Seiji Kawamoto

Effectiveness of vitamin A acetate for enhancing the production of lung cancer specific monoclonal antibodies

The antibody productivity of the human–human hybridoma cell line AE6, which produces the lung cancer specific human monoclonal antibody AE6F4, was enhanced fourfold upon stimulation with 1 μg/ml of vitamin A acetate for one day. The enhancement lasted for about two weeks, and could be repeated by another stimulation with vitamin A acetate. The enhancing effect of vitamin A acetate was influenced by the cell density. Enhancement was clearly observed when the cell density was under 106 cells/ml. However, when the cell density was over 107 cells/ml, enhancement was observed weakly or not at all. Although the enhancing effect of vitamin A acetate is not unique to AE6 cells, not all human–human hybridoma cell lines show increased productivity upon VA acetate stimulation. This study suggests that the response to vitamin A acetate may be related to the properties of a particular fusion partner which the hybridoma cell inherits. The efficacy of vitamin A acetate for production of human monoclonal antibodies using human–human hybridomas is discussed.

Cytokeratin 8 and 19 as antigens recognized by adenocarcinoma-reactive human monoclonal antibody AE6F4

The human monoclonal antibody (MAb) AE6F4 is secreted by a human-human hybridoma line established from the in vitro immunization of normal human peripheral blood lymphocytes with the human lung adenocarcinoma cell line, A549. This MAb is strongly reactive to lung cancer tissues. In the previous study, the antigens recognized by the MAb AE6F4 were purified from A549 cells and identified as 14-3-3 protein and 31 kDa cytosolic phospholipase A2 (cPLA2). The MAb AE6F4 also binds two kinds of antigens (53 kDa and 40 kDa), which are not related to 14-3-3 protein or 31 kDa cPLA2, in the human breast adenocarcinoma cell line, MCF-7. We purified a 38 kDa antigen, which is a degradation product of 53 kDa antigen from breast adenocarcinoma MCF-7 cells using ion-exchange and hydroxyapatite column chromatography. Two partial amino acid sequences of the purified 38 kDa antigen showed 95-100% homology to human cytokeratin 8 (CK8). Two-dimensional gel electrophoresis and immunoblot analysis of intermediate filament fraction separated from MCF-7 cells demonstrated that the 53 kDa and 40 kDa antigens were CK8 and CK19, respectively. Antigenic determinants on CK8 and CK19 recognized by the MAb AE6F4 were resistant to sodium periodate treatment, although antigenic determinant on 31 kDa antigen (14-3-3 protein and(or) cPLA2) was sensitive to this treatment. These results suggest that the MAb AE6F4 reacts with both carbohydrate and peptide antigenic determinants.

Molecular cloning of the 31 kDa cytosolic phospholipase A2, as an antigen recognized by the lung cancer-specific human monoclonal antibody, AE6F4

The human monoclonal antibody AE6F4 specifically reacts with human lung cancer tissues but does not with normal tissues. This monoclonal antibody recognizes a cytosolic 31 kDa antigen in the cancer cells. In a previous study, we elucidated that the 31 kDa antigen belonged to a family of proteins collectively designated as 14-3-3 proteins, which were known as protein kinase-dependent activators of tyrosine/trytophan hydroxylases, or protein kinase C inhibitor proteins. Here we report molecular cloning of the 31 kDa antigen from the human lung adenocarcinoma cell line, A549. Sequencing analysis indicates that the cloned cDNA is identical to that of previously reported human placental cytosolic phospholipase A2 (cPLA2), which is also a member of the 14-3-3 protein family. Western analysis demonstrated that a 31 kDa recombinant cPLA2 expressed in monkey COS cells was recognized by the AE6F4 monoclonal antibody. Binding of the monoclonal antibody to the recombinant cPLA2 was abolished when treated with sodium periodate, suggesting that not only are carbohydrate chains associated with the cPLA2, but they also play a crucial role in antigen recognition by the monoclonal antibody.

Lung cancer-reacting human recombinant antibody AE6F4: Potential usefulness in the sputum cytodiagnosis

Human monoclonal antibody (hMAb) AE6F4 has been shown to be potentially useful for immunocytological detection of lung cancer cells in sputum. By recombinant DNA technology, IgM type hMAb AE6F4 was switched to lgG. The IgG mimic recombinant AE6F4 antibody expression plasmid was assembled using the antibody heavy chain gene, which ligated the gene encoding VH and CH1(mu) domains of hMAb AE6F4 heavy chain to the gene encoding CH2(gamma 1) and CH3(gamma 1) domains of human IgG heavy chain, and the antibody light chain gene of hMAb AE6F4. The recombinant antibody expressed by baby hamster kidney (BHK)-21 cells showed molecular size equivalence to IgG, and consisted of human mu-gamma hybrid heavy and kappa light chains. The immunological specificity of the recombinant antibody was the same as that of hMAb AE6F4 by immunoblotting analysis to the 14-3-3 protein, the putative antigen of hMAb AE6F4, and by immunohistochemical and immunocytological analyses using tissue sections and sputa of lung cancer patients. The transfected BHK-21 cells produced the recombinant antibody persistently and the productivity was greater than 20 times that by human-human hybridoma producing hMAb AE6F4.

Production of a recombinant human monoclonal antibody using a novel hollow fiber bioreactor system

The amplified ras oncogene was used to enhance the productivity of BHK-21 cells producing a lung cancer-reactive human recombinant AE6F4 monoclonal antibody. The recombinant antibody (18 mg) was produced by the ras-amplified BHK-21 cells in a one-month high-density culture using the novel hollow fiber bioreactor system Tecnomouse.

Effect of 14-3-3 protein induction on cell proliferation of A549 human lung adenocarcinoma

We have previously shown that 14-3-3 protein, amultifunctional adaptor molecule involved in many aspects ofsignal transduction pathways, is a target antigen for thecancer-associated human monoclonal antibody. Although recentevidences suggest a crucial role of 14-3-3 family members inthe control of cell growth and differentiation, their actualcontribution toward tumor development is still controversial. Inthis article, we examined the effect of enforced 14-3-3overexpression on cell growth of the human lung adenocarcinomacell line, A549. To address this issue, we obtained14-3-3 protein-inducible A549 sublines by transfection with14-3-3 expression vector under the control ofdexamethasone-inducible promoter. We found that 14-3-3 proteininduction in some of these sublines promoted their cell proliferation. Microscopic observation revealed that morphologyof these cells became aggressive multilayer condition,suggesting that malignant phenotypes are also acquired uponectopic induction of 14-3-3 protein.

Molecular cloning of yeast cytochrome C-like polypeptide expressed in human lung carcinoma: An antigen recogizable by lung cancer-specific human monoclonal antibody

SummaryWe previously determined the amino acid sequence to the epitope (ATLFKTR) of cytochrome c fromCandida krusei, which is cross-reactive to the lung cancer-specific human monoclonal antibody HB4C5. Here we report that an antigen messenger RNA, which codes for a structure similar to the cytochrome c epitope, is expressed in the human lung adenocarcinoma A549. Sequencing analysis has revealed that this messenger RNA encodes a novel 190 amino acid polypeptide of 21-kDa containing an amino acid sequence (ALLFFT) similar to the cytochrome c epitope, although the total messenger RNA sequence is apparently different from the cytochrome c messenger RNA. Western analysis indicated that an antibody-recognizable 21-kDa antigen which has the same molecular weight as the predicted polypeptide is expressed in the A549 adenocarcinoma. Thein vitro translated product of the antigen messenger RNA and synthesized ALLFFT peptide were both shown to be reactive with the monoclonal antibody, indicating that this protein contains the epitope which enables A549 cells to specifically react with the antibody. The antigen mRNA was not expressed in non-transformed fibroblasts, suggesting that the antigen mRNA expression was associated with cellular transformation. Also in part of the antigen nucleotide sequence, there was a segment that had about 90% homology to the long terminal repeat sequence (no. 297–475) of the human endogenous retrovirus HERV-K10, which was related to the mouse mammary tumor virus.

Protein-Free Culture of Ras-Amplified Recombinant BHK-21 Cells

A ras-amplified recombinant BHK-21 cell line (ras-rBHK-IgG), which hyperproduces recombinant human monoclonal antibody, was cultured in a protein-free medium. Protein-free medium for ras-rBHK-IgG cells was found to be superior to serum containing medium in terms of viability and recombinant antibody production. In particular, antibody, production in protein-free culture was shown to be five to ten limes higher than that in serum culture. However, when culturing cells at a high density in the hollow fiber bioreactor system, the enhancement of antibody production was not observed. On the other hand, rm-rBHK-IgG cells couldbe maintained for over a month in protein-free culture using the bioreactor system in contrast with serum culture which only lasted for a half month. The total amount of antibody obtained during cultivation was about two times greater in protein-free culture than in serum culture. Therefore, protein-free culture of ras-rBHK-IgG cells was demonstrated to be effective for middle scale production of recombinant human monoclonal antibody.

Altered Expression of the CIP/KIP Family CDK Inhibitors in Tumor Phenotype-Suppressed Human Lung Cancer Cells

The cyclin-dependent kinase inhibitor (CDKI) plays pivotal roles in the control of mammalian cell cycle progression as well as in tumor suppression. However, there is relatively few evidences describing an involvement of these inhibitors in the suppression of tumor development in humans. Here we focused on the Cip/Kip family CDK inhibitors, and investigated their roles in the tumor suppressive phenotypes observed in human lung cancer-derived cells. We unexpectedly found that p21Wafl/Cipl protein level was decreased concomitant with tumor suppression, although mRNA level of p21 Wafl/Cipl was conversely up-regulated. Observed transcriptional activation of p21 Wafl/Cipl was partly attributable to increased p53 activity. These data imply that post-transcriptional p21Wafl/Cipl down-regulatory mechanism is activated in the tumor phenotype-suppressed lung cancer cells. We also found that serum starvation-triggered p27Kipl induction pathway is exclusively up-regulated concomitant with tumor suppression, whereas cell contact- or TGF-β-responsible p27Kipl induction pathway was unaffected. These data suggest that restriction point control-associated p27Kipl induction is an important pathway for tumor suppression, and that frequently observed p27Kipl down-regulation in human tumors might be attributable to inactivation of this mitogen starvation-responsible pathway.

VITAMIN A ACETATE FOR EFFICIENT PRODUCTION OF MONOCLONAL ANTIBODY BY HUMAN-HUMAN HYBRIDOMAS

Enhancing effect of vitamin A acetate on antibody productivity was examined using human-human hybridoma cell line AE6, BD9 and C5. The effect was maintained for about two weeks by treatment of A E6 hybridomas with 1 μg/ml of vitamin A acetate for a day, and thereafter recovered by its another treatment. The enhancement of antibody production was influenced by cell densities of AE6 hybridomas: two- to four-fold enhancement was observed in cell densities under 106 cells/ml, but hardly observed in those over 108 cells/ml. Therefore, vitamin A acetate might be suitable for middle scale production of human monoclonal antibody by hybridomas sustained at relatively low cell densities. In addition, enhancing effect of vitamin A acetate was also observed in BD9 hybridomas as well as AE6 hybridomas, but not in C5 hybridomas. These observations indicated that the enhancing effect might be limited by hybridoma cell lines.