Use of cell lines to produce active therapeutic proteins

Abstract

This invention relates to the production of proteins, including therapeutic plasma proteins, by virally-immortalized hepatocyte cell lines and by other eukaryotic cells, as well as to the use of such proteins for screening and therapy, as well as to nucleic acids, vectors, and transformed or transfected cells that carry the genetic information for proteins.

Description

RELATED APPLICATIONS [0001] This application claims the benefit of U.S. provisional application Ser. No. 60 / 510,509, filed on Oct. 10, 2003, which is hereby incorporated in its entirety by reference.GOVERNMENT GRANTS [0002] This invention was made in part with United States government support under grant number 70-NANB7H3070 awarded by Advanced Technology Program of the United States Department of Commerce and with support from a NIH Small Business Innovative Research grant (Grant number: R 1 43 GM66480-01). The U.S. government has certain rights in this invention.FIELD OF THE INVENTION [0003] This invention relates to the use of cell lines, particularly virally-immortalized normal human cell lines, to produce proteins, especially therapeutic proteins, including therapeutic plasma proteins (TPP), that are capable of being expressed in active form by hepatocytes and to the use of proteins, therapeutic proteins, and especially plasma proteins, produced by hepatocytes for the treatment...

AI Technical Summary

Benefits of technology

[0030] The protein can be one that is expressed by a gene that occurs naturally in the hepatocytes, in which case expression of the naturally-occurring gene encoding the protein is enhanced by introduction of a high-level promoter into the hepatocytes.

[0031] Alternatively, expression is enhanced by introducing multiple copies of the gene encoding the protein to be expressed, a subunit of the protein to be expressed, or a precursor of the protein to be expressed via the use of one or more recombinant vectors that include: (1) the gene encoding the protein to be expressed, a subunit of the protein to be expressed, or a precursor of the protein to be expressed; and (2) at least one control element affecting the transcription of the gene, the control element being operably linked to the gene.

[0041] (1) providing a eukaryotic cell, other than a human hepatocyte, that includes DNA that encodes and can express proteins forming an IαIp protein complex, the eukaryotic cell having been transformed or transfected with at least one vector that includes: (a) DNA including at least one gene for a precursor of a protein that is part of an IαIp protein complex; and (b) at least one control element operably linked to the DNA encoding at least one precursor gene in order to enhance expression of the precursor gene;

Problems solved by technology

Although, when processed correctly, blood-derived products are virtually free of transmitting viral infections, a perceived risk exists for the manufacturer, user, and patient.

Although these products have been proven safe and effective, not all behave identically to their native counterparts.

Recent findings suggest that this is the result of incomplete or inappropriate post-translational modification.

Although one strategy to circumvent these shortcomings is to use plasma-derived proteins, there are also perceived risks, as mentioned above, associated with this approach.

Currently, there is only one plasma-derived AAT licensed in the United States, which has been in very limited supply.

Despite the large body of evidence of the clinical efficacy of AAT to treat general inflammatory conditions, its use has been restricted due to the limited availability of the product.

Sepsis is a disease characterized by an overwhelming systemic response to infection, which can rapidly lead to organ dysfunction and ultimately death.

Currently, treatment for sepsis is limited to attempts to manage the underlying infection and supportive therapy if the infection leads to organ dysfunction.

Given the intensive and prolonged care necessary to treat patients with sepsis, the economic burden is profound.

Indeed, some investigators have concluded that any adjunctive therapy is destined to fail because once the clinical signs of severe sepsis are present, irreversible organ injury has already occurred.

In late 2001, Eli Lilly began marketing, Xigris, a genetically engineered version of the human activated protein C molecule; however, this drug only reduces the absolute risk of death by six percent.

Although IαIp can be purified from human serum or plasma, if proven effective there will remain a worldwide shortage of this protein to treat sepsis.

There is presently no means to produce or express highly functional, naturally-processed forms of this IαIp (e.g. naturally occurring or recombinant produced).

Further the complexity of this protein increases the difficulty of both expressing it in an active, processed form and isolating it in an active state.

However, to date, among other things, the prior art cell lines do not provide a means to safely, effectively, and cost efficiently perform the protein post-translational modifications, such as glycosylation, that are critical in the production of functional therapeutic plasma protein; produce simultaneously multiple therapeutic plasma proteins, especially Factor VIII protein or Factor IX, as well as IαIp; and maintain the continuous expression of active levels of cytochrome P450 enzyme in a serum-free media.

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