Biomedical subjects

N Hanai

Publications and source records attributed to N Hanai.

97 records · Page 6Linked to original sources

PubMedDate not supplied

Comparative studies on monoclonal antibodies raised against human gastric cancer for application to serum diagnosis of cancer.

Four murine MoAbs, KM191(IgM), KM206(IgM), KM230(IgG1) and KM231(IgG1), against human gastric cancer were generated using mice which underwent tolerance treatment to stomach tissues. They exhibited very similar high reactivities to stomach adenocarcinoma cells and low reactivities to normal cells in both membrane binding assay and immunohistochemical analysis. Their antigens were neuraminidase and protease sensitive and existed as macromolecules (1,000Kd) in body fluids. Binding of each antibody was inhibited by the others and KM231 showed the highest binding avidity. When they were used to detect the antigens shed in ascitic fluids and pleural effusions of cancer patients, KM231 allowed the most efficient detection of the antigen. The above results indicated that the four MoAbs bound to closely related epitopes on the same antigen and that the nature of its high binding avidity enabled KM231 to show the greatest efficiency in the detection of the antigen in body fluids. KM231 was applied to serum diagnosis and gave high positive percentages in pancreatic cancer(86%), hepatocarcinoma(87%), gall bladder cancer(50%), and gastric cancer(34%), whereas in healthy persons (0%) and benign diseases except for hepatitis(29%) the percentages were low. KM231 was similar to NS19-9, but quite different from NS19-9 in the high positive percentages of hepatocarcinoma in serum diagnosis.

Adenocarcinoma↗

PubMedDate not supplied

Advantage of cocktail-use of two anti-tumor monoclonal antibodies, KM-93 and KM-231, in serum diagnosis of cancer.

Two mouse monoclonal antibodies (MoAbs), KM-93 raised against human lung adenocarcinoma and KM-231 raised against human gastric cancer, were useful in serum diagnosis of several human cancer. KM-93 and KM-231 recognize sialyl Lex epitope and sialyl Lea epitope, respectively, expressed on glycoprotein and glycolipid. We established a new "cocktail" sandwich enzyme-linked immunosorbent assay system using the two MoAbs and the advantage of this assay system, which can simultaneously detect sialyl Lex and sialyl Lea antigens, is assessed in the present study. The new assay system is composed of a mixture of KM-93 and KM-231 as 1st antibodies and a mixture of biotinylated two MoAbs as 2nd antibodies. We evaluated the concentration of MoAbs and optimized it to gain high cancer-positivity. This assay system covered sialyl Lex positive and/or sialyl Lea-positive sera and gave a high rate of positive results in lung adenocarcinoma (62.3%), gastric cancer (32.5%), colon cancer (37.5%), pancreatic cancer (83.3%), bile duct and gall bladder cancer (66.7%) and hepatoma (76.9%), whereas positive results in healthy adults remained low. Positive results in benign diseases of lung (12.5%), pancreas (10.8%), gall bladder and bile duct (9.1%) were very low, but were higher in liver cirrhosis (33.3%), hepatitis and liver injury (34.8%). Simultaneous detection of two carbohydrate antigens, sialyl Lex and sialyl Lea was clearly superior to single detection.

Adenocarcinoma↗

PubMedDate not supplied

Detection of a novel lung adenocarcinoma--associated serum antigen defined by two monoclonal antibodies, KM 432 and KM 227.

Two monoclonal antibodies, KM227 (IgM) and KM432 (IgG1), were established by immunizing mice with the crude membrane fraction of lung squamous cell carcinoma and the high-molecular-weight antigen in pleural perfusion from patients with lung adenocarcinoma, respectively. They were found to recognize the carbohydrate epitope and the peptide epitope on the antigens, respectively. Both KM227 and KM432 bound to the serum antigens associated with lung adenocarcinoma and lung benign diseases. A double determinant assay in which KM432 was used as 1st antibody and biotinylated KM227 was used as 2nd antibody selectively detected the antigen, designated KA432/227, elevated in sera from patients with lung adenocarcinoma. The molecular weight of KA432/227 in pleural perfusion from patients with lung adenocarcinoma was estimated to be more than 900 kD by the gel filtration chromatography. Serum with a high KA432/227 level was most frequently found in lung adenocarcinoma (47.8%), and occasionally found in lung squamous cell carcinoma (23.1%), lung large cell carcinoma (22.2%), pancreatic carcinoma (20.0%) and carcinoma of the bile-duct and the gall bladder (25.0%). In contrast, positive percentages in benign diseases and healthy adults were very low (0 to 7.7%). No significant correlation was found between the serum level of KA432/227 and other lung adenocarcinoma associated antigens, such as sialyl Le(x). Combined detection of KA432/227 and sialyl Le(x) antigen elevated the positive percentages in lung adenocarcinoma (67.2%).

Adenocarcinoma↗

PubMedDate not supplied

Reactivity of a mouse/human chimeric anti-GM2 antibody KM966 with brain tumors.

With the aim of investigating the passive immunotherapy of brain tumors, we examined the binding of a mouse/human chimeric anti-ganglioside GM2 antibody KM966 to various organs and brain tumors. Frozen sections of 51 surgically resected brain tumors were stained with antibody KM966. Fourteen gliomas out of 16 were stained positively with antibody KM966. Eleven positive sections demonstrated homogenous staining. No specific binding to normal gray matter and white matter was observed. Some cases of meningiomas, neurinomas and metastatic brain tumors were stained with KM966 but with less frequency and intensity than gliomas. In addition, KM966 demonstrated strong antibody-dependent cellular cytotoxicity against human malignant glioma cells. These results showed that antibody KM966 will be useful for passive immunotherapy of malignant gliomas.

Adult↗

PubMedDate not supplied

Different binding properties of three monoclonal antibodies to sialyl Le(x) glycolipids in a gastric cancer cell line and normal stomach tissue.

We established a mouse monoclonal antibody (Mab) KM93 which recognized sialyl Le(x)-carbohydrate epitope determined by solid phase radioimmunoassay using a panel of authentic glycolipids. The specificity of KM93 was similar to another anti-sialyl Le(x) Mab CSLEX-1 established previously, and different from that of Mab FH6 which recognized sialyl Le(x)-i (sialyl dimeric Le(x)). In a further study, however, we found that KM93 reacted with some glycolipids much more strongly than CSLEX-1 did on thin-layer chromatography (TLC) plates. We purified two gangliosides named K-1 and K-2 from gastric cancer cell line KATOIII, and three gangliosides named H-1, H-2, and H-3 from human stomach. KM93 reacted with all of these glycolipids. CSLEX-1 reacted with K-1 and K-2 with less intensity than KM93 did, and faintly reacted with H-1, but not at all with H-2 or H-3. FH6 did not react with K-1, K-2 or H-1, while it stained H-2 and H-3. In spite of this different reactivity with Mabs, analysis by proton nuclear magnetic resonance (1H NMR) proved that carbohydrate structure of K-1 and H-2 were the same: NeuAc alpha 2-->3Ga1 beta 1-->4 [Fuc alpha 1-->3] G1cNAc beta 1-->3 Ga1 beta 1-->4G1c beta 1-->1Cer. The H1 NMR spectrum of H-3 also indicated that H-3 consisted of the same sugars as K-1. These results indicated that KM93 had wider reactivity than CSLEX-1, and that the distinct reactivity of KM93 from CSLEX-1 was not caused by sugar moiety. It was also shown that the interaction of sialyl Le(x) sugar determinant with MAb depended on the tissue origin of molecules carrying carbohydrate.

Amino Acid Sequence↗

PubMedDate not supplied

Immunocytochemical study on internalization of anti-carbohydrate monoclonal antibodies.

The fate of anti-tumor monoclonal antibodies (mAbs) after binding to the cell surface is one of the important factors for application of mAbs in therapy. For mAbs to remain on the cell surface for a long time is preferable character for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). On the other hand, quick internalization of mAbs into cytoplasm is advantageous for cytotoxic effects of toxins or cytotoxic drugs conjugated with mAbs. In this study we investigated the localization of anti-carbohydrate mAbs in vitro by immunocytochemical methods at the electron microscopic level. Anti-ganglioside GM3 mAb (mouse IgM) was localized both in cytoplasmic vesicles and on the cell surface membrane of mouse melanoma cells after incubation for 30 min at 37 degrees C. Anti-ganglioside GM2 mAb (mouse IgM), anti-ganglioside GD2 mAb (mouse IgG3) anti-ganglioside GD3 mAb (mouse IgG3) mainly existed on the cell surface of human small cell lung carcinoma cells, human neuroblastoma cells and human melanoma cells, respectively, after incubation for 30 min at 37 degrees C. On the other hand, anti-sialyl Lea mAb (mouse IgG1) was intensively incorporated into the cytoplasm of human colon tumor cells under the same conditions. Anti-ganglioside GD3 mAb and anti-sialyl Lea mAb were radiolabelled with 125Iodine and traced in in vitro culture. When the cells were incubated in the presence of the 125I-mAb for 90 min on ice, the ratio of intracellular counts to surface counts was 8/92 for anti-ganglioside GD3 mAb and 31/69 for anti-sialyl Lea mAb respectively. After the subsequent incubation for 60 min at 37 degrees C, the ratio altered to 13/87 for anti-ganglioside GD3 mAb and 54/46 for anti-sialyl Lea antigen mAb. This study suggested that internalization of anti-carbohydrate mAbs after the binding to the cell surface was different among the mAbs depending on the character of antigens. In conclusion, anti-ganglioside mAbs have beneficial characters for CDC or ADCC while anti-sialyl Lea mAb is suitable for immunoconjugates.

Animals↗

PubMedDate not supplied

Cytotoxicity of adriamycin-containing immunoliposomes targeted with anti-ganglioside monoclonal antibodies.

GM2 and GD2 have been intensely studied as tumor antigens of neuroectodermal-origin tumors. We have established several mouse or rat IgM anti-ganglioside GM2 and GD2 MoAbs and applied them in antigen-specific drug delivery. In the immunofluorescence assay, anti-GM2 MoAbs bound to neuroblastoma cells and leukemia cells, and anti-GD2 MoAb bound to neuroblastoma cells and melanoma cells. Sulfhydryl subunits of reduced IgM were directly coupled to maleimide groups on the surface of the liposomes followed by the incorporation of adriamycin by the use of Na+/K+ chemical gradient. The resultant immunoliposomes had a size of 86 nm-diameter containing approximately 400 molecules of adriamycin in its unilamellar structure and 17 molecules of the MoAb on its surface. Mouse anti-GM2 MoAb lost the binding specificity when covalently bound to the liposomes. The immunoliposome coupled with mouse anti-GD2 MoAb retained targeting activity to the antigen-positive neuroblastoma cells, IMR-32. However, it did not kill IMR-32 cells, probably because the amount of adriamycin taken up by the tumor cell was below the fatal amount. The immunoliposome coupled with rat anti-GM2 MoAbs delivered adriamycin to the neuroblastoma cells, IMR-32, and leukemia cells, TYH, in the antigen-specific manner. It also target-specifically suppressed the (3H)thymidine-uptake of the cells while the same concentration of adriamycin in the free form killed all the cell lines examined. IMR-32 cells had GM2 and GD2 in almost the same amounts, and interacted with either mouse anti-GD2 MoAb--immunoliposome or rat anti-GM2 MoAb-immunoliposome. The different cytotoxic activities of the two immunoliposomes against IMR-32 cells was probably due to the difference in the facility of internalization of the immunoliposomes after binding.

Animals↗