Sacituzumab govitecan (IMMU-132), an SN-38–conjugated antibody–drug conjugate, is showing promising therapeutic results in a phase I/II trial of patients with advanced Trop-2–expressing, metastatic, solid cancers. As members of the ATP-binding cassette (ABC) transporters confer chemotherapy resistance by active drug efflux, which is a frequent cause of treatment failure, we explored the use of known inhibitors of ABC transporters for improving the therapeutic efficacy of IMMU-132 by overcoming SN-38 resistance. Two human tumor cell lines made resistant to SN-38, MDA-MB-231-S120 (human breast cancer) and NCI-N87-S120 (human gastric cancer), were established by continuous exposure of the parental cells to stepwise increased concentrations of SN-38 and analyzed by flow cytometry for functional activities of ABCG2 and ABCB1, immunoblotting and qRT-PCR for the expression of ABCG2 at both protein and mRNA levels, and MTS assays for the potency of SN-38 alone or in combination with a modulator of ABC transporters. MDA-MB-231-S120 and NCI-N87-S120 displayed reduced sensitivity to SN-38 in vitro, with IC50 values approximately 50-fold higher than parental MDA-MB-231 and NCI-N87 cells. The increase in drug resistance of both S120 cell populations is associated with the expression of functional ABCG2, but not ABCB1. Importantly, treatment of both S120 sublines with known ABCG2 inhibitors (fumitremorgin C, Ko143, and YHO-13351) restored toxicity of SN-38, and the combination of YHO-13351 with IMMU-132 increased the median survival of mice bearing NCI-N87-S120 xenografts. These results provide a rationale for combination therapy of IMMU-132 and inhibitors of ABC transporters, such as YHO-13351. Mol Cancer Ther; 15(8); 1910–9. ©2016 AACR.

Multidrug resistance (MDR) is a common cause of treatment failure in cancer therapy (1–3). Thus, despite the notable success in treating diverse cancers, chemotherapeutics, including antibody–drug conjugates (ADC), lose clinical activity over time, as exemplified by irinotecan (4), doxorubicin (5), paclitaxel (6), cisplatin (7), gemtuzumab ozogamicin (8), inotuzumab ozogamicin (9), and others (1).

In general, the occurrence of drug resistance in cancer cells can be intrinsic or acquired, with each type resulting from a variety of factors (10), such as decreased uptake of soluble drugs, activation of drug-detoxifying systems, modulation or mutation of drug targets, defective apoptosis pathways, and above all, overexpression of one or more efflux pumps of the ATP-binding cassette (ABC) superfamily (11). The human genome comprises a total of 49 genes in the ABC superfamily (12), each assigned to one of seven subfamilies (A through G) based on the order and sequence homology of the transmembrane (TM) domain and the nucleotide-binding folds (NBF). To date, ABCB1 (also known as MDR1 or P-gp), ABCC1 (also known as MRP1), and ABCG2 (also known as BCRP, MXR, or ABC-P) account for most studies on MDR (13–15).

Members of the ABC superfamily are transmembrane proteins, expressed either as a full- or half-transporter. A full-transporter typically contains two transmembrane (TM) domains and two nucleotide-binding folds (NBFs), with the TM domains participating in substrate recognition and translocation across the membrane, while the cytosolic NBFs provide the driving force for transport via hydrolysis of the bound ATP. By contrast, a half-transporter has only one TM domain and one NBF, and must form either homodimers or heterodimers to be functional. A notable example of a full-transporter is ABCB1, whose substrates include vinca alkaloids, anthracyclines, epipodophyllotoxins, taxanes, irinotecan, and SN-38 (1). The five members of the ABCG subfamily are all half-transporters (12), of which ABCG2 has been identified for its role in mediating cellular resistance to SN-38 (16, 17), as well as to tyrosine kinase inhibitors (18).

With the molecular mechanisms of intrinsic and acquired drug resistance in cancer increasingly being delineated, multiple approaches to circumvent MDR have emerged. For example, the sensitivity of inotuzumab ozogamicin in ABCB1-expressing sublines of Daudi and Raji lymphomas could be restored effectively with PSC-833 (8), a second-generation modifier of ABCB1 (19). The use of a hydrophilic linker for conjugating DM1 to antibodies also enabled such ADCs to evade ABCB1-mediated resistance (20), presumably due to the generation of a cytotoxic metabolite that was better retained by the ABCB1-expressing cells. In addition, targeting detoxifying enzymes, such as glutathione S-transferase, with intracellularly activated prodrugs was found to be promising (21). However, the strong rationale of using inhibitors of ABC transporters to overcome MDR has met little success in clinical trials, which could be in part due to both imperfect inhibitors and inadequate study design, and is being addressed by developing newer agents with greater substrate specificity, higher potency, lower toxicity, and improved pharmacokinetic properties.

Sacituzumab govitecan, hereafter referred to as IMMU-132 (Supplementary Fig. S1), is a Trop-2–targeting ADC of SN-38, the active metabolite of irinotecan. IMMU-132 departs from most ADCs in its use of a moderately, not ultratoxic drug, its high drug-to-antibody ratio (DAR) without impairing target affinity and pharmacokinetics, and its selection of a pH-sensitive, cleavable linker to confer cytotoxicity to both tumor and bystander cells (22–24). This novel ADC is currently in clinical trials for patients with advanced triple-negative breast cancer (25), urothelial bladder cancer (26), and other solid cancers. As these patients were all heavily pretreated with chemotherapy, the presence of acquired resistance with the expression of MDR genes is highly likely, which may affect the therapeutic outcome of IMMU-132. In this study, we explored the use of known inhibitors of ABC transporters for improving the therapeutic efficacy of IMMU-132 by overcoming SN-38 resistance.

Cell lines and cultures

Human cancer cell lines (MDA-MB-231, breast; NCI-N87, stomach; A549, lung; HCT15, colon) were purchased from the ATCC with authentication by short tandem repeat profiling. Each cell line was maintained according to the recommendations of ATCC and routinely tested for mycoplasma using MycoAlert Mycoplasma Detection Kit (Lonza). The two SN-38–resistant cell lines, MDA-MB-231-S120 and NCI-N87-S120, were established by continuous exposure of the parental cells to stepwise increased concentrations of SN-38 from 6 pmol/L to 120 nmol/L over a period of approximately 2 years. In addition, a revertant cell line (NCI-N87-S120-REV) was obtained after culturing NCI-N87-S120 in SN-38–free medium for 4 months. The S120 clones were maintained in medium containing 120 nmol/L of SN-38 but cultured in drug-free medium for 7 to 14 days prior to any experiment. All cells were grown at 37°C as monolayer cultures in a humidified atmosphere of 5% CO2.

Antibodies, IMMU-132, and reagents

Polyclonal rabbit anti-ABCG2 antibody (#4477) was purchased from Cell Signaling Technology and murine anti-γH2AX-AF488 (05-636-AF488) from EMD Millipore. Pheophorbide A (PhA), fumitremorgin C (FTC), Ko143, YHO-13351, doxorubicin, paclitaxel, rhodamine 123, and verapamil were obtained from Sigma. SN-38, purchased from Biddle Sawyer Pharma, was diluted to 1 mmol/L in DMSO and stored in aliquots at −20°C. Irinotecan-HCl injection was bought from Areva Pharmaceuticals. The properties and preparation of IMMU-132 have been described previously (22–24).

Functional assays of ABC transporters

Functional assays of ABCG2, with PhA as the substrate and FTC as the inhibitor, were performed essentially as described by Robey and colleagues (27). Briefly, trypsinized cells were incubated with 1 μmol/L PhA or with a combination of 1 μmol/L PhA and 10 μmol/L FTC for 30 minutes at 37°C in 5% CO2 and washed. Subsequent incubations at 37°C for 1 hour were performed in PhA-free medium for cells treated only with PhA (to generate the efflux histogram) or in PhA-free medium containing 10 μmol/L FTC for cells treated with both PhA and FTC (to generate the FTC/efflux histogram). PhA fluorescence was measured on a FACSCanto flow cytometer equipped with a 635-nm red diode laser.

Functional assays of ABCB1 were performed as described above for ABCG2, with the substitution of rhodamine 123 for PhA and verapamil for FTC.

Western blotting

Cells were lysed in RIPA buffer (Cell Signaling Technology). The protein concentration of the lysate was quantitated by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) using BSA as the standard. Equal amounts of lysate were loaded and separated by SDS-polyacrylamide gels (4%–20%) and transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat milk powder in TBS for 1 hour and probed with appropriate primary antibodies. After washing with TBS-T, the membrane was incubated with anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology) and visualized using enhanced chemiluminescence.

In vitro cytotoxicity

Sensitivity to SN-38 or IMMU-132 was determined by an MTS-based assay using CellTiter 96 AQueous One Solution (Promega). Briefly, cells were placed into wells of 96-well plates. Working solutions of IMMU-132 (at a concentration of 2,500 nmol/L in SN-38 equivalents) and SN-38 at 2,500 nmol/L were prepared from respective stock solutions in sterile media. From these working solutions, serial 5-fold dilutions were made in sterile media to yield final concentrations between 500 and 0.0064 nmol/L in test wells. Plates were incubated at 37°C in a humidified chamber with 5% CO2 for 96 hours, after which 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium was added and returned to the incubator until cells of the untreated control had an absorbance greater than 1.0. Cytotoxicity was measured as a percent of growth relative to untreated cells. Dose–response curves were generated from the mean of triplicate determinations, and IC50 values were calculated using GraphPad Prism Software V 6.05 (Advanced Graphics Software).

The effect of ABCG2 inhibitors on the IC50 values of SN-38 for the parental and resistant cell lines was determined from the respective dose–response curves obtained for SN-38 in the presence of a nontoxic concentration of ABCG2 inhibitors.

Estimation of double-strand breaks with measurements of γH2AX-stained cells

Cells (5 × 105 cells/mL per sample) were incubated with or without SN-38 (250 nmol/L) at 37°C for the indicated times, fixed in 4% formalin for 15 minutes, then washed and permeabilized in 0.15% Triton X-100 in PBS for 15 minutes. After washing twice with 1% BSA-PBS, the cells were incubated with murine anti-γH2AX-AF488 for 45 minutes at 4°C. The signal intensity of γH2AX was measured by flow cytometry using a FACSCalibur (BD Biosciences).

Reverse transcription quantitative real-time PCR

Total RNA was extracted using RNeasy Mini Kit (Qiagen) and reverse transcribed to complementary DNA using SuperScript IV First-Strand Synthesis System (Life Technologies). qPCR was performed for each sample in triplicate on a Bio-Rad CFX96 Real-Time System using the TaqMan Gene Expression Assay (with primer sets of Hs01053790_m1 for ABCG2 and Hs99999905_m1 for GAPDH) and TaqMan Universal Master Mix II, all procured from Life Technologies. The mRNA level of ABCG2 was normalized to that of GAPDH and expressed as 2−ΔCt, where ΔCt = [Ct (ABCG2) − Ct (GAPDH)], and Ct (threshold cycle) is the number of PCR cycle corresponding to the intersection between an amplification curve and a threshold line determined for a target gene.

In vivo therapy studies

NCr female athymic nude (nu/nu) mice, 4 weeks old, were purchased from Taconic Farms. NCI-N87 and NCI-N87-S120 tumor xenografts were established by harvesting cells from tissue culture, making a 1:1 cell suspension in Matrigel (BD Biosciences), and injecting each mouse with a total of 1 × 107 cells subcutaneously in the right flank. Tumor volume (TV) was determined by measurements in two dimensions using calipers, with volumes defined as: L × w2/2, where L is the longest dimension of the tumor and w the shortest. Mice were randomized into treatment groups of 9 to 10, and therapy begun when tumor volumes were approximately 0.25 cm3. Mice bearing NCI-N87-S120 tumors were treated with irinotecan (40 mg/kg i.v., every other day for 5 times) or IMMU-132 (0.5 mg i.v. twice weekly for four weeks). YHO-13351 (0.6 mg i.v.) was administered at the same time the therapy started and again at 4 hours posttherapy. For the IMMU-132 + YHO-13351 combination group, a third injection of YHO-13351 was administered 24 hours post-IMMU-132 administration. Control mice received each agent alone. For YHO-13351 control mice, they were injected on the same schedule as when combined with irinotecan. Another group of mice bearing parental NCI-N87 tumors served as a control for efficacy of IMMU-132 and irinotecan in tumors lacking the ABCG2 pump. The lyophilized IMMU-132 was reconstituted and diluted in sterile saline as required. Irinotecan-HCl injection was diluted in sterile saline and the final dose based on body weight (40 mg/kg). Mice were euthanized and deemed to have succumbed to disease once tumors grew to >1.0 cm3 in size.

Statistical analysis

Statistical analysis for the tumor growth data was based on AUC and survival time. Profiles of individual tumor growth were obtained through linear curve modeling. An f test was employed to determine equality of variance between groups prior to statistical analysis of growth curves. A two-tailed t test was used to assess statistical significance between groups. As a consequence of incompleteness of some of the growth curves due to deaths, statistical comparisons of AUC were only performed up to the time at which the first animal within a group was sacrificed. Log-rank analysis to compare the Kaplan–Meier survival curves of two groups was performed with GraphPad Prism V6.05. Significance was set at P ≤ 0.05.

Establishment of SN-38–resistant cell lines

Table 1 summarizes the IC50 values of the parental cell lines (MDA-MB-231 and NCI-N87) and their SN-38–resistant counterparts (MDA-MB-231-S120 and NCI-N87-S120), as determined for SN-38, doxorubicin and paclitaxel. Compared with the parental cells, both S120 cells are about 50-fold more resistant to SN-38 and relatively not cross-resistant to either doxorubicin or paclitaxel. NCI-N87-S120 cells cultured for 3 weeks or longer in SN-38–free medium gradually restored their sensitivity to SN-38, resulting in a revertant cell line (NCI-N87-S120-REV) with reduced resistance to SN-38 (IC50 = 50 nmol/L) over a period of 4 months.

Table 1.

Sensitivity of the parental and SN-38–resistant cell lines to selected drugs

IC50 (nmol/L)
DrugNCI-N87NCI-N87-S120RFaMDA-MB-231MDA-MB-231-S120RFa
SN-38 4.3 ± 0.7 211 ± 39 49.1 4.8 ± 1.7 248 ± 79 51.7 
SN-38 + YHO-13351b 3.9 ± 0.5 6.4 ± 1.6 1.6 2.4 16 ± 11 6.7 
SN-38 + Ko143b ND 1.3 ± 0.4 ND ND ND ND 
Doxorubicin 32.2 ± 7.5 74.6 ± 3.3 2.3 10.9 ± 0.6 43.1 ± 4.6 4.0 
Paclitaxel 7.9 ± 4.0 8.6 ± 6.5 1.1 2.5 ± 0.1 3.2 ± 0.7 1.3 
IC50 (nmol/L)
DrugNCI-N87NCI-N87-S120RFaMDA-MB-231MDA-MB-231-S120RFa
SN-38 4.3 ± 0.7 211 ± 39 49.1 4.8 ± 1.7 248 ± 79 51.7 
SN-38 + YHO-13351b 3.9 ± 0.5 6.4 ± 1.6 1.6 2.4 16 ± 11 6.7 
SN-38 + Ko143b ND 1.3 ± 0.4 ND ND ND ND 
Doxorubicin 32.2 ± 7.5 74.6 ± 3.3 2.3 10.9 ± 0.6 43.1 ± 4.6 4.0 
Paclitaxel 7.9 ± 4.0 8.6 ± 6.5 1.1 2.5 ± 0.1 3.2 ± 0.7 1.3 

Abbreviation: ND, not determined.

aRF is the resistant factor obtained as |$\frac{{[ {{\rm{the}}\ {{\rm{IC}}_{{\rm{50}}}}\ {\rm{ mean \ of \ S120}}} ]}}{{[ {{\rm{the}}\ {{\rm{IC}}_{{\rm{50}}}}\ {\rm{ mean \ of \ parent}}} ]}}$|⁠.

bThe nontoxic concentrations used for YHO-13351 and Ko143 were 2 and 1 μmol/L, respectively.

Overexpression of functional ABCG2 in the S120 cell lines

The presence of ABCG2 in the S120 cells, but little, if any, in the parental cells is shown by Western blot analysis (Fig. 1A) and corroborated by qRT-PCR, which indicate that virtually no mRNA transcripts of ABCG2 could be detected in MDA-MB-231 and NCI-N87 cells (Supplementary Table S1). On the other hand, the mRNA levels of ABCG2 relative to GAPDH in MDA-MB-231-S120 and NCI-N87-S120 are calculated to be 27,408-fold and 167-fold higher than those in MDA-MB-231 and NCI-N87, respectively (Fig. 1B). The expression of ABCG2 was also confirmed in samples obtained from NCI-N87-S120, but not NCI-N87, xenografts (Fig. 1C).

Figure 1.

Overexpression of ABCG2 in the S120 cells. A, Western blot analysis, showing the expression of ABCG2 in the two S120 cell lines, but little or none in the parental MDA-MB-231 and NCI-N87; β-actin served as the loading control. B, qRT-PCR results showing high levels of ABCG2 mRNA (normalized to GAPDH mRNA) in the S120 cells. The fold of increase was 27,408 for MDA-MB-231-S120 and 167 for NCI-N87-S120. C, expression of ABCG2 in NCI-N87-S120, but not NCI-N87, xenografts, as confirmed by Western blot analysis.

Figure 1.

Overexpression of ABCG2 in the S120 cells. A, Western blot analysis, showing the expression of ABCG2 in the two S120 cell lines, but little or none in the parental MDA-MB-231 and NCI-N87; β-actin served as the loading control. B, qRT-PCR results showing high levels of ABCG2 mRNA (normalized to GAPDH mRNA) in the S120 cells. The fold of increase was 27,408 for MDA-MB-231-S120 and 167 for NCI-N87-S120. C, expression of ABCG2 in NCI-N87-S120, but not NCI-N87, xenografts, as confirmed by Western blot analysis.

Close modal

That ABCG2 is functionally active in the two S120 cell populations, but absent in the parental cells, is evident from the four histograms shown in Fig. 2 for PhA, which is a fluorescent substrate of ABCG2. In both parental cells, the intracellular levels of PhA, as measured by the median fluorescence intensity (MFI), are relatively high and remain practically the same with or without FTC, a potent mycotoxin (Supplementary Fig. S2) initially identified for its effective reversal of resistance to mitoxantrone, doxorubicin, and topotecan in a multidrug-selected cell line. In contrast, in either S120 cells, a high level of intracellular PhA similar to that in the parental cells could only be observed with the addition of FTC, whereas the omission of FTC resulted in a greater than 95% reduction of intracellular PhA. Similar results were obtained in human lung cancer A549 cells, known to express functional ABCG2 (28). In separate studies with rhodamine 123 and verapamil as the substrate and the inhibitor for ABCB1, respectively, activity of ABCB1 was detected in human colorectal cancer HCT15 cells, which served as a positive control for expressing ABCB1 (29), but not in either NCI-N87-S120 or NCI-N87 (Supplementary Table S2).

Figure 2.

Functional assay of ABCG2. Both parental and S120 cells were incubated with 1 μmol/L PhA alone (blue) or together with 10 μmol/L FTC (orange) to generate the PhA efflux histogram. The MFI pertaining to each signal was provided in the histograms. In both parental cells, which do not express ABCG2, the intracellular levels of PhA were high and remained practically the same with or without FTC. In the S120 cells, the active efflux by ABCG2 resulted in much lower levels of PhA, which was restored with the addition of FTC. MDA-MB-231 (left upper panel), MDA-MB-231-S120 (right upper panel), NCI-N87 (left lower panel), NCI-N87-S120 (right lower panel).

Figure 2.

Functional assay of ABCG2. Both parental and S120 cells were incubated with 1 μmol/L PhA alone (blue) or together with 10 μmol/L FTC (orange) to generate the PhA efflux histogram. The MFI pertaining to each signal was provided in the histograms. In both parental cells, which do not express ABCG2, the intracellular levels of PhA were high and remained practically the same with or without FTC. In the S120 cells, the active efflux by ABCG2 resulted in much lower levels of PhA, which was restored with the addition of FTC. MDA-MB-231 (left upper panel), MDA-MB-231-S120 (right upper panel), NCI-N87 (left lower panel), NCI-N87-S120 (right lower panel).

Close modal

The activity of ABCG2 in the two S120–resistant cell lines also was demonstrated by comparing the levels of DNA double-strand breaks (DSB) induced by SN-38 with those in the parental cells, as measured by a flow cytometric assay for quantification of γH2AX, whose signal intensities directly correspond to the number of DSBs formed (30). As shown in Fig. 3A, upon treatment with 250 nmol/L of SN-38, the levels of γH2AX rose steadily in the parental, but not the resistant S120, cells, culminating, after 3 hours, in an increase over the untreated controls that was about 2-fold for MDA-MB-231 and about 4-fold for NCI-N87 (Fig. 3B). ABCG2 is implicated in preventing the increase of γH2AX in the S120 cells treated with SN-38, as the addition of both FTC (10 μmol/L) and SN-38 (250 nmol/L) to MDA-MB-231-S120 could elevate γH2AX levels with a comparable fold-increase with that of SN-38–treated MDA-MB-231 (Fig. 3C). Similar results were obtained with NCI-N87-S120 when treated with SN-38 or IMMU-132 in the presence of FTC (Fig. 3D).

Figure 3.

γH2AX assay for DSB by flow cytometry. Cells were treated or not treated with SN-38 (250 nmol/L) for 3 hours, and the levels of γH2AX were monitored hourly by flow cytometry and shown as MFI in a bar diagram (A) or as percentage of untreated (B). The effect of FTC (10 μmol/L) to increase the formation of DSB/γH2AX was shown for MDA-MB-231-S120 treated with SN-38 (C) and for NCI-N87-S120 treated for either SN-38 or IMMU-132 (D).

Figure 3.

γH2AX assay for DSB by flow cytometry. Cells were treated or not treated with SN-38 (250 nmol/L) for 3 hours, and the levels of γH2AX were monitored hourly by flow cytometry and shown as MFI in a bar diagram (A) or as percentage of untreated (B). The effect of FTC (10 μmol/L) to increase the formation of DSB/γH2AX was shown for MDA-MB-231-S120 treated with SN-38 (C) and for NCI-N87-S120 treated for either SN-38 or IMMU-132 (D).

Close modal

Sensitizing S120 cells to SN-38 with selected ABCG2 inhibitors

The effect of two known ABCG2 inhibitors, Ko143 (31) and YHO-13351 (32), on reversing the resistance of S120 cells to SN-38 was examined in vitro at a concentration not affecting the growth of either the parental or the S120 cells. As shown in Fig. 4A–C of the representative dose–response curves, and in Table 1 of the pertaining IC50 values, the addition of either ABCG2 inhibitor, while conferring little impact on the sensitivity of parental cells to SN-38, reduced the IC50 of SN-38 by more than 90% in both resistant S120 cell lines. Limited studies also were done for other ABCG2 inhibitors, such as FTC (33), cyclosporine A (34), and GF120918 (35), with results showing comparable or less potency (data not shown). Noting the reported instability of Ko143 in rat serum (36), YHO-13351 was selected over Ko143 for in vivo evaluation.

Figure 4.

Dose–response curves of parental and S120 cells treated with different concentrations of SN-38 in the absence and presence of YHO-13351 or Ko143. Reversal of SN-38 resistance by YHO-13351 was shown for MDA-MB-231-S120 (A), NCI-N87-S120 (B), and by Ko143 for NCI-N87-S120 (C).

Figure 4.

Dose–response curves of parental and S120 cells treated with different concentrations of SN-38 in the absence and presence of YHO-13351 or Ko143. Reversal of SN-38 resistance by YHO-13351 was shown for MDA-MB-231-S120 (A), NCI-N87-S120 (B), and by Ko143 for NCI-N87-S120 (C).

Close modal

Improved efficacy of IMMU-132 when combined with YHO-13351 in SN-38–resistant NCI-N87-S120 tumors

NCI-N87-S120 tumors grew slower in the mice than did parental NCI-N87 (Fig. 5A; P = 0.005, AUC), with the median survival for untreated animals more than 2-fold longer for the mice bearing NCI-N87-S120 (P = 0.0006 vs. NCI-N87). Although treatment with IMMU-132 or irinotecan provided no significant survival benefit to mice bearing NCI-N87-S120 tumors (Fig. 5B), both of these therapies resulted in a greater than 2-fold increase in survival in mice bearing NCI-N87 (P < 0.0001; Fig. 5C). However, when IMMU-132 therapy was combined with YHO-13351 in mice bearing SN-38–resistant NCI-N87-S120, a significant 64% improvement in survival was achieved in comparison with untreated animals (P = 0.0278). Although irinotecan plus YHO-13551 likewise improved the survival of the mice, it did not reach significance (P = 0.0852). As the in vitro assay showed there was no difference between parental cell lines treated with SN-38 alone or in combination with YHO-13351, and the tumor xenograft samples of NCI-N87 were absent of ABCG2 by Western blot analysis (Fig. 1C), a combination of IMMU-132 and YHO-13351 was not examined in mice bearing NCI-N87.

Figure 5.

Efficacy of IMMU-132 in mice bearing SN-38–resistant NCI-N87-S120 gastric carcinoma xenograft. A, mean tumor growth curves for NCI-N87 and NCI-N87-S120 xenografts. B, mice bearing NCI-N87-S120 SN-38–resistant human gastric tumors were treated with IMMU-132, irinotecan, YHO-13551, or combinations as indicated on the graph and described in Materials and Methods. C, mice bearing parental NCI-N87 tumors treated with IMMU-132 or irinotecan at the same dose and schedule as used in NCI-N87-S120 tumor-bearing animals. In the survival curves of B and C, the starting day of therapy (when tumor volumes reached ∼0.25 cm3) was marked as day 0. Mice were euthanized once tumors grew to >1.0 cm3 in size.

Figure 5.

Efficacy of IMMU-132 in mice bearing SN-38–resistant NCI-N87-S120 gastric carcinoma xenograft. A, mean tumor growth curves for NCI-N87 and NCI-N87-S120 xenografts. B, mice bearing NCI-N87-S120 SN-38–resistant human gastric tumors were treated with IMMU-132, irinotecan, YHO-13551, or combinations as indicated on the graph and described in Materials and Methods. C, mice bearing parental NCI-N87 tumors treated with IMMU-132 or irinotecan at the same dose and schedule as used in NCI-N87-S120 tumor-bearing animals. In the survival curves of B and C, the starting day of therapy (when tumor volumes reached ∼0.25 cm3) was marked as day 0. Mice were euthanized once tumors grew to >1.0 cm3 in size.

Close modal

IMMU-132 is a first-in-class ADC made by conjugating the moderately toxic drug, SN-38 (nanomolar potency), with a partially stable linker, site specifically and at a high DAR of 7.6, to a humanized antibody against Trop-2 expressed in many solid cancers. Preclinical studies have demonstrated that IMMU-132, in comparison with irinotecan, protects the IgG-bound SN-38 from glucuronidation, delivers much more SN-38 (20- to 136-fold higher) to tumor xenografts, resulting in improved pharmacokinetics and pharmacodynamics (37). As such, IMMU-132 provides a paradigm change that contrasts the prevailing approach of conjugating a low level of an ultratoxic payload (picomolar potency) with a stable linker to an antibody capable of internalization upon target engagement (38, 39). Importantly, the ongoing phase II studies with IMMU-132 as a single agent in patients with metastatic triple-negative breast cancer (mTNBC) who had received a median of 5 (range = 2 to 12) prior lines of therapy have shown an interim objective response rate of 31% by RECIST 1.1 in 58 evaluable patients (40), thus extending the results obtained in phase I trials, which indicated IMMU-132 had acceptable toxicity and encouraging therapeutic activity in patients with difficult-to-treat solid cancers (25). Promising initial results have also been reported for IMMU-132 administered to patients with platinum-resistant urothelial carcinoma (26). Whereas the phase II results observed in heavily pretreated patients with mTNBC have led the FDA to grant Breakthrough Therapy Designation to IMMU-132, those who showed early progression of disease, thus failing IMMU-132, may reflect possible drug resistance resulting from one or more preexisting efflux pumps, as SN-38 is susceptible to multiple ABC transporters (1).

In the current study, NCI-N87-S120 and MDA-MB-231-S120, the two sublines made resistant to SN-38, were shown to be 50-fold less responsive to SN-38 than their parental cells. The sensitivity of NCI-N87-S120 to SN-38 could be restored to within 5-fold of NCI-N87 when propagated in vitro without SN-38 after a period of 3 weeks or longer, suggesting a nongenetic origin of such acquired resistance, which may or may not be clinically relevant. The presence of ABCG2 in the two S120 sublines, but not their parents, was supported by several lines of evidence, including the demonstration of active efflux of PhA; the detection of expressed protein by Western blotting, which was corroborated with qRT-PCR of mRNA; the increased accumulation of SN-38 by FTC using γH2AX as a surrogate marker; and the reverted resistance by YHO-13351 or Ko143. Of note, both S120 sublines were found not to carry ABCB1 and neither phenotype was cross-resistant to doxorubicin or paclitaxel, similar to a previous observation (41) that the ABCG2-expressing, SN-38–resistant human colorectal HCT116-SN50 cancer subline showed no significant cross-resistance to doxorubicin (as well as to 5-fluorouracil and oxaliplatin). In other human cancer clones selected for resistance to SN-38 via continuous exposure of parental cell lines to the drug in culture, overexpression of ABCG2 has also been reported for the sublines generated from MCF-7 (42, 43), MDA-MB-231 (43), the small-cell lung carcinoma PC-6 (17), the non–small cell lung adenocarcinoma H23 (44), and the cervical carcinoma HeLa (45). Cross-resistance of these sublines to doxorubicin varied somewhat, with the resistance ratio (IC50 in resistant subline divided by IC50 in the parental cell) being less than 1.3 for the sublines of PC-6 (17), 2.5 for the sublines of HeLa (45), and about 7.0 for the subline of H23 (44). Whereas cross-resistance to doxorubicin was not determined for the SN-38–resistant sublines derived from MCF-7 (42, 43) or MDA-MB-231 (43), these sublines remained sensitive to vincristine (42), cisplatin (42, 43), and docetaxel (43).

Although we and others have established ABCG2 as a key player in reducing SN-38 sensitivity of various SN-38–resistant cancer sublines, the potential involvement of DNA topoisomerase I (Top1), to which SN-38 specifically binds and acts as an inhibitor, in the SN-38 resistance mechanism of such cancer cells, is less defined and remains a focus of continuous research, with the current knowledge pointing to Top1 mutation (46, 47) and degradation (48) as the two main roles of Top1 underlying the molecular mechanism of resistance to camptothecin in general and SN-38 in particular.

When cultured in vitro, SN-38–resistant sublines of MCF-7 or MDA-MB-231 had longer doubling times than their parental cells (43). Thus, it is not surprising that NCI-N87-S120 xenografts grew significantly slower in the mice than NCI-N87, which is consistent with the notion that drug-resistant tumor sublines selected in vitro frequently manifest less aggressive properties than their drug-sensitive parental cell lines (49). Nevertheless, in vivo studies show that the NCI-N87-S120 xenograft retained ABCG2 expression and was resistant to IMMU-132, yet its growth could be significantly subdued by IMMU-132 in combination with YHO-13351. A parallel study shows the parental xenograft was responsive to IMMU-132 or irinotecan, but with a shorter median survival time. Together, these in vivo results suggest that suitable inhibitors that are tolerated well by the host animals can overcome ABC resistance and that the resistant tumor lines can become appreciably responsive to IMMU-132 and to a lesser extent to irinotecan. We are pursuing further work to address the feasibility of preclinical testing for such drug resistance as a predictive bioassay (43, 44) to select patients who should receive ABC-blocking therapy with IMMU-132. Meanwhile, we are examining the suitability of clinically tested tyrosine kinase inhibitors, some of which interfere with the functions of ABC transporters at nontoxic levels (50), to enhance the potency of IMMU-132 in cancer cells that are intrinsically or made resistant to SN-38.

All authors are current employees of Immunomedics, Inc., and have stocks or stock options of Immunomedics, Inc. No other potential conflicts of interest were disclosed by the authors.

Conception and design: C.-H. Chang, T.M. Cardillo, D.M. Goldenberg

Development of methodology: C.-H. Chang, D. Liu, T.M. Cardillo

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Wang, M. Zalath, D. Liu, T.M. Cardillo

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.-H. Chang, Y. Wang, M. Zalath, D. Liu, T.M. Cardillo

Writing, review, and/or revision of the manuscript: C.-H. Chang, D. Liu, T.M. Cardillo, D.M. Goldenberg

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T.M. Cardillo, D.M. Goldenberg

Study supervision: C.-H. Chang, T.M. Cardillo, D.M. Goldenberg

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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