Purpose: The purpose of this study was to evaluate the antitumor efficacy of the reduced immunogenicity anti-mesothelin immunotoxin RG7787 plus nab-paclitaxel against primary mesothelioma cell lines and tumor xenografts and the utility of mesothelin as a biomarker of tumor response.

Experimental Design: Early-passage human malignant mesothelioma cell lines NCI-Meso16, NCI-Meso19, NCI-Meso21, and NCI-Meso29 were evaluated for sensitivity to RG7787 or nab-paclitaxel alone or in combination. In addition, the antitumor activity of RG7787 plus nab-paclitaxel was evaluated using NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumor xenografts in immunodeficient mice. Serum mesothelin was measured at different time points to determine whether its levels correlated with tumor response.

Results: All four primary mesothelioma cell lines highly expressed mesothelin with 41 × 103 to 346 × 103 mesothelin sites per cell and were sensitive to RG7787, with IC50 ranging from 0.3 to 10 ng/mL. Except for NCI-Meso19, these cells were also sensitive to nab-paclitaxel, with IC50 of 10 to 25 ng/mL. In vitro, RG7787 plus nab-paclitaxel led to decreased cell viability compared with either agent alone. In NCI-Meso16 tumor xenografts, treatment with RG7787 plus nab-paclitaxel led to sustained complete tumor regressions. Similar antitumor efficacy was observed against NCI-Meso21 and NCI-Meso29 tumor xenografts. In all three tumor xenograft models, changes in human serum mesothelin correlated with response to therapy and were undetectable in mice with complete tumor regression with RG7787 and nab-paclitaxel.

Conclusions: RG7787 plus nab-paclitaxel is very active against primary human mesothelioma cells in vitro as well as in vivo, with serum mesothelin levels correlating with tumor response. These results indicate that this combination could be useful for treating patients with mesothelioma. Clin Cancer Res; 23(6); 1564–74. ©2016 AACR.

Translational Relevance

RG7787 is a reduced immunogenicity anti-mesothelin immunotoxin that could allow patients to receive multiple cycles of treatment without need for immunosuppressive agents. In this study, we show that RG7787 is very active against primary mesothelioma cell lines. Using three different mesothelioma PDX models with differing growth patterns, RG7787 given in combination with nab-paclitaxel resulted in significant antitumor efficacy, including sustained complete tumor regressions. We also demonstrate that shed human mesothelin in these PDX models is an excellent biomarker of tumor response. A clinical trial of RG7787 to treat mesothelioma patients has just opened, and combination therapy with RG7787 plus nab-paclitaxel could be more efficacious.

Malignant mesothelioma is an aggressive tumor arising from the mesothelial cells lining the lungs or abdominal cavity, and approximately 3,000 new cases are diagnosed each year in the United States. Most patients are not candidates for surgical cytoreduction at the time of their diagnosis (1). The only FDA-approved chemotherapy regimen for this tumor is combination therapy with cisplatin and pemetrexed that results in a median overall survival of approximately 12 months (2). There is clearly a need to develop more effective treatments for this disease. Our group has focused on exploiting the tumor differentiation antigen mesothelin as a target for mesothelioma therapy (3). Mesothelin is a cell-surface protein that is normally expressed on mesothelial cells lining the pleura, peritoneum, and pericardium but is highly expressed in several human cancers, including malignant mesothelioma (4–6). To target mesothelin, our group has developed an anti-mesothelin immunotoxin SS1P that is currently in clinical trials. It consists of an anti-mesothelin Fv linked to PE38, a truncated Pseudomonas exotoxin A (PE; refs. 7, 8). In clinical trials, SS1P was well tolerated and the dose-limiting toxicity was pleuritis. Significant antitumor efficacy was not seen in single-agent phase I study, most likely due to development of an anti-SS1P immune response that limited retreatment of patients (9). More recently, we have shown that combination therapy with SS1P plus pentostatin and cyclophosphamide delayed formation of anti-SS1P antibodies and resulted in major tumor regressions in some patients with treatment-refractory malignant mesothelioma (10).

Having shown proof-of-principle antitumor efficacy with SS1P, our group has now focused on developing anti-mesothelin immunotoxins that are inherently less immunogenic so there is no need to use immunosuppressive drugs (11). RG7787 (now named LMB-100) is an anti-mesothelin immunotoxin with reduced immunogenicity consisting of a humanized anti-mesothelin Fab linked to PE24. PE24 is a modified Pseudomonas exotoxin A in which part of domain II of PE toxin has been deleted, as well as seven bulky hydrophilic resides in domain III of PE toxin have been mutated to alanine to silence B-cell epitopes (12–14). Structurally, RG7787 differs from SS1P as it consists of an anti-mesothelin Fab rather than Fv and is linked to PE24 rather than PE38 and has a molecular size of 72 kDa compared with SS1P's molecular size of 62 kDa (11). RG7787 was developed in collaboration with Roche. RG7787 also can be given at much higher doses to mice than SS1P and also causes decreased vascular leak in a rat vascular leak model (15). More importantly, it has reduced antigenicity than SS1P when tested against serum from patients who made antibodies to SS1P. These characteristics make it an attractive agent for treatment of mesothelin expressing cancers, including mesothelioma.

Nab-paclitaxel is an albumin-bound paclitaxel that has recently been approved for several cancers, including pancreatic and lung cancer (16–18). However, it has not been evaluated for therapy of patients with mesothelioma. In this report, we show that nab-paclitaxel has significant activity against primary mesothelioma cell lines established from patients with mesothelioma. More importantly, we show that the treatment of mesothelioma patient-derived tumor xenografts with RG7787 plus nab-paclitaxel results in remarkable antitumor efficacy in three different tumor models. These results suggest that treatment with RG7787 and nab-paclitaxel could be effective in patients with mesothelioma. A clinical trial of single-agent RG7787 has been initiated for patients with treatment-refractory mesothelioma.

Early-passage mesothelioma cells

Early-passage mesothelioma cells were established from the ascites or pleural fluid obtained from patients with mesothelioma seen at the NCI (Bethesda, MD) on Institutional Review Board–approved protocols. We have previously described the establishment of these primary culture cells for NCI-Meso16, NCI-Meso19, and NCI-Meso21 (19). NCI-Meso29 was established from ascites of a patient with peritoneal mesothelioma following the same procedure as described previously. Cell line authentication was done in Frederick National Laboratory for Cancer Research (Frederick, MD) using short tandem repeat analysis. Cell lines NCI-Meso16, NCI-Meso19, and NCI-Meso21 were tested on July 18, 2012. Cell line NCI-Meso29 was tested on May 20, 2016.

Cell-surface mesothelin expression

Early-passage mesothelioma cells were evaluated for mesothelin cell-surface expression by flow cytometry using the procedure described earlier with minor modifications (20). In brief, cells were grown until confluent, trypsinized and washed, resuspended in FACS buffer (PBS with 5% FBS and 0.1% sodium azide), and incubated with 5 μg/mL of mouse anti-human mesothelin antibody MN (Rockland Immunochemicals Inc.) at 4°C for 1 hour. Cells were then washed and incubated with 5 μg/mL of goat anti-mouse antibody conjugated with R-PE (Invitrogen) at 4°C for 30 minutes. An isotope-matched antibody was used as a control. Geometric means were chosen as mean fluorescence intensity (MFI). The MFI of cells was compared with the MFI from a standard curve of PE conjugate calibration beads (BD QuantiBRITE PE Quantitation Kit, BD Biosciences), and the number of mesothelin sites per cell was established.

Cytotoxicity assays

The cytotoxicity of RG7787 and nab-paclitaxel (Celgene) was evaluated using cells that were within 4 to 6 passages after plating the ascites or pleural effusion. Tumor cells (1 × 104/well) were seeded in a 96-well plate. RG7787 or nab-paclitaxel were added in serial dilutions up to 100 ng/mL. Cycloheximide (Sigma) was used as a positive control at 10 ng/mL. The cells were incubated for 96 hours. For the in vitro combination study of RG7787 and nab-paclitaxel, nab-paclitaxel at a concentration of 10 ng/mL was added to the designated wells on day 1 after the cells were seeded and incubated overnight, then followed by adding different concentrations of RG7787 and incubated for 72 hours. WST-8 reagent (Dojindo) was used to quantitate the viable cells, according to the manufacturer's instructions. The final determination of color intensity by OD 450 was measured using a SoftMax M3 Microplate Reader (Molecular Devices).

Evaluation of apoptosis induced by RG7787 and nab-paclitaxel

NCI-Meso16, NCI-Meso21, and NCI-Meso29 cells (1 × 106 each) were plated in 100-mm3 culture dishes and incubated for 4 hours, followed by addition of different concentrations of nab-paclitaxel, RG7787, or both. NCI-Meso16 was treated with 10 ng/mL of both nab-paclitaxel and RG7787. NCI-Meso21 was treated with 10 and 0.1 ng/mL of nab-paclitaxel and RG7787, respectively, and NCI-Meso29 was incubated with 25 and 10 ng/mL of nab-paclitaxel and RG7787, respectively. The drug concentrations used were based on the IC50 values for these drugs obtained in prior in vitro cytotoxicity assays. Cells without any treatment were used as control. The cells were incubated with the drugs for 72 hours. After the incubation, the cells were trypsinized, washed, and stained using an Annexin V-PE and 7-AAD Apoptotic Assay Kit (BD Biosciences) according to the manufacturer's instructions. The flow cytometry analysis of stained cells was conducted on BD SORP flow cytometer (BD Biosciences). Percentage of treated cells undergoing apoptosis was determined by subtracting the percentage of apoptotic cells in control. Both early (Annexin V+ 7-AAD) and late (Annexin V+ 7-AAD+) apoptotic cells were considered for calculating the total percentage of apoptosis.

In vivo studies of RG7787 and nab-paclitaxel using primary mesothelioma xenografts

NCI-Meso16, NCI-Meso21, and NCI-Meso29 cells were grown as subcutaneous tumor xenografts in immunodeficient mice. NCI-Meso16 and NCI-Meso21 were inoculated in athymic nude mice, whereas NCI-Meso29 tumor cells were injected into NOD/SCID mice. In brief, 4- to 5-week-old female athymic nude mice (Ncr/nu/nu 01B74; NCI, Frederick, MD) or 4- to 6-week-old NOD/SCID mice were inoculated subcutaneously in the flank region with 5 × 106 tumor cells (in case of NCI-Meso16 and NCI-Meso29) or 1 × 107 tumor cells (in case of NCI-Meso21) in 200 μL RPMI containing 4 mg/mL Matrigel (BD Biosciences). Tumor volume was measured regularly by electronic caliper. The tumor volume was calculated as length × width2 × 0.4. The treatment started when tumor volume reached 100 to 110 mm3, approximately 44 days for NCI-Meso16, 82 days for NCI-Meso21, and 25 days for NCI-Meso29 after tumor cell inoculation. Both RG7787 (2.5 mg/kg) and nab-paclitaxel (100 or 75 mg/kg) were given intravenously in a volume of 200 μL. Athymic nude mice bearing NCI-Meso16 and NCI-Meso21 tumor xenografts received two courses of therapy 4 days apart (each course consisting of either single dose of nab-paclitaxel on day 1; three doses of RG7787 on days 2, 4, and 6; or single dose of nab-paclitaxel on day 1 plus three doses of RG7787 on days 2, 4, and 6). In NOD/SCID mice bearing NCI-Meso29 tumors, the second course of therapy consisted of either single dose of nab-paclitaxel, single dose of RG7787, or single dose of RG7787 given the day after nab-paclitaxel administration. All animals were hosted and cared for according to NIH guidelines, and the study was approved by the Animal Care and Use Committee of the NCI.

Pathologic analysis of mouse tumors

Representative mouse tumors from the different treatment groups were dissected after the first cycle of treatment and then fixed in 10% neutral-buffered formalin and embedded in paraffin. The paraffin sections were cut and stained with hematoxylin and eosin (H&E). Immunohistochemical stains for mesothelin and Ki67 were performed on paraffin sections with anti-mesothelin antibody (1: 200, clone MN-1, Rockland, cat# 200-301-A88) and anti-Ki67 antibody (1:1,000, Abcam, cat# ab15580) using UltraVision LP Detection System (Thermo Fisher Scientific), according to the manufacturer's instructions. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay was performed on paraffin sections using a TUNEL Apoptosis Detection Kit (Millipore, cat# 17-141) according to the manufacturer's instructions. Mesothelin expression by the tumor xenografts was evaluated for intensity of staining (0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining) as well as percentage of tumor cells that expressed mesothelin. The semiquantitative H-score was used to describe mesothelin positivity of these tumors on a scale of 0 to 300 (with the lowest score of 0 indicating no staining in the tumor, whereas maximum score of 300 indicates 100% of tumor cells with 3+ mesothelin staining; ref. 21). Pathologic evaluation was performed by a pathologist (M. Miettinen) with expertise in mesothelioma and IHC.

Mouse serum mesothelin measurement

Blood (50–100 μL) was collected from the facial vein of each study mouse before treatment and at different time points thereafter. Blood samples were centrifuged at 2,000 rpm at room temperature for 15 minutes. The sera were analyzed using MESOMARK (Fujirebio Diagnostics Inc.) according to the manufacturer's instructions.

Statistical analysis

The P values for apoptotic assays and percent cell viability quantification were calculated by performing unpaired t tests using GraphPad Prism software. P values for in vivo experiments (Fig. 4) were obtained by a two-sided t test. P values for tumor size and serum mesothelin level correlation were calculated by Spearman correlation analysis.

Early-passage mesothelioma cell lines express mesothelin and are sensitive to RG7787 and nab-paclitaxel

All four primary mesothelioma cell lines established from patients expressed mesothelin when cultured in vitro. The mesothelin sites per cell for NCI-Meso16, NCI-Meso19, NCI-Meso21, and NCI-Meso29 were 249 × 103, 41 × 103, 346 × 103, and 44 × 103, respectively (Fig. 1A). All four of these cell lines were sensitive to killing by RG7787 with IC50 of 0.3 to 10 ng/mL, with NCI-Meso21 being the most sensitive with an IC50 of 0.3 ng/mL (Fig. 1B). In the case of nab-paclitaxel, NCI-Meso16, NCI-Meso21, and NCI-Meso29 were sensitive to nab-paclitaxel with IC50 of 10, 20, and 25 ng/mL respectively, whereas NCI-Meso19 was resistant with IC50 >100 ng/mL (Fig. 1C). We also evaluated the sensitivity of these cell lines to SS1P. Except for NCI-Meso16, all other cell lines were 2 to 9-fold more sensitive to RG7787 than SS1P (Fig. 1D).

Figure 1.

Mesothelin expressions in early-passage primary mesothelioma cells and cytotoxicity of RG7787 and nab-paclitaxel. A, Primary mesothelioma cell cultures were stained with anti-mesothelin antibody MN, followed by staining with goat anti-mouse antibody conjugated with R-PE, and the binding was analyzed by flow cytometry. Results are shown in terms of histogram plots for each cell line, where open area depicts the binding of MN antibody, and the gray area shows the binding of the isotope control antibody. B, Cytotoxicity of RG7787 against the primary mesothelioma cell cultures. A total of 8,000 tumor cells were seeded in a 96-well plate, and serial dilutions of RG7787 were added. After 4 days of incubation, cell viability was measured by WST-8 assay. C, Cytotoxicity of nab-paclitaxel against primary mesothelioma cells. A total of 8,000 tumor cells were seeded in a 96-well plate, and serial dilutions of nab-paclitaxel were added. The cells were incubated for 4 days, and the percent of viable cells was determined by WST-8 assay. D, Table summarizing mesothelin expression in the primary mesothelioma cell lines as well as their sensitivity to the immunotoxins SS1P and RG7787 as well as nab-paclitaxel.

Figure 1.

Mesothelin expressions in early-passage primary mesothelioma cells and cytotoxicity of RG7787 and nab-paclitaxel. A, Primary mesothelioma cell cultures were stained with anti-mesothelin antibody MN, followed by staining with goat anti-mouse antibody conjugated with R-PE, and the binding was analyzed by flow cytometry. Results are shown in terms of histogram plots for each cell line, where open area depicts the binding of MN antibody, and the gray area shows the binding of the isotope control antibody. B, Cytotoxicity of RG7787 against the primary mesothelioma cell cultures. A total of 8,000 tumor cells were seeded in a 96-well plate, and serial dilutions of RG7787 were added. After 4 days of incubation, cell viability was measured by WST-8 assay. C, Cytotoxicity of nab-paclitaxel against primary mesothelioma cells. A total of 8,000 tumor cells were seeded in a 96-well plate, and serial dilutions of nab-paclitaxel were added. The cells were incubated for 4 days, and the percent of viable cells was determined by WST-8 assay. D, Table summarizing mesothelin expression in the primary mesothelioma cell lines as well as their sensitivity to the immunotoxins SS1P and RG7787 as well as nab-paclitaxel.

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RG7787 plus nab-paclitaxel results in increased cytotoxicity of primary mesothelioma cells

The in vitro activity of RG7787 in combination with nab-paclitaxel was evaluated using NCI-Meso16, NCI-Meso21, and NCI-Meso29 cell lines as they are sensitive to both RG7787 and nab-paclitaxel. NCI-Meso16, NCI-Meso21, and NCI-Meso29 were treated with 10, 0.3, and 10 ng/mL of RG7787, respectively, as well as 10 ng/mL (in case of NCI-Meso16 and NCI-Meso21) or 25 ng/mL (in case of NCI-Meso29) of nab-paclitaxel either alone or in combination and assessed for cell viability. As shown in Fig. 2A, the combination of RG7787 and nab-paclitaxel resulted in decreased cell viability compared with untreated cells, RG7787, or nab-paclitaxel alone–treated cells for all the three cell lines. This was especially pronounced for NCI-Meso16 cells, which had an IC50 of 0.01 for RG7787 plus nab-paclitaxel compared with their single-agent IC50 of 10 ng/mL. Figure 2B shows photomicrographs of NCI-Meso16 cells untreated and after treatment with RG7787, nab-paclitaxel, or the two drugs together. As shown, very few viable cells remained following treatment with combination therapy. The percent decrease in cell viability in the RG7787 plus nab-paclitaxel–treated cells was statistically significant compared with either RG7787 or nab-paclitaxel–treated cells. Similarly, for both NCI-Meso21 and NCI-Meso29, combination treatment with RG7787 and nab-paclitaxel resulted in decreased cell viability compared with either treatment alone.

Figure 2.

In vitro cytotoxicity of RG7787 in combination with nab-paclitaxel against primary mesothelioma cell lines NCI-Meso16, NCI-Meso21, and NCI-Meso29. A, A total of 8,000 tumor cells per well were seeded in a 96-well plate on day 0. Serial dilutions of nab-paclitaxel were added on day 1, and serial dilutions of RG7787 were added on the following day either by itself or to wells treated the day before with nab-paclitaxel. After 4 days of incubation, cell viability was determined by WST-8. B, To evaluate the effect of RG7787 plus nab-paclitaxel on cell morphology, 5 × 104 cells were seeded in a 24-well plate, and 10 ng/mL of nab-paclitaxel was added, followed by the addition of 10 ng/mL of RG7787 the following day. The morphology of tumor cells after 4 days of incubation with nab-paclitaxel alone, RG7787 alone, or the two drugs together is shown, as well as cell viability measured by WST-8.

Figure 2.

In vitro cytotoxicity of RG7787 in combination with nab-paclitaxel against primary mesothelioma cell lines NCI-Meso16, NCI-Meso21, and NCI-Meso29. A, A total of 8,000 tumor cells per well were seeded in a 96-well plate on day 0. Serial dilutions of nab-paclitaxel were added on day 1, and serial dilutions of RG7787 were added on the following day either by itself or to wells treated the day before with nab-paclitaxel. After 4 days of incubation, cell viability was determined by WST-8. B, To evaluate the effect of RG7787 plus nab-paclitaxel on cell morphology, 5 × 104 cells were seeded in a 24-well plate, and 10 ng/mL of nab-paclitaxel was added, followed by the addition of 10 ng/mL of RG7787 the following day. The morphology of tumor cells after 4 days of incubation with nab-paclitaxel alone, RG7787 alone, or the two drugs together is shown, as well as cell viability measured by WST-8.

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Increased apoptotic cell killing by RG7787 plus nab-paclitaxel varies among different mesothelioma cells

To determine whether cell death is caused by apoptosis, we evaluated NCI-Meso16, NCI-Meso21, and NCI-Meso29 for induction of apoptosis by RG7787, nab-paclitaxel, or the combination. Figure 3A is a representative example that shows the percentage of apoptotic NCI-Meso16 cells following treatment. For NCI-Meso16, the percentage of apoptotic cells (both early and late apoptotic) after 72 hours of treatment with RG7787 alone, nab-paclitaxel alone, or RG7787 plus nab-paclitaxel was 19.6%, 26.2%, and 42.9%, respectively. There was a sharp increase in the percentage of apoptotic cells when nab-paclitaxel and RG7787 were used in combination. Figure 3B, which is a summary of three separate experiments, shows that in the case of NCI-Meso16, the increase in apoptotic cells was significant with P values of 0.025 and 0.001 for combination versus nab-paclitaxel and combination versus RG7787, respectively. The fraction of NCI-Meso21 cells undergoing apoptosis with RG7787 plus nab-paclitaxel was numerically greater than seen with RG7787 or nab-paclitaxel alone, but this difference was not statistically significant. Similarly, for NCI-Meso29 cells, the percent increase in apoptotic cells when treated with RG7787 and nab-paclitaxel was significant compared with nab-paclitaxel alone–treated cells (P = 0.028) but not compared to RG7787 alone–treated cells (P = 0.449). The increase in apoptotic cell death in NCI-Meso16 cells by combination treatment with RG7787 and nab-paclitaxel is in keeping with cell viability data, as these cells are extremely sensitive to this combination.

Figure 3.

Evaluation of apoptosis induced by RG7787 and nab-paclitaxel. A, Representative Annexin V 7-AAD apoptotic assay for NCI-Meso16 cell line either untreated or treated with nab-paclitaxel, RG7787, or nab-paclitaxel plus RG7787 combination. AnnexinV+7AAD cells are in early apoptotic stage, whereas AnnexinV+7AAD+ cells are late apoptotic cells. B, Percentage of NCI-Meso16, NCI-Meso21, and NCI-Meso29 cells undergoing apoptosis after 72-hour treatment with monotherapy or combination. Of the three cell lines tested, only in the case of NCI-Meso16, the percentage of apoptotic cells in the combination group was significantly higher than either of the single treatments.

Figure 3.

Evaluation of apoptosis induced by RG7787 and nab-paclitaxel. A, Representative Annexin V 7-AAD apoptotic assay for NCI-Meso16 cell line either untreated or treated with nab-paclitaxel, RG7787, or nab-paclitaxel plus RG7787 combination. AnnexinV+7AAD cells are in early apoptotic stage, whereas AnnexinV+7AAD+ cells are late apoptotic cells. B, Percentage of NCI-Meso16, NCI-Meso21, and NCI-Meso29 cells undergoing apoptosis after 72-hour treatment with monotherapy or combination. Of the three cell lines tested, only in the case of NCI-Meso16, the percentage of apoptotic cells in the combination group was significantly higher than either of the single treatments.

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Characteristics of human mesothelioma tumor xenografts

We evaluated the antitumor efficacy of combination therapy with RG7787 and nab-paclitaxel using patient-derived NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumor xenografts in immunodeficient nude mice. NCI-Meso19 cells did not form tumors in immunodeficient mice and were therefore not evaluated. NCI-Meso16 and NCI-Meso21 were grown in athymic nude mice, whereas NCI-Meso29 cells were grown in NOD/SCID mice. NCI-Meso16 and NCI-Meso29 are rapidly growing tumors that reach 100 mm3 size by day 40 and 25, respectively, and 500 mm3 by day 70 and 45, respectively (after subcutaneous injection of 5 × 106 cells). In contrast, NC-Meso21 is a slow growing tumor and reaches 100 mm3 by day 82 and 250 mm3 by day 120 (after injection of 10 × 106 cells). All three tumor models continued to express mesothelin when grown in vivo. The H-score for NCI-Meso16, NCI-Meso21, and NCI-Meso29 was 230, 50, and 290, respectively.

Antitumor efficacy of RG7787 plus nab-paclitaxel against human mesothelioma tumor xenografts

Athymic mice bearing NCI-Meso16 tumors were randomized at day 44 when tumors reached 110 mm3 in size to four different groups (n = 8 mice/group) and received no treatment (control group): RG7787 given as 2.5 mg/kg on days 45, 47, 49, 54, 56, and 58; nab-paclitaxel 100 mg/kg given on days 44 and 53; and the combination group in which mice received RG7787 and nab-Paclitaxel at the same dose and schedule as the individual groups. In mice that received no treatment, the tumors rapidly increased in size, and the mice were sacrificed on day 100 when tumors reached 1,000 mm3 in size. In the RG7787 group, there was stabilization of tumor growth while the mice were receiving treatment but then rapidly increased in size. In the nab-paclitaxel group, there was tumor shrinkage, but by day 70, the tumors gradually increased in size and mice were sacrificed at day 140. However, in mice that received both RG7787 and nab-paclitaxel, there was complete tumor regression by day 68 in all mice, and 5 of 7 mice had no tumor regrowth when the experiment was terminated on day 142 (Fig. 4A).

Figure 4.

In vivo efficacy of RG7787 plus nab-paclitaxel against human mesothelioma tumor xenografts. A, NCI-Meso16 cells were grown as tumor xenografts in athymic nude mice. On day 44, when tumors reached 108 mm3, groups of mice (n = 8) were treated at time points indicated by vertical arrows with RG7787 (2.5 mg/kg), nab-paclitaxel (100 mg/kg), or RG7787 (2.5 mg/kg) plus nab-paclitaxel (100 mg/kg). Data points, mean tumor volumes; *, comparison in mean tumor volume between mice treated with nab-paclitaxel and RG7787 versus mice treated with nab-paclitaxel alone. B, NCI-Meso21 cells were also grown as tumor xenografts in athymic nude mice. On day 82, when tumors reached 102 mm3, groups of mice (n = 8) were treated at time points indicated by vertical arrows with RG7787 (2.5 mg/kg), nab-paclitaxel (100 mg/kg), or RG7787 (2.5 mg/kg) plus nab-paclitaxel (100 mg/kg). Data points, median tumor volumes; *, comparison in median tumor volume between mice treated with nab-paclitaxel and RG7787 versus mice treated with RG7787 alone. C, NCI-Meso29 cells were grown as tumor xenografts in NOD/SCID mice, and on day 25, when tumors were 102 mm3 in size, groups of mice (n = 7) were treated at the time points indicated by vertical arrows with RG7787 (2.5 mg/kg), nab-paclitaxel (75 mg/kg), or RG7787 (2.5 mg/kg) plus nab-paclitaxel (75 mg/kg). Data points, median tumor volumes; *, comparison in median tumor volume between mice treated with nab-paclitaxel and RG7787 versus mice treated with nab-paclitaxel alone.

Figure 4.

In vivo efficacy of RG7787 plus nab-paclitaxel against human mesothelioma tumor xenografts. A, NCI-Meso16 cells were grown as tumor xenografts in athymic nude mice. On day 44, when tumors reached 108 mm3, groups of mice (n = 8) were treated at time points indicated by vertical arrows with RG7787 (2.5 mg/kg), nab-paclitaxel (100 mg/kg), or RG7787 (2.5 mg/kg) plus nab-paclitaxel (100 mg/kg). Data points, mean tumor volumes; *, comparison in mean tumor volume between mice treated with nab-paclitaxel and RG7787 versus mice treated with nab-paclitaxel alone. B, NCI-Meso21 cells were also grown as tumor xenografts in athymic nude mice. On day 82, when tumors reached 102 mm3, groups of mice (n = 8) were treated at time points indicated by vertical arrows with RG7787 (2.5 mg/kg), nab-paclitaxel (100 mg/kg), or RG7787 (2.5 mg/kg) plus nab-paclitaxel (100 mg/kg). Data points, median tumor volumes; *, comparison in median tumor volume between mice treated with nab-paclitaxel and RG7787 versus mice treated with RG7787 alone. C, NCI-Meso29 cells were grown as tumor xenografts in NOD/SCID mice, and on day 25, when tumors were 102 mm3 in size, groups of mice (n = 7) were treated at the time points indicated by vertical arrows with RG7787 (2.5 mg/kg), nab-paclitaxel (75 mg/kg), or RG7787 (2.5 mg/kg) plus nab-paclitaxel (75 mg/kg). Data points, median tumor volumes; *, comparison in median tumor volume between mice treated with nab-paclitaxel and RG7787 versus mice treated with nab-paclitaxel alone.

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In the case of NCI-Meso21 tumor xenografts, the mice were randomized on day 80 when they reached 100 mm3 in size to four different groups (n = 8 mice per group), that is, control group, RG7787 group, nab-paclitaxel group, and RG7787 plus nab-paclitaxel group. The mice received the treatments at the same schedule and dose as described above for NCI-Meso16 tumor xenograft model. As shown in Fig. 4B, these tumors grow slowly, but treatment with RG7787 alone initially led to tumor shrinkage, followed by stabilization in tumor size, whereas treatment with nab-paclitaxel resulted in tumor stasis, followed by slow growth by day 100. However, the greatest antitumor effect was observed in the combination group, and these tumors remained small when the experiment was terminated on day 150. The decrease in tumor size in mice receiving combination treatment was statistically significant compared with RG7787 or nab-paclitaxel–treated mice.

The NCI-Meso29 tumor cells were grown in NOD/SCID mice, and our initial experiments showed that these mice are very sensitive to combination treatment with RG7787 and nab-paclitaxel at the doses that were used to treat athymic mice (data not shown). Therefore, we reduced the dose of nab-paclitaxel as well as decreased the number of RG7787 doses administered so that in the RG7787 group, mice received 2.5 mg/kg on days 26, 28, 30, and 34; in the nab-paclitaxel group, mice received 75 mg/kg on days 25 and 33; and in the combination group, mice received nab-paclitaxel 75 mg/kg on days 25 and 33 and RG7787 2.5 mg/kg on days 26, 28, 30, and 34. As shown in Fig. 4C, treatment with RG7787 alone led to tumor stasis, followed by rapid tumor growth, whereas treatment with nab-paclitaxel shrank tumors, followed by gradual increase in tumor size by day 80. However, treatment with RG7787 plus nab-paclitaxel led to marked tumor shrinkage, which was statistically significant, compared with nab-paclitaxel alone–treated mice (P < 0.05).

To evaluate the effect of the different treatments on tumor morphology, one mouse from each treatment group was sacrificed on day 33 after one cycle of therapy (one cycle of therapy consisted of one dose of nab-paclitaxel or three doses of RG7787 either alone or in combination) before major regression occurred in the combination group. There was a decrease in mesothelin-positive tumor cells in both the RG7787 and nab-paclitaxel group but much more in the combination group. Also, the combination group showed decreased Ki67 staining and increased apoptosis, as detected by TUNEL staining, compared with the other treatment groups. A representative example is shown for NCI-Meso29 tumors in Fig. 5. 

Figure 5.

Reduced cell proliferation and increased apoptosis in RG7787 plus nab-paclitaxel–treated NCI-Meso29 tumors. Mice were subcutaneously inoculated with 5 × 106 NCI-Meso29 cells, and the treatment was started when tumor size reached 100 mm3. Mice were treated with no treatment; 1 cycle of 3 × 2.5 mg/kg RG7787 every other day i.v.; 1 cycle of 1 × 75 mg/kg nab-paclitaxel; and 1 cycle of 3 × 2.5 mg/kg RG7787 combined with 1 cycle 1 × 75 mg/kg nab-paclitaxel. Representative tumor tissues from different groups were harvested after the first cycle of the indicated treatments on day 33, and representative H&E staining, immunohistochemical staining for MSLN and Ki67 expression, as well as TUNEL staining with the paraffin sections of tumor tissues were shown. Scale bars (H&E), 1,000 μm; scale bar (MSLN, Ki67, and TUNEL staining), 200 μm.

Figure 5.

Reduced cell proliferation and increased apoptosis in RG7787 plus nab-paclitaxel–treated NCI-Meso29 tumors. Mice were subcutaneously inoculated with 5 × 106 NCI-Meso29 cells, and the treatment was started when tumor size reached 100 mm3. Mice were treated with no treatment; 1 cycle of 3 × 2.5 mg/kg RG7787 every other day i.v.; 1 cycle of 1 × 75 mg/kg nab-paclitaxel; and 1 cycle of 3 × 2.5 mg/kg RG7787 combined with 1 cycle 1 × 75 mg/kg nab-paclitaxel. Representative tumor tissues from different groups were harvested after the first cycle of the indicated treatments on day 33, and representative H&E staining, immunohistochemical staining for MSLN and Ki67 expression, as well as TUNEL staining with the paraffin sections of tumor tissues were shown. Scale bars (H&E), 1,000 μm; scale bar (MSLN, Ki67, and TUNEL staining), 200 μm.

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Serum mesothelin as a biomarker of tumor response

As the NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumors highly express mesothelin, we hypothesized that these tumors could release human mesothelin in mouse blood, and therefore, measuring the levels could be used to assess response to therapy. Serum was collected before mice received any treatment (at the time of randomization) and at different time points during therapy, and mesothelin levels were measured using the MESOMARK assay (22). There was variability in the baseline serum mesothelin levels in the different tumor xenografts when treatment was started. The serum mesothelin levels in NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumor-bearing mice when the tumors were approximately 100 mm3 in size were 2.40, 0.53, and 0.25 nmol/L, respectively. In mice with NCI-Meso16 tumors, the serum mesothelin levels increased in the control group and decreased in the RG7787 and nab-paclitaxel group and became undetectable in the combination group. There was excellent correlation between tumor size and serum mesothelin levels in these mice (r = 0.89; P < 0.0001; Fig. 6). Even for the slow growing NCI-Meso21 tumor xenografts, serum mesothelin levels continued to increase in the untreated mice and nab-paclitaxel–treated mice but stayed the same in RG7787-treated mice and decreased in the combination group. The correlation between tumor size and serum mesothelin was also highly significant (r = 0.84; P < 0.0001). Similarly, in the NCI-Meso29 tumor model, there was a very good correlation between tumor size and serum mesothelin in the different treatment groups.

Figure 6.

Correlation between tumor size, tumor response, and serum mesothelin. Serum from mice bearing NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumors was collected from the control and the three treatment groups and evaluated for mesothelin using the MESOMARK assay. A, Mean tumor size of untreated mice, RG7787, nab-paclitaxel, and RG7787 plus nab-paclitaxel–treated mice for NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumors. B, Mean serum mesothelin levels of untreated mice, RG7787, nab-paclitaxel, and RG7787 plus nab-paclitaxel–treated mice for NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumors. C, Correlation between tumor size and serum mesothelin for NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumors.

Figure 6.

Correlation between tumor size, tumor response, and serum mesothelin. Serum from mice bearing NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumors was collected from the control and the three treatment groups and evaluated for mesothelin using the MESOMARK assay. A, Mean tumor size of untreated mice, RG7787, nab-paclitaxel, and RG7787 plus nab-paclitaxel–treated mice for NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumors. B, Mean serum mesothelin levels of untreated mice, RG7787, nab-paclitaxel, and RG7787 plus nab-paclitaxel–treated mice for NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumors. C, Correlation between tumor size and serum mesothelin for NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumors.

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In this article, we demonstrate antitumor efficacy for the combination of RG7787 and nab-paclitaxel against primary mesothelioma cell lines grown in vitro and sustained tumor regressions in three patient-derived mesothelioma tumor xenograft models each with different growth kinetics. In addition, we show that human mesothelin shed into the serum of mice was a predictive biomarker for tumor response in all three mesothelioma tumor xenograft models.

RG7787 is an anti-mesothelin immunotoxin with reduced immunogenicity that has several important properties that make it a potentially useful agent in the clinic (11). In this study, we wanted to evaluate its activity against primary cell lines established directly from patients with mesothelioma. Our results show that in vitro RG7787 is 2- to 9-fold more cytotoxic than SS1P against three of the four primary mesothelioma cell lines tested. We have previously shown increased antitumor efficacy of SS1P with gemcitabine and cisplatin, drugs that are commonly used to treat mesothelioma (23). Although RG7787 given in combination with these chemotherapy drugs could have increased activity, for our initial studies, we focused on combination of RG7787 with nab-paclitaxel as our prior studies have shown remarkable synergy between immunotoxins and taxanes in different tumor models (14, 24, 25). We chose to use nab-paclitaxel for our laboratory studies instead of paclitaxel as clinical trials of paclitaxel have shown no activity in patients with mesothelioma (26, 27). Nab-paclitaxel is an albumin-bound paclitaxel that has distinct pharmacologic properties compared with paclitaxel, including greater uptake by and retention within tumor, which could make it efficacious to treat solid tumors (28). Results of recent clinical trials have shown nab-paclitaxel to be effective against several human cancers, but it has not been evaluated in patients with mesothelioma (16–18). We show that primary mesothelioma cell lines are sensitive to nab-paclitaxel, with three of our four primary mesothelioma cell lines having IC50 less than 50 ng/mL. Our in vitro studies also showed that combination therapy with RG7787 plus nab-paclitaxel resulted in increased cytotoxicity against all three mesothelioma cell lines tested in vitro. However, combination treatment of only the most sensitive cell line, NCI-Meso16, produced an increased fraction of apoptotic cells compared with treatment with either agent alone.

We observed remarkable antitumor efficacy of the combination therapy using three different tumor xenograft models. This effect was seen in the fast growing NCI-Meso16 and NCI-Meso29 tumors as well as the slow growing NCI-Meso21 tumors. Also, these tumors had variable tumor mesothelin expression, with NCI-Meso16 and NCI-Meso29 having an H-score of 230 and 290, respectively, which was in keeping with high mesothelin expression present on tumor cells grown in culture. However, the NCI-Meso21 tumors had low mesothelin expression, with an H-score of 50 that was in contrast to the very high mesothelin expression (346 × 103 mesothelin sites/cell) on cells grown in vitro. This difference in mesothelin expression could be related to differences in in vivo growth conditions. For NCI-Meso16, tumor xenografts treatment with RG7787 plus nab-paclitaxel resulted in durable complete tumor regressions. In the case of NCI-Meso29 tumors, marked reduction in tumors of mice treated with RG7787 plus nab-paclitaxel was observed even though these mice received reduced doses of both nab-paclitaxel and RG7787. For the slow growing NCI-Meso21 tumor, RG7787, or nab-Paclitaxel treatment caused tumor stasis while the combination of the two agents produced sustained tumor regressions. We also show that in these tumor xenograft models, treatment with nab-paclitaxel plus RG7787 resulted in reduced mesothelin expression by tumor cells, decreased proliferative index, as well as increased apoptosis.

We also show that human mesothelin made by the mesothelioma tumor xenografts is shed into the mouse serum and that serum mesothelin correlated with tumor burden. In all the three mesothelioma tumor models, the serum mesothelin levels correlated with the tumor growth or shrinkage observed during the different treatments that the mice were receiving. These results provide preclinical evidence that serum mesothelin is a robust marker of tumor response in mesothelioma and support our earlier clinical observations where we showed that serum mesothelin levels correlated with radiologic tumor response (29–32). Given the inherent difficulties of measuring radiologic tumor response in patients with mesothelioma due to its growth characteristics, correlation with preclinical studies, like those we have performed here, provides further evidence advocating for the clinical use of serum mesothelin as a marker of tumor response in patients with elevated mesothelin levels prior to therapy. However, mesothelin levels are elevated in only about half the mesothelioma patients despite the fact that mesothelin expression on tumor cell surface is seen in the majority of epithelioid mesotheliomas (6, 33). Patients with tumor mesothelin positivity but without increased mesothelin levels in the serum could still benefit from mesothelin-directed therapies, but serum mesothelin levels cannot be used as biomarker of tumor response in these patients.

Although we show efficacy of RG7787 and nab-paclitaxel against different mesothelioma patient–derived tumor xenografts with varying growth patterns as well as mesothelin expression, our study is limited by the fact that we used a subcutaneous tumor model, rather than an orthotopic model, which may better recapitulate the tumor biology seen in patients (34). Developing intrathoracic orthotopic tumors for NCI-Meso16 and NCI-Meso21, obtained from patients with pleural mesothelioma, and an intraperitoneal model for NCI-Meso29, derived from a patient with peritoneal mesothelioma, may be advantageous. Compared with subcutaneous tumor models, these orthotopic tumors could have differences in tumor growth and spread, mesothelin expression, mesothelin shedding, as well as sensitivity to RG7787, nab-Paclitaxel, or the combination of the two agents. We plan to establish such orthotopic mesothelioma tumor models for our future studies.

In summary, our results validate the use of serum mesothelin as a biomarker of tumor response and show marked antitumor efficacy of RG7787 plus nab-paclitaxel against human mesothelioma primary cell lines as well as tumor xenografts. These results demonstrate that this combination could be potentially useful for treatment of mesothelioma. A clinical trial of RG7787 to treat mesothelioma has just opened to patient accrual (35).

No potential conflicts of interest were disclosed.

Conception and design: J. Zhang, I. Pastan, R. Hassan

Development of methodology: Q. Jiang, M. Miettinen, I. Pastan, R. Hassan

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Zhang, S. Khanna, Q. Jiang, R. Hassan

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Zhang, S. Khanna, Q. Jiang, C. Alewine, M. Miettinen, I. Pastan, R. Hassan

Writing, review, and/or revision of the manuscript: J. Zhang, S. Khanna, Q. Jiang, C. Alewine, I. Pastan, R. Hassan

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Miettinen

Study supervision: R. Hassan

This research was supported by the Intramural Research Program of the NIH, NCI, Center for Cancer Research.

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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