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Interferon Production from Human Cell Cultures
Abstract
Excerpt
Human proteins required for medical purposes, e.g., insulin, interferons, tissue plasminogen activator, and growth hormone, can now often be produced by recombinant DNA procedures. They can also be obtained by extraction from the appropriate human organ (e.g., growth hormone from pituitaries) or by stimulating human cells grown in culture to make the protein of interest. We pioneered production by the latter approach because we needed to make enough human interferon for large-scale clinical trials; we continue to use this route today, because we think it has certain advantages. To understand our reasons, some points from the history of interferons must be considered.
Discovery and Development of Interferons
Fifty years ago, Hoskins (1935) and Magrassi (1935) independently discovered the interference phenomenon: A virus replicating in an animal's organ, e.g., the brain, prevented that organ during the next few days from superinfection with any of a number of serologically unrelated viruses. Although...
We have optimized the expression of an introduced DNA coding for human erythropoietin (EPO) in stably transfected human B lymphoblastoid Namalwa cells. The results of transient expression of genomic and cDNA for EPO under the control of the SV40 early promoter-enhancer unit suggested that cDNA yields higher expression of EPO than genomic DNA. An intervening sequence was required for efficient expression, although even an intronless transcriptional unit could still express EPO. Stable Namalwa cells that secreted 1 to 2 μg of EPO/106 cells/d were then established by transfection with DNA constructs consisting of the SV40 early promoter, EPO cDNA, and the splicing junction and polyadenylation site derived from the SV40 T antigen gene or the rabbit β-globin gene. One of these transformant cell lines was easily adapted to grow in serum-free medium.
We have achieved high-level expression of human granulocyte-macrophage colony-stimulating factor (GM-CSF) in human lymphoblastoid Namalwa cells by introducing and subsequently amplifying an expression vector encoding human GM-CSF and mouse dihydrofolate reductase (DHFR). Transformants expressing elevated levels of both GM-CSF and DHFR were selected by a step-wise increase in the concentration of methotrexate (MTX). Several cell lines resistant in 800 nM MTX have been established that express GM-CSF at a rate of 10-20 micrograms/10(6) cells/day. The amplified genes are integrated and stably maintained in the chromosomes of these cell lines. Analysis of the GM-CSF produced in the amplified Namalwa cells revealed that the molecular-size distribution due to varied degrees of glycosylation was similar to that observed in the naturally-occurring molecules.
To establish a practical exogenous gene expression system in human cells, a cDNA coding for human erythropoietin (EPO) was expressed in human B-lymphoblastoid Namalwa cells. The Namalwa-derived recombinant EPO was purified from the culture fluid by a simple three-step procedure. The Namalwa EPO showed an equivalent activity in vivo to that of human urinary EPO. Oligosaccharide structure analyses suggested that almost all N-linked oligosaccharide chains of Namalwa EPO are shared by urinary EPO. The two major N-linked oligosaccharides of Namalwa EPO were fucose-containing tetraantennary and fucose-containing triantennary structures.
Recombinant plasmids for the expression of human erythropoietin (EPO) cDNA in Namalwa cells were constructed. From the results of the EPO expression efficiency in transiently transfected cells, it was found that the simian virus 40 (SV40) early promoter directs EPO synthesis more efficiently in Namalwa cells than does the long terminal repeat promoter of Rous sarcoma virus and that the 3'-noncoding sequence including splice junction and polyadenylation site derived from the rabbit beta-globin gene are more effective than those of the SV40 early gene. However, in stable transformants, no simple relationship was found between the expression level of EPO cDNA and the structure of the introduced expression vectors.
Human pro-urokinase (pro-UK) gene was engineered for expression in mammalian cells. The stability of recombinant pro-UKs produced by two kinds of cells, Chinese hamster ovary (CHO) and human lymphoblastoid Namalwa KJM-1 cells, were compared. The pro-UK expressed in CHO cells in serum-free medium was degraded by cysteine endopeptidase secreted by CHO cells. This endopeptidase was inhibited by p-chloromercuribenzonate (PCMB) and leupeptin more efficiently than by aprotinin. On the other hand, the pro-UK expressed in Namalwa KJM-1 cells was not degraded, resulting in the stable production of pro-UK at a rate of 2-3 micrograms/10(6) cells/day by use of a gene amplification method with dihydrofolate reductase (DHFR) in serum-free medium. Thus, Namalwa KJM-1 cells showed the desired characteristics as a host cell for the production of recombinant proteins. The stability of recombinant proteins in heterologous systems may vary depending on the host cells.
Butyric acid, a short chain fatty acid (SCFA), is a natural component of the animal metabolism. Physiological concentrations induce multiple and reversible biological effects. They concern regulatory mechanisms of gene expression conducing to promote markers of cell differentiation, apoptosis and cell growth control. The described hyperacetylation of histones and the induction of several immune or non-immune cell-activating mediators are consistent with the pleiotropic stimulatory effect of the agent. Butyric acid is considered as a biological response modifier (BRM) and is an interesting tool for biological studies. The history of butyric acid as a putative medication in human health is spanning since 60 years and is confusing in part because of conflicting data between exciting experimental results and clinical trials. In light of minimal impact of systemic therapy and the short half-life of the saline molecule used, it is evident that continuous infusions of butyrate are required to improve the efficacy of the treatment. Butyric acid has been viewed with skepticism because of less convenient for long-term chronic therapy. New experimental data from several studies conduced within the past decade with butyric derivatives, delivery systems, and long-acting prodrugs, have demonstrated the practical value of the therapeutic concept. To support issues regarding clinical development, it was of interest to evaluate the recent information, showing butyric acid currently considered as therapeutic purposes in the treatment of colorectal cancer and hemoglobinopathies.
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