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Published OnlineFirst July 15, 2020; DOI: 10.1158/1535-7163.MCT-20-0222

MOLECULAR CANCER THERAPEUTICS | LARGE MOLECULE THERAPEUTICS

The Role of Speciﬁc ATP-Binding Cassette Transporters in the Acquired Resistance to Pyrrolobenzodiazepine Dimer–Containing Antibody–Drug Conjugates Simon Corbett1, Shiran Huang1, Francesca Zammarchi2, Philip W. Howard3, Patrick H. van Berkel2, and John A. Hartley1,2

ABSTRACT ◥ Antibody–drug conjugates (ADC) containing pyrrolobenzodia- ADCT-502 and 4-fold for SG3199 in NCI-N87. Cross-resistance zepine (PBD) dimers are being evaluated clinically in both hema- between ADC and SG3199, and with an alternative PBD-containing tologic and solid tumors. These include ADCT-301 (camidanlumab ADC or PBD dimer was observed. The acquired resistant lines tesirine) and ADCT-402 (loncastuximab tesirine) in pivotal phase II produced fewer DNA interstrand cross-links, indicating an trials that contain the payload tesirine, which releases the PBD upstream mechanism of resistance. Loss of antibody binding or dimer warhead SG3199. An important consideration in future internalization was not observed. A human drug transporter PCR clinical development is acquired resistance. The aim was to generate Array revealed several genes upregulated in all the resistant cell and characterize PBD acquired resistant cell lines in both hemato- lines, including ABCG2 and ABCC2, but not ABCB1(MDR1). These logic and solid tumor settings. Human Karpas-299 (ALCL) and findings were confirmed by RT-PCR and Western blot, and inhi- ABCG2 ABCC2 NCI-N87 (gastric cancer) cells were incubated with increasing IC50 bitors and siRNA knockdown of and recovered doses of ADC (targeting CD25 and HER2, respectively) or SG3199 drug sensitivity. These data show that acquired resistance to PBD- in a pulsed manner until stable acquired resistance was established. ADCs and SG3199 can involve specific ATP-binding cassette drug The level of resistance achieved was approximately 3,000-fold for transporters. This has clinical implications as potential biomarkers ADCT-301 and 3-fold for SG3199 in Karpas-299, and 8-fold for of resistance and for the rational design of drug combinations.

Introduction become refractory to mAb therapy often show downregulation of the target antigen or impaired mAb–antigen complex internalization (6), a The increasing interest in pyrrolobenzodiazepine (PBD) dimers as resistance mechanism that has also been reported with trastuzumab warheads for antibody–drug conjugates (ADC) over the last decade emtansine (T-DM1; refs. 7, 8), brentuximab vedotin (BV; ref. 9), and has resulted in numerous clinical trials of PBD dimer–based ADCs in gemtuzumab ozogamicin (GO; ref. 10). both solid and hematologic tumors (1). For example, ADCT-402 Should the binding and internalization of the ADC not be signif- (loncastuximab tesirine; ref. 2) and ADCT-301 (camidanlumab tesir- icantly affected, acquired resistance to the small drug molecule released ine; ref. 3) are currently in pivotal phase II trials in diffuse large B-cell inside the tumor cell can develop due to upregulation of drug efﬂux lymphoma and Hodgkin lymphoma, respectively (1). pumps, upregulation of cellular repair mechanisms associated with the As PBD dimer–based ADCs progress through clinical development, drugs mechanism of action, or prevention of apoptosis (11, 12). The the long-term success of these drugs will depend on understanding and membrane-bound drug transporter P-glycoprotein (MDR1, ABCB1) overcoming the potential development of acquired resistance, which is one of the most commonly upregulated efﬂux pumps associated with due to the small molecule–antibody conjugate composition and small molecule drug resistance (13), as well as other members of the multistage mechanism of action of ADCs can be complex, as cells ABC transporter family (14, 15). Indeed, MDR1 has been implicated in have multiple opportunities to resist the ADC (4). Both preclinical and the resistance to T-DM1, GO, and inotuzumab ozogamicin (7, 16). The clinical studies have been carried out on currently approved ADCs, and PBD dimer SJG-136 (SG2000, Fig. 1) has been shown to be a weak the mechanism of acquired resistance generally falls into two major substrate for MDR1, so it is possible that the upregulation of this categories: related to the antibody portion of the ADC or related to the transporter could play a role in acquired resistance to PBD dimer– drug/drug-linker portion of the ADC (5). Tumor cells that have based ADCs (17). The mechanism of action of the PBD dimers involves the rapid 1 Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer formation of persistent DNA interstrand cross-links (ICL) in the Institute, London, United Kingdom. 2ADC Therapeutics (UK) Limited, QMB Innovation Centre, London, United Kingdom. 3AstraZeneca/Spirogen, QMB minor grove of the DNA, causing stalled replication forks and trig- Innovation Centre, London, United Kingdom. gering apoptosis during cell division (18). Repair of ICLs is complex and can involve a combination of repair mechanisms including Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). nucleotide excision repair and homologous recombination (HR; ref. 19). Increased repair (unhooking) of DNA ICLs has been observed Corresponding Author: John A. Hartley, UCL Cancer Institute, 72 Huntley Street, as a mechanism of clinical acquired resistance to conventional DNA London, WC1E 6BT, United Kingdom. Phone: 44-207-679-6055; E-mail: [email protected] cross-linking agents including melphalan in myeloma (20) and cisplatin in ovarian cancer (21). However, the nature of the PBD dimer Mol Cancer Ther 2020;19:1856–65 ICL, causing minimal distortion of the DNA structure, and its resulting doi: 10.1158/1535-7163.MCT-20-0222 persistence in cells, suggests that it may be refractory to some DNA 2020 American Association for Cancer Research. repair mechanisms (22).

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ABC Transporters and Acquired Drug Resistance to PBD-ADCs

Figure 1. The generation of cell lines with acquired resistance to PBD dimer or PBD dimer–based ADCs. A, General structure of the PBD dimer warheads SG3199 and SG2000, ADCs containing the drug-linker tesirine (ADCT-301, ADCT-502), and SG3560. B, Continuous exposure in vitro growth inhibition of wt and ADC- and PBD-resistant cell lines with target ADC and PBD dimer. C, ICL formation of wt and ADC- and PBD-resistant cell lines with target ADC and PBD dimer (Karpas: 130 pmol/L ADCT-301, 280 pmol/L SG3199; NCI-N97: 1 nmol/L ADCT-502, 1.7 nmol/L SG3199). D, Continuous exposure in vitro growth inhibition of wt and ADC- and PBD-resistant cell lines with SG3560 ADCs and SG2000. Each data point represents the average of at least 3 biological repeats with SD error bars. P values obtained using two-tailed, unpaired t tests.

From a clinical perspective, it is important to understand the were also generated to the free warhead SG3199 (23). Our ﬁndings mechanisms by which some tumor cells may have inherent sensitivity show that speciﬁc ABC transporter upregulation was conserved across to, or following treatment, may develop acquired resistance to, PBD tumor model and ADC or naked PBD dimer resistance. The results dimer–based ADCs. This could result in predictive biomarkers for also suggest potential treatment strategies of overcoming the resistance response to the ADC or the development of rational combination with the use of appropriate transporter inhibitors. strategies. In this study, two ADCs containing the cathepsin cleavable drug-linker tesirine (Fig. 1), which upon cleavage from the N-10 position releases the PBD dimer SG3199 (Fig. 1), were used to generate Materials and Methods cell lines with acquired resistance. One ADC, targeting CD25, was used Cell lines and reagents to develop resistance in a hematologic cancer setting, the other, The Karpas-299 (ALCL) cell line was purchased from The European targeting HER2 in a solid tumor setting. In parallel, resistant cell lines Collection of Authenticated Cell Cultures (ECACC), and the NCI-N87

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(Gastric carcinoma) cell line was purchased from the ATCC, both Antibody binding and internalization authenticate their cell lines using short tandem repeat DNA typing. Parental and resistant cells (1.5 105) were blocked on ice for Both wt and all resistant cells were grown in RPMI-1640 (Thermo 30 minutes before incubation with a serial dilution of trastuzumab Fisher) supplemented with 10% heat-inactivated FBS (Thermo Fisher) or HuMax-TAC for 1 hour on ice in triplicate. The cells were 0 and 2 mmol/L glutamine (Sigma). All cell lines were cultured at 37 Cin washed and incubated with a F(ab )2-goat anti-human IgG a humidified with a 5% CO2 atmosphere. The ADCs ADCT-301 (3), Fc-Alexa Fluor 488 secondary antibody (Thermo Fisher) 1:50 in ADCT-502 (24), PBD dimer SG3199 (23), and the CD25-specific blocking buffer. After incubation for 1 hour on ice in the dark, antibody HuMax-TAC were provided by ADC Therapeutics. Trastu- cells were washed and processed on a Fortessa X20 flow zumab was obtained from Roche. cytometer, and the maximum fluorescence intensity (MFI) measured using the B530/30 laser/filter. Resistant cell line generation Karpas-299 and NCI-N87 parental and resistant cells were Karpas-299 and NCI-N87 cells were incubated with IC50 concen- seeded in a poly-l-ornithine–coated 96-well plate and allowed to trations of ADC or PBD dimer (3 days Karpas, 6 days NCI-N87) attach at 37C. HuMax-TAC or trastuzumab was incubated with a followed by recovery without treatment, until normal cell growth had 3XmolarexcessofFabFluorpHred antibody internalization returned. This method was intended to simulate the chronic, multi- reagent (Essen Bioscience) for 15 minutes at room temperature, cycle dosing strategy used for cytotoxic therapeutics in the clinic. The and then a 3-fold serial dilution was set up with cell culture resistance of the cells was monitored every month using the MTS assay medium. The labeled antibody dilutions were added to the cells, fi and the dose of drug adjusted to match the new IC50 as the cells became and images were taken at 10x magni cation every 2 hours with increasingly resistant after multiple treatment cycles. phase contrast and red fluorescence filters in an IncuCyte Zoom (Essen BioScience). Mean red object area per well was calculated In vitro growth inhibition using the IncuCyte Zoom software. Note that 10,000 cells per well were seeded in a flat-bottom 96-well plate, and NCI-N87 cell were incubated overnight before treatment. RNA extraction and RT-PCR assays For ADC growth inhibition assays, cells were then incubated with Total RNA was extracted from Karpas-299 or NCI-N87 wt and serial dilutions of ADCT-301 or ADCT-502 in triplicate. For the resistant cells, and purified with the RNeasy Midi Kit (Qiagen). The SG3199 growth inhibition assays, cells were mixed with a serial real-time RT2 Profiler PCR Array for human drug transporters or dilution of SG3199 in DMSO before seeding. Growth inhibition was DNA damage signaling (Qiagen) was used to probe cDNA generated measured after 96 hours in Karpas-299 cells and 144 hours in NCI-N87 from Karpas-299 and NCI-N87 resistant and parental cell line lysates. cells using the CellTiter 96 AQueous One MTS Solution (Promega), CT values were normalized based on a panel of housekeeping and absorbance measured on a Multiskan Ascent plate reader (Thermo genes (HKG). The Qiagen data analysis web portal calculated fold Fisher) at 492 nm. Growth inhibition was calculated as a percentage of change/regulation using the DD CT method, in which D CT was absorbance compared with an untreated control, and IC50 values were calculated between gene of interest and an average of HKGs, followed calculated using the sigmoidal, 4PL, X is log(concentration) equation by DD CT calculations [D CT (Test Group) - D CT (Control Group)]. in GraphPad Prism. For drug transporter inhibitor combination Fold change was then calculated using 2^ (-DD CT) formula. For cytotox, the resistant cell lines were incubated overnight with either confirmation of gene upregulation, qRT-PCR was performed using 5 mmol/L MK-571 (ABCC2 inhibitor, Abcam), 10 mmol/L FTC TaqMan gene expression probes (LifeTech) for the drug transporter of (ABCG2 inhibitor, Sigma), 5 mmol/L Reversin-121 (ABCB1 inhibitor, choice or the HKG ABL-1, and fold change/regulation was calculated Cayman Chemical), or 10 nmol/L Lovastatin (SLCO2B1 inhibitor, using the DD CT method as described above. Abcam) overnight before carrying out the ADC or PBD growth inhibition assay as previously described. Immunoblot analysis Note that 1.5 to 2 106 cell lysates were collected in lysis buffer Single-cell gel electrophoresis (comet) assay (0.0625 mol/L Tris-HCL, 2% SDS, and 10% glycerol). Protein con- The formation of DNA ICLs by either ADCT-301, ADCT-502, or centrations were determined with the DC Protein Estimation Assay SG3199 was measured using a modification of the single-cell gel (BioRad) according to the manufacturer's instructions. Protein electrophoresis (comet) assay. The comet assay was carried out (20–30 mg) was loaded into a 4%–15% Mini-PROTEAN TGX gel according to the protocol described previously (25). Cells were treated (BioRad) and transferred onto a 0.2 mm nitrocellulose membrane with 130 pmol/L ADCT-301 or 280 pmol/L SG3199 (Karpas-299), or 1 (BioRad), followed by primary and secondary antibody incubation. nmol/L ADCT-502 or 1.7 nmol/L SG3199 (NCI-N87) for 2 hours, then The following antibodies were used: rabbit monoclonal anti-ABCC2 washed and incubated for 24 hours under normal cell culture condi- (Cell Signaling Technology), rabbit monoclonal anti-ABCG2 tions. All cells were irradiated with 18 Gy (5 Gy/min for 3.6 minutes). (Abcam), rabbit monoclonal anti-Calnexin (Cell Signaling Technol- ICL formation was quantitated by measuring Olive tail moment ogy), and goat anti–rabbit-horseradish peroxidase-conjugated second- (OTM) using the Komet 6 software (Andor Technology) and the ary antibody (Cell Signaling Technology). Secondary antibody was percentage reduction in OTM calculated according to the formula: detected using the ECL detection kit (Amersham). % decrease in tail moment siRNA knockdown ¼ ½1 ðÞ =ðÞ 100 TMdi TMcu TMci TMcu Silencer Select siRNA oligonucleotides targeting ABCC2 and non- targeting siRNAs were purchased from Ambion/Thermo Fisher. For TMdi ¼ Tail Moment drug irradiated; reverse transfection, Opti-MEM medium was mixed with siRNA to TMcu ¼ TM control un irradiated; give a final concentration of 25 pmol/L, and this was then combined fi TMci ¼ TM control irradiated with diluted Lipofectamine RNAiMAX (Thermo Scienti c). After 20- minute incubation at room temperature, the transfection mixture was

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ABC Transporters and Acquired Drug Resistance to PBD-ADCs

aliquoted into 6-well plates. Cells were added to each well containing Cross-resistance to other PBD-containing ADCs and siRNA and RNAiMAX complex. Forty-eight hours after transfection, DNA-interacting drugs cells were harvested and used for immunoblotting or growth inhibition To further investigate the cross-resistance in the fourcell lines, growth assays as previously described. inhibition was compared with the parental cell line when treated with the structurally similar, but less potent, PBD dimer SG2000, and a C2-linked PBD dimer–based ADC (SG3560; Fig. 1A). The cell lines were cross- Results resistant to both SG2000, and the SG3560-based ADCs (Fig. 1D). Generation of PBD-ADC and PBD dimer acquired resistant cell Comparing the IC50 values with the parental cells showed similar levels lines of resistance to SG2000 to those seen with SG3199 (Table 1). In contrast, IC50 growth inhibition concentrations of the parental cell lines to the level of resistance to the C2-linked SG3560 ADCs was greater than either the target ADC (Karpas-299: 1.5 ng/mL ADCT-301, NCI-N87: with the N10-linked (tesirine) ADCs. Indeed, no growth inhibition was 1.3 ng/mL ADCT-502) or SG3199 (Karpas-299: 70 pmol/L, NCI-N87: seen in the Karpas-299 resistant cells following treatment with the 15.9 pmol/L) were established using the MTS assay (Table 1). Cells C2-linked SG3560 ADC at the concentrations tested (Fig. 1D). The shift were incubated with the IC50 concentration of drug for approximately in IC50 was also much greater than with the N10-linked ADC in the the time required for three cell divisions, then washed and allowed to NCI-N87 ADCr (95-fold vs. 7.6-fold) and NCI-N87 PBDr cells (54-fold recover in drug-free medium until normal cell proliferation was vs. 3.7-fold, Table 1). restored. This process was repeated over a period of 12 to 18 months, Cross-resistance to three conventional DNA-interacting chemo- growth monitored, and the dose of ADC or PBD dimer increased to therapeutic drugs, cisplatin, doxorubicin, and melphalan, was also mirror the increase in IC50. This pulsatile approach resulted in the measured using the MTS assay in all resistant cell lines. No cross- generation of cell lines with an acquired resistance that was stable for resistance was seen between any of these drugs in either the Karpas-299 multiple passages in the absence of any drug. ADCr or PBDr cell lines (Supplementary Fig. S1). In contrast, there Growth inhibition assays showed an increase in the IC50 values in all was a decrease in growth inhibition in both NCI-N87 ADCr and PBDr the cell lines made resistant to either ADC (ADCr) or PBD dimer cells treated with all three of these drugs compared with the parental (PBDr) ranging from 2.9-fold to >3,000-fold (Fig. 1B, Table 1). Inter- cell line (Supplementary Fig. S1). estingly, the cell lines made resistant to ADC were also found to be cross-resistant to SG3199, and the cell lines made resistant to SG3199 Cell surface antigen expression, binding, and internalization were cross-resistant to ADC (Fig. 1B, Table 1). Because resistance to antibody-based therapies can arise from modified cell surface antigen expression, CD25 or HER2 expression Reduced DNA ICL formation in resistant cells and antibody-binding affinity were compared in resistant versus Because the mechanism of action of PBD dimers involves the parental cells by flow cytometry, and internalization kinetics and formation of DNA ICLs, a modification of the single-cell gel electro- lysosomal trafficking by the IncuCyte internalization assay. There was phoresis (comet) assay was used to measure the amount of ICL a small decrease in the MFI reached by the Karpas-299 ADCr cells formation following a single dose of either ADC or PBD dimer. All compared with the PBDr and parental cell lines (Fig. 2A); however, all the cell lines were treated with a dose previously found to give an cells continued to express high levels of antigen. In the NCI-N87 cell approximate 50% reduction in OTM in the parental cell lines after a lines, there was a small decrease in MFI reached by the PBDr cell line, 2-hour drug exposure, followed by 24-hour drug-free incubation but the ADCr cell line was unchanged compared with the parental (Karpas: 130 pmol/L ADCT-301, 280 pmol/L SG3199; NCI-N87: line (Fig. 2A). The internalization kinetics for HuMax-TAC or 1 nmol/L ADCT-502, 1.7 nmol/L SG3199). The comet assays showed trastuzumab were unchanged in the resistant cell lines treated with a significant reduction in DNA ICL formation in Karpas ADCr cells 10 or 0.3 mg/mL of antibody over a 24-hour period (Fig. 2A). treated with ADCT-301 (53.5% to 15% reduction in OTM, P value 0.0004), Karpas PBDr cells treated with SG3199 (55.5% to 25.7%, Changes in gene expression in ADCr and PBDr cell lines P value 0.003), NCI-N87 ADCr cells treated with ADCT-502 (54.7% to Another possible mechanism of resistance upstream of the DNA 29%, P value 0.004), and NCI-N87 PBDr cells treated with SG3199 ICL formation is that the PBD dimer molecules are being actively (51% to 30.7%, P value 0.004; Fig. 1C). In addition, consistent with the effluxed out of the cells when they are either released by ADC IC50 data, the level of DNA ICL formation was also significantly metabolism, or they have entered into the cell as stand-alone agents. reduced in the ADCr cell lines treated with SG3199 and PBDr cell lines To investigate this potential pathway of drug resistance, RT2 PCR treated with ADC (Fig. 1B). These data suggest mechanisms of arrays (Qiagen) were carried out comparing the gene expression of resistance in all four of the cell lines which prevent the formation of 82 known human drug transporter genes in the resistant versus DNA damage. parental cell lines. Volcano plots generated by the online analysis

Table 1. IC50 values and fold resistance of Karpas-299 and NCI-N87 resistant cell lines to PBD dimer and PBD dimer–based ADCs.

IC50 (fold resistance) Cell line ADCT-301 ng/mL ADCT-502 ng/mL SG3199 pmol/L SG2000 nmol/L mAb-SG3560 ng/mL

Karpas wt 1.5 – 70 0.9 1.3 Karpas ADCr 5,000 (3,333) – 306.5 (4.4) 2.8 (3.1) >10,000 (>7,692) Karpas PBDr 72.8 (48.5) – 204.5 (2.9) 4.7 (5.2) >10,000 (>7,692) NCI-N87 wt – 1.3 15.9 0.06 1.0 NCI-N87 ADCr – 9.9 (7.6) 60.4 (3.8) 0.46 (7.7) 95 (95) NCI-N87 PBDr – 7.2 (5.5) 58.6 (3.7) 0.47 (7.8) 54 (54)

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Figure 2. Investigating the mechanism of PBD and PBD-based ADC acquired resistance in Karpas-299 and NCI-N87 cells. A, Surface antigen expression and binding of unconjugated mAb (HuMax-TAC or trastuzumab) by ﬂow cytometry, and lysosomal internalization kinetics of 10 and 0.3 mg/mL unconjugated mAb by IncuCyte pHAb internalization assay. B, Qiagen RT2 human drug transporter PCR array of Karpas-299 and NCI-N87 ADC- and PBD-resistant cell lines. C, qRT-PCR conﬁrmation of commonly upregulated transporter genes using TaqMan probes in Karpas-299 and NCI-N87 ADC- and PBD-resistant cell lines. Each data point represents the average of at least 3 biological repeats with SD error bars, or SEM in the RT-PCR. , ABCG2; þ, ABCC2; ~, SLCO2B1; !, SLC7A7;and^, SLC22A3.

software showed a number of genes to be significantly upregulated the parental cells was confirmed in all cases by RT-PCR using TaqMan (> 2-fold change, P value <0.05) in the four acquired resistant cell lines probes (Fig. 2C). All the genes tested were found to be upregulated in (Fig. 2B). close agreement to the RT2-PCR array (fold change and SD in Table 2). Interestingly, ABCG2 was significantly upregulated in the Karpas ABCG2 and SLCO2B1 were upregulated in all four resistant cell lines, ADCr, Karpas PBDr, NCI-N87 ADCr, and NCI-N87 PBDr cell lines whereas ABCC2 was upregulated in all lines except the Karpas-299 (fold change and P values shown in Table 2). ABCC2 was also PBDr cells, and SLC22A3 and SLC7A7 were both upregulated in the upregulated in the Karpas ADCr, NCI-N87 ADCr, and NCI-N87 two solid tumor cell lines. PBDr cells. SLCO2B1 was upregulated in the Karpas PBDr, NCI-N87 In contrast to the results obtained with the human drug transporter ADCr, and NCI-N87 PBDr cells. The drug transporter genes SLC22A3 gene array, an RT2 PCR array for DNA damage signaling pathway and SLC7A7 were significantly upregulated only in the NCI-N87 showed no significantly upregulated genes in any of the acquired ADCr and NCI-N87 PBDr cell lines (Table 2). resistant lines (Supplementary Fig. S2). Overall, the data are consistent The upregulation of drug transporter genes ABCG2, ABCC2, with the mechanism of resistance being upstream of the DNA damage SLCO2B1, SLC22A3, and SLC7A7 in the resistant lines compared with produced by the PBD dimer.

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Table 2. Human drug transporter gene upregulation in Karpas-299 and NCI-N87 resistant cell lines by RT2 PCR array and TaqMan probe qRT-PCR analysis.

Fold change (P value/SD) Karpas ADCr Karpas PBDr NCI ADCr NCI PBDr Transporter gene RT2 Taq RT2 Taq RT2 Taq RT2 Taq

ABCG2 167.5 (0.0003) 134 (87) 2.4 (0.008) 3 (1.5) 107 (0.004) 111 (51) 144.4 (0.00006) 150 (40) ABCC2 3.5 (0.01) 6.5 (3.3) – 1 (0.2) 50.95 (0.0005) 55 (12) 44.8 (0.00006) 56 (0.2) SLCO2B1 – 3.1 (1.4) 13.31 (0.00004) 9.8 (10.9) 35.87 (0.01) 49 (25) 66.8 (0.0003) 60 (37) SLC22A3 ––––77.38 (0.02) 220 (134) 77.1 (0.001) 201 (48) SLC7A7 ––––8.08 (0.0003) 11 (1.1) 9.9 (0.002) 10 (4.8)

Reversal of resistance by ABC transporter inhibitors were treated with a nontoxic dose of MK-571 (5 mmol/L) or FTC In order to investigate further the contribution of ABC drug (10 mmol/L) for 24 hours before the addition of the ADC or PBD transporter upregulation in the acquired resistance to the PBD dimer dimer. Both MK-571 and FTC showed a dramatic resensitization of and PBD dimer–based ADC resistant cell lines, inhibitors of ABCG2 Karpas-299 ADCr and PBDr cells to ADCT-301 treatment (Fig. 3A). (Fumitremorgin C, FTC) and ABCC2 (MK-571) were used in com- Similarly, in the NCI-N87 ADCr and PBDr cells treated with either bination with the ADC or PBD dimer to assess the effect on growth MK-571 or FTC, the response to ADCT-502 was restored to the level of inhibition using the MTS assay. Karpas and NCI-N87 resistant cells the parental cell line (Fig. 3A). Restoration of sensitivity was observed

Figure 3. Reversing the acquired PBD or PBD-based ADC resistance with ABC drug transporter inhibitors. A, Continuous exposure in vitro growth inhibition of Karpas-299 and NCI-N87 wt and ADC- and PBD-resistant cell lines with target ADC and either 5 mmol/L MK-571 or 10 mmol/L FTC. B, Continuous exposure in vitro growth inhibition of Karpas-299 and NCI-N87 wt and ADC- and PBD-resistant cell lines with SG3199 and either 5 mmol/L MK-571 or 10 mmol/L FTC. C, ICL formation in Karpas-299 and NCI-N87 wt and ADC- and PBD-resistant cell lines with target ADC or SG3199 (Karpas: 130 pmol/L ADCT-301, 280 pmol/L SG3199; NCI-N97: 1 nmol/L ADCT-502, 1.7 nmol/L SG3199) and either 5 mmol/L MK-571 or 10 mmol/L FTC. Each data point represents the average of at least 3 biological repeats with SD error bars. P values obtained using two-tailed, unpaired t tests.

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Figure 4. ABC transporter immunoblots and ABCC2 siRNA knockdown reversed resistance in NCI-N87 resistant cells. A, Representative Western blot for ABCC2 and ABCG2 in Karpas-299 and NCI-N87 wt and ADC- and PBD-resistant cell lines. B, Representative Western blot for ABCC2 NCI-N87 ADC- and PBD-resistant cell lines with siRNA against ABCC2 or scramble control. C, ABCC2 siRNA knockdown continuous exposure in vitro growth inhibition of NCI-N87 wt and ADC- and PBD-resistant cell lines with ADCT-502 or SG3199. Each data point represents the average of at least 3 biological repeats with SD error bars.

when the ABC transporter inhibitors were combined with SG3199 in ABCG2 and ABCC2. Immunoblotting showed the ABCG2 protein Karpas ADCr and PBDr cells (Fig. 3B). Lovastatin, an inhibitor of to be upregulated in the Karpas-299 ADCr cells compared with the SLCO2B1, however failed to reverse the resistance in any of the cell parental cell line, whereas any upregulation in PBDr cells could not lines (Supplementary Fig. S3). be detected (Fig. 4A). This reﬂects the very different levels of In order to correlate the inhibition of the ABC transporters with the upregulation observed by PCR in these cells. Despite numerous reversal of ADC and PBD dimer resistance mechanistically, the comet attempts, ABCC2 protein could not be observed by immunoblot- assay was used to measure DNA ICL formation in the resistant cell ting in either parental or the resistant Karpas-299 cells. In the lines by the ADC or PBD dimer pretreated with either 5 mmol/L MK- NCI-N87 resistant cell line immunoblots, both ABCC2 and 571 or 10 mmol/L FTC. In all the resistant cell lines, treatment with ABCG2 were both clearly upregulated compared with the parental transporter inhibitor resulted in increased formation of ADC or PBD- cell line (Fig. 4A). induced DNA ICL. These data are consistent with increased retention NCI-N87 cells were transfected with siRNA against ABCC2 and of PBD dimer in cells resulting in increased DNA damage and resultant ABCG2. ABCG2 was not able to be effectively knocked out due to the cytotoxicity. very long half-life of the protein (Supplementary Fig. S4), but ABCC2 was successfully depleted in both NCI-N87 ADCr and PBDr cells Protein analysis of ABC transporters and siRNA knockout of compared with a nontarget siRNA control (Fig. 4B). The depletion of ABCC2 ABCC2 in the NCI-N87 ADCr and PBDr cells was able to restore The two drug transporters with the most consistent mRNA cytotoxic sensitivity to ADCT-502 and SG3199 to the level of the upregulation across the resistant cell lines, which also responded to parental cell line (Fig. 4C), further implicating ABCC2 in the mech- appropriate transporter inhibition to restore drug sensitivity, were anism of acquired resistance in these cell lines.

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ABC Transporters and Acquired Drug Resistance to PBD-ADCs

Discussion Two members of the ABC transporter family, ABCC2 and ABCG2, were significantly upregulated in the acquired resistance cell lines, and Understanding the acquired resistance of ADCs is not straightfor- inhibitors to these two transporters recovered the cytotoxic sensitivity ward due to their multistep mechanism of action, and multiple routes of the cells to the ADCs and SG3199. The NCI-N87 cells did not exist for the cells to avoid and escape drug-initiated apoptosis (4). We respond as well to the inhibitors in the PBD assay, most likely due to have shown that it is possible to generate cell lines with stable acquired the inability to pretreat the NCI-N87 cells with inhibitors before resistance to the highly potent PBD dimer SG3199 and also to ADCs SG3199 addition. The ABC inhibitors induced a restoration of DNA that release SG3199 as their warhead. In this study, two human cell ICL formation to levels seen in the parental cell lines. ABCC2 lines, one hematologic background (Karpas-299, ALCL) and one solid (multidrug resistance protein 2) and ABCG2 (breast cancer resistance tumor background (NCI-N87, gastric), were made resistant to either a protein) have been widely implicated in chemotherapy resis- PBD dimer–based ADC or SG3199. All four acquired resistant cell tance (12, 30). Interestingly, ABCB1 (MDR1, P-glycoprotein 1) was lines were fully characterized to understand the mechanism of resis- not significantly upregulated in any of the resistant cell lines. Previ- tance, and a conserved upregulation of specific ABC drug transporters ously, SG3199 was found to be moderately susceptible to multidrug was observed which was reversed by appropriate ABC transporter resistance mechanisms when compared in human tumor cell lines inhibitors, or siRNA. nonexpressing or expressing MDR1, and the inhibitor verapamil was The Karpas-299 cell lines were significantly more resistant to able to reverse the resistance (23). Members of the SLC transporter ADCT-301 than SG3199; therefore, the hypothesis was that more family were also upregulated in some of the acquired cell lines. than one mechanism of resistance could be present. One of the most Lovastatin, an SLC transporter inhibitor, did not reverse the resistance common ways of cells to become resistant to an antibody-based suggesting that these transporters are unlikely to be key in the therapy is the loss of antigen expression, or epitope modification, to resistance mechanism. SLC transporters mediate the influx of drugs prevent antibody binding and internalization or signaling (6). This into cells (31); therefore, it would be interesting to investigate any mechanism has not only been seen with naked antibody therapy, downregulated SLC genes, which could play a role in a resistance but also with ADCs in vitro and clinically (7–9). Our study showed phenotype by reducing the bystander effect known to play a role in that the binding and subsequent internalization of the CD25 and PBD-ADC cytotoxicity (32). HER2 antibodies was not affected in the acquired resistant cell lines, In addition to cross-resistance between the ADCs used for estab- and the apparent affinity of the antibody to all the cell lines was lishment of the resistant lines and SG3199, cross-resistance was unchanged. Although there was a small drop in maximum also observed with another, less potent, PBD dimer SG2000 and with binding of HuMax-TAC to the Karpas-299 ADCr cells, this could a C2-linked PBD-ADC (mAb-SG3560) rather than N10-linked ADCs. not account for the significant loss of growth inhibition seen with Interestingly, in the latter case, the level of resistance for mAb-SG3560 ADCT-301, or the cross-resistance observed to SG3199. Internal- was much greater than for the N10-linked ADCs. This may reflect ization assays confirmed that, in all cases, the antibody was inter- differences in the biophysical properties of the released warheads (33). nalized into the lysosomes, a process that is essential for ADC linker The PBD dimer released by mAb-SG3560 is much more hydrophobic cleavage to release the cytotoxic warhead. and may therefore be a better substrate for the ABC transporters. No Importantly, the acquired resistant cell lines were not only cross-resistance was observed in the Karpas-299 resistant cell lines resistant to the PBD dimer or ADC to which they were treated with conventional DNA cross-linking chemotherapeutic drugs cis- during establishment, but they were also cross-resistant. This platin and melphalan. This is consistent with the fact that these agents overlap in the profile of the drug resistance generated suggested are not considered to be substrates for ABCG2. In contrast, the DNA- an overlapping mechanism of resistance across the different cell intercalating drug doxorubicin is a known substrate for MDR1, but lines. Indeed, all the resistant cell lines also showed a decrease in again no cross-resistance was observed in the resistant lines, consistent DNA ICL formation when treated with the ADC or PBD dimer with the lack of upregulation of ABCB1. Some cross-resistance was, compared with the parental cell lines, which indicated that the however, observed with these agents in the NCI-N87 resistant lines, mechanism of resistance is upstream of DNA ICL formation, and which is consistent with the fact that platinum agents can be substrates not a downstream DNA repair or apoptosis signaling evasion for ABCC1/2 as well as MDR1 (34). mechanism. Taken together, the data presented in this article suggest that the Consistent with an upstream mechanism of resistance, the human ABC drug transporters ABCG2 and ABCC2 play an important role drug transporter PCR array performed on all the cell lines showed that in determining sensitivity to PBD dimers and PBD-containing specific drug transporters were upregulated in all of the cell lines, and ADCs. It remains to be determined to what extent these findings this was corroborated by qRT-PCR. In contrast, DNA damage sig- in vitro translate into clinical relevance and whether these trans- naling pathway arrays did not reveal any significant upregulation that porters become upregulated in patients that relapse on PBD-based would be relevant to PBD dimer resistance. Previously, changes in ADC therapy. Screening of tumors for expression may also be used expression of specific DNA damage signaling genes have been as a biomarker for response to treatment. Development of clinically observed following treatment of cells with conventional DNA acquired drug resistance by this mechanism would also guide cross-linking agents such as melphalan or cisplatin (26), and specific future patient management with non–PBD-based therapy, includ- DNA repair proteins such as ERCC1 have been found to be upregu- ing the use of ADCs delivering warheads of differing mechanism of lated in cells with acquired resistance to cisplatin (27, 28). DNA ICLs action. These could involve the same targeting antibody because produced by PBD dimers persist compared with those produced by significant changes in antigen expression level have not been conventional DNA cross-linking agents (23) which is likely due to poor observed in the current study. An alternative strategy could recognition by DNA repair and damage response proteins; however, involve combination therapy with specificABCtransporter the XPF-ERCC1 endonuclease and HR have been shown to contribute inhibitors, as a clinical trial in R/R Hodgkin lymphoma patients to the ultimate repair of the minor groove ICLs produced by PBD who were also previously refractory to BV, the mechanism dimers including SG3199 (29). for which is MDR1 related, has shown that combination with the

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Corbett et al.

wide-ranging ABC transporter inhibitor cyclosporine can resen- Authors’ Contributions sitize patients to BV (35). S. Corbett: Formal analysis, investigation, methodology, writing-original draft. Several PBD dimer–based ADCs are currently undergoing clinical S. Huang: Investigation. F. Zammarchi: Writing-Review and editing. P.W. Howard: development in both solid and hematologic tumors. The current study Project administration, writing-review and editing. P.H. van Berkel: Supervision, writing-review and editing. J.A. Hartley: Conceptualization, supervision, funding has shown that specific ABC transporters may be key in both selecting acquisition, project administration, writing-review and editing. patients and as a marker of acquired resistance following repeated therapy. Acknowledgments S. Corbett acknowledges ADC Therapeutics for funding a PhD studentship. J.A. Disclosure of Potential Conflicts of Interest Hartley acknowledges program grant support from Cancer Research UK (C2559A/ S. Corbett reports personal fees from ADC Therapeutics (PhD studentship) A16569). during the conduct of the study. P.W. Howard reports employment with AstraZeneca during the conduct of the study and ownership of ADCT stock The costs of publication of this article were defrayed in part by the payment of page outside the submitted work. P.H. van Berkel reports a patent for charges. This article must therefore be hereby marked advertisement in accordance WO20140571191A1 and WO2015052534A1. J.A. Hartley reports grants from with 18 U.S.C. Section 1734 solely to indicate this fact. ADC Therapeutics and Cancer Research UK during the conduct of the study; consultancy with ADC Therapeutics in which he also has an equity interest; and previous part-time employment with Spirogen/AstraZeneca. No potential Received March 25, 2020; revised May 14, 2020; accepted July 2, 2020; conflicts of interest were disclosed by the other authors. published first July 15, 2020.

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AACRJournals.org Mol Cancer Ther; 19(9) September 2020 1865

The Role of Specific ATP-Binding Cassette Transporters in the Acquired Resistance to Pyrrolobenzodiazepine Dimer−Containing Antibody−Drug Conjugates

Simon Corbett, Shiran Huang, Francesca Zammarchi, et al.

Mol Cancer Ther 2020;19:1856-1865. Published OnlineFirst July 15, 2020.

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