Endomannosidases in the modification of glycoproteins in eukaryotes

Abstract

The present invention generally relates to methods of modifying the glycosylation structures of recombinant proteins expressed in fungi or other lower eukaryotes, to more closely resemble the glycosylation of proteins from higher mammals, in particular humans. The present invention also relates to novel enzymes and, nucleic acids encoding them and, hosts engineered to express the enzymes, methods for producing modified glycoproteins in hosts and modified glycoproteins so produced.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 10 / 695,243, filed Oct. 27, 2003, which is now U.S. Pat. No. 7,332,299, and which is a continuation-in-part of U.S. application Ser. No. 10 / 371,877, filed on Feb. 20, 2003.FIELD OF THE INVENTION[0002]The present invention generally relates to methods of modifying the glycosylation structures of recombinant proteins expressed in fungi or other lower eukaryotes, to more closely resemble the glycosylation of proteins from higher mammals, in particular humans. The present invention also relates to novel enzymes and, nucleic acids encoding them and, hosts engineered to express the enzymes, methods for producing modified glycoproteins in hosts and modified glycoproteins so produced.BACKGROUND OF THE INVENTION[0003]After DNA is transcribed and translated into a protein, further post-translational processing involves the attachment of sugar residues, a process known as glycosylation. ...

AI Technical Summary

Problems solved by technology

What has not been solved, however, are the inherent problems associated with all mammalian expression systems.

Fermentation processes based on mammalian cell culture (e.g. CHO, Murine, or more recently, human cells) tend to be very slow (fermentation times in excess of one week are not uncommon), often yield low product titers, require expensive nutrients and cofactors (e.g. bovine fetal serum), are limited by programmed cell death (apoptosis), and often do not allow for the expression of particular therapeutically valuable proteins.

More importantly, mammalian cells are susceptible to viruses that have the potential to be human pathogens and stringent quality controls are required to assure product safety.

While transgenic goats and other transgenic animals (mice, sheep, cows, etc.) can be genetically engineered to produce therapeutic proteins at high concentrations in the milk, recovery is burdensome since every batch has to undergo rigorous quality control.

Whereas cells grown in a fermenter are derived from one well characterized Master Cell Bank (MCB), transgenic technology relies on different animals and thus is inherently non-uniform.

Furthermore, external factors such as different food uptake, disease and lack of homogeneity within a herd may affect glycosylation patterns of the final product.

However, high level expression of proteins in plants suffers from gene silencing, a mechanism by which highly expressed proteins are down regulated in subsequent generations.

In addition, it is known that plants add xylose and a-1,3 linked fucose, a glycosylation pattern that is usually not found in human glycoproteins, and has shown to lead to immunogenic side effects in higher mammals.

Growing transgenic plants in an open field does not constitute a well-controlled production environment.

While recombinant proteins similar to their human counterparts can be made in mammalian expression systems, it is currently not possible to make proteins with a humanoid glycosylation pattern in lower eukaryotes (e.g., fungi and yeast).

Maras and Contreras have shown recently that P. pastoris is not inherently able to produce useful quantities (greater than 5%) of GlcNAcTransferase I accepting carbohydrate.

However, this attempt resulted in little or no production of the desired Man5GlcNAc2.

As only the Man5GlcNAc2 glycans are susceptible to further enzymatic conversion to human glycoforms, this approach is very inefficient for the following reasons: In proteins having a single N-glycosylation site, at least 73% of all N-glycans will not be available for modification by GlcNAc transferase I. In a protein having two or three N-glycosylation sites, at least 93% or 98%, respectively, would not be accessible for modification by GlcNAc transferase I. Such low efficiencies of conversion are unsatisfactory for the production of therapeutic agents; given the large number of modifying steps each cloned enzyme needs to function at highest possible efficiency.

This may, in part, be due to the inefficient processing of glycans in the ER either by glucosidase I, II or resident ER mannosidase.

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