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Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Preclinical Efficacy of Anti-RON Antibody−Drug Conjugate Zt/g4MMAE for Targeted Therapy of Pancreatic Cancer Overexpressing RON Receptor Tyrosine Kinase Hang-Ping Yao,†,‡ Liang Feng,§,∥ Tian-Hao Weng,†,‡ Chen-Yu Hu,†,‡ Sreedhar Reddy Suthe,§,∥ A. G. M. Mostofa,§,∥ Ling-Hui Chen,⊥,# Zhi-Gang Wu,⊥,# Wei-Lin Wang,*,⊥,# and Ming-Hai Wang*,†,§,∥,⊥ Downloaded via UNIV OF SUSSEX on June 26, 2018 at 23:59:05 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, ‡Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, ⊥Zhejiang Provincial Key Laboratory for Precision Diagnosis & Treatment of Hepatobiliary and Pancreatic Cancers, #Division of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China § Cancer Biology Research Center and ∥Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, United States S Supporting Information *

ABSTRACT: Aberrant expression of the RON receptor tyrosine kinase, a cell surface protein, is a pathogenic feature in pancreatic cancer, which renders it a drug target for targeted therapy. Nevertheless, development of therapeutics targeting RON for pancreatic cancer therapy is hampered due to the lack of full addiction by pancreatic cancer cells to RON signaling for growth and survival. Here we describe a novel strategy using anti-RON antibody-directed drug delivery in the form of an antibody−drug conjugate for inhibition and/or eradication of pancreatic cancers. Monoclonal antibody Zt/g4 specific to the RON Sema domain was selected as the drug carrier based on its ability to induce robust RON internalization. Conjugation of Zt/g4 with monomethyl auristatin E, designated as Zt/g4-MMAE, was achieved through a protease-sensitive dipeptide linker to reach a drug to antibody ratio of 3.29:1. Zt/g4-MMAE was stable in human plasma with a dissociation rate less than 4% within a 10 day period. In vitro, Zt/g4-MMAE rapidly induced RON internalization, resulting in cell cycle arrest followed by massive cell death. The maximal effect was seen in pancreatic cancer cells with more than 10 000 receptor molecules per cell. Zt/g4-MMAE also synergized in vitro with chemotherapeutics including gemcitabine, 5-fluorouracil, and oxaliplatin to further reduce PDAC cell viability. In vivo, Zt/g4-MMAE exerts a longlasting activity, which not only inhibited but also eradicated pancreatic xenograft tumors. These finding indicate that Zt/g4directed drug delivery is highly effective for eradicating pancreatic tumors. Thus, Zt/g4-MMAE is a novel biotherapeutic with potential for therapy of RON-expressing pancreatic malignancies. KEYWORDS: receptor tyrosine kinase, antibody−drug conjugate, pancreatic cancer, therapeutic efficacy, synergism

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oncology.4−7 ADC selectively ablates cancer cells by combining the specificity of a target specific monoclonal antibody (mAb) with the delivery of a highly potent cytotoxic agent.4−7 Currently, four ADCs including ado-trastuzumab emtansine (T-DM1, Kadcyla), brentuximab vedotin (Adcetris), gemtuzumab ozogamicin (Mylotarg), and inotuzumab ozogamicin (Besponsa) have been approved for treatment of breast cancer, lymphomas, and acute lymphoblastic leukemia, respectively.8−10 The clinical application of these ADCs has provided significant benefits to patients, showing a prolonged

INTRODUCTION

Pancreatic ductal adenocarcinoma (PDAC) with local invasion or distant metastasis is one of the most aggressive cancers, with poor prognosis and high mortality.1−3 A major clinical challenge in PDAC management is the lack of effective therapeutics.1−3 Currently, only limited chemoagents are available for palliative treatment.1−3 Molecular targeting and immunoregulatory therapies have recently been applied for PDAC; however, their clinical benefits seem to be far less promising and are often disappointing.1−3 Considering these facts, it is clear that discovery of new drug targets and development of novel therapeutics are critically important to achieve acceptable clinical outcome for this deadly disease. Antibody−drug conjugates (ADC) have shown to be a promising strategy successfully applied recently in clinical © XXXX American Chemical Society

Received: March 27, 2018 Revised: June 6, 2018 Accepted: June 15, 2018

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DOI: 10.1021/acs.molpharmaceut.8b00298 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics survival period and improved quality of life.8−10 Nevertheless, ADCs approved for PDAC treatment are not available. Various ADCs using different antibodies targeting different cell surface proteins such as PSMA,11 Ephrin-A4,12 Trop-2,13 and others are under intensive study in preclinical models. Also, more than 40 ADCs are under clinical trials (www.clinicaltrials.gov). It is foreseen that ADCs aimed at unique targets may be an effective treatment modality for advanced PDAC in the near future. For the past decade, we have focused on development of RON-targeted ADCs for treatment of epithelial cancers including PDAC.14−16 As a MET proto-oncogene family member,17 aberrant RON expression contributes significantly to PDAC malignancy.18−23 In primary PDACs, RON is overexpressed in ∼30% of surgically removable tumor samples as evident by immunohistochemical staining.20,23−25 In cell models, RON overexpression transduces signals that facilitate invasive growth of PDAC cells with chemoresistant phenotypes.18−23 In addition, cell surface RON has been targeted for PDAC therapies, because it is overexpressed in these cancers.26−28 We have previously developed Zt/g4 immunoliposomes for targeted cancer killing.14−16,29 Zt/g4-directed drug delivery is effective in killing various cancer cells including chemoresistant, hypoxic, and cancer stem-like cells.14−16 These proof-of-concept studies prompted us to use Zt/g4 to develop the first anti-RON ADC, in which Zt/g4 is conjugated with maytansinoid (DM1) via a thioether linkage to form Zt/g4DM1.30−32 In preclinical models, Zt/g4-DM1 has been shown to be highly effective in inhibition of colon, breast, and lung xenograft tumor growth.30−32 However, the efficacy of Zt/g4DM1 in PDAC xenograft tumors is low.32 Among three PDAC xenograft tumor models tested, only tumors derived from BxPC-3 cells have been shown to be sensitive to Zt/g4-DM1 with significant tumor growth inhibition.32 Tumors mediated by highly malignant PDAC FG and L3.6pl cells were insensitive to Zt/g4-DM1.32 Thus, Zt/g4-DM1 is not suitable for targeted PDAC therapy. Development of novel Zt/g4based ADC with an enhanced therapeutic index is a critical step for the success of the RON-targeted PDAC therapy. The present study is the generation and therapeutic validation of a novel anti-RON Zt/g4-monomethyl auristatin E conjugate (Zt/g4-MMAE) for PDAC treatment in preclinical models. Although DM1 and MMAE share a similar mechanism of action that inhibits cell division,33,34 their antibody conjugation profiles and therapeutic activities differ significantly.33,34 By generating Zt/g4-MMAE with a desirable drug to antibody ratio (DAR), we show that Zt/g4-MMAE in vitro was effective in killing PDAC cells in a RON-specific manner. In vivo, Zt/g4-MMAE was able to completely inhibit PDAC cell-mediated xenograft tumor growth and significantly eradicate PDAC tumors. These findings lay the foundation for development of humanized Zt/g4-MMAE for potential clinical trials.

supplemented with 10% fetal bovine serum. Mouse anti-RON mAb Zt/g4, Zt/f2, and rabbit IgG antibody against the RON C-terminus (R#5029) were used as previously described.14 Goat antimouse IgG labeled with fluorescein isothiocyanate (FITC) or rhodamine was from Jackson ImmunoResearch (West Grove, PA). Generation of Zt/g4-DM1 and Zt/g4-MMAE. Conjugation of MMAE to Zt/g4 was performed under conditions for achieving a DAR of 4:1 according to manufacturer’s instruction. Synthetic maleimidocaproyl−valine−citrulline−paminobenzoyl−oxycarbonyl−monomethyl auristatin E (MC− VC−PAB−MMAE) was from Concortis (www.concortis. com). Zt/g4 at 150 mg/mL was mixed with 10 mM MC− VC−PAB−MMAE in a conjugation buffer to form Zt/g4MMAE. Zt/g4 also was conjugated with N-succinimidyl-4[maleimidomethyl]-cyclohexane-1 carboxylate (SMCC)-DM1 (www.concortis.com) to form Zt/g4-DM1 as previously described.30 In addition, control mouse IgG was conjugated with MC−VC−PAB−MMAE or SMCC-DM1 to form CmIgG−MMAE or CmIgG-DM1, respectively. All conjugates were purified using a PC10 Sephadex G25 column, sterilized through a 0.22 μM filter, and stored at 4 °C. The conjugation was verified by hydrophobic interaction chromatography (HIC) using a Varian Prostar 210 Quaternary HPLC system coupled with a TSK butyl-NPR 4.6 × 3.5 column (Tosoh Biosciences, King of Prussia, PA) as previously described.30 Assay for Zt/g4-MMAE Stability Analysis. Zt/g4MMAE at 5 μg/mL was incubated with phosphate-buffered saline (PBS) or fresh human plasma at 37 °C for various times. Changes of DARs in PBS at different time points were determined by HIC analysis. Free MMAE released into plasma from Zt/g4-MMAE was determined using the LC−MS/MS method35 with slight modifications. Methods for Analysis of ADC-Mediated Cell Surface RON Internalization. Zt/g4-MMAE-mediated cell surface RON internalization by PDAC cells was determined as previously described.30 RON remaining on the cell surface after ADC treatment was detected by the immunofluorescence assay using anti-RON mAb Zt/f2.29,30 A time required to achieve 50% reduction in cell surface RON (internalization efficacy, IE50) was calculated for individual cell lines. Detection of intracellular RON was performed using the immunofluorescence staining as previously described.30−32 Goat antimouse IgG coupled with FITC was used as the detecting antibody. Nuclear DNAs were stained with 4′,6-diamidino-2-phenylindole (DAPI). Lysosomal-associated membrane protein-1 (LAMP-1) was detected by IgG antibody specific to LAMP-1 (Cell Signaling #9091S), followed by goat antimouse IgG coupled with rhodamine. Cellular immunofluorescence was observed under an Olympus microscope equipped with DUS/ fluorescent apparatus. Assays for Analysis of Cell Cycle, Viability, and Death. Cell cycle analysis was performed by treatment of PDAC cells (1 × 106 cells per dish) with 5 μg/mL of Zt/g4-MMAE at 37 °C for 24 h. Cells were then labeled with propidium iodide and analyzed by measuring DNA contents as previously described.30 Cell viability, 96 h after Zt/g4-MMAE treatment, was determined by the MTT cell viability assay.30 Percentages of viable or dead cells were determined using the trypan blue exclusion assay as previously described.27 Western Blot Analysis. Cellular proteins (20 μg per sample) from PDAC cell lines were separated in an 8% SDS− PAGE under reduced conditions. Fragments from activated

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MATERIALS AND METHODS Cell Lines and Reagents. Human PDAC BxPC-3 and Panc-1 cell lines were from ATCC (Manassas, VA) and authenticated in 2010 with cytogenesis. PDAC FG and L3.6pl cell lines were provided by Drs. A.M. Lowy (Department of Surgery, University of California at San Diego, CA) and G.E. Gallick (University of Texas M.D. Anderson Cancer Center, Houston, TX), respectively. Cells were authenticated in 2014. Individual cell lines were cultured in their proper culture media B


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Molecular Pharmaceutics

Figure 1. Generation and characterization of anti-RON ADC Zt/g4-MMAE: (A) Schematic representation of Zt/g4-MMAE structure: Zt/g4 is a mouse mAb specific to the RON Sema domain.26 MMAE was conjugated to Zt/g4 by the valine−citruline dipeptide linker to reach a DAR of 3.29. (B) HIC analysis of the number of MMAE conjugated to Zt/g4: Individual Zt/g4-MMAEs with different numbers of MMAE (0 to 8) are marked as P0 to P8. (C) Stability of Zt/g4-MMAE in PBS. Zt/g4-MMAE was kept in PBS at 37 °C for 28 days. Samples analyzed at different time points with the average DARs are shown. (D) Stability of Zt.g4-MMAE in human plasma. Zt/g4-MMAE at 10 μg/mL was incubated with fresh human plasma for 10 days. Zt/g4-DM1 at 10 μg/mL was used as the control. The total amount of MMAE and DM1 conjugated to Zt/g4 was 180.5 and 133.8 ng/mL, respectively. Both free MMAE and DM1 dissociated from ADCs were measured using the LC−MS/MS method as previously described.32

Statistical Analysis. GraphPad Prism 7 software was used for statistical analysis. Results are shown as mean ± SD. The data between control and experimental groups were compared using Student’s t test. Chi-squared analysis was used for correlational study. Isobolograms were used for synergistic analysis in combination studies. Statistical differences at p < 0.05 were considered significant.

Poly(ADP−ribose) polymerase (PARP) or caspase 3 were detected in Western blotting using rabbit IgG antibodies specific to cleaved PARP or caspase-3 (Cell Signaling #5625S or #9664S), respectively. Membranes also were reprobed with rabbit antiactin IgG antibody (Cell Signaling #4967S) to ensure equal sample loading.30−32 Mouse Xenograft Tumor Model and Zt/g4-MMAE Treatment. The Texas Tech University Institutional Animal Care Committee approved all experiments carried out in mice. Female athymic nude mice at 6 weeks of age (Taconic, Cranbury, NJ) were injected with 5 × 106 cancer cells in the subcutaneous space of the right flank as previously described30 and randomized into different groups (five mice per group). Treatment began when tumors reached a mean tumor volume of ∼100 to 150 mm3. Zt/g4-MMAE or Zt/g4-DM1 at 20 mg/ kg in a Q12 × 2 regimen was injected through the tail vein. CmIgG−MMAE or CmIgG-DM1 served as the control and was administered using an equivalent dose and schedule. Tumor volumes were measured every 4 days and calculated as previously described.30 Mice were monitored for tumor growth up to 52 days. Animals were euthanized when tumor volumes exceeded 2000 mm3 or if tumors became necrotic or ulcerated through the skin.

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RESULTS Generation and Characterization of Zt/g4-MMAE. The inability of Zt/g4-DM1 to inhibit PDAC xenografts prompted us to conjugate Zt/g4 with MMAE, MMAF, or duocarmycin to screen a better ADC for PDAC therapy. This led us to select Zt/g4 conjugated with MMAE through cathepsin B-sensitive dipeptide linker31 as the lead candidate. Schematic representation of Zt/g4-MMAE is shown in Figure 1A. A total of 150 mg of Zt/g4 was conjugated to MMAE with conditions to achieve an average drug to antibody ratio (DAR) of 4:1. Selection of this DAR was based on published observations that one IgG molecule coupling with four MMAE molecules achieves maximal therapeutic efficacy.34 HIC analysis revealed average DARs of Zt/g4-MMAE at 3.29 C


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Figure 2. Binding and induction of RON internalization by Zt/g4-MMAE: (A) Binding of MMAE-conjugated Zt/g4 to cell surface RON. Individual PDAC cell lines at (1 x106 cells/mL) were incubated at 4 °C with 5 μg/mL of Zt/g4-DM1 for 60 min followed by flow cytometric analysis. Control mouse IgG (CmIgG), Zt/g4, and Zt/g4-DM1 were used as the control. (B) The time-dependent RON internalization. PDAC cells (1 × 106 cells per dish) were treated at 37 °C with 5 μg/mL of Zt/g4-DM1, collected at different time points, washed with acidic buffer to remove Zt/g4 bound on the cell surface,11 and then incubated with 2 μg/mL of anti-RON mAb 2F2.26 Immunofluorescence was analyzed by flow cytometer using FITC-coupled antimouse IgG. The FITC binding intensity from cells treated with Zt/g4-MMAE at 4 °C was set as 100%. The IE50 D


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Molecular Pharmaceutics Figure 2. continued

values from one representative experiment were calculated as the time required to achieve 50% reduction of cell surface RON. (C) Zt/g4-MMAEmediated reduction of RON expression. Cellular proteins (20 μg/mL) from PDAC cells treated with Zt/g4-MMAE (5 μg/mL) were analyzed in Western blotting using rabbit IgG antibody specific to RON as previously described.26 Actin was used as the loading control. (D) Immunofluorescent analysis of cytoplasmic RON: BxPC-3 cells (1 × 105 cells per chamber) were treated at 4 or 37 °C with 5 μg/mL of Zt/g4MMAE for 12 h followed by FITC-coupled antimouse IgG. After cell fixation, immunofluorescence was detected using the BK70 Olympus microscope equipped with a fluorescence apparatus. LAMP1 was used as a marker for protein cytoplasmic localization. DAPI was used to stain nuclear DNA.

Figure 3. Effect of Zt/g4-MMAE on PDAC cell cycle, survival, and death: (A) Cell cycle changes: PDAC cell lines (1 × 106 cells per dish) were treated at 37 °C with 5 μg/mL of Zt/g4-MMAE for various times, collected, stained with propidium iodide, and then analyzed by flow cytometer. Changes in cell cycle are marked with arrows. Data shown here are from one of two experiments with similar results. (B) Reduction of cell viability: PDAC cells (6000 cells per well in a 96-well plate in triplicate) were treated with different amounts of Zt/g4-MMAE for 96 h. Cell viability was determined by the MTT assay. The results shown here were from one of three experiments with similar results. The representative IC50 values from one experiment were calculated using GraphPad Prism 6 software. (C) Dose-dependent cell death. PDAC cells were cultured and treated with Zt/ g4-MMAE as described in B. The percentages of cell death from individual cell lines were determined by the trypan blue exclusion assay. The results shown here are from one of three experiments with similar results. The representative IC50 values from one experiment were calculated using GraphPad Prism 6 software. (D) Activation of cell apoptotic pathway. PDAC cells (2 × 106 cells in a 60 mm diameter dish) were treated with Zt/ g4-MMAE (5 μg/mL) as described in B. Cells were collected at different intervals. Cellular proteins (50 μg per samples) were subjected to Western blot analysis using antibodies specific to PARP or Caspase 3 fragments. Actin was used as the loading control. Images shown here are from one of three experiments with similar results.

(Figure 1B). The percentages of conjugates with different DARs from the integrated areas of the conjugates were determined (Figure 1B). The major peaks were peak 2 (33.02%) with a DAR of 2:1 and peak 4 (46.16%) with a DAR of 4:1. Zt/g4-MMAE with DARs at 2:1, 4:1, and 6:1 accounted for 87.07% of the total conjugates. DARs for Zt/g4-DM1 and

control mouse IgG−MMAE conjugates (CmIgG−MMAE) were 3.91 and 3.54, respectively. The stability of Zt/g4-MMAE was measured under the two conditions. The storage condition stability was tested by incubating Zt/g4-MMAE in phosphate-buffered saline (PBS) at 37 °C for 28 days. Changes in DARs were measured by HIC from different time points. Zt/g4-MMAE appeared to be stable E


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Figure 4. Synergism in vitro between Zt/g4-MMAE and chemotherapeutics: (A) PDAC cell lines (8000 cell per well in a 96-well plate in triplicate) were treated for 96 h with Zt/g4-MMAE at their individual IC50 doses alone or in combination with different amounts of gemcitabine, 5fluorouracil, and oxaliplatin. Cell viability was measured using the MTT assay. The results shown here were from one of three experiments with similar results. The representative data from one experiment were calculated using GraphPad Prism 6 software. (B) PDAC cells were treated for 96 h by combining 5-fluorouracil from 300 to 10 000 nM with Zt/g4-MMAE from 1 to 33.3 nM (equivalent to 0.15 to 5.0 μg/mL) to reach a fixed ratio of 300 to 1. Cell viability was measured using the MTT assay. The results shown here were from one of three experiments with similar results. The representative data from one experiment were calculated using GraphPad Prism 6 software. (C) Analysis of the synergism by Chou−Talalay plot. Percentages of cell viability from individual samples as described in B were calculated, converted, and then used for the fraction of the inhibition−combination index (CI) plot as previously described.39,40 (D) Results from B also were used for isobologram analysis to determine the IC50, IC75, and IC90 values to define the synergism as previously described.39,40 All studies were repeated three times with similar results. Data shown here are from one representative experiment.

at 37 °C in PBS (Figure 1C and Supplementary Table 1). On day 28, the average DAR was 3.06, representing only ∼7.0% reduction from the DAR of 3.29 on day 0. The major changes appeared to be peak 4 and peak 6. From day 0 to day 28, peak 4 was increased from 46.16 to 55.32% and P6 reduced from 7.89 to 0.00%. This suggests that Zt/g4-MMAE is stable at 37 °C in PBS. The stability of ADCs in human plasma was determined by measuring free MMAE dissociated from Zt/g4 using the modified LC−MS/MS method as previously described.35 As the control, the amount of free DM1 released from Zt/g4DM1 was 4.06 ng/mL (Figure 1Di), which accounted for 3.03% of DM1 dissociated from Zt/g4 (Figure 1Diii). The amount of free MMAE released from Zt/g4-MMAE within 10 days was 5.08 ng/mL of plasma (Figure 1Dii), which accounted for 2.81% of MMAE dissociated from Zt/g4 (Figure 1Div). These results suggest that the dissociation of MMAE from Zt/g4 is similar to that of DM1 from Zt/g4. Therefore, we can conclude that Zt/g4-MMAE is stable as Zt/ g4-DM1 in human plasma. Induction by Zt/g4-MMAE of Cell Surface RON Internalization. The ability of Zt/g4-MMAE to induce cell surface RON internalization was studied using a panel of PDAC cell lines known to express different levels of RON.32 The calculated RON molecules per PDAC cell were 10 214 ± 310 for BxPC-3, 16 178 ± 269 for FG, and 16 628 ± 245 for

L3.6pl cells, respectively.32 Specific binding was not observed in Panc-1 cells (cell surface RON receptors:

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