Production of a monoclonal antibody inhibiting the killer activity.

We established the hybridoma producing the monoclonal antibody (mAb) 3C3 by immunizing rats with mouse natural killer (NK)-like cells. The 3C3 mAb seemed to react mainly with T cells and T-lineage cell lines. The 3C3 antigen also seemed to be coincidentally expressed on a part of asialo GM1+ cells from nude mice, suggesting its expression on NK cells. Treatment of effector cells with 3C3 mAb markedly inhibited the killer activity against RL male-1 cells, but less so against YAC-1 cells, in vitro. It is suggested that the cell surface molecule defined by 3C3 mAb was closely associated with the killer activity of T cells and NK cells.

Establishment of hybridoma cells with natural killer(NK)-like activity against syngenic tumor cells.

The 4D1D4 hybridoma cells were derived from the fusion of spleen cells from BALB/c nude mice with NS-1 mouse myeloma cells. The surface phenotypes of 4D1D4 hybridoma cells were Thy-1.2+, L3T4 (CD4)-, Lyt-2 (CD8)-, Asialo GM1+ and p-55 interleukin-2 (IL-2) receptor (CD25)-. This phenotypic pattern was consistent with the surface phenotype of NK cells. The 4D1D4 cells showed the definite killer activity against a syngenic tumor cell line, RL male-1, but not against an allogenic YAC-1 line. The killer activity of the 4D1D4 cells was not affected by the addition of exogenous IL-2. It was, therefore, suggested that 4D1D4 cells might be representative of resting NK cells with expression of no functional IL-2 receptors. The hybridoma technology might be useful for establishment of the cloned NK cells.

Molecular cloning of the gene coding for the human T cell differentiation antigen CD7.

The CD7 molecule is a differentiation antigen found on the surface of T lymphocytes and also on a very minor fraction of acute nonlymphocytic leukemia (ANLL). To study the genomic structure of the CD7 gene, two clones (SY4 and SY22) were isolated by screening a genomic library with a CD7 cDNA probe. Restriction mapping of these two phage clones showed that both overlapped each other, covering a total length of 23 kilobases (kb). Transfection of mouse L cells demonstrated that SY22 contains the gene expressing the CD7 antigen reactive with monoclonal CD7 antibody (Tp40), while SY4 does not. Subcloning of a 10.5 kb fragment from a 14.4 kb insert of SY22 contained the structural gene for the CD7 antigen. Detailed restriction mapping and partial sequence analysis revealed the CD7 gene to consist of four exons. By RNase protection assay, multiple initiation sites -122 base pairs (bp) to -38 bp from ATG translation initiation site were demonstrated. The promoter region had high G + C content and contained two SP1 binding sites (CCGCCC) and an AP2 binding site (CCCCAGGC), but lacked CAAT and TATA motifs.

Production of interleukin-2 by YAC-1 cells stimulated with interleukin-1 and its augmentation of the natural killer activity.

The production of interleukin-2 (IL-2) by YAC-1 cells stimulated with interleukin-1 (IL-1) was examined in the in vitro culture system. The IL-2 activity was detectable in the culture supernatant of YAC-1 cells stimulated with either a mouse IL-1 preparation or human purified IL-1. This activity could be detected 1 h after stimulation with IL-1. The addition of monoclonal antibody reactive with mouse IL-2 receptor completely blocked the IL-2 activity in the culture supernatant of IL-1-stimulated YAC-1 cells. Further, the culture supernatant of IL-1-stimulated YAC-1 cells augmented the NK activity in mouse spleen cells. The role of the IL-2 activity in the culture supernatant of IL-1-stimulated YAC-1 cells on augmentation of the NK activity is discussed.

A human monoclonal antibody recognizing a surface antigen on stomach cancer cells.

Lymph-node lymphocytes of a patient with stomach cancer were fused with the mouse-human heterohybridoma, HM-5. A clone (2F9) was isolated that showed stable production of an IgM antibody reactive with NUGC-4 stomach cancer cell line. This antibody reacted predominantly with a cell surface antigen on cell lines originating from gastro-intestinal cancer and adenocarcinoma of lung, whereas it was not generally reactive with other types of cancers, or with normal kidney cells or fibroblasts. Biotin-labeled 2F9 antibody clearly stained cell smears and the nude mouse tumor of NUGC-4, but it did not show a positive reaction with stomach cancer tissues obtained from more than 10 patients, indicating that the antigen detected is very weakly expressed on tumor cells or on a limited number of stomach cancers. The antigen shed from NUGC-4 cell line was detected in the culture supernatant. 2F9 antibody precipitated a glycoprotein with a molecular weight of over 200 kilodaltons as well as a possible glycolipid, from NUGC-4 cells labeled with [3H]glucosamine or [35S]-H2SO4. Periodic acid treatment of the tissue section decreased reactivity with 2F9 antibody, but heat, neuraminidase or protease treatment did not. These results suggested that the epitope is present on a carbohydrate moiety not containing sialic acid, and that a part of the antigen molecule is sulfated.

Action of 7-ethylcamptothecin on tumor cells and its disposition in mice.

Some biological effects of camptothecin (CPT) and its new derivative 7-ethylcamptothecin (ECPT) were studied. The drugs were effective against murine leukemia; ECPT was more effective than CPT. Ip administration of ECPT or CPT gave maximum treated/control values of 325% and 232%, respectively. The drugs also inhibited the growth of KB cells in vitro, 50% effective doses of 3.5 ng/ml of ECPT and 8.6 ng/ml of CPT, indicating the stronger activity of ECPT. Pharmacokinetic studies of the drugs in mice showed that ECPT had a longer biological half-life in the terminal phase and a larger amount remained in the plasma compared with CPT. After iv administration of ECPT, the drug accumulated in the intestine, suggesting that the main route of excretion of the drug is through the biliary tract. The study on cell cycle progression by flow cytometry suggested that the main effect of both drugs on L1210 cells was the blocking of G2-M phase. These results suggest that the main reasons for the superior antitumor activity of ECPT compared with CPT are as follows: (a) ECPT had a stronger growth-inhibiting activity against tumor cells, and (b) ECPT remained in the intestinal tract for a longer time and in higher amounts when administered in vivo.

Human monoclonal antibody reactive to stomach cancer produced by mouse-human hybridoma technique.

Production of human monoclonal antibodies reactive to stomach cancer was attempted by the hybridoma technique using splenic lymphocytes from stomach cancer patients. The parental cells used were NS-1 mouse myeloma line and three human lines including RPMI-1788 6TGR, which was established in our laboratories. Ten mouse-human and two human-human (from the fusion with RPMI-1788 6TGR) hybridomas have been producing IgM antibody for over 18 months, and all the heterohybridomas yielded ascites when transplanted into nude mice. Four antibodies produced by the heterohybridomas were selected and analyzed. These 4 antibodies, 3F6, 4A10, 3H5 and 1F9, reacted predominantly to cytoplasmic antigens of stomach and other epithelial cancer lines. The reactivity against human tumors transplantable in nude mice showed that all antibodies but 3F6 were reactive with stomach and lung cancers. Smears prepared from normal and cancer tissues were also tested, and these 4 antibodies showed positive reactions not only to stomach cancer, but also to normal stomach and colon. The reactivity against fetal tissues demonstrated that 3H5 antibody was reactive with epithelium of the stomach, and 1F9 antibody was positive with epithelium of the respiratory tract and bile duct, but the other two were negative. Thus, the serological analysis showed that the antigens detected are not tumor-specific, but are differentiation antigens. Chromosome analysis of these 4 mouse-human hybridomas and another one, which seems to produce an antibody against keratin, showed that three retained human chromosome 14 on which immunoglobulin heavy chain (Ig H) gene is located, but two did not. Southern blot analysis, however, revealed that all 5 hybridomas had a human Ig H gene.

Fusion of SV40-induced endocytotic vacuoles with the nuclear membrane.

The interaction between simian virus 40(SV40)-induced endocytotic vacuoles and the nuclear membrane was investigated using cationized ferritin (CF) and concanavalin A (Con A) as cell membrane markers. These markers bound to the cell surfaces of CV-1 cells together with SV40 at 4 degrees C. Following incubation of these modified cells at 37 degrees C in serum-free medium, the cell membranes showed many invaginations. After incubation for 60 min at 37 degrees C in the same medium, many various-sized vacuoles were present that contained membrane-bound CF, Con A and SV40. After 2 h of incubation at 37 degrees C, Con A was present in some areas of the perinuclear cisterna along the nuclear membrane. The control experiment, however, showed no localization of Con A-binding on the nuclear membrane. These results provide evidence that SV40-induced endocytotic vacuoles migrate toward the nucleus and fuse with its membrane.

Formation of symbiotic complex by microenvironment-dependent mouse leukemias and thymic epithelial reticular cells.

Developing thymic leukemias of the mouse have been assumed to form symbiotic complexes with thymic microenvironments. This symbiosis is morphologically based on pseudoemperipolesis (PEMP). The mechanism of the association of microenvironment-dependent leukemia cells with thymic epithelial reticular cells (TER) was analyzed in vitro by scanning electron microscopy, microcinematography, and a quantitative assessment of PEMP. PEMP was a consequence of active locomotion of the leukemia cells, with TER passively accepting the leukemia cells "crawling" under their cytoplasm. The integrity of the cytoskeletal system of both cells was essentially required for PEMP, since cytochalasins and colchicine were highly inhibitory to PEMP. The mechanism of action of these compounds was probably dual: inhibition of the locomotive movements of the leukemia cells. A similar inhibition of PEMP was also observed with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate.

Natural antibodies to human retrovirus HTLV in a cluster of Japanese patients with adult T cell leukemia.

Human T cell lymphoma leukemia virus (HTLV) is a human retrovirus (RNA tumor virus) that was originally isolated from a few patients with leukemias or lymphomas involving mature T lymphocytes. Here we report that the serum of Japanese patients with adult T cell leukemia, but not the serum of tested normal donors, contains high titers of antibodies to HTLV. These observations, together with data from Japan showing that adult T cell leukemia is endemic in southwest Japan, suggest that HTLV is involved in a subtype of human T cell malignancy, including Japanese adult T cell leukemia.

Lack of participation of murine myeloma cells in the fusion of cocultured XC cells.

Electron microscopy of thin sections of syncytia, formed by fusion of homologous XC cells, clarified that these heterologous cells were simply invaginated by syncytia and that they were clearly separated by the limiting cytoplasmic membranes. The C particles found at the boundary showed no sign of connection with the surrounding syncytial cell membrane, while some were seen budding from the surface of the producer cells. These results strongly suggest that the XC cells, even when cocultured with the C-type virus-producing myeloma cells, fuse homologously among themselves, and the virus-producing heterologous cells do not participate in the event.

Fusion of XC cells by murine type-C viruses: cinematographic observations.

Continuous observations on the processes of multikaryon formation by purified murine type-C virus, produced by a line of murine myeloma cell cultures and Rauscher leukemia virus, were carried out by cinematography. The process first began by fusion of two mononucleated cells and additional cells continued to introduce their cytoplasm and nucleus one after another to give rise to multinucleated giant cells. These findings strongly suggested that the formation of multikaryons is due to a cellular fusion process and not to endomitosis.