Nobuki Yamaoka

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.

Expression of the cell surface antigen detected by the monoclonal antibody A7 in pancreatic carcinoma cell lines.

In a previous study, we used a murine monoclonal antibody, A7, against human colon carcinoma as a drug-carrier to treat colorectal cancer.1 In the present study, we found that MAb A7 also reacted immunohistochemically with 73% of human pancreatic carcinoma cell lines, with the A7 antigen mainly being detected on the cell surface. However, the A7 antigen was found in only 9% of the spent media of these human pancreatic carcinoma cell lines by ELISA. On the other hand, the positive incidence of CA19-9, POA, ferritin, CEA, DU-PAN-2 and SLX in those spent media was 100%, 64%, 64%, 55%, 55% and 36%, respectively. These results suggest that the A7 antigen may only rarely be shed into the sera of pancreatic cancer patients, in which case MAb A7 could be a suitable drug-carrier in targeting chemotherapy for pancreatic cancer patients.

Biodistribution of murine and chimeric Fab fragments of the monoclonal antibody A7 in human pancreatic cancer.

Much recent research has focused on the use of monoclonal antibodies (MAbs) in the immunodetection of solid tumors. Fab fragments of MAbs are more suitable for immunoscintigraphy than intact MAbs. Recently, human-mouse chimeric antibodies have been developed in an effort to reduce human antimouse antibody (HAMA) production by murine MAbs in humans. In this study125I-labeled murine and chimeric Fab fragments of the MAb A7 were injected i.v. into nude mice bearing a human pancreatic cancer (HPC-YS) xenograft. The radioactivity in tumors and in normal tissues was subsequently measured. The tumor tissue/blood ratio (TIB) of 125I-labeled murine and chimeric Fab fragments of MAb A7 increased with time in a similar manner and reached 9.68 2.54 and 10.49 ± 1.50, respectively, 24 h after injection. Moreover, the T/Bs of 125I-labeled murine and chimeric Fab fragments of MAb A7 were greater than the T/B of intact MAb A7. When mice bearing tumors that did not react with MAb A7 were studied125I-labeled murine and chimeric Fab fragments did not localize specifically to the tumors. These results suggest that chimeric Fab fragments of MAb A7 are useful carriers of radionuclides for the immunodetection of human pancreatic cancer, with equivalent activity to murine Fab fragments and less theoretical potential to induce a HAMA response.

The role of monoclonal antibody A7 as a drug modifier in cancer therapy

An anticancer antibiotic, neocarzinostatin (NCS), was covalently conjugated to the murine monoclonal antibody A7 (mAb A7), which recognizes the glycoprotein on the cell surface of human colon cancer. The biological and pharmacological properties of the conjugate (A7-NCS) were examined and compared with those of unconjugated NCS. A7-NCS exhibited a strong binding and cytotoxicity to the cell and an antigen-specific tumor accumulation. Significant tumoricidal effects in vivo were observed in the antigen-positive tumor-bearing mice treated with A7-NCS, whereas NCS mixed with mAb A7 and NCS alone were relatively ineffective. In the antigennegative tumor, the tumoricidal effect of A7-NCS was lower than in the antigen-positive tumor. The NCS concentration in blood and tumor were significantly elevated by conjugation to mAb A7. The NCS localization in tumor was higher in the antigen-positive tumor than in the antigen-negative tumor. Death due to acute toxicity was observed at a dose of 20 units (U) NCS in mice treated with unconjugated NCS, whereas toxicity was seen with a much higher dose of NCS (100 U) if the drug was conjugated to the mAb. These findings show that mAb A7 confers more favorable pharmacological properties on an anticancer drug, making it potentially more useful for cancer chemotherapy.

In vivo efficacy of neocarzinostatin coupled with Fab human/mouse chimeric monoclonal antibody A7 against human colorectal cancer.

The anticancer polypeptide neocarzinostatin (NCS) was covalently coupled to a human/mouse chimeric Fab A7 monoclonal antibody (chFabA7) and the in vivo efficacy of this conjugate was examined. NCS concentration assay was carried out, and acute toxicity and tumoricidal effects were examined. The concentration assay, using anti‐NCS monoclonal antibody, revealed that administration of the chA7Fab conjugate leads to a greater blood retention and a higher tumor accumulation of NCS, when compared to free NCS administration. The tumoricidal effect of chA7Fab‐NCS was higher than that of either free NCS or the saline control, against antigen‐positive tumors. In antigen‐negative tumors there was no difference in toxic effect among the three preparations. Values of LD50, reflecting acute toxicity, were 5050 U/kg and 3600 U/kg for the chA7Fab‐NCS and the free NCS, respectively. These results suggest that chFahA7‐NCS may be a promising tool for targeting cancer chemotherapy.

Efficacy and Specificity of a Monoclonal Antibody‐Drug Conjugate in Chemotherapy by Intratumoral Injection

The murine monoclonal antibody (Mab) A7 conjugated to neocarzinostatin (A7‐NCS) was injected intratumorally (IT) into tumor bearing nude mice. Its pharmacokinetics and tumoricidal effects were compared in the high, moderate and low antigen expressing xenograft for SW1116, WiDr and KB tumor‐bearing nude mice, respectively. When injected IT into nude mice, [125I]A7‐NCS was retained in the tumors according to the degree of antigen expression; it was also disseminated into the blood inverse proportion to the antigen expression. Addition of an excess amount of Mab A7 reduced [125I]‐A7‐NCS accumulation in SW1116 xenograft and elevated the [125I]A7‐NCS concentration in the circulation. Complete tumor reduction was found in all 5 mice with SW1116 tumor, and 2 of 5 mice with WiDr tumor. However, only incomplete tumor suppression was observed in mice with the KB tumor. The significant tumor reduction in SW1116 bearing nude mice was attenuated when excess of Mab A7 was simultaneously administered with A7‐NCS‐ These findings indicate that A7‐NCS was localized in the target tumors and exerted its tumoricidal effects depending on the degree of antigen‐antibody interaction when administered IT. Thus, A7‐NCS can be used successfully in vivo for local therapy, auguring new and promising applications for local cancer therapy.

Biodistribution of neocarzinostatin conjugated to chimeric Fab fragments of the monoclonal antibody A7 in nude mice bearing human pancreatic cancer xenografts.

In this study, we conjugated chimeric Fab fragments of the monoclonal antibody (MAb) A7, which reacts with pancreatic cancers, to the antitumor drug neocarzinostatin (chA7Fab‐NCS) and intravenously injected 125I‐labeled chA7Fab‐NCS into nude mice bearing a human pancreatic cancer xenograft. We compared the tumor localization of 125I‐labeled chA7Fab‐NCS with that of conventional 125I‐labeled A7‐NCS, which was produced by conjugation of MAb A7 and NCS. 125I‐Labeled chA7Fab‐NCS accumulated in the tumor earlier than 125I‐labeled A7‐NCS, and significantly larger amounts of 125I‐labeled chA7Fab‐NCS had accumulated in the tumor 1 hour after injection. The results suggest that chA7Fab may be a suitable carrier for NCS in immunotargeting therapy against pancreatic cancer.

In vitro reactivity and in vivo biodistribution of the monoclonal antibody A7 using human gastric carcinoma cell lines.

The monoclonal antibody (MAb) A7 has been used to treat patients with colorectal or pancreatic carcinoma with encouraging results. We therefore determined if MAb A7 would also react with gastric carcinoma cell lines. MAb A7 reacted with seven of eight gastric carcinoma cell lines tested. The intensity of the reaction, measured by flow cytometry, was equal to that of WiDr (colon) and HPC-YS (pancreas) cell lines. In nude mice bearing xenografts of the MAb A7-reactive gastric cancer line MKN45, the percentage injected dose of MAb A7 per g of tumour tissue on day 7 was 9.79; this value was 77% of that on day 1. The in vivo tumour-to-blood ratio of MAb A7 was 2.77 on day 7. Therefore, MAb A7 has long-term retention at binding sites as well as a high probability, high intensity and high specificity of reactivity against gastric cancer, which make it an ideal drug carrier for immunotargeted chemotherapy and immunodiagnosis.

Enhanced Antitumor Effect of Neocarzinostatin Conjugated to Monoclonal Antibody A7 on Human Pancreatic Carcinoma Grafted in Nude Mice

Recent research has been directed toward the use of monoclonal antibody-drug conjugates for solid tumors. In this study, we used the monoclonal antibody A7 to target the antitumor drug neocarzinostatin (NCS) to pancreatic cancer cells. The in vivo localization of the radiolabeled MAb A7 was investigated using pancreatic carcinoma grafted nude mice. MAb A7 localized in the tumor at a tissue/blood ratio of 2.04. Moreover, A7-NCS was administered to the nude mice with pancreatic carcinoma xenografts. A7-NCS showed a greater antitumor effect than free NCS.

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.