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.

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.

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).

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.

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.

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.

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.

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.