Drug Conjugate against Mutant and Wild-type c-KIT Positive ... · Clin Cancer Res; 24(17); 4297–308. 2018 AACR. Introduction The c-KIT (CD117) receptor binds the ligand stem cell

Translational Cancer Mechanisms and Therapy

Preclinical Antitumor Activity of a NovelAnti–c-KIT Antibody–Drug Conjugate againstMutant and Wild-type c-KIT–Positive SolidTumorsTinya Abrams1, Anu Connor2, Christie Fanton1, Steven B. Cohen3, Thomas Huber4,Kathy Miller1, E. Erica Hong5, Xiaohong Niu1, Janine Kline1, Marjorie Ison-Dugenny1,Sarah Harris3, Dana Walker2, Klaus Krauser3, Francesco Galimi3, Zhen Wang1,Majid Ghoddusi1, Keith Mansfield2, Si Tuen Lee-Hoeflich2, Jocelyn Holash1, Nancy Pryer1,William Kluwe6, Seth A. Ettenberg2,William R. Sellers2, Emma Lees1, Paul Kwon1,Judith A. Abraham1, and Siew C. Schleyer1

Abstract

Purpose: c-KIT overexpression is well recognized in cancerssuch as gastrointestinal stromal tumors (GIST), small cell lungcancer (SCLC), melanoma, non–small cell lung cancer(NSCLC), and acute myelogenous leukemia (AML). Treatmentwith the small-molecule inhibitors imatinib, sunitinib, andregorafenib resulted in resistance (c-KIT mutant tumors) orlimited activity (c-KIT wild-type tumors). We selected ananti–c-KIT ADC approach to evaluate the anticancer activity inmultiple disease models.

Experimental Design: A humanized anti–c-KIT antibodyLMJ729 was conjugated to the microtubule destabilizingmaytansinoid, DM1, via a noncleavable linker (SMCC). Theactivity of the resulting ADC, LOP628, was evaluated in vitroagainst GIST, SCLC, and AML models and in vivo against GISTand SCLC models.

Results: LOP628 exhibited potent antiproliferativeactivity on c-KIT–positive cell lines, whereas LMJ729 dis-

played little to no effect. At exposures predicted to beclinically achievable, LOP628 demonstrated single admin-istration regressions or stasis in GIST and SCLC xenograftmodels in mice. LOP628 also displayed superior efficacyin an imatinib-resistant GIST model. Further, LOP628 waswell tolerated in monkeys with an adequate therapeuticindex several fold above efficacious exposures. Safetyfindings were consistent with the pharmacodynamic effectof neutropenia due to c-KIT–directed targeting. Additionaltoxicities were considered off-target and were consistentwith DM1, such as effects in the liver and hematopoietic/lymphatic system.

Conclusions: The preclinical findings suggest that thec-KIT–directed ADC may be a promising therapeutic for thetreatment of mutant and wild-type c-KIT–positive cancers andsupported the clinical evaluation of LOP628 inGIST, AML, andSCLC patients. Clin Cancer Res; 24(17); 4297–308. �2018 AACR.

IntroductionThe c-KIT (CD117) receptor binds the ligand stem cell factor

(SCF), resulting in receptor homodimerization and activation of

its tyrosine kinase activity along with cKIT internalization (1).Activation of c-KIT regulates cellular functions, including apopto-sis, differentiation, and proliferation (2, 3). Gain-of-functionmutations resulting in constitutive c-KIT activation play a central

1Novartis Institutes of Biomedical Research, Emeryville, California.2Novartis Institutes of Biomedical Research, Cambridge, Massachusetts.3Genomics Institute of the Novartis Institute Foundation, San Diego, California.4Novartis Institutes of Biomedical Research, Campus Klybeckstrasse, Basel,Switzerland. 5ImmunoGen Inc., Waltham, Massachusetts. 6Novartis Pharmaceu-ticals Corporation, East Hanover, New Jersey.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Current address for T. Abrams, S.T. Lee-Hoeflich, P. Kwon, A. Conner, D. Walker,and K. Mansfield: Novartis Institutes of Biomedical Research, Cambridge,Massachusetts; current address for S. Ettenberg, Unum Therapeutics;current address for C. Fanton, Nektar Therapeutics, San Francisco, California;current address for S.B. Cohen, Sirenas Marine Discovery, San Diego, California;current address for X. Niu, Roche, Pleasanton, California; current address forS. Harris, AnaptysBio, San Diego, California; current address for K. Krauser, Shire,Cambridge, Massachusetts; current address for Z. Wang, Siemens HealthcareDiagnostics, San Francisco, California; current address for M. Ghoddusi and

K. Miller, Five Prime Therapeutics, San Francisco, California; current address forE.E. Hong, Finnegan, Henderson, Farabow, Garrett & Dunner, LLP, Boston,Massachusetts; current address for J. Holash, BioClin Therapeutics, Emeryville,California; current address for N. Pryer, BioMarin Pharmaceutical Inc., San Rafael,California; current address for E. Lees, Jounce Therapeutics, Cambridge,Massachusetts; current address for S.C. Schleyer, Novartis Institutes ofBiomedical Research, Shanghai, China; current address for J. Kline, Dynavax,Berkeley, California; current address for F. Galimi: Amgen, ThousandOaks, California; and current address for W.R. Sellers: Broad Institute,Cambridge, California.

Corresponding Author: Tinya J. Abrams, Novartis Institutes for BioMedicalResearch, 250 Massachusetts Avenue, Cambridge, MA 02139. Phone: 617-871-3246; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-3795

�2018 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 4297

on June 8, 2021. © 2018 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 15, 2018; DOI: 10.1158/1078-0432.CCR-17-3795

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-17-3795&domain=pdf&date_stamp=2018-8-9http://clincancerres.aacrjournals.org/

pathogenic role in gastrointestinal stromal tumors (GIST), semi-nomas, subsets of melanoma, acute myelogenous leukemia(AML), and acute systemicmastocytosis (ASM; refs. 4, 5). Approx-imately 95% of GISTs express c-KIT, with the majority (75%–90%)harboring activatingmutations (6), leading to uncontrolledproliferation and resistance to apoptosis. Imatinib, a small-mol-ecule inhibitor of c-KIT, significantly reduces proliferation ofGISTcell lines, supporting the importance of c-KIT as a driver ofproliferation in subsets of GIST. The small-molecule inhibitorsexhibit preferences for selective c-KIT mutations (e.g., imatinib iseffective in GIST patients harboring exon 11 mutations), whilebeing less effective against secondary c-KIT mutations (leading toresistance in�50% of patients within 2 years) and remainmostlyinactive against diseases with wild-type c-KIT (7). Beyond GIST,wild-type c-KIT is overexpressed in small cell lung cancer (SCLC),AML, non–small cell lung cancer (NSCLC), melanoma, adenoidcystic carcinoma, and Merkel cell carcinoma (8).

Antibody–drug conjugates (ADC) leverage the specificity ofmonoclonal antibodies to deliver highly potent cytotoxicagents to antigen-positive tumor cells. Here, we describe a novelc-KIT targeting ADC, LOP628, consisting of the cytotoxicmaytansinoid, DM1, covalently linked via N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) tolysine residues of the antibody LMJ729, which is the samelinker-payload utilized by the HER2-targeting ADC, Kadcyla(Trastuzumab-DM1; refs. 9, 10). Upon binding to c-KIT, LOP628becomes internalized and lysosomally processed to yield theactive catabolite Lys-Ne-MCC-DM1, resulting in cell-cyclearrest (11, 12) and potent activity against mutant and wild-typec-KIT–positive tumor models. This demonstration of compellingactivity in models of SCLC, AML, and GIST (with single anddouble c-KIT mutations) suggests that LOP628 has the potentialfor treating patients with these diseases.

Materials and MethodsMaterials

The murine anti–c-KIT antibody 9P3, humanized IgG1/kanti–c-KIT antibody LMJ729 (derived from 9P3), and the

isotype control human IgG1/k antibody (IgG1) were generatedat Novartis. The anti–c-KIT and the IgG1 isotype control anti-bodies were directly conjugated to allophycocyanin (APC). TheAPC–fluorochrome-conjugated anti-human IgG antibody wasfrom Southern Biotechnology, # 9042-11. Conjugation ofantibodies to DM1 was performed using the cross-linking agentN-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) at ImmunoGen, Inc. Membrane-permeable maytan-sine (L-DM1-Me) was kindly provided by ImmunoGen, Inc.

Cell lines were purchased from ATCC [NCI-H526 (CRL-5811),NCI-H1048 (CRL-5853), MDA-MB-468 (HTS-132), MDA-MB-453 (HTB-131), KU812 (CRL-2099), Kasumi-6 (CRL-2775),Kasumi-1 (CRL-2724), NCI-H2170 (CRL-5928)], or DSMZ[Mo7e (ACC-104)]. NCI-H1048 cells were obtained from theBroad Institute–Novartis Cancer Cell Line Encyclopedia collec-tion. The GIST-T1 line with a heterozygous KIT 52-bp deletion inexon 11 was provided by Dr. Taguchi from Kochi University,Japan (13), while GIST 882 with a homozygous missense muta-tion in KIT exon 13 (14) and GIST430 with a heterozygous KITexondeletion andaheterozygousKIT exon13V654A substitution(15) were obtained from Dr. Fletcher (Brigham and Women'sHospital, Boston, MA). Cell lines were authenticated by single-nucleotide polymorphism (SNP) fingerprinting (Sequenom) aspreviously described (16), and KIT mutations were confirmedwith RNA sequencing and deemedmycoplasma free (MycoplasmaPCR assay, IDEXX).

Internalization by flow cytometryCell lines used for internalization studies were suspended in

SF-RPMI (serum-free RPMI, 0.02% BSA, 1% PSG). Antibodieswere used at 1 mg/mL in SF-RPMI containing 75 mg/mL cyclo-heximide (Sigma-Aldrich) and mixed with cells for 45 minutesat 4�C. Cells were washed and samples resuspended in 100 mLof SF-RPMI with 75 mg/mL cycloheximide. One set of plates wasincubated at 4�C, while the other at 37�C for 30 minutes, 2, or4 hours. Cells were incubated with an APC-conjugated anti-human IgG for 45 minutes. For plates with SCF, the ligand wasadded at a final concentration of 10 ng/mL for 5 minutes atroom temperature prior to adding antibodies and transferringplates to 4�C.

c-KIT internalization and trafficking to the lysosome by AmnisKU812 cells were incubated with 1 mg/mL PE-labeled LMJ729

and an antibody against a lysosomal marker, LAMP1 (APC-conjugated CD107, BD Biosciences, 560664) for 30 minutes at4�C. Cells were washed to remove unbound antibodies. c-KITprotein levels, internalization, and colocalization with CD107were monitored for up to 40 minutes at 37�C using the AmnisImageStreamX instrument.

Western blots to determine the effect of LOP628 on c-KITdegradation

GIST-T1 or NCI-H526 cells were seeded in growth media(DMEM, 10% FBS or RPMI, 10% FBS, respectively) overnight,then treated with 100 mmol/L cycloheximide (Sigma) in methi-onine-freemedium (GIBCO:DMEM, 21013-024; RPMI, A14517-01). Additionally, cells were either treated with 5 mg/mL ADC(LOP628), 10 ng/mL rh-SCF (R&D Systems, 255-SC), or both for1, 4, or 6 hours at 37�C, at which time protein was isolated,denatured, and 5 mg loaded on a NuPAGE 4% to 12%Bis–Tris gel(Life Technologies). After protein transfer, membranes were

Translational Relevance

Constitutively activating mutations render c-KIT an onco-genic driver in diseases such as gastrointestinal stromal tumors(GIST) and acute systemicmastocytosis (ASM). Current small-molecule therapeutics are limited as they demonstrate prefer-ential activity against exon 11 mutations, resulting in second-ary c-KIT mutations that arise as the predominant resistancemechanism. Because the majority of mutations localize to theintracellular domain of c-KIT, an antibody directed against anextracellular epitope is predicted to bindmutant andwild-typec-KIT equally well. An anti–c-KIT antibody–drug conjugate(ADC), whose activity depends primarily on the cytotoxicmoiety, can potentially be efficacious against cancersexpressing mutant and wild-type c-KIT, as well as in ligand-dependent and -independent settings. This study providesproof of concept that an anti–c-KIT ADC, LOP628, can effec-tively treatmutant andwild-type c-KIT–positive cancermodelsand supports its potential clinical utility.

Abrams et al.

Clin Cancer Res; 24(17) September 1, 2018 Clinical Cancer Research4298



http://clincancerres.aacrjournals.org/

blocked inTBST-5%milk andprobedwith an anti–c-KIT antibody(Cell Signaling Technology, 3074) or GAPDH antibody (CellSignaling Technology, 3683). The secondary antibody (goat-antirabbit-HRP 1:30,000; Santa Cruz) was used.

In vitro cytotoxicity assaysAfter seeding cells overnight in standardmedia, Maytansine (L-

DM1-Me), LOP628 or a nontargeting ADC control (IgG-ADC)was added. In certain experiments, LMJ729 or IgG1 was included.The starting concentration of LOP628 and IgG-ADC was68 nmol/L. A 1:5 dilution was performed for 9 concentrationpoints. After 5 days, viability was assessed using CellTiter Glo.Luminescent counts from untreated cells (100% viability) wereused to normalize treated samples. The concentrations of treat-ment resulting in 50% of cells remaining were calculated usingnonlinear regression analysis (GraphPad Prism 6).

Receptor density (flow cytometry)Human and cynomolgusmonkeyCD34þ bonemarrowmono-

nuclear cells were evaluated for c-KIT expression by flow cyto-metry using anti–c-KIT antibody 104D2 (BD Biosciences,563856). Cells were suspended in FACS buffer (PBS, 2% FBS,0.1% sodium azide) and incubated with 104D2 at 10 mg/mL for60minutes. Sampleswere assayed using FACS (BDFACSCanto II)with a microsphere bead set (Quantum Simply Cellular anti-Mouse IgG kit; Bangs Labs) to determine the specific antibodybinding capacity (sABC) of each antibody.

c-KIT immunohistochemistryc-KIT expression in formalin-fixed, paraffin-embedded (FFPE)

tumor xenografts or human patient samples (Novartis tissuearchive) was evaluated by immunostaining (Dako, A4502)including heat and standard exposure to Ventana Cell Condi-tioning #1 antigen retrieval reagent. The primary antibody wasdiluted to 14 mg/mL and incubated for 60 minutes at roomtemperature. Subsequently, incubation with Ventana UltraMapprediluted HRP-conjugated anti-rabbit antibody (760-4315) wasperformed for 16 minutes.

An H-score of c-KIT immunostaining was generated to reflectthe expression and heterogeneity levels in tumors, using theformula: [(% of 1þ � 1) þ (% of 2þ � 2) þ (% of 3þ � 3)],where 1þ designates weak, 2þ designates moderate, and3þ designates strong staining, resulting in a range of 0 to 300.An H score >150 correlates to �50% of the cells exhibiting 2þ to3þ staining intensity.

In vivo studies in miceFemale SCID-beige mice (Harlan Laboratories) used for tumor

xenograft studies were handled in accordance with NovartisInstitutional Animal Care and Use Committee regulations andthe ILAR Guide for the Care and Use of Laboratory Animals in anAAALAC accredited facility.

GIST-T1 cellswere grownasdescribedabove.GIST430 cellsweregrown in Iscove's modified DMEM (Cellgro), 15% FBS (OmegaScientific Inc.), and 2 mmol/L L-glutamine (Cellgro). NCI-H1048andNCI-H2170 cells were grown in RPMI-1640medium (ATCC),10% FBS, and 2 mmol/L L-glutamine. Cells were subcutaneouslyimplanted in 50% Matrigel (BD Biosciences).

Pharmacodynamic studyIn the GIST T1 model, LOP628, its vehicle (10 mmol/L

Tris–hydrochloric acid, 80 mmol/L sodium chloride, 3.5%

sucrose, 0.01% Tween 20 pH7.5), or isotype control IgG-ADCwere administered as a single intravenous (i.v.) injection to micewith tumors collected at specified time points after dosing (Fig. 3Aand B) and processed to generate FFPE blocks.

The IHC protocol for assessing phospho-histone H3-positivenuclei included heat and standard exposure to Ventana CellConditioning #1 antigen retrieval reagent. The primary antibody(Cell Signaling Technology, 9701) was diluted 1:50 and incubat-ed for 60minutes. Subsequently, incubation with Jackson Immu-noResearch Laboratories goat anti-rabbit biotinylated secondaryantibody (111-065-144) was performed. For quantification ofpositive cells, tumor section images were quantified using theAperio Nuclear (version 9) image analysis algorithm on theAperio ScanScope XT system (Leica Biosystems), which includedImageScope version 11.1.2.760 software. Three tumors per timepoint were assessed.

Efficacy studiesIn vivo efficacy studies were initiated when tumors were

�200 mm3. LOP628, IgG-ADC, or LMJ729 were administeredi.v. Orally administered imatinib (Glivec) was formulated in 5%dextrose in water. Tumors were measured twice weekly, andvolumes were calculated as (length � width2)/2. Data areexpressed as the percent tumor growth inhibition (TGI) in volumeof the treated group from initial divided by the change in tumorvolume of the vehicle control group from initial (%T/C)–100%.Between-groups comparisons of final measurements were per-formed using ANOVA and a post hoc test (Sigma Plot, SystatSoftware Inc.)

In vivo studies in cynomolgus monkeysThe toxicity of LOP628 was evaluated in non-GLP and GLP

studies in cynomolgusmonkeys (Charles River Laboratories)withintravenous administration once every 3 weeks. In the dose rangefinding study, doses were 3, 10, and 30 mg/kg for a 6-weekduration and 6-week recovery period. This study included anontargeting IgG-ADC at 30 mg/kg. LOP628 doses for the GLPstudy were 3, 8, and 20 mg/kg for approximately a 3-monthduration. In addition to assessment of several standard safetyparameters, cardiovascular telemetry was incorporated usingjacketed telemetry devices.

A single-dose nonterminal cynomolgus monkey study wasconducted to investigate previously observed infusion reactionsas well as the potential for mast cell degranulation. LOP628was administered to 3 monkeys intravenously at 0.3, 1, 8, and20mg/kg. Animals were monitored for serum tryptase, cytokines,and complement analysis.

Resultsc-KIT is overexpressed in numerous tumor types, such as

melanoma, SCLC, AML, andGIST (Supplementary Fig. S1).Whileconsidered essential for many processes, such as the function ofthe interstitial cells of Cajal, in hematopoiesis andmelanogenesis(17–20), no evidence exists that suggests tumors overexpressingwild-type c-KIT are dependent on c-KIT/SCF signaling for survival.Tumors harboring c-KIT–activating mutations signal indepen-dently of SCF. Due to this, we investigated the activity of bothligand-blocking (antagonist with strong signaling inhibition) andnon–ligand-blocking anti–c-KIT ADCs (weak signaling inhibi-tion, possibly via steric hindrance; Supplementary Fig. S2).

ADC Cancer Therapeutic Targeting c-KIT

www.aacrjournals.org Clin Cancer Res; 24(17) September 1, 2018 4299




Antibodies and ADCs in this publication and their properties arelisted in Table 1. The ADCs generated were conjugated viaImmunoGen, Inc. Technology to –SMCC-DM1, the noncleavablelinker and maytansine-derived toxin.

c-KIT antibodies rapidly internalize upon binding wild-type ormutant c-KIT

To understand differences between the activities of ligand-blocking versus non-ligand-blocking anti–c-KIT antibodies, inter-nalization and degradation of c-KIT upon treatment with thedifferent antibody classes were evaluated. Internalization (mea-sured as the loss of surface c-KIT by flow cytometry) upon bindingof c-KIT antibodies was interrogated in lines expressing mutantc-KIT (GIST-T1, exon 11 mutation) or wild-type c-KIT (M07e). InGIST-T1, there was no significant difference in internalizationkinetics between LMJ451 (ligand-blocking antibody) andDYL884 or LMJ729 (non–ligand-blocking antibodies) within a4-hour time course (Fig. 1A). However, the internalization ratemediated by LMJ451 appeared diminished relative to DYL884 inthe M07e cell line, and this difference was enhanced in thepresence of SCF within a 2-hour time course (Fig. 1A). Coincuba-tion of SCF with DYL884 accelerated internalization, confirmingDYL884 did not block SCF binding to c-KIT. In contrast, theaddition of SCF did not impact internalization upon binding ofLMJ451, due to the ability of LMJ451 to inhibit binding of SCF toc-KIT. Once internalized, the c-KIT/antibody complex rapidlytrafficked to the lysosome (Fig. 1B), as seen in the costaining ofthe complex with the lysosomal marker CD107/LAMP1 in theKU812 line.

c-KIT turnover is differentially affected by treatment withligand-blocking vs. non–ligand-blocking ADCs

c-KIT is efficiently degraded both in the wild-type and mutantforms, as shown in Fig. 1C. Inhibition of new protein synthesiswith cycloheximide treatment allows for the monitoring of deg-radation of the steady-state c-KIT pool. Treatmentwith the isotypeADC was ineffective, suggesting the impact on c-KIT degradationis dependent on c-KIT ADC binding. The degradation of mutantc-KIT, detected in the GIST-T1 line, appears unaffected by SCFtreatment orby the additionof a ligand-blockingADC(GZQ167).In contrast, incubation with the non–ligand-blocking ADC(LOP628) accelerated the degradation of c-KIT and cotreatmentof SCF with LOP628 further enhanced the degradation rate. Asimilar finding was observed in the wild-type c-KIT line,NCI-H526, as the addition of LOP628, SCF, or LOP628 with SCFaugmented the c-KIT degradation rate, with the combinationbeing the most efficient. The addition of GZQ167 exhibited noimpact on its own, but when combined with SCF, the presence ofthe ligand-blocking antibody ameliorated the SCF effects. Theseresults suggest that treatment with a non–ligand-blocking ADC

may enhance degradation of the c-KIT/ADC complex similar tothat of SCF treatment, resulting in efficient trafficking to thelysosome and payload release, allowing for multiple rounds ofADC internalization. In contrast, a ligand-blocking ADC wouldutilize the intrinsic turnover rate of c-KIT and decrease the deg-radation rate mediated by the addition of SCF to wild-type c-KIT.This could cause less efficient release of the active catabolite anddecreased activity.

c-KIT ADCs demonstrate target-dependent in vitro activity in apanel of cancer cell lines

We next profiled the activity of the ligand-blocking and non–ligand-blocking ADCs in cell lines representing c-KIT expressingindications. Several ADCs from each class were tested in GIST(GIST882, GIST430, and GIST-T1), AML (Kasumi-1 andKasumi-6), and SCLC (NCI-H526 and NCI-H1048) lines. Ac-KIT–negative line, MDA-MB-453, was included as a negativecontrol. The c-KIT ADCs from both classes demonstrated similarantiproliferative activity with EC50 values in the subnanomolarrange, which represented a 3- to >1,000-fold difference in activityover the negative cell line. Similar activity was not surprising, asthese cell lines are not dependent on SCF for growth (Supple-mentary Table S1).

A ligand-blocking c-KIT ADC demonstrates suboptimal efficacyand tolerability

To extend our comparison of ligand-blocking and non–ligand-blocking c-KIT ADCs in vivo, an efficacy study (single 0.625mg/kgdose) in theGIST-T1 xenograftmodel inmice was performed. Thenon–ligand-blocking LOP628 trended toward increased efficacycompared with the ligand-blocking candidates and was the onlytreatment statistically different from the isotype control IgG-ADC(Fig. 2A). The observation of efficacy at such a low dose wasconsistent with the in vitro potency andmay relate to c-KIT's rapidinternalization and lysosomal processing.

Given the role of c-KIT in hematopoiesis (3), there was concernthat a ligand-blocking antibody may induce severe bone marrowtoxicity based on published data describing hematopoietic failurewith conditional deletion of c-KIT in adultmice (21). To assess thetolerability, we evaluated two mouse cross-reactive ADCs,LMJ451 (ligand-blocker) and LPG167 (non–ligand-blocker).After a single 10 mg/kg i.v. administration, LPG167 was welltolerated, while the mice treated with LMJ451 showed severeweight loss �13 days after treatment (Fig. 2B). Bone marrowhistology revealed severe hypocellularity due to loss of hemato-poietic cells of all lineages (Fig. 2C) as compared with that of anuntreated mouse (Fig. 2D). These findings, in addition to in vivoscreening of non–ligand-blocking ADCs and the in vitro data(Fig. 1), led to the selection of the non–ligand-blocking antibodyLMJ729, which when conjugated to SMCC-DM1 is designatedLOP628.

LOP628 induces cell death in c-KIT overexpressing cancer celllines

LOP628 demonstrates similar binding affinities to human andcynomolgus c-KIT extracellular domains, at 6.9 and 5.1 nmol/L,respectively. Examples of in vitro studies performed to address theactivity of LOP628 are shown in Fig. 3A. Cell lines devoid of c-KIT(MDA-MB-468), expressing wild-type c-KIT (NCI-H526) or asingle mutation in c-KIT (GIST-T1) were tested for inhibition ofproliferation mediated by LMJ729 or LOP628. The robust

Table 1. Nomenclature and characteristics of antibodies and ADCs used in thepaper

Ab�/ADC Ligand blocker Rodent cross-reactive AbDLY884� No NoLMS359�/DSF604 Yes NoGZQ167 Yes NoLPG166 No NoLQS721 No NoLMJ729�/LOP628 No NoLPG167 No YesLMJ451�/QYK155 Yes Yes

Abrams et al.





CGIST-T1 NCI-H526

UT CHX IgG-ADC SCFSCF +

IgG-ADC

6410 6661 1 14 4 4

UT CHX LOP628 SCFSCF +

LOP628

4610 1 1 14 4 4 666

UT CHX IgG-ADC SCFSCF +

IgG-ADC

0 1 1 1 14 4 4 46 6 6 6

GAPDH

Hoursc-KIT

SCF + LOP628SCFLOP628CHXUT

0

GAPDH

c-KIT

Hours0 111 14 4 4 46 666

Hrs

GAPDH

c-KIT

UTUT CHX GZQ167 SCFSCF +

GZQ167 CHX SCFSCF +

GZQ167

0 1 1110 4444 6 666GZQ167

1 4 6 1 4 6 1 4 6 1 4 6 Hours

LAMP1 LMJ729/LAMP1LMJ729

5−10,

40,

0,

LMJ451

LMJ729DYL884

BGIST-T1 (-SCF)

Timecourse

80

60

40

20

0

100 %

Inte

rnal

izat

ion

30 min 2 hr 4 hr

Mo7e (-/+SCF)100

80

60

40

20

0

% In

tern

aliz

atio

n

DYL884DYL88430 min 2 hr

LMJ451LMJ45130 min 2 hr

Timecourse

-SCF

+SCF

A

Figure 1.

Rapid internalization, trafficking and efficient degradation of c-KIT. A, Internalization studies were performed with a ligand-blocking Ab (LMJ451) or withnon–ligand-blocking antibodies (DYL884, LMJ729) on a mutant c-KIT cell line (GIST-T1) or a wild-type c-KIT cell line (Mo7e). The Mo7e cells were also treatedin the presence or absence of SCF. Cells were treated for 45 minutes at 4�C, washed to remove antibodies and subsequently incubated up to 4 hours at 37�C.Loss of cell surface expression of c-KITwas detected by flow cytometry.B,KU812 cells were stainedwith the anti–c-KIT Ab (LMJ729) and the anti-LAMP1 (CD107) Abat 4�C, washed to remove unbound antibodies and subsequently incubated at 37�C for up to 40 minutes. Binding and internalization were assessed at 0, 5–10,and 40minutes. C,GIST-T1 and NCI-H526 cells were treated with IgG-ADC (IgG-SMCC-DM1), LOP628 (non–ligand-blocking ADC), or GZQ167 (ligand-blocking ADC)for 0, 1, 4 or 6 hours in the absence or presence of SCF. Cycloheximide (CHX) was added in all treatment groups except for the untreated sample. c-KITlevels and a loading control (GAPDH) were detected by Western blot.






antiproliferative activity of LOP628 appears to be DM1 driven asLMJ729 exhibited no activity on NCI-H526 or GIST-T1 lines.LOP628 activity appears target dependent as there is no specificactivity on the c-KIT–negative line. To determine if theremight bea correlation between c-KIT surface expression and sensitivity toan ADC, a cell line panel was treated with the conjugated mousehybridoma antibody, 9P3-SMCC-DM1, from which humanizedLMJ729 was derived. When cell lines were sensitive to the may-tansinoid payload (IC50 < 1 nmol/L), a minimum of 20,000receptors appeared to enrich for sensitivity to a c-KIT ADC in vitro,yielding low single-digit nanomolar IC50 values (SupplementaryFig. S3).

Assessment of a mitotic arrest PD marker in a c-KIT–positivexenograft tumor model

A study was conducted to assess the degree and duration ofLOP628 treatment to induce mitotic arrest by measuringthe accumulation of cells positive for the mitotic markerphosphorylated Histone H3 (pHH3) conferred by the maytansi-noid toxin in themutant c-KIT–expressingGIST T1model inmice.

LOP628 yielded a time-dependent increase in the percentage ofpHH3-positive cells, with a peak of 30% at 48 hours after doserelative to the isotype IgG-ADC or untreated controls and decreas-ing thereafter (Fig. 3B and C). These data suggest that the highinitial exposure over the first several days drives a significantportion of the mitotic arrest and demonstrate that LOP628 iscapable of eliciting a robust PD response in tumor xenografts,consistent with the mechanism of the toxin.

Potency of LOP628 against GIST and SCLC xenograft modelsTo elucidate if the ability of LOP628 to mediate mitotic arrest

translates into in vivo efficacy, studies were conducted using GIST(high mutant c-KIT levels) and SCLC (wild-type moderate c-KITlevel) models (Supplementary Fig. S4).

LOP628 efficacy in the GIST-T1 (exon 11 mutation; imatinib-sensitive) and GIST430 (exon 11, 13 mutations; imatinib-insen-sitive) xenograft models is shown in Fig. 3D and E. In the GIST-T1study, a single 2.5 mg/kg LOP628 dose induced tumor stasis�60 days, with subsequent regrowth. Imatinib alone regressedtumors, but tumors grew back upon treatment cessation. In

DC

B

0 5 10 15 20-20

-10

0

10

20

VehicleIgG-ADCLPG167LMJ451

Days post doseDosed

10 mg/kg

A

10 20 30 40 500

200

400

600

800

1,000

1,200

1,400

VehicleIgG-ADC

LOP628*

DSF604-DM1

GZQ167-DM1

Days post-implantDosed

0.625 mg/kg

Ligand blockers

Tum

or v

olum

e (m

m3 )

mea

n ±

SEM

Chan

ge in

bod

y w

eigh

t (%

)m

ean

± SE

M

Figure 2.

Comparison of activity and toxicity between SCF ligand-blocking and non–ligand-blocking ADCs. A, Statistically significant efficacy observed with thenon–ligand-blocking LOP628, but not the ligand-blocking ADCs (DSF604 and GZQ167) as compared with the isotype control IgG-ADC after a single i.v. doseof 0.625 mg/kg in the GIST-T1 model grown subcutaneously in female SCID-beige mice. Mean tumor volumes � SEM are plotted. � , P < 0.05, ANOVA followedby post hoc Dunn method. B, The mouse-cross reactive ligand-blocking ADC LMJ451 induced significant weight loss in all mice 13 days after a single 10 mg/kg i.v.administration. The mouse cross-reactive non–ligand-blocking ADC LPG167 showed no on-target weight loss as compared with the nontargeting isotypecontrol IgG-ADC.C, Themouse cross-reactive ligand-blocking ADC LMJ451 induced significant hypocellularity in the bonemarrow 13 days after a single 10mg/kg i.v.administration, as evident from the depletion of hematopoietic cells in the bone marrow as compared with bone marrow from an untreated mouse(D; H&E stain, Sternum, 2�).

Abrams et al.





B

2 day1 day

Vehicle

3 day 5 day 7 day

LOP628 5 mg/kgUpper corners of images: % positive nuclei

p-Histone H3 Immunostain

A

VehicleIgG-ADCLOP628

20 4 6 8 10

Days post-doseDosed

0

35

30

25

20

15

10

5

% C

ells

pos

itive

for p

-His

tone

H3

MDA-MB-468

LOP628

LMJ729

IgG-ADC

IgG

GIST-T1NCI-H526%

of S

urvi

ved

cells

% o

f Sur

vive

d ce

lls

Ab or ADC (nmol/L) Ab or ADC (nmol/L)

% o

f Sur

vive

d ce

lls

Ab or ADC (nmol/L)

140

120

100

80

60

40

20

01001010.10.010.0010.00010.00001

01000.00001

01001010.10.010.0010.00010.00001

0

140

00.0010.0001 0.01 0.1 1 10

80

60

40

20

100

120

140

120

100

80

60

40

20

C

F

0200400600800

1,0001,2001,4001,6001,800

10 4015 20 25 30 35

Days post-implantation Dosed

VehicleIgG-ADC 10 mg/kg

LOP628 5 mg/kg*LOP628 10 mg/kg*

LOP628 2.5 mg/kg

VehicleIgG-ADC 10 mg/kg

LMJ729 10 mg/kg (Unconjugated LOP628)LOP628 5 mg/kgLOP628 10 mg/kg*Imatinib 80-100 mg/kg BID*

ADC

0200400600800

1,0001,2001,4001,6001,800

10 15 20 25 30 35 40 45 50Imatinib

100 mg/kgDays post-implantation

Imatinib80 mg/kg

GIST-T1

00 50 100 200150

ImatinibDays post

treatment initiationADC

Vehicles combined2.5 mg/kg IgG-ADC + 80 mg/kg Imatinib BID2.5 mg/kg LOP628 + Vehicle BID2.5 mg/kg LOP628 + 80 mg/kg Imatinib BID

400

1,800

1,6001,4001,2001,000

800600

200400T

umor

vol

ume

(mm

3 )m

ean

± S

EM

Tum

or v

olum

e (m

m3 )

mea

n ±

SE

M

Tum

or v

olum

e (m

m3 )

mea

n ±

SE

M

D

E

Figure 3.

Potent and selective activity of LOP628 in vitro and in vivo models. A, c-KIT–positive (NCI-H526, GIST-T1) and c-KIT–negative (MDA-MB-468) cell lines weretreated with a dose titration of IgG, LMJ729, IgG-ADC, or LOP628 in a 5-day proliferation assay. Representative experiments are shown here. B, Robust PDresponse with LOP628 in the GIST-T1 xenograft model. Tumors were established and a single 5 mg/kg i.v. dose of IgG-ADC or LOP628 was administered,with tumors collected at 1, 1.35, 2, 3, 4, 5, 6, 7, and 10 days after dose and immunostained to detect phospho-histone H3, representative images ofimmunostained tumors shown. C, The percentage of tumor cells positive for phospho-histone H3 in 3 tumors per time point is graphed. D, GIST-T1 xenograftmodels were established in female SCID-beige mice and when tumors reached approximately 200 mm3, mice were randomized according to tumor volumeinto treatment groups (n ¼ 8 or 9). Animals were administered a single i.v. injection of the vehicle, IgG-ADC, or LOP628; twice daily 80 mg/kg oral doses ofimatinib or the combination of LOP628 with imatinib. E, The GIST430 efficacy study was set up as with the GIST-T1 (B) and treated with the same singleagents, along with the unconjugated LOP628 antibody (LMJ729). Imatinib was dose reduced from 100 to 80 mg/kg because of weight loss (not shown).Tumor volumes of treatment groups versus days after implantation are graphed. The 10 mg/kg LOP628-treated group was statistically different from thevehicle control group (� , P < 0.05, ANOVA followed by post hoc Dunnmethod). F,NCI-H1048 SCLC xenograft model was established as with the GIST-T1 model (B).After a single LSZ102 administration at 2.5, 5, and 10 mg/kg, the treated groups at 5 and 10 mg/kg were statistically different from the vehicle-treated group(� , P < 0.05, ANOVA, Tukey test).






contrast, the single 2.5 mg/kg LOP628 administration combinedwith imatinib (80 mg/kg b.i.d. for 50 days) resulted in completeand durable regressions with no tumor regrowth 130 days afterimatinib treatment ended.

To achieve a similar efficacy level as in GIST-T1, 10 mg/kgLOP628 was required in the GIST430 model, demonstrating thedifferential sensitivities among models. There was 41% and 81%TGI (day 28) as compared with the vehicle control for the 5 and10 mg/kg LOP628 doses, respectively. The efficacy of 10 mg/kgLOP628 was superior to imatinib administered at its highesttolerated dose, which induced 53% TGI. Imatinib was reducedfrom 100 to 80 mg/kg due to weight loss at the higher dose level(Fig. 3E). LOP628 activity in the NCI-H1048 SCLC xenograftmodel (Fig. 3F), with lower and more heterogeneous c-KIT levels(Supplementary Fig, S4), required 5mg/kg to induce tumor stasis,while 15% tumor regression was observed at 10 mg/kg (day 27).LOP628 was ineffective in the NCI-H2170model expressing verylow c-KIT levels (Supplementary Fig. S5).

c-KIT expression in xenograft models and patient samplesH-score analyses of c-KIT expression by IHC in tumor

xenograft models and tissue biopsies from SCLC, melanoma,

and GIST patients demonstrate that xenograft models sensitiveto LOP628 express c-KIT at levels observed in cancer patients(Fig. 4). The efficacy data suggest that focusing on cancers withan H-score of 160 or greater, corresponding to �50% of cellsexhibiting 2þ to 3þ staining intensity, may enrich for patientsmore likely to benefit from LOP628 treatment (Fig. 4B);however, due to the limited number of c-KIT–positive modelsin these indications, the data set is small for making efficacypredictions.

LOP628 safety findings in cynomolgus monkey bone marroware consistent with c-KIT–specific targeting

c-KIT is expressed on mast cells and a subset of bone marrowhematopoietic stem cells (22). Evaluation of c-KIT levels onbone marrow mononuclear cells (IHC and FACS) and mastcells (IHC) from human and cynomolgus monkey confirmedsimilar expression levels (Supplementary Table S2) and the useof cynomolgus monkey as a relevant species for a safetyassessment of LOP628.

To understand the contribution of c-KIT–mediated toxicityto the safety profile of LOP628, a dose range finding study incynomolgus monkeys was conducted, where both LOP628 andan isotype control IgG ADC were evaluated at a matched doselevel of 30 mg/kg. Animals were dosed every 3 weeks and wereeither necropsied 1 week after last dose administration or aftera 6-week recovery period. Overall, the toxicity profiles weresimilar across both targeting and nontargeting ADCs. Therewere limited clinical signs with the exception of a single animaldosed at 30 mg/kg LOP628. After the first administration, thisanimal exhibited lethargy and hypothermia among othereffects which resolved within 48 hours. The most prominentchanges at all dose levels were noted in the bone marrowand consisted of transient, dose-dependent decreases in redcell mass (hemoglobin, hematocrit, red blood cells) withcompensatory reticulocyte response and decreased neutrophilcounts (Fig. 5B, panels B–E). Hematology changes were ofgreater magnitude with LOP628 as compared with the isotypecontrol, most likely due to the contribution of target-mediatedtoxicity in the bone marrow. The nadir for neutropenia gen-erally occurred 3 weeks after dose administration (days 35and 56), which was immediately before the next dose admin-istration followed by recovery during the week after the nextdose administration (Fig. 5B, panel C). Increased liver enzymeswere noted with both ADCs (Fig. 5B, panel A) to a similarseverity and most commonly noted preclinically as an off-target effect related to the payload, DM1. Similarly, micro-scopic changes 1-week after last dose administration in thebone marrow consisted of a mild decrease in bone marrowcellularity at 30 mg/kg LOP628 compared with vehicle(Fig. 5A). A dose level of 30 mg/kg LOP628 exceeded themaximum tolerated dose due to progressively declining hemo-globin concentrations.

In a subsequent phase I-enabling Good Laboratory Practices(GLP) cynomolgusmonkey study, LOP628was dosed at 3, 8, and20 mg/kg. LOP628-related changes were dose dependent andconsisted of effects in the bone marrow and serum chemistrychanges in the liver of similarmagnitude to the dose range findingstudy. Additional clinical monitoring implemented in the GLPstudy indicated that infusion reactions were noted in animalswithin 5minutes of dosing and were prevalent at 8 and 20mg/kgLOP628. These clinical observations included decreased blood

B

Avg.

IHC

H-S

core

SCLC

H-score rangeof cell line xenograftmodels whereLOP628/9P3* variants

AdenoCa SCC

*9P3 = parental antibody of LMJ729

MelanomaGIST

Lung

Color by % 2+/3+ IHC score:>50 160 responded to a c-KIT ADC. Asemiquantitative H-score of c-KIT immunostaining was generated using theformula: H-score ¼ [(% of 1þ � 1) þ (% of 2þ � 2) þ (% of 3þ � 3)],where 1þ designates weak staining, 2þ designates moderate staining, and3þ designates strong staining, resulting in a range of 0 to 300. B, Multiplecancer types express c-KIT at levels equivalent to those in tumor xenograftmodels in which LOP628 was efficacious.

Abrams et al.





pressure, increased heart rate, excessive scratching and facialreddening and resolved within 20 minutes after dose adminis-tration. Upon repeat dosing on day 22, clinical responses were

limited to emesis, hypoactivity, and cardiovascular changes andwere noted most prominently at 3 and 8 mg/kg LOP628, butresolved within 2 hours after dose administration. These

Figure 5.

Reversible hematotoxicity in the cynomolgusmonkey toxicity study. A, Effect of LOPtreatment on the cynomolgusmonkey liver andhematology indices after dosing LOP628 andIgG-ADC at matched dose levels (30 mg/kg).B, Impact of LOP628 on the bone marrow at 30mg/kg compared with vehicle control (inset;H&E stain). C, Serum tryptase levels afterLOP628 dose administration in monkeys.Serum tryptase was measured at predose,2, 6, and 24 hours after LOP628 dosing.






observations were consistent with the onset of anti-drug antibody(ADA) formation in all study animals starting on day 21. Car-diovascular effects related to administration of LOP628 on day 1included transient decreases in bloodpressurewith compensatoryincreases in heart rate in all animals. Subsequently, followingrepeated LOP628 administration, cardiovascular effects weremost notable at 3 and8mg/kgdose levels.Onset of cardiovascularchanges correlated with changes in clinical signs. Similar to thedose range finding monkey study, the most apparent clinicalpathology changes were limited to bone marrow changes in adose-responsive manner. At the highest non-severely toxic dose(HNSTD) of 8 mg/kg LOP628, the changes in parameters mea-suring bone marrow function were transient and minimal. Doselevels of 3 and 8mg/kgwere consideredwell tolerated inmonkeysbased on the intended end-stage cancer patient population andresulted in minimal and transient changes that were consistentwith payload effects of DM1 and the pharmacodynamic effects ofLOP628.

Coincident with ADA formation, systemic exposure to LOP628was impacted with subsequent fast clearance of the ADC. How-ever, the pharmacodynamic effect of neutropenia remained aftereach subsequent dose administration regardless of ADA forma-tion (data not shown).

To understand the infusion reactionmechanism observed afterLOP628 dosing in the GLP monkey study (cardiovascularchanges, scratching, emesis, and hypoactivity), a subsequentinvestigative single dose monkey study was conducted. Assess-ment of serum histamine and tryptase were included at severalcollection time points (up to 24 hours post dose) to determine ifinfusion reactions were related to mast cell degranulation. Basedon these analyses, LOP628 did not cause elevations in serumtryptase and histamine above baseline values (Fig. 5C). Therefore,it was concluded that infusion reactions observed in monkeyswere likely not due to mast cell degranulation.

DiscussionFor patients with advanced cancers expressing either wild-type

or mutant c-KIT, treatment options are limited, which is notablythe case in recurrent c-KITþ GIST after the approved c-KIT/RTK-targeting agents such as imatinib, sunitinib, and regorafenib havefailed, often because of the emergence of secondary mutations(23, 24). This is primarily due to the heterogeneity of c-KITresistance mutations such that even the newer targeting agentswith the potential to inhibit a broader range ofmutations, like theactivation loop inhibitor BLU-285 or switch control inhibitorDCC-2618, may still not block the full spectrum of driver muta-tions (25, 26). An alternative approach was to implementHSP90 inhibitors to reduce wild-type and c-KIT–mutant proteinlevels in GIST, but this failed to demonstrate significant clinicalbenefit (27).

Thus, we explored a novel angle to target c-KIT–expressingcancers, whereby an ADC binding to this receptor could delivera potent toxin directly to the cancer, regardless of c-KIT muta-tional status provided the extracellular domain was unper-turbed. This would broaden activity to those cancers overex-pressing wild-type c-KIT, where the target may not be a driver,such as in SCLC, because the ADC activity is driven primarily bythe DM1 cytotoxin. In selecting c-KIT as a target, we purpose-fully took advantage of its rapid internalization kinetics androute of lysosomal-mediated degradation to effectively deliver

the ADC for processing to its active catabolite, Lys-SMCC-DM1(data not shown).

However a concern was that a ligand-blocking antibody withstrong antagonism used for the ADC would be highly hemato-toxic (as observed with LMJ451 in mice) and may confer little tono efficacy benefit in cancers expressing c-KIT mutants or in thoseexpressing wild-type c-KIT where it may not be a driver. Thus wefocused on developing a c-KIT ADC with no or minimal ligand-blocking activity.

Therapeutics utilizing the ADC modality such as Kadcyla(Trastuzumab-DM1) and Adcetris have produced impressiveclinical responses while minimizing systemic toxicity. LOP628,a highly selective and potent ADC, was developed with the aim oftargeting cancers overexpressing c-KIT. Our data provides evi-dence that LOP628 selectively binds c-KIT expressing cell lines,becomes rapidly internalized and is efficiently processed to releasesufficient active catabolite to potently induce a cytotoxic responsein cell viability assays. In vitro profiling of LOP628 activity in a cellline panel revealed effective killing of c-KIT-positive tumor cellsrepresenting GIST, AML and SCLC. In vivo, LOP628 inducedmitotic arrest and was efficacious in multiple xenograft modelsexpressing wild-type c-KIT (SCLC: NCI-H1048, NCI-H526) andmutant c-KIT (GIST-T1, GIST430), with varying sensitivities thatdid not correlate with mutational status of the target, because thetwo most sensitive models tested were the GIST-T1 and NCI-H526,which regressed tumors at 2.5mg/kg (NCI-H526 graph notshown), considered a lowdose for anADC conjugated to –SMCC-DM1. Some other models required a higher 10 mg/kg dose toinduce significant efficacy, in line with preclinical observationswith Kadcyla (28). As a whole, robust antitumor responses wereobserved in tumor xenograft models representing c-KIT expres-sion in greater than 50% of the tumor at a 2þ/3þ stainingintensity. LOP628 was not effective against the NCI-H2170NSCLC xenograft model (Supplementary Figs. S4 and S5), whichexpresses little to no c-KIT, but exhibited sensitivity to the may-tansine payload, thereby supporting the ADC specificity. Thepossibility to combine LOP628with SOCor targeted therapeuticswas promising, as demonstrated by a single dose of LOP628 with50 days of imatinib resulting in GIST-T1 tumor regression for theduration of the study (180 days). The data presented here show-case the cytotoxicity of LOP628 on c-KIT overexpressing tumors(regardless of mutational status).

Being that LOP628 is not rodent cross-reactive, safety wasassessed in the cynomolgus monkey, where it was well toleratedup to 8 mg/kg with primary on-target effects noted in the bonemarrow due to c-KIT expression on hematopoietic cells and off-target (payload) effects in the liver. The therapeutic index waspredicted to be narrow based on those less sensitive preclinicalmodels requiring 10mg/kg for efficacy. Bonemarrow effects havebeennotedwithotherDM1ADCsbothpreclinically and clinically(10); however, the kinetics and severity of neutropenia in themonkey as compared with previous studies with DM1 ADCssupports contribution of both targeted c-KIT and off-targetDM1-mediated bone marrow toxicity. The bone marrow effectswere transient, and 3 and 8 mg/kg were considered to be welltolerated in monkeys. At the HNSTD in monkeys (8 mg/kg),which is commonly used to establish first-in-human (FIH) doseestimation for endstage cancer patients, there was a 6-fold safetymargin at the clinical starting dose of 0.3 mg/kg LOP628. Clin-ically, infusion reactions were noted in monkeys after each doseadministration. Due to the presence of c-KIT on mast cells,

Abrams et al.





biomarkers associated with mast cell degranulation (histamineand tryptase) were assessed as part of the investigative monkeystudy and were not elevated above baseline levels. Therefore,clinical reactions noted inmonkeys were likely not related tomastcell degranulation. The information from the GLP monkey study(HNSTD) was used to support the first-in-human (FIH) doseestimation for the LOP628 phase I clinical trial.

The monkey studies did not predict clinical acute hypersensi-tivity reactions noted at the clinical starting doses of 0.15 and 0.3mg/kg, presumably due to mast cell degranulation. This led tocessation of the phase I clinical trial. The clinical findings andmechanism behind this hypersensitivity reaction are exploredelsewhere (29). These data highlight the potential benefit oftargeting c-KIT via an ADC approach, but also the challenges oftranslating preclinical safety findings to the clinic.

Disclosure of Potential Conflicts of InterestN. Pryer, W. Kluwe, and W.R. Sellers hold ownership interest (including

patents) in Novartis Pharmaceuticals. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception anddesign: T. Abrams, A.Connor, C. Fanton, S.B. Cohen, T.Huber,K. Miller, K. Krauser, F. Galimi, K. Mansfield, N. Pryer, S.A. Ettenberg,W.R. Sellers, E. Lees, P. Kwon, S.C. SchleyerDevelopment of methodology: T. Abrams, A. Connor, C. Fanton, T. Huber,K. Miller, X. Niu, S. Harris, M. Ghoddusi, K. Mansfield

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Abrams, C. Fanton, E.E. Hong, S. Harris, K. Krauser,Z. Wang, M. Ghoddusi, W. Kluwe, S.C. SchleyerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Abrams, A. Connor, C. Fanton, K. Miller, X. Niu,S. Harris, D. Walker, K. Krauser, F. Galimi, Z. Wang, M. Ghoddusi, K. Mansfield,S.T. Lee-Hoeflich, J. Holash, W. Kluwe, S.A. Ettenberg, E. Lees, P. Kwon,S.C. SchleyerWriting, review, and/or revision of the manuscript: T. Abrams, A. Connor,S. Harris, D. Walker, K. Mansfield, S.T. Lee-Hoeflich, W. Kluwe, W.R. Sellers,E. Lees, S.C. SchleyerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Abrams, X. Niu, J. Kline, M. Ison-Dugenny,D. Walker, Z. WangStudy supervision: T. Abrams, A. Connor, K. Krauser, N. Pryer, W.R. Sellers,J.A. Abraham, S.C. Schleyer

AcknowledgmentsThe authors thank Dr. T. Tagashi for providing the GIST-T1 cell line and

Dr. Jonathan Fletcher for the GIST882 and GIST430 cell lines.All authors worked at Novartis Pharmaceuticals while this work was

conducted.

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 December 21, 2017; revised April 13, 2018; accepted May 10, 2018;published first May 15, 2018.

References1. Kindblom LG, Remotti HE, Aldenborg F, Meis-Kindblom JM.

Gastrointestinal pacemaker cell tumor (GIPACT): gastrointestinal stromaltumors show phenotypic characteristics of the interstitial cells of Cajal. AmJ Pathol 1998;152:1259–69.

2. Lennartsson J, R€onnstrand L. Stem cell factor receptor/c-Kit:from basic science to clinical implications. Physiol Rev 2012;92:1619–49.

3. Ashman LK. The biology of stem cell factor and its receptor C-kit. Int JBiochem Cell Biol 1999;31:1037–51.

4. Rubin BP, Singer S, Tsao C,Duensing A, LuxML, Ruiz R, et al. KIT activationis a ubiquitous feature of gastrointestinal stromal tumors. Cancer Res2001;61:8118–21.

5. Esposito I, Kleeff J, Bischoff SC, Fischer L, Collecchi P, Iorio M, et al. Thestem cell factor-c-kit system and mast cells in human pancreatic cancer.Lab Invest 2002;82:1481–92.

6. Hirota S, Isozaki K, Moriyama Y, Hashimoto K, Nishida T, Ishiguro S, et al.Gain-of-function mutations of c-kit in human gastrointestinal stromaltumors. Science 1998;279:577–80.

7. Kee D, Zalcberg JR. Current and emerging strategies for themanagement ofimatinib-refractory advanced gastrointestinal stromal tumors. Ther AdvMed Oncol 2012;4:255–70.

8. Miettinen M, Lasota J. KIT (CD117): a review on expression innormal and neoplastic tissues, and mutations and their clinicopath-ologic correlation. Appl Immunohistochem Mol Morphol 2005;13:205–20.

9. WiddisonWC,Wilhelm SD,Cavanagh EE,WhitemanKR, Leece BA, KovtunY, et al. Semisynthetic maytansine analogues for the targeted treatment ofcancer. J Med Chem 2006;49:4392–408.

10. Poon KA, Flagella K, Beyer J, Tibbitts J, Kaur S, Saad O, et al. Preclinicalsafety profile of trastuzumab emtansine (T-DM1): mechanism of action ofits cytotoxic component retained with improved tolerability. Toxicol ApplPharmacol 2013;273:298–313.

11. Erickson HK, Park PU, Widdison WC, Kovtun YV, Garrett LM, Hoffman K,et al. Antibody-maytansinoid conjugates are activated in targeted cancercells by lysosomal degradation and linker-dependent intracellular proces-sing. Cancer Res 2006;66:4426–33.

12. Oroudjev E, Lopus M, Wilson L, Audette C, Provenzano C, Erickson H,et al. Maytansinoid-antibody conjugates induce mitotic arrest by sup-pressing microtubule dynamic instability. Mol Cancer Ther 2010;9:2700–13.

13. Taguchi T, Sonobe H, Toyonaga S, Yamasaki I, Shuin T, Takano A, et al.Conventional andmolecular cytogenetic characterization of a new humancell line, GIST-T1, established from gastrointestinal stromal tumor. LabInvest 2002;82:663–5.

14. Tuveson DA, Willis NA, Jacks T, Griffin JD, Singer S, Fletcher CD, et al.STI571 inactivation of the gastrointestinal stromal tumor c-KIToncoprotein: biological and clinical implications. Oncogene 2001;20:5054–8.

15. Bauer S, Yu LK, Demetri GD, Fletcher JA. Heat shock protein 90 inhibitionin imatinib-resistant gastrointestinal stromal tumor. Cancer Res 2006;66:9153–61.

16. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA,Kim S, et al. The Cancer Cell Line Encyclopedia enables predic-tive modelling of anticancer drug sensitivity. Nature 2012;483:603–7.

17. Keshet E, Lyman SD, Williams DE, Anderson DM, Jenkins NA, CopelandNG, et al. Embryonic RNA expression patterns of the c-kit receptor and itscognate ligand suggest multiple functional roles in mouse development.EMBO J 1991;10:2425–35.

18. Matsui Y, Zsebo KM,Hogan BL. Embryonic expression of a haematopoieticgrowth factor encoded by the Sl locus and the ligand for c-kit. Nature1990;347:667–9.

19. Orr-Urtreger A, Avivi A, Zimmer Y, Givol D, Yarden Y, Lonai P. Develop-mental expression of c-kit, a proto-oncogene encoded by the W locus.Development 1990;109:911–23.

20. Lev S, Blechman JM, Givol D, Yarden Y. Steel factor and c-kit protoonco-gene: genetic lessons in signal transduction. Crit Rev Oncog 1994;5:141–68.

21. Kimura Y, Ding B, Imai N, Nolan DJ, Butler JM, Rafii S. c-Kit-mediatedfunctional positioning of stem cells to their niches is essential for main-tenance and regeneration of adult hematopoiesis. PLoS One 2011;6:e26918.






22. Escribano L,OcqueteauM,Almeida J,OrfaoA, SanMiguel JF. Expressionofthe c-kit (CD117)molecule in normal andmalignant hematopoiesis. LeukLymphoma 1998;30:459–66.

23. Gramza AW, Corless CL, Heinrich MC. Resistance to tyrosine kinaseinhibitors in gastrointestinal stromal tumors. Clin Cancer Res 2009;15:7510–8.

24. Liegl B, Kepten I, Le C, Zhu M, Demetri GD, Heinrich MC, et al. Hetero-geneity of kinase inhibitor resistance mechanisms in GIST. J Pathol2008;216:64–74.

25. Evans EK, Gardino AK, Kim JL, Hodous BL, Shutes A, Davis A, et al.A precision therapy against cancers driven by KIT/PDGFRA mutations. SciTransl Med 2017;9. pii: eaao1690. doi: 10.1126/scitranslmed.aao1690.

26. SchneeweissM, Peter B, Bibi S, Eisenwort G, Smiljkovic D, Blatt K, et al. TheKIT and PDGFRA switch-control inhibitor DCC-2618 blocks growth and

survival of multiple neoplastic cell types in advanced mastocytosis.Haematologica 2018. doi 10.3324/haematol.2017.179895.

27. Bendell JC, Bauer TM, Lamar R, Joseph M, Penley W, Thompson DS, et al.A phase 2 study of the Hsp90 inhibitor AUY922 as treatment for patientswith refractory gastrointestinal stromal tumors. Cancer Invest 2016;34:265–70.

28. Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, et al.Targeting HER2-positive breast cancer with trastuzumab-DM1, an anti-body-cytotoxic drug conjugate. Cancer Res 2008;68:9280–90.

29. L'Italien L, Orozco O, Abrams T, Cantagallo L, Connor A, Desai J, et al.Mechanistic insights of an immunological adverse event induced by ananti-KIT antibody–drug conjugate and mitigation strategies. Clin CancerRes 2018. pii: clincanres.3786.2017. doi: 10.1158/1078-0432.CCR-17-3786. [Epub ahead of print].


Abrams et al.




2018;24:4297-4308. Published OnlineFirst May 15, 2018.Clin Cancer Res Tinya Abrams, Anu Connor, Christie Fanton, et al. Tumors

Positive Solid−Conjugate against Mutant and Wild-type c-KITDrug−c-KIT Antibody−Preclinical Antitumor Activity of a Novel Anti

Updated version

10.1158/1078-0432.CCR-17-3795doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2018/05/15/1078-0432.CCR-17-3795.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/24/17/4297.full#ref-list-1

This article cites 26 articles, 8 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/24/17/4297.full#related-urls

This article has been cited by 3 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/24/17/4297To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-17-3795http://clincancerres.aacrjournals.org/content/suppl/2018/05/15/1078-0432.CCR-17-3795.DC1http://clincancerres.aacrjournals.org/content/24/17/4297.full#ref-list-1http://clincancerres.aacrjournals.org/content/24/17/4297.full#related-urlshttp://clincancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://clincancerres.aacrjournals.org/content/24/17/4297http://clincancerres.aacrjournals.org/

/ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages false /GrayImageMinResolution 200 /GrayImageMinResolutionPolicy /Warning /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages false /MonoImageMinResolution 600 /MonoImageMinResolutionPolicy /Warning /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 900 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False

/CreateJDFFile false /Description > /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ > /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MarksOffset 18 /MarksWeight 0.250000 /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /NA /PageMarksFile /RomanDefault /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /LeaveUntagged /UseDocumentBleed false >> > ]>> setdistillerparams> setpagedevice

Documents

Drug Conjugate against Mutant and Wild-type c-KIT Positive ... · Clin Cancer Res; 24(17); 4297–308. 2018 AACR. Introduction The c-KIT (CD117) receptor binds the ligand stem cell