Human Monoclonal Antibody HB4C5 Research Articles

Availability of oncogene activated production system for mass production of light chain of human antibody in CHO cells.

We previously established a ras-oncogene amplified Chinesehamster ovary (CHO) cell line, named ras clone I, as anuniversal host cell line for oncogene activated production(OAP) system to mass-produce recombinant protein by activationof the cytomegalovirus immediate early (CMV) promoter with ras protein. The lambda light chain(C5lambda) of human monoclonal antibody HB4C5 is expected tobe potentially useful for lung cancer targeting. We generated aC5lambda hyper-producing cell line by transfecting ras cloneI with the C5lambda gene expression plasmid regulated by theCMV promoter, of which productivity was 5.3 times greater thanthe hyper productive CHO cell line generated by using conventional CHO cells. Introduction of the adenovirus E1A geneinto the hyper-producing cell line derived from ras clone I resulted in further 9.5 times enhancement of the productivity,suggesting the synergistic effect of E1A and ras oncogenes on the recombinant protein production driven by the CMV promoter. In addition, intracellular accumulation of C5lambda andupregulation of BiP was found in hyper-producing cell lineswhich were introduced E1A and ras oncogene. This resultsuggests that excessive intracellular accumulation ofC5lambda protein, which might be caused by that the amount of produced C5lambda in ER is beyond the ability of CHO cells to secrete, might signal the BiP promoter. Our data imply that ras clone I is available as a general host cell for establishing the recombinant protein hyper-producing CHOcells by the OAP system, and suggest that further mass production of recombinant proteins in the OAP system can be possible by clarifying the accurate role of upregulated BiP protein.

Recombinant light chain of human monoclonal antibody HB4C5 as a potentially useful lung cancer-targeting vehicle

Recombinant lambda light chain of lung cancer-reacting human monoclonal antibody HB4C5 was expressed in Escherichia coli. Expression in bacteria ensured the generation of homogeneous light chain species devoid of activity-hampering N-linked glycosylation usually found in the light chain CDR-1 of HB4C5. Molecular engineering was also employed to eliminate the C-terminal two amino acid residues, i.e., Cys and Ser, to prevent the formation of lambda light chain dimers which are less reactive than the monomeric form. The lambda light chain was overexpressed in E. coli as inclusion bodies, which were solubilized, refolded, and treated with Aeromonas proteolytica aminopeptidase to remove the N-terminal Met with subsequent natural cyclization of the penultimate Gln residue to pyroglutamate, the same N-terminal end as that of naturally occurring lambda light chain in HB4C5. Monomeric recombinant lambda light chains, both before and after removal of the N-terminal Met residue, were 40 times more immunoreactive than the parent HB4C5. The immunostaining of lung cancer tissue sections with the recombinant lambda light chain indicated cancer-specific reactions to all specimens of adenocarcinoma, squamous cell carcinoma and large cell carcinoma histologies, but did not react with small cell carcinoma. Tumor radioimmunoimaging experiments in LC6 (lung squamous cell carcinoma line)--xenografted nude mice by the i.p. injection of 125I-labeled recombinant lambda light chain and 125I-labeled human lambda light chain control gave tumor-specific and recombinant lambda light chain-dependent images on day 5 postinjection, and images were also detectable on day 3. Biodistribution studies with 125I-labeled recombinant lambda light chain demonstrated that the lambda light chain could penetrate better into the tumor sites, both at the necrotic and solid parts of the xenograft, as compared to our previous results with 125I-labeled HB4C5 which could localize to the necrotic part only. These results suggest that the recombinant lambda light chain is potentially useful as a lung cancer-targeting vehicle, for such as radioimmunoimaging and radioimmunotherapy, with least possible adverse immunogenic effects.

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

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.

Activity enhancement of a lung cancer-associated human monoclonal antibody HB4C5 by N-deglycosylation : Kato M, Mochizuki K, Hashizume S, Tachibana H, Shirahata S, Murakami H. Morinaga Inst Biological Science, 2-1-1 Shimosueyoshi, Tsurumi-ku, Yokohama 230 hum. Antibodies Hybridomas 1993;4:9–14

Activity enhancement of a lung cancer-associated human monoclonal antibody HB4C5 by N-deglycosylation : Kato M, Mochizuki K, Hashizume S, Tachibana H, Shirahata S, Murakami H. Morinaga Inst Biological Science, 2-1-1 Shimosueyoshi, Tsurumi-ku, Yokohama 230 hum. Antibodies Hybridomas 1993;4:9–14

Identification of hybrid-type carbohydrate chains on the light chain of human monoclonal antibody specific to lung adenocarcinoma

The carbohydrate chains on the light chains of human monoclonal antibody HB4C5 reactive to human lung adenocarcinoma tissue have been characterized. The HB4C5 antibody consistst of two kinds of light chains (30 kDa and 32 kDa). Each chain has different carbohydrates of varying molecular masses linked to the variable regions. The 30 kDa light chain has been confirmed to be the activity species for antibody binding. The carbohydrates on the light chains were characterized by lectin blot analysis combined with glycosidase treatment. A carbohydrate chain linked to the active 30 kDa light-chain species, and one of a few carbohydrate phenotypes on the 32 kDa light-chain species is characterized as “hybrid type”. A sialylated complex-type carbohydrate could also bind to the 32 kDa light-chain species. Nucleotide sequence analysis revealed that one potential N-glycosylation site is located in the complementality determining ergion 1 of the light chain, although no such site was found in the variable region of the heavy chain.

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.

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.

Characterization of a lung cancer–associated human monoclonal antibody HB4C5

A human monoclonal antibody HB4C5, which is known to be associated with lung cancers, was characterized. Two kinds of light chains with 30-kD and 32-kD molecular mass were found to comprise the molecule of this IgM antibody. Various combinations of the two light chains could be contained in this IgM antibody molecule, which might have resulted in the heterogeneity of this antibody with different reactivities. The presence of the 30-kD light chain was indispensable for the antibody activity of cancer recognition. Monomeric 180-kD units of the antibody, having 32-kD light chains exclusively, which showed marginal activity of cancer recognition, could be removed by immunosorbent column chromatography with histone H2B-Sepharose 4B. F(ab')2 of the HB4C5 antibody containing the active 30-kD component as the dominant light-chain constituent could be purified by hydroxyapatite chromatography by HPLC, facilitating clinical applications of this lung cancer-associated monoclonal antibody of human origin.

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.

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.