Means and Methods for Generating Complex Glycans Derived From Fungal Engineered Hosts
The present application relates to the field of glyco-engineering, more specifically to glycosylation-engineered fungal cells, more specifically glycosylation-engineered yeast cells, optimized to produce highly homogenous forms of complex N-glycans on recombinant proteins. The invention specifically relates to methods to obtain pharmaceutical compositions comprising recombinant glycoproteins which have homogenous forms of complex N-glycans. In addition, the invention relates to novel pharmaceutical compositions which result from the methods of the invention.
This application is a continuation of U.S. patent application Ser. No. 16/097,951, filed Oct. 31, 2018, which is a national phase entry under 35 U.S.C. § 371 of International Patent Application PCT/EP2017/060568, filed May 3, 2017, designating the United States of America and published in English as International Patent Publication WO 2017/191208 A1 on Nov. 9, 2017, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 16168156.4, filed May 3, 2016, the entireties of which are hereby incorporated by reference.
FIELD OF THE INVENTIONThe present application relates to the field of glyco-engineering, more specifically to glycosylation-engineered fungal cells, more specifically glycosylation-engineered yeast cells, optimized to produce highly homogenous forms of complex N-glycans on recombinant proteins. The invention specifically relates to methods to obtain pharmaceutical compositions comprising recombinant glycoproteins which have homogenous forms of complex N-glycans. In addition, the invention relates to novel pharmaceutical compositions which result from the methods of the invention.
BACKGROUNDCHO cell lines are an expression system of choice for biopharmaceuticals and are used for example to make blockbuster monoclonal antibodies like Rituxan, Humira and Enbrel. However, the cost of manufacturing in CHO cell lines is very high and if one wants to make affordable medicines at a lower cost there is a need to shift to alternative host organisms. Glyco-engineered fungal organisms like for example Pichia pastoris are able to produce recombinant glycoproteins with complex N-glycosylation structures, but there is need to further engineer the glycosylation capabilities of fungal organisms. Indeed, there is still a significant background of yeast-like sugars present on recombinant proteins. Although a new full Pichia pastoris OCH1 knock-out that modifies its glycoproteins predominantly with Man8GlcNAc2 N-glycans was described recently (Krainer F W et al (2013) Sci Rep 3:3279) there was still a considerable amount of background of yeast-type high mannose glycomodifications present when recombinant glycoproteins are produced in this mutant strain. Similar observations were made in earlier reports on OCH1 knock-out strains (Davidson R C et al (2004) Glycobiology 14(5):399). In the latter study, the background was attributed mainly to phosphomannosylation and attributed to the presence in the genome of still unknown mannosyltransferases. In the present application we produced different complex glycoforms of IL-22 in complex glyco-engineered Pichia pastoris strains. Despite extensive glyco-engineering, we also observed that there was still a considerable, highly heterogeneous background present in the produced glycoforms. Although a part of the heterogeneity originates from intermediates in the sample that did not get fully processed to complex-type N-glycans, it was found that a considerable N-glycan fraction consisted of a range of other oligo-mannose- and hypermannosyl-type N-glycans. Although there exists the possibility that a fraction of cells in the strains may revert to wild-type OCH1 due to instability of the knock-in construct but it is more likely that other endogenous glycosyltransferases are responsible. We believe that still uncharacterized glycosyltransferases might have a similar activity as Och1p in Pichia pastoris and cause heterogeneity in glycoforms, even in complex glyco-engineered strains.
In addition to this, in the present application we have also characterized a novel type of neoglycan on human IL-22 produced in OCH1 mutated Pichia strains, comprising of a Man5GlcNAc2 N-glycan, with a tetra-saccharide modification that had a highly unexpected structure. This tetra-saccharide (Glcα1-2Manβ1-2Manβ1-3Glucα-) substitution is most likely attached to the innermost α-1,3 arm of the mannosyl core. We also observed similar N-glycans on glyco-engineered murine IL-22 (data not shown). Because the identified structure contained β-1,2-mannose residues, a described immunogenic epitope of C. albicans, the presence of such N-glycan would hamper the potential for therapeutic use. This particular N-glycan deviates in its structure from a previously described neoglycoform (Gomathinayagam S. et al. (2011). Glycobiology. 21(12): 1606-15) showing that our understanding of the N-glycosylation pathway and the endogenous glycosyltransferases is still limited. Moreover, it is also not understood why certain glycoproteins are prone for further modifications whereas others are not.
Despite the genome sequence of P. pastoris being available, it is not known which glycosyltransferases are responsible for generating this particular neoglycoform. Therefore, knock-out of specific additional endogenous glycosyltransferases is not straightforward. Although further engineering could partially resolve the substitution by outcompeting endogenous glycosyltransferases, in later stages of the engineering, it is likely that a number of intermediates would re-appear, including hybrid N-glycans, Man5GlcNAc2 but also oligomannose background of which the structures are difficult to determine with the current techniques. In addition, it is likely that, also neoglycoforms may form on recombinant glycoproteins, even in highly glyco-engineered strains. The only way to prevent or remedy neoglycoform formation, would be to characterize all the potential glycosyltransferases/glycosidases and knock-out these enzymes if they might show some undesired activity. The latter would be an unpractical approach and even then the effect on neoglycoform formation would be unpredictable. Because of the background, the re-appearing intermediates and the possibility of neoglycoform-formation, there exists a clear need to design strategies which allow to remove the remaining fungal-type glycosylation background from the complex N-glycans present on glycoproteins produced in glyco-engineered fungal organisms.
SUMMARY OF THE INVENTIONSo far the use of a complex glyco-engineered fungal cell system has mostly focused on exhaustive engineering processes and not on enzymatic treatment (e.g. with specific glycosidase enzymes) after the glycoprotein is produced to remove background since the aim is to keep the N-glycans and not to deglycosylate the glycoprotein. To remove the background of glycoproteins produced in complex N-glycan engineered strains of Pichia pastoris it was tested to integrate in vitro enzymatic deglycosylation using the T. reesei Endo-β-N-Acetylglucosaminidase EndoT. Recombinant EndoT has been shown to be able to release human Golgi-type oligo-mannose N-glycans but human complex N-glycans are not a substrate for this enzyme (see Stals I. et al (2012) PLOS One 7(7) e40854). However, it was unknown whether the many different types of yeast N-glycans, including some glycoforms triggered by mutation of OCH1, would be eligible substrates and form neoglycoforms. Surprisingly, EndoT performed very well in this task as more than 90% of the background consisting of hybrid N-glycans, yeast high mannose N-glycans and unexpected neoglycoforms disappeared after treatment while the bioactivity of the glycoprotein could be retained.
Moreover, it was found that the in vitro reaction was compatible with the high salt concentrations present in the ammonium sulfate fractions during purification and surprisingly this reaction could be done at 4° C. Since we observed that EndoT was able to work in these unfavorable conditions, it expands its applicability.
The use of complex N-glycosylation glyco-engineered Pichia-strains, producing a dominant human complex-type N-glycoform on glycoproteins in combination with an in vitro endoglucosaminidase clean-up step to remove undesired background, provides a powerful tool to make highly pure, customized N-glycoforms of the complex type N-glycans.
Thus where a recombinant glycoprotein, produced in a complex N-glycosylation engineered fungal organism, has one functional N-glycosylation acceptor site then a plurality of glycoforms is produced when this recombinant glycoprotein is purified from the medium and exogenously contacted with a suitable amount of endoglucosaminidase. These glycoforms occur because in the complex N-glycosylation engineered fungal organism a variety of N-glycans are formed: i) hybrid N-glycans, ii) high mannose type N-glycans, iii) unpredictable N-glycan neoglycoforms (see further in the examples section) and iv) the intended complex N-glycans as expected for the specific complex N-glycosylation engineered fungal organism. Contacting with the endoglucosaminidase (e.g. exogenously applied and added in the medium, or added during the purification conditions or added after the purification of the recombinant glycoprotein) will eliminate more than 90% of the hybrid N-glycans and high mannose type N-glycans and also eliminate unpredictable N-neoglycoforms and this will result in the formation of an N-glycan consisting of a single GlcNAc glycan structure. Off note, glycoforms having a single GlcNAc glycan will not be visualized (or cannot be determined) by the method outlined in Examples 2 and 3 but can only be detected by mass spectrometric analysis methods. The resulting complex N-glycans will not be digested upon contacting with the endoglucosaminidase. As a result, a “plurality of glycoforms” of the recombinant glycoprotein will be obtained (estimated by the visualization of the total of all N-glycans present on a recombinant glycoprotein produced in the complex N-glycosylation engineered fungal organism). Hence, plurality refers to N-glycans of the complex type, N-glycans consisting of a single GlcNAc and less than 20%, preferably less than 15%, less than 10%, less than 5% or even less than 1% N-glycans of the hybrid type N-glycans or the high mannose type N-glycans or the N-glycan neoglycoforms.
WO2010/015722 describes the co-expression of an endoglucosaminidase, mammalian glycosyltransferases and a heterologous glycoprotein. In the latter engineered cellular system the endoglycosidase enzyme is targeted to a specific compartment in the Golgi. When the latter system is applied in fungi comprising an exogenous glycoprotein then recombinant glycoproteins comprising N-glycans consisting of a single GlcNAc are produced. The latter is in contrast to the methodology of the invention which is applied in vitro and wherein the complex glyco-engineered fungal cell does not co-express an endoglucosaminidase and where recombinant glycoproteins are produced having a mixture of complex N-glycans and N-glycans consisting of a single GlcNAc. To clarify this even further when a glycoprotein, having only one N-glycosylation acceptor site, is produced in a complex glyco-engineered fungal organism and after the production the resulting glycoprotein is subsequently in vitro treated (or contacted) with an endoglucosaminidase then the glycoforms present on the resulting glycoprotein consist of a mixture of complex N-glycans and N-glycans consisting of a single GlcNAc. When a glycoprotein, having two N-glycosylation acceptor sites, is produced in a complex glyco-engineered fungal organism and after the production the resulting glycoprotein is subsequently in vitro treated (or contacted) with an endoglucosaminidase then the N-glycosylation sites present on a single glycoprotein can consist of either i) one complex N-glycan and the other one single GlcNAc, or ii) both can be a single GlcNAc or iii) both can be a complex N-glycan.
Endoglucosaminidases like EndoH are commonly used to deglycosylate glycoproteins as part of the analytics on SDS-PAGE gel similar to PNGaseF digestion. However, the latter digests are carried out on denatured proteins to determine N-glycosylation profiles on SDS-PAGE with the intent of deglycosylating the protein. Similarly, for crystallography purposes glycoproteins are also often deglycosylated. However, the digests either have to be performed without denaturation or a renaturation/refolding step has to be performed in order to determine the protein structure. The use of endoglucosaminidases as an in vitro clean-up tool (post-fermentation, e.g. during the purification or after the purification) with the aim to further use the obtained glycoprotein for pharmaceutical use has not been investigated in the art. An endoglycosidase clean-up on a complex glyco-engineered yeast platform producing glycoproteins with complex N-glycans has not been reported so far and an in vitro incubation step with an endoglucosaminidase is even considered counterintuitive.
As used herein, each of the following terms has the meaning associated with it in this section. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. The term “abnormal” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the “normal” (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term “comprising” is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are capable of operation in other sequences than described or illustrated herein. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, N.Y. (2012); and Ausubel et al., current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.
As used herein, the term “nucleotide sequence” refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleotide sequences may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of nucleotide sequences include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleotide sequence may be linear or circular.
As used herein, the term “polypeptide” refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Polypeptide sequences can be depicted with the single-letter (or one letter) amino acid code or the three letter amino acid code as depicted here below:
The term “expression vector”, as used herein, includes any vector known to the skilled person, including plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, such as adenoviral, AAV or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Expression vectors generally contain a desired coding sequence and appropriate promoter sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g. higher eukaryotes, lower eukaryotes, prokaryotes).
Typically, a vector comprises a nucleotide sequence in which an expressible promoter or regulatory nucleotide sequence is operatively linked to, or associated with, a nucleotide sequence or DNA region that codes for an mRNA, such that the regulatory nucleotide sequence is able to regulate transcription or expression of the associated nucleotide sequence. Typically, a regulatory nucleotide sequence or promoter of the vector is not operatively linked to the associated nucleotide sequence as found in nature, hence is heterologous to the coding sequence of the DNA region operably linked to. The term “operatively” or “operably” “linked” as used herein refers to a functional linkage between the expressible promoter sequence and the DNA region or gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest, and refers to a functional linkage between the gene of interest and the transcription terminating sequence to assure adequate termination of transcription in eukaryotic cells. An “inducible promoter” refers to a promoter that can be switched ‘on’ or ‘off’ (thereby regulating gene transcription) in response to external stimuli such as, but not limited to, temperature, pH, certain nutrients, specific cellular signals, et cetera. It is used to distinguish between a “constitutive promoter”, by which a promoter is meant that is continuously switched ‘on’, i.e. from which gene transcription is constitutively active.
A “glycan” as used herein generally refers to glycosidically linked monosaccharides, oligosaccharides and polysaccharides. Hence, carbohydrate portions of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan are referred to herein as a “glycan”. Glycans can be homo- or heteropolymers of monosaccharide residues, and can be linear or branched. N-linked glycans may be composed of GalNAc, Galactose, neuraminic acid, N-acetylglucosamine, Fucose, Mannose, and other monosaccharides, as also exemplified further herein.
In eukaryotes, O-linked glycans are assembled one sugar at a time on a serine or threonine residue of a peptide chain in the Golgi apparatus. Unlike N-linked glycans, there are no known consensus sequences but the position of a proline residue at either −1 or +3 relative to the serine or threonine is favourable for O-linked glycosylation.
A “glyco-engineered cell” refers to a cell that has been genetically modified so that it expresses proteins with an altered N-glycan structure and/or O-glycan structure as compared to in a wild type background. Typically, the naturally occurring modifications on glycoproteins have been altered by genetic engineering of enzymes involved in the glycosylation pathway. In general, sugar chains in N-linked glycosylation may be divided in three types: high-mannose (typically yeast), complex (typically mammalian) and hybrid type glycosylation. Besides that, a variety of O-glycan patterns exist, for example with yeast oligomannosylglycans differing from mucin-type O-glycosylation in mammalian cells. The different types of N- and O-glycosylation are all well known to the skilled person and defined in the literature. Considerable effort has been directed towards the identification and optimization of strategies for the engineering of eukaryotic cells that produce glycoproteins having a desired N- and/or O-glycosylation pattern and are known in the art (e.g. De Pourcq, K. et al., Appl Microbiol Biotechnol. 87(5), 2010). One non-limiting example of such a glyco-engineered expression system is described in patent application WO2010015722 and relates to a (higher or lower) eukaryotic cell expressing both an endoglucosaminidase and a target protein, and wherein the recombinant secreted target proteins are characterized by a uniform N-glycosylation pattern (in particular one single GlcNAc residue (in lower eukaryotes) or a modification thereof such as GlcNAc modified with Galactose (LacNAc) or sialyl-LacNAc (in mammalian cells). Also encompassed are cells genetically modified so that they express proteins or glycoproteins in which the glycosylation pattern is human-like or humanized (i.e. complex-type glycoproteins). This can be achieved by providing cells, in particular lower eukaryotic cells, having inactivated endogenous glycosylation enzymes and/or comprising at least one other exogenous nucleic acid sequence encoding at least one enzyme needed for complex glycosylation. Endogenous glycosylation enzymes which could be inactivated include the alpha-1,6-mannosyltransferase Och1p, Alg3p, alpha-1,3-mannosyltransferase of the Mnn1p family, beta-1,2-mannosyltransferases. Enzymes needed for complex glycosylation include, but are not limited to: N-acetylglucosaminyl transferase I, N-acetylglucosaminyl transferase II, mannosidase II, galactosyltransferase, fucosyltransferase and sialyltransferase, and enzymes that are involved in donor sugar nucleotide synthesis or transport. Still other glyco-engineered cells, in particular yeast cells, that are envisaged here are characterized in that at least one enzyme involved in the production of high mannose structures (high mannose-type glycans) is not expressed. Enzymes involved in the production of high mannose structures typically are mannosyltransferases. In particular, alpha-1,6-mannosyltransferases Och1p, Alg3p, alpha-1,3-mannosyltransferase of the Mnn1p family, beta-1,2-mannosyltransferases may not be expressed. Thus, a cell can additionally or alternatively be engineered to express one or more enzymes or enzyme activities, which enable the production of particular N-glycan structures at a high yield. Such an enzyme can be targeted to a host subcellular organelle in which the enzyme will have optimal activity, for example, by means of signal peptide not normally associated with the enzyme. It should be clear that the enzymes described herein and their activities are well-known in the art.
‘Glycoproteins’ as used in the application refers to proteins that, in their normal physiological context and/or their functional form, contain oligosaccharide chains (glycans) covalently attached to their polypeptide side-chains. In addition, a glycoprotein is any protein with an artificially introduced glycosylation site. Typically a glycoprotein, typically a recombinant glycoprotein, for example a heterologous recombinant glycoprotein (which does not occur normally in the fungal or yeast organism) is produced as several glycoforms when it is made in a glycosylation-engineered fungal or yeast organism. Different glycoforms (even originating from one specific functional N-glycosylation site on a (recombinant) glycoprotein) occur because of the very nature of the process of N-glycosylation. By nature the formation of complex N-glycosylation glycans (especially in a complex N-glycosylation engineered fungal organism) is never 100% efficient and hence different glycoforms occur on a specific N-glycan position present on a glycoprotein. Thus it is possible that in a glycoprotein (e.g. a glycoprotein with 2 N-glycan acceptor sites) one N-glycan is a complex N-glycan and the other N-glycan is a hybrid glycan or a high-mannose type glycan. In particular, glycoproteins as used herein are proteins that show N-glycosylation in their physiologically active form. Thus, glycoproteins typically contain a sugar chain at least on one asparagine residue. A non-limiting list of glycoproteins is provided in the specification. The term ‘glycoproteins’ is not intended to refer to the length of the amino acid chain, ‘glycopeptides’ are included within the definition of ‘glycoproteins’.
The terms ‘(glyco)protein’ and ‘enzyme’ (e.g. endoglucosaminidase, glycosyltransferase, mannosidase, mannosyltransferase) as used in the application are also intended to cover functionally active fragments and variants of the naturally occurring proteins. Indeed, for many (e.g. therapeutic) proteins, part of the protein may be sufficient to achieve an (e.g. therapeutic, enzymatic) effect. The same applies for variants (i.e. proteins in which one or more amino acids have been substituted with other amino acids, but which retain functionality or even show improved functionality), in particular for variants of the enzymes optimized for enzymatic activity. In the context of the application, a glycoprotein refers to the protein itself; a glycoprotein may be either in its glycosylated or non-glycosylated form. A ‘glycosylated’ protein is a (glyco)protein that carries at least one oligosaccharide chain. An N-glycosylated protein, particularly an N-glycosylated recombinant glycoprotein, is a glycoprotein which carries at least one oligosaccharide chain on an N-glycan.
A ‘glycoform’ as used in the present invention is a variant of a glycosylated glycoprotein wherein the variation is in the N-glycan composition present on said glycoprotein.
A ‘sugar chain’, ‘oligosaccharide chain’ or ‘carbohydrate chain’ as used herein is referred in the claims as an N-glycan (with N- referring to N-glycosylation). Sugar chains may be branched or not, and may comprise one or more types of oligosaccharide. In general, sugar chains in N-linked glycosylation may be divided in three types: high-mannose, complex and hybrid type glycosylation. These terms are well known to the skilled person and defined in the literature. Briefly, high-mannose type glycosylation typically refers to oligosaccharide chains comprising two N-acetylglucosamines with (possibly many) mannose and/or mannosylphosphate residues (but typically no other monosaccharides).
Complex glycosylation typically refers to structures with typically one, two or more (e.g. up to six) outer branches, most often linked to an inner core structure Man3GlcNAc2. For instance, a complex N-glycan may have at least one branch, or at least two, of alternating GlcNAc and optionally also galactose (Gal) residues that may terminate in a variety of oligosaccharides but typically will not terminate with a mannose residue. Several examples of complex N-glycans made in complex N-glycosylation engineered fungal organisms are shown in the appended example section. For the sake of clarity a single GlcNAc present on an N-glycosylation site of a glycoprotein is not regarded as a complex N-glycan.
Hybrid type glycosylation covers the intermediate forms, i.e. those glycosylated proteins carrying both terminal mannose and terminal non-mannose residues in addition to the two N-acetylglucosamine residues. In contrast to complex glycosylation, at least one branch of hybrid type glycosylation structures ends in a mannose residue. Hybrid N-glycans can originate from the inefficient glycosylation of the heterologous glycosyltransferase enzyme present in a complex N-glycosylation engineered fungal organism.
‘Complex N-glycosylation-engineered fungal organism’ as used in the application are fungal cells that express at least one exogenous nucleic acid sequence encoding an enzyme needed for complex N-glycosylation that is not expressed in the wild-type fungal organism, and/or that do not express at least one enzyme involved in the production of high-mannose type structures that is normally expressed in the wild type fungus. Particularly, complex N-glycosylation-engineered fungal organisms are complex N-glycosylation-engineered yeasts. Non-limiting examples of yeasts which can be engineered towards complex N-glycosylation-engineered yeasts comprise Saccharomyces species (e.g. Saccharomyces cerevisiae), a Hansenula species (e.g. Hansenula polymorpha), an Arxula species (e.g. Arxula adeninivorans), a Yarrowia species (e.g. Yarrowia lipolytica), a Kluyveromyces species (e.g. Kluyveromyces lactis), or Komagataella phaffii (Kurtzman, C. P. (2009) J Ind Microbiol Biotechnol. 36(11) which was previously named and better known under the old nomenclature as Pichia pastoris and also further used herein. According to a specific embodiment, the lower eukaryotic cells are Pichia cells, and in a most particular embodiment Pichia pastoris cells. Still other ‘complex N-glycosylation-engineered fungal organisms comprise Myceliopthora thermophila (also known as C1 by the company Dyadic), Aspergillus species (e.g. Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus japonicus), Fusarium species (e.g. Fusarium venenatum), Hypocrea and Trichoderma species (e.g. Trichoderma reesei).
According to particular embodiments, the enzyme needed for complex N-glycosylation is a mannosidase or a glycosyltransferase other than a mannosyltransferase. According to further particular embodiments, the at least one enzyme needed for complex glycosylation is selected from the group consisting of N-acetylglucosaminyl transferase I, N-acetylglucosaminyl transferase II, mannosidase II, galactosyltransferase, and sialyltransferase.
According to particular embodiments, the complex N-glycosylation yeast cell (or in short glycosylation-engineered yeast cell or glyco-engineered yeast cell) may be characterized in that at least one enzyme involved in the production of high mannose structures (high mannose-type glycans) is not expressed (or is not as functionally active in the cell as in a wild-type cell). According to further particular embodiments, at least one mannosyltransferase is not expressed in the glyco-engineered yeast cell. Typically, the mannosyltransferase that is not expressed in the glyco-engineered yeast cell is expressed in the wild-type counterpart of the yeast cell. According to yet further particular embodiments, the mannosyltransferase is a α-1, 2-mannosyltransferase, α-1, 3-mannosyltransferase, α-1, 6-mannosyltransferase, or β-1, 4-mannosyltransferase. These proteins often have specific names in yeast (e.g. Alg, Och, Mnn), but their activities are well known in the art. Alternatively or additionally, at least one mannosylphosphate transferase is not functionally active in the complex N-glycosylation-engineered yeast cell.
An ‘endoglucosaminidase’ as used herein refers to enzymes that hydrolyse the bond between the anomeric carbon of a non-terminal beta-linked N-acetylglucosamine residue in an oligosaccharide of a glycoprotein or a glycolipid, and its aglycon, thereby releasing mono- or oligosaccharides from glycoproteins or glycolipids or sugar polymers. Endoglucosaminidases are a subset of the glycosidases, and may or may not have other enzymatic activities (such as e.g. glycosyltransferase activity). A particular class of endoglucosaminidases is formed by the endo-β-N-acetylglucosaminidases or mannosyl-glycoprotein endo-β-N-acetylglucosaminidases, indicated as EC 3.2.1.96 in the International Union of Biochemistry and Molecular Biology (IUBMB) nomenclature. This particular class of enzymes are capable of catalyzing the endohydrolysis of the N,N′-diacetylchitobiosyl unit in high-mannose glycopeptides and glycoproteins containing the -[Man(GlcNAc)2]Asn- structure. One N-acetyl-D-glucosamine (GlcNAc) residue remains attached to the protein; the rest of the oligosaccharide is released intact. The result thus is a single GlcNAc-modified N-glycosylation site present on a glycoprotein. Glycoproteins with a modified GlcNAc residue will still be referred to as single GlcNAc-modified proteins, as there is no second sugar residue on position 4 of the GlcNAc residue (i.e. there is no typical sugar chain). A non-limiting list of endoglucosaminidases is provided further in the application.
Particularly with regard to the N-glycosylation-engineered fungal or yeast cells, an ‘enzyme needed for complex glycosylation’ as used herein refers to any enzyme not naturally occurring in the host fungal or yeast cell that may be involved in the synthesis of complex glycans as found in higher eukaryotes, in particular as found in mammals, more in particular as found in humans. Most particularly, such enzymes are enzymes that remove mannose residues from the sugar chain (i.e. mannosidases) or glycosyltransferases, in particular glycosyltransferases other than mannosyltransferases (i.e. glycosyltransferases that transfer monosaccharides that are not found in high-mannose glycans) and/or phosphomannosyltransferases.
A ‘glycosyltransferase’ as used in the application is any of a group of enzymes that catalyze the transfer of glycosyl groups in biochemical reactions, in particular glycosyl transfer to asparagine-linked sugar residues to give N-linked glycoproteins. Glycosyltransferases fall under EC 2.4 in the IUBMB nomenclature, a particular class of glycosyltransferases are hexosyltransferases (EC 2.4.1). Among the wide variety of these post-translational enzymes that process peptides into glycoproteins are enzymes such as, but not limited to, N-acetylglucosaminyl transferases, N-acetylgalactosaminyltransferases, sialyltransferases, fucosyltransferases, galactosyltransferases, and mannosyltransferases.
Note that exogenous mannosyltransferases are excluded for specific embodiments of N-glycosylation-engineered yeast cells described in the application. ‘Mannosyltransferases’ as used in the application refers to enzymes that catalyze the transfer of a mannosyl group to an acceptor molecule, typically another carbohydrate, in the Golgi apparatus. Mannosyltransferases are typically endogenous enzymes in fungi and yeast and involved in the synthesis of high-mannose type glycans.
Of note, an enzyme may possess glycosyltransferase activity next to its endoglucosaminidase activity. Although it may be possible to use one enzyme to exert these two activities, typically the enzymes used will fulfill only one function. Thus, it is envisaged to use enzymes that have been modified or mutated to make sure they perform only one function, or that have been modified or mutated to ensure they carry out a specific function more efficiently. Such modified enzymes are known in the art.
The present invention aims to provide compositions, particularly pharmaceutical compositions, comprising homogenous forms of N-glycans, particularly complex N-glycans present on a recombinant glycoprotein. Such recombinant glycoproteins are produced in complex N-glycosylation-engineered fungal organisms.
In one embodiment the invention provides a composition comprising a plurality of a recombinant glycoprotein, wherein the N-glycans present on said glycoforms comprise a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc and wherein said combined complex N-glycans and N-glycans consisting of a single GlcNAc are present at a level of higher than 90% of the total N-glycans in said composition.
In yet another embodiment the invention provides a composition comprising a plurality of glycoforms of a recombinant glycoprotein, wherein the N-glycans present on said glycoforms comprise a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc and wherein said complex N-glycans are present at a level of higher than 90% of the total N-glycans in said composition.
In yet another embodiment the invention provides a purified composition comprising a plurality of a recombinant glycoprotein, wherein the N-glycans present on said glycoforms comprise a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc and wherein said combined complex N-glycans and N-glycans consisting of a single GlcNAc are present at a level of higher than 90% of the total N-glycans in said composition.
In yet another embodiment the invention provides a purified composition comprising a plurality of glycoforms of a recombinant glycoprotein, wherein the N-glycans present on said glycoforms comprise a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc and wherein said complex N-glycans are present at a level of higher than 90% of the total N-glycans in said composition.
This is achieved, according to a specific aspect, by providing complex N-glycosylation-engineered fungal organisms with an exogenous nucleic acid sequence encoding a glycoprotein and contacting the secreted glycoprotein in vitro (e.g. by addition to the fermentation medium or by addition to the purified glycoprotein or by addition during the purification of the glycoprotein) with a suitable amount of an endoglucosaminidase. Typically, said endoglucosaminidase is recombinantly produced in a suitable host cell, upscaled and purified and added to the secreted glycoprotein present in the growth medium of the complex N-glycosylation engineered fungal organism. Importantly, said endoglucosaminidase is not produced by the same cell that also expresses the glycoprotein of interest. In a specific embodiment said endoglucosaminidase is applied to the recombinant glycoprotein during the purification of the recombinant glycoprotein. In yet another specific embodiment said endoglucosaminidase is applied to the recombinant glycoprotein after the purification of the recombinant glycoprotein.
In yet another embodiment the invention provides a composition comprising a plurality of glycoforms of a recombinant glycoprotein, wherein the N-glycans present on said glycoforms comprise a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc wherein said combined complex N-glycans and N-glycans consisting of a single GlcNAc are present at a level of higher than 90% of the total N-glycans in said composition wherein said composition is obtained by production of said glycoprotein in a complex N-glycosylation-engineered fungal organism comprising cultivating said recombinant complex N-glycosylation engineered fungal organism comprising an exogenous genetic construct encoding said glycoprotein under conditions wherein said glycoprotein is expressed and contacting said recombinant glycoprotein with the addition of an endoglucosaminidase.
In yet another embodiment the invention provides a composition comprising a plurality of glycoforms of a recombinant glycoprotein, wherein the N-glycans present on said glycoforms comprise a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc wherein said complex N-glycans are present at a level of higher than 90% of the total N-glycans in said composition wherein said composition is obtained by production of said glycoprotein in a complex N-glycosylation-engineered fungal organism comprising cultivating said recombinant complex N-glycosylation engineered fungal organism comprising an exogenous genetic construct encoding said glycoprotein under conditions wherein said glycoprotein is expressed and contacting said recombinant glycoprotein with the addition of an endoglucosaminidase.
The nature of the glycoprotein is not critical to the invention, but glycoproteins will typically be proteins relevant for medicine and/or industry for which homogenous N-glycosylation is important. Non-limiting examples include many hormones, growth factors, cytokines and their corresponding receptors, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), epidermal growth factor (EGF), human epidermal growth factor receptor-2 (HER-2), fibroblast growth factor-alpha (FGF-α), fibroblast growth factor-beta (FGF-β), transforming growth factor-alpha (TGF-α), transforming growth factor-beta (TGF-β), platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), nerve growth factor (NGF), nerve growth factor-beta (NGF-β); receptors of the aforementioned, growth hormones (e.g., human growth hormone, bovine growth hormone); insulin (e.g., insulin A chain and insulin B chain), proinsulin; erythropoietin (EPO); colony stimulating factors (e.g., granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF)); interleukins (e.g., IL-1 through IL-33); vascular endothelial growth factor (VEGF) and its receptor (VEGF-R); interferons (e.g., IFN-α, β, or γ); tumor necrosis factor (e.g., TNF-α and TNF-β) and their receptors, TNFR-1 and TNFR-2; thrombopoietin (TPO); thrombin; brain natriuretic peptide (BNP); clotting factors (e.g., Factor VIII, Factor IX, von Willebrands factor, and the like); anti-clotting factors; tissue plasminogen activator (TPA), e.g., urokinase or human urine or tissue type TPA; calcitonin; CD proteins (e.g., CD3, CD4, CD8, CD28, CD19, etc.); CTLA proteins (e.g., CTLA4); T-cell and B-cell receptor proteins; antibodies, bone morphogenic proteins (BMPs, e.g., BMP-1, BMP-2, BMP-3, etc.); neurotrophic factors, e.g., bone derived neurotrophic factor (BDNF); neurotrophins, e.g., NT3-6; renin; rheumatoid factor; RANTES; albumin; relaxin; macrophage inhibitory protein (e.g., MIP-1, MIP-2); viral proteins or antigens; surface membrane proteins; ion channel proteins; enzymes; alkaline phosphatase; lectins; regulatory proteins; antibodies; immunomodulatory proteins, (e.g., HLA, MHC, the B7 family); homing receptors; transport proteins; superoxide dismutase (SOD); G-protein coupled receptor proteins (GPCRs); neuromodulatory proteins; Alzheimer's Disease associated proteins and peptides, (e.g., A-beta), and others as known in the art, including fusion or chimeric proteins of the above.
The nature of the endoglucosaminidase will depend on the desired glycopopulation of the glycoproteins. For instance, endoglucosaminidases may be selected for their substrate specificity. Some endoglucosaminidases, e.g. Endo H and Endo T, hydrolyse high-mannose type sugar chains and hybrid type sugars, but leave complex carbohydrate structures intact.
Such enzymes are ideal e.g. for obtaining N-glycans consisting of complex N-glycans and for removing undesired high-mannose and/or hybrid type sugars from produced glycoproteins and as we have unexpectedly observed shown in the examples of the present invention also for the removal of N-glycan neoglycoforms on recombinant glycoproteins expressed in a complex N-glycosylation engineered fungal organism. According to particular embodiments, the endoglucosaminidase hydrolyses high mannose-type sugar chains, hybrid-type glycans, N-glycan neoglycoforms but not complex-type glycans.
Endoglucosaminidases may also have substrate specificity with regard to the glycoprotein (instead of only the sugar chain), some endoglucosaminidases are e.g. more successful in hydrolyzing sugar chains from (particularly compactly folded) proteins than other endoglucosaminidases (e.g. Endo T), others may (also) be particularly successful in hydrolyzing sugar chains from glycopeptides or not-compactly folded proteins (e.g. Endo H, Endo T). Importantly, as this typically has to do with access to or availability of the substrate rather than with the specificity of the endoglucosaminidase, this does not exclude the use of certain enzymes for specific proteins, but some endoglucosaminidases may require more time to complete the hydrolysis of all N-linked sugar structures. The hydrolysis of high-mannose N-glycans or hybrid N-glycans by Endo T or Endo H present on N-glycosylated proteins produced in complex glyco-engineered fungal cells (like Pichia pastoris) leads to N-glycans consisting of a single GlcNAc. A particular preferred class of endoglucosaminidases is formed by the mannosyl-glycoprotein endo-β-N-acetylglucosaminidases, indicated as EC 3.2.1.96 in the IUBMB nomenclature. These enzymes can remove sugar chains (hybrid N-glycans, high mannose N-glycans and neoglycoforms of N-glycans as shown herein) while leaving one GlcNAc residue on the protein. Examples of these include, but are not limited to Endo A, Endo BH, Endo CE, Endo D, Endo F1, Endo H, Endo M, Endo T (see also WO2006/050584), and ENGase. Other examples are known to the skilled person and can for instance be found on www.cazy.org, in particular under the Glycoside Hydrolase Family 85 and 18. Particularly envisaged is the use of the Endo T enzyme from Hypocrea jecorina (formerly known as Trichoderma reesei) that is described in WO2006/050584 (see e.g. SEQ IDs 9-12 therein).
‘Neoglycoforms’ can be unexpected N-glycans which may form even in highly engineered strains as a result of an intervention in the N-glycosylation pathway through heterologous expression, gene deletion or other processes. The responsible glycosyltransferases would have to be identified to initiate their knock-out.
In the present invention we show that neoglycoforms can be removed by endoglucosaminidases and their removal leads to glycoproteins which provide a more homogenous glycosylation profile or provide a higher purity while the presence of yeast-specific background is reduced.
Neoglycoforms can for example comprise a Man5GlcNAc2 N-glycan with a tetra-saccharide modification. The tetra-saccharide (Glcα1-2Manβ1-2Manβ1-3Glucα-) substitution of the Man5GlcNAc2 N-glycan can most likely be attached to the innermost α-1,3 arm of the mannosyl core. Neoglycoforms can comprise a number of intermediates that re-appear and the intermediates that re-appear can comprise Man5GlcNAc2. The Man5GlcNAc2 N-glycan can be substituted with a linear hexosyl-saccharide that contains β-mannose and/or glucose. The neoglycoforms can comprise Hex6-9GlcNAc2 N-glycans and even Hex6-11GlcNAc2 N-glycans. In addition, certain neoglycoforms may contain one or more phosphomannose residues.
In a specific embodiment the invention provides a composition comprising a plurality of glycoforms of a recombinant glycoprotein, wherein the N-glycans present on said glycoforms consist of a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc.
It can be advantageous to obtain one predominant glycoform within the mixture of complex N-glycans. Such predominant glycoforms typically result from production of a glycoprotein in a complex N-glycosylation-engineered fungal organism.
In a specific embodiment the composition comprises a plurality of glycoforms of a recombinant glycoprotein, wherein the N-glycans present on said glycoforms consist of a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc is substantially devoid of high-mannose-type N-glycan structures, devoid of hybrid glycan structures and devoid of N-glycan neoglycoforms. The wording “devoid of high-mannose-type N-glycan structures, devoid of hybrid glycan structures and devoid of N-glycan neoglycoforms” means that the N-glycans present on the recombinant glycoprotein are essentially of the complex type N-glycan. In a specific embodiment the composition comprises a plurality of glycoforms of a recombinant glycoprotein, wherein said glycoprotein is produced in a complex N-glycosylation-engineered fungal organism, wherein the N-glycans present on said glycoforms consist of a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc is substantially devoid of high-mannose-type N-glycan structures, devoid of hybrid glycan structures and devoid of N-glycan neoglycoforms.
In yet another specific embodiment the invention provides a composition comprising a plurality of glycoforms of a recombinant glycoprotein, wherein the N-glycans present on said glycoforms comprise a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc wherein the sum of said complex N-glycans and N-glycans consisting of a single GlcNAc are present at a level of higher than 90% of the total N-glycans present on said glycoprotein. The wording “the sum of said complex N-glycans and N-glycans consisting of a single GlcNAc” is equivalent to “the combined complex N-glycans and N-glycans consisting of a single GlcNAc) and refers to the fact that the process (or method) of the invention cannot exclude the complete removal of all non-complex N-glycans (id est the hybrid N-glycans and the high mannose N-glycans, originating in the complex N-glycosylation engineered fungal organism). In any case the sum of the complex N-glycans and N-glycans consisting of a single GlcNAc with respect to the total amount of N-glycans present on the recombinant glycoprotein is present at a level higher than 90%, higher than 93% and in many instances even higher than 98%.
In yet another specific embodiment the invention provides a composition comprising a plurality of glycoforms of a recombinant glycoprotein, wherein said glycoprotein is produced in a complex N-glycosylation-engineered fungal organism, wherein the N-glycans present on said glycoforms comprise a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc wherein the sum of said complex N-glycans and N-glycans consisting of a single GlcNAc are present at a level of higher than 90%, higher than 93% or even higher than 98% of the total N-glycans present on said glycoprotein.
In yet another specific embodiment the invention provides a pharmaceutical composition comprising a plurality of glycoforms of a recombinant glycoprotein, wherein the N-glycans present on said glycoforms comprise a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc wherein said complex N-glycans and N-glycans consisting of a single GlcNAc are present at a level of higher than 90%, higher than 93% or even higher than 98% in said mixture.
In yet another specific embodiment the invention provides a pharmaceutical composition comprising a plurality of glycoforms of a recombinant glycoprotein, wherein said glycoprotein is produced in a complex N-glycosylation-engineered fungal organism, wherein the N-glycans present on said glycoforms comprise a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc wherein said complex N-glycans and N-glycans consisting of a single GlcNAc are present at a level of higher than 90%, higher than 93% or even higher than 98% in said mixture.
In another specific embodiment where a recombinant glycoprotein, produced in complex N-glycosylation engineered fungal organism, has at least two functional N-glycosylation acceptor sites then also a plurality of glycoforms are produced when this recombinant glycoprotein is purified from the medium and exogenously contacted with a suitable amount of endoglucosaminidase. In this case also glycoforms of this recombinant glycoprotein will occur on the same recombinant glycoprotein consisting of a single GlcNAc and N-glycans consisting of complex N-glycans. Thus, in the case where 2 functional N-glycosylation sites are present on a glycoprotein theoretically a number of differently mixed glycoforms (e.g. mixtures of single GlcNAc N-glycans and complex N-glycans) will occur. It is understood that the sum of complex N-glycans and N-glycans consisting of a single GlcNAc with respect to the total N-glycans present on said recombinant glycoprotein will be higher than 90%.
Accordingly, in yet another embodiment the invention provides a glycoform of a recombinant glycoprotein having at least two N-glycosylation sites, wherein at least one N-glycosylation site present on said glycoprotein consists of a single GlcNAc and at least one N-glycosylation site on said same glycoprotein consists of a complex N-glycan.
In yet another embodiment the invention provides a pharmaceutical composition comprising a glycoform of a recombinant glycoprotein having at least two N-glycosylation sites, wherein at least one N-glycosylation site present on said glycoprotein consists of a single GlcNAc and at least one N-glycosylation site on said same glycoprotein consists of a complex N-glycan.
In yet another embodiment the invention provides a composition comprising a plurality of glycoforms of a recombinant glycoprotein, wherein the N-glycans present on said glycoforms comprise a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc wherein said complex N-glycans and N-glycans consisting of a single GlcNAc are present at a level of higher than 90% in said mixture wherein said composition is obtained by production of said glycoprotein in a complex N-glycosylation-engineered fungal organism comprising cultivating a recombinant complex N-glycosylation engineered fungal organism comprising an expression vector comprising a genetic construct encoding said glycoprotein under conditions where said glycoprotein is expressed and contacting said recombinant glycoprotein after it has been produced with an endoglucosaminidase.
In yet another embodiment the invention provides a pharmaceutical composition comprising a plurality of glycoforms of a recombinant glycoprotein, wherein the N-glycans present on said glycoforms comprise a mixture of complex N-glycans and an N-glycan structure consisting of a single GlcNAc wherein said complex N-glycans and N-glycans consisting of a single GlcNAc are present at a level of higher than 90% in said mixture wherein said composition is obtained by production of said glycoprotein in a complex N-glycosylation-engineered fungal organism comprising cultivating a recombinant complex N-glycosylation engineered fungal organism comprising an expression vector comprising a genetic construct encoding said glycoprotein under conditions where said glycoprotein is expressed, contacting and purifying said recombinant glycoprotein after it has been produced with an endoglucosaminidase and formulating the resulting plurality of glycoforms of the recombinant protein with an appropriate pharmaceutical excipient (or carrier).
Pharmaceutical compositions containing a composition or glycoform of a specific glycoprotein produced according to the invention can be utilized to achieve the desired pharmacological effect by administration to a patient in need thereof. A patient, for the purpose of this invention, is a mammal, including a human, in need of treatment for the particular condition or disease. Therefore, the present invention includes pharmaceutical compositions that are comprised of a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a composition or glycoform of a specific glycoprotein, or salt thereof, of the present invention. A pharmaceutically acceptable carrier is preferably a carrier that is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. A pharmaceutically effective amount of a composition or glycoform of a specific glycoprotein is preferably that amount which produces a result or exerts an influence on the particular condition being treated. The composition or glycoform of a specific glycoprotein of the present invention can be administered with pharmaceutically acceptable carriers well known in the art using any effective conventional dosage form, including immediate, slow and timed release preparations, orally, parenterally, topically, nasally, ophthalmically, intrathecally, intracerebroventricularly, sublingually, rectally, vaginally, and the like.
The complex N-glycosylation engineered fungal cells as herein may produce a plurality of glycoforms of a recombinant glycoprotein. In the case where only one functional N-glycosylation site occurs on a recombinant glycoprotein one predominant glycoform will carry a complex N-glycan while another glycoform will carry a single GlcNAc. In a specific aspect it may be advantageous to separate these two populations of glycans. For example, it can in certain instances be desirable to work with a recombinant glycoprotein which carries only a complex N-glycan. In these cases, glycoforms carrying only a single GlcNAc can easily be separated from glycoforms carrying a complex N-glycan.
According to alternative particular embodiments, said recombinant glycoprotein has one or more N-glycosylation sites presenting both a single GlcNAc and a plurality of complex N-glycans on the same N-glycosylation site of said glycoprotein.
According to particular embodiments, the recombinant glycoprotein has two or more N-glycosylation sites wherein one or more N-glycosylation sites present on said glycoprotein consist of a single GlcNAc and one or more N-glycosylation sites on said same glycoprotein consist of a plurality of complex N-glycans.
According to particular embodiments, said glycoform of said recombinant glycoprotein has at least three glycosylation sites, wherein at least one N-glycosylation site on said glycoprotein consists of a single GlcNAc and at least another N-glycosylation site consists of a plurality of complex N-glycans.
According to particular embodiments, the endoglucosaminidase enzyme exogenously added to the secreted glycoprotein after production in the complex N-glycosylation engineered fungal organism is a mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase, i.e. it has the activity of E.C. 3.2.1.96 in the IUBMB nomenclature, implying that it can remove sugar chains while leaving one GlcNAc residue on the protein. According to alternative embodiments, the endoglucosaminidase has different affinities towards different types of glycosylation structures. Typical examples of the latter are endoglucosaminidases that are able to hydrolyze hybrid type sugars and/or high-mannose sugars, but are not capable of cleaving complex type glycans. According to further particular embodiments, the endoglucosaminidase is a mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase that has different affinities towards different types of glycosylation structures. According to yet further particular embodiments, the endo-beta-N-acetylglucosaminidase is able to cleave hybrid type sugars and/or high-mannose sugars, but not complex type glycans. According to even more particular embodiments, the endoglucosaminidase is EndoH or EndoT. According to most particular embodiments, the endoglucosaminidase is Endo T.
According to particular embodiments, the at least one enzyme needed for engineering complex N-glycosylation engineered fungal (e.g. yeast) organisms is more than one enzyme. More particularly, the at least one enzyme is the number of enzymes needed to form a pathway for complex glycosylation. Most particularly, each of these enzymes needed for complex glycosylation is targeted so that they act sequentially and in the right order (typically, one enzyme will modify the sugar chain to a substrate for the next enzyme). According to a particular embodiment, the at least one enzyme needed for complex glycosylation is at least one N-acetylglucosaminyl transferase (e.g. GnT I, GnT II, GnT III, GnT IV, GnT V, GnT VI), at least one mannosidase (in particular mannosidase II), at least one fucosyltransferase, at least one galactosyltransferase, at least one sialyltransferase, or any combination of these enzymes. Examples of glyco-engineered yeasts wherein complex glycosylation pathways have been engineered are extensively described in the art (see e.g. Choi et al., 5022 2003; Hamilton et al.; Science 1244; Wildt et al., 119 2005; Hamilton et al., 387 2007; EP1211310; WO02/000879; US2006148039). In addition, a number of other genes may also be transformed in the glyco-engineered yeast cells described herein to ensure optimal production of complex-type glycosylated glycoproteins, such as ER and Golgi specific transporters (e.g. sym- and antiport transporters for UDP-galactose and other precursors), or enzymes involved in the synthesis of activated oligosaccharide precursors such as UDP-galactose and CMP-N-acetylneuraminic acid. Indeed, the contacting with the at least one enzyme needed for complex glycosylation may occur in the presence of specific glycosyl donors (e.g. sugar nucleotide donors) to ensure efficient and correct glycosylation.
The methods as described herein may be further adapted to ensure that the contact between glycoprotein and endoglucosaminidase occurs under optimal circumstances (i.e. to ensure optimal activity of the endoglucosaminidase on the glycoprotein, e.g. depending on the specific pH, temperature, salt and buffer conditions).
‘Contacted’ or ‘contacting’ as used herein does refer to physical proximity between the produced recombinant glycoprotein and the endoglucosaminidase. ‘Contacting’ in the instant invention occurs in vitro.
The methods as described herein may be further adapted to ensure that the contact between glycoprotein and endoglucosaminidase occurs under optimal circumstances (i.e. to ensure optimal activity of the endoglucosaminidase on the glycoprotein or to ensure the retention of the bioactivity of the glycoprotein itself during and after contact with the endoglucosaminidase). Contacting between the endoglucosaminidase and the glycoprotein may occur exogenously. It is possible that the contact between the endoglucosaminidase and the glycoprotein happens extracellularly after secretion of the glycoprotein. Depending on the cells and endoglucosaminidase that are used however, the optimal growth and production conditions for the cells (e.g. pH, temperature) may differ from the optimal conditions for enzymatic activity. Thus, the medium where the extracellular contact between the glycoprotein and the endoglucosaminidase takes place may be adjusted for optimal bioactivity of the glycoprotein. The temperature of the medium may be adjusted to retain optimal bioactivity of the glycoprotein. According to a particular embodiment of the invention the temperature of the medium wherein the contact between the endoglucosaminidase and the glycoprotein takes place is adjusted to 4-37° C. It may be advantageous to adjust the temperature of the medium to 4° C. In other embodiments it might be advantageous to keep temperatures above 37° C.
According to another particular embodiment, the endoglucosaminidase activity is retained at a high salt concentration of the medium. Even if the salt concentration of the medium wherein the contact between the endoglucosaminidase and the glycoprotein takes place is high, the endoglucosaminidase may be able to exert its function on the glycoprotein.
In a specific embodiment the invention provides a composition comprising recombinant N-glycosylated IL-22 or a recombinant N-glycosylated IL-22 thereof with at least 97% amino acid identity, wherein said IL-22 comprises the complex N-glycan Gal2GlcNAc2Man3GlcNAc2 which is present at more than 65% percent of the total complex N-glycans present on said recombinant IL-22.
In another embodiment the invention provides a composition comprising recombinant N-glycosylated IL-22 or a recombinant N-glycosylated IL-22 thereof with at least 97% amino acid identity, wherein said IL-22 comprises the complex N-glycan Gal2GlcNAc2Man3GlcNAc2 which is present at more than 85%, at more than 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% of the total complex N-glycans present on said recombinant IL-22.
“At least one mutated N-glycosylation acceptor site” refers to a mutation in the N-glycosylation acceptor site. It is well known in the art that potential N-glycosylation acceptor sites are specific to the consensus sequence Asn-Xaa-Ser/Thr. It must be noted that the presence of the consensus tripeptide is not sufficient to conclude that an asparagine residue (Asn) is glycosylated which is due to the fact that the folding of the protein plays an important role in the regulation of N-glycosylation. It has also been shown that the presence of proline between Asn and Ser/Thr will inhibit N-glycosylation. In the glycoprotein IL-22 there are usually (depending on the mammalian species origin) 3 different N-glycosylation acceptor sites.
In yet another embodiment the invention provides a composition comprising recombinant N-glycosylated IL-22 which has one functional N-glycosylation acceptor site or a recombinant N-glycosylated IL-22 thereof with at least 97% amino acid identity, wherein said IL-22 comprises the complex N-glycan Gal2GlcNAc2Man3GlcNAc2 which is present at more than 85% of the total complex N-glycans present on said recombinant IL-22.
In yet another embodiment the invention provides a composition comprising recombinant N-glycosylated human IL-22 which has one functional N-glycosylation acceptor site present on position N21 in the sequence depicted in SEQ ID NO: 1 or a recombinant N-glycosylated human IL-22 thereof with at least 97% amino acid identity, wherein said human IL-22 comprises the complex N-glycan Gal2GlcNAc2Man3GlcNAc2 which is present at more than 85% at more than 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% of the total complex N-glycans present on said recombinant human IL-22.
SEQ ID NO: 1 depicts the amino acid sequence of human IL-22 with one functional glycosylation site only (underlined), id est the glycosylation acceptor site N21 (hIL-22 N21 mutant). The other two glycosylation acceptor sites (marked in SEQ ID NO: 1) are mutated into non-functional N-glycosylation acceptor sites.
SEQ ID NO: 2 depicts the amino acid sequence of human IL-22 with two functional glycosylation sites (underlined), id est the glycosylation acceptor sites N21 and N35 (hIL-22 N21-N35 mutant). The other glycosylation acceptor site (marked in SEQ ID NO: 2) is mutated into a non-functional N-glycosylation acceptor site.
SEQ ID NO: 3 depicts the wild type human IL-22 (hIL-22 WT) amino acid sequence with three functional glycosylation sites (underlined)
In yet another embodiment the invention provides a method for the production of a composition comprising recombinant N-glycosylated IL-22 or a recombinant N-glycosylated IL-22 thereof with at least 97% amino acid identity, wherein said IL-22 comprises the complex N-glycan Gal2GlcNAc2Man3GlcNAc2 which is present at more than 65% percent of the total complex N-glycans present on said recombinant IL-22 as described herein before wherein said composition is produced in a complex N-glycosylation-engineered fungal organism comprising i) cultivating a recombinant complex N-glycosylation engineered fungal organism comprising a genetic construct encoding IL-22 under conditions wherein IL-22 is expressed and contacting said IL-22 after the production with the addition of an endoglucosaminidase enzyme.
In yet another embodiment the invention provides a composition comprising recombinant N-glycosylated IL-22 or a recombinant N-glycosylated IL-22 thereof with at least 97% amino acid identity, wherein said IL-22 comprises the complex N-glycan Gal2GlcNAc2Man3GlcNAc2 which is present at more than 65% percent of the total complex N-glycans present on said recombinant IL-22 wherein said composition is obtained by production of said recombinant IL-22 in a complex N-glycosylation-engineered fungal organism comprising cultivating a recombinant complex N-glycosylation engineered fungal organism comprising an expression vector comprising a genetic construct encoding IL-22 under conditions wherein said IL-22 is expressed and contacting said recombinant IL-22 with the addition of an endoglucosaminidase.
It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for cells and methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.
Materials and Methods Materials and Methods Specifically for Example 1: Endoglucosaminidase Clean-Up to Enrich Complex Glycoforms Strains and MediaPichia pastoris GS115 (his4) was used as the wild-type expression host (De Schutter, K. et al., Nat. Biotechnol. 27, 561-566 (2009)). To construct the Gal2Gn2M3-hIL-22N21 and -IL-22WT strains, N-glycan engineering was started from the M5- (Man5) and GnM5-strains (GnMan5) that modify their glycoproteins predominantly with Man5GlcNAc2 and GlcNAcMan5GlcNAc2 N-glycans respectively (Vervecken, W. et al., Appl. Environ. Microbiol. 70, 2639-2646 (2004)). The clone of the GnM5-strain with the most homogenous hybrid-type GlcNAcMan5GlcNAc2 N-glycans was transformed with the pGAPKanMnn2DmMan-II after EcoRI-linearization to express glycoproteins modified with the complex-type GlcNAcMan3GlcNAc2 N-glycans. The pGAPKanMnn2DmMan-II vector encodes a Mannosidase-II from Drosophila melanogaster that is targeted to the Golgi through the N-terminal domain of S. cerevisiae Mnn2p early-Golgi localized glycosyltransferase. The clone with the most homogenous GlcNAc3Man3 N-glycans from the GnM3-strain was used for further engineering by transforming the BgII-linearized pGAPHygMnn2rGnT-II, encoding a Golgi-localized β-N-Acetylglucosaminyltransferase-II from Rattus norvegicus, to obtain a strain expressing glycoproteins with bi-antennary, complex-type GlcNAc2Man3GlcNAc2 N-glycans (Gn2M3-strain). The clone of the Gn2M3-strain with the most homogenous GlcNAc2Man3GlcNAc2 N-glycans was then transformed with the EcoRV-linearized pGAPNorMnn2SpGal10GalT. This vector encodes a tri-partite fusion of the Mnn2p-Golgi localization domain, the Schizosaccharomyces pombe UDP-glucose/-galactose 4-epimerase and human β-1,4 galactosyltransferase-I (GalT-I). The resulting strains modifies its glycoproteins with Gal2GlcNAc2Man3GlcNAc2. The vectors and construct have been described in (Jacobs, P. P. et al., Nat. Protocols 4, 58-70 (2008)). The methodology was described extensively in Laukens, B. et al., Methods Mol. Biol. 1321, 103-22 (2015).
Strains were kept as glycerol stocks at −80° C. Prior to the experiment, a fresh culture was started up from a glycerol stock by plating on YPD supplemented with BlasticidinS-HCl (100 μg/mL), Zeocin® (100 μg/mL), G418 (500 μg/mL), Hygromycin (100 μg/mL) and Nourseothricin (100 μg/mL). All cultures were grown at 28° C. and stored at 4° C. awaiting further experimentation.
Recombinant Production of EndoTFor recombinant production of EndoT, a wild type strain (NRRL-Y11430) expressing the full size, mature EndoT under control of the AOX1-promoter was constructed. Large scale production was performed in baffled shake flasks on a level of 6 liters (24×250 ml/2 liter flask) (Schoonooghe, S., Leoen, J. & Haustraete, J., Pichia pastoris. Methods Mol. Biol. Clifton N.J. 907, 325-340 (2012)). At the end of induction, the medium was collected by centrifugation at 18,000×g for 30 min at 4° C. and diafiltered to 20 mM Tris pH 7.5. The clear supernatant was applied to a 138 ml Q sepharose FF column XK26×26 (GE Healthcare), equilibrated with 20 mM Tris pH 7.5. The column was eluted with a gradient over 5 column volumes to 1 M NaCl in the same buffer. The elution fractions were analyzed on SDS-PAGE and the EndoT containing fractions were pooled together. Finally, the protein was injected on a Superdex 75 gelfiltration column XK26×52 with PBS as running solution for formulation and to remove minor contaminants. The obtained fractions were analyzed by SDS-PAGE, the concentration was determined using the Micro-BCA assay (Pierce) and the LPS content (Endosafe-PTS) was measured (<1 EU/ml).
The final yield after purification was 0.183 g/L with >95% purity.
Production and Purification of IL-22 GlycoformsA pre-culture of the hIL-22-expression strains was inoculated in 10 mL YPD supplemented with appropriate antibiotics and grown overnight. The next day, the pre-culture was used to seed 8×250 mL BMGY (pH5.5) in 2 L baffled shake flasks. The cultures were grown and the medium was replenished with BMMY. After the cultures were induced for 48 hours, the supernatant was harvested and subjected to ammonium sulfate precipitation. Briefly, to remove aggregate proteins and remnant cells, the supernatant was saturated by adding ammonium sulfate salt up to 30%. The samples were centrifuged at 16,800 g and the resulting pellet was discarded. The supernatant was further saturated to 80% under continuous stirring. The precipitate containing hIL-22 was harvested by centrifugation. The remaining supernatant was discarded and the pellets were stored at −20° C. until further purification.
For purification, the hIL-22 ammonium sulfate pellets were dissolved in 25 mM MES pH 5.5 and filtered over a 0.22 μm bottletop filter (Millipore) to remove impurities after solubilisation. The filtrate was desalted over a Sephadex G25 XK26/80 column (GE Healthcare) running on 25 mM MES pH 5.5 and previously equilibrated with the same buffer. To remove the bulk of Pichia host proteins and to remove potential endotoxins (LPS), the desalted fractions were pooled and loaded on a Q-Sepharose XK16/32 column (GE Healthcare) equilibrated with 25 mM MES pH 5.5 as running buffer. The flowthrough containing hIL-22 was collected and the column was washed extensively prior to eluting with 1 M NaCl in 25 mM MES pH 5.5. The Q-Sepharose flowthrough was then loaded on a Source 15S column equilibrated with the same running buffer. After loading, the column was washed extensively with running buffer and hIL-22 was eluted with a stepwise linear gradient from 0-1 M NaCl in 25 mM MES pH 5.5. The fractions containing predominantly N-glycosylated hIL-22 were polished over a Superdex75 column (GE Healthcare) equilibrated with PBS set to pH 8.0. After polishing, the hIL-22 containing fractions were concentrated using Amicon spin columns (Millipore) with a 10 kDa molecular weight cut-off (MWCO), sterilized over Millex low protein binding 0.22 μm syringe filters (Millipore), and the concentration was determined by BCA (Pierce). The samples were divided in aliquots and stored at −80° C. The purification steps were performed using the Akta Explorer or Akta Pure purification platform (GE Healthcare). Chromatograms were analyzed in Unicorn 5.11 and formatted afterwards in CorelDraw 11.
Protein Analytical TechniquesProteins were analyzed on 15% Tris-Glycine SDS-PAGE gels. Prior to loading, the samples were supplemented with Laemli loading dye (200 mM Tris-HCl, pH 6.8 containing 40% glycerol, 10% SDS, 0.8% bromophenol blue with 30 mM DTT unless explicitly mentioned otherwise) and heat denatured at 98° C. for 10 minutes. As a molecular weight ladder the Precision Plus Protein All Blue Standard (Bio-Rad) was included. For visualization, the gels were Coommassie stained or transferred to nitrocellulose membranes by Semi-Dry Western Blot (1 mA/cm2). Human IL-22 was visualized using a anti-hIL-22 mouse monoclonal antibody (Abcam, ab134035) diluted 1:1000 in PBST-3% milk. Blots were revealed using anti-mouse HRP-coupled IgG (GE Healthcare) diluted 1:5000 in PBST-3% milk with Lightning ECL Enhanced Chemiluminescence Substrate (Perkin Elmer).
Protein deglycosylation using EndoH or PNGaseF (New England Biolabs) was done following the manufacturer's instructions. Briefly, 5-10 μg purified protein was heat denatured (5 minutes, 98° C.) in 1× Glycoprotein denaturation buffer (0.5% SDS, 40 mM DTT). After cooling the samples, samples for PNGaseF digestion were supplemented with 1% NP-40 and 1× buffer G7 and 1000 NEB Units PNGaseF (equivalent of 15.4 IUB mU/μl, produced in-house) were added. Samples for EndoH digestion were supplemented with 1× buffer G5 and 500 Units of EndoH (NEB). All digests were kept at 37° C. for 2-4 hours prior to loading on SDS-PAGE.
Glycosylation Analysis by Capillary ElectrophoresisFor N-glycan analysis by capillary electrophoresis, either 50 μl of the ammonium sulfate fraction or 10 μg of the purified hIL-22 was prepared following the plate-method described by Laroy, W., Contreras, R. & Callewaert, N., Nat. Protoc. 1, 397-405 (2006).
Samples for an in-solution EndoT-digest were diluted in 25 mM MES pH5.5 prior to adding 100 ng of recombinant purified EndoT. The samples were incubated overnight and dried. Labeling was done as described before. The APTS-derivatized N-glycans were analyzed on an ABI 3130 capillary DNA sequencer as described previously (Laroy, W., Contreras, R. & Callewaert, N., Nat. Protoc. 1, 397-405 (2006)). N-glycans of bovine RNase B (Man5-9 GlcNAc2, M5-9) and a dextran ladder consisting of α-1,6-linked glucose residues (Glucose Units, G.U) were both included as references. Data was analyzed with the Genemapper software (Applied Biosystems).
For exoglycosidase sequencing, exoglycosidase treatment of labeled glycans was done with Streptococcus pneumoniae β-1,4-galactosidase or β-N-Acetylhexosaminidase (Prozyme, 4 mU per digest), Trichoderma reesei α-1,2-mannosidase (produced in our laboratory, 0.33 μg per digest) and Jack Bean α-1,2/-3/-6-mannosidase (Sigma, 20 mU per digest). All the reactions were performed overnight at 37° C. in 20 mM sodium acetate (pH 5.0).
EndoT Dose-Finding for N-Glycoform Clean-UpIn order to determine the impact of EndoT treatment, a single ammonium sulfate pellet (equivalent of 250 mL culture) was re-dissolved in 25 mL of 25 mM MES pH 5.5 and filtered over a 0.22 μm SteriTop/SteriCup bottletop filter (Millipore). The total protein concentration of the filtrate was determined by BCA (Pierce). The protein was divided in two series over sterile eppendorf tubes so that each eppendorf tube contained 1 mg of total protein. A dilution series of recombinant EndoT was made by diluting recombinant EndoT in 25 mM MES pH 5.5 and spiked into the IL-22 containing ammonium sulfate fractions. Each series (from 10 μg EndoT/mg total protein to 0.001 μg/mg) was prepared in duplicate and samples were either incubated at 4° C. or 37° C. overnight (˜14 hours). The next day, the samples were evaluated for precipitation. To assess the impact of the EndoT treatment, 1 μL of each sample of both series was loaded on SDS-PAGE for transfer by Western Blot. For Coommassie analysis, 5 μl of each reaction was loaded on SDS-PAGE.
For N-glycosylation analysis, 50 μl of each sample was prepared for capillary electrophoresis (DSA-FACE) as described above. Oligo-mannose background in the N-glycan profile was revealed using an adapted Jack Bean α-mannosidase digestion. Therefore, 1 μL of APTS-labeled N-glycan sample was digested with Jack Bean α-1,2/-3/-6-mannosidase (Sigma, 10 mU per digest). Digests were performed for 2 hours at 37° C. in 20 mM sodium acetate (pH 5.0) prior to analysis by capillary electrophoresis. Longer incubation results in degradation of complex-type N-glycans due to low levels of contaminating β-N-Acetylhexosaminidase and galactosidase in the commercial jack bean preparation.
Purification of EndoT-Treated IL-22 GlycoformsTo purify hIL-22, the ammonium sulfate pellets were dissolved in 25 mM MES pH 5.5 to a final volume of 100 mL and filtered over a 0.22 μm bottletop filter. The total protein concentration of the filtrate was determined by BCA. Next, the filtrate was spiked with recombinant EndoT (0.5-1.0 μg/mg total protein). The reaction was kept at 4° C. (overnight) while gently agitating on a shaker-platform. The next day, the reaction was assessed for precipitation. Precipitate was removed over a 0.22 μm SteriTop/SteriCup bottletop filter and the filtrate was purified as described above.
In Vitro Colo-205 Assay for IL-22 BioactivityHuman Colo-205 colon carcinoma cells were ordered from the American Type Culture Collection (ATCC) and cultured according to the guidelines provided in the datasheet. Briefly, the cell line was cultured as semi-adherent cells in RPMI1640 (Gibco) supplemented with 10% Fetal Bovine Serum (FBS) at 37° C. 5% CO2. For passaging, cells growing in suspension were collected and the adherent cells were trypsinized following standard tissue culture procedures. For the Colo-205 assay, cells were seeded in 96-well U-bottom plates at 3.0×105 cells/mL (100 μl/well). Cells were allowed to adapt for 24 hours prior to stimulating the cells with a dilution series of hIL-22. All stimulations were allowed to proceed overnight. As control, a dilution series of commercially available recombinant hIL-22 (carrier-free) produced E. coli (BioLegend) was used. The next day, the plates were centrifuged at 400 g, 10 minutes at 4° C. and the supernatant was collected. The supernatant was assayed for IL-10 using the hIL-10 DuoSet ELISA (R&D systems). The data was analyzed in GraphPad Prism 6. Specific activity was determined based on the dose-response curve that was used to determine the EC50.
Materials and Methods Specifically for Example 2: Clean-Up of ProDerp1 Glycoforms Using EndoTTo obtain the different ProDerp1 glycoforms, the pPIC9ProDerp1 Pichia pastoris expression vector was transformed into the M5- (Man5) OCH1 mutated Pichia-strain that modifies its glycoproteins predominantly with Man5GlcNAc2 N-glycans (Jacobs, P. P. et al., Nat. Protocols 4, 58-70 (2008), Vervecken, W. et al., Appl. Environ. Microbiol. 70, 2639-2646 (2004)).
Next the following enzymes were consecutively transformed into this strain using their corresponding OCH1 mutated Pichia-strain vector: N-acetylglucosaminyltransferase I, Mannosidase II, N-acetylglucosaminyltransferase II and N-acetylglucosaminyltransferase IV. In between each transformation step, the N-glycan profile of ProDerp1 was analyzed using capillary electrophoresis, and the expression of ProDerp1 was analyzed. Finally a GlcNAc3Man3GlcNAc2 ProDerp1 expression strain was obtained.
After a large scale expression experiment, GlcNAc3Man3GlcNAc2 ProDerp1 was purified using a combination of hydrophobic interaction chromatography, anion exchange and gel filtration (final buffer: 50 mM Tris-HCl pH 7.4). In a next step an in vitro GalNAc transfer was performed using the following conditions: 150 μM terminal GlcNAc, 10 mM UDP-GalNAc, 50 mM Tris-HCl pH 7.4+10 mM MnCl2, 0.5 μg human beta-1,4-galactosyltransferase Y285L (specific activity >2,000 pmol/min/μg, R&D Systems) (Ramakrishnan, B. & Qasba, P. K., J. Biol. Chem. 277, 20833-20839 (2002)), overnight incubation at 37° C. To remove the high-mannose background present in GlcNAc3Man3GlcNAc2 and GalNAc3GlcNAc3Man3GlcNAc2 ProDerp1 samples, both samples were treated with EndoT (200 ng of EndoT for 10 μg of glycoprotein, overnight incubation at 37° C.).
EXAMPLES Example 1: Endoglucosaminidase Clean-Up for Complex Glycoforms of hIL-22WT and hIL-22N21 Introduction and StrategyPichia pastoris was engineered to express hIL-22WT (having 3 functional N-glycosylation sites) modified with complex type Gal2GlcNAc2Man3GlcNAc2 N-glycans. A Gal2Gn2M3-strain expressing the IL-22N21 N-glycosylation site mutant (having one functional glycosylation site) was engineered as well, all as described in the materials and methods section.
To remove the structurally heterogeneous background of N-glycans, recombinant endo-β-N-Acetylglucosaminidase from Trichoderma reesei (EndoT) was applied.
Results 1.1 EndoT Dose Finding to Clean Up Background N-GlycansTo integrate EndoT in the purification process, it was investigated if the recombinant enzyme could be added prior to purification by adding the endoglucosaminidase just after solubilizing the ammonium sulfate pellets. Since the high salt concentration might not be optimal for enzyme activity a dose-finding experiment at 4° C. was performed first to assess how much EndoT would be required in order to resolve all N-glycan background in the Gal2GlcNAc2Man3GlcNAc2-hIL-22WT sample (
Then the N-glycans are analyzed by capillary electrophoresis and an effect of EndoT on the N-glycan profile similar to samples incubated at higher temperatures (data not shown) was observed. In detail, when incubation takes place at 4° C. more EndoT is required to reach the same effect. For instance, the peak corresponding to Man5GlcNAc2 only starts decreasing from 0.01 μg EndoT/mg and up to 0.1 μg EndoT/mg is required to clear the remaining Man5GlcNAc2 completely. Similarly, the high mannose N-glycans (M9-10) persist up to 0.01 μg EndoT/mg. To remove the charged phospho-mannose containing N-glycans, up to 0.5 μg EndoT/mg is required. However, when 1 μg EndoT/mg is added, the N-glycan profile was devoid of any oligo-mannose, hybrid or phospho-mannose containing N-glycans. The N-glycans that remain are the same as for the samples incubated at higher temperatures, with no differences between peak intensities when comparing the N-glycan species across other conditions.
Capillary electrophoresis of the IL-22N21 expressing strain (
In conclusion, it was observed that the N-glycan heterogeneity correlates with the number of available N-glycosylation sites and thus, it was also determined whether there would be differences with regard to the amount of EndoT that is required to clean up the IL-22 N-glycan profiles. From the above results, it can be concluded that a decrease in N-glycan heterogeneity (e.g. IL-22N21 vs. IL-22WT) is compatible with a decrease in the amount of recombinant EndoT required to perform the clean-up, as for the current IL-22N21 sample only 0.05 μg EndoT/mg was required compared to 0.1 μg EndoT/mg total protein for IL-22WT.
1.2 Jack Bean Mannosidase Digestion Reveals and Confirms BackgroundBecause part of the background consists of elaborate high-mannose N-glycans, the signal corresponding to such N-glycans can be very diffuse, and therefore hard to distinguish using a method such as capillary electrophoresis. To circumvent this, a Jack Bean α-1,2/-3/-6-mannosidase digest on the samples that were previously incubated with EndoT at 4° C. (
To investigate the Gal2Gn2M3IL-22WT samples the control sample (not treated with EndoT or the Jack Bean mannosidase) was compared with the same sample digested with Jack bean mannosidase (
Regarding the N-glycan profile of the Gal2Gn2M3 IL-22N21 samples the control samples not treated with EndoT or Jack Bean α-mannosidase already have a relatively homogenous N-glycan profile (
1.3 Monitoring IL22 Stability after EndoT Digestion of IL-22WT and IL-22N21 by SDS-PAGE Analysis.
The impact of the overnight EndoT-digestion on IL-22 stability was investigated by analyzing the samples on SDS-PAGE (
It was possible to clearly differentiate between the bands corresponding to unglycosylated IL-22WT and the glycoforms with 1- or -2 N-glycans (
Analysis by Western Blot using an antibody reactive to IL-22 revealed that the observed diffuse smear was in fact IL-22 and that it indeed disappears with increasing concentration EndoT
(
EndoT treated IL-22N21 samples were analyzed as well by SDS-PAGE (
The signal of the hyperglycosylated background did not exceed the background signal of the blot (not shown). However, it was seen that the unglycosylated fraction increases with increasing dose of EndoT indicating the removal of some existing background that could not be detected.
1.4 Purification of EndoT-Treated Gal2GlcNac2Man3hIL-22WT.
It was investigated whether the EndoT clean-up procedure could be integrated in a purification experiment and it was tested whether the existing protocol also allowed to remove the recombinant EndoT again. In the dose dose-finding experiment, it was established that around 0.5 μg/mg EndoT (per mg of total protein) should be sufficient to clear any oligo-mannose background even when incubating at 4° C. These findings were implemented on the equivalent of a 2 L culture and after determining the total protein concentration of the solubilized ammonium sulfate fractions, EndoT was spiked accordingly. After overnight incubation at 4° C., samples were purified according to a standard protocol (
After desalting over SephadexG25 (
In the elution profile of the Gal2GlcNAc2Man3GlcNAc2-IL-22WT from the S15 Source several peaks can be discriminated (
When eluting hIL-22WT from the S15 Source column, any heterogeneity can be rapidly detected. In the elution profile of EndoT-treated Gal2GlcNAc2Man3-hIL-22WT, rather discrete peaks were observed (
The N-glycosylation profile of the crude, untreated supernatant was compared with that of the newly purified IL-22 that was treated with EndoT (
In contrast, the EndoT treated sample is more homogenous (lane 4), showing only peaks that correspond to the expected complex N-glycans (Gal2GlcNAc2Man3GlcNAc2, the GalGlcNAc2Man3GlcNAc2 isomer and residual GlcNAc2Man3GlcNAc2). Notably, α-mannosidase digestion does not reveal any background (lane 5) at all, demonstrating the efficiency of this approach.
The identity of the dominant peaks in the spectrum was also confirmed using sequential exoglycosidase digestion (
1.5 Purification of EndoT-treated Gal2GlcNac2Man3 IL-22N21.
The EndoT clean-up procedure was integrated in the purification scheme. Now this was tested to isolate clean Gal2GlcNac2Man3GlcNAc2 IL-22N21. In the dose dose-finding experiment, it was established that from 0.5 μg EndoT/(mg total protein) should be sufficient to clear any oligo-mannose background even when incubating at 4° C. For the preparative digest, the equivalent of 2 L culture was used and after determining the total protein concentration of the solubilized ammonium sulfate fractions, EndoT was spiked in at 1 μg/mg total protein to ensure complete digestion. After overnight incubation at 4° C., samples were purified using the standard protocol (
First, the samples were desalted over a SephadexG25 column (
To establish how the EndoT clean-up impacts the N-glycan profile of the purified Gal2GlcNAc2Man3GlcNAc2-hIL22N21, the N-glycosylation profile of the untreated ammonium sulfate fraction was compared with that of purified hIL-22N21 by capillary electrophoresis (
Then exoglycosidase digestion was used to further confirm the purity of the glycoforms and to confirm the identity of the dominant peaks in the spectrum (
In order to determine the bio-activity of the purified Gal2GlcNAc2Man3GlcNAc2-hIL-22WT and -hIL-22N21, the glycoforms were tested for their ability to induce IL-10 in the human Colo-205 colon carcinoma cell line. By stimulating with an escalating dose of IL-22 the EC50 of the EndoT treated IL-22 was determined and compared with a commercial recombinant IL-22 standard purified from E. coli (
During the N-glycan engineering of the various hIL-22-expression strains, an unusual glycoform was encountered that likely results from the recognition of the artificial N-glycan intermediates generated by endogenous glycosyltransferases. Previously, it was found that the Man5GlcNAc2 N-glycan of a murine IL-22 could be substituted with a linear tetra-saccharide that contains an α1,3-linked glucose, two consecutive β1,2-linked mannose residues and a capping α-1,2-glucose (data not shown). A similar observation was made for human IL-22 expressed in a Man5-strain. However, the N-glycan substitution was one hexose residue smaller. In order to evaluate whether the EndoT clean-up would also work on the neoglycoforms which were identified, an in vitro digest with EndoT on the Man5GlcNAc2hIL-22WT that was purified was tested (
The same is also seen in lane 5, therefore it could be a contaminating polymer that is present in the purified EndoT that was used in the analysis. The supernatant of the EndoT digest was analyzed using a direct labeling and a similar profile as for PNGaseF released samples was seen but the residues have shift towards the left of the profile with ˜1 GU (lane 4). This is consistent with cleavage by an Endo-β-N-Acetylglucosaminidase such as EndoT as it cleaves in between the GlcNAc residues of the chitobiose core. Except for the shift in the glycan profile, the profile was almost identical to the PNGaseF released samples, showing that in addition to Man5GlcNAc2, that is a known substrate for EndoT, the unusual N-glycan containing the potentially immunogenic β-mannosyl residues is also digested. In addition, no N-glycans on the recombinant EndoT were detected using the plate method (lane 5).
Example 2: Determining the Relative Abundance of Complex N-Glycans on IL-22N-glycan isolation and analysis was performed on a ABI3130 DNA sequencer as described by Jacobs et al. (Jacobs P P et al. (2009) Nat. Protoc. 4(1): 58-70). Peak assignment was done using the ABI GeneMapper software v3.7 (Applied Biosystems). Using the software, the peak intensity and the area under the curve (AUC) of each datapoint was calculated. N-glycan identity was assigned previously using exoglycosidase digestion. To reveal the heterogeneous background (comprising of heterogeneous oligo-mannose N-glycans), each sample was digested with Jack Bean α-mannosidase. After Jack Bean α-mannosidase, the core Man1GlcNAc2 appears as a consequence of the hydrolysis of the oligo-mannose N-glycans, allowing a more accurate estimate of the background.
The relative quantity of each glycoform within the N-glycan profiles of IL-22N21 or IL-22WT glycoform before or after endoglucosaminidase clean-up was determined. To determine the relative abundance, the AUC of peaks that were confirmed by exoglycosidase digestion was calculated over the total AUC of all assigned peaks. The background was defined as the total AUC of the core Man1GlcNAc2 peak (revealed by Jack Bean α-mannosidase digestion) and peaks that could not be confirmed by exoglycosidase digestion.
Specifically for the Gal2GlcNAc2Man3GlcNAc2 IL-22WT glycoform prior to endoglucosaminidase treatment has 62.15% complex N-glycans (against 37.85% background)—as calculated from the peaks obtained in
Specifically for the Gal2GlcNAc2Man3GlcNAc2 IL-22N21 glycoform prior to endoglucosaminidase treatment has 80.3% complex N-glycans (against 19.7% background)—as calculated from the peaks obtained in
N-glycan isolation and analysis was performed on a ABI3130 DNA sequencer as described by Jacobs et al. (Jacobs et al. (2009) Nat. Protoc. 4(1): 58-70). Peak assignment was done using the ABI GeneMapper software v3.7 (Applied Biosystems). Using the software, the peak intensity and the area under the curve (AUC) of each datapoint was calculated. N-glycan identity was assigned previously using exoglycosidase digestion. Peak calculation on the N-glycosylation profiles of IL-22WT was based on
The relative quantity of each glycoform within the N-glycan profiles of IL-22N21 or IL-22WT glycoform after endoglucosaminidase clean-up was determined. To determine the relative abundance, the AUC of peaks that were confirmed by exoglycosidase digestion was calculated over the total AUC. Peak calculation was based on the
The relative quantity of bi-antennary Gal2GlcNAc2Man3GlcNAc2 on IL-22N21 reaches 88.89% of the total complex N-glycan pool whereas 8.93% carries a single terminal galactose or up to 97% of all complex N-glycans is a bi-antennary complex N-glycan that carries at least a single terminal galactose. The calculated data are shown in
The relative quantity of bi-antennary Gal2GlcNAc2Man3GlcNAc2 on IL-22WT reaches 66.19% of the total complex N-glycan pool. In addition, 22.06% carries a single terminal galactose. Taken together, up to 88.25% is a bi-antennary complex N-glycan that carries at least a single terminal galactose. The calculated data are shown in
The aim was to produce different glycoforms of ProDerp1, the enzymatically inactive proform of dominant house dust mite allergen Derp1, containing terminal GalNAc residues.
ResultsClaims
1. A method of producing a composition comprising different glycoforms of a glycoprotein, the method comprising:
- contacting complex N-glycan glycoforms of the glycoprotein secreted from a fungal organism with an endoglucosaminidase enzyme;
- wherein the endoglucosaminidase enzyme acts on the secreted N-glycan glycoforms of the glycoprotein to produce the composition comprising different glycoforms of a glycoprotein, the composition comprising: a glycoform comprising a complex N-glycan and a glycoform comprising an N-glycan structure consisting of a single GlcNAc; and
- wherein the glycoprotein is exogenous to the fungal organism.
2. The method according to claim 1, wherein the contacting occurs during purification of the glycoprotein.
3. The method according to claim 1, wherein the contacting occurs after purification of the glycoprotein.
4. The method according to claim 1, wherein contacting with the endoglucosaminidase takes place at a high salt concentration.
5. The method according to claim 2, wherein contacting with the endoglucosaminidase takes place at a high salt concentration.
6. The method according to claim 3, wherein contacting with the endoglucosaminidase takes place at a high salt concentration.
7. The method according to claim 1, wherein the fungal organism expresses at least one exogenous nucleic acid sequence encoding an enzyme needed for complex N-glycosylation and/or wherein the fungal organism does not express an enzyme involved in the production of high-mannose type structures, which enzyme is normally expressed in a wild type from of the fungal organism.
8. The method according to claim 1, wherein the endoglucosaminidase enzyme is an endo-beta-N-acetylglucosaminidase of EC3.2.1.96.
9. The method according to claim 1, wherein the composition further comprises a glycoform comprising a complex N-glycan that is different from the glycoform comprising a complex N-glycan.
Type: Application
Filed: Feb 26, 2021
Publication Date: Sep 23, 2021
Inventors: Nico L.M. Callewaert (Nevele), Bram Laukens (Gent)
Application Number: 17/186,609
