each side ofthe disulfide bridge. Further attempts to increase the drug-to-antibody ratio (DAR) with alternative synthetic strategies wereabandoned as they failed to deliver the desired product asmost ofthe material was lost during the process due to poor solubility.Although it was commonly accepted that low DAR values corre-late with higher bioavailability and reduced toxicity (18, 19),novel findings suggest that higher DAR values can be obtainedwithout jeopardizing the ADC properties provided that the linker

is endowed with properties that compensate for the higher hydro-phobicity introducedby the payload (20). In the case ofMI130004,it is highly remarkable that, despite its low DAR, the conjugateshowedanextraordinarypotencyboth in vitro and in vivo. As alreadyreported by Strop and colleagues (21) for an auristatin-based ADCwith engineered attachment sites in a Trop-2 antibody, a likelyexplanation for the high potency obtained at such low substitutiondegree may reside in the combination of an extremely potentpayload with a high and homogeneous expression of the antigen

Figure 3.

In vivo activity of MI130004 on gastrictumor xenografts developed in mice.A, Left, evolution of tumor volumesduring treatment; right, Kaplan–Meiersurvival curves. Animals (8–10/group)were treated weekly for 5 consecutiveweeks with either MI130004 at 5 (pinkinverted triangles) and 10 (redtriangles) mg/kg, trastuzumab at30 mg/kg (blue circles), or vehicle(hollow circles) following theprocedure described in the Materialsand Methods section. B, IHC ofsamples, withdrawn 24 hours after thefirst treatment with MI130004, frommice xenografted with gastric-008tumor cells and treatedwithMI130004at different doses as shown in thepanels. Top row shows HER2 stainingwith anti–c-erb2 antibody(magnification, �20), and figuresshow percentages of HER2expression. Bottom row shows DNAstaining with Hoechst 33258, andbottom figures denote mitoticcatastrophe nuclei per five high-power fields with magnificationbeing �40.

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(HER2 in the case ofMI130004)within tumor cells and also with arapid and efficient internalization of the antibody–receptor com-plex. Likewise, it sounds reasonable that such low DAR may alsocontribute to the outstanding in vivo efficacy observed due to thelikely high bioavailability mentioned above.

The in vivo efficacy of MI130004 has been observed with severalxenografts of HER2-positive tumor cells regardless of their histo-type. Breast, gastric, and ovarian xenografts have experienced asignificant reduction in tumor growth together with improvedanimal survival in a dose-dependent manner, and these effectswere long lasting for the highest dose of MI130004 used. Histo-pathologic analysis showed that the biological effect caused in vivowas identical to that in vitro and is consistentwith themechanismofaction of PM050489. Therefore, this marine molecule has demon-strated to be an excellent payload for novel ADCs endowed withpromising features and reinforces the potential of marine naturalproducts as a source of inspiration for novel chemical scaffolds.

Disclosure of Potential Conflicts of InterestJ.F. Martínez-Leal is a department manager at and has ownership interest

(including patents) in PharmaMar SA. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: P. Avil�es, J.M. Domínguez, M.J. Guill�en, J.F. Martínez-Leal, C.M. Galmarini, C. CuevasDevelopment of methodology: J.M. Domínguez, M.J. Mu~noz-Alonso,C. Mateo, R. Rodriguez-Acebes, A. Francesch

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.J. Mu~noz-Alonso, C. Mateo, R. Rodriguez-Acebes,J.M. Molina-GuijarroAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Avil�es, J.M. Domínguez, M.J. Guill�en, J.M.Molina-Guijarro, J.F. Martínez-Leal, C.M. GalmariniWriting, review, and/or revision of themanuscript: P. Avil�es, J.M. Domínguez,M.J. Guill�en, J.M. Molina-Guijarro, J.F. Martínez-Leal, C.M. Galmarini,C. CuevasAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.F. Martínez-Leal, S. Munt, C.M. GalmariniStudy supervision: P. Avil�es, J.M. Domínguez, M.J. Guill�en, J.F. Martínez-Leal,S. Munt

AcknowledgmentsThis work was partially supported by grant IPT-2012-0198-090000

("MARINMAB" project) from Ministerio de Economía, Industria y Competi-tividad. The excellent technical skills of the in vivo Pharmacology team inPharmaMar and their critical contribution to perform the xenograft experimentsis greatly appreciated. We want to thank Patricia Rup�erez, Pablo Cobos, andCarles Celma at KYMOS for their workwith the proteolytic digestion and LC-MSanalysis of MI130004. We would also like to acknowledge the IHC workperformed by the Oncogenesis and Antitumor Drug Group at the Hospitalde Sant Pau in Barcelona.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 21, 2017; revised November 7, 2017; accepted February 1,2018; published first February 13, 2018.

Figure 4.

In vivo activity ofMI130004onovariantumor xenografts developed in mice.A, Left, evolution of tumor volumesduring treatment; right, Kaplan–Meiersurvival curves. Animals (8–10/group)were treated weekly for 5 consecutiveweeks, with either MI130004 at 5(pink inverted triangles) and 10 (redtriangles) mg/kg, trastuzumab at30 mg/kg (blue circles), or vehicle(hollow circles) following theprocedure described in the Materialsand Methods section. B, IHC ofsamples, withdrawn 24 hours after thefirst dose of MI130004, from micexenograftedwith SK-OV-3 tumor cellsand treated with 10 mg/kg MI130004,showing DNA staining with Hoechst33258, and bottom figures denotemitotic catastrophe nuclei per fivehigh-power fields with magnificationbeing �40.

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Evaluation and Characterization of the Novel ADC MI130004

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2018;17:786-794. Published OnlineFirst February 13, 2018.Mol Cancer Ther   Pablo Avilés, Juan Manuel Domínguez, María José Guillén, et al.  

Activity against HER2-Expressing TumorsIn VivoTrastuzumab with a Molecule of Marine Origin, Shows Outstanding

Drug Conjugate Combining−MI130004, a Novel Antibody

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