Hiroshi Tsuruta

Targeted chemotherapy in mice with peritoneally disseminated gastric cancer using monoclonal antibody–drug conjugate

The murine monoclonal antibody A7 (MAb A7) is reactive against most human gastric cancer cell lines. Using a nude mouse peritoneal dissemination model of human gastric cancer, we investigated targeted chemotherapy using a conjugate of neocarzinostatin (NCS) with MAb A7 (A7-NCS). After demonstrating cytotoxicity of the complex against the human gastric cancer cell line MKN45 in vitro, we intraperitoneally injected A7-NCS, NCS or saline into nude mice bearing peritoneally disseminated human gastric cancer. A7-NCS inhibited peritoneal dissemination significantly more effectively than NCS. MAb A7 may prove to be an effective carrier for antineoplastic drugs in patients with peritoneal dissemination of gastric cancer.

Effects of neocarzinostatin-chimeric Fab conjugates on the growth of human pancreatic carcinoma xenografts

Neocarzinostatin (NCS) was bound covalently to human/mouse chimeric Fab fragments of MAb A7 (chA7Fab) directed against human pancreatic carcinoma. The anti-tumour effect of chA7Fab-NCS was tested in a nude mouse model on pancreatic carcinoma and compared with A7-NCS or NCS alone. The anti-tumour effect of chA7Fab-NCS increased in a dose-dependent manner and was significantly greater than either A7-NCS or NCS. Tumour growth was completely suppressed after the administration of chA7Fab-NCS. An enzyme-linked immunosorbent assay with rabbit anti-mouse immunoglobulin was performed to examine the antigenicity of chA7Fab. ChA7Fab had less reactivity with rabbit anti-mouse immunoglobulin than either whole antibody A7 or murine Fab fragments of A7. Thus, chA7Fab-NCS can inhibit human pancreatic cancer growth in an animal and may be useful for targeting chemotherapy to pancreatic cancer in humans.

The effect of intravenous and intra-tumoural chemotherapy using a monoclonal antibody--drug conjugate in a xenograft model of pancreatic cancer

In order to investigate the efficacy of the intra-tumoural administration of an anticancer drug-monoclonal antibody conjugate in athymic nude mice bearing xenografts of a human pancreatic carcinoma, we examined the clearance of the murine monoclonal antibody A7 from the xenografts after intravenous or intra-tumoural administration and measured the antitumour effect of neocarzinostatin conjugated to MAb A7 following intravenous or intra-tumoural injection. Compared with 125I-labelled normal mouse IgG, a larger amount of 125I-labelled A7 remained in the tumour after both intravenous and intra-tumoural injection, and a significantly larger amount of 125I-labelled A7 remained in the tumour after intra-tumoural injection than that after intravenous injection. Moreover, a larger amount of 125I-labelled A7-NCS localized in the tumour after intra-tumoural injection than that after intravenous injection. Neocarzinostatin conjugated to MAb A7 showed greater activity against human pancreatic cancer than neocarzinostatin alone after both intravenous and intra-tumoural administration. Tumour growth was suppressed completely by the intra-tumoural administration of A7-NCS at a dose that did not suppress tumour growth via the intravenous route. These observations suggest that the intra-tumoural injection of neocarzinostatin conjugated to MAb A7 offers promise in treating pancreatic carcinoma.

Distribution of neocarzinostatin conjugated to biotinylated chimeric monoclonal antibody Fab fragments after administration of avidin.

We have developed chimeric Fab fragments of MAb A7 (chA7Fab) and have reported on their potential usefulness as a carrier of neocarzinostatin (NCS). However, a large amount of chA7Fab accumulates in the kidneys which might cause renal failure. This was one of the major side-effects of the chA7Fab-NCS immunoconjugate administered to humans. To decrease the kidney accumulation of chA7Fab, chA7Fab was biotinylated and administered with a subsequent injection of avidin to nude mice with pancreatic cancer. The accumulation of biotinylated chA7Fab in the blood and the kidneys decreased significantly after the injection of avidin. In a separate experiment with biotinylated chA7Fab-NCS, the blood and kidney accumulation decreased significantly after the injection of avidin. These findings suggest that the injection of biotinylated chA7Fab complexed with NCS followed by avidin may be safer and may permit the administration of larger doses of NCS without the subsequent development of renal failure.

Decreased Renal Accumulation of Biotinylated Chimeric Monoclonal Antibody-Neocarzinostatin Conjugate after Administration of Avidin

Murine monoclonal antibodies (mAbs) such as A7 administered to humans induce a human anti‐mouse antibody response. Moreover, because Fab fragments of mAbs are able to penetrate target tumors easily, they may be more suitable than intact mAb to be carriers of anticancer agents such as neocarzinostatin (NCS), which are rapidly inactivated in the blood. To address these problems, chimeric A7 Fab fragment‐NCS conjugate (chA7Fab‐NCS) was produced. However, large amounts of 12SI‐labeled chA7Fab‐NCS accumulate in the kidney and can lead to renal dysfunction. To decrease renal accumulation of chA7Fab‐NCS, chA7Fab was biotinylated and administered with a subsequent injection of avidin. Human pancreatic carcinoma‐bearing nude mice were injected with 125I‐labeled biotinylated chA7Fab‐NCS with or without subsequent administration of avidin. The accumulation of 125I‐labeled biotinylated chA7Fab‐NCS in tissue samples was measured at appropriate time intervals. 12SI‐labeled biotinylated chA7Fab‐NCS was cleared more rapidly from the blood and the kidney with the administration of avidin than without it. There was no difference between tumor accumulation in these groups. The tumor/blood ratio of radioactivity of 125I‐labeled biotinylated chA7Fab‐NCS was significantly higher with subsequent administration of avidin than without avidin. The administration of biotinylated chA7Fab‐NCS followed by avidin may enhance safety and permit the administration of larger doses of NCS without the subsequent development of renal failure. A larger amount of 12SI‐labeled biotinylated chA7Fab‐NCS was retained in the liver and spleen with the subsequent administration of avidin than without avidin

Applicability of monoclonal antibody Fab fragments as a carrier of neocarzinostatin in targeting chemotherapy

Two types of fragments of MAb A7 were produced to improve the efficacy and safety in targeting chemotherapy with neocarzinostatin. In this study, 125I‐labeled F(ab′)2 and Fab fragments of MAb A7 and 125I‐labeled MAb A7 were injected intravenously into mice with pancreatic carcinoma xenografts, and the accumulation of each antibody in the tumors was compared. A greater amount of the 125I‐labeled Fab fragments of MAb A7 localized in the tumor 2 h following the injection than was observed with the other probes. Relatively less 125I‐labeled MAb A7 localized in the tumor 2 h following the injection than was observed with the other two probes. Moreover, reaction of rabbit antimouse IgG with the Fc portion, which is the most immunopotent region of the Fab and F(ab′)2 fragments of MAb A7 and MAb A7, was determined by ELISA; the weakest reaction was observed with the Fab fragments of MAb A7. These results suggest that the Fab fragments of MAb A7 may be more suitable carriers of an anti‐cancer drug that is inactivated rapidly in the blood, such as NCS, in targeting chemotherapy than either intact MAb A7 or the F(ab′)2 fragments of MAb A7.

Reduced blood accumulation of biotinylated monoclonal antibody A7 after the subsequent administration of avidin

Clear immunoscintigraphy with radiolabeled monoclonal antibodies (MAbs) requires a high tumor tissue/blood ratio of radioactivity. In this study, we attempted to obtain a high tumor tissue/blood ratio by the active removal of radiolabeled MAb from the circulation, using the avidin-biotin system. Biotinylated 125I-labeled MAb A7 was injected intravenously into nude mice bearing a human colon cancer (WiDr) xenograft. Avidin was injected 24 h later. The tumor tissue/blood ratio of radioactivity was almost four times that of controls. These results suggest that biotinylated 125I-labeled MAb A7 and avidin are potentially useful for the rapid immunodetection of human colon cancer.

Changes in expression of the antigen recognized by monoclonal antibody A7 in human pancreatic carcinoma cells following exposure to anticancer agents

Techniques which can increase the expression level of tumor-associated antigens may improve immunotargeting therapy. We studied the reactivity of MAb A7 toward an antigen expressed on the surface of the human pancreatic cancer cell line HPC-YS after treatment with various antitumoral agents. When we applied 1 microg/ml mitomycin C (MMC) or 0.1 microg/ml neocarzinostatin (NCS) for 1 h, A7 recognizing antigen expression was enhanced until 24 h after the treatments. At a dose that completely suppressed cell growth, increased antigen expression was maintained for 96 h. Therefore, this study suggests that the combined application of an anticancer drug and MAb A7 may be useful for immunotargeting chemotherapy.