Purpose: To determine the antitumor activity of the anti–mesothelin immunotoxin SS1P in combination with gemcitabine against mesothelin-expressing tumor xenografts.

Experimental Design: The in vitro activity of SS1P in combination with gemcitabine against the mesothelin-expressing cell line A431/K5 was evaluated using cytotoxicity and apoptosis assays. The antitumor activity of this combination was evaluated in nude mice bearing A431/K5 tumor xenografts. Tumor-bearing mice were treated with different doses and schedules of gemcitabine alone, SS1P alone (0.2 mg/kg i.v. every other day × three doses), or with both agents together, and tumor volumes were measured over time.

Results:In vitro studies failed to show the synergy of SS1P plus gemcitabine against the mesothelin-expressing A431/K5 cells. In contrast, in the in vivo setting, there was a marked synergy when SS1P was combined with gemcitabine for the treatment of mesothelin-expressing tumor xenografts. This synergy was present using different doses and schedules of gemcitabine administration. In mice treated with fractionated doses of gemcitabine in combination with SS1P, complete tumor regression was observed in all mice and was long-lasting in 60% of the animals. Also, this antitumor activity was specific to SS1P because HA22, an immunotoxin targeting CD22 not expressed on A431/K5 cells, did not increase the efficacy of gemcitabine.

Conclusions: SS1P in combination with gemcitabine results in marked antitumor activity against mesothelin-expressing tumors. This combination could be potentially useful for the treatment of human cancers that express mesothelin and are responsive to gemcitabine therapy.

Mesothelin is a glycosyl-phosphatidylinositol–linked cell surface glycoprotein whose expression in normal human tissues is limited to mesothelial cells lining the pleura, pericardium, and peritoneum (1, 2). The reactivity of K1, an anti–mesothelin monoclonal antibody obtained by the immunization of mice with the human ovarian carcinoma cell line OVCAR-3, was tested by immunohistochemistry using normal human cryostat tissue sections. Most normal tissues showed no reactivity with the monoclonal antibody K1, with the exception of mesothelial cells that lined the peritoneal, pleural, and pericardial cavities. There was also weak reactivity with basal cells of the trachea and cells in the fallopian tubes (2). Mesothelin is highly expressed in several human tumors including virtually all epithelial mesotheliomas and pancreatic cancers, 70% of ovarian cancers, and 50% of lung adenocarcinomas (38). The biological function of mesothelin is not known but recent studies suggest that it may play a role in the intraperitoneal spread of ovarian cancer (9, 10). A small amount of the membrane-bound mesothelin is shed into the serum and is useful for the diagnosis of ovarian cancer and mesothelioma (11, 12). The limited expression of mesothelin on normal tissues and its high expression in several cancers makes it a good target for antibody-based therapy. SS1P is a recombinant immunotoxin consisting on an anti–mesothelin Fv, obtained by phage display from splenic mRNA of mice immunized with mesothelin linked to a truncated Pseudomonas exotoxin A that mediates cell killing (13). Preclinical studies have shown that SS1P is cytotoxic to cell lines expressing mesothelin and causes complete regression of mesothelin-expressing tumor xenografts in nude mice (3). A phase I clinical trial of SS1P given as a bolus i.v. infusion has just been completed and antitumor activity was seen in a group of heavily pretreated patients (14).

Clinical trials of monoclonal antibodies have shown that the efficacy of these antibodies is markedly increased when they are administered in combination with chemotherapy. Two notable examples include trastuzumab and rituximab administered in combination with chemotherapy for the treatment of breast cancer and non–Hodgkin's lymphoma, respectively (15, 16). Because SS1P as a single agent had activity in phase I clinical trials, we conducted preclinical studies to determine if its activity could be increased in combination with chemotherapy. We evaluated gemcitabine in combination with SS1P because gemcitabine has significant clinical activity in human cancers that express mesothelin, such as pancreatic, ovarian and lung cancer, and mesothelioma (1720). Gemcitabine, a nucleoside analogue antimetabolite, is a prodrug that undergoes phosphorylation to yield gemcitabine diphosphate and gemcitabine triphosphate. Gemcitabine diphosphate inhibits ribonucleotide reductase that decreases the cellular pool of dCTP and competes with gemcitabine triphosphate for incorporation into DNA (21). The incorporation of gemcitabine triphosphate into DNA inhibits its replication leading to apoptosis (22). SS1P is a recombinant immunotoxin that, after binding to mesothelin-expressing cells, is internalized, undergoes processing, and inhibits the activity of EF2 leading to the arrest of protein synthesis and cell death (23). Our results show the markedly increased antitumor activity of the combination of gemcitabine and SS1P against mesothelin-expressing tumor xenografts in nude mice, but this combinatorial effect was not seen in cell cultures.

Chemicals. Gemcitabine was provided by the Division of Veterinary Resources (NIH). Immunotoxins SS1P and HA22 were prepared in the Laboratory of Molecular Biology as previously described (13, 24). The Cell Counting Kit-8 used for the cytotoxicity assay was purchased from Dojindo. Assays for apoptosis were done using 4′,6-diamidino-2-phenylindole (Vector Laboratories).

Cell culture. A431/K5 cells expressing the mesothelin protein were maintained in DMEM containing 10% fetal bovine serum and 750 μg/mL of G418 as previously described (25).

In vitro cytotoxicity assays of SS1P plus gemcitabine against A431/K5 cells. A431/K5 cells were seeded in a 96-well plate at 5,000 cells per well and incubated at 37°C for 18 h. Gemcitabine was added to a final concentration of 0, 0.5, 1, 2, 4, and 8 ng/mL. Serial dilutions of SS1P in 0.2% human serum albumin were added 6 h after gemcitabine and cells were incubated at 37°C for another 48 h. The inhibition of cell growth was analyzed using the WST assay, which is based on the reduction of tetrazolium salt to formazan by the mitochondrial dehydrogenases from viable cells. Ten microliters of WST-8 solution from the Cell Counting Kit-8 were added to each well and incubated for 1 h at 37°C. Absorbance was measured at 450 nm with a reference wavelength of 650 nm using a plate reader (SPECTRAmax, Molecular Devices). Viability was expressed as a percentage of the absorbance of untreated controls.

In vitro evaluation of apoptosis induced by SS1P plus gemcitabine in A431/K5 cells. A431/K5 cells were plated in six-well plates at 50,000 cells per well. The next morning, gemcitabine and SS1P were added in varying concentrations. At 24 h, floating cells and cells detached with trypsin/EDTA were combined, centrifuged at 800 rpm for 5 min, and stained with 4′,6-diamidino-2-phenylindole (Vector Laboratories). Apoptotic cells were counted by two observers using fluorescence microscopy in a blinded fashion based on the presence of condensed, highly fluorescent nuclei (26). The data are from three separate experiments.

In vivo experiments using SS1P in combination with gemcitabine against A431/K5 xenografts in nude mice. Four- to six-week-old female athymic nude mice were purchased from the National Cancer Institute-Frederick Animal Production Area (Frederick, MD) and housed in microisolator cages for the course of the experiment. The research protocol was approved and the mice were maintained as per institutional guidelines of the NIH. Mesothelin-expressing A431/K5 cells were cultured, harvested, and inoculated s.c. into the right flank of the mouse as described previously (25). Tumor dimensions were determined using calipers and the tumor volume (mm3) was calculated by the formula: length × (width)2 × 0.4. Treatment was initiated when tumors reached ∼100 mm3 in size. Gemcitabine was reconstituted in 0.9% sodium chloride and was given as an i.p. injection. SS1P or HA22 was diluted in PBS containing 0.2% human serum albumin and given i.v. Mice were sacrificed when the tumors reached ∼1,000 mm3 according to institutional guidelines.

Statistical analyses. For the in vitro experiments, we did a mixed-model two-way factorial ANOVA on the data. Preliminary analyses indicated that an arc sin square root transformation of the data was necessary to make the residuals homogeneous. Regarding the in vivo studies, we did a one-way ANOVA for the first day of each experiment because it represented baseline measurements. For the post-baseline data, for each separate factorial treatment structure, separately by day, we did an ANOVA on the data only on days with complete data (because mice in the control or single treatment groups were sacrificed due to increasing tumor size). Residuals were examined for homogeneity and normality. Results are presented based on raw data, but preliminary analyses indicated that a cubed-root transformation of the data was necessary to normalize the residuals.

Effect of SS1P and gemcitabine against A431/K5 cells in vitro. The possible interactions between SS1P and gemcitabine in vitro against A431/K5 cells were investigated using a cytotoxicity assay. To do this, we first determined the IC50 for SS1P and gemcitabine on A431/K5 cells. For SS1P, the IC50 was 0.4 ng/mL and, for gemcitabine, it was 8 ng/mL. Next, we treated cells with various concentrations of SS1P and gemcitabine using concentrations near the IC50 of each. As shown in Fig. 1, treatment of A431/K5 cells with gemcitabine plus SS1P did not significantly alter the IC50 of SS1P, suggesting a lack of synergistic effect when the two agents were combined. For example, the IC50 of SS1P in the absence of gemcitabine was 0.4 ng/mL, and in the presence of 8 ng/mL of gemcitabine, it was 0.25 ng/mL.

Fig. 1.

Cultured A431/K5 cells were treated with various concentrations of SS1P and gemcitabine for 2 d and cell viability was measured using WST-8 assay.

Fig. 1.

Cultured A431/K5 cells were treated with various concentrations of SS1P and gemcitabine for 2 d and cell viability was measured using WST-8 assay.

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The potential interaction between SS1P and gemcitabine in vitro was also evaluated by determining the apoptosis of A431/K5 cells when treated with various concentrations of SS1P (0-1 ng/mL) and gemcitabine (0-40 μg/mL) in combination (Fig. 2). No combinatorial synergistic interaction was evident. For example, SS1P alone at the highest concentration (1 ng/mL) induced 15 ± 7% apoptosis. When SS1P was added to gemcitabine, it enhanced apoptosis by a similar amount (40 μg/mL gemcitabine, 32 ± 12% apoptosis; 1 ng/mL SS1P plus 40 μg/mL gemcitabine, 44 ± 15% apoptosis), suggesting an additive, but not a synergistic interaction.

Fig. 2.

Treatment of A431/K5 cells in vitro with gemcitabine (Gem) plus SS1P induces an additive effect on the apoptosis of A431/K5 cells. SS1P and gemcitabine were used alone or together at various concentrations for 24 h and cells were stained with 4′,6-diamidino-2-phenylindole for analysis of nuclear morphology consistent with apoptosis. Although gemcitabine alone and SS1P alone induced apoptosis, the combination of the two agents did not induce more apoptosis than expected from the addition of the two effects (n = 3 experiments; counts by observer blinded to conditions).

Fig. 2.

Treatment of A431/K5 cells in vitro with gemcitabine (Gem) plus SS1P induces an additive effect on the apoptosis of A431/K5 cells. SS1P and gemcitabine were used alone or together at various concentrations for 24 h and cells were stained with 4′,6-diamidino-2-phenylindole for analysis of nuclear morphology consistent with apoptosis. Although gemcitabine alone and SS1P alone induced apoptosis, the combination of the two agents did not induce more apoptosis than expected from the addition of the two effects (n = 3 experiments; counts by observer blinded to conditions).

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Dose-dependent effect of gemcitabine in combination with SS1P against mesothelin-expressing tumor xenografts. Athymic nude mice bearing mesothelin-expressing A431/K5 tumors were treated with SS1P alone, with gemcitabine alone, or with both agents, and the tumor growth was measured over several weeks. This experiment was done twice. In all experiments, mice were treated when the tumor size was ∼100 mm3, usually 5 to 6 days after tumor cell implantation. The dose of SS1P used (0.2 mg/kg every other day × three doses) was chosen because it prevents tumor growth during its period of administration but does not cause tumor regression which is noted when it is given at a higher dose. In our initial experiment, gemcitabine was administered on day 6 after tumor implantation as a single dose of 240 mg/kg, followed 4 h later by the first SS1P injection. SS1P was also administered on days 3 and 5 after the start of the treatment. As shown in Fig. 3, mice treated with SS1P alone or gemcitabine alone showed the slowing of tumor growth for 3 to 4 days followed by a rapid increase in tumor size. In mice treated with the combination of SS1P and gemcitabine, there was a marked reduction in tumor volume by day 3 of treatment (day 8 post-tumor inoculation, P < 0.003), which was statistically significantly different when compared with mice treated with SS1P or gemcitabine alone. This statistically significant difference in tumor volume was present at other time points of the follow-up (days 10, 13, and 15 post-tumor inoculation, P < 0.0001). Out of the six mice treated with the combination, there was complete remission of the tumor in one mouse. No complete response was noted in mice treated with SS1P or gemcitabine alone.

Fig. 3.

The treatment of mice with A431/K5 tumors showed the enhanced antitumor effect of gemcitabine plus SS1P compared with each alone and a lack of activity of the control immunotoxin HA22 in combination with gemcitabine. Mice (n = 6) were implanted with 3.0 × 106 A431/K5 cells on day 0 in each treatment group. Gemcitabine at 240 mg/kg was given as a single i.p. injection on day 6 (arrowheads). SS1P or HA22 was given i.v. at 0.2 mg/kg on days 6, 8, and 10 (thin arrows). *, days on which the tumor volume in the gemcitabine plus SS1P–treated mice were statistically significantly reduced (P < 0.003) compared with other groups.

Fig. 3.

The treatment of mice with A431/K5 tumors showed the enhanced antitumor effect of gemcitabine plus SS1P compared with each alone and a lack of activity of the control immunotoxin HA22 in combination with gemcitabine. Mice (n = 6) were implanted with 3.0 × 106 A431/K5 cells on day 0 in each treatment group. Gemcitabine at 240 mg/kg was given as a single i.p. injection on day 6 (arrowheads). SS1P or HA22 was given i.v. at 0.2 mg/kg on days 6, 8, and 10 (thin arrows). *, days on which the tumor volume in the gemcitabine plus SS1P–treated mice were statistically significantly reduced (P < 0.003) compared with other groups.

Close modal

To determine if this antitumor effect of gemcitabine in combination with SS1P was specifically due to SS1P targeting mesothelin, we also treated mice bearing A431/K5 tumors with HA22 alone or in combination with gemcitabine. HA22 is a recombinant immunotoxin that targets the antigen CD22, which is not expressed on A431/K5 cells (24). As shown in Fig. 3, HA22 had no effect on tumor growth (HA22's main effect, P ≥ 0.10 on days 8-15 post-inoculation). Similarly, in mice treated with HA22 and gemcitabine, the tumor growth was similar to that in mice treated with gemcitabine alone (HA22 × gemcitabine interaction effect, P ≥ 0.17 on days 8-15 post-inoculation). These results suggest that the synergistic effect using the combination of gemcitabine plus SS1P was due to the ability of SS1P to target mesothelin.

To determine the dose-response effect of gemcitabine, we also treated mice at a reduced dose of gemcitabine (25% and 50% of the dose given above). Figure 4 shows tumor volumes in mice treated with a single dose of gemcitabine, 60 or 120 mg/kg with or without SS1P (0.2 mg/kg every other day × three doses). Treatment was started on day 7 after tumor cell injection when the average tumor volume was ∼100 mm3. As shown in Fig. 4, by day 3 after the start of treatment, the tumor volume in mice that received combination treatment with gemcitabine plus SS1P was significantly reduced compared with mice treated with gemcitabine alone, SS1P alone, or control (120 mg/kg gemcitabine plus SS1P, P ≤ 0.0001 for days 9-16 post-tumor inoculation; 60 mg/kg gemcitabine plus SS1P, P ≤ 0.0005 on days 9-14 post-tumor inoculation). These results show that even a small dose of gemcitabine (60 mg/kg × one dose) in combination with SS1P could result in marked tumor shrinkage.

Fig. 4.

Lower doses of gemcitabine given with SS1P also show an enhanced antitumor effect against A431/K5 tumors compared with each alone. Mice with tumors (n = 5) received a single dose of gemcitabine at 120 mg/kg (Gem 120) or 60 mg/kg (Gem 60) i.p. on day 7 (arrowhead) after tumor implantation either alone or in combination with SS1P. The SS1P (0.2 mg/kg) was administered i.v. on days 7, 9, and 11 (thin arrows). *, days on which the tumor volume in the gemcitabine plus SS1P combination groups was statistically significantly less (P < 0.0005, see text for details) than with each agent alone.

Fig. 4.

Lower doses of gemcitabine given with SS1P also show an enhanced antitumor effect against A431/K5 tumors compared with each alone. Mice with tumors (n = 5) received a single dose of gemcitabine at 120 mg/kg (Gem 120) or 60 mg/kg (Gem 60) i.p. on day 7 (arrowhead) after tumor implantation either alone or in combination with SS1P. The SS1P (0.2 mg/kg) was administered i.v. on days 7, 9, and 11 (thin arrows). *, days on which the tumor volume in the gemcitabine plus SS1P combination groups was statistically significantly less (P < 0.0005, see text for details) than with each agent alone.

Close modal

Fractionated administration of gemcitabine in combination with SS1P results in sustained complete tumor remission. Our initial experiments showed a marked reduction in tumor volume when a single dose of gemcitabine was administered with SS1P, compared with gemcitabine or SS1P alone. However, complete tumor shrinkage was infrequent. Because gemcitabine in patients is commonly used in a fractionated weekly schedule, we decided to test the activity of gemcitabine given in divided doses in combination with SS1P. This experiment was done twice and the results of a typical experiment are shown in Fig. 5. Tumor-bearing mice received 80 mg/kg of gemcitabine i.p. on days 7, 9, and 11 after tumor implantation followed 4 h after each dose by 0.2 mg/kg of SS1P. In mice treated with gemcitabine or SS1P alone, there was tumor stabilization during the treatment period followed by a rapid increase in tumor size. However, in mice that received combined treatment with gemcitabine and SS1P, there was a marked reduction in tumor volume with 60% complete tumor remission by day 9 after the start of treatment. The tumor volumes in the combination treatment were statistically lower than the other three treatments (P < 0.0001, on days 9-17 post-tumor inoculation). On day 13 after the start of treatment, all mice in the combination group had tumor regression that persisted for 15 days. Of these five mice, three (60%) continued to have complete tumor remission persisting 80 days after the start of treatment, at which time they were sacrificed. These results show that a fractionated dose of gemcitabine and SS1P could result in complete regression, with no tumor regrowth in most of the treated mice.

Fig. 5.

A fractionated dose of gemcitabine given with SS1P provides effective antitumor activity against A431/K5 tumors. Mice (n = 5) bearing A431/K5 tumors received gemcitabine at 80 mg/kg i.p. on days 7, 9, and 11 (arrowheads) after tumor implantation. SS1P (0.2 mg/kg) was administered i.v. 4 h after the administration of gemcitabine on days 7, 9, and 11 (thin arrows). *, days on which the tumor volume in the gemcitabine plus SS1P combination group was statistically significantly less (P < 0.0001) than with each agent alone. Also, the percentage of mice in the gemcitabine plus SS1P–treated group with a complete response (CR; tumor no longer measurable) at different time points after the initiation of treatment. There was no complete response in the mice treated with gemcitabine or SS1P alone.

Fig. 5.

A fractionated dose of gemcitabine given with SS1P provides effective antitumor activity against A431/K5 tumors. Mice (n = 5) bearing A431/K5 tumors received gemcitabine at 80 mg/kg i.p. on days 7, 9, and 11 (arrowheads) after tumor implantation. SS1P (0.2 mg/kg) was administered i.v. 4 h after the administration of gemcitabine on days 7, 9, and 11 (thin arrows). *, days on which the tumor volume in the gemcitabine plus SS1P combination group was statistically significantly less (P < 0.0001) than with each agent alone. Also, the percentage of mice in the gemcitabine plus SS1P–treated group with a complete response (CR; tumor no longer measurable) at different time points after the initiation of treatment. There was no complete response in the mice treated with gemcitabine or SS1P alone.

Close modal

Although our in vitro studies did not show synergy between SS1P and gemcitabine, the efficacy of SS1P against mesothelin-expressing tumor xenografts was significantly increased when it was administered in combination with the cytotoxic agent gemcitabine. This positive interaction was seen even with low single doses of gemcitabine. Moreover, mice that received gemcitabine in the fractionated schedule with SS1P had complete tumor regression that was long-lasting in the majority of mice. Combination treatment with gemcitabine plus SS1P was well tolerated by the animals with no change in the mean weight of mice in the combination group compared with mice treated with SS1P alone, gemcitabine alone, or control untreated mice. In addition, the doses of SS1P and gemcitabine used in our animal experiments approximate the doses that can be safely given to humans. These calculations are based on the fact that the maximum tolerated doses in mice and humans are very similar when calculated in mg/m2 (27). The conversion of dose in mg/kg to dose in mg/m2 was done according to the methods described in Food and Drug Administration guidance for estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers. As per these guidelines, to convert an animal or human dose from mg/kg to mg/m2, the dose in mg/kg is multiplied by the conversion factor indicated as km (for mass constant), which is 3 for mice and 37 for humans.4

4

Food and Drug Administration. Guidance for industry: estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers. Available at: http://www.fda.gov/cder/guidance/5541fnl.pdf.

For SS1P, mice were given 0.2 mg/kg (0.2 mg/kg × km 3 = 0.6 mg/m2), which is much lower than the maximum tolerated dose of 0.045 mg/kg (0.045 mg/kg × km 37 = 1.6 mg/m2) noted in the phase I clinical trial of SS1P (14). The gemcitabine dose administered to mice as a single dose (240 mg/kg) or in three doses (80 mg/kg) is equal to 720 mg/m2 (240 mg/kg × 3 km). This is less than the dose of gemcitabine used in patients, which is commonly 1,000 mg/m2 given as a weekly infusion (1720).

Phase I studies of SS1P given as a single agent have been completed in patients with advanced malignancies that express mesothelin. In these phase I studies, antitumor activity was observed in a group of heavily pretreated patients (14). Prior to the initiation of phase II studies of SS1P, we wanted to determine if the combination of SS1P with chemotherapy would result in a synergistic antitumor effect. Our prior study combining SS1P with taxol showed synergy against mesothelin-expressing tumor xenografts (28). Although combination treatment with taxol and SS1P could be potentially useful for the treatment of women with ovarian cancer, it would have limited value in the treatment of tumors such as mesothelioma or pancreatic cancer because taxol has no activity against these tumors (29, 30). Therefore, we chose to conduct studies of SS1P in combination with gemcitabine, a potent cytotoxic agent with broad-spectrum activity in mesothelioma, pancreatic, ovarian, and lung cancer (1720).

The mechanism of synergy observed when combining gemcitabine with SS1P is not known. In the in vitro setting, even though the two drugs work by different mechanisms, they both ultimately lead to tumor cell killing by the induction of apoptosis. However, it is not clear that the effectiveness of the combination of SS1P and gemcitabine in vivo is via a direct effect on the tumor cell apoptosis because it could not be reproduced in vitro. This suggests that other in vivo factors play a role in the enhanced antitumor activity from using the two drugs in combination. Because gemcitabine can kill endothelial cells, it could result in enhanced permeability with increased tumor uptake of the immunotoxin (31). However, it is unlikely that this plays a major role in the increased antitumor activity of the combination of gemcitabine and SS1P because treatment of A431-K5 tumor xenografts with taxol, which is also cytotoxic to tumor endothelial cells, did not increase the uptake of radiolabeled SS1P (28). It seems likely that the synergy between chemotherapy and an immunotoxin involves a novel mechanism and efforts to understand this are under way.

Our data suggest that the combination of SS1P plus gemcitabine could be potentially useful for the treatment of patients whose tumors express mesothelin. Given the activity of single-agent gemcitabine in the treatment of malignancies that express mesothelin such as mesothelioma, and pancreatic, ovarian, and lung cancer, combining it with mesothelin-targeted therapy could potentially result in increased activity and clinical benefit.

Grant support: Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research; Cooperative Research and Development Agreements between the National Cancer Institute and Cambridge Antibody Technology. NIH RO1 CA95671 and the Mesothelioma Applied Research Foundation (V.C. Broaddus and S. Wilson).

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.

1
Chang K, Pastan I. Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers.
Proc Natl Acad Sci U S A
1996
;
93
:
136
–40.
2
Chang K, Pastan I, Willingham MC. Isolation and characterization of a monoclonal antibody, K1, reactive with ovarian cancers and normal mesothelium.
Int J Cancer
1992
;
50
:
373
–81.
3
Hassan R, Bera T, Pastan I. Mesothelin: a new target for immunotherapy.
Clin Cancer Res
2004
;
10
:
3937
–42.
4
Ordonez NG. Value of mesothelin immunostaining in the diagnosis of mesothelioma.
Mod Pathol
2003
;
16
:
192
–7.
5
Hassan R, Kreitman RJ, Pastan I, et al. Localization of mesothelin in epithelial ovarian cancer.
Appl Immunohistochem Mol Morphol
2005
;
13
:
243
–7.
6
Argani P, Iacobuzio-Donahue C, Ryu B, et al. Mesothelin is overexpressed in the vast majority of ductal adenocarcinomas of the pancreas: identification of a new pancreatic cancer marker by serial analysis of gene expression (SAGE).
Clin Cancer Res
2001
;
7
:
3862
–8.
7
Hassan R, Laszik ZG, Lerner M, et al. Mesothelin is overexpressed in pancreaticobiliary adenocarcinomas but not in normal pancreas and chronic pancreatitis.
Am J Clin Pathol
2005
;
124
:
838
–45.
8
Ho M, Bera TK, Willingham MC, et al. Mesothelin expression in human lung cancer.
Clin Cancer Res
2006
;
13
:
1571
–5.
9
Rump A, Morikawa Y, Tanaka M, et al. Binding of ovarian cancer antigen CA125/MUC16 to mesothelin mediates cell adhesion.
J Biol Chem
2004
;
279
:
9190
–8.
10
Gubbels JAA, Belisle J, Onda M, et al. Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors.
Mol Cancer
2006
;
5
:
50
–65.
11
Robinson BWS, Creaney J, Lake R, et al. Mesothelin-family proteins and diagnosis of mesothelioma.
Lancet
2003
;
362
:
1612
–6.
12
Hassan R, Remaley AT, Sampson ML, et al. Detection and quantitation of serum mesothelin, a tumor marker for patients with mesothelioma and ovarian cancer.
Clin Cancer Res
2006
;
12
:
447
–53.
13
Chowdhury PS, Viner JL, Beers R, et al. Isolation of a high affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity.
Proc Natl Acad Sci U S A
1998
;
95
:
669
–74.
14
Hassan R, Bullock S, Premkumar A, et al. Phase I study of SS1P, a recombinant anti-mesothelin immunotoxin given as a bolus I.V. infusion to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers.
Clin Cancer Res
2007
;
13
:
5144
–9.
15
Slamon DJ, Leyland-Jones B, Shak S, et al. Use of themotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2.
N Engl J Med
2001
;
344
:
783
–92.
16
Coiffier B, Lepage E, Briere J, et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma.
N Engl J Med
2002
;
346
:
235
–42.
17
Kindler HL, van Meerbeeck JP. The role of gemcitabine in the treatment of malignant mesothelioma.
Semin Oncol
2002
;
29
:
70
–6.
18
Burris HA III, Moore MJ, Andersen J, et al. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial.
J Clin Oncol
1997
;
15
:
2403
–13.
19
Pfisterer J, Plante M, Vergote I, et al. Gemcitabine plus carboplatin compared with carboplatin in patients with platinum-sensitive recurrent ovarian cancer: an intergroup trial of the AGO-OVAR, the NCIC CTG, and the EORTC GCG.
J Clin Oncol
2006
;
24
:
4699
–707.
20
Sandler AB, Nemunaitis J, Denham C, et al. Phase III trial of gemcitabine plus cisplatin versus cisplatin alone in patients with locally advanced or metastatic non-small-cell lung cancer.
J Clin Oncol
2000
;
18
:
122
–30.
21
Huang P, Chubb S, Hertel LW, Grindey GB, Plunkett W. Action of 2′,2′-difluorodeodeoxycytidine on DNA synthesis.
Cancer Res
1991
;
51
:
6110
–7.
22
Huang P, Plunkett W. Fludarabine and gemcitabine-induced apoptosis: incorporation of analogs into DNA is a critical event.
Cancer Chemother Pharmacol
1995
;
36
:
181
–8.
23
Pastan I, Hassan R, FitzGerald DJ, et al. Immunotoxin therapy of cancer.
Nat Rev Cancer
2006
;
6
:
559
–65.
24
Salvatore G, Beers R, Margulies I, Kreitman RJ, Pastan I. Improved cytotoxic activity toward cell lines and fresh leukemia cells of a mutant anti-CD22 immunotoxin obtained by antibody phage display.
Clin Cancer Res
2002
;
8
:
995
–1002.
25
Hassan R, Viner JL, Wang QC, Margulies I, Kreitman RJ, Pastan I. Anti-tumor activity of K1–38QQR, an immunotoxin targeting mesothelin, a cell-surface antigen overexpressed in ovarian cancer and malignant mesothelioma.
J Immunother
2000
;
23
:
473
–9.
26
Broaddus VC, Yang L, Scavo LM, Ernest JD, Boylan AM. Asbestos induces apoptosis of human and rabbit pleural mesothelial cells via reactive oxygen species.
J Clin Invest
1996
;
98
:
2050
–9.
27
Freireich EJ, Gehan EA, Rall DP, Schmidt LH, Skipper HE. Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man.
Cancer Chemother Rep
1966
;
50
:
219
–44.
28
Zhang Y, Xiang L, Hassan R, et al. Synergistic antitumor activity of Taxol and immunotoxin SS1P in tumor-bearing mice.
Clin Cancer Res
2006
;
12
:
4695
–701.
29
Van Meerbeeck J, Debruyne C, van Zandwijk, et al. Paclitaxel for malignant pleural mesothelioma: a phase II study of the EORTC Lung Cancer Cooperative Group.
Br J Cancer
1996
;
74
:
961
–3.
30
Whitehead RP, Jacobson J, Brown TD, Taylor SA, Weiss GR, Macdonald JS. Phase II trial of paclitaxel and granulocyte colony-stimulating factor in patients with pancreatic carcinoma: a Southwest Oncology Group study.
J Clin Oncol
1997
;
15
:
2414
–9.
31
Bocci G, Danesi R, Marangonii G, et al. Antiangiogenic versus cytotoxic therapeutic approaches to human pancreas cancer: an experimental study with a vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor and gemcitabine.
Eur J Pharmacol
2004
;
498
:
9
–18.