Katsumi Mochizuki

Activity enhancement of a lung cancer-associated human monoclonal antibody HB4C5 by N-deglycosylation.

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 Our unpublished data of DNA sequence for the light chain of this monoclonal antibody indicated that the light chain contains only one possible site for N-glycosylation, which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.

Enhanced production of human monoclonal antibodies by the use of fructose in serum-free hybridoma culture media.

It was found that the production of human monoclonal antibodies (MoAbs) by human-human hybridomas can be significantly enhanced by replacing glucose with fructose in the dish culture medium. Optimization of initial concentrations of fructose and glutamine, another influencing factor for MoAb production, enabled an enhanced production of human MoAb 2.1 times higher than that obtained using the conventional culture media employing glucose. It was shown by kinetic analysis that enhanced MoAb production at the optimum fructose concentration can be attributed to the retention of high specific antibody production rates and diminished time lag during the course of culture.These dish culture results with fructose-containing medium were successfully applied to the continuous perfusion culture with a slight modification, where 2.9- and 1.9-fold enhancements in specific antibody production rate and MoAb concentration, respectively, were attained as compared with the conventional glucose-containing medium.An inverse relationship was observed between the secreted concentrations of lactic acid and MoAb when the hybridoma was cultured in the media containing varying concentrations of fructose, i.e., the lower the lactic acid concentration, the higher the MoAb production andvice versa, suggesting that fructose at appropriate concentrations in the medium can serve as an alternative sugar for the efficient production of human MoAbs, with reduced pH shifts, for the serum-free culture of human-human hybridomas.

Histone H2B as an antigen recognized by lung cancer–specific human monoclonal antibody HB4C5

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.

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.

Class-switching of the IgM type antiadenocarcinoma human antibody HB4C5 into an IgG1 type resulted in the loss of the antigen binding ability

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.

Serodiagnosis of cancer by using Candida cytochrome c recognized by human monoclonal antibody HB4C5

Cytochrome c from various sources, such as Candida krusei, yeast, horse, and cattle, was found to be recognized by human monoclonal antibody HB4C5 specific to lung cancer. Therefore, the cytochrome c was applied to the measurement of antibody amount in patient sera with a similar reactivity to the antibody HB4C5 for serodiagnosis of cancer. The cytochrome c from Candida krusei was most valuable for the serodiagnosis of various cancers, and the yeast cytochrome c was also useful. However, horse and bovine cytochrome c did not react with antibody of the cancer patients. By using Candida cytochrome c lung, bile duct, esophagus, and liver cancers were detected at high rates of more than 50%. In the case of lung cancer, the detection rates of small-cell, squamous, large-cell and adenocarcinoma were 78%, 63%, 100%, and 34%, respectively. The rate for small-cell carcinoma was higher than that with the currently used NSE assay system, and the rate for squamous carcinoma was comparable to that with the SCC assay system, although the system using cytochrome c did not show similar reactivity to that with the SCC system. Furthermore, lung cancer was detected at early stages by using cytochrome c, and even in the case of adenocarcinoma, the rate at early stages with the cytochrome c system was higher than that with the CEA assay system. On the other hand, false positive rates of benign diseases and normal were low--8% and 2%, respectively.

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.

Serodiagnosis of cancer using porcine carboxypeptidase A as an animal antigen recognized by human monoclonal antibody HB4C5

Protein from hog which is recognized by human monoclonal antibody (HB4C5), generated from a patient with large cell lung carcinoma, was identified as carboxypeptidase A by comparison of the protein with carboxypeptidase A in enzymatic activity, immunologic reactivity, and amino acid sequence. Carboxypeptidase A activity was also found in human cancer tissue, and purified antigen from cancer tissue recognized by the antibody HB4C5 was reacted with rabbit anti-carboxypeptidase A serum, indicating that carboxypeptidase A is an antigen of HB4C5. Since large amounts of carboxypeptidase A can be obtained from porcine sources, a simple method for its purification was established. The fraction which was most reactive with HB4C5 was obtained from acetone powder of porcine pancreas by successive applications of water extraction, ammonium sulfate precipitation, trypsin treatment, and Mono Q column chromatography. Its apparent molecular weight was 40,000, according to SDS polyacrylamide gel electrophoresis. When the reactivity of IgG in sera with the purified carboxypeptidase A was measured, the detection rates for lung, ovary, larynx, uterus, and liver cancer were more than 50%, while the rates for stomach and breast cancer were around 30%, and pancreatic cancer, benign diseases, and normal controls were minimally detected.

A human monoclonal antibody to carbohydrate moiety of carcinoembryonic antigen

Hybridoma BSRF-S-97, secreting a human monoclonal antibody of IgG1 subclass reactive to the carcinoembryonic antigen, was generated by fusing the regional lymph node lymphocytes from a cervical cancer patient with RF-S1 human-mouse heteromyeloma fusion line. This monoclonal antibody was found specifically reactive to carinoembryonic antigen-producing cell lines, including those of cervical cancer (SKG-II), mucinous type ovarian cancer (RMUG-L), stomach cancer (MKN-45), and lung cancer (PC-10). The monoclonal antibody reactivity with pepsin- and periodate-treated carcinoembryonic antigen demonstrated that this monoclonal antibody recognizes the carbohydrate moiety of carcinoembryonic antigen specifically. Possibilities of the monoclonal antibody reaction with mucin and blood-group antigens were excluded by the comparative studies with a placental mucin-containing protein which reacted with carcinoembryonic antigen-specific rabbit polyclonal antibody. The monoclonal antibody conjugated with Pseudomonas exotoxin showed potent regression effects on the growth of the MKN-45 cell line in both the dish culture and xenografted nude mice, indicating potential usefulness of this human monoclonal antibody as a promising tumor targeting vehicle.