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We report a case of N 3 gastric cancer successfully treated by TS-1 followed by curative resection. The patient was a 64-year-old male. Gastrointestinal endoscopic examination showed advanced gastric cancer. Examination by computed tomography revealed gastric cancer and swollen para-aortic lymph nodes. This patient was treated by neoadjuvant chemotherapy with oral administration of TS-1 (120 mg/day, day 1-28 with 2 weeks rest). After 3 courses of TS-1, the primary lesion and swollen lymph nodes were remarkably reduced. This chemotherapy enabled pancreatoduodenectomy with D 3 lymph node dissection in curative resection. The pathological diagnosis was por, pT 2, pMP and pap, pT 1, pSM 2, pPM(-), pDM(-), pN 1, pStage II and curability A. This neoadjuvant chemotherapy regimen seems to be an effective and promising therapy for patients with advanced gastric cancer.

A 52-year-old woman underwent Auchinclass' operation for breast cancer. The histological type was papillotubular carcinoma. One year and 5 months after operation, multiple liver tumors were found on the CT scan and multiple bone metastasis on MRI. The former were treated by hepatic artery infusion chemotherapy with epirubicin and 5-FU using a subcutaneous implanted pump and the latter by 50 Gy irradiation. The patient began to complain of abdominal pain and discomfort after hepatic artery infusion, so all treatment was discontinued. Six months later the patient died of respiratory failure due to pleural dissemination. No liver mass was detected, and bone metastasis was not changed in section tissues. This suggested that the therapy for a breast cancer patient with distant metastasis must be considered according to the region of recurrence.

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.

The heavy and light chain genes (mu and lambda) of the human anti-adenocarcinoma monoclonal antibody HB4C5 (MAb-C5) were cloned from the human-human hybridoma cell line HB4C5 and co-expressed in Chinese hamster ovary (CHO) and COS-7 cells. ELISA assay and the RI imaging of the cancer tissue xenografted into nude mice showed that the recombinant MAb-C5 retained the original antigen binding activity. We then replaced the IgM constant region of the MAb-C5 heavy chain gene with the human IgG1 constant region gene and co-expressed it with the light chain gene. This recombination was confirmed by a complete DNA sequencing and Western-blot analysis. Despite the fact that the DNA sequence, the expressed protein size, and the assembly of heavy and light chains were indicated to be normal, the IgG1 type MAb-C5 could not bind to the original antigen. This result suggest that this antibody alters its antigen binding property upon class-switching.

We previously determined the amino acid sequence to the epitope (ATLFKTR) of cytochrome c from Candida 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. The in 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.

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.

Human monoclonal antibody (MAb) AE6F4, which had been shown potentially useful for the immunocytological detection of lung cancer cells in sputum, was characterized for its antigen(s). Of the three MAb-reacting materials found in A549 cells by the immunoblotting analysis, the cytoplasmic 31-kDa protein extractable with phosphate-buffered saline was evidenced as the most plausible antigen by its highest content and outstanding affinity to the MAb AE6F4-derivatized Sepharose 4B column. This 31-kDa protein was identified by the amino acid sequence analysis of the CNBr-cleaved fragment as the 14-3-3 family of proteins, the members of which are known to play important physiological roles such as in the regulation of neurotransmitter levels and intracellular signal transduction. The purified 14-3-3 protein from bovine brain showed a comparable MAb-reacting activity to that of the 31-kDa protein from A549 cells in the enzyme-linked immunosorbent assay (ELISA). The significant reactivity of bovine 14-3-3 protein by MAb AE6F4, shown by the cross inhibition of antibody binding to the coated 31-kDa antigen in ELISA as well as by the inhibition of immunostaining with lung cancer tissues, consistently demonstrated that the antigen(s) recognized by the MAb was involved in the 14-3-3 protein family. It was found that the expression of the 14-3-3 protein was significantly enhanced in lung cancer tissues compared with the neighboring normal part of the lung as examined by the immunoblotting method. These results implicated that some member(s) of the 14-3-3 protein family can be the tumor marker(s), providing a rational basis for the immunocytological diagnosis of lung cancer with this human MAb.

It has been known that the lung cancer-associated human monoclonal antibody HB4C5 comprises two lambda light chains of 30 and 32 kD and that the 30 kD species is exclusively responsible for the antibody activity. This study demonstrates that the two light chains were both N-glycosylated with glycosyl residues of different sizes, one of which was sensitive to neuraminidase and the other insensitive. Our unpublished data of DNA sequence for the light chain of this monoclonal antibody indicated that the light chain contains only one possible site for N-glycosylation, which located in the CDR-1. N-Deglycosylation of this monoclonal antibody under the denaturing condition resulted in the complete conversion of the two light chains into one identical polypeptide chain of 26 kD. Activity of this monoclonal antibody was found to be significantly enhanced by N-deglycosylation. All the facts described above consistently indicate that the activity of this monoclonal antibody is interfered with by the attachment of bulky glycosyl residues at the antigen binding site on the light chain. The N-deglycosylated antibody was stable under the storage conditions employed, suggesting that the glycosyl residues attached to the light chain do not play any important biological role except for interference with the antibody activity of binding antigen.

Histone H2B was demonstrated to be an immunoreactive material recognized by the human monoclonal antibody HB4C5, which had been already established to be specific for lung cancers. The inhibitory effect of histone H2B on the activity of HB4C5 antibody to immunostain the cytoplasmic antigen in lung adenocarcinoma tissue indicated that histone H2B at least had antigenic determinant comparable to the cytoplasmic antigen. A mouse anti-histone H2B monoclonal antibody could immunostain the cytoplasm of lung adenocarcinoma cells in sliced tissue sections in the same manner as the human monoclonal antibody HB4C5. Enzyme-linked immunosorbent assay of HB4C5 antibody on plastic immunoplates coated with histone H2B also showed specific reactivity of this antibody with histone H2B, and the reaction was effectively inhibited when extra histone H2B or mouse anti-histone H2B monoclonal antibody was added to the reaction mixture. These results consistently lead us to a conclusion that histone H2B possesses antigenicity to the human monoclonal antibody HB4C5.

Human Namalwa cells were screened in serum-free medium and in 6-thioguanine, then fused with human lymphocytes from lymph nodes of lung adenocarcinoma cancer patients. Extensive testing using 14 lung cancer cell lines, 11 other cancer cell lines and 4 normal fibroblast lines identified monoclonal antibodies produced by 4 hybridoma clones that reacted specifically with lung adenocarcinoma cells. These monoclonal antibodies also reacted with lung adenocarcinoma tissues and not normal tissues or erythrocytes of any blood type. These hybridoma clones grew and stably secreted the antibodies in serum-free medium as well as in serum-containing medium.