Identification of antibody-drug conjugate payloads which are substrates of ATP-binding cassette drug efflux transporters

Abstract

Aim: Antibody-drug conjugates (ADCs) feature an antibody recognizing a specific protein joined to a potent toxic payload. Numerous antibody-drug conjugates have received FDA approval; however, clinical resistance arises. Resistance mechanisms include decreased expression or mutation of the antibody target, impaired payload release, or increased expression of ATP-binding cassette (ABC) efflux transporters associated with multidrug resistance. We therefore sought to characterize the interactions of ABC multidrug transporters with ADC payloads.

Methods: We performed a high-throughput screen with 27 common ADC payloads using cells lines expressing ABC transporters P-glycoprotein (P-gp, encoded by ABCB1) or ABCG2 (encoded by ABCG2). Confirmatory assays were also performed using cells transfected to express P-gp, ABCG2, or MRP1 (encoded by ABCC1).

Results: Several commonly used ADC payloads were substrates of P-gp, including calicheamicin gamma1, monomethyl auristatin E, DM1, and DM4. All the pyrrolobenzodiazepines tested-SJG136, SGD-1882, SG2057, and SG3199-were substrates of P-gp, ABCG2, and MRP1. The modified anthracyclines nemorubicin and its metabolite PNU-159682 were poorly transported by both ABCB1 and ABCG2 and displayed nanomolar to picomolar toxicity. Further, we found that the efficacy of the FDA-approved ADC mirvetuximab soravtansine, with DM4 as the toxic payload, was decreased in cell lines expressing P-gp. Duocarmycin DM and PNU-159682 were exquisitely toxic to a panel of 99 cancer cell lines of varying origins.

Conclusion: Several commonly used ADC payloads can be transported by ABC transporters, potentially leading to transporter-mediated drug resistance in patients. Future ADCs should be developed using payloads that are not ABC transporter substrates.

Keywords: ABC transporter; ABCG2; P-glycoprotein; antibody-drug conjugate; drug resistance.

Figure 1:. Heatmap of ADC payload activity across multidrug-resistant and control cell lines.

(A) Unsupervised clustering of area under the curve (AUC) values from dose-response viability assays of a panel of cytotoxic agents tested against parental, multidrug-resistant, and genetically modified KB and MDR-19 cell lines. Cell lines include KB-3-1 (parental), KB-8-5-11 (ABCB1 overexpressing), MDR-19 (ABCB1-overexpressing), and their vector controls (pcDNA) or P-gp–inhibited counterparts (+TQR, tariquidar). (B) Unsupervised clustering of AUC values from the same compound panel tested in H460 (non-small cell lung cancer) parental cells, MDR sublines (H460 MX20 and H460 MX20+Ko143), and control or ABCG2-overexpressing derivatives (R5, R5+Ko143). Color scale reflects compound potency: orange indicates stronger cell killing (more cell death; greater sensitivity), while cyan indicates weaker cell killing (less cell death; more resistance). Compounds are grouped by activity outcome as indicated on the left dendrogram. Clustering was performed based on Euclidean distances of AUC profiles across cell lines.

Figure 2:. Confirmatory cytotoxicity assays with screen hits additional ADC payloads

Three-day cytotoxicity assays were performed with the noted compounds as described in the Materials and Methods using HEK-293 cells transfected with empty vector (pcDNA, black dot), or vectors containing ABCG2 (R5, red square), ABCB1 (MDR-19, blue triangle), or ABCC1 (MRP1, black box). Results from one of three independent experiments are shown. Cytotoxicity data are summarized in Table 3.

Figure 3:. P-gp overexpression confers resistance to treatment with mirvetuximab Soravtansine

Three-day cytotoxicity assays were performed with mirvetuximab soravtansine as described in the Materials and Methods using the OVCAR8/NCI/ADR-RES pair (left graph) or the KB-3-1/KB 8-5-11 pair (right graph). Results from one of three independent experiments are shown.

Figure 4:. Differential cytotoxicity of ADC payloads across cancer cell lines from diverse tissue origins.

Heatmap showing AUC values from dose–response viability assays for a panel of ADC payload compounds tested across a large set of human cancer cell lines. Each row represents a unique cancer cell line, annotated by tissue of origin (colored bar on the right-hand side), and each column corresponds to an ADC payload compound, annotated by mechanism of action (MOA) on the bottom of the heatmap. AUC values are color-coded, with red indicating higher AUC (lower sensitivity/more resistant) and blue indicating lower AUC (higher sensitivity/more cytotoxic effect). Clustering (both rows and columns) was performed using hierarchical methods based on compound response profiles.