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 cis- platin 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 Ó2020 American Association for Cancer Research. repair mechanisms (22). AACRJournals.org | 1856 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst July 15, 2020; DOI: 10.1158/1535-7163.MCT-20-0222 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 Æ 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 AACRJournals.org Mol Cancer Ther; 19(9) September 2020 1857 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst July 15, 2020; DOI: 10.1158/1535-7163.MCT-20-0222 Corbett et al. (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 Â 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) mea- sured 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.