SELECTIVE KILLING OF GASTRIC CANCER CELLS BY MONOCLONAL ANTIBODY CONJUGATED WITH A CHAIN OF RICIN

A specific cytotoxic agent against gastric cancer was constructed by covalently coupling the ricin A chain to monoclonal artibody, MGb2. MGb2 was modified by SPDP to introduce the 3-(2-pyridylthio) propionyl radical and then treated with a reduced A chain to give a disulfide linked conjugate that retained the original binding specificity of the antibody moiety. The conjugate obtained retained the activity of the antibody and the biological activity of the A chain well.

MONOCLONAL ANTIBODIES AGAINST HUMAN GASTRIC CANCER

Spleen cells from Balb/c mice immunized with five human gastric cancer cell lines in sequence were fused with murine myeloma cell line SP2/0, and hybridomas 3F4, 3G9 and 3H11, secreting monoclonal antibodiees (MoAbs) against gastric cancer, were obtained through selective culture and screening. These MoAbs have both good selectivity and a high positive rate of reaction for gastric cancer, reaching 5/5 and 84.8% to 93.5% for gastric cancer cells and tissues respectively. The reaction of MoAbs with normal cells and tissues was neglible.The corresponding antigens of the MoAbs were sensitive to digestion by trypsin and pronase and resistant to treatment with sodium periodate, indicating their nature as proteins. The antigen 3G9 could be visualized with Western blotting as two bands with molecular weights of 100KD and 70KD, however no band was found for antigens 3F4 and 3H11. There was a high expression of antigens in the majority of gastric cancer cells and tissues independent of his-topathological type of gastric cancer. A low expression of antigens was seen with other tumors and fetal gastrointestinal tissues. These could be considered as gastric cancer-associated antigens or on-cofetal antigens with a quite extensive distribution.

CYTOTOXICITY OF INDIRECT IMMUNOTOXIN MEDIATED BY ANTI-GASTRIC CANCER MONOCLONAL ANTIBODIES ON TUMOR CELLS

In the present study, an indirect assay was employed to investigate 5 anti-gastric cancer monoclonal antibodies for their cytotoxic potential as ricin A chain-containing immunotoxins. The tumor cell, were treated with dilutions of tested antibody followed by ricin A chain coupled to goat anti-mouse immunoglobulin. The cytotoxic effect was determined with tetrazolium colorimetric assay. The results showed that among the 5 antibodies chosen, MGb2 and MG7 could be well used for preparation of effective A chain immunotoxins.

LABELING McAb 3H11 AND ITS Fab FRAGMENT WITH ^（211）At AND THEIR IMMUNOREACTIVITIES AND INJURY EFFECTS ON HUMAN GASTRIC CANCER CELLS

An antigastric cancer monoclonal antibody, 3H11, and its Fab fragment, were labeled using p-(211At)-astatobenzoic acid (pAtBA) intermediate, in the yields of more than 30%. The results of in vitro experiments show that 211At-3H11 and 211At-3H11Fab are stable, and have specific immunoreactivities and cytotoxic effects to human gastric cancer cell M85. The cytotoxic effects are dependent on the concentration of 211At-3H11 or 211At-3H11 Fab and obviously stronger than that of Na211At.

THE SPECIFIC PHOTODYNAMIC EFFECTS OF MONOCLONAL ANTIBODIES CONJUGATED WITH HEMATOPORPHYRIN DERIVATIVE ON GASTRIC CANCER IN VITRO AND IN VIVO

Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated with a hematoporphyrin derivative (HPD) by using carbodiimide. The qualitative analysis of this conjugate showed that the amount of free HPD was negligible and there were no IgG aggregates among the conjugates. The conjugate retained both the antibody and photochemical activity of HPD.In vitro, the phototoxic effect of this HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The quality of selective photocytotoxicity was proven by the greater cytotoxi-city this conjugate showed than that of corresponding normal mouse IgG (NIgG) conjugated with HPD. It showed less cytotoxicity to colon cancer cell line B-80 (negative reaction to MoAb BB4.3) than to BGC825. Moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody, but not by another MoAb 3G9, which combines with BGC823 at different binding sites from MoAb BB4.3.Nude mice inoculated with 2 × 10- BGC823 cells were given HPD-BB4.3, HPD, HPD-NIgG, HPD plus BB4.3 and PBS, respectively then exposed to light. Four out of six animals treated with the HPD-BB4.3 conjugate remained tumor-free for a long period. Although two developed tumors, there was a significant difference between the HPD-BB4.3-treated group and all the control groups in tumor induction time, tumor growth rate, and survival time (p<0.001). The HPD-BB4.3 conjugate inhibited the growth of established tumors by more than 40% in comparison with control groups (p<0.05).

Gene Cloning,Expression and Immunoreactivity of a Single Chain-Fv-Fragment Antibody PS-9

The gene encoding the heavy- and light-chain Fv regions of monoclonal antibody PS-9, which recognizes a cancer-associated antigen S-Tn on the most adenocarcinoma, was cloned by PCR techniques. The light and heavy chains were connected by a flexible linker to form a single chain variable fragment (ScFv) gene with 720 bp, which was in turn fused to pCANTAB 5 phage. The single chain Fv was expressed as fusion protein displayed on the phage surface. The phagemid is used to transform competent E. Coli TG1 cells, then infected with M13K07 helper phage to rescue the phagemid and antibody ScFv gene. All randomized 12 clones were shown reacting with colon cancer cell line Ls174t, which expresses S-Tn antigen. The recombinant phage has been infected E. Coli HB2151 cells to produce soluble antibody, which can be used for immunodetection and immunotherapy for cancer.

IMMUNOELECTRON MICROSCOPIC STUDY ON THE LOCALIZATION OF MONOCLONAL ANTIBODIES ON GASTRIC CANCER CELLS

The localization of three monoclonal antibodies (MAb) against gastric cancer was studied on two human gastric cancer cell lines by immunoelectron microscopic technique. It has shown that the corresponding antigens of MAb 3G9 and 3H11 were distributed on the microvilli (M) and non-microvillus (NM) plasma membrane of target cells, with various M to NM ratios depending on the MAbs and target cells used. However, the corresponding antigens of MAb PD4 was only localized on the surface of round or finger-like bulges of target cells and never on the microvilli and non-microvillous plasma membrane. Since the nature and function of these tumor antigens have not been identified yet, the implication of the different distributions of these antigens remians to be clarifated.

RADIOIMMUNOIMAGING OF HUMAN BREAST CANCER XENOGRAFTS IN NUDE MICE BY USING HUMAN ANTI-HUMAN BREAST CANCER MONOCLONAL ANTIBODIES

In this study, the human breast cancer-bearing nude mice model has been established and the radioim-munoimaging was carried out by using human anti-human monoclonal antibodies. The results showed that the breast tumor-bearing nude mice received 131I-McAb-CM-l had a clear image of the xenograft during 4 to 6 days after injection and at the same time T/NT all over 1. 0. The highest T/NT reached 7.1. It demonstrates that McAb-CM-1 can specially combine with breast cancer tissues and hopefully it could be used clinically to improve accurate rate of the early breast cancer diagnosis.