Tatsuya Kotani

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.

Secondary Torsion of Vermiform Appendix with Mucinous Cystadenoma

Torsion of the vermiform appendix is a rare disorder, which causes abdominal symptoms indistinguishable from acute appendicitis. We report a case (a 34-year-old male) of secondary torsion of the vermiform appendix with mucinous cystadenoma. This case was characterized by mild inflammatory responses, pentazocine-resistant abdominal pain, and appendiceal tumor, which was not enhanced by the contrast medium on computed tomography presumably because of reduced blood flow by the torsion. These findings may be helpful for the preoperative diagnosis of secondary appendiceal torsion.

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.

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.

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.

Noncontrast and contrast enhanced computed tomography for diagnosing acute appendicitis: A retrospective study for the usefulness

Abdominal computed tomography (CT) provides great benefits for the differential diagnosis in patients complaining of acute abdominal pain. However, the use of diagnostic X-rays is associated with the cumulative risk of cancer development. In order to determine the relative usefulness of noncontrast and enhanced CT with intravenous contrast material for diagnosing acute appendicitis, the retrospective analysis was performed using 247 patients (46 children and 201 adults) with clinically suspected appendicitis, who were admitted to our hospital from 2002 to 2006 and underwent noncontrast or combined noncontrast and enhanced CT examination. Of 185 patients who were diagnosed to have acute appendicitis with appendiceal thickening (167 cases) or normal-sized appendix (18 cases), 73 cases underwent noncontrast CT alone and these 73 cases could be retrospectively diagnosed to have appendicitis on noncontrast CT. On the other hand, 112 cases of these 185 patients underwent noncontrast CT followed by enhanced CT, and vermiform appendix was detected in 86 cases of them (86/112, 76.8%) on noncontrast CT. These 86 cases could be retrospectively diagnosed to have acute appendicitis on noncontrast CT, whereas enhanced CT was required to detect vermiform appendix and to obtain the final diagnosis of appendicitis in the remaining 26 cases (26/112, 23.2%). Enhanced CT was superior to noncontrast CT in diagnosing appendicitis in all age and any gender groups. We suggest that enhanced, but not noncontrast, CT should be primarily performed for diagnosing acute appendicitis in all patients to minimize the radiation exposure unless intravenous administration of contrast material is contraindicated.

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.

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.