Production of modified glycoproteins having multiple antennary structures

Abstract

The present invention relates to eukaryotic host cells, especially lower eukaryotic host cells, having modified oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar and sugar nucleotide transporters to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified lipid-linked oligosaccharides are created or selected. N-glycans made in the engineered host cells exhibit GnTIII, GnTIV, GnTV, GnT VI or GnTIX activity, which produce bisected and / or multiantennary N-glycan structures and may be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, sugar, sugar nucleotide transporters, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 10 / 546,101, which is a national stage filing under 35 U.S.C. § 371 of International Application No. PCT / US04 / 005191, filed Feb. 20, 2004, which designated the United states and is a continuation-in-part (“CIP”) of U.S. Ser. No. 10 / 680,963, filed on Oct. 7, 2003, which is a CIP of U.S. application Ser. No. 10 / 500,240, filed Jun. 25, 2004, which is a national stage filing of International Application No. PCT / US02 / 41510, filed Dec. 24, 2002, which claims benefit of U.S. Provisional Application No. 60 / 344,169, filed Dec. 27, 2001. U.S. Ser. No. 10 / 680,963 is also a CIP of U.S. application Ser. No. 10 / 371,877, filed on Feb. 20, 2003, which is a CIP of U.S. application Ser. No. 09 / 892,591, filed Jun. 27, 2001, which claims the benefit of U.S. Provisional Application No. 60 / 214,358, filed Jun. 28, 2000, U.S. Provisional Application No. 60 / 215,638, filed Jun. 30, 2000, and U.S. Provi...

AI Technical Summary

Benefits of technology

[0037]The present invention thus provides a glycoprotein production method using (1) a lower eukaryotic host such as a unicellular or filamentous fungus, or (2) any non-human eukaryotic organism that has a different glycosylation pattern from humans, to modify the glycosylation composition and structures of the proteins made in a host organism (“host cell”) so that they resemble more closely carbohydrate structures found in mammalian, e.g., human proteins. The process allows one to obtain an engineered host cell which can be used to express and target any desirable gene(s), e.g., one involved in glycosylation, by methods that are well-established in the scientific literature and generally known to the artisan in the field of protein expression. Host cells with modified oligosaccharides are created or selected. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

Problems solved by technology

Improper localization of a glycosylation enzyme may prevent proper functioning of the enzyme in the pathway.

Alternatively, the host organism may not provide an adequate level of UDP-GlcNAc in the Golgi or the enzyme may be properly localized but nevertheless inactive in its new environment.

The mere presence of Man5GlcNAc2, therefore, does not assure that further in vivo processing of Man5GlcNAc2 can be achieved.

To date, there is no reliable way of predicting whether a particular heterologously expressed glycosyltransferase or mannosidase in a lower eukaryote will be (1), sufficiently translated (2), catalytically active or (3) located to the proper organelle within the secretory pathway.

Inherent problems associated with all mammalian expression systems have not been solved.

Therapeutic glycoproteins produced in a microorganism host such as yeast utilizing the endogenous host glycosylation pathway differ structurally from those produced in mammalian cells and typically show greatly reduced therapeutic efficacy.

However, N-glycans resembling those made in human cells were not obtained.

However, Man8GlcNAc2 is not a substrate for mammalian glycosyltransferases, such as human UDP-GlcNAc transferase I, and accordingly, the use of that mutant strain, in itself, is not useful for producing mammalian-like proteins, i.e., those with complex or hybrid glycosylation patterns.

While this structure has utility, the method has the disadvantage that numerous enzymatic steps must be performed in vitro, which is costly and time-consuming.

Isolated enzymes are expensive to prepare and need costly substrates (e.g., UDP-GlcNAc).

The method also does not allow for the production of complex glycans on a desired protein.

However, to date, no reports exist that show the high level in vivo trimming of Man8GlcNAc2 to Man5GlcNAc2 on a secreted glycoprotein from P. pastoris.

Moreover, the mere presence of an α-1,2-mannosidase in the cell does not, by itself, ensure proper intracellular trimming of Man8GlcNAc2 to Man5GlcNAc2.

Accordingly, mere localization of a mannosidase in the ER or Golgi is insufficient to ensure activity of the respective enzyme in that targeted organelle.

Re-engineering glycoforms of immunoglobulins expressed by mammalian cells is a tedious and cumbersome task.

Such a growth-inhibition effect complicates the ability to coexpress the target protein and GnTIII and may impose an upper limit on GnTIII overexpression.

A major shortcoming of these cells, however, is that they are incapable of producing any one homogenous glycoform in high yield.

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