The detection and localization of monocyte chemoattractant protein-1 (MCP-1) in human ovarian cancer.

Chemokines may control the macrophage infiltrate found in many solid tumors. In human ovarian cancer, in situ hybridization detected mRNA for the macrophage chemokine monocyte chemoattractant protein-1 (MCP-1) in 16/17 serous carcinomas, 4/4 mucinous carcinomas, 2/2 endometrioid carcinomas, and 1/3 borderline tumors. In serous tumors, mRNA expression mainly localized to the epithelial areas, as did immunoreactive MCP-1 protein. In the other tumors, both stromal and epithelial expression were seen. All tumors contained variable numbers of cells positive for the macrophage marker CD68. MCP-1 mRNA was also detected in the stroma of 5/5 normal ovaries. RT-PCR demonstrated mRNA for MCP-1 in 7/7 serous carcinomas and 6/6 ovarian cancer cell lines. MCP-1 protein was detected by ELISA in ascites from patients with ovarian cancer (mean 4.28 ng/ml) and was produced primarily by the cancer cells. Human MCP-1 protein was also detected in culture supernatants from cell lines and in ascites from human ovarian tumor xenografts which induce a peritoneal monocytosis in nude mice. We conclude that the macrophage chemoattractant MCP-1 is produced by epithelial ovarian cancer and that the tumor cells themselves are probably a major source. MCP-1 may contribute to the accumulation of tumor-associated macrophages, which may subsequently influence tumor behavior.

Improved antitumor effects of immunotoxins prepared with deglycosylated ricin A-chain and hindered disulfide linkages.

A monoclonal anti-Thy-1.1 antibody (OX7) was coupled to either native or chemically deglycosylated ricin A-chain (dgA) using one of two different cross-linking agents. One cross-linker, N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)tolu ene (SMPT), generates a sterically hindered disulfide bond which is relatively resistant to reduction, whereas the other, 2-iminothiolane hydrochloride, generates an unhindered disulfide bond with greater lability. A two-compartment pharmacokinetic model was used to analyze the blood levels of each immunotoxin and its breakdown product (free antibody) after i.v. injection into mice. Immunotoxins prepared with SMPT broke down in vivo 6.3-fold more slowly than those prepared with 2-iminothiolane hydrochloride, and immunotoxins containing native A-chain were cleared 2- to 3-fold more rapidly from the bloodstream than those containing dgA. As a result, 24 h after injection, 16% of the OX7-SMPT-dgA remained in the blood as compared with 0.4 to 2.5% of the other immunotoxins. Immunotoxins prepared with dgA were about 3-fold more toxic to mice than those prepared with native A-chain, whereas immunotoxins prepared with SMPT were only slightly more toxic than those prepared with 2-iminothiolane hydrochloride. When equivalent toxic doses of the immunotoxins were administered i.v. to mice which had been given injections of Thy-1.1+ AKR-A/2 lymphoma cells, the OX7-SMPT-dgA gave the best antitumor effect. A dose equivalent to one-seventh of the median lethal dose extended the survival time of the animals by the extent expected if 99.999% of the tumor cells had been eradicated. Furthermore, the tumors that did develop in the mice treated with OX7-SMPT-dgA were mutants which were resistant to all the immunotoxins. Some of the mutants were deficient in Thy-1.1 whereas others were not. In conclusion, both the use of the SMPT cross-linker and deglycosylation of the A-chain significantly improve the therapeutic index of the immunotoxins in AKR-A/2 tumor-bearing mice.

New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo.

Two new coupling agents were synthesized for making immunotoxins containing disulfide bonds with improved stability in vivo: sodium S-4-succinimidyloxycarbonyl-alpha-methyl benzyl thiosulfate (SMBT) and 4-succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)tolue ne (SMPT). Both reagents generate the same hindered disulfide linkage in which a methyl group and a benzene ring are attached to the carbon atom adjacent to the disulfide bond and protect it from attack by thiolate anions. An immunotoxin consisting of monoclonal anti-Thy-1.1 antibody (OX7) linked by means of the SMPT reagent to chemically deglycosylated ricin A-chain had better stability in vivo than an immunotoxin prepared with 2-iminothiolane hydrochloride (2IT) which generates an unhindered disulfide linkage. About 48 h after i.v. injection into mice, one-half of the SMPT-linked immunotoxin present in the blood was in intact form and one-half as released free antibody, whereas equivalent breakdown of the 2IT-linked immunotoxin was seen at about 8 h after injection. Consequently, the blood levels of the SMPT-linked immunotoxin remained higher than those of the 2IT-linked immunotoxin despite loss of immunotoxin from the blood by other mechanisms. Forty-eight h after injection, 10% of the injected dose of the SMPT-linked immunotoxin remained in the bloodstream as compared with only 1.5% of the 2IT-linked immunotoxin. The ability of immunotoxins prepared with the new reagents to inhibit protein synthesis by Thy-1.1-expressing AKR-A/2 lymphoma cells in vitro was identical to that of immunotoxins prepared with 2IT or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Clonogenic assays showed that fewer than 0.01% of AKR-A/2 cells survived exposure to high concentrations of OX7-abrin A-chain immunotoxins prepared with SMBT, 2IT, or SPDP. Twelve clones of cells which had survived treatment with the SMBT-linked immunotoxin were isolated. None of the clones was selectively resistant to the SMBT-linked immunotoxin when retested in cytotoxicity assays. In conclusion, immunotoxins prepared with the new coupling agents should have improved antitumor activity in vivo because they are longer lived and do not break down so readily to release free antibody which could compete for the target antigens.