METHOD FOR PRODUCING A MOLECULE, AND, METHOD FOR PRODUCING A PROTEIN

专利摘要:
vertebrate cell for producing a molecule, method for producing a molecule, molecule, composition, protein or lipid, expression unit, eukaryotic cell for producing a protein, and, method for producing a protein. the present invention relates to cells for producing a molecule that lacks fucose, having a reduced amount of fucose, or having other atypical sugars in their glycoportions. it also pertains to methods of producing a molecule lacking fucose, having a reduced amount of fucose, or having other atypical sugars in its glycoportions using said cells and molecules obtainable by said methods. the present invention further relates to molecules having an artificial glycosylation pattern.
公开号:BR112012006388B1
申请号:R112012006388-0
申请日:2010-09-21
公开日:2022-01-04
发明作者:Hans Henning Von Horsten；Christiane Ogorek
申请人:Probiogen Ag；
IPC主号:

专利说明:

[001] The present invention relates to cells for producing a molecule that lacks fucose, having a reduced amount of fucose, or having other atypical sugars in their glycoportions. It also pertains to methods for producing a molecule lacking fucose, having a reduced amount of fucose, or having other atypical sugars in its glycoportions that use said cells and molecules obtainable by said methods. The present invention further relates to molecules having an artificial glycosylation pattern. FUNDAMENTALS OF THE INVENTION
[002] Therapeutic glycosylated molecules intended for use in humans must have complex glycosylation patterns similar to those found in humans. Therefore, animal cells are generally used to produce therapeutic glycosylated molecules, such as proteins, or lipids, where it is desirable that the glycosylated molecules have a complex, human-equivalent pattern of glycosylation. The structure and complexity of glycans severely affect the in vivo function of the biomolecule through half-life modulation, receptor binding, induction or suppression of immune reactions.
[003] The sugar chains of glycolipids are complex and can contain a significant amount of fucose (PNAS 1985; 82: 3045-3049). The sugar chains of glycoproteins are roughly divided into two types, namely sugar chains that bind to asparagine (sugar chain linked to N-glycoside) and sugar chains that bind to another amino acid such as serine, threonine (O-glycoside-linked sugar chain), based on how it is attached to the protein moiety.
[004] N-glycoside-linked sugar chains have various structures (Biochemical Experimentation Method 23 - Method for Studying Glycoprotein Sugar Chain (Gakujutsu Shuppan Center), edited by Reiko Takahashi (1989)), but they are known to have a basic common core. The end of the sugar chain that binds to asparagine is called a reducing end, and the opposite end is called a non-reducing end. The N-glycoside-linked sugar chain includes a type of high mannose in which the mannose alone attaches to the non-reducing end of the core structure; a complex type in which the non-reducing end side of the trimanose nucleus typically has at least one galactose-N-acetyl-glucosamine (hereinafter referred to as Gal-GlcNAc) attached to each of the two branches (1, 3 and 1 .6) of mannose. The non-reducing end side of Gal-GlcNAc may contain galactose and sialic acid, halving N-acetylglucosamine or the like. In a hybrid type, the non-reducing end side of the core structure has both high-mannose and complex-type branches. In vertebrate cell glycans, fucose can be linked to antenna GlcNAc via an alpha 1,3 linkage (terminal fucose) or to GlcNAc linked to asparagine via an alpha 1,6 linkage (core fucose). Insect cells produce glycans that may contain 1,3-linked core fucose.
[005] The oligosaccharide portion of N-glycosylated proteins is initially biosynthesized from lipid-linked oligosaccharides to form a Glc3Man9GlcNAc2-pyrophosphoryl-dolichol which is then transferred to asparagine which occurs in the tripeptide sequence Asn-X-Ser or Thr, where X can be any amino acid except Pro, of a protein in the endoplasmic reticulum (ER). After that, the protein is transported to the Golgi apparatus, where the oligosaccharide moiety is further processed in the following sequence: First, all three glucose (Glc) residues are removed by glycosidases I and II to produce the Man9GlcNAc2-protein. The Man9GlcNAc2 structure can be further processed by removing several mannose (Man) residues. Initially, four α-1,2-linked mannoses are removed to give a Man5GlcNAc2-protein which is then extended by the addition of an N-acetylglucosamine (GlcNAc) residue. This new structure, the GlcNAcMan5GlcNAc2-protein, is the substrate for mannosidase II that removes α-1,3- and α-1,6- bound mannoses. After that, the other sugars, GlcNAc, galactose, fucose, and sialic acid, are added sequentially to give the complex types of structures often found in N-glycosylated proteins.
[006] An IgG molecule, for example, contains an N-linked oligosaccharide covalently linked to the conserved Asn297 of each of the CH2 domains in the Fc region. The oligosaccharides found in the Fc region of serum IgGs are mainly complex-type biantennary glycans. Variations of IgG glycosylation patterns that include terminal sialic acid binding (NeuAc), a third GlcNac branch (GlcNAc split into two parts), a terminal galactosylation (G), and α-1,6-linked core fucosylation (F) to the core structure: 2x N-Acetylglucosamine (GlcNAc) and 3x mannose (Man) (GlcNAc2Man3). The exact pattern of glycosylation depends on the structural properties of IgG subcomponents, in particular the CH2 and CH3 domains ( Lund et al. (2000) Eur. J. Biochem., 267: 7246-7257 ).
[007] Animal and human cells have fucosyltransferases that add a fucose residue to the GlcNAc residue at the reducing end of N-glycans in a protein or to other nascent glycostructures in glycolipids. Fucosylation of protein- or lipid-bound glycoportions requires a nucleotide sugar, GDP-L-fucose, as a donor and also the presence of particular fucosyl transferases, which transfer the fucosyl residue from the donor to the acceptor molecule (Becker and Lowe, 1999). In eukaryotic cells GDP-L-fucose can be synthesized via two different pathways, the more prominent de novo fucose pathway or the minor recovery pathway (Becker and Lowe, 1999). The recovery pathway or “cleaner” pathway is a minor source of GDP-L-fucose (about 10%) that can be easily blocked by omitting free fucose and fucosylated glycoproteins from the culture medium. The recovery pathway starts from extracellular Fucose that can be transported into the cytosolic compartment via fucose-specific plasma membrane transporters. Alternatively, cleaved fucose from endocytosed glycoproteins can enter the cytosol. Cytosolic L-fucose is phosphorylated by fucokinase to fucose-1-phosphate and then converted by GDP-Fucose Pyrophosphorylase to GDP-L-fucose (Fig. 1, right panel). Cell culture experiments suggest that the recovery pathway makes a relatively minor contribution to cytosolic GDP-L-fucose pools (Becker and Lowe, 1999).
[008] The most prominent de novo fucose pathway starts from GDP-D-mannose and consists of a GDP-mannose dehydratase (GMD) and GDP-keto-deoxy-mannose-epimerase/GDP-keto-deoxy-galactose-reductase ( GMER, also known as Fx in humans), both located in the cytoplasm, which by common consent convert GDP-mannose to GDP-L-fucose (Fig. 1, left panel). Later, GDP-L-fucose is transported in the Golgi complex via a GDP-fucose transporter located in the membrane of the Golgi complex. Once GDP-L-fucose has entered the luminal compartment of the Golgi apparatus, fucosyltransferases can covalently bind GDP-L-fucose to nascent glycoportions within the Golgi apparatus. In particular, Fucosyltransferase (Fut8) transfers the fucose residue via a 1,6 linkage to the 6 position of the GlcNAc residue at the reducing end of the N-glycan.
[009] The lack of fucose in glycoproteins has been shown to have specific advantages. For example, in monoclonal antibodies, immunoglobulins, and related molecules, the absence of the Asn297-linked N-glycan core fucose sugar of the Fc portion (CH2 domain) of immunoglobulins has been shown to increase or alter their binding to Fc receptors. Different types of constant regions bind different Fc receptors. Examples include binding of IgG1 Fc domains to cognate Fc receptors CD16 (FCYRIII), CD32 (FCYRII-B1 and -B2), or CD64 (FCYRI), binding of IgA Fc domains to cognate Fc receptor CD89 (FcαRI) , and binding of the IgE domains to the cognate Fc receptors FcεFR1 or CD23. Binding to FCYRIII that is present on the surface of one of the NK cells is strongly increased. (Shields et al. JBC 277 (30): 26733. (2002)).
[0010] A dominant mode of action of therapeutic antibodies is Antibody Dependent Cytotoxicity (hereinafter referred to as "ADCC activity"). Antibody binding to a target cell (a tumor cell or a cell infected with a pathogen) with its Fab portion is recognized on its Fc portion by the Fc receptor of an effector cell, typically an NK cell. Once bound, the effector cell releases cytokines such as IFN-Y, and cytotoxic granules containing perforin and granzymes that enter the target cell, inducing cell death. Binding affinity to FcyRIII is critical for antibodies that act through the ATCC. Carriers of a low affinity allele of the receptor respond insufficiently to therapeutic antibodies such as Rituximab (Cartron et al. Blood 99:754-758).
[0011] Consequently, the higher affinity for FcyRIII mediated by the absence of core fucose on the Fc glycan may increase potency or reduce the effective dose of biotherapeutic product with greater implications for clinical benefit and cost.
[0012] In order to modify the structure of the sugar chain of the produced glycoprotein, several methods were tried, such as 1) application of an inhibitor against an enzyme related to the modification of a sugar chain, 2) homozygous silencing of a gene involved in sugar synthesis or transfer 3) selection of a mutant, 3) introduction of a gene encoding an enzyme related to the modification of a sugar chain, and the like. Specific examples are described below.
[0013] Examples of inhibitors against enzymes related to the modification of a sugar chain include castanospermine and N-methyl-1-deoxynojirimycin which are glycosidase I inhibitors, bromocondulitol which is a glycosidase II inhibitor, 1-deoxynojirimycin and 1, 4-dioxy-1,4-imino-D-mannitol which are mannosidase I inhibitors, swainsonine which is a mannosidase II inhibitor and the like. Examples of a specific inhibitor for a glycosyltransferase include deoxy derivatives of substrates against N-acetylglucosamine transferase V (GnTV) and the like.
[0014] Mutants of enzymes related to the modification of sugar chains were mainly selected and obtained from a lectin resistant cell line. For example, CHO cell mutants were obtained from a lectin-resistant cell line that uses a lectin such as WGA (wheat germ agglutinin derived from T. vulgaris), ConA (cocanavalin a derivative of C. ensiformis), RIC ( a toxin derived from R. communis), L-PHA (leucoagglutinin derived from P. vulgaris), LCA (lentil agglutinin derived from L. culinaris), PSA (pea lectin derived from P. sativum) or the like.
[0015] In addition, several methods for producing recombinant antibodies that lack fucose have been reported. One of the most important enzymes that allow the core fucosylation of N-glycoportions is α-1,6-fucosyltransferase 8 (Fut8). Said enzyme catalyzes the linkage of fucose to the 6-position of N-acetylglucosamine at the reducing end through an α-bond in an N-glycoside-linked sugar chain pentanucleus of a complex-type N-glycan (WO 00/61739). Antibodies with reduced fucose content have also been obtained using a cell in which expression of fucose transporter genes is artificially suppressed (US 20090061485). The introduction of RNA capable of suppressing the function of α-1,6-fucosyltransferase has also been described to lead to the production of antibody molecules which lack fucose (EP 1 792 987 A1).
[0016] Many of the cells or methods proposed to produce molecules having a modified glycosylation pattern, which can be used for therapeutic indications, have significant drawbacks. For example, treating antibodies with enzymes that remove glycosylations, for example fucosidases to remove fucose residues, involves additional manufacturing steps that are expensive, time-consuming and have potentially significant economic and drug compatibility risks. Furthermore, molecular engineering of the cell line to silence key enzymes involved in glycoprotein synthesis is tedious, expensive and not always crowned with success. Furthermore, said cell lines have the disadvantage that they do not allow the "tunable" production of molecules with varying ADCC or CDC potency to optimize efficacy and safety for a therapeutic use. Treatment of cell lines with RNAi or antisense molecules to silence the level of key enzymes involved in glycoprotein synthesis can have non-predictable off-target effects, is expensive, and appears to be impractical for implementation at manufacturing scale.
[0017] Thus, there is a need for novel cells and advantageous methods for producing molecules with a modified glycosylation pattern having improved properties for therapeutic uses.
[0018] The present invention provides cells for the production of molecules having a modified glycosylation pattern, i.e., molecules that lack fucose, have a reduced amount of fucose, or have other atypical sugars in their glycan structures. It also provides methods for producing molecules having a modified glycosylation pattern, i.e. molecules that lack fucose, have a reduced amount of fucose, or have other artificial sugars in their glycan structures, which said cells use. Said molecules have improved properties for therapeutic uses, for example increased ADCC or CDC activity, enhanced ability to inhibit signaling events, increased ability to induce apoptosis, and/or increased ability for immune therapy. Furthermore, the cells and methods provided by the present invention allow for the tunable, reliable, inexpensive, and direct production of molecules having a modified glycosylation pattern, i.e. molecules lacking fucose, having a reduced amount of fucose, or having other artificial sugars. in their glycan structures. Furthermore, the present invention provides methods that are suitable for increasing manufacturing.
[0019] In many cases, considerable time has been invested and immense efforts have been made to generate and develop effective producer cell lines that express the transgene of interest at desirable levels. In cases where the transgene of interest is a therapeutic antibody that can benefit from enhanced ADCC effector function, it would be desirable to further engineer the producer cell line in such a way that the high producer cell is unable to bind nuclear fucose to the N -glycoportions that is capable of attaching artificial sugars to the N-glycoportions.
[0020] The present invention provides an expression unit that can be easily applied to existing genetically engineered cells in a way that renders them incapable of binding fucose to the nascent glycoprotein glycostructures or that renders them capable of binding other artificial sugars besides fucose to the nascent glycostructures of glycoproteins, for example in order to produce antibodies having improved ADCC activity. SUMMARY OF THE INVENTION
[0021] In a first aspect, the present invention relates to a vertebrate cell for producing a molecule, which naturally comprises fucose in its glycoportions, which lacks fucose or having a reduced amount of fucose in its glycoportions which comprises at least one enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate, wherein the enzyme does not catalyze the reaction that converts GDP-6-deoxy-D-junk-4-hexulose to GDP-L-fucose.
[0022] In a second aspect, the present invention relates to a method for producing a molecule, which naturally comprises fucose in its glycoportions, which lacks fucose or having a reduced amount of fucose in its glycoportions which comprises the steps of: i ) providing a vertebrate cell according to the first aspect, ii) isolating the molecule which is capable of being a substrate for a fucosyltransferase, preferably a protein or lipid, from the cell in i).
[0023] In a third aspect, the present invention relates to a molecule lacking fucose or having a reduced amount of fucose in its glycoportions obtainable by the method of the second aspect.
[0024] In a fourth aspect, the present invention relates to a molecule comprising glycoportions containing D-rhamnose, D-perosamine, deoxy-D-thalose, 6-deoxy-D-altrose, 4-keto-3,6- dideoxy-D-mannose, and/or L-collitose obtainable by the method of the second aspect.
[0025] In a fifth aspect, the present invention relates to a composition comprising glycoproteins comprising i) between 70 and 95% of N-glycans of the G0 complex type - GlcNac, G0, G1, and/or G2, and ii ) between 5 and 30% high mannose-type N-glycans, wherein the complex-type N-glycans are fucose-free or substantially fucose-free.
[0026] In a sixth aspect, the present invention relates to a protein or lipid comprising glycoportions containing D-rhamnose, D-perosamine, deoxy-D-thalose, 6-deoxy-D-altrose, 4-keto-3, 6-dideoxy-D-mannose, and/or L-colitosis.
[0027] In a seventh aspect, the present invention relates to an expression unit comprising: i) one or more vertebrate expression control sequences, and ii) a polynucleotide comprising a nucleic acid sequence encoding an enzyme which uses GDP-6-deoxy-D-junk-4-hexulose as a substrate, wherein the enzyme does not catalyze the reaction that converts GDP-6-deoxy-D-junk-4-hexulose to GDP-L-fucose.
[0028] In an eighth aspect, the present invention relates to a eukaryotic cell for producing a protein, which normally comprises fucose in its glycoportions, which lacks fucose or having a reduced amount of fucose in its glycoportions which comprises: i) a first polynucleotide comprising a nucleic acid sequence encoding an enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate, and ii) a second polynucleotide comprising a nucleic acid sequence encoding a protein, wherein the enzyme does not catalyze the reaction that converts GDP-6-deoxy-D-junk-4-hexulose to GDP-L-fucose.
[0029] In a ninth aspect, the present invention relates to a method for producing a protein, which normally comprises fucose in its glycoportions, which lacks fucose or having a reduced amount of fucose in its glycoportions which comprises: i) providing a eukaryotic cell according to the eighth aspect, ii) expressing the enzyme encoded by the first polynucleotide and the protein encoded by the second polynucleotide in said cell, and iii) isolating the protein from said cell.
[0030] In a tenth aspect, the present invention relates to a protein lacking fucose or having a reduced amount of fucose in its glycoportions obtainable by the method of the ninth aspect. DETAILED DESCRIPTION OF THE INVENTION
[0031] Before the present invention is described in detail below, it should be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by a person of ordinary skill in the art.
[0032] Preferably, terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
[0033] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise", and variations such as "comprises" and "comprises", will be understood to imply the inclusion of a established integer or step or group of integers or steps but not the exclusion of any other integer or step or integer group or step.
[0034] Several documents are cited throughout the text of this descriptive report. Each of the documents cited herein (which include all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number Sequence Submissions, etc.), whether above or below, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to predate such disclosure by virtue of the foregoing invention.
[0035] In what follows, elements of the present invention will be described. These elements are listed with the specific embodiments, however, it is to be understood that they may be combined in any way and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be interpreted as limiting the present invention to the explicitly described embodiments only. This description is to be understood to support and encompass those embodiments that combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all elements described in this application are to be considered disclosed by the description of the present application unless the context indicates otherwise.
[0036] The term "comprise" or variations such as "comprises" or "comprises" according to the present invention means the inclusion of an established integer or group of integers but not the exclusion of any other integer or group of whole numbers. The term "consisting essentially of" according to the present invention means the inclusion of an established integer or group of integers, while excluding modifications or other integers that would materially affect or alter the established integer. The term "consisting of" or variations such as "consisting of" according to the present invention means the inclusion of an established integer or group of integers and the exclusion of any other integer or group of integers.
[0037] In the context of the present invention, the term "oligopeptide" refers to a short peptide-linked chain of amino acids, for example one that is typically less than about 50 amino acids in length and more typically less than about 50 amino acids. 30 amino acids long.
[0038] The terms "polypeptide" and "protein" are used interchangeably in the context of the present invention and refer to a long peptide-linked chain of amino acids, for example one that is typically 50 amino acids in length or longer than 50 amino acids.
[0039] The term "polypeptide fragment" as used in the context of the present invention refers to a polypeptide which has a deletion, for example an amino-terminal deletion, and/or a carboxy-terminal deletion, and/or a internally compared to a full-size polypeptide.
[0040] In the context of the present invention, the term "fusion protein" refers to a polypeptide comprising a polypeptide or polypeptide fragment linked to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins.
[0041] The terms "antibody", "immunoglobulin", "Ig" and "Ig molecule" are used interchangeably in the context of the present invention. The CH2 domain of each heavy chain contains a single site for N-linked glycosylation at an asparagine residue that attaches an N-glycan to the antibody molecule, usually at residue Asn-297 (Kabat et al., Sequence of proteins of immunological interest, Fifth Ed., US Department of Health and Human Services, NIH Publication NO 91-3242). Included within the scope of the term are the classes of Igs, namely IgG, IgA, IgE, IgM, and IgD. Also included within the scope of the terms are the subtypes of IgGs, namely IgG1, IgG2, IgG3 and IgG4. The terms are used in their broadest sense and include monoclonal antibodies (which include full-length monoclonal antibodies), polyclonal antibodies, single chain antibodies, and multispecific antibodies (e.g. bispecific antibodies).
[0042] The term "antibody fragment" as used in the context of the present invention refers to a fragment of an antibody that contains at least that portion of the CH2 domain of the immunoglobulin heavy chain constant region that comprises a linked glycosylation site. in the N of the CH2 domain and is capable of specific binding to an antigen, i.e. chains of at least one VL and/or VH chain or binding part thereof.
[0043] The terms "Fc domain" and "Fc region" refer to a C-terminal portion of an antibody heavy chain that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system.
[0044] In the context of the present invention, the term "glycoprotein" refers to proteins that contain oligosaccharide chains (glycans) covalently linked to their polypeptide side chains. The carbohydrate is bound to the protein in a co-translational or post-translational modification. This process is known as glycosylation such as N-glycosylation or O-glycosylation.
[0045] "N-glycosylation" means the addition of sugar chains to the amide nitrogen in the side chain of asparagine. "O-glycosylation" means the addition of sugar chains on the hydroxyl oxygen to the side chain of hydroxylysine, hydroxyproline, serine, or threonine.
[0046] The term "glycolipid" as used in the context of the present invention refers to carbohydrate-linked lipids. They occur where a carbohydrate chain is associated with phospholipids on the exoplasmic surface of the cell membrane. Carbohydrates are found on the outer surface of all eukaryotic cell membranes. The carbohydrate structure of the glycolipid is controlled by glycosyltransferases that add the lipids and glycosylhydrolases that modify the glycan after the addition. Glycolipids also occur on the surface of enveloped viruses including those used as live attenuated vaccines.
[0047] The terms "glycan" or "glycoportion" are used interchangeably in the context of the present invention to refer to a polysaccharide or oligosaccharide. The term "oligosaccharide" means a saccharide polymer containing a small number (typically three to ten) of component sugars, also known as simple sugars or monosaccharides. The term "polysaccharide" means a polymeric carbohydrate structure, formed of repeating units (mono- or disaccharides, typically > 10) joined together by glycosidic bonds. Glycans can be found bound to proteins as in glycoproteins or bound to lipids as in glycolipids. The terms encompass N-glycans, such as high-mannose-type N-glycans, complex-type N-glycans, or hybrid-type N-glycans, O-glycans, or
[0048] In the context of the present invention, the following monosaccharides are abbreviated as follows: Glucose = Glc, Galactose = Gal, Mannose = Man, Fucose = Fuc or F, N-acetylgalactosamine = GalNAc, or N-acetylglucosamine = GlcNAc.
[0049] An "N-glycan" means an N-linked polysaccharide or oligosaccharide. An N-linked oligosaccharide is for example one that is or has been linked by an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in a protein. The predominant sugars found in N-glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and sialic acid (eg, N-acetylneuraminic acid (NANA)). Processing of sugar groups occurs co-translationally in the ER lumen and continues in the Golgi apparatus for N-linked glycoproteins. N-glycans have a pentasaccharide core of Man3GlcNAc2 core ("Man" refers to mannose; "Glc" refers to glucose; and "NAc" refers to N-acetyl; GlcNAc refers to N- acetylglucosamine). N-glycans differ with respect to the number of branches (antennas) that comprise peripheral sugars (e.g. GlcNAc, galactose, fucose and sialic acid) that are added to the Man3GIcNAc2 core structure which is also referred to as the "trimanose core". , the “pentasaccharide core” or the “paucimanose core”. N-glycans are classified according to their branched constituents (eg high mannose, complex or hybrid).
[0050] A "high mannose-type N-glycan" means an N-linked polysaccharide or oligosaccharide that has five mannose residues (Man5), or more mannose residues (e.g. Man6, Man7, or Man8).
[0051] A "Complex-type N-glycan" means an N-linked polysaccharide or oligosaccharide that typically has at least one GlcNAc linked to the 1,3 mannose branch and at least one GlcNAc linked to the 1,6 mannose branch of a nucleus. of "trimanosis". N-glycans can also have galactose ("Gal") or N-acetyl-galactosamine ("GalNAc") residues that are optionally modified with sialic acid or derivatives (e.g., "NANA" or "NeuAc", where "Neu ” refers to neuraminic acid and “Ac” refers to acetyl). Complex N-glycans can also have substitutions that comprise "splitting into two" GlcNAc and core fucose ("Fuc"). Complex-type N-glycans in the context of the present invention may contain zero (G0), one (G1), or two (G2) galactoses as well as a fucose linked to the first GlcNAc at the reducing end (indicated as G0F, G1F, G2F, respectively).
[0052] A "hybrid-type N-glycan" means an N-linked polysaccharide or oligosaccharide that has at least one GlcNAc at the terminus of the 1,3 mannose branch of the trimannose core and zero or more mannoses in the 1,6 mannose branch of the trimanose nucleus.
[0053] Abbreviations used in the context of the present invention to describe the glycostructures are defined as follows: core = Man3 GlcNAc2 G0 = GlcNAc2 Man3 GlcNAc2 G0-GlcNAc = G0 structure that misses a GlcNAc (i.e., GlcNAc Man3 GlcNAc2) G1 = structure G0 containing an additional Galactose residue (i.e. Gal GlcNAc2 Man3 GlcNAc2) G2 = G0 structure containing two additional Galactose residues (i.e. Gal2 GlcNAc2 Man3 GlcNAc2) G0F = G0 structure containing an additional fucose residue which is connected to the first pentasaccharide core GlcNAc residue (i.e. GlcNAc2 Man3 GlcNAc2 Fuc) G0F-GlcNAc = G0-GlcNAc backbone containing an additional fucose residue that is attached to the first GlcNAc residue of the pentasaccharide core (i.e. GlcNAc Man3 GlcNAc2 Fuc ) G1F = G1 structure containing an additional fucose residue that is attached to the first GlcNAc residue of the pentasaccharide core (i.e. Gal GlcNAc2 Man3 GlcNAc2 Fuc) G2F = G2 structure containing a res additional fucose residue that is attached to the first GlcNAc residue of the pentasaccharide core (i.e. Gal2 GlcNAc2 Man3 GlcNAc2 Fuc) Man4 = core structure containing an additional Mannose residue (i.e. Man3 GlcNAc2) Man5 = core structure containing two additional Mannose residues (i.e. Man2 Man3 GlcNAc2) Man6 = core structure containing three additional Mannose residues (i.e. Man3 Man3 GlcNAc2) Man7 = core structure containing four additional Mannose residues (i.e. Man4 Man3 GlcNAc2) Man8 = (core structure containing five additional Mannose residues (i.e. Man5 Man3 GlcNAc2)
[0054] An "O-glycan" means an O-linked polysaccharide or oligosaccharide. O-linked glycans are usually linked to the peptide chain through serine or threonine residues. O-linked glycosylation is a true post-translational event that occurs in the Golgi complex and does not require a consensus sequence and no oligosaccharide precursors are required for protein transfer. The most common type of O-linked glycans contain an initial GalNAc residue (or Tn epitope), these are commonly referred to as mucin-type glycans. Other O-linked glycans include glucosamine, xylose, galactose, fucose, or mannose as the initial sugar attached to Ser/Thr residues. O-linked glycoproteins are usually large proteins (> 200 kDa) that are usually biantennary with comparatively fewer branches than N-glycans.
[0055] The term "a molecule which naturally comprises fucose in its glycoportions" as used in the context of the present invention refers to any compound which upon production in a eukaryotic cell, preferably a vertebrate cell, is capable of adding fucose to the glycoportions, i.e. is with an unaltered ability to add fucose to the glycomoieties, comprises glycomoieties comprising at least one fucose residue. Such molecules comprise at least one or more sequence motifs recognized by a glycan transferring enzyme, for example comprising an Asp, Ser or Thr residue, preferably an Asn-X-Ser/Thr tripeptide sequence, wherein X is any amino acid except Pro. Preferred examples of eukaryotic cells (e.g. vertebrate cells) that produce molecules comprising fucose-containing glycoportions are CHO, AGE1.HN, AGE1.CR, AGE1.CR.PIX, or AGE1.CS. Preferably Such compounds are fusion proteins or lipids. Preferably the proteins are of eukaryotic, preferably vertebrate, more preferably mammalian origin or derivatives thereof.
[0056] The term "a molecule which lacks fucose in its glycoportions" in the context of the present invention means that in a molecule which naturally comprises fucose in its glycoportions, no detectable amount of fucose is present. Expressed in terms of purity, "a molecule lacking fucose in its glycoportions" means that a molecule which naturally comprises fucose in its glycoportions is 100% free of the sugar residue of fucose in its glycoportions.
[0057] The term "a molecule with a reduced amount of fucose in its glycoportions" refers to a molecule, in which the amount of fucose in the glycoportions is reduced by the number (n) when expressed in a cell capable of adding fucose to the glycoportions, i.e. having an unaltered ability to add fucose to the glycoportions. Thus, the reduced state is n-x, where n has the meaning given above and x is an integer from 1 to (n-1). Preferably, if n is 2 in the wild state it is reduced to 1. Similarly, if n is 3 in the wild state, it is preferably reduced to 2, or 1; if n is 4 in the wild state, it is preferably reduced to 3, 2, or 1; if n is 5 in the wild state, it is preferably reduced to 4, 3, 2, or 1; or if n is 6 in the wild state, it is preferably reduced to 5, 4, 3, 2, or 1.
[0058] The term "a composition of molecules having a reduced amount of fucose in their glycoportions" is used in the context of the present invention to indicate that molecules of a composition which naturally comprise fucose in their glycoportions have a reduced number of fucose in their glycoportions. Such a comparison is preferably carried out using molecules produced by the cell lines indicated above. Expressed in terms of reduction this means that from a given number of molecules of a composition, preferably from 1, 10, 100 or 1000 pmol of molecules of a composition, the number of fucose residues is reduced by between 10% to 95%, preferably about 15%, about 20%, about 25%, about 30%, about 35%, 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, or about 94%. The overall reduction may be due to an increase in molecules in the composition lacking fucose in their glycoportions and/or having a reduced amount of fucose in their glycoportions.
[0059] The term "a composition of molecules that is substantially free of fucose in its glycoportions" is used in the context of the present invention to indicate that molecules of a composition, which naturally comprise fucose in its glycoportions, are essentially devoid of the sugar residue. fucose in their glycoportions. Expressed in terms of purity, the term "a composition of molecules which is substantially free of fucose in its glycoportions" means that of a given number of molecules of a composition, preferably of 1, 10, 100 or 1000 pmol of molecules of a composition at least about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or at least about 99.9% of said molecules are free of residue of sugar fucose in their glycoportions.
[0060] The skilled person can readily determine experimentally the reduced amount of fucose in the glycoportions of a particular molecule, for example an antibody molecule, (i) by culturing the cells of the present invention under conditions in which the molecule of interest is produced , (ii) isolating said molecule from said cells and (iii) analyzing the sugar chain structure of said molecule with respect to the fucose residues attached to its glycoportions and calculating the average value of the fucose residues present in the sugar chain structure of said molecule, and (iv) comparing the result with the result of the same molecule, for example an antibody molecule, produced in cells, wherein the molecule is produced with an unreduced glycosylation pattern of fucose. Preferably, the cells used in the two experiments are identical but for the difference that one cell comprises at least one enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate, wherein the enzyme does not catalyze the reaction that converts GDP-6-deoxy-D-junk-4-hexulose to GDP-L-fucose. Preferably both cells are grown under identical culture conditions to exclude variations in fucosylation that might be due to differences in culture conditions.
[0061] The sugar chain structure in a molecule, for example antibody molecule, can simply be analyzed by the two-dimensional sugar chain mapping method (Anal. Biochem., 171, 73 (1988), Biochemical Experimentation Methods 23 - Methods for Studying Glycoprotein Sugar Chains (Japan Scientific Societies Press) edited by Reiko Takahashi (1989)). The structure deduced by the two-dimensional sugar chain mapping method can be determined by performing mass spectrometry such as MALDI (Matrix Assisted Laser Desorption/Ionization)-TOF-MS of each sugar chain.
[0062] Fucosylation of molecules, e.g. proteins or lipids, comprising glycoportions in eukaryotic cells (e.g. vertebrate cells) requires a nucleotide sugar, GDP-L-fucose, as a donor and also the presence of particular fucosyltransferases, that transfer the fucosyl residue from the donor molecule to the acceptor. In eukaryotic cells (eg vertebrate cells) GDP-L-fucose can be synthesized via two different pathways, the more prominent de novo fucose pathway or the minor recovery pathway.
[0063] The inventors of the present invention have discovered that the presence of an enzyme (deflector enzyme) in a eukaryotic cell (e.g. a vertebrate cell) that effectively utilizes GDP-6-deoxy-D-junk-4-hexulose as a substrate, but which does not catalyze the reaction that converts GDP-6-deoxy-D-junk-4-hexulose to GDP-L-fucose, leads to the production of a molecule, e.g. protein or lipid, that lacks fucose or has a reduced amount of fucose in their glycoportions. The term “GDP-6-deoxy-D-junk-4-hexulose” is synonymous with the term “GDP-4-keto-6-deoxy-D-mannose”. Both terms are used interchangeably herein.
[0064] Thus, in a first aspect, the present invention provides a vertebrate cell (modified) to produce a molecule, which naturally comprises fucose in its glycoportions, which lacks fucose or with a reduced amount of fucose in its glycoportions which comprises at least one enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate, wherein the enzyme does not catalyze the reaction that converts GDP-6-deoxy-D-junk-4-hexulose to GDP-L -fucose.
[0065] The enzyme present in the vertebrate cell of the first aspect of the invention may be any enzyme which uses GDP-6-deoxy-D-junk-4-hexulose as a substrate under the condition that said enzyme does not catalyze the reaction which converts GDP-6-deoxy-D-junk-4-hexulose to GDP-L-fucose. Instead said enzyme converts GDP-6-deoxy-D-junk-4-hexulose into a product that can no longer be used for the synthesis of GDP-L-fucose in a vertebrate cell.
[0066] The enzyme which is comprised in the vertebrate cell of the first aspect of the present invention is an enzyme which is normally not present in the vertebrate cell, i.e. a heterologous or artificial enzyme, for example an enzyme from one organism from another kingdom, such as prokaryotes, preferably bacteria. Alternatively said enzyme may also be an enzyme which is normally present in a vertebrate cell, but which does not convert the substrate GDP-6-deoxy-D-junk-4-hexulose into GDP-L-fucose but instead into a product different, for example due to the presence of mutations.
[0067] The enzyme, which is present in the vertebrate cell of the first aspect of the present invention, has been introduced into the vertebrate cell, for example, via protein microinjection, protein electroporation or protein lipofection. It is also possible to introduce the nucleic acid sequence encoding the enzyme, preferably integrated into an expression vector, into the vertebrate cell, for example via DNA microinjection, DNA electroporation or DNA lipofection, which is subsequently transcribed and translated. in the respective protein in the vertebrate cell. The person skilled in the art is well versed in molecular biology techniques, such as microinjection, electroporation, or lipofection, to introduce proteins or nucleic acid sequences that encode proteins into a vertebrate cell and knows how to perform these techniques.
[0068] It is preferred that two or more enzymes, i.e. 2, 3, 4, 5, 6 or 7, which use GDP-6-deoxy-D-junk-4-hexulose as a substrate and which do not catalyze the conversion of GDP-6-deoxy-D-junk-4-hexulose in GDP-L-fucose are present in a vertebrate cell to effectively block the de novo pathway of fucose in said cell.
[0069] Preferably, the enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate is selected from the group consisting of GDP-6-deoxy-D-junk-4-hexulose reductase (synonymous with GDP -4-keto-6-deoxy-D-mannose reductase, abbreviated RMD), GDP-perosamine synthetase (Per), GDP-6-deoxy-D-thalose synthetase (GTS), GDP-Fucose synthetase Cys109Ser-(GFS) mutant -Cys109Ser), GDP-4-keto-6-deoxymannose-3-dehydratase (ColD), preferably GDP-4-keto-6-deoxymannose-3-dehydratase (ColD) in combination with GDP-L-cholitose synthase (ColC) , and variants thereof, preferably the enzyme is from bacteria or derived from such a bacterial enzyme. More preferably, the enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate is a GDP-6-deoxy-D-junk-4-hexulose reductase (RMD), GDP-Fucose synthetase Cys109Ser mutant. -(GFS-Cys109Ser), and/or a GDP-perosamine synthetase (Per).
[0070] GDP-6-deoxy-D-junk-4-hexulose reductase (RMD) reduces the substrate GDP-6-deoxy-D-junk-4-hexulose to GDP-D-rhamnose. GDP-D-Rhamnose is a nucleotide sugar donor for D-rhamnosylation in bacteria and does not occur in vertebrates. Vertebrate cells also lack the specific rhamnosyltransferases so GDP-D-Rhamnose may not be incorporated into the nascent glycoprotein or glycolipid glycostructures within vertebrate cells.
[0071] The enzyme GDP-6-deoxy-D-thalose synthetase (GTS) reduces the substrate GDP-6-deoxy-D-junk-4-hexulose to GDP-deoxy-D-thalose. GDP-deoxy-D-thalose is a nucleotide donor sugar for 6-deoxy-D-thalosylation in bacteria and does not occur in vertebrates. Vertebrate cells also lack the specific deoxytalosyltransferases so GDP-deoxy-D-thalose may not be incorporated into nascent glycostructures within vertebrate cells.
[0072] In addition, the GDP-perosamine synthetase (Per) enzyme reduces and transamines the substrate GDP-6-deoxy-D-junk-4-hexulose to GDP-D-perosamine. GDP-D-perosamine is a nucleotide sugar donor for perosaminylation in bacteria, for example E. coli. GDP-D-perosamine is not normally present in vertebrate cells. Vertebrate cells also lack specific perosaminyltransferases so GDP-D-perosamine may not be bound to nascent glycostructures within vertebrate cells.
[0073] Therefore, the heterologous enzymes GTS and/or Per (i) deplete the substrate GDP-6-deoxy-D-junk-4-hexulose in the vertebrate cell, and (ii) lead to the synthesis of artificial products (i.e. GDP-deoxy-D-thalose in the case of GTS and GDP-D-perosamine in the case of Per) which can no longer be used for the synthesis of GDP-L-fucose. Consequently, molecules, which normally comprise fucose in their glycoportions, produced in the vertebrate cell comprising GTS and/or Per, lack fucose or with a reduced amount of fucose in their glycoportions.
[0074] The GDP-4-keto-6-deoxymannose-3-dehydratase (ColD) enzyme takes the substrate GDP-6-deoxy-D-junk-4-hexulose and converts it to GDP-4-keto-3,6 -dideoxy-D-mannose. Since the GDP-4-keto-3,6-dideoxy-D-mannose intermediate may be unstable in vertebrate cells, ColD is preferably used in combination with the GDP-L-colitol synthase (ColC) enzyme. The ColC enzyme belongs to the GDP-4-dehydro-6-deoxy-D-mannose epimerases/reductases class. The ColC enzyme further converts the intermediate GDP-4-keto-3,6-dideoxy-D-mannose into the unstable final product GDP-L-collitose. Both products cannot be incorporated into the nascent glycostructures within vertebrate cells as said cells lack the respective glycosyltransferase to transfer GDP-4-keto-3,6-dideoxy-D-mannose and/or GDP-L-colitose to the glycoportions of molecules present in said cells. Thus, it is preferred that ColD is present in the vertebrate cell in combination with ColC.
[0075] The GDP-Fucose synthetase (GFS) enzyme (also known as GDP-4-keto-6-deoxy-D-mannose epimerase/reductase, GMER) converts GDP-4-keto-6-deoxy-D-mannose into GDP-L-fucose in vertebrate cells. The GFS reaction involves epimerizations at both C-3” and C-5” followed by a NADPH-dependent reduction of the carbonyl at C-4. An active site mutant, preferably GFS-Cys109Ser, is used in the present invention, which converts GDP-4-keto-6-deoxy-D-mannose into a product other than GDP-L-fucose, namely GDP-6-deoxy -D-altrose (see Lau STB, Tanner, ME 2008. Mechanism and active site residues of GDP-Fucose Synthase, Journal of the American Chemical Society, Vol. 130, No. 51, pp. 17593-17602).
[0076] Preferably, two or more enzymes, i.e. 2, 3, 4, 5, 6, or 7, selected from the group consisting of GDP-6-deoxy-D-junk-4-hexulose reductase (RMD), GDP -perosamine synthetase (Per), GDP-6-deoxy-D-thalose synthetase (GTS), GDP-Fucose synthetase mutant Cys109Ser-(GFS-Cys109Ser), GDP-4-keto-6-deoxymannose-3-dehydratase (ColD ), preferably GDP-4-keto-6-deoxymannose-3-dehydratase (ColD) in combination with GDP-L-colitosis synthase (ColC), and variants thereof are present in the vertebrate cell.
[0077] An enzyme variant of RMD, Per, GTS, GFS-Cys109Ser, ColD, or ColC that is preferred in the present invention differs from the enzyme RMD, Per, GTS, GFS-Cys109Ser, ColD, or ColC from which the it is derived up to 150 (that is, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150) amino acid changes in the amino acid sequence (i.e., changes, insertions, deletions, N-terminal truncations and/or C-terminal truncations). Amino acid changes can be conservative or non-conservative. An enzyme variant RMD, Per, GTS, GFS-Cys109Ser, ColD, or ColC that is preferred in the present invention may alternatively or additionally be characterized by some degree of sequence identity to the enzymes of RMD, Per, GTS, GFS -Cys109Ser, ColD, or ColC from which it is derived. Thus, the enzyme variants of RMD, Per, GTS, GFS-Cys109Ser, ColD, or ColC, which are preferred in the present invention, have a sequence identity of at least 80%, at least 81%, at least 82%, at least at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the respective enzymes of RMD, Per, GTS, GFS-Cys109Ser, ColD , or reference ColC. Preferably, the sequence identity relates to a continuous stretch of 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 or more amino acids, preferably with respect to the entire length of the respective enzyme from RMD, Per, GTS, GFS-Cys109Ser, ColD, or ColC reference. It is particularly preferred that the sequence identity is at least 80% full-length, at least 85% full-length, at least 90% full-length, or at least 95% over the entire length, is at least 98% over the entire length, or is at least 99.5% over the entire length of the respective RMD, Per, GTS, GFS-Cys109Ser, ColD, or ColC enzyme of reference. It is also particularly preferred that the sequence identity is at least 80% with respect to at least 200 or 250 amino acids, or at least 85% with respect to at least 200 or 250 amino acids, or at least 90% with respect to at least 200 or 250 amino acids, or at least 95% of at least 200 or 250 amino acids, or at least 98% of at least 200 or 250 amino acids, or at least 99.5% of at least 200 or 250 amino acids from the respective RMD, Per, GTS, GFS-Cys109Ser, ColD, or ColC enzyme of reference.
[0078] A fragment (or deletion variant) of the enzyme of RMD, Per, GTS, GFS-Cys109Ser, ColD, or ColC preferably has a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 amino acids at its N-terminus and/or at its C-terminus and/or internally.
[0079] Additionally, an enzyme of RMD, Per, GTS, GFS-Cys109Ser, ColD, or ColC having the above indicated degree of relatedness to the reference enzyme is only considered as a variant if it exhibits the biological activity relevant to the reference enzyme. a degree of at least 30 % of the activity of the respective reference enzyme. The relevant "biological activity" in the context of the present invention is "enzyme activity", i.e. the activity of the enzyme variant of RMD, Per, GTS, GFS-Cys109Ser, ColD, or ColC to utilize the substrate GDP-6- deoxy-D-junk-4-hexulose and convert it to GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6-deoxy-D-altrose, GDP-4-keto- 3,6-dideoxy-D-mannose or GDP-L-colitosis, respectively. The skilled person can easily assess whether an enzyme variant of RMD, Per, GTS, GFS-Cys109Ser, ColD, or ColC has an enzyme activity of at least 30% of the enzyme activity of the respective enzyme RMD, Per, GTS, GFS -Cys109Ser, ColD, or ColC reference. Suitable assays, for example enzyme activity assays, for determining the "enzyme activity" of the enzyme variant compared to the enzyme activity of the respective reference enzyme are known to the person skilled in the art.
[0080] Preferably, the GDP-6-deoxy-D-junk-4-hexulose reductase (RMD) enzyme is from Pseudomonas aeruginosa (SEQ ID NO: 1). The GDP-6-deoxy-D-thalose synthetase (GTS) enzyme is preferably from Actinobacillus actinomycetemcomitans (SEQ ID NO: 2). It is preferred that the GDP-perosamine synthetase (Per) enzyme is from Vibrio cholerae (SEQ ID NO: 3). Preferably, the GDP-4-keto-6-deoxymannose-3-dehydratase (ColD) is from E. coli (SEQ ID NO: 4). The use of E. coli GDP-L-colitosis synthase (ColC) is also preferred (SEQ ID NO: 7). The wild type GDP-Fucose synthetase (GFS) is from Cricetulus griseus (Chinese hamster) (SEQ ID NO: 5). The Cricetulus griseus (Chinese hamster) GDP-Fucose synthetase mutant Cys109Ser-(GFS-Cys109Ser) has the amino acid sequence of SEQ ID NO: 6.
[0081] As mentioned above, the invention encompasses variants of the enzymes that use GDP-6-deoxy-D-junk-4-hexulose as a substrate. Thus, the present invention also encompasses variants of the sequence identifier numbers mentioned above, i.e. variants of SEQ ID NO: 1, variants of SEQ ID NO: 2, variants of SEQ ID NO: 3, variants of SEQ ID NO: 3, SEQ ID NO: 4 variants, SEQ ID NO: 5 variants, SEQ ID NO: 6 variants, and SEQ ID NO: 7 variants. As for the structural and/or functional definition of said variants, the paragraphs previously mentioned.
[0082] Preferably, the nucleic acid sequences of RMD, Per, GTS, ColD, ColC, or GFS-Cys109Ser are codon optimized. The term "codon-optimized" as used in the context of the present invention means, for example, the removal of internal Tata boxes, chi sites, ribosome entry sites, RNA instability motifs, repeat sequences, intense RNA secondary structures and cryptic junction sites as well as the use of higher-use codons in eukaryotic cells (e.g. vertebrate) or highly expressed genes in eukaryotic cells (e.g. vertebrate).
[0083] It is more preferred that the fucose recovery pathway is additionally blocked in the vertebrate cell. Thus, it is preferred to use culture medium free of fucose and fucosylated glycoproteins when culturing the cells of the present invention.
[0084] Vertebrate cells in addition to or alternatively to the enzyme comprise GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6-deoxy-D-altrose, GDP-4-keto- 3,6-dideoxy-D-mannose, and/or GDP-L-colitose to inhibit or prevent the synthesis of GDP-L-fucose as the inventors of the present invention unexpectedly reported that supplementation, particularly cytosolic supplementation, for example by intracytoplasmic injection of the artificial sugars GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6-deoxy-D-altrose, GDP-4-keto-3,6-dideoxy-D -mannose, and/or GDP-L-colitosis positively contributes to the inhibition of fucose transfer in vertebrate cells. Supplementation of the artificial sugar(s) GDP-6-deoxy-D-altrose, GDP-D-rhamnose, and/or GDP-D-perosamine is (are) particularly preferred.
[0085] It is preferred that the enzyme which uses GDP-6-deoxy-D-junk-4-hexulose as a substrate and which does not catalyze the reaction that converts GDP-6-deoxy-D-junk-4-hexulose to GDP- L-fucose is expressed from a nucleic acid sequence transiently present or stably maintained in the vertebrate cell episomally or chromosomally.
[0086] The nucleic acid sequence encoding the enzyme, preferably GDP-6-deoxy-D-junk-4-hexulose reductase (RMD), GDP-perosamine synthetase (Per), GDP-6-deoxy-D-thalose synthetase (GTS), GDP-Fucose synthetase mutant Cys109Ser-(GFS-Cys109Ser), GDP-4-keto-6-deoxymannose-3-dehydratase (ColD), or GDP-L-colitose synthase (ColC) is integrated into a expression vector, which is used to transform the cell.
[0087] Suitable expression vectors comprise plasmid cosmids, bacterial artificial chromosomes (BAC) and viral vectors. Preferably, non-viral expression vectors are used.
[0088] The expression of the nucleic acid encoding the enzyme is controlled by the expression control sequences.
[0089] The terms "expression control sequences" refer to nucleotide sequences that are affected by expression in eukaryotic cells (e.g. vertebrate cells) of coding sequences to which they are operably linked. Expression control sequences are sequences that control transcription, eg promoters, TATA box, enhancers; post-transcriptional events, for example polyadenylation, and translation of nucleic acid sequences.
[0090] Preferred promoters are constitutive promoters that include the hCMV cytomegalovirus immediate early gene promoter, the SV40 early or late promoters, or regulated promoters that include the CUP-1 promoter, the tet repressor as used, for example, in tet-on or tet-off, the promoter for 3-phosphoglycerate kinase (PGK), the acid phosphatase promoters, and the promoters of yeast α- or a-splicing factors, e.g. the immediate early gene promoter of constitutive CMV, the early or late SV 40 promoter, the polyhedrin promoter, retroviral LTRs, PGK promoter, elongation factor 1-α (EF1-α.), EF2 and phosphoenolpyruvate carboxy kinase (PEPCK). Particularly preferred promoters are promoters that only support intermediate or weak enzyme expression to avoid potential toxicity problems. The expression strength of a given promoter can be normalized by comparing the expression with the expression strength of the strong constitutive promoter that directs the expression of endogenous GAPDH. A promoter that directs the expression of the enzyme at a concentration of 10% to 1% of GAPDH is considered an intermediate promoter that directs expression, and a promoter that directs the expression of the enzyme at a concentration of less than 1% is considered a promoter. weak. Expression strength can be assessed by methods known in the art which include, for example, real-time PCR.
[0091] Marker labels may also be used in embodiments of the present invention. Preferably, the marker tag nucleic acid sequence is operably linked to the nucleic acid sequence encoding the protein (e.g. enzyme, antibody) to be labeled. Preferably the marker tag is a fluorescent protein selected from the group consisting of GFP and variants thereof; which includes, but is limited to, GFP. As used herein "operably linked" means that one nucleic acid is linked to a second nucleic acid in such a way that the expression on the array of a corresponding fusion protein can be affected by preventing array changes or stop codons. These terms also mean the binding of expression control sequences to a coding nucleic acid sequence of interest (e.g. enzyme, antibody) to effectively control expression of said sequence. These terms also refer to the binding of nucleic acid sequences encoding an affinity tag or marker tag to a coding nucleic acid sequence of interest (e.g., enzyme, antibody).
[0092] It is preferred that the nucleic acid sequence encoding the enzyme RMD, Per, GTS, GFS-Cys109Ser, ColD or ColC in the expression vector is operably linked to vertebrate-specific expression control sequences that allow for expression of the nucleic acid sequence encoding the enzyme RMD, Per, GTS, GFS-Cys109Ser, ColD or ColC in the vertebrate cell.
[0093] As a result, the enzyme(s) RMD, Per, GTS, GFS-Cys109Ser, and/or ColD, ColD preferably in combination with ColC, are expressed in the vertebrate cell of the present invention in optimal yields for the desired effect. Depending on the nature of the enzyme and the cell used for expression these yields can be high, moderate or low. It is easy for those skilled in the art to choose appropriate vertebrate-specific expression control sequences to achieve high, moderate, or low levels of expression.
[0094] The expression of the enzyme(s) RMD, Per, GTS, GFS-Cys109Ser, and/or ColD, ColD preferably in combination with ColC, in the vertebrate cell has the effect that it effectively blocks the synthesis of GDP-L-fucose and thus leads to the production of molecules, which naturally comprise fucose in their glycoportions, but which due to their expression lack fucose or have a reduced amount of fucose in their glycoportions.
[0095] For long-lasting, high-yield production of one of the recombinant proteins, stable expression is preferred. For example, eukaryotic cells (e.g. vertebrate cells) that stably express nucleic acids encoding the enzyme that use GDP-6-deoxy-D-junk-4-hexulose as a substrate, where the enzyme does not catalyze the reaction that converts GDP-6-deoxy-D-junk-4-hexulose to GDP-L-fucose and the protein, for example an antibody, can be generated. Rather than using expression vectors that contain viral origins of replication, eukaryotic cells (e.g., vertebrate cells) can be transformed with vectors controlled by appropriate expression control sequences (e.g., promoter, enhancer, transcriptional terminator, site of polyadenylation, etc.) and optionally a selectable marker. Following the introduction of foreign DNA, transformed cells can be detected by analyzing nucleic acids from such cells, by detecting the effect of expression (e.g. lack of fucose using lectin binding) or can be selected by application. of selective pressure. Suitable selection systems are well known in the art.
[0096] Preferably, the vertebrate cell further comprises at least one (acceptor) molecule which is capable of being a substrate for a fucosyltransferase. The term "(acceptor) molecule capable of being a substrate for a fucosyltransferase" as used in the context of the present invention refers to any compound of interest, for example a protein, a polypeptide, an oligopeptide, a lipid, a lipid fragment , or a fusion protein, having or comprising glycoportions to which at least one fucose residue is attached, if produced in a cell having unaltered fucosylation activity. Such a compound is a suitable substrate for a fucosyltransferase. A preferred acceptor molecule is therefore a glycoprotein, a glycopolypeptide, a glycooligopeptide, a glycolipid, a glycolipid fragment, or a glycosylated fusion protein. The term "(acceptor) molecule capable of being a substrate for a fucosyltransferase" as used in the context of the present invention also refers to any protein or lipid, provided that it is an expected glycoprotein or glycolipid to which at least one residue of fucose can be linked, i.e. a protein or lipid to which oligosaccharide structures are linked comprising a monosaccharide to which fucose can be linked by fucosyltransferase after its production by the vertebrate cell. Preferably the protein is not of prokaryotic origin. It is particularly preferred that the protein is a mammalian protein or derivative thereof.
[0097] The presence of a molecule capable of being a substrate for a fucosyltransferase, for example a protein or lipid comprising glycoportions to which at least one fucose residue can be attached, in a vertebrate cell of the invention, leads to the production of this molecule that does not comprise fucose in its glycoportions or which has a reduced amount of fucose in its glycoportions, despite the fact that fucose residues can be attached.
[0098] It is preferred that the molecule capable of being a substrate for a fucosyltransferase is a protein, preferably an endogenous or exogenous protein. The term "exogenous protein" means any protein that is coming from outside the respective cell or that is expressed inside the cell from a nucleic acid introduced into the respective cell. The term "endogenous protein" refers to any protein that is encoded by the genome of the cell. Preferably, the protein of interest, namely the expected glycoprotein, is recombinantly expressed in the vertebrate cell. It is preferred that said protein is expressed from a nucleic acid sequence transiently present or stably maintained in the vertebrate cell. Suitable expression vectors and expression control sequences have been described above with respect to the enzyme. These can also be used in the context of expressing the nucleic acid encoding the protein of interest.
[0099] Thus, in a preferred embodiment of the present invention, the vertebrate cell comprises (i) at least one polynucleotide that comprises a nucleic acid sequence encoding the enzyme GDP-6-deoxy-D-junk-4- hexulose reductase (RMD), GDP-perosamine synthetase (Per), GDP-6-deoxy-D-thalose synthetase (GTS), GDP-Fucose synthetase mutant Cys109Ser-(GFS-Cys109Ser), GDP-4-keto-6- deoxymannose-3-dehydratase (ColD), or GDP-L-colitose synthase (ColC), operably linked to vertebrate-specific expression control sequences, which allow expression of the nucleic acid sequence encoding the respective enzyme, and (ii ) at least one polynucleotide comprising a nucleic acid sequence encoding the protein of interest, namely the expected glycoprotein, for example an antibody, such as IgG1, operably linked to vertebrate-specific expression control sequences leading to expression of the nucleic acid sequence that encodes the protein of interest, for example an antibody such as IgG1, in said cell.
[00100] As a result, (i) the enzyme(s) RMD, Per, GTS, GFS-Cys109Ser, and/or ColD, ColD preferably in combination with ColC, and (ii) the protein(s)( s) of interest, namely the expected glycoprotein(s), for example an antibody, such as IgG1, is/are expressed in the vertebrate cell of the present invention.
[00101] Preferably, in the first aspect of the invention the protein is an antibody, an antibody fragment, a fusion protein, a viral protein, a viral protein fragment, an antigen, or a hormone. Most preferably, the antibody or antibody fragment is selected from the group consisting of IgG, preferably IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE. Most preferably, the fusion protein comprises the Fc region of an antibody, for example the Fc region of IgG, such as the Fc region of IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD or IgE. Most preferably, the hormone is follicle stimulating hormone (FSH), gonadotropic hormone, thyroid stimulating hormone, interleukin-2, interleukin-6, interleukin-10, interleukin-11, soluble interleukin-4, erythropoietin, or thrombopoietin . Preferably, the viral protein or viral protein fragment is comprised in the envelope membrane of an enveloped virus. It is particularly preferred that the viral protein is the Respiratory Syncytial Virus G or F protein and that the viral protein fragment is the extracellular fragment of said protein.
[00102] Another aspect of the invention is a virus comprising said viral protein or viral protein fragment.
[00103] It is preferred that the molecule capable of being a substrate for a fucosyltransferase is a lipid. Preferably, the lipid is a glyceroglycolipid, more preferably a galactolipid, a sulpholipid (SQDG), or a glycosphingolipid, most preferably a cerebroside (e.g. a galactocerebroside or a glucocerebroside), a ganglioside, a globoside, a sulphatide or a glycophosphosphingolipid. It is particularly preferred that the lipid is comprised within the envelope membrane of an enveloped virus.
[00104] Glycosphingolipid (GSL) is particularly preferred. Glycosphingolipids contain a hydrophobic N-acylsphingosine ceramide anchor and a hydrophilic tip group composed of saccharides. They are normally found on the outer surface of cell membranes. The composition of the saccharide moiety is cell type specific and depends on the developmental stage of the organism or may change with the oncogenic state of a cell.
[00105] Most preferably, the viral protein and/or lipid are comprised in the envelope of an enveloped virus.
[00106] As already mentioned above, the protein can be a viral protein that is comprised in the envelope membrane of an enveloped virus. The lipid may also be a lipid comprised in the envelope membrane of an enveloped virus.
[00107] Another aspect of the invention is a virus comprising said lipid.
[00108] The virus mentioned above can be introduced into the vertebrate cell via viral infection. The virus can also be introduced into the vertebrate cell by introducing nucleic acids encoding all or part of the virus to be produced. In this case it will be necessary to supply the proteins required for replication, assembly etc., this is usually achieved by using virus-producing cell lines capable of expressing one or more viral proteins. For example, HEK293, Per.C6 and AGE1.HN cells express adenovirus E1A proteins and are thus able to complement DNA lacking the E1 coding regions.
[00109] Preferably, the vertebrate cell is a mammalian, a fish, an amphibian, a reptile or an avian cell.
[00110] It is particularly preferred that i) the mammalian cell is a human, hamster, canine or monkey cell, preferably a Chinese hamster ovary (CHO) cell, an African green monkey kidney cell (VERO-76) (ATCC CRL-1587), a human cervical carcinoma (HeLa) cell (ATCC CCL 2), a Madin Darbin canine renal cell (MDCK) (ATCC CCL 34), a human PER.C6 cell (Crucell commercial cell, Leiden, The Netherlands), or a human AGE1.HN cell (homo sapiens) (commercial cell from ProBioGen, Berlin, Germany); ii) the fish cell is an ovarian cell (OCC) of Ictalurus punctatus (channel catfish) (ATCC CRL-2772), iii) the amphibian cell is a renal cell of Xenopus laevis (ATCC CCL-102); iv) the reptile cell is an Iguana iguana cardiac cell (IgH-2) (ATCC CCL-108); or v) the avian cell is an avian retinal cell AGE1.CR or AGE1.CR.PIX, or an avian somite cell AGE1.CS (all cells derived from the Muscovy duck, Cairina moschata). these cell lines are all commercially available from ProBioGen AG.
[00111] The cell line AGE1.CR.PIX (17a11b) was deposited by ProBioGen AG, Goethestr. 54, 13086 Berlin, Germany with DSMZ- Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany on November 24, 2005 under accession number DSM ACC2749. The AGE1.HN cell line (NC5T11#34) was deposited by ProBioGen AG, Goethestr. 54, 13086 Berlin, Germany with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany on November 4, 2005 under accession number DSM ACC2744. The cell line AGE1.CR.PIX (17a11b) is derived from embryonic retinal cells of the Muscovy duck (Cairina moschata). The AGE1.HN cell line (NC5T11#34) is derived from a periventricular neural region of a human fetus (Homo sapiens). Both cell lines are immortalized by the stable integration and expression of adenovirus E1A and E1B proteins.
[00112] Another preferred vertebrate cell is the avian EB14 cell which is a commercial cell from VIVALIS (Nantes, France). Other preferred vertebrate cells are summarized in Table 1 which follows: TABLE 1

[00113] Vertebrate cells naturally do not comprise a glycosyltransferase for GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6-deoxy-D-altrose, GDP-4-keto-3 ,6-dideoxy-D-mannose, or GDP-L-colitose since said sugars are not normally synthesized or present in vertebrate cells. Thus, even if the artificial sugars GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6-deoxy-D-altrose, GDP-4-keto-3,6-dideoxy-D -mannose, or GDP-L-colitosis are artificially produced in the vertebrate cell according to the first aspect of the present invention, they cannot be incorporated into the nascent protein or lipid glycostructures.
[00114] The inventors of the present invention, however, unexpectedly discovered that the artificial sugars GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6-deoxy-D-altrose, GDP-4 - keto-3,6-dideoxy-D-mannose, or GDP-L-colitose can be integrated into the glycan structures of proteins or lipids under the condition that the respective heterologous glycosyltransferase, and optionally a respective sugar nucleotide carrier from Heterologous GDP-Deoxyhexose is also present in the vertebrate cell.
[00115] Accordingly, in a preferred embodiment, the vertebrate cell according to the first aspect of the present invention further comprises at least one glycosyltransferase for GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D- talose, GDP-6-deoxy-D-altrose, GDP-4-keto-3,6-dideoxy-D-mannose, or GDP-L-colitose, preferably encoded by a nucleic acid comprised in the vertebrate cell and operably linked to the vertebrate-specific expression control sequences allowing expression of said nucleic acid sequence, and optionally at least one sugar nucleotide transporter from GDP-deoxyhexose to GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy -D-thalose, GDP-6-deoxy-D-altrose, GDP-4-keto-3,6-dideoxy-D-mannose, or GDP-L-colitosis, preferably encoded by a nucleic acid comprised in the vertebrate cell and operably linked to vertebrate-specific expression control sequences that allow the expression of said nucleic acid sequence. In a more preferred embodiment, the vertebrate cell according to the first aspect of the present invention further comprises a glycosyltransferase for GDP-D-rhamnose, GDP-D-perosamine, and/or GDP-6-deoxy-D-altrose .
[00116] It is particularly preferred that two or more, for example 2, 3, 4, 5, 6, or 7, of the above mentioned glycosyltransferases, and optionally two or more, for example 2, 3, 4, 5, 6, or 7, of the above mentioned GDP-deoxyhexose sugar nucleotide transporter are present in the vertebrate cell, for example eukaryotic cell.
[00117] Suitable systems for the transient or stable expression of such nucleic acids are known to the skilled person and preferred ones are described above and may similarly be used in the context of expressing glycosyltransferase, and optionally GDP-deoxyhexose sugar nucleotide transporter.
[00118] Preferably, the glycosyltransferase for GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6-deoxy-D-altrose, GDP-4-keto-3,6-dideoxy- D-mannose, or GDP-L-colitosis is recombinantly expressed in the vertebrate cell. Glycosyltransferase for GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6-deoxy-D-altrose, GDP-4-keto-3,6-dideoxy-D-mannose, or GDP-L-colitosis can be expressed from a nucleic acid sequence transiently present or stably maintained in the vertebrate cell. It is also preferred that the aforementioned GDP-deoxyhexose sugar nucleotide transporter is recombinantly expressed in the vertebrate cell. Said GDP-deoxyhexose sugar nucleotide transporter may be expressed from a nucleic acid sequence transiently present or stably maintained in the vertebrate cell.
[00119] Thus, in another preferred embodiment of the present invention, the vertebrate cell comprises (i) at least one polynucleotide comprising a nucleic acid sequence encoding the GDP-6-deoxy-D-junk-4 enzyme - hexulose reductase (RMD), GDP-perosamine synthetase (Per), GDP-6-deoxy-D-thalose synthetase (GTS), GDP-Fucose synthetase mutant Cys109Ser-(GFS-Cys109Ser), GDP-4-keto-6 -deoxymannose-3-dehydratase (ColD), or GDP-L-collitose synthase (ColC) operably linked to vertebrate-specific expression control sequences, (ii) at least one polynucleotide comprising a nucleic acid sequence encoding a protein of interest, namely an expected glycoprotein, for example an antibody, such as IgG1, operably linked to vertebrate-specific expression control sequences, which allow expression of the nucleic acid sequence encoding said protein in said cell, and ( iii) at least one polynucleotide that buys comprises a nucleic acid sequence encoding the glycosyltransferase for GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6-deoxy-D-altrose, GDP-4-keto-3,6 -dideoxy-D-mannose, or GDP-L-colitosis operably linked to vertebrate-specific expression control sequences, which allow expression of the nucleic acid sequence encoding said protein in said cell, and optionally (i) at least a polynucleotide comprising a nucleic acid sequence encoding the sugar nucleotide transporter from GDP-deoxyhexose to GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6-deoxy-D- altrose, GDP-4-keto-3,6-dideoxy-D-mannose, or GDP-L-colitosis operably linked to vertebrate-specific expression control sequences, which allow expression of the nucleic acid sequence encoding said protein in said cell.
[00120] As a result, (i) the enzyme(s), (ii) the protein(s) of interest, (iii) the glycosyltransferase(s), and optionally (iv) the GDP-deoxyhexose sugar nucleotide transporter are overexpressed in the vertebrate cell of the present invention.
[00121] Said protein(s) are further modified by the co-expressed glycosyltransferase(s), which makes use of the final product(s)( is) of the enzyme(s), namely GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6-deoxy-D-altrose, GDP-4-keto-3 ,6-dideoxy-D-mannose, and/or GDP-L-colitose, and it(s) integrate(s) within the glycostructures of the protein(s) lacking fucose or with a reduced amount of fucose in their glycoportions. This leads to the generation of new artificial protein(s) that comprise(s) D-rhamnose, D-perosamine, deoxy-D-thalose, 6-deoxy-D-altrose, 4-keto-3, 6-dideoxy-D-mannose, and/or L-colitose in their glycoportions.
[00122] Preferably, the GDP-deoxyhexose sugar nucleotide transporter mentioned above is the Golgi complex GDP-deoxyhexose sugar nucleotide transporter, i.e. the transporter that allows the transport of the aforementioned nucleotide sugars across the membrane of the Golgi complex of a vertebrate cell, for example eukaryotic cell.
[00123] The (modified) vertebrate cell according to the present invention can be used to produce enveloped viruses that comprise envelope surface glycoproteins or glycolipids having no fucose in their glycoportions or with a reduced amount of fucose in their glycoportions.
[00124] Preferably, the enveloped virus is used wholly or in part as an active component of a viral vaccine. The term "viral vaccine" means a preparation of a weakened or killed virus which upon administration stimulates the production of antibody or cellular immunity against the virus but is incapable of causing severe infections.
[00125] The (modified) vertebrate cell according to the present invention can also be used to produce enveloped viruses that comprise envelope surface glycoproteins or glycolipids that comprise D-rhamnose, D-perosamine, deoxy-D-thalose, 6 -deoxy-D-altrose, 4-keto-3,6-dideoxy-D-mannose, and/or L-colitose in their glycoportions.
[00126] In a second aspect, the present invention relates to a method for producing a molecule, which naturally comprises fucose in its glycoportions, which lacks or has a reduced amount of fucose in its glycoportions, comprising the steps of: i ) providing a vertebrate cell according to the first aspect, ii) isolating the molecule which is capable of being a substrate for a fucosyltransferase, preferably a protein or lipid, from the cell in i).
[00127] Preferably, in step i), the molecule which is capable of being a substrate for a fucosyltransferase, for example a protein, is expressed in the cell in i).
[00128] Said molecules, for example proteins or lipids lacking fucose or with a reduced amount of fucose in their glycoportions, can be easily isolated in step ii) from the vertebrate cell.
[00129] Various isolation procedures are known in the art for molecules included within eukaryotic cells (e.g. vertebrate cells) or secreted from such cells which comprise the modified molecules, e.g. proteins lacking fucose or with a reduced amount of fucose in their glycoportions. Such methods typically involve harvesting the cell, disintegration and fractionation/purification in the case of intracellular molecules and generation of a cell-free culture supernatant followed by purification of the secreted molecules.
[00130] An extraction procedure that is useful in accordance with the invention does not interfere with the modified molecules to be isolated. For example, the extraction is preferably carried out in the absence of detergents and strong reducing agents, or any agent that can induce protein denaturation.
[00131] In a third aspect, the present invention provides a molecule lacking fucose or having a reduced amount of fucose in its glycoportions obtainable by the method of the second aspect. Preferably, this molecule is a lipid or protein.
[00132] It is particularly preferred that the protein is an antibody, a hormone, an antigen or a viral protein as set forth above with respect to the first aspect.
[00133] It is particularly preferred that the lipid is a glyceroglycolipid, more preferably a galactolipid, a sulpholipid (SQDG), or a glycosphingolipid, most preferably a cerebroside (e.g. a galactocerebroside or a glucocerebroside), a ganglioside, a globoside, a sulphatide or a glycophosphosphingolipid. Preferably, the lipid, for example ganglioside, is comprised in the membrane of an enveloped virus.
[00134] Most preferably, the viral protein and/or lipid are comprised within the envelope of an enveloped virus.
[00135] In another aspect, the present invention provides a composition of molecules according to the third aspect.
[00136] In a fourth aspect, the present invention provides a molecule comprising glycoportions containing D-rhamnose, D-perosamine, deoxy-D-thalose, 6-deoxy-D-altrose, 4-keto-3,6-dideoxy- D-mannose, and/or L-collitosis obtainable by the method of the second aspect.
[00137] It is preferred that this molecule comprises no or a reduced amount of L-fucose in its glycoportions. Preferably, this molecule is a lipid or protein. Additionally vertebrate cells are provided, preferably tumor cells that express such modified molecules, preferably lipids and/or proteins. Particularly preferred are tumor cells containing glycans comprising D-rhamnose. D-rhamnose is a polysaccharide building block from Ganoderma lucidum. These polysaccharides are used in traditional Chinese medicine to enhance the activity of immune effector cells: NK cells and lymphokine-activated killer cells are activated and phagocytosis and macrophage cytotoxicity are increased (Zhu et al., 2007, Journal of Ethnopharmacology 111 (Zhu et al., 2007, Journal of Ethnopharmacology 111 ( 2), 219-226). Such cells can be used to enhance the immune response against a given tumor cell, for example as in an active immunization method.
[00138] In another aspect, the present invention provides a composition of molecules according to the fourth aspect.
[00139] In a fifth aspect, the present invention provides a composition comprising glycoproteins comprising i) between 70 and 95%, i.e. 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, and 95%, preferably 80% of G0 -GlcNac, G0, G1, and/or G2 of complex-type N-glycans, and ii) between 5 and 30%, i.e. 5%, 6%, 7 %, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, and 30%, preferably 20% high-mannose-type N-glycans, wherein the complex-type N-glycans of the glycoproteins are fucose-free or substantially free of fucose. Preferably the glycoproteins of the composition are the preferred glycoproteins indicated above, in particular antibodies.
[00140] It is particularly preferred that of the complex-type N-glycans within the composition from 70% to 80%, preferably about 75% are G0 and from 20% to 30%, preferably about 25% are G0-type N-glycans. G0 -GlcNac, G1 and/or G2 complex, and/or that of the high mannose-type N-glycans 45% to 55%, preferably about 50% are man5 glycans and 45 to 55%, preferably about 50% are N -man6, man7 and/or man8 glycans. It is understood that the numbers in each case add up to 100%.
[00141] It is also particularly preferred that of the complex-type N-glycans within the composition from 70% to 90%, preferably about 80% are G0 and from 10% to 30%, preferably about 20% are G0 N-glycans. complex type G0 -GlcNac, G1 and/or G2, and/or that of the N-glycans of the high mannose type from 60% to 85%, preferably about 70% are man5 glycans and from 15 to 40%, preferably about 30 % are man6, man7 and/or man8 N-glycans. It is understood that the numbers in each case add up to 100%.
[00142] The glycoproteins are preferably those indicated as preferred in the context of the first aspect of the invention, in particular antibodies, for example IgG1s, IgG2s, IgG3s or IgG4s, wherein these antibodies preferably have a higher ADCC activity than a composition of antibody produced in an unmodified precursor cell (e.g. eukaryotic cell, such as vertebrate cell).
[00143] ADCC is the domination mechanism by which antibodies directed against tumor (cancer) cells exhibit their effects). ADCC can also be used to kill specific immune cells to disrupt pathogenesis in autoimmune disease.
[00144] Various diseases including viral and bacterial infections can be prevented and treated by suppressing the proliferation of cells infected with a virus or bacteria using the antibody having high ADCC activity according to the present invention.
[00145] In a sixth aspect, the present invention provides a protein, preferably a non-prokaryotic protein, preferably a viral or mammalian protein, or a lipid, preferably a non-prokaryotic lipid, for example a glycosphingolipid, which comprises glycoportions containing D-rhamnose , D-perosamine, deoxy-D-thalose, 6-deoxy-D-altrose, 4-keto-3,6-dideoxy-D-mannose, and/or L-colitosis. Preferably, the viral protein and/or lipid are comprised within the envelope of an enveloped virus. Alternatively, eukaryotic cells, preferably vertebrate, more preferably mammalian, and most preferably human (allogenic or autologous tumor cells) are provided which comprise such proteins and/or lipids.
[00146] Preferably, the protein or lipid comprises D-rhamnose, D-perosamine, deoxy-D-thalose, 6-deoxy-D-altrose, 4-keto-3,6-dideoxy-D-mannose, and/or L -colitosis, more preferably D-rhamnose and/or D-perosamine, in their glycoportions and no or a reduced amount of L-fucose in their glycoportions. It is clear that proteins of the sixth aspect can also have all the properties which are the consequence of using the preferred and particularly preferred aspects of the methods of the present invention.
[00147] It is known that artificial sugars such as D-rhamnose, D-perosamine, deoxy-D-thalose, 6-deoxy-D-altrose, 4-keto-3,6-dideoxy-D-mannose, and/or L-collitosis, confer resistance to cationic peptides, e.g. polymyxin B, defensins, etc., e.g. by neutralizing the charge on the bacterial surface (see Breazeale et al., 2003, The Journal of Biological Chemistry, 278, 24731-24739 ).
[00148] Defensins are small cysteine-rich cationic proteins found, for example, in eukaryotes (eg vertebrates). They are active against bacteria, fungi and many enveloped and non-enveloped viruses. They consist of 18 to 45 amino acids that include 6 (in vertebrates) to 8 conserved cysteine residues. Eukaryotic (e.g. vertebrate) cells of the immune system contain these peptides to aid in the killing of phagocytosed bacteria, for example in neutrophilic granulocytes and almost all epithelial cells.
[00149] Thus, the establishment of enveloped viruses comprising viral proteins containing D-rhamnose, D-perosamine, deoxy-D-thalose, 6-deoxy-D-altrose, 4-keto-3,6-dideoxy-D-mannose , and/or L-collitosis, preferably the cationic amino sugar D-perosamine, in their glycoportions is highly useful, for example, in gene therapy as vectors, as these viruses would be resistant against attacks by defensins and other cationic peptides of the congenital immune defense. The production of viruses or viral glycoproteins containing one or more of the sugar residues D-rhamnose, D-perosamine, deoxy-D-thalose, 6-deoxy-D-altrose, 4-keto-3,6-dideoxy-D-mannose , and/or L-colitosis in their glycoportions would be highly useful for inducing a superior immune response. These sugar residues are naturally not present in mammalian glycoproteins. However, they are frequent in the glycoproteins and lipopolysaccharides of bacteria. Pseudomonas aeruginosa the origin of the RMD sequence is one of the most important bacterial pathogens encountered by immunocompromised hosts and cystic fibrosis (CF) patients.
[00150] Particularly, the establishment of proteins containing the D-perosamine sugar residue or enveloped viruses comprising viral proteins containing the D-perosamine sugar residue is, due to the cationicity of the D-perosamine, preferred. A virus comprising glycoproteins containing the D-perosamine sugar residue in its envelope has a cationic surface charge that can protect said virus against congenital immune defense.
[00151] In a seventh aspect, the present invention provides an expression unit comprising one or more vertebrate expression control sequences operably linked to a (first) polynucleotide comprising a nucleic acid sequence encoding an enzyme that uses GDP -6-deoxy-D-junk-4-hexulose as a substrate, where the enzyme does not catalyze the reaction that converts GDP-6-deoxy-D-junk-4-hexulose to GDP-L-fucose.
[00152] The expression unit may be any expression system known to the person skilled in the art wherein the one or more vertebrate expression control sequences and a (first) polynucleotide comprising a nucleic acid sequence encoding an enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate can be integrated, for example a plasmid, a cosmid, a virus etc.
[00153] Preferably, the one or more vertebrate expression control sequences comprised in the expression unit are operably linked to the (first) polynucleotide that comprises a nucleic acid sequence encoding an enzyme that uses GDP-6-deoxy-D- garbage-4-hexulose as a substrate to allow expression of the nucleic acid sequence encoding an enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate in the vertebrate cell, e.g. CHO or HeLa cell .
[00154] Preferably, the expression vector, for example plasmid vector, allows the transient expression of a nucleic acid sequence encoding an enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate in the cytoplasm of a vertebrate cell or allows the stable expression of a nucleic acid sequence encoding an enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate in the vertebrate cell after the integration of said sequence into the genome of the said cell.
[00155] It is particularly preferred that the enzyme using GDP-6-deoxy-D-junk-4-hexulose as a substrate is selected from the group consisting of GDP-6-deoxy-D-junk-4-hexulose reductase (RMD ), GDP-perosamine synthetase (Per), GDP-6-deoxy-D-thalose synthetase (GTS), GDP-Fucose synthetase mutant Cys109Ser-(GFS-Cys109Ser), GDP-4-keto-6-deoxymannose-3- dehydratase (ColD), preferably GDP-4-keto-6-deoxymannose-3-dehydratase (ColD) in combination with GDP-L-colitosis synthase (ColC), and variants thereof.
[00156] Preferably, the expression unit further comprises a (second) polynucleotide comprising a nucleic acid sequence encoding a glycosyltransferase for GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP- 6-deoxy-D-altrose, GDP-4-keto-3,6-dideoxy-D-mannose, or GDP-L-colitosis, preferably operably linked to one or more vertebrate expression control elements, for example a promoter, and preferably comprised in an expression vector, for example plasmid vector, to allow expression of the nucleic acid sequence encoding said glycosyltransferase in the vertebrate cell, for example the CHO cell, the HeLa cell or a tumor cell, preferably an autologous tumor cell, and optionally a (third) polynucleotide comprising a nucleic acid sequence encoding a sugar nucleotide transporter from GDP-deoxyhexose to GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy- D-thalose, GDP-6- only xi-D-altrose, GDP-4-keto-3,6-dideoxy-D-mannose, or GDP-L-colitosis, preferably operably linked to one or more vertebrate expression control elements, for example a promoter , and preferably comprised in an expression vector, for example plasmid vector, to allow expression of the nucleic acid sequence encoding said GDP-deoxyhexose sugar nucleotide transporter in the vertebrate cell, for example CHO cell, HeLa cell or a tumor cell, preferably an autologous tumor cell.
[00157] Said first, second, and optionally third polynucleotides may be separately integrated into expression vectors, for example transient expression vectors or vectors that integrate into the genome of the cell. Alternatively, said first, second, and optionally third polynucleotides may be comprised in an expression vector. Expression can be directed from one promoter, two promoters, or three promoters. In the first case it is preferred that the proteins encoded by the first, second, and optionally third polynucleotides are transcribed as a single transcript and are separated only by an IRES.
[00158] In a preferred embodiment of the invention, the expression unit comprises one or more vertebrate expression control sequences operably linked to a first polynucleotide comprising a nucleic acid sequence encoding the enzyme RMD, one or more sequences vertebrate expression control sequences operably linked to a second polynucleotide comprising a nucleic acid sequence encoding a glycosyltransferase for GDP-D-rhamnose, and optionally one or more vertebrate expression control sequences operably linked to a third polynucleotide comprising a nucleic acid sequence encoding a sugar nucleotide transporter from GDP-deoxyhexose to GDP-D-rhamnose, preferably comprised of one expression vector, two expression vectors, or three expression vectors, respectively.
[00159] In another preferred embodiment of the invention, the expression unit comprises one or more vertebrate expression control sequences that control expression of the nucleic acid sequence encoding the RMD enzyme comprised within a first polynucleotide, the nucleic acid sequence encoding a glycosyltransferase for GDP-D-rhamnose comprised within a second polynucleotide, and optionally the nucleic acid sequence encoding a GDP-deoxyhexose to GDP-D-rhamnose sugar nucleotide transporter within a third polynucleotide, wherein the first, second, and optionally third polynucleotides are separated from one another by an IRES, preferably comprised in an expression vector.
[00160] The expression unit according to the seventh aspect of the invention can also be used in medicine, in particular in the preparation of a vaccine for the therapy or prophylaxis of diseases that benefit from immune stimulation, in particular tumors.
[00161] Additionally, in the context of the seventh aspect all of the most preferred and particularly preferred embodiments of the first aspect of the invention, in particular relating to polynucleotides comprising various nucleic acids to be expressed, expression control elements, for example that targeting moderate or low expression of the enzyme using GDP-6-deoxy-D-junk-4-hexulose, are equally preferred in the context of the seventh aspect.
[00162] In many cases, considerable time has been spent and enormous efforts have been made by biopharmaceutical companies to generate and develop effective pharmaceutical producer cell lines that express the transgene of interest at desirable levels. In cases where the transgene of interest is a therapeutic antibody that can benefit from enhanced ADCC effector function, it is highly desirable to further engineer the producer cell line in such a way that the high producer cell is unable to bind nuclear fucose to the N -glycoportions that is capable of attaching artificial sugars to the N-glycoportions.
[00163] Preferably, as already mentioned above, the expression unit further comprises a second polynucleotide comprising a nucleic acid sequence encoding a glycosyltransferase for GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose , GDP-6-deoxy-D-altrose, GDP-4-keto-3,6-dideoxy-D-mannose, or GDP-L-colitosis, and optionally a third polynucleotide comprising a nucleic acid sequence encoding a transporter from GDP-deoxyhexose sugar nucleotide to GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6-deoxy-D-altrose, GDP-4-keto-3,6-dideoxy -D-mannose, or GDP-L-colitosis. It is preferred that the second polynucleotide comprising a nucleic acid sequence encoding a glycosyltransferase for GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6-deoxy-D-altrose, GDP- 4-keto-3,6-dideoxy-D-mannose, or GDP-L-colitosis is operably linked to one or more vertebrate expression control sequences to allow expression of the nucleic acid sequence encoding said glycosyltransferase in the cell from vertebrate, for example HeLa or CHO cells. It is also preferred that the third polynucleotide which comprises a nucleic acid sequence encoding a sugar nucleotide transporter from GDP-deoxyhexose to GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6 -deoxy-D-altrose, GDP-4-keto-3,6-dideoxy-D-mannose, or GDP-L-colitosis is operably linked to one or more vertebrate expression control sequences to allow expression of the nucleic acid encoding said GDP-deoxyhexose sugar nucleotide transporter in the vertebrate cell, for example HeLa or CHO cells.
[00164] The ratio of expression of nucleic acid sequences different from each other depends on both the copy number and the site of integration in the genome of the vertebrate cell. By standard screening procedures, it is possible to isolate cell clones that express the individual gene products in the desired ratio.
[00165] Said preferred expression unit can be easily applied to already existing cell lines (e.g. CHO, HeLa) in a way that makes them capable of binding artificial sugars other than fucose (e.g. D-rhamnose, D -perosamine, deoxy-D-thalose, or 6-deoxy-D-altrose) to the nascent glycostructures of glycoproteins or glycolipids. This expression unit can also be easily applied to already genetically engineered cell lines (e.g. CHO IgG1) in a way that makes them capable of binding artificial sugars other than fucose (e.g. D-rhamnose) to the nascent glycoproteins glycostructures, for example in order to produce antibodies having an artificial glycosylation structure, such as IgG1/+D-rhamnose.
[00166] Preferably, the expression unit comprises another polynucleotide (such as a second, third, or fourth polynucleotide) which comprises a nucleic acid sequence encoding a protein of interest, for example IgG1. It is preferred that the other polynucleotide, which comprises a nucleic acid encoding a protein of interest, for example IgG1, is operably linked to expression control sequences to allow expression of the nucleic acid sequence encoding said protein in a cell of vertebrate, for example HeLa or CHO cells.
[00167] In the context of the expression unit of the seventh aspect of the present invention all elements, preferably enzyme-encoding nucleic acids, expression control elements, enzyme-encoding nucleic acid sequence, which use GDP-6-deoxy-D -junk-4-hexulose as a substrate and wherein the enzyme does not catalyze the reaction that converts GDP-6-deoxy-D-junk-4-hexulose to GDP-L-fucose, disclosed in the first and second aspects of the invention are similarly favorites.
[00168] In an eighth aspect, the present invention relates to a (modified) eukaryotic cell to produce a protein, which normally comprises fucose in its glycoportions, which lacks fucose or having a reduced amount of fucose in its glycoportions which comprises: i) a first polynucleotide comprising a nucleic acid sequence encoding an enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate, and ii) a second polynucleotide comprising a nucleic acid sequence encoding a protein, in which the enzyme does not catalyze the reaction that converts GDP-6-deoxy-D-junk-4-hexulose to GDP-L-fucose.
[00169] In the context of the eukaryotic cell all of the preferred and particularly preferred embodiments described above in the context of the vertebrate cell of the first aspect of the invention are similarly preferred.
[00170] Preferably, the eukaryotic cell is a vertebrate cell, for example a mammal, a fish, an amphibian, a reptile or an avian cell, preferably as outlined above with respect to the first aspect of the invention, or an insect cell. Insect cells, for example Sf9 or Hi5 cells, used in conjunction with the baculovirus expression system are preferred to produce glycoproteins because of high yields and speed. However, insect cell glycoproteins may contain core alpha 1,3-fucose. The sugar residue contributes to a greater degree with antibodies against insect-derived glycoproteins, in particular IgE antibodies. They are the basis of allergic reactions to insect glycoproteins.
[00171] Insect cells comprising at least one enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate, wherein the enzyme does not catalyze the reaction that converts GDP-6-deoxy-D- garbage-4-hexulose in GDP-L-fucose will lack fucose or have reduced fucose in their glycans. This will reduce or eliminate allergic reactions to such glycoproteins.
[00172] The eukaryotic (modified) cell mentioned above may also comprise more than one polynucleotide that comprises a nucleic acid sequence encoding an enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate, e.g. example polynucleotides comprising nucleic acid sequences encoding different enzymes that use GDP-6-deoxy-D-junk-4-hexulose as a substrate (see first aspect of the present invention).
[00173] In a ninth aspect, the present invention relates to a method for producing a protein, which normally comprises fucose in its glycoportions, which lacks or has a reduced amount of fucose in its glycoportions, comprising the steps of: i ) providing a eukaryotic cell according to the eighth aspect, ii) expressing the enzyme encoded by the first polynucleotide and the protein encoded by the second polynucleotide in said cell, and iii) isolating the protein from said cell.
[00174] In the context of the method using the eukaryotic cell of the present invention all of the preferred and particularly preferred embodiments described above in the context of the vertebrate cell of the first aspect of the invention and the method of the second aspect of the present invention are similarly preferred in the context of the ninth aspect of the invention.
[00175] In a tenth aspect the present invention relates to a protein lacking fucose or having a reduced amount of fucose in its glycoportions obtainable by the method of the ninth aspect.
[00176] Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope of the invention. While the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention that are obvious to those skilled in the art in the relevant fields are intended to be embraced by the present invention.
[00177] The figures and examples that follow are merely illustrative of the present invention and should not be construed as limiting the scope of the invention as indicated by the appended claims in any way. BRIEF DESCRIPTION OF THE DRAWINGS
[00178] Fig. 1 shows an overview of fucose recovery and de novo pathways in eukaryotic cells (eg vertebrate cells). In the absence of fucose, cells are unable to synthesize GDP-fucose via the recovery pathway (see right panel). The de novo pathway can be blocked by the enzymatic conversion of the GDP-4-keto-6-deoxymannose intermediate to a dead end product that typically does not occur in vertebrate cells (left panel). If the derived enzyme is RMD, for example, then the dead end product is GDP-D-rhamnose. GDP-deoxyhexoses such as GDP-D-rhamnose can exert a feedback inhibition on the GMD enzyme thereby further blocking the de novo fucose pathway as well as alternative GDP-rhamnose synthesis.
[00179] Fig. 2 shows GFP fluorescence from RMD-CHO-IgG cells that stably overexpress the RMD transgene.
[00180] Fig. 3 shows an RT-PCR analysis of clones expressing the RMD Transgene. Lane M = bp-DNA-Label, Lane 1: RMD-CHO-IgG clone 1; Lane 2: RMD-CHO-IgG clone 2; Lane 3: RMD-CHO-IgG clone 3; Lane 4: RMD-CHO-IgG clone 4; Lane 5: RMD-CHO-IgG clone 5; Lane 6: RMD-CHO-IgG clone 6; Lane 7: CHO-IgG precursor clone; Lane 8: negative PCR control. The RMD band is visible in all RMD-transfected clones.
[00181] Fig. 4 shows a synoptic comparison of MALDI MS profiles of permethylated N-glycans released from IgG1 from CHO-IgG (B) and RMD-CHO-IgG (A) cells. Fc antibody oligosaccharides released by PNGase F digestion were analyzed using an UltraFlex III TOF/TOF mass spectrometer equipped with a smartbeam-II® laser. The m/z value peaks correspond to the sodium-associated oligosaccharide ion. All labeled [M + Na]+ molecular ion signals can be designated as annotated. A change of 164 Da from the main m/z peaks in the MALDI spectrum shown in panel A is indicative of fucose loss. The schematic oligosaccharide structure of each peak is illustrated above each annotated peak. N-glycostructures obtained from a therapeutic IgG1 antibody expressed in RMD-CHO-IgG cells that stably overexpress the RMD transgene are completely devoid of linked Fucose Residues and contain a comparatively greater amount of high mannose structures based on relative peak intensities. MALDI (Panel A; prominent High Mannose structures encircled) whereas N-Glycostructures obtained from a therapeutic IgG1 antibody expressed in unmodified CHO-IgG precursor cells are fucosylated in the nucleus and contain significantly lower amounts of mannose structures. high. (Fucose residues = circled black triangles; Panel B).
[00182] Fig. 5 shows a MALDI-TOF spectrum of desialylated IgG N-glycans produced using (A) CHO WT cells; (B) RMD-CHO clone H1; (C) RMD-CHO clone H2; (D) RMD-CHO clone H3. All molecular ions are present in soda [M+Na+] or potash [M+K+] (black cross) form. The dark gray circle, Man; light circle, Gal; black square, GlcNAc; black triangle, Fuc; white cross, does not contain any carbohydrate material.
[00183] Fig. 6 shows in (A) binding curves of FcgRIIIa-His (Phe158) to WT IgG and afucosylated IgG in the absence of plasma. FcYRIIIa binding was detected by an ELISA using FcYRIIIa-His as a capture reagent, peroxidase-conjugated anti-human IgG antibody as a detection reagent, and Tetramethylbenzidine (TMB) as a chromogenic substrate. Dots indicate mean absorption of TMB-reacted peroxidase at 450 nm, n = 2. Bars indicate SD. Open circles = fucosylated wild-type (WT) IgG, closed circles = non-fucosylated IgG derived from H1 clone (H1); closed squares = non-fucosylated IgG derived from clone H2 (H2); closed triangles pointing up = non-fucosylated IgG derived from clone H3 (H3). The same shows in (B) relative fold increase in binding in FCYRIII of non-fucosylated IgG relative to WT IgG. The relative fold increase in binding in FCYRIII was calculated from EC50 values. WT, wild-type fucosylated IgG; H1, non-fucosylated IgG derived from H1 clone; H2, non-fucosylated IgG derived from H2 clone; H3, non-fucosylated IgG derived from H3 clone.
[00184] Fig. 7 shows the preparation and analysis of NK Cells for ADCC activity assays. (A) Plot of dots showing the purity level of isolated primary NK cells used in ADCC assays. Primary NK cells were isolated from whole blood from a healthy human donor by magnetic bead separation. The isolated NK cells were then analyzed for purity by flow cytometry using antibodies against the NK cell markers CD16 and CD56. (B) Performance of isolated NK cells. In order to determine the cytosolic activity of the isolated NK cells, K562-targeted cells stained with calcein AM were incubated alone, with NK cells or with the cell lysing agent saponin for 4 hours. The mean fluorescence intensity (MFI) released from the incubated target cells indirectly indicates the degree of cell lysis. The MFI observed for NK-mediated cell lysis subtracted from the MFI obtained for spontaneous cell lysis indicates the maximum possible specific MFI to be observed in the ADCC assay. Note that NK-mediated lysis does not completely reach the level of MFI obtained from total lysis.
[00185] Fig. 8 shows the in vitro ADCC activity of non-fucosylated and fucosylated IgG derived from CHO and RMD-CHO. Dots indicate mean specific cell lysis (%) at a given IgG concentration (%), n = 4; bars indicate +/-SD, wild-type IgG (open squares), non-fucosylated IgG derived from clone H1 (closed circles), non-fucosylated IgG derived from clone H2 (closed triangle pointing down), and non-fucosylated IgG derived from clone H3 (closed triangle pointing up). Comparable data were obtained when SK-BR-3 cells were used as target cells (data not shown).
[00186] Fig. 9 shows a HPAEC-PAD profile of monosaccharides hydrolyzed from the cytosolic fraction of (A) unmodified CHO cells and (C) RMD-CHO clone H2. (B), (A) boosted with 10 pmol/μl of L-rhamnose and (D), (C) boosted with 10 pmol/μl of L-rhamnose. As the monosaccharides were not re-N-acetylated after hydrolysis with TFA, GlcNAc was measured as GlcNH2. Under the selected conditions, the L-rhamnose peak elutes at a retention time of approximately 15.5 minutes. Note the absence of the fucose peak from the HPAEC-PAD chromatograms of the modified H2 clone cell lysate.
[00187] Fig. 10 shows a HPAEC-PAD profile of monosaccharides released from IgG N-glycans of (A), WT CHO and (C), the modified RMD-CHO H2 clone, (B), (A) reinforced with 10 pmol/μl of L-rhamnose and (D), (C) reinforced with 10 pmol/μl of L-rhamnose. Since the monosaccharides were not re-N-acetylated after TFA hydrolysis, GlcNAc was measured as GlcNH2. Under the selected conditions, the L-rhamnose peak elutes at a retention time of approximately 15.5 minutes. Note the absence of the fucose peak from the HPAEC-PAD chromatograms of the N-glycan IgG sample derived from RMD-CHO clone H2. EXAMPLES 1. Experimental part 1.1 Materials and Methods 1.1.1 Cell lines
[00188] The recombinant CHO/DG44 cell line CHO-IgG was established earlier in our laboratory by the stable transfection of the dihydrofoliate reductase deficient CHO cell line, CHO/DG44 (Urlaub et al., 1986, Proc Natl Acad Sci USA 83(2):337-341) with an expression vector containing an antibody expression cassette comprising nucleotide sequences encoding light and heavy chains of a therapeutic monoclonal antibody (Trastuzumab (Herceptin®)). Generation of the RMD-CHO-IgG cell line started from the existing CHO-IgG cell line. Both cell lines were maintained in serum-free medium. 1.1.2 Gene Optimization and Synthesis The amino acid sequence for the oxidoreductase Rmd (Pseudomonas aeruginosa PAO1; 304 amino acids) (GenBank Accession No: AAG08839.1) was reverse translated and the resulting nucleotide sequence optimized by site silencing junction and sequence elements that destabilize RNA, optimization for increased RNA stability and adaptation of codon usage to match the requirements of CHO cells (Cricetulus griseus). 1.1.3 Construction of the RMD expression plasmid
[00189] The synthesized RMD construct was cut with EcoRI and Bgl II and dephosphorylated with calf intestinal phosphatase. The digested and dephosphorylated insert was ligated into a pre-digested bicistronic expression vector that allows coordinated co-expression of RMD and green fluorescent protein of a bicistronic message (gfp). The expression plasmid is equipped with a neomycin resistance gene to allow for direct selection of cells that have stably integrated the bicistronic expression cassette. General procedures for constructing expression plasmids are described in Sambrook, J., E.F. Fritsch and T. Maniatis: Cloning I/II/III, A Laboratory Manual New York/Cold Spring Harbor Laboratory Press, 1989, Second Edition. 1.1.4 Conversion of CHO-IgG cells that produce antibody into cells that secrete non-fucosylated antibodies
[00190] CHO-IgG cells stably expressing the therapeutic IgG1-like antibody Trastuzumab were stably transfected with the RMD-gfp transgene by electroporation according to the manufacturer's instructions (MicroPorator, PEQLAB Biotech, Germany). 24 h after electroporation transfectants were selected on alpha-MEM containing the antibiotic G418. G418 resistant clones were then isolated by cloning in limiting dilution, i.e. they were resuspended in this selective medium and seeded in 96-well plates at dilutions where their probability of obtaining a single cell colony is greater than 95% with based on Poisson statistics. To ensure monoclonality, cells grown within the 96 wells were isolated and again seeded in 96 well plates at the limiting dilution. After these two rounds of single cell cloning, a pair of isolated single cell clones were expanded into larger volumes. After that, they were adapted for suspension cultivation. Using the described electroporation protocol a transformation efficiency of approximately 2000 per 2 x 10 6 electroporated cells was obtained as assessed from the fluorescence distribution of gfp in the culture plates (Fig. 2). 1.1.5 Clone Screening by Fluorescence Microscopy
[00191] Single cell clones were seeded into 96-well plates and screened for successful RMD integration by monitoring GFP fluorescence with an Olympus IX-50 (Olympus Optical Co., Europe) fitted with a cmount adapter. For the GFP scan a 200-fold fluorescence filter was used versus phase contrast. The images were edited by the Viewfinder lite application. Additionally, mRNA expression of the RMD transgene was confirmed by RT-PCR analysis. Successful expression of the RMD transgene was confirmed by RT-PCR using an RMD-specific set of primers (Fig. 3). 1.1.6 Production of unmodified and glycoengineered IgG1 by serum-free batch culture in shaker tubes
[00192] In order to compare the glycostructures of antibodies produced by RMD-modified CHO-producing clones (RMD-CHO-IgG) with the glycostructure of IgG1-like antibodies secreted from unmodified CHO antibody-producing cells (CHO-IgG), both the cell lines were used to produce IgG1 antibodies in the culture supernatant. The antibody-producing clones RMD-CHO-IgG and CHO-IgG were inoculated at 2 x 10 5 cells/ml in a fucose-deficient culture medium. The shaker tubes were incubated at 180 rpm, 37°C, 7.5% pCO2. The culture was stopped after 7 days when the cells still showed >80% vitality. Viable cell density was measured with an automatic cell counter, Vi-CELL® XR (Beckman Coulter, Fullerton, CA), which uses trypan blue exclusion. The pattern of decline in viability and duration of the fed batch assay as well as the mean specific productivity (qp) between days 3 and 10 of the fed batch shaker assay remained comparable between the two different clones. The cells were then pelleted by centrifugation for 10' at 5000 rpm and the supernatant transferred to a separate flask. Unmodified RMD-CHO IgG and CHOIgG cells were cultured in the same way. The antibody concentration in the culture supernatants was measured at the Gyrolab Workstation (Gyros AB, Sweden) by an Interleaved Immunoassay specific for human IgG1. Both clones grew logarithmically, produced comparative IgG titers at their respective sampling dates, and had a comparative initial doubling time. Both clones retained the typical morphology of Chinese hamster ovary cells. 1.1.7 Purification of IgG1 by Protein A Affinity Chromatography
[00193] Following sterile filtration on a 0.2 μm filter, the supernatant was loaded onto a protein-A-Sepharose mini column. 0.5 ml of column support material with a total capacity of 10 mg was used. The column was equilibrated with 5 column volumes of 20 mM sodium phosphate, pH 7.0 in gravity flow. After protein binding at a slow flow rate, the column was washed twice with the equilibration buffer. Then the antibody was eluted with 4 column volumes of 0.1 M glycine buffer, pH 3.0 in gravity flow. 1 ml fractions were collected and immediately neutralized with 1 M Tris-HCl, pH 9. Integrity and purity of each purified IgG1 was confirmed by reducing SDS-PAGE analysis. Purity was >90% and the integrity of the eluted antibodies was confirmed by reductive SDS-PAGE analysis. 1.1.8 Preparation of N-linked Antibody Derivative Oligosaccharides
[00194] 100 μg of each antibody was used for mass spectrometric characterization of N-linked oligosaccharides. Proteins were digested with trypsin (0.2 mg/ml, 16 h, 37°C) before the N-Glycans were released by incubation with 1 unit of peptide-N-glycosidase F (PNGaseF, Roche Diagnostics) for 18 h at 37°C. The released N-glycans were recovered by two-step chromatography on RP-(Sep-Pak C18-) columns followed by by the clean columns of carbonic extract (Wuhrer et al., 2006, Glycobiology 16 (2006), pp. 991-1006). 50% of each pool of N-glycan was digested with 10 mU Neuraminidase 18 h at 37°C and 50% was permethylated with methyl iodide as described by Ciucanu and Kerek (Ciucanu and Kerek, 1984, Carbohidr Res. 1984: 131: 209). 1.1.9 Analysis of N-linked Oligosaccharides by MALDI-TOF-MS
[00195] The N-glycans released from each purified IgG1 were analyzed by an UltraFlex III TOF/TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) equipped with a smartbeam-II® laser. Measurements were performed in positive ion mode. Positive ions were subjected to an accelerating voltage of 25 kV and an extraction delay of 10 ns and analyzed in a reflectron mode. For analysis, desialylated glycans were dissolved in H2O and permethylated glycans were dissolved in 70% (v/v) acetonitrile. 0.5 µl of samples were mixed 1:1 (v/v) with Arabinosazone (2 mg/ml) dissolved in 80% EtOH on a target and allowed to dry at room temperature. An external calibration with a dextran ladder was used. Spectra were analyzed using Glyco-peakfinder (Maas et al., 2007, Proteomics 7, 4435-4444). The identified glycan structures were constructed with the GlycoWorkbench software (Ceroni et al., 2008, J. Proteome Res. 7(4) 1650-1659). Oligosaccharide profile analysis of products purified from the final culture medium confirmed that the Fc N-linked oligosaccharides of the products were of the high mannose type as well as of the two-antenna type complex, and that the IgG1 product secreted from the RMD clone -CHO-IgG totally lacked the core fucosylation. Glycostructure analysis showed that the ratio of non-fucosylated oligosaccharides of antibodies produced by clones co-expressing RMD increased significantly in each case (i.e., up to 99%, 95%, 97%, and 98%), compared with those of CHO clones. -IgG precursors (Fig. 4). N-Glycostructures obtained from IgG1 antibody product expressed in RMD-CHO-IgG cells are almost completely free of bound fucose residues and contain a comparatively greater amount of high mannose-like structures based on the relative peak intensities of MALDI (Fig. 4, Panel A; prominent high mannose-like structures encircled), whereas N-Glycostructures obtained from a therapeutic IgG1 antibody expressed in unmodified CHO-IgG cells are fucosylated in the nucleus and contain significantly lower amounts of structures. high mannose type (Fucose residues = circled triangles; Fig. 4, Panel B). 2. Experimental part 2.2 Materials and Methods 2.2.1 Cell lines, Gene optimization and synthesis, Construction of the RMD expression plasmid, Conversion of CHO-IgG cells that produce antibody into cells that secrete non-fucosylated antibodies, Clone screening by fluorescence microscopy and RT-PCR
[00196] As for the CHO-IgG cell line used so as to produce the RMD-CHO-IgG cell line, RMD gene synthesis and optimization, RMD expression plasmid construction, CHO cell conversion -IgG producing antibody in cells that secrete non-fucosylated antibodies, clone screening by fluorescence microscopy for successful RMD integration and RT-PCR for successful mRNA expression of RMD transgene, items 1.1 are alluded to .1 to 1.1.5 as mentioned above (1. Experimental part). 2.2.2 Fed batch culture
[00197] IgG was produced using both CHO and RMD-CHO cells (clones H1, H2 and H3) in order to compare their N-glycan structures. Cells were seeded at 4x10 5 cells/ml in 500 ml shake flasks in 100 ml of serum free medium (formulation adapted by ProBioGen; SAFC Bioscience, Lenexa, KA) supplemented with 4 mM glutamine but no antibiotics or MTX. Cultures were shaken at 180 rpm at 37°C and 7.5% CO 2 . Cells were fed 1.75 ml of PBG-Feed Mix per 100 ml of culture volume on day 4 of culture. Cell density and viability were determined by trypan blue exclusion using a ViCell cell quantitation system. automated (Beckman Coulter, Brea, CA). Aliquots of cell culture supernatant were collected on days 3, 5 and 7 in order to determine IgG concentrations, which were measured by the Gyrolab interleaved immunoassay. The culture was stopped after 7 days when the cells still showed vitality higher than 80%. Cell culture supernatant was collected and sterile filtered. 2.2.3 IgG1 Specific Gyrolab Interleaved Immunoassay
[00198] IgG1 concentration was determined by an intercalated immunoassay performed on a Gyrolab Workstation (Gyros AB, Uppsala, Sweden). The assay included the sequential addition of biotinylated capture antibody, which recognizes epitopes on the Fc part of IgG, samples of cell culture supernatant or human polyclonal IgG reference standard, and a detection antibody labeled with Alexa Fluor 647, which binds epitopes. in the Fab domain of IgG. Samples and standards were measured in triplicate. The mean OD, standard deviation (SD), and % coefficient of variation (% CV) were automatically calculated by the Gyrolab evaluator software after each round. The described IgG1 Gyrolab intercalated immunoassay was pre-validated based on the premise that the residues for each calibration standard meet an acceptance limit of 20% relative error (RE). 2.2.4 Purification of IgG using protein A affinity chromatography
[00199] The cell culture supernatant was loaded onto a 0.5 ml Protein A-Sepharose column, pre-equilibrated with 20 mM sodium phosphate, pH 7.0. After washing the column with two bed volumes of equilibration buffer, the antibody was eluted with 4 column volumes of 0.1 M glycine pH 3.0. Fractions were collected and immediately neutralized with 1 M Tris-HCl, pH 9. The integrity and purity of each purified IgG was confirmed by reducing SDS-PAGE analysis. The protein concentration of the purified IgG was determined by the Gyrolab intercalated immunoassay. 2.2.5 Processing of IgG N-glycans
[00200] IgGs (100 μg) were digested with trypsin for 16 h at 37° C. The reaction was terminated by heating the sample for 5 min at 95° C. The antibodies were further digested with 1 U of PNGase F for 18 at 37°C. The released N-glycans were isolated and desalted in reversed-phase Sep-Pak C18 cartridges followed by clean columns of carbonic extract. Each pool of N-glycan was digested with 10 mU of Neuraminidase for 18 h at 37° C. 2.2.6 Mass Spectrometry
[00201] N-glycans were analyzed in an UltraFlex III TOF/TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) equipped with a smartbeam-II® laser and a LIFT-MS/MS facility. Spectra were recorded in a reflector mode at an acceleration voltage of 25 kV and an extraction delay of 10 ns. Measurements were performed in positive ion mode. External calibration was performed using a dextran ladder. The desialylated N-glycans were dissolved in H2O. 0.5 µl samples were mixed 1:1 (v/v) with D-arabinosazone (5 mg/ml) dissolved in 70% aqueous ethanol on a steel target (Chen et al. 1997). Spectra were analyzed using Glyco-Peakfinder [Maas et al., 2007]. The identified glycan structures were constructed using the GlycoWorkbench software [Ceroni et al., 2008]. 2.2.7 Fc-gamma IIIA receptor-specific binding assay (FCYRIIIA)
[00202] The FCYRIIIA binding activity of the IgG samples was analyzed by a specific FcyRIIIA binding assay as described pr Niwa et al., 2004 with slight modifications. A histidine (HIS)-labeled FcyRIIIA (F158) (22 kDa; 158F; R&D Systems, Minneapolis, MN) was used in combination with an anti-tetraHIS monoclonal antibody (Qiagen, Hilden, Germany) for receptor binding. Immunoplates (Maxisorp, Thermo, Waltham, MA) were coated with anti-tetraHIS antibody and blocked with blocking reagent (Roche Diagnostics, Penzberg, Germany). Subsequently, HIS-labeled recombinant FcyRIIIA was added to the immunoplates. The coated plates were then incubated with serial sample dilutions and controls so that they could bind the immobilized FcyRIIIA receptor. After a washing step, bound IgGs were detected by an anti-human IgG peroxidase-conjugated mAb (Dianova, Hamburg, Germany) and the amount of bound IgG was quantified by means of peroxidase activity. After each incubation step the immunoplate was washed 3 times with PBS containing 0.2% Tween-20. Tetramethylbenzidine (TMB; Seramun, Heidesee, Germany) was used as a chromogenic substrate, the reaction was terminated using 1M sulfuric acid and finally, the absorption was detected at 450 nm (Infinite Reader F200, Tecan, Crailsheim, Germany). Based on the concentration-dependent absorption data, full curve fits were conducted using a 4-parameter logistic curve model (Magellan Software 6.1, Tecan). The intraserial precision for this FcyRIIIA binding assay was determined to be within 15% CV. 2.2.8 Antibody-dependent cellular cytotoxicity (ADCC) assay
[00203] Primary human NK cells were isolated from peripheral blood mononuclear cells (PBMCs). PBMCs were separated from whole blood from healthy human donors by density gradient centrifugation and NK cells were subsequently isolated by negative magnetic bead separation (Miltenyi, Bergisch Gladbach, Germany). The purity of the isolated NK cells was confirmed by flow cytometry (PE-conjugated CD16 and Alexa488-conjugated CD56 antibodies, BD, San Jose, CA). The BT-474 cell lines (Lasfargues et al., 1978, invasive ductal carcinoma of the breast, human, CLS, Eppelheim, Germany) and SK-BR-3 (Zabrecky et al., 1991, adenocarcinoma of the breast, human , ATCC, Manassas, VA) were used as target cells. Both cell lines were confirmed positive for the Her2/neu marker by flow cytometry (data not shown). Target cell lines were revitalized from a research cell bank 3 days prior to inoculation. Antibody-dependent NK cell-induced target cell lysis was quantified by the release of a vital strain (Calcein AM, Life Technologies, Carlsbad, CA). Target cells were stained according to the manufacturers protocol and seeded at 2 x 10 4 viable cells/wells in 50 μl of RPMI1640 (Life Technologies) + 10% FCS (Biochrom, Berlin, Germany) in 96-well microtiter plates . Serial 1:3 dilutions of antibodies in RPMI1640 + 10% FCS were prepared. 50 μL/well of each dilution was pipetted with n = 3 and pre-incubated with the target cells for 30 min at 37°C prior to NK cell inoculation. At the end of the antibody pre-incubation, effector cells were seeded at an effector to target cell (E:T) ratio of 5:1. The plates were subsequently centrifuged at 200 g for 3 min and incubated for 4 h at 37°C and 5% CO2. For each cell line, a spontaneous target cell lysis control (NK cells w/o), an antibody-independent target cell lysis control (NK cells w/), and a total target cell lysis control (induced by saponin) were induced. Total lysis in the control wells was induced by adding 15 μl/well 0.1 mg/mL saponin in RPMI1640 + 10% FCS 15 min before the end of the incubation period. In all other reservoirs, 15 μl of RPMI1640 + 10 % FCS was added. Calcein AM release was quantified by fluorescence detection in the culture supernatant. The plates were centrifuged (150 g; 3 min) and 100 μl of supernatant from each well was transferred to a 96-well black fluorescence plate (Thermo). Mean fluorescence intensity (MFI) was detected using an Infinite F200 reader (Tecan, 485/535 nm excitation/emission filter). Curve fitting was done using a 4-parameter logistic dose response model (Magellan software version 6.1). Specific cell lysis was calculated as follows: Specific cell lysis [%] = [MFI (sample) - MFI (spontaneous)] / [MFI (total) - MFI (spontaneous)] x 100. Whereas MFI (sample ) is the average fluorescence intensity released by specific target cell lysis, MFI (spontaneous) is the gradual release of the fluorescent dye by the target cells, and MFI (total) is the average fluorescence intensity obtained after total target cell lysis induced by detergent. 2.2.9 Monosaccharide analysis and rhamnose determination
[00204] Cells (3 x 10 7 ) were separated from the supernatant by centrifugation at 100 g for 5 min. These were subsequently lysed by 3 cycles of thawing-freezing. Cell membranes were then separated from cytosolic fractions at 21,000 g for 30 min at 4°C. Cell culture supernatant, cell membranes, cytosolic fractions as well as IgG N-glycans were hydrolyzed in 2N TFA for 4 h. 100°C. After evaporation under reduced atmosphere, samples were analyzed by HPAEC-PAD on a PA-1 column using a Dionex ICS-3000 and 2-deoxyribose was used as the internal standard. Neutral monosaccharides were separated by isocratic elution with 2.25 mM NaOH. Post column addition of 200 mM NaOH allowed amperometric detection. Since HPAEC-PAD does not allow for enantiomeric identity designation (Horton, D. 2004), L-rhamnose was used as a standard to determine the expected retention time for D-rhamnose. The LOD for rhamnose was determined as described in Chapter 6.3 of the ICH Tripartite Harmonized Guidelines for the Validation of Analytical Procedures (ICHQ2(R1)). 2.3 Results 2.3.1 Heterologous expression of the RMD transgene
[00205] To evaluate the effects of RMD transgene expression on fucosylation levels of secreted IgG, a vector equipped with a bicistronic expression cassette comprising the genes for GDP-6-deoxy-D-junk-4-hexulose reductase (RMD) and green fluorescent protein (gfp) was generated and introduced into a CHO/DG44 clone that was previously engineered for overexpression and secretion of a biosimilar version of the therapeutic IgG1 antibody Trastuzumab (Herceptin®, Roche). G418 resistant clones expressing the transgene were identified by their gfp-mediated green fluorescence and appeared within 2 weeks of transfection. A transformation efficiency of approximately 80% was obtained by electroporation as judged from the gfp fluorescence distribution (data not shown but comparable with data shown in Fig. 2). Successful expression of the RMD transgene was confirmed by RT-PCR using a specific RMD set of primers (data not shown but comparable to data shown in Fig. 3). Modified CHO cells that express the RMD transgene are called RMD-CHO. 2.3.2 Serum-free fed batch culture of antibody-producing CHO and RMD-CHO cells
[00206] Serum-free fed batch culture of the unmodified precursor CHO cells and the transfected RMD-CHO cell producing IgG was performed in 50 ml bioreactor tubes containing a fucose-deficient growth medium supplemented with L-glutamine. Bioreactor tubes were inoculated at a starting cell density of 4 X10 5 cells/ml and then incubated at 180 rpm, 37°C, 7.5% pCO2. The performance of the fed batch cultures was monitored for 14 days and compared side by side. Comparative analysis of parallel fed batch cultures of CHO and RMD-CHO cells showed no significant deviation over a 14 day course. initial doubling rates, proliferation rates and IgG titers at the respective sampling dates were congruent for both cell lines. Both clones retained the typical CHO cell morphology. The pattern of viability decline over the duration of the fed batch assay as well as the mean specific productivity (qp) between days 3 and 10 of the fed batch shaker assay remained comparable for the two different clones (data not shown). 2.3.3 N-glycan analysis of IgG produced from CHO and RMD-CHO cells
[00207] CHO and RMD-CHO cells were grown in a batch culture. Three different RMD-CHO clones that produce IgG were used, namely H1, H2 and H3. Cells were inoculated at a starting cell density of 4 x 10 5 cells/ml and cultured for 7 days in bioreactor tubes at 180 rpm, 37°C, 7.5% pCO2. Supernatants were harvested on day 7 and IgG samples were purified by protein A affinity chromatography. The purity and integrity of the eluted antibodies were confirmed by reducing SDS-PAGE. The N-glycans, released using PNGase F, were desialylated and subsequently analyzed by MALDI-TOF-MS. Relative quantification of N-glycan signal intensities was performed as demonstrated earlier to give reliable results when compared with chromatographic methods (Wada et al., 2007). N-glycan structures of the high mannose type as well as the double antenna complex type were detected in all samples (Fig. 5). Agalactosylated/monogalactosylated/digalactosylated monofucosylated double-antenna N-glycans were the three most abundant N-glycan structures found in wild-type (WT) IgG (Fig. 5 A). The presence of core fucose at those peaks, namely at m/z 1485.4, 1647.4 and 1809.4, was confirmed by MALDI-TOF/TOF. A diagnostic ion fragment dHex1HexNAc2 was observed in each spectrum at m/z 592.8. In the IgG samples that were produced from RMD-CHO cells, only trace amounts of fucose were observed (Fig. 5 B, C, D) representing a maximum of 2% of the total N-glycan pool (sample H2). Simultaneously, the H2 sample contained a greater amount of high mannose-like structures than wild-type IgG. 2.3.4 Binding activity of IgG1-Fc of IgG produced from CHO and RMD-CHO cells
[00208] Since the comparatively weak interaction (K ~1 μM) between IgG1 and its cognate Fc receptor FcYRIIIa is one of the main factors contributing to ADCC effector function (Sondermann et al., 2000), a binding assay of FcYRIIIa is an indirect measure to predict the ADCC activity of IgG1 monoclonal antibody samples. FcYRIIIa binding of non-fucosylated IgG secreted from RMD-CHO cells was enormously increased with equivalent binding of ~16-fold less protein to FcYRIIIa-158F when compared to fucosylated IgG secreted from wild-type CHO cells ( Fig. 6A , B). Data are reported in Fig. 6B as relative fold increase in binding of non-fucosylated IgG using fucosylated IgG as a reference. 2.3.5 ADCC activity of non-fucosylated IgG produced using RMD-CHO cells
[00209] To analyze ADCC activity, isolated NK cells and target cells expressing HER2 were co-incubated with serial dilutions of fucosylated and fucosylated IgG. As a prerequisite for the assay, NK cells were isolated from whole blood samples at a purity level of 73% (Fig. 7). The NK cell cytotoxicity control technique showed a specific lysis activity of 77% for the donor material used for the ADCC assay (Fig. 7). The target cell line expressing HER2 BT-474 (isolated from a human, ductal invasive breast carcinoma) was used in the ADCC assay. The BT-474 target cell line is also attacked by NK cells by mechanisms other than ADCC. BT-474 cells show a mean value of 16% antibody-independent cell lysis (data not shown). Data for the specific cell lysis induced by the IgG samples is demonstrated in Fig. 8. All three non-fucosylated IgG samples (H1-H3) induced an increased ADCC response compared to the WT antibody (Fig. 8). The H2 non-fucosylated IgG sample induced the highest ADCC response (Fig. 8). Similar results were obtained when SK-BR-3 (HER2-positive breast adenocarcinoma) cells were used as target cells in the assay. The calculated EC50 values for fucosylated and fucosylated IgG samples incubated with BT-474 and SK-BR-3 cells are summarized in Table 2 (see below). The observed change in EC50 between wild-type IgG and variants H1 to H3 remained comparable irrespective of the target cell line used in the ADCC assay (Fig. 8). In the presence of HER2 positive BT474 and SK-BR-3 target cell lines and purified NK cells, non-fucosylated IgG showed an average 16-fold antibody-mediated target cell depletion activity with mean EC50 values of 0.443 μg/ml and 0.00817 μg/ml which indicated much higher efficacy compared to fucosylated IgG. It should be mentioned that non-fucosylated IgG samples that showed increased FcYRIIIa binding activity also induced a higher ADCC response compared to WT IgG.
[00210] Table 2: Summary results of ADCC effector function of fucosylated (H1-3) and fucosylated (WT) IgG incubated with target cells that present different antigen (BT-474, SK-BR-3). The calculated ratio of EC50 (fucosylated) / EC50 (fucosylated) indicates enhanced ADCC effector function.
2.3.6 Monosaccharide Analysis - Absence of detectable amounts of D-rhamnose in cell lysates as well as in IgG N-glycans
[00211] Cell membranes, culture supernatants and cytosolic fractions were isolated from RMD-CHO cells. The monosaccharides were subsequently released and analyzed by the HPAEC-PAD. As expected, fucose was present in CHO cells and not in RMD-CHO cells. A rhamnose level exceeding the limit of detection (LOD) was not observed in the cytosolic fractions (Fig. 9). Based on the standard deviation of the y-intercept and slope of the calibration curves, the LODs were 1.2 pmol and 1.1 pmol per 10 μl sample injection volume for rhamnose and fucose, respectively. Similarly, N-glycans, released from purified antibodies produced using RMD-CHO cells, were hydrolyzed with TFA and the resulting monosaccharides were analyzed by HPAEC-PAD. TFA hydrolyzed N-glycans did not produce any rhamnose peaks that exceeded the LOD (Fig. 10). In conclusion, neither RMD-CHO cells nor secreted antibodies contain detectable amounts of rhamnose. 2.4 Final Comments
[00212] We evaluated a glycoengineering method to obtain the secretion of fucose-deficient mAbs from cell lines that were modified for continuous removal of a key metabolic intermediate from the de novo synthesis pathway of cytosolic fucose (Fig. 1) . Our data clearly show that a transgenic expression of a heterologous bacterial enzyme leads to the desired block in fucose synthesis in nascent glycoprotein N-glycans. The degree of fucose depletion by this method also indicates that the omission of fucose from the culture medium was sufficient to completely block the recovery pathway, which may otherwise have served as an alternative source of cytosolic GDP-fucose.
[00213] The key metabolic target in our method was GDP-4-keto-6-deoxy-D-mannose, which is a common intermediate for the synthesis of several different GDP-monodeoxyhexoses which include GDP-L-fucose, GDP-4 - deoxy-D-thalose, GDP-colitosis, and GDP-D-perosamine, for example, in bacteria. Furthermore, a Cys109Ser active site mutant of GDP-fucose synthase produces GDP-6-deoxy-D-altrose instead of GDP-fucose (Lau and Tanner, 2008).
[00214] The specific prokaryotic enzyme that we exemplarily selected for our method was GDP-6-deoxy-D-junk-4-hexulose reductase (synonymous with GDP-4-keto-6-deoxy-D-mannose reductase, abbreviated RMD ) (Kneidinger et al., 2001; Maki et al., 2002). We demonstrate here that heterologous expression of the prokaryotic enzyme GDP-6-deoxy-D-junk-4-hexulose reductase (RMD) within the cytosol can efficiently divert the fucose de novo pathway. Said enzyme uses NADH and NADPH as hydrogen donors and catalyzes the targeted reduction of the 4-keto group of the GDP-4-keto-6-deoxy-D-mannose intermediate of the fucose pathway to produce GDP-D-rhamnose. The conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose by RMD appears to progress quantitatively, because no reverse reaction was detected (Kneidinger et al., 2001).
[00215] Our data also show that GDP-rhamnose, a 6-deoxyhexose found only in glycoconjugates of certain bacteria but not in animals (Webb et al., 2004), is a product that will never produce positive results within the context of the cytosol of vertebrate. Unmodified vertebrate cells lack rhamnosyltransferases (Webb et al., 2004) and membrane transporters so that incorporation of GDP-D-rhamnose into nascent glycans is very unlikely within vertebrate cells. Our data could confirm this. They clearly show that artificial D-rhamnose was not incorporated into the secreted IgG or elsewhere in the cell (Fig. 9 and Fig. 10).
[00216] In addition to the lack of fucose in the glycan structures, the IgGs secreted from the genetically engineered clones showed a higher level of high mannose-like structures. This may arise from a feedback inhibition of GMD by GDP-D-rhamnose. The lack of GMD activity as an alternative metabolic outlet for GDP-mannose may have contributed to an elevated uptake of cytosolic GDP-D-mannose which in turn may have caused the elevation of high-mannose-like structures seen in products secreted from cells. RMD-modified hosts (Fig. 4 and Fig. 5).
[00217] Our results for the FCYRIII binding assay as well as the in vitro ADCC assay also show that non-fucosylated IgG has significantly higher binding activity for FcYRIIIa and an enhanced ADCC response. The observed EC50 change and the fold increase in target cell depletion activity (Table 2, Fig. 8) would have been even higher if the assay had been conducted in the presence of whole blood, serum or plasma. Interestingly, we observed that the H2 non-fucosylated IgG sample with the highest level of high mannose structures also showed the highest ADCC activity, regardless of the target cell used in the assay (BT474 or SK-BR-3) (Table 2).
[00218] When put together, this study demonstrates that overexpression of RMD in vertebrate host cells causes a depletion of a key intermediate important for the synthesis of cytosolic GDP-fucose, GDP-4-keto-6-deoxy-D-mannose. This method allows for the generation of metabolically engineered cell lines but also offers a highly promising new strategy to convert existing antibody-expressing cell clones in producer cells to fucose-depleted therapeutics. The ability to safely engender fucose deficiency in existing cell lines could help accelerate drug development for the next generation of monoclonal antibodies. 3. Abbreviations
[00219] ACN, Acetonitrile; CHO, Chinese Hamster Ovary; CV, coefficient of variation; dHex, deoxyhexose; EC50, half maximal effective concentration (ie the concentration of agonist that causes a response halfway between baseline and maximal response), Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; GMER, GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase; HPLC, high performance liquid chromatography; HPAEC-PAD, high pH anion exchange chromatography with pulsed amperometric detection; LOD Limit of Detection; mAb, monoclonal antibody; MALDI-TOF-MS array-assisted laser desorption ionization time-of-flight mass spectrometry; MEM, Modified Eagle's Medium; PBMCs, peripheral blood mononuclear cells; PBS, phosphate buffered saline; PNGase F, peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase F; Rha, rhamnose; RMD, GDP-6-deoxy-D-junk-4-hexulose reductase; SDS, sodium dodecyl sulfate; TFA, Trifluoroacetic acid. REFERENCES Ceroni A, Maass K, Geyer H, Geyer R, Dell A, Haslam SM 2008. GlycoWorkbench: A Tool for the Computer-Assisted Annotation of Mass Spectra of Glycans, Journal of Proteome Research, 7(4), 1650-1659. Chen, P, Baker AG, Novotny MV. 1997. The use of osazones as matrices for the matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates. Anal Biochem. 244(1): 144-51. Horton, D 2004 Advances in Carbohydrate Chemistry and Biochemistry. D. Horton, Editor, Elsevier Academic Press, Amsterdam and San Diego, Vol. 59, p.11. ICH Q2(R1): ICH Harmonized Tripartite Guideline: Validation of Analytical Procedures: Text and Methodology Q2(R1) Current Step 4 version, Parent Guideline dated 27 October 1994. Kneidinger B, Graninger M, Adam G, Puchberger M, Kosma P, Zayni S, Messner P. 2001. Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductases synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91T. J Biol Chem. Feb 23;276(8): 5577-83. Lasfargues EY, Coutinho WG and Redfield ES. 1978. Isolation of two human tumor epithelial cells from solid breast carcinomas. J Natl Cancer Inst.; 61(4): 967-78. Lau S.T.B., Tanner, M.E. 2008. Mechanism and Active Site Residues of GDP-Fucose Synthase, Journal of the American Chemical Society, vol. 130, no. 51, pp. 17593-17602 Maki M, Jarvinen N, Rabina J, Roos C, Maaheimo H, Renkonen R; Pirkko; Mattila. 2002. Functional expression of Pseudomonas aeruginosa GDP-4-keto-6-deoxy-D-mannose reductase which synthesizes GDP-rhamnose. Eur J Biochem. Jan;269(2): 593-601. Niwa R, Shoji-Hosaka E, Sakurada M, Shinkawa T, Uchida K, Nakamura K, Matsushima K, Ueda R, Hanai N, Shitara K.2004. Defucosylated anti-CC chemokine receptor 4 IgG1 with enhanced antibodydependent cellular cytotoxicity shows potent therapeutic activity to T cell leukemia and lymphoma. Cancer Res, 64: 2127-2133. Niwa R, Hatanaka S, Shoji-Hosaka E, Sakurada M, Kobayashi Y, Uehara A, Yokoi H, Nakamura K, Shitara K. 2004. Enhancement of the antibody-dependent cellular cytotoxicity of low-fucose IgG1 is independent of FcgammaRIIIa functional polymorphism . Clin Cancer Res, 10: 6248-6255. Shields RL, Lai J, Keck R, O'Connell LY, Hong K, Meng YG, Weikert SH, Presta LG. 2002. Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcgammaRIII and antibody-dependent cellular toxicity. J Biol Chem, 277: 26733-26740. Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. 2000. The 3.2-A crystal structure of the human IgG1 Fc fragment-Fc gammaRIII complex. Nature 406, 267-273. Urlaub G, Kas E, Carothers AM, Chasin LA. Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells. Cell. 1983 Jun;33(2): 405-12. Urlaub G, Mitchell PJ, Kas E, Chasin LA, Funanage VL, Myoda TT, Hamlin J. 1986. Effect of gamma rays at the dihydrofolate reductase locus: Deletions and inversions. Somatic Cell Mol Genet, 12: 555556. Wada, Y.; Azadi, P.; Costello, C.E.; Dell, A.; Dwek, R.A.; Geyer, H.; Geyer, R.; Kakehi, K.; Karlsson, N.G.; Kato, K.; Kawasaki, N.; Khoo, K.H.; Kim, S.; Kondo, A.; Lattova, E.; Mechref, Y.; Miyoshi, E.; Nakamura, K.; Narimatsu, H.; Novotny, M.V.; Packer, N.H.; Perreault, H.; Peter-Katalinic, J.; Pohlentz, G.; Reinhold, V.N.; Rudd, P.M.; Suzuki, A.; Taniguchi, N. 2007. Comparison of the methods for profiling glycoprotein glycans--HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study. Glycobiology; 17(4): 411-22. Webb NA, Mulichak AM, Lam JS, Rocchetta HL, Garavito RM. 2004. Crystal structure of a tetrameric GDP-D-mannose 4,6-dehydratase from a bacterial GDP-D-rhamnose biosynthetic pathway. Protein Sci. Feb;13(2): 529-39. Zabrecky JR, Lam T, McKenzie SJ and Carney W. 1991. The extracellular domain of p185/neu is released from the surface of human breast carcinoma cells, SK-BR-3. J Biol Chem.; 266(3): 1716-20.

权利要求:
Claims (11)
[0001]
1. Method for producing a molecule that naturally comprises fucose in its glycoportions, lacking fucose or with a reduced amount of fucose in its glycoportions, characterized in that it comprises the steps of: i) providing a vertebrate cell comprising at least one enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate, wherein the at least one enzyme is selected from the group consisting of GDP-6-deoxy-D-junk-4-hexulose reductase (RMD), GDP-perosamine synthetase (Per), GDP-6-deoxy-D-thalose synthetase (GTS), GDP-Fucose synthetase Cys109Ser- mutant (GFS-Cys109Ser), and GDP-4-keto-6-deoxymannose -3-dehydratase (ColD), preferably GDP-4-keto-6-deoxymannose-3-dehydratase (ColD) in combination with GDP-L-cholitose synthase (ColC), and ii) isolating the molecule that is capable of being a substrate for a fucosyltransferase, preferably a protein or lipid, of the cell in i).
[0002]
2. Method according to claim 1, characterized in that GDP-6-deoxy-D-junk-4-hexulose reductase (RMD) is from Pseudomonas aeruginosa (SEQ ID NO: 1).
[0003]
3. Method according to claim 1 or 2, characterized in that the cell comprises GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-thalose, GDP-6-deoxy-D-altrose, GDP-4-keto-3,6-dideoxy-D-mannose, and/or GDP-L-colitosis.
[0004]
4. Vertebrate method according to claims 1 to 3, characterized in that the cell further comprises at least one molecule that is capable of being a substrate for a fucosyltransferase.
[0005]
5. Method according to claim 4, characterized in that the molecule is a protein or a lipid.
[0006]
6. Method according to claim 5, characterized in that the protein is an endogenous or an exogenous protein.
[0007]
Method according to claims 5 or 6, characterized in that the protein is an antibody, preferably an IgG1 antibody, an antibody fragment, preferably an antibody fragment comprising the Fc region of an antibody, a protein of fusion, preferably a fusion protein comprising the Fc region of an antibody, a viral protein, viral protein fragment, an antigen, or a hormone.
[0008]
Method according to claims 1 to 7, characterized in that the vertebrate cell is a mammalian cell, a fish cell, an amphibian cell, a reptile cell or an avian cell.
[0009]
9. Method according to claim 8, characterized in that i) the mammalian cell is a human, hamster, canine or monkey cell, preferably a Chinese hamster ovary (CHO) cell, a kidney cell of African green monkey (VERO-76) (ATCC CRL-1587), a human cervical carcinoma (HELA) cell (ATCC CCL 2), a Madin Darbin canine renal cell (MDCK) (ATCC CCL 34), a PER cell human .C6, or a human AGE1.HN cell; ii) the fish cell is an ovarian cell (OCC) of Ictalurus punctatus (channel catfish) (ATCC CRL-2772); iii) the amphibian cell is a renal cell of Xenopus laevis (ATCC CCL-102); iv) the reptile cell is an Iguana iguana cardiac cell (IgH-2) (ATCC CCL-108); or v) the avian cell is an avian retinal cell, preferably an AGE1.CR.PIX cell, or an avian somite cell.
[0010]
10. Method for producing a protein that normally comprises fucose in its glycoportions, lacking fucose or with a reduced amount of fucose in its glycoportions, characterized in that it comprises the steps of: i) providing an insect cell comprising: a) a first polynucleotide comprising a nucleic acid sequence encoding an enzyme that uses GDP-6-deoxy-D-junk-4-hexulose as a substrate, wherein the enzyme is selected from the group consisting of GDP-6-deoxy -D-garbage-4-hexulose reductase (RMD), GDP-perosamine synthetase (Per), GDP-6-deoxy-D-thalose synthetase (GTS), GDP-Fucose synthetase Cys109Ser- mutant (GFS-Cys109Ser), and GDP-4-keto-6-deoxymannose-3-dehydratase (ColD), preferably GDP-4-keto-6-deoxymannose-3-dehydratase (ColD) in combination with GDP-L-cholitose synthase (ColC), and b) a second polynucleotide comprising a nucleic acid sequence encoding a protein, ii) expressing the enzyme encoded by the first the polynucleotide and the protein encoded by the second polynucleotide in said cell, and iii) isolating the protein from said cell.
[0011]
Method according to claim 10, characterized in that the protein is an antibody, preferably an IgG1 antibody, an antibody fragment, preferably an antibody fragment comprising the Fc region of an antibody, a fusion protein, preferably a fusion protein comprising the Fc region of an antibody, a viral protein, viral protein fragment, an antigen, or a hormone.

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法律状态:
2018-07-03| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2020-04-28| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-05-12| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-10-06| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-03-09| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-06-01| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-09-21| B25A| Requested transfer of rights approved|Owner name: PROBIOGEN AG (DE) |

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2021-10-13| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-01-04| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 21/09/2010, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

优先权:

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申请号 | 申请日 | 专利标题

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US24462409P| true| 2009-09-22|2009-09-22|

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US61/244624|2009-09-22|

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PCT/EP2010/005772|WO2011035884A1|2009-09-22|2010-09-21|Process for producing molecules containing specialized glycan structures|

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