Immunotoxin and Taxol synergy results from a decrease in shed mesothelin levels in the extracellular space of tumors

Abstract

Recombinant immunotoxins are chimeric proteins composed of the Fv portion of a tumor-specific antibody fused to a toxin. SS1P (CAT-5001) is an immunotoxin composed of an antimesothelin Fv fused to a 38-kDa portion of Pseudomonas exotoxin A. Immunotoxins have been shown to be active in lymphomas and leukemias, but are much less active against solid tumors. We recently reported that Taxol and other chemotherapeutic agents show striking synergistic antitumor activity in mice when immunotoxin SS1P, which targets the mesothelin antigen on solid tumors, is given with Taxol. Using a pair of Taxol-sensitive and Taxol-resistant KB tumors equally sensitive to immunotoxin SS1P, we examined the mechanism of synergy. We show that synergy is only observed with Taxol-sensitive tumors, ruling out an effect of Taxol on endothelial cells. We also show that the KB tumors have high levels of shed mesothelin in their extracellular space; these levels increase with tumor size and, after Taxol treatment, dramatically fall in the drug-sensitive but not the drug-resistant tumors. Because the mesothelin levels in the tumor exceed the levels of SS1P in the tumor, and because shed mesothelin is being continuously released into the circulation at a high rate, we propose that synergy is due to the Taxol-induced fall in shed antigen levels.

Antibody-based therapies now play an important role in cancer treatment. These therapies include humanized or fully human unmodified antibodies, as well as antibodies used to deliver toxic payloads such as radioisotopes, toxins, and low-molecular-weight cytotoxic drugs (1, 2). One well recognized impediment to effective antibody-based therapy is tumor penetration. Several factors contribute to this poor penetration, including a defective vasculature characterized by abnormal blood vessels, a lack of functional lymphatics so that proteins can only enter slowly by diffusion, and elevated interstitial fluid pressure generating an outward flow of fluid. There also is a site barrier limiting the entry of antibodies and other antibody-based therapeutics caused by the binding of antibodies to the first layer of cells they encounter as they leave capillaries within the tumors (3, 4). Another factor is a collagen-rich and stiff extracellular matrix (5). Some of these barriers also can affect the entry of standard low-molecular-weight chemotherapeutic agents (6). One way to improve the uptake of antibodies by tumors is to administer large amounts, achieving high blood levels. However, this strategy cannot be used with immunoconjugates and immunotoxins because the cytotoxic payload produces side effects not observed with naked antibodies (7, 8). To improve the efficacy of antibody-based therapies, they have been combined with chemotherapy, and striking increases in antitumor activity have been observed (9–12).

Recombinant immunotoxins are genetically engineered proteins composed of the Fv portion of an antibody fused to a bacterial or plant toxin. They kill cells by binding to a cell-surface receptor, which carries them into the cell interior, where they arrest protein synthesis and induce apoptosis (13). Although smaller than antibodies, the immunotoxins share properties that limit their entry into tumors. Ontak (denileukin diftitox) is a fusion of IL-2 with a diphtheria toxin fragment; it is approved by the Food and Drug Administation for the treatment of cutaneous T cell lymphoma and has shown activity on other T cell malignancies (14). BL22 (CAT-3888) contains the Fv portion of an anti-CD22 antibody fused to a 38-kDa fragment of Pseudomonas exotoxin A. It has shown a high complete response rate in drug-resistant hairy cell leukemia (15), but is much less effective in other B cell malignancies (16). In contrast to results in hairy cell leukemia, the treatment of solid epithelial tumors with immunotoxins has been less successful, although minor tumor regressions have been observed in some trials with immunotoxins containing PE38 (17, 18).

To improve the efficacy of immunotoxins on solid tumors, we combined immunotoxin therapy with chemotherapy. Using a mouse xenograft model in which A431/K5 cells expressing mesothelin were grown as solid tumors, we showed that the combination of an immunotoxin and a chemotherapeutic drug caused a synergistic antitumor response (19). In those studies, we used the SS1P immunotoxin (CAT-5001) that targets the mesothelin protein, which is present on mesotheliomas as well as on ovarian, pancreatic, and lung cancers. Mesothelin is a 42-kDa cell-surface protein that is present in large amounts on tumor cells and also is shed in small amounts into the blood (20). SS1P was tested in combination with paclitaxel (Taxol), cisplatin (cis-diamminedichloroplatinum), and cyclophosphamide (Cytoxan), and significant synergistic effects were seen with all these combinations. To investigate the mechanism of synergy, we measured immunotoxin uptake, but failed to find that immunotoxin uptake was increased by Taxol (19).

To identify the cells in the tumor responsible or required for synergy, we focused on the tumor cells' role in the synergy process. We used two closely related cell lines, KB-3-1 and KB-8-5, which differ in their response to Taxol (and several other hydrophobic cytotoxic agents) because of overexpression of the MDR1 gene (21, 22). The cell lines express mesothelin and are equally sensitive to the immunotoxin. We show here that synergy is only observed with drug-sensitive tumors. We further show that, within the extracellular space of tumors, the concentration of shed mesothelin is high and falls dramatically after Taxol treatment of drug-sensitive but not drug-resistant tumors. On the basis of these findings, we propose that a decrease in shed antigen contributes to the synergy observed. Because many of the targets for immunotherapy are shed into the circulation, we also propose that high-antigen concentrations within tumors may diminish the efficacy of other antibody-based therapies.

Results

We previously showed that Taxol and SS1P (CAT-5001) have synergistic effects on A431/K5 tumors (19). To gain information about the mechanism of synergy, we chose to investigate a pair of human KB (HeLa) cell lines that: (i) are derived from a human cervical cancer that expresses mesothelin constitutively, (ii) are equally sensitive to killing by immunotoxin SS1P, and (iii) differ in their sensitivity to Taxol. As shown in Table 1, KB-3-1 cells are sensitive to Taxol, whereas KB-8-5 cells, which overexpress the MDR1 gene, are 10-fold resistant (21, 22). To examine the effect of Taxol and SS1P on these tumors, we implanted the two cell lines in nude mice. Seven days later, when the tumors reached ≈120 mm³ in volume, the animals were treated with one 20 mg/kg dose of Taxol alone, three 0.3 mg/kg doses of immunotoxin SS1P every other day, or both Taxol and SS1P. When both agents were used, Taxol was given 1 day before the first dose of immunotoxin as in previous experiments (19).

Table 1.

IC₅₀ (nanograms per milliliter) of cytotoxic agents

Cell line	Taxol	SS1P	ATFP
A431/K5	4	0.4	0.3
KB-3–1	3	5	0.4
KB-8–5	30	3	0.4

The calculation of IC₅₀ is based on the in vitro cytotoxicity assays of these drugs on several cell lines with the WST-8 assay.

The data in Fig. 1 show that a 0.3 mg/kg dose of SS1P significantly slows the growth of KB-3-1 tumors, compared with the control group (P < 0.05), on day 12. Taxol also significantly retarded growth (P < 0.001) on day 12. When the tumors were treated with both SS1P and Taxol, tumor shrinkage was significantly greater than with either agent alone (P < 0.001). Furthermore, when analyzed for synergy, as described in Materials and Methods, a strong synergistic interaction was demonstrated (P < 0.0001) on days 9, 11, and 14. These data demonstrate that the synergy between SS1P and Taxol is not limited to A431/K5 tumors.

Fig. 1.

Combined treatment of Taxol and SS1P on KB-3-1 and KB-8-5 tumors. Mice in the Taxol group were injected i.p. with a single dose of 20 mg/kg Taxol on day 0. The SS1P group received 0.3 mg/kg SS1P on days 1, 3, and 5. For the combination group, mice were given Taxol at 20 mg/kg i.p. on day 0, followed by three 0.3 mg/kg injections of SS1P i.v. on days 1, 3, and 5. The control group received three injections of saline i.v. on days 1, 3, and 5. Each group contained 13 mice for KB-3-1 tumors and 10 for KB-8-5 tumors. For KB-3-1 tumors, the control group had a volume of 899 ± 235 (SD) mm³ on day 12; Taxol, 490 ± 124 mm³; SS1P, 706 ± 189 mm³; and combination, 115 ± 84 mm³. For KB-8-5 tumors, the tumor volume of the control group on day 12 was 978 ± 190 mm³; Taxol, 844 ± 102 mm³; SS1P, 830 ± 170 mm³; and combination, 737 ± 157 mm³.

We next evaluated the effect of Taxol and SS1P on drug-resistant KB-8-5 tumors. SS1P had a small, but significant, growth-inhibitory effect comparable to that on KB-3-1 tumors (P < 0.05). However Taxol had a much smaller growth-inhibitory effect than it did with KB-3-1 tumors, and on day 12 the effect was barely statistically significant (P = 0.04). When Taxol and SS1P were combined, the antitumor effect was additive, but not synergistic (P = 0.79). These data show that the tumor cells must be quite sensitive to killing by Taxol for synergy with SS1P to be observed. Because synergy was not observed with the Taxol-resistant tumors, the data indicate that Taxol is not acting on endothelial cells or other cell types in the tumor matrix to cause synergistic tumor regressions.

Shed Mesothelin in Tumors.

Mesothelin, the target of SS1P, is a GPI-anchored membrane protein that is shed from the surface of cancer cells (20) and is present at low concentrations in the blood of patients with tumors expressing mesothelin (23–25). We hypothesized that the concentrations of mesothelin in the extracellular space of tumors must be much higher than concentrations in the blood and perhaps high enough to significantly block the action of SS1P. We also hypothesized that killing tumor cells by Taxol treatment might lower mesothelin levels in the tumor, increasing SS1P activity.

To examine these hypotheses, we used an ELISA kit to measure mesothelin levels in the extracellular fluid (ECF) from KB-3-1 tumors. In the initial experiment, we collected tumors of different sizes, prepared the ECF, and measured the mesothelin levels as described in Materials and Methods. The fluid was collected by using the nylon mesh basket method (26, 27). As shown in Fig. 2A, mesothelin is present in the ECF of KB-3-1 tumors, and the concentration of mesothelin increases dramatically to high concentrations as the tumors get larger. In small tumors (≈120 mm³), the mesothelin concentration in the ECF is ≈26 nM. In larger tumors (600 mm³), the ECF of mesothelin levels are ≈60 nM. The correlation between the ECF and tumor weight is significant, with a coefficient of 0.799 (P < 0.01).

Fig. 2.

Presence of shed mesothelin in tumor ECF. Mice with KB-3-1 tumors were killed on different days, and tumor ECF and serum were collected. (A) Mesothelin levels in the ECF. (B) Mesothelin levels in serum. (C) Correlation of mesothelin level in the ECF and serum. x axis, mesothelin level in the ECF; y axis, mesothelin level in serum. (D) Western blot analysis of shed mesothelin in the ECF. Lane 1, 2 μl of KB-3-1 ECF with a shed mesothelin level of 5 μg/ml (determined by Mesomark assay); lane 2, 2 μl of A431/CAPC ECF (negative control); lane 3, 10 ng of meso-Fc (positive control) with 2 μg of BSA; lane 4, 2 μl of normal mouse serum (negative control).

We also measured mesothelin concentrations in the blood of the tumor-bearing mice and found that mesothelin was present in animals with small tumors and that blood levels rose with tumor size (Fig. 2B). Animals with 120-mm³ tumors had serum levels of ≈0.7 nM that rose to 8–10 nM in mice with large tumors. There was a good correlation of blood levels with tumor volume (coefficient of 0.893; P < 0.01). There also was a significant correlation of mesothelin in the serum with mesothelin concentrations in tumor ECF (coefficient of 0.905; P < 0.01) (Fig. 2C).

To determine the characteristics of mesothelin in the ECF, we performed a Western blot by using biotinylated SS1P to detect mesothelin. As shown in Fig. 2D, a distinct band with an apparent molecular weight of 42 kDa was detected in the ECF of KB-3-1 tumors, which indicates that the mature, processed form of mesothelin is present and not the high-molecular-weight precursor of ≈92 kDa (28). The ECF from A431 tumors, which do not express mesothelin, did not show a band indicating specificity. As expected, the biotinylated SS1P reacted with a 150-kDa mesothelin-rabbit Fc fusion protein.

Free mesothelin is able to block the killing of mesothelin-expressing cells by SS1P. The data in Fig. 3 show that the ECF from KB-3-1 tumors and serum from mice with KB-3-1 tumors blocked the cytotoxic effects of SS1P. In these experiments, we used a 1:100 dilution of the ECF and a 1:50 dilution of serum to prevent nonspecific effects of tissue or serum factors on the assay. The addition of a mesothelin-Fc standard protein shifted the IC₅₀ from 5 ng/ml to 12 ng/ml with 20 ng/ml mesothelin (0.27 nM), to 50 ng/ml with 200 ng/ml (2.7 nM), and to 250 ng/ml with 1,000 ng/ml (13.5 nM). Using this standard curve, we calculated that the diluted ECF sample used in the blocking experiment contains 9 ng/ml (0.29 nM), and the diluted serum contains 11 ng/ml (0.35 nM). These values are close to the 6 ng/ml levels measured directly by ELISA.

Fig. 3.

Shed mesothelin in the ECF and serum blocks the cytotoxic effect of SS1P on KB-3-1 cells. Blocking experiments were conducted by determining the cytotoxicity of SS1P in the presence of serum mesothelin or ECF mesothelin. Mesothelin-Fc fusion protein served as a positive control. (A) Blocking by mesothelin-Fc fusion protein. Cytotoxicity of SS1P was done in the presence of various concentrations of mesothelin-Fc (0–1,000 ng/ml). (B) Blocking by serum mesothelin. Serum from normal mice with same dilution was used as a negative control. (C) Blocking by ECF mesothelin. The ECF from CAPC-transfected A431 tumor xenograft is used as a negative control.

Effect of Taxol Treatment on Mesothelin Levels in Serum and the ECF.

Having verified that the ECF of tumors contains authentic mesothelin, we determined the effect of Taxol treatment on mesothelin levels in tumors. When KB-3-1 tumors reached 120 mm³ in volume (day 0), Taxol was administrated at 20 mg/kg as in the combination study. The control group received i.p. saline injections. Tumors and blood were collected on days 0, 1, 3, and 5, which corresponded to the days SS1P was given in the treatment regimen. As shown in Fig. 4A, Taxol treatment arrested tumor growth for at least 5 days, whereas tumor volume in the control group increased from 120 mm³ on day 0 to 400 mm³ on day 5. The data in Fig. 4 also show that mesothelin levels in the tumor and blood of the control group rose over 5 days, but in the Taxol group there was a sustained decrease in mesothelin levels in the tumor and serum. On day 5, there was an ≈10-fold difference in mesothelin levels in the tumors and serum comparing Taxol-treated and control groups (P < 0.001).

Fig. 4.

The effect of Taxol treatment on mesothelin level in the ECF and serum of KB-3-1 and KB-8-5 tumors. Nude mice bearing KB-3-1 (A) and KB-8-5 tumors (B) were treated with a single 20-mg/kg dose of saline or Taxol i.p. on day 0, when tumors reached 120 mm³. Mice bearing KB-3-1 tumors were killed on days 0, 1, 3, and 5, whereas mice bearing KB-8-5 tumors were killed on days 0, 3, and 5. The ECF and serum mesothelin levels were measured at these time points. Tumor volume was recorded just before death. Filled circle, saline-treated group; open circle, Taxol-treated group.

We then did a similar experiment with drug-resistant KB-8-5 tumors (Fig. 4B). Taxol at 20 mg/kg was given when the tumor volume reached 120 mm³ (day 0). Mesothelin levels in the ECF and serum were measured on days 0, 3, and 5. In the untreated tumors, the levels of mesothelin in the ECF and serum increased with time. The data show that Taxol caused a small but not significant decrease in the growth rate of KB-8-5 tumors over 5 days. The data also show that Taxol treatment did not produce a decrease in mesothelin levels in the ECF or serum. The levels of mesothelin were not different from those of the saline treatment group on days 3 and 5 (P > 0.05).

Effect of Tumor Removal on Mesothelin Levels in the Blood.

To determine how rapidly mesothelin is being removed from the circulation, we took three mice with KB-3-1 tumors, removed the tumors when they reached 300 mm³ in size, and collected blood for mesothelin assays at several time periods. The data in Fig. 5 show that by 1 h there is a dramatic decrease in mesothelin levels in all three animals to ≈30% of the initial levels, indicating mesothelin is rapidly removed from the blood. Because mesothelin has a molecular weight of ≈42 kDa, it is small enough to be removed by glomerular filtration in the kidney. These data indicate that large amounts of mesothelin must be constantly released by the tumor to maintain levels in the blood.

Fig. 5.

Half-life of shed mesothelin in mouse circulation. KB-3-1 tumors were surgically removed, and blood was collected at 0, 1, 4, and 7.5 h. Mesothelin levels were determined by ELISA. Curves are plotted with percentage of remaining mesothelin as the y axis and time of blood collection as the x axis.

Synergistic Effect of Taxol with an Immunotoxin Targeting the Human Transferrin Receptor.

It is known that many cell-surface antigens are shed into the blood, including the transferrin receptor (29). We previously made an immunotoxin targeting the human transferrin receptor, HB21(Fv)-PE40 (ATFP) (30). Because ATFP does not react with the mouse transferrin receptor, it can be safely used to target human tumors growing in mice. We decided to use it in synergy experiments with Taxol because it is equally toxic to KB-3-1 and KB-8-5 tumor cells (Table 1). Mice with 120-mm³ tumors were treated with one 20 mg/kg dose of Taxol alone, three 0.2 mg/kg doses of ATFP, or both agents as used in a previous study. The data in Fig. 6 show that either Taxol or ATFP alone significantly inhibited the growth of the KB-3-1 tumors (P < 0.001 on day 12). When the two agents were combined, there was striking synergy (P < 0.0001 on days 12 and 14). The results were different with the KB-8-5 tumors. The effect of Taxol treatment was not significant (P = 0.46) on day 12. ATFP produced a significant antitumor effect (P < 0.002) on day 12, but when the two agents were combined, no synergy was observed. Thus, synergy was observed in Taxol-sensitive tumors with two different immunotoxins, and no synergy was observed with drug-resistant tumors.

Fig. 6.

Treatment of mice with KB-3-1 and KB-8-5 tumors with ATFP in combination with Taxol. Mice in the Taxol group were injected i.p. with 330 μl of 20 mg/kg Taxol on day 0 as a single dose. Mice in ATFP group received 200 μl of 0.2 mg/kg ATFP three times i.v. on days 1, 3, and 5. For the combination group, mice were given 330 μl of 20 mg/kg Taxol i.p. on day 0 and then three injections of 200 μl of 0.2 mg/kg ATFP i.v. on days 1, 3, and 5. The control group received three injections of 200 μl of saline i.v. on days 1, 3, and 5. Each group included 15 mice, except for the combination group of KB-3-1 tumors, which contained 13 mice. The control group of KB-3-1 tumors had a tumor volume of 971 ± 282 mm³ on day 12; Taxol, 455 ± 268 mm³; ATFP, 358 ± 160 mm³; and combination, 15 ± 16 mm³. For KB-8-5 tumors, the tumor volume of the control group on day 12 was 1,083 ± 276 mm³; Taxol, 1,014 ± 230 mm³; ATFP, 718 ± 302 mm³; and combination, 419 ± 216 mm³.

Discussion

The goal of the current study was to determine the mechanism of synergy between immunotoxins and chemotherapy. We treated Taxol-sensitive and Taxol- resistant tumors with two different immunotoxins and only observed synergy with the drug-sensitive tumors. This finding rules out the possibility that synergy is due to a direct effect of Taxol on capillaries or other components making up the stroma of the tumor. This result focused our attention on the tumor cell and the tumor environment. It is well accepted that one barrier to the entry of antibodies into tumors is created by the binding to receptors on the cells nearest the blood vessels (4). We realized that many receptors are shed into the circulation, and that the concentration within the ECF of the tumor must be much higher than that in the blood. Using a specific ELISA for mesothelin, we demonstrated the presence of mesothelin in the ECF and its capacity to reduce killing by the immunotoxin SS1P. Finally, we observed that mesothelin levels in the tumor rapidly decreased in drug-sensitive tumors after Taxol treatment and not in Taxol-resistant tumors. We conclude that this decrease in mesothelin levels is an important factor in synergy. Thus, in addition to a site barrier (4, 31, 32), defective capillaries, high interstitial pressure, and a lack of functional lymphatics (3), the concentration of soluble shed antigen should be added to the list of factors that limit antibody entry into tumors. Taxol treatment reduces interstitial pressure (33) and kills many cells within the tumor mass, thereby reducing the packing of cells, an important factor in antibody entry.

Mouse and Human Tumor Calculations.

In mice, SS1P is rapidly removed from the circulation with a 20-min half-life (34). Initial plasma levels at a 0.3 mg/kg (6 μg) dose used in the Taxol synergy experiments are ≈6 μg/ml (≈100 nM) in plasma (Fig. 7). Assuming a total uptake at 6 h of 4% of the injected dose per gram of tumor (19), 4% of 6 μg (0.24 μg) of SS1P would accumulate in a 1-g tumor. If the SS1P were all in the ECF (15% of total tumor volume), the concentration would be ≈1.6 μg per ml (24 nM) in the ECF (Fig. 7). This concentration of SS1P is less than the mesothelin concentrations in large tumors shown in Figs. 2 and 7, which are ≥60 nM. As a consequence, shed mesothelin would significantly lower the free SS1P level in the tumor's ECF. Furthermore, mesothelin is continuously produced and released into the circulation with a half-life of <1 h, which would increase its ability to inactivate SS1P. In humans, the blood levels of SS1P at the maximum tolerated dose are ≈500 ng/ml (10 nM) (18). Human tumors with a high mesothelin concentration in the ECF would have substantial SS1P inactivation. Measurements of soluble mesothelin levels in tumors would be of great interest.

Fig. 7.

Concentrations of mesothelin and SS1P in the extracellular space of tumors and in the sera of mice. Filled bar, concentration of shed mesothelin; open bar, concentration of SS1P.

Clinical Implications.

The current study has several important clinical implications. Immunotoxin therapy of solid tumor masses should be combined with chemotherapy to achieve the best response. Because of the high-shed antigen levels in tumors, it seems unwise to do single-agent phase 2 trials against protein targets that are known to be shed into the blood. We will soon open a phase 2 trial in mesothelioma, in which SS1P (CAT-5001) will be combined with pemetrexed/cisplatin to see whether synergy can be observed in clinical studies. In the phase 1 trial with SS1P (CAT-5001), several minor antitumor responses were observed (18). If synergy occurs with human tumors, we should observe much better responses in the phase 2 trial. Human tumors are much larger than the mouse tumors we have studied and might be expected to have even higher mesothelin levels because mesothelin levels in tumors increase with tumor size (Fig. 2).

Because many tumor antigens that are now used as tumor targets are shed into the blood, their concentrations within the tumor are likely to be high and may diminish the effectiveness of antibody-based therapies unless combined with chemotherapy. These therapies include Her2/neu, EGF receptor, CD25, and CD30 (31, 35–37). If other antigens are released from tumors at a high rate, free shed antigen in the tumor also may significantly lower the concentrations of unconjugated antibodies within tumors. Although in our study we did not measure transferrin receptor levels, they have been measured by Bjerner et al. (29) and were found to be quite high in the serum of patients with lymphomas. When combining chemotherapy with immunotoxin therapy, the timing of drug administration seems to be important. In our animal studies, we started immunotoxin therapy 1 day after chemotherapy and used chemotherapeutic agents that rapidly kill tumor cells. Thus, on days 3 and 5, when the second and third doses of immunotoxin are given, soluble mesothelin levels are low. We plan to explore other treatment schedules.

In summary, KB tumors contain high amounts of shed mesothelin in their ECF that diminishes the ability of the immunotoxin SS1P to reach cells distant from the blood vessels feeding the tumor. Taxol kills some tumor cells and alters the tumor environment so that mesothelin levels decrease and access of the immunotoxin to tumor cells increases, leading to improved tumor responses.

Materials and Methods

Chemicals.

Taxol was provided by the Division of Veterinary Resources (National Institutes of Health). Immunotoxins SS1P, HA22, and HB21(Fv)-PE40 (ATFP) were prepared as previously described (19), and 6 mg/ml Taxol was diluted 1:5 with 0.9% NaCl.

Cell Culture and In Vitro Assays of Immunotoxin and Taxol on Tumor Cells.

KB-3-1 and KB-8-5 cells were grown in DMEM with 10% FBS. Cells were seeded in 96-well plates at 5,000 per well and incubated at 37°C overnight. Serial dilutions of Taxol and immunotoxin in 0.2% human serum albumin were added 24 h after plating, and cells were incubated at 37°C for another 48 h. Inhibition of cell growth was determined by using WST-8 assays from Dojindo (Kumamoto, Japan). Viability was expressed as the percentage of the absorbance value of untreated controls.

Blocking of the Cytotoxic Effect of SS1P by Shed Mesothelin.

KB-3-1 cells were seeded in 96-well plates at 5,000 per well and incubated at 37°C overnight. The concentrations of mesothelin in the serum from KB-3-1 tumor-bearing mice or in the ECF from KB-3-1 tumors were measured by ELISA with a Mesomark kit. Using these values, serum was diluted 1:50 and the ECF was diluted 1:100 with cell culture medium and added to wells used for cytotoxicity assays. The final concentration of mesothelin in the wells was 0.18 nM (≈6 ng/ml). Serial dilutions of SS1P were added 30 min later. Cells were incubated at 37°C for another 48 h. Inhibition of cell growth was determined by using WST-8 assays. Mesothelin-Fc fusion protein at concentrations of 20, 200, and 1,000 ng/ml were used to produce a standard inhibition curve. Mesothelin-Fc is a fusion of human mesothelin with the Fc of rabbit IgG and was produced in HEK293T cells (38).

Tumor Experiments.

Suspensions of KB-3-1 or KB-8-5 cells (4.0 × 10⁶ cells per 200 μl) were implanted s.c. into the thigh area of the rear leg of 5- to 6-week-old 18- to 20-g athymic nude mice. Tumor dimensions were determined every other day by using calipers. Tumor volume (mm³) was calculated by the formula: (a) × (b²) × 0.4, where a is tumor length and b is tumor width in millimeters.

Treatment was started when tumors reached ≈120 mm³. Taxol (6 mg/ml) diluted 1:5 with 0.9% NaCl was given i.p. SS1P or other immunotoxins were diluted with 0.2% human serum albumin, and 0.2 ml was given i.v. The animal protocol was approved by the National Cancer Institute Animal Care and Use Committee. All animal experiments were stopped when the tumors reached 1,000 mm³.

Isolation of the ECF from Tumors.

Tumor ECF was obtained by a previously described centrifugation method with minor modifications (26, 27). Tumors were excised from killed mice and blotted gently with tissue paper to remove fluid on the surface after cutting into four pieces by two cross-wise incisions in the middle of the tumor. The fragments were transferred into a 1.5-ml centrifuge tube, which contained a nylon mesh basket to retain the tumor fragments, with the cut part of the tumor facing the bottom. The tube was sealed with parafilm and centrifuged at 424 × g for 10 min at 4°C. The fluid at the bottom was transferred to a fresh tube, diluted 1:10 with PBS, and centrifuged at 100,000 × g in a TLA45 rotor (Beckman, Palo Alto, CA) for 30 min to remove any remaining cell debris. The supernatant was stored frozen and used for assays.

Determination of the Half-Life of Mesothelin in the Circulation of Mice.

Mice with KB-3-1 tumors were anesthetized and their tumors surgically removed, and 50 μl of blood was collected at 0, 1, 4, and 7.5 h. The mesothelin level in the serum was determined by using the Mesomark kit according to the manufacturer's protocol (Fujirebio Diagnostics, Malvern, PA).

Characterization of Shed Mesothelin in the ECF of Tumors by Western Blot.

Two microliter ECF samples from KB-3-1 tumors were separated on SDS/4–12% PAGE and transferred to PVDF membranes. To detect mesothelin, SS1P was labeled with Sulfo-NHS-Biotin (Pierce Chemical, Rockford, IL) according to the manufacturer's protocol. Biotinylated-SS1P at 1 μg/ml was incubated with the membrane for 1 h. After washing with TBS/T, the membrane was incubated with streptavidin-HRP (1:10,000) for 30 min at room temperature. Membranes were washed with TBS/T and visualized by ECL (GE Healthcare, Chalfont St. Giles, U.K.), and 10 ng of mesothelin-Fc fusion protein, with 2 μg of BSA as a carrier, was used as the positive control. The ECF from an A431 tumor (39) and 1 μl of normal mouse serum were negative controls.

Statistical Analysis.

Statistical analysis on synergy was done by David Venzon (Biostatistics and Data Management Section/Center for Cancer Research/National Cancer Institute). Repeated measures ANOVA was applied to the changes in successive tumor spherical diameters. Synergy was defined as an interaction effect significantly greater than the sum of the Taxol and SS1P effect. Results of cytotoxicity assay and correlation test were analyzed by using SigmaPlot 9.0 software (SPSS, Chicago, IL). To analyze the effect of Taxol on mesothelin levels in serum and the ECF, the analysis of a covariance model with tumor volume as a covariate was used. Data are reported as the mean ± SEM, along with P values. A value of P < 0.05 was considered statistically significant. Statistical analyses were performed by using version 9 of PC SAS (SAS Institute, Cary, NC).

Abbreviation

ECF

extracellular fluid.