AU2013201263B2 - System and method for the production of recombinant glycosylated proteins in a prokaryotic host - Google Patents

Abstract

Abstract The present invention relates generally to a system and a method for the production of recombinant N-glycosylated target proteins. In one aspect, the system of the present invention comprises a prokaryotic organism (e.g. Escherichia coli) into which is introduced a genetic information encoding for a metabolic apparatus capable of carrying out the requested N-glycosylation of the target protein. Said prokaryotic organism also contains the genetic information required for the expression of one or more recombinant target proteins. The metabolic apparatus preferably comprises specific glycosyltransferases for the assembly of the oligosaccharide on a lipid carrier and an OTase that covalently links this oligosaccharide to specific residues of the desired protein.

Description

AUSTRALIA Patents Act COMPLETE SPECIFICATION (ORIGINAL) Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: ETH ZUrich Actual Inventor(s): Markus Aebi, Michael Wacker Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: SYSTEM AND METHOD FOR THE PRODUCTION OF RECOMBINANT GLYCOSYLATED PROTEINS IN A PROKARYOTIC HOST Our Ref: 964515 POF Code: 125692/490246 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): - 1- 1A SYSTEM AND METHOD FOR THE PRODUCTION OF RECOMBINANT GLYCOSYLATED PROTEINS IN A PROKARYOTIC HOST The present application is a divisional application from Australian patent application 5 number 2008201902, the entire disclosure of which is incorporated herein by reference. Field of Invention The present invention relates to an expression system and a method for the 10 production of recombinant human and/or animal and/or plant and/or prokaryotic and/or fungal glycoproteins. Such glycoproteins may serve as nutrition or medical drugs for human or animals or plants because of their identical structure to the glycoproteins normally produced in these organisms. 15 Technical Background Glycosylation constitutes one of the most important of all post-translational protein modifications in eukaryotic cells and may have numerous effects on function, structure, physical properties and targeting of particular proteins. Generally, the cNenafmo WO 03/074687 PCT/CH03/00153 -2 carbohydrate moiety is to be regarded as having significant effects on both the structure and on the physicochemical features of a protein and may affect its en zymatic activity, antigenicity or thermal stability. The sugars can be linked via the e-amine group of an asparagine (N-glycosidic bond) or the hydroxyl group of a 5 serine or threonine (0-glycosidic bond) residue. The N-linked protein glycosylation is by far the most common protein modifica tion found in eukaryotes. The complex glycosylation process starts at the cyto plasmic face of the endoplasmatic reticulum (ER) with the assembly of an ollgo 10 saccharide on the lipid carrier dolichylpyrophosphate [Burda, P. and Aebi, M. (1999) The dolichol pathway of N-linked glycosylation. Blochim Biophys Acta, 1426, 239-257]: 2 N-acetylglucosamine and 5 mannose residues are attached to this lipid in a stepwise fashion. The lipid linked oligosaccharide (LLO) is then flipped into the lumen of the ER, where by addition of 4 mannose and 3 glucose 15 residues full length LLO is obtained. In the central reaction of the process, this oligosaccharide Is transferred to selected asparagine residues of newly synthe sized polypeptides. This reaction is catalyzed by the oligosaccharyl transferase (OTase) in the lumen of the ER. The OTase is a complex of at least 8 subunits and this enzyme is responsible for the formation of the N-glycosidic bond. While 20 still in the ER, three glucose and one mannose residue are quickly removed from the oligosaccharide of the glycoprotein. Glycoproteins are then transported to the Golgi apparatus where further trimming and addition of sugar moieties occurs before they are targeted to their final destinations [Varki, A., Cummings, R., Esko, J., Freeze, H., Hart, G. and Marth, J. (1999) Essent/als of Glycobiology. 25 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York]. Whereas the LLO synthesis is a highly conserved process in eukaryotic cells, the modifica tions in the Golgi are not only species specific but also cell-type specific and lead to a high degree of diversity with respect to the structure of the N-linked oligo saccharides. 30 2a Throughout the description and the claims of this specification the word 'comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps. 5 The discussion of documents, acts, materials, devices, articles and the like is Included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention before the priority date of each claim of this application. 10 WIwk amt1 70009472m 5 9 2 a? I Is"" 15.1.00 doc WO 03/074687 PCT/CH03/00153 -3 Prior art The production of human proteins in a variety of heterologous expression sys tems has become an important technique to generate recombinant proteins for research purposes as well as pharmaceutical applications. It is generally recog 5 nized that there is no universal expression system available for production. Fur thermore, the selection of a cell type for expression of heterologous proteins de pends on many factors. These include criteria such as cell growth characteristics, expression levels, intracellular or extracellular expression, and biological activity of the protein of Interest as well as its intended use. But one of the most impor 10 tant criteria to be considered is whether a protein needs to be glycosylated for its application. Many human therapeutics are glycoproteins, and the importance of the posttranslational modification of polypeptides with defined oligosaccharides is well documented by their implication in numerous biological phenomena. 15 In mammalian glycoproteins, the majority of N-glycans are of the complex type, i.e. they consist of a pentasaccharide core of two N-acetylglucosamine and three mannose residues. This core Is the remaining structure of the oligosaccharide that was originally transferred from dolichylpyrophosphate to proteins. In the Golgi it is further modified with antennae comprising additional N-acetylglucosa 20 mine, galactose, sialic acid and often fucose residues. An enormous diversity of impressively complex oligosaccharide structures is thereby possible. The impor tance of the complex N-glycans and the essential role of these structures are shown with experiments using knock-out mice unable to synthesize these com plex-type N-glycans. These mice die before birth. Recently the congenital disor 25 der of glycosylation (CDG) has been described in humans. This is a group of con genital multi-systemic diseases characterized by deficiency in the generation of N-glycans. These disorders are manifested by a wide variety of clinical features, such as disorders of the nervous system development, psychomotor retardation, dysmorphic features, hypotonia, coagulation disorders, and immunodeficiency. 30 The broad spectrum of features reflects the critical role of N-glycoproteins during embryonic development, differentiation, and maintenance of cell functions and emphasizes the importance of the correct oligosaccharlde structure.

WO 03/074687 PCT/CH03/00153 -4 Since the majority of therapeutically relevant proteins are glycosylated in their natural forms, they should also be glycosylated as recombinant proteins in order to get the correct biological activity. Thus, monitoring of the glycosylation pattern in quality control of recombinant proteins to assure product safety, efficiency and 5 consistency has become Increasingly important. Systems for the expression of glycosylated proteins have been developed. The most commonly used are Chi nese hamster ovary (CHO) cell lines [Grabenhorst, E., Schlenke, P., Pohl, S., Nimtz, M. and Conradt, H.S. (1999) Genetic engineering of recombinant glyco proteins and the glycosylation pathway in mammalian host cells. Glycoconjugate 10 Journal, 16, 81-97], insect cells [Altmann, F., Staudacher, E., Wilson, I.B. and Marz, L. (1999) Insect cells as hosts for the expression of recombinant glyco proteins. Glycoconjugate Journal, 16, 109-123] or fungal cells [Malissard, M., Zeng, S. and Berger, E.G. (1999) The yeast expression system for recombinant glycosyltransferases. Glycoconjugate Journal, 16, 125-139. or Maras, M., van 15 Die, I., Contreras, R. and van den Hondel, C.A. (1999) Filamentous fungi as pro duction organisms for glycoproteins of bio-medical interest. Glycoconjugate Jour nal, 16, 99-107]. These cell lines have all the capability to glycosylate proteins, but they exhibit major differences in the production of recombinant glycopro teins. As mentioned above, the synthesis of the LLO and the transfer of the oli 20 gosaccharide to polypeptides is a highly conserved mechanism in all eukaryotes, whereas further processing and trimming of the N-glycans In the Golgi vary be tween organisms and cell type. Therefore, the final structure of the recombinant glycoprotein Is defined by the production cell used. CHO cells are the mammalian cell lines commonly used for the expression of recombinant glycoproteins. They 25 are able to synthesize complex type N-glycans, but some human tissue-specific terminal sugar residues are not synthesized by these cells since they do not ex press the proper glycosyltransferases. Therefore, the host cell lines must be Im proved by genetic engineering with the introduction of these glycosyltransfer ases. 30 Insect cells (see also WO 00/52135] are also widely used to produce recombinant proteins, as they can synthesize large quantities of a protein of Interest when Infected with powerful baculovirus-based gene expression vectors, and they can WO 03/074687 PCT/CH03/00153 -5 provide post-translational modifications similar to those provided by mammalian cells. The N-glycosylation pathway parallels the mammalian pathway until the formation of the core pentasaccharide. But normally insect cells do not express additional transferases in the Golgi and therefore the N-glycans produced are 5 truncated (paucimannosidic) instead of a complex type as found In mammalian cells. Fungi, in particular Saccharomyces cerevislae or Pichla pastoris, are suitable host organisms for the production of eukaryotic heterologous proteins. These systems 10 combine well-known techniques for the molecular and genetic manipulations, the cells are easy to grow and they have the capability for complex post-translational modifications such as protein glycosylation. In contrast to animal cells, fungi do not further trim the ollgosaccharide in the Golgi but instead elongate it directly by the addition of mannose residues to form mannanes with up to 200 mannose 15 units. Some glycoproteins escape these modifications and their maturation is more limited, yielding short core type oligosaccharides with up to 13 mannose residues. These three examples of N-glycosylation in eukaryotes emphasize the differences 20 In the structure of N-glycans. The implication on the function reveals that exact analysis of the structure Is essential. Significant advances in carbohydrate struc tural analyses have been achieved during the past years. Especially In mass spectrometry (on-line ESI-MS, nanospray tandem mass spectrometry (ESI MS/MS) and improved MALDI/TOF techniques), very sensitive instrumentation 25 for glycosylation analysis has been made available. Problems in prior art The importance of a highly defined oligosaccharlde structure on recombinant gly 30 coproteins contrasts sharply to the inability of presently used biotechnological processes to generate glycoproteins. This is due to the fact that the structure of a protein-linked oligosaccharlde is determined directly by the cell type used and all of the eukaryotic production systems exhibit this specificity. It might be possi- WO 03/074687 PCT/CH03/00153 ble to genetically engineer eukaryotic production cell lines in such a way that a defined oligosaccharlde structure Is produced. However, the plethora of glycosyl transferases active in the Golgi compartment of eukaryotes makes such an ap proach very difficult. Additional problems with the use of eukaryotic expression 5 systems are the following: In general, the mammalian expression system has its drawbacks in the use of growth medium, which contains calf serum. This raises concern about blosafety because of possible contamination with bovine spongi form encephalopathy (BSE). Furthermore human cell line cultures are much more difficult to keep sterile, these cells grow slowly and require expensive process 10 control. As mentioned before, the specific glycoprotein synthesized depends di rectly on the cell line or cell type used. In other words, a recombinant glycopro tein only gets modified with its original N-glycan if the heterologous system ex presses the same enzymes of the N-glycosylation pathway as in its origin. Oth erwise host cells must be adapted by genetic engineering of the glycosylation 15 pathway In the Golgi (Fig. 1B), and this represents the major drawback of human cell lines In the expression of recombinant glycoproteins. The difficulties of the production of recombinant proteins In insect cells with the help of the baculoviruses expression system are the following: Baculoviruses es 20 sentially have a lytic Infection mode, i.e. when the product is harvested, a large proportion of the host cells is lysed and releases degradative enzymes. In addi tion, the protein synthesis is maximal near death of Infected cells and It is possi ble that the overall processing of the protein is suboptimal at that time. Particu larly proteins destined for the plasma membrane or for secretion are affected by 25 the depletion of components of the post-translational machinery of the secretory pathway. Furthermore, large scale insect cell culture offers particular challenges to the blotechnologist due to the higher oxygen consumption and higher shear sensitivity of the cells as compared to mammalian cells. Like in mammalian cells, the major drawback in the heterologous expression of glycoproteins resides in 30 the different structure of the N-glycan as described before. Especially the lack of terminal slalic acid residues Is detrimental, because these sugars play important roles In glycoprotein biology.

-7 To summarize, the three main eukaryotic expression systems mostly fail to produce glycans of a desired structure. In contrast to eukaryotic systems, the gram-negative bacterium Escherichia coli offers several technical advantages for the production of heterologous proteins. It is the oldest and most productive system used. However, the 5 inability of E, coli cells to exert post-translational modifications of proteins remains the strongest drawback for its use as the preferred host for the production of human proteins. 10 Summary of the Invention To overcome the problem of production of recombinant glycoproteins in E co/i, a metabolic machinery capable to obtain protein glycosylation is introduced into this bacterium. Accordingly, the present invention provides an expression system with which recombinant proteins, in particular N-glycosylated proteins are producible. 15 The present invention provides a system for the production of recombinant human, human-like, or animal, or plant, or fungal, or bacterial N-glycosylated target proteins, the system comprising a prokaryotic organism into which is introduced a genetic information encoding for a metabolic apparatus capable of carrying out the requested N 20 glycosylation of the target protein. The system according to the invention is characterized in that said prokaryotic organism also contains the genetic information required for the expression of one or more recombinant target proteins. In one aspect, the present invention provides a prokaryotic host cell comprising (i) a 25 target protein comprising the N-glycosylation consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro, and wherein the target protein can be glycosylated at the Asn residue of the consensus sequence; and (ii) a heterologous oligosaccharyl transferase that links the consensus sequence to an oligo- or polysaccharide. 30 In another aspect, the present invention provides a prokaryotic host cell comprising (i) a nucleic acid that encodes a target protein comprising the N-glycosylation consensus -8 sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro, and wherein the target protein can be glycosylated at the Asn residue of the consensus sequence; and (ii) a nucleic acid that encodes a heterologous oligosaccharyl transferase that links the consensus sequence to an oligo- or polysaccharide. 5 Advantages over prior art Since E coil is easier to handle and to grow and its genetics are very well known, the production of human, human-like, animal or plant or fungal or bacterial glycoproteins in E coli is a breakthrough in biotechnology. 10 As mentioned before, recombinant glycoproteins to date have to be produced in less suited eukaryotes. But although the first steps in the synthesis of N-glycoproteins are highly conserved in all organisms the further trimming and processing differs quite significantly between eukaryotes. Therefore the N-glycans of recombinant glycoproteins 15 depend on the glycosylation genes present in the expression system used. This could give rise to production of recombinant glycoproteins where the N-glycans differ in their structure compared to the original one. 20 In contrast, the introduction of a genetic information encoding for a metabolic apparatus capable of carrying out the requested glycosylation of the protein, e.g. an operon, into an organism that normally does not glycosylate proteins offers the opportunity to manipulate the structure of the N-glycan by introducing specific glycosyltransferases. 25 Brief Description of the Drawings Some of the problems known from prior art as well as the method according to the invention are explained in more detail referring to schematic drawings that WO 03/074687 PCT/CH03/00153 are exemplary embodiments of the invention and are not Intended to narrow the scope of protection of the present invention. There is shown in Fig. 1 the expression of recombinant glycoproteins in eukaryotes, whereas Fig. 1A shows the expression of a target glycoprotein, and 5 Fig. 1B shows genetic engineering of existing glycosylation path ways in the Golgi; Fig. 2 the Escherlchla col expression system, with the expression of a re combinant target protein and the introduction of a specific glyco 10 sylation pathway according to the Invention; and in Fig. 3 the legend for the signs representing individual elements of the oli gosaccharldes residues of the glycoproteins in Fig. 1 and Fig. 2. 15 Description of the Invention Figure 1 shows the expression of recombinant glycoproteins in eukaryotic ex pression systems. Figure 1A shows the expression of a target glycoprotein, wherein the assembly of the lipid linked oligosaccharide (LLO; step I) and the 20 transfer of the oligosaccharide to the protein by means of an OTase (step II) is a highly conserved process in the Endoplasmatic Reticulum (ER). In contrast, the modifications in the Golgi are cell type specific (step III). Figure 1B again shows the expression of a target glycoprotein, wherein the as 25 sembly of the lipid linked oligosaccharide (LLO; step I) and the transfer of the oligosaccharide to the protein by means of a OTase (step II) is a highly con served process in the ER. In addition, an attempt to carry out genetic engineer ing of existing glycosylation pathways in the Golgi is shown. To produce a recom binant protein with a specific structure in eukaryotic cells, the host cells have to 30 be adapted by genetic engineering of this glycosylation pathway in the Golgi. "X" signs mark deletions required to exclude undesired pathways. "Asn" indicates an asparagine, "PP" a pyrophosphate, and "SO 4 " a sulfate group.

WO 03/074687 PCT/CH03/00153 - 10 Both figures 1A and 1B show that the expression of the recombinant protein is carried out outside the ER (step Ib) and that this target protein then is imported into the ER (step I1b), The explanation of the signs representing the individual elements of the oligosaccharides derives from the legend in Fig. 3. 5 To obtain a recombinant glycoprotein with a specific oligosaccharlde structure in eukaryotic cells requires the tailoring of highly complex, essential pathways and this might possibly interfere with the viability of the production cell. This is not 10 the case In the E. col/ system. Here, the tailoring is obtained by the introduction of specific components of the glycosylation machinery that lead to the desired glycoprotein (Fig. 2). 15 Since all the basic components monosaccharidess) required for the assembly of oligosaccharides are present in E. coll cells, the above mentioned solution re quires the introduction: a) of specific glycosyltransferases for the assembly of the oligosaccharide on a 20 lipid carrier, and b) an OTase that covalently links this oligosaccharide to specific residues of the desired protein. This solution offers the possibility to design the oligosaccharide structure by the 25 expression of specific glycosyltranferases and does not affect vital functions of the production cell. Figure 2 shows the Escher/chla coil expression system according to the invention with the expression of a recombinant target protein (step Ib), which then is in 30 troduced to the glycoprotein synthesis (step NIb). To obtain a specific glycopro tein in E. col, specific glycosyltransferases for the assembly of the lipid-linked oligosaccharide (LLO"; step I) are introduced into the host. The OTase covalently links this oligosaccharide to specific residues of the desired protein (step II).

WO 03/074687 PCT/CH03/00153 - 11 - In another attempt, the oligosaccharide, that Is attached to the desired protein as described In Figure 2, can be exchanged using a different oligosaccharlde as a 5 substrate in a enzymatic reaction in vitro. It was shown that the immobilized endo--N-acetylglucosaminidase (Endo-A) from Arthrobacter protophormiae could transfer an oilgosaccharide to ribonuclease B that contained a covalently linked N-acetylglucosamine [Fujita, K., Tanaka, N., Sano, M., Kato, I., Asada, Y. and Takegawa, K. (2000) Synthesis of neoglycoenzymes with homgogenous N 10 linked oligosaccharides using immobilized endo-p-N-acetylglucosaminidase A. Biochemical and Biophysical Research Communications, 267, 134-138]. Thus the invention gives the possibility to produce a glycoprotein in E. coi/ and then, in a second step, to modify the ollgosaccharide that is covalently linked to the protein by exchanging it with a different oligosaccharlde of defined structure with the 15 Immobilized Endo-A in vitro. The Invention encompasses the production of glycosylated glycoproteins. There are many benefits derived from the glycosylation of such target proteins. Such benefits include, but are not limited to, increased in vivo circulatory half life of a 20 protein; increased yields of recombinant proteins; increased biological activity of the protein including, but not limited to, enzyme activity, receptor activity, bind ing capacity; altered antigenicity; improved therapeutic properties; increased capacities as a vaccine or a diagnostic tool, and the like. Examples of mammalian glycoproteins that can be produced with this invention and that can serve as me 25 dicaments for humans, animals or plants, include but are not limited to, erythro poletin, transferrin, Interferons, immunoglobulines, interleukins, plasminogen, and thyrotropin. Also prokaryotic and/or fungal glycoproteins can be produced with the Invention and can serve as medicaments for humans, animals and plants, e.g. glycoproteins from C. fejun/ and from fungi. Further applications for 30 glycoproteins produced with this invention include, but are not limited to, indus trial enzymes, functional food, cosmetics, packaging materials, and textiles.

WO 03/074687 PCT/CH03100153 - 12 Example 1 The present Invention bases on the finding, that Campylobacterjejuni, a gram 5 negative bacterium, produces glycoproteins. Utilizing methods known per se, we have introduced the C. jejuni gene encoding AcrA, a glycoprotein, into E. coll. This results in the expression of non-glycosylated AcrA protein (see Fig. 2, step Ib). Subsequently and again utilizing known methods, an operon of C. jejuni en coding a) specific glycosyltransferases and b) an OTase was introduced Into E. 10 coll. This resulted in the production of specifically glycosylated AcrA protein ac cording to the invention (see Fig. 2, steps I and II), as verified - always using methods known to skilled persons - by the binding of a highly specific lectin and glycosylation specific antibodies to the heterologously produced AcrA protein [Mi chael Wacker et al. (2002) N-linked glycosylation in Campylobacterjejuni and its 15 functional transfer into E. coll (SCIENCE, Vol 298: 1790-1793]. In addition, the structure of the oligosaccharide linked to AcrA was verified by mass spectros copy. Next it was shown, that the oligosaccharide was only transferred to the C amino group of the asparagine within the consensus sequence Asn-X-Ser/Thr where X can be any amino acid except Pro [Gavel, Y. and Von Heijne, G. (1990). 20 Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering. Protein Eng, 3, 433-442]. When the consensus sequence was mutated, the oligosaccharide was not trans ferred to the protein anymore. Therefore, it was verified - always using methods known to skilled persons - that the OTase of C. jejuni recognized the same con 25 sensus sequence as the OTase of eukaryotes and archaea and transferred the oligosaccharlde by the same proposed mechanism to the protein [Wacker, M., Linton, D., Hitchen, P.G., Nita-Lazar, M., Haslam, S.M., North, S.J., Panico, M., Morris, H.R., Dell, A., Wren, B.W. and Aebi, M. (2002). N-linked glycosylation in Campylobacterjejuni and its functional transfer into E. coli. Science, 298: 1790 30 1793].

WO 03/074687 PCT/CH03/00153 - 13 Specific glycosyl transferases and oligosaccharyl transferases utilized to geneti cally modify E. col can be of prokaryotic or eukaryotic origin as glycosyl transfer ases are ubiquiteous and oligosaccharyl transferases are known from archaea.

Claims (10)

1. A prokaryotic host cell comprising: (i) a target protein comprising the N-glycosylation consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro, and 5 wherein the target protein can be glycosylated at the Asn residue of the consensus sequence; and (ii) a heterologous oligosaccharyl transferase that links the consensus sequence to an oligo- or polysaccharide.

2. A prokaryotic host cell comprising: (i) a nucleic acid that encodes a target protein comprising the N-glycosylation consensus sequence Asn-X-Ser(Thr), wherein X can be any 10 amino acid except Pro, and wherein the target protein can be glycosylated at the Asn residue of the consensus sequence; and (ii) a nucleic acid that encodes a heterologous oligosaccharyl transferase that links the consensus sequence to an oligo- or polysaccharide.

3. The prokaryotic host cell of claim 1 or claim 2, wherein said oligosaccharyl transferase is from Campylobacterjejuni. 15

4. The prokaryotic host cell of any one of claims 1 to 3, wherein said prokaryotic host cell is Escherichia coli,

5. The prokaryotic host cell of any one of claims 1 to 4, wherein said target protein is a bacterial protein.

6. The prokaryotic host cell of any one of claims 1 to 4, wherein said target protein is a ?0 mammalian protein.

7. The prokaryotic host cell of any one of claims 1 to 6, wherein said target protein is heterologous to the host cell.

8. A method of producing a recombinant N-glycosylated target protein, the method comprising culturing the prokaryotic host cell of any one of claims 1 to 7 under conditions 25 suitable for the production of proteins and isolating the N-glycosylated recombinant target protein from said culture.

9. A vaccine produced by the method of claim 8.

10. A prokaryotic host cell of claim I or claim 2, substantially as herein described with reference to the Example and/or any one or more of the Figures. 30