Cancer Therapy: Preclinical

Anti ^ Mesothelin Immunotoxin SS1P in Combination with Gemcitabine Results in Increased Activity against Mesothelin-Expressing Tumor Xenografts Raffit Hassan,1 V. Cour tney Broaddus, 3 Shannon Wilson,3 David J. Liewehr,2 and Jingli Zhang1

Abstract 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 SS1Pin combination with gemcitabine against the mesothelin-expressingcell line A431/K5 was evaluated usingcytotoxicity and apoptosis assays. The antitumor activity of this combination was evaluated in nude mice bearingA431/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-expressingA431/K5 cells. In contrast, in the in vivo setting, there was a marked synergy when SS1Pwas 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 SS1Pbecause HA22, an immunotoxin targeting CD22 not expressed on A431/K5 cells, did not increase the efficacy of gemcitabine. Conclusions: SS1Pin combination with gemcitabine results in marked antitumor activity against mesothelin-expressingtumors. 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 cells of the trachea and cells in the fallopian tubes (2). surface glycoprotein whose expression in normal human tissues Mesothelin is highly expressed in several human tumors is limited to mesothelial cells lining the pleura, pericardium, including virtually all epithelial mesotheliomas and pancreatic and peritoneum (1, 2). The reactivity of K1, an anti– cancers, 70% of ovarian cancers, and 50% of lung adenocarci- mesothelin monoclonal antibody obtained by the immuniza- nomas (3–8). The biological function of mesothelin is not tion of mice with the human ovarian carcinoma cell line known but recent studies suggest that it may play a role in the OVCAR-3, was tested by immunohistochemistry using normal intraperitoneal spread of ovarian cancer (9, 10). A small human cryostat tissue sections. Most normal tissues showed no amount of the membrane-bound mesothelin is shed into the reactivity with the monoclonal antibody K1, with the exception serum and is useful for the diagnosis of ovarian cancer and of mesothelial cells that lined the peritoneal, pleural, and mesothelioma (11, 12). The limited expression of mesothelin pericardial cavities. There was also weak reactivity with basal 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 Authors’ Affiliations: 1Laboratory of Molecular Biology, and 2Biostatistics and Fv, obtained by phage display from splenic mRNA of mice Data Management Section, Center for Cancer Research, National Cancer Institute, immunized with mesothelin linked to a truncated Pseudomonas 3 and the Clinical Center, NIH, Bethesda, Maryland, and LungBiologyCenter, San exotoxin A that mediates cell killing (13). Preclinical studies Francisco General Hospital, University of California, San Francisco, California Received 6/28/07; revised 8/30/07; accepted 9/6/07. have shown that SS1P is cytotoxic to cell lines expressing Grant support: Intramural Research Program of the NIH, National Cancer Institute, mesothelin and causes complete regression of mesothelin- Center for Cancer Research; Cooperative Research and Development Agreements expressing tumor xenografts in nude mice (3). A phase I clinical between the National Cancer Institute and Cambridge Antibody Technology. trial of SS1P given as a bolus i.v. infusion has just been NIH RO1 CA95671 and the Mesothelioma Applied Research Foundation completed and antitumor activity was seen in a group of (V.C. Broaddus and S.Wilson). The costs of publication of this article were defrayed in part by the payment of page heavily pretreated patients (14). charges. This article must therefore be hereby marked advertisement in accordance Clinical trials of monoclonal antibodies have shown that the with 18 U.S.C. Section 1734 solely to indicate this fact. efficacy of these antibodies is markedly increased when they are Requests for reprints: Raffit Hassan, Laboratory of Molecular Biology, National administered in combination with chemotherapy. Two notable Cancer Institute, 37 Convent Drive, Room 5116, Bethesda, MD 20892-4264. Phone: 301-451-8742; Fax: 301-402-1344; E-mail: [email protected]. examples include trastuzumab and rituximab administered in F 2007 American Association for Cancer Research. combination with chemotherapy for the treatment of breast doi:10.1158/1078-0432.CCR-07-1592 cancer and non–Hodgkin’s lymphoma, respectively (15, 16).

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Because SS1P as a single agent had activity in phase I clinical institutional guidelines of the NIH. Mesothelin-expressing A431/K5 trials, we conducted preclinical studies to determine if its cells were cultured, harvested, and inoculated s.c. into the right flank of the mouse as described previously (25). Tumor dimensions were activity could be increased in combination with chemotherapy. 3 We evaluated gemcitabine in combination with SS1P because determined using calipers and the tumor volume (mm ) was calculated by the formula: length  (width)2  0.4. Treatment was initiated when gemcitabine has significant clinical activity in human cancers tumors reached f100 mm3 in size. Gemcitabine was reconstituted in that express mesothelin, such as pancreatic, ovarian and lung 0.9% sodium chloride and was given as an i.p. injection. SS1P or HA22 cancer, and mesothelioma (17–20). Gemcitabine, a nucleoside was diluted in PBS containing 0.2% human serum albumin and given analogue antimetabolite, is a prodrug that undergoes phos- i.v. Mice were sacrificed when the tumors reached f1,000 mm3 phorylation to yield gemcitabine diphosphate and gemcitabine according to institutional guidelines. triphosphate. Gemcitabine diphosphate inhibits ribonucleotide Statistical analyses. For the in vitro experiments, we did a mixed- reductase that decreases the cellular pool of dCTP and competes model two-way factorial ANOVA on the data. Preliminary analyses with gemcitabine triphosphate for incorporation into DNA indicated that an arc sin square root transformation of the data was in vivo (21). The incorporation of gemcitabine triphosphate into DNA necessary to make the residuals homogeneous. Regarding the inhibits its replication leading to apoptosis (22). SS1P is a studies, we did a one-way ANOVA for the first day of each experiment because it represented baseline measurements. For the post-baseline recombinant immunotoxin that, after binding to mesothelin- data, for each separate factorial treatment structure, separately by day, expressing cells, is internalized, undergoes processing, and we did an ANOVA on the data only on days with complete data inhibits the activity of EF2 leading to the arrest of protein (because mice in the control or single treatment groups were sacrificed synthesis and cell death (23). Our results show the markedly due to increasing tumor size). Residuals were examined for homoge- increased antitumor activity of the combination of gemcitabine neity and normality. Results are presented based on raw data, but and SS1P against mesothelin-expressing tumor xenografts in preliminary analyses indicated that a cubed-root transformation of the nude mice, but this combinatorial effect was not seen in cell data was necessary to normalize the residuals. cultures. Results

Materials and Methods Effect of SS1P andgemcitabine against A431/K5 cells in vitro. The possible interactions between SS1P and gemcitabine Chemicals. Gemcitabine was provided by the Division of Veterinary in vitro against A431/K5 cells were investigated using a cyto- Resources (NIH). Immunotoxins SS1P and HA22 were prepared in the toxicity assay. To do this, we first determined the IC50 for SS1P Laboratory of Molecular Biology as previously described (13, 24). The and gemcitabine on A431/K5 cells. For SS1P, the IC was Cell Counting Kit-8 used for the cytotoxicity assay was purchased from 50 0.4 ng/mL and, for gemcitabine, it was 8 ng/mL. Next, we Dojindo. Assays for apoptosis were done using 4¶,6-diamidino-2- phenylindole (Vector Laboratories). treated cells with various concentrations of SS1P and gemcita- Cell culture. A431/K5 cells expressing the mesothelin protein bine using concentrations near the IC50 of each. As shown in were maintained in DMEM containing 10% fetal bovine serum and Fig. 1, treatment of A431/K5 cells with gemcitabine plus SS1P 750 Ag/mL of G418 as previously described (25). did not significantly alter the IC50 of SS1P, suggesting a lack of In vitro cytotoxicity assays of SS1P plus gemcitabine against A431/K5 synergistic effect when the two agents were combined. For cells. A431/K5 cells were seeded in a 96-well plate at 5,000 cells per example, the IC50 of SS1P in the absence of gemcitabine was well and incubated at 37jC for 18 h. Gemcitabine was added to a final 0.4 ng/mL, and in the presence of 8 ng/mL of gemcitabine, it concentration of 0, 0.5, 1, 2, 4, and 8 ng/mL. Serial dilutions of SS1P was 0.25 ng/mL. in 0.2% human serum albumin were added 6 h after gemcitabine and The potential interaction between SS1P and gemcitabine cells were incubated at 37jC for another 48 h. The inhibition of cell in vitro was also evaluated by determining the apoptosis of growth was analyzed using the WST assay, which is based on the reduction of tetrazolium salt to formazan by the mitochondrial A431/K5 cells when treated with various concentrations of SS1P A dehydrogenases from viable cells. Ten microliters of WST-8 solution (0-1 ng/mL) and gemcitabine (0-40 g/mL) in combination from the Cell Counting Kit-8 were added to each well and incubated for (Fig. 2). No combinatorial synergistic interaction was evident. 1 h at 37jC. Absorbance was measured at 450 nm with a reference For example, SS1P alone at the highest concentration (1 ng/mL) 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 Fig. 1. Cultured A431/K5 cells were treated with various concentrations of SS1P protocol was approved and the mice were maintained as per and gemcitabine for 2 d and cell viability was measured using WST-8 assay.

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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. To determine if this antitumor effect of gemcitabine in combination with SS1P was specifically due to SS1P targeting Fig. 2. Treatment of A431/K5 cells in vitro with gemcitabine (Gem)plusSS1P mesothelin, we also treated mice bearing A431/K5 tumors with induces an additive effect on the apoptosis of A431/K5 cells. SS1Pand gemcitabine HA22 alone or in combination with gemcitabine. HA22 is a were used alone or together at various concentrations for 24 h and cells were recombinant immunotoxin that targets the antigen CD22, stained with 4¶,6-diamidino-2-phenylindole for analysis of nuclear morphology consistent with apoptosis. Although gemcitabine alone and SS1P alone induced which is not expressed on A431/K5 cells (24). As shown in apoptosis, the combination of the two agents did not induce more apoptosis than Fig. 3, HA22 had no effect on tumor growth (HA22’s main expected from the addition of the two effects (n = 3 experiments; counts by P z observer blinded to conditions). effect, 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 induced 15 F 7% apoptosis. When SS1P was added to (HA22  gemcitabine interaction effect, P z 0.17 on days 8-15 gemcitabine, it enhanced apoptosis by a similar amount post-inoculation). These results suggest that the synergistic (40 Ag/mL gemcitabine, 32 F 12% apoptosis; 1 ng/mL SS1P effect using the combination of gemcitabine plus SS1P was due plus 40 Ag/mL gemcitabine, 44 F 15% apoptosis), suggesting to the ability of SS1P to target mesothelin. an additive, but not a synergistic interaction. To determine the dose-response effect of gemcitabine, we Dose-dependent effect of gemcitabine in combination with SS1P also treated mice at a reduced dose of gemcitabine (25% and against mesothelin-expressing tumor xenografts. Athymic nude 50% of the dose given above). Figure 4 shows tumor volumes mice bearing mesothelin-expressing A431/K5 tumors were in mice treated with a single dose of gemcitabine, 60 or 120 treated with SS1P alone, with gemcitabine alone, or with both mg/kg with or without SS1P (0.2 mg/kg every other day  three agents, and the tumor growth was measured over several weeks. doses). Treatment was started on day 7 after tumor cell This experiment was done twice. In all experiments, mice were injection when the average tumor volume was f100 mm3. treated when the tumor size was f100 mm3, usually 5 to As shown in Fig. 4, by day 3 after the start of treatment, the 6 days after tumor cell implantation. The dose of SS1P used tumor volume in mice that received combination treatment (0.2 mg/kg every other day  three doses) was chosen because with gemcitabine plus SS1P was significantly reduced com- it prevents tumor growth during its period of administration pared with mice treated with gemcitabine alone, SS1P alone, but does not cause tumor regression which is noted when it is or control (120 mg/kg gemcitabine plus SS1P, P V 0.0001 for given at a higher dose. In our initial experiment, gemcitabine days 9-16 post-tumor inoculation; 60 mg/kg gemcitabine plus was administered on day 6 after tumor implantation as a single SS1P, P V 0.0005 on days 9-14 post-tumor inoculation). These

Fig. 3. The treatment of mice with A431/K5 tumors showed the enhanced antitumor effect of gemcitabine plus SS1Pcompared 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 Â 10 6 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.

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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.

results show that even a small dose of gemcitabine (60 mg/kg  mice in the combination group had tumor regression that one dose) in combination with SS1P could result in marked persisted for 15 days. Of these five mice, three (60%) continued tumor shrinkage. to have complete tumor remission persisting 80 days after the Fractionatedadministrationof gemcitabine in combination start of treatment, at which time they were sacrificed. These with SS1P results in sustainedcomplete tumor remission. Our results show that a fractionated dose of gemcitabine and SS1P initial experiments showed a marked reduction in tumor could result in complete regression, with no tumor regrowth in volume when a single dose of gemcitabine was administered most of the treated mice. with SS1P, compared with gemcitabine or SS1P alone. However, complete tumor shrinkage was infrequent. Because Discussion gemcitabine in patients is commonly used in a fractionated weekly schedule, we decided to test the activity of gemcitabine Although our in vitro studies did not show synergy between given in divided doses in combination with SS1P. This SS1P and gemcitabine, the efficacy of SS1P against mesothe- experiment was done twice and the results of a typical lin-expressing tumor xenografts was significantly increased experiment are shown in Fig. 5. Tumor-bearing mice received when it was administered in combination with the cytotoxic 80 mg/kg of gemcitabine i.p. on days 7, 9, and 11 after tumor agent gemcitabine. This positive interaction was seen even implantation followed 4 h after each dose by 0.2 mg/kg of with low single doses of gemcitabine. Moreover, mice that SS1P. In mice treated with gemcitabine or SS1P alone, there was received gemcitabine in the fractionated schedule with SS1P tumor stabilization during the treatment period followed by a had complete tumor regression that was long-lasting in the rapid increase in tumor size. However, in mice that received majority of mice. Combination treatment with gemcitabine combined treatment with gemcitabine and SS1P, there was a plus SS1P was well tolerated by the animals with no change in marked reduction in tumor volume with 60% complete tumor the mean weight of mice in the combination group compared remission by day 9 after the start of treatment. The tumor with mice treated with SS1P alone, gemcitabine alone, or volumes in the combination treatment were statistically lower control untreated mice. In addition, the doses of SS1P and than the other three treatments (P < 0.0001, on days 9-17 post- gemcitabine used in our animal experiments approximate the tumor inoculation). On day 13 after the start of treatment, all doses that can be safely given to humans. These calculations

Fig. 5. A fractionated dose of gemcitabine given with SS1P provides effective antitumor activity against A431/K5 tumors. Mice (n = 5) bearingA431/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 SS1Palone.

www.aacrjournals.org 7169 Clin Cancer Res 2007;13(23) December 1, 2007 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Cancer Therapy: Preclinical are based on the fact that the maximum tolerated doses in limited value in the treatment of tumors such as mesothelioma mice and humans are very similar when calculated in mg/m2 or pancreatic cancer because taxol has no activity against these (27). The conversion of dose in mg/kg to dose in mg/m2 was tumors (29, 30). Therefore, we chose to conduct studies of SS1P done according to the methods described in Food and Drug in combination with gemcitabine, a potent cytotoxic agent with Administration guidance for estimating the maximum safe broad-spectrum activity in mesothelioma, pancreatic, ovarian, starting dose in initial clinical trials for therapeutics in adult and lung cancer (17–20). healthy volunteers. As per these guidelines, to convert an The mechanism of synergy observed when combining animal or human dose from mg/kg to mg/m2, the dose in gemcitabine with SS1P is not known. In the in vitro setting, mg/kg is multiplied by the conversion factor indicated as km even though the two drugs work by different mechanisms, (for mass constant), which is 3 for mice and 37 for humans.4 they both ultimately lead to tumor cell killing by the  For SS1P, mice were given 0.2 mg/kg (0.2 mg/kg km 3=0.6 induction of apoptosis. However, it is not clear that the mg/m2), which is much lower than the maximum tolerated effectiveness of the combination of SS1P and gemcitabine  2 dose of 0.045 mg/kg (0.045 mg/kg km 37 = 1.6 mg/m ) in vivo is via a direct effect on the tumor cell apoptosis noted in the phase I clinical trial of SS1P (14). The gemcitabine because it could not be reproduced in vitro. This suggests dose administered to mice as a single dose (240 mg/kg) or in that other in vivo factors play a role in the enhanced three doses (80 mg/kg) is equal to 720 mg/m2 (240 mg/kg  antitumor activity from using the two drugs in combination. 3 km). This is less than the dose of gemcitabine used in Because gemcitabine can kill endothelial cells, it could result patients, which is commonly 1,000 mg/m2 given as a weekly in enhanced permeability with increased tumor uptake of infusion (17–20). the immunotoxin (31). However, it is unlikely that this Phase I studies of SS1P given as a single agent have been plays a major role in the increased antitumor activity of completed in patients with advanced malignancies that express the combination of gemcitabine and SS1P because treatment mesothelin. In these phase I studies, antitumor activity was of A431-K5 tumor xenografts with taxol, which is also observed in a group of heavily pretreated patients (14). Prior cytotoxic to tumor endothelial cells, did not increase the to the initiation of phase II studies of SS1P, we wanted to uptake of radiolabeled SS1P (28). It seems likely that the determine if the combination of SS1P with chemotherapy synergy between chemotherapy and an immunotoxin would result in a synergistic antitumor effect. Our prior study involves a novel mechanism and efforts to understand this are combining SS1P with taxol showed synergy against mesothelin- under way. expressing tumor xenografts (28). Although combination Our data suggest that the combination of SS1P plus treatment with taxol and SS1P could be potentially useful for gemcitabine could be potentially useful for the treatment of the treatment of women with ovarian cancer, it would have 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,

4 Food and DrugAdministration. Guidance for industry: estimatingthe maximum ovarian, and lung cancer, combining it with mesothelin- safe startingdose in initial clinical trials for therapeutics in adult healthy volunteers. targeted therapy could potentially result in increased activity Available at: http://www.fda.gov/cder/guidance/5541fnl.pdf. and clinical benefit.

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Raffit Hassan, V. Courtney Broaddus, Shannon Wilson, et al.

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