U.S. patent application number 14/271475 was filed with the patent office on 2014-08-21 for methods for producing modified glycoproteins.
This patent application is currently assigned to GlycoFi, Inc.. The applicant listed for this patent is GlycoFi, Inc.. Invention is credited to Tillman U. Gerngross.
Application Number | 20140234902 14/271475 |
Document ID | / |
Family ID | 27395978 |
Filed Date | 2014-08-21 |
United States Patent
Application |
20140234902 |
Kind Code |
A1 |
Gerngross; Tillman U. |
August 21, 2014 |
METHODS FOR PRODUCING MODIFIED GLYCOPROTEINS
Abstract
Cell lines having genetically modified glycosylation pathways
that allow them to carry out a sequence of enzymatic reactions,
which mimic the processing of glycoproteins in humans, have been
developed. Recombinant proteins expressed in these engineered hosts
yield glycoproteins more similar, if not substantially identical,
to their human counterparts. The lower eukaryotes, which ordinarily
produce high-mannose containing N-glycans, including unicellular
and multicellular fungi are modified to produce N-glycans such as
Man.sub.5GlcNAc.sub.2 or other structures along human glycosylation
pathways. This is achieved using a combination of engineering
and/or selection of strains which: do not express certain enzymes
which create the undesirable complex structures characteristic of
the fungal glycoproteins, which express exogenous enzymes selected
either to have optimal activity under the conditions present in the
fungi where activity is desired, or which are targeted to an
organelle where optimal activity is achieved, and combinations
thereof wherein the genetically engineered eukaryote expresses
multiple exogenous enzymes required to produce "human-like"
glycoproteins.
Inventors: |
Gerngross; Tillman U.;
(Hanover, NH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GlycoFi, Inc. |
Lebanon |
NH |
US |
|
|
Assignee: |
GlycoFi, Inc.
Lebanon
NH
|
Family ID: |
27395978 |
Appl. No.: |
14/271475 |
Filed: |
May 7, 2014 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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13934551 |
Jul 3, 2013 |
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14271475 |
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13461111 |
May 1, 2012 |
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13934551 |
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11981408 |
Oct 30, 2007 |
8211691 |
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13461111 |
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11240432 |
Sep 30, 2005 |
7326681 |
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11981408 |
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09892591 |
Jun 27, 2001 |
7029872 |
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11240432 |
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60279997 |
Mar 30, 2001 |
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60215638 |
Jun 30, 2000 |
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60214358 |
Jun 28, 2000 |
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Current U.S.
Class: |
435/69.1 ;
435/254.2; 435/254.21; 435/254.22; 435/254.23; 435/254.3;
435/254.6; 506/10 |
Current CPC
Class: |
C12Y 302/01 20130101;
C12N 9/2488 20130101; C12N 9/1048 20130101; A61P 37/00 20180101;
C12N 15/79 20130101; A61P 3/10 20180101; C12N 15/80 20130101; C12N
15/81 20130101; C12N 1/14 20130101; A61P 29/00 20180101; C12P
21/005 20130101; C12Y 302/01113 20130101; C07K 2319/05 20130101;
C07K 2319/04 20130101 |
Class at
Publication: |
435/69.1 ;
506/10; 435/254.23; 435/254.21; 435/254.2; 435/254.22; 435/254.3;
435/254.6 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C12N 15/80 20060101 C12N015/80; C12N 15/81 20060101
C12N015/81 |
Claims
1. A method for producing glycoproteins having carbohydrate
structures similar to those produced by human cells in a lower
eukaryote comprising providing a unicellular or multicellular
fungal host, which does not express one or more enzymes involved in
production of high mannose structures, and introducing into the
host one or more enzymes for production of a carbohydrate structure
selected from the group consisting of Man.sub.5GlcNAc.sub.2,
Man.sub.8GlcNAc.sub.2 and Man.sub.9GlcNAc.sub.2, wherein the
enzymes are selected to have optimal activity at the pH of the
location in the host where the carbohydrate structure is produced
or which are targeted to a subcellular location in the host where
enzyme will have optimal activity to produce the carbohydrate
structure.
2. The method of claim 1 wherein the host is deficient in the
activity of one or more enzymes selected from the group consisting
of mannosyltransferases and phosphomannosyltransferases.
3. The method of claim 2 wherein the host does not express an
enzyme selected from the group consisting of 1,6
mannosyltransferase, 1,3 mannosyltransferase, and 1,2
mannosyltransferase.
4. The method of claim 1 wherein the host is selected from the
group consisting of Pichia pastoris, Pichia finlandica, Pichia
trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia
opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia
guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica,
Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula
polymorpha, Kluyveromyces sp., Candida albicans, Aspergillus
nidulans, and Trichoderma reesei.
5. The method of claim 2 wherein the host is an OCH1 mutant of P.
pastoris.
6. The method of claim 1 comprising introducing into the host a
nucleotide molecule encoding one or more mannosidases involved in
the production of Man.sub.5GlcNAc.sub.2 from Man.sub.8GlcNAc.sub.2
or Man.sub.9GlcNAc.sub.2.
7. The method of claim 6 where the at least one mannosidase has a
pH optimum within 1.4 pH units of the average pH optimum of other
representative enzymes in the organelle in which the mannosidase is
localized, or having optimal activity at a pH between 5.1 and
8.0.
8.-9. (canceled)
10. The method of claim 1 comprising providing a host that is able
to form Man.sub.5GlcNAc.sub.2 structures, displaying GnT I activity
and having UDP-Gn transporter activity.
11. The method of claim 1 comprising providing a host which has a
UDP specific diphosphatase activity.
12. The method of claim 1 comprising introducing into the host one
or more enzymes selected from the group consisting of mannosidases,
glycosyltransferases and glycosidases, wherein the enzymes are
targeted to the endoplasmic reticulum, the early, medial, late
Golgi or the trans Golgi network.
13. The method of claim 12 wherein the mannosidase enzyme is
predominantly localized in the Golgi apparatus or the endoplasmic
reticulum.
14. The method of claim 12 wherein the enzymes are localized by
forming a fusion protein between a catalytic domain of the enzyme
and a chimeric localization region encoded by at least one genetic
construct formed by the in-frame ligation of a DNA fragment
encoding a cellular targeting signal peptide with a DNA fragment
encoding a glycosylation enzyme or catalytically active fragment
thereof.
15.-18. (canceled)
19. The method of claim 1 wherein the glycoprotein includes
N-glycans of which greater than 27 mole percent comprise fewer than
six mannose residues.
20. The method of claim 1 wherein the glycoprotein comprises one or
more sugars selected from the group consisting of galactose, sialic
acid, and fucose.
21. The method of claim 1 wherein the glycoprotein comprises at
least one oligosaccharide branch comprising the structure
NeuNAc-Gal-GlcNAc-Man.
22. The method of claim 1 wherein the glycoprotein comprises
N-glycans having fewer than four mannose residues.
23. The method of claim 1 wherein subsequent to isolation from the
host, the glycoprotein is subjected to at least one further
glycosylation or carboxylation reaction in vitro.
24. The method of claim 1 comprising the steps of (a) providing a
DNA library comprising at least two genes encoding exogenous
glycosylation enzymes; (b) transforming the host with the library
to produce a genetically mixed population expressing at least two
distinct exogenous glycosylation enzymes; and (c) selecting from
the population a host producing the desired glycosylation
phenotype.
25.-31. (canceled)
32. The host produced by the method of claim 1.
33.-34. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] Priority is claimed to U.S. Provisional Application Ser. No.
60/214,358, filed on Jun. 28, 2000, U.S. Provisional Application
Ser. No. 60/215,638, filed on Jun. 30, 2000, and U.S. Provisional
Application Ser. No. 60/279,997, filed on Mar. 30, 2001.
FIELD OF THE INVENTION
[0002] The present invention is directed to methods and
compositions by which fungi or other eukaryotic microorganisms can
be genetically modified to produce glycosylated proteins
(glycoproteins) having patterns of glycosylation similar to
glycoproteins produced by animal cells, especially human cells,
which are useful as human or animal therapeutic agents.
BACKGROUND OF THE INVENTION
Glycosylation Pathways
[0003] De novo synthesized proteins may undergo further processing
in cells, known as post-translational modification. In particular,
sugar residues may be added enzymatically, a process known as
glycosylation. The resulting proteins bearing covalently linked
oligosaccharide side chains are known as glycosylated proteins or
glycoproteins. Bacteria typically do not glycosylate proteins; in
cases where glycosylation does occur it usually occurs at
nonspecific sites in the protein (Moens and Vanderleyden, Arch.
Microbiol. 1997 168(3):169-175).
[0004] Eukaryotes commonly attach a specific oligosaccharide to the
side chain of a protein asparagine residue, particularly an
asparagine which occurs in the sequence Asn-Xaa-Ser/Thr/Cys (where
Xaa represents any amino acid). Following attachment of the
saccharide moiety, known as an N-glycan, further modifications may
occur in vivo. Typically these modifications occur via an ordered
sequence of enzymatic reactions, known as a cascade. Different
organisms provide different glycosylation enzymes
(glycosyltransferases and glycosidases) and different glycosyl
substrates, so that the final composition of a sugar side chain may
vary markedly depending upon the host.
[0005] For example, microorganisms such as filamentous fungi and
yeast (lower eukaryotes) typically add additional mannose and/or
mannosylphosphate sugars. The resulting glycan is known as a
"high-mannose" type or a mannan. By contrast, in animal cells, the
nascent oligosaccharide side chain may be trimmed to remove several
mannose residues and elongated with additional sugar residues that
typically do not occur in the N-glycans of lower eukaryotes. See R.
K. Bretthauer, et al. Biotechnology and Applied Biochemistry, 1999,
30, 193-200; W. Martinet, et al. Biotechnology Letters, 1998, 20,
1171-1177; S. Weikert, et al. Nature Biotechnology, 1999, 17,
1116-1121; M. Malissard, et al. Biochemical and Biophysical
Research Communications, 2000, 267, 169-173; Jarvis, et al. 1998
Engineering N-glycosylation pathways in the baculovirus-insect cell
system, Current Opinion in Biotechnology, 9:528-533; and M.
Takeuchi, 1997 Trends in Glycoscience and Glycotechnology, 1997, 9,
S29-S35.
[0006] The N-glycans that are produced in humans and animals are
commonly referred to as complex N-glycans. A complex N-glycan means
a structure with typically two to six outer branches with a
sialyllactosamine sequence linked to an inner core structure
Man.sub.3GlcNAc.sub.2. A complex N-glycan has at least one branch,
and preferably at least two, of alternating GlcNAc and galactose
(Gal) residues that terminate in oligosaccharides such as, for
example: NeuNAc-; NeuAc.alpha.2-6GalNAc.alpha.1-;
NeuAc.alpha.2-3Gal.beta.1-3GalNAc.alpha.1-;
NeuAc.alpha.2-3/6Gal.beta.1-4GlcNAc.beta.1-;
GlcNAc.alpha.1-4Gal.beta.1-(mucins only);
Fuc.alpha.1-2Gal.beta.1-(blood group H). Sulfate esters can occur
on galactose, GalNAc, and GlcNAc residues, and phosphate esters can
occur on mannose residues. NeuAc (Neu: neuraminic acid; Ac: acetyl)
can be O-acetylated or replaced by NeuGl (N-glycolylneuraminic
acid). Complex N-glycans may also have intrachain substitutions of
bisecting GlcNAc and core fucose (Fuc).
[0007] Human glycosylation begins with a sequential set of
reactions in the endoplasmatic reticulum (ER) leading to a core
oligosaccharide structure, which is transferred onto de novo
synthesized proteins at the asparagine residue in the sequence
Asn-Xaa-Ser/Thr (see FIG. 1A). Further processing by glucosidases
and mannosidases occurs in the ER before the nascent glycoprotein
is transferred to the early Golgi apparatus, where additional
mannose residues are removed by Golgi-specific 1,2-mannosidases.
Processing continues as the protein proceeds through the Golgi. In
the medial Golgi a number of modifying enzymes including
N-acetylglucosamine transferases (GnT I, GnT II, GnT III, GnT IV
GnT V GnT VI), mannosidase II, fucosyltransferases add and remove
specific sugar residues (see FIG. 1B). Finally in the trans Golgi,
the N-glycans are acted on by galactosyl tranferases and
sialyltransferases (ST) and the finished glycoprotein is released
from the Golgi apparatus. The protein N-glycans of animal
glycoproteins have bi-, tri-, or tetra-antennary structures, and
may typically include galactose, fucose, and N-acetylglucosamine.
Commonly the terminal residues of the N-glycans consist of sialic
acid. A typical structure of a human N-glycan is shown in FIG.
1B.
Sugar Nucleotide Precursors
[0008] The N-glycans of animal glycoproteins typically include
galactose, fucose, and terminal sialic acid. These sugars are not
generally found on glycoproteins produced in yeast and filamentous
fungi. In humans, the full range of nucleotide sugar precursors
(e.g. UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine,
CMP-N-acetylneuraminic acid, UDP-galactose, GDP-fucose etc.) are
generally synthesized in the cytosol and transported into the
Golgi, where they are attached to the core oligosaccharide by
glycosyltransferases. (Sommers and Hirschberg, 1981 J. Cell Biol.
91(2): A406-A406; Sommers and Hirschberg 1982 J. Biol. Chem.
257(18): 811-817; Perez and Hirschberg 1987 Methods in Enzymology
138: 709-715.
[0009] Glycosyl transfer reactions typically yield a side product
which is a nucleoside diphosphate or monophosphate. While
monophosphates can be directly exported in exchange for nucleoside
triphosphate sugars by an antiport mechanism, diphosphonucleosides
(e.g. GDP) have to be cleaved by phosphatases (e.g. GDPase) to
yield nucleoside monophosphates and inorganic phosphate prior to
being exported. This reaction is important for efficient
glycosylation; for example, GDPase from S. cerevisiae has been
found to be necessary for mannosylation. However the GDPase has 90%
reduced activity toward UDP (Berninsone et al., 1994 J. Biol. Chem.
269(1):207-211.alpha.). Lower eukaryotes typically lack
UDP-specific diphosphatase activity in the Golgi since they do not
utilize UDP-sugar precursors for Golgi-based glycoprotein
synthesis. Schizosaccharomyces pombe, a yeast found to add
galactose residues to cell wall polysaccharides (from
UDP-galactose) has been found to have specific UDPase activity,
indicating the requirement for such an enzyme (Berninsone et al.,
1994). UDP is known to be a potent inhibitor of
glycosyltransferases and the removal of this glycosylation side
product is important in order to prevent glycosyltransferase
inhibition in the lumen of the Golgi (Khatara et al., 1974). See
Berninsone, P., et al. 1995. J. Biol. Chem. 270(24): 14564-14567;
Beaudet, L., et al. 1998 Abc Transporters: Biochemical, Cellular,
and Molecular Aspects. 292: 397-413.
Compartmentalization of Glycosylation Enzymes
[0010] Glycosyltransferases and mannosidases line the inner
(luminal) surface of the ER and Golgi apparatus and thereby provide
a catalytic surface that allows for the sequential processing of
glycoproteins as they proceed through the ER and Golgi network. The
multiple compartments of the cis, medial, and trans Golgi and the
trans Golgi Network (TGN), provide the different localities in
which the ordered sequence of glycosylation reactions can take
place. As a glycoprotein proceeds from synthesis in the ER to full
maturation in the late Golgi or TGN, it is sequentially exposed to
different glycosidases, mannosidases and glycosyltransferases such
that a specific N-glycan structure may be synthesized. The enzymes
typically include a catalytic domain, a stem region, a membrane
spanning region and an N-terminal cytoplasmic tail. The latter
three structural components are responsible for directing a
glycosylation enzyme to the appropriate locus.
[0011] Localization sequences from one organism may function in
other organisms. For example the membrane spanning region of
.alpha.-2,6-sialyltransferase (.alpha.-2,6-ST) from rats, an enzyme
known to localize in the rat trans Golgi, was shown to also
localize a reporter gene (invertase) in the yeast Golgi
(Schwientek, et al., 1995). However, the very same membrane
spanning region as part of a full-length of
.alpha.-2,6-sialyltransferase was retained in the ER and not
further transported to the Golgi of yeast (Krezdorn et al., 1994).
A full length GalT from humans was not even synthesized in yeast,
despite demonstrably high transcription levels. On the other hand
the transmembrane region of the same human GalT fused to an
invertase reporter was able to direct localization to the yeast
Golgi, albeit it at low production levels. Schwientek and
co-workers have shown that fusing 28 amino acids of a yeast
mannosyltransferase (Mnt1), a region containing an N-terminal
cytoplasmic tail, a transmembrane region and eight amino acids of
the stem region, to the catalytic domain of human GalT are
sufficient for Golgi localization of an active GalT (Schwientek et
al. 1995 J. Biol. Chem. 270(10):5483-5489). Other
galactosyltransferases appear to rely on interactions with enzymes
resident in particular organelles since after removal of their
transmembrane region they are still able to localize properly.
[0012] Improper localization of a glycosylation enzyme may prevent
proper functioning of the enzyme in the pathway. For example
Aspergillus nidulans, which has numerous .alpha.-1,2-mannosidases
(Eades and Hintz, 2000 Gene 255(1):25-34), does not add GlcNAc to
Man.sub.5GlcNAc.sub.2 when transformed with the rabbit GnT I gene,
despite a high overall level of GnT I activity (Kalsner et al.,
1995). GnT I, although actively expressed, may be incorrectly
localized such that the enzyme is not in contact with both of its
substrates: the nascent N-glycan of the glycoprotein and
UDP-GlcNAc. Alternatively, the host organism may not provide an
adequate level of UDP-GlcNAc in the Golgi.
Glycoproteins Used Therapeutically
[0013] A significant fraction of proteins isolated from humans or
other animals are glycosylated. Among proteins used
therapeutically, about 70% are glycosylated. If a therapeutic
protein is produced in a microorganism host such as yeast, however,
and is glycosylated utilizing the endogenous pathway, its
therapeutic efficiency is typically greatly reduced. Such
glycoproteins are typically immunogenic in humans and show a
reduced half-life in vivo after administration (Takeuchi,
1997).
[0014] Specific receptors in humans and animals can recognize
terminal mannose residues and promote the rapid clearance of the
protein from the bloodstream. Additional adverse effects may
include changes in protein folding, solubility, susceptibility to
proteases, trafficking, transport, compartmentalization, secretion,
recognition by other proteins or factors, antigenicity, or
allergenicity. Accordingly, it has been necessary to produce
therapeutic glycoproteins in animal host systems, so that the
pattern of glycosylation is identical or at least similar to that
in humans or in the intended recipient species. In most cases a
mammalian host system, such as mammalian cell culture, is used.
Systems for Producing Therapeutic Glycoproteins
[0015] In order to produce therapeutic proteins that have
appropriate glycoforms and have satisfactory therapeutic effects,
animal or plant-based expression systems have been used. The
available systems include: [0016] 1. Chinese hamster ovary cells
(CHO), mouse fibroblast cells and mouse myeloma cells
(Arzneimittelforschung. 1998 August; 48(8):870-880); [0017] 2.
transgenic animals such as goats, sheep, mice and others (Dente
Prog. Clin. Biol. 1989 Res. 300:85-98, Ruther et al., 1988 Cell
53(6):847-856; Ware, J., et al. 1993 Thrombosis and Haemostasis
69(6): 1194-1194; Cole, E. S., et al. 1994 J. Cell. Biochem.
265-265); [0018] 3. plants (Arabidopsis thaliana, tobacco etc.)
(Staub, et al. 2000 Nature Biotechnology 18(3): 333-338) (McGarvey,
P. B., et al. 1995 Bio-Technology 13(13): 1484-1487; Bardor, M., et
al. 1999 Trends in Plant Science 4(9): 376-380); [0019] 4. insect
cells (Spodoptera frugiperda Sf9, Sf21, Trichoplusia ni, etc. in
combination with recombinant baculoviruses such as Autographa
californica multiple nuclear polyhedrosis virus which infects
lepidopteran cells) (Altmans et al., 1999 Glycoconj. J.
16(2):109-123).
[0020] Recombinant human proteins expressed in the above-mentioned
host systems may still include non-human glycoforms (Raju et al.,
2000 Annals Biochem. 283(2):123-132). In particular, fraction of
the N-glycans may lack terminal sialic acid, typically found in
human glycoproteins. Substantial efforts have been directed to
developing processes to obtain glycoproteins that are as close as
possible in structure to the human forms, or have other therapeutic
advantages. Glycoproteins having specific glycoforms may be
especially useful, for example in the targeting of therapeutic
proteins. For example, the addition of one or more sialic acid
residues to a glycan side chain may increase the lifetime of a
therapeutic glycoprotein in vivo after administration. Accordingly,
the mammalian host cells may be genetically engineered to increase
the extent of terminal sialic acid in glycoproteins expressed in
the cells. Alternatively sialic acid may be conjugated to the
protein of interest in vitro prior to administration using a sialic
acid transferase and an appropriate substrate. In addition, changes
in growth medium composition or the expression of enzymes involved
in human glycosylation have been employed to produce glycoproteins
more closely resembling the human forms (S. Weikert, et al., Nature
Biotechnology, 1999, 17, 1116-1121; Werner, Noe, et al 1998
Arzneimittelforschung 48(8):870-880; Weikert, Papac et al., 1999;
Andersen and Goochee 1994 Cur. Opin. Biotechnol. 5: 546-549; Yang
and Butler 2000 Biotechnol. Bioengin. 68(4): 370-380).
Alternatively cultured human cells may be used.
However, all of the existing systems have significant drawbacks.
Only certain therapeutic proteins are suitable for expression in
animal or plant systems (e.g. those lacking in any cytotoxic effect
or other effect adverse to growth). Animal and plant cell culture
systems are usually very slow, frequently requiring over a week of
growth under carefully controlled conditions to produce any useful
quantity of the protein of interest. Protein yields nonetheless
compare unfavorably with those from microbial fermentation
processes. In addition cell culture systems typically require
complex and expensive nutrients and cofactors, such as bovine fetal
serum. Furthermore growth may be limited by programmed cell death
(apoptosis).
[0021] Moreover, animal cells (particularly mammalian cells) are
highly susceptible to viral infection or contamination. In some
cases the virus or other infectious agent may compromise the growth
of the culture, while in other cases the agent may be a human
pathogen rendering the therapeutic protein product unfit for its
intended use. Furthermore many cell culture processes require the
use of complex, temperature-sensitive, animal-derived growth media
components, which may carry pathogens such as bovine spongiform
encephalopathy (BSE) prions. Such pathogens are difficult to detect
and/or difficult to remove or sterilize without compromising the
growth medium. In any case, use of animal cells to produce
therapeutic proteins necessitates costly quality controls to assure
product safety.
[0022] Transgenic animals may also be used for manufacturing
high-volume therapeutic proteins such as human serum albumin,
tissue plasminogen activator, monoclonal antibodies, hemoglobin,
collagen, fibrinogen and others. 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, the process is costly since every batch has to undergo
rigorous quality control. Animals may host a variety of animal or
human pathogens, including bacteria, viruses, fungi, and prions. In
the case of scrapies and bovine spongiform encephalopathy, testing
can take about a year to rule out infection. The production of
therapeutic compounds is thus preferably carried out in a
well-controlled sterile environment, e.g. under Good Manufacturing
Practice (GMP) conditions. However, it is not generally feasible to
maintain animals in such environments. Moreover, whereas cells
grown in a fermenter are derived from one well characterized Master
Cell Bank (MCB), transgenic animal 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 effect glycosylation patterns of the
final product. It is known in humans, for example, that different
dietary habits result in differing glycosylation patterns.
[0023] Transgenic plants have been developed as a potential source
to obtain proteins of therapeutic value. However, high level
expression of proteins in plants suffers from gene silencing, a
mechanism by which the genes for highly expressed proteins are
down-regulated in subsequent plant generations. In addition, plants
add xylose and/or .alpha.-1,3-linked fucose to protein N-glycans,
resulting in glycoproteins that differ in structure from animals
and are immunogenic in mammals (Altmann, Marz et al., 1995
Glycoconj. J. 12(2); 150-155). Furthermore, it is generally not
practical to grow plants in a sterile or GMP environment, and the
recovery of proteins from plant tissues is more costly than the
recovery from fermented microorganisms.
Glycoprotein Production Using Eukaryotic Microorganisms
[0024] The lack of a suitable expression system is thus a
significant obstacle to the low-cost and safe production of
recombinant human glycoproteins. Production of glycoproteins via
the fermentation of microorganisms would offer numerous advantages
over the existing systems. For example, fermentation-based
processes may offer (a) rapid production of high concentrations of
protein; (b) the ability to use sterile, well-controlled production
conditions (e.g. GMP conditions); (c) the ability to use simple,
chemically defined growth media; (d) ease of genetic manipulation;
(e) the absence of contaminating human or animal pathogens; (f) the
ability to express a wide variety of proteins, including those
poorly expressed in cell culture owing to toxicity etc.; (g) ease
of protein recovery (e.g. via secretion into the medium). In
addition, fermentation facilities are generally far less costly to
construct than cell culture facilities.
[0025] As noted above, however, bacteria, including species such as
Escherichia coli commonly used to produce recombinant proteins, do
not glycosylate proteins in a specific manner like eukaryotes.
Various methylotrophic yeasts such as Pichia pastoris, Pichia
methanolica, and Hansenula polymorpha, are particularly useful as
eukaryotic expression systems, since they are able to grow to high
cell densities and/or secrete large quantities of recombinant
protein. However, as noted above, glycoproteins expressed in these
eukaryotic microorganisms differ substantially in N-glycan
structure from those in animals. This has prevented the use of
yeast or filamentous fungi as hosts for the production of many
useful glycoproteins.
[0026] Several efforts have been made to modify the glycosylation
pathways of eukaryotic microorganisms to provide glycoproteins more
suitable for use as mammalian therapeutic agents. For example,
several glycosyltransferases have been separately cloned and
expressed in S. cerevisiae (GalT, GnT I), Aspergillus nidulans (GnT
I) and other fungi (Yoshida et al., 1999, Kalsner et al., 1995
Glycoconj. J. 12(3):360-370, Schwientek et al., 1995). However,
N-glycans with human characteristics were not obtained.
[0027] Yeasts produce a variety of mannosyltransferases e.g.
1,3-mannosyltransferases (e.g. MNN1 in S. cerevisiae) (Graham and
Emr, 1991 J. Cell. Biol. 114(2):207-218), 1,2-mannosyltransferases
(e.g. KTR/KRE family from S. cerevisiae), 1,6-mannosyltransferases
(OCH1 from S. cerevisiae), mannosylphosphate transferases (MNN4 and
MNN6 from S. cerevisiae) and additional enzymes that are involved
in endogenous glycosylation reactions. Many of these genes have
been deleted individually, giving rise to viable organisms having
altered glycosylation profiles. Examples are shown in Table 1.
TABLE-US-00001 TABLE 1 Examples of yeast strains having altered
mannosylation N-glycan (wild N-glycan Strain type) Mutation
(mutant) Reference S. pombe Man.sub.>9GlcNAc.sub.2 OCH1
Man.sub.8GlcNAc.sub.2 Yoko-o et al., 2001 FEBS Lett. 489 (1): 75-80
S. Man.sub.>9GlcNAc.sub.2 OCH1/MNN1 Man.sub.8GlcNAc.sub.2
Nakanishi-Shindo et al,. cerevisiae 1993 J. Biol. Chem. 268 (35):
26338-26345 S. Man.sub.>9GlcNAc.sub.2 OCH1/MNN1/
Man.sub.8GlcNAc.sub.2 Chiba et al., 1998 J. Biol. cerevisiae MNN4
Chem. 273, 26298-26304
[0028] In addition, Japanese Patent Application Public No. 8-336387
discloses an OCH1 mutant strain of Pichia pastoris. The OCH1 gene
encodes 1,6-mannosyltransferase, which adds a mannose to the glycan
structure Man.sub.8GlcNAc.sub.2 to yield Man.sub.9GlcNAc.sub.2. The
Man.sub.9GlcNAc.sub.2 structure is then a substrate for further
mannosylation in vivo, leading to the hypermannosylated
glycoproteins that are characteristic of yeasts and typically may
have at least 30-40 mannose residue per N-glycan. In the OCH1
mutant strain, proteins glycosylated with Man.sub.8GlcNAc.sub.2 are
accumulated and hypermannosylation does not occur. However, the
structure Man.sub.8GlcNAc.sub.2 is not a substrate for animal
glycosylation enzymes, such as human UDP-GlcNAc transferase I, and
accordingly the method is not useful for producing proteins with
human glycosylation patterns.
[0029] Martinet et al. (Biotechnol. Lett. 1998, 20(12), 1171-1177)
reported the expression of .alpha.-1,2-mannosidase from Trichoderma
reesei in P. pastoris. Some mannose trimming from the N-glycans of
a model protein was observed. However, the model protein had no
N-glycans with the structure Man.sub.5GlcNAc.sub.2, which would be
necessary as an intermediate for the generation of complex
N-glycans. Accordingly the method is not useful for producing
proteins with human or animal glycosylation patterns.
[0030] Similarly, Chiba et al. 1998 expressed
.alpha.-1,2-mannosidase from Aspergillus saitoi in the yeast
Saccharomyces cerevisiae. A signal peptide sequence
(His-Asp-Glu-Leu) was engineered into the exogenous mannosidase to
promote retention in the endoplasmic reticulum. In addition, the
yeast host was a mutant lacking three enzyme activities associated
with hypermannosylation of proteins: 1,6-mannosyltransferase
(OCH1); 1,3-mannosyltransferase (MNN1); and
mannosylphosphatetransferase (MNN4). The N-glycans of the triple
mutant host thus consisted of the structure Man.sub.8GlcNAc.sub.2,
rather than the high mannose forms found in wild-type S.
cerevisiae. In the presence of the engineered mannosidase, the
N-glycans of a model protein (carboxypeptidase Y) were trimmed to
give a mixture consisting of 27 mole % Man.sub.5GlcNAc.sub.2, 22
mole % Man.sub.6GlcNAc.sub.2, 22 mole % Man.sub.7GlcNAc.sub.2, 29
mole % Man.sub.8GlcNAc.sub.2. Trimming of the endogenous cell wall
glycoproteins was less efficient, only 10 mole % of the N-glycans
having the desired Man.sub.5GlcNAc.sub.2 structure.
[0031] Since only the Man.sub.5GlcNAc.sub.2 glycans would be
susceptible to further enzymatic conversion to human glycoforms,
the method is not efficient for the production of proteins having
human glycosylation patterns. In proteins having a single
N-glycosylation site, at least 73 mole % would have an incorrect
structure. In proteins having two or three N-glycosylation sites,
respectively at least 93 or 98 mole % would have an incorrect
structure. Such low efficiencies of coversion are unsatisfactory
for the production of therapeutic agents, particularly as the
separation of proteins having different glycoforms is typically
costly and difficult.
[0032] With the object of providing a more human-like glycoprotein
derived from a fungal host, U.S. Pat. No. 5,834,251 to Maras and
Contreras discloses a method for producing a hybrid glycoprotein
derived from Trichoderma reesei. A hybrid N-glycan has only mannose
residues on the Man.alpha.-6 arm of the core and one or two complex
antennae on the Man.alpha.-3 arm. 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 maintain, may need unusual and
costly substrates (e.g. UDP-GlcNAc), and are prone to loss of
activity and/or proteolysis under the conditions of use.
[0033] It is therefore an object of the present invention to
provide a system and methods for humanizing glycosylation of
recombinant glycoproteins expressed in Pichia pastoris and other
lower eukaryotes such as Hansenula polymorpha, Pichia stiptis,
Pichia methanolica, Pichia sp, Kluyveromyces sp, Candida albicans,
Aspergillus nidulans, and Trichoderma reseei.
SUMMARY OF THE INVENTION
[0034] Cell lines having genetically modified glycosylation
pathways that allow them to carry out a sequence of enzymatic
reactions, which mimic the processing of glycoproteins in humans,
have been developed. Recombinant proteins expressed in these
engineered hosts yield glycoproteins more similar, if not
substantially identical, to their human counterparts. The lower
eukaryotes, which ordinarily produce high-mannose containing
N-glycans, including unicellular and multicellular fungi such as
Pichia pastoris, Hansenula polymorpha, Pichia stiptis, Pichia
methanolica, Pichia sp., Kluyveromyces sp., Candida albicans,
Aspergillus nidulans, and Trichoderma reseei, are modified to
produce N-glycans such as Man.sub.5GlcNAc.sub.2 or other structures
along human glycosylation pathways. This is achieved using a
combination of engineering and/or selection of strains which: do
not express certain enzymes which create the undesirable complex
structures characteristic of the fungal glycoproteins, which
express exogenous enzymes selected either to have optimal activity
under the conditions present in the fungi where activity is
desired, or which are targeted to an organelle where optimal
activity is achieved, and combinations thereof wherein the
genetically engineered eukaryote expresses multiple exogenous
enzymes required to produce "human-like" glycoproteins.
[0035] In a first embodiment, the microorganism is engineered to
express an exogenous .alpha.-1,2-mannosidase enzyme having an
optimal pH between 5.1 and 8.0, preferably between 5.9 and 7.5. In
an alternative preferred embodiment, the exogenous enzyme is
targeted to the endoplasmic reticulum or Golgi apparatus of the
host organism, where it trims N-glycans such as
Man.sub.8GlcNAc.sub.2 to yield Man.sub.5GlcNAc.sub.2. The latter
structure is useful because it is identical to a structure formed
in mammals, especially humans; it is a substrate for further
glycosylation reactions in vivo and/or in vitro that produce a
finished N-glycan that is similar or identical to that formed in
mammals, especially humans; and it is not a substrate for
hypermannosylation reactions that occur in vivo in yeast and other
microorganisms and that render a glycoprotein highly immunogenic in
animals.
[0036] In a second embodiment, the glycosylation pathway of an
eukaryotic microorganism is modified by (a) constructing a DNA
library including at least two genes encoding exogenous
glycosylation enzymes; (b) transforming the microorganism with the
library to produce a genetically mixed population expressing at
least two distinct exogenous glycosylation enzymes; (c) selecting
from the population a microorganism having the desired
glycosylation phenotype. In a preferred embodiment, the DNA library
includes chimeric genes each encoding a protein localization
sequence and a catalytic activity related to glycosylation.
Organisms modified using the method are useful for producing
glycoproteins having a glycosylation pattern similar or identical
to mammals, especially humans.
[0037] In a third embodiment, the glycosylation pathway is modified
to express a sugar nucleotide transporter enzyme. In a preferred
embodiment, a nucleotide diphosphatase enzyme is also expressed.
The transporter and diphosphatase improve the efficiency of
engineered glycosylation steps, by providing the appropriate
substrates for the glycosylation enzymes in the appropriate
compartments, reducing competitive product inhibition, and
promoting the removal of nucleoside diphosphates.
DESCRIPTION OF THE FIGURES
[0038] FIG. 1A is a schematic diagram of typical fungal
N-glycosylation pathway.
[0039] FIG. 1B is a schematic diagram of a typical human
N-glycosylation pathway.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The methods and recombinant lower eukaryotic strains
described herein are used to make "humanized glycoproteins". The
recombinant lower eukaryotes are made by engineering lower
eukaryotes which do not express one or more enzymes involved in
production of high mannose structures to express the enzymes
required to produce human-like sugars. As used herein, a lower
eukaryote is a unicellular or filamentous fungus. As used herein, a
"humanized glycoprotein" refers to a protein having attached
thereto N-glycans including less than four mannose residues, and
the synthetic intermediates (which are also useful and can be
manipulated further in vitro) having at least five mannose
residues. In a preferred embodiment, the glycoproteins produced in
the recombinant lower eukaryotic strains contain at least 27 mole %
of the Man5 intermediate. This is achieved by cloning in a better
mannosidase, i.e., an enzyme selected to have optimal activity
under the conditions present in the organisms at the site where
proteins are glycosylated, or by targeting the enzyme to the
organelle where activity is desired.
[0041] In a preferred embodiment, eukaryotic strains which do not
express one or more enzymes involved in the production of high
mannose structures are used. These strains can be engineered or one
of the many such mutants already described in yeasts, including a
hypermannosylation-minus (OCH1) mutant in Pichia pastoris.
[0042] The strains can be engineered one enzyme at a time, or a
library of genes encoding potentially useful enzymes can be
created, and those strains having enzymes with optimal activities
or producing the most "human-like" glycoproteins, selected.
[0043] Lower eukaryotes that are able to produce glycoproteins
having the attached N-glycan Man.sub.5GlcNAc.sub.2 are particularly
useful since (a) lacking a high degree of mannosylation (e.g.
greater than 8 mannoses per N-glycan, or especially 30-40
mannoses), they show reduced immunogenicity in humans; and (b) the
N-glycan is a substrate for further glycosylation reactions to form
an even more human-like glycoform, e.g. by the action of GlcNAc
transferase I to form GlcNAcMan.sub.5GlcNAc.sub.2.
Man.sub.5GlcNAc.sub.2 must be formed in vivo in a high yield, at
least transiently, since all subsequent glycosylation reactions
require Man.sub.5GlcNAc.sub.2 or a derivative thereof. Accordingly,
a yield is obtained of greater than 27 mole %, more preferably a
yield of 50-100 mole %, glycoproteins in which a high proportion of
N-glycans have Man.sub.5GlcNAc.sub.2. It is then possible to
perform further glycosylation reactions in vitro, using for example
the method of U.S. Pat. No. 5,834,251 to Maras and Contreras. In a
preferred embodiment, at least one further glycosylation reaction
is performed in vivo. In a highly preferred embodiment thereof,
active forms of glycosylating enzymes are expressed in the
endoplasmic reticulum and/or Golgi apparatus.
Host Microorganisms
[0044] Yeast and filamentous fungi have both been successfully used
for the production of recombinant proteins, both intracellular and
secreted (Cereghino, J. L. and J. M. Cregg 2000 FEMS Microbiology
Reviews 24(1): 45-66; Harkki, A., et al. 1989 Bio-Technology 7(6):
596; Berka, R. M., et al. 1992 Abstr. Papers Amer. Chem. Soc. 203:
121-BIOT; Svetina, M., et al. 2000 J. Biotechnol. 76(2-3):
245-251.
[0045] Although glycosylation in yeast and fungi is very different
than in humans, some common elements are shared. The first step,
the transfer of the core oligosaccharide structure to the nascent
protein, is highly conserved in all eukaryotes including yeast,
fungi, plants and humans (compare FIGS. 1A and 1B). Subsequent
processing of the core oligosaccharide, however, differs
significantly in yeast and involves the addition of several mannose
sugars. This step is catalyzed by mannosyltransferases residing in
the Golgi (e.g. OCH1, MNT1, MNN1, etc.), which sequentially add
mannose sugars to the core oligosaccharide. The resulting structure
is undesirable for the production of humanoid proteins and it is
thus desirable to reduce or eliminate mannosyl transferase
activity. Mutants of S. cerevisiae, deficient in mannosyl
transferase activity (e.g. och1 or mnn9 mutants) have shown to be
non-lethal and display a reduced mannose content in the
oligosacharide of yeast glycoproteins. Other oligosacharide
processing enzymes, such as mannosylphophate transferase may also
have to be eliminated depending on the host's particular endogenous
glycosylation pattern. After reducing undesired endogenous
glycosylation reactions the formation of complex N-glycans has to
be engineered into the host system. This requires the stable
expression of several enzymes and sugar-nucleotide transporters.
Moreover, one has to locate these enzymes in a fashion such that a
sequential processing of the maturing glycosylation structure is
ensured.
Target Glycoproteins
[0046] The methods described herein are useful for producing
glycoproteins, especially glycoproteins used therapeutically in
humans. Such therapeutic proteins are typically administered by
injection, orally, pulmonary, or other means.
[0047] Examples of suitable target glycoproteins include, without
limitation: erythropoietin, cytokines such as interferon-.alpha.,
interferon-.beta., interferon-.gamma., interferon-.omega., and
granulocyte-CSF, coagulation factors such as factor VIII, factor
IX, and human protein C, soluble IgE receptor .alpha.-chain, IgG,
IgM, urokinase, chymase, and urea trypsin inhibitor, IGF-binding
protein, epidermal growth factor, growth hormone-releasing factor,
annexin V fusion protein, angiostatin, vascular endothelial growth
factor-2, myeloid progenitor inhibitory factor-1, and
osteoprotegerin.
Method for Producing Glycoproteins Comprising the N-Glycan
Man.sub.5GlcNAc.sub.2
[0048] The first step involves the selection or creation of a lower
eukaryote that is able to produce a specific precursor structure of
Man.sub.5GlcNAc.sub.2, which is able to accept in vivo GlcNAc by
the action of a GlcNAc transferase I. This step requires the
formation of a particular isomeric structure of
Man.sub.5GlcNAc.sub.2. This structure has to be formed within the
cell at a high yield (in excess of 30%) since all subsequent
manipulations are contingent on the presence of this precursor.
Man.sub.5GlcNAc.sub.2 structures are necessary for complex N-glycan
formation, however, their presence is by no means sufficient, since
Man.sub.5GlcNAc.sub.2 may occur in different isomeric forms, which
may or may not serve as a substrate for GlcNAc transferase I. Most
glycosylation reactions are not complete and thus a particular
protein generally contains a range of different carbohydrate
structures (i.e. glycoforms) on its surface. The mere presence of
trace amounts (less than 5%) of a particular structure like
Man.sub.5GlcNAc.sub.2 is of little practical relevance. It is the
formation of a particular, GlcNAc transferase I accepting
intermediate (Structure I) in high yield (above 30%), which is
required. The formation of this intermediate is necessary and
subsequently allows for the in vivo synthesis of complex
N-glycans.
[0049] One can select such lower eukaryotes from nature or
alternatively genetically engineer existing fungi or other lower
eukaryotes to provide the structure in vivo. No lower eukaryote has
been shown to provide such structures in vivo in excess of 1.8% of
the total N-glycans (Maras et al., 1997), so a genetically
engineered organism is preferred. Methods such as those described
in U.S. Pat. No. 5,595,900, may be used to identify the absence or
presence of particular glycosyltransferases, mannosidases and sugar
nucleotide transporters in a target organism of interest.
[0050] Inactivation of Fungal Glycosylation Enzymes Such as
1,2-.alpha.-mannosidase
[0051] The method described herein may be used to engineer the
glycosylation pattern of a wide range of lower eukaryotes (e.g.
Hansenula polymorpha, Pichia stiptis, Pichia methanolica, Pichia
sp, Kluyveromyces sp, Candida albicans, Aspergillus nidulans,
Trichoderma reseei etc.). Pichia pastoris is used to exemplify the
required manipulation steps. Similar to other lower eukaryotes, P.
pastoris processes Man.sub.9GlcNAc.sub.2 structures in the ER with
a 1,2-.alpha.-mannosidase to yield Man.sub.8GlcNAc.sub.2. Through
the action of several mannosyltransferases, this structure is then
converted to hypermannosylated structures
(Man.sub.>9GlcNAc.sub.2), also known as mannans. In addition, it
has been found that P. pastoris is able to add non-terminal
phosphate groups, through the action of mannosylphosphate
transferases to the carbohydrate structure. This is contrary to the
reactions found in mammalian cells, which involve the removal of
mannose sugars as opposed to their addition. It is of particular
importance to eliminate the ability of the fungus to
hypermannosylate the existing Man.sub.8GlcNAc.sub.2 structure. This
can be achieved by either selecting for a fungus that does not
hypermannosylate, or by genetically engineering such a fungus.
[0052] Genes that are involved in this process have been identified
in Pichia pastoris and by creating mutations in these genes one is
able to reduce the production of "undesirable" glycoforms. Such
genes can be identified by homology to existing
mannosyltransferases (e.g. OCH1, MNN4, MNN6, MNN1), found in other
lower eukaryotes such as C. albicans, Pichia angusta or S.
cerevisiae or by mutagenizing the host strain and selecting for a
phenotype with reduced mannosylation. Based on homologies amongst
known mannosyltransferases and mannosylphosphate transferases, one
may either design PCR primers, examples of which are shown in Table
2, or use genes or gene fragments encoding such enzymes as probes
to identify homologues in DNA libraries of the target organism.
Alternatively, one may be able to complement particular phenotypes
in related organisms. For example, in order to obtain the gene or
genes encoding 1,6-mannosyltransferase activity in P. pastoris, one
would carry out the following steps. OCH1 mutants of S. cerevisiae
are temperature sensitive and are slow growers at elevated
temperatures. One can thus identify functional homologues of OCH1
in P. pastoris by complementing an OCH1 mutant of S. cerevisiae
with a P. pastoris DNA or cDNA library. Such mutants of S.
cerevisiae may be found at
http://genome-www.stanford.edu/Saccharomyces/and are commercially
available at http://www.resgen.com/products/YEASTD.php3. Mutants
that display a normal growth phenotype at elevated temperature,
after having been transformed with a P. pastoris DNA library, are
likely to carry an OCH1 homologue of P. pastoris. Such a library
can be created by partially digesting chromosomal DNA of P.
pastoris with a suitable restriction enzyme and after inactivating
the restriction enzyme ligating the digested DNA into a suitable
vector, which has been digested with a compatible restriction
enzyme. Suitable vectors are pRS314, a low copy (CEN6/ARS4) plasmid
based on pBluescript containing the Trp1 marker (Sikorski, R. S.,
and Hieter, P., 1989, Genetics 122, pg 19-27) or pFL44S, a high
copy (2.mu.) plasmid based on a modified pUC19 containing the URA3
marker (Bonneaud, N., et al., 1991, Yeast 7, pg. 609-615). Such
vectors are commonly used by academic researchers or similar
vectors are available from a number of different vendors such as
Invitrogen (Carlsbad, Calif.), Pharmacia (Piscataway, N.J.), New
England Biolabs (Beverly, Mass.). Examples are pYES/GS, 2.mu.
origin of replication based yeast expression plasmid from
Invitrogen, or Yep24 cloning vehicle from New England Biolabs.
After ligation of the chromosomal DNA and the vector one may
transform the DNA library into strain of S. cerevisiae with a
specific mutation and select for the correction of the
corresponding phenotype. After sub-cloning and sequencing the DNA
fragment that is able to restore the wild-type phenotype, one may
use this fragment to eliminate the activity of the gene product
encoded by OCH1 in P. pastoris.
[0053] Alternatively, if the entire genomic sequence of a
particular fungus of interest is known, one may identify such genes
simply by searching publicly available DNA databases, which are
available from several sources such as NCBI, Swissprot etc. For
example by searching a given genomic sequence or data base with a
known 1,6 mannosyltransferase gene (OCH1) from S. cerevisiae, one
can able to identify genes of high homology in such a genome, which
a high degree of certainty encodes a gene that has 1,6
mannosyltransferase activity. Homologues to several known
mannosyltransferases from S. cerevisiae in P. pastoris have been
identified using either one of these approaches. These genes have
similar functions to genes involved in the mannosylation of
proteins in S. cerevisiae and thus their deletion may be used to
manipulate the glycosylation pattern in P. pastoris or any other
fungus with similar glycosylation pathways.
[0054] The creation of gene knock-outs, once a given target gene
sequence has been determined, is a well-established technique in
the yeast and fungal molecular biology community, and can be
carried out by anyone of ordinary skill in the art (R. Rothsteins,
(1991) Methods in Enzymology, vol. 194, p. 281). In fact, the
choice of a host organism may be influenced by the availability of
good transformation and gene disruption techniques for such a host.
If several mannosyltransferases have to be knocked out, the method
developed by Alani and Kleckner allows for the repeated use of the
URA3 markers to sequentially eliminate all undesirable endogenous
mannosyltransferase activity. This technique has been refined by
others but basically involves the use of two repeated DNA
sequences, flanking a counter selectable marker. For example: URA3
may be used as a marker to ensure the selection of a transformants
that have integrated a construct. By flanking the URA3 marker with
direct repeats one may first select for transformants that have
integrated the construct and have thus disrupted the target gene.
After isolation of the transformants, and their characterization,
one may counter select in a second round for those that are
resistant to 5'FOA. Colonies that able to survive on plates
containing 5'FOA have lost the URA3 marker again through a
crossover event involving the repeats mentioned earlier. This
approach thus allows for the repeated use of the same marker and
facilitates the disruption of multiple genes without requiring
additional markers.
[0055] Eliminating specific mannosyltransferases, such as 1,6
mannosyltransferase (OCH1), mannosylphosphate transferases (MNN4,
MNN6, or genes complementing lbd mutants) in P. pastoris, allows
for the creation of engineered strains of this organism which
synthesize primarily Man.sub.8GlcNAc.sub.2 and thus can be used to
further modify the glycosylation pattern to more closely resemble
more complex human glycoform structures. A preferred embodiment of
this method utilizes known DNA sequences, encoding known
biochemical glycosylation activities to eliminate similar or
identical biochemical functions in P. pastoris, such that the
glycosylation structure of the resulting genetically altered P.
pastoris strain is modified.
TABLE-US-00002 TABLE 2 Target Gene(s) in PCR primer A PCR primer B
P. pastoris Homologues ATGGCGAAGGCAGA TTAGTCCTTCCAAC 1,6- OCH1
S.cerevisiae, TGGCAGT TTCCTTC mannosyltransferase Pichia albicans
TAYTGGMGNGTNGA GCRTCNCCCCANCK 1,2 KTR/KRE family, RCYNGAYATHAA
YTCRTA mannosyltransferases S.cerevisiae Legend: M = A or C, R = A
or G, W = A or T, S = C or G, Y = C or T, K = G or T, V = A or C or
G, H = A or C or T, D = A or G or T, B = C or G or T, N = G or A or
T or C.
[0056] Incorporation of a Mannosidase into the Genetically
Engineered Host
[0057] The process described herein enables one to obtain such a
structure in high yield for the purpose of modifying it to yield
complex N-glycans. A successful scheme to obtain suitable
Man.sub.5GlcNAc.sub.2 structures must involve two parallel
approaches: (1) reducing endogenous mannosyltransferase activity
and (2) removing 1,2-.alpha.-mannose by mannosidases to yield high
levels of suitable Man.sub.5GlcNAc.sub.2 structures. What
distinguishes this method from the prior art is that it deals
directly with those two issues. As the work of Chiba and coworkers
demonstrates, one can reduce Man.sub.8GlcNAc.sub.2 structures to a
Man.sub.5GlcNAc.sub.2 isomer in S. cerevisiae, by engineering the
presence of a fungal mannosidase from A. saitoi into the ER. The
shortcomings of their approach are twofold: (1) insufficient
amounts of Man.sub.5GlcNAc.sub.2 are formed in the extra-cellular
glycoprotein fraction (10%) and (2) it is not clear that the in
vivo formed Man.sub.5GlcNAc.sub.2 structure in fact is able to
accept GlcNAc by action of GlcNAc transferase I. If several
glycosylation sites are present in a desired protein the
probability (P) of obtaining such a protein in a correct form
follows the relationship P.dbd.(F).sup.n, where n equals the number
of glycosylation sites, and F equals the fraction of desired
glycoforms. A glycoprotein with three glycosylation sites would
have a 0.1% chance of providing the appropriate precursors for
complex and hybrid N-glycan processing on all of its glycosylation
sites, which limits the commercial value of such an approach.
[0058] Most enzymes that are active in the ER and Golgi apparatus
of S. cerevisiae have pH optima that are between 6.5 and 7.5 (see
Table 3). All previous approaches to reduce mannosylation by the
action of recombinant mannosidases have concentrated on enzymes
that have a pH optimum around pH 5.0 (Martinet et al., 1998, and
Chiba et al., 1998), even though the activity of these enzymes is
reduced to less than 10% at pH 7.0 and thus most likely provide
insufficient activity at their point of use, the ER and early Golgi
of P. pastoris and S. cerevisiae. A preferred process utilizes an
.alpha.-mannosidase in vivo, where the pH optimum of the
mannosidase is within 1.4 pH units of the average pH optimum of
other representative marker enzymes localized in the same
organelle(s). The pH optimum of the enzyme to be targeted to a
specific organelle should be matched with the pH optimum of other
enzymes found in the same organelle, such that the maximum activity
per unit enzyme is obtained. Table 3 summarizes the activity of
mannosidases from various sources and their respective pH optima.
Table 4 summarizes their location.
TABLE-US-00003 TABLE 3 Mannosidases and their pH optimum. pH Source
Enzyme optimum Reference Aspergillus saitoi 1,2-.alpha.-mannosidase
5.0 Ichishima et al., 1999 Biochem. J. 339 (Pt 3): 589- 597
Trichoderma reesei 1,2-.alpha.-mannosidase 5.0 Maras et al., 2000
J. Biotechnol. 77 (2-3): 255-263 Penicillium citrinum
1,2-.alpha.-D-mannosidase 5.0 Yoshida et al., 1993 Biochem. J. 290
(Pt 2): 349- 354 Aspergillus nidulans 1,2-.alpha.-mannosidase 6.0
Eades and Hintz, 2000 Homo sapiens 1,2-.alpha.-mannosidase 6.0 IA
(Golgi) Homo sapiens IB 1,2-.alpha.-mannosidase 6.0 (Golgi)
Lepidopteran insect Type I 1,2-.alpha.-Man.sub.6- 6.0 Ren et al.,
1995 Biochem. cells mannosidase 34 (8): 2489-2495 Homo sapiens
.alpha.-D-mannosidase 6.0 Chandrasekaran et al., 1984 Cancer Res.
44 (9): 4059-68 Xanthomonas 1,2,3-.alpha.-mannosidase 6.0 manihotis
Mouse IB (Golgi) 1,2-.alpha.-mannosidase 6.5 Schneikert and
Herscovics, 1994 Glycobiology. 4 (4): 445-50 Bacillus sp.
(secreted) 1,2-.alpha.-D-mannosidase 7.0 Maruyama et al., 1994
Carbohydrate Res. 251: 89-98
[0059] When one attempts to trim high mannose structures to yield
Man.sub.5GlcNAc.sub.2 in the ER or the Golgi apparatus of S.
cerevisiae, one may choose any enzyme or combination of enzymes
that (1) has/have a sufficiently close pH optimum (i.e. between pH
5.2 and pH 7.8), and (2) is/are known to generate, alone or in
concert, the specific isomeric Man.sub.5GlcNAc.sub.2 structure
required to accept subsequent addition of GlcNAc by GnT I. Any
enzyme or combination of enzymes that has/have shown to generate a
structure that can be converted to GlcNAcMan.sub.5GlcNAc.sub.2 by
GnT I in vitro would constitute an appropriate choice. This
knowledge may be obtained from the scientific literature or
experimentally by determining that a potential mannosidase can
convert Man.sub.8GlcNAc.sub.2-PA to Man.sub.5GlcNAc.sub.2-PA and
then testing, if the obtained Man.sub.5GlcNAc.sub.2-PA structure
can serve a substrate for GnT I and UDP-GlcNAc to give
GlcNAcMan.sub.5GlcNAc.sub.2 in vitro. For example, mannosidase IA
from a human or murine source would be an appropriate choice.
[0060] 1,2-mannosidase Activity in the ER and Golgi
[0061] Previous approaches to reduce mannosylation by the action of
cloned exogenous mannosidases have failed to yield glycoproteins
having a sufficient fraction (e.g. >27 mole %) of N-glycans
having the structure Man.sub.5GlcNAc.sub.2 (Martinet et al., 1998,
and Chiba et al., 1998). These enzymes should function efficiently
in ER or Golgi apparatus to be effective in converting nascent
glycoproteins. Whereas the two mannosidases utilized in the prior
art (from A. saitoi and T. reesei) have pH optima of 5.0, most
enzymes that are active in the ER and Golgi apparatus of yeast
(e.g. S. cerevisiae) have pH optima that are between 6.5 and 7.5
(see Table 3). Since the glycosylation of proteins is a highly
evolved and efficient process, it can be concluded that the
internal pH of the ER and the Golgi is also in the range of about
6-8. At pH 7.0, the activity of the mannosidases used in the prior
art is reduced to less than 10%, which is insufficient for the
efficient production of Man.sub.5GlcNAc.sub.2 in vivo.
TABLE-US-00004 TABLE 4 Cellular location and pH optima of various
glycosylation- related enzymes of S. cerevisiae. pH Gene Activity
Location optimum Author (s) Ktr1 .alpha.-1,2 Golgi 7.0 Romero et
al., mannosyltransferase 1997 Biochem. J. 321 (Pt 2): 289-295 Mns1
.alpha.-1,2-mannosidase ER 6.5 CWH41 glucosidase I ER 6.8 --
mannosyltransferase Golgi 7-8 Lehele and Tanner, 1974 Biochim.
Biophys. Acta 350 (1): 225-235 Kre2 .alpha.-1,2 Golgi 6.5-9.0
Romero et al., mannosyltransferase 1997
[0062] The .alpha.-1,2-mannosidase enzyme should have optimal
activity at a pH between 5.1 and 8.0. In a preferred embodiment,
the enzyme has an optimal activity at a pH between 5.9 and 7.5. The
optimal pH may be determined under in vitro assay conditions.
Preferred mannosidases include those listed in Table 3 having
appropriate pH optima, e.g. Aspergillus nidulans, Homo sapiens IA
(Golgi), Homo sapiens IB (Golgi), Lepidopteran insect cells
(IPLB-SF21AE), Homo sapiens, mouse IB (Golgi), and Xanthomonas
manihotis. In a preferred embodiment, a single cloned mannosidase
gene is expressed in the host organism. However, in some cases it
may be desirable to express several different mannosidase genes, or
several copies of one particular gene, in order to achieve adequate
production of Man.sub.5GlcNAc.sub.2. In cases where multiple genes
are used, the encoded mannosidases should all have pH optima within
the preferred range of 5.1 to 8.0, or especially between 5.9 and
7.5. In an especially preferred embodiment mannosidase activity is
targeted to the ER or cis Golgi, where the early reactions of
glycosylation occur.
[0063] Formation of Complex N-Glycans
[0064] A second step of the process involves the sequential
addition of sugars to the nascent carbohydrate structure by
engineering the expression of glucosyltransferases into the Golgi
apparatus. This process first requires the functional expression of
GnT I in the early or medial Golgi apparatus as well as ensuring
the sufficient supply of UDP-GlcNAc.
[0065] Integration Sites
[0066] Since the ultimate goal of this genetic engineering effort
is a robust protein production strain that is able to perform well
in an industrial fermentation process, the integration of multiple
genes into the fungal chromosome involves careful planing. The
engineered strain will most likely have to be transformed with a
range of different genes, and these genes will have to be
transformed in a stable fashion to ensure that the desired activity
is maintained throughout the fermentation process. Any combination
of the following enzyme activities will have to be engineered into
the fungal protein expression host: sialyltransferases,
mannosidases, fucosyltransferases, galactosyltransferases,
glucosyltransferases, GlcNAc transferases, ER and Golgi specific
transporters (e.g. sym and antiport transporters for UDP-galactose
and other precursors), other enzymes involved in the processing of
oligosaccharides, and enzymes involved in the synthesis of
activated oligosaccharide precursors such as UDP-galactose,
CMP-N-acetylneuraminic acid. At the same time a number of genes
which encode enzymes known to be characteristic of non-human
glycosylation reactions, will have to be deleted.
[0067] Targeting of Glycosyltransferases to Specific
Organelles:
[0068] Glycosyltransferases and mannosidases line the inner
(luminal) surface of the ER and Golgi apparatus and thereby provide
a "catalytic" surface that allows for the sequential processing of
glycoproteins as they proceed through the ER and Golgi network. In
fact the multiple compartments of the cis, medial, and trans Golgi
and the trans-Golgi Network (TGN), provide the different localities
in which the ordered sequence of glycosylation reactions can take
place. As a glycoprotein proceeds from synthesis in the ER to full
maturation in the late Golgi or TGN, it is sequentially exposed to
different glycosidases, mannosidases and glycosyltransferases such
that a specific carbohydrate structure may be synthesized. Much
work has been dedicated to revealing the exact mechanism by which
these enzymes are retained and anchored to their respective
organelle. The evolving picture is complex but evidence suggests
that stem region, membrane spanning region and cytoplasmic tail
individually or in concert direct enzymes to the membrane of
individual organelles and thereby localize the associated catalytic
domain to that locus.
[0069] Targeting sequences are well known and described in the
scientific literature and public databases, as discussed in more
detail below with respect to libraries for selection of targeting
sequences and targeted enzymes.
Method for Producing a Library to Produce Modified Glycosylation
Pathways
[0070] A library including at least two genes encoding exogeneous
glycosylation enzymes is transformed into the host organism,
producing a genetically mixed population. Transformants having the
desired glycosylation phenotypes are then selected from the mixed
population. In a preferred embodiment, the host organism is a
yeast, especially P. pastoris, and the host glycosylation pathway
is modified by the operative expression of one or more human or
animal glycosylation enzymes, yielding protein N-glycans similar or
identical to human glycoforms. In an especially preferred
embodiment, the DNA library includes genetic constructs encoding
fusions of glycosylation enzymes with targeting sequences for
various cellular loci involved in glycosylation especially the ER,
cis Golgi, medial Golgi, or trans Golgi.
[0071] Examples of modifications to glycosylation which can be
effected using method are: (1) engineering an eukaryotic
microorganism to trim mannose residues from Man.sub.8GlcNAc.sub.2
to yield Man.sub.5GlcNAc.sub.2 as a protein N-glycan; (2)
engineering an eukaryotic microorganism to add an
N-acetylglucosamine (GlcNAc) residue to Man.sub.5GlcNAc.sub.2 by
action of GlcNAc transferase I; (3) engineering an eukaryotic
microorganism to functionally express an enzyme such as an
N-acetylglucosamine transferase (GnT I, GnT II, GnT III, GnT IV,
GnT V, GnT VI), mannosidase II, fucosyltransferase, galactosyl
tranferase (GalT) or sialyltransferases (ST).
[0072] By repeating the method, increasingly complex glycosylation
pathways can be engineered into the target microorganism. In one
preferred embodiment, the host organism is transformed two or more
times with DNA libraries including sequences encoding glycosylation
activities. Selection of desired phenotypes may be performed after
each round of transformation or alternatively after several
transformations have occurred. Complex glycosylation pathways can
be rapidly engineered in this manner.
[0073] DNA Libraries
[0074] It is necessary to assemble a DNA library including at least
two exogenous genes encoding glycosylation enzymes. In addition to
the open reading frame sequences, it is generally preferable to
provide each library construct with such promoters, transcription
terminators, enhancers, ribosome binding sites, and other
functional sequences as may be necessary to ensure effective
transcription and translation of the genes upon transformation into
the host organism. Where the host is Pichia pastoris, suitable
promoters include, for example, the AOX1, AOX2, DAS, and P40
promoters. It is also preferable to provide each construct with at
least one selectable marker, such as a gene to impart drug
resistance or to complement a host metabolic lesion. The presence
of the marker is useful in the subsequent selection of
transformants; for example, in yeast the URA3, HIS4, SUC2, G418,
BLA, or SH BLE genes may be used.
[0075] In some cases the library may be assembled directly from
existing or wild-type genes. In a preferred embodiment however the
DNA library is assembled from the fusion of two or more
sub-libraries. By the in-frame ligation of the sub-libraries, it is
possible to create a large number of novel genetic constructs
encoding useful targeted glycosylation activities. For example, one
useful sub-library includes DNA sequences encoding any combination
of enzymes such as sialyltransferases, mannosidases,
fucosyltransferases, galactosyltransferases, glucosyltransferases,
and GlcNAc transferases. Preferably, the enzymes are of human
origin, although other mammalian, animal, or fungal enzymes are
also useful. In a preferred embodiment, genes are truncated to give
fragments encoding the catalytic domains of the enzymes. By
removing endogenous targeting sequences, the enzymes may then be
redirected and expressed in other cellular loci. The choice of such
catalytic domains may be guided by the knowledge of the particular
environment in which the catalytic domain is subsequently to be
active. For example, if a particular glycosylation enzyme is to be
active in the late Golgi, and all known enzymes of the host
organism in the late Golgi have a certain pH optimum, then a
catalytic domain is chosen which exhibits adequate activity at that
pH.
[0076] Another useful sub-library includes DNA sequences encoding
signal peptides that result in localization of a protein to a
particular location within the ER, Golgi, or trans Golgi network.
These signal sequences may be selected from the host organism as
well as from other related or unrelated organisms. Membrane-bound
proteins of the ER or Golgi typically may include, for example,
N-terminal sequences encoding a cytosolic tail (ct), a
transmembrane domain (tmd), and a stem region (sr). The ct, tmd,
and sr sequences are sufficient individually or in combination to
anchor proteins to the inner (lumenal) membrane of the organelle.
Accordingly, a preferred embodiment of the sub-library of signal
sequences includes ct, tmd, and/or sr sequences from these
proteins. In some cases it is desirable to provide the sub-library
with varying lengths of sr sequence. This may be accomplished by
PCR using primers that bind to the 5' end of the DNA encoding the
cytosolic region and employing a series of opposing primers that
bind to various parts of the stem region. Still other useful
sources of signal sequences include retrieval signal peptides, e.g.
the tetrapeptides HDEL or KDEL, which are typically found at the
C-terminus of proteins that are transported retrograde into the ER
or Golgi. Still other sources of signal sequences include (a) type
II membrane proteins, (b) the enzymes listed in Table 3, (c)
membrane spanning nucleotide sugar transporters that are localized
in the Golgi, and (d) sequences referenced in Table 5.
TABLE-US-00005 TABLE 5 Sources of useful compartmental targeting
sequences Gene or Location of Gene Sequence Organism Function
Product MnsI S. .alpha.-1,2-mannosidase ER cerevisiae OCH1 S. 1,6-
Golgi (cis) cerevisiae mannosyltransferase MNN2 S. 1,2- Golgi
(medial) cerevisiae mannosyltransferase MNN1 S. 1,3- Golgi (trans)
cerevisiae mannosyltransferase OCH1 P. pastoris 1,6- Golgi (cis)
mannosyltransferase 2,6 ST H. sapiens 2,6-sialyltransferase trans
Golgi network UDP-Gal T S. pombe UDP-Gal transporter Golgi Mnt1 S.
1,2- Golgi (cis) cerevisiae mannosyltransferase HDEL at C- S.
retrieval signal ER terminus cerevisiae
[0077] In any case, it is highly preferred that signal sequences
are selected which are appropriate for the enzymatic activity or
activities which are to be engineered into the host. For example,
in developing a modified microorganism capable of terminal
sialylation of nascent N-glycans, a process which occurs in the
late Golgi in humans, it is desirable to utilize a sub-library of
signal sequences derived from late Golgi proteins. Similarly, the
trimming of Man.sub.8GlcNAc.sub.2 by an .alpha.-1,2-mannosidase to
give Man.sub.5GlcNAc.sub.2 is an early step in complex N-glycan
formation in humans. It is therefore desirable to have this
reaction occur in the ER or early Golgi of an engineered host
microorganism. A sub-library encoding ER and early Golgi retention
signals is used.
[0078] In a preferred embodiment, a DNA library is then constructed
by the in-frame ligation of a sub-library including DNA encoding
signal sequences with a sub-library including DNA encoding
glycosylation enzymes or catalytically active fragments thereof.
The resulting library includes synthetic genes encoding fusion
proteins. In some cases it is desirable to provide a signal
sequence at the N-terminus of a fusion protein, or in other cases
at the C-terminus. In some cases signal sequences may be inserted
within the open reading frame of an enzyme, provided the protein
structure of individual folded domains is not disrupted.
[0079] The method is most effective when a DNA library transformed
into the host contains a large diversity of sequences, thereby
increasing the probability that at least one transformant will
exhibit the desired phenotype. Accordingly, prior to
transformation, a DNA library or a constituent sub-library may be
subjected to one or more rounds of gene shuffling, error prone PCR,
or in vitro mutagenesis.
[0080] Transformation
[0081] The DNA library is then transformed into the host organism.
In yeast, any convenient method of DNA transfer may be used, such
as electroporation, the lithium chloride method, or the spheroplast
method. To produce a stable strain suitable for high-density
fermentation, it is desirable to integrate the DNA library
constructs into the host chromosome. In a preferred embodiment,
integration occurs via homologous recombination, using techniques
known in the art. For example, DNA library elements are provided
with flanking sequences homologous to sequences of the host
organism. In this manner integration occurs at a defined site in
the host genome, without disruption of desirable or essential
genes. In an especially preferred embodiment, library DNA is
integrated into the site of an undesired gene in a host chromosome,
effecting the disruption or deletion of the gene. For example,
integration into the sites of the OCH1, MNN1, or MNN4 genes allows
the expression of the desired library DNA while preventing the
expression of enzymes involved in yeast hypermannosylation of
glycoproteins. In other embodiments, library DNA may be introduced
into the host via a chromosome, plasmid, retroviral vector, or
random integration into the host genome. In any case, it is
generally desirable to include with each library DNA construct at
least one selectable marker gene to allow ready selection of host
organisms that have been stably transformed. Recyclable marker
genes such as ura3, which can be selected for or against, are
especially suitable.
[0082] Selection Process
[0083] After transformation of the host strain with the DNA
library, transformants displaying the desired glycosylation
phenotype are selected. Selection may be performed in a single step
or by a series of phenotypic enrichment and/or depletion steps
using any of a variety of assays or detection methods. Phenotypic
characterization may be carried out manually or using automated
high-throughput screening equipment. Commonly a host microorganism
displays protein N-glycans on the cell surface, where various
glycoproteins are localized. Accordingly intact cells may be
screened for a desired glycosylation phenotype by exposing the
cells to a lectin or antibody that binds specifically to the
desired N-glycan. A wide variety of oligosaccharide-specific
lectins are available commercially (EY Laboratories, San Mateo,
Calif.). Alternatively, antibodies to specific human or animal
N-glycans are available commercially or may be produced using
standard techniques. An appropriate lectin or antibody may be
conjugated to a reporter molecule, such as a chromophore,
fluorophore, radioisotope, or an enzyme having a chromogenic
substrate (Guillen et al., 1998. Proc. Natl. Acad. Sci. USA 95(14):
7888-7892). Screening may then be performed using analytical
methods such as spectrophotometry, fluorimetry, fluorescence
activated cell sorting, or scintillation counting. In other cases,
it may be necessary to analyze isolated glycoproteins or N-glycans
from transformed cells. Protein isolation may be carried out by
techniques known in the art. In cases where an isolated N-glycan is
required, an enzyme such as endo-.beta.-N-acetylglucosaminidase
(Genzyme Co., Boston, Mass.) may be used to cleave the N-glycans
from glycoproteins. Isolated proteins or N-glycans may then be
analyzed by liquid chromatography (e.g. HPLC), mass spectroscopy,
or other suitable means. U.S. Pat. No. 5,595,900 teaches several
methods by which cells with desired extracellular carbohydrate
structures may be identified. Prior to selection of a desired
transformant, it may be desirable to deplete the transformed
population of cells having undesired phenotypes. For example, when
the method is used to engineer a functional mannosidase activity
into cells, the desired transformants will have lower levels of
mannose in cellular glycoprotein. Exposing the transformed
population to a lethal radioisotope of mannose in the medium
depletes the population of transformants having the undesired
phenotype, i.e. high levels of incorporated mannose. Alternatively,
a cytotoxic lectin or antibody, directed against an undesirable
N-glycan, may be used to deplete a transformed population of
undesired phenotypes.
Methods for Providing Sugar Nucleotide Precursors to the Golgi
Apparatus
[0084] For a glycosyltransferase to function satisfactorily in the
Golgi, it is necessary for the enzyme to be provided with a
sufficient concentration of an appropriate nucleotide sugar, which
is the high-energy donor of the sugar moiety added to a nascent
glycoprotein. These nucleotide sugars to the appropriate
compartments are provided by expressing an exogenous gene encoding
a sugar nucleotide transporter in the host microorganism. The
choice of transporter enzyme is influenced by the nature of the
exogenous glycosyltransferase being used. For example, a GlcNAc
transferase may require a UDP-GlcNAc transporter, a
fucosyltransferase may require a GDP-fucose transporter, a
galactosyltransferase may require a UDP-galactose transporter, or a
sialyltransferase may require a CMP-sialic acid transporter.
[0085] The added transporter protein conveys a nucleotide sugar
from the cytosol into the Golgi apparatus, where the nucleotide
sugar may be reacted by the glycosyltransferase, e.g. to elongate
an N-glycan. The reaction liberates a nucleoside diphosphate or
monophosphate, e.g. UDP, GDP, or CMP. As accumulation of a
nucleoside diphosphate inhibits the further activity of a
glycosyltransferase, it is frequently also desirable to provide an
expressed copy of a gene encoding a nucleotide diphosphatase. The
diphosphatase (specific for UDP or GDP as appropriate) hydrolyzes
the diphosphonucleoside to yield a nucleoside monosphosphate and
inorganic phosphate. The nucleoside monophosphate does not inhibit
the glycotransferase and in any case is exported from the Golgi by
an endogenous cellular system. Suitable transporter enzymes, which
are typically of mammalian origin, are described below.
EXAMPLES
[0086] The use of the above general method may be understood by
reference to the following non-limiting examples. Examples of
preferred embodiments are also summarized in Table 6.
Example 1
Engineering of P. pastoris with .alpha.-1,2-mannosidase to Produce
Insulin
[0087] An .alpha.-1,2-mannosidase is required for the trimming of
Man.sub.8GlcNAc.sub.2 to yield Man.sub.5GlcNAc.sub.2, an essential
intermediate for complex N-glycan formation. An OCH1 mutant of P.
pastoris is engineered to express secreted human interferon-.alpha.
under the control of an aox promoter. A DNA library is constructed
by the in-frame ligation of the catalytic domain of human
mannosidase IB (an .alpha.-1,2-mannosidase) with a sub-library
including sequences encoding early Golgi localization peptides. The
DNA library is then transformed into the host organism, resulting
in a genetically mixed population wherein individual transformants
each express interferon-.beta. as well as a synthetic mannosidase
gene from the library. Individual transformant colonies are
cultured and the production of interferon is induced by addition of
methanol. Under these conditions, over 90% of the secreted protein
includes interferon-.beta.. Supernatants are purified to remove
salts and low-molecular weight contaminants by C.sub.18 silica
reversed-phase chromatography. Desired transformants expressing
appropriately targeted, active .alpha.-1,2-mannosidase produce
interferon-.beta. including N-glycans of the structure
Man.sub.5GlcNAc.sub.2, which has a reduced molecular mass compared
to the interferon of the parent strain. The purified supernatants
including interferon-.beta. are analyzed by MALDI-TOF mass
spectroscopy and colonies expressing the desired form of
interferon-.beta. are identified.
Example 2
Engineering of Strain to Express GlcNAc Transferase I
[0088] GlcNAc Transferase I activity is required for the maturation
of complex N-glycans. Man.sub.5GlcNAc.sub.2 may only be trimmed by
mannosidase II, a necessary step in the formation of human
glycoforms, after the addition of GlcNAc to the terminal
.alpha.-1,3 mannose residue by GlcNAc Transferase I (Schachter,
1991 Glycobiology 1(5):453-461). Accordingly a library is prepared
including DNA fragments encoding suitably targeted GlcNAc
Transferase I genes. The host organism is a strain, e.g. a yeast,
that is deficient in hypermannosylation (e.g. an OCH1 mutant),
provides the substrate UDP-GlcNAc in the Golgi and/or ER, and
provides N-glycans of the structure Man.sub.5GlcNAc.sub.2 in the
Golgi and/or ER. After transformation of the host with the DNA
library, the transformants are screened for those having the
highest concentration of terminal GlcNAc on the cell surface, or
alternatively secrete the protein having the highest terminal
GlcNAc content. Such a screen is performed using a visual method
(e.g. a staining procedure), a specific terminal GlcNAc binding
antibody, or a lectin. Alternatively the desired transformants
exhibit reduced binding of certain lectins specific for terminal
mannose residues.
Example 3
Engineering of Strains with a Mannosidase II
[0089] In another example, it is desirable in order to generate a
human glycoform in a microorganism to remove the two remaining
terminal mannoses from the structure GlcNAcMan.sub.5GlcNAc.sub.2 by
action of a mannosidase II. A DNA library including sequences
encoding cis and medial Golgi localization signals is fused
in-frame to a library encoding mannosidase II catalytic domains.
The host organism is a strain, e.g. a yeast, that is deficient in
hypermannosylation (e.g. an OCH1 mutant) and provides N-glycans
having the structure GlcNAcMan.sub.5GlcNAc.sub.2 in the Golgi
and/or ER. After transformation, organisms having the desired
glycosylation phenotype are selected. An in vitro assay is used in
one method. The desired structure GlcNAcMan.sub.3GlcNAc.sub.2 (but
not the undesired GlcNAcMan.sub.5GlcNAc.sub.2) is a substrate for
the enzyme GlcNAc Transferase II. Accordingly, single colonies may
be assayed using this enzyme in vitro in the presence of the
substrate, UDP-GlcNAc. The release of UDP is determined either by
HPLC or an enzymatic assay for UDP. Alternatively radioactively
labeled UDP-GlcNAc is used.
[0090] The foregoing in vitro assays are conveniently performed on
individual colonies using high-throughput screening equipment.
Alternatively a lectin binding assay is used. In this case the
reduced binding of lectins specific for terminal mannoses allows
the selection of transformants having the desired phenotype. For
example, Galantus nivalis lectin binds specifically to terminal
.alpha.-1,3-mannose, the concentration of which is reduced in the
presence of operatively expressed mannosidase II activity. In one
suitable method, G. nivalis lectin attached to a solid agarose
support (available from Sigma Chemical, St. Louis, Mo.) is used to
deplete the transformed population of cells having high levels of
terminal .alpha.-1,3-mannose.
Example 4
Engineering of Organisms to Express Sialyltransferase
[0091] The enzymes .alpha.-2,3-sialyltransferase and
.alpha.-2,6-sialyltransferase add terminal sialic acid to galactose
residues in nascent human N-glycans, leading to mature
glycoproteins. In human the reactions occur in the trans Golgi or
TGN. Accordingly a DNA library is constructed by the in-frame
fusion of sequences encoding sialyltransferase catalytic domains
with sequences encoding trans Golgi or TGN localization signals.
The host organism is a strain, e.g. a yeast, that is deficient in
hypermannosylation (e.g. an OCH1 mutant), which provides N-glycans
having terminal galactose residues in the trans Golgi or TGN, and
provides a sufficient concentration of CMP-sialic acid in the trans
Golgi or TGN. Following transformation, transformants having the
desired phenotype are selected using a fluorescent antibody
specific for N-glycans having a terminal sialic acid.
Example 5
Method of engineering strains to express UDP-GlcNAc Transporter
[0092] The cDNA of human Golgi UDP-GlcNAc transporter has been
cloned by Ishida and coworkers. (Ishida, N., et al. 1999 J.
Biochem. 126(1): 68-77. Guillen and coworkers have cloned the
canine kidney Golgi UDP-GlcNAc transporter by phenotypic correction
of a Kluyveromyces lactis mutant deficient in Golgi UDP-GlcNAc
transport. (Guillen, E., et al. 1998). Thus a mammalian Golgi
UDP-GlcNAc transporter gene has all of the necessary information
for the protein to be expressed and targeted functionally to the
Golgi apparatus of yeast.
Example 6
Method of Engineering Strains to Express GDP-Fucose Transporter
[0093] The rat liver Golgi membrane GDP-fucose transporter has been
identified and purified by Puglielli, L. and C. B. Hirschberg 1999
J. Biol. Chem. 274(50):35596-35600. The corresponding gene can be
identified using standard techniques, such as N-terminal sequencing
and Southern blotting using a degenerate DNA probe. The intact gene
can is then be expressed in a host microorganism that also
expresses a fucosyltransferase.
Example 7
Method of Engineering Strains to Express UDP-Galactose
Transporter
[0094] Human UDP-galactose (UDP-Gal) transporter has been cloned
and shown to be active in S. cerevisiae. (Kainuma, M., et al. 1999
Glycobiology 9(2): 133-141). A second human UDP-galactose
transporter (hUGT1) has been cloned and functionally expressed in
Chinese Hamster Ovary Cells. Aoki, K., et al. 1999 J. Biochem.
126(5): 940-950. Likewise Segawa and coworkers have cloned a
UDP-galactose transporter from Schizosaccharomyces pombe (Segawa,
H., et al. 1999 Febs Letters 451(3): 295-298).
[0095] CMP-Sialic Acid Transporter
[0096] Human CMP-sialic acid transporter (hCST) has been cloned and
expressed in Lec 8 CHO cells by Aoki and coworkers (1999).
Molecular cloning of the hamster CMP-sialic acid transporter has
also been achieved (Eckhardt and Gerardy Schahn 1997 Eur. J.
Biochem. 248(1): 187-192). The functional expression of the murine
CMP-sialic acid transporter was achieved in Saccharomyces
cerevisiae by Berninsone, P., et al. 1997 J. Biol. Chem.
272(19):12616-12619.
TABLE-US-00006 TABLE 6 Examples of preferred embodiments of the
methods for modifying glycosylation in a eukaroytic microorganism,
e.g. Pichia pastoris Suitable Suitable Suitable Transporters
Desired Catalytic Suitable Sources of Gene and/or Structure
Activities Localization Sequences Deletions Phosphatases
Man.sub.5G1cNAc.sub.2 .alpha.-1,2- Mns1 (N-terminus, OCH1 none
mannosidase S. cerevisiae) MNN4 (murine, Och1 (N-terminus, MNN6
human, S. cerevisiae, P. pastoris) Bacillus sp., Ktr1 A. nidulans)
Mnn9 Mnt1 (S. cerevisiae) KDEL, HDEL (C-terminus)
GlcNAcMan.sub.5G1cN GlcNAc Och1 (N-terminus, OCH1 UDP-GlcNAc
Ac.sub.2 Transferase I, S. cerevisiae, P. pastoris) MNN4
transporter (human, murine, KTR1 (N-terminus) MNN6 (human, murine,
rat etc.) KDEL, HDEL K. lactis) (C-terminus) UDPase (human) Mnn1
(N-terminus, S. cerevisiae) Mnt1 (N-terminus, S. cerevisiae) GDPase
(N-terminus, S. cerevisiae) GlcNAcMan.sub.3GlcN mannosidase Ktr1
OCH1 UDP-GlcNAc Ac.sub.2 II Mnn1 (N-terminus, MNN4 transporter S.
cerevisiae) MNN6 (human, murine, Mnt1 (N-terminus, K. lactis) S.
cerevisiae) UDPase (human) Kre2/Mnt1 (S. cerevisiae) Kre2 (P.
pastoris) Ktr1 (S. cerevisiae) Ktr1 (P. pastoris) Mnn1 (S.
cerevisiae) G1cNAc.sub.(2-4)Man.sub.3G1cNAc.sub.2 GlcNAc Mnn1
(N-terminus, OCH1 UDP-GlcNAc Transferase S. cerevisiae) MNN4
transporter II, III, IV, V Mnt1 (N-terminus, MNN6 (human, murine,
(human, murine) S. cerevisiae) K. lactis) Kre2/Mnt1 (S. cerevisiae)
UDPase (human) Kre2 (P. pastoris) Ktr1 (S. cerevisiae) Ktr1 (P.
pastoris) Mnn1 (S. cerevisiae) Gal.sub.(1-4)GlcNAc.sub.(2-4)-
.beta.-1,4- Mnn1 (N-terminus, OCH1 UDP-Galactose
Man.sub.3GlcNAc.sub.2 Galactosyl S. cerevisiae) MNN4 transporter
transferase Mnt1 (N-terminus, MNN6 (human, (human) S. cerevisiae)
S. pombe) Kre2/Mnt1 (S. cerevisiae) Kre2 (P. pastoris) Ktr1 (S.
cerevisiae) Ktr1 (P. pastoris) Mnn1 (S. cerevisiae) NANA.sub.(1-4)-
.alpha.-2,6- KTR1 OCH1 CMP-Sialic acid
Gal.sub.(1-4)GlcNAc.sub.(2-4)- Sialyltransferase MNN1 (N-terminus,
MNN4 transporter Man.sub.3GlcNAc.sub.2 (human) S. cerevisiae) MNN6
(human) .alpha.-2,3- MNT1 (N-terminus, Sialyltransferase S.
cerevisiae) Kre2/Mnt1 (S. cerevisiae) Kre2 (P. pastoris) Ktr1 (S.
cerevisiae) Ktr1 (P. pastoris) MNN1 (S. cerevisiae)
TABLE-US-00007 TABLE 7 DNA and Protein Sequence Resources 1.
European Bioinformatics Institute (EBI) is a centre for research
and services in bioinformatics: http://www.ebi.ac.uk/ 2. Swissprot
database: http://www.expasy.ch/spr 3. List of known
glycosyltransferases and their origin. .beta.1,2 (GnT I) EC
2.4.1.101 4. human cDNA, Kumar et al (1990) Proc. Natl. Acad. Sci.
USA 87: 9948-9952 5. human gene, Hull et al (1991) Biochem.
Biophys. Res. Commun. 176: 608-615 6. mouse cDNA, Kumar et al
(1992) Glycobiology 2: 383-393 7. mouse gene, Pownall et al (1992)
Genomics 12: 699-704 8. murine gene (5' flanking, non-coding), Yang
et al (1994) Glycobiology 5: 703-712 9. rabbit cDNA, Sarkar et al
(1991) Proc. Natl. Acad. Sci. USA 88: 234-238 10. rat cDNA, Fukada
et al (1994) Biosci.Biotechnol.Biochem. 58: 200-201 1,2 (GnT II) EC
2.4.1.143 11. human gene, Tan et al (1995) Eur. J. Biochem. 231:
317-328 12. rat cDNA, D'Agostaro et al (1995) J. Biol. Chem. 270:
15211-15221 13. (.beta.1,4 (GnT III) EC 2.4.1.144 14. human cDNA,
Ihara et al (1993) J. Biochem.113: 692-698 15. murine gene, Bhaumik
et al (1995) Gene 164: 295-300 16. rat cDNA, Nishikawa et al (1992)
J. Biol. Chem. 267: 18199-18204 .beta.1,4 (GnT IV) EC 2.4.1.145 17.
human cDNA, Yoshida et al (1998) Glycoconjugate Journal 15:
1115-1123 18. bovine cDNA, Minowa et al., European Patent EP 0 905
232 .beta.1,6 (GnT V) EC 2.4.1.155 19. human cDNA, Saito et al
(1994) Biochem. Biophys. Res. Commun. 198: 318-327 20. rat cDNA,
Shoreibah et al (1993) J. Biol. Chem. 268: 15381-15385 .beta.1,4
Galactosyltransferase, EC 2.4.1.90 (LacNAc synthetase) EC 2.4.1.22
(lactose synthetase) 21. bovine cDNA, D'Agostaro et al (1989) Eur.
J. Biochem. 183: 211-217 22. bovine cDNA (partial), Narimatsu et al
(1986) Proc. Natl. Acad. Sci. USA 83: 4720-4724 23. bovine cDNA
(partial), Masibay & Qasba (1989) Proc. Natl. Acad. Sci. USA
86: 5733-5377 24. bovine cDNA (5' end), Russo et al (1990) J. Biol.
Chem. 265: 3324 25. chicken cDNA (partial), Ghosh et al (1992)
Biochem. Biophys. Res. Commun. 1215-1222 26. human cDNA, Masri et
al (1988) Biochem. Biophys. Res. Commun. 157: 657-663 27. human
cDNA, (HeLa cells) Watzele & Berger (1990) Nucl. Acids Res. 18:
7174 28. human cDNA, (partial) Uejima et al (1992) Cancer Res. 52:
6158-6163 29. human cDNA, (carcinoma) Appert et al (1986) Biochem.
Biophys. Res. Commun. 139: 163-168 30. human gene, Mengle-Gaw et al
(1991) Biochem. Biophys. Res. Commun. 176: 1269-1276 31. murine
cDNA, Nakazawa et al (1988) J. Biochem. 104: 165-168 32. murine
cDNA, Shaper et al (1988) J. Biol. Chem. 263: 10420-10428 33.
murine cDNA (novel), Uehara & Muramatsu unpublished 34. murine
gene, Hollis et al (1989) Biochem. Biophys. Res. Commun. 162:
1069-1075 35. rat protein (partial), Bendiak et al (1993) Eur. J.
Biochem. 216: 405-417 2,3-Sialyltransferase, (ST3Gal II) (N-linked)
(Gal-1,3/4-GlcNAc) EC 2.4.99.6 36. human cDNA, Kitagawa &
Paulson (1993) Biochem. Biophys. Res. Commun. 194: 375-382 37. rat
cDNA, Wen et al (1992) J. Biol. Chem. 267: 21011-21019
2,6-Sialyltransferase, (ST6Gal I) EC 2.4.99.1 38. chicken, Kurosawa
et al (1994) Eur. J. Biochem 219: 375-381 39. human cDNA (partial),
Lance et al (1989) Biochem. Biophys. Res. Commun. 164: 225-232 40.
human cDNA, Grundmann et al (1990) Nucl. Acids Res. 18: 667 41.
human cDNA, Zettlmeisl et al (1992) Patent EP0475354-A/3 42. human
cDNA, Stamenkovic et al (1990) J. Exp. Med. 172: 641-643 (CD75) 43.
human cDNA, Bast et al (1992) J. Cell Biol. 116: 423-435 44. human
gene (partial), Wang et al (1993) J. Biol. Chem. 268: 4355-4361 45.
human gene (5' flank), Aasheim et al (1993) Eur. J. Biochem. 213:
467-475 46. human gene (promoter), Aas-Eng et al (1995) Biochim.
Biophys. Acta 1261: 166-169 47. mouse cDNA, Hamamoto et al (1993)
Bioorg. Med. Chem. 1: 141-145 48. rat cDNA, Weinstein et al (1987)
J. Biol. Chem. 262: 17735-17743 49. rat cDNA (transcript
fragments), Wang et al (1991) Glycobiology 1: 25-31, Wang et al
(1990) J. Biol. Chem. 265: 17849-17853 50. rat cDNA (5' end),
O'Hanlon et al (1989) J. Biol. Chem. 264: 17389-17394; Wang et al
(1991) Glycobiology 1: 25-31 51. rat gene (promoter), Svensson et
al (1990) J. Biol. Chem. 265: 20863-20688 52. rat mRNA (fragments),
Wen et al (1992) J. Biol. Chem. 267: 2512-2518
[0097] Additional methods and reagents which can be used in the
methods for modifying the glycosylation are described in the
literature, such as U.S. Pat. No. 5,955,422, U.S. Pat. No.
4,775,622, U.S. Pat. No. 6,017,743, U.S. Pat. No. 4,925,796, U.S.
Pat. No. 5,766,910, U.S. Pat. No. 5,834,251, U.S. Pat. No.
5,910,570, U.S. Pat. No. 5,849,904, U.S. Pat. No. 5,955,347, U.S.
Pat. No. 5,962,294, U.S. Pat. No. 5,135,854, U.S. Pat. No.
4,935,349, U.S. Pat. No. 5,707,828, and U.S. Pat. No.
5,047,335.
[0098] Appropriate yeast expression systems can be obtained from
sources such as the American Type Culture Collection, Rockville,
Md. Vectors are commercially available from a variety of sources.
Sequence CWU 1
1
6121DNAArtificial SequencePrimer A for target gene in P. pastoris
(1,6-mannosyltransferase) 1atggcgaagg cagatggcag t
21221DNAArtificial SequencePrimer B for target gene in P. pastoris
(1,6-mannosyltransferase) 2ttagtccttc caacttcctt c
21326DNAArtificial SequencePrimer A for target gene in P. pastoris
(1,2 mannosyltransferases) 3tantggngng tngancnnga natnaa
26420DNAArtificial SequencePrimer B for target gene in P. pastoris
(1,2 mannosyltransferases) 4gcntcncccc ancnntcnta 2054PRTArtificial
SequenceSignal tetrapeptide 5His Asp Glu Leu 1 64PRTArtificial
SequenceSignal tetrapeptide 6Lys Asp Glu Leu 1
* * * * *
References