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Cancer Therapy: Preclinical

Synthetic Lethality Exploitation by an Anti–Trop-2-SN-38 Antibody–Drug Conjugate, IMMU-132,Plus PARP Inhibitors in BRCA1/2–wild-typeTriple-Negative Breast CancerThomas M. Cardillo, Robert M. Sharkey, Diane L. Rossi, Roberto Arrojo,Ali A. Mostafa, and David M. Goldenberg

Abstract

Purpose: Both PARP inhibitors (PARPi) and sacituzumabgovitecan (IMMU-132) are currently under clinical evaluation intriple-negative breast cancer (TNBC).We sought to investigate thecombined DNA-damaging effects of the topoisomerase I (TopoI)–inhibitory activity of IMMU-132 with PARPi disruption ofDNA repair in TNBC.

Experimental Design: In vitro, human TNBC cell lines wereincubated with IMMU-132 and various PARPi (olaparib, ruca-parib, or talazoparib) to determine the effect on growth, double-strandedDNA (dsDNA) breaks, and cell-cycle arrest. Mice bearingBRCA1/2-mutated or –wild-type human TNBC tumor xenograftswere treated with the combination of IMMU-132 and PARPi(olaparib or talazoparib). Study survival endpoint was tumorprogression to >1.0 cm3 and tolerability assessed by hematologicchanges.

Results: Combining IMMU-132 in TNBC with all threedifferent PARPi results in synergistic growth inhibition,

increased dsDNA breaks, and accumulation of cells in the S-phase of the cell cycle, regardless of BRCA1/2 status. A combi-nation of IMMU-132 plus olaparib or talazoparib producessignificantly improved antitumor effects and delay in time-to-tumor progression compared with monotherapy in mice bear-ing BRCA1/2-mutated HCC1806 TNBC tumors. Furthermore,in mice bearing BRCA1/2–wild-type tumors (MDA-MB-468 orMDA-MB-231), the combination of IMMU-132 plus olaparibimparts a significant antitumor effect and survival benefit abovethat achieved with monotherapy. Most importantly, this com-bination was well tolerated, with no substantial changes inhematologic parameters.

Conclusions: These data demonstrate the added benefit ofcombining Topo I inhibition mediated by IMMU-132 with syn-thetic lethality provided byPARPi in TNBC, regardless ofBRCA1/2status, thus supporting the rationale for such a combinationclinically. Clin Cancer Res; 23(13); 3405–15. �2017 AACR.

IntroductionSynthetic lethality is a concept where the simultaneous

mutational loss of function of two different genes results incell death, whereas loss of just one gene is still compatible withcellular viability (1). This concept has been applied to cancertherapy in which a cell carrying a genetic mutation is targetedwith a chemotherapeutic that blocks the function of anothergene used by the cell to overcome this first mutation. In thiscontext, the drug will be more potent in cells carrying themutation than it would be in others that are genetically intact.One class of defects susceptible to synthetic lethality is those

that affect homologous recombination repair (HRR) of double-stranded DNA (dsDNA) breaks. BRCA1 and BRCA2 are twosuch genes involved in HRR, and mutational loss of BRCA1/2makes a cell more susceptible to drugs that block other DNArepair mechanisms (2). Another protein involved in maintain-ing the integrity of DNA is PARP (3). PARP is a family ofenzymes whose primary function is to repair single-strandedDNA breaks before they advance to double-stranded breaks.PARP inhibitors (PARPi) have been developed to treat multiplecancer types with BRCA1/2 mutations, thereby creating syn-thetic lethality in the BRCA1/2-defective cells (4).

Clinically, therapy with PARPi has resulted in sustained antitu-mor responses in ovarian (5–8), prostate (5), pancreatic (9), andtriple-negative breast cancers (TNBC; refs. 7, 10). In patients withTNBC, approximately 25% carry germline mutations of BRCA1/2(11). In one clinical trial, TNBC patients with germline BRCA1/2mutations were treated with the PARPi, olaparib. Although thistherapy demonstrated a higher disease stabilization rate inBRCA1/2-mutant compared with nonmutant patients, there wereno sustained responses achieved in either cohort (7). This is incontrast to Tutt and colleagues (10), in which 60% of TNBCpatients with germline BRCA1/2 mutations had a partial responseand 35% stable disease. This discrepancy is thought to be due, inpart, to the large heterogeneity of TNBC and BRCA mutations.

Efforts to improve the effect of PARPi focus mainly on stressingDNA repair pathways by increasing dsDNA breaks with such

Immunomedics, Inc., Morris Plains, New Jersey.

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

Prior presentation: Presented in part at the annual meetings of the AmericanAssociation for Cancer Research 2016, and the San Antonio Breast CancerSymposium 2015.

Corresponding Authors: D.M. Goldenberg, Immunomedics, Inc., 300 AmericanRd., Morris Plains, NJ 07950. Phone: 973-605-8200; Fax: 973-605-8282; E-mail:[email protected]; or T.M. Cardillo, Phone: 973-605-8200; Fax: 973-605-1340; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-2401

�2017 American Association for Cancer Research.

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agents as ionizing radiation or platinum-based therapeutics,while these repair pathways are being blocked with PARPi (12,13). In addition to agents that directly interact with DNA to causebreaks, agents that inhibit topoisomerase I (Topo I), includingirinotecan, have been shown to synergize with PARPi to deter thegrowth of a range of human tumor cell lines, including those oflung, ovarian, colon, and breast cancers (14, 15). These effortsdemonstrate that by combining a DNA-damaging agent with anHRR synthetic lethality–based therapy, improved antitumoreffects can be achieved compared with each single modality.

Sacituzumab govitecan (IMMU-132) is an antibody–drug con-jugate (ADC) composed of the active metabolite of irinotecan,SN-38, conjugated to an anti–Trop-2 antibody (drug:Ab ratio ¼7.6; ref. 16). Preclinically, IMMU-132 has significant efficacyacross a broad range of Trop-2–positive human cancer xenograftmodels, including non–small cell lung cancer (NSCLC), pancre-atic, colon, gastric cancers, and TNBC (16–18). In both gastric andTNBC tumor cells, IMMU-132 specifically mediated dsDNAbreaks in Trop-2–expressing cells (16, 17). In a current clinicaltrial (ClinicalTrials.gov, NCT01631552), IMMU-132 hasachieved objective responses against a range of solid tumors,including small-cell lung carcinoma (19),NSCLC (20), and TNBC(21). In TNBC patients, with a median of five prior therapies,IMMU-132 treatment achieved a confirmed objective responserate of 31%. This therapy was well-tolerated over multiple cycleslasting as long as 1 year, with neutropenia being themajor adverseevent (21).

Because PARPi can enhance the activity of DNA-damagingagents, such as SN-38, the current study was undertaken toexamine the effect of combining IMMU-132–mediated DNAdamage with PARPi in BRCA1/2-mutated and –wild-type humanTNBC cell lines and xenografts. Three different PARPi wereassessed for their interaction with IMMU-132 in terms of cyto-toxicity,DNA stability, and in vivo efficacy. In addition, tolerabilitywas determined in vivo for such combinations. Results from thesestudies provide the rationale for using IMMU-132 and PARPi

combinations clinically for the treatment of TNBC, regardless,surprisingly, of BRCA1/2 mutational status.

Materials and MethodsCell lines, ADCs, and PARPis

All human cancer cell lines were purchased from the AmericanType Culture Collection (ATCC). Each cell line was maintainedaccording to the recommendations of the ATCC, routinely testedfor mycoplasma using MycoAlert Mycoplasma Detection Kit(Lonza), and authenticated by short-tandem repeat assay by theATCC. Cells were in culture less than 6monthswhen employed inexperiments. IMMU-132 and a control ADC (anti-CD20 hA20-SN-38) were prepared at Immunomedics, Inc., as described pre-viously (18). PARPi (olaparib, talazoparib, and rucaparib) werepurchased and dissolved in DMSO according to the manufac-turer's recommendations (Selleck Chemicals).

In vitro combination cytotoxicity assays, Western blotting, andcell-cycle analysis

Cytotoxicity studies were conducted as described previously(18) and are presented in Supplementary Data. Cell lysates wereprepared, and immunoblotting for phospho-H2A.X, poly(ADP-ribose) (PAR), PARP, FACCD2, BARD1, Rad51, ERCC1, andb-actin was done as described in Supplementary Data. Concen-trations, timing, and primary antibodies are shown in the figurelegends. Asynchronistic cells were used for cell-cycle analysis, asdescribed in Supplementary Data.

In vivo therapeutic studiesAll animal studies were approved by Rutgers School of

Biomedical and Health Sciences Institutional Animal Care andUse Committee. NCr female athymic nude (nu/nu) mice, 4 to 8weeks old, were purchased from Taconic Farms. Xenograftswere established by harvesting cells from tissue culture andmixing 1:1 with Matrigel, such that each mouse received a totalof 1 � 107 cells s.c. in the right flank. Tumor volume wasdetermined by measurements in two dimensions using calipers,with volumes defined as: L x w2/2, where L is the longestdimension of the tumor and w the shortest. Mice were ran-domized into treatment groups, and therapy begun whentumor volumes were approximately 0.3 cm3. Treatment regi-mens, dosages, and number of animals in each experiment aredescribed in the Results section and in the figure legends. Thelyophilized IMMU-132 and control ADC (anti-CD20, hA20-CL2A-SN-38) were reconstituted and diluted as required insterile saline. Olaparib was diluted in 2-hydroxy-propyl-b-cyclodextran/PBS (10% w/v) and administered i.p. to themice. Talazoparib was diluted in 10% DMAc/5% KolliphorHS15/85% PBS and administer p.o. to the animals.

Mice were deemed to have succumbed to disease progressionand euthanizedonce tumors grew to greater than1.0 cm3 in size. Apartial response is defined as shrinking the tumor >30% frominitial size. Stable disease is when the tumor volume remainsbetween 70% and 120% of initial size. Time-to-tumor progres-sion (TTP) was determined as time when tumor grew more than20% from its nadir.

Hematologic toxicity of combined IMMU-132 and olaparibwas assessed in female BALB/c mice, as described in Supplemen-tary Data. Dosages and timing are described in the Results sectionand in the figure legends.

Translational Relevance

Synthetic lethality is an approach gaining interest as atherapy of tumors with defects in DNA homologous recom-bination repair pathways. PARP inhibitors (PARPi) are cur-rently under clinical evaluation based on their syntheticlethality potential in tumors with BRCA1/2 mutations. Saci-tuzumab govitecan (IMMU-132), composed of a topoisom-erase I inhibitor, SN-38, conjugated to an anti–Trop-2 anti-body, is also under clinical investigation and has achievedobjective responses in a range of solid tumors, includingrelapsed/refractory triple-negative breast cancer (TNBC;NCT01631552). We demonstrate preclinically that combin-ing IMMU-132 with PARPi in TNBC results in increased DNAdamage above that achieved with single-agent exposure,regardless of BRCA1/2 status. Furthermore, IMMU-132 plusPARPi, at clinically relevant doses, produce significantlyimproved antitumor effects compared with monotherapy inmice bearing both BRCA1/2-mutated and –wild-type TNBCtumors. This combination is well tolerated by the animals.These data provide the rationale for combining IMMU-132and PARPi clinically against TNBC.

Cardillo et al.

Clin Cancer Res; 23(13) July 1, 2017 Clinical Cancer Research3406




Statistical analysis of in vivo dataA critical-Z test was performed on the survival data of treatment

and control groups with P � 0.05 for any mouse deemed anoutlier. Such mice were removed from further statistical analysisand are noted in the Results section. Statistical analysis of tumorgrowth was based on AUC. Profiles of individual tumor growthwere obtained through linear-curve modeling. An F-test wasemployed to determine equality of variance between groups priorto statistical analysis of growth curves. A two-tailed t test was usedto assess statistical significance between the various treatmentgroups and controls, except for the saline control, where a one-tailed t test was used in the comparison. Survival studies wereanalyzed using Kaplan–Meier plots (log-rank analysis), using thePrism GraphPad Software (v6.05) package (Advanced GraphicsSoftware, Inc.). Significance was set at P � 0.05.

ResultsChanges in expression of various HRR proteins mediated byIMMU-132

It had previously been found that upregulation of severaldifferent proteins involved inHRRuponDNAdamage (FANCD2,BARD1, Rad51, and ERCC1) plays an important role in resistanceto DNA-damaging treatment in TNBC, particularly in BRCA1/2–wild-type tumor cells (22–24). To determine the effect IMMU-132–mediated DNA damage has on these other repair proteins,BRCA1/2–wild-type TNBC cell lines (MDA-MB-231, MDA-MB-468, and BT-20) were exposed to various concentrations ofIMMU-132 (25, 50, and 100 nmol/L SN-38 equivalents) for 24hours. In two of the cell lines (MDA-MB-231 and BT-20), levels ofall four HRR proteins examined were elevated, suggesting that thecells were activating these DNA-repair pathways in response toIMMU-132–mediated damage (Supplementary Fig. S1). In aBRCA1/2-defective cell line (HCC1806), levels of these proteinsremained unchanged in response to IMMU-132 exposure. Inter-estingly, MDA-MB-468 (BRCA1/2–wild-type, PTEN defective),FANCD2, Rad51, and ERCC1 levels dropped relative to basallevels upon IMMU-132 exposure. These data suggest thatHCC1806 and MDA-MB-468 TNBC tumors would be the mostsensitive to the combination of IMMU-132 and PARPi, becausethey do not activate these HRR rescue pathways.

In vitro synergistic growth inhibition of TNBCwhen IMMU-132is combined with various PARPi

Changes in IC50 values for IMMU-132 when combined withvarious PARPi were determined in human TNBC cell lines withBRCA1/2 mutations (HCC38 and HCC1806) and those withwild-type BRCA1/2 (MDA-MB-468, MDA-MB-231, and BT-20).

Isobolograms for IMMU-132 were clearly enhanced when com-bined with olaparib, rucaparib, or talazoparib, demonstrating asynergistic interaction in all four cell lines (Supplementary Fig.S2). IC50 values for IMMU-132 alone were 1.9 to 82.5 nmol/L,whereas olaparib and rucaparib IC50 values were 5.4 to 256.7mmol/L (Table 1). Although talazoparib was more potent (4.7 to322 nmol/L) than either olaparib or rucaparib, IC50 values forIMMU-132 indicated itwas>2-foldmorepotent than talazoparib.Calculated combinatorial index (CI) values demonstrate thateven when combined with IC10 concentrations, an additive effectwas achievable (i.e., CI ¼ 1.0), but this was further enhanced tosynergy (i.e., CI < 1.0) when as little as IC20 concentrations wereused (Table 1). Importantly, this synergy occurred in bothBRCA1/2-mutated and –wild-type cell lines, despite the upregulation ofHRR proteins shown in MDA-MB-231 and BT-20 in response toIMMU-132–mediated DNA damage.

IMMU-132 plus PARPi mediated increases in dsDNA breaksAssessment of dsDNA breaks was measured in TNBC cell

lines for IMMU-132 and PARPi combinations (24-hourincubation; Fig. 1). Four TNBC cell lines were tested withIMMU-132 and olaparib (Fig. 1A). Not unexpectedly, PARwas not detected in any of the olaparib-treated cells, whereasIMMU-132 alone had only a minor effect on PAR levels.When combined, PAR was still not detected, which was dueto inhibition, not loss of PARP, as indicated by equivalentlevels of full-length PARP (FL-PARP) present in all samples.Increased dsDNA breaks, as evidenced by increases in p-H2A.X(25), were shown in all four cell lines treated with olaparibalone, although the level in MDA-MB-231 was only 30% aboveuntreated controls, which were set to 1.0, whereas MDA-MB-468 cells showed a 2.5-fold increase (highest response). Inthree of the four cell lines, IMMU-132 treatment alone alsoresulted in increased p-H2A.X levels with 9 nmol/L SN-38equivalents of IMMU-132 (187 ng/mL of the ADC), demon-strating a >2.5-fold increase (range, 2.5–13.9-fold). Only theBRCA1/2–wild-type MDA-MB-231 was seemingly resistant tosingle-agent IMMU-132 or olaparib. However, when IMMU-132 (9 nmol/L) was combined with olaparib, there was >5-foldincrease in p-H2A.X in all four cell lines, including MDA-MB-231, which exhibited a 5.7-fold increase in p-H2A.X levels.Even at 3 nmol/L SN-38 equivalents of IMMU-132 (62 ng/mLof the ADC), when combined with olaparib, levels of p-H2A.Xrose >76% above that achieved by each agent alone in all fourcell lines, with the highest response in MDA-MB-231 (171%),followed by HCC1806 (136%), MDA-MB-468(116%), andHCC38 (76%).

Table 1. CI values for IMMU-132 plus olaparib, rucaparib, or talazoparib in four TNBC cell lines

Single-agent IC50 values (mean � SD) CI valuesIMMU-132 Olaparib Rucaparib Talazoparib Olaparib þ IMMU-132 Rucaparib þ IMMU-132 Talazoparib þ IMMU-132

Cell Line (nmol/L) (mmol/L) (mmol/L) (nmol/L) IC10 IC20 IC30 IC10 IC20 IC30 IC10 IC20 IC30

HCC1806 1.9 � 0.4 7.3 � 1.4 5.4 � 0.3 4.7 � 1.0 0.81 0.59 0.29 1.09 0.80 0.46 1.21 0.80 0.50HCC38 3.4 � 0.5 28.5 � 18.7 16.9 � 0.7 104.9 � 44.7 0.46 0.25 0.16 1.07 0.91 0.47 0.49 0.20 0.11MDA-MB-468 3.5 � 0.4 9.8 � 1.0 10.3 � 0.8 322.3 � 27.7 0.87 0.49 0.22 1.01 0.63 0.34 0.37 0.15 0.07MDA-MB-231 7.5 � 1.2 21.7 � 6.0 7.1 � 1.0 36.3 � 8.0 0.89 0.42 0.28 0.69 0.40 0.22 0.40 0.17 0.08BT-20 82.5 � 22.8 256.7 � 40.4 n.d. n.d. 0.91 0.54 0.27 n.d. n.d. n.d. n.d. n.d. n.d.

NOTE: CI values, CI values determined as described in Materials and Methods.IC10, IC20, and IC30, CI values determined when IMMU-132 or PARPi used at concentrations calculated to cause 10%, 20%, and 30% growth inhibition when usedalone, respectively.Abbreviation: n.d., not done.

Synergy and Efficacy of Combined IMMU-132 Plus PARPi in TNBC

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Figure 1.

Western blot assessment of effects on PAR, PARP, and dsDNA breaks mediated by IMMU-132 plus PARPi in TNBC tumor lines. Cells were plated overnight in 6-wellplates before the addition of chemotherapeutics. After a 24-hour incubation, cells were harvested and cell lysates resolved and transferred for Westernanalysis as described in Materials and Methods. PAR and FL-PARP levels were determined on the same gel. Assessment of dsDNA breaks (p-H2A.X) wascalculated as ratios relative to untreated control (Unt) normalized to b-actin protein loading control (Dp-H2A.X). A, All four TNBC cell lines (HCC38, HCC1806,MDA-MB-468, and MDA-MB-231) were exposed to olaparib (Olap), IMMU-132, or the combination of both. IMMU-132 concentrations are expressed in terms ofSN-38 equivalents. B, HCC1806 cells exposed to rucaparib (Ruc) and IMMU-132 or to (C) talazoparib (Tala) and IMMU-132.

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Rucaparib and talazoparib were used at 10- and 100-fold lowerconcentrations, respectively, when combined with IMMU-132 inHCC1806 (Fig. 1B and C, respectively). Likewise, the amount ofIMMU-132 required to demonstrate increases in dsDNA breaks,when combined with these two PARPi, was lower (0.1, 1, and 3nmol/L SN-38 equivalents). Rucaparib alone resulted in a1.6-foldincrease in dsDNA breaks, whereas 0.1 nmol/L SN-38 equivalentsof IMMU-132 (2 ng/mL of the ADC) resulted in a 2.2-foldincrease. Together, IMMU-132 plus rucaparib further increasedthe amount of dsDNA breaks 3.9-fold, which is 77% higher thaneither single agent. Talazoparib and IMMU-132 combined dem-onstrated a dose-response with the amount of dsDNA breaksincreasing by 78% at the lowest IMMU-132 concentration to216% at the highest concentration. These data demonstrate thatthree different PARPi, when combined with IMMU-132, result inincreased DNA damage in the form of double-stranded breaksabove that observed with single-agent treatments, which is con-sistent with the in vitro cytotoxicity assays demonstrating syner-gistic growth inhibition when IMMU-132 was combined withthese PARPi.

Changes in cell cycle mediated by IMMU-132 plus olaparibCell-cycle changes were assessed in all four TNBC cell lines

when IMMU-132 and olaparib were incubated with asynchronis-tic cells for 24hours (Supplementary Fig. S3).Greater than 80%ofthe cells in the untreated groups were in theG1-phase. Cells beganto accumulate in S-phase when exposed to either olaparib orIMMU-132 alone. However, the combination demonstrated aneven greater percentage of cells in S-phase, with a concomitantdecrease in the percentage of cells in G1-phase and little change inG2–M. For both BRCA1/2-mutated cells and BRCA1/2–wild-typeMDA-MB-231, the combination of IMMU-132 and olaparibresulted in a greater than 2-fold increase in the percentage of cellsin the S-phase relative to each agent alone. MDA-MB-468 dem-onstrated the least increase in the percentage of cells in S-phaserelative to single-agent treatment, but thiswas dueprimarily to thegreater effect each agent alone had, withmore than 32% and 50%of the cells in S-phase upon treatmentwitholaparib or IMMU-132alone, respectively. Even so, the combination increased the num-ber of cells in S-phase by 12% over IMMU-132 alone. Similarresults were observed when IMMU-132 was combined withrucaparib (1 mmol/L) or talazoparib (0.2 mmol/L), in which thecombination of IMMU-132 plus either PARPi increased the per-centage of cells in S-phase for all four cell lines, relative to single-agent treatment (Supplementary Tables S1 and S2, respectively).

Improved efficacy of IMMU-132 in mice bearing TNBC tumorswhen combined with olaparib or talazoparib

Therapeutic efficacy of combining IMMU-132 with olaparibwas tested in HCC1806 and MDA-MB-468 tumor xenografts(Fig. 2). Importantly, in both experiments, the dose and sched-ule of IMMU-132 administered to the animals were chosen togive only a modest therapeutic effect in order to detect apotential interaction between IMMU-132 and olaparib.

In mice bearing HCC1806 tumors (Fig. 2A), the combinationtherapy resulted in a significant antitumor effect when comparedwith all other treatment groups (P < 0.0017, AUC; SupplementaryTable S3). In this group, all of the mice exhibited a partialresponse, with 30% being tumor-free (i.e., complete response)when the experiment endedon therapyday108.MeanTTP for thisgroup was 36.9 � 8.1 days (Table 2). In contrast, tumors in mice

given IMMU-132or olaparibmonotherapy progressedwithmeanTTP's of 17.6 � 3.9 and 9.1 � 4.8 days, respectively. Between thetwomonotherapy groups, IMMU-132 significantly delayed tumorprogression when compared with olaparib therapy (P¼ 0.0009).Overall, the combination had a significantly greater delay in TTPwhen compared with either single-agent therapy group (P <0.0001). Of note, this combination proved to be well-toleratedby the mice, with no appreciable loss in body weight (Supple-mentary Fig. S4A). Evenmice treatedwith 4-foldmore IMMU-132(500 mg twice weekly x 4 weeks) combined with olaparib dem-onstrated no significant loss in weight (Supplementary Fig. S4B).

In mice bearing MDA-MB-468 tumors, the combination ofIMMU-132 plus olaparib had a significant antitumor effect whencompared with all other treatment groups, including IMMU-132monotherapy (Fig. 2B; P < 0.0040; AUC; Supplementary TableS4). In terms of response rate, 30% of mice treated with onlyIMMU-132 exhibited apartial response, as did 20%of the animalstreated with olaparib (Table 2). In contrast, all of the mice in thecombination group exhibited a partial response, with a TTP of55.2 � 6.5 days, which was significantly better than all the othergroups (P < 0.0004) except IMMU-132 alone, which approachedsignificance at a TTP of 45.1 � 13.6 days (P ¼ 0.0604). As in theHCC1806 tumor model, the mice tolerated the combination ofIMMU-132 and olaparib without loss of body weight (Supple-mentary Fig. S4C).

Given the ability of IMMU-132 and olaparib to improveefficacy significantly compared with single-agent therapy inMDA-MB-468 tumors, which carry wild-type BRCA1/2, anothertherapy study utilized mice bearing MDA-MB-231 tumors thatlikewise are wild-type for BRCA1/2, but demonstrated synergywhen IMMU-132 and PARPi were combined in vitro. MDA-MB-231 tumors progressed very rapidly, reaching experimental end-points (i.e., tumor volume >1.0 cm3) within 14 days of startingtherapy. Given this rapid disease progression, antitumor effectsare represented as the ability to provide survival benefit and nottumor regressions. As noted in the previous two therapy experi-ments, olaparib alone had no antitumor effects in mice bearingMDA-MB-231 tumors, with median survival no different thansaline controlmice (Fig. 2C).Not unexpectedly, IMMU-132 alonedid not significantly improve the overall survival of the treatedanimals, since it was shown previously to be resistant to thistherapy (16). However, when mice were treated with the combi-nation of IMMU-132 plus olaparib, there was a significantincrease in median survival from 14 days for saline controlanimals to 21 days for those mice treated with the combination(P¼ 0.0005). Likewise, treatment with olaparib also resulted in asignificant survival benefit when combined with the nonspecificcontrol ADC [median survival time (MST)¼17days;P¼ 0.0257],suggesting that the olaparib does sensitize this resistant tumor toSN-38. These results show, unexpectedly, that even in tumorsresistant to PARPi and IMMU-132 monotherapies, the combina-tion is able to overcome this resistance and impart a survivalbenefit when compared with saline control animals.

Because talazoparib's potency was greater than olaparib in vitro,both alone and when combined with IMMU-132, mice bearingHCC1806 tumors were treated with the combination of IMMU-132 and talazoparib (Fig. 2D). Whereas mice in the olaparibexperiments were treated with 50 mg/kg, talazoparib-treatedanimals were given only 0.33 mg/kg. As observed in the olaparibexperiments, IMMU-132 plus talazoparib had a significant anti-tumor effect when compared with all other treatments, including






Figure 2.

Therapeutic efficacy of IMMU-132 plus PARPi in BRCA1/2-deficient and –wild-type TNBC tumor xenograft disease models. Tumors were established, and diseaseendpoints are described in Materials and Methods. All the ADCs and controls were administered in the amounts indicated (green arrows, PARPi injections;purple arrows, ADC injections). A, Mice bearing HCC1806 tumors (N ¼ 9–10) were injected with IMMU-132 weekly for 4 weeks and with olaparib daily(Monday through Friday) for 4 weeks. B, MDA-MB-468 tumor–bearing mice (N ¼ 9–10) were injected weekly with IMMU-132 for 2 weeks with 1 week off beforerepeating. Likewise, olaparib was administered daily (Monday to Friday) for 2 weeks with 1 week off before repeating. C, Mice bearing MDA-MB-231 tumors(N ¼ 10) were treated with IMMU-132 and olaparib under the same schedule as the HCC1806 mice. D, Mice bearing HCC1806 tumors (N ¼ 9–10) were treatedwith IMMMU-132 weekly for 4 weeks and talazoparib daily (Monday to Friday) for 4 weeks. All IMMU-132 and control ADC injections were administered as i.v.Olaparib was administered i.p. and talazoparib p.o.

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monotherapy with IMMU-132 or talazoparib (P < 0.0088, AUC).Mean TTP was 21.8 � 9.6 days, which was >2.8-fold longer thanany of the other treatment groups (P < 0.0021; Table 3). Similar tothe combinations with olaparib, IMMU-132 plus talazoparib didnot result in any observable toxicity in the mice (SupplementaryFig. S4D).

Hematologic tolerability of IMMU-132 plus olaparib in na€�veBALB/c mice

Clinical studies indicate that there is no off-tumor, on-targettoxicities in patients (19), and therefore an experiment wasperformed to determine if the combination of a PARP inhibitorwith IMMU-132's SN-38 would have increased toxicities at ther-apeutically active doses. Because normal tissues of mice are nottargeted by the anti–Trop-2 antibody comprising IMMU-132,toxicity studies were conducted in mice to determine possibleoff-target toxicities at doses of IMMU-132 and olaparib shown tohave therapeutic activity when combined in tumor-bearing mice(i.e., 250 mg IMMU-132 plus 50 mg/kg olaparib). The scheduleused mimics the clinical regimen of IMMU-132 (i.e., weekly for 2weeks with 1 week off per cycle) with olaparib administered alsoover a 2-week period. This same dose schedule does demonstrateantitumor activity over this 2-week period, as shown in theefficacy studies described above (Fig. 2A and B). Na€�ve BALB/cmice were administered a 2-week course of IMMU-132 plusolaparib therapy to ascertain any possible myelotoxicity. On day15 (i.e., 7 days after the last IMMU-132 injection and 3 days afterlast dose of olaparib), there was no evidence of hematologictoxicity in any of the mice (Fig. 3). In particular, the combinationdid not result in any significant drop in total white blood cell(WBC) or lymphocyte counts. Most importantly, there was noevidence of neutropenia or thrombocytopenia in any of thesemice. Onlymice treated with olaparibmonotherapy demonstratea significant drop in lymphocytes relative to vehicle controlanimals (P ¼ 0.0481), although the mean (3.92 � 1.13 K/mL)remained within the normal range (3.8 to 8.9 K/mL). After afurther 7 days (i.e., day 22), the assessment of toxicity was

determined in the remaining mice. At this time-point, all threetreatments showed a significant drop in WBC and lymphocytecounts relative to control animals (P < 0.0012). However, mostof these values still remained within an acceptable range, withthe lowest values found in those mice treated with the combina-tion, which fell just below the lower end of the normal range(mean lymphocyte count of 3.62�0.54K/mL vs. 7.04�1.17K/mLfor control, P ¼ 0.003). These results indicate that the combina-tion of IMMU-132 and olaparib, which has been shown to elicitantitumor effects in TNBC tumor-bearing mice, is not associatedwith any significantmyelotoxicity, and suggests a potentially hightherapeutic window.

DiscussionExploitation of defects to a cell's HRR pathways has gained

widespread interest as a means to induce synthetic lethality invarious human cancers, including TNBC (1, 2). Of particularinterest are those drugs that target PARP (26, 27). Although someclinical studies have suggested that use of PARPi can providemeaningful responses, other trials have shown no significantimprovements in clinical outcome (7, 10). Given these mixedresults, new approaches seek to combine PARPi with other DNA-damaging agents, such as platinum-based drugs, microtubuleinhibitors, or irinotecan (14, 22, 28–31). Here, we examined theeffect of combining several PARPi (olaparib, rucaparib, andtalazoparib) with our Topo I–inhibiting ADC, anti–Trop-2/SN-38-ADC, and IMMU-132. All three PARPi synergized with IMMU-132 to inhibit the growth of four human TNBC cell lines. Thisoccurred independently of BRCA1/2 status, as evidenced by thesynergistic growth inhibition in vitro of two cell lines with wild-type BRCA1/2. IMMU-132 combined with these PARPi increasedthe dsDNAbreaks above that observedwith each single agent, andthe accumulation of cells in the S-phase of the cell cycle. Mostimportantly, in mice bearing either a BRCA1/2-mutated TNBCtumor (HCC1806) or ones with wild-type BRCA1/2 (MDA-MB-468 and MDA-MB-231), the combination of IMMU-132 plus a

Table 2. TTP for TNBC tumor–bearing mice treated with IMMU-132 plus olaparib

HCC1806 MDA-MB-468

% PR TTPIMMU-132 vs.controls

Combination vs.controls % PR TTP

IMMU-132vs. controls

Combination vs.controls

Treatment N (TF) (Days) (P value) (P value) N (TF) (days) (P value) (P value)

IMMU-132 þ Olaparib 10 100 (3) 36.9 � 8.1 N.A. N.A. 10 100 (1) 55.2 � 6.5 N.A. N.A.Control ADC þ Olaparib 10 0 (0) 12.1 � 5.8 0.0290 <0.0001 10 20 (2) 16.6 � 14.0 0.0007 <0.0001IMMU-132 9 22 (0) 17.6 � 3.9 N.A. <0.0001 10 30 (1) 45.1 � 13.6 N.A. 0.0604Olaparib 9 11 (0) 9.1 � 4.8 0.0009 <0.0001 10 20 (2) 10.5 � 3.7 <0.0001 <0.0001Control ADC 10 0 (0) 6.3 � 4.5 <0.0001 <0.0001 10 0 (0) 20.3 � 20.7 0.0073 0.0004Saline 10 0 (0) 7.7 � 5.0 <0.0001 <0.0001 9 0 (0) 8.6 � 3.1 <0.0001 <0.0001Abbreviations: N, number of mice per group; N.A., not applicable; TF, number of mice tumor-free when experiment ended; TTP, time-to-tumor progression for micenot tumor-free as defined in Materials and Methods; % PR, percentage of mice that exhibited a positive response to treatment.

Table 3. TTP for HCC1806 tumor–bearing mice treated with IMMU-132 plus talazoparib

TTP IMMU-132 vs. control Combination vs. controlTreatment N (days) (P value) (P value)

IMMU-132 þ Talazoparib 10 21.8 � 9.6 N.A. N.A.Control ADC þ Talazoparib 10 6.8 � 5.4 0.6979 0.0004IMMU-132 9 7.9 � 6.6 N.A. 0.0021Talazoparib 9 5.9 � 5.9 0.5099 0.0005Control ADC 10 4.6 � 2.1 0.1866 0.0003Saline 10 4.2 � 1.9 0.0709 0.0001

Abbreviations: N, number of mice per groups; N.A., not applicable; TTP, TTP as defined in Materials and Methods.






PARPi resulted in significant antitumor effects above thoseobserved with monotherapy. These results clearly illustrate thepotential clinical benefit that may be derived by combiningIMMU-132 with a PARPi in TNBC.

Topo I is an enzyme utilized by the cell to allow unwindingof the DNA strand during transcription and replication (32).PARP-1, the most abundant of the PARP proteins, has beenfound to colocalize with Topo I throughout the cell cycle.However, upon DNA damage, PARP-1 dissociates from TopoI, resulting in reduced activity of this enzyme (33). We hypoth-esized that by combining the Topo I inhibitor carried byIMMU-132 (i.e., SN-38) with a PARPi, there would be anaccumulation of dsDNA breaks due to the inability of theremaining HRR pathways in the cell to repair this damagewith high fidelity, resulting in apoptosis and cell death. Inaddition, in cells lacking functional BRCA1/2 or are otherwisedeficient in HRR, the more error-prone, nonhomologous end-joining (NHEJ) pathway is utilized, further compromising thecell toward irreparable DNA damage and apoptosis (34).Others have found that in both BRCA1–wild-type and -mutatedTNBC cell lines, the combination of CPT-11 (the prodrugof SN-38) and a PARPi could result in synergistic growth

inhibition in vitro (22). Here, too, we demonstrated thatIMMU-132, when combined with olaparib, rucaparib, or tala-zoparib, mediates synergistic tumor cell growth inhibition andincreased dsDNA breaks in both BRCA1/2-mutated and –wild-type cell lines. These data confirm the ability of IMMU-132–mediated inhibition of Topo I, when combined with PARPi, tosynergize growth inhibition in human TNBC cells regardless ofBRCA1/2 mutational status.

HRR pathways are more active during the late S–G2-phase ofthe cell cycle, whereas the more error-prone NHEJ repair pathwayis most evident throughout the S-phase (35). Cells with HRRdefects, as well as those exposed to PARPi, will arrest at G2–M-phase (23, 36). Although we found that PARPi (olaparib, ruca-parib, and talazoparib) and IMMU-132 exposure resulted in smallincreases in the number of cells in the G2–M-phase of the cellcycle, it was also evident that therewas greater accumulation in theS-phase. Most importantly, the combination demonstrated aneven greater percentage of cells in S-phase, with a concomitantdecrease in the percentage of cells in G1- and little change in theG2–M-phase. Interestingly, the interaction of PARP-1 and Topo Iwas found to be most concentrated during the S-phase (33). Ourdata are consistent with this and suggest that when using a PARPi

Figure 3.

Tolerability of IMMU-132 plus olaparibin na€�ve BALB/cmice. Female BALB/cmice were placed into four differenttreatment groups and administeredIMMU-132 (250 mg), olaparib(50 mg/kg), or both. Control micereceive only saline and olaparibdiluent on the same schedule asIMMU-132 and olaparib, respectively.Whole blood was removed from5 mice on day 15 (7 days after finalIMMU-132 injection and 3 days afterfinal olaparib injection) and 5 mice onday 22 (14 days after final IMMU-132injection and 10 days after finalolaparib injection) for automated CBCdeterminations. Dotted linesrepresent upper and lower end of thenormal range for each parameter,respectively. A one-tailed t test wasused to assess statistical differencesafter an F-test to determine equalityof variance. � , P < 0.05 versus day 15control values. �� , P <0.012 versus day22 control values.

Cardillo et al.





combinedwith IMMU-132,we are disrupting this PARP-1/Topo 1interaction, resulting in accumulation in the S-phase and thustoward the more error-prone NHEJ repair pathway that predo-minates during this phase of the cell cycle.

Combining IMMU-132 therapy with a PARPi (olaparib ortalazoparib) in mice bearing both BRCA1/2-mutated and–wild-type tumors demonstrated significant antitumor effectsgreater than that observed with monotherapy, thus extendingour in vitro observations to in vivo TNBC models. Likewise,others have demonstrated improved efficacy when a PARPi iscombined with a DNA-damaging chemotherapeutic in micebearing human TNBC xenografts (22, 30). In one example,CPT-11 was used in combination with veliparib to improve thetherapeutic outcome significantly in mice bearing s.c. MX-1TNBC tumors (22). Similar to our experience, the PARPi alonehad no effect on tumor growth, whereas the combinationof CPT-11 and veliparib demonstrated a significantly enhancedantitumor effect. It should be noted that CPT-11 is moreeffectively converted to its active SN-38 form in mice than inhumans, and therefore similar findings may not be reproduc-ible in the clinical setting (37). However, by using IMMU-132with its SN-38 payload attached, we are not dependent onthe efficiency of prodrug-to-drug conversion by the patient,and therefore the antitumor effects we observed with thecombination of IMMU-132 plus PARPi are more clinicallyrelevant.

Approximately 80% of breast cancer patients carrying heredi-taryBRCA1mutations present with TNBC (38). Although BRCA1/2 status is an important marker in TNBC, it is not the only one.Many other BRCA1/2--wild-type TNBCs occur as sporadic tumorsthat share traits with germline BRCA–mutated tumors and aretermed to have BRCAness (39, 40). For example, analysis ofgermline mutations in 158 TNBC patients demonstrated that inaddition toBRCA1mutations (17%of patients), mutations of theCHEK2 checkpoint kinase 2 gene, nibrin NBN gene, and ATMgenes also were found (3.9%), and are themselves involved invarious aspects of the HRR pathway (41–43). Overall, the evi-dence suggests that besides BRCA1/2 status, many other genescontribute to the BRCAness of TNBC. In accordance with theseobservations, our ability to synergize and demonstrate signifi-cantly improved efficacy in the MDA-MB-468 tumor could beexplained by the PTENmutation it carries and its loss of function.Loss of PTEN expression results in Rad51 dissociation from DNAreplication forks, and subsequent destabilization and stalledreplication. Cells lacking PTEN have deficient HRR functions,likely due to reduced Rad51 recruitment to the replicating DNA,culminating in loss of fidelity during DNA synthesis (44). As withBRCA1/2mutations, cells lackingPTEN function are prone toHRRdeficiencies and, subsequently, have been shown to be sensitive toPARPi (45, 46). Our results demonstrate that IMMU-132 com-bined with a PARPi will significantly improve efficacy in bothtraditional BRCA1/2-defective TNBC tumors and in tumors dem-onstrating BRCAness in the form of deficiencies in other proteinsinvolved in HRR pathways (e.g., PTEN).

Because not all TNBC tumors exhibit BRCAness, we evalu-ated the ability of IMMU-132 plus a PARPi to inhibit tumorgrowth in such tumor types. MDA-MB-231 carries nomutationsto PTEN, BRCA1/2, or other known HHR proteins, and as suchis expected to be resistant to PARPi treatment (22, 46). Previousin-vivo studies demonstrated that mice bearing MDA-MB-231tumor xenografts were resistant to IMMU-132 therapy (16).

One reason for this resistance may be over-expression of Rad51in MDA-MB-231 (47). Cells exposed to SN-38 and olaparibhave been found to increase Rad51 recruitment in the HRRpathway (48). In fact, we found that IMMU-132 exposure doesresult in an increase in several different proteins involved inHRR, including Rad51, BARD1, FANCD2, and ERCC1 inBRCA1/2–wild-type cell lines. Of note was the observation thatwhile these proteins are upregulated in some cell lines (MDA-MB-231 and BT-20), they are actually downregulated in MDA-MB-468, which may account for its sensitivity to both IMMU-132 monotherapy and the combination therapy. At the sametime, it is likely that this increased expression of these HRRproteins in MDA-MB-231 allows for a more efficient repair ofdamaged DNA caused by IMMU-132 than would otherwiseoccur in cells that had low expression. A second possible reasonfor MDA-MB-231 resistance may be that it has low expressionof Trop-2 on its surface, which could limit how much SN-38can be delivered to the tumor by IMMU-132 (17). However,when IMMU-132 and its SN-38 payload were combined witholaparib, we achieved a significant survival benefit in MDA-MB-231 tumor–bearing animals. Moreover, although the amountof SN-38 delivered by IMMU-132 alone may be insufficientto retard tumor growth, enough must accrete in the tumor sothat when combined with olaparib, we are able to slow tumorprogression enough to provide a survival benefit. We arecurrently studying various MDA-MB-231 clones that were trans-fected with Trop-2 to increase expression levels in order tobetter determine what role Trop-2 surface expression plays in atumor's sensitivity to IMMU-132 therapy, either alone or whencombined with PARPi.

In a current phase I/II clinical trial, IMMU-132 has dem-onstrated an objective response rate (confirmed completeresponse þ partial response) of 31% in 58 patients withmetastatic TNBC (21). At the MTD, grade 3–4 toxicitiesincluded neutropenia, febrile neutropenia, diarrhea, fatigue,anemia, leukopenia, and dyspnea. All of these toxicities weremanageable, with the incidence of severe diarrhea being lessthan that reported for the parental prodrug, irinotecan. Single-agent olaparib toxicities include grade 3–4 fatigue and throm-bocytopenia (5). In clinical studies evaluating the combina-tion of either olaparib or veliparib with irinotecan, dose-limiting toxicities included fatigue, anorexia, diarrhea, nausea,vomiting, febrile neutropenia, neutropenia, and thrombocy-topenia (31, 49). A hallmark in these clinical trials was thatthe amount of irinotecan and PARPi administered was reducedbelow the single-agent MTDs in order to gain acceptabletolerability. Based on our experience with IMMU-132 in theclinic, as well as those reported with PARPi plus irinotecan, wedo not expect to observe any different dose-limiting toxicitywith a combination of IMMU-132 plus PARPi's. The doses ofIMMU-132 administered to mice in all our preclinical studieswere chosen based on their ability to produce a minimaleffect, so that we could better determine if the combinationswere beneficial and therefore indicated a good therapeuticwindow when IMMU-132 was combined with a PARPi. Impor-tantly, in all our combination studies, the mice tolerated boththe PARPi and IMMU-132 well, with no significant loss inbody weight or substantial hematologic toxicity during thecourse of treatment. Given such a potential for a high ther-apeutic window, we speculate that even at lower IMMU-132dosages, combinations with a PARPi in this subset of breast






cancer patients will achieve improved therapeutic responseswithout unmanageable toxicity.

In summary, through the utilization of PARPi to target TNBCand create synthetic lethality combined with Topo I inhibitionmediated by IMMU-132, a synergistic growth–inhibitory out-come was achieved in human TNBC tumor lines, regardless ofBRCA1/2 status. Although BRCAness is an important element inthis combined effect, it is not limiting. Indeed, PARPi are underinvestigation in a variety of cancers involving breast, ovarian,pancreatic, NSCLC, gastric cancer, glioblastoma, melanoma, andothers. Our results indicate that the combination of PARPi andIMMU-132 could broaden the range of tumors that are usuallytreated with the former. It is interesting that IMMU-132 doestarget all of these cancer types (17), which may make this com-bination a rational approach. However, combination studieswithPARPi have been problematic because of hematologic toxicity,thus requiring dosemodification. The studies described here haveshown tolerability for the combination in terms of hematologicparameters and animal body weight, but the ultimate clinicalexperience will of course be critical. Given the promising resultsobtained thus far with IMMU-132 in patients with TNBC, thelogical next step is to combine this therapy with PARPi to furtherimprove clinical outcome.

Disclosure of Potential Conflicts of InterestR.M. Sharkey holds ownership interest (including patents) in, and is a

consultant/advisory board member for, Immunomedics, Inc. D.M. Goldenberg

holds ownership interest (including patents) in Immunomedics, Inc. Nopotential conflicts of interests were disclosed by the other authors.

Authors' ContributionsConception and design: T.M. Cardillo, R.M. Sharkey, D.L. Rossi,D.M. GoldenbergDevelopment of methodology: D.L. RossiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T.M. Cardillo, D.L. Rossi, A.A. MostafaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T.M. Cardillo, R.M. Sharkey, D.L. Rossi, R. Arrojo,A.A. Mostafa, D.M. GoldenbergWriting, review, and/or revision of the manuscript: T.M. Cardillo,R.M. Sharkey, D.L. Rossi, D.M. GoldenbergAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T.M. Cardillo, R.M. Sharkey, D.L. Rossi,D.M. GoldenbergStudy supervision: T.M. Cardillo, D.M. Goldenberg

AcknowledgmentsWe thank Serengulam V. Govindan, Nalini Sathyanarayan, ChristineMazza-

Ferreira, Jing Xia, and Jennifer Donnell for contributions to synthetic andconjugation chemistries, Maria Zalath for assistance with the animal studies,and Rongxiu Li for technical assistance in performing the cell-cycle FACSanalysis.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 27, 2016; revised December 21, 2016; accepted Decem-ber 28, 2016; published OnlineFirst January 9, 2017.

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2017;23:3405-3415. Published OnlineFirst January 9, 2017.Clin Cancer Res Thomas M. Cardillo, Robert M. Sharkey, Diane L. Rossi, et al. wild-type Triple-Negative Breast Cancer

−BRCA1/2Drug Conjugate, IMMU-132, Plus PARP Inhibitors in −Trop-2-SN-38 Antibody−Synthetic Lethality Exploitation by an Anti

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