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

Abstract

Antibody-drug conjugates (ADC) containing pyrrolobenzodiazepine (PBD) dimers are being evaluated clinically in both hematologic and solid tumors. These include ADCT-301 (camidanlumab tesirine) and ADCT-402 (loncastuximab tesirine) in pivotal phase II trials that contain the payload tesirine, which releases the PBD dimer warhead SG3199. An important consideration in future clinical development is acquired resistance. The aim was to generate and characterize PBD acquired resistant cell lines in both hematologic and solid tumor settings. Human Karpas-299 (ALCL) and NCI-N87 (gastric cancer) cells were incubated with increasing IC₅₀ doses of ADC (targeting CD25 and HER2, respectively) or SG3199 in a pulsed manner until stable acquired resistance was established. The level of resistance achieved was approximately 3,000-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 upstream mechanism of resistance. Loss of antibody binding or internalization 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 drug transporters. This has clinical implications as potential biomarkers of resistance and for the rational design of drug combinations.

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, antibody-drug conjugates containing the drug-linker tesirine (ADCT-301, ADCT-502), and SG3560. B. Continuous exposure in vitro 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 in vitro 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. p-values obtained using two-tailed, unpaired t-tests.

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 internalisation kinetics of 10 and 0.3 μg/mL unconjugated mAb by IncuCyte pHAb internalisation 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; +: ABCC2; ▴: SLCO2B1; ▾: SLC7A7; ♦: SLC22A3.

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, ADC and PBD resistant cell lines with target ADC and either 5 μM MK-571 or 10 μM FTC. B. Continuous exposure in vitro growth inhibition of Karpas-299 and NCI-N87 wt, ADC and PBD resistant cell lines with SG3199 and either 5 μM MK-571 or 10 μM FTC. C. Interstrand cross-link formation in Karpas-299 and NCI-N87 wt, ADC and PBD resistant cell lines with target ADC or SG3199 (Karpas: 130 pM ADCT-301, 280 pM SG3199; NCI-N97: 1 nM ADCT-502, 1.7 nM SG3199) and either 5 μM MK-571 or 10 μM 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.

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, 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, 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.