A DNA-InteractingPayload Designedto Eliminate Cross ... · Large Molecule Therapeutics A DNA-InteractingPayload Designedto Eliminate Cross-Linking Improves the Therapeutic Index of

Large Molecule Therapeutics

A DNA-Interacting Payload Designed to EliminateCross-Linking Improves the Therapeutic Index ofAntibody–Drug Conjugates (ADCs)Michael L. Miller, Manami Shizuka, Alan Wilhelm, Paulin Salomon, Emily E. Reid,Leanne Lanieri, Surina Sikka, Erin K. Maloney, Lauren Harvey, Qifeng Qiu,Katie E. Archer, Chen Bai, Dilrukshi Vitharana, Luke Harris, Rajeeva Singh,Jose F. Ponte, Nicholas C. Yoder, Yelena Kovtun, Katharine C. Lai, Olga Ab,Jan Pinkas, Thomas A. Keating, and Ravi V.J. Chari

Abstract

Tumor-selective delivery of cytotoxic agents in the form ofantibody–drug conjugates (ADCs) is now a clinically validatedapproach for cancer treatment. In an attempt to improve theclinical success rate of ADCs, emphasis has been recentlyplaced on the use of DNA–cross-linking pyrrolobenzodiaze-pine compounds as the payload. Despite promising earlyclinical results with this class of ADCs, doses achievable havebeen low due to systemic toxicity. Here, we describe thedevelopment of a new class of potent DNA-interacting agents

wherein changing the mechanism of action from a cross-linkerto a DNA alkylator improves the tolerability of the ADC. ADCscontaining the DNA alkylator displayed similar in vitropotency, but improved bystander killing and in vivo efficacy,compared with those of the cross-linker. Thus, the improvedin vivo tolerability and antitumor activity achieved in rodentmodels with ADCs of the novel DNA alkylator couldprovide an efficacious, yet safer option for cancer treatment.Mol Cancer Ther; 17(3); 650–60. �2018 AACR.

IntroductionAntibody–drug conjugates (ADCs) represent a promising, and

potentially less toxic, alternative to conventional chemotherapy.Ideally, the antibody component of the ADC will selectivelydeliver the linked cytotoxic agent ("payload") to the cancercell, effectively minimizing the amount of off target toxicity tocells lacking the antigen (1–4). Despite over 30 years of ADCresearch, there are currently only four FDA-approved ADCs,ado-trastuzumab emtansine (T-DM1, Kadcyla), brentuximabvedotin (Adcetris), inotuzumab ozogamicin (BESPONSA), andjust recently, gemtuzumab ozogamicin (MYLOTARG; refs. 5–7).Only one (T-DM1) of the four approved ADCs is for the treatmentof solid tumors. These recent successes have revitalized thisapproach for cancer treatment, and about 65 ADCs are underclinical evaluation. The majority of ADCs currently in the clinicuse microtubule inhibitors, such as the auristatins and maytansi-noids, as the payload (8). Because DNA-interacting agents arewidely used in cancer therapy, and twoof the four approved ADCsuse aDNA-interacting compound (calicheamicin), there has been

a recent shift in emphasis toward using such compounds aspayloads in ADCs (9–15). Indeed, the first ADCof a highly potentDNA-interacting agent to be evaluated in the clinic was gemtu-zumab ozogamicin, which incorporated calicheamicin, a com-pound that causes DNA double strand breaks. This ADC wasapproved by the FDA, but subsequently withdrawn from themarket due to toxic side effects, notably veno-occlusive disease(16, 17). This ADC was re-approved recently after a new, lowerdose regimen in combination with chemotherapy displayedtherapeutic benefit. Recently, a number of ADCs that incorporatepyrrolobenzodiazepine dimers (PBDs), which are potent DNAcross-linkers, have been entered into clinical evaluation andemerging data from human trials indicate that doses achievablewith these ADCs are very low (e.g., 18-fold and 90-fold lowerdose than that of T-DM1 for the PBD ADCs rovalpituzumabtesirine, which targets DLL3, and the CD33-targeting conjugatevadastuximab talirine, respectively; refs. 11, 18). While encour-aging antitumor activity has been reported for rovalpituzumabtesirine, adverse effects, such as pleural effusion have been doselimiting (18). In addition, despite promising early clinical results,the phase III clinical trial of vadastuximab talirine was recentlydiscontinued due to an unfavorable safety profile. Thus, there is aneed for novel DNA-interacting agents with an alternative mech-anism of action in order to provide ADCs with good in vivo anti-tumor activity, coupled with improved tolerability.

We recently reported on a new chemical class of cytotoxicpayload, indolinobenzodiazepine dimers (termed IGNs), forADCs (12). The IGN class has two distinct structural features thatdistinguish it from the PBDs (based on the SJG-136 core; refs. 19,20). First, the pyrrolidine ring was replaced with an indolinomoiety, which led to tighter DNA binding and resultant

ImmunoGen, Inc., Waltham, Massachusetts.

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

Present address for M. Shizuka: Pharmaron, Inc., Waltham, Massachusetts.

Corresponding Author: Ravi V.J. Chari, ImmunoGen, Inc., 830 Winter Street,Waltham, MA 02451. Phone: 781-895-0692; Fax: 781-895-0614; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-17-0940

�2018 American Association for Cancer Research.

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approximately 10-fold increase in in vitro potency. Second, theintroduction of a phenyl spacer to connect the two IGN mono-mers was found to further improve the in vitro potency and alsoprovide a site for linker attachment. Within this IGN class, wediscovered that a diimine-containing IGN ADC that, like thePBDs, cross-links DNA had an unfavorable toxicity profile inmice. The animals displayed prolonged body weight loss, as wellas delayed lethality (12). We then studied the effects of changingthe mechanism of action of the IGN by selective reduction of oneof the imines, to provide a monoimine IGN payload. In contrastto the diimine in the PBD dimer, which is known to act by cross-linking DNA in a preferred GATC sequence selective manner, theIGN monoimine lacks the structural feature necessary for cross-linking DNA, and thus is only capable of DNA alkylation, pro-viding a distinct mode of action compared with that of the PBDs(19). In vivo testing revealed that themonoimine IGNADC,whichalkylates DNA, did not display any signs of prolonged or delayedtoxicity (12). For those studies, the comparison was made withADCs in which the IGN diimine or monoimine was linked to theantibody via a noncleavable linker.

The primary goals of the present study were to (i) design IGNADCs with cleavable linkers with favorable pharmacokineticproperties and improved potency against tumors with both lowand/or heterogeneous antigen expression and (ii) compare theeffects of the mechanistic change from DNA cross-linking toDNA alkylation on the in vitro activity and in vivo tolerabilityand efficacy of these ADCs. This report describes the design,development, and preclinical characterization of novel IGNsthat are linked via a protease cleavable linker to create ADCswith either DNA alkylator or cross-linker activity. The datasuggest that ADCs of the monoimine IGN alkylator representa more efficacious and potentially safer therapeutic option forthe treatment of cancer.

Materials and MethodsCell lines, antibodies, and reagents

The human cancer cell lines KB (cervical adenocarcinoma),JEG-3 (choriocarcinoma), T47D (epithelial ductal carcinoma),NCI-H2110 (lung adenocarcinoma), Namalwa (Burkitt lym-phoma), OV-90 (ovarian adenocarcinoma), MV4-11 (AML),COLO 205, and HCT-15 (colorectal adenocarcinoma) wereobtained from the ATCC. Ishikawa (human endometrial ade-nocarcinoma) cells were purchased from the European Collec-tion of Cell Cultures. The SHI-1 (AML) cell line was procuredfrom DMSZ. The cell lines were purchased within the period of2000 to 2014, and characterized by the vendor. No further cellline authentication was conducted by the authors. COLO 205MDR was generated by transfection of COLO 205 cells withrecombinant retrovirus carrying the human MDR1 cDNA aspreviously described (21). KB-GRC1-MDR (human cervicaladenocarcinoma) was a gift from Dr. Igor Roninson (Universityof Illinois, College of Medicine, Chicago, IL). Upon receipt,each cell line was expanded by passaging two to three times,aliquoted, and frozen. For use in experiments, cell lines werecultured in media recommended by the vendor in a humidifiedincubator at 37�C, 6% CO2 for no longer than 2 months.Humanized antifolate receptor-a (FRa) antibody, humanizedanti-CD123, chimeric mouse-human anti-EpCAM antibody,and chimeric nontargeting control antibody to the Kunitzsoybean protease inhibitor (KTI) were all constructed with the

human IgG1 isotype and generated at ImmunoGen, Inc(21, 22). The anti-CD123 antibody was engineered to possessa cysteine residue at position 442 of the heavy chain (HC442).None of the antibodies used in the studies cross-react withantigen on mouse tissues. The control anti-KTI antibody doesnot bind to any human antigen. Additional chemical reagentswere purchased from Sigma-Aldrich.

Preparation of IGN ADCsStructures of the IGNpayloads are shown in Fig. 1. Conjugation

of IGN dimers to an antibody was accomplished by linking viaamide bonds to lysine residues, or thioether bonds to engineeredcysteines (see Supplementary Methods) of the antibody. IGNmolecules bearing an N-hydroxysuccinimide (NHS) ester werereversibly sulfonated at the imine moiety by pretreatment witheither a 5-fold (for monoimine) or 13-fold (for diimine) molarexcess of sodium bisulfite and then added in an approximately5-foldmolar excess to the antibody in aqueous buffer [15mmol/LHEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid),pH8.5], containing 10%to15%N,N-dimethylacetamide (DMA).Upon completion of conjugation, the reaction mixtures werepurified and buffer exchanged into 10 mmol/L succinate,50mmol/L sodium chloride, 8.5%w/v sucrose, 0.01%Tween-20,and 50 mmol/L sodium bisulfite pH 6.2 formulation bufferusing NAP desalting columns (Illustra Sephadex G-25 DNAGrade, GE Healthcare). Dialysis was performed in the samebuffer for 4 hours at room temperature and then overnight at4�C utilizing Slide-a-Lyzer dialysis cassettes (ThermoScientific30,000 MWCO) to remove any unreacted small molecules. Thepurified conjugates were found to have an average of 2.5 to3 IGNmolecules linked per antibody (lysine-linked) or approx-imately 2 IGN molecules linked per antibody (cysteine-linked),as determined by UV-Vis spectroscopy using molar extinc-tion coefficients e325 nm ¼ 15,231/cm/M and e280 nm ¼26,864/cm/M for IGN, and e280 nm ¼ 201,400/cm/M for theantibody. The ADCs were >95% monomeric (by size exclusionchromatography), and contained <0.4% unconjugated IGN(by acetone precipitation, reverse-phase HPLC analysis). Theprocedure for conjugation to engineered cysteines is describedin the Supplementary section.

In vitro cytotoxicityCytotoxic potencies were assessed using a WST-based cell

viability assay (Dojindo, Molecular Technologies, Inc.) as pre-viously described (21). Briefly, human tumor cells (1,000–5,000 cells/well, depending on the cell line), in the appropriateculture medium were incubated with conjugates � 1 mmol/L ofthe corresponding unconjugated antibody for 5 days, at 37�C.Cell viability was determined from background-corrected WSTabsorbance. Antibody bound per cell (ABC) values were deter-mined as previously described (23).

Bystander cell killing assayFRa-positive (transfected with FOLR1-encoding cDNA)

300.19 cells (1,000 cells/well) and FRa-negative 300.19(2,000 cells/well) were treated with ADCs separately, or as amixed cell population where the ADC concentration was variedin a range where it was nontoxic for the FRa-negative cells buttoxic for the receptor-positive cells. After 4 days at 37�C, cellviability was measured by addition of Cell Titer Glow (Promega)reagent followed by luminescence measurement (22).

ADCs with Potent DNA-Alkylating Agents

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Processing of IGN ADCs by target cancer cells and assay of IGNcatabolites

OV-90 cells (4 � 104 ABC) were incubated with 2 mg/mL ofanti-FRa ADC 2a or 2b, without or with a 100-fold excess ofunconjugated antibody to demonstrate antigen specificity.After incubation with ADC for 1 hour at 37�C, the cells werewashed with media to remove unbound conjugate. Freshmedia were added, and the cells were incubated for 23 hoursat 37�C, followed by harvest of cells and media supernatant.The IGN catabolite in the supernatant was extracted using asolid-phase cartridge (Oasis HLB; Waters), eluted with aceto-nitrile, and concentrated under vacuum. The extract was ana-lyzed for IGN catabolite level using ELISA (see SupplementaryMethods).

In order to analyze the export of IGN catabolites from cancercells, JEG-3, NCI-H2110, and OV-90 cells (�1–5 � 106) wereincubated with unconjugated IGN 1a or 1b (200 nmol/L) for2 hours at 37�C. After incubation, the cells were washed, and thenincubated with fresh media for an additional 22 hours at 37�C.The amount of exported IGN was determined by ELISA (seeSupplementary Methods).

In vivo antitumor activity and tolerability experimentsThe level of antigen expression in xenografts (H-score) was

determined using immunohistochemical analysis as previouslydescribed (see also Supplementary Methods; ref. 24).

Female CB.17 SCID mice at 6 weeks of age were received fromCharles River Laboratories. All in vivo procedures were performedin strict accordance with the NIH Guide for the Care and Use ofLaboratory Animals. Ishikawa, NCI-H2110 or OV-90 tumor cells(1� 107 cells/animal) were subcutaneously implanted into mice.When the tumors were established and reached a size of approx-imately 100mm3(typically 7–18dayspostinoculation, dependingupon the model) the animals were randomized into treatmentgroups of 6 and treated with a single i.v. administration of vehicle(0.2mL/mouse) or IGNADC. Tumor volumes (V)were calculatedby caliper measurements in three dimensions using the formulaV¼ Length�Width�Height� 1/2. Tumor sizewasmeasured 2 to3 times weekly and tumor growth inhibition (T/C value) wasdeterminedusing the following formula: T/C(%)¼median tumorvolumeof the treated/median tumor volumeof the control� 100.

For tolerability studies, all toxicity, changes in body weight, anddetermination of maximum tolerated dose (MTD) studies wereconducted in nontumor bearing CD-1 mice (Charles River Labora-tories) after a single i.v. administrationof theADC.MTDisdefinedasthe dosewhere no animals died orwere required tobe sacrificed dueto >20% body weight loss or signs of distress or morbidity (hunch-ing, lack of movement, inability to eat or drink, or signs of pain).

Pharmacokinetic studiesThe anti-FRa antibody and ADC 2b were radiolabeled by

treatment with N-succinimidyl-[2,3,3H]propionate as previously

Figure 1.

Structural representations. Chemical structures of IGN catabolites (1a, 1b), and structural representation of lysine-linked ADCs of IGN cross-linker (2a, 3a, 4a)and alkylator (2b, 3b, 4b) or cysteine-linked (site-specific) ADCs of IGN cross-linker (5a) and alkylator (5b).

Miller et al.

Mol Cancer Ther; 17(3) March 2018 Molecular Cancer Therapeutics652




described (25). For tritium labelingon the IGNcomponentofADC2b, one of the imine moieties of the IGN dimine was selectivelyreduced with [3H]NaBH4, followed by conjugation to the anti-body. Nontumor bearing CD1 mice were randomly distributedinto 3 groups of 6 animals/group. Mice received a single intrave-nous injection of the test article at a dose of 0.27mg/kg. Bloodwassampled via the retro-orbital sinus at time points ranging from 2minutes to 840 hours. The concentration of the test articles inplasma was determined from the measured radioactivity.

ResultsDesign of the payload/linker

The initial goal was to design an IGN compound that woulddisplay high potency (IC50 values in the picomolar range) across abroad panel of tumor cell lines, and also possess a functionalgroup suitable for incorporation of a cleavable linker. From ourstructure-activity relationship studies, we identified the DNAcross-linker 1a, containing an anilino group that could be usedto incorporate a protease-labile linker (Fig. 1). In an appropriatelydesigned peptide linker, protease-mediated cleavage of the amidebond between the anilino group and the peptidewould efficientlyrelease the payload 1a inside the cell (26). Because we wanted tocompare ADCs of a DNA-cross-linking IGNwith ADCs of a DNA-alkylating IGN that would be incapable of forming cross-links, weselectively reduced one of the imines in 1a to provide 1b. The twocompounds displayed a similar level of in vitro cytotoxicity towarda panel of six solid tumor cell lines (IC50 for 1a ¼ 3–20 pmol/L,and for 1b¼ 4–21 pmol/L), indicating that, for the IGN class, thecross-linking mechanism was not required to achieve high poten-cy (Table 1). In addition, the potency of diimine IGN 1a andmonoimine IGN 1b was similar against the three multidrugresistant (MDR) cell lines tested, albeit these lines were lesssensitive to both compounds (Table 1). After screening a panelof di- and tri-peptides, and using the criteria of good plasmastability and efficient cleavage upon incubation with tumor celllysosomal extracts, L-Ala-L-Ala was chosen as the linker. Thepeptide linker was synthesized with a terminal N-hydroxysucci-nimide ester to enable conjugation to lysine residues of theantibody, or a maleimido group to facilitate conjugation toengineered cysteines of the antibody. Attachment of these linkersto 1a and 1b, via amide bonds, provided the final compoundssuitable for linkage to antibodies (Supplementary Fig. S1).

For conjugation via lysine residues, three antibodies werechosen: (a) humanized antifolate receptor-a (FRa) antibody;(b) a chimeric anti-EpCAM antibody; and (c) a nontargeting

control chimeric antibody to the Kunitz soybean protease inhib-itor (KTI). Both chimeric antibodies consisted of murine Fab andhuman IgG1 Fc. Reaction of these antibodies with IGNs bearingN-hydroxysuccinimide esters resulted in ADCs 2a, 2b, 3a, 3b, 4aand 4b, with an average drug to antibody ratio (DAR) of approx-imately 2.7 (Fig. 1). For site-specific conjugation, an anti-CD123antibody bearing engineered cysteine residues at HC442 wascoupled with the maleimido-containing IGNs to generate ADCs5a and 5b with a DAR of approximately 2 (Fig. 1).

In vitro cytotoxicityThe in vitro cytotoxicity of anti-FRa (2a, 2b) and anti-EpCAM

(3a, 3b) ADCs was tested using a panel of antigen-positive celllines, with receptor expression ranging from low (2� 104) to high(3 � 106) molecules of antibody bound per cell (ABC). Bothdiimine andmonoimine IGN ADCs displayed a similar degree ofhigh potency (IC50 values, 3–300 pmol/L; Table 1). In all cases,these IGN ADCs, conjugated via peptide linkers, displayed highantigen specificity as their cytotoxic activity was significantlydiminished upon addition of excess (1 mmol/L) unconjugatedantibody (Supplementary Fig. S2A). Additionally, antigen-specif-ic potencywas also demonstrated by an approximately 1,000-foldlower activity of the nontargeting ADCs 4a and 4b, with IC50

values between 4 and 6 nmol/L (Supplementary Fig. S2B). Asimilar trend was noted in the three MDR tumor cell lines tested,with ADCs of the IGN alkylator being equipotent to that of thecross-linker version (Table 1). Thus, diimine and monoimineanti–FRa-IGN ADCs (2a, 2b) effectively killed the MDR-positiveKB-GRC1 cervical cell line with IC50 values of 50 and 70 pmol/L,respectively. Likewise, the anti–EpCAM-IGN ADCs (3a, 3b) dis-played high cytotoxicity in MDR-positive colon cancer cell lines,HCT-15 and COLO 205-MDR, with IC50 values of approximately50 to 145pmol/L for both IGNdiimine andmonoimine ADCs. Inthe case of site-specific conjugates, the anti-CD123 ADC of theIGN cross-linker (5a) was approximately 2-foldmore potent thanthat of the alkylator (5b) toward a panel of CD123-expressing celllines. Thus, the IC50 values toward MV4-11 and SHI-1 cell lineswere 0.4 and 2 pmol/L for the cross-linker ADC 5a comparedwith1 and 5 pmol/L, respectively, for the alkylator ADC 5b.

In vivo efficacy and tolerabilityAn in vivo efficacy study comparing anti-FRa ADCs of the cross-

linker and alkylator IGN payloads was conducted in a subcuta-neous Ishikawa endometrial mouse xenograft model. In thismodel, the antigen is expressed in a heterogeneous manner, with

Table 1. Comparative in vitro cytotoxicity of IGNs and IGN ADCs with either a diimine or monoimine toward cancer cell lines

ABCa IC50, pmol/LAb Cell line Indication (1,000s) 1a 1b 2a 2b

Anti-FRa KB Cervical 3,000 3 4 3 4KB-GRC1-MDR Cervical, PGPþ 700 100 100 50 70T47D Breast 100 20 20 20 20NCI-H2110 NSCLC 50 4 6 60 100OV-90 Ovarian 40 10 10 300 200Ishikawa Endometrial 20 9 5 50 10

1a 1b 3a 3bAnti-EpCAM COLO 205 Colon 1,100 19 21 12 9

COLO 205-MDR Colon, PGPþ 1,300 52 51 75 53HCT-15 Colon, PGPþ 700 191 257 145 126

aNumber of antibody molecules bound per cell.






anH-score of 120, and approximately 30%of the cells are antigen-negative (Fig. 2A). Both ADCs (2a and 2b) displayed dose-dependent antigen-specific antitumor activity following a singlei.v. dose. However, the ADC 2b of theDNA alkylator was found tobe more active than the cross-linker ADC 2a (Fig. 2B). Treatmentwith ADC 2b at the 0.29mg/kg dose (containing�5 mg/kg-linkedIGN) resulted in complete regressions (CR) of the tumors in 4 of 6mice, with 2 animals remaining tumor-free at end of study (day75). In contrast, the cross-linker ADC 2a caused no tumor regres-sions at the same dose (Fig. 2C). The level of tumor growthinhibition of 2a at this dose was similar to that seen with halfthe dose (0.14 mg/kg) of 2b. Thus, the alkylator ADC was 2-foldmore active than the cross-linker variant in this model.

In a second study, the activities of the two anti-FRa ADCs(2a, 2b), and corresponding nontargeting control ADCs 4a and4b, were compared using NCI-H2110 non–small cell lungcancer xenografts. In this model, antigen expression was alsoheterogeneous, with an H-score of 110, and approximately20% of the cells were antigen negative (Supplementary Fig.S3). Both ADCs were highly active at a single 0.29 mg/kg dose(�5 mg/kg-linked IGN), with all animals tumor-free at the endof the study (day 100; Fig. 2D). In contrast, the two nontarget-ing conjugates were inactive. At a lower dose of 0.14 mg/kg, theDNA alkylator ADC 2b was slightly more active than the cross-linker ADC 2a, with 3 of 6 CRs for 2b and 1 of 6 tumor-freesurvivors, versus 1 of 6 CRs and no tumor-free survivors for 2a(Supplementary Fig. S4).

Next, a tolerability study in nontumor bearing CD-1 micewas conducted in order to assess the therapeutic index [TI, theratio of maximum tolerated dose (MTD) to minimally effectivedose, (MED)] for the anti-FRa ADCs 2a, 2b. The anti-FRaantibody does not cross-react with normal mouse tissue, andthus the toxicity readout does not account for any target-mediated effects. The alkylator ADC was tolerated at an approx-imately 2-fold higher dose, with an MTD of approximately6 mg/kg compared with approximately 2.8 mg/kg for the cross-linker ADC. As a result, a greater therapeutic window wasachieved with the ADC of the DNA-alkylating monoimine. Inorder to determine if the difference in tolerability is influencedby the site of conjugation, the MTDs of the site-specificallylinked anti-CD123 ADCs were compared. The anti-CD123antibody also does not cross-react with normal mouse tissue.At a dose of 6 mg/kg, the alkylator ADC was well tolerated, withminimal differences in body weights seen relative to vehicle-treated control animals for the duration of the study (day 90,Fig. 3A and B). In contrast, the cross-linker ADC was toxic at thisdose with 5 of 8 animals dead by day 30 and the remainingthree animals displaying prolonged body weight loss leading todeath by day 60 (Fig. 3C). Even at a lower 4 mg/kg dose, thecross-linker ADC displayed prolonged body weight loss anddelayed deaths (Fig. 3D). The MTD of this ADC was approx-imately 2.8 mg/kg, although delayed onset of weight loss wasstill observed (Supplementary Fig. S5).

Bystander killing and catabolite exportADCs with cleavable linkers generate intracellular catabolites

that can diffuse out of the targeted cells into proximal antigen-negative cells, to induce killing of these "bystander" cells, pro-vided that the released catabolite is sufficiently hydrophobic topenetrate theneighboring cell (27–29). Inorder tounderstand thepossible cause of the improved antitumor activity of the ADC of

the alkylator relative to cross-linker, we compared the in vitrobystander killing activities of the two ADCs. In this assay, acoculture of antigen-negative and antigen-positive cells weretreated with the ADCs at concentrations that are not cytotoxicfor the antigen negative cells (0% killing) and highly potent(100% killing) for antigen-positive cells, when these two celllines were cultured separately. Thus, a mixed culture of FRa-transfected 300.19 cells (FRa-positive) and FRa-negative par-ent 300.19 cells were exposed to ADCs at concentrationsranging from 0.05 to 0.4 nmol/L, at which the FRa-negativecells were not affected when cultured alone. For both ADCs, itwas found that the level of bystander killing of proximalantigen-negative cells in mixed cell culture was augmentedwith increasing ADC concentration. However, the DNA-alky-lating ADC 2b displayed a significantly higher level of bystand-er activity as compared with the cross-linking ADC 2a at allconcentrations tested. ADC 2b killed 100% of FRa-negativecells at a concentration as low as 0.2 nmol/L, whereas approx-imately 30% of the FRa-negative bystander cells remainedwhen treated with ADC 2a at this concentration (Fig. 4A).Similar results were obtained using another cell line pair,wherein antigen-negative Namalwa cells were cocultured withFRa-positive NCI-H2110 cells (Supplementary Fig. S6).

In order to identify the cellular catabolites of ADC 2b,a radiolabeled IGN molecule was prepared by controlledreduction of one of the imines with [3H]NaBH4 and conju-gated with the anti-FRa antibody to provide the radiolabeledADC (Supplementary Fig. S7). Treatment of target cells withthis ADC, followed by extraction of metabolites from themedia with organic solvent and LC/MS analysis, revealed that1b was the major catabolite. Similar experiments with unla-beled ADC 2a, identified 1a as the major catabolite in themedia. In another experiment, the cellular catabolites from thetwo ADCs were isolated as DNA adducts as previouslydescribed (30). Treatment with nuclease to digest the DNA,followed by thermal treatment released the catabolites, whichwere then separated and characterized by LC/MS (Supplemen-tary Fig. S8). Compounds 1a and 1b were again identified ascatabolites from cells treated with 2a and 2b, respectively,indicating that the active, DNA-binding compounds are thesame as those found diffused into the media and responsiblefor bystander killing

As the two catabolites 1a and 1b were equipotent in vitro(Table 1), a similar level of bystander killing would be expectedwith the corresponding ADCs. In order to understand the reasonfor the improved bystander killing with the alkylating ADC 2b,we next compared the relative efficiency of diffusion of uncon-jugated IGNs 1a and 1b from the cells into the media usingthree different FRa-expressing cell lines, OV-90, JEG-3, and NCI-H2110. After a short exposure to 1a or 1b, cells were washedto remove nonbound compound, incubated in fresh media for22 hours, and the amount of each compound exported out ofthe cell determined by ELISA. In each of the lines examined, a 2-to 3-fold greater amount of the alkylator compound was foundin the media compared with the cross-linker (Fig. 4B). Forexample, treatment of NCI-H2110 cells with an equivalentamount of 1a or 1b led to the export of 1.4 pmol of the cross-linker compared with 3.9 pmol of the alkylator per million cells.To extend thesefindings,OV-90 cellswere treatedwithADCs2aor2b for 1 hour, washed to remove any nonbound conjugate, andincubated for 23 hours to allow time for intracellular catabolism.

Miller et al.





Figure 2.

In vivo antitumor activity comparing ADCs of an IGN DNA cross-linker and DNA alkylator. Day of dosing indicated by arrow. A, IHC staining for FRaexpression on Ishikawa xenograft; H score of 120. B, In vivo antitumor activity of the anti-FRa ADCs 2a and 2b in human endometrial adenocarcinoma Ishikawaxenografts in SCID mice. C, Tumor growth in individual mice treated with ADC 2a or ADC 2b at 0.28 mg/kg. D, In vivo antitumor activity of the anti-FRaADCs 2a and 2b in comparison with nontargeting ADCs (4a, 4b) in human lung adenocarcinoma NCI-H2110 xenografts in SCID mice.






Isolation of the small molecule catabolites frommedia by extrac-tion with organic solvent, and quantitation by ELISA revealedthat the catabolite of the alkylator ADC was exported to anapproximately 4-fold greater extent than the cross-linker (0.32and 0.075 pmol per million cells, respectively; Fig. 4C).

Preclinical studies of DNA alkylator ADCsBased on the superior TI and greater bystander killing activity of

ADCs bearing the DNA alkylator form of IGN, this class wasselected for additional preclinical studies. The antitumor activityof ADC 2b was evaluated using OV-90 subcutaneous xenografts.In this model, FRa expression is heterogeneous, with predomi-nantly only low to moderate intensity staining (H-score of 45)and approximately 65% of the cells being antigen-negative.Treatment with a single dose of 0.14 mg/kg ADC 2b resulted insignificant tumor growth delay. At two higher single doses (0.28and 0.56 mg/kg) the ADC was highly active causing CRs thatlasted 100 days in all animals (Fig. 5A). At thematching high dosetested (0.56 mg/kg), the nontargeting ADC 4b showed onlymodest tumor growth delay. These studies were extended toinclude the anti-EpCAM ADC 3b, using NCI-H2110 xenografts(Fig. 5B). EpCAM antigen expression in this model was alsoheterogeneous, with an H score of 160. The ADC displayeddose-dependent antitumor activity with 6 of 6 CRs, and allanimals being tumor-free at the end of the study (75 days), at

a dose of 0.15 mg/kg, which is approximately 40-fold lower thanthe MTD.

In order to assess the stability of the peptide linkage used inthe ADCs, the pharmacokinetics of ADC 2b were comparedwith that of the unconjugated anti-FRa antibody using radi-olabeled materials. In the conjugate, either the antibody com-ponent was [3H] labeled or the IGN molecule was [3H] labeled(Supplementary Fig. S7). The clearance of the ADC was com-pared with that of radiolabeled unconjugated antibody at adose of 0.27 mg/kg. The ADC was stable in circulation, as thepharmacokinetic profile of the ADC (measured by the [3H]IGNor [3H]-Ab) of the ADC was comparable with that of theunconjugated antibody for about 400 hours, after which timepoint the conjugate displayed more rapid clearance than theantibody (Fig. 6). Separately, the ADC was administered at ahigher dose (2.5 mg/kg) and the concentrations of the antibodyand IGN components were determined by ELISA. The clearancecurves were overlapping, thus providing further evidence ofhigh in vivo stability (Supplementary Fig. S9).

DiscussionA majority of ADCs currently in clinical evaluation use tubu-

lin-interacting compounds, such as the maytansinoids and aur-istatins, as their cytotoxic moieties (8). However, a recent shift in

Figure 3.

Single-dose ADC tolerability in CD-1 mice (8 animals mice per group). A, Vehicle control. B, A single intravenous injection of 6 mg/kg of alkylator ADC 5b.C, A single intravenous injection of 6 mg/kg of cross-linker ADC 5a. D, A single intravenous injection of 4 mg/kg of cross-linker ADC 5a.

Miller et al.





ADC design toward the use of payloads with alternative modesof action, such as topoisomerase 1 inhibition (camptothecins)and DNA cross-linking [pyrrolobenzodiazepines (PBDs)], hasbeen spurred in part by the emergence of promising earlyclinical data (18, 31–33). ADCs using PBD payloads aregenerally more potent in vitro and in vivo than those carrying

camptothecins or tubulin-interacting compounds (34, 35).This higher potency, however, comes at a cost of greatertoxicity, and doses achieved in the clinic have been consider-ably lower than that of the FDA-approved maytansinoid-containing ADC T-DM1. A low achievable dose of the ADCmay be insufficient to saturate the antigens in solid tumors,

Figure 4.

Bystander killing assessment. A,Comparison of bystander cell killingactivity of the anti-FRa ADC 2b (alkylator)with the corresponding cross-linking ADC2a, toward cocultured FRaþ and FRa�cells.B, Export of IGN catabolites 1a (cross-linker) and 1b (alkylator) from variouscell types. C, Export of catabolites fromOV-90 cells treated with ADCs 2a or 2b.

Figure 5.

In vivo antitumor activity IGN DNA alkylator ADCs. Day of dosing indicated by arrow. A, In vivo antitumor activity of the anti-FRa ADC 2b (single i.v. injection,0.14, 0.28, or 0.56 mg/kg) in comparison with the nontargeting ADC 4b in human ovarian adenocarcinoma OV-90 xenografts. B, Dose-dependent antitumoractivity of the anti-EpCAM ADC 3b (single i.v. injection, 0.075 and 0.15 mg/kg) in human lung adenocarcinoma NCI-H2110 xenografts.






particularly if there is normal tissue expression of the target(36, 37). In addition, because of intolerable side effects,treatment with PBD ADCs in clinical trials to date has beenlimited to doses 6 weeks apart (18).

We have previously reported the development of a new classof highly cytotoxic molecules, termed IGNs, consisting ofindolinobenzodiazepine dimers for use as effector moleculesin ADCs. These compounds share the same mechanism ofaction as the PBDs, yet exhibit greater potency in vitro (12).Our first set of ADCs prepared with the "diimine" cross-linkingIGN compound conjugated via a noncleavable linker exhibiteddelayed onset of prolonged body weight loss in mice (12).Accordingly, changing the mechanism of action from cross-linking to DNA alkylation, prevented this delayed toxicity. Inthe current study, we sought to develop ADCs that maintainhigh potency and good tolerability, but also display antitumoractivity toward tumors that express the antigen in a heteroge-neous manner, a common feature of tumors in patients. InADCs wherein the payload is linked via a cleavable linker, thereleased catabolite (if sufficiently hydrophobic) can diffuseinto neighboring cells and cause bystander killing of low ornonantigen expressing cells, or cells not directly accessible tothe ADC due to poor tumor vascularization. Thus, designingan ADC with good bystander killing is important for effectivekilling of heterogeneous or poorly vascularized tumors.

With this in mind, we designed a peptide linker that could becleaved by proteases to release a potent, hydrophobic, and cell-permeable IGNmolecule. An IGNmolecule that had an anilinogroup was selected for several reasons: (i) the IGN moleculebearing the anilino group displayed high potency toward apanel of cancer cell lines in vitro, with IC50 values in thepicomolar range; (ii) the anilino nitrogen of the IGN moleculewill be uncharged at physiological pH, thereby facilitatingdiffusion across cell membranes; (iii) the amino group allowsfor incorporation of a peptide linker; and (iv) the amide bondbetween the aromatic amine and the peptide linker is expectedto undergo cleavage by proteases in cells to release the parentIGN molecule, while being stable in circulation (26). From anin vitro screen of a panel of di- and tri-peptides, L-Ala-L-Ala wasfound to be most efficiently cleaved at the aromatic amide site.ADCs of cross-linking IGN and alkylator IGN payloads were

prepared with a cleavable peptide linker, and a head to headcomparison of the two versions was conducted. An ADC usingthis linker was found to be highly stable in vivo. Unexpectedly,the payload form capable of DNA alkylation displayed similarpotency to that of the cross-linker form. Even more striking wasour finding that the alkylator ADC showed a significantlyhigher level of bystander killing than the cross-linker versionat equivalent doses, which led to superior in vivo antitumoractivity in heterogeneous tumor models. Cellular processingstudies with the two ADCs revealed that the catabolite (iden-tified as the parent IGN anilino compound) from the alkylatorADC diffused out of the cell to a greater degree than the cross-linker, sufficient to account for the observed improvement inbystander killing and in vivo efficacy. It is reasonable to suggestthat the IGN alkylator binds less tightly to DNA in cells andthus can diffuse out more easily, and experiments to examinethis postulate are ongoing. Prominent bystander activity of IGNADCs may be beneficial for eradication of tumors with hetero-geneous target expression and/or with impaired ADC penetra-tion (low vascularization, high interstitial pressure etc.). Uponbinding to the target receptor, IGN-ADCs internalize andundergo lysosomal processing to generate IGN catabolites.These catabolites either bind to DNA, triggering cell-cycle arrestfollowed by apoptosis, or diffuse out of the cell into proximalcells causing bystander killing. Additional sources of IGN cata-bolites that can induce bystander killing maybe derived fromIGN molecules that have dissociated from DNA of live cells orcells that have undergone apoptosis. Thus, an IGN catabolitethat binds less tightly to DNA, such as the DNA alkylatormonoimine that can form only one covalent bond, is expectedto efflux more readily to induce better bystander killing activitythan the cross-linker that can form two covalent bonds.

Finally, it was found that the ADCbearing the cross-linker formof IGNwas approximately 2- to 3-foldmore toxic inmice than thealkylator version. When linked in a site-specific manner to engi-neered cysteines, the cross-linker ADC displayed delayed andprolonged toxicity at a dose where the alkylator ADC was welltolerated, similar to that previously observed for lysine-linkedADCs, indicating that the site and nature of the antibody–payloadlinkage does not influence the toxicity pattern of the IGN cross-linker ADCs.

Based on these findings, we conclude that the IGN alkylatorform provides a more efficacious and tolerable option forincorporation into ADCs, compared with cross-linking. In vivostudies in additional xenograft models with this form showeddose-dependent antitumor activity at doses well below theMTD. Overall, ADCs of the linkable IGN alkylator formrepresent a promising new therapeutic class for clinicalevaluation.

Disclosure of Potential Conflicts of InterestM.L. Miller and K.C. Lai have ownership interest (including patents) in

ImmunoGen. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: M.L. Miller, M. Shizuka, L. Harvey, D. Vitharana,J.F. Ponte, J. Pinkas, R.V.J. ChariDevelopment of methodology:M.L. Miller, M. Shizuka, P. Salomon, E.E. Reid,S. Sikka, L. Harvey, Q. Qiu, K.E. Archer, C. Bai, D. Vitharana, R. Singh, K.C. Lai,O. Ab, T.A. Keating

Figure 6.

Pharmacokinetic profiles. Comparison of pharmacokinetics in mice treatedwith 0.27 mg/kg of unconjugated anti-FRa [3H] antibody or anti-FRa ADC 2b,[3H]-labeled on the antibody or on the IGN component.


Miller et al.




Acquisition of data (provided animals, acquired and managed patients, pro-vided facilities, etc.): M. Shizuka, A. Wilhelm, P. Salomon, E.E. Reid, L. Lanieri,E.K. Maloney, Q. Qiu, K.E. Archer, C. Bai, L. Harris, N.C. Yoder, J. PinkasAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.L. Miller, M. Shizuka, A. Wilhelm, P. Salomon,E.E. Reid, L. Lanieri, E.K. Maloney, L. Harvey, K.E. Archer, D. Vitharana,N.C. Yoder, Y. Kovtun, O. Ab, T.A. KeatingWriting, review, and/or revision of the manuscript: M.L. Miller, M. Shizuka,P. Salomon, J.F. Ponte, Y. Kovtun, K.C. Lai, T.A. Keating, R.V.J. ChariAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M.L. Miller, M. Shizuka, E.K. Maloney,L. Harvey, C. Bai, D. Vitharana, K.C. LaiStudy supervision:M.L. Miller, M. Shizuka, A. Wilhelm, J. Pinkas, T.A. Keating,R.V.J. Chari

AcknowledgmentsWe thank Richard Bates for carefully editing the manuscript, and Richard

Gregory, Thomas Chittenden, Joseph Kenny, David Pleynet, and Sarah Kiely fortheir helpful suggestions, and Ashley Morneault for the IHC support. All workwas funded by ImmunoGen, Inc.

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.

Received September 22, 2017; revised November 15, 2017; accepted Decem-ber 28, 2017; published OnlineFirst February 13, 2018.

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Miller et al.




2018;17:650-660. Published OnlineFirst February 13, 2018.Mol Cancer Ther Michael L. Miller, Manami Shizuka, Alan Wilhelm, et al. (ADCs)

Drug Conjugates−Improves the Therapeutic Index of Antibody A DNA-Interacting Payload Designed to Eliminate Cross-Linking

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