For ADC growth inhibition assays, cells were then incubated with serial dilutions of ADCT-301 or ADCT-502 in triplicate

For ADC growth inhibition assays, cells were then incubated with serial dilutions of ADCT-301 or ADCT-502 in triplicate. level of resistance achieved was ~3000-fold for ADCT-301 and 3-fold for SG3199 in Karpas-299, and 8-fold for ADCT-502 and 4-fold for SG3199 in NCI-N87. Cross-resistance between ADC and SG3199, and with an alternative PBD-containing ADC or PBD dimer was observed. The acquired resistant lines produced fewer DNA interstrand cross-links indicating an Ctsl up-stream mechanism of resistance. Loss of antibody binding or internalisation was not observed. A human drug-transporter PCR Array revealed several genes upregulated in all the resistant cell lines including ABCG2 and ABCC2, but not ABCB1(MDR1). These findings were confirmed by RT-PCR and western blot, and inhibitors and siRNA knockdown of ABCG2 and ABCC2 recovered drug sensitivity. These data show that acquired resistance to PBD-ADCs and SG3199 can involve specific ATP-binding cassette (ABC) drug transporters. This has clinical implications as potential biomarkers of resistance and for the rational design of drug combinations. S(-)-Propranolol HCl Keywords: Acquired drug resistance, PBD dimer, antibody-drug conjugate, ABC transporter Introduction The increasing desire for pyrrolobenzodiazepine (PBD) dimers as warheads for antibody-drug conjugates (ADCs) over the last decade has resulted in numerous clinical trials of PBD dimer-based ADCs in both solid and hematological tumors [1]. For example, ADCT-402 (loncastuximab S(-)-Propranolol HCl tesirine) [2] and ADCT-301 (camidanlumab tesirine) [3] are currently in pivotal phase 2 trials in Diffuse Large B-Cell Lymphoma (DLBCL) and Hodgkin Lymphoma (HL), respectively [1]. As PBD-dimer based ADCs progress through clinical development, the long-term success of these drugs will depend on understanding and overcoming the S(-)-Propranolol HCl potential development of acquired resistance, which due to the small molecule-antibody conjugate composition and multi-stage mechanism of action of ADCs can be complex, as cells have multiple opportunities to resist the ADC [4]. Both pre-clinical and clinical studies have been carried out on currently approved ADCs and the mechanism of acquired resistance generally falls into two major categories; related to the antibody portion of the ADC, or related to the drug/drug-linker portion of the ADC [5]. Tumor cells that have become refractory to monoclonal antibody (mAb) therapy often show downregulation of the target antigen or impaired mAb-antigen complex internalisation [6], a resistance mechanism that has also been reported with trastuzumab emtansine (T-DM1) [7, 8], brentuximab vedotin (BV) [9] and gemtuzumab ozogamicin (GO) [10]. Should the binding and internalisation of the ADC not be significantly affected, acquired resistance to the small drug molecule released inside the tumor cell can develop due to upregulation of drug efflux pumps, upregulation of cellular repair mechanisms associated with the drugs mechanism of action, or prevention of apoptosis [11, 12]. The membrane bound drug transporter P-glycoprotein (MDR1, ABCB1) is one of the most commonly upregulated efflux pumps associated with small molecule drug resistance [13], as well as other members of the ABC transporter family [14, 15]. Indeed, MDR1 has been implicated in the resistance to T-DM1, GO and inotuzumab ozogamicin [7, 16]. The PBD dimer SJG-136 (SG2000, Physique 1) has been shown to be a poor substrate for MDR1 so it is possible that this upregulation of this transporter could play a role in acquired resistance to PBD dimer-based ADCs [17]. Open in a separate window Physique 1 The generation of cell lines with acquired resistance to PBD S(-)-Propranolol HCl dimer or PBD dimer-based ADCs. A. General structure of the PBD dimer warheads SG3199 and SG2000, antibody-drug conjugates made up of the drug-linker tesirine (ADCT-301, ADCT-502), and SG3560. B. Continuous exposure growth inhibition of wt, ADC and PBD resistant cell lines with target ADC and PBD dimer. C. Interstrand cross-link formation of wt, ADC and PBD resistant cell lines with target ADC and PBD dimer (Karpas: 130 pM ADCT-301, 280 pM SG3199; NCI-N97: 1 nM ADCT-502, 1.7 nM SG3199). D. Continuous exposure growth inhibition of wt, 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. growth inhibition 10,000 cells per well were seeded in a flat bottom 96-well plate, and NCI-N87 cell were incubated overnight before treatment. For ADC growth inhibition assays, cells were then incubated with serial dilutions of ADCT-301 or ADCT-502 in triplicate. For the SG3199 growth inhibition assays, cells were mixed with a serial dilution of SG3199 in DMSO before seeding. Growth inhibition was measured after 96 hours in Karpas-299 cells and 144 hours in NCI-N87 cells using the CellTiter 96 AQueous One MTS Answer (Promega), and absorbance measured on a Multiskan Ascent plate reader (ThermoFisher) at 492 nm. Growth inhibition was calculated as a percentage of absorbance compared to an untreated control, and IC50 values were calculated using the sigmoidal, 4PL, X is usually log(concentration) equation in GraphPad Prism. For drug transporter inhibitor.